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### Working for a better futur e



## HFCPE

Bacterial Reverse Mutation Test

HLS study number: VHJ0008

Version ID: Final

Issue date: 23 October 2009Huntingdon Life Sciences VHJ0008



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# Sponsor and Test Facility Details

Sponsor Zeon Corporation Shin Marunouchi Center Building 1-6-2 Marunouchi Chiyoda-ku Tokyo 100-8246 JAPAN

Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences VHJ0008



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# Table of Contents

Sponsor and Test Facility Details ........................................................................................... 2 Compliance with Good Laboratory Practice............................................................................ 5 Quality Assurance Statement ................................................................................................. 6 Contributing Scientists ........................................................................................................

.... 7 Summary........................................................................................................................

......... 8

1. Introduction ...................................................................................................................

. 9

2. Experimental Procedure............................................................................................... 11

2.1 Test substance................................................................................................. 11 2.2 Bacterial strains................................................................................................ 11 2.3 Positive controls ............................................................................................... 12 2.3.1 In the absence of S9 mix ...................................................................... 12 2.3.2 In the presence of S9 mix..................................................................... 13 2.4 S9 metabolizing system ................................................................................... 13 2.4.1 Preparation of S9 fraction..................................................................... 13 2.4.2 Preparation of S9 mix ........................................................................... 13 2.5 Selection of vehicle and formulation of test substance .................................... 13 2.6 Mutation test procedure.................................................................................... 14 2.6.1 First test................................................................................................ 14 2.6.2 Second test........................................................................................... 14 2.7 Stability, homogeneity and formulation analysis .............................................. 14

3. Assessment of Results................................................................................................. 15

3.1 Acceptance criteria........................................................................................... 15 3.2 Analysis of data ................................................................................................ 15 3.3 Criteria for assessing mutagenic potential ....................................................... 15

4. Deviations from Protocol.............................................................................................. 15

5. Maintenance of Records .............................................................................................. 16

6. Results ........................................................................................................................

. 16

6.1 First test............................................................................................................ 16 6.2 Second test ...................................................................................................... 17

7. Conclusion ................................................................................................................... 17

8. References................................................................................................................... 18



# List of Tables

Table 1 Results obtained in the absence of metabolic activation: test 1.................... 19

Table 2 Results obtained in the presence of metabolic activation: test 1................... 21

Table 3 Viability counts obtained in test 1 .................................................................. 23

Table 4 Results obtained in the absence of metabolic activation: test 2.................... 24

Table 5 Results obtained in the presence of metabolic activation: test 2................... 26

Table 6 Viability counts obtained in test 2 .................................................................. 28Huntingdon Life Sciences VHJ0008



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# List of Appendices

Appendix 1 Historical Control Data .................................................................................. 29 Appendix 2 Eye Research Centre GLP Compliance Statement....................................... 30 Appendix 3 S9 Quality Control Certificate ........................................................................ 31Huntingdon Life Sciences VHJ0008



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# Contributing Scientists

HFCPE: Bacterial Reve rse Mutation Test


## Study management

Kenneth May BSc CBiol MSB Study Director

Alica Šoltésová Study SupervisorHuntingdon Life Sciences VHJ0008



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# Summary

In this in vitro assessment of the mutage nic potential of HFCPE, histidine-dependent auxotrophic mutants of Salmonella typhimurium , strains TA1535, TA1537, TA98 and TA100, and a tryptophan- dependent mutant of Escherichia coli , strain WP2 uvr A (pKM101), were exposed to HFCPE diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a negative control.

Two independent mutation tests were perfor med in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Both tests included a pre-incubation stage.

Concentrations of HFCPE up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory gui delines that this assay follows. Other concentrations used were a series of ca half-log 10 

### dilutions of the highest concentration. Toxicity (observed as a slight ly thin background lawn of non -revertant colonies, together with a reduction in re vertant colony numbers) was seen in all strains following exposure to HFCPE at 5000 μg/plate in both tests.

No evidence of mutagenic act ivity was seen at any concentration of HFCPE in either mutation test.

The concurrent positive contro ls demonstrated the sensi tivity of the assay and the metabolising activity of the live r preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

It is concluded that HFCPE showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.Huntingdon Life Sciences VHJ0008



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# 1. Introduction

This report describes a study designed to assess HFCPE for its ability to cause point (gene) mutation in Salmonella typhimurium and Escherichia coli . The study was conducted in compliance with the following guidelines:

OECD Guidelines for the Testing of Chemicals (1997). Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471.

