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nt 
UNITED 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, PESTICIDES AND 

TOXIC SUBSTANCES 

Subject: Follow-up to fax transmission of 4/13/12 (ePMA removal ofCBI claims regarding 

identified studies) 

Dear Mr. Merritt: 

This is sent to you pursuant our conversation of several minutes ago. In Larry Robinson's fax of 

April 13th while there is a waiver of confidential business information (CBI) claims in the 

identified studies there is a an assertion of copyright protections and some additional text that 

provides in part ... "Reproduction, reference, summarization, retransmission or other use of this 

copyrighted document. .. including electronic transmission, using the internet or email .... is 

strictly prohibited .... " 

While we understand the basis for the copyright protections, we have a concern that the language 

in the fax might be interpreted to mean that ePMA is asserting that the studies submitted per the 

TSCA activity are not to be made available in an Agency e-docket (electronic docket) or be 

included in EPA websites among the collection of TSCA health 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 of non-CBI materials submitted by entities subject to TSCA 

regulations (including the posting of such materials on government websites) and submitted by 

such entities in order to comply with a TSCA requirement, but are rather intended to limit 

entities outside the government from the unauthorized use or appropriation of the studies. 

Can you please confirm CPMA's intent? Many thanks. 

Scott M. Sherlock, Attorney Advisor 

Environmental Assistance Division 

Office of Pollution Prevention and Toxics 


DISCLAIMER

This report is theproperty of Clarinet Its emateat is

Confidential and not fir ginwal release. ft bs not

allowed to s"t and reveal the rioen ort Wcotent to a

Ghird party withent prier writs approval of Clarinet.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: 34
ROC 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 ANIMAL WELFARE............................................................... 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 3 H-METHYL THYMIDINE ................... 16

6.1.3 DETERMINATION OF INCORPORATED 3 HTDR................... 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 VIABI LITY / MORTALITY ....................................................... 18

7.3 CLINICAL SIGNS ................................................................ 18

7.4 BODY WEIGHTS *....................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
ROC 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.................................................................. 34
RCC 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) in Mice

(identification of Contact Allergens)

Sponsor Cla riant G mbH

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~ithrich
ROC STUDY NUMBER 846498

P., B.61, Vem-BE-1675--

1.3 SCHEDULE

Experimental Starting Date 06-NOV-2002

Experimental Completion Date 20-NO V-2002

Delivery of Animals 06-NO V-2002

Acclimatization *06-NO V-2002 to 12-NO V-2002

Treatment (epicutaneous) 13-NOV-2002 to 1 5-NO V-2002

Treatment (intravenous) 18-NOV-2002

Observation **06-NO V-2002 to 18-NOV-2002

Termination 20-NO V-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) 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 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 -NO V-2002 Study Plan 11 -NO V-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.

Quality Assurance: S. Wyttenbach

d ae: 3c, 'Z-7C.)
RCC STUDY NUMBER 546498

P.- 8.61, Vers. -BE 1675

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:

local Lymph Node Assay (LLNA) in Mice

(Identification of Contact Allergens)

A non-GLP pretest was performed and is excluded 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 in compliance with the Swiss Ordinance relating to Good

Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance is based

on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted

November 26rn 1997 by decision of the OECD Council tC(97) 186/Final].

Study Director: Dr. W. Wang-Fan

date: uiCW " (49
ROC STUDY NUMBER 846498

P. B- 61, Vers.-BE 1675

1.8 TEST GUIDELINES

The study procedures described in this report are based on the following guidelines:

OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitizationo adopted by the

Council on July 17, 1992 (reported Paris, April 29, 1993).

OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitization:

Local Lymph Node Assay (adopted 24 June 2002).

1.9 ANIMAL WELFARE

This study was performed in an AAALAC-approved laboratory in accordance 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. A commentary on

collaborative studies and new directions. Food and Chemical Toxicology, , 165-169.

Basketter D.A., Gerbrick G.F., Kimber 1. and Loveless S.E. (1996). The local lymph node

assay: a viable 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 of validation.

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 in the Local Lymph Node

Assay: A Statistical 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

In order to study a possible 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) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe

(left and right) on three consecutive days. A control group of four mice was treated with 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 (

3 H-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 3 H-methyl thymidine measured in a ~

scintillation counter.

No test item-related clinical signs were observed.

All treated 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 in an incorporation of 3 HTdR at least 3-fold or greater

than that recorded in control mice, as indicated by the 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.

