# Final Report

Study Title : Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium Data Requirement

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## - 2 - STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA Sections § 10(d)(1)(A), (B) or (C). Company Company Agent: Date:

These data are the property of and as such are considered to be confidential for all purposes other than compliance with FIFRA § 10. Submission of these data in compliance with FIFRA does not constitute a waiver of any right to confidentiality, which may exist under any statute or in any other country.

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## - 3 - GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium

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## - 4 - FLAGGING STATEMENTS Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium

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## - 5 - STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium

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## - 6 - QUALITY ASSURANCE STATEMENT Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium

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## - 7 - REVIEWING SCIENTIST’S STATEMENT Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium

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## - 8 - RESPONSIBLE PERSONNEL Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium The following personnel were responsible for key elements of the study: Study Director M Johnson BSc PhD Study Supervisor K Masters BSc

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## - 9 - ARCHIVE STATEMENT Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium All primary data, or authenticated copies thereof, specimens and the final report will be retained in the archives for one year after issue of the final report. At this time the Sponsor will be contacted to determine whether the data should be returned, retained or destroyed on their behalf. Sponsors will be notified of the financial implications of each of these options at that time. Specimens requiring storage deep frozen are specifically excluded from the above. These will be retained for as long as the quality of the material permits evaluation, but for no longer than three months after issue of the final report. The Sponsor will be notified of the intent to destroy samples and any financial implications before specimens are destroyed on their behalf.

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## - 10 - CONTENTS STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS
................................ ................................ ........... 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
................................ ................................ . 3 FLAGGING STATEMENTS
................................ ................................ ................................ ................................ ..... 4 STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT
................................ ... 5 QUALITY ASSURANCE STATEMENT
................................ ................................ ................................ ............... 6 REVIEWING SCIENTIST’S STATEMENT
................................ ................................ ................................ ........... 7 RESPONSIBLE PERSONNEL
................................ ................................ ................................ ................................ ... 8 ARCHIVE STATEMENT
................................ ................................ ................................ ................................ ......... 9 CONTENTS
................................ ................................ ................................ ................................ .............................. 10 SUMMARY
................................ ................................ ................................ ................................ .............................. 11 INTRODUCTION
................................ ................................ ................................ ................................ .................... 13 MATERIALS
................................ ................................ ................................ ................................ ........................... 15 Test article
................................ ................................ ................................ ................................ ........................ 15 Controls
................................ ................................ ................................ ................................ ............................ 16 Metabolic activation system
................................ ................................ ................................ ......................... 17 Bacteria
................................ ................................ ................................ ................................ ............................. 18 METHODS
................................ ................................ ................................ ................................ ............................... 19 Toxicity Range-Finder Experiment
................................ ................................ ................................ ................ 19 Mutation experiments
................................ ................................ ................................ ................................ ..... 19 Colony counting
................................ ................................ ................................ ................................ .............. 20 Analysis of results
................................ ................................ ................................ ................................ .......... 21 RESULTS
................................ ................................ ................................ ................................ ................................ .. 23 Toxicity, solubility and dose selection
................................ ................................ ................................ ........ 23 Mutation
................................ ................................ ................................ ................................ ........................... 24 CONCLUSION
................................ ................................ ................................ ................................ ......................... 25 REFERENCES
................................ ................................ ................................ ................................ ........................... 26 APPENDICES
................................ ................................ ................................ ................................ ........................... 27 Appendix 1 Raw plate counts and calculated mutagenicity data Experiment1
................................ ..... 27 Appendix 2 Raw plate counts and calculated mutagenicity data Experiment2
................................ ..... 43 Appendix 3 Historical negative (solvent) control values for S. typhimurium strains
.......................... 55 Appendix 4 Historical positive control values for S. typhimurium strains
................................ ............ 56 Appendix 5 Quality control statements for S-9
................................ ................................ ........................... 57 Appendix 6 Sponsor's Certificate of Analysis
................................ ................................ ............................ 59

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## - 11 - SUMMARY was assayed for mutation in five histidine-requiring strains of Salmonella typhimurium(TA98, TA100, TA1535, TA1537 and TA102), both in the absence and in the presence of metabolic activation by an Aroclor 1254 -induced rat liver post-mitochondrial fraction (S -9), in two separate experiments. An initial toxicity Range-Finder Experiment was carried out in strain TA100 only in the absence and presence of S-9, using final concentrations of 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (solvent) and positive controls. No evidence of toxicity was observed following any of these treatments. These data were considered to be acceptable for mutation assessment and are presented in this report as the TA100 mutagenicity data for Experiment 1. Experiment 1 treatments of the remaining test strains in the absence and presence of S-9 retained the same test doses as employed for the Range -Finder Experiment. Evidence of toxicity was observed following the top dose treatment of strain TA1535 in the absence of S -9 and in strains TA98, TA1535 and TA1537 in the presence of S -9. Follow ing treatments of strain TA102 in the absence of S-9, evidence of toxicity was observed at the highest four doses tested. Because of this unexpected excessive toxicity, repeat treatments were performed in strain TA102 in the absence of S -9 using the same d

oses. Following these repeat treatments, evidence of toxicity was again observed at the highest four doses tested. In order to provide a thorough assessment of potential mutagenicity in this strain, additional treatments were performed with a dose range of 0.0128-200μg/plate. Evidence of toxicity was observed following the top two dose treatments. Experiment 2 treatments of all the tester strains in the absence and presence of S -9 were performed with the maximum test dose of 5000 μg/plate, or up to an esti

mate of the lower limit of toxicity for strain TA102 in the absence of S -9. Narrowed dose ranges were employed (0.3227-200μg/plate for strain TA102 in the absence of S -9 or 8.192- 5000 μg/plate

for all other treatments), in order to examine more closely th ose concentrations of approaching the maximum test dose and therefore considered most likely to provide evidence of any mutagenic activity. In addition,all treatments in the presence of S-9 were further modified by the inclusion of a preincubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Evidence of toxicity was observed following the

