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## - ntUNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D

.C., 20460

VIA FACSIMILE: 201-437-4801 April 19,2012 Attention: Glenn Merritt (201-437-4000) OFFICE OF PREVENTION, PESTICIDESAND TOXIC SUBSTANCES

Subject: Follow-upto fax transmission of4/13/12 (ePMAremoval ofCBI claimsregarding identified studies) 

### DearMr. Merritt: This is sent to you pursuant ourconversation ofseveral minutes ago. In Larry Robinson's fax of April 13th while there is a waiver ofconfidential business information (CBI) claims in the identified studies there is a an assertion ofcopyrightprotections and some additional text that provides in part
... "Reproduction, reference, summarization, retransmission orotheruse ofthis copyrighted document.
..including electronic transmission, using the internet oremail
....is strictlyprohibited
...." While we understand the basis for the copyrightprotections, we have a concernthat the language in the fax might be interpreted to mean that ePMA is asserting that the studies submitted perthe TSCA activity are not tobemade available in an Agencye-docket (electronic docket) orbe included in EPA websites among the collection ofTSCAhealth and safety studies. We are thinking that the identified limitations noted in the fax are not intended to apply to EPA's normal procedures for the handling ofnon-CBI materials submitted by entities subject to TSCA regulations (including the posting ofsuch materials ongovernment websites) and submittedby such entities in orderto complywith a TSCA requirement, but are rather intended to limit entities outside the government from the unauthorized use or appropriation ofthe studies. Canyou please confirm CPMA's intent? Manythanks.

Scott M. Sherlock, Attorney Advisor Environmental Assistance Division Office ofPollution Prevention and Toxics

### DISCLAIMER Thisreportis theproperty ofClarinet Its emateat is Confidential and not firginwal release. ft bs not allowedtos"t andreveal therioen ort Wcotent to a Ghirdparty withent prier writs approval ofClarinet.j



## RCC Study Number 846498

r 

## P. B. 61, Vers. BE 1675: Local Lymph Node Assay (LLNA) in Mice (identification of Contact Allergens) Report


### Author: Dr. W. Wang-Fan Sponsor: Clariant GmbH Division PA, TS AZO Industriepark Hbchst, Geb. D787 D-65926 Frankfurt am Main/ Germany Study Completion Date: 28 January 2003

Total Number of Pages: 34ROC STUDY NUMBER 846498 P.B.61, Vers. BE 1675 -
TABLE OF CONTENTS 1 PREFACE ............................................................................... 4 1.1 GENERAL ......................................................................... 4 1.2 RESPONSIBILITIES .............................................................. 4 1.3 SCHEDULE........................................................................ 5 1.4 ARCHIVING ....................................................................... 5 1.5 SIGNATURES..................................................................... 6 1.6 QUALITY ASSURANCE UNIT ................................................... 7 1.7 STATEMENT OF COMPLIANCE................................................ 8 1.8 TEST GUIDELINES............................................................... 9 1.9 ANIMALWELFARE............................................................... 9 1.10 REFERENCES................................................................... 2 SUMMARY.............................................................................. 10 3 CONCLUSION.......................................................................... 11 4 OBJECTIVE ............................................................................ 12 4.1 PURPOSE........................................................................ 12 5 MATERIALS AND METHODS......................................................... 12 5.1 TEST SYSTEM................................................................... 12 5.2 ALLOCATION.................................................................... 13 5.3 HUSBANDRY .................................................................... 13 5.4 CHEMICALS...................................................................... 14 5.5 TEST ITEM ....................................................................... 14 5.6 TEST ITEM PREPARATION.................................................... 14 5.7 RATIONALE...................................................................... 15 6 STUDY CONDUCT..................................................................... 16 6.1 TREATMENT PROCEDURES .................................................. 16 6.1.1 TOPICAL APPLICATION................................................ 16 6.1.2 ADMINISTRATION OF 3H-METHYL THYMIDINE ................... 16 6.1.3 DETERMINATION OF INCORPORATED 3HTDR................... 16 6.1.4 INTERPRETATION OF RAW DATA.................................... 17 6.2 OBSERVATIONS ................................................................ 17 6.3 STATISTICAL ANALYSIS....................................................... 17 6.4 DATA COMPILATION ........................................................... 17 7 RESULTS .............................................................................. 18 7.1 CALCULATION AND RESULTS OF INDIVIDUAL DATA ................... 18 7.2 VIABILITY / MORTALITY ....................................................... 18 7.3 CLINICAL SIGNS ................................................................ 18 7.4 BODY WEIGHTS *....................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18ROC STUDY NUMBER 846498 .B.61,Vrs. BE 1675
APPENDIX A Calculation and Results of Individual Data........................................... 20 APPENDIX B Individual/ Summary Clinical Signs................................................... 22 APPENDIX C Individual / Summary Body Weights.................................................. 22 APPENDIX D Results of Positive Control............................................................. 26 APPENDIX E GOOD LABORATORY PRACTICE Statement of Compliance (Principal Investigator) ................................... 31 Quality Assurance Unit (Principal Investigator) ...................................... 32 APPENDIX F GLP
- Certification..................................................................... 34 LAST PAGE OF REPORT.................................................................. 34RCC STUDY NUMBER 846498 P.B. 61, Vers.-BE 1675


