# Huntingdon Life 

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### REPORT 

### 3-Phenylpropyl benzoate: Assessment of Skin Sensitization

Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach)

HLS study number: Version ID: Issue date: 6 February 2014

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## Sponsor and Test Facility Details Sponsor

Testfacility Emerald Performance Materials LLC 2020 Front Street Suite 100 Cuyahoga Falls OH 44221 USA Huntingdon Life Sciences Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambridgeshire PE284HS 

### UK

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## Table of Contents Sponsor and Test Facility Details..........................................................................................2 Compliance with Good Laboratory Practice .......................................................................... 5 Quality Assurance Statement................................................................................................6 Contributing Scientists ..........................................................................................................7 Summary ..............................................................................................................................8 1. Introduction ..................................................................................................................9 1.1 Objective...........................................................................................................9 1.2 Principle ofthe locallymph node assay ............................................................ 9 1.3 Regulatory compliance ..................................................................................... 9 1.4 Test system ......................................................................................................9 2. Experimental Procedure ............................................................................................. 1 0 2.1 Study schedule and structure.......................................................................... 10 2.1.1 Time schedule..................................................................................... 10 2.1.2 Identity oftreatment groups................................................................. 1 0 2.2 Test substance and formulation ...................................................................... 11 2.2.1 Test substance.................................................................................... 11 2.2.2 Vehicle selection ................................................................................. 11 2.2.3 Formulation ......................................................................................... 11 2.3 Animal management....................................................................................... 12 2.3.1 Animal housing, diet and water supply ................................................ 12 2.4 Concentration selection .................................................................................. 13 2.4.1 Preliminary investigations.................................................................... 13 2.4.2 Main study........................................................................................... 13 2.5 Administration oftest substance ..................................................................... 14 2.5.1 Preliminary investigation ..................................................................... 14 2.5.2 Main phase ......................................................................................... 14 2.5.3 Administration of 3H-methyl Thymidine ............................................... 14 2.6 Observations .................................................................................................. 14 2.6.1 Clinical signs ....................................................................................... 14 2.6.2 Dermal reactions ................................................................................. 14 2.6.3 Measurement of ear swelling............................................................... 15 2.6.4 Body weight ........................................................................................ 15 2.7 Terminal studies ............................................................................................. 15 2.7.1 Termination ......................................................................................... 15 2.7.2 Preparation of single cell suspensions................................................. 15 2.7.3 Determination of incorporated 3H-methyl Thymidine ........................... 15 2.8 Data treatment................................................................................................ 16 2.8.1 Stimulation Index................................................................................. 16 2.8.2 Estimated Concentration ofThree....................................................... 16 2.9 Computer systems.......................................................................................... 16 2.10 Quality Assurance and archiving procedures .................................................. 16 2.10.1 Quality Assurance ............................................................................... 16 2.10.2 Archives .............................................................................................. 17 2.11 Deviations from protocol ................................................................................. 17 3. Results ....................................................................................................................... 18 3.1 Preliminary investigation ................................................................................. 18 3.1.1 Mortality and clinical signs................................................................... 18
3Huntingdon Life Sciences: L 3.1.2 Dermal reactions ................................................................................. 18 3.1.3 Measurement of ear thickness ............................................................ 18 3.1.4 Body weight ........................................................................................ 18 3.2 Main phase ..................................................................................................... 18 3.2.1 Mortality and clinical signs................................................................... 18 3.2.2 Dermal reactions ................................................................................. 18 3.2.3 Body weight ........................................................................................ 19 3.3 Stimulation Index and Estimated Concentration ofThree................................ 19 4. Conclusion .................................................................................................................20 5. References.................................................................................................................21 

## List ofTables Table 1 Group dpm/node and Stimulation Index.......................................................22 

## List of Appendices Appendix 1 Preliminary phase- dermal reactions (erythema) ........................................23 Appendix 2 Preliminary phase- Measurement ofear thickness .....................................24 Appendix 3 Preliminary and Main phases- Individual and group mean body weights and individual body weight change (g) ................................................................25 

