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 

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 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 ........................................................................................................................... 14
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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........................................................................................................ 22
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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 .............................................................................................................. 67 
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LIST OF TABLES 

Table 1 Toxicity Test in the Absence of S9 Mix (4 h Exposure) .................................. 33 

Table 2 Toxicity Test in the Presence 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) .................................................................................. 39 
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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 Historical 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 Mutant Selection Data..................................................................... 59 

Appendix 10 List of Abbreviations........................................................................................ 67 
1 COMPLIANCE STATEMENT 

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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 Director 
2 QUALITY ASSURANCE STATEMENT 

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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. 
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 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 BSc 
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4 SUMMARY 

Alcohols, C18-22 Distn. Residues was assayed for mutagenic potential in the mouse 

lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine 

kinase locus: tk+tk- to tktk-. Alcohols, C18-22 Distn. Residues was prepared as a part 

solution/part suspension in dimethylsulphoxide. Tests were conducted both in the absence 

and in the presence of a post-mitochondrial supernatant fraction obtained from 

Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function 

oxidase activity (S9 mix). The study was designed 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, Alcohols, C18-22 Distn. Residues was shown to be nontoxic at the highest practical concentration of 200 μg/mL. Precipitation of the 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 (μg/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 ‘small’ type colonies. Duplicate 

cultures were carried through the experiments for each treatment point. Vehicle control 

cultures were also included and were 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 the vehicle controls and each concentration of 

Alcohols, C18-22 Distn. Residues. In addition, all the experiments were tested for doserelated trends. 

No evidence of mutagenic activity was obtained with Alcohols, 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 presence of S9 mix, when tested up to and beyond 

its limit of solubility in the test system. 
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5 INTRODUCTION 

The objective of the study was to determine the 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 States 40 CFR § 798.5300 

(updated 2001). The study also meets Japanese requirements. 

5.1.2 Quality Compliance 

The study was conducted in accordance with the OECD Principles of Good Laboratory 

Practice as set forth by the United Kingdom Department 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 detailed 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 Statement page for date of Study 

Director's signature 

5.4 Archiving of Data 

All raw data generated and recorded during this study will be stored in the Scientific Archives 

of Charles River Preclinical Services Edinburgh for 2 years after the issue of the final report. 

After the 2 year period, the Sponsor will be consulted regarding the disposal, transfer or 

continued storage of the raw data. 
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 Test Facility Study No. 787739 

The original signed copy of the final report will be stored indefinitely 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, distn. 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 dark 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. Residues was found to have limited solubility in 

all solvents compatible with the test system. Dimethylsulphoxide (DMSO) was chosen as the 

solvent giving the best solubility/dispersal characteristics. 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 milky 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 precipitate compared. There was a greater level 

of precipitate/undissolved material present in the water sample than in the DMSO sample and 
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 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 immediately prior to dosing (within 1 h). Detailed 

records of preparation of the dosing solutions were maintained to allow checking of 

procedures. Chemical analysis of the test item formulations was not conducted; however 

Alcohols, C18-22 Distn. Residues was tested up to and beyond its limit 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 tests can be used, for example, to confirm results 

obtained with bacterial cell tests. A positive result 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 reversing 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 induced in cultured mammalian cells (Fischer 

and Sartorelli (1964); Chu and Malling (1968); Kao and Puck (1968)). The thymidine kinase 

heterozygote system, where tk+

tk- is mutated to tktk-, 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 tktk- are resistant 

to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine 

kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism 

and halts further cell division. Thus, mutant cells are able to proliferate in the presence of 

TFT, whereas normal cells, which contain 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 validation studies of the L5178Y cell system are 

those of Clive et al (1979); Amacher et al (1980); Jotz and Mitchell (1981); McGregor et al

(1987, 1988a, 1988c, 1991a, 1991b); Myhr et al (1990); Honma et al (1999a, 1999b). 
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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 mutant cells divide at the normal rate, producing 

large colonies, while other cells divide at a distinctly slower rate, producing small colonies. A 

high proportion of large type colonies are associated with small chromosomal deletions or 

point mutations, while a large proportion of the small type colonies are associated with large 

chromosomal deletions. Assessment of the relative numbers of both colony types can provide 

information to support results obtained in bacterial mutation and 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 number and have a high cloning efficiency in 

serum-enriched cloning medium. 

