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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 August 15 to 21, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed equivalent or similar to OECD test guideline No. 429 with deviations: no ear thickness measurement, no range-finding test performed. However, no sign or irritation or systemic toxicity were observed at the concentration giving a SI above 3.
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:
temperature and humidity of the animal room were outside the target range; individual weights of animals at start of dosing and at scheduled kill not reported; no ear thickness measurement, no range-finding test 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
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Indianapolis, Indiana.
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 18.0-23.3 g
- Housing: Animals were individually housed in plastic shoebox-style cages.
- Diet: Purina Rodent Chow 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.1-25.1 °C
- Humidity: 38- 89 %
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: August 15, 2002 To: August 21, 2002
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.25, 0.5, 1.0, 2.5 and 5.0 % in 4:1 acetone/olive oil
No. of animals per dose:
5 females/dose (except vehicle control group where 8 females/dose)
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 sensitiser if at least one concentration of the test material results in a 3-fold or greater stimulation index (SI).

TREATMENT PREPARATION AND ADMINISTRATION:
The vehicle and dosing solutions were prepared on the bench top daily prior to dosing. All suspensions were mixed by vortexing. 25 µL of control or test material was applied to the dorsum of each ear using a calibrated Finnpipet daily for three consecutive days. Animals were not treated on Days 4 and 5. On Day 6, animals were injected in the lateral tail vein with 0.25 mL containing 2 µCi of 125I-labelled Iododeoxyuridine and 10^-5 M FuDR in phosphate buffered saline (PBS). Approximately 5 h later, animals 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 % tricholoroacetic acid (TCA) and refrigerated at approximately 4 °C. Approximately 16 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 treatment group mean DPM by the control (vehicle) group mean DPM.
Positive control substance(s):
other: Isoeugenol (CAS No. 97-54-1)- at 0.5 %, 1 % and 5 % in 4:1 acetone/olive oil
Statistics:
- To test if the compound was 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 than 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. 0.25, 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 (specifically a Kruskal-Wallis test) were 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 three concentrations, 0.5, 1 and 5 % using the above methods.
- A fitted linear equation was used to determine the concentration of test material required to elicit a stimulation index of 3 (EC-3).
- All calculations were performed using Microsoft Excel and SAS, version 8. PROCs GLM, FREQ, NPAR1WAY and MEANS were utilized.
Positive control results:
Only 1 and 5 % concentrations of isoeugenol resulted in group SI statistically significantly greater than 3 (one-sample t tests). A fitted linear regression yielded an EC-3 of 0.69 % for isoeugenol
Parameter:
SI
Remarks on result:
other: Mean Stimulation index for 0.25, 0.5, 1.0, 2.5 and 5.0 % were 0.6, 1.6, 3.5, 6.2 and 7.9, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM for vehicle, 0.25, 0.5, 1.0, 2.5 and 5.0 % were 26.9, 16.9, 42.8, 95.2, 167.6 and 213.9, 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.

- There was some weight loss (0.1-1.1 g) was observed in 11/48 animals over the 6 day period from dosing to lymph node harvest. This weight loss occurred across dose and treatment groups. The animals appeared healthy otherwise with a final body weight range of 19.8-22.9 g

- The highest doses (1, 2.5 and 5 %) of the test article had SI of 3.5, 6.2 and 7.9. A one-sample t test on the untransformed SI values indicated that the SI value for 2.5 and 5 % were statistically significant. All the other doses tested (0.25 and 0.5 %) had SI values less than 3.0.

- The calculated EC-3 for test material using a fitted quadratic equation was 1.22% and potency value was 305 µg/cm2.
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 sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

In a Local Lymph Node Assay (LLNA) conducted similarly to the OECD test guideline No 429 and in compliance with GLP, groups of female CBA/J mice (5 females/group) were topically applied with test material at the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 % final concentration in 1:4 acetone:olive oil to the dorsal surface of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group (8 females/group) was treated with 1:4 acetone:olive oil alone and a positive control group (5 females/group) was treated with isoeugenol at the dose concentration of 0.5, 1.0 and 5.0 % in acetone:olive oil (4:1) in same manner to confirm the sensitivity and reliability of the test method. The animals were allowed to rest without dosing on Days 4 and 5. On Day 6, animals were injected intravenously with 125I- labelled luDR to label proliferating cells. 125I-incorporation was quantified using a gamma counter and disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.

 

The positive control, Isoeugenol gave SI of 4.2 and 18.4, when tested at 1 and 5 % in 4:1 acetone/olive oil, respectively. The test system was therefore considered to be valid.

 

No mortality, irritation or other adverse toxic effects were noted in any of the animals. Mean DPM for 0 (vehicle), 0.25, 0.5, 1.0, 2.5 and 5.0 % were 26.9, 16.9, 42.8, 95.2, 167.6 and 213.9, respectively. Stimulation Index (SI Value) calculated for test material was found to be 0.6, 1.6, 3.5, 6.2 and 7.9 for the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 %, respectively. The highest doses (1, 2.5 and 5 %) of the test article had SI of 3.5, 6.2 and 7.9. A one-sample t test on the untransformed SI values indicated that the SI value for 2.5 and 5 % were statistically significant. All the other doses tested (0.25 and 0.5 %) had SI values less than 3.0. The calculated EC-3 for test material using a fitted quadratic equation was 1.22% and potency value was 305 µg/cm2.

 

Under the test conditions, test material is classified asa skin sensitiser 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. Groups of six female animals were treated with the test material as a solution in acetone/olive oil 4:1 at concentrations of 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 4.2 and 18.4, when tested at 1 and 5 % in 4:1 acetone/olive oil, respectively. 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 26.9, 16.9, 42.8, 95.2, 167.6 and 213.9, respectively.

Stimulation index for 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 0.6, 1.6, 3.5, 6.2 and 7.9, respectively.

The highest doses (1, 2.5 and 5 %) of the test article had SI of 3.5, 6.2 and 7.9, respectively. A one-sample t test on the untransformed SI values indicated that the SI values of 2.5 and 5% were statistically significant. All the other doses tested (0.25 and 0.5 %) had SI values less than 3.0.

The calculated EC-3 for test material using a fitted quadratic equation was 1.22 %.

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

Deviations from the guideline were reported for this study: ear thickness measurements and preliminary screening tests were not performed. However, no sign of irritation or systemic toxicity were observed at the concentration giving a SI above 3.

The substance was only tested up to 5% without jutification, however it could be explained by the classification of the substance as skin irritant. 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.


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). Deviations from the guideline were reported for this study: 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. The substance was only tested up to 5% without jutification, however it could be explained by the classification of the substance as skin irritant. 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.

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. 1A, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP), since EC3 is < 2% (Potency = Strong)

No data was available regarding respiratory sensitisation.