### REPORT 3-PPB Bacterial Reverse Mutation Test

HLS study number: Version 10: Issue date:
Page 1 of 37 Final Report 10 February 2014 

# I I 

# I 

# IHuntingdon 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

2Huntingdon 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 .............................................................................................. 10 2.3 Positive controls.............................................................................................. 11 2.3.1 In the absence ofS9 mix..................................................................... 11 2.3.2 In the presence of S9 mix.................................................................... 11 2.4 S9 metabolizing system .................................................................................. 12 2.4.1 Preparation ofS9 fraction .................................................................... 12 2.4.2 Preparation ofS9 mix.......................................................................... 12 2.5 Selection ofvehicle and formulation oftestsubstance .................................... 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 ofdata .............................................................................................. 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

3Huntingdon 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 ofAnalysis ..................................................................................36

4Huntingdon Ufe Sciences: 

## Compliance with Good Laboratory Practice 3-PPB: Bacterial Reverse Mutation Test 

### Tite studydescribed in this report was conducted in compliance with the following Good Laboratory Practice standards, with the exception stated below, and Iconsider the data generated to be valid. The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended 

### by Slatutory Instrument2004 No. 994}. 

### OECD Principles ofGood 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 ofGood Laboratory Practice are accepted by the regulatory authorities of the United States ofAmerica and Japan on the basis ofintergovernmental agreements. In line with normal practice in this type ofshort-term study, the protocol did not require analysis ofthe dose form.

Kenneth May BSc CBiol MSB Study Director Huntingdon Life SciencesHuntlngdon 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 

### DateofReporting 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 ofReporting to

**Man~gement** 23 

### Aug 2013 

### 26 Sep 2013 21 

### Nov 2013 

### In addition, an inspection ofthe facility where this study was conducted was carried out on an annual basis. These inspections were reported to Company Management.

Tim 

## WiilltlingMRQ Group Manager DepartmentofQuality Assurance Huntingdon Life Sciences

## Contributing Scientists 3-PPB: Bacterial Reverse Mutation Test Study management Kenneth May BSc CBiolMSB StudyDirector 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 forthe Testing ofChemicals (1997). Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471. EC Commission RegulationNo. 440/2008. Method B.l3/14: Mutagenicity- Reverse mutation test using bacteria. OJ 

### L 142/248. US EPA. Health Effects TestGuidelines (1998). OPPTS 870.5100 Bacterial reverse mutation test. EPA 712-C-98-247.

9Huntingdon Life Sciences: 

## 2. Experimental Procedure 2.1 Test substance

Identity: Chemical name: Commercial/othername: Lotnumber. Expiry: Appearance: Storage conditions: Purity/ Assay: Date received: Certificate ofanalysis: 

### 2.2 Bacterial strains 3-PPB 3-phenylpropyl benzoate K-Flex® 613; X-613 EH101912A 19 October 2014 Clear, colourless liquid Room temperature, undernitrogen 99.1% 22 October 2013 Appendix 4

10Huntingdon Ufe Sciences: 

### Foruse in tests, an aliquotoffrozen culture was added to 

### 25 mL ofnutrient broth and incubated, with shaking, at 37°C for I0hours. These cultures were intended to 

### provide a viable cell density ofat least I09 

### per mL, which was confirmed by performing viability counts, in 

### which aliquots (0.1 mL) ofa 10.,; dilution ofthe 10-hourcultures were spread on the surface ofplates ofnutrientagar. After incubation at 37°C for 24 hours, the total number ofresultant 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

11Huntingdon Life Sciences: 

### 2.4 59 metabolizing system 2.4.1 Preparation of 59fraction

2.4.2 Preparation of 59 mix


## 2.5 Selection ofvehicle and formulation oftest substance

2.6 Mutation test procedure 2.6.1 Firsttest

12Huntingdon Life Sciences: .