6.6.2 Culture Medium 

The basic culture medium (R0P) was RPMI 1640 medium, supplemented with penicillin 

(100 units/mL), streptomycin (100 μg/mL), sodium 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 R0P to 

give R10P. 

The medium used during treatment for 4 h was R0P supplemented with 5% v/v 

heat-inactivated horse serum (R5P). The medium used during treatment for 24 h was R10P. 

For colony formation, cloning medium was used, consisting of R0P supplemented with 

heat-inactivated horse serum (20% v/v), sodium pyruvate (1.9 mM), and amphotericin B 

(fungizone) (2.5 μg/mL). 

For selection of tktk- cells, cloning medium was supplemented 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. 
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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 principally 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 prove the reproducibility 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 significant 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 nucleoside analogues, base analogues and aneuploidy 

inducers. The study was designed, therefore, to include a 24 h exposure period in the second 

experiment in the absence of S9 mix, should the results of the first experiment (using a 

standard 4 h exposure) be negative. 

6.8 Justification of Dose Levels 

Concentrations in mutation experiments should extend into the toxic range, the maximum 

usable limit allowing a relative total growth value of at least 10% of the concurrent vehicle 

control. It was necessary, therefore, to perform initial toxicity tests in the absence and 

presence of S9 mix. 

The final concentrations of Alcohols, C18-22 Distn. 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 maximum 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. 
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 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 mix1): 25, 50, 100 and 200 

Assay 2 (in the presence of S9 mix1): 25, 50, 100 and 200 

Assay 3 (in the absence of S9 mix2): 25, 50, 100 and 200 

Assay 4 (in the presence of S9 mix1): 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 based 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 vehicle control cultures. These cultures were 

subjected to the same experimental manipulations as the treated cultures. In this study, the 

vehicle control cultures were treated with DMSO. Single cultures were treated in the toxicity 

tests. Four vehicle control cultures were treated in each mutation experiment. 

6.9.2 Positive Control Materials 

All mutation assays included duplicate positive 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 inducer; 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 period, 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 experimental 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 tests. In the mutation 

assays, duplicate cultures were treated for all Alcohols, C18-22 Distn. Residues 

concentrations. 
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6.10 Experimental Procedure 

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

All water used in the preparation of reagents 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 adult 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 characterised by testing selected pre-mutagens 

in an Ames test with S. typhimurium TA 1538. The results of the characterisation and 

preparation details of the batches used in the mutation experiments 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, R0P was added to pre-weighed cofactors: nicotinamide adenine 

dinucleotide phosphate (NADP) disodium salt and glucose-6-phosphate (G-6-P) disodium 

salt, giving final concentrations in the ‘S9 mix’ of: 

NADP Na2 4 mM (= 3.150 mg/mL) 

G-6-P Na2 25 mM (= 7.605 mg/mL) 

This solution was immediately filter-sterilised by passage through 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 similar to that described below for the mutation assays with the 

exception that only one culture was prepared for each treatment. 

The selection in the mutation experiments of the treatments for final assessment is dependent 

on suspension growth following treatment. The measure used to assess toxicity in the 

Toxicity Test was therefore relative suspension growth. The cell population densities were 

recorded over 2 days (following treatment) using a haemocytometer, then the total suspension 
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 Test Facility Study No. 787739 

growths were expressed as percentages of the vehicle control group (= relative suspension 

growth, or RSG). 

The toxicity test was performed using the standard 4 h exposure period in the absence and 

presence of S9 mix. An additional toxicity test was performed in the absence of S9 mix with 

24 h exposure to Alcohols, C18-22 Distn. Residues, as a contingency against the later 

requirement for a full experiment using this extended exposure period. 

