Ctfa_microbiology_guidelines.pdf

CTFA Microbiology Guidelines

CTFA technical guidelines

CTFA Microbiology Guidelines

CTFA

Cosmetic, Toiletry, and Fragrance Association

Phone: 202/331-1770

280416 Cover.indd 1

Fax: 202/331-1969

www.ctfa.org

2007

The Cosmetic, Toiletry, and Fragrance Association 1101 17th Street, N.W., Suite 300 Washington, D.C. 20036

CTFA

Cosmetic, Toiletry, and Fragrance Association

3/6/08 9:31:54 PM

CTFA Microbiology Guidelines 2007

EDITORS John F. Krowka, Ph.D. John E. Bailey, Ph.D.

PRODUCTION Natasha Clover

PUBLISHED BY The Cosmetic, Toiletry, and Fragrance Association 1101 17th Street, N.W., Suite 300 Washington, D.C. 20036 Phone: 202/331-1770 Fax: 202/331-1969 www.ctfa.org

Copyright © 2007 The Cosmetic, Toiletry, and Fragrance Association No portion of the CTFA Microbiology Guidelines may be reproduced in whole or in part, in any form or by any electronic or mechanical means, including information exchange and retrieval systems (except for the purpose of official, nonpublic use by the United States Government), without prior written permission from The Cosmetic, Toiletry, and Fragrance Association, Inc., 1101 17th Street, N.W., Suite 300, Washington, DC 20036-4702. Printed in the United States of America

Table of Contents (Bold are new in 2007)

Acknowledgements.......................................................................................................v Foreword ....................................................................................................................vii Introduction .................................................................................................................1 1.

Microbiological Quality Assurance for the Cosmetic Industry ...............................3

2.

Microbiological Evaluation of the Plant Environment* .......................................11

3.

Cleaning and Sanitization ...................................................................................29

4.

Microbiology Staff Training* ...............................................................................55

5.

Handling, Storage and Analysis of Raw Materials ................................................69

6.

Microbiological Sampling....................................................................................73

7.

Microbiological Quality for Process Water...........................................................81

8.

Microbiology Laboratory Audit* .........................................................................95

9.

Microbial Validation and Documentation .........................................................109

10. Maintenance and Preservation of Test Organisms ..............................................129 11. Raw Material Microbial Content.......................................................................139 12. Establishing Microbial Quality of Cosmetic Products* ......................................143

* These guidelines and methods were newly written or substantively revised for the 2005 CTFA Microbiology Guidelines.

Table of Contents continued (Bold are new in 2007)

13. Determination of Preservative Adequacy in Cosmetic Formulations ..................149 14. Preservation Testing of Eye Area Cosmetics .......................................................151 15. Microbiological Assessment of Product Quality After Use*................................155 16. Microbiological Risk Factor Assessment of Atypical Cosmetic Products* ...........163 17. Determination of Preservation Efficacy in Nonwoven Substrate Products .. 173 18. M-1 Determination of the Microbial Content of Cosmetic Products ................179 19. M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa ..........................................................................................................183 20. M-3 A Method for Preservation Testing of Water-Miscible Personal Care Products** .........................................................................................................189 21. M-4 Method for Preservation Testing of Eye Area Cosmetics ............................197 22. M-5 Methods for Preservation Testing of Nonwoven Substrate Personal Care Products* ..........................................................................................................207 23. M-6 A Method for Preservation Testing of Atypical Personal Care Products* ............................................................................................ 217 24. M-7 A Rapid Method for Preservation Testing of Water-Miscible Personal Care Products.............................................................................................. 229 25. Glossary of Microbiological Terms ....................................................................237

* These guidelines and methods were newly written or substantively revised for the 2005 CTFA Microbiology Guidelines. ** Method M-3 underwent substantive revisions and review for the 2007 CTFA Microbiology Guidelines.

Acknowledgements

The Guidelines presented in this volume were developed by the CTFA Microbiology Committee, with assistance from many members of the CTFA Scientific Advisory Committee. As the development and updating effort has been a continuing one, listing all of the experts involved from CTFA member companies would be beyond the capabilities of the current editors. Therefore, to all who had a part, a very warm and sincere thank you. The editors also would like to thank Michelle Duelley at CTFA and Don English at Avon for their assistance.

Foreword

In 1969, CTFA began publishing its Technical Guidelines in the CTFA Cosmetic Journal. These guidelines were developed by the newly organized CTFA Microbiology Committee and were concerned with microbiological issues. The benefits of having the Guidelines available in a single volume, and presented in a standardized format, were recognized, and in 1974, the first independent compilation of the Technical Guidelines was published. In 1993, after several major revisions and additions to the Guidelines, CTFA responded to requests made by the users and split the Guidelines into separate volumes so that individuals might purchase sets relating specifically to their areas of responsibility. The Guidelines are now published by CTFA in three volumes: Microbiology, Quality Assurance, and Safety Evaluation. The CTFA Technical Guidelines are dynamic documents that undergo extensive development and review prior to publication by CTFA technical committees and staff, as well as public review by CTFA members and nonmember companies, federal government agencies, and scientific professional societies. Comments from individuals are welcome at any time. While CTFA has sought to ensure that these Guidelines generally satisfy applicable U.S. federal statutory and regulatory requirements as of the date they were drafted, CTFA can assume no responsibility for their adequacy, nor does it purport to advise as to the necessity for their use in any particular situation. In those Guidelines that address regulatory requirements, decisions such as when a report must be filed and what information must be included in it can be made only by those individuals responsible for making such submissions. With regard to all of the areas covered by CTFA Guidelines, each company must independently assume responsibility to ensure that their conduct is consistent with all current, applicable federal, state and local laws and regulations. It must be emphasized to the user that these Guidelines are intended only to aid manufacturers in developing programs that meet their individual needs. The Guidelines must not be considered either minimum or maximum requirements of effective programs. Alternative ways to reach the goals of the Guidelines may well exist and may be equally useful. Guidelines on any topic must, of course, be adapted to the particular operations of the manufacturer using them. Pamela G. Bailey President & CEO

John E. Bailey, Ph.D. Executive Vice President – Science

INTRODUCTION

Introduction

The production of quality personal care products requires a commitment from the manufacturer to establish and maintain a total quality program. The microbiological component of such a program is designed to ensure: (1) the product that reaches the consumer is free of microorganisms that could affect the product quality and consumer health, and (2) during normal product use, the quality of the product will not be affected by microbial activity. The CTFA Microbiology Guidelines are intended to provide manufacturers with guidance regarding establishing and maintaining a microbiological quality program within their companies. The Guidelines are also recommended for contract packagers and suppliers of raw materials. Sections of the Guidelines will vary as to applicability for different sectors of the industry and for individual companies. The Guidelines are organized into separate sections. The major provisions for an effective microbiological quality program are outlined in the basic guideline “Microbiological Quality Assurance for the Cosmetic Industry” (Section 1). More specific information on building and equipment design, personnel training, cleaning, sanitization and housekeeping immediately follows in a general section. The quality of raw materials used in cosmetic products is addressed in guidelines that cover handling, storage, analysis and sampling. Since process water is a major raw material in cosmetics and toiletries, a separate guideline focuses on process water systems and quality. Because of the increasing dependency on microbiological laboratories to provide supportive data related to product safety and quality, guidelines are offered for evaluating laboratory practices both in-house and in contract laboratories. “Microbial Validation and Documentation” of methods under Section 9 offers guidance for use in the laboratory as well as the plant environment. As an alternative to manufacturing sterile products, the consideration of rational limits to microbiological content based on the best available information is practical and proper. Microbiological limits for finished products as well as raw materials are covered in separate guidelines. “Establishing Microbial Quality of Cosmetic Products” (Section 12) is the result of the international harmonization efforts of Colipa, CTFA, and JCIA. “Microbiological Risk Factor Assessment of Atypical Cosmetic Products” (Section 16) offers advice on conducting risk assessment and testing of atypical and non-aqueous cosmetic formulations. An extensive glossary defines terms used in the Guidelines.

CTFA MICROBIOLOGY GUIDELINES

|

INTRODUCTION

|

1

INTRODUCTION

These Guidelines are not intended to establish minimum industry standards for all cosmetic, toiletry and fragrance products. Also, the Guidelines do not cover all areas that might be addressed under a specific category. CTFA intends to include additional topics in future updates to the Guidelines. In the interim, cosmetic companies are encouraged to refer to other microbiology resources. While these Guidelines can help ensure that products are microbiologically acceptable, they cannot substitute for day-to-day familiarity with the principles of microbial control. The Guidelines must never be taken to restrict additional activities when circumstances dictate. Sections of these Guidelines that are new or substantively updated in 2005 or in this edition of the CTFA Microbiology Guidelines are indicated in the Table of Contents. References including website addresses for all sections have been updated for the 2007 edition. On November 28, 2007 the Cosmetic Toiletry and Fragrance Association changed its name to the Personal Care Products Council. The new, broader and more contemporary name for the assocition reflects our increasingly diverse membership. The Microbiology Guidelines will not be printed with the new name until the next edition.

2

|

INTRODUCTION

|

CTFA MICROBIOLOGY GUIDELINES

SECTION 1

INTRODUCTION Adequate control of the microbiological quality of finished cosmetic products depends upon the implementation of an effective microbiological quality assurance program. Although the applicability of some aspects of such a program will vary for different types of products, processes, and facilities, the major areas described below should be reviewed. The reader is directed to review the “Glossary of Microbiological Terms” at the end of this document to ensure a proper understanding of the Guidelines. Note that these guidelines do not apply to products that have been defined as drugs or pharmaceuticals by regulatory agencies. The Food and Drug Administration’s (FDA) Current Good Manufacturing Procedures (CGMPs) for Finished Pharmaceuticals should be consulted for the manufacture of drug products.1

QUALITY ASSURANCE Quality assurance is defined as “those planned and systematic activities necessary to provide confidence that a product satisfies given acceptance criteria.”2 The goal of an effective microbiological quality assurance program is to assure that the finished product consistently meets established microbiological standards. The microbiological quality assurance program can be viewed as having several major components: • Personnel, including qualifications, functions, and training • Physical environment, including plant, grounds, equipment and sanitary procedures • Materials, including storage, raw materials, packaging, and finished goods • Procedures, including sampling, testing, laboratory practices, and auditing The CTFA Quality Assurance Guidelines provides information for establishing quality assurance programs within cosmetic manufacturing facilities, as well as establishing the control systems designed to assure product quality and consumer safety.3

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

|

CTFA MICROBIOLOGY GUIDELINES

|

3

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

Microbiological Quality Assurance for the Cosmetic Industry

PERSONNEL AND TRAINING All personnel should have the necessary training or experience to perform their assigned functions in manufacturing and quality control.4

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

Quality Assurance Microbiology Laboratory The personnel responsible for microbiological quality control should be of adequate number and have the necessary training and/or experience to ensure that cosmetics meet established control limits. A Microbiologist should have acquired by education and/or experience the expertise needed to supervise operations and be capable of: • Sampling raw materials, process water, bulk and finished goods • Developing and performing test methods • Performing sanitation inspection of plant facilities • Performing environmental studies • Performing documentation and record keeping • Interpreting and reporting test results • Developing and implementing hygiene action plans • Participating in investigation of out of specification microbiological results A Technician should be qualified by education and/or experience in microbiological technique.

Manufacturing/Operations A Supervisor should be qualified by training and/or experience to properly ensure maintenance of the microbiological integrity of the product being manufactured. Compounders, Filling Line Operators, etc. should have an understanding of causes of microbiological contamination, common contamination sources and their prevention.

Education Program In order to maintain microbiological quality, it is important to instill general microbiological awareness and to train operating employees in hygienic practices. Examples of microbiological and hygiene training to be emphasized are listed below. • Potential sources for product contamination by the following avenues: − Physical contact with manufacturing equipment and formulation ingredients, especially following poor personal hygiene − Gross contamination from process and/or rinse water; condensation on standing; dust and particulate matter laden with microorganisms, including airborne spores and vegetative cells − Unsanitary or dirty equipment − Contaminated raw materials

4

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

PHYSICAL ENVIRONMENT Plant and Grounds Buildings and equipment should be designed for ease of cleaning and sanitization. They should be clean and maintained in an orderly manner. The manufacturing area should be designed to minimize the risk of contaminating raw materials, packaging components, or products. These areas should have walls and floors that are easy to clean and sanitize. Overhead repositories for dust, such as piping and ductwork, should be kept to a minimum and cleaned when necessary. Building openings, including doors, should be designed, operated, and maintained to protect the manufacturing areas and to minimize environmental contamination. Windows should be properly screened and each manufacturing facility should have an effective rodent and insect control system. Ventilation systems should include, where appropriate, changeable filters properly maintained to restrict entry of particulate matter, insects, microorganisms, and other contaminants. Positive air pressure should be available in areas containing easily contaminated materials. Water used for humidifying should be of acceptable microbiological quality. Hand-washing facilities should be provided near the production area. Signs reminding personnel to wash hands should be prominently displayed at the washing facilities. Hand cleansers and disposable towels should be available. Eating and smoking should not be permitted in the manufacturing areas. Clean containers, utensils and microbiologically acceptable water used with a disinfectant-type cleaner should be available for general environmental cleaning. Clean containers appropriately labeled should be provided for collecting waste and scrap materials. Designated areas should be provided for storing raw materials and finished goods.

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

|

CTFA MICROBIOLOGY GUIDELINES

|

5

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

• Training on proper cleaning and sanitizing procedures (See “Cleaning and Sanitization” under Section 3) • Encouraging employees to report plant conditions that could affect product integrity • Personal Hygiene: − No person with any health condition that could adversely affect products should have direct contact with raw materials, packaging products, or product contact surfaces − Personnel should store personal belongings and eat, drink, or use tobacco only in designated areas

Machinery and Equipment

SECTION ORGANZIATION 1: QUALITY ASSURANCE OF THE SAFETY FOR THE EVALUATION COSMETIC INDUSTRY GUIDELINES

It is desirable that equipment be constructed for effective cleaning and sanitization and designed to protect products from contamination. The CTFA Quality Assurance Guidelines “Annex 3: Equipment, Part II-Processing” gives important pointers on construction, cleanability, and related items.5 Possible Sources of Contamination: • Pipes may contain crevices, pits, sharp turns, dead ends, connections, unsanitary welded joints. • Equipment may contain pits, crevices, poorly sealed lids, leaking pump shaft seals, defective sight glasses. • Utensils - Plastic is difficult to clean. Wood is not acceptable for most cosmetic manufacturing applications. • Personnel - The human body is a reservoir of large numbers of microorganisms. Protective apparel should be worn whenever appropriate. Infected cuts or abrasions on the hands should be covered. • Atmosphere - Dust is laden with airborne microorganisms. • Other - General condensation, standing water, reused filter pads, cleaning rags and compressed air can be sources of microbial contamination. Recommendations: • Pipes - Stainless steel, glass and plastic hose are the best materials; sanitary snap joint fittings are preferred. Pipes should be graded to drain with no dead legs. • Equipment - Lids on compounding tanks should be tight fitting and vented to minimize condensate formation. Drains should be at the lowest point. Equipment should be designed to minimize backwash contamination potential. Hard to clean equipment should be dismantled and cleaned out of place between product changeovers. • Utensils should be made of stainless steel and be thoroughly cleaned, rinsed, air dried, and properly protected from contamination between uses. • Personnel should be properly trained in personal hygiene (e.g., washing hands after restroom use, contact with food, etc., and prior to contact with product) and the sanitary use of equipment. • Atmosphere - Introduction of clean air and the exclusion of particulate matter will help. • Other - Single-service towels should be used where possible. Compressed air lines associated with product contact equipment should be protected with appropriate point of use filters.

6

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

SANITARY PROCEDURES Cleaning and sanitization procedures are essential to ensure good microbial quality in the manufacture of cosmetics and personal care products. Written cleaning and sanitizing procedures should be established, distributed and implemented by responsible personnel. These procedures should be validated in order to consistently meet hygienic manufacturing requirements. Refer to the CTFA “Cleaning and Sanitization” guideline in Section 3 for further detail.

Care should be taken to prevent introducing microbes when storing materials. The following are desirable conditions of storage: dry; protected from airborne contaminants; maintained within reasonable temperature limits (ideally above freezing and below 100°F); located within low traffic areas; and large enough to segregate incoming materials from material already received and approved. Materials should be stored in a manner that allows for sufficient cleaning and inspection. Raw material containers and storage areas should be protected from contamination by air, dust, water, and personnel. Storage areas and raw material containers should be cleaned on schedule. For more specific guidance for storing raw materials consult “Handling, Storage and Analysis of Raw Materials” under Section 5. Bulk storage of raw materials, process intermediates, and finished products should be protected from microbial contamination. Bulk material should be properly labeled. Bulk subjected to extended storage should be sampled and retested before use in accordance with established procedures. A program should be established for cleaning and sanitizing bulk storage containers. (See Section 3 “Cleaning and Sanitization”).

RAW MATERIALS Specifications Cosmetic manufacturers should evaluate the microbiological quality of their raw materials and establish appropriate specifications based on the best available scientific information. Microbiological specifications should be established for all raw materials susceptible to contamination. (See Section 11 “Raw Material Microbial Content”). The microbiological quality assurance program should include provisions that: • The material is sampled immediately upon receipt from manufacturer. • Material is held in a clean quarantine area until testing is completed. • Rejected materials are clearly marked for prompt disposition. • Accepted materials are so marked. • Procedures are in place to re-sample and test susceptible raw materials stored for prolonged periods prior to use.

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

|

CTFA MICROBIOLOGY GUIDELINES

|

7

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

STORAGE

Identifying Materials All material should be clearly marked to identity.6

Sampling Plan

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

For sampling plans see “Microbiological Sampling” (Section 6) in the CTFA Microbiology Guidelines and “Annex 17: Sampling” 7 in the CTFA Quality Assurance Guidelines.

PACKAGING MATERIALS AND OTHER COMPONENTS Packaging materials (tubes, jars, bottles, caps, brushes, applicators and other components) should be properly controlled (e.g., handling, storage, testing and proper documentation of results) to minimize contamination and to maintain microbiological standards and specifications. A program should be established to ensure that appropriate packing materials and product containers conform to in-house microbiological specifications.

FINISHED GOODS Finished goods should be sampled and tested to assure that products meet established microbiological specifications and should not be released for distribution until the satisfactory completion of the testing. See “Microbiological Sampling” (Section 6) and “Establishing Microbial Quality of Cosmetic Products” (Section 12) for guidance.

LABORATORY PRACTICES Microbiological Quality Assurance Laboratory Several key functions of the Microbiological Quality Assurance Laboratory are: • Analyze raw materials for microbial content and determine if microbiological specifications are met. • Check to ensure that plant hygiene procedures are implemented and effective • Ensure that the microbial status of finished product meets established specifications. • Investigate and resolve contamination problems. • Establish a program to routinely monitor critical control points, including cleaning, sanitization and storage of processing and filling equipment. • Establish appropriate documentation and record-keeping procedures for laboratory testing.

8

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

Microbiological testing should be conducted in a laboratory specifically designed for this purpose. Alternatively, microbiological quality assurance may be subcontracted. For additional guidance see the “Microbiology Laboratory Audit” under Section 8.

Procedures

It is recommended that completed records be maintained after distribution of the batch of manufactured product.

MONITORING The CTFA Quality Assurance Guidelines recommends periodic self-audits.8 Periodic surveillance or inspection of facilities, operations, practices, housekeeping and sanitation is an excellent adjunct to a microbiological quality assurance program. Such monitoring helps to verify consistent compliance with established procedures, confirm that the systems continue to be adequate for provision of safe and effective products, and identify areas that may require improvement. Appropriate measures should be taken where undesirable trends become evident or when conditions are noted that may cast doubt on product or process integrity.

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

|

CTFA MICROBIOLOGY GUIDELINES

|

9

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

The following are the main suggested procedures for the microbiological laboratory: • Sampling - It is recommended that the procedure as outlined in “Microbiological Sampling” (Section 6) be followed. • Testing - It is recommended that raw materials, bulk in-process, and finished goods be tested for microbial content. See “Raw Material Microbial Content” (Section 11) and “Establishing the Microbial Quality of Cosmetic Products” (Section 12). • Water - Particular attention should be given to water, as it is the most important raw material as well as a solvent for cleaning, disinfecting and rinsing. See “Microbiological Quality for Process Water” (Section 7). • Preservation - The inability of microorganisms to survive in packaged products should be verified during product development. See “Determination of Preservative Adequacy in Cosmetic Formulations” (Section 13). • Monitoring - Control and monitor sanitization procedures by the use of swabs, direct contact plates, air samplers, and other means. Refer to “Cleaning and Sanitization” (Section 3). • Documentation/Record Keeping - Maintain accurate, detailed records providing the history of a material. A central file should be maintained for periodic review by a microbiologist and should be the prime record for all testing performed. (See “Microbial Validation and Documentation” under Section 9).

SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY

REFERENCES 1. U.S. Food and Drug Administration. 2005. “Current Good Manufacturing Procedures (CGMPs) for Finished Pharmaceuticals.” Title 21. Code of Federal Regulations, Part 211 (21 CFR 211). http://www.fda.gov.

4. Bailey and Nikitakis. “Annex 1: Personnel and Training.”

2. Bailey, John E., and Nikitakis, Joanne M. 2007. (Ed). “Glossary of Terms and Definitions”. In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

6. Bailey and Nikitakis. “Annex 6: Finished Products/Lot Identification & Control.”

3. Bailey and Nikitakis.

10

|

CTFA MICROBIOLOGY GUIDELINES

|

5. Bailey and Nikitakis. “Annex 3: Equipment, Part II - Processing.”

7. Bailey and Sampling.”

Nikitakis.

“Annex

17:

8. Bailey and Nikitakis. “Annex 14: Internal Audits.”

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

SECTION 2

Microbiological Evaluation of the Plant Environment INTRODUCTION The cosmetic manufacturing plant environment may directly or indirectly affect the microbiological quality of cosmetic and personal care finished products. Environmental assessment of the plant primarily employs air and surface monitoring techniques. Evaluation of monitoring results takes into consideration the intrinsic factors that affect the microbial environmental quality within the facility. Changes in environmental data (i.e., trend analysis) can serve as useful indicators of the need for investigation and possible corrective actions.

Since each manufacturing plant is unique, manufacturers have the responsibility of determining what type of program is most suitable for their facilities. This guideline provides general and specific information to aid manufacturers in designing environmental monitoring programs suited to their own needs. Some information offered may not be directly applicable to the operations of every facility. However, an established environmental monitoring program can provide the data, tools, and procedures needed to maintain a well-functioning facility. An effective program can provide information about areas of the plant that may affect the microbial quality of the finished product. Manufacturers may want to consider the Food and Drug Administration’s (FDA) Hazard Analysis and Critical Control Point (HACCP), which was recently mandated for the food industry. This program is a systematic approach for evaluating hazards and risks of various parts of a process and places controls and systems at critical points.6 Good communication between microbiologists and facility engineers is essential in maintaining awareness of changing environmental factors that could alter the microbiological quality of the plant environment.

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

11

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

For facilities that manufacture products regulated as over-the-counter (OTC) drug products in the United States, refer to the United States Pharmacopeia (USP),1 Parenteral Drug Association (PDA),2 and Current Good Manufacturing Practices (cGMPs)3, 4, 5 for guidance on how to conduct environmental monitoring for a manufacturing plant.

MANUFACTURING ENVIRONMENT The quality of the manufacturing plant environment is largely influenced by five basic factors: facilities, equipment, personnel, housekeeping, and cleaning and sanitization. Understanding these factors is essential when developing an environmental control program. Additional guidance on these topics is given in “Annex 2- Premises” in the CTFA Quality Assurance Guidelines7 and in “Cleaning and Sanitization” in Section 3 of this document.

Manufacturing Facility

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Design A well-designed, well-constructed manufacturing facility can contribute to a high-quality finished product. Proper design can minimize cross-contamination and contamination from the surrounding environment. Contamination of the plant environment by microorganisms, dust, and dirt can be controlled by the use of vent filters, drain traps, and tight-fitting doors and windows. Airborne dust contamination can be minimized by the use of filtered air-handling systems that provide adequate ventilation, temperature, and humidity controls to prevent cross-contamination. Materials used for building interiors should be durable, easily cleaned, and adequately maintained. Overhead utilities (pipe work) can be designed so that they do not adversely affect the manufacturing environments. Duct work for these utility systems should be composed of nonporous and nonflaking material. General building design should include suitable barriers to separate manufacturing and packaging areas from warehouses, offices, locker rooms, and washrooms. In particular, a good building design provides separate areas for material receipt, storage, weighing, compounding, filling, packaging, etc. Traffic flow of both personnel and materials (e.g., raw ingredients, packaging components, and finished stock) can be minimized in processing and packaging areas. When considering building design concepts, some decisions on the desired level of control may be based on present and anticipated requirements of products and manufacturing.

Operational Influences Both internal and external conditions are important factors that can affect the microbiological quality of the plant environment. These diverse factors should be taken into consideration when determining facility design as well as the frequency of microbiological monitoring. Examples of internal influences: • Start up of air conditioning or heating systems • Construction • Duct and vent cleaning • Modifications to equipment • Plant alterations • Equipment maintenance • Change in activity level

12

|

CTFA MICROBIOLOGY GUIDELINES

|

ANNEX 2

PREMISES (FACILITY)

Examples of external influences: • Construction • Surrounding environment (farm lands, wet lands) • Climate (temperature, humidity) • Seasonal changes

Manufacturing and Filling Equipment Equipment constructed for effective cleaning and sanitization and designed to protect products from contamination is recommended. When designing or purchasing new manufacturing or filling equipment, microbiology, quality, engineering, and manufacturing personnel should evaluate this equipment for its ease of cleaning and sanitization in addition to cost and efficiency.

The CTFA Quality Assurance Guidelines Annex 3, “Part I – Packaging Equipment” and “Part II – Processing Equipment” give important direction on construction, cleanability, and related items. 8,9

Personnel Personnel are encouraged to practice good personal hygiene. Wearing clean uniforms and, where appropriate, head covers, beard covers, clean gloves, or finger cots will help prevent contamination. Adequate locker room facilities, washrooms, and eating areas should be physically separate from the manufacturing, filling, and packaging areas of the plant. It is recommended that employees responsible for sanitation and housekeeping be thoroughly trained in all pertinent procedures as part of an ongoing training program. All employees involved with manufacturing and packaging should be trained to follow cosmetic good manufacturing practices through a regular training program.10 Training programs are most effective if documented and conducted periodically according to a pre-planned schedule. For more information, refer to “Cleaning and Sanitization” in Section 3.

Housekeeping The general plant environment should be kept in a clean and orderly state. For example, cleaning and/or sanitization of floors, walls, ceilings, vents, pipes, fixtures, and equipment exteriors should be conducted on a regular schedule according to written operating procedures. Equipment, SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

13

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Some useful factors that influence sanitary maintenance of this equipment include: • Equipment drawings and/or schematics that can provide helpful guidance for cleaning and sanitization protocols (refer to “Cleaning and Sanitization” in Section 3) • Equipment manufactured from smooth, nonporous materials (e.g., 316L stainless steel) • Valves and gauges that are easily disassembled for cleaning • Equipment made from materials that are compatible with products and cleaning and sanitization solutions

hoses, tools, and other items not in use should be stored in a clean state and protected from contamination. Cosmetic facilities should have effective programs for control of rodents and other pests, and for proper refuse disposal. For additional guidance, refer to “Annex 2 - Premises” in the CTFA Quality Assurance Guidelines. 7

Cleaning and Sanitization Equipment cleaning and sanitization is carried out on an established schedule, usually between batches of different products, according to written procedures. It is important to assure that cleaning and sanitization is documented and validated, equipment is identified as to sanitary status, and cleaned and/or sanitized equipment is kept dry and covered. Refer to Section 3 “Cleaning and Sanitization” for cleaning and sanitization procedures, frequency, expiration times, and validation processes. 8 See “Annex 3-Part II - Processing Equipment”9 in the CTFA Quality Assurance Guidelines and “Microbial Validation and Documentation” in Section 9 for additional guidance.

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

ENVIRONMENTAL MONITORING PROGRAM The following are among the elements to be considered when establishing an environmental monitoring plan.

Training Personnel involved with environmental sampling should be properly trained according to a written procedure applicable to such testing. Training materials should at least address the following: • Methods and materials for collecting and processing samples • Appropriate areas for monitoring • Frequency of monitoring • Interpretation of test results • Determination of alert and action levels • Proper documentation and communication of results • Corrective action procedures

Documentation Documentation provides an organized record of the microbiological evaluation. It is recommended that the following information be included for proper documentation of an environmental monitoring program: Procedural Information • Physical location (manufacturing area, warehouse, etc.) • Sampling site • Sampling method 14

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

• Collection/recovery medium used • Incubation time and temperature • Sampling frequency Reported Data • • • • • • • •

Specific site sampled Media quality control information Date and time sample was collected Weather conditions Activities occurring near the sampling site at time of sampling Results Date and time Signature of investigator (a microbiologist or suitably trained individual)

Additional information may be included based on in-house needs or company policies.11

Baseline Data

Statistical analysis of environmental historical microbiological test data may be used to set the alert and action level criteria for deciding when to investigate a shift in the trend. It is common practice to periodically reevaluate the alert and action levels. There are several statistical methods for evaluating the data.12, 13

SURFACE SAMPLING Surfaces 14,15 The microbiological quality of physical surfaces within a manufacturing environment can directly or indirectly affect the microbial quality of finished cosmetic products. Physical surfaces coming into direct contact with finished products may include: • Bulk raw material storage vessels or containers • Intermediate and finished product storage vessels • Processing equipment • Filling equipment

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

15

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

The objective of an environmental monitoring program is to obtain microbiological data that can serve as indicators of change in the environment. Monitoring these indicators can help identify the need for investigation and possible corrective actions to reduce or eliminate the contamination source in the environment. Criteria are determined based on in-house needs. Prior to the setting of microbial environmental monitoring criteria, periodic microbiological monitoring of physical surfaces is performed to determine the baseline levels of the microbial flora within the different areas of the manufacturing environment. There may be baseline variations in the microbial levels depending on internal and external conditions. Operational influences are summarized in the discussion on the “Manufacturing Environment.”

• • • • •

Transfer pumps and lines Pumps Valves Utensils Ancillary equipment and other working contact surfaces

Surfaces not coming in direct contact with the product that could affect microbiological quality may include: • Walls • Floors • Ceilings • Overhead lighting and piping • Vertical and horizontal support beams • Overhead walkways • External processing and filling equipment surfaces • External packaging materials

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Monitoring Frequency The type and frequency of microbiological monitoring of physical surfaces depend on the susceptibility of the finished product to microbial contamination as well as other factors. Additional factors to consider are the scale of manufacturing, condition, and design of the plant, type of process, local environmental factors, and company policies. Areas in direct contact with finished products are usually monitored more frequently than areas that are not. In areas directly contacting a product, any increase from predetermined microbial alert and action levels may indicate a potential microbiological problem that could affect product quality. Increased frequency of testing, adjustments to cleaning and sanitization protocols, or changes in other processes may be indicated if a potential microbiological problem is found to potentially affect product quality. Supervisory personnel should routinely review the microbial test data generated and, if required, adjust the frequency of environmental monitoring. Methods 16,17 Sampling by means of swabs, direct contact devices, or contact plates are the most common methods of monitoring surfaces for microbial contamination. Note that swabs and contact plates will not recover total microbial bioburden from a surface. Exit monitoring of rinse water can be used to evaluate interior surfaces of manufacturing and filling equipment. However, rinse water testing may not be useful in detecting the presence of biofilm bacteria.18 See Table 2-1 for different surface sampling methods. Swabbing Sterile swabs can be used to sample environmental surfaces for the presence of microbial contamination. The sterile swab is wetted in sterile buffer, saline solution, or broth and rubbed

16

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

over a measured portion of the surface to be monitored. The swab is then either streaked across an agar plate or placed into a sterile broth tube. The plate or tube is incubated for the appropriate length of time. Swabs Swabs can be used on flat, irregular surfaces and on hard-to-reach areas. The three most common composition materials for swabs are Dacron, cotton wool, and calcium alginate. Calcium alginate is a fibrous material that dissolves in sodium citrate or sodium hexametaphosphate, a characteristic that facilitates the total release of microorganisms that have been recovered on the swab from the surface. This allows for a quantitative analysis.19 Leachables from cotton wool swabs, such as fatty acids, may be inhibitory or detrimental to microbial growth.20 Whichever type of swab is used, all on-going testing should be performed with the same type of swab and be processed as soon after collection as feasible. In cases where there is a delay (e.g., swab samples need to be shipped to a laboratory for processing and analysis), transport swabs may be used. Transport swabs are designed to maintain the viability and numbers of microbes present at the time of sampling until the time of processing. The swab manufacturer should be consulted for storage and temperature conditions to determine how long after use a transport swab can maintain the viability of microorganisms.21 Sampling

If sanitizer or disinfectant residues are present on the surfaces being sampled, they may interfere with the test results. All of the solutions used to wet swabs should contain a neutralizer if disinfectant or sanitizer residues are expected. An appropriate cleaner, such as 70% alcohol, should be used after sampling to remove swab residue. A sterile template (e.g., 2”x 2” area) may be used to standardize the size of the surface area sampled. Processing Swabs Three basic techniques are commonly used to process swabs after sampling a surface: Direct Swab Methods After a swab has been used to sample a test surface, it can either be streaked directly onto an agar surface in a Petri dish or it can be added to an enrichment broth as described below. A variety of media, both general and selective, may be used, e.g., Trypticase Soy Agar, Pseudomonas Isolation Agar, MacConkey, Sabouraud Dextrose, etc. If general and selective media are used, the general media should be inoculated first. If the selective media is inoculated first, inhibitory ingredients may be carried over to the general media and prevent growth. A neutralizer should be included in the media if there is a concern that sanitizer/disinfectant residues on the sampled surface may interfere with the test results.

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

17

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Using aseptic technique, the sample site is sampled by rubbing a premoistened swab over the surface. Moistened swabs are essential for recovery of the highest possible numbers of bacteria, molds, or yeasts. Solutions used to wet the swab may include, but are not limited to, sterile buffer, saline, or broth.

This technique may be used for testing those surface areas on which low numbers of microorganisms are expected and for which a quantifiable result is needed. Streaked Petri dishes or enrichment broth are incubated for the appropriate period and temperature. Note: The use of selective agars when directly plating swabs can be inhibitory to injured microorganisms. The results, either microbial growth or no growth, may not be representative of the types of microorganisms actually present on the surface. Unless looking for specific types of microorganisms, the use of general microbial growth media or enrichment techniques may give more useful information in an environmental monitoring plan. Test results may be recorded as: • Growth or no growth per swab • Growth or no growth per unit area (e.g., per square inch or square centimeter) If low numbers are recovered, individual colonies may be counted and recorded as the number of microorganisms per swab or unit area

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Swab Enrichment Methods This technique may be used in areas where low numbers of microorganisms are expected. After sampling a test surface, aseptically transfer the used swab directly into a test tube of enrichment broth. Include a neutralizer in the enrichment broth if there is a concern that sanitizer/disinfectant residues on the sampled surface may interfere with the test results. Incubate the test tube with swab for the appropriate period and temperature. Test results may be recorded as: • Growth or no growth per swab, based on the presence or absence of turbidity in a general enrichment broth • Growth or no growth per unit area (e.g., per square inch or square centimeter)

Standard Plate Count Methods This technique can be used in those areas in which there could be either high or low numbers of microorganisms. After sampling the test surface, aseptically transfer the swab into a sterile test tube containing 10 milliliters of enrichment broth. Enrichment broth may include neutralizer(s) for sanitizer/disinfectant residues. Vortex the test tube to release microorganisms from the swab into the broth. If sampling with calcium alginate swabs, sodium citrate or sodium hexametaphosphate may be used to dissolve the swab and aid in releasing recovered microorganisms from the swab. Remove aliquot(s) of the broth and plate onto a general microbial growth agar medium and/or onto selective/differential agar media. Agar media may include a neutralizer(s) for sanitizer/disinfectant residues that may have been picked up by the swab in sampling test surfaces. If high 18

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

numbers of microorganisms are expected, further dilute the original swab dilution sample and plate these aliquots. To record results, count the colonies on countable plates, multiply by the dilution factor, and record as number of microorganisms per swab or per unit area (e.g., per square inch or square centimeter).

Contact Sampling

Rinse Water Method This technique is generally used to sample either interior surfaces of equipment that cannot be reached using a swab technique (e.g., kettles, tanks, etc.) or other hard-to-access surfaces that come into direct contact with the finished product. The rinse water method consists of flushing the selected surface with a suitable volume of sterile rinse water, collecting a sample of the rinse water, and then quantitatively determining the number of microorganisms in the sample. Membrane filtration, pour plates, spread plates, or Most Probable Number (MPN) procedures can be used to quantify the microbial recovery. Factors to be considered include: • Surfaces can be selectively tested using this technique • Chance of introducing testing contamination is minimal • Allows testing of otherwise inaccessible areas • Can be used to monitor the efficacy of equipment-sanitizing procedures • Rinse water monitoring may not detect biofilm bacteria that may adhere to the interior equipment and transfer line surfaces

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

19

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Contact sampling may be performed using modified Petri dishes (i.e., RODAC™ plates), paddles, or flexible films, which contain a solid agar culture medium whose convex surface extends above the carrier. Selective and non-selective agar media may be used. The sterile agar surface is applied to the test surface so that the agar makes total contact with the area being sampled. An appropriate cleaner such as 70% alcohol is used after sampling to remove any remaining agar residue. The sampling devices are incubated, after which the degree of microbial contamination per unit area can be determined. Factors to be considered when choosing one of these methods are: • Suitability for flat surfaces only • Usefulness in remote areas under field conditions • Commercial availability of disposable units • Suitability for qualitative/quantitative analysis of environmental cleaning and sanitization procedures • Limited shelf life • Cost • Problem of confluence, with certain microorganisms, especially if agar surface is wet

General Applications Physical surfaces coming into direct contact with the product should be examined for the presence of bacteria and fungi that are known to cause product spoilage or harm to the consumer. Indirect surfaces such as walls and floors should also be monitored to determine background levels of microorganisms that are intrinsic to the manufacturing environment. In general, all equipment (processing and filling), valves, traps, and working surfaces should be monitored on a defined and periodic basis. Transfer lines should be taken apart and tested. Viable microbial counts should be performed to determine the levels of microorganisms present in these areas.6

AIR SAMPLING 22-28

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

The selection of sites for air sampling is based primarily on the potential for adverse microbiological effects on the finished product. Routine air monitoring may include selected environmental sites within the facility as well as sources of compressed air used in manufacturing. Factors to take into consideration when developing an environmental air monitoring program include room design, airflow patterns, proximity to vents, and potential for product exposure.

Monitoring Frequency The air-monitoring program establishes the frequency of routine sampling at each location based on in-house needs, with the areas of greater microbiological concern monitored more frequently. The schedule for air monitoring in each designated area is based upon previously determined microbial baseline levels. Monitoring frequency is determined in part by the type of activities in each area, such as machine operation, personnel, physical cleaning, construction, etc. Seasonal changes and climate are also important considerations when establishing an air-sampling program. Areas of greater microbiological concern, such as exposed product and raw materials, are usually monitored more frequently. Supervisory personnel, the plant microbiologist, or another suitably trained individual should review and analyze the microbial test data generated during air sampling. These data can be used for trend analysis and to provide a history of the plant environment, which can be used to evaluate sampling frequency or investigate shifts in microbiological quality. Methods 26-28 A variety of methods may be employed for environmental and compressed air sampling. Each is designed to meet specific needs. Some sampling methods measure all particulates, including viable and non-viable microorganisms. Others only measure viable organisms. Consider the following factors when choosing an air-sampling method: • Ability to determine change of air contamination over time • Anticipated bioburden (quantity, viability, type) • Collection medium 20

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

• Quantitative vs. qualitative measurement • Ability to determine the number of colony-forming units per unit of time or volume sampled The monitoring method(s) chosen will be influenced by the requirements of the individual facility. The requirement or need to measure only viable microorganisms versus all other particulate matter should be determined.

Viable Methods The most commonly used methods for measuring viable organisms, many of which are available commercially, are listed below. Also see Table 2-2. Settling Plate A Petri dish containing Trypticase Soy Agar or other suitable general microbial growth agar is directly exposed in the sampling area (i.e., placed upright in the area with the lid off ). Particles in the air settle onto the agar surface. After a specified exposure time, the Petri dish is collected, covered with the lid, and incubated. The number of microbial colonies is counted directly from the plate.

Air is collected via centrifugation through impeller blades, and microorganisms are deposited onto the surface of a nutrient agar medium in a strip. The growth agar strip is incubated and the number of organisms per volume of air is calculated. Sieve Impaction Sampler Air is drawn into the unit through a sieve and over the surface of an agar plate. After incubation of the plate, the number of viable microorganisms per unit of time or volume of air may be determined. Slit-to-Agar Sampler Microorganisms are impinged directly onto a microbial growth agar surface in a Petri dish that rotates beneath a slit opening. Air is drawn through this slit with a vacuum. The speed of the Petri dish rotation and the volume of air sampled can be adjusted. After incubation of the plate, the number of viable microorganisms per unit of time or volume of air can be calculated. Liquid Impinger Air is drawn through a sampler tube and particles are collected in a liquid medium. The air rises and is removed from the system. Serial dilutions of the liquid medium are made, and duplicate aliquots are plated into empty Petri dishes to which a sterile melted microbial growth agar is added. The Petri dishes are allowed to solidify and are incubated. After incubation, the number of microbial colonies is counted per Petri dish and an average is calculated for each duplicate serial dilution. SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

21

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Centrifugal Air Sample

Multi-Stage Particle Sizing Sampler The sampler contains up to six plates, arranged vertically. A measured volume of air is drawn through successively smaller holes in the sieve plates, resulting in acceleration of the particles at each stage. Viable particle size distribution is calculated from the plate counts at each stage. Membrane Filter Air to be sampled is impinged on a gelatin membrane filter, which is then removed from the filter holder and placed in a dish containing a general microbial growth medium. After an appropriate incubation period, the number of microbial colonies on the membrane surface is counted. This unit can also filter for phage and is most commonly used in clean rooms and isolators.27

Non-Viable Methods

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Monitoring of non-viable airborne particulates is outside the scope of the guideline. Standards for air based on particulate matter counts are addressed elsewhere.29, 30 A method that uses optical particle counts is commonly employed. Compressed Air Sampling Compressed air that comes into direct contact with the product process or that could adversely affect the manufacturing environment may be monitored. The Slit-to-Agar method has been modified for sampling compressed air. This instrumentation, adapted for sampling compressed gas up to pressures of 125 psi, is based on the impingement principle of particle capture. The circular sweeping of an agar plate surface is controlled at a critical distance beneath a laser cut air intake slit and creates a radial undulation over the area of impingement. The speed of the plate rotation and sampling are precisely controlled so that, after a period of incubation, the growth on the agar surface can be used to quantitatively measure microbial contamination.

EVALUATION OF RESULTS There are no set criteria for microbiological monitoring of the environment in plants manufacturing cosmetics. Trend analysis performed on the data from microbiological monitoring of surfaces and air in the plant offers a useful evaluation tool. Shifts from established data patterns may indicate changes in the environment or work practices that may have the potential to affect the microbial quality of the finished product. In the evaluation of environmental test results, alert and action levels should be established for each manufacturing area. Levels set will depend on the areas monitored, historical trend data from the area, type of monitoring, and potential effects on finished product quality.

22

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

Factors to Be Considered in Setting Alert and Action Levels Many factors influence the microbial alert and action levels established for each production area. These include, but may not be limited to: • Objective of the Sampling – Whether, for example, to measure seasonal changes in air quality to determine effectiveness of a sanitization procedure, or to monitor changes in work practices. • Area Sampled – Microbial criteria for air in a warehouse differ from the criteria for air over a filling machine. Criteria for the surface of cleaned and sanitized compounding equipment differ from those in areas that do not directly contact product. • Type of Product – The type of finished product manufactured, i.e., hostile vs. microbiologically susceptible, is an important aspect. Susceptible products likely to be more sensitive to microbial contamination by environmental influences are expected to require more stringent controls in processing and packaging areas.

Documentation

Once a program is in place, the results generated should be documented by keeping an organized record system. Records of environmental monitoring should be maintained for an appropriate length of time. These data can be used to establish a normal range of results. Supervisory personnel, a microbiologist, or other qualified individual should routinely review all results. Based on these results, a feedback mechanism can be established whereby all departments involved are informed when the environmental quality is outside of the normal range. The responsibility for documentation and communication should be addressed by internal standard operating procedures. Refer to “Microbial Documentation and Validation” in Section 9 and to “Annex 5 - Production Control” in the CTFA Quality Assurance Guidelines.”31

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

23

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Documentation is important to establish the historical trend data in the plant. Trend analysis of microbial recovery levels in the environment may be useful when: • Investigating potential sources of microbial contamination in a product • Identifying sources of microbial contamination in the plant • Identifying potential seasonal trends • Setting and adjusting cleaning and housekeeping schedules • Choosing sanitizers

Interpretation Once all the data have been collected, consideration of the following information will help in its interpretation: • Total number of microorganisms recovered (quantitative analysis) • Percent of positive results as compared to the total number of areas tested (qualitative analysis); for example, this might be useful when using qualitative swabs • Presence or absence of objectionable organisms

Alert/Action Level Response Regardless of the assessment method used, once a normal range of microbial recovery has been established, the manufacturer should set alert and action levels for all areas that are routinely monitored. If microbiological test results reach an alert level, a manufacturer can take a number of actions that may include collecting additional samples, observing the manufacturing area, evaluating the process, increasing the testing of finished goods, reviewing practices, etc.

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Investigation If microbiological test results reach an action level, a complete investigation is indicated, followed by corrective actions. At a minimum, these include: • Confirmation of specification results • Microbiology laboratory interview/investigation • Review of product logs/records • Review of cleaning/sanitization procedures • Review of product and area history • Interview personnel (production and laboratory areas) Corrective Actions Depending on the outcome of an investigation, corrective actions may include: • Retesting of the affected areas (depending on the extent of the problem, manufacturing or filling may be delayed until all environmental testing is complete.) • Recleaning and resanitization of equipment, floors, walls, etc. • Additional testing of the finished product to ensure its quality • Retraining/reinforcement of cleaning and sanitization procedures • Modification of engineering controls • Modification of practices or processes • Documentation of corrective action taken

24

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

CONCLUSION A microbiological monitoring program for the plant environment is a tool to help assure the microbiological quality of products manufactured for the cosmetic industry. An environmental monitoring program includes the microbiological monitoring of surfaces, air, raw materials, and finished products. The value of the program depends on adequately trained testing personnel, documentation of test data, assessment of results, and appropriate response. Once a microbiological profile of the plant has been established, a trend analysis can be used to develop alert and action criteria. Changes in the trend may indicate the need for an investigation, after which a correction action plan may be implemented. Each step of the process should be accompanied by appropriate documentation. Table 2-1 Surface Sampling Methods Description

Advantages

Disadvantages

Swabs

An environmental swab is a sampling device composed of a synthetic (e.g., Dacron, calcium alginate) or cotton tip affixed to a wood or plastic stick. It is used to sample discreet areas in difficult-to-reach locations or irregular surfaces.

1. Inexpensive. 2. Convenient. 3. Suitable for irregular surfaces. 4. Calcium alginate tips can be dissolved in media to release all microorganisms collected. 5. Can be used for highly contaminated areas. 6. Can be utilized in remote areas under field conditions. 7. Can be qualitative or quantitative.

1. Leachables from cotton may inhibit fastidious microorganisms 2. Microorganisms may become trapped in the swab head. 3. May leave a residue or microbial growth agar on the surface after being sampled; the agar media residue needs to be removed.

Contact Plates

A contact plate may be modified Petri dishes, paddles, or flexible films containing a solid microbial growth agar whose convex surface extends above the carrier. The sampling device may contain any of a number of various types of microbial growth agar with or without a disinfectant/ sanitizer neutralizing agent.

1. Can be used in remote areas. 2. Samples the same size area each time as defined by the size of the device. 3. Can be qualitative or quantitative.

1. Expensive. 2. Short shelf life under field conditions. 3. Not suitable for irregular surfaces.. 4. Microbial overgrowth may be a problem. 5. May leave a residue or microbial growth agar on the surface after being sampled; the agar media residue needs to be removed.

Rinse Water

The rinse water technique consists of flushing the surface to be tested with a sterile rinse solution such as water.

1. Can be used to test otherwise inaccessible areas such as the interior equipment surfaces of manufacturing equipment. 2. Larger surface areas may be sampled.

1. Quantitative. May not detect the presence of biofilm. Bacteria. 2. Not suitable for many applications. 3. Extensive manipulation may be required. 4. Sample processing may affect test results.

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Method

Table 2-1 SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

|

CTFA MICROBIOLOGY GUIDELINES

|

25

Table 2-2 Air Sampling Methods32 Method

Description

Advantages

Disadvantages

Sedimentation (i.e., Settling Plates)

An open Petri dish filled with a microbial growth agar that is exposed for a specified time period (i.e., 15 to 60 minutes) that is left exposed to the air.

1. Easy. 2. Least expensive. 3. Constant surveillance not necessary. 4. Large numbers of areas can be monitored in a short amount of time. 5. Any type of microbial growth agar media can be used. 6. No sampling device required.

1. Cannot determine the amount of air sampled. 2. Microbial count cannot be correlated with air volume. 3. Disposition of colonies is affected by size of particles, temperature, and flow/volume of air passing across surface. 4. Affected by air movement in area. 5. If left exposed for a long period of time, plates can desiccate.

Provides a rough estimate of airborne contaminants

Lightweight, portable unit that measures a quantifiable volume of air (1 to 1,000 liters). The sampling media are agar strips.

1. High recovery efficiency. 2. Portable. 3. Fast and easy to operate. 4. Excellent for areas that are difficult to access. 5. Measures concentration of viable particles as function of time and unit volume of air. 6. Selective/differential agar strips are available.

1. Requires special agar strips that are expensive and available only from the manufacturer. 2. Strips have a limited shelf life. 3. Strips susceptible to over growth in heavily contaminated areas.

Sieve Impaction Sampler

Air is drawn through a uniformly perforated surface and is distributed over an agar surface.

1. Colony overlapping minimal. 2. Large air volumes possible. 3. Portable. 4. Air flow can be calibrated. 5. May be used to sample compressed air when used with a vacuum. 6. Choice of agar dish size and media is flexible.

May be cumbersome if used with a vacuum.

Slit to Agar Sampler

Air is pulled through a slit over a revolving plate.

1. Measures concentration of viable particles as function of volume of air. 2. No serial dilution or plating required. 3. Wide application in surveillance of ambient air contamination. 4. Volume and speed adjustable. 5. Constant surveillance not necessary. 6. Remote sampling probe can be used.

1. Vacuum source required. 2. Not easily portable. 3. Large numbers of sampling areas are needed, very time consuming. 4. Electrical connection required. 5. Best suited for clean rooms 6. Some systems require 150 millimeter (mm) agar plates.

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Centrifugal Impactor

Table 2-2 26

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

Table 2-2 Air Sampling Methods32 continued Description

Advantages

Disadvantages

Liquid Impingement

Air is drawn through a sampler tube and particles are collected in liquid medium. Microbial counts are determined in the liquid.

1. Samples with high viable counts can be diluted for enumeration. 2. Quantitation is good for spores and vegetative cells. 3. Inexpensive.

1. Low sampling rate. 2. Vacuum source required. 3. Time consuming. 4. Requires dilution and plating. 5. Breaks up bacterial particles. 6. Device may consist of breakable glass components.

Sieve Multi-Stage Particle Sizing Sampler

A specific volume of air is drawn through a series of sieve plates, resulting in particle size separation. This allows plate counts at each stage.

1. Determines size of particles. 2. Particles. 3. Measures concentration of viable particles as a function of time. 4. No serial dilution or plating required. Comparable to the impingers.

1. Limited sampling duration does not provide entire picture. 2. Requires many plates. 3. Vacuum required. 4. Not well adapted for heavily contaminated areas. 5. Agar desiccation.

Membrane Filter

Air is drawn through a gel filter disc, which is then placed on an agar surface for enumeration of microorganisms.

