Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

- LLNA: skin sensitiser (OECD 429, GLP, K, Rel. 2);

- HRIPT: not a skin sensitiser at 0.5%

Respiratory sensitisation:

No data available.

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:
19-25 September 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed equivalent or similar to OECD test guideline No. 429 with deviations: no ear thickness measurement, no range-finding test performed.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Age: 6-7 weeks old instead of 8-12 weeks old; Individual weights of animals at start of dosing and at scheduled kill not reported; animal room humidity level was outside the target range; 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 (beginning of acclimatization)
- Weight at study initiation: 17.1-22.7 g
- Housing: Animals were housed individually in plastic shoebox-style cages.
- Diet: Purina Rodent Chow 5002, ad libitum
- Water: Raleigh tap water, ad libitum
- Acclimation period: 8 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature: 17.9-24.8 °C
- Humidity: 43-79 %
- Photoperiod: 12 h dark / 12 h light

- IN-LIFE DATES: 19-25 September 2001
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1.0%, 5.0%, 10%, 20%, and 40% in acetone/olive oil (4:1)
No. of animals per dose:
5
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 test and control items were prepared on the bench top daily, prior to dosing. The test and control items were soluble in the vehicle at the doses prepared. All suspensions were mixed by vortexing.
- 25 µL of control or test item 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) and refrigerated at approximately 4°C. Approximately 19 h later the cells were centrifuged and resuspended in fresh 5 % TCA. The radioactivity was measured using a gamma counter (Packard Instruments).
Disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.
Positive control substance(s):
other: isoeugenol (CAS No 97-54-1)
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. If this was found to be significant, then a Dunnett's t test was performed using an alpha of 0.05.
- If the Barlett'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 three concentrations, 0.5%, 1 %, and 5% using the above methods.
- The fitted quadratic model of the ST 22 C 01 data was used for determination of the EC3.
- A fitted quadratic equation was used to determine the concentration of isoeugenol required to elicit a stimulation index of 3 (EC3).
- All calculations were performed using Microsoft Excel and SAS, version 6.12. PROCs GLM, FREQ, NPARI WAY, and MEANS were utilized.
Positive control results:
The 5% concentration of isoeugenol (SI = 14.7) resulted in a group SI greater than 3. The calculated EC3 for isoeugenol was 1.54%, corresponding to an EC3 potency value of 385 µg/cm^2. Based on the results from this study, the positive control isoeugenol would be classified as a moderate sensitizer (potency value range of 100-1000 µg/cm^2. This is consistent with previously reported results (Gerberick et aI., 2001).
Key result
Parameter:
SI
Value:
0.8
Variability:
SE 0.2
Test group / Remarks:
1% acetone/olive oil (4:1 v/v)
Key result
Parameter:
SI
Value:
11.3
Variability:
SE 2.8
Test group / Remarks:
5% acetone/olive oil (4:1 v/v)
Key result
Parameter:
SI
Value:
11.1
Variability:
SE 1.1
Test group / Remarks:
10% acetone/olive oil (4:1 v/v)
Key result
Parameter:
SI
Value:
7
Variability:
SE 0.9
Test group / Remarks:
20% acetone/olive oil (4:1 v/v)
Key result
Parameter:
SI
Value:
2.3
Variability:
SE 1.1
Test group / Remarks:
40% acetone/olive oil (4:1 v/v)
Key result
Parameter:
EC3
Value:
0.94
Remarks on result:
other: corresponds to an EC3 potency value of 235 µg/cm2
Cellular proliferation data / Observations:
STIMULATION INDEX
Stimulation index (as mean ± SE) for 1, 5, 10, 20 and 40 % in acetone/olive oil (4:1 v/v) were 0.8 ± 0.2, 11.3 ± 2.8, 11.1 ± 1.1, 7.0 ± 0.9 and 2.3 ± 1.1, respectively.

DISINTEGRATIONS PER MINUTE (DPM)
DPM (as mean ± SE) for 0 (vehicle), 1, 5, 10, 20 and 40 % in acetone/olive oil (4:1 v/v) were 25.6 ± 6.0, 19.4 ± 4.6, 288.4 ± 72.7, 283.8 ± 28.8, 178.9 ± 22.3 and 59.9 ± 27.8, respectively.
Analyses of the natural logarithm of DPM values for test item had the following results: Bartlett's Chi Square test for homogeneity was found to be non-significant (p = 0.2484), and a one-way analysis of variance was used using dose (p=0.0001). The Dunnett's t test was performed using an alpha of 0.05 and confirmed that test item at 5%, 10%, and 20% were significantly different from control.