EC Commission Directive 2000/32/EC Annex 4D -B.13/14. Mutagenicity - Reverse mutation test in bacteria. No. L 136/57.

US EPA Health Effects Test Guidelines (1998). OPPTS 870.5100 Bacterial reverse mutation test. EPA 712-C-98-247. Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No . 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000.
Official notice of J MHLW, METI and ME (21 November 2003):

YAKUSHOKUHATSU No.1121002 SEIKYOKU No.2 KANPOKIHATSU No. 031121002 Official Notice of J MOL (8 February 1999) ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444. ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

The in vitro technique describe d by Ames and his co-workers ( Ames , McCann and Yamasaki 1975 ; Maron and Ames 1983 ) enables the mutagenic effect of a test substance to be determined by exposing specially selected strains of Salmonella typhimurium to the test substance.

Normally S. typhimurium is capable of synthesising the essential amino acid, histidine, but the mutant strains used in this test are incapab le of this function. When these strains are exposed to a mutagen, reverse mutation to the or iginal histidine-independent form takes place in a proportion of the population. These are referred to as revert ants, and are readily detected by their ability to grow and form colonies on a histidine-deficient medium (supplemented with biotin, since these strains are also incapable of biotin synthesis).

A technique based on similar princi ples has also been described by Green (1984) . This system employs mutant strains of Escherichia coli that are incapable of synthesising the amino acid, tryptophan, which is required for growth.

The strains used carry additional mutations that render them more sensitive to mutagens ( Gatehouse et al, 1990 ). The S. typhimurium strains have a defective cell coat, which allows greater permeability of test substances into th e cell. All the strains are deficient in normal DNA repair processes. In addition, three of them possess a plasmid (pKM101) which introduces an error-prone repair process, resulting in increased sensitivity to some mutagens.Huntingdon Life Sciences VHJ0008



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### Some substances do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell. Th erefore, the bacteria and test substance are incubated in both the absence and presence of a supplemented rat liver homogenate fraction (S9 mix).

The study comprised two independent tests to assess the mutagenic po tential of the test substance.

The protocol was approved by Huntingdon Life Sciences Management and by the Study Director on 7 August 2009, and by the Sponsor on 17 August 2009.

Experimental start date: 17 August 2009. Experimental completion date: 2 September 2009.Huntingdon Life Sciences VHJ0008



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# 2. Experimental Procedure

2.1 Test substance

Identity: HFCPE

Chemical name: 1,3,3,4,4,5,5-Heptafluorocyclopentene

Appearance: Colourless liquid

Storage conditions: Room temperature

Batch number: 20090601

Expiry date: End of June 2010

Purity: 99.3%

Date received: 22 June 2009



## 2.2 Bacterial strains

The genotype of each bacterial strain used in this study is outlined in the following table:

Species Strain Genotype 

### Salmonella typhimurium TA1535 his G46 rfa ∆ uvr B Salmonella typhimurium TA1537 his C3076 rfa ∆ uvr B Salmonella typhimurium TA98 his D3052 rfa ∆ uvr B pKM101 Salmonella typhimurium TA100 his G46 rfa ∆ uvr B pKM101
Escherichia coli WP2 uvr A (pKM101) trp E ochre uvr A pKM101
Note: The deletion ( ∆ ) through uvr B also affects the nitrate reductase ( chl A) and biotin ( bio ) genes



### These strains were used to detect base ch anges and frameshift mutations as follows:

base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvr A (pKM101). frameshift mutagens: S. typhimurium TA1537 and TA98.Huntingdon Life Sciences VHJ0008



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### The strains of S. typhimurium were obtained from the Nationa l Collection of Type Cultures, London, England.

The strain of E. coli was obtained from the National Colle ctions of Industrial and Marine Bacteria, Aberdeen, Scotland.

Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were stored as aliquots of nutrient broth cultures at a pproximately -80oC. Dimethyl sulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreserva tive. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability ( rfa mutation), sensitivity to UV light, a nd the pKM101 plasmid, which confers resistance to ampicillin. The responses of the st rains to a series of reference mutagens were also assessed.

For use in tests, an aliquot of frozen cultu re was added to 25 mL of nutrient broth and incubated, with shaking, at 37 ° C for 10 hours. These cultures were intended to provide a viable cell density of at least 10 9 

### per mL, which was confirmed by performing viability counts, in which aliquots (0.1 mL) of a 10 -6 

### dilution of the 10-hour cultures were spread on the surface of plates of nutrient agar. After incubation at 37°C for 24 hours, the total number of resultant colonies was counted.