1. Large volume of air. 2. 3μm pore size retains Coli-phages. 3. Gelatin overcomes desiccation.

1. Equipment cumbersome. 2. Requires additional manipulation of membranes.

Table 2-2

REFERENCES 1. United States Pharmacopeia. 2007. United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD. http://www.usp.org/. 2. Parenteral Drug Association, Inc. 2001. “Fundamentals of Environmental Monitoring Program.” PDA Journal of Pharmaceutical Science and Technology. Supplement TR13. 55(5). http://www. pda.org. 3. U.S. Food and Drug Administration. 2006. “Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs, General.” 21 CFR, Part 210. 4. U.S. Food and Drug Administration. 2005. “Current Good Manufacturing Practice for Finished Pharmaceuticals.” 21 CFR, Part 211. 5. U.S. Food and Drug Administration. July 2000. “Good Manufacturing Practice Guide for Active Pharmaceutical SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

Ingredients.” Draft ICH Consensus Guideline. http://www.fda.gov/cder/ guidance/4011dft.pdf. 6. Pierson, M.D., and D.A. Corlett. 1992. HACCP Principles and Applications. Originally published by Chapman & Hall. (Norwell, MA: Kluwer Academic Publishers, 1992). 7. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. “Annex 2 – Premises”. In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association. 8. Bailey and Nikitakis. “Annex 3-Part I Packaging Equipment.” 9. Bailey and Nikitakis. “Annex 3-Part II Processing Equipment.” 10. Bailey and Nikitakis. “Annex 1-Personnel and Training.” 11. Parenteral Drug Association, Inc. 2001. “Fundamentals of Environmental Monitoring Program.” PDA Journal of |

CTFA MICROBIOLOGY GUIDELINES

|

27

SECTION 2: EVALUATION OF THE PLANT ENVIRON MENT

Method

EVALUATION SECTION 2: EVALUATION OF PRIMARY DERMAL OF THE IRRITATION PLANT ENVIRON POTENTIAL MENT

Pharmaceutical Science and Technology. Supplement TR 13 55(5):12. 12. Parenteral Drug Association, Inc. Supplement TR 13 55(5): 8-9. 13. Reich, R.R., and M.J. Miller. 2003. “Developing a Viable Environmental Monitoring Program for Nonsterile Pharmaceutical Operations.” Pharmaceutical Technology: 92-100. 14. American Society for Testing Materials. ASTM Standards on Materials and Environmental Microbiology. 2000. Philadelphia, PA. 15. Block, Seymour Stanton. 2000. Disinfection, Sterilization, and Preservation. Philadelphia, PA: Lippincott Williams & Wilkins. 16. Hickey, P.J., C.E. Beckelheimer, and T. Parrow. 1992. “Microbiological Tests for Equipment, Containers, Water, and Air“. In Standard Methods, For the Examination of Dairy Products. Edited by Robert T. Marshall, Ph.D. (Ed.), Washington, D.C. 17. Lemmen, S. W., Hafner, H., Zolldan, D., Amedick, G., Lutticken, R. 2001. “Comparison of two sampling methods for the detection of Gram-positive and Gram-negative bacteria in the environment: moistened swabs versus RODAC plates.” International Journal of Hygiene and Environmental Health. 203, 245-248. 18. U.S. Food and Drug Administration. 1993. Inspection References. Guide to Inspections Validation of Cleaning Processes. http://www.fda.gov/ora/inspect_ref/igs/ valid.html. 19. Parenteral Drug Association, Inc. Supplement TR 13 55(5): 20. 20. Betty A. Forbes, Daniel F. Sahm, and Alice S. Weissfeld. 1978. Bailey and Scott’s Diagnostic Microbiology. St. Louis, MO: The C.V. Mosby Company. 21. Wilson, D.A., M.S. Tuohy, and G.W. Propcop. 2001. “Effects of storage temperature on the recovery of bacteria from three swab transport system: BD CultureSwab and BD Culturette (BD

28

|

CTFA MICROBIOLOGY GUIDELINES

|

Diagnostics System, Sparks, MD) and Starplex Starswab II (Etobicoke, Ontario, Canada).” Paper presented at The American Society of Microbiology General Meeting. Orlando, FL. 22. Hering, S.V. 1989. “Air Sampling Instruments for Evaluation of Atmospheric Contaminants.” Paper presented at the American Conference of Governmental Industrial Hygienists, Cincinnati, OH. 23. Kundsin, R. B. (Ed). 1980. Airborne Contagion. New York: NY Academy of Sciences. 24. Parenteral Drug Association, Inc. Supplement TR 13 55(5): 15-17. 25. Powitz, R. W. 2002. “Sampling for Airborne Biological Contaminants: A Rational Approach.” Advancing Applications in Contamination Control. 17-19. 26. Mehta, S.K., et al. 2000. “Evaluation of Portable Air Samplers for Monitoring Airborne Culturable Bacteria.” AIHA Journal 61. 27. Shelby, S., et al. 1995. “Effect of Impact Stress on Microbial Recovery on an Agar Surface.” Applied and Environmental Microbiology 61(4): 1232-1239. 28. Sartorius. http://www.sartorius.com. Click on “For Your Laboratory” and then “Air Monitoring.” 29. International Organization for Standardization. 1999. “Cleanrooms and associated controlled environments – Part 1: Classification of air cleanliness.” ISO 14644-1. Geneva, Switzerland. http:// www.iso.org. 30 International Organization for Standardization. 2000. “Cleanrooms and associated controlled environments – Part 2: Specifications for testing and monitoring to prove continued compliance with ISO 14644-1.” ISO 14644-1. Geneva, Switzerland. http://www.iso.org. 31. Bailey and Nikitakis. “Annex 5 Production Control.”. 32. Hess, Kathleen. 1996. Environmental Sampling for Unknowns. Boca Raton, FL: CRC Lewis Publishers.

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

SECTION 3

Cleaning and Sanitization

INTRODUCTION Cleaning and sanitization procedures are essential to ensure microbial quality in the manufacture of cosmetics and personal care products. These procedures should be validated in order to consistently meet hygienic manufacturing requirements. The design of these procedures should take into account the product formulation and all aspects of manufacturing.

GENERAL CONSIDERATIONS Specific internal programs for cleaning and sanitization should be established. These programs are essential to: • Assure the microbiological quality of the product • Meet legal regulations where required • Minimize the microbial load contributed by processing, filling, and storage equipment • Avoid the cost associated with microbial failure • Help maintain the company commitment to quality

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

29

SECTION 3 CLEANING AND SANITIZATION

If cosmetics and over-the-counter (OTC) drugs are manufactured with the same equipment, refer to FDA guidelines for the manufacture of OTC drugs.1, 2, 3 • Cleaning is the process of removing product residue and contaminants such as dirt, dust, and grease from the surface and is the essential first step in any cleaning and sanitization procedure. • Sanitization is the process utilized to reduce viable microbial contaminants to an acceptable level. All surfaces must be clean for the sanitization procedure to be effective. • Validation is the process of substantiating and verifying that the process does what it purports to do. • Documentation is the process of organizing all relevant information in an orderly and easily understood format. This documentation is required to validate a process and maintain an historical record of the process and equipment usage.

Guidance for the development of operating procedures is addressed in each section below. Written protocols are required prior to attempting to validate any process. For more information, see the “Microbial Validation and Documentation Guidelines for the Cosmetic Industry.” (Section 9).

TRAINING Personnel should be properly trained and supervised in the cleaning and sanitization of the facility and equipment. A document should be written to outline the training process. Ongoing training should be conducted according to a pre-planned schedule. Performance should be monitored to verify that the training is effective and proper procedures are followed.

Purpose Training should be used to: • Bring new employees to the required level of competency • Introduce new cleaning and sanitization methods and products to all employees • Reinforce existing programs Re-training should be conducted according to a predetermined schedule, with more frequent training if needed.

SECTION 3 CLEANING AND SANITIZATION

Content A training program should impart an understanding of the elements of cleaning and sanitization and their effect on product quality. 4, 5 Training should include: • Overall microbiological awareness and basic microbiology • Basic concepts of microbial contamination, common contamination sources, and their avoidance • Consequences of microbial contamination • Sanitary practices • The risks associated with not following appropriate sanitary practices • Good housekeeping • Personal hygiene • Basic equipment operation and design • Importance of cleaning and sanitization and a clear understanding of each process • Product type and proper procedure based on product formula ingredients • Proper and safe use of cleaning and sanitizing agents • Concentration, dilution, and contact time of cleaners and sanitizers • Product and chemical residues, including cleaners and sanitizers

30

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Training should also include: • A review of the procedure for proper cleaning and sanitization of the equipment and areas in the employees’ care • Effects of changes to process, formula or equipment changes on cleaning and sanitization requirements (i.e., change control) • Recordkeeping of cleaning and sanitization performed • A mechanism for reporting to appropriate personnel any observations that indicate a potential for contamination

Documentation Training should be documented. At a minimum, this written record should include: • Name of the trainer • Attendees • Date/time of training Additional items such as training materials and any tools used to measure comprehension and understanding may also be included.

DOCUMENTATION Documentation is the keeping of all essential records of cleaning and sanitization. All these records should be complete, clear, and concise. In addition to the training documentation discussed above, manufacturing facilities should document validation and ongoing operations.

Validation

Protocol The cleaning or sanitization procedure protocol consists of: • A written description of the objective of the validation study and acceptance criteria • A written explanation of the process This documentation should include a detailed description of the product, process, and equipment involved, as well as the protocol and test procedures to be used.

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

31

SECTION 3 CLEANING AND SANITIZATION

All cleaning and sanitization procedures should be validated. Validation documentation consists of two components, a protocol and a summary document.

Summary The summary document consists of: • A report summarizing the raw data supporting conformance to acceptance criteria, with raw data either attached or available • A conclusion to include at least a statement of acceptance, any recommendations, and revalidation requirements Cosmetic Microbiology: A Practical Handbook, edited by Daniel K. Brennan provides a useful reference for further guidance.4

Routine Documentation Routine or ongoing documentation includes routine logs necessary to maintain a history of the equipment usage and are an essential part of any investigation. This information can also serve as part of a validation information package. It can be used for trend analysis, evaluating cycle reduction, and improving efficiency.

SECTION 3 CLEANING AND SANITIZATION

Logs The routine log should include the following information for each cleaning, sanitizing or changeover activity: • Date, start and end times of the cleaning • Date, start and end times of the sanitization, including expiration time • Product and batch preceding the cleaning and sanitization • Operating procedure, SOP, or procedure number for the cleaning and sanitization being carried out • Any variation from the established operating procedure • Sign off by operator • Review, approval and sign off by verifier/reviewer • Time, date and identity of next batch start up • Date and description of any repairs or equipment down time

Status In addition to permanent logs, current cleaning and sanitization status should be clearly displayed on equipment. Examples of status designation labels are: • Contents and Batch or Lot Number • Empty Needs Cleaning • Needs Cleaning • Clean Needs Sanitizing • Sanitized Information on equipment status should also note the date sanitized and the expiration time and date. 32

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

MANUFACTURING FACILITY The environment of the manufacturing facility strongly influences the microbial quality of the finished product. Appropriate building design and maintenance are critical. Standard procedures for facility cleaning and sanitization should be written and a record of their implementation should be maintained.5, 6

Design and Maintenance Buildings should be designed for ease of cleaning and sanitization to allow for the sanitary manufacture of product. This design should minimize cross-contamination and contamination from the surrounding environment. Maintenance of the building should maintain the integrity of the sanitary design. The building layout should be organized to accommodate a rational flow of materials, clean operations, and adequate supporting activities in the facility. Separate areas should be maintained for material receipt, storage, weighing, compounding, filling, packing, etc., to prevent crosscontamination. It is critical that there be access to all surfaces for cleaning and sanitizing. These surfaces include equipment, walls, storage cupboards, piping, under stairs, behind tanks, etc.

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

33

SECTION 3 CLEANING AND SANITIZATION

The following are important points to be considered in the design of a facility: • Exterior walls, entryways and other building openings should be designed to prevent access to pests, vermin, birds, insects, and other potential sources of contamination. They should be kept in good repair. Options may include automatic closing doors, screens on windows, screened vents, sealed pipe entries, and loading bays designed to minimize environmental contamination. • Building systems, including heat, air conditioning, and compressed air, should be monitored to assure that they do not contribute to contamination. Scheduled preventative maintenance such as filter changes should be performed. • Building leaks should be repaired immediately. • Adequate drainage should be provided to get rid of wastewater effectively because stagnant or standing water allows for microbial growth. • Positive air pressure can be used to reduce the risk of contamination in areas where product is openly exposed to the environment, as in compounding and filling operations. • Equipment should be raised from the floor or otherwise constructed so that floors can be kept clean. • To avoid extraneous material from contaminating product, piping, wiring, transport belts and other potential sources of contamination should not be positioned above tanks or filling lines. • Floor, wall and ceiling surfaces should be free from cracks, crevices and open joints. • Finishes should be smooth and non-porous to allow for easy cleaning and sanitization. Peeling paint should be removed.

• There should be access to all wall areas to allow cleaning and eliminate conditions that contribute to buildup of debris. • Adequate storage should be provided for items not in use to minimize clutter. • Areas where equipment is washed should be of sanitary design and properly maintained. • Adequate lavatories with hand-washing facilities should be provided. • Locker areas and lunchrooms should be separate from the manufacturing area.

Manufacturing and Production Areas The frequency of cleaning and sanitization is determined by the types of activities conducted in any given area. Qualified personnel should visually monitor each area routinely. Cleaning and sanitizing schedules can be adjusted, and remedial action taken as needed. Precautions should be taken to minimize airborne dust during all cleaning. Any spills of raw materials, product, or packaging components should be cleaned up promptly. Recommendations are given below for some specific areas.

Walls, ceilings, pipes, and fixtures Clean and/or sanitize on a scheduled basis (for example, monthly, quarterly or more frequently, if needed). Vacuum to remove loose material.

Floors

SECTION 3 CLEANING AND SANITIZATION

Clean floors on a scheduled basis and include the following: • Vacuum and/or sweep frequently • Wet-mop or machine scrub on a predetermined schedule • Sanitize as appropriate

Cleaning equipment and supply storage Store cleaning equipment and supplies properly in a clean area. Maintain the supply area in an orderly manner. Separate supplies and equipment for lavatory cleaning from other cleaning supplies.

Warehouse Areas Raw materials, packaging components, finished products and equipment should be stored in warehouse areas under acceptable environmental conditions. Precautions should be taken to prevent contamination from any source.

34

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

General guidance for the warehouse area includes the following: • Aisles should be kept neat and clean by sweeping, damp mopping or machine scrubbing. An appropriate, freshly prepared cleaner should be used. • An established, monitored, and documented insect and rodent control program should be in place. • Stored materials and containers should be kept clean, orderly, protected and correctly identified. • Container exteriors should be cleaned before transferring material into manufacturing areas.

Waste Disposal Area Provisions should be made for regular, timely removal and disposal of waste from the proximity of finished products, components, and manufacturing areas to minimize the risk of microbial contamination. Suggestions for handling waste include: • Place refuse for disposal in designated containers using plastic liners. Construct containers so they are leakproof and rustproof, and cover whenever possible. • Empty refuse containers a minimum of once daily and clean when necessary prior to reuse. • Clean spills immediately and remove debris from the manufacturing areas. • Use disposable towels and discard immediately after single use. Avoid the use of rags. • Isolate waste disposal areas from manufacturing, and routinely clean and sanitize to minimize odors. • Dispose of product or process waste in accordance with current government regulations. • Handle all hazardous waste, including spills, per the facility hazardous waste management plan.

In general, the written procedures for the cleaning and sanitization of the floors, walls, ceilings, pipes, and general building environment should include the following: • Type(s) of cleaner and/or sanitizer • Instructions for the preparation of the proper concentration of cleaner and/or sanitizer • Instructions for the proper use of cleaning and sanitizing equipment • Written schedule for the routine cleaning/sanitizing of each area including: − Areas to be treated − Method(s) of treatment − Frequency of treatment

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

35

SECTION 3 CLEANING AND SANITIZATION

Documentation

The cleaning and sanitizing procedures performed should also be documented. The record should be signed by the individual doing the work as it is completed and routinely checked and initialed by a qualified individual.

MANUFACTURING AND FILLING EQUIPMENT Manufacturing and filling equipment has direct contact with product. The following are important considerations with this equipment:7, 8 • Documentation of cleaning and sanitization of equipment • Sanitary equipment design • Frequency and monitoring of cleaning and sanitization • Procedure for cleaning and sanitization • Special equipment and procedures • Criteria for cleaning and sanitization

Documentation of Equipment Cleaning and Sanitization

SECTION 3 CLEANING AND SANITIZATION

Operating Procedures Operating procedures for each piece of equipment should be in place and include the following: • Equipment identification • Equipment disassembly instructions, where necessary • Product-specific instructions where applicable • Type of cleaners and sanitizers to be used • Instructions for preparation of the proper concentrations of cleaning and/or sanitizing solutions • Proper application technique, rinse procedure, contact times, and temperature for cleaning and sanitizing solutions • Proper storage, labeling and protection of equipment once it has been cleaned and/or sanitized • Time limit between sanitization of equipment and use • Safety considerations

Equipment status log For each piece of equipment, a cleaning and sanitizing log should be prepared, maintained, and made readily available. This log should include the product to be made, name of the equipment, and the cleaning and sanitization date. In addition, the log should be signed and dated after each procedure. It should be routinely checked and initialed by the operator and a qualified individual. Equipment status tags are recommended to assure proper identification. See “Routine Documentation” under Section 3.

36

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Sanitary Equipment Design It is essential that process and filling equipment have good drainage and be designed for ease of cleaning and sanitization. In addition, the equipment should be durable enough to withstand sanitizing chemicals and/or physical agents.

General equipment design The following guidance on overall equipment design is intended to minimize conditions that may lead to microbial growth in the equipment. It also offers suggestions to reduce the potential degradation of the equipment by the effects of the sanitizers and cleaners used. • Design process and filling equipment to minimize the retention of residual product and/or wash water. Residual water will dilute product and/or sanitizer which can lead to microbial growth and the development of adaptable microorganisms. • Minimize condensation in equipment. This can dilute product and create an environment for microbial growth. • Design equipment so that internal and external surfaces are accessible and easily cleanable. All surfaces should be as free as possible of crevices that can harbor product or microorganisms. • Choose equipment and its surface finish that are easily cleanable and durable. 316 or 316L stainless steel with a 140 grit or better finish, a type 2B finish, or equivalent quality, is recommended for susceptible products. • Choose materials of construction that are not easily degraded, etched, or reacted when in contact with the product or sanitizers. • Routinely inspect and replace gaskets when necessary, as they are easily contaminated. • Use sanitary welding techniques to avoid creating crevices or rough surfaces that are difficult to clean. Orbital welding and/or gas tungsten arc welding are recommended.

Tanks/Vessels • • • • • • • •

Minimize sharp corners because they are difficult to clean. Avoid narrow recesses that could trap product and water. Design tanks with a domed head to minimize condensation. Choose tanks and vessels with conical or dish shaped bases if possible, with a center drain, to allow for complete draining. Design vessel openings and surfaces to be cleanable. Design and maintain covers to fit well and close easily. Design vents to minimize debris. Eliminate unused drop leg pipes.

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

37

SECTION 3 CLEANING AND SANITIZATION

Common sanitary design practices for specific types of equipment are listed in the following sections.

Transfer Pipes • Minimize the length of pipe runs to make cleaning easier and reduce the risk of biofilm formation. • Slope pipe runs to be self-draining and cleanable with no dead legs/ends. • Design piping systems to have a minimal number of T-fittings. • Use sanitary welding techniques to avoid the creation of difficult-to-clean crevices and rough surfaces. • Use sanitary fittings for all connections. • Avoid screw-threaded piping that comes in contact with the product.

Valves Valves should be easily cleanable with no dead spaces to collect product residue or water. An example of a sanitary valve is a diaphragm valve.

Pumps Sanitary pumps are recommended. The design and installation should allow for complete drainage. Pumps should be easily accessible for inspection, cleaning, and sanitization.

Filling Equipment Fillers should be designed to be easily cleaned and sanitized. Avoid drip pans and water-lubricated belts. If positive air is used in filling equipment, microbial air filters and air line dryers should be monitored to prevent air line condensate from contaminating finished products.

SECTION 3 CLEANING AND SANITIZATION

Gaskets Gaskets are potential sites for contamination. Gasket materials should be compatible with the product as well as the cleaning and sanitizing solutions. Non-porous, chemically inert materials are recommended. Care should be taken to assure that gaskets are properly installed.

Hoses Transfer hoses should be of a material that is compatible with product, cleaners and sanitizers used. They should have sanitary fittings. Cleaned and sanitized hoses should be drained to dry and capped when not in use.

38

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Schedule

Frequency All equipment should have a regular cleaning and sanitization schedule. The frequency should be determined based on several factors: • Product vulnerability to contamination • Type of equipment used • Difficulty in removing product from the equipment • Whether continuous process batching is being performed

Validation Cleaning and sanitization schedules should be validated. Ideally, cleaning and sanitization between batches of products and/or at the end of the day’s production is preferable. Continuous process of the same product may alter this frequency. An additional determination of an effective time interval between equipment sanitization and start-up should also be made. This is achieved by validating the process.

Expiration limit A validated time or expiration limit should be set for each equipment-sanitizing procedure. This will depend on equipment and method of sanitization. This expiration limit reflects the allowable time a piece of sanitized equipment can stand before requiring resanitization.

Monitoring

A microbiologist or suitably trained individual should review the entire processing system to determine potential areas for microbial contamination.

Areas Areas may include processing lines, storage and mixing vessels, fillers, pumps, pipe connections, flexible hoses, pressure relief valves, pigging systems, strainers, utensils, and other related equipment. Most probable areas to be monitored include low-point drains, internal seams and gaskets, internal filler nozzles, and the interior pump.

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

39

SECTION 3 CLEANING AND SANITIZATION

Personnel

Procedures Visually inspect dismantled equipment for residual product or water. Determine the presence of microbial contamination by a method appropriate to the equipment. This monitoring should be documented. If a chemical sanitizer is used for sanitization, a neutralizing medium specific for that sanitizer must be employed in the equipment monitoring procedure. Methods used to determine the presence of microbial contamination may include swabbing, direct contact, or testing the final rinse water. If steam or hot water is used for sanitization, temperature recording devices or thermal strips should be employed in the equipment monitoring procedure.

Methods Sampling by means of swabs or contact plates is used to monitor surfaces. Exit monitoring of rinse water can be used to sample interior surfaces. Swabbing A sterile cotton or calcium alginate swab is wetted in sterile buffer, saline solution or broth and rubbed over a measured portion of the surface of the sanitized equipment. The swab is then either streaked across an agar plate or placed into a sterile broth tube. The plate or tube is incubated for the appropriate length of time. Examination of the plate will give an organism count, and the individual colonies can be lifted from the plate and identified. Tubes are examined for turbidity; this is a pass/fail test. Swabbing is very useful for irregular surfaces or curved equipment.

SECTION 3 CLEANING AND SANITIZATION

Direct Contact Contact plates contain agar, which has a convex surface. These plates are pressed against the surface of equipment, then incubated. Examination of the plate will give an organism count and individual colonies can be lifted from the plate and identified. The surface of the equipment touched by the contact plate must be cleaned of any agar residue after sampling. Contact plates cannot be used for irregular surfaces and are practical only for flat surfaces.

Final Rinse Test Water of known microbiological quality and volume is rinsed through the equipment. The water is recovered and filtered via membrane filtration technique. The membrane is placed onto a plate and incubated. Examination of the plate will give an organism count and individual organisms can be identified. Note that rinse water analysis may not detect the presence of biofilm on equipment surfaces. 40

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Validation Validation of equipment sanitization efficacy can be done via swabbing onto hardened agar plates. Monitoring frequency may be based on the manufacturing history of the product or at the discretion of the microbiologist or hygienist, e.g., it can be done after each sanitization or on a periodic basis. If a chemical sanitizer is used, analytical testing may be used to detect residues.

General Procedures Water Water utilized in the cleaning and sanitizing processes may be described as: • Make water - The water used to make up cleaners and sanitizers should have a low microbial bioburden to avoid contaminating the cleaner and to avoid consuming the sanitizer. • Rinse water for cleaned equipment - Water used to rinse cleansers from cleaned equipment should be fresh, potable water that has a microbiological quality that meets EPA drinking water quality standards.9 • Rinse water for sanitized equipment - Water used to rinse chemical sanitizers from sanitized equipment must have no higher microbial bioburden than the microbial specifications of the product to be made in that equipment.4 When water is used to rinse equipment, the equipment must be drained and used within a validated expiration time.

Equipment cleaning and sanitization

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

41

SECTION 3 CLEANING AND SANITIZATION

Clean and sanitize all lines; processing, storage and filling equipment; pumps; pipe connections; flexible hoses and utensils, as well as plant facilities in the immediate processing and filling areas as follows: 1. Remove product residue from all contact surfaces by thoroughly rinsing with water or water/detergent solution between 120° F (49 C) and 180° F (82 C). Temperature is dependent on product type and equipment compatibility. Note: Non-aqueous-type product residues should be removed by appropriate predetermined methods. 2. Assure thorough cleaning and sanitization of pigging equipment and of the pig itself. When not in use, pigs must be handled and stored under sanitary, dry conditions. Note: Pipeline pigs are devices made of non-porous materials used for recovery of product, product separation, and cleaning of manufacturing pipelines. The pig launcher and receiving station must be sanitary in design as this equipment can easily harbor microbial contaminants. 3. Circulate a cleaning solution for a period of time and at a temperature capable of effectively removing soil residue in the circuit and/or equipment. All surfaces not accessible by this cleaning procedure should be cleaned manually and/or by using special equipment or methods.

4. Rinse the cleaning solution thoroughly from the system with microbiologically acceptable water, as determined by in-house standards. 5. Before use, equipment should be sanitized according to the written procedure for the piece of equipment involved. See “Special Equipment and Methods” below. 6. It is suggested that rinse water for sanitized equipment contain no higher microbial content than the limits established for the formulated products. Used process equipment should be cleaned as soon after processing as possible to facilitate removal. Product dried and hardened on equipment surfaces can be difficult to remove thoroughly. Cleaned/sanitized equipment should be properly stored before use to prevent recontamination. In general, equipment should be drained dry with open ends covered to prevent recontamination.

Special Equipment and Procedures Special cleaning and sanitizing equipment and methods may be employed for processing and filling apparatus. The equipment and methods are generally designed to fit the individual needs of each manufacturing facility. There are several methods for cleaning and/or sanitizing. Manual Manual methods involve the preparation of cleaning solution and the scrubbing of equipment or parts using a brush, single-use cloth or pad. It is an effective but highly time consuming method. Soak This method involves immersing utensils or equipment parts in containers of detergent solution for extended periods of time. Generally, this method is used in combination with manual cleaning. SECTION 3 CLEANING AND SANITIZATION

Spray Low or high-pressure sprays are used to remove soil. In most cases, the cleaning action of the pressure sprays is enhanced by the use of detergents. High-pressure spray nozzles such as spray balls or injectors may be permanently installed in mixing or storage tanks. High-pressure spray wand equipment is also widely used. This type of equipment is mobile and/or portable. It is used for general surface cleaning. Spray pressures developed should range from 200 to 1000 pounds per square inch (psi). Fog Fogging is a method of generating a mist for the application of sanitizers. Large areas of equipment surfaces can be treated by fogging in a very short time, using small amounts of sanitizers. This method should only be used in closed systems by properly trained personnel. 42

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Clean In Place (CIP) CIP is a semi- or fully automated, self-contained system for cleaning and sanitizing equipment. Cleaning and sanitizing solutions are circulated for a specific time at specified temperatures. No disassembly of equipment is necessary. Each system is unique and to work well, should be properly designed, evaluated and controlled. Factors to consider when using CIP are: detergent/sanitizer type; detergent/ sanitizer concentration; temperature; and design of equipment. Some equipment design factors include type, number, positioning of spray balls, type of pump, velocity rates, baffles that may shadow areas of a tank, etc.

Acceptance Criteria Prior to validation of the cleaning and sanitization process for each piece of equipment, acceptance criteria should be selected. Criteria should take into account the types of products processed by the equipment. Typically, criteria include microbial bioburden that meet specific requirements or limits of the products, the absence of pooled water, limitations on product residue, absence of objectionable organisms. Alert and action levels for microorganisms should be established by quality assurance based on finished product specifications.

CLEANERS (See Table 3-1) A cleaner can be defined as a chemical or blend of chemicals formulated to remove undesirable soils from a contact surface. These chemicals may be solvents, acids, bases, detergents, and/or water-based chemical blends. Industry has focused on aqueous cleaners because of concerns for the environment and employee exposure.

Characteristics of an Efficient Cleaner Aqueous cleaners are typically formulated to contain several ingredients to allow for maximum cleaning effectiveness. The ingredient requirements depend on the intended use of the cleaner. Efficient aqueous cleaners utilize surfactants (anionic, nonionic, cationic and/or amphoteric), dispersants, emulsifiers, wetting agents, builders, chelating agents, sequestering agents, corrosion-inhibiting agents and stabilizers. The surfactants are used for emulsification, wetting and penetration; builders for neutralizing hard water interferences; chelating inorganic soils and saponification of natural oils; and additives for corrosion inhibition, anti-redisposition and good rinseability. See Table 3-1 for information on specific chemical cleaners. For additional information, see References 7, 8 & 10 at the end of this section.

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

43

SECTION 3 CLEANING AND SANITIZATION

Aqueous cleaners are defined as blends of water-soluble chemicals designed to remove soils into a water-based solution with a water continuous phase during cleaning. These consist of surface active ingredients and other cleaning chemicals that use detergency to lift soils from surfaces by displacing the soil with the surface active materials. This occurs because the surface active ingredients have a higher affinity for the surface than they do for the soil.7,10

Characteristics essential to a good cleaner include: • Compatibility with equipment, i.e., non-corrosive • Quick solubility • Good wetting action • Good penetration properties • Good emulsification and soil-dispersion properties • Good rinsing properties • Economical and readily available • Environmentally friendly and non-hazardous Table 3-1 gives examples, descriptions and advantages/disadvantages of several different cleaners.

Selection of a Cleaner

SECTION 3 CLEANING AND SANITIZATION

Although the characteristics of an efficient cleaner may be more general, the selection of a particular cleaner for a particular cleaning task requires specific information. The most important considerations include knowledge of the type of substrate to be cleaned and the type of soil to be removed. The cleaner type should be matched to the surface to be cleaned (metal, glass, plastic, etc.), the soil type (organic, inorganic, oils, heavy soils, light soils) and the desired cleaning method (manual, soaking, CIP, power spray wand, etc.). The cleaner should also be widely available and economical. Information on the level of cleanliness required (acceptance criteria) should also be known. Several questions can be asked prior to the selection of a cleaning system: • Does the cleaner have good detergency on the type of soil to be removed? • Is the cleaner recommended for the cleaning process to be used? • Is the cleaner free-rinsing? • Is the cleaner hazardous or environmentally unfriendly? • Is the cleaner economically priced at the use level and widely available?

Variables Affecting Efficiency Besides the selection of an efficient cleaner, several other factors are extremely relevant to the success of a cleaning process. Beyond the cleaner itself, cleaning efficiency is influenced by cleaner concentration, agitation, temperature, cleaning/contact time, rinse method and drying method. These process variables must be considered, specified, and controlled to ensure a consistent and optimized cleaning process.

Cleaner Concentration The concentration of the cleaner should be selected through consultation with the manufacturer followed by in-house validation. Optimizing cleaning temperature, time or agitation may reduce the concentration of cleaner required.

44

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Temperature Usually the higher the cleaning process temperature, the more efficient the cleaning. However, this depends on the soil type (melt-point issues). Temperature should be optimized for the soil being cleaned and validated using in-house methods. Safety considerations should be included when personnel exposure is possible.

Time In general, the longer the cleaning process, the more thorough the cleaning. Cleaning time is dependent on the other factors of the process, including agitation, temperature and cleaner aggressiveness. Soaking may take hours, whereas high-pressure sprays may require from seconds to minutes. Cleaning time should be considered in the validation of the entire cleaning process/ system.

Agitation Depending on the product/soil to be cleaned, the range of applied mechanical fluid energy required for effective cleaning will vary. Equipment can simply be soaked or immersed in a cleaner solution, manually scrubbed, or cleaned with direct impingement using dynamic spray balls or jet-spray devices. In general, increased agitation and turbulence improves cleaning efficiency.

Rinse It is important that the rinse procedure completely removes debris detached from the equipment during cleaning. The specified volume of rinse water should be validated for each particular rinse program. A general recommendation is that the rinse water volume be at least three times the volume of the cleaner solution used. There should be no cleaner residue.7

Drying

Use of 70% alcohol as a finishing step can aid in the evaporation of water. Alcohol can be used as a dryer/sanitizer and is especially useful for anhydrous products where it is essential that no moisture remain on the equipment. Caution should be used when using alcohol on equipment that could present a fire hazard.

Testing to Measure Cleaning Process for Efficacy The development of a testing and measurement system is key to optimizing and validating the effectiveness of a specific cleaning process. The method selected for measuring the effectiveness

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

45

SECTION 3 CLEANING AND SANITIZATION

To reduce the potential for corrosion, eliminate the opportunity for microbial regrowth, and prevent dilution of chemical sanitizers, equipment should be properly drained and dried after rinsing. Evaporation is the simplest and least-expensive drying method. Other methods include circulated hot air, vacuum-drying, and forced-air blow drying. For these methods, the air source may be filtered to provide high-quality air for drying.

of the cleaning process should provide information needed to determine that key criteria are met. Testing of the cleaning process initially requires the development of a baseline level of cleanliness and an effective method to measure cleanliness. In many cases, visual assessments of equipment or simple gravimetric analysis will suffice. Alternatively, video scopes, chemical tracer measurements (fluorescent whiteners, total organic carbon (TOC) in residual water, or conductivity) may be used. Various methods involve the extraction of the contaminating soil from the surface followed by quantitative chemical analysis.7 The simplest method that provides appropriately sensitive results should be used. After the cleaning system has been selected, it should be validated against the targeted product and on the equipment where the production will occur. Either a quantitative or qualitative method may be used to judge the cleaning process, and then acceptance criteria should be established. Experimentation may occur initially on a smaller bench or pilot-plant scale; however, the cleaning system should be validated on the actual equipment due to concerns with scale-up. Each variable of the cleaning process (cleaner concentration, time, temperature, agitation, etc.) should be considered to determine the optimal conditions.

SANITIZERS Definition A sanitizer is either a chemical or physical agent that is effective in reducing microbial contamination on product contact surfaces. A sanitizer should achieve a 99.9% (3 log10) reduction of pathogenic or unacceptable microorganisms and reduce other organisms to a minimal acceptable level. A sanitizer may be considered effective if it reduces microorganisms to acceptable levels, with no detectable objectionable microorganisms, as determined by the cleaning and sanitization protocol. 7, 8, 11, 12

SECTION 3 CLEANING AND SANITIZATION

Characteristics and Selection of an Efficient Sanitizer The following are desirable characteristics of a sanitizer: • Effective against a broad range of microorganisms • Provides adequate microbial reduction; 99.9% effective against organisms of concern • Effective in a relatively short contact time • Stable and efficacious over time, both in concentrate form and at use levels • Economical to use • Non-toxic at use levels • Non-corrosive at use levels • Compatible with products and equipment • Free from objectionable odors and residue • Meets regulatory requirements • Biodegradable

46

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Chemical Sanitizers Combined cleaner/sanitizer agents are available. However, these agents can have reduced detergent and/or disinfectant activity compared to each agent alone. Additionally, cleaners have a high optimal pH, whereas most chemical sanitizers are more effective at neutral or acidic pH. Some useful chemical sanitizing agents are chlorine, iodophores, quaternary ammonium compounds, ethyl alcohol, phenolic compounds, formalin, phosphoric acid, hydrogen peroxide, peracetic acid, and ozone. See Table 3-2 for information on frequently used chemical sanitizers.

Physical Sanitizers The most common physical sanitizer is thermal energy, either in the form of steam or hot water (180°F or 82°C minimum). A major advantage of heat is its ability to penetrate into small cracks and crevices. Heat is also non-corrosive, cost-effective, measurable with recording devices or thermal strips, efficient, effective against a broad range of microorganisms, and leaves no residue. See Table 3-3 for information on frequently used physical sanitization methods.

Factors Affecting Efficacy Cleaning must always precede sanitization. In-house validation of each specific piece of equipment is needed to assure sanitizer efficacy. Roughness of surface, bad welds or other defects can make the equipment difficult to sanitize. Care should always be taken to follow label directions and manufacturer instructions and recommendations. Water incorporated into sanitizers should be of acceptable microbial quality.

The following process variables should be considered, specified, and controlled to ensure consistent sanitizer performance: • Condition of equipment surfaces • Materials of construction • Concentration of sanitizer • Contact time • Temperature • Optimal pH range • Mechanical energy (pressure and flow rate)

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

47

SECTION 3 CLEANING AND SANITIZATION

Operators should be properly trained. Improper use may give ineffective results, release toxic fumes, or corrode equipment.

Measurement and Validation of Sanitization Effectiveness Prior to validation of the sanitization process, the acceptance criteria should be selected for specific equipment and products. Typical or suggested criteria include microbial bioburden that meets specific requirements or limits and the absence of pooled water or product residue. A suggested approach for validating the sanitization procedure effectiveness: 1. Sanitize the equipment. 2. Break down the equipment. 3. Evaluate microbial bioburden and organism type on product flow surfaces including difficult-to-reach areas such as gaskets, valves, pumps, etc. 4. Check both microbial levels and organism types. The validation of a sanitization procedure should not be performed immediately after cleaning but at the longest potential time the equipment will stand before use. This gives an expiration time for sanitization after which the equipment must be resanitized. See the section above on “Monitoring” under MANUFACTURING AND FILLING EQUIPMENT.

SUMMARY The selection and effective use of a cleaning or sanitizing agent and/or method is dependent on several factors: the manufacturing facility, the type of product processed, and the design and layout of the equipment. All cleaning and sanitizing procedures should be properly designed and their use documented and validated. Personnel should receive adequate instruction and training in these areas.

SECTION 3 CLEANING AND SANITIZATION

With attention to these details, a cleaning and sanitizing program will positively contribute to achieving a sanitary manufacturing facility.

48

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Table 3-1 Chemical Cleaners Refer to manufacturer’s use directions and material safety data sheets (MSDS) Cleaner Type

pH Range

Soils Removed

Examples

Advantages/Disadvantages

Mineral-Acid and Mild Acid Cleaners

0.2 - 5.5

Heavy scales to inorganic salts

1. Strong acids: Hydrochloric acid Sulfuric acid Phosphoric acid

1. Good for acid-soluble soils

Soluble metal complexes

2. Weak acids (dilute solutions of organic acids): Acetic acid Citric acid Neutral Cleaners

5.5 - 8.5

Light oils Small particulate

2. Efficient for metal oxide removal 3. May be harsh on hands 4. May have toxicity, environmental and handling issues

Mild, unbuilt surfactant solutions (may include water-miscible solvents such as alcohols or glycol ethers)

1. Rely on dissolution and emulsification, rather than aggressive chemical attack 2. Lowered toxicity and corrosivity concerns

Mild Alkaline and Alkaline

8.5 - 12.5

Oils Fats Grease Particulates Films

Ammonium hydroxide Sodium carbonate Sodium phosphate Borax solutions

1. Alkalinity promotes a. saponification b. solubilization of alkalinesoluble soils c. hydrolysis

Corrosive Alkaline

12.5 - 14

Heavy grease and oils

Sodium hydroxide Potassium hydroxide Sodium silicates

1. Work best when soil can be hydrolyzed; i.e. saponification of fatty soils 2. Harsh on hands 3. Some exposure hazards and product toxicity hazards 4. Corrosivity Table 3-1

SECTION 3 CLEANING AND SANITIZATION

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

49

Table 3-2 Chemical Sanitizers

SECTION 3 CLEANING AND SANITIZATION

General types and uses are listed below. Refer to manufacturer’s use directions and material safety data sheets (MSDS). Suggested Concentration & Contact Times

Type

Description

Advantages

Disadvantages

Chlorine

Sodium hypochlorite Calcium hypochlorite Lithium hypochlorite Chlorine gas Chloramines Chlorocyanurates

200 ppm as free chlorine 30 minutes

1. Excellent activity 2. Readily available 3. Can be used alone in cold water on clean equipment 4. Rapid, sensitive test available to determine concentration during sanitization and to verify removal of residual after rinsing

1. Odor 2. Chlorine is less reactive as pH increases 3. Inactivated by organics 4. Reactive with metal surfaces - corrosive if misused; must carefully regulate exposure time 5. Sensitive to light and temperature 6. NIOSH recommended employee exposure limit 0.5 ppm ceiling for 15 minutes13

Cationic surfactants

Quaternary ammonium compounds (normally in combination with nonionics)

200 ppm at time recommended by manufacturer

1. Cleans (has excellent detergent properties) 2. Excellent activity 3. Noncorrosive 4. Can be used alone in water 5. Deodorizes 6. Residual activity 7. Odorless 8. Very stable

1. Not sporicidal 2. Most effective against microorganisms at neutral or slightly alkaline pH 3. Hard-water tolerance may vary 4. Residue may be incompatible with product 5. Inactivated by anionic cleaners 6. May not be compatible with non- ionics 7. Exit monitoring requires titration

Iodophors

Iodine in nonionic surfactants with H3PO4

12.5 - 25 ppm 10 minutes

1. Cleans as formulated 2. Excellent activity 3. Residual activity 4. Non-toxic at use concentrations 5. Stable at use concentrations

1. Poor sporicidal activity 2. May stain 3. Usually formulated 4. Rinsing required

Alcohols

Isopropyl Ethyl

60-70% isopropyl alcohol for 15 minutes

1. No rinsing 2. Readily available 3. Fast-drying 4. Used alone

1. Not effective against bacterial spores

Somewhat temperaturedependent (higher temperature increases biocidal effect) Chlorine-releasing compounds may require other conditions of use, e.g., pH contact time, concentration

60-95% ethyl alcohol for 15 minutes; some applications to 30%

Table 3-2 50

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Table 3-2 Chemical Sanitizers

continued

General types and uses are listed below. Refer to manufacturer’s use directions and material safety data sheets (MSDS). Suggested Concentration & Contact Times

Description

Phenols (Phenolic derivatives)

Phenyl and/or 1:200 solution chlorinated phenols

1. Cleans 2. Excellent activity 3. Deodorizes

1. Must be formulated 2. Rinsing required 3. Used solution may be unstable (use within 2-3 hours) 4. Worker exposure limits 5. Activity reduced by presence of organic matter

Pine

Pine oils formulated with soap or surfactants

1. Cleans 2. Excellent activity 3. Deodorizes 4. Degreases

1. Must be formulated 2. Odor may be incompatible with certain products

Formalin

1% (as formaldehyde) 37% w/v 30 minutes solution (aqueous, as free formaldehyde)

1. Excellent activity 2. Readily available 3. Can be used alone

1. Odor 2. Highly reactive 3. Toxicity 4. Should be used cold in a closed system 5. Skin protection required 6. NIOSH/OSHA exposure limit to formaldehyde is airborne concentration ceiling of 0.1 ppm, 15 minute contact time13

Phosphoric acid

H3PO4 solution

Varies, refer to manufacturer’s use directions

1. Good activity 2. Stainless steel 3. Used cold 4. Short contact time

1. Used under acidic conditions to be effective. 2. Most used in combination with iodophors

Hydrogen peroxide

Purchased as a stabilized solution

1.5% of a 35% solution for 30 minutes

Effective vs. organics

1. Explosive at high levels 2. Reactive 3. Minimal disinfection capacity

Chlorine dioxide

Mixture of oxychloro species: (chlorite/ chlorate/ oxychloro species, chlorine dioxide)

1-10 ppm Cl02 100200 ppm expressed as chlorine dioxide

1. Strong oxidizing chemical 2. More tolerant of organic matter than chlorine 3. Less corrosive to stainless steel 4. Less pH sensitive

1. Sensitive to light and temperature 2. NIOSH recommended employee exposure limit to chlorine is 0.5 ppm ceiling for 15 minutes13

Per manufacturer’s use directions

Advantages

Disadvantages

SECTION 3 CLEANING AND SANITIZATION

Type

Table 3-2 SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

51

Table 3-2 Chemical Sanitizers

continued

General types and uses are listed below. Refer to manufacturer’s use directions and material safety data sheets (MSDS).

Description

Peroxyhydrogen peroxide

Peroxyacetic acid Peracetic acid

Acid anionics

Ozone14

Suggested Concentration & Contact Times

Advantages

Disadvantages

Refer to manufacturer’s instructions

1. Low residue 2. Environmentally responsible 3. Broad spectrum bacteria 4. Generally non-corrosive to stainless steel and aluminum 5. Relative tolerance to organic soil 6. Active at up to pH 7.5 7. Good activity against biofilms

1. Metal ion sensitivity 2. Corrosive to soft metals 3. Odor of concentrate 4. Varied activity against fungi 5. Corrosive and toxic only in concentrated solutions (>40%) 6. Potential of fire hazard

Anionic surfactants and acids

Minimum 100 ppm

1. Stable 2. Generally non-corrosive 3. Non-staining 4. Low odor 5. Not affected by hardwater minerals 6. Removes and controls mineral films

1. pH sensitive (optimal pH 2-3) 2. Limited and varied antimicrobial activity (poor vs. mold & yeast) 3. High foaming

Oxidizing gas

1-3 ppm 30 minutes

1. Powerful oxidizing gas 2. Broad-spectrum activity 3. Fast acting 4. Deodorizes 5. Minimal handling

1. Unstable 2. pH sensitive (optimal pH 6-8.5) 3. Temperature sensitive 4. Corrosive 5. No residual 6. Must be generated at site 7. OSHA airborne exposure limit 0.1 ppm ozone13

SECTION 3 CLEANING AND SANITIZATION

Type

Table 3-2

52

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 3: CLEANING AND SANITIZATION

Table 3-3 Physical Sanitization Methods Suggested Concentration & Contact Times

Type

Description

Steam Heat

Water at 100°C 30 minutes Temperature must be reached at furthest point in system

1. High product compatibility 2. Easy availability 3. Efficacious 4. Breaks down biofilm 5. Non-selective

1. Possible residues (boiler/pipes) 2. Excessive dwell time 3. High energy consumption 4. Condensation 5. High humidity

Hot Water

80° - 100°C

30 minutes

(70° - 80°C)

(2 hours)

1. High product compatibility 2. Easy availability 3. Effective over long distances of pipes 4. Exit monitoring simple 5. Not corrosive 6. No residue 7. Non-selective to all microbial genera

1. Volume required 2. High energy consumption 3. High humidity 4. Condensation 5. Excessive dwell time

Electrical heat tape

In combination with other methods

Effective for hard-to-reach equipment or piping (specialized or limited use)

Not for general use

Direct Heat

Advantages

Disadvantages*

* Heat may cause equipment damage by expansion of close-fitting and/or moving parts. Heat must be used with thermally stable materials. Scalding water poses a potential hazard. Table 3-3

SECTION 3 CLEANING AND SANITIZATION

SECTION 3: CLEANING AND SANITIZATION

|

CTFA MICROBIOLOGY GUIDELINES

|

53

REFERENCES 1. U.S. Food and Drug Administration. 2006. “Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs; General.” In 21 CFR, Part 210. http://www.fda. gov. 2. U.S. Food and Drug Administration. 21 CFR, Part 211. 3. U.S. Food and Drug Administration. 2000. “Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients.” In Draft ICH Consensus Guideline. http:// www.fda.gov/cder/guidance/4011dft.pdf. 4. Brannan, Daniel K. (Ed.). 1997. Cosmetic Microbiology: A Practical Handbook. Florida: CRC Press. 5. Bloomfield, S. F., and R. M. Baird. (Ed). 1996. Microbial Quality Assurance in Cosmetics, Toiletries and Non-Sterile Pharmaceuticals. Philadelphia: Taylor & Francis.

SECTION 3 CLEANING AND SANITIZATION

6. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. “Annex 2 – Premises”. In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association. 7. Block, Seymour Stanton. 2000. Disinfection, Sterilization, and Preservation. Philadelphia: Lippincott Williams & Wilkins. 8. Russell, A. D., W. B., Hugo, G. A., Ayliffe. 1999. Principle and Practices of Disinfection, Preservation and Sterilization. Vermont: Blackwell Scientific Publications.

54

|

CTFA MICROBIOLOGY GUIDELINES

|

9. U.S. Environmental Protection Agency. 1998. “National Primary Drinking Water Regulations: Disinfectants and Disinfection Byproducts Notice of Data Availability.” 40 CFR Part 141. http:// www.epa.gov/safewater/standard/v&efrn.pdf. 10. McLaughlin, Malcolm C., and Alan S. Zisman. 1998. The Aqueous Cleaning Handbook: A Guide to Critical-Cleaning Procedures, Techniques and Validation. Rosemont, NJ: Morris-Lee Publishing Group. 11. American Society for Testing and Materials. 2000. “E1153-94 Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate Non-Food Contact Surfaces,” In: ASTM Standards: Biological Effects and Environmental Fate; Biotechnology; Pesticides, vol. 11.05. West Conshohocken, PA. 12. Ecolab Inc. Food and Beverage Division. 2003. “Making the Right Choices Sanitizers”. 13. U.S. Department of Health and Human Services (NIOSH). 2005. NIOSH Pocket Guide to Chemical Hazards. http://www. cdc.gov/niosh/npg/. 14. Olsen, Wayne P. “Ozone.” 1999. In: PDA Journal of Pharmaceutical Science and Technology 53: 125. Bethesda, MD: Parenteral Drug Association.

SECTION 3: CLEANING AND SANITIZATION

SECTION 4

Microbiology Staff Training

INTRODUCTION The staff of the microbiology department has an essential role in maintaining product quality that meets development specifications, marketing design, and customer expectations. The knowledge and skills of this group are crucial. Microbial test results must be accurate and reliable so that decisions based on the test data can be made with confidence. Training of the microbiology laboratory staff should cover the following general areas: • Following documented procedures • Qualifying staff to perform the analysis • Adhering to aseptic technique • Checking equipment function • Performing routine equipment maintenance • Laboratory controls and documentation This training provides confidence that test results are accurate and can be relied upon during the decision-making process. Many different types of microbiological tests may be performed in a cosmetic microbiology laboratory. These can include content testing of microbiologically susceptible raw ingredients and finished products, preservative challenge testing of product formulations, and the analysis of environmental test samples such as cleaning and sanitization swabs, air, or water samples from a cosmetic manufacturing facility. If cosmetics and over-the-counter (OTC) drugs are tested in the same laboratory, refer to FDA guidelines for the manufacture of OTC drugs and to relevant chapters in the United States Pharmacopeia (USP).1,2,3

SECTION 4: MICROBIOLOGY STAFF TRAINING

|

CTFA MICROBIOLOGY GUIDELINES

|

55

SECTION 4: MICROBIOLOGY STAFF TRAINING

There are two goals in having a training program for the employees in a cosmetic microbiology laboratory: First, to provide an in-depth, well-rounded program in how and why a certain microbiological test is to be conducted on a particular test sample; and second, to ensure that the microbiological testing for a particular type of sample will be performed exactly the same way by each employee every time a sample is received for testing. The purpose of this guideline is to provide information regarding requirements for a microbiology staff training program.

ESTABLISHING A PROGRAM The establishment of a training program should include, but not be limited to, the understanding of microbiological concepts, review of Standard Operating Procedures (SOPs), and review of test methods or procedures. It is important that the individual have full understanding of the principles of aseptic technique. Internal or external training classes can be provided as part of the training program for an employee. It is recommended that hands-on training be included to demonstrate proficiency in using laboratory equipment and conducting microbiological test methods. It is recommended that a knowledgeable, qualified individual possessing appropriate academic and work experience should train new employees to the laboratory.

Training Frequency All new laboratory employees should receive training prior to beginning work in the laboratory. In addition, it is recommended that all current staff employees receive periodic re-training at intervals appropriate to keep them current and proficient in performing the various procedures for which they are responsible. It is the responsibility of management to ensure that each staff member is updated or trained according to the company’s policy or Standard Operating Procedures.

Documentation For each employee in a cosmetic microbiology laboratory, a training record or log should be established. The documentation should include, but not be limited to, training and dates when proficiency has been demonstrated for each particular test method, technique, and policy or procedure used by that individual during a workday. It is important that no laboratory staff member be allowed to perform any laboratory task until documentation is established indicating sufficient training was received and proficiency was demonstrated. The trainer should either initial or sign and date the training record or log to verify that the training was received and completed for that task. Each training record or log should be periodically reviewed and initialed or signed and dated by the supervisor of the testing laboratory. Records should be kept for an appropriate length of time. It is also important that proper documentation exists verifying that the trainer has the necessary experience and knowledge to conduct a particular microbial test method or use a particular piece of laboratory equipment.

Topics

SECTION 4: MICROBIOLOGY STAFF TRAINING

The training topics will often depend upon the laboratory equipment utilized, testing methods performed, laboratory function, and individual job responsibilities. The tables in the sections that follow suggest topics and elements that should be included in a microbiology staff training program.

