In Vitro Chromosomal Aberrations

Final Report Study Title

Screening Assay for Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells with Argentyn 23

Author(s)

Hemalatha Murli, PhD

Sponsor

Natural Immunogenics Corporation 7440 S.W. 50th Terrace, Unit 107 Miami, Florida 33155

Test Facility

Covance Laboratories Inc. 9200 Leesburg Pike Vienna, Virginia 22182-1699

Covance Study Number

24742-0-437SC

Report Issued

08 April 2003

Page Number

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Covance 24742-0-437SC

STUDY INFORMATION 1. Sponsor:

Natural Immunogenics Corporation

2. Test Article:

Argentyn 23, A Professional Colloidal Silver Formulation 23ppm concentration Ultra-fine Ag+dispersion, Lot No.:86HT 30 January 2003 Transparent, colorless liquid Room temperature

Date Received: Physical Description: Storage Conditions: 3. Type of Assay: Protocol Number: Covance Study No.:

Screening Assay for Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells 437SC, Edition 1, (+/-S9) 24742-0-437SC

4. Study Dates: Study Initiation Date: 27 January 2003 Experimental Start Date: 04 February 2003 Experimental Termination Date: 20 March 2003 INTRODUCTION At the request of Natural Immunogenics Corporation, Covance investigated the ability of Argentyn 23 to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation. The assay was initiated both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from AroclorTM-induced rat liver (S9). Most known chemical clastogens (chromosome-breaking agents) require a period of DNA synthesis to convert initial DNA damage into chromosome alterations visible at mitosis. At predetermined intervals after exposure to the test article, the cells were treated with a metaphase-arresting substance, Colcemid , then harvested and stained, and metaphase cells were analyzed microscopically for the presence of chromosomal aberrations. Many mutagenic chemicals do not act directly on DNA but do so after being converted to active inter-mediates by enzymes found in liver. CHO cells have little or no capacity to metabolize test articles, so an exogenous metabolic activation system (rat liver S9 homogenate) was included with a series of treatments to enhance the degree of conversion and the ability of the assay to detect clastogenic, metabolic intermediates. This study evaluated structural chromosomal aberrations (defined as structural chromosome damage expressed as breakage, or breakage followed by reunion, of both sister chromatids at an identical site). Numerical aberrations (a change in the number of chromosomes from the modal number of 21 for the CHO cell line used in this assay) are

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not determined by this protocol. However, the occurrence of polyploidy or endoreduplication, which was scored, may indicate that the test article has the potential to induce numerical aberrations. The in vitro metabolic activation system (Maron and Ames, 1983) consisted of S9 and an energy-producing system (NADP plus isocitric acid). Various hepatic P450 isoenzyme levels were increased by treatment of the rats with AroclorTM 1254 (single concentration of 500 mg/kg) and sacrificed 5 days later (Molecular Toxicology, Inc., Lot No. 1393). The S9 fraction, prepared in potassium chloride, was retained frozen at ≤-60°C until use. Aliquots of S9 were thawed immediately before use and added to the other components to form the activation system described as follows: S9 Activation System Component Concentration in Cultures NADP (sodium salt) 1.5 mg/mL (1.8 mM) Isocitric acid 2.7 mg/mL (10.5 mM) Homogenate (S9 fraction) 15.0 µL/mL* (1.5%) *This concentration of rat S9, obtained from Molecular Toxicology Inc., Boone, NC, has consistently caused cyclophosphamide to be highly clastogenic. The CHO cell line was derived from an ovarian biopsy of a female Chinese hamster. The Chinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent cell line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells were subsequently subcloned in this laboratory, and stock cultures stored in liquid nitrogen. The CHO-WBL subclone is a permanent cell line with an average cycle time of 12 to 14 hours and a modal chromosome number of 21. The CHO cells were grown in McCoy’s 5a culture medium which was supplemented with .10% heat-inactivated fetal bovine serum (FBS), L-glutamine (2mM), penicillin G (100 units/mL), and streptomycin (100 µg/mL). Single cultures were used for each dose of the test article. Cultures were incubated with loose caps in a humidified incubator at 37°C ± 2°C in an atmosphere of 5% ± 1.5% CO2 in air. The dose rangefinding assay was conducted with a ~3.0-hour treatment period in the presence of S9, and ~20.0-hour treatment period without S9. All cultures were harvested ~20.0 hours from the initiation of treatment. This harvest time corresponds to 1.5 times the cell cycle time of approximately 13 hours (Galloway et al., 1994). The chromosomal aberrations assay was conducted with a ~3.0-hour treatment period in the presence of S9, and ~20.0-hour treatment period without S9. All cultures were harvested ~20.0 hours from the initiation of treatment. This harvest time corresponds to 1.5 times the cell cycle time of approximately 13 hours (Galloway et al., 1994).

