FINAL REPORT

Test Facility Study No. 787739, Report No. 30896



### Alcohols, C18-22 Distn. Residues (CAS No. 1160164-88-4): Mouse Lymphoma Mutation Study

TEST FACILITY: Charles River Tranent Edinburgh EH33 2NE UK

SPONSOR: SASOL Germany GmbH Anckelmannsplatz 1 D-20537 Hamburg Germany

Page 1 of 67Page 2 Test Facility Study No. 787739

TABLE OF CONTENTS

1 COMPLIANCE STATEMENT................................................................................ 7

2 QUALITY ASSURANCE STATEMENT ............................................................... 8

3 RESPONSIBLE PERSONNEL ................................................................................ 9

4 SUMMARY .............................................................................................................. 10

5 INTRODUCTION.................................................................................................... 11

5.1 Regulatory Citations ................................................................................................ 11

5.1.1 Test Guideline........................................................................................................... 11

5.1.2 Quality Compliance ................................................................................................. 11

5.2 Study Location.......................................................................................................... 11

5.3 Study Dates ............................................................................................................... 11

5.4 Archiving of Data..................................................................................................... 11

5.5 Deviations from Protocol......................................................................................... 12

6 MATERIALS AND METHODS ............................................................................ 12

6.1 Test Item ................................................................................................................... 12

6.2 Test Item Characterisation ..................................................................................... 12

6.3 Dose Formulations ................................................................................................... 12

6.3.1 Preparation of Dose Formulations ......................................................................... 12

6.3.2 Analysis of Dose Formulations................................................................................ 13

6.4 Test System ............................................................................................................... 13

6.5 Justification of the Test System .............................................................................. 13

6.5.1 Colony Sizing ............................................................................................................ 14

6.6 Cells and Cell Culture.............................................................................................. 14

6.6.1 Cells ..........................................................................................................................

. 14Page 3 Test Facility Study No. 787739

6.6.2 Culture Medium....................................................................................................... 14

6.6.3 Cell Culture .............................................................................................................. 14

6.7 Experimental Design................................................................................................ 15

6.8 Justification of Dose Levels ..................................................................................... 15

6.9 Administration of Test/Control Items.................................................................... 16

6.9.1 Vehicle Controls ....................................................................................................... 16

6.9.2 Positive Control Materials....................................................................................... 16

6.9.3 Test Item ................................................................................................................... 16

6.10 Experimental Procedure.......................................................................................... 17

6.11 S9 Mix........................................................................................................................ 17

6.12 Toxicity Test ............................................................................................................. 17

6.13 Mutation Tests.......................................................................................................... 18

6.13.1 Treatment (4 h exposure period) ............................................................................ 18

6.13.2 Extended Treatment (24 h exposure) ..................................................................... 18

6.13.3 Expression of Genetic Damage ............................................................................... 19

6.13.4 Plate Reading............................................................................................................ 19

7 CALCULATIONS AND DATA ACCEPTANCE................................................. 20

7.1 Calculations .............................................................................................................. 20

7.1.1 Assessment of Relative Suspension Growth .......................................................... 20

7.1.2 Assessment of Relative Total Growth .................................................................... 21

7.1.3 Assessment of Mutant Fractions............................................................................. 21

7.1.4 Colony Size Fractions .............................................................................................. 22

7.2 Historical Control Data ........................................................................................... 22

7.3 Analysis of Data........................................................................................................ 22Page 4 Test Facility Study No. 787739

7.3.1 Acceptance Criteria ................................................................................................. 22

8 STATISTICAL ANALYSIS AND DATA EVALUATION ................................. 23

8.1 Statistical Analysis ................................................................................................... 23

8.2 Data Evaluation........................................................................................................ 23

8.2.1 Interpretation of Toxicity........................................................................................ 23

8.2.2 Interpretation of Mutagenic Activity ..................................................................... 24

9 RESULTS AND DISCUSSION .............................................................................. 26

9.1 Toxicity Tests (Tables 1 - 3) .................................................................................... 26

9.2 Mutation Assays (Tables 4 - 7)................................................................................ 26

9.2.1 Vehicle Control Groups........................................................................................... 26

9.2.2 Positive Control Groups .......................................................................................... 26

9.2.3 Alcohols, C18-22 Distn. Residues............................................................................ 26

10 CONCLUSION......................................................................................................... 28

11 REFERENCES......................................................................................................... 29

12 TABLES.................................................................................................................... 33

13 APPENDICES .......................................................................................................... 40

Final Page of Report ........................................................................................................... ... 67Page 5 Test Facility Study No. 787739

LIST OF TABLES

Table 1 Toxicity Test in the Abse nce of S9 Mix (4 h Exposure) .................................. 33 Table 2 Toxicity Test in the Pr esence of S9 Mix (4 h Exposure).................................. 34 Table 3 Toxicity Test in the Absence of S9 Mix (24 h Exposure) ................................ 35 Table 4 Mutation Test in the Absence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 1) .................................................................................. 36 Table 5 Mutation Test in the Presence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 2) .................................................................................. 37 Table 6 Mutation Test in the Absence of S9 Mix (24 h Exposure) Summary of Means of Data (Assay 3) .................................................................................. 38 Table 7 Mutation Test in the Presence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 4) .................................................................................. 39Page 6 Test Facility Study No. 787739

LIST OF APPENDICES

Appendix 1 Certificate of Analysis ...................................................................................... 40 Appendix 2 Cell Culture....................................................................................................... 41 Appendix 3 In vitro Activation Preparation Form ............................................................... 43 Appendix 4 Worked Examples of Calculations ................................................................... 44 Appendix 5 Mouse Lymphoma L5178Y Cell Hi storical Control Data from July 2003 to December 2008.................................................................................... 47 Appendix 6 Statistical Method............................................................................................. 48 Appendix 7 Individual Suspension Growth Data................................................................. 51 Appendix 8 Individual Cloning Efficiency Data (Non-mutants) ......................................... 55 Appendix 9 Individual Mu tant Selection Data..................................................................... 59 Appendix 10 List of Abbreviations........................................................................................ 671 COMPLIANCE STATEMENT Page 7 Test Facility Study No. 787739

I, the undersigned, hereby declare that this study was performed in accordance with the OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for GLP and as accepted by Regulatory Authorities throughout the European Community, United States of America (FDA and EPA) and Japan (MHLW, MAFF and METI).

The study was conducted according to the procedures herein described and this report represents a true and accurate record of the results obtained.

Colin <?'Riach BSc Study Director2 QUALITY ASSURANCE STATEMENT Page 8 Test Facility Study No. 787739

Study title: Alcohols, C18-22 Distn. Residues: Mouse Lymphoma Mutation Study

The Charles River Quality Assurance Unit conducted a protocol review, process-based inspections and report audits relevant to this short-term study, as detailed below.

Date of QA Activity

05 October 2009 20 October 2009

21 October 2009 22 October 2009 04 November 2009 29/30 December 2009 19 January 2010 Activity

Protocol Review Dose Preparation/Dosing/ Protocol Compliance Cell Counting and Dilutions Cloning Scoring Draft Report Audit Final Report Audit Date of Report to Management

05 October 2009 20 October 2009

21 October 2009 22 October 2009 04 November 2009 05 January 2009 19 January 2010

The protocol review and report audits were reported to the Study Director on the same date as management. The outcome of each process-based inspection is also reported to the Study Director, where relevant.

Facilities relevant to this study are included in Charles River's annual facility inspection
programme. The outcome of each inspection is reported to Management.

This report is considered to describe accurately and completely the procedures used in the study and the results obtained.Page 9 Test Facility Study No. 787739

3 RESPONSIBLE PERSONNEL

Study Director: Colin G Riach BSc

Report Compilation: Colin G Riach BSc

Project Leader: Wanda C Zajac

Technical Assistance: Pawel Jurczak

Statistician: Marion Thom MSc

Quality Assurance: Alison McNaughton HNC Sarah L Drake BScPage 10 Test Facility Study No. 787739

4 SUMMARY

Alcohols, C18-22 Distn. Residues was assaye d for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scori ng for forward mutations at the thymidine kinase locus: tk + tk - to tk tk - . Alcohols, C18-22 Distn. Residu es was prepared as a part solution/part suspension in dimethylsulphoxide. Tests were conducted both in the absence and in the presence of a post-mitochondr ial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors requ ired for mixed-function oxidase activity (S9 mix). The study was desi gned to be consistent with ICH Guidelines, OECD Guideline No. 476 and EC Directive 2000/32/EC B.17. The study also meets the requirements of the United States and Japan.

In preliminary cytotoxicity tests, Alcohol s, C18-22 Distn. Residues was shown to be nontoxic at the highest practical concentration of 200 μg/mL. Pr ecipitation of th e test item occurred at this concentration.

Four independent mutation assays were conducted, as follows:

Assay No. Presence or absence of S9 Treatment time (hours) Final concentrations ( μ g/mL)
1 Absence 4 25, 50, 100, 200 2 Presence 4 25, 50, 100, 200 3 Absence 24 25, 50, 100, 200 4 Presence 4 25, 50, 100, 200

Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘sma ll’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and we re tested in quadruplicate.

Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, the results were analysed for comparison of the log mutant fraction between th e vehicle controls and each concentration of Alcohols, C18-22 Distn. Residues. In additi on, all the experiments were tested for doserelated trends.

No evidence of mutagenic act ivity was obtained with Alcoho ls, C18-22 Distn. Residues in any of the 4 assays.

It was concluded that Alcohols, C18-22 Distn. Residues is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the pres ence of S9 mix, when tested up to and beyond its limit of solubility in the test system.Page 11 Test Facility Study No. 787739

5 INTRODUCTION

The objective of the study was to determine th e potential of Alcohols, C18-22 Distn. Residues to induce forward mutations at the tk + tk - locus of mouse lymphoma L5178Y cells.

5.1 Regulatory Citations

5.1.1 Test Guideline

This study was designed to comply with ICH Guidelines, OECD Guideline No. 476 (Adopted 1997), EC Directive 2000/32/EC B.17 (Adopted 2000) and United St ates 40 CFR § 798.5300 (updated 2001). The study also meets Japanese requirements.