### I 

## PREFACE 1.1 GENERAL Title P.B.61, Vers. BE 1675: local lymph -Node Assay (LLNA) inMice (identification of Contact Allergens) Sponsor Clariant GmbH Division PA, TS AZO Industriepark Hbchst, Geb. D787 D-65926 Frankfurt am Main /Germany Study Monitor Dr. Reinhard Jung Test Facility RCC Ltd a)Toxicology Division CH

- 4452 Itingen /Switzerland Test Site b)Environmental Chemistry &Pharmanalytics Division CH

- 4452 Itingen /Switzerland 

### 1.2 RESPONSIBILITIES Study Director Dr. W.Wang-Fan (a) Deputy Study Director L.G. Ullmann (a) Technical Coordinator /Necropsy N.Schaifer (a) Principal Investigator Dr. R.Burni (b) 3HTdR Determination Head of RCC Quality Assurance 1. W~ithrichROC STUDY NUMBER 846498 P.,B.61, Vem-BE-1675-- 

### 1.3 SCHEDULE Experimental Starting Date 06-NOV-2002 Experimental Completion Date 20-NOV-2002 Delivery of Animals 06-NOV-2002 Acclimatization *06-NO V-2002 to 12-NOV-2002 Treatment (epicutaneous) 13-NOV-2002 to 1 5-NOV-2002 Treatment (intravenous) 18-NOV-2002 Observation **06-NO V-2002 to 18-NOV-2002 Termination 20-NOV-2002 Report 28-JAN-2003

*Non-GLP and therefore was excluded from the Statement of Compliance.

The observation during acclimatization period was Non-GLP and therefore excluded from the Statement of Compliance. 

### 1.4 ARCHIVING ROC Ltd (CH-4452 Itingen / Switzerland) will retain the study plan, raw data, sample of test item(s) and the final report of the present study for at least ten years. No data will be discarded without the Sponsor's consent.ROC STUDY NUMBER 846498 P.B.3 61,-Vers. BE 1675 

### 1.5 SIGNATURES Study Director: Dr. W. Wang-Fan

date: 

## .2C'zc3 Management: S. Corney

date: -ROC STUDY NUMBER 846498 P. B.61,- Vers-.-BE 1675----- 

### 1.6 QUALITY ASSURANCE UNIT RCC Ltd,Toxicology Division,CH-4452 Itingen ISwitzerland STATEM ENT RCC Study Number 846498 Test Item P.B.61, Vers. BE 1675 Study Director Dr. W.Wang-Fan Title P.B.61, Vers. BE 1675: local L~ymph N~ode Assay(LLNA) inMice (Identification of Contact Allergens)
The general facilities and activities are inspected periodically and the results are reported to the responsible person andthe management. Study procedures were periodically inspected. The study plan and this report were audited by the RCC Quality Assurance Unit. The dates are given below: Dates and Types of QAU Inspections Dates of Reports to the Study Director and to Management 11-NOV-2002 Study Plan 11-NOV-2002 09-DEC-2002 Process Based 09-DEC-2002 21-JAN-2003 Draft Report 21-JAN-2003 27-JAN-2003 Report 27-JAN-2003
This statement also confirms that this final report reflects the raw data. In addition this final report includes a QAU-Statement issued by the Test Site Quality Assurance Unit. QualityAssurance: S.Wyttenbach