Observations on the precipitation of Alcohols, C18-22 Distn. Residues were made after 

dosing and at the end of the exposure period. Observations 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 R10P) were dispensed to sterile 

tubes containing 3.9 mL R0P. Freshly prepared S9 mix or R0P (1 mL) was added to each tube 

followed by 0.1 mL of test solution. Vehicle 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 105 cells/mL, in R5P 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 R10P 

medium (20 mL). This step was repeated to give a cell density of ca 3 x 105

/mL. 

The cells were returned to the rotating drum and allowed to express their genetic lesions at ca

37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 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 exposure to the test item through >1 cell cycle. 

On the day of the test (Day 0), samples of cell culture (in 10 mL R10P) were dispensed to 

sterile tubes containing 7.8 mL R0P. R50P (R0P:serum, 50:50) (2 mL) was added to each tube 

followed by 0.2 mL of the test solution. Vehicle control cultures received 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 105 cells/mL, in R10P medium. 
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(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 density. The lower density is required to allow cell 

growth during the exposure period. The serum concentration is not lowered, as some 

essential nutrients can become exhausted 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 R10P medium (20 mL). This step was repeated. Cell counts were made and the densities 

adjusted (where higher) to give ca 3 x 105 cells/mL. The cells were returned to the rotating 

drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were 

adjusted, after counting, to ca 3 x 105 cells/mL on Day 2. 

6.13.3 Expression of Genetic Damage 

On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined. The cell 

counts over the 2 or 3 days of the experiments 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. This was determined by performing two parallel 

cloning assays: the cloning efficiency assay 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 solution was added to cloning medium to give a 

final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an 

estimated 1 x 104

 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, 

so giving an estimated 2000 cells per well. 

All dishes were incubated at ca 37°C in an atmosphere of 5% CO2:95% air (v/v) until the 

colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for 

mutant selection assay). 

6.13.4 Plate Reading 

The plates were scored using a dissecting microscope fitted to a light box with dark field 

illumination. The number of empty wells in each plate in the cloning efficiency assay was 
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counted. When scoring the mutant selection assay, separate counts were made of the numbers 

of wells containing large type and small type colonies. 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 morphological 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 examples 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 each 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 and 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: 

FinalConcentration on Day 1

Day 2 Count

x 

Concentration on Day 0

Day 1Count
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 Test Facility Study No. 787739 

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

FinalConc.on Day 2

Day 3Count

x 

FinalConc.on Day 1

Day 2 Count

x 

Conc.on Day 0

Day 1Count

The total suspension growth values are then expressed 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 cytotoxicity 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 percentages of the vehicle control mean. The CE is 

calculated from the zero term of the Poisson distribution using the formula: 

number of cells per well

ln(P(o)) CE − =

total wells

empty wells where P(o) =

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 the 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 106

 viable cells. 

CE of non - mutants

CE of mutants

CE in non -selectivemedium

CE in medium containing TFT Mutantfraction per viable cell = =

1

100

 x 

mean vehicle control total growth

individual total growth RelativeTotal Growth (%) =
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 Test Facility Study No. 787739 

7.1.4 Colony Size Fractions 

The ratio of small type mutant colonies to large type mutant colonies was expressed for each 

culture. 

7.2 Historical Control Data 

The historical ranges of vehicle and positive controls 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 al (2007)). 

Errors in cell dilution or very high mutant fractions may result in plates with no negative 

wells. The Poisson distribution analysis does not generate data for such scores and so any 

pair of plates containing 0% empty wells was rejected. Plates 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 results, and therefore such instances were 

accepted.) 

Results for any one treatment/concentration were inadequate if there were less than 2 

acceptable cultures. Where results were obtained from a single culture, they may have been 

included as supporting evidence. 

Analysable results should have been obtained from at least 4 concentrations 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 Alcohols, C18-22 Distn. Residues should have been limited by 

solubility or toxicity, or, in the absence of these, should have been the maximum practical 

concentration of test item, based on the recommendations in current guidelines, ie

5000 μg/mL or 10 mM, whichever is lower. 
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 Test Facility Study No. 787739 

The negative control mean mutant fraction range recommended by the IWGT mouse 

lymphoma workgroup for the microwell assay 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 mutants per million were accepted, providing the 

positive control results were unequivocally acceptable. 

