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Working for a better future

HFCPE 

Bacterial Reverse Mutation Test 

HLS study number: VHJ0008 

Version ID: Final 

Issue date: 23 October 2009 
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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 

 UK 
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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 .................................................................. 28
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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 ........................................................................ 31


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Contributing Scientists 

HFCPE: Bacterial Reverse Mutation Test 

Study management 

Kenneth May BSc CBiol MSB 

Study Director 

Alica Šoltésová 

Study Supervisor 
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Summary 

In this in vitro assessment of the mutagenic 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 uvrA (pKM101), 

were exposed to HFCPE diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a 

negative control. 

Two independent mutation tests were performed 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 guidelines that this assay follows. Other 

concentrations used were a series of ca half-log10 dilutions of the highest concentration. 

Toxicity (observed as a slightly thin background lawn of non-revertant colonies, together 

with a reduction in revertant colony numbers) was seen in all strains following exposure to 

HFCPE at 5000 μg/plate in both tests. 

No evidence of mutagenic activity was seen at any concentration of HFCPE in either 

mutation test. 

The concurrent positive controls demonstrated the sensitivity of the assay and the 

metabolising activity of the liver 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. 
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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 described 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 incapable of this function. When these strains are 

exposed to a mutagen, reverse mutation to the original histidine-independent form takes place 

in a proportion of the population. These are referred to as revertants, 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 principles 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 the 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. 
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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. Therefore, 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 potential 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. 
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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 hisG46 rfa ∆ uvrB

Salmonella typhimurium TA1537 hisC3076 rfa ∆ uvrB

Salmonella typhimurium TA98 hisD3052 rfa ∆ uvrB pKM101 

Salmonella typhimurium TA100 hisG46 rfa ∆ uvrB pKM101 

Escherichia coli WP2 uvrA (pKM101) trpE ochre uvrA pKM101 

Note: The deletion (∆) through uvrB also affects the nitrate reductase (chlA) and biotin (bio) genes 

These strains were used to detect base changes and frameshift mutations as follows: 

base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvrA 

(pKM101). 

frameshift mutagens: S. typhimurium TA1537 and TA98. 
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The strains of S. typhimurium were obtained from the National Collection of Type Cultures, 

London, England. 

The strain of E. coli was obtained from the National Collections 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 approximately -80oC. Dimethyl 

sulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of 

frozen strain was tested for amino acid requirement and, where applicable, for cell membrane 

permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers 

resistance to ampicillin. The responses of the strains to a series of reference mutagens were 

also assessed. 

For use in tests, an aliquot of frozen culture 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 109

 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 uvrA (pKM101) 
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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 uvrA (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 male Sprague-Dawley derived rats, dosed with phenobarbital and 

5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a 

commercial source and stored at approximately -80°C. The quality control statement relating 

to each batch of S9 preparation 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), MgCl2 (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 soluble in dimethyl sulphoxide (DMSO). DMSO 

(A.C.S. spectrophotometric grade) was, therefore, 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 the regulatory guidelines that this assay follows. The highest 

concentration in each test was diluted with DMSO to produce a series of lower 

concentrations, separated by approximately half-log10 intervals. 

All concentrations cited in this report are expressed in terms of the HFCPE sample as 

received. 
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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 concentrations 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 for 30 minutes with shaking. After this preincubation, 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan 

(0.05 mM) were added. The mixture was thoroughly shaken and overlaid onto previously 

prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually 

labelled with a unique code, identifying the contents of the dish. Three Petri dishes were 

used for each treatment. Plates were also prepared 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 selected for use in the second test would be the 

same as that used in the first. If toxic effects were observed, a lower concentration might be 

chosen, ensuring that signs of bacterial inhibition were present at this maximum 

concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If 

precipitate were observed on the plates at the end of the incubation period, at least four nonprecipitating concentrations should be obtained, unless otherwise 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 and 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. 
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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 limits of the current historical 

control range of the laboratory unless otherwise justified by the Study Director. The 

historical range is maintained as a rolling record over a maximum of five years. Also, the 

positive control compounds must induce an increase in mean revertant colony numbers of at 

least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle 

controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 109

/mL. 

3.2 Analysis of data 

The mean number and standard deviation of revertant colonies were calculated for all groups. 

The “fold-increases” relative to the vehicle controls were calculated in order to compare the 

means for all treatment groups with those obtained for the vehicle control groups. 

3.3 Criteria for assessing mutagenic potential 

If exposure to a test substance produces a reproducible 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 positive dose-response relationship, it is considered 

to exhibit mutagenic activity in this test system. No statistical analysis is performed. 

If exposure to a test substance does not produce a reproducible increase 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 criteria 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 general, treatment-associated increases in 

revertant colony numbers below two or three times the vehicle controls (as described above) 

are not considered biologically important. It should be noted that it is acceptable to conclude 

an equivocal response if no clear results can be obtained. 

Occasionally, these criteria may not be appropriate to the test data and, in such cases, the 

Study Director would use his/her scientific judgement. 

4. Deviations from Protocol 

There were no deviations from the protocol. 
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5. Maintenance of Records 

Records and documentation relating to this study (including electronic records) will be 

maintained in the archives of Huntingdon Life Sciences 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 positive 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, buffer and test substance formulation. 

The viability counts (see Table 3 and Table 6) confirmed that the viable cell density of the 

cultures of the individual organisms exceeded 109/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 historical control range of the laboratory (Appendix 1). 

Appropriate positive control chemicals (with S9 mix where required) induced substantial 

increases in revertant colony numbers with all strains in all reported tests, confirming 

sensitivity of the cultures and activity of the S9 mix. 

6.1 First test 

Toxicity, observed as a slightly thin background lawn of non-revertant colonies and reduction 

in the number of revertant colonies, was obtained in all strains following exposure to HFCPE 

at 5000 μg/plate. A maximum exposure concentration of 5000 μg/plate was, therefore, 

selected for use in the second test. 

No substantial increases in revertant colony numbers over control counts were obtained with 

any of the tester strains following exposure to HFCPE at any concentration up to 

5000 μg/plate in either the presence or absence of S9 mix. 
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6.2 Second test 

Toxicity, observed as a thin or absent background lawn of non-revertant 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 revertant colony numbers over control counts were obtained with 

any of the tester strains following exposure to HFCPE at any concentration up to 

5000 μg/plate in either the presence 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. 
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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. (1983) Revised methods for the Salmonella mutagenicity 

test. Mutation Res. 113, 173-215. 
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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 lawn 
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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 lawn 
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Table 2 Results obtained in the presence of metabolic activation: 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 lawn 
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Table 2 Results obtained in the presence of metabolic activation: 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 lawn 
 Huntingdon Life Sciences 

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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 Individual 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, 230 
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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 lawn 
 Huntingdon 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 lawn 
 Huntingdon Life Sciences 

 VHJ0008 

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Table 5 Results obtained in the presence of metabolic activation: 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 lawn 
 Huntingdon Life Sciences 

 VHJ0008 

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Table 5 Results obtained in the presence of metabolic activation: 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 lawn 
 Huntingdon 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 Individual 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, 229 

 
 Huntingdon Life Sciences 

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Appendix 1 Historical Control Data 

Presented below are the historical control data (mean revertant 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 - -++ - -++

NaN3 NaN3 B[a]P AAN NaN3 NaN3 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

NaN3 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-oxide
 Huntingdon Life Sciences 

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Appendix 2 Eye Research Centre GLP Compliance Statement 
 Huntingdon Life Sciences 

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Appendix 3 S9 Quality Control Certificate