d

ae: 3c, 'Z-7C.)RCC STUDY NUMBER 546498 P.-8.61, Vers. -BE1675 GOOD LABORATORY PRACTICE 

### 1.7 STATEMENT OF COMPLIANCE RCC Study Number 846498 Test Item P.B.61, Vers. BE 1675 Study Director Dr. W.Wang-Fan Title P.B.61, Vers. BE 1675: localLymph NodeAssay (LLNA)inMice (Identification of Contact Allergens) Anon-GLP pretest was performed and isexcluded from this Statement of Compliance. Stability of test item formulation was excluded from the Statement of Compliance. The data concerning Delivery of Animals, Age, Body weights, Mortality / Viability and Clinical signs (local / systemic) during acclimatization period were excluded from this Statement of Compliance. This study has been performed incompliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance isbased on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26 rn 1997 by decision of the OECD Council tC(97) 186/Final]. Study Director: Dr. W.Wang-Fan

date: " uiCW (49ROC STUDY NUMBER 846498 P.B- 61, Vers.-BE 1675 

### 1.8 TEST GUIDELINES The study procedures described inthis report are based on thefollowing guidelines: OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitizationo adopted bythe Council on July 17, 1992 (reported Paris, April 29, 1993). OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitization: Local Lymph NodeAssay (adopted 24 June 2002). 

### 1.9 ANIMAL WELFARE This study was performed inan AAALAC-approved laboratory inaccordance with the Swiss Animal Protection Law under license no. 114. 

### 1.10 REFERENCES Kimber I., Hilton J.and Weisenberger C. (1989). The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis, 21, 215-220. Kimber 1.and Basketter D.A. (1992). The murine local lymph node assay. Acommentary on collaborative studies and newdirections. Food and Chemical Toxicology, , 165-169. Basketter D.A., Gerbrick G.F., Kimber 1.and Loveless S.E. (1996). The local lymph node assay: aviable alternative to currently accepted skin sensitization tests. Food and Chemical Toxicology, 34, 985-997. Chamberlain M.and Basketter D.A. (1996). The local lymph node assay: status ofvalidation. Food and Chemical Toxicology,34, 999-1002. Basketter D.A., Lea L.J., Cooper K., Stocks J., Dickens A., Pate L., Dearman R.J. and Kimber 1.(1999). Threshold for Classification as a Skin Sensitizer inthe Local Lymph Node Assay: AStatistical Evaluation. Food and Chemical Toxicology, 37,1-8. Steiling W., Basketter D.A., Berthold K., Butler M., Garrigue J-L., Kimber I., Lea L.J., Newsome C., Roggeband R., Stropp G., Waterman S. and Wiemann C. (2001): Skin Sensitisation Testing

- New Perspectives and Recommendations. Food and Chemical Toxicology, 39, 293-301.RCC STUDY NUMBER 846498 

## 2 SUMMARY Inorder to study apossible allergenic potential of P.B.61, Vers. BE 1675 three groups of four female mice were each treated with the test item at concentrations of 5%, 10 %and 25 %(wlw) inacetone:olive oil, 4:1 (v/v) bytopical application to the dorsum of each ear lobe (left and right) on three consecutive days. Acontrol group of four mice was treatedwith the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine ( 3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed,the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ~ scintillation counter. No test item-related clinical signs were observed. Alltreated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted inan incorporation of 3HTdR at least 3-fold or greater than that recorded incontrol mice, as indicated bythe STIMULATION INDEX (S.1.).
Test Item concentration S.I. Grou 2 1.3 Group3 101.8 Group4 251.2 No dose-response relation was observed. An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.I. value of 3.