In any experiment, MMS and at least one concentration of 3-MC should have yielded an 

induced mutant fraction (IMF = No. mutants per 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 historical control database. Variations 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 absence 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 subjected 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 biological 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 normal, 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 RTG values down to 20%, if a conclusion of non-
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 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 dealing 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 the 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, biologically irrelevant increases in mutant fraction may arise that 

prove statistically significant (Moore et al (2006)). The IWGT recommends 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 plus 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 concentrations were biologically 

significant and there was a significant linear trend. An experiment may also have been 

classed as positive in the absence of a linear trend if there was mitigating evidence. This may 

have been, for example, the presence of a similar level of toxicity at all concentrations 

assessed. In such a case, the confirmatory experiment 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 with the historical maximum for difference 

between vehicle controls (No Effect 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 negative response in the standard exposure in the 

absence of S9 mix, but gave a positive response in the extended exposure, were liable to a 

confirmatory experiment with 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 other criteria for a positive response described in 

8.2.2.1 above, a test item was defined as non-mutagenic, 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 concentration of 5000 μg/mL or 10 mM, whichever is 

lower 

• the highest practical concentration 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-mutagenic 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 data 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 colonies was stated for all cultures. These ratios 

provide evidence of adequate recovery 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 toxic 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 concentration of 200 μg/mL was 

considered to be a precipitating dose level. 

9.2 Mutation Assays (Tables 4 - 7) 

The summarised results giving the mean values for the duplicate cultures (and quadruplicate 

vehicle controls) are presented in Tables 4 - 7. The original data 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 presented in Appendix 8, and the Day 2/3 Mutant 

Selection data are presented 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 L5178Y cell line (Mitchell et 

al (1997)). It was noted that the value obtained 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 (see Section 7.3.1) and therefore the value was 

accepted. All other values met the acceptance criteria in Section 7.3.1 in full. 

9.2.2 Positive Control Groups 

All positive control log mutant fractions were significantly higher than the vehicle controls at 

P<0.05. The IMF values and colony size ratios 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 - Table 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 and presence of S9 mix (4 h exposure). 

None of the assessed concentrations tested significant 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 
 Page 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 either 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 therefore 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 significant 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 either 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 treatment conditions and 

concentrations of test item were identical) was made. There were no statistically significant 

differences between the Alcohols, C18-22 Distn. Residues-treated groups and the vehicle 

controls. The test for linear trend was not significant (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 limit of solubility in the test system. 
 Page 29 

 Test Facility Study No. 787739 

11 REFERENCES 

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the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test Validation and 

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Ames B N, McCann J and Yamasaki E (1975). Methods for detecting carcinogens and 

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Chu E H Y and Malling H V (1968). Mammalian cell genetics. II. Chemical induction of 

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Clive D, Flamm W G, Machesko M R and Bernheim N J (1972). A mutational assay system 

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mutations in L5178Y cells in culture. Mutation Research, 115, 225-251. 

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Honma M, Hayashi M, Shimada H, Tanaka N, Wakuri S, Awogi T, Yamamoto K I, NorikoUshio K, Nishi Y, Nakadate M and Sofuni T (1999a). Evaluation of the mouse lymphoma tk
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assay (microwell method) as an alternative to the in vitro chromosomal aberration test. 

Mutagenesis, 14, 5-22. 

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Sofuni T (1999b). The need for long-term treatment in the mouse lymphoma assay. 

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Serres F J and Ashby J (Eds). Elsevier, Amsterdam, 580-593. 

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Responses of the L5178Y tk+

/tk- Mouse Lymphoma Cell Forward Mutation Assay to Coded 

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/tk- Mouse Lymphoma Cell Forward Mutation Assay II: 18 

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(1988b). Studies of an S9-based metabolic activation system used in the mouse lymphoma 

L5178Y cell mutation assay. Mutagenesis, 3, 485-490. 