## 2.3 Positive controls

2.3.1 In the absence of S9 mix

Identity: Sodium azide CAS No.: 26628-22-8 Solvent: DMSO (ACS spectrophotometric grade) Concentration: 2 μg/plate for strains TA100 and TA1535

Identity: 9-Aminoacridine CAS No.: 90-45-9 Solvent: DMSO (ACS spectrophotometric grade) Concentration: 50 μg/plate for strain TA1537

Identity: 2-Nitrofluorene CAS No.: 607-57-8 Solvent: DMSO (ACS spectrophotometric grade) Concentration: 2 μg/plate for strain TA98

Identity: 4-Nitroquinoline-1-oxide CAS No.: 56-57-5 Solvent: DMSO (ACS spectrophotometric grade) Concentration: 2 μg/plate for strain WP2 uvr A (pKM101)Huntingdon Life Sciences VHJ0008



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### 2.3.2 In the presence of S9 mix

Identity: 2-Aminoanthracene CAS No.: 613-13-8 Solvent: DMSO (ACS spectrophotometric grade) Concentration: 5 μg/plate for strains TA100 and TA1535 10 μg/plate for strain WP2 uvr A (pKM101)

Identity: Benzo[a]pyrene CAS No.: 50-32-8 Solvent: DMSO (ACS spectrophotometric grade) Concentration: 5 μg/plate for strains TA98 and TA1537



## 2.4 S9 metabolizing system

2.4.1 Preparation of S9 fraction

S9 fraction, prepared from ma le Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxi dases in the liver, was purchased from a commercial source and stored at approximately -80°C. The qua lity control statement relating to each batch of S9 prepar ation used is included in the raw data and a copy is included in this report as Appendix 3

. Lot No.: 2416 (Date of preparation: 6 May 2009)

2.4.2 Preparation of S9 mix

The S9 mix contained: S9 fraction (10% v/v – test 1, 20% v/v – test 2), MgCl 2 

### (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.



## 2.5 Selection of vehicle and formulation of test substance

The Sponsor indicated that HFCPE was solubl e in dimethyl sulphoxide (DMSO). DMSO (A.C.S. spectrophotometric grade) was, theref ore, used as the vehicle for this study.

The highest concentration of HFCPE tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in th e regulatory guidelines that this assay follows. The highest concentration in each test was diluted w ith DMSO to produce a series of lower concentrations, separated by approximately half-log 10 

### intervals.

All concentrations cited in this report are expressed in terms of the HFCPE sample as received.Huntingdon Life Sciences VHJ0008



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## 2.6 Mutation test procedure

2.6.1 First test

Aliquots of 0.1 mL of the test substance solutions (seven con centrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels, sealed with a suitable septum in order to prevent loss of volatile material. The negative control was the chosen vehicle, DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture. The mixtures of bacteria, buffer, S9 mix and test solution were incubated at 37°C fo r 30 minutes with shaking. After this preincubation, 2 mL of agar c ontaining histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM) were added. The mixture was t horoughly shaken and ove rlaid onto previously prepared Petri dishes containing 25 mL minima l agar. Each Petri dish was individually labelled with a unique code, identifying the cont ents of the dish. Three Petri dishes were used for each treatment. Plates were also prepar ed without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. Treated plates were sealed in polythene bags in groups so that vapour was not lost, and there was no crosscontamination between different concentrations or between plates treated in the presence or absence of S9 mix. All plates were incubated at approximately 37 ° C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer ).

Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn . In the absence of any toxic effects, the maximum concentration sel ected for use in the second test would be the same as that used in the first. If toxic eff ects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were presen t at this maximum concentration. Ideally, a minimu m of four non-toxic concentrati ons should be obtained. If precipitate were observed on the plates at the end of the incubation pe riod, at least four nonprecipitating concentrations s hould be obtained, unless othe rwise justified by the Study Director.

2.6.2 Second test

As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was increased concentration of S9 in the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.



## 2.7 Stability, homogeneity an d formulation analysis

The stability of HFCPE and the stability and homogeneity of HFCPE in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.Huntingdon Life Sciences VHJ0008



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# 3. Assessment of Results

3.1 Acceptance criteria

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence lim its of the current historical control range of the laboratory unless otherwise jus tified by the Study Director. The historical range is maintained as a rolling reco rd over a maximum of five years. Also, the positive control compounds must induce an increa se in mean revertant colony numbers of at least twice (three times in th e case of strains TA1535 and TA 1537) the concurrent vehicle controls. Mean viable cell c ounts in the 10-hour bacterial cultures must be at least 10 9 

### /mL.


## 3.2 Analysis of data

The mean number and standard deviation of revert ant colonies were calculated for all groups. The “fold-increases” relative to th e vehicle controls were calculated in order to compare the means for all treatment groups with thos e obtained for the vehicle control groups.


