REPORT 

3-PPB 

Bacterial Reverse Mutation Test 

HLS study number: 

Version 10: 

Issue date: 

Page 1 of 37 

Final Report 

10 February 2014 

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Huntingdon Life Sciences: ' 

Sponsor and Test Facility Details 

Sponsor 

Test facility 

Emerald Kalama Chemical, LLC 

1296 Third Street NW 

Kalama 

WA98625 

USA 

Huntingdon Life Sciences 

Eye Research Centre 

Eye 

Suffolk 

IP23 7PX 

UK 

2 
Huntingdon Life Sciences: 

Table of Contents 

Sponsor and Test Facility Details .......................................................................................... 2 

Compliance wHh Good Laboratory Practice .......................................................................... 5 

Quality Assurance Statement ................................................................................................ 6 

Contributing Scientists .......................................................................................................... 7 

Summary .............................................................................................................................. 8 

1. Introduction .................................................................................................................. 9 

2. Experimental Procedure ............................................................................................. 10 

2.1 Test substance ............................................................................................... 10 

2.2 Bacterial strains .............................................................................................. 1 0 

2.3 Positive controls .............................................................................................. 11 

2.3.1 In the absence of S9 mix ..................................................................... 11 

2.3.2 In the presence of S9 mix .................................................................... 11 

2.4 S9 metabolizing system .................................................................................. 12 

2.4.1 Preparation of S9 fraction .................................................................... 12 

2.4.2 Preparation of S9 mix .......................................................................... 12 

2.5 Selection of vehicle and formulation oftest substance .................................... 12 

2.6 Mutation test procedure .................................................................................. 12 

2.6.1 First test .............................................................................................. 12 

2.6.2 Second test ......................................................................................... 13 

2. 7 Stability, homogeneity and formulation analysis .............................................. 13 

3. Assessment of Results ............................................................................................... 14 

3.1 Acceptance criteria ......................................................................................... 14 

3.2 Analysis of data .............................................................................................. 14 

3.3 Criteria for assessing mutagenic potential.. ..................................................... 14 

4. Deviations from Protocol ............................................................................................ 15 

5. Maintenance of Records ............................................................................................. 16 

6. Results ....................................................................................................................... 17 

6.1 First test.. ........................................................................................................ 17 

6.2 Second test. .................................................................................................... 17 

7. Conclusion ................................................................................................................. 18 

8. References ................................................................................................................. 19 

3 
Huntingdon Life Sciences: 

List of Tables 

Table 1 

Table2 

Table 3 

Table 4 

Table 5 

Table 6 

Resu~s obtained in the absence of metabolic activation: test 1 .................... 20 

Resu~s obtained in the presence of metabolic activation: test 1 ................... 22 

Viabiltty counts obtained in test 1 ................................................................. 24 

Results obtained in the absence of metabolic activation: test 2 .. : ................. 25 

Resu~ obtained in the presence of metabolic activation: test 2 ................... 27 

Viability counts obtained in test 2 ................................................................. 29 

List of Appendices 

Appendix 1 Historical Control Data ................................................................................. 30 

Appendix 2 GLP Compliance Statement. ........................................................................ 32 

Appendix 3 59 Quality Control Certificate ....................................................................... 34 

Appendix 4 Certificate of Analysis .................................................................................. 36 

4 
Huntingdon Ufe Sciences: 

Compliance with Good Laboratory Practice 

3-PPB: Bacterial Reverse Mutation Test 

Tite study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards, with the exception stated below, and I consider the data 

generated to be valid. 

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 

No. 3106, as amended by Slatutory Instrument 2004 No. 994}. 

OECD Principles of Good Laboratory Practice (as revised in 1997}, 

ENV/MC/CHEM (98) 17. 

EC Commission Directive 2004/10/EC of II February 2004 (Official Journal 

No. L 50/44). 

These principles of Good Laboratory Practice are accepted by the regulatory authorities of 

the United States of America and Japan on the basis of intergovernmental agreements. 

In line with normal practice in this type of short-term study, the protocol did not require 

analysis of the dose form. 

