RCC - CCR Study Number 1058800 Final Report Page 2 of 21 Ciba EFKA 8530



## 1 COPY OF GLP-CERTIFICATERCC - CCR Study Number 1058800 Final Report Page 3 of 21 Ciba EFKA 8530



## 2 CONTENTS

1 COPY OF GLP-CERTIFICATE 2 2 CONTENTS 3 3 PREFACE 4 3.1 General 4 3.2 Responsibilities 4 3.3 Schedule 4 3.4 Project Staff Signatures 5 3.5 Good Laboratory Practice 5 3.6 Guidelines 5 3.7 Archiving 5 3.8 Deviations from the Study Plan 6 4 STATEMENT OF COMPLIANCE 7 5 STATEMENT OF QUALITY ASSURANCE UNIT 8 6 SUMMARY 9 7 OBJECTIVE 10 7.1 Aims of the Study 10 7.2 Outline of the Performed Study 10 8 MATERIALS AND METHODS 11 8.1 Test Item 11 8.2 Test Item Preparation 11 8.3 Rationale 12 8.4 Chemicals 12 8.5 Vehicle 12 9 TEST SYSTEM 13 9.1 Animal Species 13 9.2 Allocation 14 9.3 Experimental Design and Procedures 15 9.4 Observations 16 9.5 Statistical Analysis 16 10 RESULTS 17 10.1 Calculation and Results of Individual Data 17 10.2 Viability / Mortality 17 10.3 Clinical Signs 17 10.4 Body Weights 17 11 DISCUSSION 18 11.1 CONCLUSION 18 12 REFERENCES 19 13 DISTRIBUTION OF THE REPORT 19 14 ANNEX 1 20 14.1 Tables of Body Weights 20 15 ANNEX 2 21 15.1 Results of the GLP Positive Control 21RCC - CCR Study Number 1058800 Final Report Page 4 of 21 Ciba EFKA 8530



## 3 PREFACE 3.1 General Title: Local Lymph Node Assay (LLNA) in Mice with Ciba EFKA 8530 Sponsor: Ciba Specialty Chemicals Inc. Segment Coating Effects P.O. Box WRO 1045.1.30 CH-4002 Basel Switzerland Study Monitor: Mr. Henry Su Test Facility: RCC Cytotest Cell Research GmbH (RCC-CCR) In den Leppsteinswiesen 19 64380 Rossdorf Contracting Institute: RCC Ltd CH-4452 Itingen Reference Number: B04814 

## 3.2 Responsibilities Study Director: Dipl. Biol. Krista Meurer Deputy Study Director: Dr. Naveed Honarvar Management: Dr. Wolfgang Völkner Head of Quality Assurance Unit: Frauke Hermann



## 3.3 Schedule Date of the Study Plan: January 16, 2007 Date of 1 st Amendment to Study Plan: March 23, 2007 Experimental Starting Date (non-GLP Pre-Experiment): December 07, 2006 Experimental Starting Date (GLP Main Experiment): January 24, 2007 Experimental Completion Date: January 30, 2007 Date of Draft Report: February 06, 2007 Date of Final Report: April 13, 2007RCC - CCR Study Number 1058800 Final Report Page 9 of 21 Ciba EFKA 8530



## 6 SUMMARY
In the study the test item Ciba EFKA 8530 dissolved in acetone: olive oil (4+1) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100 %. The animals did not show any clinical signs of toxicity after the first and second application. After the third application all doses induced redness of the of the ear skin of all four animals of the group. No cases of mortality were observed. In this study Stimulation Indices (S.I.) of 14.54, 16.09, and 16.74 were determined with the test item at concentrations of 25, 50, and 100 % in acetone: olive oil (4+1), respectively. The test item Ciba EFKA 8530 was found to be a skin sensitizer

. No EC3 value could be calculated, since all S.I.’s were above 3.RCC - CCR Study Number 1058800 Final Report Page 10 of 21 Ciba EFKA 8530



## 7 OBJECTIVE 7.1 Aims of the Study The purpose of this Local Lymph Node assay was to identify the contact allergenic potential of Ciba EFKA 8530 when administered to the dorsum of both ear lobes of mice. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man. 

## 7.2 Outline of the Performed Study In order to study a possible allergenic potential of Ciba EFKA 8530, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine ( 3 H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and 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 subsequently washed 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.RCC - CCR Study Number 1058800 Final Report Page 11 of 21 Ciba EFKA 8530



## 8 MATERIALS AND METHODS 8.1 Test Item
Internal RCC-CCR Test Item Number: S 690911 The test item and the information concerning the test item were provided by the sponsor. Identity: Ciba EFKA 8530 Batch No.: 21531 FE6 Aggregate state at room temperature: Liquid Colour: Yellowish Purity: 100 % Solubility in water: Slightly soluble Stability in solvent: Not indicated by the sponsor Storage: At room temperature Expiration Date: March 15, 2011 