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## - 12 - top dose treatment of strain TA102 in the presence of S -9 and at the top two dose treatments of strain TA102 in the absence of S -9 and TA1537 in the presence of S-9. The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No statistically significant, dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any

It was concluded that did not induce mutation in five histidinerequiring strains of

Salmonella typhimurium(TA98, TA100, TA1535, TA1537 and TA102) when tested under the conditions of this study. These conditions included treatments at concentrations up to either 5000 μ g/plate or the lower limit of toxicity in the absence and in the presence of a rat liver metabolic activation system (S -9).

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## - 13 - INTRODUCTION The Ames test is a rapid, reliable and economical method of evaluating the mutagenic potential of a test article by measuring genetic activity in one or more histidine-requiring strains of Salmonella typhimuriumin the absence and presence of a liver metabolising system (1). A large database has been accumulated with this assay, confirming its ability to detect genetically active compounds of most chemical classes with around 80-90% sensitivity and specificity (2). The following bacterial strains were used in this study:
Organism Strain Type of mutation in the histidine gene S. typhimurium TA98 frame-shift S. typhimurium TA100 base-pair substitution S. typhimurium TA1535 base-pair substitution S. typhimurium TA1537 frame-shift S. typhimurium TA102 base-pair substitution 

## With the exception of strain TA102, these strains require biotin as well as histidine for growth. In strain TA102 the critical mutation in the histidine gene is located on a multicopyplasmid pAQ1. This strain is particularly sensitive to the activities of oxidative and crosslinking mutagens. The pKM101 plasmid derivatives (TA98, TA100 and TA102) have increased sensitivity to certain mutagens as the pKM101 plasmid codes for an error-prone DNA repair system (3). When exposed to a mutagen, some of the bacteria in the treated population undergo genetic changes which revert them to a non-histidine-requiring state, and they can then grow without exogenous histidine. Different tester strains are used because each is mutated by particular chemical classes of compound. A compound that is mutagenic in one strain need not be so in another (4). The objective of this study was therefore to evaluate the mutagenic activity of by examining its ability to revert five histidine-requiring strains of Salmonella typhimuriumin the absence and in the presence of a rat liver metabolising system (S -9). The procedures used in this study were in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Tests B 13/14 (2000), the requirements of Japanese MOHW (1999) and MAFF (2000), ICH Harmonised Tripartite Guideline (1997) and EPA-OPPTS Guideline 870.5100 (1998).

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## - 14 - This study was performed according to the protocol. The study was initiated on 1 April 2005. Experimental work started on 20 May 2005 and was completed on 27 June 2005. The study completion date is considered to be the date on which the Study Director signs the final report.

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## - 15 - MATERIALS

Test article batch number 8488/035, was an orange liquid, which was received on 12May

2005. It was stored at room temperature in the dark. The test article was stated to contain only high purity (>99.5%) tall oil fatty acid and The Sponsor provided information that the test article was stable for the duration of this study. A copy of the Sponsor's Certificate of Analysis is presented in Appendix 6 . The test article information and Certificate of Analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of the stability and characteristics of the test article were the responsibility of the Sponsor. Test article solutions were prepared by formulating in ethanol, with the aid of vortex mixing where required, immediately prior to assay to give the maximum required treatment solution concentration. This solution was not filter-sterilised and further dilutions were made using ethanol. The test article solutions were protected from light and used within approximately 63⁄4 hours of the initial formulation of the test article. Solutions were used as follows overleaf:

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## - 16 - Experiment S-9 Concentration of treatment solution (mg/mL) Final concentration (μg/plate)
Range-Finder Experiment and Mutation Experiment 1 − and + 0.016 0.08 0.40 2.00 10.00 50.00 1.6 8 40 200 1000 5000 Mutation Experiment 1 TA102 additional treatments − 0.000128 0.00064 0.0032 0.016 0.08 0.40 2.00 0.0128 0.064 0.32 1.6 8 40 200 Mutation Experiment 2 (strains TA98, TA100, TA1535 and TA1537 in the absence and presence of S-9 and strain TA102 in the presence of S-9) − and + 0.08192* 0.2048* 0.512* 1.28* 3.20* 8.00* 20.00* 50.00* 8.192 20.48 51.2 128 320 800 2000 5000 Mutation Experiment 2 (strain TA102) − 0.003277 0.008192 0.02048 0.0512 0.128 0.32 0.80 2.00 0.3277 0.8192 2.048 5.12 12.8 32 80 200

* Concentration of treatments solutions used for the Experiment 2 pre-incubation treatments were twice that stated above, in order to permit treatments at the final concentration stated, whilst volume additions were reduced to 0.05 mL. 