## 10RCC STUDY NUMBER 846498 P.B.61,Vers.-BE 1675 

## 3 CONCLUSION Inthis study STIMULATION INDICES of 1.3, 1.8 and 1.2 were determined with the test item at concentrations of 5%,10 %and 25 %(w/w) inacetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted inan incorporation of 3 HTdR at least 3-fold or greater than that recorded incontrol mice, as indicated by the STIMULATION INDEX. Based on these criteria, the test item P. B.61, Vers. BE 1675 was found to be a nonsensitizerwhen tested up to 25 %(w/w) inacetone:olive oil, 4:1 (v/v).ROC STUDY NUMBER 846498 P.B. 61, Ver'sl BE 1675 

### 4 OBJECTIVE 4.1 PURPOSE The purpose of this Local Lymph Node Assay was to identify the contact allergenic potential of P.B.61, Vers. BE 1675when administered tothe dorsumn of both ear lobes of mice. This study should provide arational basis for risk assessment to the sensitizing potential of the test item inman. 

### 5 MATERIALS AND METHODS 5.1 TEST SYSTEM Test system Mice, CBA/CaOlaHsd Rationale Recognized as the recommended test system. Source Harlan Netherlands B.V. Postbus 6174 NL

- 5960 AD Horst/ The Netherlands Number of animals for 2females the pre-test (non-GLP) Number of animals for 16 females (reserve animals were used as needed and the main study listed inthe raw data and the report) Numberof animals per group 4females (nuiliparous and non-pregnant) Number of test groups 3 Number of control (vehicle) group 1 Age 8-12 weeks (beginning of acclimatization) Body weight (beginning of acclimatization) * * Non-GLP and therefore was excluded from Statement of Compliance. Identification Each cage by unique cage card, inevery cage each animal by individual code marked at tail with a permanent pen. Randomization Randomly selected by computer algorithm at time of delivery. Acclimatization Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.



### 12RCC STUDY NUMBER 846498 P.B.61, Vers.BE 1675 

### 5.2 ALLOCATION The animals were distributed as follows:

Groups CONCENTRATION") NUMBER OFANIMALS CAGE NUMBER %(wiw) PER GROUP (Group housing) 1(Control Groupa)
- 4 1 2 5 4 2 3 10 4 3 4 25 4 4 Svehicle group: acetone:olive oil, 4:1 (v/v) b)in a non-GLP conform pre-test intwo mice, test item concentrations of 1%,2.5 %,5% and 10 % (wlw) were tested on one ear each. No irritation effects were observed at the concentrations applied. The sensitivity and reliability of the experimental technique employed was assessed by use of asubstance which isknown to have skin sensitization properties inmice, CBA/CaOlaHsd. The validation- / positive control study was performed with ALPHA-HEXYLCINNAMALDEHYDE (RCC Study Number 844357) between 12-JUN-2002 and 26-JUN-2002, see Appendix C.


### 5.3 HUSBANDRY Room no. E21 / ROC Itingen Conditions Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 + 3 0C,relative humidity 30

- 70 %and 10

- 15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at ROC. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8hours music during the light period. Accommodation In groups of four in Makrolon type-3 cages with standard softwood bedding ( T Lignocel", Schill AG, CH-4132 Muttenz). Diet Pelleted standard Kliba 3433, batch no. 67/02 mouse maintenance diet (Provimi Klba AG, CH-4303 Kaiseraugst) available adlibitum. Results of analyses for contaminants are archived at RCC. Water Community tap water from Itingen, available adlibitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.