## 8.2 Test Item Preparation Before each dosing occasion the high and mid dose was freshly prepared using a magnetic stirrer. The test item was placed into a volumetric flask glass beaker on a tared balance. The mid dose was diluted with acetone: olive oil (4+1) to achieve the low dose. The test item concentrations were prepared serially. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice (pretest excluded from Statement of Compliance). The data showed that the highest test item concentration, which can be used is 100 %. At this concentration the treated mouse did not show any signs of irritation. The test item in the main study was, therefore, assayed at 25, 50, and 100 %. Concentrations were in terms of material as supplied.RCC - CCR Study Number 1058800 Final Report Page 12 of 21 Ciba EFKA 8530


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

## 8.4 Chemicals

3 H-methyl Thymidine GE Healthcare TRA 310, aqueous solution, sterilised 74 GBq/mmol (2 Ci/mmol), 37 MBq/ml (1 mCi/ml) quantities: 9.25 MBq (250 μ Ci), 37 MBq (1 mCi) Trichloroacetic acid Merck 1.00807.0250 (min. 99.5 %) Phosphate buffered saline Fluka no. 79382 (1 tablet solved in 200 ml deionised water) 

## 8.5 Vehicle Acetone:olive oil (4+1) 1) Acetone Supplier VWR International GmbH (D-64295 Darmstadt) Catalogue number 1.00014.1011 Purity 99.6% Storage conditions In the original container at room temperature (20 °C ± 3 °C), away from direct sunlight. 2) Olive oil Supplier Sigma/Aldrich Chemicals (D-82024 Taufkirchen) Catalogue Number O1514 Storage conditions In the original container at room temperature (20 °C ± 3 °C), away from direct sunlight.RCC - CCR Study Number 1058800 Final Report Page 13 of 21 Ciba EFKA 8530



## 9 TEST SYSTEM 9.1 Animal Species Test system Mice, CBA/CaOlaHsd Rationale Recognised as the recommended test system Source Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands Number of animals for the pre-test (non-GLP) 2 females Number of animals for the main study 16 females Number of animals per group 4 females (nulliparous and non-pregnant) Number of test groups 3 Number of control (vehicle) groups 1 Age 7 - 8 weeks (beginning of acclimatisation) Identification Single caging. The animals will be distributed into the test groups at random and identified by cage number. Acclimatisation Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.RCC - CCR Study Number 1058800 Final Report Page 14 of 21 Ciba EFKA 8530 

### 9.1.1 Husbandry The animals were kept conventionally. The experiment was conducted under standard laboratory conditions. Housing: single Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen) Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen) Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf) Environment: temperature 22 + 3°C relative humidity 30 – 72 % artificial light 6.00 a.m. - 6.00 p.m.


## 9.2 Allocation
The animals were distributed as follows: Group Concentration b %) Number of Animals per Group Cage Numbers (Single Housing) 1 (Control Group a )

 4 1

- 4 2 (Low Dose) 25 4 5 - 8 3 (Mid Dose) 50 4 9 - 12 4 (High Dose) 100 4 13 - 16 a) vehicle group = acetone: olive oil (4+1) b) In a non-GLP conform pre-test in two mice, test item concentrations of 12.5, 25, 50, and 100 % were tested on one ear each. No irritation effects were observed at these concentrations after a single application. The sensitivity and reliability of the experimental technique employed was assessed by use of a substance, which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. The validation- / positive control study was performed with  - Hexylcinnamaldehyde in acetone:olive oil (4+1) using CBA/CaOlaHsd mice in December 2006, see Annex 2.RCC - CCR Study Number 1058800 Final Report Page 15 of 21 Ciba EFKA 8530 

## 9.3 Experimental Design and Procedures 9.3.1 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 25, 50, and 100 % (w/v) in acetone: olive oil (4+1). The application volume, 25 R l, was spread over the entire dorsal surface (   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).



### 9.3.2 Administration of 3 

### H-Methyl Thymidine

3 H-methyl thymidine ( 3 HTdR) was purchased from GE Healthcare International (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 R l of 81.2 R Ci/ml 3 HTdR (corresponds to 20.3 R Ci 3 HTdR per mouse) by intravenous injection via a tail vein. 

### 9.3.3 Determination of Incorporated 3 

### HTdR Approximately five hours after treatment with 3 HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz)

. The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 R m 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 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3 HTdR incorporation was then measured on a -scintillation counter. Similarly, background 3 HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3 HTdR incorporation as the number of radioactive disintegrations per minute (DPM).RCC - CCR Study Number 1058800 Final Report Page 16 of 21 Ciba EFKA 8530 

### 9.3.4 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 DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser 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 (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.


## 9.4 Observations In addition to the sensitising reactions the following observations and data were recorded during the test and observation period: Mortality / Viability once daily (week day) from experimental start to necropsy. Body weights prior to the first application and prior to treatment with 3 HTdR. Clinical signs (local / systemic) once daily (week day) during the application of the test item. Especially the treatment sites were observed carefully. 