10
RCC STUDY NUMBER 846498

P. B. 61, Vers.- BE 1675

3 CONCLUSION

In this 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) in acetone: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 in an incorporation of 3 HTdR at least 3-fold or greater

than that recorded in control 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 nonsensitizer when tested up to 25 % (w/w) in acetone: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 1675 when administered to the dorsumn of both ear lobes of mice.

This study should provide a rational basis for risk assessment to the sensitizing potential of

the test item in man.

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 2 females

the pre-test (non-GLP)

Number of animals for 16 females (reserve animals were used as needed and

the main study listed in the raw data and the report)

Number of animals per group 4 females (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, in every 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.

12
RCC STUDY NUMBER 846498

P. B.61, Vers.BE 1675

5.2 ALLOCATION

The animals were distributed as follows:

Groups CONCENTRATION") NUMBER OF ANIMALS CAGE NUMBER

% (wiw) PER GROUP (Group housing)

1 (Control Grou pa) - 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 in two 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

a substance which is known to have skin sensitization properties in mice, 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 8 hours 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 ad libitum. Results of

analyses for contaminants are archived at RCC.

Water Community tap water from Itingen, available ad libitum.

Results of representative bacteriological, chemical and

contaminant analyses are archived at RCC.

13
ROC STUDY NUMBER 846498

P. B. 61 , Vers. BE 1,675

5.4 CHEMICALS

3 H-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 (1 tablet solved in 200 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

Stability of test item formulation Excluded from the Statement of Compliance.

Storage conditions In the original container at room temperature

(20 0C ± 3 OC), away from direct sunlight.

Safety precautions Routine hygienic procedures (gloves, goggles, face

mask)

The test item information was supplied by the 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 in the

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 in 2 mice with concentrations of 1 %, 2.5 %, 5 % and 10 %

(w/w) (pretest excluded from Statement of Compliance).

The test item in the 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.

14
ROC 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.

15
ROC 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) in acetone:olive oil, 4:1 (vlv). The application volume, 25 p1, was spread over the entire

dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further

group of mice was treated with an equivalent volume of the relevant vehicle alone (control

animals). A hair 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, 2 Ci/mmol; concentration, 1 mCi/mI).

Five days after the first topical application, all mice were administered with 250 p1 of

78.3 PCi/mI 3 HTdR (equal to 19.6 pCi 3 HTdR) by intravenous injection via a tail vein.

6.1.3 DETERMINATION OF INCORPORATED 3 HTDR*

Approximately five hours after treatment with 3 HTdR 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

(8 nodes 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. 3 ml) and

incubated at approximately +4 OC for at least 18 hours for precipitation of macromolecules.

The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to

glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.

The level of 3 HTdR incorporation was then measured on a f-scintillation counter. Similarly,

background 3 HTdR levels were also measured in two 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

16
AGO STUDY NUMBER 846498

P. B. 61, Vers. BE 1675 --

6.1.4 INTERPRETATION OF RAW DATA

The proliferative response of lymph node cells is expressed as the number of radioactive

disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3 HTdR

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.

A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:

- First, that exposure to at least one concentration of the test item resulted in an

incorporation of 3 HTdR at least 3-fold or greater than that recorded in control mice,

as indicated by the 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 a stimulation index of 3 as an arbitrary indication of sensitizing

activity was made on the basis of investigations performed with a wide range of

chemicals.

6.2 OBSERVATIONS

In addition to the sensitizing reactions the following observations and data were recorded

during the test and observation period:

Mortality / Viability Twice daily from 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 in the body weight tables.

6.4 DATA COMPILATION

Body weights were recorded on-line.

17
RCC 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

3 H-methyl thymidine measured on a P-scintiilation counter. A test item is regarded as a

sensitizer if the exposure to at least one concentration resulted in an incorporation of 3 HTdR

at least 3-fold or greater than that recorded in control mice, as indicated by the

STIMULATION INDEX.

Test item concentration SI

% (wlw)

Group 2 5 1.3

Group 3 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

18
ROC STUDY NUMBER 846498

P. B. 6l7,Vers. BE 1675

APPENDIX A

CALCULATION AND RESULTS OF INDIVIDUAL DATA

19
RCC 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) ______ ___ BG a) lymph nodes lymph node b)

- BG 1 3 --- -

-- BG 11 0 -- -- - -

- CG 1 4127 4125 8 516 -

5 TG 2 5537 5535 8 692 1.3

10 TG 3 1 7611 1 7609 8 951 1.8s

25 TG 4 5019 5017 8 6271.

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.l. = Stimulation Index

a) =The mean value was taken from the figures BG I and BG 11

b) Since the lymph nodes of the animals of a dose group were pooled, DPMINode was

determined by dividing the measured value by the number of lymph nodes pooled

20
ROC STUDY NUMBER 846498

P. B. 61-, Vers. BE 1675

APPENDIX B

INDIVIDUAL ISUMMARY BODY WEIGHTS

21
RCC 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

ro
RCC 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 0
RCC 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