## 0.1 mL volume additions of test article formulations were used for plate-incorporation treatments and 0.05 mL volume additions were used for pre-incubation treatments. Controls Control treatments were performed using the same addition volumes per plate as the test article treatments, 0.1 mL for plate incorporation treatments and 0.05 mL for pre -incubation treatments . Negative controls comprised treatments with the solvent ethanol. The positive control chemicals were supplied and used as tabulated overleaf:

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## - 19 - METHODS
Toxicity Range-Finder Experiment The test article was tested in strain TA100, at the concentrations detailed previously. Triplicate plates without and with S-9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1 ° C:

• 

## 0.1 mL bacterial culture
• 

## 0.1 mL test article solution or control
• 

## 0.5 mL 10% S-9 mix or buffer solution followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1 ° C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony counting).
Mutation experiments The test article was tested for mutation in five strains of Salmonella typhimurium(TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9. Experiment 1 mutagenicity data for strain TA100 were provided by the Range-Finder Experiment treatments. Negative (solvent) controls were included in each assay, in quintuplicate without and with S-9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S -9 mix used in each experiment was confirmed by AAN or B[a]P treatments (again in triplicate) of the strains in the presence of S-9. Platings were achieved as described above. Following Experiment 1 treatments of TA102 in the absence of S-9, unexpected excessive toxicity was observed, and it was considered that there were insufficient doses unaffected by toxicity to allow a thorough assessment of potential mutagenicity to be performed. Repeat Experiment 1 treatments of strain TA102 in the absence of S-9 were therefore performed. Excessive toxicity was also observed following these repeat treatments therefore additional Experiment 1 treatments of strain TA102 in the absence of S -9 were performed using a

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## - 20 - modified dose range. Following these initial additional treatments, the solvent controls for strain TA102 in the absence of S -9 fell sufficiently below the normal historical control range that the data were considered to be uncharacteristic for the strain, therefore these data were invalidated are not reported. Repeat additional treatments were performed and are presented as Experiment 1 additional treatment data for strain TA102 in the absence of S -9. As the results of the first experiment were negative, treatments in the presence of S -9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S -9 mix detailed above, were mixed together and incubated for 1 hour at 37±1 ° C, with shaking, before the addition of 2.5 mL molten agar at 46±1 ° C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay. Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the solvent (

ethanol ) employed in this study. This, and some other organic solvents, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the solvent that may have otherwise occurred. Following initial Experiment 2 treatments of strain TA98 in the absence of S -9, no data were obtained for the test article treated plates due to contamination. Following initial Experiment 2 treatments of strain TA100 in the presence of S-9 and strain TA102 in the absence and presence of S -9, the solvent controls fell sufficiently below the historical ranges such that the data were considered to be uncharacteristic for the strains and are therefore not reported. Repeat treatments of strains TA98 in the absence of S -9, TA100 in the presence of S -9 (together with diagnostic controls in the absence of S-9, data not reported), and TA102 in the absence and presence of S -9 were performed and are reported as the Experiment 2 data for these strains.
Colony counting Colonies were counted electronically using a Seescan Colony Counter (Seescan plc) or manually where confounding factors such as split agar affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.

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## - 21 - Analysis of results Treatment of data Individual plate counts from all experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. The accepted normal ranges for mean numbers of spontaneous revertants on solvent control plates for this laboratory are presented in Appendix 3 . Data for this laboratory are consistent with ranges of spontaneous revertants per plate considered acceptable elsewhere (6). The accepted normal ranges for mean numbers of induced revertants on positive control plates for this laboratory are presented in Appendix 4 . The ranges quoted are based on a large volume of historical control data accumulated from experiments where the correct strain and assay functioning were considered to have been confirmed. For evaluation of data there are many statistical methods in use, and several areacceptable (7,8). The m-statistic was calculated to check that the data were Poissondistributed (8), and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis (8). Acceptance criteria The assay was considered valid if the following criteria were met: 1. the negative control counts fell within the normal ranges as defined in Appendix 3 2. the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S -9 preparation 3. no more than 5% of the plates were lost through contamination or some other unforeseen event.

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## - 22 - Evaluation criteria The test article was considered to be mutagenic if: 1. the assay was valid (see above) 2. Dunnett's test gave a significant response (p ≤ 0.01) and the data set(s) showed a significant dose correlation 3. the positive responses described above were reproducible.

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## - 23 - RESULTS
Toxicity, solubility and dose selection Details of all treatment solution concentrations and final test article doses are provided in the Test article section. An initial toxicity Range-Finder Experiment was carried out in strain TA100 only in the absence and presence of S-9, using final concentrations of test article at 1.6, 8, 40, 200, 1000 and 5000μg/plate, plus negative (solvent) and positive controls. No evidence of toxicity as would normally be manifest as a thinning of the background bacterial lawn and/or a marked reduction in revertant numbers was observed following any of these treatments. These data were considered to be acceptable for mutation assessment and are presented in this report as the TA100 mutagenicity data for Experiment 1. Experiment 1 treatments of the remaining test strains in the absence and presence of S-9 retained the same test doses as employed for the Range-Finder Experiment. Evidence of toxicity in the form of a marked reduction in revertant numbers was observed following the top dose treatment of strain TA1535 in the absence of S-9 and in strains TA98, TA1535 and TA1537 in the presence of S -9. For treatments of strain TA102 in the absence of S -9 a slight thinning of the background bacterial lawn and/or a marked reduction in revertant numbers was observed following the highest four dose treatments. Because of this unexpected excessive toxicity, repeat treatments were performed using strain TA102 in the absence of S