56

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

LABORATORY ORIENTATION A general orientation should be given to any new individual as an introduction for entering the microbiology laboratory. The topics covered during orientation should remain general in scope, give an overview of Standard Operating Procedures (SOPs), and cover guidelines within the laboratory as an introduction. The topics listed in Table 4-1 may be included in a general orientation. Other topics may be added at the discretion of the person developing the training. More specific topics are discussed in detail in sections that follow.

MICROBIOLOGY LABORATORY Equipment

SECTION 4: MICROBIOLOGY STAFF TRAINING

|

CTFA MICROBIOLOGY GUIDELINES

|

57

SECTION 4: MICROBIOLOGY STAFF TRAINING

Equipment availability and usage will vary depending on the testing performed in each laboratory. Most laboratories will contain many of the instruments listed below. Employees should be trained in the safe and effective use of each piece of equipment needed to fulfill their job function. The list below is not exhaustive; however, it does contain many of the basic pieces of equipment requiring calibration. Each laboratory will need a customized list depending on their particular testing requirements. Common microbiology laboratory equipment includes: • Balances • Sterilizers/Autoclaves • pH Meters • Water Baths • Incubators • Refrigerators • Low temperature freezers • Automatic pipetting/dispensing devices (e.g. pipettors, micropipettors, dispensing pumps, etc.) • Laminar flow hoods/biological safety cabinets • Microscopes • Stereoscopes • Laboratory water system • Bunsen burners • Colony counters • Sample mixing devices (e.g., vortexes, Waring® Blenders, etc.) • Laboratory shakers • Centrifuges • Laboratory ovens • Air samplers • Stopwatches • Spectrophotometers • Lyophilizers

• • • • •

Automated microbial identification systems Automated microbial counting devices Water activity instruments Dishwashers Automated data collection systems

Calibration of Laboratory Equipment Every testing laboratory has pieces of equipment that require periodic calibration to verify that they are maintained and operated in accordance with the manufacturer’s specifications. Training in verification of the operational status of the equipment, including its calibration, is important. Besides learning how to use a piece of laboratory equipment, an employee should be trained in how to recognize when an instrument is not operating correctly. The list below is not exhaustive; however, it contains the basic equipment that needs periodic calibration and is found in most microbiology laboratories. Additional information on calibration of microbiological equipment is given in Table 8-1 in “Microbiology Lab Audit” in Section 8. • Balances (e.g., weight checks) • pH Meters (e.g., daily) • Micropipettors • Thermometers (e.g., test and standard) • Temperature recorders • Water activity instruments • Sterilizers/Autoclaves • Timers • Temperature recorders • Chamber pressure gauges • Heat distribution and penetration of chamber and chamber loads • Stopwatches • Spectrophotometers • Laminar/Biological safety cabinets • Air samplers • Automated microbial identification systems • Automated microbial counting devices

SECTION 4: MICROBIOLOGY STAFF TRAINING

LABORATORY TECHNIQUES Common Techniques Table 4-2 contains common key elements to be included in a training program for an individual responsible for conducting tests in a microbiology laboratory. The list contains key microbiological

58

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

techniques that may be employed in the laboratory; however, it is not inclusive of all the different types of techniques that might be used in every laboratory. Additional techniques performed in your laboratory should be added to your training program. Specific tests are discussed in detail in sections that follow.

Microbial Content Testing Microbial content testing is performed on raw ingredients, packaging components, and finished goods that are susceptible to microbial contamination. It is important that an individual performing these types of tests be trained and have demonstrated proficiency in using the techniques listed in Table 4-3.

Preservative Effectiveness Testing With the exceptions of the preparation of microbial challenge inocula and inoculated test samples, many techniques used for conducting preservative effectiveness tests are common to the routine analysis of test samples for microbial content. In addition to these laboratory manipulations, training should include the calculation of percentage or logarithmic reduction and the interpretation of acceptance criteria.

ENVIRONMENTAL MONITORING General To effectively monitor the quality of the cosmetic manufacturing plant environment, laboratory employees with the responsibility for conducting environmental monitoring should be trained in all methods currently in use. Environmental testing comprises three major categories: surface sampling, air sampling, and water analysis. Refer to “Microbiological Evaluation of the Plant Environment” (Section 2) in these guidelines for information on conducting environmental monitoring in a manufacturing plant. Training should be based on written procedures which include: • Methods and materials • Suggested sites to monitor • Frequency of testing • Interpretation of results to include specification levels, where applicable • Determination of alert and action levels, documentation • Communication of results • Corrective action procedures

|

CTFA MICROBIOLOGY GUIDELINES

|

59

SECTION 4: MICROBIOLOGY STAFF TRAINING

SECTION 4: MICROBIOLOGY STAFF TRAINING

Environmental Monitoring Test Methods Surfaces For monitoring the microbial content of surfaces in a manufacturing plant, a laboratory employee should be trained in how to use one or more of the following surface sampling test methods: • Swab • Contact plate (e.g., RODAC plates) • Flexible films or contact slides • Final rinse water Air For monitoring the microbial content of air in different locations of a manufacturing plant, a laboratory employee should be trained in how to use one or more of the following air sampling methods: • Settling plate (sedimentation plate) • Centrifugal air sampler • Sieve impaction sampler • Slit-to-agar sampler • Liquid impinger • Multi-stage particle sizing sampler • Membrane filter • Compressed air Water For determining the microbial content of water samples in a manufacturing plant, a laboratory employee should be trained on how to perform the activities listed in one or more of the areas in Table 4-4.

SECTION 4: MICROBIOLOGY STAFF TRAINING

IDENTIFICATION OF MICROBIAL ISOLATES Microorganisms are often isolated in environmental, microbial content or preservative challenge test samples. At times, there may be a need to identify microbial isolate to either the genus or species level. Results may determine whether a sample passes or fails and may provide information on the source of the contamination. It is expected that the individual conducting the testing has the necessary educational background and proper training to correctly identify a microbial isolate to the genus or species. It is strongly recommended that this individual demonstrate proficiency in performing microbial identifications. Table 4-5 contains key microbiological tests that may be employed to identify microbial isolates. The list may not be all inclusive. Additional microbial identification tests performed on isolates in different laboratories should be added to the laboratory training program.

60

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

CLEANING AND SANITIZATION Proper cleaning and sanitization of the manufacturing equipment and facility are vital to ensure microbial quality in the manufacture of cosmetic and personal care products. Refer to “Cleaning and Sanitization” (Section 3) for detailed information for a cosmetic manufacturing facility. Depending upon the structure of the company, the role of the microbiologist and laboratory staff may include the following: • Advising on hygienic equipment design • Cleaning and sanitization procedures and validation • Performing equipment monitoring to analyze for microbial bioburden • Auditing cleaning and sanitization procedures • Interpreting test results • Advising on action steps The microbiology department is, by function, an integral part of the cleaning and sanitization program. It is recommended that training include the following: • Aseptic sampling • Testing methods such as: − Swabbing − Direct contact − Final rinse water • Validation protocol • Ongoing environmental monitoring procedures • Documentation − Documentation of validation and qualification of cleaning and sanitization procedures − Logs for equipment cleaning and sanitization history • Basic understanding of: − Cleaning ♦ Chemicals ♦ Physical methods − Sanitizers ♦ Physical methods ♦ Chemical (including pH range, soil effects, concentration, and contact time)

|

CTFA MICROBIOLOGY GUIDELINES

|

61

SECTION 4: MICROBIOLOGY STAFF TRAINING

SECTION 4: MICROBIOLOGY STAFF TRAINING

• Basic Understanding of sanitary equipment design and equipment function − Process water system − Processing equipment ♦ Mixers/kettles ♦ Transfer pumps ♦ Transfer pipes and hoses ♦ Valves and gaskets ♦ Storage tanks and vessels ♦ Ancillary and associated equipment (including scoops, pitchers, funnels) ♦ Packaging equipment

CONCLUSION It must be realized that the topics listed above and the suggested elements for a training program for a microbiology laboratory cannot be all-inclusive. These elements are only for guidance on the components of a microbiology staff training program. If a microbial technique, procedure, or a piece of laboratory equipment is not listed here and is being performed or used in a microbiology laboratory, then it should be included in the training record or log for each employee whose job duties include using the equipment or performing the procedure.

SECTION 4: MICROBIOLOGY STAFF TRAINING

Proficiency testing, as a means of demonstrating competence, is an integral part of a training program.

62

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

Table 4-1 General Orientation to the Microbiology Laboratory Topics

Areas to be Covered

Organizational Structure

• Vice-president • Director • Lab manager • Microbiologists • Technicians • Contract temporary staff • Customers

Introduction to Laboratory

• Director

Personnel and Customers

• Laboratory manager • Microbiologists • Technicians • Contract temporary staff • Relevant customer staff

Types of Microbiological

• Environmental monitoring

Testing Conducted

• Microbial content testing • Preservative challenge testing • Selective media • Culture identification • Other tests

Laboratory Rules and Safety4

• Laboratory safety manual • Occupational safety training (29 CFR)

Introduction to Current Cosmetic Good Manufacturing Practices (GMPs)5

• Documentation of methods and test results • Record keeping rules • Out-of-Specification investigations and documentation • Labeling • Dating • Signatures • Expiration dates • Lot numbers • Other items as appropriate

Good Laboratory Housekeeping

• General organization and cleanliness • Cleaning schedule • Cleaning checklist • Others where applicable

Table 4-1 SECTION 4: MICROBIOLOGY STAFF TRAINING

|

CTFA MICROBIOLOGY GUIDELINES

|

63

SECTION 4: MICROBIOLOGY STAFF TRAINING

• Waste handling and disposal

Table 4-2 Key Microbiological Techniques Technique

Detailed LIst

General

• Aseptic technique • Use of controls (e.g., positive and negative) • Check expiration dates of media, reagents, etc.

Media and Broth Preparation

• Medium/broth selection for application

Diluent Preparation

• Ingredient/component weighing • Water selection • Equipment selection • Sterilization • Sterility/growth promotion controls • Shelf life • Documentation • Isolation

Organism Identification

• Gram stain • Spore stain • Lacto phenol cotton blue – mold • Automated methods (as applicable) • Catalase, oxidase, coagulase testing Maintenance of Microbial

• Lyophilized or frozen culture reconstitution

Culture Stocks

• Rotation and generation criteria, including identification criteria • Preparation of slants • Documentation for traceability of stocks

Sample Preparation and Testing

• Inocula preparation and enumeration • Sample weighing • Pour plates • Streaking • Broth enrichment • Incubation temperatures and times • Colony counting

Waste Disposal

• Autoclaving/sterilization • Labeling

SECTION 4: MICROBIOLOGY STAFF TRAINING

• Chemical and biological hazardous waste handling

Table 4-2 64

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

Table 4-3 Microbial Content Testing Technique

Detailed LIst

How to Sample

• Liquid or granular/solid raw ingredients • Packaging components (e.g., applicators, sponges, etc.) • Bulk finished products • Finished products

Sample Preparation

• Water-miscible/dispersible raw ingredients and finished products • Water immiscible raw ingredients and finished products • Packaging components

Enumeration

• Bacterial and yeast/mold plate count procedures — Membrane filtration method — Pour plate method — Automated methods

Detection

• Enrichment testing

Microbiological Acceptance Criteria

• Release test specifications/reject procedures — Raw ingredients — Packaging components — Finished goods

Verification of Test Methods

• Demonstrate that enumeration and detection methods are capable of recovering microorganisms from test samples Table 4-3

Table 4-4 Water Monitoring Activities Area of Training

Detailed LIst

Sample Collection

• Key sites in applicable water system: — Non-circulating hot/cold (with or without chlorine) — Circulating hot or cold — Other • Importance of timing – holding of sample

Use of Chlorine Inactivators

• Where Required

Test Methods:

• Pour plate method

Total Count

• Membrane filtration method • Paddle Testers (e.g., Membrane Dip Samplers, Agar Dip Slides)

Coliform Screening

• Membrane filtration • Presence/Absence Enrichment • Differential/Selective Agar

Pseudomonas detection

• Pour Plate Method • Membrane Filtration Method Table 4-4

SECTION 4: MICROBIOLOGY STAFF TRAINING

|

CTFA MICROBIOLOGY GUIDELINES

|

65

SECTION 4: MICROBIOLOGY STAFF TRAINING

• Most Probable Number (MPN)

Table 4-5 Microbial Identification Tests Identification of Bacteria

Test

Staining

• Gram stain (e.g., Gram-negative and Gram-positive) • Potassium Hydroxide (KOH) Test - for use on inconclusive Gram stain results for a bacterial isolate to separate them into Gram-negative and Gram-positive bacteria groups. • Morphology (e.g., bacillus or cocci) • Spore Stain

Biochemical Tests for Gram-negative Bacilli

• Cytochrome Oxidase Test -to separate Gram-negative bacilli into fermentor and non-fermentor groups. • Oxidation/Fermentation Test -Glucose for Gram-negative bacilli • Catalase Test - to separate Gram-positive cocci into Catalase Positive and Negative groups.

Biochemical Tests for Gram-positive Cocci

• Coagulase Test - to separate Catalase Positive Grampositive cocci into Coagulase Positive and Negative groups. • Hemolytic Reactions - to identify the various members of Catalase-negative Gram-positive cocci species. Specific Biochemical Reactions

• Assimilation/Utilization of Specific Chemicals • Fatty Acid Cell Wall Analysis

Genotypic Methods

• DNA Analysis System

Identification of Yeast

Test

Staining

• Morphology

Morphology

• Germ Tube Test

Use of Specific Biochemical Reactions

• Assimilation of Specific Chemicals

Identification of Mold

Test

Examination

• Morphology/Slide Preparation

SECTION 4: MICROBIOLOGY STAFF TRAINING

Table 4-5

66

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

ADDITIONAL INFORMATION Akers, M. 1993. “cGMP Education and Instruction: A Corporate Approach to Employee Training Worldwide.” Pharmaceutical Technology. 17: 51-60. Beauchemin, K., D. Gallup, and M. Gillis. 2001. “Read and Understand vs. a CompetencyBased Approach to Designing, Evaluation, and Validating SOP Training.” PDA Journal of Pharmaceutical Science and Technology, 55 (1): 10-15. Deluca, P.P. 1983. “Microcontamination Control: A Summary of an Approach to Training.” PDA Journal of Pharmaceutical Science and Technology, 37(6): 218-224. Gallup, D., K. Beauchemin, and M. Gillis. 1999. “A Comprehensive Approach to Compliance Training in a Pharmaceutical Manufacturing Facility.” PDA Journal of Pharmaceutical Science and Technology, 53(4): 163-167. Gallup, D., K. Beauchemin, and M. Gillis. 1999. “Competency-Based Training Program Design.” PDA Journal of Pharmaceutical Science and Technology, 53(5): 240-246. Levechck, J.W. 1991. “Training for GMPs.” Journal of Parenteral Science and Technology, 45(6): 270-275. Parenteral Drug Association, Inc. 2001. “Technical Report No. 35, A Proposed Training Model for the Microbiological Function in the Pharmaceutical Industry.” PDA Journal of Pharmaceutical Science and Technology, 55(6).

REFERENCES 1. U.S. Food and Drug Administration. 2006. “Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs General.” 21 CFR, Part 210. 2. U.S. Food and Drug Administration. July 2000. “Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients.” Draft ICH Consensus Guideline. http://www.fda.gov/cder/ guidance/4011dft.pdf.

4. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Institutes of Health. 2007. Biosafety in Microbiological and Biomedical Laboratories (BMBL): 5th Edition.” Washington, DC. http://www. cdc.gov. 5. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

SECTION 4: MICROBIOLOGY STAFF TRAINING

|

CTFA MICROBIOLOGY GUIDELINES

|

67

SECTION 4: MICROBIOLOGY STAFF TRAINING

3. United States Pharmacopeia. 2007. United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD. http://www.usp.org.

SECTION 4: MICROBIOLOGY STAFF TRAINING

68

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 4: MICROBIOLOGY STAFF TRAINING

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIAL

SECTION 5

Handling, Storage and Analysis of Raw Material INTRODUCTION Raw materials used by the cosmetic industry are not expected to be sterile as received. Some commodities, especially those of natural origin, may contain large microbial populations. The incorporation of such raw materials into product formulations is undesirable because the organisms introduced could: • Contaminate equipment and environment • Present a health hazard • Produce undesirable changes in products • Reduce preservative effectiveness

CATEGORIES A program to control organisms in raw materials should consider the physical and chemical nature of the raw materials as well as the subsequent processing involved in the manufacture of quality products. In general, raw materials may be categorized as: • Hostile - A raw material that will not support and may inhibit the growth of microorganisms. • Inert - A raw material that may act as a carrier of microorganisms but ordinarily will not promote microbial proliferation. • Supportive - A raw material that serves as a nutritional substrate and supports microbial growth. • Preserved - A raw material to which antimicrobial substances have been added to inhibit microbial growth.

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS

|

CTFA MICROBIOLOGY GUIDELINES

|

69

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIAL

STABILITY Raw materials have various degrees of stability throughout their shelf life, which may be affected by the presence of microorganisms. To monitor changes in microbial content, raw materials should be examined upon receipt and on a regular basis by acceptable microbiological procedures.

EXPIRATION Expiration dates should be established as determined by history and in-house experience. An appreciable change from the normal microbial profile indicates a possible problem, which should be investigated.

RECEIPT Raw materials received should be properly labeled, placed on quarantine status, and held until released by Quality Assurance. For further guidance, refer to “Annex 17 - Sampling” in the CTFA Quality Assurance Guidelines.1

STORAGE Raw materials should be stored under conditions that minimize the possibility for microbial contamination. Among the various factors to consider are: • Control of environmental factors such as temperature, humidity, ventilation and light • Proper housekeeping practices • Rodent, small animal and insect-control programs • Quarantine systems • Special storage conditions where indicated Procedures for the control of raw materials should be adequately outlined for the department responsible and reviewed and approved by qualified personnel. Once established, the procedures should be reviewed on a periodic basis.

TRANSFER Transfer systems for raw materials include sanitized containers, transfer lines, pumps, and related equipment. These systems should be evaluated on an individual basis depending on the specific raw material involved. The raw material categories listed above will aid in this evaluation. For example, a supportive raw material will require greater consideration (i.e., stringent, sanitary handling) and more monitoring than a hostile one.

70

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS

Appropriate control procedures are required for sampling raw materials. • Personnel - Personnel responsible for sampling raw materials should be trained in aseptic sampling techniques, preferably by a qualified microbiologist. Individuals with contagious illnesses or open lesions should not touch or otherwise contact materials being sampled.2 • Containers - All sampling containers should be sterile and of suitable size. • Utensils - All sampling utensils should be sterile and suited to the particular raw material. Long-handled dippers, syringes, sampling tubes, “thieves,” spatulas, spoons, and pipettes are all examples of sampling utensils. Some of these are commercially available as presterilized items. • Techniques - To ensure that samples are representative of the lot or batch, a logical sampling plan should be developed.1 When samples are obtained for microbiological analysis the following procedures should be observed. • Sanitize sample sites externally. • Obtain subsurface samples of dry raw materials. • Mix liquids for homogeneity. • Where possible, take representative samples from the top, middle and bottom of bulk tanks. • Properly seal containers.

TESTING Microbiological testing of raw materials can be accomplished according to “M-1 Determination of the Microbial Content of Cosmetic Products” (Section 18) or other appropriate method. The nature of the raw material will determine the method used. This method or any departure from it must be validated through appropriate testing.

REFERENCES 1. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. “Annex 17 – Sampling”. In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

2. Bailey and Nikitakis. “Annex 1 - Personnel and Training.”

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS

|

CTFA MICROBIOLOGY GUIDELINES

|

71

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIAL

SAMPLING

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIAL

72

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS

SECTION 6

Microbiological Sampling

Appropriate microbiological techniques are needed for sampling raw materials, bulk in-process, packaging components, and finished goods to ensure cosmetic product quality. Although each area has its own specific needs, there are basic similarities that are vital to all. From the time raw materials arrive until the finished product emerges, product history and proper identification are essential. In general, aseptic techniques should be followed for valid evaluations of samples. The frequency, sampling and screening methods may vary, but the need for monitoring by qualified personnel is of utmost importance.

CATEGORIES OF SUSCEPTIBILITY All raw materials, packaging components, bulk in-process, and finished goods differ in susceptibility to microbial growth. In order to assess the risk of growth occurring in a material, it is helpful to establish categories of susceptibility. These categories of susceptibility influence the extent of sampling and testing required for each material.

Category 1 (High Susceptibility) High-susceptibility materials include: • Eye products (aqueous and semi-aqueous) • Emulsions • Geriatric and pediatric preparations • Cream lip preparations (water-based emulsions) • Water-based products • Raw materials of natural origin

SECTION 6: MICROBIOLOGICAL SAMPLING

|

CTFA MICROBIOLOGY GUIDELINES

|

73

SECTION 6: MICROBIOLOGICAL SAMPLING

INTRODUCTION

Category 2 (Medium Susceptibility)

SECTION 6: MICROBIOLOGICAL SAMPLING

Medium-susceptibility materials include: • Pressed powders (compact powders, blushing powders) • Stick preparations, make-up sticks • Loose powders (face) • Bath powders (dusting talc) • Some aerosol products • Eye powders, pressed and loose, and stick preparations

Category 3 (Low Susceptibility) Low-susceptibility materials include: • Alcoholic preparations (≥ 20%) • Deodorants and anti-perspirants • Bath salts • Many aerosol products • Raw materials with antimicrobial activity

Category 4: (Nonsusceptible) A nonsusceptible material is one that by nature of its components, exclusive of preservatives, will not support the survival of vegetative organisms. NOTE: The susceptibility of packaging components and other raw materials should be evaluated based on their composition and the nature of the product with which they are used. The above categories are based on the following: • History - Necessity and frequency of testing a material are based on past microbiological profiles. Determining the microbial content of a designated number of batches over a period of time helps to establish the susceptibility category. • Susceptibility Tests - Materials may be challenged with microorganisms and tested for susceptibility.

SAMPLING DEVICES The following devices for sampling, available from scientific supply houses, may be used: • Sterile Thief can be used for liquid and/or powder. Glass is not recommended. • Sterile Scoops are used for powders. Glass is not recommended. • Sterile Cups can be used for liquid and/or powder. Avoid contact of hands with products.

74

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 6: MICROBIOLOGICAL SAMPLING

PERSONNEL The individual(s) responsible for sampling should be trained in aseptic sampling technique by a microbiologist or other qualified person and should be familiar with visual characteristics of containers and/or materials to be sampled.

RAW MATERIALS

Sampling Technique Aseptic technique should be followed at all times by trained personnel. Sampling should be performed with sterile equipment, which can be of stainless steel, plastic, or any other microbiologically acceptable material. Devices for sampling include ladles, cups, spatulas, scoops, and spoons. In general, glass devices should be avoided because of the danger of breakage. Each container should be sampled with a separate sterile device.

Techniques for Specific Containers Bags 1. Place the bag in a flat position. 2. Clean and sanitize the area to be opened. 3. Aseptically make an opening in the bag. 4. Aseptically remove the sample, transfer to a sterile container, and cap the container. 5. Close the opening of the bag carefully and seal it. 6. Label, initial and date the sample container. 7. Identify each bag sampled.2 Tank Car and Storage Tanks Tank cars and storage tanks present unique problems in sampling. As it is imperative to obtain a representative sample, it is necessary to sample top and bottom. For example, a “thief,” such as a sterile plastic bottle (of unreactive material such as polypropylene) held in a modified water sampler holder, may be used to transfer the sample to a sterile, properly labeled container.

SECTION 6: MICROBIOLOGICAL SAMPLING

|

CTFA MICROBIOLOGY GUIDELINES

|

75

SECTION 6: MICROBIOLOGICAL SAMPLING

Designated personnel should be notified of the receipt of each shipment of raw materials. The shipment should be inspected for physical damage as indicated by leakage of liquids or powders, rusty or dented containers, and broken or torn containers that expose the contents to outside contamination. Tank car shipments may be inspected through the top for gross contamination. Any container damaged in such a manner that the contents could be contaminated should be rejected and the supplier notified.1 Each container should be properly labeled.2

SECTION 6: MICROBIOLOGICAL SAMPLING

Drums Containing Liquids 1. Clean and sanitize the cover. 2. If the entire lid is removable, a ladle-type device may be employed. When there is only a small opening, a dipper-like device is preferred. 3. Transfer the sample to a sterile, properly labeled container, and cap the container. 4. Identify each container sampled (label, initial and date).2 Drums Containing Dry Materials 1. Clean and sanitize the cover. 2. Sample from the container using a sterile sampling device. 3. Transfer the sample to a sterile, properly labeled container and cap the container. 4. Identify each container sampled (label, initial and date).2

Sample Properties The intrinsic properties and microbiological history (internal monographs developed from previous microbiological assessments) of a raw material are of prime importance in ascertaining sampling frequency. The type and homogeneity of the material will also play a role in this determination. A microbiologically nonhomogeneous material generally requires a greater number of samples. Raw material lots, depending on the type of material, amount ordered, and/or the supplier, are received in various forms: boxcars, truckloads, bags, boxes and drums. A determination of the number of samples per lot to be taken (whether the lot is in the form of a single boxcar or in the form of many bags) should be made. Typical sampling plans can be found in the CTFA Quality Assurance Guidelines.2 In most cases, 30-100 grams of sample are aseptically taken from each container or area of the container chosen by one of the above methods. It is also feasible and practical to test composite samples of the same lot number of certain raw materials, which are by previous analysis and/ or nature considered microbiologically homogeneous. If composites of a lot are shown to be unacceptable by in-house standards, then all previously sampled containers should be reassayed.1 More extensive sampling and testing may also be necessary.

Stability Raw materials should be investigated to determine susceptibility to microbial proliferation, including the effect of storage conditions. Retest intervals should be scheduled to determine the continued adherence to microbial content specifications.

Stored Unopened Containers “Low susceptibility” raw materials should be sampled as necessary. “High susceptibility” raw materials should be sampled on a defined periodic basis or prior to use. If no history is available, the raw materials should be retested just prior to use in manufacturing. 76

|

CTFA QUALITY ASSURANCE GUIDELINES

|

ANNEX 6

FINISHED PRODUCTS/LOT ID AND CONTROL

Partially Used Containers Partially used and resealed raw materials, especially of “high susceptibility,” which have been stored for a defined, preestablished period of time, should be retested just prior to use in manufacturing.

PACKAGING COMPONENTS

Sampling Upon receipt of a shipment of components, a trained quality-assurance sampler should check for proper identification. The quality-assurance sampler randomly samples the shipment. The shipment is then sent to a designated area until it has been released. A visual examination should be done for obvious defects, such as mold, dust, dirt, insects, or other extraneous materials. If there is any evidence of these, a microbiological examination should be done. As a rule, most components are considered microbiologically safe and not routinely tested except for applicators, brushes and puffs and, in predetermined cases, those that are in direct contact with “highly susceptible” products. Only surface areas that come in direct contact with the product are tested.

Sampling Techniques 1. Clean and sanitize area of carton or containers to be opened. 2. Aseptically remove a sufficient number of pieces to ensure that a representative sample is submitted. 3. Place samples in a suitable bag or container and properly seal to prevent contamination. 4. Place identification label on the outside of the sample containing the following information: − Name of item − Supplier − Date received − Date submitted to microbiology department − Lot number − All other pertinent information needed for the identification of the sample. 5. Properly identify, initial and date each carton or container sampled.

SECTION 6: MICROBIOLOGICAL SAMPLING

|

CTFA MICROBIOLOGY GUIDELINES

|

77

SECTION 6: MICROBIOLOGICAL SAMPLING

Components should be inspected before shipment by the supplier. The burden of correcting problems and minimizing defects should be the responsibility of the supplier; however, it is still the cosmetic manufacturer’s responsibility to have an acceptable component to give the consumer.

Storage When the sampling of components meets all inspecting criteria, the shipment is then accepted. The warehouse is notified so that the balance of the shipment can be stored in the proper place. Components should be kept elevated from the ground in a relatively clean dust-free area with a good rotation system.

SECTION 6: MICROBIOLOGICAL SAMPLING

BULK IN PROCESS Bulk products should be sampled and tested to ensure acceptability of the product before filling, as a secondary check on sanitary manufacturing practices, to build a product profile, and as an economic control to save on labor and component cost. If at any time after manufacture an adjustment is made to the batch, samples should be resubmitted for microbiological testing. This applies to both hot and cold mixes.

Types of Mixes 1. Cold Mix - No heat is applied at any time during manufacture. Sample in accordance with the procedures stated previously. 2. Hot Mix - Sample after cooling. 3. Aerosols - Sample the concentrate in the same manner as for hot and cold mixes. In the above three types of mixes, approximately the same sample size should be obtained. NOTE: If composites of a lot of bulk mixed products are shown to be unacceptable by in-house standards, then all samples should be retested individually and if necessary all previously sampled containers should be reassayed. More extensive sampling and testing may also be necessary. Tanks should be sampled from the top and bottom before mixing or stirring the contents. Special attention should be given to the interface of the possible moisture layer on the top of the material. Low-susceptibility materials (Category 3) should be sampled on a defined periodic basis or prior to use.

Sampling of Bulk Product Because bulk products are usually stored in tanks, drums, carboys and cartons, a representative sample should be taken regardless of the container size and shape. A representative sample may be 50-100 grams of a well-mixed product collected in a sterile container. • These samples should be sufficient in size to perform all necessary tests, including confirmatory tests.

78

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 6: MICROBIOLOGICAL SAMPLING

• Drums and all sub-units of a manufactured batch of high- and mediumsusceptibility materials may be sampled according to typical plans as outlined in CTFA Quality Assurance Guidelines, “Annex 17 - Sampling.”2 • Stored bulk material should be resampled and retested on a defined periodic basis or prior to use.

FINISHED GOODS

Samples for testing should be taken at the beginning, middle and end of each shift. If more than one shift fills a batch, samples from each shift should be submitted. In determining the number of samples taken, consideration should be given to multiple filling lines, container size and extended downtime and product susceptibility. It is suggested that for possible future reference at least two unopened samples per batch and/or lot be retained. Retention time should be comparable to that normally required for other quality control purposes.2

Composites Composite samples of products from each sampling time may be made; i.e., equal portions of samples at the beginning, at the middle, and at the end of the run would be combined to provide three composite samples.

Frequency of Testing Samples should be tested as soon as possible after manufacture. In general, the frequency of testing is determined by the nature of the preparation, efficacy of any antimicrobial agent present, manufacturing process, and experience gained as a result of previous microbiological evaluation. In practice, it is recommended that, with few exceptions, all susceptible finished products be tested with the same frequency. This will permit the detection of microbiological problems resulting from formula changes, errors in compounding or failure of good manufacturing practices.

Microbial Limits The microbial content for products should follow “Establishing Microbial Quality of Cosmetic Products” (Section 12), in-house specifications, or other appropriate criteria.

Product Release Finished products should not be released for consumer use until all microbiological testing has been completed and products are approved for release.

SECTION 6: MICROBIOLOGICAL SAMPLING

|

CTFA MICROBIOLOGY GUIDELINES

|

79

SECTION 6: MICROBIOLOGICAL SAMPLING

Sampling Intervals and Quantities

REFERENCES 1. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. “Annex 8 - Discrepant Materials Control.” In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

SECTION 6: MICROBIOLOGICAL SAMPLING

2. Bailey and Nikitakis. “Annex 17 – Sampling.”

80

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 6: MICROBIOLOGICAL SAMPLING

SECTION 7

Microbiological Quality for Process Water

INTRODUCTION This overview of process water system design, treatment and sanitization methods also includes concepts about process water validation. It is intended for use by microbiologists and other technical personnel involved with the installation, qualification, and maintenance of process water systems for cosmetic manufacture.

• It is a major raw material in cosmetics and toiletries. • It can be a major source of contamination for the entire manufacturing system. • The presence of specific microorganisms can pose a potential health hazard. • Microbial contaminants in process water can produce physical changes in odor, color and clarity in product formulations. These effects may be present even after the organisms are destroyed. • A high microbial load introduced by process water may reduce preservative activity or cause preservative failure in the final product. The microbiological quality of cosmetic process water varies and can be influenced by conditions of manufacture such as pH, temperature, equipment, and the presence of chemicals. It should be noted that seasonal variation in feed water may also alter process water quality. In general, it is suggested that process water contain no higher microbial load than the limits established for the finished product. At a minimum, the microbiological quality of process water should meet EPA drinking water quality standards.1, 2 A manufacturer should be familiar with the microbiological profile of the water purification and distribution system. Alert and action levels should be established for the microbiological acceptability of process water.3, 4, 5

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

81

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Process water is purified water that has been obtained by distillation, ion-exchange treatment, reverse osmosis, or other suitable means to remove chemical and physical impurities. The microbiological content of process water should be defined and controlled because:

DESIGN AND MAINTENANCE Water supply, storage and distribution systems can be major sources of microbial contamination. Bacterial growth and subsequent biofilm formation can occur as a result of poor design, inadequate maintenance, and improper cleaning and sanitization procedures.6 Note: Bacterial species of the genus Pseudomonas tend to be the most problematic in contaminating process water systems. Common sources of microbiological problems are deionization beds, water softeners, carbon or sand filters, storage tanks, water meters, valves, lines, dead ends, and return lines. The water quality should be monitored downstream from these points. The system should be cleaned and sanitized routinely.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

The product manufacturer is responsible for making sure the system is designed for ease of maintenance and sanitization. It is extremely important that the microbiologist be involved in the design, installation, and modification of new or existing systems. The entire system should be designed for proper drainage. Elbows, tees and bends should be kept to a minimum. U-bends should be avoided unless they can be inverted so as not to form a pocket where water can stagnate. Unused valves or branch lines should be removed, since these could result in “dead legs” where microbial proliferation could occur. Areas that restrict or reduce the flow of water, such as water meters and filter housings, are frequently sites of microbiological contamination. Fittings such as unions and valves should be of the sanitary type for ease of cleaning and accessible to facilitate their removal when necessary. Long runs of welded pipe can be incorporated into a water system if hygienic design, materials and construction have been used. Consideration should be given to the materials of construction. Not all materials are compatible with certain sanitizing agents. As examples, hypochlorite reacts with silver solder; glutaraldehyde and quaternaries react with rubber. In addition, some plumbing materials are capable of supporting microbial growth, especially certain plastic tubing, packing and jointing compounds.7 It has been determined that Teflon® is better than unplasticized polyvinyl chloride (PVC), which is better than high-density polyethylene (HDPE), which is better than plasticized PVC for inhibiting the development of mold and bacterial biofilms on plumbing material.8 To minimize biofilm formation and for ease of sanitization, 316 stainless steel should be used whenever possible. Service and maintenance of system components should be assessed. When deionizing systems are used, regeneration and general maintenance should be the responsibility of a competent individual who is familiar with the equipment. The equipment vendor could be consulted for advice. In-line filters, ultraviolet lamps, and other equipment requiring special attention should be serviced according to the vendor’s specifications to maintain them at peak operating efficiency. Microbial growth can build up on a filter and be a source of contamination, as a result of bacterial grow-through, long before water flow rate is affected.9 When process water is held in a storage tank, steps should be taken to control the growth of microorganisms. This can be accomplished by circulating the water through an ultraviolet light source. Another effective method is the use of heat (65-80°C or 150-176°F) to control microbial levels in storage tanks.2 Other methods are listed below under “Sanitization of Process Equipment and Lines.”

82

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

It is the responsibility of the manufacturer, with the cooperation of the microbiologist and the engineering department, to establish and document methods and procedures for maintaining the microbial purity in water storage tanks and water distribution systems. If the system is also designed to remove minerals, it should be examined at regular intervals to insure maximum efficiency in removal of inorganic materials. Further information on design and maintenance of process water systems can be found in References 10-13.

VALIDATION AND MONITORING The water system is a critical part of any cosmetic manufacturing facility. Appropriate validation and operation of the system is necessary to maintain product quality. Refer to “Microbial Validation and Documentation” (Section 9) for additional information. Validation is the process that shows the system can consistently produce water of the quality required. It is an overall program which yields documented evidence that the system does what it purports to do.5 For water systems, this may take the form of one of three conceptual approaches: prospective, concurrent, or retrospective validation.

Concurrent validation is accomplished during the actual implementation of the water treatment process. Concurrent validation shows a system to be in a state of control through the use of validated methods to evaluate representative samples taken at strategic sites in the water system. It involves ongoing monitoring of water microbial quality over an extended time. Retrospective validation, the third approach, entails the collection and documentation of key historic data to prove the system performs as specified. Once a system is validated and its typical microbiological profile is established, a control plan can be implemented to maintain the performance of the water treatment system.14 If ongoing monitoring of water quality reveals microbial counts beyond specified quality levels, appropriate remedial action should be taken. Monitoring usually includes the identification of alert and action levels.14 Alert levels are those microbial levels that, when exceeded, signal a potential drift from usual operating conditions, but not sufficient to compromise the quality of water as an ingredient. In contrast, action levels signal an excessive drift that requires remedial action. The control plan should also include a change control system that evaluates the effects of subsequent changes to the water system and provides for the implementation of appropriate action or revalidation, if necessary, to maintain water system quality.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

83

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Prospective validation involves the execution of an experimental plan, the validation protocol, before the process is implemented. This approach typically consists of three elements. First, data are developed to support standard operating procedures (installation qualification or “IQ”). Next, procedures show how water of the specified quality is consistently produced (operational qualification or “OQ”). Finally, data are developed to demonstrate that seasonal variations in feed water do not adversely affect process water quality.2

WATER TREATMENT METHODS (See Table 7-1) Depending on the chemical and microbial content of incoming water, many different methods or combinations of methods may be used to produce microbiologically acceptable process water. Selection of the appropriate methods and the point of application should be based on a thorough knowledge of the composition of the raw water and the applicability of each process for correction of each problem. It should be noted that bacterial tolerance or resistance to chemical agents can occur. Furthermore, biofilm formation in the process water system can reduce the efficacy of water treatment methods. Refer to “Cleaning and Sanitization” (Section 3) for additional information. The water treatment method may be selected based on factors other than microbiological quality, as for example, the removal of chemical impurities by distillation.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Chlorination Chlorine and chlorine-donating compounds are widely used because they are relatively inexpensive, easily monitored, and effective at low concentrations (2-10 ppm).15 The biocidal effects of gaseous chlorine and chlorine compounds are inactivated in the presence of organic or inorganic residuals, and at pH levels above 8.5. Chlorine compounds provide residual activity for transport and storage conditions, but residual chlorine may have to be removed from the water prior to its use in manufacturing. Chlorine is a respiratory irritant and is corrosive at high concentrations. The National Institute for Occupational Safety and Health (NIOSH) occupational health guidelines recommend an exposure limit for employees of a 0.5 ppm chlorine ceiling for 15 minutes.16

Ozonation Ozone has a strong oxidizing effect which is rapidly lost over time as it decomposes to oxygen. Validation system control should take this into account. A dissolved ozone level of less than 0.5 mg/l effectively kills bacteria and viruses.17 It is less affected by temperature and pH changes than is chlorine. In addition, ozone provides residual activity for transport and storage conditions. Dissolved ozone in water must be removed prior to use of the water for manufacturing purposes. Ultraviolet lights or granular-activated carbon are commonly used for this. Routine maintenance of these systems will be required Ozone cannot be used successfully in water containing more than 0.5 ppm of soluble manganese because manganese is converted to an insoluble form. The NIOSH/OSHA occupational health guidelines stipulate that the permissible exposure limit for employees is 0.1 ppm (0.2 mg/m3) ozone.16

84

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Filtration Submicron filtration, reverse osmosis, and ultrafiltration methods are mechanical means of removing microorganisms from water. These methods introduce no chemical residues and do not pose a safety hazard. Filtration methods can be highly effective. However, if not properly maintained, a microbial build-up can occur on filter membranes that can lead to downstream contamination. Submicron Filtration Submicron filters (pore size 0.22-0.45 micron) are nominally rated to remove all microorganisms and are most effective at points of use. These filters should be tested for integrity to assure absence of defects. Extensive pretreatment of water may be necessary to reduce the replacement frequency of submicron filters. Reverse Osmosis

Ultrafiltration Ultrafiltration, like reverse osmosis, removes most microorganisms.19 Unlike reverse osmosis, the ultrafiltration membrane may be regenerated, and operating pressures are much lower. Costs are lower than are those for reverse osmosis methods, but are still considerably higher than for chemical methods.

Recirculation Recirculation of water is a process that controls microbial levels because constant motion eliminates stagnation and markedly diminishes microbial proliferation on surfaces. However, it should not be considered alone as a method for treatment of contaminated water. Recirculation should be used in conjunction with other procedures such as ultraviolet irradiation or heat. Flow rates of 1-2 m/sec are recommended for water systems to minimize microbial adhesion.20

Distillation Distillation effectively removes microorganisms without introducing residual chemicals. This method requires a substantial initial capital investment with continued need for large amounts of energy. The distillation process is more efficient for products that are formulated at high temperatures. Distillation results in considerable loss of water yields, and the high temperatures that are generated pose a potential safety hazard.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

85

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Reverse osmosis removes greater than 99% of microbial contamination.18 In an appropriate position in the process water lines, it can replace deionization beds that are often a source of microorganisms. Initial reverse osmosis costs are higher than chemical treatment but lower than distillation. Reverse osmosis membranes are somewhat fragile; the high operating pressures render them susceptible to rupture. Care should be taken to control the pH of incoming water so as not to destroy the membrane.

Heat Treatment Heat treatment (80°C or 175°F), like distillation, represents a large initial capital investment with continued needs for large amounts of energy. There is some loss of water through evaporation, and contact time should be carefully controlled so as to assure biocidal effects. As with distillation, the high temperatures generated could also represent a potential safety problem.

Ultraviolet Irradiation Ultraviolet irradiation (UV) in the range of 250-260 nm16 destroys most vegetative microorganisms if the absorbed dose is sufficient. The absorbed dose depends on depth and turbidity of water, flow rate, lamp intensity, and temperature. UV irradiation becomes less effective as the bioburden increases. UV irradiation does not introduce chemical residues. It can be used at various points in the system as well as at the point of water use.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

To maintain maximum effectiveness of the UV light source, the UV cell housing must be cleaned regularly. Lamp intensity must be monitored to ensure sufficient energy output. Personnel must be shielded from irradiation to prevent eye damage. Manufacturer’s recommendations should be followed when designing a maintenance schedule.

SANITIZATION OF PROCESS EQUIPMENT AND LINES (See Table 7-2) These methods are guidelines and should be used according to the particular need and compatibility with the process water system. For final selection, the efficacy of the concentration and contact time should be experimentally verified. Any sanitization method must be validated for the intended process. Suggested concentrations reflect percent active ingredient. For certain methods, rinsing may be required to assure that there is no residual sanitizer remaining in equipment or lines.

Chemical Methods - Most Commonly Used Sanitizers Chlorine Chlorine and chlorine compounds are readily available, inexpensive and easily monitored. Target concentration of 200-500 ppm of available chlorine for 10 minutes contact time has generally been found effective over a pH range of 6-8.5. Chlorine is rapidly inactivated by trace organic residuals and at pH levels above 8.5. With prolonged contact, stainless steel and other metals are attacked by chlorine or chlorine compounds. Deionization resins can be oxidized in the presence of free chlorine. At full strength, chlorine and chlorine compounds are respiratory irritants and appropriate protective measures for personnel must be provided.16 Ozone Although the main application of ozone is in water treatment, it may be used as a sanitizer at 10 to 50 ppm for a contact time of approximately ten minutes.21 Its activity is not as pH-dependent as is chlorine, and operating and maintenance costs are low. 86

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

It is not suitable where water contains more than 0.5 ppm of manganese. Residual ozone can be removed by granular activated carbon (GAC) filters and UV lights. GAC filters, if used, may be a source of microbial growth and should be monitored. Ozone requires electric energy and cooling water, and the large initial capital costs may make it impractical if used only occasionally as a sanitizer. For personnel protection, NIOSH/OSHA guidelines stipulate a permissible exposure limit of 0.1 ppm ozone.16 Peroxygen Compounds Peroxide and peroxyacetic acid are two strong oxidizers employed as process water equipment sanitizers. A 1.5% solution of hydrogen peroxide for a one-hour contact time provides excellent sanitization of process water equipment. Hydrogen peroxide has recently gained wide acceptance since the innocuous end products, oxygen and water, can be readily disposed of via the sewer without adversely affecting the environment. The presence of organic matter can decrease the antimicrobial activity of hydrogen peroxide.

When compared to hydrogen peroxide, peracetic acid vapor has a potential fire and explosion hazard when heated (56°C or 133°F). At high temperatures, peracetic acid decomposes and is corrosive and toxic. Concentrated solutions of peroxygen compounds should be treated with caution as they are irritating to skin, mucous membranes, and eyes. Adequate protection should be employed during handling.

Less Commonly Used Sanitizers Iodine or Iodophors At a concentration of 25 ppm with a contact time of 10 minutes, elemental iodine and most iodophors destroy microorganisms.15 Iodine, like chlorine, is easily monitored, but unlike chlorine, it is less susceptible to inactivation by organic residuals. Elemental iodine is effective over a wide pH range, but some iodophors should be used under the controlled conditions of pH concentration and contact time as specified by the supplier. Elemental iodine is a respiratory irritant and corrosive at high concentrations. For personnel protection, NIOSH/OSHA guidelines stipulate a permissible exposure ceiling of 0.1 ppm iodine vapor.16 Iodophors are generally easier to handle and much less toxic and irritating. Iodophors can also be used as residual sanitizers at slightly higher concentrations. Formalin (37% Formaldehyde) Formalin at a concentration of 1-2% formaldehyde for a contact tome of 2-3 hours is an effective sanitizing agent. Deionization resin beds can be sanitized simultaneously provided the particular resin is compatible with formaldehyde.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

87

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Peracetic or peroxyacetic acid is an effective biocide with no toxic residuals. Peroxyacetic acid solutions show broad spectrum effectiveness at low concentrations, even in the presence of organic matter. At room temperature, solutions of 0.05% to 3.0% peroxyacetic acid demonstrate excellent sporicidal activity at 15 minutes to 15 seconds contact, respectively.15 Peracetic acid is commercially available as a 15% aqueous solution and poses no environmental hazards since it decomposes into acetic acid and water.

Since formaldehyde is a respiratory irritant and skin sensitizer, personnel must be adequately protected during its use. The current NIOSH recommendation is 0.016 ppm averaged over an 8-hour day with a 0.1 ppm 15-minute ceiling. OSHA stipulates an exposure limit of 0.75 ppm formaldehyde.16 Questions about formaldehyde toxicity have caused many manufacturers to discontinue use of this chemical. Quaternary Compounds Quaternary surfactants are active over a wide pH range at concentrations of 200-300 ppm. They are less sensitive to organic residuals than are halogenated compounds. Quaternary compounds may be mixed with cationic or nonionic detergents for cleaning action. They are incompatible with anionics. Their compatibility with deionization resins varies and should be ascertained for particular resins. At very high concentrations, quaternaries are toxic. Detergent-Sanitizers The detergent-sanitizer combinations provided by various manufacturers require careful adherence to the instructions for use. If sanitizing effects are satisfactory, a significant saving of time is possible for combining washing and sanitizing in one operation. Toxicity and safety hazards vary and must be considered individually. SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Glutaraldehyde Glutaraldehyde can be used as a sanitizer at diluted concentrations of 0.1% to 0.25% for a fiveminute contact time.22 Glutaraldehyde is effective against a broad spectrum of microorganisms over a wide temperature range on a variety of surfaces. It is most effective, although not very stable, in an alkaline solution.15 It is compatible with common materials of construction that can tolerate exposure to water. It is an effective sanitizer even in extremely hard water. Closed systems are recommended. Glutaraldehyde is inactivated by ammonia, primary amines, bisulfites and proteins. It is a respiratory irritant and personal protective equipment is required when handling.

Physical Methods Hot Water Sanitizing with hot water requires temperatures of 80°C or 175°F for at least 30 minutes contact time. Lower temperatures may be used, but longer times would be required. Although this method leaves no residual material, energy requirements are high. Care should be taken to be sure that high temperatures are attained throughout the process water system. Also, scalding water temperatures create a potential personnel hazard. Steam Steam, like hot water, has the advantage of not requiring rinsing or cleaning to remove residual material. However, a large energy output is needed and high temperatures should be present throughout the system to destroy microorganisms in remote or less accessible areas. Filtration may be required to remove foreign materials from the steam source prior to use. If the addition of boiler treatment chemicals is warranted, potential residual effects should be assessed.

88

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

SAMPLING AND TESTING METHODS To assure that water treatment methods and sanitization procedures are effective, routine sampling and testing for microbiological quality should be conducted. A sampling program should be established which takes into consideration the purification system, water consumption rate, size of the system, validation data, and any other factors that can affect water quality. Sample Collection Method Prior to obtaining a sample, flush the valve for a sufficient period of time to remove any stagnant water (minimum of two to three minutes).23 Where warranted, rinse or sanitize the surface of the valve with 70% alcohol (or other suitable sanitizer), flush the valve to remove residual sanitizer, and obtain a sample. Use sterile wide-mouth glass or plastic containers having a suitable volume capacity. Sampling must be thorough and representative of the system and done under strict aseptic conditions. Some specific critical sampling areas are: points of use, storage tanks, before and after deionization beds, before and after UV lights, water meters, filters, and return water. In general, sampling areas should also include any area in which the flow of water is reduced and microorganisms might proliferate. Testing Procedure

Acceptability Criteria Alert and action levels should be established for purified water, taking into consideration such factors as processing conditions, susceptibility of the product to contamination, and intended use of the product. It is the responsibility of the manufacturer to assure that microbial limits for process water are appropriate and achievable. Microbial limits that have been defined should be documented.4, 5, 14

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

89

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Samples should be tested within one hour after collection if not refrigerated, or within 24 hours if refrigerated. The microbiological testing of process water can be accomplished by different methods that can be selected on an individual basis. Plate count, most probable number, and membrane filtration are methods commonly employed in most laboratories. The method employed when establishing a microbiological history of a water system should be validated to assure the accuracy of results due to technique or media. “Standard Methods for the Examination of Water and Wastewater” is recommended for guidance in establishing test procedures.23

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Table 7-1 Microbiological Control Methods - Water Treatment Methods Method

Advantages

Restrictions/Disadvantages

Toxicity/Safety Hazards

Chlorination (as provided by aqueous OCI, CI2, HOCI solutions and CIO2 )

1. Low level residual required (target 2-10 ppm) 2. Sensitive test available to monitor concentration 3. Relatively inexpensive and readily available 4. Relatively short contact time 5. pH range 6.0-8.5 6. Not affected by hard water 7. Residual activity useful for transport/storage conditions

1. Rapidly inactivated by trace 1. Respiratory irritant and organic residuals corrosive liquid at full strength. 2. Chlorine is less reactive as pH increases 2. Reaction with trace organic residuals can form 3. Chlorine is more reactive as trihalomethanes which are temperature increases human carcinogens. 4. Most frequently designed 3. NIOSH recommended to precede deionization, employee exposure limit consequently water will be 0.5 ppm ceiling for 15 min. susceptible to contamination 5. May require method to remove residual (e.g., carbon filters). 6. Chlorine can be corrosive to gaskets and stainless steel processing equipment.

Ozonation

1. Not as pH-dependent as chlorine. 2. Low operating and maintenance costs. 3. Half-life of ozone is 20 minutes-decomposes rapidly to oxygen. 4. Residual activity useful for transport/storage conditions.

1. Not applicable to waters possessing high (>0.5 ppm) Mn++ concentration. 2. Residual organic materials may be biodegraded which could lead to biofilm formation. 3. Dissolved ozone must be removed from prior water use (e.g., UV or granulated carbon).

Filtration 1. Submicron

1. Submicron filters at point of use are most effective in removing all microorganisms. 2. Integrity tests (Bubble Point) can be performed.

1. Development of defects can None permit microbial passage. 2. Replacement of filters should be regularly performed and carefully monitored. 3. Water quality most often requires extensive pretreatment to minimize replacement frequency.

2. Reverse Osmosis

1. Removes >99% microorganisms. 2. No chemical interference. 3. Can replace deionization beds which often represent a potential site for microbial proliferation.

1. Initial capital outlay and maintenance is higher than chemical treatment. 2. Loss in water yield (approximately 50%). 3. Regeneration of membrane is limited. 4. Water quality may require pretreatment to control pH.

None

3. Ultra Filtration

1. Removes most microorganisms. 2. No chemical interference. 3. Can be used at point of use

1. Initial capital outlay and maintenance is higher than chemical treatment. 2. Loss in water yield (approximately 20%).

None

OSHA airborne exposure limit 0.1 ppm

Table 7-1 90

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Table 7-1 Microbiological Control Methods - Water Treatment Methods

continued

Method

Advantages

Restrictions/Disadvantages

Toxicity/Safety Hazards

Distillation

No chemical interference.