5.1.2 Quality Compliance

The study was conducted in accordance with th e OECD Principles of Good Laboratory Practice as set forth by the United Kingdom De partment of Health and as accepted by Regulatory Authorities throughout the European Community, United States of America (FDA and EPA) and Japan (MHLW, MAFF and METI).

All routine activities conducted during the course of this study are detail ed in Charles River’s Standard Operating Procedures.

5.2 Study Location

The study was conducted at Charles River, Tranent, Edinburgh, EH33 2NE, UK.

5.3 Study Dates

Study Initiation Date: 01 October 2009 Experimental Start Date: 13 October 2009 Experimental Completion Date: 01 December 2009 Study Completion Date: See Compliance St atement page for date of Study Director's signature

5.4 Archiving of Data

All raw data generated and record ed during this study will be stored in the Scientific Archives of Charles River Preclinical Serv ices Edinburgh for 2 years after th e issue of the final report. After the 2 year period, the Sponsor will be co nsulted regarding the disposal, transfer or continued storage of the raw data.Page 12 Test Facility Study No. 787739

The original signed copy of th e final report will be stored i ndefinitely in the Scientific Archives of Charles River Preclinical Services Edinburgh.

5.5 Deviations from Protocol

There were no deviations from the protocol.

6 MATERIALS AND METHODS

6.1 Test Item

The test item was received on 11 September 2009. An archive sample was retained. A copy of the Certificate of Analysis is shown in Appendix 1.

6.2 Test Item Characterisation

Active ingredient: Alcohols C18-22, di stn. Residues (CAS No. 1160164-88-4) Lot No: 03585/MA (product code: 5901RN2) Description: Beige waxy solid Purity: 100% by definition Storage Conditions: Stored in the da rk at ambient room temperature Supplier: SASOL Germany GmbH Expiry date: 31 August 2013

6.3 Dose Formulations

6.3.1 Preparation of Dose Formulations

In preliminary tests, Alcohols, C18-22 Distn. Re sidues was found to have limited solubility in all solvents compatible with th e test system. Dimethylsulphoxide (DMSO) was chosen as the solvent giving the best solubility/dispersal ch aracteristics. A partial solution was obtained using the following technique.

DMSO was added to a pre-weighed sample of Alcohols, C18-22 Distn. Residues to give a concentration of 20 mg/mL. The mixture was vortex-mixed for several minutes, and then placed in an incubator at 37°C for ca 45 min. After this, the mixture was vortex-mixed for a further ca 10 minutes. This process produced a m ilky suspension with fine particles that could be drawn up a wide-bore pipette tip.

To check whether the DMSO was carrying dissolved test item above the saturation limit in the aqueous test conditions, 0.1 mL of the above 20 mg/mL preparation was added to 2.5 mL samples of water and DMSO and the level of pr ecipitate compared. There was a greater level of precipitate/undissolved material present in the water sample than in the DMSO sample andPage 13 Test Facility Study No. 787739

it was therefore concluded that a concentration of 20 mg/mL in DMSO (being greater than the saturation level in aqueous conditions) was a suitable maximum concentration to use in the study. After a 1 in 100 dilution into the cell cultures, the 20 mg/mL solution gave a high concentration of 200 μg/mL.

6.3.2 Analysis of Dose Formulations

The test item formulations were prepared immedi ately prior to dosing (within 1 h). Detailed records of preparation of the dosing soluti ons were maintained to allow checking of procedures. Chemical analysis of the test item formulations was not conducted; however Alcohols, C18-22 Distn. Residues wa s tested up to and beyond its lim it of solubility in the test system in all experiments.

6.4 Test System

Specific-locus mutation tests with mammalian cells in vitro can be used to demonstrate and quantify genetic damage in these cells. Such test s can be used, for example, to confirm results obtained with bacterial cell tests. A positive re sult with the mammalian cell test as well as a bacterial cell assay increases the need for careful evaluation of the toxic potential of the test chemical. A negative result, while not revers ing the interpretation of a positive result with bacteria, does reduce the value of that result and reduces the concern for the genotoxicity of the chemical.

6.5 Justification of the Test System

Since 1964, mutations have been knowingly indu ced in cultured mammalian cells (Fischer and Sartorelli (1964); Chu and Malling (1968); Ka o and Puck (1968)). The thymidine kinase heterozygote system, where tk + tk - is mutated to tk tk - , was described by Clive et al (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). In this assay, cells deficient in thymidine kinase (TK) due to the mutation tk + tk - to tk tk - are resistant to the cytotoxic effects of the pyrimidine anal ogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sens itive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferat e in the presence of TFT, whereas normal cells, which co ntain thymidine kinase, are not.

A more detailed description of the system as a test for mutagens was published later (Clive and Spector (1975)). Among the published valida tion studies of the L5178Y cell system are those of Clive et al (1979); Amacher et al (1980); Jotz and Mitc hell (1981); McGregor et al (1987, 1988a, 1988c, 1991a, 1991b); Myhr et al (1990); Honma et al (1999a, 1999b).Page 14 Test Facility Study No. 787739

6.5.1 Colony Sizing

It has been widely shown (Hozier et al (1981) and others) that genetic damage involving the TK locus results in 2 phenotypes. Some muta nt cells divide at the normal rate, producing large colonies, while other cells divide at a dist inctly slower rate, producing small colonies. A high proportion of large type colonies are asso ciated with small chromosomal deletions or point mutations, while a large proportion of the sm all type colonies are associated with large chromosomal deletions. Assessment of the rela tive numbers of both colony types can provide information to support results ob tained in bacterial mutation a nd chromosome aberration tests.

6.6 Cells and Cell Culture

6.6.1 Cells

The cells used were from the tk + tk - -3.7.2C mouse lymphoma L5178Y cell line obtained from Dr D Clive, Burroughs Wellcome & Company, Research Triangle Park, NC27709, USA, in December 1982. The cells grow in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome numb er and have a high cloning efficiency in serum-enriched cloning medium.

6.6.2 Culture Medium

The basic culture medium (R 0 P) was RPMI 1640 medium, su pplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), sodi um bicarbonate (1.125 g/L) and pluronic acid (0.05% w/v). For cell growth, heat-inactivated horse serum (10% v/v) was added to R 0 P to give R 10 P.

The medium used during treatment for 4 h was R 0 P supplemented with 5% v/v heat-inactivated horse serum (R 5 P). The medium used during treatment for 24 h was R 10 P.

For colony formation, cloning medium was used, consisting of R 0 P supplemented with heat-inactivated horse serum (20% v/v), s odium pyruvate (1.9 mM), and amphotericin B (fungizone) (2.5 μg/mL).

For selection of tk tk - cells, cloning medium was supplemen ted with trifluorothymidine (TFT) at 3 μg/mL.

6.6.3 Cell Culture

Cell stocks for use in the mutation assays were prepared in suitable volumes according to the procedures detailed in Appendix 2.Page 15 Test Facility Study No. 787739

6.7 Experimental Design

Two principal methods of performing the mouse lymphoma assay exist: the soft agar cloning method originally developed by Clive and the microwell method (Cole et al (1983)). The assay performed in this laboratory is based pr incipally on the methods of Clive, with cloning performed according to the method of Cole.

Although some guidelines e.g. , ICH, do not dictate an automatic repetition of experiments, it is considered appropriate to pr ove the reproducib ility of all findings in this assay system. Consequently, 2 experiments were conducted in both the absence and the presence of an exogenous enzyme supplement (S9 mix).

A validation study commissioned by the ICH (Honma et al (1999b)) has shown that the detection (by the L5178Y assay) of a significa nt number of substances positive in the chromosome aberration assay was enhanced by an extended exposure period in the absence of S9 mix. Such substances included nucleosi de analogues, base analogues and aneuploidy inducers. The study was designed, therefore, to include a 24 h exposur e period in the second experiment in the absence of S9 mix, should the results of the firs t experiment (using a standard 4 h exposure) be negative.

6.8 Justification of Dose Levels

Concentrations in mutation experiments shoul d extend into the toxic range, the maximum usable limit allowing a relative total growth valu e of at least 10% of the concurrent vehicle control. It was necessary, ther efore, to perform initial toxi city tests in the absence and presence of S9 mix.

The final concentrations of Alcohols, C18-22 Dist n. Residues used in the toxicity test were as follows:

0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 μg/mL

The highest concentration represents the maximu m attainable in the test system, when the 20 mg/mL formulation in DMSO (see Section 6.3.1) was dosed 1 in 100 into the cell cultures. Subsequently, four mutation assays were performed, as detailed on the next page.Page 16 Test Facility Study No. 787739

The concentrations of Alcohols, C18-22 Distn. Residues tested were as follows (μg/mL):

Assay 1 (in the absence of S9 mix 1 ): 25, 50, 100 and 200 Assay 2 (in the presence of S9 mix 1 ): 25, 50, 100 and 200 Assay 3 (in the absence of S9 mix 2 ): 25, 50, 100 and 200 Assay 4 (in the presence of S9 mix 1 ): 25, 50, 100 and 200

1 Experiment using a 4 h exposure period
2 Experiment using a 24 h exposure period

Each dose range selection was ba sed on all available results at the time the selection was made.

6.9 Administration of Test/Control Items

6.9.1 Vehicle Controls

All toxicity and mutation assays included vehi cle control cultures. These cultures were subjected to the same experimental manipulations as the treated cultures. In this study, the vehicle control cultures were treat ed with DMSO. Single cultures were treated in the toxicity tests. Four vehicle control cultures we re treated in each mutation experiment.

6.9.2 Positive Control Materials

All mutation assays included duplicate positiv e control cultures. The positive control materials used in the absence of S9 mix (4 h exposure period) were: 250 μg/mL ethyl methanesulphonate (EMS), a large colony induc er; and 10 μg/mL methyl methanesulphonate (MMS), which usually induces greater numbers of small colonies. When performing an experiment using the extended 24 h exposure pe riod, these concentrations were reduced to 150 μg EMS/mL and 5 μg MMS/mL. In the presence of S9 mix, 3-methylcholanthrene (3-MC), a large and small colony inducer, was used at concentrations of 2.5 and 10 μg/mL. EMS and 3-MC were dissolved in DMSO, while MMS was dissolved in water. The positive control cultures were subjected to the same e xperimental manipulations as the Alcohols, C18- 22 Distn. Residues-treated cultures.