-9 using the same doses. Following t hese repeat treatments, evidence of toxicity in the form of a slight thinning of the background lawn was again observed at the highest four doses tested. It was considered that there were too many doses affected by toxicity to allow a thorough assessment of potential mutagenicity to be performed, therefore additional treatments were performed using strain TA102 in the absence of S -9 with final concentrations of 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200μg/plate. Following these additional treatments a slight thinning of the background bacterial lawn was observed at the highest two doses tested. Experiment 2 treatments of all the tester strains in the absence and presence of S -9 were performed with the maximum test dose of 5000 μg/plate or up to an estimate of the lower limit of toxicity for strain TA102 in the absence of S -9. Narrowed dose ranges were employed (0.3227-200μg/plate for strain TA102 in the absence of S -9 and 8.192- 5000 μg/plate

for all other treatments), in order to examine more closely those

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## - 24 - concentrations of test article approaching the maximum test dose and therefore considered most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre -incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Evidence of toxicity in the form of a slight thinning of the background bacterial lawn and/or a marked reduction in revertant numbers was observed following the top dose treatment of strain TA102 in the presence of S -9 and the highest two dose treatments of strain TA102 in the absence of S -9 and strain TA1537 in the presence of S-9.

Mutation The individual plate counts were averaged to give mean values, which are presented in Appendix 1 and Appendix 2 . From the data it can be seen that mean solvent control counts fell within the normal historical ranges ( Appendix 3 ). The positive control chemicals all induced large increases in revertant numbers in the appropriate strains, which fell within the normal historical ranges (

Appendix 4 ). Less than 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study therefore demonstrated correct strain and assay functioning and was accepted as valid. The mutation data were evaluated as follows: No statistically significant, dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any test article mutagenic activity.

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## - 25 - CONCLUSION It was concluded that did not induce mutation in five histidinerequiring strains of

Salmonella typhimurium(TA98, TA100, TA1535, TA1537 and TA102) when tested under the conditions of this study. These conditions included treatments at concentrations up to either 5000 μ g/plate or the lower limit of toxicity in the absence and in the presence of a rat liver metabolic activation system (S -9).

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## - 26 - REFERENCES 1. Ames B N, McCann J and Yamasaki E (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31 , 347-364. 2. Gatehouse D G, Wilcox P, Forster R, Rowland I R and Callander R D (1990) Bacterial mutation assays. In "Basic Mutagenicity Tests UKEMS Recommended Procedures". Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part I Revised. Ed D J Kirkland. Cambridge University Press, pp 13-61. 3. Maron D M and Ames B N (1983) Revised methods for the Salmonellamutagenicity test. Mutation Research 113 , 173-215. 4. Garner R C (1979) Carcinogen prediction in the laboratory: a personal view. Proc Roy Soc Ser B 205 , 121-134. 5. Booth S C, Welch A M and Garner R C (1980) Some factors affecting mutant numbers in the Salmonella/microsome assay. Carcinogenesis 1 , 911 -923. 6. De Serres F J and Shelby M D (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Research 64 , 159-165. 7. Venitt S, Forster R and Longstaff E (1983) Bacterial Mutation Assays. In "Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Part I. Basic Test Battery." Ed B J Dean. United Kingdom Environmental Mutagen Society, Swansea, pp 5-40. 8. 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 "Statistical Evaluation of Mutagenicity Test Data". Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Part III. Ed D JKirkland. Cambridge University Press, pp 26-65.

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## - 27 - APPENDICES Appendix 1 Raw plate counts and calculated mutagenicity data

Experiment 1 Summary of mean revertant colonies (-S-9) - Experiment 1

TA98 TA100 TA1535 TA1537 TA102 Substance Dose Level

μg/plate Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Ethanol 100 μ l 33 ±5 98 ±5 16 ±2 14 ±3 205 ±25

1.6 35 ±5 105 ±19 14 ±4 18 ±4 139 ±5

8 28 ±6 98 ±8 11 ±1 16 ±2 118 ±3

40 32 ±7 102 ±6 20 ±0 15 ±6 80 ±21

200 29 ±4 90 ±14 16 ±5 10 ±3 81 ±16 (S) 1000 27 ±2 84 ±12 10 ±6 17 ±1 76 ±12 (S) 5000 21 ±2 80 ±15 8 ±2 13 ±5 65 ±11 (S) Positive controls Compound 2NF NaN 3 NaN 3 AAC MMC

Dose Level 5 μg 2 μg 2 μg 50 μg 0.2 μg
Mean ±SD 1124 ±156 628 ±10 649 ±56 230 ±64 498 ±52

SD Standard deviation 2NF 2 -Nitrofluorene NaN 3 Sodium azide AAC 9 -Aminoacri dine MMC Mitomycin C S Slight thinning of background bacterial lawn

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## - 28 - Summary of mean revertant colonies (+S-9) - Experiment 1
TA98 TA100 TA1535 TA1537 TA102 Substance Dose Level

μg/plate Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Ethanol 100 μ l 57 ±7 98 ±15 26 ±8 15 ±4 195 ±16

1.6 44 ±2

(M) 88 ±16 14 ±5 15 ±3 164 ±23

8 53 ±6 82 ±2 20 ±9 14 ±8 192 ±5

40 46 ±7 81 ±5 23 ±6 12 ±2 204 ±5

200 48 ±7 103 ±8 18 ±6 15 ±4 208 ±10

1000 39 ±5 84 ±2 24 ±2 18 ±4 188 ±19

5000 20 ±4 80 ±12 10 ±2 7 ±2 126 ±18

Positive controls Compound B[a]P AAN AAN AAN AAN

Dose Level 10 μg 5 μg 5 μg 5 μg 20 μg
Mean ±SD 361 ±86 1303 ±330 150 ±18 53 ±30 652 ±114