## 9.5 Statistical Analysis
The mean values and standard deviations were calculated in the body weight tables.RCC - CCR Study Number 1058800 Final Report Page 17 of 21 Ciba EFKA 8530



## 10 RESULTS 10.1 Calculation and Results of Individual Data Vehicle: acetone: olive oil (4+1) Calculation Result

Test item concentration % (w/v) Group Measurement DPM DPM-BG a) number of lymph nodes DPM per lymph node b) S.I. --- BG I 83.11 --- --- --- --- --- BG II 336.98 --- --- --- --- --- CG 1 7315.07 7105.0 8 888.1 25 TG 2 103496.00 103286.0 8 12910.7 14.54 50 TG 3 114555.00 114345.0 8 14293.1 16.09 100 TG 4 119128.00 118918.0 8 14864.7 16.74 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 II 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 The EC3 Value could not be calculated, since all SI ́s are above 3. 

## 10.2 Viability / Mortality No deaths occurred during the study period. 

## 10.3 Clinical Signs The animals did not show any clinical signs of toxicity after the first and second application. After the third application all doses induced redness of the of the ear skin of all four animals of the group. 

## 10.4 Body Weights The body weight of the animals, recorded prior to the first application and prior to treatment with 3 HTdR, was within the range commonly recorded for animals of this strain and age. The individual body weight values are included in Annex 1.RCC - CCR Study Number 1058800 Final Report Page 18 of 21 Ciba EFKA 8530



## 11 DISCUSSION
In order to study a possible contact allergenic potential of Ciba EFKA 8530, three groups each of four female mice were treated daily with the test item at concentrations of 25, 50, and 100 % (w/v) in acetone: olive oil (4+1) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone: olive oil (4+1)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine ( 3 Hmethyl 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 subsequently washed 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. All treated animals survived the scheduled study period. They did not show any clinical signs of toxicity after the first and second application. After the third application all doses induced redness of the of the ear skin of all four animals of the group. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3 HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 14.54, 16.09, and 16.74 were determined with the test item at concentrations of 25, 50, and 100 % in acetone: olive oil (4+1). The EC3 value could not be calculated, since all obtained SI ́s were above 3.


## 11.1 CONCLUSION
The test item Ciba EFKA 8530 was found to be a skin sensitiser under the described conditions.RCC - CCR Study Number 1058800 Final Report Page 19 of 21 Ciba EFKA 8530



## 12 REFERENCES
1) 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. 2) Kimber I. and Basketter D.A. (1992). The murine local lymph node assay. A commentary on collaborative studies and new directions. Food and Chemical Toxicology, 30 , 165-169. 3) Basketter D.A., Gerbrick G.F., Kimber I. 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. 4) Chamberlain M. and Basketter D.A. (1996). The local lymph node assay: status of validation. Food and Chemical Toxicology, 34 , 999-1002. 5) Basketter D.A., Lea L.J., Cooper K., Stocks J., Dickens A., Pate I., Dearman R.J. and Kimber I. (1999). Threshold for Classification as a Skin Sensitizer in the Local Lymph Node Assay: a Statistical Evaluation. Food and Chemical Toxicology, 37 , 1-8. 6) 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.



## 13 DISTRIBUTION OF THE REPORT Sponsor 1x copy Study Director 1x originalRCC - CCR Study Number 1058800 Final Report Page 20 of 21 Ciba EFKA 8530



## 14 ANNEX 1 14.1 Tables of Body Weights

Animal No. Dose Group Initial Weight (g) weight prior to treatment with 3HTdR (g) 1 1 18.4 18.4 2 1 16.9 18.1 3 1 19.1 20.7 4 1 18.4 20.0 5 2 20.5 20.8 6 2 18.5 19.8 7 2 19.3 20.1 8 2 18.9 19.8 9 3 21.9 20.9 10 3 20.3 20.3 11 3 18.4 19.1 12 3 20.0 20.0 13 4 19.6 20.5 14 4 19.1 18.6 15 4 19.0 19.7 16 4 18.7 19.5
Mean 19.2 19.8 Standard Deviation 1.1 0.8RCC - CCR Study Number 1058800 Final Report Page 21 of 21 Ciba EFKA 8530



## 15 ANNEX 2 15.1 Results of the GLP Positive Control Study performed in December 2006. Positive control substance:  -Hexylcinnamaldehyde Vehicle: acetone:olive oil (4+1) Calculation Result

Test item concentration % (w/v) Group Measurement DPM DPM-BG a) number of lymph nodes DPM per lymph node b) S.I.

--- BG I 197.50 --- --- --- --- --- BG II 22.03 --- --- --- --- --- CG 1 4049.44 3939.7 8 492.5 5 TG 1 8152.94 8043.2 8 1005.4 2.04 10 TG 2 24974.40 24864.6 8 3108.1 6.31 25 TG 3 49155.30 49045.5 8 6130.7 12.45 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 II 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 % (w/v) S.I.

Group 1 5 (a) 2.04(b) Group 2 10 (c) 6.31(d) EC3 = (a-c) [(3-d)/(b-d)] + c = 6.1% (w/v)