## List ofAnnexes Annex 1 Annex2 Certificate ofanalysis ...................................................................................26 GLP Compliance Statement... ......................................................................28

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## Compliance with Good Laboratory Practice 3-Phenylpropyl benzoate: Assessment ofSkin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach)

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## QualityAssurance Statement 3-Phenylpropyl benzoate: Assessment ofSkin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach) The following inspections and audits have been carried out in relation to this study: StudyPhase
Protocol Audit Protocol Amendment No. 1 Report Audit Date oflnspection
11 Dec 2013 03 Jan 2014 05 Feb 2014 Date ofReporting to Study Director and Management 11 Dec 2013 03 Jan 2014 05 Feb 2014 Process based inspections: At or aboutthe time this study was in progress inspections of procedures employed on this type ofstudy were carried out. These were conducted and reported to appropriate Company Management as indicated below: Process Based Inspections
Clinical Signs Preparation and Administration ofRadiolabel TerminalProcedures Bodyweights StudyManagement and Conduct Assay Procedures Dose Administration Formulation Procedures Date(s) oflnspection
05 Nov 2013 18 Nov 2013 18 Nov 2013 21 Nov2013 17 Dec 2013 31 Dec 2013 14 Jan 2014 16 Jan 2014 Date ofReporting to Management 05Nov 2013 18Nov2013 18Nov 2013 21 Nov2013 17 Dec 2013 31 Dec2013 15 Jan 2014 17 Jan 2014 In addition, an inspection ofthe facility where this study was conducted was carried outon an annual basis. These inspections were promptly reported to Company Management.

Dilys SmithMRQA Lead Auditor Department ofQuality Assurance Huntingdon Life Sciences Date

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## Contributing Scientists 3-Phenylpropyl benzoate: Assessment ofSkin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach) Study management David G CoIeman BSc (Hons) Study Director Kerstin Pryke (BSc Hons) Trainee Study Manager

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## Summary

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## 1. Introduction 1.1 Objective The objective ofthis studywas to assess the skin sensitization potential of 3-Phenylpropylbenzoate. 

### 1.2 Principle ofthe local lymph node assay The theorybehindthe local lymph node assay (LLNA) is that a sensitizing substance induces proliferation oflymphocytes inthe lymph nodes draining the site oftestsubstance exposure. The lymphocyte proliferation is proportional to the dose and potency ofthe sensitizing substance and is quantified by measuring the incorporation ofradiolabelled Thymidine. Assessment ofsensitization potential is made by comparingthe mean proliferation in each test group to the mean proliferation in the vehicle control group, withthe ratio between treated and control groups termedthe StimulationIndex (SI). A test substance is regarded as a sensitizer ifthe SI is 3 ormore in any ofthe concentrations tested. 

### 1.3 Regulatory compliance The study was designed to meetthe requirements ofthe following guidelines: OECD Guideline for Testing ofChemicalsNo. 429 "Skin Sensitization: Local LymphNode Assay". 22 July 2010. EEC Methods forthe determination oftoxicity and otherhealth effects. Commission Regulation No. 440/2008. PartB, Method B.42. Skin sensitisation: LocalLymph Node Assay. 30 May 2008, as amended in Commission Regulation No. 640/2012 dated 6 July 2012. 

### 1.4 Test system The female CBA/Camouse is chosen as the test species as it has been shownto be a suitable model for this study and is the species/strain/sexrecommended by the test guidelines.