6.9.3 Test Item

Single cultures were treated for each concentration in the initial toxicity te sts. In the mutation assays, duplicate cultures were treated for all Alcohols, C18-22 Distn. Residues concentrations.Page 17 Test Facility Study No. 787739

6.10 Experimental Procedure

All experimental procedures were conducted using aseptic technique and under amber light.

All water used in the preparation of reagen ts was produced in-house, by reverse osmosis followed by mixed-bed deionisation and sterilisation by autoclaving.

6.11 S9 Mix

As a general reference, see McGregor et al (1988b).

Aroclor 1254-induced S9 enzymes (the supernatant of the post-mitochondrial 9000 g fraction) were prepared in-house from the livers of a dult male Fischer rats, as described by Ames et al (1975).

S9 was stored in sterile plastic tubes immersed in liquid nitrogen ( ca -196°C) and used within 6 months of preparation.

The enzymic activity of each batch of S9 was ch aracterised by testing selected pre-mutagens in an Ames test with S. typhimurium TA 1538. The results of the characterisation and preparation details of the ba tches used in the mutation e xperiments are presented in Appendix 3. S9 batches used must also have shown, within each mouse lymphoma test, a satisfactory mutagenic response in cells treated with 3-methylcholanthrene.

To prepare S9 mix, R 0 P was added to pre-weighed cofactors: nicotinamide adenine dinucleotide phosphate (NADP) disodium salt and glucose-6-phosphate (G-6-P) disodium salt, giving final concentrat ions in the ‘S9 mix’ of:

NADP Na 2 4 mM (= 3.150 mg/mL) G-6-P Na 2 25 mM (= 7.605 mg/mL)

This solution was immediately filter-sterilised by passage th rough a 0.2 μm disposable filter assembly and mixed 9:1 (v/v) with the S9.

6.12 Toxicity Test

The exposure of the cells was sim ilar to that described below for the mutation assays with the exception that only one culture wa s prepared for each treatment.

The selection in the mutation experiments of the treatments for final assessment is dependent on suspension growth following treatment. Th e measure used to a ssess toxicity in the Toxicity Test was therefore relative suspen sion growth. The cell popul ation densities were recorded over 2 days (followi ng treatment) using a haemocytometer, then the total suspensionPage 18 Test Facility Study No. 787739

growths were expressed as percentages of th e vehicle control group (= relative suspension growth, or RSG).

The toxicity test was performed using the st andard 4 h exposure peri od in the absence and presence of S9 mix. An additi onal toxicity test was performed in the absence of S9 mix with 24 h exposure to Alcohols, C18-22 Distn. Resi dues, as a contingency against the later requirement for a full experiment using this extended exposure period.

Observations on the precipitati on of Alcohols, C18-22 Distn. Residues were made after dosing and at the end of the exposure period. Ob servations of pH change (colour change in indicator in RPMI medium) were made and if any change was noted, pH measurements were made.

6.13 Mutation Tests

6.13.1 Treatment (4 h exposure period)

On the day of the test (Day 0), samples of cell culture (in 5 mL R 10 P) were dispensed to sterile tubes containing 3.9 mL R 0 P. Freshly prepared S9 mix or R 0 P (1 mL) was added to each tube followed by 0.1 mL of test solution. Vehicl e control cultures received 0.1 mL DMSO. Positive control cultures received 0.1 mL of the appropriate solution. The final reaction mixture in all cultures contained 10 mL of cells, at a population density of

ca 6.0 x 10 5 cells/mL, in R 5 P medium.

All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 4 h. After this, the cells were gently sedimented by centrifugation at ca 200 g for 5 min and resuspended in R 10 P medium (20 mL). This step was repeated to give a cell density of ca 3 x 10 5 /mL.

The cells were returned to the rotating drum and allowed to express th eir genetic lesions at ca 37°C for 2 days. Cell numbers we re adjusted, after counting, to ca 3 x 10 5 cells/mL on Day 1.

6.13.2 Extended Treatment (24 h exposure)

An experiment is conducted using an extended 24 h exposure period, when the results of the first experiment in the absence of S9 mix are negative. The extended exposure period facilitates continuous ex posure to the test item through >1 cell cycle.

On the day of the test (Day 0), sa mples of cell culture (in 10 mL R 10 P) were dispensed to sterile tubes containing 7.8 mL R 0 P. R 50 P (R 0 P:serum, 50:50) (2 mL) was added to each tube followed by 0.2 mL of the test solution. Vehi cle control cultures re ceived 0.2 mL DMSO. Positive control cultures received 0.2 mL of the appropriate solution. The final reaction mixture in all cultures contained 20 mL of cells, at a population density of

ca 3 x 10 5 cells/mL, in R 10 P medium.Page 19 Test Facility Study No. 787739

(The larger volumes allow the same numbers of cells to be treated as in the experiments conducted at 4 h exposure, but at half the densit y. The lower density is required to allow cell growth during the exposure period. The seru m concentration is not lowered, as some essential nutrients can become e xhausted during the exposure period.)

All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R 10 P medium (20 mL). This step was repeate d. Cell counts were made and the densities adjusted (where higher) to give ca 3 x 10 5 cells/mL. The cells were returned to the rotating drum and allowed to expre ss their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 10 5 cells/mL on Day 2.

6.13.3 Expression of Genetic Damage

On Day 2 (4 h exposure) or Day 3 (24 h expos ure), cell counts were determined. The cell counts over the 2 or 3 days of the experiment s provided a measure of suspension growth. This in turn provided a measure of RSG (see Section 7.1.1). This was used when choosing dose levels to carry through to final assessment, as no other measures of toxicity were known at the time the decision was required.

In this study all treated cultures were selected for assessment. The cultures were then assessed for expression of genetic damage. Th is was determined by performing two parallel cloning assays: the cloning efficiency a ssay and the mutant selection assay.

For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well.

For the mutant selection assay, TFT stock solu tion was added to cloning medium to give a final concentration of 3 μg/mL. Into this me dium, the cell cultures were diluted to give an estimated 1 x 10 4 cells/mL. Two 96-well dishes were f illed with 200 μL ce ll culture per well, so giving an estimated 2000 cells per well.

All dishes were incubated at ca 37°C in an atmosphere of 5% CO 2 :95% air (v/v) until the colonies were fully developed (at least 9 days for cloning efficiency assa y, at least 12 days for mutant selection assay).

6.13.4 Plate Reading

The plates were scored using a dissecting micr oscope fitted to a light box with dark field illumination. The number of empty wells in eac h plate in the cloning efficiency assay wasPage 20 Test Facility Study No. 787739

counted. When scoring the mutant selection as say, separate counts were made of the numbers of wells containing large type and small type co lonies. Large colonies are defined as covering greater than 1⁄4 of the floor of the well, while small colonies cover less than 1⁄4 of the well (Moore et al (2000)). In addition, there are morphologi cal differences. Large colonies tend to be similar to those found on the cloning efficiency plates, being generally flat. Small colonies tend to look dense in comparison. See exampl es on next page. Any wells containing both colony types were scored as a large type. (The total number of empty wells is required for the calculation of mutant fraction, so ea ch well can only be scored once).

Empty Well Large Colony Small Colony

7 CALCULATIONS AND DATA ACCEPTANCE

7.1 Calculations

Worked examples of the calculations made in the current study are given in Appendix 4.

7.1.1 Assessment of Relative Suspension Growth

For treated cultures, this value includes initial cell loss to toxicity a nd subsequent recovery during the expression period. The measure is used to assess the results of the preliminary toxicity test, and to determine acceptable dose levels for assessment in the mutation tests.

Total suspension growth (SG) is calculated as follows, for experiments with a 4 h exposure period:

1 Day on ion Concentrat Final Count 2 Day
x 0 Day on ion Concentrat Count 1 DayPage 21 Test Facility Study No. 787739

For experiments with a 24 h e xposure period, the calculation is:

2 Day on Conc. Final Count 3 Day
x 1 Day on Conc. Final Count 2 Day
x 0 Day on Conc. Count 1 Day

The total suspension growth values are then e xpressed as percentages of the vehicle control mean value to give the relative suspension growth (RSG).

7.1.2 Assessment of Relative Total Growth

The recommended endpoint for assessing cytotoxi city in mouse lymphoma mutation tests is Relative Total Growth (RTG) (Moore et al (2002)). RTG combines the suspension growth above with the cloning efficiency (CE) of the non-mutants at the end of the expression period, again expressing individual values as percenta ges of the vehicle control mean. The CE is calculated from the zero term of the Po isson distribution using the formula:

per well cells of number ln(P(o)) CE −

=

wells total s empty well P(o) where =

Total Growth = SG x CE (non-mutants)

7.1.3 Assessment of Mutant Fractions

The number of empty wells from the non-mutant cloning efficiency assay and the number of empty wells from the TFT-resistance assay were used to calculate th e mutant fraction, as below.

The CE of both cell types was calculated as in Section 7.1.2. The mutant fraction per viable cell was calculated as below:

Each mutant fraction was expressed per 10 6 viable cells. mutants

- non of CE mutants of CE
medium selective

- non in CE TFT containing medium in CE cell viable per fraction Mutant = = 1 100 x growth total control vehicle mean growth total individual (%) Growth Total Relative =Page 22 Test Facility Study No. 787739

7.1.4 Colony Size Fractions

The ratio of small type mutant colonies to la rge type mutant colonies was expressed for each culture.

7.2 Historical Control Data

The historical ranges of vehicle and positive cont rols are continually updated and are used as guides in the acceptance of each experiment. The summarised historical data are presented in Appendix 5.

7.3 Analysis of Data

7.3.1 Acceptance Criteria

For 4 h experiments, the mean vehicle control suspension growth was required to be between 8 and 32 (Moore et al (2003)). For experiments with a 24 h exposure period, a value between 32 and 180 was required (Moore et a l (2007)).