1. Large initial capital investment and ongoing requirement for large amount of hear energy. 2. Considerable loss of water in water yields.

Scalding water poses potential hazard.

Heat

No chemical interference.

Scalding water poses 1. Large initial capital potential hazard. investment. 2. Ongoing requirement for large amount of heat energy. 3. Some loss of water in yields. 4. Should have sufficient contact time and temperature to assure biocidal activity.

Ultraviolet Irradiation

1. The absorbed dose depends 1. No chemical interference. on depth and turbidity 2. Relatively easy installation of water, flow rate, lamp and low maintenance. intensity, and temperature. 3. Thin films units more 2. UV lamps need to be cleaned efficient than older at the end of their rated designs. hours, as the UV output 4. Most vegetative organisms decays with time to an destroyed by irradiation. ineffective level. 5. Can be used at point of 3. High bioburden will reduce use. effectiveness.

UV light can cause eye damage. Eye protection is required when lamps are replaced or inspected.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

91

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Table 7-1

Table 7-2 Sanitization Methods for Process Water Equipment and Lines Method

Advantages

Restrictions/Disadvantages

Toxicity/Safety Hazards

Active chlorine compounds (includes liquid CI2, HCIO3, CIO2, organic and nonorganic chloramines)

1. Rapid, sensitive test available to determine concentration during sanitization and to verify removal of residual after rinsing. 2. Material is commonly available and relatively inexpensive.

1. Rapidly inactivated by trace organic residuals. 2. Chlorine is less reactive as pH increases. 3. Efficacy is somewhat temperature-dependent (higher temperature increases biocidal effect). 4. Will attack 316 stainless steel and other metals with prolonged contact; carefully regulate exposure time. 5. Capable of oxidizing resins at low concentrations of free chlorine. 6. Chlorine-releasing compounds may require other conditions of use; e.g., pH contact time, concentration.

1. Respiratory irritant and corrosive liquid at full strength. 2. Reaction with trace organic residuals can form trihalomethanes which are human carcinogens. 3. NIOSH exposure limit of 0.5 ppm ceiling for 15 min.

Ozone Target concentration 10-50 ppm for about 10 minutes to sanitize.

1. Not as pH-dependent as chlorine. 2. Low operating and maintenance costs. 3. Half-life of ozone is 20 minutes, decomposes rapidly to oxygen.

1. Not suitable for water supplies containing greater than 0.5 ppm manganese. 2. Activated carbon filters or ultra-violet light are necessary to remove residual ozone. 3. Electric energy is required; cooling water may be needed after treatment. 4. Initial capital outlay makes it more practical as continuous process water treatment system than as occasional sanitizing agent.

NIOSH/OSHA exposure limit is airborne concentration ceiling of 0.1 ppm, 15 minute contact time.

Peroxygen compounds Target concentrations 1.5% H2O2 for 60 minutes.

1. Innocuous by-products.

1. Organic matter may decrease efficiency. 2. Unstable. 3. Gas formation in pumps could cause pumps to seize.

1. Strong oxidizers are respiratory and skin irritants. 2. Corrosive and toxic only in concentrated solutions (>40%). 3. Potential of fire hazard.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Target concentration 200-500 ppm available CI2; 10-minute contact time; pH range of 6.0-8.5.

Table 7-2

92

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Table 7-2 Sanitization Methods for Process Water Equipment and Lines continued Advantages

Restrictions/Disadvantages

Iodine (elemental iodine or iodophors) Target concentration of 25 ppm 10-minute contact time.

1. Rapid, sensitive test is available to determine the concentration during sanitization and to verify removal of residual after rinsing. 2. Less sensitive than chlorine to inactivation by residual organics. 3. Material is relatively inexpensive and generally available. 4. Elemental iodine is effective over a wide pH and temperature range. 5. Can have a residual effect.

1. Exposure of resins is not 1. Elemental iodine is recommended. respiratory irritant and corrosive liquid at full 2. Iodophors may require other strength. conditions of use-contact time, concentration, etc. 2. Iodophors represent very low toxicity. 3. Iodophors require acid pH. 3. Reaction with trace 4. Unstable in the presence of organic residuals can form hard water, heat, and organic trihalomethanes which are soil. human carcinogens. 4. NIOSH/OSHA permissible exposure ceiling 0.1 ppm.

Formalin (37% formaldehyde)

1. Generally resins will not be attacked, and beds can be sanitized at the same time as piping holding systems.

1. Should verify compatibility with resin prior to use. 2. Large amounts of water are necessary to flush formaldehyde residues from the system.

1. Respiratory and skin irritant sensitizer. 2. NIOSH/OSHA exposure limit is airborne concentration ceiling of 0.1 ppm, 15 minute contact time.

DetergentSanitizers

By combining washdown and sanitization steps, significant time savings are possible.

As described by vendor.

As described by vendor.

Glutaraldehyde Target concentration of 0.1% to 0.25%; 5minute contact time

1. Effective broad-spectrum activity over wide pH and temperature range. 2. Compatible with common construction materials; effective on wide variety of surfaces; sanitizes even in very hard water.

1. Inactivated by ammonia, primary amines, bisulfites and proteins. 2. Surfaces must be thoroughly cleaned prior to treatment.

Respiratory and contact irritant; avoid inhalation of vapors. Personal protective equipment required.

Hot water Target 80°C (175°F); contact time 30 minutes.

1. May be readily available. 2. No chemical interference.

Energy supplies may be expended to produce temperatures required.

Scalding water poses potential work hazard.

Steam Flowing steam contact time 30 minutes.

1. No chemical interference.

1. Large energy supplies may be expended to produce temperatures required throughout the entire system. 2. Significant temperature drops at remote points may limit efficacy. 3. Filtration of steam may be required to remove foreign material.

Scalding steam poses potential work hazard.

Target concentration of 1-2% formalin; 2-3 hours contact time.

Toxicity/Safety Hazards

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

Method

Table 7-2 SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

|

CTFA MICROBIOLOGY GUIDELINES

|

93

REFERENCES

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

1. U.S. Environmental Protection Agency. 1998. “National Primary Drinking Water Regulations: Disinfectants and Disinfection.” 40 CFR Parts 141 and 142. http:// www.epa.gov/safewater/standard/v&efrn.pdf.

11. Cross, J. 1993. Designing Water Systems that Comply With GMP. Manufacturing Chemist. 64: 31-33. 12. Brown, J., N. Jayawardena, and Y. Zelmanovich. 1991. “Water Systems for Pharmaceutical Facilities.” Pharmaceutical Engineering. 11: 15-23.

2. U.S. Food & Drug Administration. 1993. “FDA Guide to Inspections of High Purity Water Systems.” http://www.fda.gov.

13. Lorch, W., (Ed) 1987. Handbook of Water Purification. New York: John Wiley & Sons.

3. United States Pharmacopeia. 2007. <1231>. “Water for Pharmaceutical Purposes.” United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD. 687-706.

14. Pharmaceutical Manufacturing Association Deionized Water Committee. 1985. “Validation and Control System Concepts for Water Treatment Systems.” Pharmaceutical Technology, 8, 52-56.

4. Anon. 1984. “Use of Alert and Action Levels in Pharmaceutical Manufacturing.” Pharm. Manuf. 1: 24-26.

15. Block, S.S. 2000. Disinfection, Sterilization and Preservation. Philadelphia: Lea & Febiger.

5. Pharmaceutical Manufacturing Association’s Deionized Water Committee. 1984. “Protection of Water Treatment Systems, Part III: Validation and Control.” Pharmaceutical Technology. 8: 54-68.

16. U.S. Department of Health and Human Services (NIOSH). 2005. NIOSH Pocket Guide to Chemical Hazards. http://www. cdc.gov/niosh/npg/.

6. Duddridge, J. J. 1988. “Biofilm Growth in Water for Cosmetics.” Manufacturing Chemist. 59: 42-44. 7. Burman, N. P. and J. Colvourne. 1977. “Techniques for the Assessment of Growth of Microorganisms on Plumbing Materials Used in Contact with Potable Water Supplies.” Journal of Applied Bacteriology 43: 137-144. 8. Van der Kooij, D. and H.R. Veenendall. “Assessment of the Biofilm Formation: Potential of Synthetic Material in Contact with Drinking Water During Distribution,” paper presented at the American Water Works Association, 1993 Water Quality Technology Conference. 9. Meltzer, T. H. et. al. 1979. ”Adsorptive Retention of Pseudomonas dimunuta by Membrane Filters.” J. Parenteral Drug Association 33: 40-51. 10. Avalone, H. L. 1988. “Microbiological Control of Topicals.” Pharmaceutical Technology. 12: 55-62. 94

|

CTFA MICROBIOLOGY GUIDELINES

|

17. “Ozone, the Process Water Sterilant.” 1984. Pharmaceutical Manufacturing. 1623. 18. Cross, J. R. 1987. “Contemporary Techniques for the Production of Ultrapure Water in the Pharmaceutical Industry.” Drug-Dev-Ind-Pharm. 13: 9-11. 19. Marcus, D. L. and R. Pastrick. 1988. “Ultrafiltration - Its Role in Today’s Water Purification Systems.” Ultrapure Water, 40-45. 20. Cross, J. 1995. “Water Purification.” Chemical Engineering, 983: 15-16. 21. Mittelman, Marc W. 1986. “Biological Fouling of Purified-Water Systems: Part III, Treatment.” Microcontamination 4, 30-40. 22. Union Carbide Corporation. Technical literature provided by supplier. 23. American Public Health Association 1995. Standard Methods for the Examination of Water and Wastewater 20th Edition. Washington, DC.

SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

SECTION 8

Microbiology Laboratory Audit

INTRODUCTION Within the cosmetic industry, quality assurance, development, and contract microbiology testing laboratories provide supportive microbiological data related to product safety and quality. Microbiological aspects of formula development, from product conception to finished goods, need to be addressed to determine if microbiological standards are met. Examples include the development and evaluation of preservative systems and the examination of the microbial content of raw materials, intermediates, finished goods, and the manufacturing environment. Laboratory practices should be evaluated at regular intervals to ensure that the generation of data is reproducible, accurate, and reliable. An audit serves to review existing practices, systems, and equipment to ensure that they perform as expected. Microbiology lab audits should be conducted by individuals familiar with the functions and processes that typically occur in a microbiology laboratory. This document provides guidance for conducting an audit of both in-house and contract cosmetic microbiology testing laboratories. If cosmetics and over-the-counter (OTC) drugs are tested in the same laboratory, refer to Food and Drug Administration (FDA) guidelines for the manufacture of OTC drugs, and to relevant chapters in the USP.1,2,3

PERSONNEL Supervisory personnel have the responsibility of ensuring that operating systems are consistent with existing cosmetic regulations and current cosmetic industry good manufacturing practices (GMPs).4 Laboratory supervisors should be familiar with applicable regulations and current industry practices. Initial and continuing training programs are a valuable means of ensuring that employees are qualified for their roles and responsibilities and informed about all laboratory procedures.

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

95

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Based on the information presented in this document, an example of a Cosmetic Microbiology Laboratory Audit Checklist can be found in Table 8-1.

The following documentation may be considered in recording an employee’s qualifications and is most useful when maintained on file for reference: • Job descriptions for all laboratory positions, which may describe the qualifications, primary and secondary responsibilities, and job functions. • Organizational charts showing each staff position and each incumbent identified by position, name, and reporting relationship. • Skills checklist or applicable training documentation for each employee detailing the microbiological techniques and procedures that an individual has been trained and certified to perform on a routine basis (see Table 8-2). • Records of initial and continuing training for each employee.

SECTION 8:\ MICROBIOLOGY LABORATORY AUDIT

LABORATORY FACILITIES Adequate laboratory facilities should be provided to minimize errors in test results due to contamination, inaccuracy in data interpretation, equipment failure, or sampling mistakes. It is recommended that rooms used for microbiological testing be of suitable size, construction, and location to facilitate proper operation. In general, the attributes of an adequate microbiology laboratory facility include, but are not limited, to the following: • The laboratory contains separate areas for microbiological analysis, support functions (e.g., media preparation, sample preparation, sample login, clerical work), and storage of personal belongings. • The area for microbiological analysis is used exclusively for testing aspects such as sample handling, performing procedures, transfer of cultures, counting of colonies, etc. • All surfaces in the laboratory are nonporous, cleanable, and sanitizable. • The facility design minimizes exposure of laboratory areas to air currents. • Laboratory access is restricted to minimize foot traffic by non-laboratory personnel. • Microbiological quality of the laboratory environment is controlled by appropriate mechanical and physical means such as use of positive-pressure room air, germicidal lamps, high-efficiency particulate air filters, and/or laminar flow stations. • Workspaces such as laboratory benches and laminar flow hoods are sanitized at the beginning and end of the workday, as well as between individual procedures. • Adequate ventilation and lighting is provided, especially over work areas. • Electrical service is appropriate for the equipment used in the laboratory and associated areas to avoid power outages and equipment failure. • Adequate sink areas with hot and cold water service are provided. • Sufficient counter and shelf space are available so that all procedures can be performed while preventing overcrowding, safety hazards, or any cross contamination.

96

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

• Clean uniforms or lab coats are provided. • Microbiologically contaminated materials are decontaminated prior to disposal. • Floors are kept clean by regular mopping and sanitized by using a disinfectant detergent. • Adequate containers that are appropriately labeled are provided for microbiological waste, non-hazardous waste, and general trash disposal. General trash is removed from the laboratory each working day.

SAFETY The microbiology laboratory should have a written safety policy that addresses laboratory personnel, equipment, and processes. This policy may reference a laboratory safety manual.5,6

LABORATORY EQUIPMENT It is recommended that standard operating procedures as well as supporting documentation (e.g., equipment manuals, calibration data) be readily available to personnel for each piece of equipment. Equipment is to be maintained in accordance with the manufacturer’s guidelines and routinely calibrated, with clearly visible calibration stickers containing calibration date, calibrator’s name, and next scheduled calibration date. Personnel should be adequately trained on each piece of equipment necessary to their function prior to their routine use of that equipment. A summary of calibration recommendations can be found in Table 8-3. The following specific attributes for some common laboratory equipment are recommended as a minimum guideline:

Incubators should maintain a uniform and constant temperature within predetermined limits; ±2.0ºC is recommended for most laboratory applications. An accurate thermometer with a bulb continuously immersed in liquid (water, mineral oil, or propylene glycol) is maintained at appropriate location(s) (e.g., hot and cold spots detected through temperature mapping) in the incubator. Daily temperature readings are recorded on laboratory workdays. Daily humidity readings are recorded for humidity-controlled incubators. Temperature recording devices or maximum and minimum registering thermometers within the incubator are recommended to record temperature variations. Provision is made for humidity control (e.g., placing a beaker of water in the incubator). The temperature setting is appropriate for the types of organisms being incubated.

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

97

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Incubators

Hot-Air Sterilizing Ovens Hot-air sterilizing ovens maintain a uniform and constant temperature of up to 200ºC, within predetermined limits; ±5.0ºC is suggested for most laboratory applications. Ovens are equipped with thermometers capable of accurate measurement between 160º to 200ºC. Ovens are of sufficient size to prevent crowding of the interior.

Autoclaves Laboratory autoclaves are capable of maintaining a uniform and constant temperature of 121º -123ºC and reach target temperature within 30 minutes. In order to allow sufficient heat distribution and penetration for sterilization, autoclaves are properly sized and loaded to prevent crowding of items. Autoclaves are equipped with accurate thermometers or temperature recorders, pressure gauges, and properly adjusted safety valves. If a time-temperature recorder is not available, an alternative temperature monitoring device or bioindicator is used. Autoclave tape is not an indicator of sterility. A maintenance program is in place, which includes an annual certification of temperature and pressure gauges, timers, and temperature recording devices. In addition, individual run records (e.g., time-temperature recording) are maintained. Records are routinely reviewed for deviation. An autoclave log is maintained detailing run conditions and allowing traceability of autoclave run documentation with specific loads. Temperature controllers, temperature recording devices, pressure gauges, and timers must be certified for accuracy to insure proper operation.

Colony Counters A standard colony counter or comparable instrument may be used. Automated colony counters may be used where accuracy and reliability have been validated. Automated colony counter accuracy is checked each day of use and a log-in book is maintained.

ANNEX 8 DISCREPANT MATERIALS CONTROL

pH Meters pH meters are capable of accurately measuring the hydrogen ion concentration to 0.1 pH units. The pH meter should be calibrated each day of use by using at least two certified pH buffer solutions that bracket the pH range of the sample. Buffers should not be used past their expiration dates. Probes for taking the pH of a sample should be appropriate for the material being analyzed.

Balances Balances used for routine weighing (e.g., media, samples, etc.) are accurate for the utilized task. Balances and weights are calibrated according to a regular preventative maintenance schedule with weight (mass) standards traceable to NIST Reference standards. Weight checks should be performed with calibrated weights.

98

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Labware Disposable or reusable labware is inert to the materials with which it may be used. For glassware items, borosilicate glass is recommended because of its ability to withstand high temperatures. The following are recommendations for selected examples of commonly used labware: • Pipettes - sterile glass or disposable plastic pipettes may be used. The accuracy of the pipettes is ± 5%. If used, micropipettors should be calibrated at least annually. • Dilution bottles and tubes - bottles and tubes are single use or are made of autoclavable material. Screw caps are equipped with inert liners. All reusable items are thoroughly cleaned and rinsed using a protocol that assures no detectable detergent residue. • Media preparation utensils -clean borosilicate glass, stainless steel, or other suitable inert labware are recommended. • Petri dishes - sterile borosilicate glass or disposable plastic Petri dishes are recommended.

Microscopes A monocular or binocular microscope suitable for the intended purpose is recommended. The microscope should be capable of a minimum total magnification (combined ocular and objective lenses) of 1000x. A dissecting scope may be used for lower magnification. An annual maintenance check is recommended by which lenses, alignment, and mechanism aspects are checked.

Refrigerators/Freezers Commercially available refrigerators are suitable unless there is a need for an explosion-proof model. Refrigerator temperature should be maintained at 5±3ºC and routinely checked. No food should be stored in laboratory refrigerators or freezers. Standard freezers have autodefrost. Ultrafreezers (e.g., -80ºC) are maintained 5ºC within designed temperature.

Water baths have thermostatic controls to deliver temperatures from ambient to 100ºC ±2.0ºC. An accurate, calibrated thermometer should be placed in the water bath to monitor the temperature. Temperature readings are recorded daily when in use and also after equilibrium has been reached following any adjustment of the temperature controls. Biocide may be added to the water to prevent/control microbial growth.

Laminar Flow Hoods Laminar flow hood operation is certified at least annually to verify adequacy of airflow, condition of pre-filters, HEPA filter integrity, and particle size exclusion limit. An appropriate sanitizer is recommended for use to disinfect interior hood surfaces before and after use. UV lights (if installed) are maintained and operated according to manufacturer’s recommendations and replaced or verified annually. SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

99

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Water Baths

Biological Safety Cabinets A biological safety cabinet is used when a material is suspected of being highly contaminated, when manipulating high concentrations of microorganisms considered to be a biohazard, or when there is a potential for aerosolization of microorganisms.5 Operation is certified at least annually, or after 5,000 hours of use, to verify the cabinet performance for airflow, condition of prefilters, HEPA filter efficiency, and particle size exclusion limit. UV lights (if installed) should be maintained and operated according to manufacturer’s recommendations, and replaced or verified annually.

Water Treatment Systems The laboratory has an appropriate system for producing the desired grade of water (e.g., deionized, distilled) for use in microbial growth media and other applications. The water treatment unit is maintained and operated consistent with manufacturer’s recommendations. Water is periodically monitored to ensure it meets chemical and microbiological quality. If a UV light is part of the water system, it should be maintained and operated according to the manufacturer’s directions. Note: If cosmetics and over-the-counter (OTC) drugs are tested in the same laboratory, refer to FDA reference materials and the USP Guidelines for Water.1, 2, 3

Lyophilizers Vacuum and temperature records are maintained for each lyophilization operation. Temperature, vacuum, and timing systems should be calibrated every six months.

Thermometers

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Thermometers are of appropriate range for the application. Measurement accuracy is initially established and rechecked at least annually by using a NIST-certified thermometer.

Spectrophotometers Calibration is performed against standard solutions every six months or as recommended by the manufacturer.

Centrifuges Centrifuges are calibrated for temperature and rotor speed annually.

Water Activity Devices Devices for the measurement of water activity should be calibrated each day of use against known standards.

100

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Media Dispensers The dispensing volume should be verified on a regularly scheduled basis. For more detailed information on calibration of microbiological equipment, see “Annex 16 Calibration Systems” in the CTFA Quality Assurance Guidelines.4

DOCUMENTATION The purpose of documentation is to provide the written record of a laboratory operation. A history of operation is maintained through the retention of documents (e.g., logbooks, worksheets, calibration records, etc.). Establishment of a document retention program is highly recommended since it defines the policy of the company, retention time, form (microfilm, etc.), place of storage, etc. Any documentation should describe the function of the laboratory operation with properly prepared and regularly updated procedures. Scheduled reviews of laboratory procedures should be performed to ensure that procedures are as described. Discrepancies should be corrected and procedures amended when appropriate. Laboratory personnel should document any investigations and procedure changes. For additional information, refer to “Microbial Validation and Documentation” (Section 9), “Determination of the Microbial Content of Cosmetic Products” (Section 18), “Establishing Microbial Quality of Cosmetic Products” (Section 12) and “Raw Material Microbial Content” (Section 11), in this document. The following quality control checks are recommended as part of laboratory procedures: Methods Validation All microbiological methods should be validated and documented to ensure the accuracy of the test response.

Media performance should be documented via positive and negative controls on each prepared batch of media.

Equipment Checks (see Table 8-3) Calibration, performance, and maintenance logs should be maintained for each piece of laboratory equipment. All entries should be dated and initialed.

Personnel Documentation of microbiology laboratory personnel training should be maintained as well as the documentation described in Section 4 of this guideline. A current signature list conforming to signatures issued in the laboratory and retraining documentation should also be maintained. SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

101

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Media

Forms Forms used to document test results should be periodically reviewed and updated to reflect current procedures.

Procedures Written standard operating procedures and test methods should be reviewed and updated on a regular basis to document changes. All procedures should include an effective date and supersedes date to identify the current version. All changes should be communicated to laboratory personnel.

Investigations and Discrepancies

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Laboratories should have in place an out-of-specification investigation procedure that includes the steps outlining the process, detailing the findings, and reporting the results. Any discrepancies in the microbiological test results and/or procedures should be investigated, documented, and evaluated by the appropriate personnel.

102

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Table 8-1 Cosmetic Microbiology Laboratory Audit Checklist (Example) This list gives examples of points to consider when performing an audit or preparing to receive an audit. It is not intended to be all-inclusive or to infer that all points are applicable to all laboratories. General 1. Is there an organizational chart showing each incumbent and reporting relationship? 2. Is the signature list up-to-date? 3. Are there written job descriptions for each laboratory position? 4. Are individual qualifications for laboratory personnel on file and updated regularly? 5. Is there a training and development program for laboratory staff members? 6. Is there formal training documentation? Laboratory Facilities 1. Is access to the microbiology laboratory controlled? 2. Are laboratory facilities clean and orderly? 3. Is the laboratory free of dust, drafts, and temperature extremes? 4. Does the laboratory have adequate workspace, ventilation, and light? 5. Are there adequate facilities for cold storage, microbial media, and storage of samples? 6. Is the staff regularly reviewing sanitization and cleaning records? Laboratory Safety 1. Are laboratory coats worn only in laboratory areas? 2. Are proper shoes and clothing worn in laboratory areas? 3. Is a safety committee and/or advisor established and functional? 4. Are all laboratory staff members provided with, or do they have access to, the laboratory safety manual? 5. Are the members of the janitorial staff provided appropriate training for the microbiology area? 6. Does the laboratory provide: a. Designated containers for broken glass, sharp objects, etc.? b. First aid kits that are easily accessible and well maintained? c. Conveniently located and operational eyewash and deluge shower stations? d. Up-to-date and readily accessible fire extinguishers and blankets? e. Clearly marked emergency exits? f. Emergency telephone numbers that are widely distributed and conveniently located? SECTION 8: MICROBIOLOGY LABORATORY AUDIT

7. Has mouth pipetting been eliminated? 8. Is microbiologically contaminated waste decontaminated before disposal? Laboratory Equipment 1. Review records for equipment calibration and preventive maintenance. 2. Review records for calibration of standards and equipment daily calibration check logs (evaluate what corrective actions were taken when calibration failed). 3. Review cleaning log records (e.g., incubators, water baths, etc.). 4. Review sterilization cycle log records for autoclaves. 5. Ensure that autoclave controls are present (e.g., biological indicator test results). 6. Identify and tag each piece of laboratory equipment along with its calibration, preventive maintenance, and operational status. 7. Review temperature monitoring log records for incubators, refrigerators, and controlled areas. 8. Review laminar flow hood HEPA filter certification records. 9. Make sure that up-to-date equipment operating instructions are available. 10. Ensure that appropriate laboratory/instrumentation is available for use in accordance with required methodology. 11. Keep track of service contracts for equipment maintenance for each type of equipment.

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

103

Table 8-1 Cosmetic Microbiology Laboratory Audit Checklist (Example) continued This list gives examples of points to consider when performing an audit or preparing to receive an audit. It is not intended to be all-inclusive or to infer that all points are applicable to all laboratories. Environmental Monitoring 1. Is there a monitoring program for equipment cleaning (e.g., swab tests)? 2. Are records for environmental monitoring being reviewed? 3. Is there a periodic evaluation of trended environmental monitoring data? 4. Are environmental isolates from swabs and air samples characterized (e.g., gram stain) and/or identified? Microbial Media, Buffers, and Reagents 1. Are expiration dates for microbial media, buffers, and reagents assigned and updated? 2. Are microbial media growth promotion test results being reviewed? 3. After sterilization, is the pH recorded for each prepared lot/batch of microbial growth media? 4. Are microbial growth media, buffers, and reagents traceable to preparation records? 5. Are quality control and quarantine practices for purchased and laboratory prepared microbial growth media being reviewed? 6. Are records for receipt, storage, preparation, sterilization, and storage of microbial growth media, buffers, and reagents being reviewed? Microbial Content Testing 1. At the time of testing, are negative controls performed to verify the sterility of media, buffers, and materials used as well as aseptic technique? 2. Whenever appropriate, are positive test controls (e.g., media) performed? 3. Are inoculum counts verified at the time of test (e.g., positive controls and validations)? 4. Is work performed in a laminar flow hood or biological safety cabinet? 5. Is test data analyzed for aberrant/out-of-specification (OOS) results? 6. Are test methods followed as written? 7. Are isolated microorganisms characterized as needed? 8. Has microbial growth media used in testing been released from quarantine? 9. Have the microbiological test methods for determining microbial content been validated? 10. Have the microbiological test method data been properly documented? 11. Is microbiological testing performed on susceptible raw ingredients? SECTION 8: MICROBIOLOGY LABORATORY AUDIT

12. Is in-process bulk batch testing performed? 13. Are sampling requirements for finished products being followed? Culture Maintenance 1. Are ATCC microbial cultures verified upon receipt (prior to use)? 2. Are record keeping and traceability of microorganisms used in testing in place? 3. Is the seed lot technique/control of number of passages (e.g., not more than five passages from original) being evaluated? Water 1. Is there a source of distilled or deionized water? 2. Is water monitored routinely for chemical and microbiological quality? 3. Are review data generated to include evaluation of corrective action plans when test results are aberrant? 4. Are sanitization and preventive maintenance log records for laboratory water system reviewed? 5. Are procedures for taking microbiological test samples of water reviewed? 6. Are representative water isolates identified?

Table 8-1 104

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Table 8-1 Cosmetic Microbiology Laboratory Audit Checklist (Example) continued This list gives examples of points to consider when performing an audit or preparing to receive an audit. It is not intended to be all-inclusive or to infer that all points are applicable to all laboratories. Sample Handling 1. Are there adequate written procedures for receipt, storage, and handling of test samples? 2. Are there established sample turnaround times/targets? 3. Are sample submission forms stamped with the sample’s date and time of receipt? 4. Are samples given an unambiguous sample number when logged? 5. Does a permanent record exist for sample log-in data? 6. Are appropriate chain-of-custody procedures documented and followed when required? 7. Are there established operating procedures available for the disposal of samples? Quality Assurance/Quality Control (QA/QC) System 1. Is the quality assurance manual readily available to all staff members? 2. Is the quality assurance manual updated regularly? 3. Does the quality assurance officer operate independently of analyses? 4. Does the laboratory have periodic system audits? 5. Does the laboratory evaluate its performance through proficiency testing? 6. Do proficiency testing programs have feedback and corrective action programs, procedures, and protocols in place? 7. Does the laboratory provide in-house training on quality? 8. Are QA policies, protocols, and procedures documented for the following: a. Methods? b. Sample collection and handling? c. Quality control for each type of test performed? d. Procurement and inventory control? e. Operation and calibration of laboratory equipment? f. Preventive maintenance? g. Records management? 9. Is data retrievable and identifiable for each test result? 10. Are applicable computer software programs documented and back-up copies secured? SECTION 8: MICROBIOLOGY LABORATORY AUDIT

11. Are any analyses subcontracted? 12. Are subcontracted laboratories evaluated for QA? Are they audited, when and how often? Records Management 1. Is a system in place that provides for retrievability and traceability of sample source, methodology of analyses, results, person performing analysis, and date? 2. Are records and reports adequately secured and retained for the required length of time to ensure their integrity? 3. Are all laboratory notebooks, when completed, filed in a secure, controlled archive area from which they can be easily retrieved? 4. Are all laboratory equipment/instrument maintenance logs uniquely identified and stored for easy retrieval? 5. If the laboratory operates a computerized data/information management system (LIMS), are there backups to ensure integrity and availability of data/information in the event of a system/power failure?

Table 8-1 SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

105

Table 8-1 Cosmetic Microbiology Laboratory Audit Checklist (Example) continued This list gives examples of points to consider when performing an audit or preparing to receive an audit. It is not intended to be all-inclusive or to infer that all points are applicable to all laboratories. Test Reports 1. Do the laboratory’s reports accurately and clearly present test results and all other relevant information? 2. Does each test report include the following: a. Identification of laboratory issuing the report b. Identification of client, if applicable c. Sample identification and description (e.g., sample name and lot number) d. Dates/times of sample collection or receipt. Receipt and types of testing performed e. Identification of microbiological test methods used in the analysis f. Description of sampling procedure, where relevant g. Any deviations, additions, or exclusions from a test method h. Disclosure of any subcontractor used i. Results and any failures identified j. Identity of person accepting responsibility for the testing k. Laboratory reports in an understandable format l. Corrections or additions made to test reports after they were issued m. A policy/protocol for handling inquiries and complaints about test reports and results. n. A policy/protocol in place outlining the checking and authorization for data release to clients. Table 8-1

Table 8-2 Microbiology Laboratory Skills Checklist (Example)

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

This Microbiology Skills Checklist provides examples of training areas for the skills needed and the basic duties and responsibilities assigned to staff in the microbiology laboratory. The need for proficiency in specific tasks is dependent on an employee’s work level and area of responsibility. The skills trainer/assessor should be proficient in all areas being reviewed. This list may be modified as needed based on equipment and function of the lab.

Techniques and Analyses Techniques and Analyses

Equipment Use and Operation

❏ Aseptic technique

❏ Microscope

❏ Pipetting – traditional and micropipettor

❏ Water bath

❏ Streak for Isolation

❏ Autoclave

❏ Gram stain/KOH string test

❏ Colony counter

❏ Catalase test

❏ Spectrophotometer

❏ Oxidase test

❏ Water activity meter

❏ Microbial identification – traditional and rapid

❏ Lyophilizer

❏ Water testing and sample collection

❏ Anaerobic chamber and jar

❏ Individual test method proficiency Microorganisms Media

❏ Inoculum preparation

❏ Preparation and storage

❏ Organism maintenance techniques

❏ Media quality control ❏ Media remelt in autoclave/microwave ❏ Use of differential media Table 8-2 106

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Table 8-3 Microbiology Lab - Equipment Calibration and Performance Daily/Continuously Temperature controlled devices including incubators, waterbaths, refrigerators, freezers, centrifuges

Verify temperature control

Humidified chambers

Verify humidity control

Autoclave

Sterility indication – each day of use

pH meters

Calibration – each day of use

Water activity device

Calibration – each day of use

Weekly Treated water

Chemical and microbial testing

Six months Balances

Calibration

Spectrophotometers

Calibration

Dispensers

Calibrate dispensing volume

Lyophilizer

Vacuum, temperature, timing calibration

Annually Temperature calibration

Centrifuge

Calibration (temperature, rotor speed)

Laminar flow/biohazard hood

Certify airflow and operation

Thermometers

Accuracy vs. NIST thermometer

Pipettors

Dispensing accuracy and precision calibration

Stomachers

Efficacy

Sonicators

Efficacy

Microscope

Inspection – alignment, lenses, mechanical aspects

UV lamps

Certification or replacement Table 8-3

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

|

CTFA MICROBIOLOGY GUIDELINES

|

107

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

Autoclave

REFERENCES 1. U.S. Food and Drug Administration. 2006. “Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs, General.” 21 CFR, Part 210. 2. U.S. Food and Drug Administration. July 2000. “Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients.” Draft ICH Consensus Guideline. http://www.fda.gov/cder/guidance/ 4011dft.pdf. 3. United States Pharmacopeia. 2007. United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD.

6. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Institutes of Health. 2007. “Biosafety in Microbiological and Biomedical Laboratories (BMBL)”. http://www.cdc.gov. 7. Davis, R.S. 1989. “NIST Measurement Services: Mass Calibrations.” National Institute Standardized Technology, Spec. Publ., 250–31. http://ts.nist.gov/MeasurementServices/calibrations/upload/ SP250-31.pdf. 8. Wise, J.A. 1988. “NIST Measurement Services: Liquid-in-Glass Thermometer Calibration Service.” National Institute Standardized Technology, Spec. Publ., 25023. http://ts.nist.gov/MeasurementServices/calibrations/upload/SP250-23.pdf.

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

4. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

5. Fleming, D.O., and D. L. Hunt, (Ed). 2000. Biological Safety: Principles and Practices. Washington, DC: ASM Press.

108

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 8: MICROBIOLOGY LABORATORY AUDIT

SECTION 9

Microbial Validation and Documentation INTRODUCTION Validation assures that the methods and procedures used by the cosmetic microbiology laboratory are accurate and reproducible. Documentation provides the record of the validation. In order for results to be meaningful, methods and procedures must be validated. Otherwise, the conclusions drawn could be erroneous. For example, failure to properly neutralize the preservative system during testing can result in false negative results where lack of organism recovery may be due to inhibition of the organism. A variety of techniques for validating and documenting methods and procedures is available. In addition to the test methods and procedures used in the laboratory, the microbiological aspects of process water systems and of cleaning and sanitizing procedures can also be validated by the microbiologist. The results of a validation should be documented in an organized record keeping system. Once these methods and procedures are validated and documented, there is a high degree of confidence that they can consistently produce accurate and reliable results. This completes the full circle of quality assurance and is necessary to assure product quality. Personnel responsible for any aspect of the validation must be adequately trained by education and/or experience.

GENERAL CONSIDERATIONS

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

109

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Validation is a process designed to establish documented evidence that a method or procedure does what it purports to do. A validation or revalidation should be performed: • When a new method or procedure is developed. • If there is a significant change in procedure, supplier, product formulation or equipment. • As part of a recommended revalidation schedule.

Validation Format The validation format should contain the following elements: Scope The scope identifies the area being validated, describes the purpose of the validation, and tells what it encompasses. This should include the types of validation that will be performed. Three types of validation include: Prospective Prospective validation establishes documented evidence that a process does what it purports to do based on a preplanned validation protocol. All information and results are gathered before implementation. Concurrent Concurrent validation establishes documented evidence that a process does what it purports to do based on information generated during actual implementation of the process. The test methods, procedures (such as cleaning and sanitizing), systems (such as deionized water system) and equipment are being used while data are gathered to support the validation. Retrospective Retrospective validation consists of presenting documented evidence that, based on a review and analysis of historical data and information, a process does what it was meant to do and that, all things being equal, can be expected to continue performing properly. Concurrent and retrospective validation can be performed simultaneously.

Description A detailed step by step description of the procedure or method is provided.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Requirements The requirements and acceptance criteria for the areas to be validated are described. For example: • Equipment cleaning and sanitization requirements include specific sites, number of swabs before and after, and defined performance criteria. • Laboratory autoclave requirements include number of heat penetration studies, defined acceptable results, and negative controls acceptability criteria. • Test methods criteria include the number of replicate platings and the allowable variance.

Protocol A written protocol for each method or procedure to be validated should be included.

110

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Documentation A validation protocol should include appropriate documentation of all methods, procedures and results.

Conclusion Each validation protocol should include a conclusion that indicates the results of the validation. If the results are satisfactory, a statement may be made that the validation is satisfactory and the method or procedure can produce accurate and reliable results. If the results are unsatisfactory, the next steps or modification required to complete a satisfactory validation should be indicated. A final written report is prepared once the validation has been completed. Any revalidation information can be added as it is generated.

Documentation Once a procedure or method is validated, all results generated during its use should be documented by keeping an organized record system. They can be organized by product or type of test and can include: • Material identification/description • Identification/code number • Vendor/in-house lot number • Reference to the validated procedure used • Acceptance criteria • Tested by • Date tested • Reviewed and approved by All results should be routinely reviewed by supervisory personnel. Records should be kept for an appropriate length of time. Refer to “Annex 5 – Production Control” in the CTFA Quality Assurance Guidelines.1 Documented results can be maintained as part of a product or raw material profile in the development of an historical data base. Validation procedure results should be included as part of this data base. Results compiled in an historical data base prior to validation may be useful as part of a retrospective validation. The data base can also be used as a guide in interpreting test results and establishing test guidelines and requirements.

An autoclave is an instrument that uses moist heat supplied by steam under pressure to sterilize materials. The contents, whether liquid or solid, are exposed to saturated steam at the required temperature and period of time. Pressure serves as a mechanism for obtaining higher temperatures than otherwise could be obtained. SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

111

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

LABORATORY AUTOCLAVE

Validation Preliminary Considerations The accurate measurement of both time and temperature are necessary to confirm the sterilization of the autoclave chamber contents. Before an autoclave cycle can be validated, temperature controllers, temperature recording devices, pressure gauges, and timers of the autoclave must be certified for accuracy to insure proper operation. The autoclave load configuration and contents are important elements. The autoclave cycle will vary based on volume of containers, number of containers, container type, media type, etc.

Heat Measuring Devices Biological indicators and/or chemical and/or mechanical heat measuring devices can be used to validate an autoclave cycle. The use of biological indicators (BIs) for certain media cycles may be problematic if minimal sterilization cycles are required. BIs are available with different populations. Select the most appropriate population. Mechanical Devices These include calibrated autoclave thermometers or thermocouples.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Biological Indicators (BI) Due to their high resistance to moist heat, Bacillus (now called Geobacillus) stearothermophilus spores on a paper strip or as a spore suspension in a glass ampule are the most commonly used biological indicator for monitoring autoclave performance. At the end of the autoclave cycle, the Bacillus stearothermophilus biological indicators are removed and incubated per manufacturer’s directions. These are usually incubated at 56.0-60.0°C for 7 days to account for the possibility of slower growth following exposure to sublethal heat. Use an appropriate media if spore strips are used. If BI are used, they should be certified against the label claim. Negative and positive controls should be incubated along with the autoclaved biological indicator samples. An unautoclaved biological indicator should be incubated as a positive control. If spore strips are used, a negative media control should be also included. Chemical/Physical Indicators Chemical and physical indicators are used as a secondary check to monitor a validated cycle. They are not intended to be used as primary indicators to validate a cycle. Autoclave tape indicates it has been exposed to an autoclave cycle when, for example, black stripes or the word “autoclaved” appear on the tape. Ampules which contain a material that melts and changes color when exposed to the proper temperature may also be used to monitor an autoclave cycle.

112

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Heat Distribution and Penetration Heat penetration and/or distribution studies should be performed on empty (or minimum) and maximum load configurations of the autoclave chamber by using temperature measuring devices and biological/chemical indicators. The biological/chemical indicators and/or the heat measuring devices should be evenly distributed throughout the autoclave chamber for the monitoring of representative areas. At minimum, the monitoring devices should be placed at the four corners and center of the autoclave. The actual number may vary based on the size of the chamber and the load pattern. Heat Distribution The purpose of a heat distribution study of an autoclave is to determine the uniformity of temperature in the load. Heat distribution studies should be performed on load configurations of the autoclave chamber by using temperature measuring devices. It is suggested that a minimum of three heat distribution studies be done on the load configuration being validated. A mean chamber temperature is taken from all the distributed temperature readings. Chamber temperature uniformity can be considered acceptable if individual temperature readings deviate less than ±1.0°C from the mean chamber temperature. A mean temperature deviation greater than ±2.5°C versus the set temperature may indicate equipment malfunction. The chamber uniformity range should be based on the manufacturer’s recommendation for the autoclave capability. Heat Penetration The purpose of a heat penetration study is to assure that all the containers within a loading pattern will consistently be exposed to a sufficient amount of heat for sterilization. A minimum of three heat penetration studies is suggested. Heat penetration studies may be performed by placing chemical/biological indicators in containers of media distributed in a load pattern throughout the autoclave. Biological and/or chemical indicators should be placed throughout the chamber including areas considered to be the most difficult to sterilize.

Documentation

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

113

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

The following information should be recorded for each validation run: • Date of validation • Autoclave identification or number • Run number • Sterilization time • Sterilization temperature • Load contents (media, broth or agar) • Number of containers, configuration • Indicators used and the lot number • Placement of indicators, temperature measuring devices • Results of indicators, measured temperatures • Control results.

A cycle is considered to be validated when all the chemical and/or biological indicators show appropriate reactions or no growth after the process and all heat measuring devices read within acceptable parameters.2

Routine Monitoring Each autoclave cycle should be monitored for the following: • Temperature and time - Confirm temperature and length of cycle on recording charts or cycle printouts. • Pressure - Observe pressure on gauge and confirm if recorded on cycle printouts. • Biological indicators - Monitor using BIs at least quarterly where applicable for monitoring a validated cycle. • Chemical/Physical indicators - Temperature-sensitive indicators, such as autoclave tape, should be used with each load. Preventive maintenance should be performed on a routine basis by the manufacturer or other qualified personnel. Records of maintenance should be retained.

MEDIA General Microbiological culture media contain growth-promoting substances such as available sources of carbon, nitrogen and inorganic salts. The quality of the growth characteristics of microorganisms in culture media depends upon the care taken in the preparation of the medium. To insure satisfactory microbial culture media for use in the microbiology laboratory, a validation and quality control program should be established for freshly prepared or received media as well as to establish the shelf life of the media.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

This applies whether the culture media are prepared from commercially dehydrated products or purchased from a supplier in prepared form. This program is needed to ensure that the media can consistently perform as expected over its shelf life. This program should include the identification and control of those factors which affect the performance of the media. Some of these factors include: • Preparation of media • Temperature • pH • Storage conditions • Shelf life

114

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Validation and Quality Control Program Elements of a media validation and quality control program include: Inventory Control and Storage of Dehydrated Media • Write the date received and the date opened on the containers of dehydrated media. • Follow the manufacturers’ expiration dates and storage conditions. Most dehydrated microbial culture media can be stored in a cool, dry place, preferably at a temperature below 30°C. Manufacturers recommend storing certain types of dehydrated microbial culture media at a refrigerated temperature (2-8°C). • Rotate laboratory stock of culture media so that the oldest container is used first. This practice will allow a turnover in the dehydrated media containers. It will help keep media stock fresh and within the manufacturer’s expiration dates. Media with outdated expiration dates should be discarded. • Protect laboratory culture media that is in dehydrated form from absorbing additional moisture from the environment during storage. A high moisture content could possibly cause the degradation of various ingredients of the media. • Supplements and additives, where used, should be stored per manufacturer’s recommendations. For example, store under refrigeration if the material is sensitive to heat degradation, or protect from light until use if the material is light sensitive.

Preparation of Dehydrated Media

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

115

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Preparation Follow manufacturer’s directions for the preparation of dehydrated media. Factors in preparing dehydrated media which may affect its performance include: • Water for reconstitution - Use purified water, such as distilled, deionized or reverse osmosis, to reconstitute dehydrated culture media. At a minimum, the water should meet the Environmental Protection Agency guidelines for drinking water standards.3 Microbiological and chemical evaluation of purified water is recommended. The quality of purified water may be checked for specific chemical parameters, for example using current USP. or in-house requirements, on a regular basis.4,5 • Agar temperature - Monitor temperature when pouring agar plates for streak plates. The correct temperature should be employed since an incorrect agar temperature can result in the alteration of the final water content of the medium by excessive evaporation and medium shrinkage or excessive condensate. • Additive temperature - Temperature of additives or supplements which are required to be added after sterilization are important. They should be added at the correct temperature to avoid the chemical degradation or destruction of the additive or supplement. • pH - The pH of the microbial culture media is critical. There should be a laboratory procedure that lists the acceptable pH ranges for a liquid or agar

medium. In general, the pH reading should be taken after autoclaving and subsequent cooling to room temperature and meet established requirements. Media Records Establish a batch record for each lot of culture medium that is prepared in the laboratory. The following information should be included on the batch sheet: • Medium name • Date of preparation • Manufacturer’s lot or batch number • Quantity prepared • Signature of preparer • Method of preparation • pH after autoclaving • pH adjustment, if required • Sterilization time and temperature • Volume dispensed • Number of units dispensed • General comments (e.g., appearance of the medium during preparation) • Performance test results and disposition (acceptable/unacceptable). Where available and/or appropriate obtain a certificate of analysis from the supplier of prepared media.

Labeling and Storage of Prepared Media

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Labeling Label all microbial culture media, whether commercially prepared or prepared in the laboratory. Include the following information: • Date of preparation or date received of commercially prepared media • Type of media • Shelf life or an expiration date, either on the label or on a separate list. Storage Establish a shelf life, an expiration date for the culture media. In general, the shelf life of a particular medium is dependent upon how well that medium will continue to support the growth of test microorganisms. The shelf life can be determined by growth promotion studies over time and storage conditions. This can be accomplished by comparing the growth promotion abilities of the stored versus freshly prepared microbial culture media. Factors which affect the shelf life of prepared media include: • Form - The formulation and packaging of the medium will decide its basic susceptibility to deterioration during storage. In most cases, the shelf life of an agar medium in a Petri dish will be shorter than the same dehydrated agar in a sealed container or bottle.

116

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

• Storage temperature - The optimum storage temperature for the majority of prepared microbial culture media is about 2-8°C. Most liquid media will keep for many months at 2-8°C, but have a tendency to form deposits. This is especially true for culture media at double strength. If stored at room temperature, shelf life may be shortened. • Light exposure - Dye-containing media may fade if exposed to light. • Evaporation - A volume check on liquid media should be made on older stocks which may change due to evaporation. • Dehydration and contamination - Prepared solid media can be stored for many months in an airtight container. The storage of agar Petri dishes presents two main problems: dehydration and contamination. The length of storage time that agar Petri dishes can be kept for use will depend upon the ability to avoid microbial contamination and the loss of moisture. To prevent moisture loss, agar Petri dishes can be wrapped in plastic bags for storage at 2-8°C.

Performance Testing of Microbial Culture Media Performance testing is conducted to confirm that a given media yields expected results when inoculated with applicable microorganisms. Usually on the same batch preparation sheet or on a separate coordinated sheet, the following performance testing details are listed: • Medium • Batch/preparation date • Date tested • Sterility evaluation • Specific microorganisms • Growth promoting, differential, and inhibitory ability • Pass/Fail • Operator’s signature and date A sterility check should be performed for each lot of prepared microbial culture medium. The tester should incubate a sufficient number of units at the temperature and time for which the medium is going to be used in the laboratory testing. The sterility test for each lot of prepared medium will document that it was sterilized properly. This test will ensure that any microbial contamination detected during a test procedure using this lot of prepared medium was not caused by a lot of improperly sterilized media.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

117

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Each lot of dehydrated microbial culture medium or commercial or laboratory lot should be tested, as appropriate, for its ability to support, differentiate, or inhibit the growth of microorganisms. For general selection media, the choice of which microorganisms used to validate the ability of that medium to support growth is up to the individual laboratory. It is preferable to use representative strains of different types of organisms. However, selective or differential media should utilize microorganisms that will demonstrate the characteristics of that medium. A negative control organism may be included to determine that there are no false positive reactions.

If all control parameters of the preparation process are validated and monitored, testing may be done less frequently, but on a regularly scheduled basis. Two methods used by industry to validate the growth promotion ability of a microbial culture medium are described below. • One method involves inoculating a culture medium by streaking solid media or pipetting into liquid media with a dilution yielding 10-100 colony forming units (CFU) prepared from a 24-hour culture of a known microbiological strain. • The second method consists of inoculating by streaking or pipetting and spreading a solid culture medium surface with a 10-3 dilution of a 24-hour culture of a known microorganism(s). The choice of method is up to the individual laboratory. There are many factors to consider including the type of test and the sensitivity and/or detection limits of the test procedures for which the media will be used. The known microbiological strains used may be American Type Culture Collection (ATCC) strains of a particular microorganism. In either case, the inoculated media should be incubated at the same temperature and time for which it will be used in laboratory testing. After incubation, the inoculated liquid media is examined for the presence of turbidity and general solid media for the presence of growth. Selective and enrichment agars are examined for typical color and colony morphology of the strain used to inoculate them. If the lot is within the correct pH range, passes the sterility test, and passes the growth promotion test, it can be used for testing in the microbiology laboratory. If the medium’s pH is out of range, if the medium does not pass the sterility test, or if it fails to support the growth of the test microorganism(s), the medium fails the criteria for use in the laboratory. There may be times when performance testing is conducted concurrently with the use of the media for laboratory testing. If a lot of media fails the performance test and was used in the laboratory, all laboratory testing should be repeated with acceptable media. Results of performance testing should be documented and reviewed for accuracy.

TEST METHODS

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

General Considerations Validation of a microbiological method is the process by which it is established through laboratory studies that the performance characteristics of the method meet the requirements for the intended application. Protocols should be designed to generate reliable, accurate and reproducible results. Microbiological methods are designed to detect and/or identify microorganisms when present. These methods include conventional plate count/streak plate procedures, as well as rapid or automated methods. These methods are intended to perform within a wide range of variables, some of which are: • Different types of product • Different types of media • Incubation conditions • Organisms • Detection limits 118

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Elements of a Validation Protocol A protocol should be designed to incorporate all variables inherent to the method. Each step of the protocol or procedure should be documented and include (when applicable): • Reference to protocol or test procedure number • Incubation conditions (time or temperatures) • Neutralizing agents for preservatives or other growth-inhibiting agents • Confirmation of preservative neutralization • Identity of personnel performing each step • Type of media • Media lot numbers • Test organisms - known profile (biochemical, morphological, etc.) • Test organism inoculum level • Dilutions tested • Acceptance criteria which includes test and control sample. For example, one acceptance criterion is recovery of artificially introduced microorganisms into a test and control sample. Data is analyzed comparing test and control sample results. • Test personnel’s signature upon test completion • Reviewer’s signature • Test organisms used should be maintained, as recommended, by the supplier of the organism. • Subculturing should exhibit unique, demonstrable characteristics of the organisms to ensure a pure and known culture. • Organism storage, transfers and subcultures should be documented. Modifications to the protocol should be documented. The accuracy of a test method can be validated by performing repeated tests on the same sample. Its reproducibility is usually confirmed by designing a collaborative test whereby a number of different laboratories perform the same procedure on material from the same batch. A file should be maintained for all data generated by the test procedures. In addition to the above elements and general considerations, the following items apply to specific test procedures.

Microbial Content Testing

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

119

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

• Microbial content testing may be performed on raw materials, components, bulk product, in-process material and finished goods. • Microbial limit guidelines, sample size and frequency of testing should be established. • The established testing guidelines should have appropriate documentation.

Refer to “Determination of the Microbial Content of Cosmetic Products” (Section 18) “Establishing the Microbial Quality of Cosmetic Products,” (Section 12) and “Microbiological Limit Guidelines for Raw Materials” (Section 11).

Preservative Efficacy Testing Method The method of preservative challenge testing adopted for use should predict preservative efficacy under routine good manufacturing practice and normal consumer use conditions. The preservative system is not intended to compensate for poor GMPs.

