Textile Chemicals In Ea

KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 241 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 241

KAISA KLEMOLA

Textile Toxicity Cytotoxicity and Spermatozoa Motility Inhibition Resulting from Reactive Dyes and Dyed Fabrics

Doctoral dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium ML1, Medistudia building, University of Kuopio on Friday 17 th October 2008, at 12 noon

Department of Biosciences University of Kuopio

JOKA KUOPIO 2008

Distributor :

Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html

Series Editors:

Professor Pertti Pasanen, Ph.D. Department of Environmental Science



Professor Jari Kaipio, Ph.D. Department of Physics

Author’s address:

Savonia University of Applied Sciences Kuopio Academy of Design P.O. Box 98 FI-70101 KUOPIO FINLAND Tel. +358 17 308 111 Fax +358 17 308 222 E-mail: [email protected]

Supervisors:

Docent Pirjo Lindström-Seppä, Ph.D. Faculty of Medicine University of Kuopio



Professor Jyrki Liesivuori, Ph.D. Department of Pharmacology, Drug Development and Therapeutics University of Turku

Reviewers:

Professor Hanna Tähti, Ph.D. Faculty of Medicine, Medical School University of Tampere



Professor Pertti Nousiainen, Ph.D. Department of Materials Science Tampere University of Technology

Opponent:

Docent Eero Priha, Ph.D. Finnish Institute of Occupational Health Tampere

ISBN 978-951-27-0979-3 ISBN 978-951-27-1094-2 (PDF) ISSN 1235-0486 Kopijyvä Kuopio 2008 Finland

Klemola, Kaisa. Textile toxicity: Cytotoxicity and spermatozoa motility inhibition resulting from reactive dyes and dyed fabrics. Kuopio University Publications C. Natural and Environmental Sciences 241. 2008. 67 p. ISBN 978-951-27-0979-3 ISBN 978-951-27-1094-2 (PDF) ISSN 1235-0486 ABSTRACT The textile industry utilises chemicals in the production of fibres, to refine materials in different processes and to produce better quality textile products. Although the chemical itself may be toxic, there is limited data relating to the toxicity of the final textile product. This information is of clear importance for consumers. The aim of this study was to investigate the toxicity of textile substances by using cell tests in vitro. These tests have been found to be useful when materials containing unknown chemicals need to be evaluated. Boar semen, mouse hepatoma cell line (hepa-1) and a human keratinocyte cell line (HaCaT cells) were exposed to different concentrations of three reactive dyes (Reactive Yellow 176, Reactive Red 241 and Reactive Blue 221) and to the extracts of cotton fabrics dyed with these dyes. The viability of the cell cultures was evaluated. The concentrations IC50 and IC20 to decrease cell protein concentrations in Hepa-1 and HaCaT cell cultures were calculated. These values represent the concentration of the test sample where the protein content in the wells is 50% (IC50) or 80 % (IC20) compared to that of non-exposed cells. The IC20 values were taken to represent the limit of toxicity for fabric extracts. The IC50 and IC20 values were estimated when the dyes were studied. The spermatozoa motility inhibition test was considered to show evidence of toxicity, if at least 25% of the cells were not motile by microscopic observation (50% was set as maximal value of viability). Thus in the spermatozoa test only IC50 value was estimated. After 24 hours exposure of spermatozoa cells to reactive dyes, the IC50 values were 135 µg/ml (yellow), 124 µg/ml (red) and 127 µg/ml (blue). After 72 hours exposure, the blue dye was most toxic to the spermatozoa cells. In hepa-1 cells, no statistical significant difference in the toxicity between blue, red and yellow was found, the IC50 values being as follows: 392 µg/ml (yellow), 370 µg/ml (red), 361 µg/ml (blue). The IC20 values were 176 µg/ml (yellow), 108 µg/ml (red), 158 µg/ml (blue). In HaCaT cells, IC50 values were 237 µg/ml (yellow), 155 µg/ml (red), 278 µg/ml (blue). HaCaT cells exhibited toxicity with low concentrations of the dyes, with the red dye being the most toxic. The IC20 values in the HaCaT cell line were 78 µg/ml (yellow), 28 µg/ml (red), 112 µg/ml (blue). However, the dyed fabrics were not toxic to all studied cells. The fabric extracts were not toxic to hepa-1 and HaCaT cells since the measured protein content was over 80% of control. In the spermatozoa test compared to control, more than 50% of the test spermatozoa cells showed motility. In addition to reactive dyes and dyed fabrics, the effects of industrial dyed and finished cotton fabrics were investigated in cell tests. All of the studied raw fabric materials (untreated) were non- toxic. The reactive dyed and press shrunk fabric was not toxic. The flame retarded cotton fabric caused little toxicity to the spermatozoa cells. Most of the knitted cotton fabrics were toxic to hepa-1 and HaCaT cells with the exception that the yellow fabric extract was not toxic to HaCaT cells neither was the red fabric extract toxic to the hepa-1 cells. The other knitted fabric extracts affected the viability of the cells less than 80% compared to control. These results show that cell tests are suitable for studies into the toxicity of textile dyes and fabrics but different cell models should be used in these evaluations. The in vitro bioassays provide information which will help in the development of less harmful textile processes and products. Universal Decimal Classification: 667.281, 677.027.423.5 National Library of Medicine Classification: QV 235, QV 602, QV 627, QV 663, WA 465, QY 95 Medical Subject Headings: Textiles/toxicity; Cotton Fiber; Coloring Agents/toxicity; Azo Compounds/toxicity; Flame Retardants/toxicity; Spermatozoa; Sperm Motility; Toxicity Tests; Cell Line; Cells, Cultured; Cell Survival; Inhibitory Concentration 50; Biological Assay; In Vitro

ACKNOWLEDGEMENTS This study was carried out in the Department of Biosciences, University of Kuopio during 20022008. I am deeply indebeted for her kindness, all her advice and support to Docent Pirjo Lindström-Seppä, the principal supervisor of my work. My sincere thanks are also due to my supervisor Professor Jyrki Liesivuori, for his advice and encouragement. I owe my thanks to Professor Atte von Wright, Head of the Department of Biosciences, for providing the facilities and position for my work in his department. I am delighted to have had the change to enjoy such a pleasant working atmosphere. I wish to express my gratitude to Professor Hanna Tähti and Professor Pertti Nousiainen, the referees of this thesis, for their constructive comments on my work. I am deeply grateful to my co-author Professor John Pearson. I greatly appreciate his efforts in scientific research of textiles and for his pleasant collaboration. I thank Professor Osmo Hänninen for giving encouragement and his belief to me. I express my sincere thanks to Ewen MacDonald, Ph.D., for revising the language of this thesis. I am particularly grateful to Ulla Honkalampi-Hämäläinen, M.Sc., for discussions, encouragement and her friendship. I owe my warmest thanks to Virve Kärkkäinen, M.Sc., and Mrs. Riitta Venäläinen for guiding me with cell cultures. I wish to thank all those persons who have made this series of studies possible by helping me either in the field of laboratory work or by providing technical assistance. I express my thanks to Mrs. Mirja Rekola, Mr. Jouni Heikkinen, Mr. Tuomo Jalkanen and Mr. Väinö Klemola. I thank warmly my colleagues in the Kuopio Academy of Design, Mrs. Marke Iivarinen, Mrs. Riitta Junnila-Savolainen, Mrs. Helena Kauttonen, Mrs. Eeva Kontturi and Mrs. Raili Mähönen. During this work, their patience and friendship has been valuable. The encouragement and support of my friends and relatives are deeply appreciated. My warmest thanks belong to my family, my husband Paavo and our son Väinö, for their care, patience and understanding. This work was conducted mainly with the support of Finnish Concordia Fund, Magnus Ehrnrooth Foundation and Juho Vainio Foundation. This work was also supported by a grant from the Lisa Andström Fund (International Zonta District 20). Kuopio, September 2008 Kaisa Klemola

ABBREVIATIONS ASA

ATP BSA C of V CI CMC CYP1A DDT DMDHEU DMEM DMSO DNF EC50 ECVAM EPA EROD FDA GLP HaCaT Hepa-1 IARC IC20 IC50 INVITTOX ISO LD50 LOAEL MAP MEIC MEM MTT NOAEL OECD PBDE PBS REACH

Syöpäsairauden vaaraa aiheuttaville aineille ja menetelmille ammatissaan altistuvien rekisteri. Vuosittainen tilasto. Työterveyslaitos, Helsinki. The Finnish Register of occupational exposure to carcinogens. Finnish Institute of Occupational Health. adenosine triphosphate bovine serum albumin coefficient of variation Colour Index carboxymethylcellulose a subfamily of cytochrome P450 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane dimethylolhydroxyethyleneurea Dulbecco`s Modified Eagle`s Medium dimethylsulphoxide 2,4-dinitrophenol effective concentration for 50% of maximal effect The European Centre for the Validation of Alternative Methods Environmental Protection Agency 7-ethoxyresorufin O-deethylase Food and Drug Administration Good Laboratory Practice human keratinocyte cell line hepa-1 mouse hepatoma cell line International Agency for Research on Cancer inhibitory concentration decreasing response to 80% compared to control inhibitory concentration decreasing response to 50 % compared to control data bank on the use of in vitro techniques in toxicology and toxicity testing International Standard Organization lethal dose, required to kill 50% of animals in the acute toxicity test lowest adverse effect level mitogen-activated protein kinase The Multicenter Evaluation of In Vitro Cytotoxicity Minimum Essential Medium (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test no adverse effect level Organization for Economic Cooperation and Development polybromide diphenylether phosphate buffered saline The Registration, Evaluation and Authorisation of Chemicals

SD THP THPC THPS VOC WHO WST-1

standard deviation tetrakis-hydroxymethyl-phosphonium tetrakis-hydroxymethyl- phosphonium- chloride tetrakis-hydroxymethyl-phosphonium- sulphate volatile organic compounds World Health Organization Water-soluble tetrazolium assay

LIST OF ORIGINAL PUBLICATIONS This thesis is based on following original publications referred to in the text by their Roman numerals I-IV. I

II

III

IV

Klemola K, Honkalampi-Hämäläinen U, Liesivuori J, Pearson J, Lindström-Seppä P. Evaluating the toxicity of reactive dyes and fabrics with the spermatozoa motility inhibition test. AUTEX Research Journal 2006 6(3), 182-190. Klemola K, Pearson J, von Wright A, Liesivuori J, Lindström-Seppä P. Evaluating the toxicity of reactive dyes and dyed fabrics with the hepa-1 cytotoxicity test. AUTEX Research Journal 2007 7(3), 224-230. Klemola K, Pearson J, Lindström-Seppä P. Evaluating the toxicity of reactive dyes and dyed fabrics with the HaCaT cytotoxicity test. AUTEX Research Journal 2007 7(3), 217-223. Klemola K, Pearson J, Liesivuori J, Lindström-Seppä P. Evaluating the toxicity of fabric extracts using the hepa-1 cytotoxicity test, the HaCaT cytotoxicity test and the spermatozoa motility inhibition test. The Journal of Textile Institute, in press.

The original papers in this thesis have been reproduced with the permission of the publishers.

