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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 13 to December 19, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted similarly to OECD 429 Guideline with deviations: no ear thickness measurement; no range-finding test was performed.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
no ear thickness measurement; no range-finding test was performed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J Hsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN
- Age at study initiation: 6-8 weeks
- Weight at randomization: 15.9-22.6 g (18.4 g)
- Housing: Animals were housed individually in plastic shoebox-style cages
- Diet: Purina Rodent Chow 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 66-75 °F
- Humidity: 26-83 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: December 13, 2000 To: December 19, 2000
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.1, 0.25, 0.5, 1, 2.5 and 5 %
No. of animals per dose:
6
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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 statistically significant 3-fold or greater stimulation index (SI).
TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of control or test item were applied to the dorsum of each ears on Days 1, 2 and 3. Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 2 μCi of 125lododeoxyuridine (IODR) on Day 6. After approximately five hours, all animals were killed by Co2 asphyxiation and the draining auricular lymph node of each ear was excised. The nodes were pooled for each animal in Hank’s Balanced salt solution with HEPES (HBSS/H). The cell suspension was washed with HBSS and then with PBS, prior to being resuspended in 5 % tricholoroacetic acid (TCA) and refrigerated at approximately 4 °C. Approximately 18 h later the cells were centrifuged and resuspended in fresh 5 % TCA. The radioactivity was measured using a gamma counter (Packard instruments).

OTHERS:
- The results from each cell suspension counted on the gamma counter were recorded in counts per minute (CPM). The CPM were converted to disintegrations per minute (DPM) by dividing by the gamma counter efficiency and multiplying by 100. After the DPM values had been calculated, the mean "blank" DPM was subtracted from each mouse DPM to obtain corrected DPM values. The mean corrected DPM and standard error of the mean (SE) were determined for each group. The stimulation index (SI) was then calculated by dividing the treated group mean DPM by the control (vehicle) group mean DPM.
Positive control substance(s):
other: Isoeugenol - at 0.5 % and 5 % in 4:1 acetone/olive oil
Statistics:
- To test if the compound is a sensitizer, a one-sample t test was performed for testing if the individual untransformed SI values of each dose level of each compound were different from 3.0. The natural log transformed dpm values for each compound were compared against vehicle by first performing a Bartlett's Chi-Square test for variance homogeneity. If this was found to be non-significant, a one-way analysis of variance was used using dose (concentration) - i.e., 0.1, 0.25, 0.5, 1.0, 2.5 and 5 %. If this was found to be significant then a Dunnett’s t test was performed using an alpha of 0.05.
- If the Bartlett's Chi-Square was found to be significant non-parametric analyses were performed. Specifically, a Kruskal-Wallis test was performed. If this was found to be significant, then a Jonckheere’s-Terpera test was performed for dose-dependent trends.
- A confirmatory analysis was performed against the known standard isoeugenol at two concentrations, 0.5 and 5 % using the above methods.
- A fitted quadratic equation was used to determine the concentration required to elicit a stimulation index of 3 (EC3). The data from the concentrations tested were fit using a quadratic equation (a linear term and a square term of the concentration).
- A fitted linear equation was used to determine the concentration of isoeugenol required to elicit a stimulation index of 3 (EC3) since only 3 dose levels (0% - vehicle, 0.5 and 5 %) of isoeugenol were tested.
- All calculations were performed using Microsoft Excel and SAS, version 6.12. PROCs GLM, FREQ, NPARIWAY and MEANS were utilized.
Positive control results:
Although both the 0.5 and 5 % concentrations of isoeugenol resulted in group SI greater than 3, statistical analysis (one-sample t tests) showed only the 5 % concentration of isoeugenol to have SI values statistically significantly greater than 3.0 (p ≤ 0.05). A fitted linear regression yielded an EC-3 of 0.50 % for isoeugenol.
Parameter:
SI
Remarks on result:
other: Stimulation index for 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 1.5, 2.3, 2.7, 2.4, 2.7 and 4.5, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM for vehicle, 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 5.2, 7.6, 11.9, 14.4, 12.8, 14.0 and 23.5, respectively.