### 13ROC STUDY NUMBER 846498 P.B. 61,Vers. BE 1,675 

### 5.4 CHEMICALS 3H-methyll Thymidine Amersham TRA 310, batch no. 304 , aqueous solution, sterilized 74 GBq/mmol (2 Ci/mmol), 37 MBq/ml (1 mCi/mi) quantities: 9.25 MBq (250 ~ICi), 37 MBq (1 mCi) Trichioroacetic acid Baker no. 0344 (min. 99.5 %) Phosphate buffered saline Fluka no. 79382 (1tablet solved in200 ml bi-distilled water) 

### 5.5 TEST ITEM Identity P.B.61, Vers. BE 1675 Description dark blue powder Batch number Versuch Be 1675 Purity 100% Stability of test item Stable under storage conditions Expiry date 20-SEP-2007 Stabilityof test item formulation Excluded from the Statement of Compliance. Storage conditions In the original container at room temperature (20 0 C±3OC), away from direct sunlight. Safety precautions Routine hygienic procedures (gloves, goggles, face mask) The test item information was supplied bythe Sponsor. 

### 5.6 TEST ITEM PREPARATION The test item was placed into a glass beaker on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was added. The weight/weight dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item inthe vehicle was maintained during treatment with the magnetic stirrer. The preparations were made shortly before each dosing occasion. To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in2mice with concentrations of 1%,2.5 %,5%and 10 % (w/w) (pretest excluded from Statement of Compliance). The test item inthe main study was assayed at three consecutive concentrations that were selected by the Sponsor. Concentrations were in terms of material as supplied unless otherwise stated by the Sponsor.



### 14ROC STUDY NUMBER 846498 P.B. 61, Vers. BE 1675 

### 5.7 RATIONALE The study procedure was used to detect a possible contact allergenic potential of the test item applied.



### 15ROC STUDY NUMBER 8-46498 P.-B.-6-1-,-Vers. BE 1675 

### 6 STUDY CONDUCT 6.1 TREATMENT PROCEDURES 6.1.1 TOPICAL APPLICATION Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %,10 %and 25 % (wlw) inacetone:olive oil, 4:1 (vlv). The application volume, 25 p1, was spread over the entire dorsal surface (0

- 8mm) of each ear lobe once daily for three consecutive days. Afurther group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Ahair dryer was used to dry the ears surface as quickly as possible to avoid loss of test item applied. 6.1.2 ADMINISTRATION OF 3H-METHYL THYMIDINE 3

H-methyl thymidine ( 3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2Ci/mmol; concentration, 1mCi/mI). Five days after the first topical application, all mice were administered with 250 p1 of 78.3 PCi/mI 3HTdR (equal to 19.6 pCi 3HTdR) by intravenous injection via atail vein. 6.1.3 DETERMINATION OF INCORPORATED 3HTDR* Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zcjrich). The draining lymph nodes were rapidly excised and pooled for each experimental group (8nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3ml) and incubated at approximately +4OC for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in5%trichloroacetic acid (1ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a f-scintillation counter. Similarly, background 3HTdR levels were also measured intwo 1 mI-aliquots of 5%trichloroacetic acid. The P-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Measurements at RCC Environmental Chemnistry &Pharmanalytics Division


### 16AGO STUDY NUMBER 846498 P.B. 61, Vers. BE 1675 -- 6.1.4 INTERPRETATION OF RAW DATA The proliferative response of lymph node cells isexpressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM1NQDE values were determined, mean scintillation-background DPM was subtracted from test and control raw data. Atest item isregarded as asensitizer inthe LLNA ifthe following criteria are fulfilled:

- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded incontrol mice, as indicated bythe STIMULATION INDEX.

- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. The decision to select astimulation index of 3as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals. 

### 6.2 OBSERVATIONS Inaddition to the sensitizing reactions the following observations and data were recorded during thetest and observation period: Mortality/Viability Twice dailyfrom acclimatization start to the termination of in-life phase.* Body weights prior to necropsy. Clinical signs (local/systemic) Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.* The procedures during acclimatization period were Non-GLP and therefore excluded from the Statement of Compliance. 