SD Standard deviation B[a]P Benzo[a]pyrene AAN 2 -Aminoanthracene M Plates counted manually

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## - 29 - Experiment 1 Table 1 Test strain: TA98 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 25 34 33 35 39 1.6 30 M 40 34 8 21 33 30 40 32 26 39 200 33 29 26 1000 25 27 29 5000 23 20 19 Positive 1244 947 1180 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 33.20 5 5.12 1.6 34.67 3 1.04^ 5.03 0.16 NS 0.92 0.41 NS 8 28.00 3 0.84 6.24 0.45 NS -0.71 -1.52 NS 40 32.33 3 0.97 6.51 0.06 NS -0.02 -0.25 NS 200 29.33 3 0.88 3.51 0.22 NS -0.02 -1.07 NS 1000 27.00 3 0.81 2.00 0.37 NS -0.01 -1.75 NS 5000 20.67 3 0.62 2.08 0.64 NS -0.00 -3.82 NS Positive 1123.67 3 33.85 156.31 M Statistic = 0.724 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: M Plate counted manually

^ Maximum increase above control

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## - 30 - Experiment 1 Table 2 Test strain: TA98 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 58 46 63 56 62 1.6 41 M 45 M 45 M 8 59 52 48 40 38 49 50 200 50 40 53 1000 42 41 33 5000 17 19 25 Positive 405 261 416 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 57.00 5 6.78 1.6 43.67 3 0.77 2.31 0.79 NS -8.33 -2.97 NS 8 53.00 3 0.93 5.57 0.09 NS -0.19 -0.84 NS 40 45.67 3 0.80 6.66 0.37 NS -0.18 -2.53 NS 200 47.67 3 0.84 6.81 0.23 NS -0.02 -2.06 NS 1000 38.67 3 0.68 4.93 0.54 NS -0.01 -4.23 NS 5000 20.33 3 0.36 4.16 0.83 NS -0.01 -9.70 NS Positive 360.67 3 6.33 86.49 M Statistic = 0.718 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: M Plate counted manually

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## - 31 - Experiment 1 Table 3 Test strain: TA100 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 99 105 94 99 92 1.6 98 127 91 8 92 107 95 40 107 105 95 200 105 77 87 1000 78 97 76 5000 92 85 64 Positive 622 640 622 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 97.80 5 5.07 1.6 105.33 3 1.08^ 19.09 0.34 NS 4.71 0.80 NS 8 98.00 3 1.00 7.94 0.05 NS -0.16 0.02 NS 40 102.33 3 1.05 6.43 0.10 NS 0.06 0.52 NS 200 89.67 3 0.92 14.19 0.37 NS -0.05 -1.00 NS 1000 83.67 3 0.86 11.59 0.51 NS -0.02 -1.73 NS 5000 80.33 3 0.82 14.57 0.50 NS -0.00 -2.18 NS Positive 628.00 3 6.42 10.39 M Statistic = 1.441 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes:

^ Maximum increase above control

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## - 34 - Experiment 1 Table 6 Test strain: TA1535 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 14 26 22 M 31 35 1.6 14 M 19 10 8 19 29 11 40 26 27 17 200 14 25 15 1000 22 25 26 5000 9 12 10 Positive 130 159 162 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 25.60 5 8.14 1.6 14.33 3 0.56 4.51 0.66 NS -7.04 -2.49 NS 8 19.67 3 0.77 9.02 0.21 NS -0.49 -1.29 NS 40 23.33 3 0.91 5.51 0.09 NS 0.04 -0.39 NS 200 18.00 3 0.70 6.08 0.16 NS -0.02 -1.59 NS 1000 24.33 3 0.95 2.08 0.15 NS 0.00 -0.15 NS 5000 10.33 3 0.40 1.53 0.47 NS -0.00 -3.57 NS Positive 150.33 3 5.87 17.67 M Statistic = 1.812 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: M Plate counted manually

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## - 35 - Experiment 1 Table 7 Test strain: TA1537 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 13 M 16 10 15 18 1.6 14 M 22 19 8 16 14 18 40 9 18 19 M 200 6 11 12 1000 16 18 17 5000 18 12 9 Positive 167 229 294 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 14.40 5 3.05 1.6 18.33 3 1.27^ 4.04 0.54 NS 2.46 1.36 NS 8 16.00 3 1.11 2.00 0.12 NS 0.11 0.61 NS 40 15.33 3 1.06 5.51 0.05 NS -0.01 0.25 NS 200 9.67 3 0.67 3.21 0.57 NS -0.03 -1 .96 NS 1000 17.00 3 1.18 1.00 0.10 NS 0.00 0.97 NS 5000 13.00 3 0.90 4.58 0.16 NS -0.00 -0.58 NS Positive 230.00 3 15.97 63.51 M Statistic = 0.894 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: M Plate counted manually