CONTENTS 1. INTRODUCTION

13

2. REVIEW OF LITERATURE

15

2.1 2.1.1 2.1.2

Textile fibres Classification of textile fibres Cellulose in cotton

15 15 16

2.2 2.2.1 2.2.2 2.2.3

Textile dyes Dye molecule Classification of textile dyes Reactive dyes

17 17 19 20

2.3

Finishing of cellulosic textiles

22

2.4 2.4.1 2.4.2 2.4.3

Adverse effects of textile substances Adverse effects of chemicals caused by the production of cellulosic fibres Adverse effects of reactive dyes Adverse effects of finishing chemicals used for cellulosic textile materials

23 23 24 25

2.5 2.5.1 2.5.2 2.5.3 2.5.4

Toxicity tests Mechanisms of toxicity Testing for toxicity The use of cells in vitro Endpoints used in the evaluation of toxicity in in vitro cell tests

26 26 27 29 30

3. OBJECTIVES

32

4. MATERIALS AND METHODS

33

4.1 4.1.1 4.1.2 4.1.3

Dyes and fabric samples Reactive dyes Fabric Commercial fabrics

33 33 33 33

4.2

Origin of the cells

33

4.3 4.3.1

Procedures for sample preparation and toxicity testing Procedure for dyeing the fabrics

34 34

4.3.2 4.3.3 4.3.4 4.3.5

Preparation of fabric extracts The spermatozoa motility inhibition test Cytotoxicity test with hepa-1 mouse hepatoma cells and with human keratinocyte HaCaT cells Statistical methods

34 34 35 36

5. RESULTS

37

5.1 5.2 5.3 5.4

37 39 40 41

The IC50 and IC20 values for three reactive dyes The toxicity of the fabric extracts Reliability of the results Statistical significance

6. DISCUSSION

42

6.1 6.2 6.3 6.4 6.5

42 44 45 46 47

Reactive dyes in the cell tests The fabric extracts in the cell tests In vitro cell tests for assaying textile substances The reliability of the tests The possibilities for utilizing cell-based tests for studying textile substances

7. CONCLUSIONS

49

8. REFERENCES

50

Introduction 1. INTRODUCTION The manufacture and processing of textiles utilises many different chemical reagents, such as acids, bases, water softeners, salts, organic solvents, dyes and a range of finishes (Trotman 1984). A significant number of these are harmful to the environment, to the people working in textile processing and potentially to consumers. There is some information available about the toxic and other effects of the individual reagents on textile workers. However, there is limited information about the overall toxicity of dyed and finished materials. Although a reagent itself may be toxic, its presence in the finished material may cause no adverse effects. Official patient organizations concerned with asthma and allergies as well as consumer organisations provide some information about the safety of different consumer products (http://www.efanet. org/, http://www.allergia.com, http://www.kuluttajavirasto.fi). However, there is little information available about possible toxic effects of textile products, although the toxic effects of many of the reagents used in their manufacture are known. Allergic reactions and irritation to the skin and respiratory tract have been found to be the most common occupational diseases in workers in the textile industry (Hatch 1984, Nilsson et al. 1993, Zuskin et al. 1996, 1998, Niven et al. 1997, Järvholm 2000). In a study of 72 textile workers in North Carolina, contact dermatitis developed in 24 of them after five years’ exposure to textile chemicals (Soni and Sheretz 1996). In contrast, in Finland, a mere 26 work-related diseases were recorded in the 25,000 workers in the textile industry during 2001 (Karjalainen 2002, ASA): this may be due to good working conditions. Some textile dyes have been assessed for potential mutagenicity (Przybojewska et al. 1989, Jäger et al. 2004, Schneider et al. 2004, Mathur and Bhatnagar 2007,) and genotoxicity (Sharma and Sobti 2000). For example, a high incidence of bladder cancer was detected in Mataro, Spain among the textile workers using reactive dyes (Gonzales et al. 1988). In a European Union EU-funded research project, 281 textile dyes were assessed for potential mutagenic properties using Salmonella typhimurium strains TA98 and TA100. The study revealed positive results for about 28% of the dye products investigated (Schneider et al. 2004). Currently, the EU has set the limiting values for the amounts of carcinogenic aromatic amines (30 ppm) allowed to evaporate from textiles. (2002//61/ EY, 2003/3/EY, VNa694/2003). It is well-known that certain textile finishing compounds are able to release formaldehyde, which can cause adverse effects (IARC 2004). Finland has set the limiting values (100mg – 300mg/kg) for the amounts of formaldehyde permitted in textiles (KTMa 210/1988). The United Kingdom Health and Safety Executive has 2 ppm workplace exposure limit for formaldehyde. There have been many studies conducted on environmental problems of wastewaters due to the presence of toxic textile chemicals and techniques for decolourisation of dyes and removal of textile chemicals are under development (Choudhary et al. 2004, Khan and Husain 2007, Dincer et al. 2007). However, many textile chemicals are organic compounds and not easily extracted from water. It has been shown that some surface waters in India, e.g. in Jaipur, have a high mutagenic activity due to the presence of chemicals released from the textile industry (Mathur et al. 2005a). There is some information about the possible toxic effects of textile chemicals. A globally used

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Kaisa Klemola: Textile Toxicity Öko-Tex-100 textile standard assesses whether textile products with this eco-label contain harmful amounts of certain compounds, for instance heavy metals (Öko-Tex Standard 100, 1997). Many chemical analyses have been performed on these eco-labelled fabrics. However, this standard does not require any biological tests to evaluate the adverse effects of textile materials. Information about product safety is still limited. In the present study, in vitro cell tests were used to evaluate the potential toxicity of textile dyes and dyed fabrics. Cell tests were selected because of their sensitivity to chemicals. Three different types of cells were used: boar spermatozoa cells, mouse hepatocyte cell line (Hepa-1) and human keratinocyte cell line (HaCaT). Reactive dyes used to dye cotton were used as the test material. Cotton was selected because it is the most widely used natural textile fibre and reactive dyes since they are widely used for dying cotton. These cell tests may be useful in the development of safer working conditions in the textile industry and healthier textile products for consumers.

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Review of the literature 2. REVIEW OF THE LITERATURE 2.1 Textile fibres 2.1.1 Classification of textile fibres Textile fibres contain natural and man-made fibres. The natural fibres include cellulose fibres and protein fibres. The man-made fibres consist of raw oil based synthetic fibres and natural polymers regenerated fibres. The fibres can be classified according to their chemistry or according to their origin, the latter being the most commonly used (ISO 6938 1984, ISO 2076 1999, Sundquist 1987, 1988). (Table1: a and b). Table 1: a and b. The classification of the textile fibres (modified from ISO 6938 1984, ISO 2076 1999); a= natural fibres, b= man-made fibres.

a. mineral plant fibres seed fibres

animal fibres

bast fibres

fruit fibres

leaf fibres

cotton

flax

coconut

abaca

alpaca

kapok

hemp

sisal

angora

jute

henequen

camel’s hair

fibres

wool, fur

ramie

silk

asbestos

cashmere lama mohair vicuna wool

b. man-made fibres regenated fibres

synthetic fibres

inorganic fibres

viscose

polyester

glass

modal

polyamide

metallic

lyocell

acrylic

ceramic

acetate

modacrylic

triacetate

elasthane

About 49 million tonnes of all textile fibres were produced during 1994 in the whole world about 38 per cent of this being cotton. In fact, cotton is the mostly produced natural fibre in the world (Worldwide Textile Production 1980-2003). Since the production of cellulose fibres is so extensive, the need for chemicals for the industrial treatments of the cellulose fibres is also widespread.

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Kaisa Klemola: Textile Toxicity 2.1.2 Cellulose in cotton Cotton cellulose is a linear, cellulose polymer, the repeat unit (monomer) being cellobiose which consists of two glucose units (Figure 1). The degree of polymerisation (DP) is about 5000 and the polymer is about 5000 nm long and about 0,8 nm thick. The DP in flax is higher and lower in viscose. The important reactive groups on cellulose are the highly polar hydroxyl (–OH) and methylol (–CH2OH) groups: reactive dye molecules react with these groups. Hydrogen bonds are formed between the polar groups on adjacent polymer chains in crystalline areas of the fibres. Van der Waals` bonds are also present but compared with hydrogen bonding, they are of little significance.

Figure 1. Cotton cellulose consists of cellobiose units.

The cotton fibre is hygroscopic owing to the polar –OH groups in its polymers. This enables cotton to avidly absorb the polar dye and pigment molecules. However, water and dye molecules can only enter the polymer system in its amorphous regions since the inter-polymer spaces in the crystalline regions are too small to accommodate these molecules (Gohl & Vilensky 1983, Gordon 2006). In cotton, the polymer system is about 65 to 70% crystalline and, correspondingly, about 30-35% amorphous (Figure 2). Cellulose fibres are weakened and destroyed by strong acids. Acidic conditions hydrolyse the polymer at the glucoside oxygen atom, which links the two glucose units to form the cellobiose unit. Cellulose fibres are relatively resistant to alkalis. Oxidising bleaches leave the fibre polymer system largely intact if the process is done carefully.

Figure 2. Amorphous and crystalline regions of the polymer system (Gohl & Vilensky 1983).

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Review of the literature 2.2 Textile dyes 2.2.1 Dye molecules Organic molecules become coloured, and are thus useful dye molecules, if they contain at least one of the following radicals called chromophores (which provide colour) and auxochromes (which intensify and deepen the colour) which can selectively absorb and reflect incident light (Tables 2-3), (Gohl & Vilensky 1983, Broadbent 2001). Chromophores are unsaturated organic radicals. Their specific state of unsaturation enables them to absorb and reflect incident electromagnetic radiation within a very narrow band of visible light. Loosely held electrons in the conjugated system of the chromophores are able to absorb certain incident light waves (Gohl & Vilensky 1983). The auxochromes influence the orbitals of the loosely held electrons of the chromophores, which causes these electrons to absorb and reflect incident light energy only of specific wavelengths. This also intensifies and deepens the hue of the dye molecule. Auxochromes also increase the overall polarity of the dye molecule and make it more readily soluble in water and more readily attracted to the fibre polymer (Figure 3) which improves the colour- fastness properties of the dyed fibre (Gohl & Vilensky 1983). Table 2. Chromophores, (Gohl & Vilensky 1983). *In the formula of NO2 the bond should be drawn as -O-, not as

.

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Kaisa Klemola: Textile Toxicity Table 3 Auxochromes (Gohl & Vilensky 1983).

Figure 3. Structural formula of a textile dye molecule. C.I. Acidic Blue 86, 44075 – an acid dye. (Gohl and Vilensky 1983).

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Review of the literature The majority of dyes can be regarded as resonance hybrids with the colours obtained depending on the energy states of the orbitals. Lengthening the conjugated chains increases the number of double bonds and decreases the energy gaps between the π-orbitals. Therefore, the longer the conjugated chain, the less energy will be required to excite the electrons and the greater will be the wavelength of the absorbed light. This can be seen in the properties of carbocyanine dye (Trotman 1984) (Figure 4). As the number (n) of double bonds increases, there is a clearly defined shift of the light absorbed towards red, with a corresponding relative increase in the proportion of blue reflected (Trotman 1984).

Figure 4. Carbocyanine dye. When n = 0 (yellow), n=1 (red), n=2 (greenish blue), n=3 (blue) (Trotman 1984).

2.2.2 Classification of textile dyes There are over 13,000 different compounds classified as dyes in the Colour Index (CI) 2001. About 8000 compounds of them are textile dyes and they give rise to about 40000 commercial names which are used as textile dyes. The CI classifies the dyes according to their application class and to their chemical structure (Table 5). For the textile dyer, the classification according to application class is more significant. The classes are: acidic dyes, azoic (naphthol) dyes, metal-complex dyes, developed dyes, disperse dyes, mordant dyes, reactive dyes, direct dyes, cationic (basic) dyes, sulphur dyes and vat dyes as well as pigments and fluorescent brighteners (Sundquist 1985, modified from Aalto et al. 1994, Talvenmaa 1998, Colour Index 2001). Each dye has a five-digit CI-number.

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Kaisa Klemola: Textile Toxicity Table 5. Textile dyes according to their chemistry. (Colour Index 2001, modified from Aalto et al. 1994) nitroso dyes

indamine- and indophenol dyes

nitro dyes

azine dyes

azoic dyes

oxanthine dyes

azoic developed dyes

thiazine

stilbene dyes

sulfur dyes

carotenoid dyes

amino developed dyes

diphenylmethane dyes

hydroxyketone dyes

triarylmethane dyes

anthracinone dyes

xanthene dyes

indigo dyes

arcidine dyes

phtalocyanine dyes

cinoline dyes

organic natural dyes

methine and polymethine dyes

oxidation developed dyes

thiazole dyes

inorganic pigments

Synthetic textile dyestuffs contain many different chemicals with different applications, for instance to increase shelf life, to improve water solubility and to reduce dusting. Thus commercial dyestuffs contain many other molecules in addition to dye molecules themselves (Talvenmaa 1997, Broadbent 2001). 2.2.3 Reactive dyes Reactive dyes are named according to their chemical reactivity with fibre polymers. These dyes are widely used in dyeing cotton and other cellulose-based fibres. Around 120,000 tonnes are produced per year accounting for over 60% of all dyes for cellulosic fibres (Holme 2004). Reactive dyes form a covalent bond between the dye molecule and the fibre polymer (Figures 5-7). This produces a stable covalent bond which results in excellent colour fastness. This property as well as the simplicity of the dyeing process, means that reactive dyes, despite their relatively high cost, are widely used on cellulosic fibres.