Kenneth May BSc CBiol MSB 

Study Director 

Huntingdon Life Sciences 
Huntlngdon Life Sciences: 

Quality Assurance Statement 

3·PPB: Bacterial Reverse Mutation Test 

The following inspections and audits have been carried out in relation to this study: 

Study Phase 

Protocol Audit 

Report Audit 

Date(s) oflnspection 

04 Nov 2013 

30 Jan 2014-31 Jan 2014 

Date of Reporting to Study 

Director and Management 

04 Nov 2013 

31 Jan 2014 

Process based inspections: At or about the time this study was in progress inspections of 

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below: 

Process Blllied Inspections 

Plate reading (In Vitro) 

Formulation procedures, 

sampling and treatment (In 

Vitro) 

Study management & 

conduct (In Vitro) 

Date(s) oflnspection 

23 Aug2013 

23 Sep 2013-24 Sep 2013 

18 Nov 2013-21 Nov 2013 

Date of Reporting to 

Man~gement 

23 Aug 2013 

26 Sep 2013 

21 Nov 2013 

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were reported to Company Management. 

Tim WiilltlingMRQ 

Group Manager 

Department of Quality Assurance 

Huntingdon Life Sciences 
Contributing Scientists 

3-PPB: Bacterial Reverse Mutation Test 

Study management 

Kenneth May BSc CBiol MSB 

Study Director 

Alica Soltesova 

Study Scientist 

7 

Huntingdon Ufe Sciences; 
Summary 

8 

Huntingdon Life S ctences: . 
Huntingdon Life Sciences: 

1. Introduction 

This report describes a study designed to assess 3-PPB for its potential to cause point (gene) 

mutation in Salmonella typhimurium and Escherichia coli. The study was conducted in 

compliance with the fOllowing regulations or guidelines: 

OECD Guidelines for the Testing of Chemicals (1997). Genetic Toxicology: 

Bacterial Reverse Mutation Test, Guideline 471. 

EC Commission Regulation No. 440/2008. Method B.l3/14: 

Mutagenicity- Reverse mutation test using bacteria. OJ L 142/248. 

US EPA. Health Effects Test Guidelines (1998). OPPTS 870.5100 

Bacterial reverse mutation test. EPA 712-C-98-247. 

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Huntingdon Life Sciences: 

2. Experimental Procedure 

2.1 Test substance 

Identity: 

Chemical name: 

Commercial/other name: 

Lot number. 

Expiry: 

Appearance: 

Storage conditions: 

Purity/ Assay: 

Date received: 

Certificate of analysis: 

2.2 Bacterial strains 

3-PPB 

3-phenylpropyl benzoate 

K-Flex® 613; X-613 

EH101912A 

19 October 2014 

Clear, colourless liquid 

Room temperature, under nitrogen 

99.1% 

22 October 2013 

Appendix 4 

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Huntingdon Ufe Sciences: 

For use in tests, an aliquot of frozen culture was added to 25 mL of nutrient broth and 

incubated, with shaking, at 37°C for I 0 hours. These cultures were intended to provide a 

viable cell density of at least I 09 per mL, which was confirmed by performing viability 

counts, in which aliquots (0.1 mL) of a 10.,; dilution of the 10-hour cultures were spread on 

the surface of plates of nutrient agar. After incubation at 37°C for 24 hours, the total number 

of resultant colonies was counted. 

2.3 Positive controls 

2.3.1 In the absence of 59 mix 

I 

2.3.2 In the presence of 59 mix 

11 
Huntingdon Life Sciences: 

2.4 59 metabolizing system 

2.4.1 Preparation of 59 fraction 

2.4.2 Preparation of 59 mix 

2.5 Selection of vehicle and formulation of test substance 

2.6 Mutation test procedure 

2.6.1 First test 

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Huntingdon Life Sciences: . 

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2.6.2 Second test 

2.7 Stability, homogeneity and formulation analysis 

13 
Huntingdon Life Sciences: 

3. Assessment of Results 

3.1 Acceptance criteria 

3.2 Analysis of data 

3.3 Criteria for assessing mutagenic potential 

14 
Huntingdon Life Sciences: 

4. Deviations from Protocol 

15 
Huntingdon Life Sciences: 

5. Maintenance of Records 

16 
6. Results 

17 

Huntingdon Life S c1ences: . 
Huntingdon Life Sciences: 

7. Conclusion 

18 
Huntingdon Life Sciences: 

8. References 

Published Literature 

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

mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Res. 31, 

347-364. 