Errors in cell dilution or very high mutant fractions may resu lt in plates with no negative wells. The Poisson distribution analysis does no t generate data for such scores and so any pair of plates containing 0% empty wells was rejected. Plat es containing 100% empty wells may arise from either errors in cell dilution or severe toxicity. Any such data was given consideration before acceptance.

The mean vehicle control cloning efficiency was required to be greater than 65% and less than 120%. (Experience shows that the acceptance or rejection of an occasional culture giving >120% CE makes no difference to the overall re sults, and therefore su ch instances were accepted.)

Results for any one treatment/concentration were inadequate if there were less than 2 acceptable cultures. Where results were obtaine d from a single culture, they may have been included as supporting evidence.

Analysable results should have b een obtained from at least 4 c oncentrations of Alcohols, C18- 22 Distn. Residues in any experiment. These may have included results extending into the toxic range of less than 10% RTG.

The highest concentration of Al cohols, C18-22 Distn. Residues should have been limited by solubility or toxicity, or, in the absence of these, should ha ve been the maximum practical concentration of test item, based on th e recommendations in current guidelines, ie 5000 μg/mL or 10 mM, whichever is lower.Page 23 Test Facility Study No. 787739

The negative control mean mutant fracti on range recommended by the IWGT mouse lymphoma workgroup for the microwell a ssay is 50-170 mutants per million (Moore et al (2006)). At Charles River, the range is just slightly lower, being ca 40-160 mutants per million. Concern over accepting results in the range 40-50 mutants per million centres on the possibility of reduced recovery of the less viable mutants. Any experiments with a vehicle mean mutant fraction between 40 and 50 muta nts per million were accepted, providing the positive control results were unequivocally acceptable.

In any experiment, MMS and at least one con centration of 3-MC should have yielded an induced mutant fraction (IMF = No. mutants pe r million for treated group, minus No. mutants per million for vehicle control) of at least 300 mutants per million, with a ratio of small/large colonies of greater than 0.67 (>40% small). The mean RTG values for all positive control treatments should have been >10%. EMS, a large colony inducer, was included at a low dose to monitor the sensitivity of the system to weak mutagens.

Appendix 5 reflects the current hi storical control database. Va riations between batches of positive control chemicals, media, serum and S9 preparations can all result in large differences in positive control responses. There is therefore no requirement for the positive control mutant fractions obtained in this study to fall within the historical ranges.

There should have been an absen ce of confounding technical problems, eg , contamination, outliers, excessive toxicity, osmolality and pH changes.

8 STATISTICAL ANALYSIS AND DATA EVALUATION

8.1 Statistical Analysis

The results for each experiment were subjecte d to statistical analysis by the recommended UKEMS method. The methods used are detailed in Appendix 6.

8.2 Data Evaluation

8.2.1 Interpretation of Toxicity

Mutagenic responses that occur only at RTG values below 10%, while being statistically significant, are recognised as having questionable bi ological significance (Clive et al (1983), Scott et al (1991)). Furthermore, results arising from an initial high cell kill are also prone to high divergence and thus tend to be statistically less robust. As genetic damage naturally reduces cell viability, it is norma l, however, for mutagenic responses to occur only at reduced levels of survival ( ie , lowered RTG). If the test item was toxic, results should have been obtained from concentrations resulting in RT G values down to 20%, if a conclusion of non-Page 24 Test Facility Study No. 787739

mutagenic was to be reached with confidence. It is therefore important to obtain results in the RTG range 10 to 20%, when dea ling with toxic test items.

Any data obtained for treatments resulting in an RTG value below 10%, have been reported, but have not been included in th e statistical analysis. Any such data were not included in the general assessment of the results unless they were required to support a conclusion of non-mutagenicity (see Section 8.2.2.2).

8.2.2 Interpretation of Mutagenic Activity

8.2.2.1 Criteria for a Positive Result

It is recognised that small, bi ologically irrelevant increases in mutant fraction may arise that prove statistically significant (Moore et al (2006)). The IWGT reco mmends that biological significance be attached to increases in IMF that exceed a value based on the global background mutant fraction. This value, the global evaluation factor (GEF), is defined as the mean of the global vehicle control distribution pl us one standard deviation. For the microwell cloning version of the assay, this value is 126 mutants per million. Biological significance was therefore assumed to apply to treatments that gave an IMF value >126 mutants per million.

An experiment was considered positive if one or more concentrati ons were biologically significant and there was a significant linear tr end. An experiment may also have been classed as positive in the absence of a linear tr end if there was mitigating evidence. This may have been, for example, the presence of a sim ilar level of toxicity at all concentrations assessed. In such a case, the confirmatory ex periment would have been expected to assess concentrations covering different levels of toxicity, to establish a linear trend.

Additional comparisons that can aid interpretation of results include:

a comparison of the induced mutant fraction wi th the historical maximum for difference between vehicle controls (No E ffect Maximum - see Appendix 5)

b comparison of the mutant fraction of a treated group with the historical range of vehicle control values (see Appendix 5)

A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition. Test items that gave a ne gative response in the st andard exposure in the absence of S9 mix, but gave a positive response in the extended exposure, were liable to a confirmatory experiment w ith the extended exposure.Page 25 Test Facility Study No. 787739

8.2.2.2 Criteria for a Negative Result

In the absence of any significant findings or ot her criteria for a positive response described in 8.2.2.1 above, a test item was defined as non-mu tagenic, provided data were obtained in both the absence and the presence of S9 mix that accompanied one or more of the following:

• the predetermined maximum concentrati on of 5000 μg/mL or 10 mM, whichever is lower

• the highest practical concentr ation limited by the solubility or pH of the test item

• RTG in the range 10-20%

It is acknowledged (Moore et al (2002)) that there are some circumstances under which a chemical may be determined to be non-muta genic when there was no treatment showing an RTG value between 10 and 20%. These situations are as follows:

a there was no evidence of mutagenic activity in a series of data points within 100% to 20% RTG and there was at least one da ta point between 20% and 25% RTG. b there was no evidence of mutagenic activity in a series of data points between 100% to 25% RTG and there was also a data point between 10% and 1% RTG.

8.2.2.3 Colony Sizing

The ratio of small to large type mutant coloni es was stated for all cultures. These ratios provide evidence of adequate rec overy of small type colonies from control cultures, and may provide additional information regarding the type of genetic damage being induced by positive test items.Page 26 Test Facility Study No. 787739

9 RESULTS AND DISCUSSION

9.1 Toxicity Tests (Tables 1 - 3)

Alcohols, C18-22 Distn. Residues was not toxi c to the cells under any of the exposure conditions (4 h exposure in the absence and presence of S9 mix and 24 h exposure in the absence of S9 mix). As stated in Section 6. 3.1, the highest concen tration of 200 μg/mL was considered to be a pr ecipitating dose level.

9.2 Mutation Assays (Tables 4 - 7)

The summarised results giving the mean values for the duplicate cultu res (and quadruplicate vehicle controls) are presented in Tables 4 - 7. The original da ta obtained in the generation of the Relative Suspension Growth values for each culture in the 4 mutation assays are transcribed in Appendix 7. Similarly, the Day 2/3 Cloning Efficiencies of the non-mutants and the Relative Total Growth values are pres ented in Appendix 8, and the Day 2/3 Mutant Selection data are pres ented in Appendix 9.

9.2.1 Vehicle Control Groups

The solvent control mean mutant fractions were within the normal ranges experienced in this laboratory (Appendix 5) and reported in the literature with the L 5178Y cell line (Mitchell et al

(1997)). It was noted that the value obtai ned in Assay 2 (47 mutants per million) was slightly below the recommended IWGT range of 50-170. However, the concurrent positive control results were unequivocally acceptable (s ee Section 7.3.1) and therefore the value was accepted. All other values met the accepta nce criteria in Sect ion 7.3.1 in full.

9.2.2 Positive Control Groups

All positive control log mutant fr actions were significantly higher than the vehicle controls at P<0.05. The IMF values and colony size rati os for MMS and 3-MC were all acceptable by the criteria stated in Section 7.3.1.

9.2.3 Alcohols, C18-22 Distn. Residues

9.2.3.1 Assays 1 (in the Absence of S9 Mix - Ta ble 4) and 2 (in the Presence of S9 Mix - Table 5 )

Alcohols, C18-22 Distn. Residues was assessed for mutagenic activity at concentrations of: 25, 50, 100 and 200 μg/mL in the absence a nd presence of S9 mix (4 h exposure).

None of the assessed concentrations tested si gnificant for increase in log mutant fraction in either experiment. In the absence of S9 mix, the test for linear trend was not reported as thePage 27 Test Facility Study No. 787739

slope was negative, while in the presence of S9 mix, the test for linear trend was not significant (P= 0.79). There was no toxicity at any concentration in ei ther experiment. The highest concentration of 200 μg /mL was considered to be a precipitating dose level (See Section 6.3.1).

Both experiments were classed negative, and th erefore the second experiment in the absence of S9 mix was conducted with the extended, 24 h exposure period.

9.2.3.2 Assays 3 (in the Absence of S9 Mix - Table 6) and 4 (in the Presence of S9 Mix - Table 7)

Alcohols, C18-22 Distn. Residues was assessed for mutagenic activity at concentrations of: 25, 50, 100 and 200 μg/mL in the absence of S9 mix (24 h exposure) and in the presence of S9 mix (4 h exposure).

None of the assessed concentrations tested si gnificant for increase in log mutant fraction in either experiment. In the absence of S9 mix, the test for linear trend was not reported as the slope was negative, while in the presence of S9 mix, the test for linear trend was not significant (P= 0.36). There was no toxicity at any concentration in ei ther experiment. The highest concentration of 200 μg /mL was considered to be a precipitating dose level (See Section 6.3.1).

Both experiments were classed negative.

9.2.3.3 Combined Statistical Analysis

A combined statistical analysis of Assays 2 and 4 (where all tr eatment conditions and concentrations of test item were identical) wa s made. There were no statistically significant differences between the Alcohols, C18-22 Dist n. Residues-treated groups and the vehicle controls. The test for linear tre nd was not signifi cant (P = 0.51).Page 28 Test Facility Study No. 787739

10 CONCLUSION

It was concluded that Alcohols, C18-22 Distn. Residues is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide up to and beyond its lim it of solubility in the test system.Page 29 Test Facility Study No. 787739

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McGregor D B, Martin R, Ca ttanach P, Edwards I, McBride D and Caspary W J (1987). Responses of the L5178Y tk + /tk - Mouse Lymphoma Cell Forward Mutation Assay to Coded Chemicals. I: Results for Nine Compounds . Environmental Mutagenesis, 9, 143-160.

