Final Report

Study Title : Reverse

Mutation in Five Histidine-requiring Strains of

Salmonella typhimurium

Data Requirement
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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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GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium
Final Report

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FLAGGING STATEMENTS

Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium
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STUDY DIRECTOR AUTHENTICATION

AND GLP COMPLIANCE STATEMENT

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

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

Reverse Mutation in Five Histidine-requiring Strains of Salmonella typhimurium
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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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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.
Final Report

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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 Experiment 1.....................................27

Appendix 2 Raw plate counts and calculated mutagenicity data Experiment 2.....................................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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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. Following

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 doses. 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 0.0128-200 μg/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 estimate of the lower

limit of toxicity for strain TA102 in the absence of S-9. Narrowed dose ranges were

employed (0.3227-200 μg/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 those

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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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 μg/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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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 typhimurium in 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 multicopy

plasmid 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 typhimurium in 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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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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MATERIALS

Test article

batch number 8488/035, was an orange liquid, which was

received on 12 May 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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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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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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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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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 are

acceptable (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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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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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 5000 μg/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 these 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 200 μg/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 (0.3227-200 μg/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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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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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 μg/plate or the lower limit of toxicity in the

absence and in the presence of a rat liver metabolic activation system (S-9).
Final Report

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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 Salmonella mutagenicity

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 J Kirkland. Cambridge University

Press, pp 26-65.
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APPENDICES

Appendix 1

Raw plate counts and calculated mutagenicity data

Experiment 1

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

Substance Dose Level TA98 TA100 TA1535 TA1537 TA102

μg/plate

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

Ethanol 100 μl 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 NaN3 NaN3AAC 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

NaN3 Sodium azide

AAC 9-Aminoacridine

MMC Mitomycin C

S Slight thinning of background bacterial lawn
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Summary of mean revertant colonies (+S-9) - Experiment 1

Substance Dose Level TA98 TA100 TA1535 TA1537 TA102

μg/plate

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

Ethanol 100 μl 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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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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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
Final Report

- 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


Final Report

- 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
Final Report

- 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

Substance Dose Level TA102

μg/plate

Mean ± SD

Ethanol 100 μl 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 Mitomycin C

S Slight thinning of background bacterial lawn

Final Report

- 41 -

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

Substance Dose Level TA102

μg/plate

Mean ± SD

Ethanol 100 μl 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

(μg/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

Substance Dose Level TA98 TA100 TA1535 TA1537 TA102

μg/plate

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

Ethanol 50 μl 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 above 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. typhimurium strains

 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 NaN3508 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 NaN3511 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 Mitomycin C

B[a]P Benzo[a]pyrene NaN3 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 -