^ Maximum increase above control

### Final Report



## - 36 - Experiment 1 Table 8 Test strain: TA1537 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 11 19 12 13 19 M 1.6 17 11 17 8 17 21 5 40 12 13 10 200 19 12 15 1000 22 18 14 5000 6 10 6 Positive 27 85 46 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 14.80 5 3.90 1.6 15.00 3 1.01 3.46 0.03 NS 0.13 0.08 NS 8 14.33 3 0.97 8.33 0.05 NS -0.07 -0.39 NS 40 11.67 3 0.79 1.53 0.30 NS -0.08 -0.93 NS 200 15.33 3 1.04 3.51 0.06 NS 0.00 0.18 NS 1000 18.00 3 1.22^ 4.00 0.32 NS 0.00 0.92 NS 5000 7.33 3 0.50 2.31 0.48 NS -0.00 -2.57 NS Positive 52.67 3 3.56 29.57 M Statistic = 1.289 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: M Plate counted manually

^ Maximum increase above control

### Final Report



## - 37 - Experiment 1 Table 9 Test strain: TA102 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 203 163 218 217 223 1.6 144 135 139 8 118 121 116 40 68 105 68 200 65 S 96 S 81 S 1000 83 S 84 S 62 S 5000 61 S 57 S 77 S Positive 441 509 544 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 204.80 5 24.52 1.6 139.33 3 0.68 4.51 0.88 NS -40.92 -4.46 NS 8 118.33 3 0.58 2.52 0.78 NS -9.62 -6.12 NS 40 80.33 3 0.39 21.36 0.77 NS -2.49 -9.64 NS 200 80.67 3 0.39 15.50 0.59 NS -0.42 -9.57 NS 1000 76.33 3 0.37 12.42 0.49 NS -0.07 -9.99 NS 5000 65.00 3 0.32 10.58 0.46 NS -0.01 -11.20 NS Positive 498.00 3 2.43 52.37 M Statistic = 2.309 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: S Slight thinning of background bacterial lawn

### Final Report



## - 38 - Experiment 1 Table 10 Test strain: TA102 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 190 202 217 173 194 1.6 189 160 143 8 190 198 188 40 199 209 203 200 206 199 218 1000 169 188 206 5000 142 129 106 Positive 522 699 736 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 195.20 5 16.15 1.6 164.00 3 0.84 23.26 0.68 NS -19.50 -2.72 NS 8 192.00 3 0.98 5.29 0.06 NS 0.35 -0.25 NS 40 203.67 3 1.04 5.03 0.39 NS 0.47 0.71 NS 200 207.67 3 1.06^ 9.61 0.43 * 0.11 1.03 NS 1000 187.67 3 0.96 18.50 0.02 NS -0.00 -0.63 NS 5000 125.67 3 0.64 18.23 0.77 NS -0.01 -6.42 NS Positive 652.33 3 3.34 114.38 M Statistic = 1.394 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes:

^ Maximum increase above control

### Final Report



## - 39 - Summary of mean revertant colonies (-S-9) - Experiment 1 repeat

TA102 Substance Dose Level

μg/plate Mean ±SD Ethanol 100 μ l 306 ±20

1.6 253 ±18

8 261 ±8

40 180 ±17

(S) 200 209 ±16

(S) 1000 181 ±13

(S) 5000 160 ±20

(S) Positive controls Compound MMC

Dose Level 0.2 μg
Mean ±SD 569 ±38

SD Standard deviation MMC Mi tomycin C S Slight thinning of background bacterial lawn

### Final Report



## - 41 - Summary of mean revertant colonies (-S-9) - Experiment 1 additional treatments

TA102 Substance Dose Level

μg/plate Mean ±SD Ethanol 100 μ l 245 ±10

0.0128 248 ±19

0.064 263 ±6

0.32 273 ±8

1.6 233 ±14

8 211 ±18

40 149 ±22

(S) 200 86 ±22

(S) Positive controls Compound MMC

Dose Level 0.2 μg
Mean ±SD 559 ±42

SD Standard deviation MMC Mitomycin C S Slight thinning of background bacterial lawn

### Final Report



## - 42 - Experiment 1 additional treatments Table 12 Test strain: TA102 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 240 232 242 258 253 0.0128 257 261 226 0.064 270 260 258 0.32 267 271 282 1.6 234 247 219 8 230 210 194 40 137 S 175 S 136 S 200 100 S 97 S 60 S Positive 511 583 584 Treatment ( μ g/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 245.00 5 10.44 0.0128 248.00 3 1.01 19.16 0.12 NS 234.38 0.19 NS 0.064 262.67 3 1.07 6.43 0.57 * 277.68 1.19 NS 0.32 273.33 3 1.12^ 7.77 0.69 *** 83.23 1.89 NS 1.6 233.33 3 0.95 14.01 0.38 NS -11.00 -0.81 NS 8 211.33 3 0.86 18.04 0.70 NS -5.48 -2.40 NS 40 149.33 3 0.61 22.23 0.89 NS -2.61 -7.40 NS 200 85.67 3 0.35 22.28 0.88 NS -0.83 -13.83 NS Positive 559.33 3 2.28 41.86 M Statistic = 1.581 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: S Slight thinning of background bacterial lawn