McGregor D B, Brown A, Catta nach P, Edwards I, McBride D and Caspary W J (1988a). Responses of the L5178Y tk + /tk - Mouse Lymphoma Cell Forward Mutation Assay II: 18 Coded Chemicals. Environmental and Molecular Mutagenesis, 11, 91-118.

McGregor D B, Edwards I, Riach C G, Cattanach P, Martin R, Mitchell A and Caspary W J (1988b). Studies of an S9-based metabolic ac tivation system used in the mouse lymphoma L5178Y cell mutation assay. Mutagenesis, 3, 485-490.

McGregor D B, Brown A, Catta nach P, Edwards I, McBrid e D, Riach C and Caspary W (1988c). Responses of the L5178Y tk + /tk - Mouse Lymphoma Cell Forward Mutation Assay: III. 72 Coded Chemicals. Environmental and Molecular Mutagenesis, 12, 85-154.

McGregor D B, Brown A G, Ho wgate S, McBride D, Riach C G and Caspary W J (1991a). Responses of the L5178Y Mouse Lymphoma Ce ll Forward Mutation Assay. V: 27 Coded Chemicals. Environmental and Mo lecular Mutagenesis, 17, 196-219.

McGregor D B, Brown A, Cattana ch P, Edwards I, McBride D, Riach C G, Shepherd W and Caspary W J (1991b). Responses of the L5178Y Mouse Lymphoma Forward Mutation Assay: V. Gases and Vapors. Environmen tal and Molecular Mutagenesis, 17, 122-129.Page 31 Test Facility Study No. 787739

Mitchell A D, Auletta A E, Clive D, Kirby P E, Moore M M and Myhr B C (1997). The L5178Y/tk +/- mouse lymphoma specific gene and chro mosomal mutation assay. A phase III report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research, 394, 177-303.

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Moore M M, Honma M, Clements J, Bolcsfoldi G, Cifone M, Delongchamp R, Fellows M, Gollapudi B, Jenkinson P, Kirby P, Kirchner S, Muster W, Myhr B, O’Donovan M, Oliver J, Ornori T, Ouldelhkim M-C, Pant K, Preston R, Riach C, San R, Stankowski Jr L F, Thakur A, Wakuri S, Yoshimura I (2003). Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Te sts Workgroup Report—Plymouth, UK 2002. Mutation Research, 540, 127-140.

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12 TABLES

Table 1 Toxicity Test in the Absence of S9 Mix (4 h Exposure)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations Day 1 Day 2 Total Suspension

Growth Relative Suspension

Growth %

DMSO (100 μL added) - 15.0 18.9 31.5 100.0 Alcohols, C18-22

Distn. Residues 0.78 - 18.8 15.0 31.3 99.5

1.56 - 19.1 13.6 28.9 91.6

3.13 - 18.1 13.0 26.1 83.0

6.25 - 17.7 14.3 28.1 89.3

12.50 - 17.6 14.0 27.4 86.9

25.00 - 16.8 15.4 28.7 91.3

50.00 - 18.4 15.2 31.1 98.7

100.00 - 13.6 16.9 25.5 81.1

200.00 pptn 14.2 14.5 22.9 72.6

pptn = PrecipitationPage 34 Test Facility Study No. 787739

Table 2 Toxicity Test in the Presence of S9 Mix (4 h Exposure)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations Day 1 Day 2 Total Suspension

Growth Relative Suspension

Growth %

DMSO (100 μL added) - 15.8 14.0 24.6 100.0 Alcohols, C18-22

Distn. Residues 0.78 - 14.0 14.2 22.1 89.9

1.56 - 15.0 12.0 20.0 81.4

3.13 - 16.7 12.6 23.4 95.1

6.25 - 14.2 14.7 23.2 94.4

12.50 - 14.7 14.7 24.0 97.7

25.00 - 15.6 12.9 22.4 91.0

50.00 - 12.3 15.7 21.5 87.3

100.00 - 15.6 12.0 20.8 84.6

200.00 pptn 14.3 13.6 21.6 87.9

pptn = PrecipitationPage 35 Test Facility Study No. 787739

Table 3 Toxicity Test in the Absence of S9 Mix (24 h Exposure)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations Day 1 Day 2 Day 3 Total Suspension

Growth Relative Suspension

Growth

%

DMSO (200 μL added) - 11.2 17.7 13.0 95.4 100.0 Alcohols, C18-22

Distn. Residues 0.78 - 11.2 18.7 12.9 100.1 104.8

1.56 - 11.0 15.9 13.5 87.5 91.6

3.13 - 11.7 13.6 16.0 94.3 98.8

6.25 - 12.2 14.1 13.3 84.7 88.8

12.50 - 11.9 17.7 11.0 85.8 89.9

25.00 - 12.8 13.9 13.9 91.6 96.0

50.00 - 13.1 15.4 13.5 100.9 105.7

100.00 - 14.2 15.0 13.0 102.6 107.4

200.00 pptn 14.1 15.1 11.2 88.3 92.5

pptn = PrecipitationPage 36 Test Facility Study No. 787739

Table 4 Mutation Test in the Absence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 1)

Chemical Concentration (μg/mL) Relative Total Growth

% Mutant Fraction (x 10 -6 ) IMF (Induced Mutant Fraction x10 -6 ) Ratio of Small to

Large Colonies Statistical Comparison

DMSO (100 μL added) 100 54 N/A 1.28 N/A

EMS 250 66 539 484 0.47 ∗

MMS 10 37 708 654 2.55 ∗ Alcohols, C18-22

Distn. Residues 25 111 52 - 1.72 NS

50 98 58 3 1.93 NS

100 107 37 - 2.17 NS

pptn 200 114 35 - 1.25 NS

IMF = Mutant fraction of tr eatment minus mutant fracti on of vehicle control group N/A = Not Applicable

∗ = Significant difference in log mutant fraction compared with vehicle control (P<0.05) NS = Not Significant Test for linear trend of mutant fraction with concn. of Al cohols, C18-22 Distn. Residues = not reported – slope negative - = IMF ≤ 0 pptn = PrecipitationPage 37 Test Facility Study No. 787739

Table 5 Mutation Test in the Presence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 2)

Chemical Concentration (μg/mL) Relative Total Growth

% Mutant Fraction (x 10 -6 ) IMF (Induced Mutant Fraction x10 -6 ) Ratio of Small to

Large Colonies Statistical Comparison

DMSO (100 μL added) 100 47 N/A 1.18 N/A

3-MC 2.5 71 403 355 1.08 ∗

10 65 576 528 1.20 ∗ Alcohols, C18-22

Distn. Residues 25 104 66 19 2.43 NS

50 101 56 9 1.24 NS

100 92 60 13 0.61 NS

pptn 200 122 53 6 1.78 NS

IMF = Mutant fraction of tr eatment minus mutant fracti on of vehicle control group N/A = Not Applicable

∗ = Significant difference in log mutant fraction compared with vehicle control (P<0.05) NS = Not Significant Test for linear trend of mutant fraction with concn. of Alcohols, C18-22 Distn. Residues = not significant (P = 0.79) pptn = PrecipitationPage 38 Test Facility Study No. 787739

Table 6 Mutation Test in the Absence of S9 Mix (24 h Exposure) Summary of Means of Data (Assay 3)

Chemical Concentration (μg/mL) Relative Total Growth % Mutant Fraction (x 10 -6 ) IMF (Induced Mutant Fraction x10 -6 ) Ratio of Small to

Large Colonies Statistical Comparison

DMSO (200 μL added) 100 134 N/A 2.94 N/A

EMS 150 11 3741 3608 0.32 ∗

MMS 5 31 1912 1778 1.53 ∗ Alcohols, C18-22

Distn. Residues 25 86 107 - 3.29 NS

50 78 141 8 3.94 NS

100 86 144 10 2.82 NS

pptn 200 101 120 - 2.15 NS

IMF = Mutant fraction of tr eatment minus mutant fracti on of vehicle control group N/A = Not Applicable

∗ = Significant difference in log mutant fraction compared with vehicle control (P<0.05) NS = Not Significant Test for linear trend of mutant fraction with concn. of Al cohols, C18-22 Distn. Residues = not reported – slope negative - = IMF ≤ 0 pptn = PrecipitationPage 39 Test Facility Study No. 787739

Table 7 Mutation Test in the Presence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 4)

Chemical Concentration (μg/mL) Relative Total Growth

% Mutant Fraction (x 10 -6 ) IMF (Induced Mutant Fraction x10 -6 ) Ratio of Small to

Large Colonies Statistical Comparison

DMSO (100 μL added) 100 114 N/A 1.51 N/A

3-MC 2.5 73 935 822 1.29 ∗

10 66 1143 1029 1.21 ∗ Alcohols, C18-22

Distn. Residues 25 94 149 36 1.35 NS

50 117 117 4 1.56 NS

100 91 143 29 2.34 NS

pptn 200 89 135 22 2.21 NS

IMF = Mutant fraction of tr eatment minus mutant fracti on of vehicle control group N/A = Not Applicable

∗ = Significant difference in log mutant fraction compared with vehicle control (P<0.05) NS = Not Significant Test for linear trend of mutant fraction with concn. of Alcohols, C18-22 Distn. Residues = not significant (P = 0.36) pptn = PrecipitationPage 40 Test Facility Study No. 787739

13 APPENDICES

Appendix 1 Certificate of AnalysisPage 41 Test Facility Study No. 787739

Appendix 2 Cell Culture

Cell Storage

Cells in logarithmic phase of growth were collected by centrifugation (1000 r.p.m. x 5 min (200 g )) and resuspended in freezing medium (10% DMSO in culture medium) to a final cell density of ca 10 x 10 6 cells/mL. Samples (2 mL) were frozen slowly before being stored in liquid nitrogen. New cultures are establis hed from frozen stocks every 3 months.