## 3.3 Criteria for assessing mutagenic potential

If exposure to a test substance produces a repr oducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positiv e dose-response relationship, it is considered to exhibit mutagenic activity in this test syst em. No statistical analysis is performed.

If exposure to a test substance does not produ ce a reproducible increas e in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

If the results obtained fail to satisfy the crit eria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In ge neral, treatment-asso ciated increases in revertant colony numbers below two or three ti mes the vehicle controls (as described above) are not considered biologically im portant. It should be noted that it is acceptable to conclude an equivocal response if no cl ear results can be obtained.

Occasionally, these criteria may not be appropria te to the test data a nd, in such cases, the Study Director would use his/ her scientific judgement.



# 4. Deviations from Protocol

There were no deviations from the protocol.Huntingdon Life Sciences VHJ0008



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# 5. Maintenance of Records

Records and documentation relating to this st udy (including electronic records) will be maintained in the archives of Huntingdon Life Sc iences for a period of one year from the date on which the Study Director signs the final report . This will include but may not be limited to the Study Protocol, raw data, a copy of the final report and other samples and specimens generated during the course of this study. If requested, Huntingdon Life Sciences will continue to retain the materials at additional cost.

Samples and specimens that no longer afford evaluation will be discarded in accordance with Standard Operating Procedures.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the final report in its archive indefinitely.



# 6. Results

The results obtained with HFCPE and positiv e control compounds are presented in Table 1 and Table 2 (first test) and in Table 4 and Table 5 (second test).

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffe r and test substance formulation.

The viability counts (see Table 3 and Table 6 ) confirmed that the viab le cell density of the cultures of the individual organisms exceeded 10 9 

### /mL in all cases, and therefore met the acceptance criteria.

The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current histor ical control range of the laboratory ( Appendix 1 ). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with al l strains in all repor ted tests, confirming sensitivity of the cultures and activity of the S9 mix.



## 6.1 First test

Toxicity, observed as a slight ly thin background lawn of non-re vertant colonies and reduction in the number of revertant coloni es, was obtained in all strains following exposure to HFCPE at 5000 μg/plate. A maximum exposure concentr ation of 5000 μg/plate was, therefore, selected for use in the second test.

No substantial increases in re vertant colony numbers over control counts were obtained with any of the tester strains following exposur e to HFCPE at any concentration up to 5000 μg/plate in either the pres ence or absence of S9 mix.Huntingdon Life Sciences VHJ0008



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## 6.2 Second test

Toxicity, observed as a thin or absent b ackground lawn of non-re vertant colonies and reduction in the number of revertant colonies, was obtained in all strains following exposure to HFCPE at 5000 μg/plate.

No substantial increases in re vertant colony numbers over control counts were obtained with any of the tester strains following exposur e to HFCPE at any concentration up to 5000 μg/plate in either the pres ence or absence of S9 mix.



# 7. Conclusion

It is concluded that HFCPE showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.Huntingdon Life Sciences VHJ0008



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# 8. References

AMES, B.N., McCANN, J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella /mammalian microsome mutagenicity test. Mutation Res

. 31 , 347-364.

GATEHOUSE, D.G., ROWLAND, I.R., WILCOX, P., CALLANDER, R.D. and FORSTER, R. (1990) Bacterial mutation assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part I. Basic Mutagenicity Tests , pp.13-61. Cambridge University Press, Cambridge.

GREEN, M.H.L. (1984) Mutagen testing using trp + 

### reversion in Escherichia coli in KILBEY, B.J., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook of Mutagenicity Test Procedures. Second edition , pp.161-187. Elsevier Science Publishers BV, Amsterdam.

MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I. de G., ROBINSON, W.D. and TWEATS, D.J. (1989) Analysis of data from microbial colony assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data , pp.26-65. Cambridge University Press, Cambridge.