McGregor D B, Brown A, Cattanach P, Edwards I, McBride D, Riach C and Caspary W 

(1988c). Responses of the L5178Y tk+

/tk- Mouse Lymphoma Cell Forward Mutation Assay: 

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Responses of the L5178Y Mouse Lymphoma Cell Forward Mutation Assay. V: 27 Coded 

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McGregor D B, Brown A, Cattanach P, Edwards I, McBride D, Riach C G, Shepherd W and 

Caspary W J (1991b). Responses of the L5178Y Mouse Lymphoma Forward Mutation 

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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 chromosomal mutation assay. A phase III 

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Harrington-Brock K, Mitchell A, Muster W, Myhr B, O'Donovan M, Ouldelhkim M-C, San 

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Collard D, Fellows M, Flanders K, Gollapudi B, Jenkinson P, Kirby P, Kirchner S, Kraycer J, 

McEnaney S, Muster W, Myhr B, O'Donovan M, Oliver J, Ouldelhkim M-C, Pant K, Preston 

R, Riach C, San R, Shimada H, Stankowski Jr. L F (2002). Mouse lymphoma thymidine 

kinase gene mutation assay: Follow-up International Workshop on Genotoxicity Test 

Procedures, New Orleans, Louisiana, April 2000. Environmental and Molecular Mutagenesis, 

40, Issue 4, 292-299. 

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: 

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Delongchamp R, Durward R, Fellows M, Gollapudi B, Hou S, Jenkinson P, Lloyd M, 

Majeska J, Myhr B, O'Donovan M, Omori T, Riach C, San R, Stankowski Jr. L F, Thakur A 

K, Van Goethem F, Wakuri S, Yoshimura I (2006). Mouse lymphoma thymidine kinase gene 

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Doppalapudi R, Fellows M, Gollapudi B, Hou S, Jenkinson P, Muster W, Pant K, Kidd D A, 

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Myhr B, McGregor D, Bowers L, Riach C G, Brown A G, Edwards I, McBride D, Martin R 

and Caspary W J (1990). L5178Y Mouse Lymphoma Cell Mutation Assay Results With 41 

Compounds. Environmental and Molecular Mutagenesis, 16 (Suppl 18), 138-167. 

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Mutation Research, 257, 147-205. 
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12 TABLES 

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

Suspension Count 

(x 105

/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 = Precipitation 
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 Test Facility Study No. 787739 

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

Suspension Count 

(x 105

/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 = Precipitation 
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 Test Facility Study No. 787739 

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

Suspension Count 

(x 105

/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 = Precipitation 
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 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 treatment minus mutant fraction 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 reported – slope negative 

- = IMF ≤ 0 

pptn = Precipitation 
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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 treatment minus mutant fraction 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 = Precipitation 
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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 treatment minus mutant fraction 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 reported – slope negative 

- = IMF ≤ 0 

pptn = Precipitation 
 Page 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 treatment minus mutant fraction 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 = Precipitation 
 Page 40 

 Test Facility Study No. 787739 

13 APPENDICES 

Appendix 1 Certificate of Analysis 
 Page 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 106

 cells/mL. Samples (2 mL) were frozen slowly before being stored in 

liquid nitrogen. New cultures are established from frozen stocks every 3 months. 

Initially, a new culture was seeded into a T-75 flask containing 20 mL R10P gassed with 5% 

CO2 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 established by seeding 3 x 105

cells/mL in fresh R10P 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 105

 cells/mL to maintain the cells in logarithmic growth. For weekend 

maintenance, the cultures were diluted back to ca 1 x 104

 cells/mL. 

Cell ‘Cleansing’ 

The tk+

tk- heterozygote cells grown in suspension spontaneously mutate to tktk- at a rate of 

2 x 10-6 mutations/generation. These homozygous mutants were removed before testing 

began. 

The week before an experiment, Thymidine/Hypoxanthine/Glycine/Methotrexate (THGM) 

stock solution (1 mL) was added to a sub-population of the stock cell suspension (100 mL). 