Test Procedure The type of test procedure used should be determined. For example: • Semi-quantitative - Swab or Streak plate Method • Quantitative - Total Plate Count

Protocol The elements of the protocol should include: • Length of the test • Test organism(s) • Media • Sampling frequency • Confirmation of the neutralization of the preservative • Test parameters (incubation time, temperature, etc.) • Sample size • Acceptance criteria. Refer to “M-3 The Determination of Preservation Adequacy of Water-Miscible Cosmetic and Toiletry Formulations” (Section 20), “M-4 Method for the Preservation Testing of Eye Area Cosmetics” (Section 21), and “M-6 A Method for the Preservation Testing of Atypical Personal Care Products.” (Section 23).

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Environmental Ambient air, compressed air, and equipment surfaces in the manufacturing plant are monitored for the presence of microorganisms using apparatus such as settling plates, air samplers, swabs, and contact plates. Equipment should be evaluated by reviewing manufacturer’s technical information. Surface monitoring procedures can be validated in the laboratory by applying known types and levels of organisms to sample surfaces simulating production surface materials. Recovery using

120

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

the various surface monitoring techniques can then be compared to initial types and levels of organisms to determine the acceptability of the procedure. • Maintain equipment maintained per manufacturer’s specifications and or recommendations. • Validate that the media used supports organism growth. • Ensure that media neutralizes any sanitizer present. • Establish action and alert levels based on historical data. Refer to “Microbiological Evaluation of the Plant Environment” (Section 2).

ORGANISM IDENTIFICATION Identification Systems Microorganisms may be identified using classical methods. Identification via commercial identification kits and/or automated systems is common practice in cosmetic microbiology laboratories. Systems used for microbial identification should be validated to ensure they consistently and accurately identify microorganisms tested. A validated system should show equivalence with a known method. Introduction of an alternate identification system requires a validation vs. the current system to ensure performance is comparable or better than the current system(s). Some of the factors to include: • Side-by-side comparison testing of the standard routine cultures used in the laboratory and/or ATCC cultures. • Side-by-side comparison testing of unknown organisms such as those isolated from the plant product. Standard cultures of microorganisms, such as ATCC cultures, should be used. A performance check should be done on each new lot of kits received. It is important to follow the directions supplied with the systems. The manufacturer of the identification systems may recommend a series of QA microorganisms to test reliability and accuracy. QA test organisms should be chosen to give a positive response to each of the tests. Organisms used for the QC test should include the following: • Positive Control - Inoculate with organisms the manufacturer claims are identifiable by the kit. • Negative Control/Identification System Control - If applicable, inoculate with sterile saline or appropriate diluent. Do not inoculate with any organisms. Follow manufacturer’s recommendations. A system can be considered valid if the known organisms are properly and consistently identified and the negative controls/identification system controls, as applicable, do not demonstrate any reactions.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

121

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Records should be kept specifying the following: • System tested • Manufacturer • Lot number

• • • • • •

Date tested Organisms tested Results Acceptance criteria - action taken if system or performance check not valid Initials or signature of test performer Initials or signature of reviewer, as appropriate

Refer to “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa” (Section 19).

MANUFACTURING, EQUIPMENT CLEANING AND SANITIZING PROCEDURES Introduction Validation of cleaning and sanitization methods is a procedure to establish performance characteristics of a cleaning and sanitizing process and to document the process will consistently yield equipment that meets microbiological, physical and analytical chemistry requirements. The microbiological validation process basically consists of: • Determining requirements • Writing a protocol • Following the written protocol • Testing and documenting the results Results should consistently demonstrate that the process is in control and all steps should be carefully documented. The protocol should be approved by the appropriate personnel including the Microbiology Department. Performing the tests and evaluating the results should be the responsibility of the Microbiology Department. This activity should be coordinated with analytical chemistry, manufacturing, engineering and key personnel in other departments.

Validation Protocol Components of the validation protocol include:

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

1. Determine specification or outcome desired • What results are required to indicate that the procedure will result in equipment that meets requirements? 2. Outline cleaning and sanitizing procedure to be validated • Identify specific equipment and testing locations. • What pumps, valves, filters, tanks, lines will be looked at and where? • List cleaning methods and agents, concentration, contact time, temperature. • Equipment must be clean before it can be sanitized. • List method(s) of sanitization. • Be sure that the sanitizer has itself been evaluated. 122

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

• Map the cleaning and sanitization process; for example: − Identify Tank #XXX and associated pipes, pumps & lines. − Rinse with hot DI (deionized) water for XXX minutes to remove product residue. Disassemble pumps/equipment when necessary to remove product residue. − Drain rinse water. − Fill tank with ?% solution XX. − Recirculate/mix for XXX minutes. − Drain wash solution. − Rinse to remove wash solution. − Sanitize (follow directions for use of sanitizing agent). • Establish a time limit for use of prepared sanitizing solution. • Establish frequency of sanitization. • Establish a time limit for resanitization. 3. Determine what steps should be evaluated, document test procedure (be specific) and determine what data is required. 4. Determine test methods used to provide the data and acceptance criteria required in item 3 (above) to confirm the efficacy of the cleaning and sanitizing procedures. Several types of methods include: • Visual examination • Visual inspection to detect product residue • Examination for “odor” • Inspection to detect the odor of residual perfume or sanitizer • Swabs • Swabs to detect microorganisms • RODAC plate • Contact plates to detect microorganisms • Rinse water • Tests on rinse water to detect microorganisms and/or chemical residue 5. Compare results of tests performed before and after the cleaning and sanitizing procedures to confirm the efficacy of the process. 6. Validation testing should be performed a minimum of three times.

After a process is validated, routine testing, such as equipment monitoring, can be used to monitor the process. Refer to “Annex 3, Part 1 - Packaging Equipment” and “Annex 3, Part II - Processing Equipment,” CTFA Quality Assurance Guidelines.1 SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

123

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Once the validation for a piece of equipment is completed, it need only be repeated if there is a change in process, equipment or any parameter that may effect the outcome of the process, such as product type. To remain aware of any changes, a Process Audit should be routinely done and communication with key personnel maintained. There should be a routine review, for example, annually, to insure and document that there have been no changes.

PROCESS WATER SYSTEM Validation The purpose of validating a process water system is to generate documented evidence to provide a high degree of confidence that the process water system will consistently produce water that meets established guidelines. This is especially important with a water system since microbiological test results may not be available before the water is used in manufacturing. The following elements should be included: System description Describe the system and give an explanation of the purpose of each element. These should include: Type of system • Deionizing (DI) - carbon beds, deionizing beds (separate, mixed or both) • Reverse Osmosis • Distillation Unit Flow rate of the system Description of water flow system • Type of piping • Number of outlets • Points of use • Recirculation route if applicable. Description of the storage tanks, if applicable Description and location of any filters present • Micron size • Prefilter • Final filter Description and location of any treatment element • UV light with rated throughput • Chlorination with concentration • Heat with temperature requirements • Ozonization with concentration. Description and location of sampling and use points.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

A schematic of the system should also be included.

124

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

System Maintenance Describe the system maintenance. Include the following elements: • Frequency of regeneration, backflush of carbon beds and/or changes of the DI bed and/or filters • Frequency of cleaning and sanitizing the system • Method of cleaning and sanitizing, such as heat treatment or use of cleaning or sanitizing solutions • Description of cleaning and/or sanitizing solution(s) used

The Validation Protocol Type of validation Type of validation performed may be: • Prospective • Concurrent • Retrospective • Combination Conditions The conditions under which a revalidation will be performed should be listed, whether periodically, after a significant system change, or under other circumstances. Suggested revalidation requirements include: • Perform a complete revalidation if there is a significant change such as in equipment, procedure, etc. • Perform a reduced or modified revalidation at a preset interval, for example, annually, to ensure that the system is still performing as expected. A periodic revalidation may also be accomplished by a formal review of the data and system to document that there are no significant changes, and the system continues to perform as intended. Time period covered Samples should be tested more frequently, a minimum of two or three times per week during this period. The time period covered should include seasonal changes so all variables are part of the validation.

Test criteria Include the criteria used and any action limits that are established. Also specify what actions are to be taken. SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

125

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Test methods Include a detailed step-by-step description of the methods used or a reference to established test methods. For example, tests commonly performed are: • Chemical, as per USP4 or internal requirements. • Microbiological, including total plate count and any other tests needed to support the criteria as per established internal or compendial methods.

Results The validation protocol should include copies of all test results during the validation test period.

Discussion of Results A discussion of the test results should include any investigations or actions taken if results do not meet expected criteria.

Conclusion A determination based on an analysis of the results of whether or not the system can consistently produce water which meets requirements if the key parameters are followed.

Documentation Once established as part of the validation, critical system parameters, such as frequency of regeneration, frequency of cleaning/sanitizing, names and concentrations of cleaners/sanitizers used, and filter changes, should also be checked on a routine basis and documented to ensure the proper operation of the system. This information is usually recorded as part of the system maintenance record. This record should include: • Dates the system was cleaned and sanitized • Names and concentrations or conditions of the cleaners/sanitizers used • Maintenance of UV light if applicable • Dates the system was regenerated, backflushed and/or filters or resin beds were changed • Dates and explanation of any repairs to the system.

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

These parameters should be reviewed as part of an investigation if results do not conform to the guidelines. After the water system has been validated, samples should be tested on a routine basis to monitor its performance. These results should be recorded and trends noted. This information should include: • Sampling points • Reference to the validated test procedure/method • Acceptance guidelines • Any investigation results and/or actions taken if results do not conform to guidelines • Tested by and date tested • Reviewed and approved by A record tracing the use of the water (batch numbers, product) should also be maintained. This is usually maintained as part of the manufacturing record. Refer to “Microbiological Quality for Process Water” (Section 7). 126

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

LABORATORY EQUIPMENT AND INSTRUMENTATION Diverse laboratory equipment and instrumentation is utilized in the microbiology laboratory to assist in preparing and performing microbiological analyses. All instrumentation should be regularly monitored to ensure its proper performance and reliability. A master logbook of equipment requiring monitoring and the observed results should be maintained. In addition, a file should be kept for each piece of equipment including: • Name of instrument • Model number • Serial number • Purchase date • Manufacturer and/or distributor • Maintenance and operational manuals • Service representative information

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

|

CTFA MICROBIOLOGY GUIDELINES

|

127

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

A list of suggested equipment and instrumentation to be monitored includes but is not limited to: • Laboratory autoclaves (as above) • Laminar flow hoods/biological safety cabinets − Air flow − Integrity of HEPA filter − Particle size • Balances − Standard weights • Centrifuges − Rotor speed (rpm) − Timer • Incubators/refrigerators − Temperature • Water baths − Temperature • Microscopes − Optical alignment − Calibration for scale − Cleaning and maintenance • Automatic colony counters − Standard template • pH meters − Standard pH buffers • Spectrophotometers − Standard solution • Water activity devices − Standardized salt solution

• Thermometers − ASTM, NIST Information pertaining to proper control steps for carrying out performance checks and preventive maintenance should be found in the operator’s manual. To maintain equipment accuracy and reliability, these tests and maintenance should be performed at regularly scheduled intervals. The documentation for such testing should include the following: • Frequency of performance (monitoring schedule) • Criteria for acceptance/rejection • Performance against standards • Results • Noted deficiencies • Corrective action if necessary • Documentation of corrective action and “fit for purpose” performance should be reviewed and signed • Initials or signature of test performer and reviewer. During the validation of standard operation, all results should be recorded accurately including deviations and corrective action initiated, if necessary. Records should be reviewed periodically by supervisory personnel. All equipment should receive regular preventative maintenance. Specialized checks, such as autoclave servicing, centrifuge calibration and laminar flow hood maintenance should be performed by trained personnel or an authorized representative of the equipment manufacturer. Refer to “Annex 16 - Calibration Systems,” CTFA Quality Assurance Guidelines.1

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

REFERENCES 1. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

4. United States Pharmacopeia. 2007. <1231>. “Water for Pharmaceutical Purposes.” United States Pharmacopeia and the National Formulary. USP30 NF25. Rockville, MD. 687-706.

2. Parenteral Drug Association. 1978. Validation of Steam Sterilization Cycles, Technical Monograph No. 1.

5. U.S. Food & Drug Administration, 1993. “FDA Guide to Inspections of High Purity Water Systems.” http://www.fda.gov.

3. EPA 40 CFR Part 141.1998. National Primary Drinking Water Regulations: Disinfectants and Disinfection Byproducts Notice of Data Availability. http://www. epa.gov/safewater/standard/v&e-frn.pdf.

128

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

SECTION 10

Maintenance and Preservation of Test Organisms INTRODUCTION Cosmetic microbiology laboratories require maintenance and preservation of stock cultures for research, educational, and industrial testing purposes. The conservation of viable, uncontaminated cultures without variation or mutation of their original characteristics is essential for preservation of product isolates, challenge testing of antimicrobial agents, efficacy and stability testing, quality control evaluations, etc. Various short- and long-term methods are available for preservation of microorganisms. Culture maintenance implies viability and purity, whereas preservation involves retention of phenotypic and genotypic characteristics over a period of time. All preservation methods have advantages and disadvantages that must be considered in view of the needs of the user. Laboratories that stock few cultures that are frequently used may select an inexpensive, short-term method such as subculturing to agar slants, immersing in mineral oil, ordinary freezing or various drying techniques. Others who have large culture collections may primarily use long-term methods such as freeze-drying or ultra-freezing. Species of microorganisms, ease of manipulation, and financial restraints are additional selection criteria.

SHORT-TERM METHODS Subculturing Maintaining organisms on artificial media with periodic transfer is the most common and simplest method of preserving a culture line. It has the advantage of not requiring any advanced skills or equipment. However, it is also the method that has the greatest propensity for causing strain variation. Therefore, the microbiologist should carefully consider which organisms are to be maintained in this manner. Media used for subculturing should be nutritionally minimal. By slowing metabolism, accumulation of toxic metabolites is kept to a minimum, and the possibility of culture death or mutation is reduced. Experience is perhaps the best determinant of how frequently subculturing is done. Kept under refrigeration, most cultures will survive a month’s storage. However, some fastidious strains may require shorter intervals between subculturing, while others may be more tolerant of prolonged storage. In general, length of storage should be the maximum time that ensures a viable, unaltered culture.

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

|

CTFA MICROBIOLOGY GUIDELINES

|

129

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

Frequent checks of unique characteristics (e.g., pigment, colonial morphology, biochemical traits) should be performed in addition to periodic extensive identification procedures, since this method may create variations in strains of organisms.

Overlay Techniques As an extension of common short-term subculture methods, an overlay of mineral oil or liquid paraffin is often used. This has the effect of reducing availability of air to the organism, thereby slowing the metabolic rate. After growth of the culture in an appropriate agar, as a slant or a stab, or in broth, add sterile oil to a depth of 1-2 cm above the highest point of growth. The cultures may then be stored at room temperature or refrigerated. A common source of contamination as a result of this method is nonsterile oil. To prevent this, the oil should be heated to 170ºC for 1-2 hours. Recovery of the organism is accomplished by removing a small amount of the growth with an inoculating needle or loop and transferring to an appropriate growth medium. This technique, used extensively over the years, has been very successful in fungal preservation. Its chief disadvantage is that it is a messy procedure and therefore a poor method when frequent retrieval is required.

LONG-TERM METHODS Drying The preservation of organisms by drying or desiccation is accomplished by the removal of water and prevention of rehydration. There are a wide variety of drying methods that have been extensively used.

Sand and Soil Sand and soil have been found acceptable for preserving sporulating fungi. Sand or garden loam having a water content of approximately 20% is put into glass bottles and filled to between half and two-thirds capacity. The sand/soil is then sterilized by autoclaving at least twice at 121ºC for 20 minutes, allowing the contents to cool between runs. The inoculum or spore suspension is prepared by adding 5 mL of sterile water to the culture and gently scraping the colony to release the spores. If the isolate does not sporulate, then a suspension of mycelia can be used. The suspension is then dispersed in 1.0-mL amounts into the sterile soil/sand bottles. The inoculated bottles are allowed to stand at room temperature for a period of 3-14 days depending on the growth of the organism. This allows the fungi to utilize the moisture and for growth to slow down for storage. The soil/sand culture bottles are stored with loose caps in the refrigerator (4-7 ºC). The fungi can be revived by sprinkling a few grains of soil onto a suitable medium. 130

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

Silica Gel The silica gel method has been found to be a successful drying method for fungi and yeasts as well as bacteria. In this method, bottles one-quarter filled with silica gel (6-22 mesh) are sterilized in a hot-air oven at 180 ºC for 2-3 hours. The bottles are then cooled by placing the bottles in trays of water and freezing in a deep freeze (-17 ºC to -24 ºC). The organism suspension is prepared in sterile 5% nonfat skimmed milk. The suspension is cooled and added to the silica gel bottles in an ice bath. Only three-fourths of the silica gel crystals are wetted to avoid oversaturation. The bottles are left in the ice bath for 20 minutes until the ice is melted. The bottles are kept at 25 ºC until the crystals readily separate when shaken, between 1 and 2 weeks. After this time, the viability is checked by sprinkling a few crystals onto medium for growth. If growth is good, the bottle caps are tightened and the bottles are stored over indicator silica gel in an airtight container at 4ºC. The indicator gel must be replenished from time to time. Note: It is very important to keep all apparatus very cold during inoculation to minimize the effect of the heat generated when the gels are hydrated.

Porcelain Penicylinders and Silica Gel Crystals Porcelain penicylinders are inoculated with the microorganism suspension, dried on silica gel crystals and stored in the refrigerator at 0-4ºC. Test tubes, one-third filled with silica gel crystals (6-16 mesh) topped with a layer of glass wool, are sterilized in a hot-air oven for 2 hours. Rubber stoppers are sterilized by autoclaving at 121ºC for 30 minutes. Two sterile porcelain penicylinders are placed in a 48-hour microorganism suspension and allowed to absorb for 15 minutes, then aseptically removed and placed in a sterilized silica gel tube. The stopper is applied and the tube stored at 0-4ºC. Organisms are revived by culturing a penicylinder into a tube of appropriate nutrient medium and incubating for growth. Inoculated cylinders can be stored for 6 months or longer depending on the organism.

Paper Strips or Discs Yeasts have been successfully stored on paper. The paper replica method, developed by Basel et al. (1977)1, has been found to maintain several hundred strains of yeasts for 3-6 years. A mature yeast colony is suspended in a drop of sterile evaporated milk and mixed thoroughly. Sterile filter paper discs (Watman #2 filter paper) cut into 1 cm2 sections are immersed in the suspension for a short time. They are then transferred to sterile aluminum foil packets. Three edges of the foil are left open to allow the discs to dry. The packets with discs are placed in a desiccator and allowed to dry for 2-3 weeks at 4ºC. Once dry, all edges of the packets are sealed and packets are then stored in a dry area at 4ºC. SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

|

CTFA MICROBIOLOGY GUIDELINES

|

131

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

Studies have shown isolates of Fusarium and other genera to survive by this method 10-20 years. No data are available on the preservation of bacteria and yeasts by the soil/sand method.

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

For revival, the disc is streaked across a plate containing appropriate medium for recovery. The disc can remain in one corner of the plate. The plate is incubated at the appropriate temperature for growth.

Predried Plugs A number of types of bacteria that are difficult to preserve by freeze-drying have been successfully preserved on predried plugs. Predried plugs can be made of cotton-wool cellulose, starch, peptone or dextran. Suspensions of organisms are dropped onto the plug. Preserving agents, such as mixtures of 5% peptone and 5% glucose or 5% peptone and 5% sorbitol, can be added to improve recovery at various storage temperatures. The plug is then dried in a desiccator and stored in ampules under vacuum either at 4ºC or at room temperature. Organisms can be recovered by rinsing the plugs with 0.5 mL broth and plating out a few drops onto appropriate solid media. Descriptions of several modifications in technique and apparatus have been documented. Microorganisms such as several species of Salmonella, Escherichia coli, Staphylococcus aureus and Vibrio sp. have been successfully recovered after 3 to 6 years of storage on predried cellulose plugs. In contrast, other species showed very poor recovery. Consequently, this method may be limited to preservation of particular groups of microorganisms.

Gelatin Discs Many species of bacteria have been successfully preserved by the gelatin disc method. Bacterial growth is suspended in melted nutrient gelatin. Drops of the suspension are delivered to a Petri dish with the aid of a ropping pipette delivering 0.02 mL. The Petri dish is covered and placed in a deep freeze at -20ºC to -40ºC until the drops are frozen, indicated by a change from transparent to opaque. The Petri dishes are transferred to the freeze-dryer containing desiccant trays with phosphorous pentoxide. The freeze-dryer is turned on and cultures dried overnight. After drying, the gelatin discs are transferred to sterile vials containing silica gel and cotton-wool and stored at 5ºC. To revive the organisms, the gelatin disc is placed in 1.0 mL of nutrient broth and allowed to melt by warming in a 37ºC water bath. Once melted, a loopful of broth is transferred to a suitable solid medium for growth.

Advantages and Disadvantages of Drying Techniques Viability of cultures preserved by drying techniques can extend to years. Capital equipment costs are low and the methods are not labor-intensive. Many of the methods are suitable for storing large numbers and frequently used cultures, since aliquots of dried material can be removed from containers without great risk of contamination. On the other hand, these methods cannot be universally applied. Most are limited to use with particular groups of organisms. Since there is not enough data available on the reliability of these methods, users are advised to gain experience first, giving careful consideration to the criteria outlined in the introduction.

132

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

Freezing of microorganisms as a method of culture preservation is successful because water is made unavailable to the cell. Final storage temperature and cooling and rewarming rates are critical variables in the operation, as is selection of a suitable cryoprotective agent. In general, the lower the storage temperature, the greater the rate and longevity of survival. Low-temperature freezing (-70ºC to -140ºC) is better than temperatures of 0ºC to -20ºC, which do not preserve well because the cells are exposed to high salt concentrations during the freezing period.

Frozen Suspension A cell suspension of the microorganism is prepared, routinely as an overnight broth culture, utilizing a minimal medium, so as to lower the metabolic rate of the organism. After centrifugation, the pellet is resuspended in fresh sterile broth, to which has been added an appropriate cryoprotective agent. The most frequently used are glycerol (10-15% vol/vol) and DMSO (5-10% vol/vol). If agar slants are used as the source of cells, the growth is washed from the slant with sterile broth containing the cryoprotective agent. The selection of cryoprotective agent and its concentration may be determined in advance by examining for any toxic effects on the organism. This may vary depending on the species of microorganism. Aliquots of the resuspended cells (0.5-5.0 mL) are placed in sterile vials, allowing sufficient space for expansion of the liquid during freezing. Vials are placed in a bed of crushed dry ice until frozen solid, then removed and transferred to an ultra-low-temperature freezer at -70ºC to 90ºC. Duplicate vials of each strain should be prepared to have one set of cultures remain frozen at all times as a permanent collection. To recover the organisms, the vial is removed from the freezer, opened, and a small amount of material scraped from the surface with a sterile stick or needle, inoculated into appropriate media and incubated at specified growth conditions. The vial should not be allowed to thaw or re-warm. This can be minimized by keeping it in a bed of dry ice when working with it. Freezing cell suspensions at low temperatures is a reliable, efficient means of long-term preservation. It is not labor-intensive, and large numbers of cultures can be maintained. The high initial equipment costs and the possibility of associated mechanical freezer malfunctions or power failures may be of concern. A contingency plan should be developed in case of such emergencies.

Glass Bead Storage An alternate ultra-low-temperature storage method is to coat the microbial suspension onto glass beads. This eliminates the problem of repeated freezing and warming of frozen suspensions when subcultures are made, because each bead may be removed without any warming of the remaining beads. The individual bead is then used as the inoculum. Glass beads, approximately 2 mm in diameter, are washed with detergent and rinsed with dilute HCl to neutralize the pH, followed by a distilled-water rinse. After drying, the beads are placed in screw-cap glass vials, the quantity per vial dependent on intended use and the vial size. The vials with the beads are sterilized by autoclaving at 121ºC for 15 minutes. The organism is grown on SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

|

CTFA MICROBIOLOGY GUIDELINES

|

133

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

Freezing

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

the surface of an appropriate nonselective agar plate as an overnight culture, then inspected for any contaminants. Approximately 1 mL of an appropriate sterile broth with cryoprotective agent (see Frozen Suspension) is added to the agar plate and mixed with the growth to form a thick suspension. This suspension is then added to the vial containing the beads. After the beads are wetted thoroughly, excess suspension is removed from the bottom of the vial. The vials are then placed in an ultra-low-temperature freezer maintained at -70ºC to -90ºC. To recover the organism, a bead is aseptically removed from the vial and placed on the surface of a plate of solid medium and rubbed over the surface to release organisms. Alternately, the bead may be placed in a tube of sterile broth medium. The plate or tube is then incubated at appropriate growth parameters. This method has the same advantages and disadvantages as frozen suspension techniques and is easier for frequently used organisms. However, it is slightly more time-consuming due to the extra steps of preparation and coating of the glass beads.

Ultra-Freezing (-140ºC to -196ºC) Ultra-freezing, using liquid nitrogen, is a method that offers long-term storage of organisms, preservation of their original characteristics, and relative simplicity in processing. Storage in liquid nitrogen provides a stable culture collection of identical sub-units that are easily accessible when needed or preserved indefinitely providing the storage temperature is maintained below -130ºC. Organisms can be stored using nitrogen in either the liquid phase at -196ºC or in the vapor phase at -140ºC and below. Most organisms used in routine laboratory work do not require programmable cooling rate equipment with its high capital costs. A liquid nitrogen storage container is sufficient for maintaining a typical organism collection. It is recommended to include a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO) in the culture suspension to enhance freeze/thaw survival. Generally, bacterial broth cultures should be harvested in the mid- to late-log phase, agar fungus cultures when spores are mature. Cultures should be centrifuged, supernatant decanted, the pellet reconstituted with broth plus cryoprotectant, and the desired aliquot distributed aseptically to ampules. The ampules are then capped and placed on aluminum canes for storage. To revive a frozen culture after withdrawal from liquid nitrogen, place the ampule in a container and then rapidly place the container into a 35ºC water bath. When the culture has thawed and warmed to 35ºC, transfer it aseptically to an appropriate growth medium. Cultures that will be used for challenge testing should be held a minimum of two hours in a nutritive medium to allow resuscitation before exposure to a hostile environment. The use of liquid nitrogen requires some precaution. Ampules that are stored in the liquid phase can present the risk of explosion if liquid nitrogen penetrates an imperfect seal and expands rapidly when warmed. Polypropylene screw-cap ampules reduce the hazard that glass ampules pose, as well as eliminating several steps of preparation. Safety glasses or a plastic face shield and protective gloves should be worn when depositing or withdrawing cultures from liquid nitrogen. Failure to maintain a minimal level of liquid nitrogen can result in the loss of an entire culture collection. To prevent this, liquid nitrogen levels must be monitored regularly.

134

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

Freeze-drying, or lyophilization, is widely used to successfully preserve a broad range of bacteria, fungi, and viruses. In each case, the process for each particular microorganism should be optimized to provide for the greatest recovery of the original population. Several factors in the process should be considered in reaching this goal: • Growth phase of the culture • Temperature of growth • Composition of the growth and suspending medium • Freezing temperature • Rate and duration of the freeze-drying process • Final moisture content • Rehydration processes Freeze-drying is a process where water is removed by sublimation. The organisms are first suspended in a suitable medium, usually a cryoprotectant. They are then placed in glass ampules and a vacuum is applied to remove the water as it sublimes. After drying, the ampule is flamesealed under vacuum or an inert gas.

Culture Preparation and Cryoprotectants The culture should be grown in a medium particularly suited for maintaining preservative resistance characteristics while not decreasing viability. Use of preservative or product as a supplement to ordinary nutrient media can be a successful means of obtaining this goal. Cells are generally grown to late logarithmic phase. If grown on agar slants or plates, the cells are washed off with aid of a sterile policeman or pasteur pipette and resuspended in a minimal amount of broth media. If grown in liquid suspension, they are harvested by centrifugation and the pellet is resuspended in a small amount of broth. To each milliliter of broth-suspended culture, one milliliter of sterile 12% sucrose can be added, mixed, and then placed into the ampules. Other suspending media include a mixture of inositol (5 gm) and horse serum (100 mL) or a mixture of nutrient broth (2.5 gm), inositol (5 gm), and distilled water (100 mL). Skim milk has also been used with success.

General Procedure The microbial suspensions are pipetted into glass ampules, cotton plugged, frozen, and a vacuum (0.05 to 0.2 torr) applied. The ampule can remain in a brine-ice bath or dry ice/methanol bath while under the vacuum to control the rate of sublimation. Once dry, the ampules are flamesealed under vacuum. The vacuum of the sealed ampules may be checked by use of a highfrequency spark tester. Other variations include manifold batch preparation, centrifugal drying, or shelf drying. Vials used may be single or double vials.

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

|

CTFA MICROBIOLOGY GUIDELINES

|

135

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

Freeze-Drying (Lyophilization)

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

Recovery Rehydration of the dried cultures should be done with a medium of the same composition as the suspension broth. The vials are scored, wiped clean with ethanol, broken into, and sterile medium introduced to rehydrate the culture. The rehydrated culture is then transferred to a broth or agar medium. Once growth occurs, a check to ensure all the type characteristics including product/preservative resistance should be done. If the checks prove stable and viable, the lyophilization and storage conditions can be considered validated for the culture under those conditions of freeze-drying and storage.

Advantages and Disadvantages In general, stability of a microbial population’s characteristics is not adversely affected by freezedrying. However, some selection, particularly loss of plasmids, can occur in some bacterial species. Also, a significant drop in viability can occur in sensitive species. A major advantage of freeze-drying is that once the culture is lyophilized, it is stable over periods up to 50 years without the need for special storage conditions. A major disadvantage is the high initial capital cost for equipment.

SPECIAL CONSIDERATIONS Microorganisms are used by the cosmetic industry to obtain a variety of important data. Consequently, the microbiologist must have some assurance that the organisms used will retain their original genotype. This is especially true of organisms isolated from contaminated product or organisms known to have resistance to certain materials. In many instances, conventional methods for maintaining such organisms may not be adequate. Spontaneous reversion may occur if mutants are removed from their original environment to artificial media or subjected to physical stress. Occasionally, the organism may not survive even minimal storage on artificial media. The microbiologist should give special attention to these organisms and store them in such a manner that their original attributes are not lost and viability is maintained. This may mean periodic subculturing into material from which the original isolate was obtained or devising an artificial medium that will ensure a culture that has retained the desired trait. These organisms should also have well-documented profiles. In addition to periodic screening, certain key traits unique to the isolate should be monitored frequently to provide an added measure of assurance of strain stability. Any deviation from the expected biological profile may indicate that the organism is no longer suited for use.

136

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

The aforementioned methods are an overview of those techniques currently acknowledged as satisfactory for maintaining and storing microorganisms. There are perhaps several other methods less commonly employed that may be equally reliable; their absence here does not imply otherwise. Whether published or internally developed methods are used, it is the responsibility of the microbiologist to carefully monitor the variables in the method, periodically ascertain the purity and integrity of each strain of microorganism, and maintain any applicable documentation relating to a culture collection. Adherence to sound microbiological practices will ensure that data obtained from procedures using microorganisms are accurate, reproducible, and meaningful.

ADDITIONAL INFORMATION Brown, Michael R.W. and Peter Gilbert. 1995. Microbiological Quality Assurance A Guide Towards Relevance and Reproducibility of Inocula. Boca Raton, FL: CRC Press. Downes, Frances Pouch and Keith Ito, (Eds). 2001. Compendium of Methods for the Microbiological Examination of Foods. Washington, DC: American Public Health Association. Gerhardt, Philipp, R.G.E. Murray, Willis A. Wood, and Noel R. Krieg, (Eds). 1994. Methods for General and Molecular Bacteriology. Washington, DC: American Society of Microbiology. Gherna, R.L. 1989. Practical Handbook of Microbiology. Edited by W.M. O’Leary. Boca Raton, FL: CRC Press. 249-250. Hill, L.R. 1981. Essays in Applied Microbiology. Edited by J.R. Norris and M.H. Richmond. Chichester: John Wiley & Sons. Kirsop, B.E. and A. Doyle. 1991. Maintenance of Microorganisms and Cultured Cells: A Manual of Laboratory Methods. Burlington, MA: Academic Press. Lapage, S.P., K. F. Redway, and R. Rudge. 1978. CRC Handbook of Microbiology. Edited by A.I. Laskin and H.A. Lechevalier. Florida: Chemical Rubber Press. 743-758. Simione, F.P. and E.M. Brown, (Eds). 1991. American Type Culture Collection Preservation Methods: Freezing and Freeze-drying. Manassas, VA: ATCC. http://www.atcc.org.

REFERENCES 1. Basel, J., R. Contopoulou, R. Mortimer and S. Fogel. 1977. UK Federation for Culture Collections Newsletter. 4: 7.

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

|

CTFA MICROBIOLOGY GUIDELINES

|

137

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

CONCLUDING REMARKS

SECTION 10: MAINTENANCE AND PRESERVATION OF TEST ORGANISMS

138

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

SECTION 11

Raw Materials Microbial Content

In order to minimize the chance of contaminated finished product, it is necessary to control the microbial content of cosmetic raw materials along with other physical and chemical attributes. Cosmetic manufacturers should evaluate the microbiological quality of their raw materials and establish appropriate specifications based on the best available scientific information. Water and water supplies are addressed in the CTFA “Microbiological Quality for Process Water” (Section 7). Water systems should be properly validated and controlled. Quality specifications for water should be set, including alert and action levels.

GENERAL CONSIDERATIONS When establishing acceptable levels for raw material microbial content, the following criteria should be considered: • Chemical composition • Physical nature • Origin and availability • Lot uniformity • Intended use of the product • Concentration of raw material used in the product • Manufacturing process • Raw material history • Storage conditions • Water activity Many synthetic raw materials currently used by the industry contain low microbial counts, due to extremes in pH, low water content, or inherent antimicrobial properties. Others may be supplied as aqueous dispersions or solutions, and may be susceptible to microbial proliferation. Therefore, it is important to evaluate susceptible synthetic materials upon receipt to ensure that they have not been contaminated during the manufacturing process, packaging, transportation and storage.

SECTION 11: RAW MATERIALS MICROBIAL CONTENT

|

CTFA MICROBIOLOGY GUIDELINES

|

139

SECTION 11: RAW MATERIALS MICROBIAL CONTENT

INTRODUCTION

SECTION 11: RAW MATERIALS MICROBIAL CONTENT

Naturally occurring raw materials are likely to contain a high level of microorganisms that may pose a contamination risk to the finished product if not reduced or eliminated during processing. The microbial content may vary depending upon the type and source of the raw material. It may be necessary to treat such materials to reduce microbial levels before use or to purchase already treated materials. The criteria set by the manufacturer for the microbial content of a raw material should take into consideration the release criteria established for each finished product. For example, the absence of Salmonella is significant if a raw material is used in an oral product. A raw material microbial content specification is usually not greater than that for the finished product, especially when it is used at greater than 1% in the formulation. A raw material with a microbial count greater than that set for the finished product may be acceptable if its use does not compromise the safety and stability of the formulation and its concentration in the finished product is low.

SPECIFIC CRITERIA This guideline recognizes the importance of using raw materials of the highest quality in the manufacture of cosmetics. Special conditions may allow or necessitate acceptance criteria that vary from those recommended below. It is recommended that the minimum test portion be 1 g or 1 mL of sample. The following are recommended guidelines: • All Synthetic and Natural Raw Materials not more than 102 CFU per g or mL Note: Interpretation of results The inherent variability of a plate count should be taken into account, thus the interpretation may be as follows: • 10² - may be interpreted as 5 x 10² In addition to these recommended numerical guidelines, no raw material should have a microbial content recognized as either harmful to the user or able to compromise integrity of the finished product as recovered by standard plate count, specific pathogen test, or an equivalent automated procedure.

GENERAL RECOMMENDATIONS As cosmetics and toiletries need not be manufactured from sterile raw materials, it is important that raw materials are obtained from qualified suppliers and handled, stored, and used under conditions designed to deter microbial proliferation or subsequent contamination. The CTFA Quality Assurance Guidelines are a useful guide for the storage and handling of raw materials1 as well as microbiological sampling techniques.

140

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 11: RAW MATERIAL MICROBIAL CONTENT

Sampling techniques are located in “Microbiological Sampling” (Section 6) and in “Annex 17: Sampling” in the CTFA Quality Assurance Guidelines2. Validated microbiological analytical methods should permit the detection of microorganisms and ensure the inactivation of the preservative (See Section 18: “M-1 Determination of the Microbial Content of Cosmetic Products” and the Annual Book of ASTM Standards3). The presence of objectionable organisms can be determined by identification of isolates using procedures such as described in “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa” (Section 19).

It is recommended4 that a qualified microbiologist or independent microbiology laboratory be engaged to: • Design procedures for the examination of specific raw materials • Examine the manufacturer’s raw materials for microbial content on a continuing basis • Interpret assay data on a routine basis • Periodically review and update procedures, when applicable

REFERENCES 1. Bailey, John E., and Nikitakis, Joanne M. 2007. (Ed). “Annex 4 – Raw Materials and Packaging Materials”. In CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

3. ASTM E 1054-91. 1999. “Standard Practices for Evaluating Inactivators of Antimicrobial Agens Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” Annual Book of ASTM Standards 11.05.

2. Bailey and Nikitakis. “Annex 17 – Sampling.”

4. Bailey and Nikitakis. “Annex 10 – Subcontractor Quality Assurance.”

SECTION 11: RAW MATERIALS MICROBIAL CONTENT

|

CTFA MICROBIOLOGY GUIDELINES

|

141

SECTION 11: RAW MATERIALS MICROBIAL CONTENT

If raw materials are found to have a microbial content greater than specified, an investigation to identify and eliminate the source of the contamination can assist in implementing preventative measures 4.

SECTION 11: RAW MATERIALS MICROBIAL CONTENT

142

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 11: RAW MATERIAL MICROBIAL CONTENT

SECTION 12

Establishing Microbial Quality of Cosmetic Products INTRODUCTION Control over the microbial content of cosmetics is consistent with the control of other aspects of quality, and is necessary for ensuring consumer safety and product stability.

It should be recognized that the application of microbial limits alone will not guarantee product quality, and that a microbial quality management process must be implemented to ensure that manufacturers produce products that conform to specifications. This quality management process encompasses correct product development to ensure consumer safety, supplier quality management, and adherence to Good Manufacturing Practices (GMPs)1 to prevent microbiological problems from occurring. Essential to the implementation of effective microbial quality management is a microbial awareness education and training program for all levels of employees.

GENERAL CONSIDERATIONS Product Development Product Preservation Cosmetic and toiletry formulations that can support microorganisms or are susceptible to microbial contamination should contain preservatives to retard microbial growth. The manufacturer has the responsibility of producing an effectively preserved product. For products conducive to microbial growth that can result in contamination, preservation efficacy should be determined by appropriate tests during the development phase. The function of preservation is to protect consumers and prevent product spoilage during normal and reasonably foreseeable product use. Preservatives should not be used in lieu of good

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

143

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF COSMETIC PRODUCTS

Because cosmetics are applied to bacteria-populated skin, microorganisms need to be controlled in, but not necessarily eliminated from, cosmetics. Therefore, it is appropriate to assign rational limits to microbial content based on the best available information. Criteria such as the product’s intended use, route of administration, and target population should be taken into account when establishing microbial guidelines for safety and quality. The microbial quality guidelines presented here are intended to assist manufacturers in judging the microbiological quality of their products.

production hygiene. Understanding the causes of microbial growth and eliminating them during production will lead to control and prevention of microbial contamination. It must be emphasized that preservation systems cannot be chosen satisfactorily on theoretical grounds and they require in situ determination of their efficacy by microbiological challenge tests or other appropriate test systems during product development.

Development of Suitable Formulations Whenever possible, the formulator should be encouraged to develop formulations that are incapable of supporting microbial growth, hence reducing the need for the addition of a preservative. However, if a preservative is shown to be necessary, it should be selected at an early stage in product development and considered as an integral part of the formulation.

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF COSMETIC PRODUCTS

Water is essential for microbial growth. A preservative system should have solubility and partition characteristics such that it is available at effective concentration in the aqueous phase of a multiphase system. A preservative system should be effective against a broad spectrum of microorganisms and safe at the concentration used. Combinations of preservatives can sometimes be more effective than individual compounds. Storage temperature, light exposure, and prolonged storage stability are important. The concentration of the preservative system in a product formulation should be at levels necessary to allow for antimicrobial activity sufficient to ensure adequate preservation. The preservative system should be compatible with other product constituents and effective at the pH of the formulation. These determinations can all be obtained from the testing of formulations.

Raw Materials Cosmetics and toiletries produced for use by the general public are not required to be manufactured from sterile raw materials or under aseptic conditions. Therefore, microorganisms found in the general environment, in raw materials, and in formulation components may be introduced into the product during manufacture. It is important that raw materials and components be handled and stored under conditions designed to deter microbial contamination and proliferation. Because raw materials can contribute a significant level of microbial contamination to the finished product, it is important to monitor and control them. The monitoring of raw materials should be appropriate to their susceptibility to microbial contamination, as determined by a risk assessment. If practical, manufacturers should use raw material suppliers whose products yield the lowest population of microbial contamination. Water is one of the major raw materials used in the formulation of cosmetic and toiletry products and one that can be populated by large numbers of microorganisms. This may present a distinct hazard to the microbiological stability of the finished products. Therefore, steps must be taken to ensure that water used as an ingredient or for processing is regularly monitored and, where necessary, appropriately treated.

144

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 12: ESTABLILSHING MICROBIAL QUALITY OF PRODUCTS

Good Manufacturing Practices Good manufacturing practices (GMPs) are necessary to avoid accidental human or environmental microbial contamination during product manufacture. The manufacturing equipment should be designed for ease of cleaning and sanitizing, as well as for processing capability. It is recommended that, wherever possible, the physical plant of the manufacturing establishment be designed and constructed to facilitate the conduct of manufacturing operations (making, packaging, storage, and quality control) in accordance with current GMPs. To comply with GMP, manufacturers of cosmetics and toiletries must define and follow specific cleaning, sanitization, and control procedures. This process should include procedures to control microorganisms in susceptible raw materials, bulk and finished products, as well as on personnel, equipment, and premises. Adequate records should be maintained for all aspects of microbiological testing during the development and manufacture of each product and for all control procedures used at the manufacturing facility. In particular, documentation during the manufacture of products is an essential component of GMP.

Control and Assessment of Bulk and Finished Products

The manufacturer should take into account the specific nature of the product when establishing microbiological criteria for safety. The manufacturer is responsible for assuring that: • Any microorganisms present are incapable of growing in the product. • The species and quantity of microbes do not present a hazard to the consumer when using the product as directed. • Any microorganisms present do not compromise the stability of the product. • The packaging (including cap or closure) does not promote microbial contamination throughout the anticipated life of the product. A sampling plan developed for formulations that are susceptible to microbial contamination during the manufacturing process should be appropriate to the formulation and take into consideration the manufacturing process history. It is further recommended that a qualified microbiologist or independent microbiology laboratory be engaged to develop validated microbiological procedures to analyze specific products, periodically examine manufacturing procedures, and interpret assay data.

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

145

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF COSMETIC PRODUCTS

Susceptible formulations should be tested for microbial content on a routine basis, with product data periodically reviewed for trends. The testing program should take into account microbiologically susceptible raw materials, processing steps, and storage conditions.

SPECIFIC CRITERIA Acceptance criteria for the various classes of cosmetic products should be established when necessary. For microbiologically susceptible products, conformance to the criteria is determined by using a suitable technique, such as a plate count procedure. A risk assessment of products that are not considered microbiologically susceptible may either support a decision not to test some products or indicate reduced testing levels for others. An enumeration method for a particular product should be qualified for the type of product being tested. The method must permit the detection at a minimum, or the growth of any relevant microorganisms present. The test method used should adequately inactivate microbial growth inhibitors present in the product. It is recommended that the minimum test portion be 1 g or 1 ml of sample.

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF COSMETIC PRODUCTS

No product should have a microbial content recognized as either harmful to the user or able to compromise product integrity, as recovered by standard plate count, test for specified organism, or an equivalent alternative procedure. Specified organisms can be determined and identified from colonies recovered on suitable microbial growth media. Alternative procedures can also be used to detect the presence of specified organisms. The conditions under which cosmetics are manufactured, marketed, and used by the consumer vary throughout the world. Alternative microbial limits, such as those set by compendia, governmental bodies, or other recognized authorities, may be applicable in some cases. The application of specific criteria under this guideline should be decided by each national agency, whether government or association. Where applicable, additional criteria for microbial content may be listed in an annex that is specific to the country, region, or other area. Some suggested criteria for microbial content are listed below: • Baby products - not more than 102 CFU per g or ml • Eye area products - not more than 102 CFU per g or ml • All other products - not more than 103 CFU per g or ml Note: Interpretation of results: The inherent variability of a plate count should be taken into account, thus the criteria recommended should be interpreted as follows: 10² - maximum limit of acceptance is 5 x 10². 10³ - maximum limit of acceptance is 5 x 10³. Any microbial content exceeding acceptance criteria should be investigated to identify and eliminate the source of the contamination. Preventive measures should then be implemented. A variety of different methods and procedures that may be used to make sure that cosmetic products are in conformance with the recommended limits are available2-7.

146

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 12: ESTABLILSHING MICROBIAL QUALITY OF PRODUCTS

REFERENCES 1. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association. 2. The European Cosmetic Toiletry and Perfumery Association. 1997. “Colipa Guidelines on Microbial Quality Management (MQM).” Brussels. http://www. colipa.com. 3. Krowka, John F. and Bailey, John E. (Ed). 2007. CTFA Microbiology Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association, 2007. http://www.ctfa.org. 4. “The SCCP’s Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation”, 6th Revision. Adopted

by SCCP during 10th Plenary Meeting. December 19, 2006. http://ec.europa. eu/health/ph_risk/committees/04_sccp/ docs/sccp_s_04.pdf. 5. Department for Business, Enterprise & Regulatory Reform. April, 2005. “Guidance on the Implementation of the Cosmetic Products (Safety) Regulations 2004.” London. http://www.dti.gov.uk/ files/file25422.pdf. 6. Japan Cosmetic Industry Association (JCIA), 1997. “Microbial Test Methods for Cosmetics.” Tokyor. http://www.jcia. org. 7. U. S. Food and Drug Administration, FDA/Industry Activities Staff Booklet, 1992. Cosmetics Handbook. http://www. fda.gov. SECTION 12: ESTABLISHING MICROBIAL QUALITY OF COSMETIC PRODUCTS

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

147

SECTION 12: ESTABLISHING MICROBIAL QUALITY OF COSMETIC PRODUCTS

148

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 12: ESTABLILSHING MICROBIAL QUALITY OF PRODUCTS

SECTION 13

Determination of Preservative Adequacy in Cosmetic Formulations INTRODUCTION The design of preservation tests and the subsequent interpretation of results is a complex process. The technical personnel responsible for preservation testing should, therefore, be professionally educated and experienced in conducting test procedures and evaluating the data generated. It is important to remember that microorganisms are ubiquitous and capable of adaptation and selection. No method can guarantee adequate microbial control under all conditions. In addition, the importance of adhering to good manufacturing procedures in the production of cosmetics and toiletries cannot be overstated.

GENERAL CONSIDERATIONS Developmental Formulations Formulations that differ considerably from each other should be tested during the developmental stage. Where the only variable in experimental design is the preservative system, it is essential that each formulation be prepared from microbiologically acceptable raw materials. An unpreserved formulation should be included in the test procedure as a control to determine the need for preservation. In order to evaluate the stability of preservative systems under consideration, developmental formulations should also be tested after storage at temperatures simulating warehouse, shipping, and shelf-life conditions.

SECTION 13: DETERMINATION OF PRESERVATIVE ADEQUACY

|

CTFA MICROBIOLOGY GUIDELINES

|

149

SECTION 13: DETERMINATION OF PRESERVATIVE ADEQUACY

The following factors should be considered when designing a preservation test or recommending a preservative system for a new formulation: • The nature of the raw materials in the formulation • Information on preservation of similar formulations • Manufacturing procedures • Types of packaging used to contain product • Information on the product's intended use, including area of application, frequency of use, shelf-life, etc. • Anticipated storage and/or shipping conditions

Pilot Batches Preservation tests should be performed on individual pilot batches to confirm the effectiveness of the preservative system. Tests may be run on either bulk material prior to filling or filled samples. Where possible, these tests should be accompanied by analytical verification of the preservative(s) concentration. Final Package Preservation tests should be conducted on the product in the final package to ensure package compatibility with the preservative system. A decrease in preservative effectiveness over a period of time can result when the preservative system is altered by the final package, reacts chemically with it, or is absorbed into the packaging material.

RECOMMENDATIONS Since many cosmetic and toiletry products are used on a regular basis, an effective preservative system should ensure the reduction of microorganisms to a low and steadily decreasing level, even after severe microbial insult. The following minimal criteria are recommended: Bacteria There should be at least a 99.9% reduction of vegetative bacteria within 7 days following each challenge and no increase for the duration of the test period. Yeasts and Molds

SECTION 13: DETERMINATION OF PRESERVATIVE ADEQUACY

There should be at least a 90% reduction of yeasts and molds within 7 days following each challenge and no increase for the duration of the test period.

CONDITIONS The tests should be carried out for a minimum of 28 days. At least one rechallenge is recommended. If a product does not meet these criteria, additional evaluations should be considered. It is the responsibility of the manufacturer to select an appropriate methodology and appropriate criteria. Methods for determining the adequacy of preservation of cosmetic and toiletry formulations are described in the Methods section. In addition, AOAC INTERNATIONAL official method 998.101 offers a validated method that may be referenced.

REFERENCE 1. AOAC INTERNATIONAL. 2000. “Efficacy of Preservation of Non-Eye Area WaterMiscible Cosmetic and Toiletry Formulations,” Official Method 998.10. In: Official Methods of Analysis of AOAC INTERNATIONAL. Gaithersburg, MD.

150

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 13: DETERMINATION OF PRESERVATIVE ADEQUACY

SECTION 14

Preservation Testing of Eye Area Cosmetics

INTRODUCTION It is recognized that cosmetic products may be environments in which microorganisms can adapt and proliferate unless proper precautions are taken during formulation and manufacture. The intended use of eye area cosmetics makes it imperative that these products be prepared with preservative systems that remain effective.1 The alleged incidence of corneal ulceration due to the periocular use of bacteria-laden cosmetics2 has led the Food and Drug Administration (FDA) to specifically address the adequate preservation of these eye area cosmetics3 and the CTFA to recommend the same microbial limits as those indicated for baby products. (Section 12: “Establishing the Microbial Quality of Cosmetic Products”). Eye area products are normally free of microbial contamination when purchased. However, some products may contain organisms representative of human skin flora after use by the consumer.4 Microorganisms may be introduced into the product from the environment or by the consumer, who may, for example, add tap water to a product to make it less viscous. In evaluating the adequacy of preservation of eye area cosmetics, it is important to point out that there is no substitute for judgment by knowledgeable microbiologists. It must also be recognized that the addition of preservatives to cosmetics is an adjunct to, but not a substitute for, good manufacturing practices.

GENERAL CONSIDERATIONS Developmental Formulations Formulations that differ in at least one ingredient, e.g., binder or surfactant, should be tested during the developmental stage using appropriate test microorganisms. If several preservative systems are to be evaluated, each test formulation should be prepared concurrently from the same microbiologically acceptable raw materials.

|

CTFA MICROBIOLOGY GUIDELINES

|

151

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

Pilot Batches It may be desirable to perform a preservation test on individual pilot batches of product to verify the effectiveness of the preservative system. If feasible, these tests should be accompanied by analytical determinations of preservative presence and concentration. Tests may be performed on bulk material or on the filled samples.

Stability Product should be evaluated for preservative stability in commercial packaging by testing after storage that simulates warehouse, shipping and shelf-life conditions.

RECOMMENDATIONS Since eye area cosmetics are usually applied daily, an effective preservation system will help ensure a low level of microorganisms even after severe microbial insult acquired during product use or misuse. There are many references recommending preservative efficacy in sterile ophthalmics4, 6, 7, 8 and several in aqueous eye cosmetics.5, 7 Given the daily use of eye area cosmetics, it is recommended that multiple challenges be made to fully ensure adequacy of preservation. 9 The following are recommended as minimal criteria for preservative performance.

Aqueous Liquid and Semi-Liquid Eye Cosmetics Vegetative Bacteria There should be greater than 99.9% reduction of vegetative bacteria by aerobic plate count or quantitative spread plate methods within 7 days following each challenge and continued reduction to a less-than-detectable level by the end of the test period. Yeast and Molds There should be greater than 90% reduction of yeasts and molds by aerobic plate count or quantitative spread plate methods within 7 days following each challenge and continued reduction for the duration of the test period. Spore-Forming Bacteria There should be bacteriostatic activity against spore-forming bacteria throughout the entire test period.