24
4k

RCC STUDY NUMBER 846498

P. B. 61, Vers. BE 1675 - -

APPENDIX C

RESULTS OF POSITIVE CONTROL

25
RCC 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

276
RCC STUDY NUMBER 846498

P. B. 617 Vers. BE 1675

SUMMARY

In order to study a possible 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) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of

each ear lobe (left and right) on three consecutive days. A control 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 a tail 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 in a f3-scintillation counter.

No test item-related clinical signs were observed.

All treated 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 in an incorporation of 3 HTdR at least 3-fold or greater

than that recorded in control mice, as indicated by the STIMULATION INDEX (S.1.).

Test Item concentration S.l.

% (Wlv) _____

Group 2 5 2.9

Group 3 10* 2.6*

Group 4 _25* -17.1 .

EC3 = 11.3 %(wtv)

The value was used in calculation of EC3.

CONCLUSION

In this 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) in acetone: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 in an incorporation of 3HTdR at least 3-old or greater

than that recorded in control mice, as indicated by the 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) in acetone:olive oil, 4:1 (v/v).

ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at

concentration of 25 % (wlv).

EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this 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).

27
ROC ST UDY 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

3 H-methyl thymidine measured on a P-scintillation counter. A test item is regarded as a

sensitizer if the exposure to at least one concentration resulted in an incorporation of 3 HTdR

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

Group 3 10* 2.6 *

Group 4 125' 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,P 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 -BG number of dpmn per b) S.I.

% (w/v) _______lymph nodes lymph nodeb

- BGI1 3 - --

-BG 11 3 -- -- -- -

- CG 1 4232 4229 8 529 --

5 TG 2 12169 12166 8 1521 2.9

10 TG 3 10977 10974 8 1372 2.6

25 TG 4 29856 29853 8 3732 7.1

BG = Background (1 ml 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 I and 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) _____

Group 2 5 2.9

Group 3 10 (a) 2.6 (b)

Group 4 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 3 on the LLNA dose response plot.

-29
RCC 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 3 HTd1R Determination: Dr. R. Burni, Environmental Chemistry &

Pharmanalytics Division

Phase to: P. B. 61, Vers. BE 1675:

Local Lymph Node Assay (LLNA) in Mice

(identification of Contact Allergens)

The preparation of the [methyl- 3

H]Thymidine solution and determination of radioactivity

content were conducted in compliance with the Swiss Ordinance relating to Good Laboratory

Practice, adopted February 2 nd , 2000 [RS 813.016.5]. This Ordinance is based on the OECD

Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26 th, 1997

by decision of the OECD Council [C(97)186/Finall.

Principal Investigator 3 HTdR 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 3 HTdR Determination: Dr. R. Burn, Environmental Chemistry &

Pharmanalytics; Division

Phase to: 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 is given below.

Dates and Types of QAU Inspections Dates of Reports to the Principal Investigator and to the

Management

November 08, 2002 Process based November 08, 2002

Quality Assurance: Mr. J. L-Otte

Date: ~ ~ r2z
ROC STUDY NUMBER 846498

P. B. 61, Vers. BE 1675

APPENDIX E

GLP - CERTIFICATION
RCC STUDY NUMBER 846498

P. B. 61, Vers. BE 1675

The Swiss GLP Monitoring Authorities

(%~ Fedral Swss ASwiss Agency for the , Intercantonial Ofie

Offi9 of" Environment Forests /A ifor the Contro of

Public Health A and Landscape \ VMedicines

Statement of GLP Compliance

It is 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 of expertise*

- 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. It was 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 they were inspected.

Federal Office of Public Health

The Director

Bern, May 2002 Prof. Th. Zeltner

* TOX = Toxicciogy: ACC aAnalytical and Clinical Chemistry: ECT = Envirorsnental toxicity on aquatic and terrestrial

organisms ; ENF = Behaviour in water, *oil and air. Etloeccumulatiori; EMN - Studies on effects on mosocosms. and natural

ecosystems; MUT =Muteaenicijy; PCT = Physical-cheical testing ; RES = Residue studies ;0Th - Other, to be specfed.