Figure 5. The vinyl sulphate radical of a reactive dye molecule and its bonding to the cellulose polymer. (Gohl & Vilensky 1983)

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Review of the literature

Figure 6. A dichlorotriazinyl reactive dye and its bonding to the cellulose polymer. (Gohl & Vilensky 1983)

Figure 7. A monochlorotriazinyl reactive dye. (Trotman 1984)

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Kaisa Klemola: Textile Toxicity Typically reactive dyes are applied using salt and subsequently fixation is brought about by the addition of soda ash to the dye bath. The reaction between the fibre and the dye molecule takes place under alkaline conditions. The dye can also undergo hydrolysis with water and this decreases the colour yield of the dye as well as lessening the colour fastness due to reduced number of covalent bonds within the fibres (Gohl & Vilensky 1983, Trotman 1984, Broadbent 2001). 2.3 Finishing of cellulosic textiles Cellulose fibres ignite and burn easily and therefore it has been important to develop flame-retardant treatments for those materials. Many of these compounds are based on water-soluble inorganic salts that are easily removed by water, rain and perspiration: they provide only temporary protection. Boron (polybromide diphenylether PBDE) and phosphorous compounds are widely used. Durable flame retardancy is typically obtained by the use of organophosphorous compounds (Lewin and Sello 1983). Tetrakis-hydroxymethyl-phosphonium-salts such as Proban (Rhodia; - tetrakis-hydroxymethyl phosphonium chloride (THPC)-urea) are used as durable flame retardants for cotton, cellulose and cellulose-blend clothing fabrics. Pyrovatex CP (Ciba Speciality Chemicals) is a phosphonoalkylamide used to confer durable flame retardancy on cellulosic fabrics being used in furnishings (Gohl & Vilensky 1991, WHO 2000, Schindler and Hauser 2004). There are numerous chemicals used to make fabrics water repellent. These include aluminium and zirconium soaps, waxes, wax like substances, metal complexes, and pyridinium- and methylol- compounds. The use of methylol stearamide with partially formed urea-formaldehyde provides a good water-repellent finishes with adequate fastness properties. Fabrics can also be made water-repellent by incorporation of thermo-setting silicone resins. Water-repellency combined with oil- and soilrepellency can also be obtained by the application of fluoropolymers such as Scotchguard (3M) and Teflon (Du Pont) (Kissa 1984, Schindler and Hauser 2004). Modern developments include the use of nanotechnology. Cotton textiles can be protected against attack by micro-organisms with quaternary ammonium compounds, such as cetyl trimethyl ammonium chloride being used to achieve this goal. Chemical modification of the cellulose polymers is possible: reaction of fibres with acrylonitrile produces durable protection (Gohl and Vilensky 1983, Schindler and Hauser 2004). Chun & Gamble (2007) reported the use of silver nanoparticles, graft polymerisation of N-halamide monomers, chloromelamine derivatives and cross-linked chitosan to produce durable effects. A number of durable easy-care finishes have been developed to reduce creasing of cellulose fibres with most of these based on dimethyloldihydroxy-ethyleneurea (DMDHEU) (Priha 1988, Schindler and Hauser 2004). Many finishing formulations require the use of catalysts, residues of which may be found in treated fabrics, and non-ionic softeners.

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Review of the literature 2.4 Adverse effects of textile substances Asthma, rhinitis and dermatitis are three of the most common adverse effects evoked by textile processing (Eskelson and Goodman 1963, James 1985, Docker et al. 1987, Jahkola et al. 1987, Jolanki et al. 1999, Piipari and Keskinen 2003). Textile manufacture is a global activity and it is difficult for consumers to obtain information about the production and chemicals of textile products. It is known that harmful chemicals may have been used, but potential adverse effects of textile products are not widely appreciated. However, information about the chemicals of textile products can be important, especially for sensitized consumers. The Austrian Textile Research Institute and the German Hohenstein Research Institute jointly developed the Öko-tex-100 standard and eco-label (www.oeko-Tex.com) which is now used globally to indicate that the textile product has been tested for the presence of harmful substances. However, the overall toxic effects of end products are not included and information about overall toxicity is not available. 2.4.1 Adverse effects of chemicals caused by the production of cellulosic fibres Many chemicals are used in the cultivation of the cotton plant, for instance chlorinated phenols, zinc and copper salts and residues may still be present in cotton-based textiles (Suojanen 1995, Öko-Tex Standard 100, 1997). Some fertilizers contain high concentrations of cadmium (about 138 mg/kg) in phosphoric fertilizers (Malm & Louekari 2000) and some insecticides e.g. methoxyethylmercurium (Komulainen et al. 1992). Synthetic pyrethroids cause irritation effects (Priha 1988). Serious effects on the central nervous system have been detected following the use of organophosphate insecticides (Minton et al. 1988, Marrs 1993, Lopez-Carillo and Lopez-Cervantes 1993, Stevens et al. 1995). It has been stated that women working with organophosphates are at risk of developing non-Hodgkins lymphoma (Zahm et al. 1993). It has been found that patients who were exposed to organophosphates and carbamates had low levels of cholinesterase activity in their blood (De Peyester 1993). For products with the Öko-Tex Standard 100 label, limiting values have been set for the amounts of recognized harmful chemicals allowed to be present (Öko-Tex Standard 100 1997). Much work has been done in Western Europe and Scandinavia to ensure that textile products do not contain chemicals that might be harmful to the consumer and as consequence many textile chemicals have been banned. However, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) was found in England and Sweden not only in raw fabrics but also in clothes (Ahonen 1994, Suojanen 1995). Although most of the harmful chemicals are washed away during industrial processing, some may still remain in the textile end products. It has been recognized for over 200 years that prolonged exposure to cotton dust can produce byssinosis. This results in the weakening of the lungs and chronic pulmonary inflammation (Duffell 1985, Sigsgaard 1992, Beckett et al. 1994, Hayes et al. 1994, Christiani & Wang 2003). It has also been claimed to be a risk factor in the development of nose- and skin cancer (Lund 1991, Luce et al. 1992). There is less large-scale use of chemicals in the production of other natural cellulosic fibres. How-

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Kaisa Klemola: Textile Toxicity ever, the production of regenerated cellulosics such as viscose, utilises harmful chemicals and these can cause problems. For example, adverse effects on the eyes and respiration have been detected in persons who have been exposed to carbon disulfide which is a known neurotoxic compound (Aaserud et al. 1990, Vanhoorne et al. 1991). The most widely used chemicals after fibre formation are surfactants and auxiliaries, which may act as irritants. Acidic detergents can cause eczema and contact dermatitis. Enzymes, bleaches and brighteners can evoke respiratory allergic reactions. Aromatic, and chlorinecontaining organic solvents are known irritants. Other auxiliaries, including strong acids, mineral oils and salts can cause irritation and allergic reactions (Estlander and Jolanki 1980, Virtanen and Hannuksela 1999). In conclusion, the production of cellulosic fibres utilises chemicals that can evoke adverse effects including allergic reactions and irritation to the skin and the respiratory system. Some of these harmful substances may remain in the textile end products. However, there is minimal information for consumers about these adverse effects. Although cellulosic fibre processing has been extensively studied, the actual end products remain to be more thoroughly investigated. 2.4.2 Adverse effects of reactive dyes Symptoms of asthma, rhinitis and dermatitis have been frequently detected in workers exposed to reactive dyes (Hatch 1984, Thoren et al. 1986, Nilsson et al.1993, Manzini et al. 1996, Park et al. 2006, 2007). Dyes containing anthraquinone or azo structures are known to cause contact dermatitis (Estlander 1988, Wilkinson and McGechaen 1996). The result of a clinical and immunological investigation of respiratory disease indicated that about 15% of 400 workers handling reactive dyes experienced work-related respiratory and nasal symptoms (Docker et al. 1987). Many studies have also found statistically significant relationships between reactive dyes and increasing immunoglobulin blood values in workers who have been contact with these dyes (Alanko et al. 1978, Topping et al. 1989, Park et al. 1991). However, it should be noted that dermatological problems associated with dyes in textiles are relatively rare (Maurer et al. 1995), although Hatch et al. (2003) have noted such effects and have given the name ‘colored clothing allergic contact dermatitis (ACD)’ to these symptoms. Moreau & Goossens (2005) reported similar effects and stated that reactive dyes should be classified as potential allergens, even their presence in clothes. The cause of skin reactions is difficult to trace because the dye usually acts as a delayed sensitizer and as such does not cause an immediate response (Hatch and Maibach 1995, Wang et al. 2002). It has been assumed that since the properties that enable the dyes to react with textile fibres also allow them to bind to body protein, the health hazard resulting from exposure to such substances is significant (Keneklis 1981). Since reactive dyes are chemically very active, they can cause harmful effects, especially when in their powdered form. It was noticed as early as 1981 that some sulphonyl ethyl sulphate derivatives were carcinogenic (Keneklis 1981). The international register of cancer-causing chemicals includes many textile dyes (including those based on benzidine and o-toluidine) and their raw chemicals for synthesis (IARC 1987, TMp 838/1993, Aalto et al. 1994). However, in Finland, the register of

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Review of the literature individuals exposed to carcinogens contains little information about textile industry workers (Kauppinen 1999, Vuorela 2003). The UK Health and Safety Executive warns industrial workers who have contact with reactive dyes that they may become sensitized, stressing the potential for respiratory sensitization. In many European countries, the use of textile dyes releasing certain aromatic amines at concentrations above 30 ppm is forbidden (VNa 694/2003, 2002/61/EY and 2003/3/EY). If one wishes to detect any adverse effects of textile dyes, then it is important to conduct tests for mutagenicity and genotoxicity (Przybojewska et al. 1989, Schneider et al. 2004, Mathur et al. 2005a, Mathur et al. 2005b, Dogan et al. 2005), carcinogenicity (De Roos et al. 2005) and teratogenicity (Birhanli and Ozmen 2005). All these studies have revealed adverse effects of dyes, although not all the dyes tested were reactive dyes. Due to the problems associated with textile dyes, the dyeing process is under constant development, with increasing attention being paid to the ecological effects of these chemicals. 2.4.3 Adverse effects of finishing chemicals used for cellulosic textile materials Many finished fabrics may release formaldehyde. This problem is associated especially with the permanent press, flame proofing and antimicrobial treatments. Formaldehyde has been demonstrated to cause harmful effects (James 1985, Priha et al. 1986, 1988, Priha 1992,1995, Priha et al. 1996, Jahkola et al. 1987, WHO 1989, Garcia et al. 1995, IARC 2004, Carlson et al. 2004). Specific effects include irritation to skin, eyes and the respiratory tract (James 1995) and it may also cause asthma (Piipari and Keskinen 2003). In Finland, limiting values have been set for the amount of formaldehyde allowed in fabrics (Priha 1995; Jolanki et al. 1999; Suomen säädöskokoelma 210/88). The effects of formaldehyde in textile work places have been widely studied (Nousiainen 1979, 1982, 1983,1984:a and b, Roberts and Rossano 1984, Priha et al. 1996, Scheman et al. 1998). Since high concentrations of formaldehyde may cause cancer, the international register of cancer causing chemicals classifies formaldehyde to The Group 1: carcinogenic to humans (IARC 2004). With regard to flame retarding agents, polybromide biphenyls have been found to cause adverse effects and have been removed from sale (WHO 1997). Polybromide diphenylethers (PBDE) may form polychloride dioxins and furans in a fire and their use is now greatly restricted. PBDE and polychlorinated PCBs can interact and enhance developmental neurobehavioral effects when the exposure occurs during a critical stage of neonatal brain development (Eriksson et al. 2006). Neonatal exposure to brominated flame retardant, 2,2´,4,4`,5-pentabromodiphenyl ether has been shown to cause altered susceptibility in the cholinergic neurotransmitter system in the adult mouse (Vikberg et al. 2002). In addition, it has been noted that PBDE disrupts spontaneous behaviour, impairs learning and memory in adult mice (Vikberg et al. 2003). An increase in the amounts of PBDE has been found in the environment and this chemical is even present in mother`s milk. However, in the USA, the measured amounts of brominated flame reagents in the environment have been claimed to pose no threat to the health of children (Hays and Pyatt 2006). According to IARC (1998) it is not certain whether the widely used phosphonium salt as flame retardants are carcinogens. Tetrakis-hydroxymethyl-phosphoniumchloride (THPC) was tested and treated fabrics did not cause skin irritation to humans. THPC was not shown to be carcinogenic in