### ' 2.6.2 Second test



### 2.7 Stability, homogeneity and formulation analysis

13Huntingdon Life Sciences: 

## 3. Assessment of Results 3.1 Acceptance criteria

3.2 Analysis ofdata
3.3 Criteria for assessing mutagenic potential

14Huntingdon Life Sciences: 

## 4. Deviations from Protocol

15Huntingdon Life Sciences: 

## 5. Maintenance of Records

16

## 6. Results

17 Huntingdon Life S . c1ences:Huntingdon Life Sciences: 

## 7. Conclusion

18Huntingdon 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 mutagenicitytest. 

### 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.). 

### UKEMSSub-committee on Guidelinesfor Mutagenicity Testing. Report. Part1. 

### BasicMutagenicity 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, SandZeiger, 

### E. (1994) Reportfrom the working group on bacterial mutation assays: InternationalW01kshop on Standardisation ofGenotoxicityTest Procedures. MutationResearch, 312,217-233. Green, M.H.L. (1984) Mutagentesting using 

### trp+ reversion in 

### Escherichia coli in Kilbey, 

### B.J., Legator, M., Nichols, W. andRamel, C. (Eds.). 

### Handbookof 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 ofdatafrom microbial colony assays in: Kirkland, D.J. (Ed.). 

### UKEMSSub-committee on Guidelines forMutagenicity Testing. Report. Part Ill. 

### Statistical Evaluation of Mutagenicity TestData, pp.26-65. Cambridge University Press, Cambridge. Maron, D.M. and Ames, B.N. (1983) Revised methods forthe 

### Salmonella mutagenicitytest. 

### Mutation Res. 113, 173-215.

19Huntingdon Life Sciences: ' 

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

20

### Table 1 Huntingdon Ute Sciences· 

### Results obtained in the absence of metabolic activation: test 1 (continued)

21Huntingdon 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)

23Huntingdon Life Sciences: Table3 Viability counts obtained in test 1

24Huntingdon 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)

26Huntingdon 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)

28Huntingdon Life Sciences: Table 6 Viability counts obtained in test 2

29Huntingdon Life Sciences:: 

### Appendix 1 Historical Control Data

30Huntingdon Life Sciences: 

### IITSTORICAL CONTROL DATA

31Huntingdon Life Sciences:, 

### Appendix 2 GLP Compliance Statement

32Huntingdon Life Sciences: 

### EYERESEARCH 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 TESTTYPE(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. AndrewJ. Gray Head, UK GLP Monitoring Auth9rlty

33Huntingdon Life Sciences: Appendix 

### 3 59 Quality Control Certificate

34Huntingdon Life Sciences: '



# ~~-.**------~-~0-·=-~-:-!** GmbH. 

# MOLTOX" ••!•>1,..,.11 L1r "fu..**1~,11t~!;\',** '"':.. POST MITOCHONDRIAL SUPERNATANT (89) QUALTIY CONTROL&:: PRODUCflON CERTIFICA'l'E Anl11111 lltanullll SPECI:tS:&II. sntAlN: Spmtuc Dawley SEXI,Ma!A

**AGII~** WIUGHTI.l11.::..12i.&

**nssU~JJm** Part Nun!bu h ton,.tiGI · LOT NO.Ilillt 1'ARTNO,t.ll.:l..l1

**VOLUME:~** lJUFFIRl !W.M.KCi STORAGE! AI !X b;Jqw -7P:C RZFERBMCit Matluablnla, u al., /lt'l'llN-MIMIKHkAdlwll/fn.llf · **PUPt~ IXPIR'/1~**  INDUCING AGENTI PbGmbufllh\='! 6, ' 

# ............ M!Ua(!IJQ/6 TUtliJ{{fJ. de Sems, ed.), Ebt<kr, 1!716, pI' For.

**R11w:n~ Pa!]W~£!:~**  0R!r

**~IOCHIUO,USJllYI** . A:su)'lld .cootding:10tM JMtllod ofLoW!)

**.tal~** JIJC193;26!1, 19SI

• PR.DTEIN: ftll\&"1111 •UIIGf; **~~album[!\** II
**Ill&~**
.- A.LXOXYRESORUF(N.()-DEALXYl.ASE'1.CfiVITJ£S' fold• 