^ Maximum increase above control

### Final Report



## - 44 - Summary of mean revertant colonies (+S-9) - Experiment 2
TA98 TA100 TA1535 TA1537 TA102 Substance Dose Level

μg/plate Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Ethanol 50 μ

l 28 ±3 111 ±14 20 ±6 13 ±6 185 ±33
8.192 37 ±3 105 ±12 14 ±4 12 ±2 184 ±6

20.48 37 ±3 98 ±7 14 ±4 13 ±5 261 ±37

51.2 37 ±11 101 ±1 12 ±5 11 ±6 241 ±40

128 41 ±6 102 ±7 15 ±2 15 ±1 213 ±33

320 34 ±2 134 ±34 17 ±4 10 ±3 252 ±27

800 30 ±9 91 ±10 13 ±6 9 ±5 255 ±10

2000 27 ±1 81 ±5 9 ±2 8 ±5 (S) 240 ±28

5000 33 ±6 90 ±13 13 ±2 9 ±1 (S) 197 ±27

(S) Positive controls Compound B[a]P AAN AAN AAN AAN

Dose Level 10 μg 5 μg 5 μg 5 μg 20 μg
Mean ±SD 242 ±54 995 ±83 221 ±21 120 ±31 923 ±19

SD Standard deviation B[a]P Benzo[a]pyrene AAN 2 -Aminoanthracene S Slight thinning of background lawn

### Final Report



## - 45 - Experiment 2 Table 13 Test strain: TA98 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 20 31 28 23 27 8.192 29 24 28 20.48 19 18 18 51.2 32 28 25 128 16 27 23 320 16 23 23 800 28 20 22 2000 28 23 24 5000 8 22 17 Positive 875 788 694 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 25.80 5 4.32 8.192 27.00 3 1.05 2.65 0.17 NS 0.15 0.36 NS 20.48 18.33 3 0.71 0.58 0.66 NS -0.36 -2.24 NS 51.2 28.33 3 1.10^ 3.51 0.12 NS 0.03 0.72 NS 128 22.00 3 0.85 5.57 0.18 NS -0.02 -1.15 NS 320 20.67 3 0.80 4.04 0.33 NS -0.01 -1.53 NS 800 23.33 3 0.90 4.16 0.16 NS -0.00 -0.71 NS 2000 25.00 3 0.97 2.65 0.02 NS 0.00 -0.20 NS 5000 15.67 3 0.61 7.09 0.44 NS -0.00 -3.39 NS Positive 785.67 3 30.45 90.52 M Statistic = 0.860 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes:

^ Maximum increase above control

### Final Report



## - 48 - Experiment 2 Table 16 Test strain: TA100 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 90 125 121 110 108 8.192 112 92 112 20.48 97 105 92 51.2 101 100 101 128 94 107 106 320 108 M 172 121 800 89 101 82 2000 84 84 76 5000 103 89 77 Positive 1075 909 1001 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 110.80 5 13.66 8.192 105.33 3 0.95 11.55 0.23 NS -0.67 -0.54 NS 20.48 98.00 3 0.88 6.56 0.47 NS -0.63 -1.28 NS 51.2 100.67 3 0.91 0.58 0.37 NS -0.20 -1.00 NS 128 102.33 3 0.92 7.23 0.22 NS -0.05 -0.83 NS 320 133.67 3 1.21^ 33.83 0.51 * 0.08 2.08 NS 800 90.67 3 0.82 9.61 0.13 NS -0.01 -2.09 NS 2000 81.33 3 0.73 4.62 0.45 NS -0.01 -3.13 NS 5000 89.67 3 0.81 13.01 0.38 NS -0.00 -2.21 NS Positive 995.00 3 8.98 83.16 M Statistic = 1.732 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: M Plate counted manually

^ Maximum increase above control

### Final Report



## - 51 - Experiment 2 Table 19 Test strain: TA1537 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 17 10 7 21 22 8.192 11 14 11 20.48 10 15 16 51.2 20 17 6 128 9 13 11 320 16 18 14 800 16 7 4 2000 8 11 15 5000 11 12 17 Positive 111 80 112 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 15.40 5 6.66 8.192 12.00 3 0.78 1.73 0.33 NS -0.42 -0.76 NS 20.48 13.67 3 0.89 3.21 0.17 NS -0.09 -0.32 NS 51.2 14.33 3 0.93 7.37 0.03 NS -0.01 -0.32 NS 128 11.00 3 0.71 2.00 0.24 NS -0.02 -1.06 NS 320 16.00 3 1.04^ 2.00 0.09 NS 0.00 0.31 NS 800 9.00 3 0.58 6.24 0.29 NS -0.01 -1.91 NS 2000 11.33 3 0.74 3.51 0.23 NS -0.00 -1.00 NS 5000 13.33 3 0.87 3.21 0.07 NS -0.00 -0.41 NS Positive 101.00 3 6.56 18.19 M Statistic = 1.736 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes:

^ Maximum increase above control

### Final Report



## - 52 - Experiment 2 Table 20 Test strain: TA1537 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 9 11 9 23 15 8.192 14 M 11 10 20.48 18 11 9 51.2 7 17 8 128 15 15 14 320 7 11 13 800 6 14 6 2000 5 S 13 S 5 S 5000 9 S 10 S 8 S Positive 96 110 155 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 13.40 5 5.90 8.192 11.67 3 0.87 2.08 0.19 NS -0.21 -0.42 NS 20.48 12.67 3 0.95 4.73 0.08 NS -0.04 -0.17 NS 51.2 10.67 3 0.80 5.51 0.20 NS -0.05 -0.89 NS 128 14.67 3 1.09^ 0.58 0.13 NS 0.01 0.52 NS 320 10.33 3 0.77 3.06 0.15 NS -0.01 -0.91 NS 800 8.67 3 0.65 4.62 0.33 NS -0.01 -1.60 NS 2000 7.67 3 0.57 4.62 0.40 NS -0.00 -2.02 NS 5000 9.00 3 0.67 1.00 0.32 NS -0.00 -1.34 NS Positive 120.33 3 8.98 30.83 M Statistic = 1.645 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: S Slight thinning of background lawn M Plate counted manually