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Kaisa Klemola: Textile Toxicity rats and mice in a 2-year bioassay. However, dermal studies with rabbits have shown that THP salts are promoters but not initiators of skin cancer (WHO 2000). Antimicrobial treatments are not widely used on clothing: their use is limited mainly to tent fabrics and woollen carpets. Many antimicrobial agents are relatively toxic and have been found to sensitize humans (Kanerva 1998, Kalimo and Lahti1999, Yazdankhah et al. 2006). Other finishing agents: water repellents, stain repellents, dirt repellents and antistatic agents have seldom caused skin or other health problems (Priha et al. 1988). However, these reagents are also available in aerosol forms where they are combined with carbohydrates and fluorochemicals, and in these situations they have caused pulmonary inflammation (Wright and Lee 1986, von Essen 1996, Vernez et al. 2006). These aerosol products are available for home use by consumers. Indigo-dyed denim fabrics were shown to be mutagenic, showing the importance of analysing the finished fabrics in addition to the pure chemicals. Since the mutagenicity of indigo was low, the genotoxicity of denim extracts must have been due either to some unknown chemicals or to some unknown reactions (Rannung et al. 1992). Knasmuller et al. (1993) showed that 18 of 196 fabric samples examined were mutagenic, 16 of them only after metabolic activation. Though many azo dyes have been shown to be mutagenic (Chung and Cerniglia 1992, Kaur 1993), it seems that azodyed fabrics are not toxic (Kaur et al. 1993). Nonetheless, when azo-dyed fabrics for clothing were tested with the Ames bacterial assay, almost 20% of the silk fabrics and over 10% of the cotton fabrics gave positive results in this test for mutagenesis (Pfitzenmeier 1990). The textile industry utilises different washing, dyeing and finishing chemicals which can contain nonylphenol and nonylphenolethoxylates. These chemicals and their metabolites are strongly corrosive and have been found to be toxic to the aqueous environment and to humans. These chemicals have been claimed to cause hormonal changes and therefore the European Union has recommended that their use should be limited (2001/838/EY, VNa 596/2004). The causes of toxicity of a fabric can be difficult to trace since they may be due to the combined effects of several of the chemicals present in the textile. Although the problems caused by many textile chemicals are recognized, potential problems in fabrics have not been widely studied and, as with the production of cellulosic fibres, there is limited information available to consumers. 2.5 Toxicity tests 2.5.1 Mechanisms of toxicity The toxicity is of a compound becomes apparent when a toxicant is delivered to its target and reacts with it and the resultant cellular dysfunction manifests itself in toxicity. Sometimes a xenobiotic does not react with a specific target molecule but rather adversely influences the biological (micro) environment causing molecular, organellar, cellular or organ dysfunction and leads to deleterious effects. (Gregus and Klaassen 2001) The most complex path to toxicity involves four steps. First, the toxicant is delivered to its target or targets, after which the ultimate toxicant interacts with endogenous target molecules, triggering perturbations in cell function and/or structure, which initiate repair mechanisms at the molecular, cel-

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Review of the literature lular and/or tissue level. When the perturbations induced by the toxicant exceed the repair capacity or when repair becomes malfunctional, then toxicity occurs. The examples of chemically induced toxicities followed by this four-step course are tissue necroses, cancer and fibroses. (Gregus and Klaassen 2001) One chemical may yield several ultimate toxicants, one ultimate toxicant may react with several types of target molecules, and the reaction with one type of target molecule may have a number of different consequences. Thus, the toxicity of a chemical may involve several mechanisms which can interact with and influence each other in an intricate manner. (Gregus and Klaassen 2001) A number of xenobiotics such as heavy-metal ions, strong acids and bases, nicotine, and ethylene oxide are directly toxic, whereas the toxicity of other compounds is largely due to metabolites. Biotransformation of a compound to a harmful compound is called metabolic activation. However, the conversion of a bioactive parent compound to a less bioactive or inactive metabolite(s) that is/are efficiently eliminated is mostly usual. This conversion is called metabolic inactivation, or detoxification. (Parkinson 2001) Non-polar xenobiotics accumulate into lipid-containing tissues or are metabolised to a more water soluble compounds. The first step is xenobiotic metabolism, the so-called phase I reactions that consisting of non-synthetic reactions like oxidation, reduction and hydrolysis. Phase II reactions in the second phase are conjugation reactions with compounds having hydroxyl -OH, amine –NH2 or carboxylic –COOH groups. The functional groups may be present in the parent compound or may have been formed during phase I reactions that lead to toxicity. (Bend and James 1978, Sijm and Opperhuizen 1989, Parkinson 2001) Benzene, similar to other aromatic compounds, is oxidised into a variety of reactive metabolites which are normally more toxic than the original compounds. For instance, benzene can be oxidized to a variety of quinines and semiquinones that can cause hematopoietic toxicities and leukaemia. Benzene and many other volatile organic compounds (VOCs) are converted via multiple metabolic pathways to products with varying toxicities. Some of these competing pathways are considered as bioactivation, others as detoxification pathways. A variety of factors (for example differences between species, functions of enzymes) can influence the prominence of the different pathways and hence alter toxicity outcomes. When different cell signalling pathways become disrupted, the cell has typically become exposed to some toxic substance (Alberts et al. 2002, Gregus and Klaassen 2001). 2.5.2 Testing for toxicity Toxicity tests are not designed to demonstrate that a chemical is safe but to characterize the toxic effects it can produce. Currently there is a new set of regulations and toxicity tests that have to be performed on chemicals intended for commercial use (www.ymparisto.fi-REACH). During the past years, some regulations have been introduced, for instance, the Food and Drug Administration (FDA), Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) have issued good laboratory practise standards (GLP). These guidelines are expected to be followed when toxicity tests are conducted in support of the introduction of a chemical

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Kaisa Klemola: Textile Toxicity to the market. (Eaton and Klaassen 2001) The typical in vivo acute toxicity test (LD50) is estimated by determining the number of animals that die in a 14-day period after treatment of a single dosage of the chemical. Clinical chemistry and histopathology are performed after 14 days of exposure. However, this test has been removed from the guidelines (in 2002), and replaced by three ethically more acceptable refinement tests, which use much less animals and in which the lethal dose is not determined any more (Worth and Balls 2002). Subacute toxicity tests are performed to obtain information on the toxicity of a chemical after repeated administration and as an aid in deciding how to conduct subchronic studies. Long-term or chronic exposure studies are performed similarly to subchronic studies except that the period of exposure is longer than three months, usually from six months to two years. (Eaton and Klaassen 2001) Numerous in vivo and in vitro procedures have been devised to test chemicals for their ability to cause mutations. Some genetic alterations can be visualized with the light microscope. The test for mutagens that has been most widely used in toxicology is the Salmonella/microsome test developed by Ames et al. (1975). In vitro tests for mutagens are in widespread use. In addition, many in vitro tests based on cell cultures have been developed, each with its own particular advantage in the area of toxicological research. In addition to ethical benefits, these tests are cheaper and quicker to carry out and generate smaller quantities of toxic waste than other tests (Baksi and Frazier 1990). Cytotoxicity can include changes in the integrity of membranes and cytoskeleton, cellular metabolism, energy metabolism and synthesis and degradation of cellular constituents, ion transport and cell division. In addition, compounds can be selectively cytotoxic or cause cytotoxicity by interference with cell-specific functions (Seibert et al. 1992). The most important results obtained from toxicity tests are the values of LOAEL (the lowest dose that causes adverse effects) and NOAEL (the dose that does not cause any adverse effect) which set the limiting values of toxicity. The toxic value from an in vitro test is typically stated as the effective concentration EC50 which represents the dose that causes 50% of cells to die. Another value often used in in vitro studies is the IC50 value, e.g. when the inhibition of enzyme activity is measured as an endpoint to indicate the viability of the cells in culture. In the present study, IC50 values are used to describe the inhibition of the viability of the cells in culture and the inhibition of the movements of spermatozoa. In the area of toxicology, LD50 is no longer in use. NOAEL is the most important value and is helpful when the limiting values for chemicals need to be set. At present, in all chemical safety testing animal experiments must be applied. In the OECD and EU guidelines there are in vitro replacement tests only for skin absorption, skin corrosion, phototoxicity, severe eye irritation and mutagenicity. In addition, several tests have been validated by the European Centre for the Validation of Alternative Methods ECVAM, but they have not yet been accepted for regulatory purposes (http://ecvam. jrc/it). Nonetheless, in vitro tests can provide mechanistic data and also these kinds of tests are useful for screening of chemicals. The safety data sheets still include results of LD50 assays. (Eaton and Klaassen 2001, Liesivuori, oral communication). It is known that in vivo animal tests do not always predict toxicity in humans. However, it is often possible to calculate relatively safe doses for humans. It has also become increasingly evident that

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Review of the literature chemicals causing carcinogenicity in animals are not necessarily carcinogenic to humans (Grisham 1997, Dybing and Sanner 1999, Hengstler et al. 1999). However, for regulatory and risk assessment purposes, positive carcinogenicity tests in animals are usually interpreted as being indicative of potential human carcinogenicity (Eaton and Klaassen 2001). 2.5.3 The use of cells in vitro A large range of cell lines is available for studying toxicity: human and animal cells, lines from pathological cases, tumour lines, normal lines and transformed lines. These cell lines have many uses e.g. when studying the effects of various anticancer drugs during the early phase of the drug development. Retinal pigment epithelial cell lines have been exposed to anti-oestrogenic drugs in studies of eye toxicity (Toimela et al. 1995, Mäenpää et al. 2004). The effects of hyperoxia have been studied with cervical cancer cells (Campian et al. 2004) and the effects of radiation with malignant pleural mesothelioma cell lines (Häkkinen et al. 1996). Cells can be useful for analysing different kinds of materials to reveal acute toxicity. In addition, it is possible to study the overall toxicity of materials with unknown chemical compositions. Information about the toxicity of individual chemicals may be available, but the combined effects are difficult to study and are not often available in the literature. In different studies, boar spermatozoa cells, hepa-1 mouse hepatoma cells and human keratinocyte HaCaT cells have been found to be useful. Boar spermatozoa cells have a simple metabolism compared to somatic cells. They are completely dependent on their surrounding environment for nutrients and they are also not able to detoxify toxic end products due to the low concentrations of detoxifying enzymes in the cell cytosol. Many physiological processes in spermatozoa are controlled by membrane potentials and ion fluxes (Mann and Lutwak-Mann 1982). The motility of semen can be measured in several ways e.g. both hyperactivation and inhibition. Inhibition of motility is a consequence of membrane depolarisation (Gao et al. 1997). Hyperactivation of motility is associated with membrane hyperpolarisation (Zeng et al. 1995). Bacterial toxins have been found to affect the cell membrane and the mitochondrial membrane potential in the sperm cells at very low concentrations (Hoonstra et al. 2004). Bovine spermatozoa have been used in studying cytotoxicity (Seibert et al. 1989, 1992, Seibert and Gosch 1990, INVITTOX Protocol 21 1991). The spermatozoa in vitro test has been found to be useful when detecting the presence of hazardous substances in indoor building materials subjected to moisture damage and containing complex microbial communities of bacteria and fungi (Andersson 1999). In addition, paper materials have been studied (Severin et al. 2005). Hepa-1 mouse hepatoma cells (Hepa-1c1c7) are a commonly used cell line to assess the potential toxicity of dioxin and dioxin-like compounds, materials such as laboratory animal beddings and feeds, fly ash samples and paper products (Kopponen et al. 1991, 1992a,b, 1993, 1994a,b,c, 1994, Kärenlampi and Törrönen, 1990, Törrönen et al. 1989, 1991, 1994, Severin et al. 2005). Kopponen et al. (1997) conducted some preliminary studies with textile dyes and fabrics using the hepa-1 cytotoxicity test. Since 1989, the Hepa-1 cytotoxicity test has been included as a part of the Multicenter Evaluation

