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Ciba EFKA 8530 

1 COPY OF GLP-CERTIFICATE 
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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 21
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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 1st 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, 2007 




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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. 
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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. 
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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. 
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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 μCi), 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. 
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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. 
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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 Concentrationb %) Number of Animals per 

Group 

Cage Numbers 

(Single Housing) 

1 (Control Groupa)  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. 
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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 Rl, 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 3H-Methyl Thymidine 

3

H-methyl thymidine (3HTdR) 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 Rl of 

81.2 RCi/ml 3

HTdR (corresponds to 20.3 RCi 3HTdR per mouse) by intravenous injection 

via a tail vein. 

9.3.3 Determination of Incorporated 3HTdR 

Approximately five hours after treatment with 3HTdR 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 Rm

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 3HTdR 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). 
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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 3HTdR 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. 
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Ciba EFKA 8530 

10 RESULTS 

10.1 Calculation and Results of Individual Data 

Vehicle: acetone: olive oil (4+1) 

Test item Calculation Result 

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. 
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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. 
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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 original 
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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 0.8 
RCC - 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)