Gatehouse, D.G., Rowland, l.R., Wilcox, P., Callander, R.D. and Forster, R. (1990) Bacterial 

mutation assays in: Kirkland, D.J. (Ed.). UKEMS Sub-committee on Guidelines for 

Mutagenicity Testing. Report. Part 1. Basic Mutagenicity Tests, pp.l3-61. Cambridge 

University Press, Cambridge. 

Gatehouse, D., Haworth, S., Cebula, D., Gocke, E., Kier, L., Matsushima, T., Melcion, C., 

Nohmi, T., Ohta, T., Venitt, Sand Zeiger, E. (1994) Report from the working group on 

bacterial mutation assays: International W01kshop on Standardisation ofGenotoxicity Test 

Procedures. Mutation Research, 312,217-233. 

Green, M.H.L. (1984) Mutagen testing using trp+ reversion in Escherichia coli in Kilbey, 

B.J., Legator, M., Nichols, W. and Ramel, C. (Eds.). Handbook of Mutagenicity Test 

Procedures. Second edition, pp.16l-l87. Elsevier Science Publishers BV, Amsterdam. 

Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I. de G., Robinson, W.D. and 

Tweats, D.J. {1989) Analysis of data from microbial colony assays in: Kirkland, D.J. (Ed.). 

UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part Ill. Statistical 

Evaluation of Mutagenicity Test Data, pp.26-65. Cambridge University Press, Cambridge. 

Maron, D .M. and Ames, B .N. ( 1983) Revised methods for the Salmonella mutagenicity test. 

Mutation Res. 113, 173-215. 

19 
Huntingdon Life Sciences: ' 

Table 1 Results obtained in the absence of metabolic activation: test 1 

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Table 1 

Huntingdon Ute Sciences· 

Results obtained in the absence of metabolic activation: test 1 

(continued) 

21 
Huntingdon Life Sciences: 

Table2 Results obtained in the presence of metabolic activation: test 1 

22 
Table 2 

Huntingdon Life Sciences: 

Results obtained in the presence of metabolic activation: test 1 

(continued) 

23 
Huntingdon Life Sciences: 

Table3 Viability counts obtained in test 1 

24 
Huntingdon Life Sciences: 

Table 4 Results obtained in the absence of metabolic activation: test 2 

25 
Table4 

Huntingdon Life Sciences: 

Results obtained in the absence of metabolic activation: test 2 

(continued) 

26 
Huntingdon Life Sciences: 

Table 5 Results obtained in the presence of metabolic activation: test 2 

27 
Table 5 

Huntingdon Life Sciences: 

Results obtained in the presence of metabolic activation: test 2 

(continued) 

28 
Huntingdon Life Sciences: 

Table 6 Viability counts obtained in test 2 

29 
Huntingdon Life Sciences:: 

Appendix 1 Historical Control Data 

30 
Huntingdon Life Sciences: 

IITSTORICAL CONTROL DATA 

31 
Huntingdon Life Sciences:, 

Appendix 2 GLP Compliance Statement 

32 
Huntingdon Life Sciences: 

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2012 

f n-;;, Department 

C · ofHea/th 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY 

Huntingdon Life Sciences 

Eye Research Centre 

Oecoid 

Eye 

Suffolk 

IP237PX 

DATE OF INSPECTION 

18th- 20th June 2012 

TEST TYPE(S) 

Analytical/Clinical 

Chemistry 

Environmental Fate 

Environmental Toxicity 

Ecosystems 

Phys.Chem. Testing 

Residue studies 

Mutagenicity 

Toxicology 

An inspection for compliance with the Principles of Good Laboratory Practice was 

carried out at the above test facility as part of the UK Good Laboratory Practice 

Compliance Monitoring Programme. 

This statement confirms that, on the date of issue, the UK Good Laboratory 

Practice Monitoring Authortty were satisfied that the above test facility was 

operating In compliance with the OECD Principles of Gcod Laboratory Practice. 

This statement constitutes a Good Laboratory Practice Instrument (as defined In the 

UK Good Laboratory Practice Regulations 1999). 

Dr. Andrew J. Gray 

Head, UK GLP Monitoring Auth9rlty 

33 
Huntingdon Life Sciences: 

Appendix 3 59 Quality Control Certificate 

34 
Huntingdon Life Sciences: ' 

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Huntingdon Life Sciences: 

Appendix 4 Certificate of Analysis 

36 
iErn~aldPerformance. ~aterials"'" 

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Huntingdon Life Sciences: 

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