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Kaisa Klemola: Textile Toxicity of In Vitro Cytotoxicity (MEIC) programme, in which the overall toxic potencies of a wide range of chemicals relevant to human toxicity have been studied (Clemedson et al. 1996, INVITTOX Protocol 112 1995). The programme revealed that the various cell lines and growth end point measurements gave similar cytotoxicity results in most cases. The Hepa-1 cytotoxicity test has given satisfactory values for practically all chemicals tested and these mouse cells are, in general, better indicators of human toxicity than rat cells. Human keratinocyte HaCaT cells possess some metabolic activity, but are not as versatile as hepa-1 cells. HaCaT cells are human skin cells and for this reason they have been considered as relevant when human toxicity has been studied. HaCaT cells have been widely used for instance in evaluating skin irritation (Wilhelm et al. 1994), skin cancer (Merryman et al. 1999), genotoxicity and mutagenicity of textile dyes (Wollin et al. 2004) and the cytotoxicity caused by potential contact with nickel and chromium (Little et al. 1996). In particular, HaCaT cells have been used for investigating adverse effects of UV-radiation (Isoherranen et al. 1999, O`Reilly and Mothersill 1997). The cells have also been reported as being useful in clarifying cell signalling pathways (Assefa et al. 1997; Shimizu et al. 1999). 2.5.4 Endpoints used in evaluation of toxicity in in vitro cell tests The total protein content of the cells is a widely measured and validated endpoint (INVITTOX Protocol 112 1995). In this test, some dead cells may also be measured and may cause variation in the viability results. However, the content of the total protein provides valuable information about viability. The neutral red (NR) cytotoxicity test determines the number of living cells in the culture. In living cells, neutral red penetrates cell membranes and accumulates in lysosomes and can be measured photometrically (INVITTOX, Protocol 64). The MTT-test (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) measures the activity of mitochondrial succinate-tetrazolium reductase system of cells (Kwang-Mahn et al. 2005) Immunotoxicological activity has been tested by exposing mouse macrophages and measuring the endpoints by the MTT-test when indoor air of moisture-damaged buildings has been studied ( Huttunen et al. 2008). Apoptosis and cell viability have also been measured with the MTT-test when anticancer drugs have been evaluated (Giovagnini et al. 2008). In addition, in the studies where the skin models have been validated, the MTT-test has been used (Kidd et al. 2007). A water-soluble tetrazolium assay, the WST-1 test, which is similar to the MTT test, has been performed to measure the mitochondrial synthesis and cellular proliferation rate (Kwang-Mahn et al. 2005). For example, the test has been used when the proliferation rate of human osteoblast-like cells has been studied (Weibrich et al. 2002). The rate of apoptosis has been measured when the antidepressant drug, desipramine has been evaluated in human PC3 prostate cancer cells (Chang et al. 2008). Mitochondrial viability and apoptosis induced by aluminium, mercuric mercury and methylmercury have been assessed with the WST-1 test (Toimela and Tähti 2004). In addition, pyrethroid compounds in neural cell cultures have been examined with the WST-1 test (Kakko et al. 2004). In that study, the energy of the exposed cells was also investigated by measuring their ATP content.

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Review of the literature Lactate dehydrogenase leakage is coupled to energy production of the cells. The enzyme catalyzes the final reaction of anaerobic glycolysis, the reduction of pyruvate to lactate (Campbell and Farrell 2006). LDH has been used as an indicator of cellular damage of membrane when the effects of mercury, methylmercury and aluminium on gial fibrillary acidic protein expression in rat cerebellar astrocyte cultures have been studied (Toimela and Tähti 1995). If one wishes to understand the mechanisms of toxic effects, several types of cells are available for use and several endpoints are available for measurement.

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Kaisa Klemola: Textile Toxicity 3. OBJECTIVES There is limited information about the toxicity of textile substances when they are present in finished textile articles. The aim of this study was to evaluate the toxicity of textile reactive dyes and fabric extracts with cell tests in vitro. The detailed aims were as follows: 1.

To assess the usefulness of two cell lines and bovine spermatozoa for the determination of possible acute toxicity of textile reactive dyes (I-III).

2.

To examine whether dyed materials differ in their toxicity from the pure reactive dyes. The aim was to study the overall toxicity of the dyes in fabrics (I-III).

3.

To evaluate the toxicity of common commercially available fabrics. The aim was to determine if the materials after finishing and dyeing differ in their toxicity compared to the raw materials (IV).

4.

To define which tests are useful in providing information about the toxicity of reactive dyes. In addition, the purpose was to study whether data from cell tests could provide useful information about potential toxicity or safety of textile products. It was also of interest to examine if these tests could be further developed into routine tests for use in the textile industry.

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Materials and methods 4. MATERIALS AND METHODS 4.1 Dyes and fabric samples 4.1.1 Reactive dyes The three reactive dyes studied were monchlorotriazinyl dyes commonly used in cloth-making and by workers in the crafts industry. They are also the basic three dyes used when mixing different colour combinations. Colour Index numbers for the dyes were: Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221 (Drimarene red CL-5B, Drimarene yellow CL-2R and Drimarene blue CL-2RL respectively). The dyes were obtained from Clariant Ltd. The samples of the dye powder were dissolved in the appropriate cell medium solution. 4.1.2 Fabric A typical sheeting fabric was used in this study: plain weave bleached cotton (white), obtained from a commercial fabric shop. It was observed that the material contained some brightener. One sample was self dyed with an unknown reactive brilliant yellow dye. 4.1.3 Commercial fabrics In this study, those fabrics dyed in industrial processes are called commercial fabrics. Two qualities of 100% cotton fabrics for working clothes were investigated. One fabric was vat dyed and treated with flame retardant: blue, twill woven (175g/m2). The other fabric was reactive dyed and press shrunk: brown and blue, twill woven (260g/m2). In addition, there were two different types of knitted fabrics: 100% cotton 160g/m2 and 50% /50% cotton/modal 145g/m2. Knitted fabrics were reactive dyed with yellow, red and blue dyes respectively. The fabrics were finished in textile factories in Finland. More detailed information about these fabrics was not available. All commercial fabrics were available to study in the form of raw fabric material. However, the chemical content of the raw fabrics was not known. 4.2 Origin of the cells Boar semen for testing was available from the Insemination Center of Pieksämäki, Finland. Hepa-1 mouse hepatoma cells (the wild type Hepa-1c1c7) were obtained from The Department of Physiology, University of Kuopio, Finland. The cells were originally obtained from Dr. D.W. Nebert, Department of Environmental Health, University of Cincinnati, Ohio, USA in 1982. Human keratinocyte HaCaT cells were obtained as a gift from the Department of Anatomy, University of Kuopio, Finland.

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Kaisa Klemola: Textile Toxicity 4.3 Procedures for sample preparation and toxicity testing 4.3.1 Procedure for dyeing the fabrics Fabric samples (10g) were washed gently without soap. The amount of dye used was 3% on each fabric. The dye bath contained 400 ml water with 50g Na2SO4/l H2O and 20g Na2CO3/l H2O. Dyeing continued for one hour at 55ºC. Na2CO3 was added to the dye bath ten minutes after the beginning of the dyeing process to adjust the pH. After dyeing, the fabrics were spooled in cool and warm water baths and kept in pure boiling water for 10 minutes. The dyeing process in the present study was based on the common procedures used in industry and by crafts workers. A total of 3% dye represents a strong colour on fabrics. 4.3.2 Preparation of fabric extracts The fabric pieces (1cm x 1cm) were extracted with sterilized water (1g fabric/20ml H2O) in laboratory test tubes. The tubes were shaken at room temperature for two hours and incubated at 37ºC for 18 hours. The samples were shaken thoroughly before centrifugation for 5min at 4500 rpm. The fabric extracts were sterile filtered through polyester and cell growth medium compounds were added to the extracts before exposure to the cells. The fabric extracts contained the same concentration of cell growth compounds as in the controls of pure growth medium. There were 3-4 parallel samples. However, there was only one extract sample for each commercial fabric extract. 4.3.3 The spermatozoa motility inhibition test The spermatozoa test based on that used by Andersson (1999) and it was modified for use with textile samples. Valinomycin dissolved in dimethylsulphoxide (DMSO) in several concentrations was used as the positive control: 2ng, 4ng, 8ng and 16ng/2ml respectively. Plain semen and water were used as negative controls to determine the level of normal values. A solution of valinomycin in DMSO with no semen was also tested. The samples were added to semen, using 2ml of semen with 40µl of the dye samples or fabric extracts. All samples were compared to the control sample of plain semen. Exposure continued for 24 hours and/or 72 hours at room temperature. The tubes were inverted once a day. Before analysis, the tubes were gently mixed manually. All processing was carried out under sterile conditions. After 24 hours and/or 72 hours of exposure, sperm motility was measured and compared to the motility of sperm in the plain semen controls. The effects of water and DMSO were also tested. After gently mixing the tubes, 200µl samples were taken to small plastic tubes for incubation at 37ºC for five minutes before microscopic analysis. The temperature of the pre-warmed objective glasses used in the microscopic analyses was also 37ºC. The samples were gently mixed and the amount of living spermatozoa cells was qualitatively observed by light microscopy (100 – 400 x magnification). The results reflected the motility capacity percentages of living cells. The optimal value of the plain