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RESULTS Test Article Handling The dosing solutions were prepared in dimethylsulfoxide (DMSO; Acros Organics, Lot No. A017325001). Argentyn 23 was solubilized in DMSO at a stock concentration 100-fold higher than the dose in tissue culture medium. Lower doses were obtained by serial dilutions of the stocks with DMSO. A dose volume of 10.0 µL/mL was used. A summary of the treatment times is given below. Summary of Rangefinding/Chromosomal Aberrations Assay Treatment Schedule in Hours (Approximate) Activation Condition − S9 + S9

Test Article Added 0 0

Wash --3.0

Colcemid Added 18.0 18.0

Harvest Started 20.0 20.0

In the dose rangefinding assay, concentrations of 7.81, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 µg/mL were tested with and without S9. No toxicity was observed in the cultures treated with 1000 µg/mL tested with and without S9 (Tables 1 and 2). The chromosomal aberrations assay was conducted with concentrations of 2000, 3500, and 5000 µg/mL with and without S9. In the chromosomal aberrations assay without S9, no toxicity was observed in any of the test cultures (Table 3). Structural chromosomal aberrations were evaluated at 5000 µg/mL (Table 2). No significant increase in the number of cells with structural chromosomal aberrations, polyploidy, or endoreduplication was observed. In the chromosomal aberrations assay with S9, no toxicity was observed in any of the test cultures (Table 5). Structural chromosomal aberrations were evaluated at 5000 µg/mL (Table 6). No significant increases in the number of cells with structural chromosomal aberrations, polyploidy, or endoreduplication was observed. CONCLUSION Argentyn 23 was considered negative for inducing structural chromosomal aberrations, polyploidy, or endoreduplication with and without metabolic activation.

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REFERENCES Evans, H.J., Chromosomal aberrations produced by ionizing radiation. International Review of Cytology, 13:221-321 (1962). Evans, H.J., Cytological Methods for Detecting Chemical Mutagens. Chemical Mutagens, Principles and Methods for their Detection, Hollaender, A. (ed.), Vol. 4, pp. 129, Plenum Press: New York and London (1976). Galloway, S.M., Aardema, M.J., Ishidate, M., Jr., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., and Sofuni, T., Report from working group on in vitro tests for chromosomal aberrations. Mutation Research, 312(3):241-261 (1994) . Maron, D.M., and Ames, B.N., Revised methods for the Salmonella mutagenicity test. Mutation Research, 113:173-215 (1983). Thakur, A.J., Berry, K.J., and Mielke, P.W., Jr., A FORTRAN program for testing trend and homogeneity in proportions. Computer Programs in Biomedicine, 19:229-233 (1985).