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## 2. Experimental Procedure 2.1 Study schedule and structure 2.1.1 Time schedule Study initiation: (protocol signed by Study Director) Experimental start date: (Initial animal allocation) Treatment commenced: Completion oflive phase: Experimental completion date: (date ofscintillation counting) Study completion: 

### 2.1.2 Identity of treatment groups 05 December 2013
05 December 2013 11 December 2013 20 January 2014 21 January 2014
When Study Director signs fmalreport

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### 2.2 Test substance and formulation 2.2.1 Test substance Identification: Alternative Identities:
Chemical name: Alternative Chemical names:

CAS registry number: Intended use: Appearance: Storage conditions: Supplier: Lot number: Expiry date: Purity: Sample receipt: 3-Phenylpropylbenzoate X-613, K-Flex 613 Kalama K-Flex 613 Kalama K-Flex 613 Plasticizer Benzenepropano

**~** !-benzoate !-Propanol, 3-phenyl-, benzoate 3-Phenyl-1-propanolbenzoate 3-PPB 60045-26-3 Plasticizer/coalescent Clear, colourless liquid Room temperature under nitrogen Sponsor EH101912A 19 October 2014 99.1% 22 October 2013 The Sponsor was responsible for the characterisation ofthetest substance andthe documentation ofthe methods ofsynthesis, fabrication or derivation and stability. 

### 2.2.2 Vehicle selection

2.2.3 Formulation

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### 2.6.3 Measurement of ear swelling

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### 2.8 Data treatment

2.9 Computer systems The computer systemsthatwere used on this study to acquire and quantifY datainclude:

System name* System function Pristima Pharmacy data management 

# • All version numbers ofthe systems are maintained by Huntingdon Life Sciences 

## 2.10 Quality Assurance and archiving procedures 2.10.1 Quality Assurance Details ofthe Quality Assurance inspections and audits are presented onthe Quality Assurance Statement.

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### 3.2.3 Body weight

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## 4. Conclusion

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## 5. References

21Huntingdon Life Sciences: Table 1 Group dpm/node and Stimulation Index

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### Appendix 1 Preliminary phase- dermal reactions (erythema)

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### Appendix 2 Preliminary phase- Measurement ofear thickness

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### Appendix 3 Preliminary and Main phases- Individual and group mean body weights and individual bodyweightchange (g)

25' " Huntingdon Life Sciences: 1 Annex 1 Certificate ofanalysis

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# i Emerald Performance Materials'"



# ~ Kalama Chemical Huntingdon Life Sciences:

CERTIFICATE OF ANALYSIS Emerald Kalama Chemical, LLC 1296Third Street

**North~-.es~** Kalama, WA986"'..5

Phone: (360) 673-2550 Fax: (360) 673-3564

PRODUCT: Kalama® K-Fiex® 613 Plasticizer
This is to certify that the product Identified below has been representatively sampled and analyzed according to our quality control programs. It meets or exceeds the specifications for

**K~Ftex** 613.
Lot Number: Date of Manufacture: CAS# 60045-26-3

Appearance, visual /v;say,% Acidity,% Color, APHA

Cherise Hytton Quallty Manager EH101912A October 2012

SPECIFICATIONS Clear and free of particulates 99.0, Minimum 0.3, Maximum 100, Maximum

Certificate computer generated thus not requiring a signature.

27 ANALYSIS Pass 99.1 0.1 42Huntingdon Life Sciences: Annex 2 GLP Compliance Statement

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### HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2012 

# i

# nH) Department



### ~ ofHea/th
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004191EC
TEST FACILITY Huntingdon Life Sciences Woolley Road Alconbury Huntingdon Cambridgeshire PE284HS TESTTYPE(S) AnalytlcaUCIInical Chemistry Environmental Fate Environmental Toxicity Ecosystems

Toxicology Residue Studies ReissuedFebruary 2013 to include·Residue Studies

DATE OF INSPECTION 20 to22 November2012
An inspection for compliance with the Prtnciples of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practice Compliance Monitortng Programme. · This statement confirms that, on the date of issue, the UK'Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliancewith the OECD Principles of Good Laboratory Practice. This statementconstitutes a GoOd Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999).



### ~fl. {



### ,_[.~ Or. Andrew J. Gray 

## ~ Head, UK GLP MonHorlng Authority 

## ~

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