The following day the culture was pelleted and resuspended in R10P 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 strongly to dihydrofolate 

reductase, hence thymidylate synthetase-mediated thymidylate production (TMP) stops. TMP 

can be maintained by TK phosphorylation of exogenous thymidine (THG). Purine 
 Page 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 source to replace the blocked folate metabolic 

pathway. Hence tk+

tk- cells can survive the methotrexate treatment while tktk- cells die. 
 Page 43 

 Test Facility Study No. 787739 

Appendix 3 In vitro Activation Preparation Form 
3M 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 methyl 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 105

/mL. On Day 2, the cells 

had reached 11.0 x 105

/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: 

FinalConcentration on Day 1

Day 2 Count

x 

Concentration on Day 0*

Day 1Count

* Routinely 3.0 x 105

/mL for all cultures 

In this instance, 

Note: if toxicity causes the cell density to be below 3.0 x 105

/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, rather than the more usual 3.0 x 105

/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 Relative Total Growth (Appendix 8, page 55)

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

the formula: 

number of cells per well

ln(P(o)) CE − =

total wells

empty wells where P(o) =

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 

mean vehicle control total growth

individual total growth RelativeTotal Growth (%) =

 57.1%

1

100

 x 

25.7

14.7 =

 0.40

192

41 36 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 was 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 subtracting the small colony wells and the 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 compensate 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 = =

- 6 745 x 10

0.57

- 6 425.39 x 10 Mutant fraction per viable cell = =

CE of non - mutants

CE of mutants Mutant fraction per viable cell = 
3M Ref. No. 09-188 

Appendix 5 Mouse Lymphoma L5178Y Cell Historical Control Data from July 2003 to De

Vehicle Control Data 

Mutant Fraction x 10-6 

Mean Colony 

Size Ratio 

(Small/Large

Vehicle 

Control S9 Exposure Time n†

Mean SD Range 

No-effect 

Maximum 

(Induced Mutant 

Fraction) Mean SD 

All, pooled - 4 h 71 71 16 49-121 42 1.28 0.47 All, pooled - 24 h 50 78 21 51-129 49 1.09 0.43 All, pooled + 4 h 120 78 22 45-148 70 1.24 0.44 

† = Each value is the mean of 4 replicate cultures The No-effect Maximum represents the maximum difference recorded between the 2 pairs of vehicle control cultures in any experimeis, the lower mean mutant fraction (x 10-6) is subtracted from the higher. This difference, when applied to the response from a mutage

termed the induced mutant fraction (IMF). 

Positive Control Data 

Mutant Fraction x 10-6 RTG % 

Me

S

(Sm

Positive 

Control S9 

Concentration 

(μg/mL) 

Exposure 

Time 

n†

Mean SD Range Mean Range Mean S

EMS - 250 4 h 69 601 127 431-910 64 38-94 0.51 0

MMS - 10 4 h 71 908 238 429-1659 40 22-78 2.34 0

EMS - 150 24 h 50 2096 608 1324-3801 31 15-99 0.38 0

MMS - 5 24 h 50 1880 471 1026-3023 31 17-82 1.78 0

3-MC + 2.5 4 h 120 1061 338 570-2277 49 16-96 1.37 0

EMS = Ethyl methanesulphonate MMS = Methyl methanesulphonate 3-MC = 3-Methylcholanthrene 

† = Each value is the mean of 2 replicate cultureAudited by: Alison McNaughton (Quality Assu

09 February 2009 
3M 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 the historical control. Any dose levels with 

heterogeneity factor statistically higher than the historical control were excluded from all 

statistical analysis. 

3. Determination of the heterogeneity factor for the experiment. 

4. Calculation of a new historical control heterogeneity factor. 

5. Calculation of the log mutant fraction. 

6. Comparison of the log mutant fraction between the control and each treatment dose (at 

P<0.05). 

7. Test for linear increasing trend of mutant fraction with increasing dose of test item (at 

P<0.05). 

Unless otherwise stated, the term 'heterogeneity factors' is defined 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, assays were analysed in chronological order. If 

the new value was exceptionally large (as determined by significance testing below), the 

experimental results were considered unacceptably variable and were discarded. Otherwise, 

the new heterogeneity factors were calculated for use in the analysis and the historical value 

was updated. 