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Table 1: Assessment of Toxicity for Chromosome Aberrations Assay - Without Metabolic Activation ~20 Hour Treatment, ~20 Hour Harvest Assay No.: 24742 Test Article: Argentyn 23

Vehicle Control Test Article

Trial No.: A1

Treatment DMSO

Date: 02/25/03

10.0 µL/mL 1000 µg/mL

a

Lab No.: CY010803

Confluencea % Vehicle Control

% Mitotic Index

% Mitotic Reduction

100

10.1

0

100

10.8

0

This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells. DMSO = dimethylsulfoxide

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Table 2: Assessment of Toxicity for Chromosome Aberrations Assay - With Metabolic Activation ~3 Hour Treatment, ~20 Hour Harvest Assay No.: 24742 Test Article: Argentyn 23

Vehicle Control Test Article

Trial No.: A1

Treatment DMSO

Date: 02/25/03

10.0 µL/mL 1000 µg/mL

a

Lab No.: CY010803

Confluencea % Vehicle Control

% Mitotic Index

% Mitotic Reduction

100

12.1

0

100

12.6

0

This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells. DMSO = dimethylsulfoxide

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Table 3: Assessment of Toxicity for Chromosomal Aberrations Assay - Without Metabolic Activation ~20 Hour Treatment, ~20 Hour Harvest Assay No.: 24742 Test Article: Argentyn 23

Trial No.: B1

Date: 02/25/03

Confluencea

Vehicle Control Test Article

Treatment DMSO

Lab No.: CY012803

% Vehicle Control

% Mitotic Index

% Mitotic Reduction

100

10.0 µL/mL 2000 µg/mL 3500 µg/mL

10.4

0

100

14.4

0

100

16.2

0

5000 µg/mL

100

14.2

0

a

This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells. DMSO = dimethylsulfoxide

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Table 4: Chromosome Aberrations in Chinese Hamster Ovary Cells - Without Metabolic Activation ~20 Hour Treatment, ~20 Hour Harvest Assay No.: 24742

Trial No.: B1

Date: 02/25/03

% # MITOTIC ENDOINDEX REDUPLICELLS REDUC- CATED SCORED TION a CELLS CONTROLS VEHICLE:

DMSO

POSITIVE:

MMC

TEST ARTICLE

10.0µL/mL

A 100 B 100 TOTAL 200 AVERAGE %

0 0

2 7

0.0

4.5

0 0

6 4

-

0.0

5.0

0

0 0.0

5 5.0

0

0.400 µg/mL

A 50 B 50 TOTAL 100 AVERAGE %

100 5000µg/mL AVERAGE %

# POLYPLOID CELLS

Lab No.: CY012803

JUDGEMENT (+/-) b

Test Article: Argentyn 23

NUMBERS AND PERCENTAGES (%) OF CELLS SHOWING STRUCTURAL CHROMOSOME ABERRATIONS Simple TOTALS c Gaps chte chre mab -g +g Breaks 2 2 4 2.0

-

3 2 5 5.0

15 15 30 30.0

20 20 40 40.0

-

chte: chromatid exchange chre: chromosome exchange mab: multiple aberrations, greater than 4 aberrations a % Mitotic index reduction as compared to the vehicle control. b Significantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01. c -g = # or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps. d Significantly greater in -g than the vehicle control, p ≤ 0.01. DMSO = Dimethylsulfoxide MMC = Mitomycin C

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13 3 16 16.0

JUDGEMENT (+/-) d

0 0 0 0.0

2 2 4 2.0

38 30 68 68.0

39 31 70 70.0

+

0 0.0

0 0.0

-

Covance 24742-0-437SC

Table 5: Assessment of Toxicity for Chromosomal Aberrations Assay - With Metabolic Activation ~3 Hour Treatment, ~20 Hour Harvest Assay No.: 24742 Test Article: Argentyn 23

Trial No.: B1

Date: 02/25/03

Confluencea

Vehicle Control Test Article

Treatment DMSO

Lab No.: CY012803

% Vehicle Control

% Mitotic Index

% Mitotic Reduction

100

10.0 µL/mL 2000 µg/mL 3500 µg/mL

14.0

0

100

14.1

0

100

15.4

0

5000 µg/mL

100

15.2

0

a

This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells. DMSO = dimethylsulfoxide