^ Maximum increase ab ove control

### Final Report



## - 53 - Experiment 2 Table 21 Test strain: TA102 -S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 207 242 298 205 206 0.3277 202 200 203 0.8192 232 211 223 2.048 235 308 273 5.12 251 293 239 12.8 183 159 178 32 175 136 157 80 84 S 108 S 106 S 200 122 S 100 S 114 S Positive 444 357 380 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 231.60 5 40.27 0.3277 201.67 3 0.87 1.53 0.45 NS -91.34 -1.60 NS 0.8192 222.00 3 0.96 10.54 0.15 NS -12.65 -0.46 NS 2.048 272.00 3 1.17^ 36.51 0.51 * 23.15 2.12 NS 5.12 261.00 3 1.13 28.35 0.47 * 9.02 1.58 NS 12.8 173.33 3 0.75 12.66 0.38 NS -3.45 -3.30 NS 32 156.00 3 0.67 19.52 0.62 NS -2.62 -4.43 NS 80 99.33 3 0.43 13.32 0.80 NS -1.78 -8.56 NS 200 112.00 3 0.48 11.14 0.70 NS -0.70 -7.54 NS Positive 393.67 3 1.70 45.08 M Statistic = 2.875 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: S Slight thinning of background lawn

^ Maximum increase above control

### Final Report



## - 54 - Experiment 2 Table 22 Test strain: TA102 +S-9 Treatment ( μg/plate) Revertant numbers/plate
Solvent 157 161 212 167 228 8.192 184 179 190 20.48 258 226 299 51.2 226 286 210 128 249 184 206 320 275 259 222 800 247 267 252 2000 212 241 268 5000 199 S 169 S 222 S Positive 901 934 934 Treatment ( μg/plate) Mean (of) N Fold Increase Standard

Deviation Correlation

Coefficient Slope of

best fit Dunnett's

t value Solvent 185.00 5 32.64 8.192 184.33 3 1.00 5.51 0.01 NS -0.08 0.02 NS 20.48 261.00 3 1.41^ 36.59 0.73 ** 3.67 3.59 * 51.2 240.67 3 1.30 40.07 0.56 * 1.24 2.68 NS 128 213.00 3 1.15 33.06 0.20 NS 0.18 1.41 NS 320 252.00 3 1.36 27.18 0.39 * 0.14 3.22 * 800 255.33 3 1.38 10.41 0.42 * 0.06 3.38 * 2000 240.33 3 1.30 28.01 0.29 NS 0.02 2.69 NS 5000 196.67 3 1.06 26.58 0.10 NS -0.00 0.62 NS Positive 923.00 3 4.99 19.05 M Statistic = 3.883 Key to significance: * p ≤0.05 ** p ≤0.01 *** p ≤0.005 NS not significant Key to postfixes: S Slight thinning of background lawn

^ Maximum increase above control

### Final Report



## - 56 - Appendix 4 Historical positive control values for S. typhimuriumstrains Induced numbers for individual plates Reference ranges (1) Strain S-9 No. of studies Chemical Mean Median Observed range 95% 90%
TA98

- 36 2NF 1042 992 465.6 - 2457.7 627.4 - 1712.6 689.2 - 1492.2 TA98 + 36 B[a]P 292 292 92.0 - 642.3 125.5 - 470.2 140.2 - 441.0
TA100

- 36 NaN 3 508 524 30.0 - 858.0 182.7 - 743.5 256.3 - 699.8 TA100 + 36 AAN 1168 1062 95.2 - 2472.3 255.8 - 2155.0 340.2 - 2054.0
TA1535

- 36 NaN 3 511 511 254.3 - 948.0 294.7 - 739.8 306.6 - 705.5 TA1535 + 36 AAN 223 208 30.6 - 510.5 75.0 - 436.5 86.8 - 412.2
TA1537

- 36 AAC 174 116 18.2 - 1976.4 33.5 - 986.4 44.2 - 378.3 TA1537 + 36 AAN 188 172 9.0 - 676.0 35.0 - 424.2 45.2 - 374.6
TA102

- 4 MMC 458 389 111.6 - 1077.6 124.6 - 1042.6 153.6 - 1014.6 TA102 + 29 AAN 1004 962 75.4 - 2068.2 112.8 - 1939.4 339.2 - 1820.2
(1) Reference ranges are calculated from percentiles of the observed distributions. Ranges calculated between Nov 04 and Jan 05 by CLEH Statistics, using data from studies # selected without bias over the periods given below: S.typhimurium strains (except TA102) Jan 04 to Apr 04 S.typhimurium strain TA102 Apr 04 to Oct 04 # With the exception of TA102, all studies had been audited prior to data collection. 2NF 2-Nitrofluorene MMC Mitomyc in C B[a]P Benzo[a]pyrene NaN 3 Sodium azide AAN 2-Aminoanthracene AAC 9-Aminoacridine All revertant numbers used to derive the above data are induced revertant numbers, i.e. the number of revertant colonies on the positive control plates minus the mean number of spontaneous revertant colonies on concurrent negative control plates.

### Final Report



## - 57 - Appendix 5 Quality control statements for S-9

### Final Report



## - 58 -