MARON, D.M. and AMES, B.N. (1 983) Revised methods for the Salmonella mutagenicity
test. Mutation Res

. 113 , 173-215.Huntingdon Life Sciences VHJ0008



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### Table 1 Results obtained in the absence of metabolic activation: test 1

Study number: VHJ0008 Experiment: Test 1 Assay conditions: Pre-incubation assay

Without metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA98 DMSO 35.0 2.6 33, 38, 34
HFCPE 5 μg 37.0 4.4 1.1 32, 39, 40 15 μg 36.0 5.6 1.0 31, 42, 35 50 μg 32.7 8.1 0.9 40, 34, 24 150 μg 30.0 8.7 0.9 20, 35, 35 500 μg 31.3 2.5 0.9 31, 34, 29 1500 μg 30.3 5.7 0.9 24, 35, 32 5000 μg 27.7 1.5 0.8 26 S, 28 S, 29 S

TA100 DMSO 111.0 7.0 108, 119, 106
HFCPE 5 μg 104.0 18.5 0.9 103, 86, 123 15 μg 96.0 9.2 0.9 86, 104, 98 50 μg 102.7 6.7 0.9 101, 97, 110 150 μg 101.3 18.1 0.9 94, 88, 122 500 μg 114.0 2.6 1.0 111, 115, 116 1500 μg 108.7 2.5 1.0 111, 109, 106 5000 μg 39.7 6.5 0.4 40 S, 33 S, 46 S

TA1535 DMSO 21.3 0.6 22, 21, 21
HFCPE 5 μg 21.7 3.2 1.0 24, 18, 23 15 μg 20.3 0.6 1.0 20, 21, 20 50 μg 22.7 4.2 1.1 24, 26, 18 150 μg 21.3 0.6 1.0 21, 21, 22 500 μg 18.3 2.5 0.9 21, 18, 16 1500 μg 18.0 2.0 0.8 20, 16, 18 5000 μg 0.3 0.6 0.0 1 S, 0 S, 0 S

Key to Plate Postfix Codes S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 20 

### Table 1 Results obtained in the absence of metabolic activation: test 1 (continued)

Study number: VHJ0008 Experiment: Test 1 Assay conditions: Pre-incubation assay

Without metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA1537 DMSO 14.3 2.1 16, 12, 15
HFCPE 5 μg 13.0 4.6 0.9 9, 18, 12 15 μg 12.7 2.5 0.9 10, 13, 15 50 μg 12.0 5.0 0.8 7, 12, 17 150 μg 15.0 3.0 1.0 18, 12, 15 500 μg 14.3 3.2 1.0 18, 12, 13 1500 μg 11.3 5.0 0.8 6, 16, 12 5000 μg 0.0 0.0 0.0 0 S, 0 S, 0 S

WP2 uvrA DMSO 114.0 2.6 115, 116, 111 (pKM101) HFCPE 5 μg 81.0 13.9 0.7 65, 89, 89 15 μg 95.0 7.5 0.8 88, 103, 94 50 μg 111.0 12.2 1.0 117, 97, 119 150 μg 117.0 17.6 1.0 104, 110, 137 500 μg 105.7 8.0 0.9 114, 105, 98 1500 μg 100.7 10.4 0.9 89, 109, 104 5000 μg 46.0 3.0 0.4 43 S, 46 S, 49 S

TA98 2NF 2 μg 204.7 2.1 5.8 204, 203, 207
TA100 NaN3 2 μg 839.7 157.1 7.6 1017, 718, 784
TA1535 NaN3 2 μg 225.0 5.2 10.5 231, 222, 222
TA1537 AAC 50 μg 397.3 58.5 27.7 340, 395, 457
WP2 uvrA (pKM101) NQO 2 μg 1812.3 263.5 15.9 1549, 2076, 1812

Key to Positive Controls Key to Plate Postfix Codes

2NF NaN3 AAC NQO 2-Nitrofluorene Sodium azide 9-Aminoacridine 4-Nitroquinoline-1-oxide S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



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### Table 2 Results obtained in the presen ce of metabolic act ivation: test 1

Study number: VHJ0008 Experiment: Test 1 Assay conditions: Pre-incubation assay

With metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA98 DMSO 45.7 6.7 53, 40, 44
HFCPE 5 μg 57.0 13.2 1.2 42, 67, 62 15 μg 38.3 10.2 0.8 34, 50, 31 50 μg 44.0 8.7 1.0 40, 54, 38 150 μg 41.7 6.7 0.9 46, 45, 34 500 μg 44.3 9.0 1.0 49, 50, 34 1500 μg 45.3 7.4 1.0 48, 51, 37 5000 μg 25.3 2.1 0.6 26 S, 27 S, 23 S