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Table 6: Chromosome Aberrations in Chinese Hamster Ovary Cells - With Metabolic Activation ~3 Hour Treatment, ~20 Hour Harvest Assay No.: 24742

Trial No.: B1

Date: 02/25/03

% # MITOTIC ENDOINDEX REDUPLICELLS REDUC- CATED SCORED TION a CELLS CONTROLS VEHICLE:

POSITIVE:

DMSO

CP

TEST ARTICLE

10.0µL/mL

A 100 B 100 TOTAL 200 AVERAGE %

0 0

6 9

0.0

7.5

0 0

4 6

-

0.0

5.0

0

1 1.0

5 5.0

0

12.5µg/mL

A 50 B 50 TOTAL 100 AVERAGE %

100 5000µg/mL AVERAGE %

# POLYPLOID CELLS

Lab No.: CY012803

JUDGEMENT (+/-) b

Test Article: Argentyn 23

NUMBERS AND PERCENTAGES (%) OF CELLS SHOWING STRUCTURAL CHROMOSOME ABERRATIONS Simple TOTALS c Gaps chte chre mab -g +g Breaks 1 1 0.5

-

6 8 14 14.0

14 10 24 24.0

13 19 32 32.0

1 1 1.0 1 1.0

chte: chromatid exchange chre: chromosome exchange mab: multiple aberrations, greater than 4 aberrations a % Mitotic index reduction as compared to the vehicle control. b Significantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01. c -g = # or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps. d Significantly greater in -g than the vehicle control, p ≤ 0.01. DMSO = Dimethylsulfoxide CP = Cyclophosphamide

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15 15 30 30.0

JUDGEMENT (+/-) d

0 0 0 0.0

1 0 1 0.5

36 38 74 74.0

40 42 82 82.0

+

1 1.0

1 1.0

-

Covance 24742-0-437SC

HISTORICAL CONTROL DATA CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS, ~20 HOUR HARVEST - 1/2002 THROUGH 06/2002