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

Non-Aqueous Eye Products Vegetative Bacteria There should be a 99.9% or greater reduction of vegetative bacteria by aerobic plate count or quantitative spread plate methods within 7 days following each challenge and continued reduction to a less-than-detectable level by the end of the test period. 152

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

Yeasts and Molds There should be at least a 90% reduction of yeasts and molds by aerobic plate count or quantitative spread plate methods within 7 days following each challenge and the level should remain at or below that level for the duration of the test. Spore-Forming Bacteria There should be bacteriostatic activity against spore-forming bacteria throughout the entire test period. These minimal criteria are suggested to aid manufacturers in evaluating the adequacy of preservation of eye area cosmetics. Ultimately, it is the responsibility of the manufacturer to select appropriate criteria that will ensure product integrity.

REFERENCES 1. Wilson, L.A., J. W. Kuehne, S. W. Hall, and D. G. Ahearn. 1971. “Microbial Contamination in Ocular Cosmetics.” American Journal of Ophthalmology. 71(6):1298-1302. 2. Wilson, L.A. and D. G. Ahearn. 1977. “Pseudomonas-Induced Corneal Ulcers Associated with Contaminated Eye Mascaras.” American Journal of Ophthalmology. 84:112-119. 3. Madden, J.M. and G. J. Jackson. 1981. “Cosmetic Preservation and Microbes: Viewpoint of the Food and Drug Administration.” Cosmetics & Toiletries 96:75-77. 4. Wilson, L.A., A. I. Julian, and D. G. Ahearn. 1975. “The Survival and Growth of Microorganisms in Mascara During Use.” American Journal of Ophthalmology. 79(4):596-601.

5. Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” Journal of the Society of Cosmetic Chemistry. 18: 797-807. 6. Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” Journal of the Society of Cosmetic Chemistry. 23: 703-720. 7. Cosmetics, Toiletry & Perfumers Association, 1983. Appendix III - CTPA Recommended Microbiological Limits and Guidelines to Microbiological Quality Control. London. 8. United States Pharmacopeia. 2007. United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD. 9. “A Study of the Use of Re-challenge in Preservation Testing of Cosmetics.” 1981. CTFA Cosmet. Journal. 13:19-22.

|

CTFA MICROBIOLOGY GUIDELINES

|

153

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

154

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

SECTION 15

Microbiological Assessment of Product Quality after Use INTRODUCTION Every cosmetic manufacturer has a responsibility to establish the microbiological safety of its finished products. Doing this is a two-step process. The first is to assure consumers that each cosmetic product is free from the numbers and types of objectionable microorganisms that could affect product quality and/or the health of the consumer. (In addition to the information provided in this publication, users should also refer to the CTFA Quality Assurance Guidelines1.) Secondly, cosmetic manufacturers should ensure that each cosmetic product is not affected by the introduction of microorganisms during normal or reasonably anticipated use by the consumer. To prevent this, manufacturers may add preservatives to cosmetic product formulations. For most cosmetic products (e.g., water miscible), microbial challenge testing is performed to verify that the preservative system of a formulation can prevent the growth of microorganisms. For additional guidance on testing preservation efficacy, refer to “M-3 A Method for Preservation Testing of Water-Miscible Personal Care Products” (Section 20), “M-4 Method for Preservation Testing of Eye Area Cosmetics” (Section 21), and “M-6 A Method for Preservation Testing of Atypical Personal Care Products” (Section 23). In addition, the analyses of used test samples for microbial content may provide added assurance in the adequacy of the preservative system during use. Used samples may be collected for microbial analyses as part of another study being conducted, such as a clinical or sensory study. Furthermore, there are atypical cosmetic products (e.g., anhydrous gels, waxed-based sticks, loose or pressed powders, etc.) for which the traditional preservative challenge test may not yield the appropriate information regarding either the microbial integrity or susceptibility to contamination of the product by the consumer. For these types of products, analysis of samples after an in-use study should be considered.

ESTABLISHING A PROGRAM When developing an adequately preserved product, microbiologists should consider the nature of the product, directions for its application, the microbial quality of the raw materials in the product, and the manufacturing process. While the microbiologist can exercise control over these factors, a cosmetic company cannot control how a consumer will use a finished product.

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

|

CTFA MICROBIOLOGY GUIDELINES

|

155

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

To establish a program for evaluating the microbiological integrity or susceptibility to contamination of a cosmetic product during consumer use, it is necessary to generate information relating to the way the product will or should be used. This information can be obtained from the product manager or through questionnaires, consumer letters, or consumer market tests. To determine how to structure an in-use study for a cosmetic product, the following information should be obtained: application, handling, length-of-use, and storage. This information will aid the investigator in selecting the appropriate test panel members, defining usage instructions, and setting a timetable. Study design should reflect actual product usage as closely as possible.*

PANEL SELECTION Selection of panel test members should be based on consumer habits and practices. The following elements may be considered in structuring the panel: • Typical consumer age, sex, geographical distribution, product usage patterns, etc. • Panel size should be a function of the degree of statistical sensitivity desired, with larger panels yielding increased sensitivity. An exact size-versus-sensitivity determination may be made by consulting an appropriate sampling table. After the panel structure has been determined, the study director will forward a request for participation to potential panelists. It is recommended that this request include at least 20% more individuals than are required to participate as test panel members to allow for attrition and the initial inability of some people to participate. The request for participation should include the test panel’s start and termination dates and a concise, comprehensive outline of what the test panelists would be expected to do during the study.

PRODUCT EVALUATION Before being submitted for evaluation, any formulation should be reviewed and approved for application and the microbial content of unused test samples tested to ensure the product is microbiologically safe under the prescribed conditions of use. In addition, the formulation being tested should have been evaluated for safety and cleared for usage by the appropriate product safety department. Product identification should be recorded, including any pertinent history, product age, and lot or batch number. To determine actual usage by a test panel member, it is recommended that each test sample be weighed prior to distribution as well as after it is returned.

*Note: If test samples of a cosmetic formulation are to be used in a clinical study for the establishment of proposed product claims or to determine actual product safety, the study director is responsible for incorporating applicable aspects of Good Clinical Practice (see http://www. fda.gov/oc/gcp/ default.htm) where appropriate. 156

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

EVALUATION OF USED PRODUCT When used test samples are returned to the test site, each test panelist should complete a questionnaire or hand in a diary. In designing a questionnaire, the following points should be considered: • How often was the product used? • When was the product used? • Where was the product stored between uses? • Were there any problems associated with the product’s use? The questionnaire should be designed to generate information that might be helpful in pinpointing the reasons for an aberrant test result. It may provide the investigator or microbiologist with information regarding the product’s ability to withstand either inappropriate or normal consumer usage. A sample questionnaire is presented in Table 15-1. If a test panelist uses a diary, the following information should be recorded: time at which the product was applied each day, amount of product used at each application, and the location of the product between applications. When evaluating returned test samples, microbiological content testing should be conducted before any other testing is performed. This is to ensure that recovered microbial contaminants from a test sample were introduced during consumer usage and not from subsequent handling. The microbiological evaluation of the used product should be conducted within a reasonable time after the last use of the product by test panelists (i.e., seven days or less). All or a portion of the returned samples will be tested for microbiological content, the number chosen based upon some type of statistical sampling plan (e.g., the square root plus one). If aberrant microbiological test results are obtained, the microbiologist has the option of analyzing additional returned test samples to confirm the result.

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

|

CTFA MICROBIOLOGY GUIDELINES

|

157

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

When the test samples are distributed, a comprehensive set of use instructions should be prepared and given to each test panel member. The instructions for using a test sample should include the following: • Approximate quantity of material to apply • Method of application • Frequency of use • Handling of the product between uses • Instructions for returning the product (intermittent evaluation or after final use) • Any other instructions pertinent to the product under review • Name of a person to contact if any questions should arise

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

MICROBIAL CONTENT OF PRODUCT The method used for determining the microbial content of used product samples depends on the nature of the product. Several methods may be used. While the aerobic plate count method is considered a quantitative method, many of the others are considered semi-quantitative. Whenever possible, quantitative recovery is preferable to semi-quantitative recovery. Semi-quantitative results should be reported as an estimate of the microbial content of the unit. Where feasible, an aliquot of the used test sample may be aseptically removed from each container and analyzed for microbial content using an aerobic plate count method (e.g., “M1 Determination of the Microbial Content of Cosmetic Products” (Section 18) and “M-2 Examination for Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa” (Section 19) or in-house method). For water immiscible products (e.g., oils or emulsions), a suitable solubilizing agent may be incorporated into the test diluent or broth to make the sample aliquot miscible with water in order to recover microorganisms present in the test sample. For products where microorganisms would only be recovered from the product surface (e.g., sticks, pressed powders, hot pour products in compacts), only the surface of the product sample should be tested. For these “atypical products,” the following methods of recovery may be considered. • A sterile moistened applicator may be used to sample the product surface, and then streaked onto a Petri dish containing solid culture media. • The product may be sampled by a direct contact method using a contact plate (a modified Petri dish containing a solid culture medium whose convex surface extends above the carrier), paddles, or flexible film containing solid culture media. Alternative test methods to those described above may also be used. Appropriate preservative neutralizers should be incorporated into product diluents, liquid or solid media. Whatever method is chosen, it should be verified for the recovery of microorganisms. The same method should be applied to the control sample.

MICROBIAL CONTENT OF APPLICATORS/UNIQUE PACKAGING ELEMENTS Product applicators or unique packaging elements (natural or synthetic) that come into direct contact with the product may be evaluated for estimated microbial content, as these items are the vectors of microbial contamination into the product. The following semi-quantitative methods may be used to determine estimated microbial content: • Aseptically transfer the applicator to a container of sterile diluent or liquid culture medium. After vigorously shaking or vortexing for a set period of time, perform anaerobic plate count on an aliquot of the diluent or liquid culture medium. • The applicator may be sampled using a direct contact method (see “Microbial Content of Product” above). Alternative test methods to those described above may be used. Appropriate preservative neutralizers, as required, should be incorporated into diluents, liquid or solid media. Whatever method is chosen, it should be verified for recovery of microorganisms. 158

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

It is recommended that recovered microorganisms from test samples be identified. If multiple types of microbial colonies are obtained, representative microbial colonies may be selected for identification.

INTERPRETATION OF RESULTS For convenience, it is recommended that all test results be summarized. The interpretation of microbiological in-use test results is largely a matter of in-house specifications. The extent to which microorganism recoveries can be considered significant must be viewed in light of what the ultimate effect would be on the consumer, the type of product (e.g., typical or atypical product formulation), and the manner in which the product would typically be used by the consumer (e.g., eye versus lip). Recovery of low levels of microorganisms may be of some significance, including both waterin-oil and oil-in-water formulations, especially in water-based products where the potential for proliferation may exist. Thus, the acceptance criteria for samples of water-based products returned from in-use studies normally reflect the specification for end product release. For these products, further investigation into recovery of low levels of microorganisms may be warranted. However, for products with a water activity of less than 0.6, the potential for proliferation does not exist. For these types of products, recovery of normal skin flora may be expected. Therefore, the acceptance criteria for atypical products may differ significantly from those of water-based products. Higher microbial counts may be acceptable in atypical products used in areas of the body that contain higher microbial populations and where there is less potential risk to the consumer. To ensure that the number of organisms recovered does not increase over time, initial samples or additional samples may be retested to confirm stasis or reduction in recoverable levels of microorganisms. If levels of microorganisms recovered do increase, then the formulation and/or package design should be reviewed. Aberrant results may warrant further investigation including performance of non-microbiological testing. In-use studies of test samples cannot give a complete picture of how well a product will withstand consumer handling and use. However, an in-use study for a proposed product may provide a margin of added assurance to the manufacturer, as well as alert them to potential problems that could occur in the field. Regardless of the nature of the test data generated, consumer in-use studies can provide meaningful information as to how a product may behave during repeated microbiological insult during consumer usage.

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

|

CTFA MICROBIOLOGY GUIDELINES

|

159

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

IDENTIFICATION OF ISOLATES

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

Table 15-1: Sample Consumer Evaluation Questionnaire (Information needed by microbiologist) Product: __________________________________________________________________________________

Panelist ID: ________________________________________________________________________________

1.

How often did you apply the product? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

2.

When did you apply the product (e.g., after bath/shower, after housework, before retiring, etc.)? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

3.

Did you use the product on areas other than the area specified in the instructions? If so, where? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

4.

Where was the product kept when not in use? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

5.

When was the product last used? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

6.

List any comments you may have. ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

Table 15-1

160

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association. Brannan, D.K., J.C. Dille, and D.J. Kaufman. 1987. “Correlation of In-Vitro Challenge Testing with Consumer Use Testing for Cosmetic Products.” Applied and Environmental Microbiology, 58(3):1827-1832. Brannan, D.K., and J.C. Dille. 1990. “Type of Closure Prevents Microbial Contamination of Cosmetics During Consumer Use.” Applied and Environmental Microbiology, 56(5):1476-1479. CTFA Microbiology Committee. December 1985. “CTFA Survey: Correlation of the In-Vitro Preservative Challenge Test with Consumer Use Testing,” Presented by R. Spielmaker at the Society for Cosmetic Chemists Scientific Conference. Farrington, J.K., et al. 1994. “Ability of Laboratory Methods to Predict In-Use Efficacy of Antimicrobial Preservatives in an Experimental Cosmetic.” Applied and Environmental Microbiology, 60(12): 4553-4558. U.S. Food and Drug Administration. Guidances, Information Sheets, and Important Notices on Good Clinical Practice in FDA-Regulated Clinical Trials. http://www.fda.gov/oc/gcp/guidance. html. Larson, E.L., et al. 2003. “Microbial flora of hands of homemakers.” American Journal Infect. Control, 31(2): 72-79. Lindstrom, S.M. 1986. “Consumer Use Testing: Assurance of Microbial Product Safety.” Cosmetics and Toiletries, 101: 73-74. Lindstrom, S.M., and J.D. Hawthorne. 1986. “Validating the Microbiological Integrity of Cosmetic Products through Consumer-Use Testing.” J. Soc. Cosmet. Chem., 37: 481-428. Orth, D.C., R.F. Barlow, and C.A. Gregory. 1992. “The Required D-Value: Evaluating Product Preservation in Relation to Packaging and Consumer Use/Abuse.” Cosmetics and Toiletries, 107(12): 39-43. Passaro, D.J., et al. 1997. “Postoperative Serratia marcescens Wound Infections Traced to an Out-of-Hospital Source.” J. Infect. Diseases, 175: 992-995. Ryan, G.M., D.J. Floumoy, and P. Schlagertre. 1994. “Microbiological Flora and Nail Polish: A Brief Report.” J. Okla. State Med. Assoc., 87: 504-505. Singer, S. 1987. “The Use of Preservative Neutralizers in Diluents and Plating Media.” Cosmetics and Toiletries, 112(12): 55-60. Trick, W.E., et al. 2002. “Impact of Ring Wearing on Hand Contamination and Comparison of Hand Hygiene Agents in a Hospital.” Clinical and Infectious Diseases, 36:1383-1390. Wilson, L.A., A.I. Julian, and D.G. Ahearn. 1975. “The Survival and Growth of Microorganisms in Mascara During Use.” American Journal of Ophthalmology, 79(4): 596-601.

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

|

CTFA MICROBIOLOGY GUIDELINES

|

161

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

ADDITIONAL INFORMATION

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

Yablonski, J.I., and S.E. Mancuso. 2002. “Preservation of Atypical Cosmetic Systems.” Cosmetics and Toiletries, 117(4): 31-40. United States Pharmacopeia. 2007. United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD: http://www.usp.org.

REFERENCES 1. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines. Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.

162

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE

SECTION 16

INTRODUCTION Every cosmetic manufacturer has a dual responsibility relative to the microbiological quality of its products. The first is to ensure that the product, as purchased, is free from the numbers and types of microorganisms that could affect product quality and consumer health. The second is to ensure that microorganisms introduced during normal product use will not adversely affect the quality or safety of the product.* During product development, the microbiologist may use several tools to evaluate the ability of a product to prevent the growth of microorganisms introduced during product use. The challenge test, which involves introducing a known quantity of microorganisms into a formula and monitoring the rate of kill over time, is frequently used. A second method of evaluating product quality during consumer use is by evaluating the product after a use test that simulates “real life” situations.** Finally, the microbiologist may perform a microbiological risk assessment of the product. The risk assessment process is based on a number of factors generally accepted as important in evaluating the spoilage potential of a product. It is intended to guide the microbiologist and formulator in determining what level of testing is necessary to assure the quality of the product during manufacturing and consumer use. This guideline serves as an aid to the cosmetic microbiologist in assessing the microbiological quality of formulations for which the normally recommended method of challenge testing, developed for water-based formulations may not yield appropriate information. These include anhydrous formulations, formulations with low-water content, or those products where water is the internal phase.

* For examples, see “M-3 The Determination of Preservation Adequacy of Water-Miscible Cosmetic and Toiletry Formulations” (Section 20), “M-4 Method for the Preservation Testing of Eye Area Cosmetics” (Section 21), and “M-6 A Method for Preservation Testing of Atypical Personal Care Products” (Section 23). For guidance on the methods, see “The Determination of Preservation Adequacy of Cosmetic and Toiletry Formulations” (Section 13) and “Preservation Testing of Eye Area Cosmetics” (Section 14). **For guidance, see “Microbiological Assessment of Product Quality after Use” (Section 15).

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

163

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

Microbiological Risk Factor Assessment of Atypical Cosmetic Products

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

This guideline also serves as a tool to aid the microbiologist in recommending ways of reducing product susceptibility to microbial growth. Certain cosmetic products, depending on their composition and presentation (packaging), may have negligible potential for microbial proliferation during use. Microbial contamination of a cosmetic product during use is a function of the physico-chemical characteristics of the product and the way in which it is packaged (i.e., its presentation).

PRODUCT TYPES Common examples of atypical products are listed below. In each example, water is not readily available to provide an environment that supports the growth of microorganisms. Water in the product may be surrounded by oil or silicone as the external phase, with the water being present as small droplets and influenced by other water-soluble formula ingredients. Also, the water activity may be too low to support growth. In some cases, the product might be totally anhydrous.1 Common examples of atypical products: • Wax-based products • Products with high oil/low water content • Siloxane and siloxane derivative-based products • Lip balms • Pomades • Ointments • Powders • Cream-to-powder makeup In addition to products with low-water activity, products with the physico-chemical characteristics below do not allow the proliferation of harmful microorganisms: • Products with an alcohol content equal to or greater than 20% (vol/vol)2 • Products with a pH of less than 3 or greater than 103,4,5

PRODUCT SUSCEPTIBILITY Atypical products may contain raw ingredients that do not support the growth of microbial contaminants and, therefore, may prevent microorganisms from proliferating when the product is subjected to normal consumer use. In these types of products, organisms may survive, but cannot reproduce. This may be due to low water activity or low water activity in combination with pH and/or antagonistic formula ingredients that are water soluble. Water droplet size may also be critical in the water phase. If the water activity reading is low in a product formulation, or if the formula is anhydrous, studies have shown that microorganisms will not proliferate. In fact, this is the basis for the use of water activity as an assessment tool in determining the risk for microbiological proliferation for food products, like cereals.6, 7 In some atypical products, microbial survival may occur on the outside of the product without ever permeating and spreading through the product. This observation is also due to the low “free water” content. 164

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

Because most atypical products will not support microbial proliferation, the method of product delivery may be the vector for transferring microbial contamination from the product back to the consumer.

RISK FACTOR ASSESSMENT

Water Activity The metabolism and reproduction of microorganisms demand the presence of water in an available form. The most useful measurement of water availability in a product formulation is water activity (aw). Water activity is defined as the ratio of the water vapor pressure of the product to that of pure water at the same temperature:6 aw = p/po = (n2/(n1 + n2)) where, p is the vapor pressure of the solution, po is the vapor pressure of pure water, n1 is the number of moles of solute, and n2 is the number of moles of water. \ When a solution becomes more concentrated, vapor pressure decreases and the water activity falls from a maximum of 1.00 (aw) for pure water. As the water activity level falls below the optimum value for each microorganism, the length of the lag phase in the microbial growth cycle will increase toward infinity unless rehydration occurs. Listed below are examples of the minimum water activity levels required for the growth of selected microorganisms.3,6,8 Approximate Water Activity (aw) Required for the Growth of Selected Microorganisms 1,10 • • • • • •

Most bacteria 0.94 – 1.00 Enterbacteraciae >0.93 Pseudomonas species >0.96 Staphylococcus aureus >0.86 Most spoilage yeast >0.70 Most spoilage mold >0.60

The water activity values listed on the previous page should be considered as reference points since microbial growth may occur at lower values depending on differences in temperature or nutrient content of the product formulation. Even though water activity values are important in assisting in the risk analysis for microbial contamination, water activity should not be used as the sole indicator in determining whether product testing is necessary for a particular product formulation. SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

165

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

A number of factors need to be evaluated when performing a microbial risk assessment to determine what type of testing or preservative system may be needed for a particular formulation. Listed below are several factors to be considered in determining a product’s potential risk of microbial contamination during consumer use.

Formula Review Every formula contains raw materials that have an impact on the susceptibility of the formula to microbiological contamination. Raw materials can be classified as susceptible, hostile, or neutral to microbial growth, and their concentration will affect the susceptibility of the formulation to microbial contamination.

ANNEX 16 CALIBRATION SYSTEMS

It is recommended that a microbiologist review formulas to determine their potential susceptibility to microbial contamination. If a formulation tends to absorb moisture, samples of these types of atypical products should be microbiologically evaluated (including aw) after exposure to high humidity conditions (i.e., 50–75% for about three weeks or until equilibrium is demonstrated). Anhydrous products may contain binders or other hygroscopic materials that can absorb moisture. In addition, consideration of water on the surface of products may occur under high humidity conditions. Consumers may introduce water during normal use or misuse. The physical product form will affect whether microbial contaminants will be introduced at the product surface or mixed throughout the product during consumer use. Factors to be considered are: • Raw material susceptibility • Raw material microbial load • Percent concentration of raw material • Presence of preservative inactivators • Presence of preservative potentiators • Presence of fragrance and other ingredients that may act as preservatives • Binder level in powders (the higher the binder percentage, the more hydrophobic the product will be) • Product's physical form (solid or liquid; melting point)

Site of Application Risk assessment needs may vary since the site of a product’s application is an important risk factor in determining the level and type of microbiological testing required for a cosmetic product. For example, an eye area product poses a much greater risk of microbiological contamination to the consumer than a product applied to other areas of the body. Lip products, under normal conditions, generally come into contact with higher numbers of microorganisms that are part of the normal microflora present in the consumer’s lip area, but do not pose a health risk. Some points to consider when determining the risk in relation to the site of a product’s application include: • Is the site on the body to which the formulation is to be applied an area where microbial levels are high (lip) or low (eye)? • Is the product applied under moist (higher risk) or dry (lower risk) conditions? • What is the mode of application (brush, sponge, or finger)? • How frequently is the product used?

166

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

Applicators and the Mode of Application The mode of application plays a large role in determining the risk factor of a product. Even though the product may not support growth, the method of delivery may be a vector of microbial bacterial contamination.

Some typical questions need to be asked before microbiological testing is conducted on applicators: • Are applicators such as puffs, brushes, or pads used with the products? • Could these applicators act as a breeding ground for microorganisms or a vector for microbial contamination of the product and/or consumer? • Do the product and component come in direct contact with the consumer (lips, eyes, fingers)? • Do these applicators contain an antimicrobial agent? • Is the applicator stored in direct contact with the product? • Are there directions given on how to store or clean applicators between uses? When evaluating and determining the risk factors for microbiological contamination in product applicators, the following additional factors are to be considered: • Type of applicator • Type of material used • Treated or not treated with an antimicrobial agent • The efficacy of a treated applicator should be tested via a zone of inhibition test or other appropriate method.9, 10 • Wet/dry application of product

Packaging The type of packaging used for a finished product is a critical risk factor in determining the overall potential for microbial contamination during consumer usage. The use of packaging can provide additional protection by restricting direct access to the product. The following factors are among those that should be taken into consideration when assessing product risk in regards to packaging: • Is packaging for single or multiple use? • What is the size of the package? • What is the mode of dispensing?

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

167

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

Applicators could provide an environment that might be suitable for microbial proliferation. For example, porous sponge applicators may be a concern due to their ability to absorb moisture and retain sebum and dirt from the skin. With the presence of water, sebum, and dirt from the skin, there maybe enough water and nutrients present in a used applicator to allow for microbial proliferation. In those applicators that are to be used in conjunction with a wet/dry product, the incorporation of an antimicrobial agent may be considered to prevent microbial proliferation. The preservatives system of a product must not be expected to inhibit microbial growth in or on a product applicator.

• What is the predicted use-up rate? • Does the package type allow for direct consumer contact? • Is the package pressurized?

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

Confirmation of the microbial integrity of the applicator and finished product can be determined by conducting an in-use study.

Manufacturing Process Certain aspects of the manufacturing process (e.g., high temperature) may affect the microbiological contamination susceptibility for a cosmetic product. It is useful for both the microbiologist and cosmetic chemist to review the manufacturing process to determine the potential risk of microbial contamination to the formulation. Factors to be considered: • Are there processing factors that could affect the efficacy of the preservative system? • What is the temperature of the manufacturing process? • What is the microbial content of the raw materials? • Are hostile raw materials used to make the product? • What is the order of the addition of raw materials?

PRODUCT TESTING General After evaluating the above factors, the microbiologist can determine what level and type of microbiological testing is necessary. A risk assessment may indicate that a challenge test is not needed for some atypical products. If it is determined that microbiological testing is necessary, the following information can be used to assist in selecting the most appropriate test method:

Challenge Tests General Considerations The recommended preservative challenge test methods that are used for determining the preservative adequacy of aqueous-based products may not be suitable for evaluating certain atypical product formulations. When testing and assessing preservative challenge test data for atypical products, the following factors are important points to consider: • A test in which an aqueous-based inoculum is introduced into an anhydrous sample may change the physical dynamics of the product and, therefore, may not predict its microbial stability. • Most preservatives are water soluble. In emulsions, preservatives are used in the water phase because contaminating microorganisms require water to proliferate. 168

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

• For an emulsion in which the external phase is water immiscible and an aqueous challenge inoculum is used, the water-soluble preservatives will be unable to penetrate the water-immiscible phase. In these cases, the preservatives will not be available to either inhibit proliferation or have acidal activity against each of the challenge microorganisms.

Possible Test Method Modifications

Reduction in Inoculum Concentration For some atypical products, (e.g., anhydrous products), a reduction in the challenge inoculum size to 103 to 104 Colony-Forming Units (CFU) per gram or milliliter may be used instead of the inoculum concentration of 105 to 106 CFU per gram or milliliter that is recommended in aqueous-based challenge test methods. By reducing the inoculum size, it is easier to measure stasis or quantify an increase in the microbial count. Reduction in Inoculum Volume Reduction of the ratio of microbial inoculum suspension to the volume of product may also be considered. The recommended ratio of inoculum suspension for aqueous-based products is no more than 1.0% for a challenge sample. For atypical product formulations, the ratio of inoculum suspension to product may need to be reduced to 0.1% to minimize changes in the physical dynamics of the product. Surface Inoculation and Sampling For solid atypical products, such as anhydrous sticks and powders, inoculation and sampling of the product surface instead of the whole product more closely simulates potential consumer contamination. This modification also maintains the physical product integrity. In these types of products, the microorganisms are not able to penetrate into the interior and will always be found on the outermost layer of the product after consumer usage. Note: If performing challenge testing on a solid anhydrous stick or powder product, inoculate a sufficient number of samples to obtain a unique sample for each sampling time point. Inoculum Delivery Systems For liquid, anhydrous, atypical product formulations, one may consider using an oil-soluble carrier system, such as light mineral oil or other suitable oil carrier, to deliver and disperse the microbial challenge inoculum into liquid atypical product formulations to form a homogeneous mixture. If using this technique, the absence of inhibitory or toxic properties of the oilsoluble carrier system should be verified for each of the challenge organisms.11

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

169

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

If preservative challenge testing is performed on an atypical product formulation, aqueous-based challenge protocols may need to be modified to take into account a number of factors. For specific test methods see M-3 and M-4 of the CTFA Microbiology Guidelines (Sections 20 and 21). A number of possible modifications to these methods are discussed on the next page.

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

Sampling Diluents The recovery procedure for determining the microbial counts from inoculated challenge samples of an atypical product may need to be modified from those that are commonly used in aqueous-based challenge test methods. For example, water-in-oil emulsions and anhydrous products are not readily miscible with water. It has been demonstrated that 1.0-gram aliquots of an anhydrous product solubilizer in a 1.0-gram aliquot of sorbitan monostearate (Tween 80) and this mixture were dispersed further by increasing the volume with an aqueous diluent to make a 1:10 dilution.12

Challenge Test Acceptance Criteria It is the responsibility of the manufacturer to set the challenge test criteria to the product type and form. In the performance of challenge testing of atypical products, the pass/fail criteria may need to be modified in comparison to the preservative challenge test criterion that is commonly used for aqueous-based products. For example, the challenge acceptance criteria for anhydrous atypical products may be stasis for certain types of challenge microorganisms, because these organisms do not need a source of water to survive. If criteria other than the aqueous-based challenge criteria are used to show adequate preservation for an atypical product formulation, a risk assessment needs to be conducted by the microbiologist to justify the use of these alternate preservative challenge test criteria.

In-Use Studies General In addition to or in place of a product challenge test, an in-use study may provide sufficient data to conduct a risk assessment on some products. An in-use study may be used to evaluate the microbiological integrity of a product during consumer use. The study design should reflect actual product use as closely as possible. For further information, refer to “Microbiological Assessment of Product Quality after Use” in Section 15.

Testing When samples are returned from an in-use study, an aerobic plate count must be conducted before any other tests are performed to ensure that any microbial contaminants recovered were introduced by the panelists and did not arise from subsequent handling in the laboratory. Useful microbial content information may be obtained by a similar evaluation of the components (such as applicators) that come into direct contact with the product during use. Microbiological analysis of these samples can be conducted by using either standard in-house methods or the CTFA methods, “M-1 Determination of the Microbial Content of Cosmetic Products” and “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa” (Sections 18 and 19). It is recommended that microbiological evaluation take place within seven days after the last consumer use.

170

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

Criteria

ADDITIONAL INFORMATION Brannan, D.K., J.C. Dille, and D.J. Kaufman. 1987. “Correlation of In-Vitro Challenge Testing with Consumer Use Testing for Cosmetic Products.” Applied and Environmental Microbiology, 58(3):1827-1832. CTFA Microbiology Committee. December 1985. “CTFA Survey: Correlation of the In-Vitro Preservative Challenge Test with Consumer Use Testing,” Presented by R. Spielmaker at SCC Scientific Conference. Farrington, J.K., et al. 1994. “Ability of Laboratory Methods to Predict In-Use Efficacy of Antimicrobial Preservatives in an Experimental Cosmetic.” Applied and Environmental Microbiology, 60(12):4553-4558. Kabara, J.J., and D. S. Orth. 1996. Preservative-Free and Self-Preserving Cosmetics and Drugs. New York: Marcel Dekker, 45-73. Lindstrom, S.M. 1986. “Consumer Use Testing: Assurance of Microbial Product Safety.” Cosmetics and Toiletries, 101:73-74. Lindstrom, S.M., and J.D. Hawthorne. 1986. “Validating the Microbiological Integrity of Cosmetic Products through Consumer-Use Testing.” J. Soc. Cosmet. Chem. 37: 481-428. Orth, D.S. 1993. Handbook of Cosmetic Microbiology. New York: Marcel Dekker, 151. Orth, D.S. and S.R. Milstein. 1989. “Rational development of preservative systems for cosmetic products.” Cosmetics and Toiletries, 104(10): 91-103. Orth, D.S., R.F. Barlow, and C.A. Gregory. 1992. “The Required D-Value: Evaluating Product Preservation in Relation to Packaging and Consumer Use/Abuse.” Cosmetics and Toiletries, 107(12): 39-43. Wilson, L.A., A.I. Julian, and D.G. Ahearn. 1975. “The Survival and Growth of Microorganisms in Mascara During Use.” American Journal of Ophthalmology. 79(4): 596-601. Yablonski, J. I. 2002. “Preservation of Atypical Skin Care and Related Cosmetic Product Systems.” Cosmetics and Toiletries, 117.

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

171

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

The pass/fail criteria for in-use return samples normally reflect the microbiological test specifications that are used for quality control end product release. The pass/fail criteria for atypical products or for water-based products may vary significantly depending on the product type and area of use. For example, it may be acceptable that products that have been used in the lip area may contain a higher microbial level than products used in the eye when evaluated after use. To ensure that the number of microorganisms recovered after usage does not increase over time, these types of atypical products should be tested at a prescribed time interval. If microbial counts do increase over time, the formulation and/or package design should be reviewed to determine what steps could be taken to prevent them from increasing.

REFERENCES

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL COSMETIC PRODUCTS

1. Yablonski, J.I. and S.E. Mancuso. 2002. “Preservation of Atypical Cosmetic Product Systems.” Cosmetics and Toiletries, 117(4):31-40. 2. Block, S.S. 1997. “Alcohol.” In: Disinfection, Sterilization, and Preservation, editors Ali, Y., et al. 2001: 229-253, especially p. 234. 3. Brannan, D.K., 1997. Cosmetic Microbiology. New York: CRC Press, 47-50. 4. Kabara, J.J. 1984. “Food grade chemicals in a systems approach to cosmetic preservation.” In: Cosmetic and Drug Preservation: Principles and Practice. New York: Marcel Dekker, 339-356. 5. Kabara, J.J. and D.S. Orth. 1996. Preservative-Free and Self-Preserving Cosmetics and Drugs. New York: Marcel Dekker, 245-246. 6. Silliker, J.H. et al., editors. 1980. “International Commission on Microbiological Specifications for Food.” Microbial Ecology of Food. Orlando, FL: Academic Press, 76-91.

172

|

CTFA MICROBIOLOGY GUIDELINES

|

7. Doyle, Michael P., Larry R. Beuchat, and Thomas J. Montville, (Ed). 1997. Food Microbiology Fundamentals and Frontiers. Washington, DC: ASM Press, ISBN 155581-117-5. 8. Jay, J.M., (Ed). 2000. Modern Food Microbiology, Sixth Edition. Gaithersburg, MD: Aspen Publishers, 38-44, especially p. 42. 9. Hartman, P.A. 1968. Miniaturized Microbiological Methods. Orlando, FL: Academic Press. 10. Curry, J. 1985. “Water Activity and Preservation.” Cosmetic and Toiletries, 100: 53-54. 11. ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents. http://www.astm.org. 12. McConville, J.F., C.H. Anger, and D.W. Anderson. 1974. “Method for Performing Aerobic Plate Counts of Anhydrous Cosmetics Utilizing Tween 60 and Arlacel 80 as Dispersing Agents.” Applied Microbiology, 27, No.1: 5-7.

SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS

SECTION 17

Determination of Preservation Efficacy in Nonwoven Substrate Personal Care Products INTRODUCTION

In view of the differences between wipes and other types of personal care products, the standard preservation efficacy tests for aqueous-based (See “M-3 A Method for Preservation Testing of Water-Miscible Personal Care Products” in Section 20) or atypical (“M-6 A Method for Preservation Testing of Atypical Personal Care Products” in Section 23) personal care products are not suitable for testing these product forms. The two major test method differences have to do with the procedure for inoculum introduction and the procedure for the recovery of introduced microorganisms. It is recommended that, when developing preservation efficacy methods and testing protocols, the cosmetic microbiologist be aware of the factors listed below under “General Considerations” and how they may affect the reliability of the test method under development. This document is intended to be used in conjunction with “M-5 Methods for Preservation Testing of Nonwoven Substrate Personal Care Products” in Section 22).

GENERAL CONSIDERATIONS Training Those who develop methods and test protocols for wipes are expected to have training and experience in conducting and verifying the procedures (See “Microbial Validation and Documentation” in Section 9) in evaluating the data, and interpreting the results obtained. Standard laboratory safety procedures for microbiological testing should be followed.

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

|

CTFA MICROBIOLOGY GUIDELINES

|

173

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

Nonwoven substrate personal care products, commonly called wipes, constitute a wide and expanding variety of items that differ significantly from other types of personal care products in their composition, intended use, and physical characteristics.1 The nonwoven matrix or substrate is composed of fibers or filaments that are bonded together mechanically, thermally, or chemically and is used for the delivery of cosmetics or other product systems.

Components A nonwoven personal care product is composed of the following components: • Substrate - nonwoven carrier including coatings or finishes applied to that carrier • Add-ons - personal care formulation applied to a substrate; liquids and lotions are the most common • Packaging - final container for delivering the finished product Any change to the composition or nature of any of these components may affect the overall preservation efficacy of the final product and may require retesting.

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

Substrate The nature and composition of substrates can have a decided effect upon preservative-substrate interaction as well as subsequent preservative system performance. A substrate2,3 is a nonwoven web of long and short fibers held together by some means of bonding other than weaving. Fibers can be natural or synthetic. The substrate functions as the carrier for a variety of productspecific add-ons. Generally, substrates are composed of any one or a combination of various fiber types including cellulose or wood pulp, rayon or viscose, polyester and polypropylene polymer extrusions or bicomponent materials. Bonding technologies include mechanical entanglement, chemical or adhesive binding, thermal melting and hydrogen bonding. Binders can significantly affect the preservative system.4,5 For example, anionic binders, commonly used in some substrates, have the potential to inactivate or seriously disrupt most cationic preservative systems. Alternatively, binders may contain preservatives that may result in a more robust product. Other substrate issues that can affect preservation efficacy may include the fiber type and composition, the web forming process, the web bonding process, the proportion of pulp to binder, the composition and ionic nature of the chemical binding agent, and the presence and nature of substrate finishes or coatings. Depending on the nature and degree of reactivity of fiber surfaces, preservatives may become chemically or physically bound, reducing their antimicrobial activity. This may also be the case with certain finishes, coatings and other substrate surface treatments that can react with preservatives.

Add-Ons An add-on is the formulation applied to a nonwoven substrate. The add-on can be of varied composition and may be in the form of a liquid, lotion, emulsion, powder, cream, ointment, oil or other material. Although preservation efficacy demonstrated for the add-on may provide useful information, it may not be predictive of the preservation efficacy of the final nonwoven substrate product. Substrate and packaging may also influence preservation efficacy.

Packaging The packaging size and type, e.g. tubs, canisters, soft packages, single pack, etc., should be taken into consideration in developing the most appropriate protocol for the preservation efficacy

174

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

testing of the final product. How the product changes over time may be dependent on the type of package chosen, e.g. evaporation of the add-on through the package or adsorption of the addon to the package can occur, potentially affecting preservative stability.

Intended Use and Delivery of Product Intended use and delivery of the product may influence the test procedure. The type of packaging, e.g. open tub or pack, may influence the number of inoculations in the test method. The length of the test should be representative of packaging design, with consideration given to how long the consumer may use the product after it has been opened; e.g., a single use package versus one containing multiple substrates. The selection of challenge organisms ideally reflects the final use of the product. For example, challenge organisms for baby wipes (coliforms) may differ from those for an eye area product (Pseudomonads - See Section 14). End use and delivery of the product may determine acceptance criteria. For example, there may be different acceptance criteria for single pack versus multi pack products.

Preservative Stability

PRODUCT TESTING Preliminary Considerations It is recommended that the microbial content of the substrate, the add-on, and the finished product be determined prior to starting the preservation efficacy test. Due to the unique nature of non-woven products, molds are organisms of particular concern due to their ability to degrade cellulose fibers and the exposure of the large surface area of the wipe to the environment during manufacture and use. A high microbial load may reduce preservative activity or cause preservative failure in the final product. A sedimentation study8 to determine fluid migration through a vertical stack of wipes may be useful information for some test protocols, for instance if the inoculum is delivered by filter carrier. This data is useful in determining the distribution, or degree of sedimentation, of the add-on within packages of saturated wipe products packaged in stacks. A period of time for equilibration of the add-on and the substrate before testing is recommended. This allows time for total saturation of the add-on and distribution of the preservative. It is recommended that all aspects of product testing, such as organism recovery, neutralization, inoculation, etc, be verified for method suitability. (See Section 9: “Microbial Validation and Documentation”). SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

|

CTFA MICROBIOLOGY GUIDELINES

|

175

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

It is recommended that preservative stability be evaluated in the finished product packaging because of possible interactions of preservative, add-on, substrate, and package. The stability of the preservative system in the add-on does not necessarily reflect its stability in the finished product. It is recommended that accelerated aging studies on finished wipe products be confirmed with real time studies. Additional information on stability testing of personal care products is available from COLIPA6 and from the IFSCC7.

Organisms Organism strains recognized by United States Pharmacopeia (USP)9 and/or used in the industry for preservation efficacy testing are recommended. Additional reference strains and/or organisms appropriate to the product, including spores, may be used where deemed necessary. Pure or mixed culture inocula may be used. However, if different microorganisms are pooled, antagonism among microbes may occur or it may be difficult to differentiate between types of survivors. Some products may not require a full preservation efficacy test. For example, dry wipes, as defined by water activity measurement, may require limited or alternative testing. (See Section 16: “Microbiological Risk Factor Assessment of Atypical Cosmetic Products”).

Inoculation Procedures The choice of inoculation site, inoculum volume, and distribution of the inoculum onto the product should be determined with the anticipated consumer use and packaging of the product in mind. The procedure used should be representative of product use.

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

There are several aspects to consider when choosing an inoculation procedure: Packaging Although testing in the final product packaging is preferred, there are situations where it is impractical or impossible to do so. In these cases, testing outside of the final package is an acceptable alternative. If the same product is delivered in different packaging, i.e. tubs, canisters, soft packages, etc., it is recommended that each package type be tested.

Inoculum Volume It is recommended that a consistent inoculum volume be chosen to achieve a set organism level. This volume is dependent on the method of inoculation (Section 17). Keeping the inoculum volume to a minimum will avoid dilution of the add-on.

Inoculation Site / Distribution The nonwoven substrate can be inoculated using a variety of methods. It is important to verify that the inoculum site, distribution, and inoculum recovery is appropriate to the final packaging and use.

Reinoculation Reinoculation of the nonwovens during the preservation efficacy test is determined based on how the product is used by the consumer and whether the nonwovens are tested in or out of the final package. If a reinoculation is performed, ensure that there are adequate numbers of wipes to complete assays for the required test period.

176

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

Recovery Procedures Neutralization Verification It is recommended that neutralization studies be conducted with all challenge organisms used in the test. See ASTM 1054 for details10.

Recovery Verification The nonwoven substrate material is likely to entrap some microorganisms, resulting in a less than complete recovery of the inoculum. The level of recovery may change from product to product, depending on the combination of substrate and add-on. In most cases, mechanical or other action (Section 17) is necessary to release microorganisms from the substrate. Addition of a surfactant and/or multiple extractions from the same sample may be necessary to optimize recovery. It is recommended that the interpretation of results take into account the established recovery efficiency which is based on a time zero count.

RECOMMENDATIONS

Whatever the product, an effective preservative system for a personal care wipe should prevent proliferation of introduced microorganisms, even after repeated microbial insult.

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

|

CTFA MICROBIOLOGY GUIDELINES

|

177

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

It is recommended that a risk assessment be used to establish acceptance criteria for a specific product (Section 16). The risk assessment should reflect the final product and its intended use and may take into consideration many factors including, but not limited to, the following: • Test method and microorganisms • Nature of the add-on • Nature of the substrate • Degree of saturation • Degree of microbial recovery from the substrate • Design and size of the packaging • Intended use and target consumer • Performance and history of similar products

REFERENCES 1. Lochhead, Robert Y. 2006. “Emerging Technologies for Cosmetic and Personal Care Wipes.” Cosmetics and Toiletries 121: 47-52. 2. International Nonwovens & Disposables Association (INDA) website. http://www. inda.org. 3. European Disposables and Nonwovens Association (EDNA) website. http:// www.edana.org.

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

4. Sutton, S. 1996. “Neutralizer Evaluations as Control Experiments for Antimicrobial Efficacy Tests.” In: Handbook of Disinfectants and Antiseptics. Edited by J. M. Ascenzi. 43-62. Marcel Dekker, Inc. 5. McCarthy, Terrence J. 1984. “Formulated Factors Affecting Activity of Preservatives.” In: Cosmetic and Drug Preservation Principles and Practice. Edited by Jon J. Kabara. 359-388. Marcel Dekker, Inc. 6. The European Cosmetic, Toiletry, and Perfumery Association (COLIPA) and CTFA. 2004. Guidelines on Stability Testing of Cosmetic Products. http://www.colipa.com.

178

|

CTFA MICROBIOLOGY GUIDELINES

|

7. International Federation of Societies of Cosmetic Chemists. 1992. “Fundamentals of Stability Testing.” IFSCC Monograph Number 2. Cranford, NJ: Micelle Press. 8. Cremieux, A., S. Cupferman, and C. Lens. 2005. “Method for the Evaluation of the Efficacy on Antimicrobial Preservatives in Cosmetic Wet Wipes.” International Journal of Cosmetic Science 27: 223-236. 9. United States Pharmacopeia. 2007. <51> “Antimicrobial Effectiveness Testing.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD. 79-81. 10. ASTM International. 2003. “ASTM 1054 E 1054 - Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” In: Annual Book of ASTM Standards 11.05. West Conshohocken, PA.

SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

SECTION 18

M-1 Determination of the Microbial Content of Cosmetic Products 1. Scope 1.1 This is an acceptable plate count procedure for determining the microbial content of cosmetic products.

2. Applicable Documents 2.1 “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa” (Section 19).

3. Suggested Materials 3.1 Media for the enumeration of bacteria or fungi 3.1.1

Media for the enumeration of bacteria or fungi* Letheen Agar (BBL, Difco) SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

Microbial Content Agar (Difco) Nutrient Agar (BBL, Difco, Oxoid) Standards Methods Agar with Lecithin and Polysorbate 80 (BBL) Trypticase Soy Agar (BBL) Trypticase Soy Agar with Lecithin and Polysorbate 80 (BBL) Tryptic Soy Agar (Difco) Tryptone Soya Agar (Oxoid) or equivalent

*It must be demonstrated that the test method adequately inactivates the microbial growth inhibitors present in the product. It is recommended that a neutralizer be present in the diluent or agar or both. 1

SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

|

CTFA MICROBIOLOGY GUIDELINES

|

179

3.1.2

Media for the enumeration of fungi* Mycophil Agar with low pH (BBL) Potato Dextrose Agar (BBL, Difco, Oxoid) Sabouraud Dextrose Agar (BBL, Difco, Oxoid) or equivalent

3.1.3

Media used as diluents.* Acto Tryptone (1%) (Difco). Letheen Broth (BBL, Difco). Nutrient Broth (BBL, Difco, Oxoid). Trypticase Azolectin Tween Broth Base (BBL). D/E Neutralizing Media (Difco) or equivalent. 3.1.3.1

Prepare dilution bottles containing 80 mL of diluent for waterimmiscible products and 90 mL for water-miscible products.

3.1.3.2

Sterile wide-mouth dilution bottles containing 10 mL of Polysorbate 80.

ANNEX 18 SPECIFICATIONS

3.2 Equipment 3.2.1

Autoclave

3.2.2

Sterile Petri dishes, 15 × 100 mm

3.2.3

20 mL sterile syringes, 10 mL pipettes, 1.0 mL pipettes, spatulas and other sampling devices

3.2.4

Water bath capable of maintaining a temperature range of 45°-50°C

3.2.5

Microbiological incubators at 20°-25°C and 30°-34°C

3.2.6

Colony counter

3.2.7

Compound light microscope with 1000X oil immersion lens

3.2.8

Stereo microscope

4. Procedure for Aerobic Plate Count 4.1 Use sterile materials, equipment and aseptic techniques. 4.2 For water-miscible products, transfer by means of a syringe, pipette or spatula 10mL or g of the well-mixed product to a dilution bottle containing 90 mL of diluent (this is a 1:10 dilution). *It must be demonstrated that the test method adequately inactivates the microbial growth inhibitors present in the product. It is recommended that a neutralizer be present in the diluent or agar or both. 1 180

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

4.3 For water-immiscible products, transfer by means of a syringe, pipette or spatula 10 mL or g of the well-mixed product to a dilution bottle containing 10 mL of Polysorbate 80. Disperse the product within the Polysorbate 80 with a spatula. Volume to 100 mL with diluent (this is a 1:10 dilution). 4.4 The precise volume or weight of sample and diluent may be varied. A dilution ratio of 1:10 with a minimum sample size of 10 mL or g is recommended. 4.5 When the same agar is used for bacterial and fungal assays, dispense 1 mL of the dilution into each of three Petri dishes and 0.1 mL into three additional Petri dishes (to give triplicate plates of 1:10 and 1:100 dilutions). Add 15 to 10 mL melted agar medium kept at 44°-48°C and rotate plates sufficiently to disperse the product. Allow the agar to solidify and invert plates. Incubate one plate of each dilution as follows: a)

At 30°-35°C for a minimum of 48 hours for the bacterial assay

b)

At 20°-25°C for a minimum of 5 days for the fungal assay

c)

In a refrigerator to prevent growth. Or: Dispense 1 mL of the dilution into two Petri dishes and 0.1 mL into two additional Petri dishes (to give duplicate plates of 1:10 and 1:100 dilutions). Add melted agar medium (as above) and incubate one plate of each dilution as follows: (1) At 30°-35°C for a minimum of 48 hours followed by a minimum of 48 hours at 20°-25°C. (2) In a refrigerator to prevent growth.

a)

Bacterial assay medium at 30°-35°C for a minimum of 48 hours

b)

Fungal assay medium at 20°-25°C for a minimum of 5 days

c)

Remaining bacterial and fungal medium plates in a refrigerator to prevent growth

4.7 Include a laboratory control using apparatus, dilution blank (without product), media and appropriate incubation. Concurrent contamination on test and control plates invalidates the test. Find and eliminate the source of contamination. Repeat both control and product tests. 4.8 Count the colonies. If there is difficulty in distinguishing colonies from material, compare to the refrigerated plates, or examine the colonies under a stereo microscope. (With experience, the refrigerated plates can be eliminated from the procedure.) 4.9 The number of colony forming units (CFU) per mL or g is the colony count multiplied by the appropriate dilution factor (10 or 100).

SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

|

CTFA MICROBIOLOGY GUIDELINES

|

181

SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

4.6 When separate agars are used for bacterial and fungal assays, dispense 1 mL of the dilution into each of four Petri dishes and 0.1 mL into four additional Petri dishes (to give quadruplicate plates of 1:10 and 1:100 dilutions). Add 15-20 mL of agar medium for bacterial assay kept at 44°-48°C to two plates of each dilution. Add 15-20 mL of agar medium for fungal assay kept at 44°-48°C to two plates of each dilution. Rotate all plates sufficiently to disperse the product. Allow the agar to solidify and invert the plates. Incubate one plate of each dilution as follows:

4.10 Neutralization of preservatives should be validated for each product tested. This may be accomplished by streaking a 10–4 dilution of an appropriate organism onto the surface of test plates at the end of the incubation period. Failure of the inoculum to grow invalidates the test. Repeat the test using greater dilution of the test material. 4.11 Morphologically suspect colonies can be further identified by the methods described in “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.”1

REFERENCES

SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

1. 1999. “ASTM E 1054-91 – Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” Annual Book of ASTM Standards 11.05.

182

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT

SECTION 19

M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa 1. Scope 1.1 This is an acceptable procedure for determining whether or not bacteria isolated from cosmetics are Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa.

2. Applicable Documents 2.1 “M-1 Determination of the Microbial Content of Cosmetic Products” (Section 18). 2.2 “Microbial Limit Tests” <61>. The United States Pharmacopeia, 30th edition (2007)1

3. Suggested Materials 3.1 Media Eosin-Methylene Blue Agar plates (EMB)

3.1.2

EC Broth

3.1.3

Pseudomonas Isolation Agar

3.1.4

Motility Agar

3.1.5

Trypticase Soy Broth/TSB (BBL), Tryptic Soy Broth (Difco), Tryptone Soya Broth (Oxoid) or equivalent

3.1.6

Pseudomonas F Agar

3.1.7

Pseudomonas P Agar

3.1.8

MacConkey Agar

3.1.9

Vogel-Johnson Agar Plates (V-J)

SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.

|

CTFA MICROBIOLOGY GUIDELINES

|

183

SECTION 19: M-2 EXAMINATION FOR S.a., E.c., AND P.a.