TA100 DMSO 133.0 5.0 128, 138, 133
HFCPE 5 μg 150.7 14.6 1.1 137, 166, 149 15 μg 130.7 9.0 1.0 125, 141, 126 50 μg 141.0 12.5 1.1 128, 142, 153 150 μg 116.3 8.5 0.9 125, 116, 108 500 μg 121.0 14.8 0.9 138, 111, 114 1500 μg 137.7 23.8 1.0 159, 142, 112 5000 μg 88.7 8.1 0.7 98 S, 84 S, 84 S

TA1535 DMSO 22.7 6.5 16, 29, 23
HFCPE 5 μg 22.3 6.5 1.0 29, 22, 16 15 μg 22.7 4.2 1.0 18, 24, 26 50 μg 19.7 3.2 0.9 22, 21, 16 150 μg 20.7 4.9 0.9 23, 24, 15 500 μg 19.0 3.6 0.8 23, 18, 16 1500 μg 16.3 0.6 0.7 16, 17, 16 5000 μg 3.7 0.6 0.2 4 S, 4 S, 3 S

Key to Plate Postfix Codes S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 22 

### Table 2 Results obtained in the presen ce of metabolic act ivation: test 1 (continued)

Study number: VHJ0008 Experiment: Test 1 Assay conditions: Pre-incubation assay

With metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA1537 DMSO 27.0 5.6 32, 21, 28
HFCPE 5 μg 26.3 8.5 1.0 35, 18, 26 15 μg 24.0 2.6 0.9 23, 22, 27 50 μg 31.3 8.3 1.2 34, 22, 38 150 μg 27.0 5.6 1.0 33, 22, 26 500 μg 26.3 5.5 1.0 26, 21, 32 1500 μg 29.0 5.3 1.1 31, 33, 23 5000 μg 18.7 2.9 0.7 22 S, 17 S, 17 S

WP2 uvrA DMSO 124.0 3.5 122, 128, 122 (pKM101) HFCPE 5 μg 152.7 13.8 1.2 163, 137, 158 15 μg 175.0 18.5 1.4 154, 189, 182 50 μg 128.0 16.6 1.0 116, 147, 121 150 μg 121.0 34.4 1.0 159, 112, 92 500 μg 124.3 14.2 1.0 134, 131, 108 1500 μg 112.3 4.5 0.9 112, 117, 108 5000 μg 74.3 3.2 0.6 72 S, 73 S, 78 S

TA98 B[a]P 5 μg 245.3 23.5 5.4 219, 264, 253
TA100 AAN 5 μg 2057.0 150.5 15.5 2230, 1956, 1985
TA1535 AAN 5 μg 219.0 11.1 9.7 229, 221, 207 TA1537 B[a]P 5 μg 106.0 2.6 3.9 104, 105, 109
WP2 uvrA (pKM101) AAN 10 μg 652.7 103.9 5.3 738, 683, 537

Key to Positive Controls Key to Plate Postfix Codes B[a]P AAN Benzo[a]pyrene 2-Aminoanthracene S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 23 

### Table 3 Viability counts obtained in test 1

Study number: VHJ0008 Experiment: Test 1 Assay conditions: Pre-incubation assay

Viability plate summary

Strain Mean counts per plate Standard Deviation I ndividual colony counts (100 μL aliquots of 10 -6

dilution of 10-hour culture)

TA98 Viability 206.3 7.5 202, 215, 202
TA100 Viability 227.7 16.9 247, 216, 220
TA1535 Viability 280.0 19.5 287, 295, 258
TA1537 Viability 208.7 33.6 246, 181, 199
WP2 uvrA (pKM101) Viability 228.7 9.1 237, 219, 230Huntingdon Life Sciences VHJ0008



### 24 

### Table 4 Results obtained in the absence of metabolic activation: test 2

Study number: VHJ0008 Experiment: Test 2 Assay conditions: Pre-incubation assay

Without metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA98 DMSO 30.0 1.7 32, 29, 29
HFCPE 50 μg 26.0 5.2 0.9 23, 32, 23 150 μg 23.3 2.3 0.8 26, 22, 22 500 μg 28.3 5.5 0.9 32, 22, 31 1500 μg 23.7 3.8 0.8 21, 22, 28 5000 μg 21.7 0.6 0.7 22 S, 22 S, 21 S