Initially, a new culture was seeded into a T-75 flask containing 20 mL R 10 P gassed with 5% CO 2 in air. The cell suspension was gently layered on the bottom of the flask without disturbing the cells and incubated for ca 24 h at ca 37°C. When the cells were determined to be growing well (3-5 generations in 24 h) a stock culture was estab lished by seeding 3 x 10 5
cells/mL in fresh R 10 P into a polypropylene centrifuge tube (Corning 250 mL), where it was maintained on a 10 r.p.m. roller at ca 37°C. Stock cultures were examined for mycoplasma and shown to be uninfected.

Cell Dilution

With a generation time of 10-12 h, there is a 5-fold increase in cell number in 24 h and 25-fold in 48 h. Cultures were subcultured daily (except at weekends) to a density of approximately 3 x 10 5 cells/mL to maintain the cells in logarithmic growth. For weekend maintenance, the cultures were diluted back to ca 1 x 10 4 cells/mL.

Cell ‘Cleansing’

The tk + tk - heterozygote cells grown in suspen sion spontaneously mutate to tk tk - at a rate of 2 x 10 -6 mutations/generation. These homozygous mutants were removed before testing began.

The week before an experiment, Thymidin e/Hypoxanthine/Glycine/Methotrexate (THGM) stock solution (1 mL) was added to a sub-populat ion of the stock cell suspension (100 mL). The following day the culture was pelleted and resuspended in R 10 P supplemented with THG (THGM without methotrexate). This treatment of the cells was carried out once only before their use in an experiment. The culture was ready for use the following week. Unused cells were disposed of at the end of that week.

Mechanism: Methotrexate blocks folate metabolism by binding str ongly to dihydrofolate reductase, hence thymidylate synthetase-mediate d thymidylate production (TMP) stops. TMP can be maintained by TK phosphorylation of exogenous thymidine ( T HG). PurinePage 42 Test Facility Study No. 787739

Appendix 2 (continued)

metabolism is similarly affected and this block is surmounted by added hypoxanthine. Glycine is added to provide a single carbon sour ce to replace the blocked folate metabolic pathway. Hence tk + tk - cells can survive the methotrexate treatment while tk tk - cells die.Page 43 Test Facility Study No. 787739

Appendix 3 In vitro Activation Preparation Form3M Ref. No. 09-188 Page 44 Test Facility Study No. 787739

Appendix 4 Worked Examples of Calculations

The calculations involved in the result analysis for the first me thyl methanesulphonate-treated culture in Assay 1 are described below. The calculations may contain rounding errors.

Method of calculating the Suspension Growth (Appendix 7, page 51)

Suspension growth (SG) represents the potential of the culture to multiply itself over the 2 day expression period (or 3 day, in the case of experiments with 24 h exposure periods), by accounting for the concentration to which the cells are diluted after counting:

On Day 1, after counting, the cells were diluted to 3.0 x 10 5 /mL. On Day 2, the cells had reached 11.0 x 10 5 /mL.

The calculation for SG from Day 1 to Day 2 would be:

The cells therefore had multiplied 3.7-fold between Day 1 and Day 2.

The calculation for the full period therefore, is:

1 Day on ion Concentrat Final Count 2 Day
x * 0 Day on ion Concentrat Count 1 Day

* Routinely 3.0 x 10 5 /mL for all cultures

In this instance,

Note: if toxicity causes the cel l density to be below 3.0 x 10 5 /mL on any day, the culture is not diluted. In such an instance, the denominator in the above calculations for the day in question will be the actual cell density, rath er than the more usual 3.0 x 10 5 /mL.

The cells therefore had multiplied 14.7-fold over the 2 day expression period.

This figure is then expressed as a percentage of the mean vehicle control value to give the Relative Suspension Growth (RSG). 14.7 3.0 11.0
x 3.0 12.0

= 3.7 3.0 11.0

=3M Ref. No. 09-188 Page 45 Test Facility Study No. 787739

Appendix 4 (continued) Worked Examples of Calculations

In this instance,

Assessment of Cloning Efficiency and Re lative Total Growth (Appendix 8, page 55)

The cloning efficiency (CE) is calculated from the zero term of the Poisson distribution using the formula:

per well cells of number ln(P(o)) CE −
=

wells total s empty well P(o) where =

In this instance,

Thus 57% of the cells in the MMS-treated culture were clonable at the end of the expression period.

Relative Total Growth (RTG) combines SG with CE.

Total Growth = SG x CE (non-mutants)

In this instance,

14.7 x 0.57 = 8.38

1 100 x growth total control vehicle mean growth total individual (%) Growth Total Relative = 57.1% 1 100
x 25.7 14.7

=

0.40 192 36 41 P(o) = +

=

0.57 1.6 ln(0.40)

- CE = =3M Ref. No. 09-188 Page 46 Test Facility Study No. 787739

Appendix 4 (continued) Worked Examples of Calculations

In this instance,

The total growth of the culture therefore wa s 33% of the vehicle control mean value.

Method of calculating the Mutant Fraction (Appendix 9, page 59)

This figure gives the number of mutant cells per million clonable cells. The number of empty wells is obtained by subtracti ng the small colony wells and th e large colony wells from the total wells plated.

In this instance,

192 – (12 + 45 +17 + 36) = 82

The CE of the mutant cells is calculated as for the non-mutants.

In this instance,

This figure is finally adjusted to compensa te for the CE of the whole population, thus:

In this instance: 33 1 100 x 25.12 8.38

=

0.43 192 82 P(o) = =

425.39 2000 ln(0.43)

- CE = =

6

- 10 x 745 0.57 6

- 10 x 425.39 cell per viable fraction Mutant = = mutants

- non o f CE mutants of CE cell per viable fraction Mutant =3M Ref. No. 09-188Page 47Test Facility Study No. 787739Appendix 5Mouse Lymphoma L5178Y Cell Historical Control Data from July 2003 to December 2008Vehicle Control DataMutant Fraction x 10-6Mean ColonySize Ratio(Small/Large)VehicleControlS9ExposureTimen†Mean SD RangeNo-effectMaximum(Induced MutantFraction)Mean SD RangeAll, pooled - 4 h 71 71 16 49-121 42 1.28 0.47 0.45-2.73All, pooled - 24 h 50 78 21 51-129 49 1.09 0.43 0.35-2.11All, pooled + 4 h 120 78 22 45-148 70 1.24 0.44 0.35-2.75† = Each value is the mean of 4 replicate culturesThe No-effect Maximum represents the maximum difference recorded between the 2 pairs of vehicle control cultures in any experiment. Thatis, the lower mean mutant fraction (x 10-6) is subtracted from the higher. This difference, when applied to the response from a mutagen, istermed the induced mutant fraction (IMF).Positive Control DataMutant Fraction x 10-6RTG %Mean ColonySize Ratio(Small/Large)PositiveControlS9Concentration(μg/mL)ExposureTimen†Mean SD Range Mean Range Mean SD RangeEMS - 250 4 h 69 601 127 431-910 64 38-94 0.51 0.13 0.25-0.85MMS - 10 4 h 71 908 238 429-1659 40 22-78 2.34 0.55 1.46-3.65EMS - 150 24 h 50 2096 608 1324-3801 31 15-99 0.38 0.10 0.22-0.63MMS - 5 24 h 50 1880 471 1026-3023 31 17-82 1.78 0.41 1.07-3.073-MC + 2.5 4 h 120 1061 338 570-2277 49 16-96 1.37 0.37 0.93-3.61EMS = Ethyl methanesulphonateMMS = Methyl methanesulphonate3-MC = 3-Methylcholanthrene† = Each value is the mean of 2 replicate culturesAudited by: Alison McNaughton (Quality Assurance)09 February 20093M Ref. No. 09-188 Page 48 Test Facility Study No. 787739

Appendix 6 Statistical Method

Survival and mutant data were available for 4 experiments, two without S9 mix (Assays 1 and 3) and two with S9 (Assays 2 and 4). Assays were measured over an exposure period of 4 hours or 24 hours. Data were analysed using methods outlined in Robinson et al (1989). The analyses comprised the following:

1. Determination of the heterogeneity factor for each dose level. 2. Comparison of the heterogeneity factor with th e historical control. Any dose levels with heterogeneity factor statistically higher than the historical control were excluded from all statistical analysis. 3. Determination of the heterogene ity factor for the experiment. 4. Calculation of a new historical control heterogeneity factor. 5. Calculation of the l og mutant fraction. 6. Comparison of the log mutant fraction betw een the control and each treatment dose (at P<0.05). 7. Test for linear increasing trend of mutant fr action with increasing dose of test item (at P<0.05).

Unless otherwise stated, the term 'heterogeneity factors' is define d separately for survival (Hs) and mutant (Hm) data.

When each assay was analysed, a new value for the heterogeneity factor was calculated and compared with the historical value. As such, a ssays were analysed in chronological order. If the new value was exceptionally large (as de termined by significance testing below), the experimental results were considered unacceptabl y variable and were discarded. Otherwise, the new heterogeneity factors were calculated fo r use in the analysis and the historical value was updated.

Beginning with Assay 1, the data were anal ysed using the methods outlined in Robinson et al (1989). The dose-specific heterogeneity f actors for each dose level were determined.

The dose-specific heterogeneity factors were co mpared with the historical controls for consistency using a one-sided F-test (Pearson and Hartley (1989)) at th e 0.1% level. Any dose levels where either the mutant or the su rvival heterogeneity factors were significantly higher than their respective hist orical controls were excluded fr om all of the analyses for the
assay.

The overall heterogeneity factors for the assay were determined.

The heterogeneity factors were compared with the historical controls for consistency using a one-sided F-test at the 1% level. If either the mutant or the survival heterogeneity factors3M Ref. No. 09-188 Page 49 Test Facility Study No. 787739

Appendix 6 (continued)

were significantly higher than th eir respective historical contro ls the assay was discarded and the old historical controls remained in place. Otherwise, new historical control heterogeneity factors were calculated, using weighting of 1/20 of the curre nt estimate and 19/20 of the historical estimate.