3.1.1

3.2 Reagents 3.2.1

Cytochrome Oxidase Test - Use filter paper impregnated with 1% tetraethyl phenylenediaminedihydrochloride

3.2.2

Materials for Coagulase Test - Mammalian plasma, preferably rabbit or horse, with or without suitable additives. (see USP 301)

3.3 Equipment 3.3.1

Water baths at 37°C, 42°C and 45.5°C

3.3.2

Gram-Stain materials

3.3.3

Ultraviolet light

3.3.4

Fermentation tubes

3.3.5

Compound light microscope with 1000X, oil immersion lens

3.3.6

Microbiological incubator: 35°-37°C

4. Procedure 4.1 The following procedures should be performed only with isolated colonies. 4.2 Gram Stain 4.2.1

If colonies are observed with 24 hours of testing, Gram Stains of representative colonies should be performed. Colonies appearing after 24 hours should be streaked onto plates of the same medium, incubated 18-24 hours and Gram Stained.

4.3 Test for Staphylococcus aureus 4.3.1

Gram-positive cocci appearing in clusters should be streaked onto V-J Agar Plates and incubated at 35° ± 2°C for 24 hours.

SECTION 19: M-2 EXAMINATION FOR S.a., E.c., AND P.a.

After 24 hours, transfer characteristic growth (see Table 19-1) from the surface of V-J medium to individual tubes each containing 0.5 mL of a mammalian plasma. Simultaneously assay coagulase positive and negative cultures. Incubate in a water bath at 37°C, examining the tubes at 3 hours and at subsequent intervals up to 24 hours. If no coagulation in any degree is observed, the colonies are not S. aureus. If the reactions of the controls are not correct, the assay results are invalid. If the test is positive and a quantitative level is desired, count the type colonies on the primary isolation medium corresponding to the positive colony selected from that medium and planted on V-J agar. Express as CFU of S. aureus colonies per mL or g of product. 4.4 Test for Pseudomonas aeruginosa 4.4.1

184

|

Gram-negative rods should be tested for cytochrome oxidase activity. If oxidase positive, streak colonies onto Pseudomonas Isolation Agar, Pseudomonas

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.

Agar F for detection of fluorescein, and Pseudomonas Agar P for detection of pyocyanin. Incubate at 35° ± 2°C for not less than 3 days. Examine the streaked surfaces under ultraviolet light to determine whether colonies having the characteristics of pseudomonads are present (see Table 19-1). Perform either a microscopic motility test or stab motility agar with growth from selective Pseudomonas agar and observe for motility after incubating the stab culture for 24 hours. Transfer oxidase and motility positive colonies to TSB and incubate at 42°C for 24-48 hours. Growth at 42°C indicates P. aeruginosa. Lack of characteristic pigmentation of colonies and failure to grow at 42°C indicates other pseudomonads. If the test is positive, count the type colonies on the primary isolation medium corresponding to the P. aeruginosa colony selected from that medium and planted on selective Pseudomonas Isolation Agar. Express as CFU of P. aeruginosa colonies per mL or g of product. 4.5 Test for Escherichia coli 4.5.1

Oxidase-negative, Gram-negative rods should be tested to determine whether or not they are E. coli. Streak growth onto EMB and MacConkey Agar Plates and incubate at 35° ± 2°C for 24 hours. Transfer characteristic growth (see Tables 19-1 and 19-2) from MacConkey and/or EMB agars to EC Broth containing fermentation tubes. Incubate at 45.5°C in a water bath for 24-48 hours. Production of gas is characteristic of E. coli. If the test is positive, count the type colonies on the primary isolation medium corresponding to the positive colony selected from that medium and planted on MacConkey and EMB agars. Express as the number of E. coli colonies per mL or g of product.

4.6 Positive Controls 4.6.1

With each test for S. aureus, E. coli or P. aeruginosa a test should be conducted simultaneously with known cultures as positive controls. 4.6.1.1

Staphylococcus aureus Check each negative V-J plate after incubation by streaking with 10-4 dilution of S. aureus from a 24-hour broth culture. Failure to grow invalidates the test.

4.6.1.2

Pseudomonas aeruginosa

SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.

|

CTFA MICROBIOLOGY GUIDELINES

|

185

SECTION 19: M-2 EXAMINATION FOR S.a., E.c., AND P.a.

Check each negative Pseudomonas Isolation Aagar plate, Pseudomonas F Plate, Pseudomonas P Plate and TSB tube, after incubation with a streak or inoculum of a 10-4 dilution of P. aeruginosa from a 24-hour broth culture. A positive check requires that P. aeruginosa grow in a specific medium and produce pigment as specified in Tables 19-1 and 19-2. Failure to do so invalidates that portion of the examination.

4.6.1.3

Escherichia coli Check each negative MacConkey and EMB plate and each negative EC broth tube after incubation with a streak of inoculum of a 10-4 dilution of E. coli from a 24-hour broth culture. Failure of E. coli to grow in a specific medium as specified in Tables 19-1 and 19-2 invalidates that portion of the examination.

4.7 Alternative Methods 4.7.1

The identification of S. aureus, P. aeruginosa or E. coli may be confirmed by further suitable cultural and biochemical tests or by the use of rapid identification kits.

Table 19-1: Identification of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli: Presumptive Tests PRESUMPTIVE TEST S. aureus

E. coli

P. aeruginosa

Vogel-Johnson Agar

Black, surrounded by yellow zone

ND

ND

MacConkey Agar

ND

Brick red may have surrounding zone of precipitated bile

ND

Pseudomonas Isolation Agar

ND

ND

Generally greenish

Pseudomonas Agar F In Normal Light

ND

ND

Colorless to yellow

Pseudomonas Agar F In Ultraviolet Light

ND

ND

Yellowish

Pseudomonas Agar P In Normal Light

ND

ND

Greenish

Pseudomonas Agar P In Ultraviolet Light

ND

ND

Blue

Gram Stain

Positive clusters of cocci

Negative rods (cocco-bacilli)

Negative rods (slender)

SECTION 19: M-2 EXAMINATION FOR S.a., E.c., AND P.a.

ND = Not done Table 19-1

186

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.

Table 19-2: Identification of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli: Completed Test COMPLETED TEST S. aureus

E. coli

P. aeruginosa

Coagulase Test

Positive

ND

ND

Cytochrome Oxidase Test

ND

Negative

Positive

E.M.B. Agar

ND

Metallic sheen under Purple reflected light and a blue-black appearance under transmitted light

Fermentation E.C. Broth at 45.5°C

ND

Positive

ND

Growth at 42°C, TSB

ND

Growth

Growth

ND = Not done Table 19-2

REFERENCES 1. United States Pharmacopeia. 2007. <61> “Microbial Limit Tests.” United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD. 83-88.

|

CTFA MICROBIOLOGY GUIDELINES

|

187

SECTION 19: M-2 EXAMINATION FOR S.a., E.c., AND P.a.

SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.

SECTION 19: M-2 EXAMINATION FOR S.a., E.c., AND P.a.

188

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

SECTION 20

M-3 A Method for Preservation Testing of WaterMiscible Personal Care Products 1. Scope 1.1 This is an acceptable procedure for determining the preservative efficacy of watermiscible cosmetic and toiletry formulations1-4. 1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents 2.1 “Determination of Preservative Adequacy in Cosmetic Formulations” (Section 13).

3. Materials 3.1 Selection of Challenge Microorganisms The microbial strains listed in Table 20-1 may be considered for use in developing preservation data of Personal Care products. Either pure or mixed microbial culture suspensions may be used to challenge test formulations. Inocula consisting of only pure microbial cultures will yield specific data on each test microorganism employed in the challenge study. When conducting mixed culture challenge studies, it is recommended that closely related types of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be pooled separately. 3.2 Maintenance of Challenge Microorganisms Refer to the ATCC culture maintenance recommendations, available on their website5, and to other sources6-8. Storage of other organisms relevant to the product in the original product or incorporation of product into maintenance medium is often the only way to retain its unique characteristics. This method is especially appropriate where the isolate is subsequently inoculated into a similar material. SECTION 20: M-3 TESTING WATER-MISCIBLE PERSONAL CARE

|

CTFA MICROBIOLOGY GUIDELINES

|

189

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

3.3 Test Media 3.3.1

Inocula Suspending Fluids Suspending fluids are used to prepare the bacterial and fungal suspensions for inoculating the test product. The following may be used: •

Phosphate Buffer (pH 7.0)

•

0.85% Sodium Chloride Solution (Normal Saline)

•

Sodium Chloride Peptone Solution (1% peptone in normal saline)

Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate 80 or other surfactant to the suspending fluid is recommended to aid in dispersion of mold spores. 3.3.2

Microbial Plate Count Diluents Plating diluents serve to disperse the sample and dilute it to levels that permit recovery of surviving microorganisms from an inoculated product formulation. The choice of diluent depends on its ability to meet the requirements of preservative neutralization (Section 4.1). The following are examples of diluents that may be used: •

Buffered Sodium Chloride Peptone Solution

•

Dey/Engley (D-E) Neutralizing Broth

•

Eugon Broth

•

Letheen Broth

•

Modified Letheen Broth

•

Phosphate Buffer, pH 7

•

Soybean Casein Digest Medium (Tryptic Soy Broth)

•

Trypticase Azolectin™ Tween™ (TAT) Broth

•

Saline-Tween-Lecithin Diluent

Addition of neutralizers may be necessary to demonstrate adequate preservative neutralization. If neutralizing systems other than those listed above are used, absence of neutralizer toxicity should be verified. 3.3.3

Recovery Agars Many factors affect organism viability. Therefore, it is important for the agar to provide optimum nutritional support for the recovery of the challenge organisms. The following have been found suitable for preservation studies:

190

|

•

Eugon Agar

•

Letheen Agar

•

Microbial Content Agar

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 20 M-3 TESTING WATER-MISCIBLE PERSONAL CARE

Modified Letheen Agar

•

Plate Count Agar

•

Soybean Casein Digest Agar (Tryptic soy agar)

•

Microbial Content Agar with Tween

Addition of neutralizers may be necessary to demonstrate adequate preservative neutralization. Other suitable agars and neutralizing agents may be used. If the above agars do not support the growth of fungi, one of the following agars may be considered: •

Malt Agar

•

Malt Extract Agar

•

Mycological Agar

•

Potato Dextrose Agar

•

Sabouraud Dextrose Agar

Other suitable agars and neutralizing agents may be used.

4. Preliminary Tests 4.1 Preservative Neutralization Carryover of antimicrobial activity from the product formulation into the plate count diluent and recovery growth agar may occur. This may inhibit the growth of surviving challenge test microorganisms, resulting in a false negative microbial count. To avoid a false negative result, neutralization of the antimicrobial properties of the formulation must take place in the plate count diluent and/or the recovery growth agar. Antimicrobial neutralization may normally be accomplished by use of chemical neutralizing agents, physical dilution, or a combination of both. Verification of neutralization is generally performed by inoculating the product dilution with a low level of challenge microorganisms and performing the enumeration method Side-by-side dilutions with and without a product formulation are made. Enumeration of the microorganisms from these dilutions is performed. Neutralization is verified if microbial recoveries are similar. If one or more challenge microorganisms cannot be recovered, the use of a higher dilution and/or the investigation of additional chemical neutralizers may be considered9,10. 4.2 Microbial Content Test It is recommended that that a microbial content test11, 13 be performed on the test sample prior to performing the preservative efficacy test. Verification of neutralization of the antimicrobial properties of the test sample should be demonstrated (See Section 4.1 above and References 9 and 10) for the microbial content test.

SECTION 20: M-3 TESTING WATER-MISCIBLE PERSONAL CARE

|

CTFA MICROBIOLOGY GUIDELINES

|

191

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

•

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

5. Inoculation Procedures 5.1 Preparation of Inocula Freshly prepared cultures should be used for inoculating test samples. In general, culture conditions in Table 20-2 should be considered when preparing the inocula. Refer to the ATCC website5 for optimal growth media and conditions for specific microorganisms. Inclusion of cellulose degrading molds may necessitate longer incubation periods and require a paper source for growth. 5.1.1

Preparation of Initial Bacteria and Yeast Suspensions Broth cultures or cultures grown on solid agar media are acceptable for use. For reference strains such as the ATCC strains, no more than five transfers from the stock culture are recommended12. Broth cultures should be centrifuged and then re-suspended in the chosen suspending fluid. (See 3.3.1) Microbial growth on a solid medium is transferred to the chosen suspending fluid.

5.1.2

Preparation of Initial Mold Suspensions The mold inoculum is prepared by washing the sporulating agar culture with the chosen suspending fluid (See 3.3.1) and filtering the spore suspension through sterile gauze or glass wool. Sterile glass beads can be used as an aid in the dispersion of spores in the suspending fluid.

5.1.3

Preparation of Bacterial Spore Suspensions If spore-forming bacteria are to be included in the test, the inocula may be prepared as indicated in the AOAC Sporicidal Test3. Some strains are commercially available as prepared spore suspensions.

5.1.4

Preparation of Challenge Inocula 5.1.4.1

Inoculum levels

The recommended inoculation levels for challenge testing are: •

1×106 Colony-Forming Units (CFU) of bacteria pergram of product

•

1×105 CFU of yeast per gram of product

•

1×105 CFU of mold spores per gram of product The inoculum level for the challenge microorganisms should be verified by standard microbiological techniques such as pour plate methods.

5.2 Product Challenge 5.2.1

Pure and mixed-culture challenge Either pure or mixed cultures may be used to challenge test formulations. Pure culture challenge, although more time-consuming, will yield specific data on each microorganism employed in the study. Mixed-culture challenge, on the other hand, can be used to obtain rapid pass-or-fail decisions on preservative adequacy and reduce the workload. However, antagonism among organisms

192

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 20 M-3 TESTING WATER-MISCIBLE PERSONAL CARE

All products should be thoroughly mixed manually or mechanically after inoculation to distribute the challenge microorganisms uniformly. It is recommended that the volume of the inoculum be < 1% of the sample weight and should not alter the character of the product being challenged. Challenged formulations should then be stored at ambient temperature for the duration of the test. 5.3 Sampling the Challenged Product 5.3.1

Sampling interval Challenged formulations should be sampled for viable microorganisms at selected time intervals after inoculations. The frequency of sampling should follow a set pattern to facilitate future comparison of test results between different product formulations or samples, for example, weekly up to 28 days after inoculation.

5.3.2

Sampling and plating methods The inoculated product should be thoroughly mixed just prior to sampling to ensure that the sample is representative. In some cases, the inoculum can thrive in “pockets” of growth in the formulation while other areas are relatively free of microorganisms. Many aerobic microorganisms grow especially well at the formulation-air interface. Often it is very difficult to break up the “pockets” of growth, and special procedures are needed. The following mixing methods have been used to overcome this problem: •

Vigorous mixing with a stirring rod

•

Capping and shaking vigorously by hand

•

Mixing in a vortex mixer

•

Mixing with a magnetic stirrer

•

Mixing with a propeller stirrer

•

Mixing with a non-aerating stirrer

•

Mixing in a micro blender

•

Mixing in a stomacher

•

Gentle mixing in a tissue grinder Sample size will in part determine the minimum detectable level. A sample sizeofatleastonegramoronemilliliterofproductforthequantitativepourplatemethodis recommended. Aseptic techniques must be employed.

SECTION 20: M-3 TESTING WATER-MISCIBLE PERSONAL CARE

|

CTFA MICROBIOLOGY GUIDELINES

|

193

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

may occur. It is recommended that broadly related types of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, or molds be pooled separately when conducting mixed-culture challenge.

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

5.3.2.1

Quantitative pour plate method Serial dilutions are prepared from the aliquot recovered from the challenged sample unit. Each serial dilution is thoroughly mixed, and an aliquot is transferred to a Petri dish. Melted agar maintained at 44-48°C is added to the Petri dish, and the dish is rotated to uniformly disperse the product dilution. The agar plates are allowed to solidify, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 20-2). After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of microorganisms per sample unit.

5.3.2.2

Quantitative spread plate method The quantitative spread plate method is performed in a manner similar to the pour plate method; however, an aliquot of each dilution is transferred directly onto the surface of solidified microbial growth agar. The sample aliquot is then evenly spread over the agar surface. The agar plates are allowed to dry, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 20-2). After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of microorganisms per sample unit.

6. Other Considerations 6.1 Length of Test Procedure It is recommended that preservation tests be carried out for a minimum of 28 days. In some cases, numbers of challenge microorganisms may be reduced below detectable levels during the early stages of the test only to adapt to the preservative system and later proliferate. A final judgment of preservative adequacy should not be made until all the data are obtained. 6.2 Rechallenge Consideration may be given to rechallenge. A rechallenge is useful for determining if a formulation is marginally preserved and identifying which types of microorganisms may be potential problems for that particular formulation. 6.3 Storage Stability sIt is important that challenge tests also be conducted on samples aged in the final container in order to determine the stability of the preservative system.

194

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 20 M-3 TESTING WATER-MISCIBLE PERSONAL CARE

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

Table 20-1: Suggested Challenge Microorganisms Type

Microorganism (ATCC5 Number)

Recommendation

Gram-Positive Cocci

Staphylococcus aureus (6538)* Staphylococcus epidermidis (12228)

Select at least one

Fermentative Gram-Negative Bacilli

Klebsiella pneumoniae (10031) Enterobacter cloacae (13047)) Escherichia coli (8739)* Enterobacter gergoviae (33028)

Select at least one

Non-Fermentative Gram-Negative Bacilli

Pseudomonas aeruginosa (9027)* Burkholderia cepacia (25416) Pseudomonas fluorescens (13525) Pseudomonas putida (31483)

Select at least one

Yeasts

Candida albicans (10231)*

Recommended

Molds

Aspergillus niger (16404)* Penicillium species

Select at least one

Spore-Forming Bacilli

Bacillus subtilis (6051)

Optional

Other

Other organisms relevant to product

Optional

*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231) and Aspergillus niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing method4. Table 20-1

Table 20-2: Culture Conditions for Preparation of Inocula Cultures

Media**

Temperature

Time

Bacteria

Soybean Casein Digest (Tryptic Soy) Broth/Agar Nutrient Broth/Agar Eugon Agar/Broth

30-37°C

18-48 hours

Yeasts

Sabouraud Dextrose Agar Soybean Casein Digest (Tryptic Soy) Broth/Agar Mycophil (Mycological) Broth/Agar

25-35°C

24-48 hours

Molds

Sabouraud Dextrose Agar Potato Dextrose Agar Mycophil (Mycological) Agar Malt Extract Agar

20-30°C

7-28 days

** Available in dehydrated forms from commercial sources. Table 20-2

SECTION 20: M-3 TESTING WATER-MISCIBLE PERSONAL CARE

|

CTFA MICROBIOLOGY GUIDELINES

|

195

SECTION 20 M-3: TESTING WATER-MISCIBLE PERSONAL CARE

REFERENCES 1. AOAC INTERNATIONAL. 2000. “Official Method 998.10 - Efficacy of Preservation of Non-Eye Area Water-Miscible Cosmetic and Toiletry Formulations.” In: Official Methods of Analysis of AOAC INTERNATIONAL. Gaithersburg, MD. 2. American Society for Testing and Materials. 2001. “ASTM E 640-78 - Standard Test Method for Preservatives in WaterContaining Cosmetics.” In: American Society for Testing and Materials. West Conshohocken, PA. 3. AOAC INTERNATIONAL. 2000. “Official Method 966.04 - Sporicidal Activity of Disinfectants.” In: Official Methods of Analysis of AOAC INTERNATIONAL. Gaithersburg, MD. 4. United States Pharmacopeia. 2007. <51> “Antimicrobial Effectiveness Testing.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD. 79-81. 5. The American Type Culture Collection (ATCC) website: http://www.atcc. org recommends appropriate media for the microbial strains it provides and lists formulations for these media on its website: http://www.atcc.org/common/catalog/media/mediaIndex.cfm. The media formulations listed are not ready-to-use products for sale by the ATCC but in some cases other commercial suppliers are listed. 6. Brown, M.R.W. and P. Gilbert, (Ed). 1995. Microbiological Quality Assurance: A Guide Towards Relevance and Reproducibility of Inocula. Boca Raton, FL: CRC Press.

196

|

CTFA MICROBIOLOGY GUIDELINES

|

7. Kirsop, B.E. and A. Doyle, (Ed). 1991. Maintenance of Microorganisms and Cultured Cells, Second Edition. New York, NY: Academic Press. 8. Simione, F. P. 1998. “Cryopreservation Manual.” Nalgene Nunc International, http://www.nalgenelabware.com/techdata/technical/manual.asp 9. United States Pharmacopeia. 2007. <1227> “Validation of Microbial Recovery from Pharmacopeal Articles.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD, 684-686. 10. American Society for Testing and Materials. 1999. ASTM E 1054-91, “Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” In: Annual Book of ASTM Standards, 11.05. West Conshohocken, PA. ASTM. http://www.astm.org. 11. Krowka, John F., and Bailey, John E. 2007. “M-1 Determination of the Microbial Content of Cosmetic Products.” In: CTFA Microbiology Guidelines. Washington, DC: The Cosmetic Toiletry and Fragrance Association. 12. Reichgott, M. 2003. “Reference Strains: How Many Passages Are Too Many?” ATCC Connection, 23, No. 2, http://www. atcc.org/common/documents/pdf/tb06. pdf 13. United States Pharmacopeia. 2007. <61> “Microbial Limit Tests.” United States Pharmacopeia and the National Formulary. USP30 - NF25. Rockville, MD. 83-88.

SECTION 20 M-3 TESTING WATER-MISCIBLE PERSONAL CARE

SECTION 21

1. Scope 1.1 This is an acceptable procedure for determining the preservative efficacy of eye area cosmetic products.1, 2 1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents 2.1 “Preservation Testing of Eye Area Cosmetics” (Section 14).

3. Materials 3.1 Selection of Challenge Microorganisms The following types of microorganisms should be given consideration in developing preservation data: Additional microorganisms should also be included in the test procedure if preservation problems have been encountered with such microorganisms. 3.2 Maintenance of Challenge Microorganisms See also Section 10 and Reference 3. Table 21-2 shows conditions recommended for culture maintenance: Alternatively, cultures may be freeze-dried, frozen or grown on a slant and overlaid with sterile mineral oil. Although initially more time-consuming, these methods eliminate the necessity of frequent transfers and help ensure better culture stability. The viability of cultures must be checked regularly. In-house isolates may present unique maintenance problems. Storage in the original product or incorporation of product in the maintenance medium is often the only way to retain viability and continued resistance. This method is especially appropriate where the isolate is subsequently inoculated into a similar product.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS |

CTFA MICROBIOLOGY GUIDELINES

|

197

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

M-4 Method for Preservation Testing of Eye Area Cosmetics

3.3 Test Media 3.3.1

Plating diluents Plating diluents serve to disperse the sample and dilute it to levels that permit better recovery of the microbial population of a challenged formulation. Ideally, the diluent should contain both neutralizing agents and a biologically inert surface-active agent.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

The following diluents have been found suitable for preservation studies: •

Williamson Buffer Suspending Fluid (modified)4

•

Letheen Broth*

•

Thioglycolate Broth*

•

TAT Broth*

•

Dey/Engley (D-E) Neutralizing Broth

The addition of lecithin and an appropriate polysorbate to commercially available dehydrated broth formulations is also acceptable. 3.3.2

Recovery media It is important that the recovery medium provide adequate nutritional support for the growth of damaged cells. It is recommended that neutralizing agents be incorporated into the agar to counteract preservative carry-over from the diluent to the recovery medium. Letheen agar is an example of a commercially available medium containing neutralizers. It can be used in the recovery of bacteria, yeasts and molds. Thioglycolate agar should be considered for inactivation of mercury and other heavy metals, while D-E Medium (DIFCO) is useful when the preservative system is unknown or when several different types of preservatives are present. In most cases, the addition of lecithin and an appropriate polysorbate to any nutritionally adequate growth medium for bacteria, yeasts or molds is sufficient to achieve preservative neutralization.

3.3.3

Evaluating preservative neutralization5, 6 The presence of active preservatives carried over from the challenged formulation into the plating diluent and recovery medium may inhibit viable organisms and result in false-negative readings. Neutralizing agents should be incorporated into the plating diluent and/or recovery medium in order to inactivate preservatives and permit accurate enumeration of the microbial content. Methods to evaluate neutralizer effectiveness are as follows: If growth is not obtained on the dilution plates after incubation, inoculate the surface of the 10-1 and 10-2 plates with approximately 100 CFU of a mixed culture of Gram-positive bacteria. Perform the same procedure with a mixed culture of Gram-negative bacteria, a mixed culture of yeasts and a mixed culture of molds. If growth is not apparent on any one of the streaks after incubation, neutralization of the preservative system is inadequate and an

198

|

CTFA MICROBIOLOGY GUIDELINES

| SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

appropriate neutralizer must be found. Where neutralizers are not available or effective, physical dilution or membrane filtration to recover surviving microorganisms from the sample may be necessary.

4. Procedure 4.1 Aseptic technique should be practiced, and all test materials and media should be sterile.

4.2.1

Inoculum preparation Fresh cultures should be used for challenging preservative systems. Either broth cultures or cultures grown on solid media are recommended. After two or three consecutive daily transfers, bacterial and yeast cultures may be used to challenge the product. If log-phase cells are desired, an incubation period of 18-24 hours is usually adequate. Broth cultures may be used directly or centrifuged and resuspended in phosphate buffer (pH 7.0) or 0.85% saline. Growth on a solid medium is transferred to phosphate buffer prior to use. The mold inoculum is prepared by washing the 7- to 14-day-old agar slants with phosphate buffer or 0.85% saline and filtering the spore suspension through gauze. Low concentrations of polysorbates (e.g., 0.05% polysorbate 80) can be added to the saline to aid in spore dispersal. In addition, harvesting spore suspensions with glass beads in the saline aids in their dispersal. In-house isolates should be inoculated into the test formulation directly from contaminated product. If this is not feasible, in-house isolates may be cultured in broth or on solid media as described above. Table 21-3 shows conditions that should be considered when preparing the inoculum: If spore-forming bacteria are to be employed, inocula can be prepared as indicated in the AOAC sporicidal test.7

4.2.2

Sample Preparation The amount of product required to perform a preservation study should be a minimum of 20 grams for each test microorganism or pool of microorganisms. Sufficient product is needed to sample at each evaluation time and to have some material remaining in the event that additional platings are required. When rechallenges8 are used, it will be necessary to increase the amount of product to be tested. During the early developmental stage, a formulation may be challenged in glass containers. Subsequent preservation tests should be conducted on product in the final package to ensure its compatibility with the preservative system. It is recommended that all formulations be examined for microbial content prior to initiating preservation studies.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS |

CTFA MICROBIOLOGY GUIDELINES

|

199

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

4.2 Aqueous Liquid and Semi-liquid Eye Products

4.2.3

Product challenge 4.2.3.1

Pure and mixed-culture challenge

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

Either pure or mixed cultures may be used to challenge test formulations. Pure-culture challenge, although more time consuming, will yield specific data on each microorganism employed in the study. Mixed-culture challenge, on the other hand, can be used to obtain rapid pass-or-fail decisions on preservative adequacy and reduce the workload. However, antagonism among organisms may occur. It is recommended that closely related types of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, or yeasts and molds be pooled separately when conducting mixed culture challenge. 4.2.3.2

Inoculum levels Challenge levels ranging from 1 × 104 to 1 × 108 CFU per gram of product have been reported in the literature for preservative system evaluation.9, 10, 11 A reasonable challenge should be larger than the total challenge expected during consumer use. On this basis, the following levels are recommended: •

1×106 CFU of bacteria per gram of product

•

1×105 CFU of yeast per gram of product

•

1×105 CFU of mold spores per gram of product

After inoculation, all products should be thoroughly mixed manually or mechanically to uniformly distribute the challenge microorganisms. The volume of the inoculum should not alter the character of the product being challenged. Challenged formulations should then be incubated at controlled room temperature under appropriate conditions of humidity for the duration of the test. 4.2.4

Sampling the challenged product 4.2.4.1

Sampling intervals Challenged formulations should be sampled for viable microorganisms at selected time intervals after inoculations. The frequency of sampling should follow a set pattern to facilitate future comparison of results. Sampling is recommended immediately after inoculation (0 hour) and at 1-3, 7, 14, 21 and 28 days. When rechallenged, sampling should be once a week thereafter for at least 3 weeks.

4.2.4.2

Sampling methods The inoculated product should be thoroughly mixed just prior to sampling to ensure that the sample is representative. In some cases, the inoculum can thrive in “pockets” of growth in the formulation while other areas are relatively free of microorganisms. Many

200

|

CTFA MICROBIOLOGY GUIDELINES

| SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

aerobic microorganisms grow especially well at the formulationair interface. Often it is very difficult to break up the “pockets” of growth, and special procedures are needed. The following mixing methods have been used to overcome this problem: Vigorous mixing with a stirring rod

•

Capping and shaking vigorously by hand

•

Mixing in a vortex mixer

•

Mixing with a magnetic stirrer

•

Mixing with a propeller stirrer

•

Mixing with a non-aerating stirrer

•

Mixing in a micro blender

•

Gentle mixing in a tissue grinder

•

Mixing in a stomacher

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

•

Sample size will in part determine the minimum detectable level. A sample size of one-gram or one-milliliter of product for the quantitative pour plate method is recommended. 4.2.4.3

Plating (enumeration) methods Quantitative pour plate method Serial tenfold dilutions are prepared from the one-gram or onemilliliter aliquot withdrawn from the challenged formulation. Each dilution is thoroughly mixed, and one milliliter from each dilution is transferred by pipette to a Petri dish. Melted agar maintained at 43-46°C is added to the Petri dish, and the dish is rotated to uniformly disperse the product. Solidified plates for bacteria and yeast are incubated at 30-37°C for 48-72 hours. Mold plates are incubated at 20-25°C for 5-7 days. After incubation, the colonies are counted, and the resulting figure is multiplied by the dilution factor to obtain the number of microorganisms per gram or milliliter of product. Quantitative spread plate method The quantitative spread plate method is performed in a manner similar to the pour plate method; however, 0.1-0.2 ml of each dilution is pipetted directly onto the surface of solidified agar. The sample is then evenly spread over the surface of the agar with a glass “hockey stick.” The plates are allowed to dry and then incubated, and colonies are counted as described above.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS |

CTFA MICROBIOLOGY GUIDELINES

|

201

4.3 Non-Aqueous Eye Cosmetics 4.3.1

Inoculum preparation Aqueous inoculum - See 4.2.1. Emulsified aqueous inoculum - This is an aqueous inoculum emulsified with not more than 1% of dispersing agent such as polysorbate, sorbitan oleate or glycerol.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

Oil inoculum - Challenge cultures may be resuspended in light mineral oil. 4.3.2

Inoculation methods 4.3.2.1

Mixing See 4.2.4.2 above. A glass rod, tongue depressor, or mechanical mixer may be necessary to uniformly disperse test microorganisms. Inoculated product that has collected on the mixing device or on the container’s inner surfaces or edges must be worked back into the sample to prevent excessive loss of product.

4.3.2.2

Surface inoculation Swabbing - A swab is dipped into an inoculum of known concentration and swabbed across the entire product surface. Spreading - A known volume of inoculum is pipetted onto the surface of the product and uniformly spread using a glass rod or other instrument. Dipping - The product in its container is dipped into an inoculum of known concentration for a predetermined length of time. Spraying - The product is sprayed with a suspension of inoculum using an atomizer. Appropriate safety precautions should be taken.

4.3.3

Product challenge 4.3.3.1

Oils and water-in-oil emulsions Procedure Prepare enough of the formulation to permit adequate sampling at each test interval. At least 20 mL or 20 grams of the product should be challenged with each test microorganism or mixture of test microorganisms. Use containers that can be sealed to prevent excessive evaporation and are large enough to allow for adequate mixing. The containers should not react with the product. Inoculum The inoculum volume should be 0.1% to 1.0% of the sample volume to keep the sample as water-free as possible. The inoculum may be an aqueous or oil suspension added as a liquid or spray.

202

|

CTFA MICROBIOLOGY GUIDELINES

| SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

4.3.3.2

Loose powders Procedure At least 20 grams of product should be challenged with each test organism or mixture of test microorganisms. This sample size is usually large enough to permit numerous samplings. Standardized containers that can be loosely capped and are large enough to allow for adequate mixing should be used. The containers should not react with the product.

A fine spray or a liquid inoculum (volume 1% to 5% of the test sample) should be added to the product and thoroughly mixed. 4.3.3.3

Pressed powders Pressed powders treated as loose powders Procedure Pressed powders may be removed from containers, ground (e.g., mortar and pestle) into fine particles, and processed as described above. A minimum sample size of 20 grams should be prepared for each challenge microorganism or pool of microorganisms. Inoculum See 4.3.3.2. Pressed powders in pans or cakes Procedure For pressed powders inoculated on the surface, a suitable number of pans or cakes should be prepared for adequate sampling for each sampling interval. It is suggested that each pan or cake contain one gram of sample. Inoculum These samples are surface inoculated using any of the methods under “Surface Inoculation” above.

4.3.3.4

Wax-based products Bulk samples Procedure For bulk samples, a minimum size of 20 grams should be prepared for each challenge microorganism or pool of microorganisms. Briefly warming the bulk product to no more than 45°C may aid in the dispersion of the challenge inoculum.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS |

CTFA MICROBIOLOGY GUIDELINES

|

203

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

Inoculum

Inoculum An oil or emulsified aqueous inoculum is suggested. It may be applied as a liquid or a spray and mixed as in “Mixing” above. Pan, cake or stick samples Procedure SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

For pans, cakes or sticks that are surface inoculated, see 4.3.3.3. Pressed powders in pans or cakes Inoculum See 4.3.2.2. 4.3.4

Sampling the challenged product See 4.2.4.

Table 21-1: Suggested Challenge Microorganisms Type

Microorganism

Recommendation

In-house Isolates

As appropriate

one or more

Gram-Positive Cocci

Staphylococcus aureus Staphylococcus epidermidis

at least one

Fermentative Gram-Negative Rod

Klebsiella pneumoniae Enterobacter cloacae Escherichia coli Proteus species Enterobacter gergoviae

at least two

Non-Fermentative Gram-Negative Rod

Pseudomonas aeruginosa Burkholderia cepacia Pseudomonas fluorescens Pseudomonas putida Flavobacterium species Acinetobacter species

at least one in addition to P. aeruginosa

Yeasts

Candida albicans Candida parapsilosis

at least one

Molds

Aspergillus niger Penicillium luteum

at least one

Spore-Forming Bacteria

Bacillus subtilis

optional Table 21-1

204

|

CTFA MICROBIOLOGY GUIDELINES

| SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

Table 21-2: Suggested Culture Conditions Maintenance Media*

Storage Conditions

Transfer Frequency

Bacteria

Nutrient Agar Tryptic (Trypticase) Soy Agar Eugon Agar

Refrigeration 4-8°C

Weekly, Biweekly, or Monthly

Yeasts

Tryptic (Trypticase) Soy Agar Potato Dextrose Agar Mycophil (Mycological) Agar

Refrigeration 4-8°C

Biweekly or Monthly

Molds

Sabouraud Dextrose Agar Potato Dextrose Agar Mycophil (Mycological) Agar

Refrigeration 4-8°C

Biweekly or Monthly

* Available in dehydrated forms from Becton Dickinson Microbiology Systems, BBL Division (Cockeysville, MD 21030), DIFCO (Detroit, MI 28401) and other commercial sources

Table 21-2

Table 21-3: Suggested Inoculum Conditions Cultures

Media*

Temperature

Time

Bacteria

Tryptic (Trypticase) Soy Broth/Agar Nutrient Broth/Agar Eugon Agar/Broth

30-37°C

18-48 hours

Yeasts

Tryptic (Trypticase) Soy Broth/Agar Mycophil (Mycological) Broth/Agar

30-37°C

24-48 hours

Molds

Sabouraud Dextrose Agar Potato Dextrose Agar Mycophil (Mycological) Agar

20-25°C

7-14 days

* Available in dehydrated forms from Becton Dickinson Microbiology Systems, BBL Division (Cockeysville, MD 21030), DIFCO (Detroit, MI 28401) and other commercial sources.

Table 21-3

ADDITIONAL INFORMATION Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” Journal of the Society of Cosmetic Chemistry 23:703-720. 1972. Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” Journal of the Society of Cosmetic Chemistry 18:797-807. Wilson, L.A., Kuehne, J.W., Hall, S.W. and Ahearn, D.G. 1971. “Microbial Contamination in Ocular Cosmetics,” American Journal of Ophthalmology, 71(6):1298-1302.

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS |

CTFA MICROBIOLOGY GUIDELINES

|

205

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

Cultures

SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

REFERENCES 1. American Society for Testing and Materials. 2001. “Standard Test Method for Preservatives in Water-Containing Cosmetics.” ASTM E 640-78. West Conshohocken, PA.

6. Wilson, L.A., Julian, A.J. and Ahearn, D.G. April 1975. “The Survival and Growth of Microorganisms in Mascara During Use”, American Journal of Ophthalmology 79(4): 596-601.

2. AOAC INTERNATIONAL. 2000. “Efficacy of Preservation of Non-Eye Area Water-Miscible Cosmetic and Toiletry Formulations” Official Method 998.10, Official Methods of Analysis of AOAC INTERNATIONAL, 17th Ed. Gaithersburg, MD.

8. AOAC INTERNATIONAL. 2000. “Sporicidal Activity of Disinfectants.” Official Method 966.04, Official Methods of Analysis of AOAC INTERNATIONAL, 17th Ed. Gaithersburg, MD.

3. Brown, M.R.W. and P. Gilbert, (Ed). 1995. Microbiological Quality Assurance: A Guide Towards Relevance and Reproducibility of Inocula. CRC Press. 4. P. Williamson and A. Klingman. 1965. “A New Method for the Quantitative Investigation of Cutaneous Bacteria.” Journal of Invest Dermatology 45(6): 498-503. 5. “Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products. ASTM E 1054-91.

206

|

CTFA MICROBIOLOGY GUIDELINES

9. Preservation Subcommittee of The Cosmetic, Toiletry, and Fragrance Association. 1981. “A Study of the Use of Rechallenge in Preservation Testing of Cosmetics”. CTFA Cosmet. Journal 13:19-22. 10. Wilson, L.A. and Ahearn, D.G. 1977. “Pseudomonas-induced Corneal Ulcers Associated with Contaminated Eye Mascaras” American Journal of Ophthalmology 84: 112-119. 11. Madden, J.M. and Jackson, G.J. 1981. “Cosmetic Preservation and Microbes: Viewpoint of the Food and Drug Administration”. Cosmetics and Toiletries 96:7577.

| SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS

SECTION 22

M-5 Methods for Preservation Testing of Nonwoven Substrate Personal Care Products 1. Scope 1.1 These methods cover a variety of procedures currently used within the cosmetics industry to evaluate preservative efficacy of different types of nonwoven substrate, wipe, or towelette products.

It is recommended that the method chosen reflect consideration of the manufacturing process, the type of packaging used, and the end use of the product. 1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents 2.1 “Determination of Preservative Adequacy in Nonwoven Substrate Personal Care Products” (Section 13).

3. Materials 3.1 Selection of Challenge Microorganisms The microbial strains listed in Table 22-1 may be considered for use in developing preservation data of nonwoven substrate, wipe, or towelette products. 3.2 Maintenance of Challenge Microorganisms Refer to the American Type Culture Collection (ATCC) culture maintenance recommendations, available on the ATCC Web site,1 and other sources.2,3,4

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

207

EVALUATION SECTION 22: OFM-5 PRIMARY TESTING DERMAL OF NONWOVEN IRRITATION SUBSTRATE POTENTIAL PC PRODUCTS

These methods apply to nonwoven substrate personal care products that contain an aqueous-based add-on solution. For nonwoven personal care products containing nonaqueous add-on materials or concentrates, it is important that critical consideration be given to the typical use of the finished product and risk assessment and testing be completed as detailed in “Microbiological Risk Factor Assessment of Atypical Cosmetic Products” (Section 16).

3.3 Test Media 3.3.1

Inocula Suspending Fluids Suspending fluids are used to prepare the bacterial and fungal suspensions for inoculating the test product. The following may be used: •

Phosphate Buffer (pH 7.0)

•

0.85% Sodium Chloride Solution (Normal Saline)

•

Sodium Chloride Peptone Solution (1% Peptone in Normal Saline)

Other suitable fluids may be used. To aid in dispersion of mold spores, adding 0.05% – 0.1% polysorbate 80 or other surfactant to the suspending fluid is recommended. 3.3.2

Microbial Plate Count Diluents

EVALUATION SECTION 22: OFM-5 PRIMARY TESTING DERMAL OF NONWOVEN IRRITATION SUBSTRATE POTENTIAL PC PRODUCTS

Plating diluents serve to disperse the sample and dilute it to levels that permit recovery of surviving microorganisms from an inoculated product formulation. The choice of diluent depends on its ability to meet the requirements of preservative neutralization (Under “Preliminary Tests” later in this section). The following are examples of diluents that may be used: •

Buffered Sodium Chloride Peptone Broth

•

Dey/Engley (D-E) Neutralizing Broth

•

Eugon Broth

•

Letheen Broth

•

Modified Letheen Broth

•

Phosphate Buffer, pH 7

•

Soybean Casein Digest Medium (Tryptic Soy Broth)

•

Trypticase Azolectin™ Tween™ (TAT) Broth

•

Saline-Tween-Lecithin Diluent

Other suitable diluents may be used. The addition of neutralizers may be necessary to demonstrate adequate preservative neutralization (See 4.2). 3.3.3

Recovery Agars Many factors affect organism viability. Therefore, it is important for the agar to provide optimum nutritional support for the recovery of the challenge organisms. The following have been found suitable for preservation studies:

208

|

•

Eugon Agar

•

Letheen Agar

•

Microbial Content Agar

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

•

Modified Letheen Agar

•

Plate Count Agar

•

Soybean Casein Digest Agar (Tryptic Soy Agar)

•

Microbial Content Agar with Tween

The following media are specifically recommended for the recovery of yeasts and molds during preservation studies: •

Malt Agar

•

Malt Extract Agar

•

Mycological Agar

•

Potato Dextrose Agar

•

Sabouraud Dextrose Agar

Other suitable agars may be used. The addition of neutralizers may be necessary to demonstrate adequate preservative neutralization.

4.1 Initial Count It is recommended that all formulations be examined for microbial content prior to initiation of preservation studies. 4.2 Preservative Neutralization Carryover of antimicrobial activity from the product formulation into the plate count diluent and recovery growth agar may occur. This may inhibit the growth of surviving challenge test microorganisms resulting in a false negative microbial count. To avoid a false negative result, neutralization of the antimicrobial properties of the formulation must take place in the plate count diluent and/or the recovery growth agar. Antimicrobial neutralization may normally be accomplished by the use of chemical neutralizing agents, physical dilution, or a combination of both. Verification of neutralization is generally performed by inoculating the product dilution with a low level of challenge microorganisms and performing the enumeration method (See Section 6.2). Side-by-side dilutions with and without a product formulation are made. Enumeration of the microorganisms from these dilutions is performed. Neutralization is verified if microbial recoveries are similar. If one or more challenge microorganisms cannot be recovered, the use of a higher dilution and/or the investigation of additional chemical neutralizers may be considered. Refer to ASTM E1054-025 or USP6 for additional detail. In some cases, low recovery of organisms may be due to poor recovery efficacy rather than failure to neutralize the preservative system.

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

209

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

4. Preliminary Tests

4.3

Recovery Efficiency Recovery of microorganisms from the nonwoven substrate is a separate issue from antimicrobial neutralization. The substrate may entrap the microorganisms resulting in incomplete recovery of the microbial population by the use of conventional dilution and plating techniques. Therefore, additional techniques may be used to verify the consistent recovery of microorganisms from the substrate material (Section 6.1.3).

5. Inoculation Procedures 5.1 Preparation of Inocula Freshly prepared cultures should be used for inoculating test samples. In general, culture conditions in Table 22-2 should be considered when preparing the inocula. Refer to the ATCC Web site3 for optimal growth media and conditions for specific microorganisms. Inclusion of cellulose degrading molds may necessitate longer incubation periods and require a paper source for growth. 5.1.1

Preparation of Initial Bacteria and Yeast Suspensions

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

Either broth cultures or cultures grown on solid agar media are acceptable for use. For reference strains such as the ATCC strains, no more than five transfers from the stock culture are recommended.7 Broth cultures should be centrifuged and then re-suspended in the chosen suspending fluid (See 3.3.1). Microbial growth on a solid medium is transferred to the chosen suspending fluid. 5.1.2

Preparation of Initial Mold Suspensions The mold inoculum is prepared by washing the sporulating agar culture with the chosen suspending fluid (See 3.3.1) and filtering the spore suspension through sterile gauze or glass wool. Sterile glass beads can be used as an aid in the dispersion of spores in the suspending fluid.

5.1.3

Preparation of Bacterial Spore Suspensions If spore-forming bacteria are to be included in the test, the inocula may be prepared as indicated in the AOAC Sporicidal Test.8 Some strains are commercially available as prepared spore suspensions.

5.1.4

Preparation of Challenge Inocula 5.1.4.1

Inoculum Levels The recommended inoculation levels for challenge testing are:

210

|

•

1x106 Colony-Forming Units (CFU) of bacteria per sampling unit of product

•

1x105 CFU of yeast per sampling unit of product

•

1x105 CFU of mold spores per sampling unit of product

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

The inoculum level for the challenge microorganisms should be verified by standard microbiological techniques such as pour plate methods. 5.1.4.2

Culture Suspensions Either pure or mixed microbial culture suspensions may be used to challenge test formulations. Inocula consisting of only pure microbial cultures will yield specific data on each test microorganism employed in the challenge study. When conducting mixed culture challenge studies, it is recommended that closely related types of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be pooled separately. These suspensions may be used directly for inoculation or dried onto filter carriers as described below.

5.1.4.3

Dried Inoculum on Filter Carriers

5.2 Sample Preparation In addition to the quantity required for the test, it is recommended that extra sample units be prepared in the event they are needed. An unpreserved control should be included if possible. If product rechallenge is desired, sufficient sample units must be prepared prior to the start of the test. The sample reporting unit used depends on the inoculation and sampling method chosen. If applicable, determine and record the weight and/or the average area of the nonwoven substrate product sample, e.g., 1 g or 1 cm2 (See Section 7). If possible, preservative challenge testing should be conducted on product in the final package to ensure compatibility with the preservative system and to represent the marketed product. Where the product does not lend itself to testing in the container/ package, other approaches may be employed, as detailed below in Section 5.2.4. 5.2.1

Tubs Aseptically open packages and inoculate the product as received according to the inoculation procedure (Section 5.3). Reseal the packages and follow the sampling procedure under Section 6.1.

5.2.2

Soft Packages Aseptically open packages, and inoculate the product as received according to the inoculation procedure (Section 5.3). Some inoculation techniques may

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

211

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

Dried inoculum carriers are prepared by filtering the culture suspensions onto 13 mm 0.45 micron membrane filters (such as cellulose ester membranes) to achieve inoculum levels recommended after drying. The filters are placed in a covered Petri dish and dried at 37°C for 20 to 30 minutes. The number of viable microorganisms on dried carriers must be equivalent to the recommended levels. If necessary, the volume of filtered culture suspension may be increased to take into account mortality due to desiccation.

allow for the aseptic introduction of the inocula directly into the package. Reseal the packages and follow the sampling procedure (Under “Sampling the Challenged Product” in Section 6.1 below). 5.2.3

Canisters Aseptically open canister, remove the roll, and inoculate the top sheets of the product according to an appropriate inoculation procedure (Section 5.3). Reinsert the roll into the canister. Seal the canisters and follow the sampling procedure (Section 6.1).

5.2.4

Transferred Samples Aseptically open packages and transfer an appropriate number of nonwoven substrate units to sterile, resealable containers for inoculation. Follow the inoculation procedure (Section 5.3). Seal the containers and follow the sampling procedure (Section 6.1).

5.3 Methods for Inoculation

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

Inoculation of nonwoven substrates can be accomplished in a variety of ways. Methods for inoculating product are described below. In each case, verification of microorganism recovery (described below) is an important component of the method verification.

212

|

5.3.1

A specific volume of an inoculum suspension is delivered by pipette using a point delivery over the sample unit in a predetermined pattern. (For example, place 0.1 ml in five different areas of the substrate such as the four corners and the center.) After inoculation, the package is sealed. This inoculation method can be used to inoculate one or a series of multiple sample units in one package.

5.3.2

A specific volume of an inoculum suspension is delivered by multi-channel pipette using a point delivery over the sample unit in a predetermined pattern. After inoculation, the package is sealed. This inoculation method can be used to inoculate one or a series of multiple sample units in one package.

5.3.3

A specific volume of an inoculum suspension is aseptically introduced onto the substrate in a straight line down the center of the substrate sample. The inoculum must be applied to the substrate so that uniform cross sections may be cut off of the substrate(s) for sampling. After inoculation, the package is sealed. This inoculation method can be used to inoculate one or a series of multiple sample units in one package.

5.3.4

By means of a syringe, a specific volume of an inoculum suspension is aseptically introduced into the package containing a sample unit. After inoculation, the package is sealed, and the inoculum is well mixed by massaging the package. This inoculation method is quantitative for single unit soft packages and qualitative for multiple unit soft packages. This method is not suitable for tubs or canisters.

5.3.5

In a Class 2 (or greater) biological safety cabinet, the inoculum is sprayed evenly over the entire surface of a pre-determined area of the substrate, (e.g., a 9 cm2 sample in a 10 cm2 Petri dish), by using an airbrush or other suitable

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

spraying device. The substrate sample is sprayed for an appropriate time to deliver the target inoculum. The quantity of inoculum delivered must be calculated for the specific spray device. The inoculated sample should be sealed in a plastic bag or other suitable container to prevent drying. 5.3.6

Dried inocula on membrane filter carriers are placed between two substrate layers in the package. Placement of the filter may be determined by conducting a sedimentation study. After inoculation, the package is resealed.

5.4 Storage of Inoculated Samples Challenged formulations can be stored at controlled or ambient temperature underconditions of humidity considered appropriate for the final product packaging for the duration of the test.

6. Recovery Procedures 6.1 Sampling the Challenged Product 6.1.1

Sampling Intervals

6.1.2

Sampling Sites The method of sampling chosen will depend upon several factors including the method of inoculation. Below are several sampling methods that may be used. 6.1.2.1

For most inoculation methods, the top nonwoven substrate in the stack or the outer most nonwoven substrate in the roll may be sampled from a product package.

6.1.2.2

For a product challenge method where multiple nonwoven substrates in a product package are inoculated, the inoculated substrate units per package should be sampled at the appropriate time.

6.1.2.3

For a product challenge method where an inoculated nonwoven substrate is aseptically transferred to a secondary package (See “Transferred Samples” in Section 5.3), one product package per sampling interval may be sampled.

6.1.2.4

For a product challenge method where the inoculum is evenly distributed across the nonwoven substrate, a uniform cross section (e.g., 1 g) of the substrate may be sampled at each sampling interval.

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

213

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

Challenged formulations should be sampled for viable microorganisms at selected time intervals after inoculations. The frequency of sampling should follow a set pattern to facilitate future comparison of test results between different product formulations or samples, for example, weekly up to 28 days after inoculation.

6.1.2.5

6.1.3

For a product challenge method using dried inocula, a membrane filter carrier is sampled at each interval. Additionally, one or two substrates above and one or two below the filter may be sampled separately at each sampling interval to evaluate migration of organisms through the sample.

Recovery Methods Aseptically remove the inoculated product or membrane filter carrier from the container(s) and thoroughly mix with the preservative neutralizing diluent. Care must be taken to sample the areas of the product that have been inoculated.

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

Organisms may be recovered from the sample using the following processing techniques: •

Mixing with diluent and glass beads by means of a mechanical wrist shaker or reciprocal shaker for a predetermined period.

•

Mixing with diluent in a vortex mixer is recommended when sample sizes are small, e.g., 1 g or less.

•

Mixing in a Stomacher™ with a diluent, e.g., 1 to 2 minutes at medium speed.

Other methods that may be employed include manual shaking or the use of an orbital mixer or a blender. The addition of glass beads may improve recovery of microorganisms, although they are not recommended for use in plastic bags or with a blender. 6.2 Enumeration Methods 6.2.1

Quantitative Pour Plate Method Serial dilutions are prepared from the aliquot recovered from the challenged sample unit. Each serial dilution is thoroughly mixed and an aliquot is transferred to a Petri dish. Melted agar maintained at 44-48°C is added to the Petri dish, and the dish is rotated to uniformly disperse the product dilution. The agar plates are allowed to solidify, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 22-2). After incubation, the number of microbial colonies is counted and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of microorganisms per sample unit.