Activation Negative Control 3 Hour Treatment

% -g

% +g

% Polyploid Cells

% Endoreduplicated Cells

Without

MIN MAX AVG SD (±) N

0.0 2.0 0.8 0.69 23

0.0 5.0 2.0 1.38 23

0.0 8.0 2.4 2.23 23

0.0 1.0 0.1 0.27 23

Without

MIN MAX AVG SD (±) N

0.0 1.5 0.4 0.43 23

0.0 6.5 2.0 1.71 23

0.0 7.0 2.2 2.19 23

0.0 1.0 0.1 0.31 23

Positive Control - MMC 3 Hour Treatment

Without

MIN MAX AVG SD (±) N

41.0 97.0 70.9 14.14 23

44.0 97.0 74.4 13.11 23

0.0 7.0 2.3 2.18 23

0.0 4.5 0.5 0.98 23

Negative Control Continuous Treatment

Without

MIN MAX AVG SD (±) N

0.0 2.0 0.6 0.60 25

0.5 7.5 2.4 1.50 25

0.0 12.0 2.2 2.80 25

0.0 2.0 0.2 0.50 25

Without

MIN MAX AVG SD (±) N

0.0 3.0 0.8 0.66 25

0.0 6.0 2.5 1.46 25

0.0 6.0 1.7 1.86 25

0.0 2.0 0.3 0.48 25

Positive Control - MMC Continuous Treatment

Without

MIN MAX AVG SD (±) N

30.0 100.0 60.3 19.59 25

33.0 100.0 63.9 18.73 25

0.0 8.5 2.2 2.01 25

0.0 1.0 0.2 0.31 25

Negative Control 3 Hour Treatment

With

MIN MAX AVG SD (±) N

0.0 3.0 0.8 0.87 42

0.0 8.0 2.4 1.99 42

0.0 12.0 2.2 2.82 42

0.0 2.5 0.5 0.78 42

With

MIN MAX AVG SD (±) N

0.0 3.0 0.6 0.80 43

0.0 7.0 2.0 1.64 43

0.0 10.5 2.2 2.31 43

0.0 2.0 0.3 0.46 43

With

MIN MAX AVG SD (±) N

15.6 74.0 42.7 13.59 43

19.4 75.0 47.9 12.78 43

0.0 9.5 1.8 1.99 43

0.0 3.5 0.8 0.92 43

Vehicle Control (Pooled) 3 Hour Treatment

Vehicle Control (Pooled) Continuous Treatment

Vehicle Control (Pooled) 3 Hour Treatment

Positive Control - CP 3 Hour Treatment

N = Number of trials MMC = Mitomycin C CP = Cyclophosphamide -g = % of cells with chromosome aberrations +g = % of cells with chromosome aberrations + % of cells with gaps

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DEFINITIONS OF CHROMOSOMAL ABERRATIONS FOR GIEMSA STAINED CELLS g (GAPS) chtg Chromatid Gap:

("tid gap"). An achromatic (unstained) region in one chromatid with clear discontinuity with no visible connecting material, the size of which is equal to or less than the width of a chromatid.

chrg Chromosome Gap:

("isochromatid gap, IG"). Same as chromatid gap but at the same locus in both sister chromatids.

SIMPLE BREAKS chtb Chromatid Break:

An achromatic region in one chromatid, larger than the width of a chromatid with clear partial or complete displacement.

chrb Chromosome Break:

Chromosome has a clear break, forming an abnormal (deleted) chromosome with an acentric fragment that is dislocated.

ace Acentric Fragment:

ace is different from the chrb only in that it is not apparently related to any specific chromosome.

COMPLEX EXCHANGES chte (chromatid exchange) id Interstitial Deletion:

tr Triradial:

Length of chromatid "cut out" from midregion of a chromatid resulting in a small fragment or ring lying beside a shortened chromatid or a gap in the chromatid. An exchange between two chromosomes, or one chromosome and an acentric fragment, which results in a three-armed configuration.

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COMPLEX EXCHANGES (CONTINUED) trf Triradial Fragment:

A three-armed configuration with a fragment.

qr Quadriradial:

As triradial, but resulting in a four-armed configuration.

ci Chromosome Intrachange:

Exchange within a chromosome; e.g., a ring that does not include the entire chromosome.

su Sister Union:

Intra-arm interchange with sister union of broken ends.

nud Non-union Distal:

Intra-arm interchange with non-union of broken ends distally.

nup Non-union Proximal:

Intra-arm interchange with non-union of broken ends proximally.

cx Complex Rearrangement:

An exchange among more than two chromosomes or fragments, which is the result of several breaks.

chre (chromosome exchange) dic Dicentric:

An exchange between two chromosomes, which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as dicf.

dicf Dicentric Fragment:

Dicentric with fragment.

r Ring:

A chromosome, which forms a circle, containing a centromere. This is often associated with an acentric fragment in which case it is classed as RF.

rf Ring Fragment:

Ring with associated acentric fragment.

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OTHER ab Abnormal:

Abnormal monocentric chromosome. This is a chromosome whose morphology is abnormal for the karyotype, and often the result of a translocation, pericentric inversion, etc. Classification used if abnormality cannot be ascribed (e.g., a reciprocal translocation).

mab Multiple Aberrations:

A cell, which contains more than 4 aberrations. A heavily damaged cell should be analyzed to identify the types of aberrations and may not actually have >4 (e.g., multiple fragments such as those found associated with a tricentric).

POLYPLOID CELLS poly Polyploid cell:

A cell containing multiple copies of the haploid number (n) of chromosomes.

endo Endoreduplication:

4n cell in which separation of chromosome pairs has failed.

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