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Materials and methods control sample was set at 50% because the semen always contains dead and damaged cells. In the evaluation, the following observations of sperm activity were conducted: speed of movement, ability to move forward, damage that can be detected by microscopy, rotation, vibration and dead cells. The toxicity limit was considered to be 25% of unexposed cells: levels of 25% and lower represent toxicity. The limiting value of 25% therefore represents the IC50 (inhibitory concentration) which means the concentration where 50% of the original living spermatozoa (optimal value of control was set 50%) were dead, nonmotile, weakly moving or rotating at the end of the exposure time. The toxic effects on spermatozoa were qualitatively evaluated in the field of vision. The crude categories were: 50% - cells move forward, have strong vibration and high activity; 40 - 45% - cells move forward, have strong vibration but less activity than controls; 30 - 35% - some vibration remaining, many dead cells, most of the cells are rotating, only some are moving forward very slowly; 20 – 25% - most of the cells are dead, some are rotating; 10 - 15% - only some cells are moving slowly, most of the cells dead and 5%- isolated cells are vibrating slowly. The results were recorded within these 5% boundaries. 4.3.4 Cytotoxicity test with hepa-1 mouse hepatoma cells and human keratinocyte HaCaT cells In order to assess the potential toxicity of dyes and fabric extracts, the hepa-1 cell cytotoxicity test was used (modified from INVITTOX [data bank on the use of in vitro techniques in toxicology and toxicity testing] protocol number 112). The same modified method was used for human keratinocyte HaCaT cells. The cells were grown as a monolayer at 37ºC in an atmosphere of 5% CO2; hepa-1 cells were grown in -MEM (Minimum Essential Medium) and HaCaT-cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium). Both medium solutions were supplemented with 1% glutamine, 10% foetal calf serum and 1% penicillin/streptomycin solution. The test was carried out in 96-well plastic microplates seeded with a 200µl cell suspension per well (5x104 cells/ml). After growing for 24 hours, the culture was about 60% confluent. The cells were exposed to the dye samples or to the samples of the sterilized filtered (0.2µm pore size) fabric extracts. Non-exposed cells with medium were used as a negative control. All results were compared to these controls. 2,4dinitrophenol was used as a positive control at three concentrations: 0.5 mg 2,4-dinitrophenol/ml DMSO was used as one control and diluted to concentrations of 0.05mg/ml and 0.005mg/ml medium respectively. After 72 hours of exposure, the cells were washed twice with PBS-buffer (phosphate buffered saline, pH 7.2). Before the addition of sodium phosphate buffer it was possible to observe the viability of the cells by light microscopy to obtain preliminary information. Subsequently, 50µl of sodium phosphate buffer (0.05mM, pH 8.0) was added to each well before freezing the plates for at least 15 min at -70ºC. After breaking the frozen cells, the plates were thawed for 15 min and cell growth was detected by assaying the total protein content in the cultures (Kennedy et al 1993, 1995). 150µl of sodium phosphate-buffer was added to the wells followed by 50µl of cold fluorescamine (1.08mM in acetonitrile). The plates were allowed to stand at room temperature for 15 min before being stirred in a microtitration plate shaker for one minute. The total protein content in each well was measured using a plate reading fluorometer at a wavelength of 405/460nm. The protein (bovine serum albumin BSA) standard curves were measured in each bioassay. All processing, except for the

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Kaisa Klemola: Textile Toxicity protein assays, was carried out under sterile conditions. The inhibitory concentration value, the IC50 value is the concentration of the sample in which the wells with exposed cells have only 50% of the protein content compared to that of the non-exposed cells (100%): the level of the protein content is a reflection of the viability of the cells. The inhibitory concentration IC20 denotes the sample concentration where the protein content is 80% compared to the protein content of non-exposed cells. In such a case, the studied dye or fabric extract has inhibited the cell proliferation by 20 %. The IC20 value was considered to represent the value of LOAEL (lowest observed adverse effect level) which is the lowest concentration value of the sample showing adverse effects. The sample was considered to have low toxicity if the protein content was less than 80%, but more than 50% compared to that of non-exposed cells. A reduction of more than 50% in the protein content would represent a clearly toxic effect. 4.3.5 Statistical methods The coefficients of variation (C of V % were used as evidence of reliability with lower values than 10%) and standard deviations (SDs) were calculated for all measurements to evaluate the reliability. The number of parallel samples was in the range of 3-11 when the spermatozoa cells were used as the test cells. In the hepa-1 and HaCaT cell tests, the number of the parallel samples was 3-4. The number of the samples represented the number of independent measurements and the number of plates. Every plate contained 3-4 parallel dye or fabric extract samples. Statistical significance of differences between three reactive dyes was performed for each cell test by variance analyse, ANOVA. Values of p < 0.05 were considered as statistically significant.

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Results 5. RESULTS 5.1 The IC50 and IC20 values for three reactive dyes The three reactive dyes studied were Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221. The dyes were assayed with boar spermatozoa cells, hepa-1 mouse hepatoma cell line and human keratinocyte cell line, HaCaT-cells. The inhibitory concentrations measured as IC50 and IC20 values were mathematically calculated from the functions which were drawn from the mean values of different dye concentrations. Cell growth was assessed as the amount of cellular protein after the exposure (Figures 8-10).

Figure 8. IC50 value for the yellow dye when boar spermatozoa cells were used, exposed for 24 hours (I); *n=3-10;SD = ±( 2-5,2); *The number of the samples (n) represents the number of the plots at the various concentrations of the dye on the curve.

Figure 9. IC50 and IC20 values for the yellow dye when hepa-1 cells were used, exposed for 72 hours (II); n = 2-7, SD = ±(2,1-9).

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Figure 10. IC50 and IC20 values for the yellow dye when HaCaT-cells were used, exposed for 72 hours (III); n = 2-6; SD = ±(3,5-9,8).

Table 6. Summary of the mean concentration values of IC50 and IC20 (µg/ml) for the dyes studied.

HaCat

Hepa-1

spermatozoa

72 h

72 h

24 h

72 h

µg/ml (n)

µg/ml (n)

µg/ml (n)

µg/ml (n)

155 (5)

370 (4-6)

124 (4-9)

46 (3-9)

IC50

Reactive Red 241

IC50

Reactive Yellow 176

237 (6)

392 (4-7)

135 (4-9)

60 (4-9)

IC50

Reactive Blue 221

278 (2-4)

361 (2-4)

127 (4-10)

- (3-9)

IC20

Reactive Red 241

28 (5)

108 (4-6)

IC20

Reactive Yellow 176

78 (6)

176 (4-7)

IC20

Reactive Blue 221

112 (2-4)

158 (2-4)

According to the IC50 mean values, hepa-1 cells tolerated the highest dye concentrations. The spermatozoa cells were most sensitive and displayed toxicity with the lowest dye concentrations. Of the three cells used, HaCaT cells were of intermediate sensitivity also showing somewhat different responses to the different dyes. The results for all three dyes studied showed the same trends (I-III: Table 6). The IC20 mean values showed that with the reactive dyes studied, the hepa-1 cells had the highest tolerance (II-III: Table 6). The red dye was the most toxic to HaCaT cells and this value was even lower than the IC50 value for spermatozoa cells after the same 72 hours of exposure (Table 6). However, after 72 hours of exposure, the blue dye evoked such high toxicity to the spermatozoa cells that it was not possible to determine the IC50 value (I. Table 6). However, in HaCaT cells the blue dye was less toxic than the yellow dye (I-III: Table 6). Thus, the relative toxicity of the dye studied depended on which cells were used.

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Results 5.2 The toxicity of the fabric extracts In the present study, the plain cotton fabric was dyed with the same reactive dyes as above: Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221. In addition, a commercially obtained fabric dyed with an unknown brilliant yellow reactive dye was studied. The plain weave bleached cotton fabric samples which were used as controls were toxic when studied with hepa-1 and HaCaT cells. The protein contents of the samples measured as an indication of cell growth were found to be 35 - 50% of the non-exposed cells (100%) when studied with hepa-1 and HaCaT cells. However, the dyed fabrics were not toxic. In addition to the dyed fabrics, a commercial flame retarded fabric was studied. It was not toxic to these cell lines (IV: Figure 11).

Figure 11. The cytotoxicity of the dyed plain weave and commercial flame retardant fabrics. (IV).

Thus, according to the spermatozoa motility inhibition test and the cytotoxicity tests with hepa1 and HaCaT cells, none of the untreated fabrics studied were considered toxic. Over 50% of the spermatozoa cells retained motility (n=3-6). The protein contents in the extract samples indicated cell growth levels of over 80% compared to the non-exposed cells (100%) when hepa-1 and HaCaT cells were used (n=3: IV). The same fabrics after industrial dyeing and finishing were then analyzed. The spermatozoa cells did not show toxicity with the fabric extracts apart from the flame retardant fabric, which displayed low toxicity (n=3-11: IV). The fabric extracts from the commercial woven fabrics dyed with reactive dyes resulted in decrease in measured protein contents to less than 20% compared to the nonexposed cells when hepa-1 and HaCaT cells were used (IV). The extracts from the two types of untreated knitted fabrics were not toxic when assayed with the three different cell tests. The studied knitted fabrics did not display any toxicity in the spermatozoa inhibition test after commercial dyeing and finishing (n= 3-11, Figure 12: IV). However, after industrial dyeing and finishing, fabric extracts were toxic to the hepa-1 and HaCaT cells (n=3: Figure 12) though the yellow and red cotton fabrics were exceptions (IV). The yellow cotton was not toxic

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Kaisa Klemola: Textile Toxicity in the HaCaT- and the red cotton was not toxic in the hepa-1-cytotoxicity tests (IV). When the blue knitted fabrics were studied in hepa-1 cells the extracts evoked strong toxic effects compared to the situation with the same extracts in HaCaT cells. In addition, the blue knitted fabrics were most toxic to the hepa-1 cells (IV).

Figure 12. The percentage of protein in cell exposure by knitted fabric extracts compared to the nonexposed cells when hepa-1 and HaCaT cells were used (n=3: IV). * CO=cotton, CMD=modal

5.3 Reliability of the results The commercial fabrics in the study were industrially dyed and finished. These woven fabrics were not toxic when tested with hepa-1 and HaCaT cells. Before and after dyeing and finishing, the coefficients of variation (C of V) were 3 – 13% (n=3) (IV). The industrial untreated knitted fabrics showed C of V values of 4-13%. When the materials were industrially dyed and finished, most knitted fabrics were toxic to hepa-1 mouse cells and on HaCaT cells (n=3-4). The C of V values ranged from 2 –24% (IV). The spermatozoa motility test showed only the flame retardant fabric to be toxic. The industrial fabrics had C of V values of 0 – 18 % (n=3-11) (IV). Table 7. Coefficient of variation values (C of V %) for dye samples and fabric extracts. The fabrics were dyed with Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221 (I-III). toxic results

non-toxic results

spermatozoa (dye)

after 24h exposure

31-69

0-18

spermatozoa (dye)

after 72h exposure

0-58

9-37

hepa-1 (dye)

9-46

<10

HaCat (dye)

7-21

2-14

spermatozoa (fabric)

after 24h exposure

10-16

spermatozoa (fabric)

after 72h exposure

8-35

hepa-1 (fabric)

10-17

HaCat (fabric)

9-13

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Results For assays using spermatozoa cells, valinomycin was used as a positive control. The values covered the toxic and non-toxic areas. Two concentrations, 2ng/2ml and 4ng/2ml of valinomycin had no adverse effects after 24 hours exposure but 8 ng/2ml and 16 ng/2ml concentrations did cause toxicity. DMSO was not toxic at the concentrations used in the tests (I, IV). In the HaCaT and hepa-1 assays, non-exposed cells with medium were used as negative controls and all results were compared to them. As a positive control, 2,4-dinitrophenol (DNF) was used giving C of V values of 2 – 25%. High C of V values were noted in the toxic range. (II-IV). DNF was used as a positive control and the mean value of the protein content measured from cell suspensions after exposure at low concentrations was 80% of the protein content of the non-exposed cells (II-IV). The low control concentration of DNF (0,05mg/ml) resulted in 60% protein content (C of V 11-18%), while the highest control concentration of DNF caused severe inhibition of cell growth, only 10 % remaining of protein content of the control (C of V 2-25%: II-IV). 5.4 Statistical significance In the spermatozoa 24-h test, the difference in the results between the three studied reactive dyes was not statistically significant; for the high concentration of the dyes (196µg/ml), p = 0.763; for the low concentration (39µg/ml), p = 0.122. Hepa-1 cell test showed statistical significant differences compared to control at a concentration of 150µg/ml, p = 0.022, but not at the high concentration of the dyes (600µg/ml), p = 0.225. With HaCaT cells, the differences between the three studied dyes were statistically significant; for the concentration of 100µg/ml, p = 0.003; for 190 µg/ml, p = 0.002; for 380 µg/ml, p = 0.000.