### 6.3 STATISTICAL ANALYSIS The mean values and standard deviations were calculated inthe body weight tables. 

### 6.4 DATA COMPILATION Body weights were recorded on-line.



### 17RCC STUDY NUMBER 846498 P.B.61,-Vers. BE 1675 

### 7 RESULTS 7.1 CALCULATION AND RESULTS OF INDIVIDUAL DATA The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a P-scintiilation counter. A test item is regarded as a sensitizer ifthe exposure to at least one concentration resulted inan incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX. Test item concentration SI %(wlw) Group2 5 1.3 Group3 10 1.8 Group 4 25 1.2 No dose-response relation was observed. An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.1. value of 3. See p.20 

### 7.2 VIABILITY/I MORTALITY No deaths occurred during the study period. 

### 7.3 CLINICAL SIGNS No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 

### 7.4 BODY WEIGHTS * The body weight of the animals, recorded prior to necropsy, was within the range commonly recorded for animals of this strain and age. See pp. 22-24



### 18ROC STUDY NUMBER 846498 P. B. 6l7,Vers. BE 1675



### APPENDIX A CALCULATION AND RESULTS OF INDIVIDUAL DATA

19RCC STUDY NUMBER 846498 P.B.61, Vers.-BE 1675 

### CALCULATION AND RESULTS OF INDIVIDUAL DATA

The following results were obtained:
Vehicle: acetone:olive oil, 4:1 (v/v)
Test item Measurement Calculation Result concentration dpm dpm
- number of dpm per 

### S.I. % (w/w)

______ ___ BGa) lymph nodes lymph nodeb)

- BG 1 3 --- -

-- BG 11 0 -- -- - -

- CG1 4127 4125 8 516

- 5 TG2 5537 5535 8 692 1.3 10 TG3 1 7611 1 7609 8 951 1.8s 25 TG4 5019 5017 8 6271.
BG = Background (1ml 5%trichloroacetic acid) induplicate CG = Control Group TG = Test Group S.l. = Stimulation Index a) =The mean valuewas taken from the figures BG Iand BG 11

b) Since the lymph nodes of the animals of adose group were pooled, DPMINode was determined by dividing the measured value bythe number of lymph nodes pooled



### 20ROC STUDY NUMBER 846498 P.B.61-, Vers. BE 1675



## APPENDIX B INDIVIDUAL ISUMMARY BODY WEIGHTS



## 21RCC STUDY NUMB~ER 846498 EW-InD - 1 P.B.61, VERB-. BE 1675 12-DEC-02
BODY WEIGHTS (GRAM) FEMALES

ACCLIMATISATXON TREATMENT DAYS1 WEEKS11 ANIMAL

GROUP I (CONTROL GROUP) 1 --- 20.3 2

-- 20.0 3 --- 21.6 4 --- 22.9 GROUP 2 (TEST GROUP 5%) 5 21.0 6 21.0 7
-- 20.1
-- 20.5 GROUP 3 (TEST GROUP 10%) 9 1S. 10 --- 19.:3 11 --- 23.5 12 --- 19.8 GROUP 4 (TEST GROUP 25%)

13 --- 20.1 14 -- 19.3 is-- 19.7 16 --- 19.9

roRCC STUDY NUMBER 846498 BW- SU14 - 1 P.B.61, VERS. BE 1675 12 -DEC- 02
BODY WEIGHTS (GRAM) SUMMARY FEMALES

ACCLZMATZSATION GROUP 1 GROUP 2 GROUP 3 GROUP 4 CONTROL GROUP TEST GROUP 5% TEST GROUP 10% TEST GROUP 25%

DAY I MEA - - - --- - -- WEEK I ST.DEV. --- --- ----- K 0 0 0 0RCC STUDY NUMBER 846498 EW-STIM - 2 P.B.61, VERS. BE 1675 12 -DEC-02 BODY WEIGHTS (GRAM) SUM1MARY FEMALES

TREATMENT GROUP 1 GROUP 2 GROUP 3 GROUP 4 CONTROL GROUP TEST GROUP 5% TEST GROUP 10% TEST GROUP 25%
DAY 6 MEAN 21.2 20.7 20.3 19.8 WEEK 1 ST.0EV. 1.3 0.4 2.1 0.4 N 4 4 4 4



## 244k

RCC STUDY NUMBER 846498 P.B.61, Vers. BE 1675

- -



### APPENDIX C RESULTS OF POSITIVE CONTROL



## 25RCC STUDY NUMBER 846498 P.-B. 6l7,Vers. BE 1675



## RCC Study Number 844357

ALPHA-HEXYLCI NNAMALDEHYDE: Local Lymph Node Assay (LLNA) in Mice (identification of Contact Allergens) 