6.2.2

Quantitative Spread Plate Method The quantitative spread plate method is performed in a manner similar to the pour plate method; however, an aliquot of each dilution is transferred directly onto the surface of solidified microbial growth agar. The sample aliquot is then evenly spread over the agar surface. The agar plates are allowed to dry, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 22-2).

214

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of microorganisms per sample unit.

7. Reporting Calculate and report the percent reduction of inoculum counts per substrate, per gram of product, or per unit area for each organism or organism pool.9

Table 22-1: Suggested Challenge Microorganisms Microorganism (ATCC Numbers)

Recommendation

Gram-Positive Cocci

Staphylococcus aureus (6538)* Staphylococcus epidermidis (12228)

Select at least one

Fermentative GramNegative Bacilli

Klebsiella pneumoniae (10031) Enterobacter cloacae (13047) Escherichia coli (8739)* Enterobacter gergoviae (33028)

Select at least one

Non-Fermentative GramNegative Bacilli

Pseudomonas aeruginosa (9027)* Burkholderia cepacia (25416) Pseudomonas fluorescens (13525) Pseudomonas putida (31483)

Select at least one

Yeasts

Candida albicans (10231)* Candida parapsilosis (22019)

Select at least one

Molds

Aspergillus niger (16404)* Chaetomium globosum (6205)** Trichoderma reesei (13631)** Cladosporium oxysporum (76499)** Penicillium species

Select at least one

Spore-Forming Bacilli

Bacillus subtilis (6051)

Optional

Other

In-house isolates

Optional

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

Type

*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231), and Aspergillus niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing Method.5 10 **Inclusion of cellulose degrading molds may necessitate longer incubation periods and require a paper source for growth.6 Table22-1

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

215

Table 22-2: Culture Conditions for Preparation of Inocula Cultures

Media*

Temperature

Time

Bacteria

Soybean Casein Digest (Tryptic Soy) Broth/Agar Nutrient Broth/Agar Eugon Broth/Agar

30-37°C

18-48 hours

Yeasts

Sabouraud Dextrose Agar Soybean Casein Digest (Tryptic Soy) Broth/Agar Mycophil (Mycological) Broth/Agar

25-35°C

24-48 hours

Molds

Sabouraud Dextrose Agar Potato Dextrose Agar Mycophil (Mycological) Agar Malt Extract Agar

20-30°C

7-28 days

* Available in dehydrated forms from commercial sources.

Table 22-2

SECTION 22: M-5 TESTING OF NONWOVEN SUBSTRATE PC PRODUCTS

REFERENCES 1. The American Type Culture Collection (ATCC; website: www.atcc.org ) recommends appropriate media for the microbial strains it provides and lists formulations for these media on its website (http:// www.atcc.org/common/catalog/media/ mediaIndex.cfm) . The media formulations listed are not ready-to-use products for sale by the ATCC but in some cases other commercial suppliers are listed. 2. Brown, M.R.W. and P. Gilbert, (Ed). 1995. Microbiological Quality Assurance: A Guide Towards Relevance and Reproducibility of Inocula. Boca Raton, FL: CRC Press. 3. Kirsop, B.E. and A. Doyle, (Ed). 1991. Maintenance of Microorganisms and Cultured Cells. Orlando, FL: Academic Press. 4. Simione, F. P. 1998. Cryopreservation Manual. Rochester, NY: Nalgene Nunc International. http://www.nalgenelabware.com/techdata/technical/manual.asp. 5. ASTM E 1054. 2003. “Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” Annual Book of ASTM Standards, 11.05. Washington, DC: ASTM. www.astm.org. 216

|

CTFA MICROBIOLOGY GUIDELINES

|

6. United States Pharmacopeia. 2007. <1227> “Validation of Microbial Recovery from Pharmacopeal Articles.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD. 684-686. 7. Reichgott, M. Winter 2003. “Reference Strains: How Many Passages Are Too Many?” ATCC Connection. Vol 23, No. 2, available at http://www.atcc.org/common/documents/pdf/tb06.pdf. 8. AOAC INTERNATIONAL. 2000. Official Method 966.04, “Sporicidal Activity of Disinfectants.” Official Methods of Analysis of AOAC INTERNATIONAL. Gaithersburg, MD. www.aoac.org . 9. AOAC INTERNATIONAL. 2000. Official Method 998.10, “Efficacy of Preservation of Non-Eye Area Water Miscible Cosmetic and Toiletry Formulations.” Official Methods of Analysis of AOAC INTERNATIONAL. Gaithersburg, MD. www.aoac.org. 10. United States Pharmacopeia. 2007. <51> “Antimicrobial Effectiveness Testing.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD. 79-81.

SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS

SECTION 23

M-6 A Method for Preservation Testing of Atypical Personal Care Products 1. Scope 1.1 This general method reflects a variety of approaches currently used within the cosmetics industry and serves as an acceptable procedure for determining the preservative efficacy of atypical personal care products, such as oils, powders, or other formulations that have low water content and/or are not miscible with water. The recommended preservative challenge test methods used for determining the preservative adequacy of aqueous-based products (See References 1 and 2, and Section 20) may not be suitable for evaluating certain atypical product formulations. When testing and assessing preservative challenge test data for atypical products, the following factors are important points to consider (See “Microbiological Risk Factor Assessment of Atypical Cosmetic Products” in Section 16). A test in which an aqueous-based inoculum is introduced into an hydrous product may change the physical dynamics of the product and, therefore may not predict its microbial stability.

•

Most preservatives are water soluble. In emulsions, preservatives are used in the water phase because contaminating microorganisms require water to proliferate.

•

For an emulsion in which the external phase is water immiscible (emulsions in which water is not the external or continuous phase) and an aqueous challenge inoculum is used, the water-soluble preservatives may not be able to migrate into the aqueous phase. In these cases, the preservatives may not be available to inhibit proliferation or have cidal activity against each of the challenge microorganisms.

1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents 2.1 “Microbiological Risk Factor Assessment of Atypical Cosmetic Products” (Section 16). 2.2 “Determination of Preservative Adequacy in Cosmetic Formulations” (See Section 13).

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

217

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

•

3. Materials 3.1 Selection of Challenge Microorganisms. The microbial strains listed in Table 23-1 may be considered for use in developing preservation data of Personal Care products. Either pure or mixed microbial culture suspensions may be used to challenge test formulations. Inocula consisting of only pure microbial cultures will yield specific data on each test microorganism employed in the challenge study. When conducting mixed culture challenge studies, it is recommended that separate pools of closely related types of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be maintained. 3.2 Maintenance of Challenge Microorganisms Refer to the ATCC culture maintenance recommendations, available on their website, and to other sources.3,5,6,7 Organisms appropriate to the product under test may be best stored in the original product matrix to retain their unique characteristics. Periodic testing may be employed to verify retention of these characteristics. 3.3 Test Media 3.3.1

Inocula Suspending Fluids Suspending fluids are used to prepare the bacterial and fungal suspensions for inoculating the test product. The following may be used: •

Phosphate Buffer (pH 7.0)

•

0.85% Sodium Chloride Solution (Normal Saline)

•

Sodium Chloride Peptone Solution (1% peptone in normal saline)

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate 80 or other surfactant to the suspending fluid is recommended to aid in dispersion of mold spores. 3.3.2

Microbial Plate Count Diluents Plating diluents serve to disperse the sample and dilute it to levels that permit recovery of surviving microorganisms from an inoculated product formulation. The choice of diluent depends on its ability to meet the requirements of preservative neutralization (See Section 4.1 “Preservative Neutralization”). The following are examples of diluents that may be used:

218

|

•

Buffered Sodium Chloride Peptone Solution

•

Dey/Engley (D-E) Neutralizing Broth

•

Eugon Broth

•

Letheen Broth

•

Modified Letheen Broth

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

•

Phosphate Buffer, pH 7

•

Soybean-Casein Digest Medium (Tryptic Soy Broth)

•

Tryptone-Azolectin-Tween® (TAT) Broth

Addition of neutralizers may be necessary to demonstrate adequate preservative neutralization. Other suitable diluents may be used. 3.3.3

Recovery Agars Many factors affect organism viability. Therefore, it is important for the agar to provide optimum nutritional support for the recovery of the challenge organisms. The following have been found suitable for preservation studies: •

Eugon Agar

•

Letheen Agar

•

Microbial Content Agar

•

Modified Letheen Agar

•

Plate Count Agar

•

Soybean-Casein Digest Agar Medium (Tryptic Soy Agar) The addition of neutralizers may be necessary to demonstrate adequate preservative neutralization. Other suitable agars may be used. If the above agars do not support the growth of fungi, one of the following agars may be considered: Malt Agar

•

Malt Extract Agar

•

Mycological Agar

•

Potato Dextrose Agar

•

Sabouraud Dextrose Agar

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

•

Other suitable agars may be used.

4. Preliminary Tests 4.1 Preservative Neutralization Carryover of antimicrobial activity from the product formulation into the plate count diluent and recovery growth agar may occur. This may inhibit the growth of surviving challenge test microorganisms resulting in a false negative microbial count. To avoid a false negative result, neutralization of the antimicrobial properties of the formulation must take place in the plate count diluent and/or the recovery growth agar. Antimicrobial neutralization may normally be accomplished by use of chemical neutralizing agents, physical dilution, or a combination of both. SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

219

Verification of neutralization is generally performed by inoculating the product dilution with a low level of challenge microorganisms and performing the enumeration method7. Side-by-side dilutions with and without a product formulation are made. Enumeration of the microorganisms from these dilutions is performed as described7. Neutralization is verified if microbial recoveries are similar. If one or more challenge microorganisms cannot be recovered, the use of a higher dilution and/or the investigation of additional chemical neutralizers may be considered. 4.2 Microbial Content Test It is recommended that that a microbial content test (Section 18) be performed on the test sample prior to performing the preservative efficacy test. Verification of neutralization of the antimicrobial properties of the test sample should be demonstrated (See Section 4.1 and Reference 7) at the same time as the microbial content test.

5. Inocula Preparation 5.1 Preparation of Inocula Freshly prepared cultures should be used for inoculating test samples. In general, culture conditions in Table 23-2 should be considered when preparing the inocula. 5.1.1

Preparation of Initial Bacteria and Yeast Suspensions Either broth cultures or cultures grown on solid agar media are acceptable for use. For reference strains such as the ATCC3 strains, no more than five transfers from the stock culture are recommended8. 5.1.1.1

Aqueous Inoculum Broth cultures should be centrifuged and then re-suspended in the chosen suspending fluid. (See Section 20) Microbial growth on a solid medium is transferred to the chosen suspending fluid.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

5.1.1.2

Emulsified Aqueous Inoculum An aqueous emulsified inoculum may be prepared by adding not more than 1% of dispersing agent such as polysorbate, sorbitan oleate or glycerol to the aqueous inoculum.

5.1.1.3

Oil Inoculum Challenge cultures may be resuspended in light mineral oil. Note: If using this technique, the absence of inhibitory or toxic properties of the dispersing agent or oil soluble carrier system should be verified for each of the challenge organisms.

220

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

5.1.2

Preparation of Initial Mold Suspensions 5.1.2.1

Aqueous Mold Inoculum The mold inoculum is prepared by washing the sporulating agar culture with the chosen suspending fluid and filtering the spore suspension through sterile gauze or glass wool. Sterile glass beads can be used as an aid in the dispersion of spores in the suspending fluid.

5.1.2.2

Emulsified Aqueous Mold Inoculum An aqueous emulsified inoculum may be prepared by adding not more than 1% of dispersing agent such as polysorbate, sorbitan oleate or glycerol to the aqueous inoculum.

5.1.2.3

Oil Inoculum Challenge cultures may be resuspended in light mineral oil.

5.1.3

Preparation of Bacterial Spore Suspensions If spore-forming bacteria are to be included in the test, the inocula may be prepared as indicated in the AOAC Sporicidal Test9. Some strains are commercially available as prepared spore suspensions.

5.1.4

Inoculum Levels Inoculum challenge levels ranging from 1 × 104 to 1 × 108 CFU per gram or ml of product have been reported in the literature for preservative system evaluation.10-12 For some atypical products, (e.g., anhydrous products), a reduction in the challenge inoculum size to 103 to 104 Colony-Forming Units (CFU) per gram or milliliter may be used instead of the inoculum concentration of 105 to 106 CFU per gram or milliliter that is recommended in the aqueous based challenge test methods.

6. Inoculation Procedures for Test Samples Depending on the product form, the following carriers for inocula may be considered. The volume of the inoculum should not alter the character of the product being tested. •

Aqueous inoculum (See Section 20)

•

Emulsified aqueous inoculum

•

Oil inoculum

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

221

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

Note: By reducing the inoculum size, it is easier to measure stasis or quantify an increase in the microbial count.

6.1 Inoculum Dispersed into Product 6.1.1

Oils, water-in-oil emulsions, water in silicone and semisolid products (<20% water) Procedure Prepare enough of the formulation to permit adequate sampling at each test interval. At least 20 mL or 20 grams of the product should be challenged with each test microorganism or mixture of test microorganisms. Inoculum The inoculum volume should be 0.1% to 1.0% of the sample volume in order to keep the sample as water-free as possible. The inoculum may be an aqueous or oil suspension.

6.1.2

Loose Powders Procedure At least 20 grams of product should be challenged with each test organism or mixture of test microorganisms. This sample size is usually large enough to permit numerous samplings. Inoculum The inoculum (volume 0.1% to 1% of the test sample) should be added to the product and thoroughly mixed.

6.1.3

Pressed Powders Procedure Pressed powders can be inoculated on the surface or removed from containers, ground (e.g., mortar and pestle) into fine particles, and processed.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

A minimum sample size of 20 grams should be prepared for each challenge microorganism or pool of microorganisms. For wet dry pressed powders, up to 5% water may first be added to the product, prior to inoculation. 6.1.4

Mixing A glass rod, tongue depressor, or mechanical mixer may be necessary to uniformly disperse test microorganisms. Inoculated product that has collected on the mixing device or on the container’s inner surfaces or edges must be worked back into the sample to prevent excessive loss of product (See Section 20).

6.2 Surface inoculation Swabbing A swab is dipped into an inoculum of known concentration and swabbed across the entire product surface. 222

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

Spreading A known volume of inoculum is pipetted onto the surface of the product and uniformly spread using a glass rod or other instrument. Dipping The product in its container is dipped into an inoculum of known concentration for a predetermined length of time. Spraying The product is sprayed with a suspension of inoculum using an atomizer. Appropriate safety precautions should be taken. 6.3 Wax based solid products For solid atypical products, such as anhydrous sticks or pans, inoculation and sampling of the product surface instead of the whole product more closely simulates potential consumer contamination. This modification also maintains the physical product integrity. In these types of products, the microorganisms are not able to penetrate into the interior and will always be found on the outer-most layer of the product after consumer usage. Although this type of product is not usually susceptible to microbial contamination, surface inoculation may be used. Note: If performing challenge testing on a solid anhydrous stick or powder product, inoculate a sufficient number of samples to obtain a unique sample for each sampling time-point. 6.4 Storage of Inoculated Samples Inoculated samples should be stored under ambient conditions 6.5 Sampling the Challenged Product 6.5.1

Sampling interval

•

Water in oil and/or silicone emulsions Three sampling points, such as 7, 14, 28 day

•

Semisolid products (<20% water) Three sampling points, such as 7, 14, 28 day

•

Oil or silicone based products (anhydrous) Three sampling points, such as 2,7,14 days.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

223

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

Challenged formulations should be sampled for viable microorganisms at selected time intervals after inoculations. The frequency of sampling should follow a set pattern to facilitate future comparison of test results between different product formulations. Sampling intervals should be based on microbial contamination risk as demonstrated by product water activities and other microbiological related attributes.

6.5.2

•

Loose, wet dry and pressed powders Three sampling points, such as 2,7,14 days

•

Wax based and other solid products Three sampling points, such as 2,7,14 days.

Sampling and plating methods The inoculated product should be thoroughly mixed just prior to sampling to ensure that the sample is representative. For water-immiscible products (e.g. oils and emulsions), a suitable solubilizing agent may be incorporated into the test diluent or broth to make the sample aliquot miscible with water in order to recover microorganisms present in the test sample. For solid products, the surface may be sampled by removing the top layer. For products where microorganisms would only be recovered from the product surface (e.g., sticks, pressed powders, hot pour products in compacts), only the surface of the product sample should be tested. For these “atypical products”, the following methods of recovery may be considered. •

A sterile moistened applicator may be used to sample the product surface, and then streaked onto a Petri dish containing solid culture medium.

•

The product may be sampled by a direct contact method using a contact plate (a modified Petri dish containing a solid culture medium whose convex surface extends above the carrier), paddles, or flexible film containing solid culture media.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

In some cases, the inoculum can thrive in “pockets” of growth in the formulation while other areas are relatively free of microorganisms. Many aerobic microorganisms grow especially well at the formulation-air interface. Often it is very difficult to break up the “pockets” of growth, and special procedures are needed. The following mixing methods have been used to overcome this problem: •

Hand mixing with a stirring rod

•

Capping and shaking vigorously by hand

•

Mixing in a vortex mixer

•

Mixing with a magnetic stirrer

•

Mixing with a propeller stirrer

•

Mixing with a non-aerating stirrer

•

Mixing in a micro blender

•

Mixing in a stomacher

Sample size will in part determine the minimum detectable level. A sample size of at least one gram or one milliliter of product for the quantitative pour plate method is recommended. Aseptic techniques must be employed.

224

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

6.5.2.1

Quantitative pour plate method Serial dilutions are prepared from the aliquot recovered from the challenged sample unit. Each serial dilution is thoroughly mixed, and an aliquot is transferred to a Petri dish. Melted agar maintained at 45-48°C is added to the Petri dish, and the dish is rotated to uniformly disperse the product dilution. The agar plates are allowed to solidify, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 23-3). After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of CFU per gram or milliliter of sample.

6.5.2.2

Quantitative spread plate method The quantitative spread plate method is performed in a manner similar to the pour plate method; however, an aliquot of each dilution is transferred directly onto the surface of solidified microbial growth agar. The sample aliquot is then evenly spread over the agar surface. The agar plates are allowed to dry, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 23-2). After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of CFU per gram or milliliter of sample.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

225

Table 23-1: Suggested Challenge Microorganisms Type

Microorganism (ATCC Number3)

Recommendation

Gram-Positive Cocci

Staphylococcus aureus (6538)* Staphylococcus epidermidis (12228)

Select at least one

Fermentative Gram-Negative Bacilli

Klebsiella pneumoniae (10031) Enterobacter cloacae (13047)) Escherichia coli (8739)* Enterobacter gergoviae (33028)

Select at least one

Non-Fermentative GramNegative Bacilli

Pseudomonas aeruginosa (9027)* Burkholderia cepacia (25416) Pseudomonas fluorescens (13525) Pseudomonas putida (31483)

Select at least one

Yeasts

Candida albicans (10231)*

Recommended

Molds

Aspergillus niger (16404)* Penicillium species

Select at least one

Spore-Forming Bacilli

Bacillus subtilis (6051)

Optional

Other organisms relevant to the product

Optional

*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231) and Aspergillus niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing method4.

Table23-1

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

Table 23-2: Culture Conditions for Preparation of Inocula Cultures

Media*

Temperature

Bacteria

Soybean-Casein Digest Medium/Soybean30-37°C Casein Digest Agar Medium (Tryptic Soy Broth/Agar ) Nutrient Broth/Agar Eugon Broth/Agar

18-48 hours

Yeasts

Sabouraud Dextrose Agar 25-35°C Soybean-Casein Digest Medium/SoybeanCasein Digest Agar Medium (Tryptic Soy Broth/Agar ) Mycophil (Mycological) Broth/Agar

24-48 hours

Molds

Sabouraud Dextrose Agar Potato Dextrose Agar Mycophil (Mycological) Agar Malt Extract Agar

7-28 days

20-30°C

Time

* Available in dehydrated forms from commercial sources.

Table 22-2

226

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

Table 23-3: Incubation Conditions for Recovery of Microorganisms Cultures

Media

Bacteria Yeasts

For recovery agars, see Section 3.3.3

Molds

Temperature

Time

30-37°C

24-72 hours

25-35°C

48-72 hours

20-30°C

3-7 days Table 23-3

ADDITIONAL INFORMATION Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” J. of Soc. Cosmet. Chem. 23:703-720. Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” J. of Soc. Cosmet. Chem. 18:797-807. Wilson, L.A., J.W. Kuehne, S.W. Hall, and D.G. Ahearn. 1971. “Microbial Contamination in Ocular Cosmetics,” American Journal of Ophthalmology. 71(6):1298-1302. Yablonski, J.I. and S.E. Mancuso. 2002. “Preservation of Atypical Cosmetic Systems.” Cosmetics & Toiletries 2: 41.

REFERENCES 4. United States Pharmacopeia. 2007. <51> “Antimicrobial Effectiveness Testing.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD. 79-81.

2. AOAC International. 2000. “Official Method 998.10 - Efficacy of Preservation of Non-Eye Area Water-Miscible Cosmetic and Toiletry Formulations.” In: Official Methods of Analysis of AOAC International. Gaithersburg, MD.

5. Brown, M.R.W. and P. Gilbert (Ed). 1995. Microbiological Quality Assurance: A Guide Towards Relevance and Reproducibility of Inocula. Boca Raton, FL: CRC Press.

3. The American Type Culture Collection (ATCC) website: http://www.atcc. org recommends appropriate media for the microbial strains it provides and lists formulations for these media on its website: http://www.atcc.org/common/catalog/media/mediaIndex.cfm. The media formulations listed are not ready-to-use products for sale by the ATCC but in some cases other commercial suppliers are listed.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

6.

Kirsop, B.E. and A. Doyle (Ed). 1991. Maintenance of Microorganisms and Cultured Cells. Second edition. New York, NY: Academic Press.

7. ASTM International. 1999. ASTM E 1054-91, “Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” In: Annual Book of ASTM Standards, 11.05. West Conshohocken, PA.

|

CTFA MICROBIOLOGY GUIDELINES

|

227

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

1. ASTM International. 2007. ASTM E 640-78,” Standard Test Method for Preservatives in Water-Containing Cosmetics.” In: Annual Book of ASTM Standards. West Conshohocken, PA.

8. Reichgott, M. 2003. “Reference Strains: How Many Passages Are Too Many?” In: ATCC Connection, 23, No. 2, http://www. atcc.org/common/do cuments/pdf/tb06. pdf

11. Madden, J. M. and G.J. Jackson. 1981. “Cosmetic Preservation and Microbes: Viewpoint of the Food and Drug Administration.” Cosmetics & Toiletries 96:7577.

9. AOAC International. 2000. Official Method 966.04 “Sporicidal Activity of Disinfectants.” In: Official Methods of Analysis of AOAC International. Gaithersburg, MD.

12. Wilson, L.A., A.J. Julian and D.G. Ahearn. 1975. “The Survival and Growth of Microorganisms in Mascara During Use.” Am J. Ophthal 79(4): 596-601.

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

10. Wilson, L.A. and D.G. Ahearn. 1977. “Pseudomonas-Induced Corneal Ulcers Associated with Contaminated Eye Mascaras.” Am J. Ophthal. 84:112-119.

228

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS

SECTION 24

M-7 A Rapid Method for Preservation Testing of Water-Miscible Personal Care Products Scope 1. Introduction 1.1 This procedure allows the rapid determination of preservative performance in watermiscible personal care products. This procedure is intended to be used as a screening test during product development to quickly differentiate between preservative systems that may be capable of providing adequate preservation and those which have insufficient anti-microbial activity to protect the product. It is not intended to provide the definitive information on the adequacy of preservation of the final formulation. This information is obtained through standard tests, such as those described in Methods M-3 (Section 20), M-5 (Section 22), and M-6 (Section 23). This procedure may also be used to rapidly qualify products to which minor formulation changes have been made. However, to assure that the product is adequately preserved, more stringent criteria for the elimination of microorganisms over the course of the test than those used in conventional tests should be adopted. 1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents 2.1 “Determination of Preservative Adequacy in Cosmetic Formulations” (Section 13).

3. Materials 3.1 Selection of Challenge Microorganisms

3.2 Maintenance of Challenge Microorganisms Refer to the ATCC culture maintenance recommendations, available on their website1, and to other sources3-5. SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

229

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

The microbial strains listed in Table 24-1 may be considered for use in developing preservation data on Personal Care products.

Storage of other organisms appropriate to the product under test in the original product or incorporation of product into maintenance medium is often the only way to retain its unique characteristics. This method is especially appropriate where the isolate is subsequently inoculated into a similar material. 3.3 Test Media 3.3.1

Inocula Suspending Fluids Suspending fluids are used to prepare the bacterial and fungal suspensions for inoculating the test product. The following may be used: •

Phosphate Buffer (pH 7.0)

•

0.85% Sodium Chloride Solution

•

Sodium Chloride Peptone Solution (1% peptone in 0.85% saline)

Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate 80 or other surfactant to the suspending fluid is recommended to aid in dispersion of mold spores. 3.3.2

Microbial Plate Count Diluents Plating diluents serve to disperse the sample and dilute it to levels that permit recovery of surviving microorganisms from an inoculated product formulation. The choice of diluent depends on its ability to meet the requirements of preservative neutralization (Section 4.1). The following are examples of diluents that may be used: •

Buffered Sodium Chloride Peptone Solution

•

Dey/Engley (D-E) Neutralizing Broth

•

Eugon Broth

•

Letheen Broth

•

Modified Letheen Broth

•

Phosphate Buffer, pH 7

•

Soybean-Casein Digest Medium (Tryptic Soy Broth)

•

Tryptone-Azolectin-Tween® (TAT) Broth

Addition of neutralizers may be necessary to demonstrate adequate preservative neutralization6. Other suitable diluents may be used.

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

3.3.3

Recovery Agars Many factors affect organism viability. Therefore, it is important for the agar to provide optimum nutritional support for the recovery of the challenge organisms. The following have been found suitable for preservation studies:

230

|

•

Eugon Agar

•

Letheen Agar

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

•

Microbial Content Test Agar

•

Modified Letheen Agar

•

Plate Count Agar

•

Soybean-Casein Digest Agar Medium (Tryptic Soy Agar)

Addition of neutralizers may be necessary to demonstrate adequate preservative neutralization6. Other suitable agars may be used. If the above agars do not support the growth of fungi, one of the following agars may be considered: •

Malt Agar

•

Malt Extract Agar

•

Mycological Agar

•

Potato Dextrose Agar

•

Sabouraud Dextrose Agar

Other suitable agars may be used.

4. Preliminary Tests 4.1 Preservative Neutralization Carryover of antimicrobial activity from the product formulation into the plate count diluent and recovery growth agar may occur. This may inhibit the growth of surviving challenge test microorganisms resulting in a false negative microbial count. To avoid a false negative result, neutralization of the antimicrobial properties of the formulation must take place in the plate count diluent and/or the recovery growth agar6. Antimicrobial neutralization may normally be accomplished by use of chemical neutralizing agents, physical dilution, or a combination of both. Verification of neutralization is generally performed by inoculating the product dilution with a low level of challenge microorganisms and performing the enumeration method Side-by-side dilutions with and without a product formulation are made. Enumeration of the microorganisms from these dilutions is performed. Neutralization is verified if microbial recoveries are similar. If one or more challenge microorganisms cannot be recovered, the use of a higher dilution and/or the investigation of additional chemical neutralizers may be considered6. 4.2 Microbial Content Test

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

231

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

It is recommended that that a microbial content test (See Section 18) be performed on the test sample prior to performing the preservative efficacy test. Verification of neutralization of the antimicrobial properties of the test sample should be demonstrated (See Section 4.1 and Reference 6) at the same time as the microbial content test.

5. Inoculation Procedures 5.1 Preparation of Inocula Freshly prepared cultures should be used for inoculating test samples. In general, culture conditions in Table 24-2 should be considered when preparing the inocula. Refer to the ATCC website1 for optimal growth media and conditions for specific microorganisms. 5.1.1

Preparation of Initial Bacteria and Yeast Suspensions Either broth cultures or cultures grown on solid agar media are acceptable for use. For reference strains such as the ATCC strains, no more than five transfers from the stock culture are recommended7. Broth cultures should be centrifuged and then re-suspended in the chosen suspending fluid. Microbial growth on a solid medium is transferred to the chosen suspending fluid.

5.1.2

Preparation of Initial Mold Suspensions The mold inoculum is prepared by washing the sporulating agar culture with the chosen suspending fluid (See Section 3.3.1 above) and filtering the spore suspension through sterile gauze or glass wool. Sterile glass beads can be used as an aid in the dispersion of spores in the suspending fluid.

5.1.3

Preparation of Bacterial Spore Suspensions If spore-forming bacteria are to be included in the test, the inocula may be prepared as indicated in the AOAC Sporicidal Test8. Some strains are commercially available as prepared spore suspensions.

5.1.4

Preparation of Challenge Inocula 5.1.4.1

Inoculum levels The recommended inoculation levels for challenge testing are: •

1x105 to 1×106 Colony-Forming Units (CFU) of bacteria per gram of product

•

1-5×105 CFU of yeast per gram of product

•

1-5×105 CFU of mold spores per gram unit of product

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

The inoculum level for the challenge microorganisms should be verified by standard microbiological techniques such as pour plate methods. It is recommended that the volume of the inoculum be < 1% of the sample weight/volume and should not alter the character of the product being challenged. 5.2 Product Challenge Either pure or mixed microbial culture suspensions may be used to challenge test formulations. Inocula consisting of only pure microbial cultures will yield specific data on each test microorganism employed in the challenge study. When conducting mixed

232

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

culture challenge studies, it is recommended that closely related types of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be pooled separately. All products should be thoroughly mixed manually or mechanically after inoculation to distribute the challenge microorganisms uniformly. The volume of the inoculum should not alter the character of the product being challenged. Challenged formulations should then be stored at ambient temperature for the duration of the test. 5.3 Sampling the Challenged Product 5.3.1

Sampling interval To obtain rapid results, it is suggested that challenged formulations be sampled for viable microorganisms at 1, 2 or 3 and 7 days following inoculation.

5.3.2

Sampling and plating methods The inoculated product should be thoroughly mixed just prior to sampling to ensure that the sample is representative. In some cases, the inoculum can thrive in “pockets” of growth in the formulation while other areas are relatively free of microorganisms. Many aerobic microorganisms grow especially well at the formulation-air interface. Often it is very difficult to break up the “pockets” of growth, and special procedures are needed. The following mixing methods have been used to overcome this problem: •

Vigorous mixing with a stirring rod

•

Capping and shaking vigorously by hand

•

Mixing in a vortex mixer

•

Mixing with a magnetic stirrer

•

Mixing with a propeller stirrer

•

Mixing with a non-aerating stirrer

•

Mixing in a micro blender

•

Mixing in a stomacher

•

Gentle mixing in a tissue grinder

Sample size will in part determine the minimum detectable level. A sample size of at least one gram or one milliliter of product for the quantitative pour plate method is recommended. 5.3.2.1

Quantitative pour plate method

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

233

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

Serial dilutions are prepared from the aliquot recovered from the challenged sample unit. Each serial dilution is thoroughly mixed, and an aliquot is transferred to a Petri dish. Melted agar maintained at 45-48°C is added to the Petri dish, and the dish is rotated to uniformly disperse the product dilution. The agar plates are allowed to solidify, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 24-2).

After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of microorganisms per gram. 5.3.2.2

Quantitative spread plate method The quantitative spread plate method is performed in a manner similar to the pour plate method. However, an aliquot of each dilution is transferred directly onto the surface of solidified microbial growth agar. The sample aliquot is then evenly spread over the agar surface. The agar plates are allowed to dry, then inverted and incubated under conditions appropriate for the test microorganisms (see Table 24-2).

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

After incubation, the number of microbial colonies is counted, and the resulting figure is multiplied by the appropriate dilution factor to obtain the number of microorganisms per gram.

234

|

CTFA MICROBIOLOGY GUIDELINES

|

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

Table 24-1: Suggested Challenge Microorganisms Type

Microorganism* (ATCC1 Number)

Recommendation

Gram-Positive Cocci

Staphylococcus aureus (6538)* Staphylococcus epidermidis (12228)

Select at least one

Fermentative Gram-Negative Bacilli

Klebsiella pneumoniae (10031) Enterobacter cloacae (13047)) Escherichia coli (8739)* Enterobacter gergoviae (33028)

Select at least one

Non-Fermentative GramNegative Bacilli

Pseudomonas aeruginosa (9027)* Burkholderia cepacia (25416) Pseudomonas fluorescens (13525) Pseudomonas putida (31483)

Select at least one

Yeasts

Candida albicans (10231)*

Recommended

Molds

Aspergillus niger (16404)* Penicillium species

Select at least one

Spore-Forming Bacilli

Bacillus subtilis (6051)

Optional

Other organisms relevant to the product

Optional

*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231) and Aspergillus niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing method2.

Table 24-1

Table 24-2: Culture Conditions for Preparation of Inocula Cultures

Media**

Temperature

Bacteria

Soybean-Casein Digest Medium/Soybean30-37°C Casein Digest Agar Medium (Tryptic Soy Broth/Agar ) Nutrient Broth/Agar Eugon Broth/Agar

18-48 hours

Yeasts

Sabouraud Dextrose Agar 25-35°C Soybean-Casein Digest Medium/SoybeanCasein Digest Agar Medium (Tryptic Soy Broth/Agar ) Mycophil (Mycological) Broth/Agar

24-48 hours

Molds

Sabouraud Dextrose Agar Potato Dextrose Agar Mycophil (Mycological) Agar Malt Extract Agar

7-28 days

20-30°C

Time

** Available in dehydrated forms from commercial sources.

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

|

CTFA MICROBIOLOGY GUIDELINES

|

235

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

Table 24-2

Table 24-3: Incubation Conditions for Recovery of Microorganisms Cultures

Media

Bacteria Yeasts

For recovery agars, see Section 3.3.3

Molds

Temperature

Time

30-37°C

24-72 hours

25-35°C

48-72 hours

20-30°C

3-7 days Table 24-3

REFERENCES 1. ATCC recommends many different media in order to provide optimal conditions for growing its microbial cultures. The formulations for these media are part of their catalog database, which can be searched for any medium recommended in an ATCC strain description. Formulations for recommended cell culture media are not included in the database, but the on-line catalog description for each cell line has details about the appropriate medium. Media formulations found via the internet search are not ready-to-use products for sale by the ATCC. The catalog is no longer published in hard copy. Visit http://www.atcc.org; the search page for media formulations in the on-line catalog is http://www.atcc.org/common/catalog/ media/mediaIndex.cfm .

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

2. United States Pharmacopeia. 2007. <51> “Antimicrobial Effectiveness Testing.” United States Pharmacopeia and the National Formulary. USP30 – NF25. Rockville, MD. 79-81. 3. Brown, M.R.W., and P. Gilbert, (Ed). 1995. Microbiological Quality Assurance: A Guide Towards Relevance and Reproducibility of Inocula. Boca Raton, FL: CRC Press.

236

|

CTFA MICROBIOLOGY GUIDELINES

|

4. Kirsop, B.E., and A. Doyle, (Ed). 1991. Maintenance of Microorganisms and Cultured Cells, 2nd edition. New York, NY: Academic Press. 5. Simione, F. P. 1998. “Cryopreservation Manual.” Nalgene Nunc International, http://www.nalgenelabware.com/techdata/technical/manual.asp 6. ASTM International. 2007. ASTM E 1054-91, “Standard Practices for Evaluating Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” In: Annual Book of ASTM Standards. West Conshohocken, PA. 7. Reichgott, Michael. 2003. “Reference Strains: How Many Passages Are Too Many?” ATTCC Connection 23, No. 2, http://www.atcc.org/common/documents/pdf/tb06.pdf 8. AOAC International. 2000. Official Method 966.04, “Sporicidal Activity of Disinfectants.” In: Official Methods of Analysis of AOAC International. Gaithersburg, MD.

SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS

This glossary provides definitions for microbiological and associated terms used throughout the cosmetic industry. Its purpose is to assist individuals in understanding the terms used in the CTFA Microbiology Guidelines.

A Action Level level or range that, when exceeded, indicates that a process has deviated from its normal operating condition, and that requires corrective action to be taken to bring the process back into its normal operating condition. Adaptation a change or changes in an organism or population of organisms, through which the organism(s) become more suited to the prevailing environmental conditions. Add-on in a non-woven substrate based product, the formulation added to the substrate, e.g., a lotion, solution, emulsion, oil, or other material. Aerobic requiring oxygen for growth.

GLOSSARY

Glossary of Microbiological Terms

Agar a gelatinous colloidal extract from algae consisting of agarose and agaropectin that is used in microbial growth media to make it a semi-solid at room temperature. Air Sampling a technique by which the quantity of viable microorganisms or particulate matter present in a volume of air is determined or isolated.

Alert Level a level or range that, when exceeded, warns that a process may have deviated from its normal operating condition. Ambient Air environmental or room air. Anaerobic able to grow in the absence of oxygen. Anhydrous without water. Antimicrobial a chemical agent, either produced by a microorganism or by synthetic means, that is capable of killing or suppressing the growth of microorganisms. Antimicrobial Preservative Efficacy Test See Preservation Test.

GLOSSARY

|

CTFA MICROBIOLOGY GUIDELINES

|

237

GLOSSARY

Antiseptic a substance for use on living tissue that either destroys or inhibits the growth of microorganisms. Aqueous containing water. Aseptic free of microorganisms that are capable of causing infection or contamination. Aseptic Technique precautionary measures taken in microbiological work to prevent the contamination by extraneous microorganisms. Atypical Product a product in which water is not readily available to provide an environment that supports growth of microorganisms, a product in which the water activity is too low to support growth, or a product having other physico-chemical characteristics that do not allow growth of microorganisms.

Biofilm a complex structure consisting of diverse microcolonies of various microorganisms embedded in a matrix of extracellular organic polymers adhering to moist surfaces. Biological Indicator a characterized preparation of a specific microorganism resistant to a particular sterilization process. Biostatic Activity the ability of a chemical agent or a physical condition that inhibits the growth of microorganisms. Bulk in Process a product in a partial state of completion or finished goods before filling.

Bulk Product product that has completed the processing steps up to final packaging but has not been placed in the final package.

B

C

Bactericide a chemical or physical agent that destroys viable bacteria.

CFU See Colony-forming Unit

Bacteriostat an agent that inhibits the growth of bacteria.

Bacterium (pl. bacteria) a single-celled, prokaryotic microorganism that multiplies by cell division. Biochemical Characteristics biochemical reactions that are indicative for a particular microbial species. Biocide a chemical or physical agent that destroys all viable microorganisms.

238

|

CTFA MICROBIOLOGY GUIDELINES

|

Calibration the set of operations and conditions that establishes the relationship between values produced by a measuring instrument or system, or values obtained from a material measure, and the corresponding values from a known reference standard. Challenge Organisms microorganisms used in preservative challenge tests. Challenge Test See Preservation Test.

GLOSSARY

Contact Time the time during which a microorganism or microbial growth medium is in the presence of a test surface or chemical agent.

Cleaner a chemical or blend of chemicals formulated to remove undesirable soils from a surface; may be a solvent, acid, base, detergent, and/or water-based mixture.

Contamination the presence of undesirable organisms.

Cleaning the process of separating and eliminating generally visible dirt from a surface, accomplished using, in variable proportions, chemical action, mechanical action, temperature and duration of application. Cleaning Agent an agent designed to remove visible and non-visible foreign matter from surfaces. Coliform Organisms gram-negative, nonspore-forming bacteria of intestinal origin that ferment lactose with gas formation. Colony a macroscopically visible growth of microorganisms on a solid culture medium. Colony-forming Unit (CFU) an organism or cluster of organisms that causes a visible colony. Compressed Air air under pressure greater than that of the atmosphere. Concurrent Validation the generation of current test data that will be used to document that a process or procedure does what it is intended to do. Contact Plate See RODAC Plate.

GLOSSARY

Culture, Fresh a population of a single species of a microorganism that has been recently cultivated either in a liquid medium or on an agar medium. Culture Maintenance a process that keeps a microorganism alive, uncontaminated, and without variation or mutation, so that it is as close as possible to the original isolate. Culture Medium a nutrient-containing liquid (broth) or solid (agar) that supports the growth of microorganisms. Culture, Mixed the presence of more than one species of microorganism in a culture medium. Culture Slant an inclined agar medium in a test tube used for growth of microorganisms. Culture Stability maintenance of biochemical or other desirable characteristics of bacteria or fungi over time and through repeated subcultures.

D Dead End See Dead Leg. Dead Leg any area within a piping system that allows material to accumulate and then stagnate.

|

CTFA MICROBIOLOGY GUIDELINES

|

239

GLOSSARY

Chlorination the use of chlorine or chlorine-donating compounds to effect sanitization or to control microbial levels.

GLOSSARY

Deionization a water-treatment process that removes ions from water by passing it through cationic and anionic resin beds.

Filtration the removal of particulates from a fluid by an appropriately sized filter.

Diluent a medium or vehicle used to reduce a material to a less concentrated form.

Finished Goods any manufactured cosmetic product or component that is suitable for use, whether or not it is packaged or labeled.

Disinfection the destruction of disease-causing or objectionable microorganisms, with the exception of spores, on inanimate surfaces by chemical or physical means. Distillation a process that consists of driving gas or vapor from a liquid or solid by heating and then condensing to liquid. Documentation records containing all relevant information organized in an orderly and easily understood format, usually applied to a process, method, or equipment usage.

E Enteric Organisms microorganisms associated with the intestinal tract. Eucaryotic a type of cell that has a well-defined nuclear membrane.

F Fecal Contamination adulterated by feces, often inferred from the presence of coliform bacteria, but may be present whether or not coliforms are present. Filamentous Fungus an organism that exhibits mycelial or threadlike morphology.

240

|

CTFA MICROBIOLOGY GUIDELINES

|

Finished Product a manufactured cosmetic product that has undergone all stages of production, including packaging in its final container as placed on the market and made available to the consumer. Formalin a clear 37% aqueous solution of formaldehyde. Fungicide a chemical or physical agent that kills fungi. Fungistat an agent that inhibits the growth of fungi. Fungus a saprophytic, symbiotic, or parasitic, heterotrophic, eukaryotic microorganism, i.e., a yeast or mold

G Genotype genetic material contained in the entire complement of alleles (chromosomes). Genus (pl. genera) a specific biological classification of very closely related species of organisms, ranking between a family and a species. Glutaraldehyde a saturated dialdehyde that is chemically related to formaldehyde.

GLOSSARY

Gram-positive bacteria that retain a violet color after the Gram stain procedure. Gram Stain differential staining procedure used for bacterial classification. Growth an increase in the number of microorganisms. Growth Phase See Log Phase.

H Halogenated Compounds chemical compounds containing either chlorine, bromine, iodine, or fluorine. Heat Distribution measurement of temperature uniformity within an autoclave chamber with empty and loaded chamber configurations. Heat Penetration measurement of temperature uniformity within items for sterilization of a loaded autoclave chamber configuration.

I Indigenous occurring naturally within a particular environment. Innocuous microorganisms commonly considered harmless. Inoculation the introduction of microorganisms into a product formulation or onto a substrate. Inoculum material containing living microorganisms used for inoculation. Installation Qualification (IQ) a description of the physical characteristics, drawings, specifications, operating manuals, calibration of instrumentation, and verification of the proper installation of utilities for a piece of equipment. Iodophore a chemical complex of iodine and a surface active agent that functions as a disinfectant through slow release of iodine. Isolate (verb) to separate a mixed population of microorganisms to obtain pure cultures.

Homogeneous uniform throughout, in structure or composition.

Isolate (Noun) a microorganism cultured from an ingredient, finished product, or the environment

Hostile Environment any material or condition unfavorable for survival or growth of microorganisms.

L Log Phase a pattern of microbial growth in which there is an exponential increase in the number of viable cells.

GLOSSARY

|

CTFA MICROBIOLOGY GUIDELINES

|

241

GLOSSARY

Gram-negative bacteria that retain a red color after the Gram stain procedure.

GLOSSARY

Lyophilized freeze-dried.

Motility the movement of a bacterial cell in any medium.

M

N

Maintenance support and verification operations, either periodic or unplanned, intended to keep facility and equipment in proper working condition.

Natural Raw Materials substances of plant, animal, or mineral origin that may be minimally processed before use.

Membrane Filter a pliable filter or filter unit containing pores of a known size used to separate out microorganisms from a fluid.

Negative Control a sample in a test series without the test agent used as a standard of comparison in judging experimental effects.

Membrane Filtration removal of particulates (including microorganisms, depending on the filter=s rating) using a membrane.

Neutralizing Agent a chemical substance added to a medium to inactivate an antimicrobial agent.

Microbes microorganisms, including bacteria, yeasts and molds.

O

Microbial Content the number of viable microorganisms present in a specific volume or quantity of material. Microbial Proliferation Rate reproduction rate of microorganisms.

Operational Qualification (OQ)

Microbiological Limits maximum microbial content and specific microbial type restrictions established for raw materials or finished products. Microbiological Specification a statement of microbial content limits. Mold filamentous fungus. Most Probable Number a counting technique that utilizes a multiple Adilution@ to extinction approach in microbial growth medium and a mathematical formula to estimate the number of microorganisms present in a sample. 242

|

CTFA MICROBIOLOGY GUIDELINES

Objectionable Organism an organism that can be harmful to the user based upon the nature of the product, its intended use and its potential hazard, or is able to compromise the physical integrity or appearance of the product.

|

a list of critical components, operating ranges, as defined by the specification, and actual performance for a piece of equipment. Ozone a highly reactive allotropic triatomic form of oxygen used in disinfection and deodorization. Ozonation the addition of ozone to a water system to reduce microbial levels.

GLOSSARY

P

Pathogen a disease-causing organism. Peroxygen Compounds chemical compounds that contain the bivalent group O-O and are used as sanitizing agents. Pipeline Pig a device made of non-porous materials that is used to remove and clean product from manufacturing pipelines; it fits the internal radius of a pipe, is inserted, launched and moved through the pipe length pushed by air, product or water. Plate Count the number of viable colony-forming units (CFU) per plate; a method of determining how many CFU per measure (usually per gram or ml) of the sample being evaluated. Positive Control a sample in a test series with a test agent that has a known observable effect for use as a standard of comparison. Potable Water water that is suitable for drinking. Pour Plate a plate count method in which a test material is introduced and dispersed uniformly after the addition of molten agar medium to a Petri dish. Preservation Test a method in which a material is inoculated with selected microorganisms to determine the antimicrobial effectiveness of a preserved or an unpreserved formulation.

GLOSSARY

Preservative Efficacy Testing See Preservation Test. Preservative Failure deterioration in the effectiveness of the preservative system in a product that allows for microbial growth or survival. Preservative System the agent(s) incorporated into a product to reduce or prevent microbial growth. Procaryotic a type of cell without a nuclear membrane. Process Water treated water used as a raw material in the manufacture of a product. Process Water System a manufacturing system used to make process water. Proliferation microbial growth. Prospective Validation the establishment of documented evidence that a process does what it purports to do, based on a preplanned validation protocol. Purified Water process water obtained by distillation, ion-exchange treatment, reverse osmosis, or other suitable means, the quality of which may be checked for specific chemical parameters, for example using current USP or in-house requirements.

|

CTFA MICROBIOLOGY GUIDELINES

|

243

GLOSSARY

Package Compatibility the absence of a detrimental interaction between a product and its package.

Preservative a chemical agent that kills microorganisms or prevents microbial growth

GLOSSARY

Q Quality Assurance those planned and systematic activities necessary to provide confidence that a product satisfies given acceptance criteria Quaternary Compound a quaternary ammonium compound in which the ammonium hydrogen atoms have been replaced with organic radicals, generally used as a surface active agent and/or a biocide.

R Raw Material any ingredient used in the manufacture of a product.

Sampling Devices tools and equipment used to aseptically sample materials, surfaces, or an environment. Sampling Personnel individuals trained in proper sampling techniques to prevent extraneous microbial contamination. Sanitary hygienic. Sanitary Manufacturing Practice guidelines to maintain clean and sanitary conditions within the manufacturing environment.

Resistance the ability of microorganisms to maintain viability in adverse environmental conditions.

Sanitization the process utilized to reduce viable microorganisms on a surface to an acceptable level; surfaces must be clean for the sanitization procedure to be effective.

Retrospective Validation the use of historical test data to document that a process does what it is intended to do.

Sanitizer a chemical agent used for sanitization of clean surfaces.

RODAC Plate Replicate Organism Detection and Counting (RODAC); an agar plate used for determining surface contaminants by direct contact.

Selective medium a medium that allows the growth of certain types of microorganisms in preference to others. Semi-quantitative ess than quantitative measurement, often referring to a measurement on an arbitrary scale, e.g., 0 to ++++.

S Sample one or more representative portions or items selected from a set to obtain information about that set. Sampling operations relating to the taking and preparation of samples.

244

|

CTFA MICROBIOLOGY GUIDELINES

|

Shelf Life a period of time during which a raw material or finished product may be stored and remain suitable for use. Species one kind of microorganism; a subdivision of a genus.

GLOSSARY

Suitable Medium a medium used for optimal growth of microorganisms, or one that will suppress the growth of certain organisms, while allowing the growth of Atarget@ organisms.

Spore a highly resistant form of a microorganism, e.g., mold or bacilli.

Surface Monitoring periodic determination of the microbial content of a surface.

Spread Plate a plate count method in which a small volume of test material is dispersed, by means of a sterile spreader, over the entire surface of a solid agar medium in a Petri dish.

Susceptible subject to microbial contamination.

Stabile Product a finished product that maintains acceptable characteristics over a specified time and under defined environmental conditions. Standard Operating Procedures written procedures detailing how to operate equipment or execute a process. Sterile free from viable microorganisms and spores. Sterilization the use of either physical or chemical agents to destroy all viable microorganisms and spores from a material or equipment. Streak Plate a method in which inoculum is spread across the surface of a prepared solid agar medium in a Petri dish, to produce isolated colonies. Subculture preparation of a fresh culture from an existing culture. Submicron Filtration a filtration process that uses a filter with a pore size smaller than 1 micron.

GLOSSARY

Swab a wad of absorbent material usually wound around or attached to the end of a small stick or applicator and used for removing material or microorganisms from a surface area. See also Transport Swab. Swabbing the process of wiping a surface with a moist, sterile applicator, in order to collect viable microorganisms.

T Taxonomy the classification of organisms, based on mutual similarities. Total Plate Count See Plate Count. Transport Swab a swab designed to maintain, under specified conditions of time and temperature, the viability and numbers of microorganisms recovered from a sampling procedure. Trend Analysis review and analysis of routine data for patterns and variations.

|

CTFA MICROBIOLOGY GUIDELINES

|

245

GLOSSARY

Specification clear and accurate description of the essential technical requirements for items, materials, or services; in the microbiology laboratory, often refers to a statement of microbial limits.

GLOSSARY

U Ultrafiltration a water-treatment process in which a selective permeable membrane filter is used to separate dissolved molecules based on size. Ultraviolet referring to electromagnetic radiation with wavelengths shorter than visible light and longer than X-rays, generally between 200 and 400 nanometers. Ultraviolet lamp a light that produces ultraviolet radiation for disinfection.

V Validation substantiation and verification that a specific process or test method consistently does what it is intended to do.

Verification evidence that establishes or confirms that the specified application of a process or test method consistently and accurately does or measures what it is intended to do or measure; evidence that establishes or confirms the accuracy of a procedure or criterion. Viable capable of living.

W Waste any material or product that its holder intends for disposal. Water Activity the ratio of the vapor pressure in a product to that of pure water; this ratio is used to evaluate the susceptibility of a water-based product to microbial contamination.

Validation Protocol an approved written plan that details the means by which validation will be achieved and defines the acceptance criteria for a process or test method.

Wipes products consisting of a nonwoven matrix or substrate that is composed of fibers or filaments that are bonded together mechanically, thermally, or chemically and used for the delivery of cosmetics or other product systems

Vectors carriers of microorganisms.

Y

Vegetative Bacteria viable bacterial cells not in a resting state.

Yeast a single-cell fungus that reproduces primarily by budding.

246

|

CTFA MICROBIOLOGY GUIDELINES

|

GLOSSARY

CTFA Microbiology Guidelines

CTFA technical guidelines

CTFA Microbiology Guidelines

CTFA

Cosmetic, Toiletry, and Fragrance Association

Phone: 202/331-1770

280416 Cover.indd 1

Fax: 202/331-1969

www.ctfa.org

2007

The Cosmetic, Toiletry, and Fragrance Association 1101 17th Street, N.W., Suite 300 Washington, D.C. 20036

CTFA

Cosmetic, Toiletry, and Fragrance Association

3/6/08 9:31:54 PM