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Kaisa Klemola: Textile Toxicity 6. DISCUSSION 6.1 Reactive dyes in the cell tests When the reactive dyes were studied in vitro (Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221), toxicity was detected with the spermatozoa cells at low dye concentrations. Spermatozoa are dependent on their surrounding environment for nutrients and also for removal of toxic end products since they have low levels of detoxifying enzymes in their cytosol. The physiological processes in spermatozoa are controlled by membrane potentials and ion fluxes (Mann and LutwakMann 1982) and therefore the spermatozoa cells may be useful for indicating acute toxicity: they can be assumed to give relevant information about toxicity (Seibert 1992, Hoonstra 2004). The cell lines have been widely used and different tests have been developed as an alternative for animal tests (http://www.ecvam.jrc.it). The IC50 and IC20 values for the above mentioned reactive dyes were higher with hepa-1 cells than with HaCaT cells. This can be explained by the ability of hepa-1 hepatoma mouse cells to metabolise xenobiotics (INVITTOX Protocol 112 1995). Induction of CYP1A1 (detected as arylhydrocarbon hydroxylase and 7-ethoxyresorufin o-deethylase, EROD activity) has been shown to be useful for the assessment of the biological potencies of pure chemicals like PCBs and for the estimation of dioxin-like compounds in extracts of environmental samples (Kennedy et al. 1993, Kennedy et al. 1995). Hepa-1 cells have been widely used as a model for studying the induction mechanism of CYP1A1 (Kärenlampi and Törrönen 1990, Kopponen et al. 1994a). Though metabolism of xenobiotics usually facilitates the excretion of chemicals, certain steps in the biotransformation pathway can be responsible for the activation of foreign chemicals to reactive intermediates that ultimately result in toxicity, carcinogenicity and other adverse effects. This has been clearly detected in aquatic species (Varanasi et al. 1987, Stegeman and Lech 1991). In this thesis, no enzyme activities were measured as the aim was simply to assess cell viability to reveal acute toxicity of the studied substances. Human keratinocytes have some ability to metabolise xenobiotics. However, the phase I metabolism in the skin is usually only a small fraction of that found in the liver. The enzyme cytochrome P4501A1 is inducible in the epidermis by agents that are inducers also in other tissues. Thus exposure to this kind of agent could influence skin biotransformation and even sensitize epidermal cells to other agents that are not good inducers themselves, a phenomenon observable in cell culture (Walsh et al. 1995). In addition, the enzymes participating in phase II metabolism are expressed in the skin. In general, this activity occurs at a much lower rate than that observed in the liver, but exceptions are evident, as in the case of quinone reductase (Khan et al. 1987). In this study, the aim was to obtain information about acute toxicity and therefore only the viability of the human keratinocyte cells was evaluated. The cytotoxicity tests with hepa-1 and HaCaT cells provided information about the toxicity of the substances studied, not information about their exact metabolic effects. When the dyes were studied, it seemed that hepa-1 cells tolerated higher dye concentrations than HaCaT cells, as reflected by the higher IC50 and IC20 values with hepa-1 cells than with HaCaT cells. This may be attributable to differences in the metabolic abilities of the cell lines. Since the various cells used in the present study possess different capabilities it is not unexpected

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Discussion that they exhibit some differences in the results obtained. However, when the dyes were studied similarities were noted in the end points and the results supported each other. This was evaluated via the determination of the IC20 and IC50 values. It is possible to determine which concentration of the particular dye evokes the first level of adverse effects and when the material is clearly toxic to the cells. Kopponen et al. (1997) studied reactive textile dyes using hepa-1 cells, but these dyes were not the same as those used in the present study. The IC50 values were in the range 53 –825µg/ml. In the Safety Data Sheets, these dyes had LD50 values of 2000 – 9000mg/kg. In the present study, all IC50 values with hepa-1 cells were in the range 158 - 392µg/ml (I-III). The Chemical Safety Data Sheets for the reactive dyes in this study indicate the blue and red dyes were more toxic than the yellow dye. In the Safety Data Sheet, the LD50 value (rats, orally) for the yellow dye is 5000mg/kg, and for the red and blue dyes 2000mg/kg. According to toxicity tests conducted with activated sludge, the toxicity of the blue dye measured as EC50 (the molar concentration of an agonist, which produces 50% of the maximum possible response for that agonist) was greater than 100µg/ml. In the present study, the IC50 values for the blue dye were 127µg/ml (spermatozoa cells, exposed for 24 hours), 361µg/ml (hepa-1 cells, exposed for 72 hours), 278µg/ml (HaCaT cells, exposed for 72 hours). When the spermatozoa cells were exposed to the blue dye for 72 hours, all cells displayed no motility. The IC20 values in the present study for the blue dye were between 112158µg/ml with HaCaT and hepa-1 cells. According to the OECD 209 method (an acute toxicity test for testing sludge), the red and yellow dyes had IC50 values higher than 1000µg/ml. According to the OECD 203 method (an acute fish toxicity test), the LC50 values were claimed to be in excess of 100µg/ml. In the present study, the IC50 mean values from the spermatozoa test and hepa-1 test for the yellow dye pointed to higher values than those obtained for the red and the blue dyes. The IC20 mean values with HaCaT cells were lower than 100 µg/ml for the red and the yellow dyes (exposed for 72 hours). In the present study, the IC20 mean values from the HaCaT test for the red and the yellow dyes indicated that these compounds may be more toxic than the corresponding values for toxicity published in the Chemical Safety Data Sheets. The dyestuffs used in the present study are, in fact, mixtures of different chemicals (I-IV). They contain, for instance, carboxymethylcellulose CMC, calcium stearate and other chemicals, but it is not possible to obtain information from the manufacturers about what other chemicals are present. It is possible that not only the dye molecules, but also the other chemicals may evoke adverse effects. In addition, it is possible that the dye formulations which were studied are not consistent. The dyes are monochlorotriazinyl dyes e.g. containing a sulphonyl-group, chlorine, fluorine and nitrogen, and the blue dye contains copper, but the precise chemical structures of the dye molecules are not available. This information would be useful in assessing the toxicity of dye molecules. The discussion of the results of this study relates to the toxicity of a mixture of different chemicals and not to the pure molecules. This study was not designed to evaluate the effects of a pure dye. The pH of the dyes is in the range 4,5 – 6,5 and it is evident that these pH values do not cause toxic effects to the cells. When hepa-1 and HaCaT cells were used, the growth medium was buffered (pH 7,1-7,2) and the samples diluted. The sample concentrations during the tests were low (less than 400µg/ml). When the spermatozoa cells were used, the concentrations of the samples were lower

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Kaisa Klemola: Textile Toxicity than those incubated with the hepa-1 and HaCaT cells. In the textile dyeing process, the dye bath is alkaline because reactive dyes demand a high pH for good reactivity. It can therefore be assumed that the dyes may be more toxic during the dyeing process than during the conditions of the present series of studies. 6.2 The fabric extracts in the cell tests Reactive dyes bind to the fibre covalently (Trotman 1984). The binding is very stable and during the process, the dye molecule loses its reactivity. Although the extracts of some fabric samples contained colour, the extracts were not toxic. This is because dye molecules are easily hydrolysed in water. In the present study, the plain cotton fabric (bleached) became less toxic after the dyeing process. It can be assumed that washing and dyeing must have removed harmful chemicals (I-IV). Although reactive dyes themselves cause adverse effects like asthma, rhinitis and dermatitis (Estlander 1988, Nilsson et al. 1993, Hatch and Maibach 1995, Manzini et al. 1996, Wilkinson and McGechaen 1996), after reacting with fibre molecules, the dyes are stable and should not be toxic within the fabric. However, any unbounded dye remaining in the fabric could cause allergic reactions. Since reactive dyes and their hydrolysis products are water-soluble, unbounded dye can be washed off. Therefore, washing of newly dyed products is recommended (Moreau and Goossens 2005). However, more information is needed about the overall toxicity of reactive dyes and dyed fabrics. The fabrics to be dyed in the present study were more toxic in the HaCaT cell tests than they were in the hepa-1 cells. The fabric itself, before it was dyed, was toxic. The protein contents of the samples taken as indications of cell growth were in the range 35-50% of control. However, after dyeing, the material was no longer toxic with cells demonstrating over 80% protein content of control. This indicates that for these reactive dyes studied, the end products of the dyeing process do not cause toxic effects in cell tests: i.e. the fabric became non-toxic. In addition, all data obtained in the spermatozoa motility tests confirmed these non-toxic responses (IV). The commercial untreated industrial woven and knitted fabrics were not toxic (the protein content of the cells did not decline to any significant degree, i.e. less than 20 %). After the industrial dyeing and finishing processes, the flame retardant fabric displayed low toxicity when incubated with the spermatozoa cells. The other commercial woven fabrics were not toxic. However, after industrial dyeing and finishing, the knitted fabrics evoked adverse effects in hepa-1 and HaCaT cells (protein contents of cells were considerably below 80% of control with the exception of the yellow cotton in HaCat cells and the red cotton in hepa cells), evidence that some harmful chemicals were present. The knitted fabrics were coloured with reactive dyes. According to the present study, these agents should not produce adverse effects in the fabrics. It has been observed that some fabric softeners may cause adverse effects in fish (Wester and Roghain 1992). It is also well known that free formaldehyde in textiles can cause toxicity (Priha 1992, 1995, Priha et al. 1988, 1996). However, in the present study there was no information available about what kind of different chemicals were used when the knitted fabric was manufactured. It is usual to treat knitted fabrics with softeners, so it can be assumed that the softeners may have caused some toxic effects. In addition, it is possible that the

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Discussion studied material contains chemicals for binding the extra dye to the fibre to improve the wet-fastness. In addition, residues of harmful nonylphenols (surfactants; 2001/838/EY) have been found. However, in the present study with the knitted fabrics, it remained unclear which chemical or combinations of chemicals produced the adverse effects on the cells. In this study, it was interesting to note that hepa-1 mouse cells were more sensitive to toxic knitted fabrics than human keratinocyte HaCaT cells. This was opposite to the anticipated results. The knitted fabrics produced a particularly clear toxic effect on hepa-1 cells, although these cells would have been expected to be resistant since they possess better metabolic capabilities than HaCaT cells. 6.3 In vitro cell tests for assaying textile substances The cells in culture usually grow well, and in the present tests the quality of the cells was standardized. However, there were differences in the quality of the spermatozoa cells used in the present assays and this affected their motility. In some assays, a substantial number of cells were broken, while in others, a significant proportion had good motility. In the cell viability experiments, it was noted that the cell growth at the edges of the plates was often less impressive than in the middle. However, the exposure results in every cell culture assay as in the spermatozoa motility assay were compared to those of the non-exposed cells, avoiding possible errors due to differences in the quality of the cells. Since the evaluation of the spermatozoa motility inhibition is subjective, this test is a qualitative test, and variations may occur in the results. However, in the present study, several researchers studied the same samples and the spread of their results was within 5%. The results of the spermatozoa test are read within 5% bands and this can have an extra effect on the variation inherent in the results. However, despite these limitations, the test can be used reliably for screening samples and when combined with other tests: the inhibition of boar spermatozoa motility has proved valuable when evaluating the toxicity of food constituents (Salkinoja-Salonen et al. 1999). In addition, the test has demonstrated its value as a cell toxicity assay in detecting hazardous substances in products without the need for whole-animal exposure or foetal calf serum for cell cultures (Hoornstra et al. 2004). The present study indicated that the spermatozoa test is also valuable for studying the potential toxicity of textile substances. However, further development will be necessary when fabric extracts are going to be studied. When hepa-1 mouse cell line and human keratinocyte HaCaT cell line were used, the total protein contents were measured to indicate the viability and proliferation capacity of the cells. However, this is not a very exact method for assessing the viability of the cells, since the protein content of some dead cells may be also measured even though the washing procedure should remove the dead (unattached) cells. There is also the possibility that some living cells are lost before protein measurements during the washing procedures of the plates. However, protein measurement is a widely used procedure and it does provide some information about the toxicity of the studied material. The hepa1 cell test has been found to be sensitive and reliable (INVITTOX Protocol 112 1995, Kopponen et al. 1992, Törrönen et al. 1994, Kopponen et al. 1997). In the present study, these cell cultures were sensitive to the toxic effects of the compounds in the textiles under study. The NR test is useful if one wishes more exact information about the cells (INVITTOX Protocol 64

