[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 13 January 2004 to 5 February 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similarly to OECD test guideline No. 429 and in compliance with GLP. However the maximum concentration tested was not justified and a screening study was not performed.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Maximum concentration tested 40% without justification

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

GLP compliance statement

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): ST 30 C 03
- Substance type: pure active substance
- Physical state: liquid
- Storage condition of test material: Approximately 4 °C under nitrogen in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: The Jackson Laboratory (Bar Harbor, ME - USA)
- Age at study initiation: > 6-7 weeks
- Weight at study initiation: 17.4 - 22.6 g
- Housing: mice were housed individually in plastic shoebox-style cages at the North Carolina State University College of Veterinary Medicine.
- Diet (e.g. ad libitum): ad libitum (Purina Rodent Chow 5002)
- Water (e.g. ad libitum): City of Raleigh tap water (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 - 27.6 °C
- Humidity (%): 13 % - 43 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 January 2004 To: 26 January 2004

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

1%, 5%, 10%, 20%, and 40%

No. of animals per dose:

5 (excepted for vehicle control: 8)

Details on study design:

RANGE FINDING TESTS: Not performed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: individual method
- Criteria used to consider a positive response: A substance is considered a sensitizer if at least one concentration of the test material results in a
stimulation index (SI) greater than or equal to 3.

TREATMENT PREPARATION AND ADMINISTRATION: The test article, ST 30 C 03, was tested at 1%, 5%, 10%, 20%, and 40% final concentrations in acetone/olive oil (AOO; 4:1). The control articles included the vehicle (AOO) and a known sensitizer, isoeugenol, at 0.5 %, 1.0%, and 5.0%. The mice were restrained by hand and 25 µL of test or control article was applied daily for three consecutive days to the dorsum of each ear using a calibrated Finnpipette. The animals were allowed to rest without dosing on Days 4 and 5. The mice were observed daily for signs of toxicity or irritation.
On Day 6, individually numbered mice (labelled by tail markings) were injected in the lateral tail vein with 0.25 mL containing 2 µCi of I-125 labelled luDR and 10.5 M FuDR (Sigma) in phosphate buffered saline (PBS). Approximately 5 hours later, the mice were euthanized by CO2 asphyxiation, and the auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA; VWR Scientific) and refrigerated at approximately 4°C. Approximately 21 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter (Packard Instruments).

Positive control substance(s):

other: Isoeugenol (0.5%, 1.0%, and 5.0%)

Statistics:

The naturaI log transformed DPM (Desintegration per Minut) values for each compound were compared against vehicle with a Bartlett's Chi-Square test for variance homogeneity. If the Bartlett's Chi-Square resuIts were found to be non-significant, a one way analysis of variance (ANOVA) was performed using dose (concentration). If the ANOVA was found to be significant, then a Dunnett's test was performed using an alpha of 0.05.
If the Barlett's Chi-Square was found to be significant, nonparametric analyses were conducted, specifically a Kruskal-Wallis (KW) test. If the nonparametric analyses were found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.

Results and discussion

Positive control results:

An EC-3 of 1.28 % was determined for the positive control isoeugenol in the current study using a linear regression method with an excellent fit. This is in agreement with the mean EC-3 value of isoeugenol of 1.2 +/- 0.6% (Basketter & Cadby, Contact Dermatitis 2004 Jan;50(1):15-7). A potency value of 320 µg/cm² was calculated. These data would classify isoeugenol as a moderate sensitizer (potency value between 100 and 1000 µg/cm²) and are consistant with previously reported results; this demonstrate the capability of the test laboratory to identify positive dermal sensitizers.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 0.6, 0.8, 1.1, 1.1 and 2.4 for 1%, 5%, 10%, 20% and 40% respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 27.0, 17.3, 22.3, 28.4, 29.6 and 65.9 for 0%, 1%, 5%, 10%, 20% and 40% respectively.

Any other information on results incl. tables

Table 7.4.1: DPM measurements:

Treatment (% of test material)	N° of animal	Mean DPM	Standard Deviation	Standard error of the mean
1	5	17.3	11.8	5.3
5	5	22.3	18.7	8.4
10	5	28.4	9.8	4.4
20	5	29.6	28.4	12.7
40	5	65.9	23.6	10.6
AOO	7	27.0	13.0	4.9
Isoeugenol 0.5 %	5	16.9	19.3	8.6
Isoeugenol 1.0 %	5	28.7	32.1	14.3
Isoeugenol 5.0 %	5	326.7	141.5	63.3
AOO= Acetone- Olive Oil (4:1) = vehicle

DPM measurements for the four first different concentrations of test material (1%, 5%, 10%, 20%) were similar to DPM measurement of vehicle. DPM measurements for the 5th and last concentration tested (40%) was roughly 2 times higher than vehicle.

 

Table 7.4.2: Stimulation Index Following Exposure to Test & Control Material

Treatment (% of test material)	Mean Stimulation Index (SI)	Standard error of the mean
1	0.6	0.2
5	0.8	0.3
10	1.1	0.2
20	1.1	0.5
40	2.4	0.4
Isoeugenol 0.5 %	0.6	0.3
Isoeugenol 1.0 %	1.1	0.5
Isoeugenol 5.0 %	12.1	2.3

All the calculated SI values for the test material were smaller than 3, for that reason no reliable EC-3 could be calculated (EC-3 > 40 %). Calculation of the EC-3 potency value assumed a conversion of 1 mL = 1 g and was based on an exposure area of 1 cm² per mouse ear and the application of 25 µL. The EC-3 potency value for isoeugenol was determined to be 320 µg/cm². With an EC-3 greater than 40% for ST 30 C 03, the potency value of the test article can only be estimated to be greater than 10000 µg/cm².       

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, the test material (1%-40%) did not produce evidence of skin sensitization (delayed contact hypersensitivity).

Executive summary:

The sensitization potential of the test material was evaluated in a Local Lymph Node Assay performed according to the OECD test guideline No. 429 and in compliance with GLP, by measuring its ability to stimulate proliferation of lymphocytes within the auricular lymph nodes of female CBA/J mice.

Mice were treated daily for three consecutive days by direct epicutaneous application of 25 µL of test or control article to the dorsum of each ear. The test material was tested at 1%, 5%, 10%, 20%, and 40% final concentrations in acetone/olive oil. The control articles included the vehicle, acetone/olive oil (AOO), and a known sensitizer, isoeugenol. Isoeugenol was tested at 0.5%, 1.0%, and 5.0% concentrations in AOO (4:1). The mice were observed daily. Three days after the final auricular application, the animals were injected intravenously with I-125 labeled Iododeoxyuridine to label proliferating cells. I-125 incorporation was quantified using a gamma counter.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 and 3.

None of the mice assigned to this study experienced visible irritation or other adverse toxic effects.

A significant lymphoproliferation was noted in the positive control group, the study was therefore considered valid.

The test material (1%-40%) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Under the test conditions, the test material is not classified according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.