TA100 DMSO 155.7 6.8 158, 148, 161
HFCPE 50 μg 136.7 23.2 0.9 143, 111, 156 150 μg 178.0 61.9 1.1 110, 193, 231 500 μg 180.0 40.4 1.2 210, 196, 134 1500 μg 153.3 15.0 1.0 169, 152, 139 5000 μg 119.0 20.2 0.8 111 S, 104 S, 142 S

TA1535 DMSO 25.3 1.2 26, 26, 24
HFCPE 50 μg 22.0 5.0 0.9 27, 17, 22 150 μg 16.7 6.4 0.7 24, 13, 13 500 μg 19.7 8.3 0.8 17, 13, 29 1500 μg 21.3 2.3 0.8 24, 20, 20 5000 μg 16.7 3.5 0.7 20 S, 17 S, 13 S

Key to Plate Postfix Codes S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 25 

### Table 4 Results obtained in the absence of metabolic activation: test 2 (continued)

Study number: VHJ0008 Experiment: Test 2 Assay conditions: Pre-incubation assay

Without metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA1537 DMSO 13.7 2.1 16, 12, 13
HFCPE 50 μg 8.7 2.3 0.6 10, 6, 10 150 μg 11.7 1.2 0.9 11, 13, 11 500 μg 15.0 5.3 1.1 21, 11, 13 1500 μg 8.3 2.1 0.6 10, 6, 9 5000 μg 2.7 2.1 0.2 2 S, 5 S, 1 S

WP2 uvrA DMSO 114.3 8.6 116, 105, 122 (pKM101) HFCPE 50 μg 130.3 15.0 1.1 131, 145, 115 150 μg 124.0 7.8 1.1 119, 120, 133 500 μg 83.3 3.2 0.7 81, 82, 87 1500 μg 94.3 32.8 0.8 79, 72, 132 5000 μg 70.7 10.7 0.6 65 S, 64 S, 83 S

TA98 2NF 2 μg 165.7 3.8 5.5 170, 164, 163
TA100 NaN3 2 μg 739.7 50.3 4.8 793, 733, 693
TA1535 NaN3 2 μg 526.7 33.1 20.8 561, 524, 495
TA1537 AAC 50 μg 619.7 258.4 45.3 477, 464, 918
WP2 uvrA (pKM101) NQO 2 μg 1754.7 319.0 15.3 1433, 2071, 1760

Key to Positive Controls Key to Plate Postfix Codes

2NF NaN3 AAC NQO 2-Nitrofluorene Sodium azide 9-Aminoacridine 4-Nitroquinoline-1-oxide S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 26 

### Table 5 Results obtained in the presen ce of metabolic act ivation: test 2

Study number: VHJ0008 Experiment: Test 2 Assay conditions: Pre-incubation assay

With metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA98 DMSO 39.3 1.2 40, 38, 40
HFCPE 50 μg 46.0 5.2 1.2 49, 40, 49 150 μg 43.3 8.4 1.1 39, 53, 38 500 μg 43.3 5.5 1.1 38, 43, 49 1500 μg 39.7 2.9 1.0 38, 43, 38 5000 μg 34.7 0.6 0.9 35 S, 34 S, 35 S

TA100 DMSO 163.7 7.4 161, 172, 158
HFCPE 50 μg 159.7 13.3 1.0 145, 171, 163 150 μg 167.0 10.8 1.0 176, 155, 170 500 μg 171.0 19.1 1.0 169, 191, 153 1500 μg 155.3 34.0 0.9 142, 130, 194 5000 μg 134.3 2.5 0.8 132 S, 134 S, 137 S

TA1535 DMSO 30.7 1.5 29, 32, 31
HFCPE 50 μg 24.3 11.0 0.8 28, 33, 12 150 μg 26.7 6.0 0.9 26, 33, 21 500 μg 22.7 5.0 0.7 22, 18, 28 1500 μg 29.0 0.0 0.9 29, 29, 29 5000 μg 15.7 1.2 0.5 17 S, 15 S, 15 S

Key to Plate Postfix Codes S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 27 

### Table 5 Results obtained in the presen ce of metabolic act ivation: test 2 (continued)

Study number: VHJ0008 Experiment: Test 2 Assay conditions: Pre-incubation assay

With metabolic activation

Strain Addition Concentration per plate Mean
revertants per plate Standard Deviation Fold increase relative to

vehicle Individual revertant colony counts

TA1537 DMSO 29.7 3.2 32, 26, 31
HFCPE 50 μg 25.3 1.2 0.9 26, 24, 26 150 μg 28.7 3.8 1.0 26, 27, 33 500 μg 26.7 5.9 0.9 31, 20, 29 1500 μg 24.3 3.8 0.8 27, 20, 26 5000 μg 10.7 4.5 0.4 6 S, 15 S, 11 S

