Huntingdon 

Life Sciences 

Working for a bett(~r future 

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 

Page 1 of 29 
Huntingdon Life Sciences· 

Sponsor and Test Facility Details 

Sponsor 

Test facility 

Emerald Performance Materials 

LLC 

2020 Front Street 

Suite 100 

Cuyahoga Falls 

OH 44221 

USA 

Huntingdon Life Sciences 

Huntingdon Research Centre 

Woolley Road 

Alcon bury 

Huntingdon 

Cambridgeshire 

PE284HS 

UK 

2 
Huntingdon Life Sciences: 

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 of the locally mph 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 of treatment 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 of test 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 

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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 of Tables 

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 of ear thickness ..................................... 24 

Appendix 3 Preliminary and Main phases- Individual and group mean body weights and 

individual body weight change (g) ................................................................ 25 

List of Annexes 

Annex 1 

Annex2 

Certificate of analysis ................................................................................... 26 

GLP Compliance Statement... ...................................................................... 28 

4 
Huntingdon Life Sciences: 

Compliance with Good Laboratory Practice 

3-Phenylpropyl benzoate: Assessment of Skin Sensitization Potential 

using the Local Lymph Node Assay in the Mouse (Pooled treatment 

group approach) 

5 
Huntingdon Life Sciences: 

Quality Assurance Statement 

3-Phenylpropyl benzoate: Assessment of Skin 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: 

Study Phase 

Protocol Audit 

Protocol Amendment No. 1 

Report Audit 

Date oflnspection 

11 Dec 2013 

03 Jan 2014 

05 Feb 2014 

Date of Reporting to Study 

Director and Management 

11 Dec 2013 

03 Jan 2014 

05 Feb 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 Based Inspections 

Clinical Signs 

Preparation and Administration 

ofRadiolabel 

Terminal Procedures 

Bodyweights 

Study Management 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 of Reporting to 

Management 

05Nov 2013 

18Nov2013 

18Nov 2013 

21 Nov2013 

17 Dec 2013 

31 Dec2013 

15 Jan 2014 

17 Jan 2014 

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

annual basis. These inspections were promptly reported to Company Management. 

Dilys Smith MRQA 

Lead Auditor 

Department of Quality Assurance 

Huntingdon Life Sciences 

Date 

6 
Huntingdon Life Sciences: .' 

Contributing Scientists 

3-Phenylpropyl benzoate: Assessment of Skin Sensitization Potential 

using the Local Lymph Node Assay in the Mouse (Pooled treatment 

group approach) 

Study management 

David G Co Ieman B Sc (Hons) 

Study Director 

Kerstin Pryke (BSc Hons) 

Trainee Study Manager 

7 
Huntingdon Life Sciences: 

Summary 

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Huntingdon Ufe Sciences: 

1. Introduction 

1.1 Objective 

The objective of this study was to assess the skin sensitization potential of 

3-Phenylpropyl benzoate. 

1.2 Principle of the local lymph node assay 

The theory behind the local lymph node assay (LLNA) is that a sensitizing substance induces 

proliferation oflymphocytes in the lymph nodes draining the site oftest substance exposure. 

The lymphocyte proliferation is proportional to the dose and potency of the sensitizing 

substance and is quantified by measuring the incorporation of radio labelled Thymidine. 

Assessment of sensitization potential is made by comparing the mean proliferation in each 

test group to the mean proliferation in the vehicle control group, with the ratio between 

treated and control groups termed the Stimulation Index (SI). A test substance is regarded as 

a sensitizer if the SI is 3 or more in any of the concentrations tested. 

1.3 Regulatory compliance 

The study was designed to meet the requirements of the following guidelines: 

OECD Guideline for Testing of Chemicals No. 429 "Skin Sensitization: Local 

Lymph Node Assay". 22 July 2010. 

EEC Methods for the determination of toxicity and other health effects. 

Commission Regulation No. 440/2008. Part B, Method B.42. Skin sensitisation: 

Local Lymph Node Assay. 30 May 2008, as amended in Commission Regulation 

No. 640/2012 dated 6 July 2012. 

1.4 Test system 

The female CBA/Ca mouse is chosen as the test species as it has been shown to be a suitable 

model for this study and is the species/strain/sex recommended by the test guidelines. 

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Huntingdon Life Sciences: 

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 of scintillation 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 fmal report 

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Huntingdon Life Sciences: 

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-Phenylpropyl benzoate 

X-613, K-Flex 613 

Kalama K-Flex 613 

Kalama K-Flex 613 Plasticizer 

Benzenepropano ~ !-benzoate 

!-Propanol, 3-phenyl-, benzoate 

3-Phenyl-1-propanol benzoate 

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 of the test substance and the 

documentation of the methods of synthesis, fabrication or derivation and stability. 

2.2.2 Vehicle selection 

2.2.3 Formulation 

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Huntingdon Life Sciences: 

12 
Huntingdon Life Sciences: 

13 
Huntingdon Life Sciences: · 

14 
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2.6.3 Measurement of ear swelling 

15 
Huntingdon Life Sciences: 

2.8 Data treatment 

2.9 Computer systems 

The computer systems that were used on this study to acquire and quantifY data include: 

System name* System function 

Pristima Pharmacy data management 

• All version numbers of the systems are maintained by Huntingdon Life Sciences 

2.10 Quality Assurance and archiving procedures 

2.1 0.1 Quality Assurance 

Details of the Quality Assurance inspections and audits are presented on the Quality 

Assurance Statement. 

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Huntingdon Life Sciences: 

• r ( J .I 

17 
Huntingdon Life Sciences: l I 

18 
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3.2.3 Body weight 

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Huntingdon Life Sciences: ' 

4. Conclusion 

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Huntingdon Life Sciences: ' 

5. References 

21 
Huntingdon Life Sciences: 

Table 1 Group dpm/node and Stimulation Index 

22 
Huntingdon Life Sciences: 

Appendix 1 Preliminary phase- dermal reactions (erythema) 

23 
Huntingdon Life Sciences: ' 

Appendix 2 Preliminary phase- Measurement of ear thickness 

24 
' ' . 

Huntingdon Life Sciences: 

Appendix 3 Preliminary and Main phases- Individual and group mean 

body weights and individual body weight change (g) 

25 
' " 

Huntingdon Life Sciences: 1 

Annex 1 Certificate of analysis 

26 
i Emerald Performance Materials'" 

~ Kalama Chemical 

Huntingdon Life Sciences: 

CERTIFICATE OF ANALYSIS 

Emerald Kalama Chemical, LLC 1296 Third Street North~-.es~ Kalama, WA 986"'..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 

42 
Huntingdon Life Sciences: 

Annex 2 GLP Compliance Statement 

28 
Huntingdon Life Sciences: ' 

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 

Alcon bury 

Huntingdon 

Cambridgeshire 

PE284HS 

TEST TYPE(S) 

AnalytlcaUCIInical Chemistry 

Environmental Fate 

Environmental Toxicity 

Ecosystems 

Toxicology 

Residue Studies 

Reissued February 2013 to include ·Residue 

Studies 

DATE OF INSPECTION 20 to 22 November 2012 

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 compliance with the OECD Principles of Good Laboratory Practice. 

This statement constitutes a GoOd Laboratory Practice Instrument (as defined in the 

UK Good Laboratory Practice Regulations 1999). 

~fl. { ,_[.~ 

Or. 

Head, 

Andrew 

UK GLP 

J. Gray 

MonHorlng Authority 

~ 

~ 

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