For each assay not discarded under the above, the following were performed:

The log mutant fraction and its variance were calculated for each dose level.

Log mutant fraction was compared between the control and each treatment dose at the 5% level using a one-sid ed Dunnett's test (Dunnett (1955)) for an increase in mutant fraction, if and only if the treatment dose mutant fraction was greater than the control.

A one sided χ 2 test (Pearson and Hartley (1989)) with 1 degree of freedom was performed at the 5 % level to test for linea r increasing trend of mutant fraction with increasing dose of test item, if and only if the direction of the slope parameter was positive.

In addition, Assay 2 and Assay 4 had equal exposu re periods and equal dose levels were used. A two-sided χ 2 test (Pearson and Hartley (1976)), with (M-1) degrees of freedom (M = number of assays) was therefore used to test for consistency at each dose level between the assays at the 1% level. At each dose level the assays were considered consistent and therefore it was appropriate to report a combined result.

In Assays 2 and 4, the combined differences in log mutant frequency between the control and each remaining treatment dose were tested at the 5% level using a one-sided Dunnett's test (Dunnett (1955)) for an increase in mutant fr equency. (This was only performed if the treatment dose mutant frequency was greater than the control.)

A one-sided χ 2 test with 1 degree of freedom was perf ormed for the combined data at the 5% level to test for linear increasing trend of mu tant frequency with increasing dose of test material. (This was only performed if the di rection of the combined slope parameter was positive.)3M Ref. No. 09-188 Page 50 Test Facility Study No. 787739

Appendix 6 (continued)

References:

Robinson W D, Green M H L, Healy M J R, Ga rner R C and Gatehouse D: (1989). Statistical Evaluation of Bacterial/Mammalian Fluctuation Tests, in: Kirkland D J (Ed.), Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge.

Pearson, E S & Hartley, H O (1989) Biometrika Tables for Statisticians Volume 1 3 rd edition, Griffin, London

Dunnett, C W (1955). A multiple comparison pr ocedure for comparing several treatments with a control. Journal of American St atistical Association , 1096-1121.3M Ref. No. 09-188 Page 51 Test Facility Study No. 787739

Appendix 7 Individual Suspension Growth Data

Mutation Test in the Absence of S9 Mix (4 h Exposure) Suspension Growth (Assay 1)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations Da y 1 Da y 2 Total Suspension Growth (%) Relative Suspension

Growth

Vehicle Control Mean →

- - - 25.7

-
DMSO (100 μL added) - 12.5 18.8 26.1 101.6

13.7 18.0 27.4 106.6

14.9 14.0 23.2 90.2

14.7 16.0 26.1 101.7

EMS 250 - 11.9 14.4 19.0 74.1

12.7 12.4 17.5 68.1

MMS 10 - 12.0 11.0 14.7 57.1

12.0 14.0 18.7 72.6 Alcohols, C18-22 Distn. Residues 25 - 12.0 16.0 21.3 83.0

13.8 18.8 28.8 112.1

50 - 15.8 17.6 30.9 120.2

15.7 14.4 25.1 97.7

100 - 12.1 17.0 22.9 88.9

13.1 17.8 25.9 100.8

200 - 13.7 18.4 28.0 109.0

13.2 18.6 27.3 106.13M Ref. No. 09-188 Page 52 Test Facility Study No. 787739

Appendix 7 (continued)

Mutation Test in the Presence of S9 Mix (4 h Exposure) Suspension Growth (Assay 2)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations Da y 1 Da y 2 Total Suspension Growth (%) Relative Suspension

Growth

Vehicle Control Mean →

- - - 25.5

-
DMSO (100 μL added) - 14.3 20.6 32.7 128.5

12.0 17.6 23.5 92.1

13.7 15.0 22.8 89.6

13.2 15.6 22.9 89.8

3-MC 2.5 - 11.3 15.2 19.1 74.9

9.9 18.0 19.8 77.7

10 - 9.9 14.6 16.1 63.0

11.3 14.0 17.6 69.0 Alcohols, C18-22 Distn. Residues 25 - 14.9 18.0 29.8 117.0

15.5 16.2 27.9 109.5

50 - 15.6 16.0 27.7 108.9

12.4 18.0 24.8 97.3

100 - 13.9 16.0 24.7 97.0

12.4 15.0 20.7 81.1

200 - 15.1 16.0 26.8 105.4

13.0 15.0 21.7 85.03M Ref. No. 09-188 Page 53 Test Facility Study No. 787739

Appendix 7 (continued)

Mutation Test in the Absence of S9 Mix (24 h Exposure) Suspension Growth (Assay 3)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations
Da y 1 Da y 2 Da y 3 Total Suspension Growth (%) Relative Suspension

Growth Vehicle Control Mean →

- - - - 55.8

-

DMSO (200 μL added) - 8.2 11.4 17.0 58.9 105.5

8.0 13.1 16.0 62.1 111.4

8.1 10.8 16.2 52.5 94.1

7.9 10.6 16.0 49.6 89.0

EMS 150 - 5.8 8.8 7.5 14.2 25.4

6.0 6.3 7.0 9.8 17.6

MMS 5 - 6.9 10.1 9.4 24.3 43.5

6.0 11.1 9.0 22.2 39.8 Alcohols, C18-22

Distn. Residues 25 - 6.8 12.0 17.0 51.4 92.1

5.6 14.3 14.6 43.3 77.6

50 - 5.9 14.1 15.4 47.4 85.1

5.7 14.7 17.4 54.0 96.8

100 - 6.0 15.0 14.6 48.7 87.3

5.7 15.7 14.6 48.4 86.8

200 - 6.2 13.9 16.6 53.0 95.0

6.1 11.0 16.6 41.3 74.03M Ref. No. 09-188 Page 54 Test Facility Study No. 787739

Appendix 7 (continued)

Mutation Test in the Presence of S9 Mix (4 h Exposure) Suspension Growth (Assay 4)

Suspension Count (x 10 5 /mL) Chemical Concentration (μg/mL) Observations Da y 1 Da y 2 Total Suspension Growth (%) Relative Suspension

Growth

Vehicle Control Mean →

- - - 17.8

-
DMSO (100 μL added) - 10.0 14.8 16.4 92.4

10.4 17.6 20.3 114.3

9.0 17.6 17.6 98.9

9.1 16.6 16.8 94.3

3-MC 2.5 - 8.1 13.6 12.2 68.8

9.1 12.6 12.7 71.6

10 - 7.2 15.0 12.0 67.4

7.7 14.0 12.0 67.3 Alcohols, C18-22 Distn. Residues 25 - 10.2 14.0 15.9 89.2

11.0 15.0 18.3 103.0

50 - 10.2 14.0 15.9 89.2

11.1 15.0 18.5 104.0

100 - 10.0 18.0 20.0 112.4

8.9 18.0 17.8 100.0

200 - 7.7 17.8 15.2 85.6

9.3 17.6 18.2 102.23M Ref. No. 09-188 Page 55 Test Facility Study No. 787739

Appendix 8 Individual Cloning Efficiency Data (Non-mutants)

Mutation Test in the Absence of S9 Mix (4 h Exposure) Day 2 Cloning Efficiency (Non-mutants) and Relative Total Growth (Assay 1)

Day 2 Cloning Efficiency Plates Chemical Concentration (μg/mL) Plate 1 Empty Wells Plate 2 Empty Wells Wells Lost Total Wells Scored Cloning Efficiency

(nonmutants) Total Growth Relative Total Growth

%

Vehicle Control Mean → - - - - 0.98 25.12 -

DMSO (100 μL added) 30 15 0 192 0.91 23.68 94

23 22 0 192 0.91 24.85 99

16 17 0 192 1.10 25.51 102

20 18 0 192 1.01 26.46 105

EMS 250 27 25 0 192 0.82 15.54 62

15 24 0 192 1.00 17.43 69

MMS 10 41 36 0 192 0.57 8.38 33

38 43 0 192 0.54 10.07 40 Alcohols, C18-22

Distn. Residues 25 14 10 0 192 1.30 27.73 110

23 17 0 192 0.98 28.26 112

50 25 20 0 192 0.91 28.02 112

28 21 0 192 0.85 21.44 85

100 13 19 0 192 1.12 25.59 102

14 20 0 192 1.08 28.03 112

200 20 20 0 192 0.98 27.46 109

20 13 0 192 1.10 30.02 1203M Ref. No. 09-188 Page 56 Test Facility Study No. 787739

Appendix 8 (continued)

Mutation Test in the Presence of S9 Mix (4 h Exposure) Day 2 Cloning Efficiency (Non-mutants) and Relative Total Growth (Assay 2)

Day 2 Cloning Efficiency Plates Chemical Concentration (μg/mL) Plate 1 Empty Wells Plate 2 Empty Wells Wells Lost Total Wells Scored Cloning Efficiency

(nonmutants) Total Growth Relative Total Growth

%

Vehicle Control Mean → - - - - 1.05 26.16 -

DMSO (100 μL added) 23 31 0 192 0.79 25.95 99

17 17 0 192 1.08 25.39 97

13 13 0 192 1.25 28.53 109

17 17 0 192 1.08 24.76 95

3-MC 2.5 20 21 0 192 0.96 18.42 70

26 16 0 192 0.95 18.81 72

10 22 16 0 192 1.01 16.26 62

17 22 0 192 1.00 17.51 67 Alcohols, C18-22

Distn. Residues 25 20 16 0 192 1.05 31.18 119

28 22 1 191 0.84 23.37 89

50 20 19 0 192 1.00 27.63 106

19 19 0 192 1.01 25.11 96

100 15 21 0 192 1.05 25.85 99

18 16 2 190 1.08 22.23 85

200 13 9 0 192 1.35 36.35 139

9 16 0 192 1.27 27.61 1063M Ref. No. 09-188 Page 57 Test Facility Study No. 787739

Appendix 8 (continued)

Mutation Test in the Absence of S9 Mix (24 h Exposure) Day 3 Cloning Efficiency (Non-mutants) and Relative Total Growth (Assay 3)

Day 3 Cloning Efficiency Plates Chemical Concentration (μg/mL) Plate 1 Empty Wells Plate 2 Empty Wells Wells Lost Total Wells Scored Cloning Efficiency