WP2 uvrA DMSO 165.0 11.1 155, 177, 163 (pKM101) HFCPE 50 μg 106.7 13.6 0.6 105, 121, 94 150 μg 104.7 1.5 0.6 106, 105, 103 500 μg 93.3 14.4 0.6 77, 99, 104 1500 μg 93.0 30.5 0.6 72, 79, 128 5000 μg 74.3 14.6 0.5 72 S, 61 S, 90 S

TA98 B[a]P 5 μg 180.7 23.2 4.6 196, 192, 154
TA100 AAN 5 μg 1174.3 300.3 7.2 867, 1467, 1189
TA1535 AAN 5 μg 176.0 11.0 5.7 187, 176, 165 TA1537 B[a]P 5 μg 126.7 10.8 4.3 139, 119, 122
WP2 uvrA (pKM101) AAN 10 μg 419.3 63.0 2.5 356, 420, 482

Key to Positive Controls Key to Plate Postfix Codes B[a]P AAN Benzo[a]pyrene 2-Aminoanthracene S Slight thinning of background lawnHuntingdon Life Sciences VHJ0008



### 28 

### Table 6 Viability counts obtained in test 2

Study number: VHJ0008 Experiment: Test 2 Assay conditions: Pre-incubation assay

Viability plate summary

Strain Mean counts per plate Standard Deviation I ndividual colony counts (100 μL aliquots of 10 -6

dilution of 10-hour culture)

TA98 Viability 303.0 34.8 286, 343, 280
TA100 Viability 219.3 6.1 218, 226, 214
TA1535 Viability 271.3 16.5 271, 288, 255
TA1537 Viability 310.0 17.8 304, 330, 296
WP2 uvrA (pKM101) Viability 228.3 34.0 262, 194, 229Huntingdon Life Sciences VHJ0008



### 29 

### Appendix 1 Historical Control Data

Presented below are the histor ical control data (mean reve rtant colony counts) from the period 1 May 2004 to 30 April 2009.

DMSO

S9 Mix -+-+-+-+-+

Maximum 288 310 43 36 295 323 75 85 50 53 Minimum 75 56 11 10 54 59 17 17 6 12 Mean 160 179 22 22 154 172 38 51 14 29 No. of values 678 687 658 656 591 586 685 684 650 650 Standard deviation 26 28 4 4 33 33 6 8 4 5 Upper 99% limit 226 250 33 32 238 257 54 73 25 41 Lower 99% limit 93 107 10 12 70 88 22 30 3 18 TA100 TA1535 TA98 TA1537 WP2 uvr A (pKM101)



### Positive Controls

S9 Mix - -++ - -++ NaN 3 NaN 3 B[a]P AAN NaN 3 NaN 3 AAN AAN (0.5 μg) (2 μg) (5 μg) (5 μg) (0.5 μg) (2 μg) (2 μg) (5 μg)

Maximum 1008 2440 1180 4910 673 2306 350 994
Minimum 281 367 212 424 134 193 54 52 Mean 671 969 652 2205 456 1101 141 320 No. of values 52 884 51 890 55 858 55 858 Standard deviation 185 254 231 818 96 271 71 101 

### TA100 TA1535



### S9 Mix - -+--+-+ AF-2 NQO AAN 2NF 2NF B[a]P AAC B[a]P (0.05 μg) (2 μg) (10 μg) (1 μg) (2 μg) (5 μg) (50 μg) (5 μg)

Maximum 3749 5221 1286 805 792 1287 2153 766
Minimum 233 494 192 190 114 93 121 73 Mean 1093 2247 532 292 328 280 688 175 No. of values 54 812 839 55 885 940 900 900 Standard deviation 762 874 164 110 114 118 307 73 WP2 uvr A (pKM101) 

### TA98 TA1537

NaN 3 Sodium azide AF-2 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 2NF 2-Nitrofluorene AAC 9-Aminoacridine B[a]P Benzo[a]pyrene AAN 2-Aminoanthracene NQO 4-Nitroquinoline-1-oxideHuntingdon Life Sciences VHJ0008



### 30 

### Appendix 2 Eye Research Cent re GLP Compliance StatementHuntingdon Life Sciences VHJ0008



### 31 

### Appendix 3 S9 Quality Control Certificate