- No irritation or other adverse toxic effects were noted in any of the mice used in this study, and there were no animal deaths in any of the groups.

 - The highest dose (5 %) of the test article had an SI = 4. 5. A one-sample t test on the untransformed SI values indicated that this SI of 4.5 was statistically significant. All the other doses tested (0.1, 0.25, 0.5, 1 and 2.5 %) had SI values less than 3.0.

- The calculated EC-3 for test material using a fitted quadratic equation was 2.4 %.
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test material is classified as a skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/J Hsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted similarly to the OECD test guideline No 429 and in compliance with GLP.

Six groups, each of six female animals, were treated with 50 μl (25 μl per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 0.1, 0.25, 0.5, 1, 2.5 and 5 % for 3 consecutive days. A further group of six animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on Days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 125lododeoxyuridine. The irritant potential of the test item was assessed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Animals were observed for mortality and clinical signs during the study.

The positive control, Isoeugenol gave a SI of 16.7, when tested at 5 % in 4:1 acetone/olive oil. The test system was therefore considered to be valid.

No irritation or other adverse toxic effects were noted in any of the mice used in this study, and there were no animal deaths in any of the groups. Mean DPM for vehicle, 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 5.2, 7.6, 11.9, 14.4, 12.8, 14.0 and 23.5, respectively. Stimulation index for 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 1.5, 2.3, 2.7, 2.4, 2.7 and 4.5, respectively. The highest dose (5 %) of the test article had an SI = 4. 5. A one-sample t test on the untransformed SI values indicated that this SI of 4.5 was statistically significant. All the other doses tested (0.1, 0.25, 0.5, 1 and 2.5 %) had SI values less than 3.0. The calculated EC-3 for test material using a fitted quadratic equation was 2.4 %.

 

Under the test conditions, test material is classified as a skin sensitizer in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A key study was identified (BRT, 2001). This Local Lymph Node Assay (LLNA) was performed similarly to the OECD test guideline No 429 and in compliance with GLP. Deviations from the guideline were reported: ear thickness measurements and preliminary screening tests were not performed. However, no sign or irritation or systemic toxicity were observed at the concentration giving a SI above 3. Moreover, extensive data are available on the members of the rose ketones family which support the skin sensitisation study result. The substance is expected to interact with skin proteins via a Michael addition mechanism.

In the key study, groups of six female animals were treated with the test material as a solution in acetone/olive oil 4:1 at concentrations of 0.1, 0.25, 0.5, 1, 2.5 and 5 % for 3 consecutive days. A further group of six animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on Days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of125lododeoxyuridine. The irritant potential of the test item was assessed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Animals were observed for mortality and clinical signs during the study.

The positive control, Isoeugenol gave a SI of 16.7, when tested at 5 % in 4:1 acetone/olive oil. The test system was therefore considered to be valid.

No irritation or other adverse toxic effects were noted in any of the mice used in this study, and there were no animal deaths in any of the groups.

Mean DPM for vehicle, 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 5.2, 7.6, 11.9, 14.4, 12.8, 14.0 and 23.5, respectively.

Stimulation index for 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 1.5, 2.3, 2.7, 2.4, 2.7 and 4.5, respectively.

The highest dose (5 %) of the test article had an SI = 4. 5. A one-sample t test on the untransformed SI values indicated that this SI of 4.5 was statistically significant. All the other doses tested (0.1, 0.25, 0.5, 1 and 2.5 %) had SI values less than 3.0.

The calculated EC-3 for test material using a fitted quadratic equation was 2.4 %.

Under the test conditions, the test item is classified as a skin sensitizer.


Migrated from Short description of key information:
LLNA, sensitising (similar to OECD 429, GLP, K, Rel. 2)

Justification for selection of skin sensitisation endpoint:
Only one study available, GLP-compliant and of good quality (Klimisch score = 2).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP6.

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP), since EC3 is > 2%.

No data was available regarding respiratory sensitisation.