Beginning with Assay 1, the data were analysed using the methods outlined in Robinson et al 

(1989). The dose-specific heterogeneity factors for each dose level were determined. 

The dose-specific heterogeneity factors were compared with the historical controls for 

consistency using a one-sided F-test (Pearson and Hartley (1989)) at the 0.1% level. Any 

dose levels where either the mutant or the survival heterogeneity factors were significantly 

higher than their respective historical controls were excluded from 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 factors 
3M Ref. No. 09-188 Page 49 

 Test Facility Study No. 787739 

Appendix 6 

(continued) 

were significantly higher than their respective historical controls 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 current 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-sided 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 χ2

 test (Pearson and Hartley (1989)) with 1 degree of freedom was 

performed at the 5 % level to test for linear 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 exposure periods and equal dose levels were used. 

A two-sided χ2

 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 frequency. (This was only performed if the 

treatment dose mutant frequency was greater than the control.) 

A one-sided χ2

 test with 1 degree of freedom was performed for the combined data at the 5% 

level to test for linear increasing trend of mutant frequency with increasing dose of test 

material. (This was only performed if the direction 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, Garner 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 3rd edition, 

Griffin, London 

Dunnett, C W (1955). A multiple comparison procedure for comparing several treatments 

with a control. Journal of American Statistical 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 105 Chemical /mL) Concentration 

(μg/mL) Observations 

Day 1 Day 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 27.3 106.1 
3M 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 105 Chemical /mL) Concentration 

(μg/mL) Observations 

Day 1 Day 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 85.0 
3M 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 105 Chemical /mL) Concentration 

(μg/mL) Observations 

Day 1 Day 2 Day 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 74.0 
3M 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 105 Chemical /mL) Concentration 

(μg/mL) Observations 

Day 1 Day 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 18.2 102.2 
3M 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 30.02 120 
3M 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 27.61 106 
3M 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 52.56 101 
3M 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 88 
3M Ref. No. 09-188 

Appendix 9 Individual Mutant Selection Data 

Mutation Test in the Absence of S9 Mix (4 h Exposure) 

Day 2 Mutant Selection (Assay 1) 

Day 2 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Total 

Empt

Well

Vehicle Control 

Mean → - - - - - - - 

DMSO 

(100 μL 

added) 4 3 4 5 0 1.00 192 17

 3 3 3 3 0 1.00 192 18 4 9 6 11 0 2.00 192 16 4 6 6 5 0 1.10 192 17

EMS 250 42 20 38 19 0 0.49 192 7

 37 22 45 15 0 0.45 192 7

MMS 10 12 45 17 36 0 2.79 192 8

 13 35 17 34 0 2.30 192 9

Alcohols, C18-22 

Distn. Residues 25 3 3 3 7 0 1.67 192 17

 4 7 5 9 0 1.78 192 16

 50 1 4 1 2 0 3.00 192 18 8 6 7 7 0 0.87 192 16
3M Ref. No. 09-188 

Appendix 9 (continued) 

Day 2 Mutant Selection (Assay 1) Continued 

Day 2 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Total 

Empt

Well

Alcohols, C18-22 

Distn. Residues 100 4 3 2 5 0 1.33 192 17

 3 5 1 7 0 3.00 192 17

 200 3 5 3 4 0 1.50 192 17 2 1 4 5 0 1.00 192 18
3M Ref. No. 09-188 

Appendix 9 (continued) 

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

Day 2 Mutant Selection (Assay 2) 

Day 2 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Total 

Empt

Well

Vehicle Control 

Mean → - - - - - - - 

DMSO 

(100 μL 

added) 2 3 5 4 0 1.00 192 17

 5 6 5 8 0 1.40 192 16 5 7 5 3 0 1.00 192 17 2 5 4 3 0 1.33 192 17

3-MC 2.5 25 28 22 23 0 1.09 192 9

 21 34 31 22 0 1.08 192 8 10 29 43 29 34 0 1.33 192 5 31 32 31 34 0 1.06 192 6

Alcohols, C18-22 

Distn. Residues 25 4 9 5 6 0 1.67 192 16

 2 8 3 8 0 3.20 192 17

 50 6 6 2 8 0 1.75 192 17 5 4 6 4 0 0.73 192 17
3M Ref. No. 09-188 

Appendix 9 (continued) 