# Validation- / Positive Control Study

276RCC STUDY NUMBER 846498 P.B. 617Vers. BE 1675 

## SUMMARY Inorder to study apossible allergenic potential of ALPHA-HEXYLCINNAMALDEHYDE three groups of four female mice each were treated with the test item at concentrations of 5 %, 10 % and 25 % (w/v) inacetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. Acontrol group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (vlv)) only. Five days after the first topical application the mice were injected intravenously into atail vein with radio-labelled thymidine (H-methyll thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichioroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured inaf3-scintillation counter. No test item-related clinicalsigns were observed. Alltreated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted inan incorporation of 3HTdR at least 3-fold or greater than that recorded incontrol mice, as indicated by the STIMULATION INDEX (S.1.). Test Item concentration S.l. % (Wlv)

_____ Group 2 5 2.9 Group3 10* 2.6* Group 4 _25* -17.1 . EC3= 11.3%(wtv) Thevaluewas used incalculation of EC3. 

## CONCLUSION Inthis study STIMULATION INDICES of 2.9, 2.6 and 7.1 were determined with the test item at concentrations of 5 %,10 %and 25 %(w/v) inacetone:olivebil, 4:1 (vlv). A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted inan incorporation of 3HTdR at least 3-old or greater than that recorded incontrol mice, as indicated bythe STIMULATION INDEX. Based on these criteria, the test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a non-sensitizer when tested up to 10 %(w/v) inacetone:olive oil, 4:1 (v/v). ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at concentration of 25 %(wlv). EC3 isthe estimated concentration for a STIMULATION INDEX of 3. Inthis study EC3 of 11.3 %(w/v) was theoretically calculated with STIMULATION INDICES of 2.6 and 7.1 at test item concentrations of 10 %and 25 %(w/v).



## 27ROC STUDY NUMBER 846498 P.B.61-, Vers. BE 1675 

### RESULTS CALCULATION AND RESULTS OF INDIVIDUAL DATA The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a P-scintillation counter. A test item is regarded as a sensitizer ifthe exposure to at least one concentration resulted inan incorporation of 3HTdR at least 3-old or greater than that recorded in control mice, as indicated by the STIMULATION INDEX. Test item concentration S.I. % (wlv) Group 2 5 2.9 Group3 10* 2.6* Group4 

### 125' 17.1* EC3= 11.3%(wlv)

*The value was used in calculation of EC3. 

### VIABILITY / MORTALITY No deaths occurred during the study period. 

### CLINICAL SIGNS No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 

### BODY WEIGHTS The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.ROC STUDY NUMBER 846498 P.B. -61,PVers. BE 1675 -- 

### CALCULATION AND RESULTS OF INDIVIDUAL DATA
The following results were obtained: Vehicle: acetone:olive oil, 4:1 (v/v)
Test item Measurement Calculation Result concentration dpm') dpm -BG number of dpmn per b) S.I. % (w/v) _______lymph nodes lymph nodeb

- BGI1 3

- --

-BG 11 3 -- -- -- -

- CG1 4232 4229 8 529

-- 5 TG 2 12169 12166 8 1521 2.9 10 TG3 10977 10974 8 1372 2.6 25 TG 4 29856 29853 8 3732 7.1 BG = Background (1ml 5 %trichloroacetic acid) in duplicate CG = Control Group TG = Test Group S.I. = Stimulation Index a) - The mean value was taken from the figures BG Iand BG 11 b) =- Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was determined by dividing the measured value by the number of lymph nodes pooled Test item concentration S.l. %(w/v)

_____ Group2 5 2.9 Group3 10(a) 2.6(b) Group4 25(c) 17.1 (d) I EC3 = (a-c) [(3-d)I(b-d)] + c = 11.3 %(wI',)I EC3 = Estimated concentration for a STIMULATION INDEX of 3. a,b,c,d= Co-ordinates of the two pair of data lying immediately above and below the S.l. value of 3on the LLNA dose response plot.