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Kaisa Klemola: Textile Toxicity 1992). Mitochondrial activity can be studied by MTT and WST-1 assays (Toimela and Tähti 2004, Kwang-Mahn et al. 2005). In addition, cell energy and membrane effects have been studied by ATP and LDH measurements (Toimela and Tähti 1995, Kakko et al. 2004). However, in the present study, the methods to study overall toxicity of textile substances were the focus of interest and more detailed information about mechanism of toxicity in the cells was not the target of the study. The fabric sample extracts were filtered through polyester. Polyester cannot bind covalently to dye molecules. Some extracts were coloured, since they contained free dye molecules that had passed through the filter. However, the results did not show any adverse effects. The dye molecules are hydrolysable and hence lose their reactivity in aqueous solutions (Trotman 1984). The fabric extracts were not concentrated. The concentrations of the samples were the same for the hepa-1 and HaCaT cell assays, but the spermatozoa cell concentration was lower, as this was a requirement of the assay method. The fabric extracts were not concentrated because that process may have produced chemical reactions during the procedure, possibly affecting their toxicity. The extraction method for the spermatozoa motility test needs further development. The fabrics may contain compounds that are lipid soluble. The physical property that commonly enables xenobiotics to be absorbed through the cell membrane is the lipophilicity (Gregus and Klaassen 2001). Therefore, in the future, it will be important also to use lipophilic solvents when extracting fabric materials. 6.4 The reliability of the tests The coefficient of variation was typically high when cell toxicity was present. This is understandable when living material is being analysed. However, the limiting values for toxicity and non-toxicity were clearly identified. When the three reactive dyes were compared to each other, the results of the spermatozoa test (exposed for 24 h) did not reach statistical significance (p > 0.05). Statistical significance in hepa-1 cells was observed when the concentrations of the dyes were low. In HaCaT cells, all concentrations of the tested dyes resulted in statistically significant changes. It is clear that there is a need to develop further the spermatozoa test, for instance by studying a fuller range of fabric extracts. It has to be emphasised that the spermatozoa motility test is a qualitative test. In addition, the quality of the spermatozoa cells can lead to variations in the final results. The spermatozoa motility inhibition test is only suitable for crude screening of chemical samples. According to the C of V values in this study (0-18%), it seems that the optimal exposure time is 24 hours since this results in the lowest C of V values. In the hepa-1 and HaCaT tests, further development may lead to better reproducibility; e.g. it may be useful to expose cells in the medium that contains a lower concentration of serum. The high concentrations of the serum used may cause mistakes since it may be able to bind certain molecules. All three methods must therefore be developed and validated further if they are to be used routinely. For the standardization of the tests, the range of substances should be extended and the validation of the test should be made in co-operation with other laboratories. However, the present study shows that these tests are promising novel methods for studying the toxicity of components on textiles.

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Discussion 6.4 The possibilities for utilizing cell-based tests for studying textile substances Improvements have occurred not only in the working conditions in the textile industry but also in the environmental-friendliness of industrial dyeing-finishing processes (Nousiainen 1997). In addition, the ecology of laundry services (Kalliala 1997) has been studied. Some information about the impacts of waste waters from textile processing is available. However, more advanced studies in the area of toxicology are needed, especially using a range of cell tests, since there is a scarcity of research providing information for the consumer about the potential toxicity of textile end-products. The Öko-Tex-100 textile standard assesses the amounts of certain harmful compounds in textile products related to prescribed limits, for instance heavy metals (Öko-Tex Standard 100 1997). However, this standard does not require that common biological tests have to be carried out to detect any adverse effects of the products, nor have the combined effects of different chemicals been studied. In addition, fabrics may contain chemicals that are not assessed in the Öko-Tex-100 standard, so the cell tests represented here are useful in confirming the safety of the product. Safety Data Sheets show information about the toxicity of individual chemicals. The EU new chemical legislation (REACH, The Registration, Evaluation and Authorisation of Chemicals) demands that the safety of 30000 industrial chemicals should be evaluated by the year 2018. The evaluations of 20000 of these chemicals have been planned to be performed without animal experiments by using all information available and also by increasing the use of validated alternative tests. The cell tests can provide extra information to the standard tests. It is also recommended that more tests that do not involve the use of animals should be devised, although subchronic and chronic effects with animals may still needed in the foreseeable future. Cell tests can be used not only for pure chemicals but also for testing materials containing different chemical components. In vitro cytotoxicity tests provide preliminary information about acute toxicity and other parameters. One of the most sensitive biochemical responses is the induction of specific cytochrome P450 (CYP) enzymes. It would be interesting to study further in more detail the specific effects of textile dyes and fabric extracts, for instance their effects on mitogen activated protein kinase pathways and other cell signalling pathways in human keratinocytes. The effects of organochlorine pesticides have been studied using HaCaT cells (Ledirac et al. 2005) and it would be useful to use HaCaT cells in toxicity tests with different textile chemicals. Another approach would be to use macrophages to evaluate the effects of the compounds on immunological activity. In addition, it would be of interest to measure the energy metabolism of the cells, e.g. the ATP balance in cells exposed to different textile chemicals. Cell proliferation studied by MTT and WST-1 assays may be useful when more detailed information about the effects of textile substances needs to be obtained. In the European Centre for the Validation of Alternative Methods, ECVAM, skin models have been validated and endorsed as suitable for testing skin corrosion and irritation in vitro. Two skin models have been validated: the EPISKIN model and the EpiDerm model (EU, ECVAM 2007, Grindon et al. 2007, El Ghalbzouri et al. 2008). The EPISKIN model is a three-dimensional human skin model, formed from a bovine collagen matrix, the surface with a type of human collagen (Valerie et al. 2005). The EpiDerm model consists of a multi-layered, differentiated, in vitro model of the human epidermis (Valerie et al. 2005). Textile materials are often in contact with the skin. Thus, in the fu-

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Kaisa Klemola: Textile Toxicity ture studies of textile substances it is recommended that these skin models should be used. In vitro cytotoxicity tests can also be used if wastewaters need to be studied. There have been many investigations for developing cleaning techniques in wastewaters emerging from the textile industry: e.g. the ability of microorganisms to carry out dye decolorization (Banat et al. 1996, Marquez and Costa 1996, Khan and Husain 2007, Asgher et al. 2007), aerobic/anaerobic treatments (Liakou et al. 1997, Ciardelli et al. 2001, Frijters et al. 2006), ion exchange methods (Lin and Chen 1997) and coalabsorption (Santhy and Selvapathy 2005, Dincer et al. 2007). The toxicity of wastewaters has been evaluated with different techniques, for instance, with the luminescent bacterium Vibrio Fischeri (Wang et al. 2002). The cell tests used in the present study represent assays that could also provide information about the toxicity of wastewater. In vitro cell tests are useful when one wishes to study overall cell toxicity. The manufacturing of textiles is a global activity and often virtually nothing is known about the chemicals used. The data is also difficult to obtain since in most cases this information is regarded as a trade secret. However, some residues of the chemicals used may remain in the textile products causing health problems for some consumers. Cell tests are effective if materials with unknown chemical contents need to be studied and thus they are suitable for studying textile materials. The toxicity of the textile material which was found in this study may be attributable to some individual chemical or to the combined effects of several chemicals. In quality control laboratories, biological cell tests can provide simple and rapid methods to evaluate the potential toxicity of textile products. In the future, it could be possible to develop a new labelling scheme for textile materials, related to the Öko-Tex standard 100, based on measured biological effects. For consumers, such labelling could provide information about the safety of the product. In addition, cell tests could be useful in developing ecologically sound textile processes, resulting in a labelling scheme analogous to the Öko-Tex standard 100 used today by some textile manufacturers.

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Conclusions 7. CONCLUSIONS In the present series of studies, three in vitro cell tests were used to assess the acute toxicity or potential adverse effects of textile reactive dyes and reactive dyed fabrics. All cell tests showed clearly that the three reactive dyes (Reactive Red 241, Reactive Yellow 176, Reactive Blue 221) were toxic, but extracts from reactive dyed fabrics were not toxic even when they had been coloured with a dye which on its own was toxic. The dye in the extracts had been apparently hydrolysed and therefore had not the same level of reactivity as the intact dye. After dyeing with reactive dyes, a previously toxic textile material was no longer toxic apparently because during the dyeing process, harmful chemicals must have been removed or the material had become inactivated in some other way, for instance by the chemicals losing their reactivity. Before industrial finishing, the commercial knitted fabrics studied did not evoke any adverse effects in the cells. After dyeing and finishing, some commercial fabrics did cause toxicity. Since these fabrics had been dyed with reactive dyes (which are not toxic in the dyed fabric) it is hypothesized that the toxicity is caused by some unknown chemical(s) used in finishing. In the hepa-1 and HaCaT cell tests, the same sample concentrations were used, whereas the concentration was lower in the spermatozoa test. This latter test needs further refinement to be of practical use for analysing fabrics. The spermatozoa inhibition test is still a qualitative, rapid test and may be used for screening of samples. Human keratinocyte HaCaT cells were more sensitive than hepatoma hepa-1 liver cells when incubated with the dyes and reactive dyed fabric extracts were. This might be attributable to the fact that liver cells have a good capability to metabolise different substances. There were no major differences in the toxicities of the three studied reactive dyes when the dye concentration was low with hepa-1 cell line. However, all tested concentrations of these dyes evoked statistically significant effects on the viability of HaCaT cells. These facts support the need for using a combination of different cell lines in parallel to provide comprehensive information about toxicity. The in vitro cell tests can clearly reveal acute toxicity. However, caution is required not to overestimate the capabilities of these cells to display all responses which can be extrapolated to humans. These tests were found to be suitable when materials such as textiles with unknown components need to be analysed and this study shows that they may be valuable in forming the basis for developing routine tests for use by the textile industry.

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Kuopio University Publications C. Natural and Environmental Sciences

C 218. Madetoja, Elina. Novel process line approach for model-based optimization in papermaking. 2007. 125 p. Acad. Diss. C 219. Hyttinen, Marko. Formation of organic compounds and subsequent emissions from ventilation filters. 2007. 80 p. Acad. Diss. C 220. Plumed-Ferrer, Carmen. Lactobacillus plantarum: from application to protein expression. 2007. 60 p. Acad. Diss. C 221. Saavalainen, Katri. Evaluation of the mechanisms of gene regulation on the chromatin level at the example of human hyaluronan synthase 2 and cyclin C genes. 2007. 102 p. Acad. Diss. C 222. Koponen, Hannu T. Production of nitrous oxide (N2O) and nitric oxide (NO) in boreal agricultural soils at low temperature. 2007. 102 p. Acad. Diss. C 223. Korkea-aho, Tiina. Epidermal papillomatosis in roach (Rutilus rutilus) as an indicator of environmental stressors. 2007. 53 p. Acad. Diss. C 224. Räisänen, Jouni. Fourier transform infrared (FTIR) spectroscopy for monitoring of solvent emission rates from industrial processes. 2007. 75 p. Acad. Diss. C 225. Nissinen, Anne. Towards ecological control of carrot psyllid (Trioza apicalis). 2008. 128 p. Acad. Diss. C 226. Huttunen, Janne. Approximation and modellingerrors in nonstationary inverse problems. 2008. 56 p. Acad. Diss. C 227. Freiwald, Vera. Does elevated ozone predispose northern deciduous tree species to abiotic and biotic stresses? 2008. 109 p. Acad. Diss. C 228. Semenov, Dmitry. Distance sensing with dynamic speckles. 2008. 63 p. Acad. Diss. C 229. Höytö, Anne. Cellular responses to mobile phone radiation: proliferation, cell death and related effects. 2008. 102 p. Acad. Diss. C 230. Hukkanen, Anne. Chemically induced resistance in strawberry (Fragaria × ananassa) and arctic bramble (Rubus arcticus): biochemical responses and efficacy against powdery mildew and downy mildew diseases. 2008. 98 p. Acad. Diss. C 231. Hanhineva, Kati. Metabolic engineering of phenolic biosynthesis pathway and metabolite profiling of strawberry (Fragaria × ananassa). 2008. 80 p. Acad. Diss. C 232. Nissi, Mikko. Magnetic resonance parameters in quantitative evaluation of articular cartilage: studies on T₁ and T₂ relaxation time. 2008. 83 p. Acad. Diss.