Registration Dossier

Administrative data

Description of key information

LLNA, sensitising (similar to OECD 429, GLP, K, Rel. 2)

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 February 14 to March 24, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted similarly to OECD 429 Guideline with deviations: no ear thickness measurement; no range-finding test was performed. However, no sign or irritation or systemic toxicity were observed at the concentration giving a SI above 3.
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 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
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 study initiation: 15.5-20.9 g (mean body weight: 18.6 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: 66-78 °F
- Humidity: 22-66 %
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: February 14, 2001 To: March 24, 2001.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.1, 0.25, 0.5, 1.0, 2.5, and 5 % in 4:1 acetone/olive oil
No. of animals per dose:
6 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 statistically significant 3-fold or greater stimulation index (SI).

TREATMENT PREPARATION AND ADMINISTRATION:
- The vehicle and dilutions of the control and test materials 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 i.v. 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; LabChem lot # 0322-11) 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 animal 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 (Sl) 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 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 test material concentration 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 (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 two concentrations, 0.5 and 5 % using the above methods.
- A fitted quadratic equation was used to determine the concentration of test material 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) as only 3 doses (0, 0.5 and 5 %) of isoeugenol were tested.
- All calculations were performed using Microsoft Excel and SAS, version 6.12. PROCs GLM, FREQ, NPAR1WAY and MEANS were utilized.
Positive control results:
Only 5 % concentrations of isoeugenol resulted in group SI statistically significantly greater than 3 and statistical analysis (one-sample t tests). A fitted linear regression yielded an EC-3 of 1.8 % for isoeugenol.
Parameter:
SI
Value:
0.4
Test group / Remarks:
0.1 % test group
Parameter:
SI
Value:
0.8
Test group / Remarks:
0.25 % test group
Parameter:
SI
Value:
1.1
Test group / Remarks:
0.5 % test group
Parameter:
SI
Value:
1.5
Test group / Remarks:
1 % test group
Parameter:
SI
Value:
1.9
Test group / Remarks:
2.5 % test group
Key result
Parameter:
SI
Value:
4.4
Test group / Remarks:
5 % test group
Key result
Parameter:
EC3
Value:
3.3

- 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 = 4.4. A one-sample t test on the untransformed SI values indicated that this SI of 4.4 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.

- Mean disintegrations per minute (DPM) for vehicle, 0.1, 0.25, 0.5, 1, 2.5 and 5 % were 8.6, 3.7, 6.9, 9.53, 13, 16.3 and 37.9, respectively.

- The calculated EC-3 for test material using a fitted quadratic equation was 3.3 %.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified in Category 1B for skin sensitisation according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
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 Hsd mice (6 females/group) were topically applied with test material at the dose concentrations of 0.1, 0.25, 0.5, 1, 2.5 and 5 % 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 was treated with 1:4 acetone:olive oil alone and a positive control group was treated with isoeugenol at the dose concentration of 0.5 and 5 % 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- labeled 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 a SI of 7.2, when tested at 5 % in 4:1 acetone/olive oil. 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 vehicle, 0.1, 0.25, 0.5, 1, 2.5 and 5 % were 8.6, 3.7, 6.9, 9.5, 13, 16.3 and 37.9, respectively. Stimulation Index (SI Value) calculated for test material treated groups was found to be 0.4, 0.8, 1.1, 1.5, 1.9 and 4.4 for the dose concentrations of 0.1, 0.25, 0.5, 1, 2.5 and 5 %, respectively. The highest dose (5 %) of the test article had an SI = 4.4. A one-sample t test on the untransformed SI values indicated that this SI of 4.4 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 3.3 %.

Under the test conditions, test material is classified as a skin sensitiser (Category 1B) according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

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 0.1, 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 7.2, 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 8.6, 3.7, 6.9, 9.5, 13, 16.3 and 37.9, respectively.

Stimulation index for 0.1, 0.25, 0.5, 1, 2.5 and 5 % was 0.4, 0.8, 1.1, 1.5, 1.9 and 4.4, respectively.

The highest dose (5 %) of the test article had an SI = 4. 4. A one-sample t test on the untransformed SI values indicated that this SI of 4.4 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 3.3 %.

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 structural alerts identified are the following:

- interaction with skin proteins via a Michael addition mechanism (alpha, beta-unsaturated ketones).

- formation of an antigenic adducts with skin proteins after their activation to hydroperoxides by air oxidation, which can react with protein directly via a radical thiol-ene reaction, or indirectly after their secondary oxidation to alpha,beta-unsaturated epoxides or alpha,beta unsaturated carbonyl derivatives (Terpenoid).

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.

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) and to the GHS, since EC3 is > 2%.

No data was available regarding respiratory sensitisation.