# """""'"" 

# - DROD 281,281 69.Z EROD MROO PROD BID.Us.\Y: IAI,l.U !AI. fAl 281,282 51.1 !7.4 11'.1 ·TeST FOJ. THE PRESENCE OP ADVENTITIOUS AOENTS Sampk:Jots-9 wt:JC assayed !'or Ills pl'tSIIII'K" **oftz:~~WlllnJJinJ** mkrollorJI by platla1 1.0 ml vol11111t11 on NWiont A,_ .00 Mlntmal 01- (VO&Ci·1-l'ftllll' B, **~cmfttld** wilh 0.05 mM L·histidiM IN![). blolln) media. Dl!plka\1 Jlaiu

**wen~** lUll il\or4t-4811"111C\iktion 1135 :l:l"C. Tbo klled 111111pJ. n>d a=t:p(IIICII

**~;~'(tala.** • PROMUTAODI ACTIVATION l>lo. Hlsr
**~atllll$** IA?J TAJm )OJ lll4 Thlblllty of!bc SllmpiiiO acdwi,uthlllum (EIBrj M4 . cydophospllan.lda (CPA) to **kr.te~:mdlatM** mutapnlo 10 TMI ftlld TAl»,,
**~ly,** w..decannltocd

**~** 10 Lesca, ct ill, . Ullltllhln Rn IU: 299, 19U."Dllf. 'Ntftl
**~** UI'CVIIWit( per· PIEtiir or pH IPS CPA. OUIIIIOMofthe nmplo S9,

**n.nal~t;** fto111 Q.2- fO% In S9 mb, l'tiR latod Air their lblllty to acli't'ftC. bcnm(u)pyrcnc (BI') and **2-llfll~lhn=lc** ('2.-AA) \.lllnlem!t:dliiN lllUII&CIIk: 10 TAlGO. A-)'1 Wlll't cooduelrd .s
**desai~** by

**~** .t.

**~** (Jh/DI Rul 13: 173, tnt · · ut &ownt.l!lm.!I!b.hfl l'tllt!1a"" l"t

**p~,!f**
**~·D.** J. 1 ijP{SJII)) '·1.11: 111 300 l•M

**{1~** PI) 111. J 11 702 Approved:f,:U;

**~',.,;:~.** 0&109/13 MOLr.ct!LAR TOXICOl..OClY, INC. lll 

# '" "" _.. 

# "' :im

RldhanJtJ str. 2 35394 Glellen

**~any.· ron:+-~9**  (0) 6+1·94390-0 fix: -+-48 (tl) 6of1 - 94391).22 !nfo@.binoVLdo "WWt.r.tinova.de 

# l

35Huntingdon Life Sciences: 

### Appendix 4 Certificate ofAnalysis

36

## iErn~aldPerformance.**~aterials"'"**

**~** 

## KalamaChemu:al' Huntingdon Life Sciences:

CE!mf1Cl\TE OF.<\NAI.VSlS

**~mtl~lcl** K:atmwa
**Cf~~mit::i~** U£

**~2"A1'h-m~-~~- ¥~3«Q.** WA:t'Z$25

**i~ ~~p:;r$-~**  l:mr.
**-ir:M)tn~<-'5M** PRODUCT: 

### Kalama®

**K-Fiex~** 

### 613 Plasticizer TllisIsto<orlifyttm!theproduaic!!lntifieclllelow

**hal-~** rep119en!alively

**~** onda""J:,!zodac:«>rddng lD ourqualty<mlrolprogrlllllll- Jt .-tBoo: ""'"'""" !fleopecilic.alioo&f<lrIt-flax613.

!\ppeenlnce,- Auay,% Mdity,% Color,.APHA EH11l1612A
seegAQATIQNS

**Clear~ndmoe of~**  99.0, Millimurn 6".3f MB:imwn 100,Malcimum

37 ,WALYSIS p.,.. 9!U 0.1 42