(nonmutants) Total Growth Relative Total Growth

%

Vehicle Control Mean → - - - - 0.94 52.29 -

DMSO (200 μL added) 24 26 0 192 0.84 49.49 95

19 26 0 192 0.91 56.31 108

18 21 0 192 1.00 52.29 100

22 15 0 192 1.03 51.07 98

EMS 150 37 43 0 192 0.55 7.76 15

52 51 0 192 0.39 3.81 7

MMS 5 41 29 0 192 0.63 15.30 29

28 29 0 192 0.76 16.85 32 Alcohols, C18-22

Distn. Residues 25 23 18 0 192 0.96 49.58 95

23 21 0 192 0.92 39.87 76

50 29 22 0 192 0.83 39.31 75

35 20 0 192 0.78 42.19 81

100 28 18 0 192 0.89 43.46 83

24 17 0 192 0.96 46.69 89

200 16 23 0 192 1.00 52.78 101

9 16 0 192 1.27 52.56 1013M Ref. No. 09-188 Page 58 Test Facility Study No. 787739

Appendix 8 (continued)

Day 2 Cloning Efficiency (Non-mutants) and Relative Total Growth (Assay 4)

Day 2 Cloning Efficiency Plates Chemical Concentration (μg/mL) Plate 1 Empty Wells Plate 2 Empty Wells Wells Lost Total Wells Scored Cloning Efficiency

(nonmutants) Total Growth Relative Total Growth

%

Vehicle Control Mean → - - - - 1.04 18.42 -

DMSO (100 μL added) 12 18 0 192 1.16 19.08 104

23 18 0 192 0.96 19.62 107

20 14 0 192 1.08 19.04 103

19 23 0 192 0.95 15.94 87

3-MC 2.5 23 22 0 192 0.91 11.10 60

11 16 0 192 1.23 15.62 85

10 14 26 0 192 0.98 11.76 64

18 18 0 192 1.05 12.53 68 Alcohols, C18-22

Distn. Residues 25 18 14 0 192 1.12 17.77 96

21 23 0 192 0.92 16.88 92

50 13 14 0 192 1.23 19.45 106

11 14 0 192 1.27 23.57 128

100 27 30 0 192 0.76 15.18 82

23 14 0 192 1.03 18.32 99

200 17 17 0 192 1.08 16.48 89

29 17 0 192 0.89 16.24 883M Ref. No. 09-188Page 59Test Facility Study No. 787739Appendix 9Individual Mutant Selection DataMutation Test in the Absence of S9 Mix (4 h Exposure)Day 2 Mutant Selection (Assay 1)Day 2 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Vehicle ControlMean→- - - - - - - - - 54DMSO(100 μLadded)4 3 4 5 0 1.00 192 176 43.51 483 3 3 3 0 1.00 192 180 32.27 364 9 6 11 0 2.00 192 162 84.95 774 6 6 5 0 1.10 192 171 57.92 57EMS 250 42 20 38 19 0 0.49 192 73 483.52 59237 22 45 15 0 0.45 192 73 483.52 485MMS 10 12 45 17 36 0 2.79 192 82 425.39 74513 35 17 34 0 2.30 192 93 362.45 672Alcohols, C18-22Distn. Residues25 3 3 3 7 0 1.67 192 176 43.51 334 7 5 9 0 1.78 192 167 69.75 7150 1 4 1 2 0 3.00 192 184 21.28 238 6 7 7 0 0.87 192 164 78.81 923M Ref. No. 09-188Page 60Test Facility Study No. 787739Appendix 9(continued)Day 2 Mutant Selection (Assay 1) ContinuedDay 2 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Alcohols, C18-22Distn. Residues100 4 3 2 5 0 1.33 192 178 37.86 343 5 1 7 0 3.00 192 176 43.51 40200 3 5 3 4 0 1.50 192 177 40.67 412 1 4 5 0 1.00 192 180 32.27 293M Ref. No. 09-188Page 61Test Facility Study No. 787739Appendix 9(continued)Mutation Test in the Presence of S9 Mix (4 h Exposure)Day 2 Mutant Selection (Assay 2)Day 2 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Vehicle ControlMean→- - - - - - - - - 47DMSO(100 μLadded)2 3 5 4 0 1.00 192 178 37.86 485 6 5 8 0 1.40 192 168 66.77 625 7 5 3 0 1.00 192 172 55.00 442 5 4 3 0 1.33 192 178 37.86 353-MC 2.5 25 28 22 23 0 1.09 192 94 357.10 37021 34 31 22 0 1.08 192 84 413.34 43510 29 43 29 34 0 1.33 192 57 607.22 60031 32 31 34 0 1.06 192 64 549.31 551Alcohols, C18-22Distn. Residues25 4 9 5 6 0 1.67 192 168 66.77 642 8 3 8 0 3.20 192 171 57.92 6950 6 6 2 8 0 1.75 192 170 60.85 615 4 6 4 0 0.73 192 173 52.10 513M Ref. No. 09-188Page 62Test Facility Study No. 787739Appendix 9(continued)Day 2 Mutant Selection (Assay 2) ContinuedDay 2 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Alcohols, C18-22Distn. Residues100 5 5 6 3 0 0.73 192 173 52.10 506 5 12 4 0 0.50 192 165 75.77 70200 2 9 7 6 0 1.67 192 168 66.77 493 11 6 6 0 1.89 192 166 72.75 573M Ref. No. 09-188Page 63Test Facility Study No. 787739Appendix 9(continued)Mutation Test in the Absence of S9 Mix (24 h Exposure)Day 3 Mutant Selection (Assay 3)Day 3 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Vehicle ControlMean→- - - - - - - - - 134DMSO(200 μLadded)10 13 3 18 0 2.38 192 148 130.14 1557 18 6 16 0 2.62 192 145 140.38 1552 10 5 15 0 3.57 192 160 91.16 926 19 5 16 0 3.18 192 146 136.94 133EMS 150 73 21 68 21 0 0.30 192 9 1530.14 279670 23 69 25 0 0.35 192 5 1824.03 4686MMS 5 35 51 36 52 0 1.45 192 18 1183.56 187739 53 31 59 0 1.60 192 10 1477.46 1947Alcohols, C18-22Distn. Residues25 5 16 2 14 0 4.29 192 155 107.04 1113 10 7 13 0 2.30 192 159 94.30 10250 2 17 7 14 0 3.44 192 152 116.81 1415 17 2 14 0 4.43 192 154 110.27 1413M Ref. No. 09-188Page 64Test Facility Study No. 787739Appendix 9(continued)Day 3 Mutant Selection (Assay 3) ContinuedDay 3 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Alcohols, C18-22Distn. Residues100 6 15 3 21 0 4.00 192 147 133.53 1507 18 10 10 0 1.65 192 147 133.53 138200 4 9 9 16 0 1.92 192 154 110.27 1117 20 9 18 0 2.38 192 138 165.12 1303M Ref. No. 09-188Page 65Test Facility Study No. 787739Appendix 9(continued)Mutation Test in the Presence of S9 Mix (4 h Exposure)Day 2 Mutant Selection (Assay 4)Day 2 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Vehicle ControlMean→- - - - - - - - - 114DMSO(100 μLadded)10 12 8 11 0 1.28 192 151 120.11 1048 10 5 15 0 1.92 192 154 110.27 11410 13 9 12 0 1.32 192 148 130.14 1209 11 6 12 0 1.53 192 154 110.27 1163-MC 2.5 40 40 31 50 0 1.27 192 31 911.75 100538 43 35 53 0 1.32 192 23 1061.00 86510 42 44 48 39 0 0.92 192 19 1156.53 118033 53 36 51 0 1.51 192 19 1156.53 1105Alcohols, C18-22Distn. Residues25 15 18 10 15 0 1.32 192 134 179.83 1618 9 10 16 0 1.39 192 149 126.77 13850 7 10 7 12 0 1.57 192 156 103.82 859 19 15 18 0 1.54 192 131 191.15 1503M Ref. No. 09-188Page 66Test Facility Study No. 787739Appendix 9(continued)Day 2 Mutant Selection (Assay 4) ContinuedDay 2 Mutant ColoniesPlate 1 Plate 2ChemicalConcentration(μg/mL)LargeColonyWellsSmallColonyWellsLargeColonyWellsSmallColonyWellsWellsLostRatio ofSmall toLargeColoniesTotalWellsScoredTotalEmptyWellsCloningEfficiency(mutants)(x10-6)MutantFraction(x10-6)Alcohols, C18-22Distn. Residues100 7 16 4 12 0 2.55 192 153 113.53 1508 16 7 16 0 2.13 192 145 140.38 136200 9 22 6 12 0 2.27 192 143 147.33 1366 16 7 12 0 2.15 192 151 120.11 1343M Ref. No. 09-188 Page 67 Test Facility Study No. 787739

Appendix 10 List of Abbreviations

3-MC 3-methylcholanthrene CCF Cell Count Factor CE Cloning Efficiency DMSO dimethylsulphoxide EMS ethyl methanesulphonate GEF global evaluation factor G-6-P glucose 6-phosphate ICH International Committee on Harmonisation IMF Induced Mutant Fraction IWGT International Work shop on Genetic Toxicology MMS methyl methanesulphonate NADP nicotinamide adenine dinucleotide phosphate NPS Not plated, as surplus to requirement NPT Not plated, as too toxic for assessment OECD Organisation for Economic Co-operation and Development P (0) Zero term of the Poisson distribution RPMI Roswell Park Memorial Instit ute (developers of RPMI 1640 medium) R 0 P RPMI 1640 medium, supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), sodium bicar bonate (1.125 g/L) and pluronic acid (0.05% w/v) R 10 P as R 0 P with Heat –inactivated horse serum (10% v/v) R 50 P as R 0 P with Heat –inactivated horse serum (50% v/v) R 5 P as R 0 P with Heat –inactivated horse serum (5% v/v) RSG Relative Suspension Growth RTG Relative Total Growth S survival SG Suspension Growth TFT trifluorothymidine TK thymidine kinase UKEMS United Kingdom Enviro nmental Mutagen Society