Day 2 Mutant Selection (Assay 2) Continued 

Day 2 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Total 

Empt

Well

Alcohols, C18-22 

Distn. Residues 100 5 5 6 3 0 0.73 192 17

 6 5 12 4 0 0.50 192 16

 200 2 9 7 6 0 1.67 192 16 3 11 6 6 0 1.89 192 16
3M Ref. No. 09-188 

Appendix 9 (continued) 

Mutation Test in the Absence of S9 Mix (24 h Exposure) 

Day 3 Mutant Selection (Assay 3) 

Day 3 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Total 

Empt

Well

Vehicle Control 

Mean → - - - - - - - 

DMSO 

(200 μL 

added) 10 13 3 18 0 2.38 192 14

 7 18 6 16 0 2.62 192 14 2 10 5 15 0 3.57 192 16 6 19 5 16 0 3.18 192 14

EMS 150 73 21 68 21 0 0.30 192 

 70 23 69 25 0 0.35 192 

MMS 5 35 51 36 52 0 1.45 192 1

 39 53 31 59 0 1.60 192 1

Alcohols, C18-22 

Distn. Residues 25 5 16 2 14 0 4.29 192 15

 3 10 7 13 0 2.30 192 15

 50 2 17 7 14 0 3.44 192 15 5 17 2 14 0 4.43 192 15
3M Ref. No. 09-188 

Appendix 9 (continued) 

Day 3 Mutant Selection (Assay 3) Continued 

Day 3 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Tota

Empt

Well

Alcohols, C18-22 

Distn. Residues 100 6 15 3 21 0 4.00 192 1

 7 18 10 10 0 1.65 192 1

 200 4 9 9 16 0 1.92 192 1 7 20 9 18 0 2.38 192 1
3M Ref. No. 09-188 

Appendix 9 (continued) 

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

Day 2 Mutant Selection (Assay 4) 

Day 2 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Tota

Emp

Wel

Vehicle Control 

Mean → - - - - - - - 

DMSO 

(100 μL 

added) 10 12 8 11 0 1.28 192 1

 8 10 5 15 0 1.92 192 1 10 13 9 12 0 1.32 192 1 9 11 6 12 0 1.53 192 1

3-MC 2.5 40 40 31 50 0 1.27 192 

 38 43 35 53 0 1.32 192  10 42 44 48 39 0 0.92 192  33 53 36 51 0 1.51 192 

Alcohols, C18-22 

Distn. Residues 25 15 18 10 15 0 1.32 192 1

 8 9 10 16 0 1.39 192 1

 50 7 10 7 12 0 1.57 192 1 9 19 15 18 0 1.54 192 1
3M Ref. No. 09-188 

Appendix 9 (continued) 

Day 2 Mutant Selection (Assay 4) Continued 

Day 2 Mutant Colonies 

Plate 1 Plate 2 

Chemical Concentration (μg/mL) Large Colony 

Wells 

Small 

Colony 

Wells 

Large 

Colony 

Wells 

Small 

Colony 

Wells 

Wells 

Lost 

Ratio of 

Small to 

Large 

Colonies 

Total 

Wells 

Scored 

Tota

Emp

Wel

Alcohols, C18-22 

Distn. Residues 100 7 16 4 12 0 2.55 192 1

 8 16 7 16 0 2.13 192 1

 200 9 22 6 12 0 2.27 192 1 6 16 7 12 0 2.15 192 1
3M 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 Workshop 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 Institute (developers of RPMI 1640 medium) 

R0P RPMI 1640 medium, supplemented with penicillin (100 units/mL), 

streptomycin (100 μg/mL), sodium bicarbonate (1.125 g/L) and pluronic acid 

(0.05% w/v) 

R10P as R0P with Heat –inactivated horse serum (10% v/v) 

R50P as R0P with Heat –inactivated horse serum (50% v/v) 

R5P as R0P 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 Environmental Mutagen Society