## -29RCC STUDY NUMBER 846498 P. B.,61,Vers. BE 1675



### APPENDIX D

GOOD LABORATORY PRACTICE

- 

### STATEMENT OF COMPLIANCE (PRINCIPAL INVESTIGATOR)

- 

### QUALITY ASSURANCE UNIT (PRINCIPAL INVESTIGATOR)RCC STUDY NUMBER 846089 P.B.61, Vers. BE 1675
GOOD LABORATORY PRACTICE 

## STATEMENT OF COMPLIANCE

RCC Study Number: 846089 Study Director: Dr. W.Wang-Fan, Toxicology Division Test Item: P.B.61, Vers. BE 1675 Principal Investigator 3HTd1R Determination: Dr. R.Burni, Environmental Chemistry & Pharmanalytics Division Phase to: P.B.61, Vers. BE 1675: Local Lymph Node Assay (LLNA) in Mice (identification ofContact Allergens)

The preparation of the [methyl-3H]Thymidine solution and determination of radioactivity content were conducted incompliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance isbased on the OECD Principles ofGood Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision ofthe OECD Council [C(97)186/Finall.

Principal Investigator 3HTdR Determination: Dr. R.Burni

Date:RCC STUDY NUMBER 846089 P.B.61, Vers. BE 1675 

## QUALITY ASSURANCE UNIT

RCC Ltd, Environmental Chemistry &Pharmanalytics Division, CH-4452 Itingen/Switzerland STATEME NT RCC Study Number: 846089 Study Director: Dr. W.Wang-Fan, Toxicology Division Test Item: P.B.61, Vers. BE 1675 Principal Investigator 3HTdR Determination: Dr. R.Burn, Environmental Chemistry & Pharmanalytics; Division Phaseto: P.B.61, Vers. BE 1675: Local Lymph Node Assay (LLNA) in Mice (Identification of Contact Allergens) The general facilities and activities are inspected periodically and the results are reported to the responsible person and the management. Study procedures were periodically inspected. The date isgiven below.

Dates and Types of QAU Inspections Dates of Reports tothe Principal Investigatorand to the Management November 08, 2002 Process based November 08, 2002
QualityAssurance: Mr. J.L-Otte



### Date: ~ ~ 

## r2zROC STUDY NUMBER 846498 P. B.61, Vers. BE 1675



### APPENDIX E GLP

- 

### CERTIFICATIONRCC STUDY NUMBER 846498 P. B.61, Vers. BE 1675
The Swiss GLP Monitoring Authorities (%~ Swss Fedral ASwiss Agency for the , Intercantonial Ofie Offi9 of" Environment Forests /A iforthe Contro of PublicHealth A and Landscape \ VMedicines 

# Statement of GLP Compliance

Itis hereby conifirmed that during the period of August 15- 17, 2000 August 28-29,.2001 and April 15, 2002 the following Test Facilities of RCC Ltd 4452 Itingen Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for the Environment, Forests and Landscape and the Intercantonal Office for the Control of Medicines with respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities areas ofexpertise*

-Toxicology Division TOX, ACC, MUT

- Environmental Chemistry and ACC, ECT, ENF, EMN,

Pharmanalytics Division PCT,RES,OTH (Animal metabolism)

- Microbiological Diagnostics by

Biotechnology &Animal Breeding Division 0TH (Microbiology)
The inspection was performed in agreement with the OECD Guidelines for National GLP Inspections and Audits. Itwas found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time theywere inspected. Federal Office of Public Health The Director

Bern, May 2002 Prof. Th. Zeltner

*TOX = Toxicciogy:ACC aAnalytical and Clinical Chemistry: ECT = Envirorsnental toxicityon aquatic and terrestrial organisms ;ENF =Behaviour inwater, *oil and air. Etloeccumulatiori; EMN - Studieson effects on mosocosms.and natural ecosystems; MUT =Muteaenicijy; PCT =Physical-cheical testing ; RES = Residue studies ;0Th - Other, to be specfed.