EC3 CALCULATION
A substance is considered a sensitizer if at least one concentration of the test item results in a statistically significant 3-fold or greater stimulation index (SI). Three doses (5%, 10%, and 20%) of the test item had an SI that was statistically significantly greater than 3. Test item at 1% and 40% had SI < 3. The reason for the abrogation of the elevated SI response at 20% and 40% is not known.
A quadratic regression model of the data for test item, resulted in an EC3 = 0.94%, using the predicted regression equation SI =2.107 + (0.981 x Dose) - (0.0248 x Dose^2).

CLINICAL OBSERVATIONS:
All animals appeared healthy and there were no signs of irritation at the dosing site.

BODY WEIGHTS
There was no body weight loss in any of the animals dosed with test item.

None

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test item is classified as “Category 1A” strong skin sensitiser according to the Regulation EC No. 1272/2008 (CLP) and GHS since the EC3 value is < 2%.
Executive summary:

In a Local Lymph Node Assay (LLNA), groups of female CBA/J Hsd mice (5 females/group) were topically applied with test item at the dose concentrations of 1.0%, 5.0%, 10%, 20%, and 40% final concentration in acetone/olive oil (4:1) to the dorsum of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group was treated with 4:1 acetone:olive oil alone and a positive control group was treated with isoeugenol at the dose concentration of 0.5%, 1% and 5% in acetone/olive oil (4:1 v/v) in same manner to confirm the sensitivity and reliability of the test method. Three days after the final auricular application (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.

No mortality, irritation or other adverse toxic effects were noted in any of the animals.

 

Stimulation index (as mean ± SE) for 1, 5, 10, 20 and 40 % in acetone/olive oil (4:1 v/v) were 0.8 ± 0.2, 11.3 ± 2.8, 11.1 ± 1.1, 7.0 ± 0.9 and 2.3 ± 1.1, respectively. DPM (as mean ± SE) for 0 (vehicle), 1, 5, 10, 20 and 40 % in acetone/olive oil (4:1 v/v) were 25.6 ± 6.0, 19.4 ± 4.6, 288.4 ± 72.7, 283.8 ± 28.8, 178.9 ± 22.3 and 59.9 ± 27.8, respectively.

 

A substance is considered a sensitizer if at least one concentration of the test item results in a statistically significant 3-fold or greater stimulation index (SI). Three doses (5%, 10%, and 20%) of the test item had an SI that was statistically significantly greater than 3. Test item at 1% and 40% had SI < 3. The reason for the abrogation of the elevated SI response at 20% and 40% is not known. The calculated EC3 for test item was 0.94%.

 

In the case of the positive control, the 5% concentration of isoeugenol resulted in a group SI that was statistically significantly greater than SI=3.The calculated EC3 for isoeugenol was 1.54%.

 

Under the test conditions, test item is classified as “Category 1A” strong skin sensitiser according to the Regulation EC No. 1272/2008 (CLP) and 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, Rel.2). This Local Lymph Node Assay was conducted similarly to the OECD test guideline No 429 and in compliance with GLP.

Groups of female mice (5/group) were topically applied with test item at the dose concentrations of 1.0%, 5.0%, 10%, 20%, and 40% final concentration in acetone/olive oil (4:1) to the dorsum of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group was treated with 4:1 acetone:olive oil alone and a positive control group was treated with isoeugenol at the dose concentration of 0.5%, 1% and 5% in acetone/olive oil (4:1 v/v) in same manner to confirm the sensitivity and reliability of the test method.

The positive control, isoeugenol gave a calculated EC3 for isoeugenol was 1.54%. The test system was therefore considered to be valid. Stimulation index (as mean ± SE) for 1, 5, 10, 20 and 40 % test material in acetone/olive oil (4:1 v/v) were 0.8 ± 0.2, 11.3 ± 2.8, 11.1 ± 1.1, 7.0 ± 0.9 and 2.3 ± 1.1, respectively. DPM (as mean ± SE) for 0 (vehicle), 1, 5, 10, 20 and 40 % in acetone/olive oil (4:1 v/v) were 25.6 ± 6.0, 19.4 ± 4.6, 288.4 ± 72.7, 283.8 ± 28.8, 178.9 ± 22.3 and 59.9 ± 27.8, respectively. No mortality, irritation or other adverse toxic effects were noted in any of the animals. Test item at 1% and 40% had SI < 3. The reason for the abrogation of the elevated SI response at 20% and 40% is not known.

The calculated EC3 for test item was 0.94%.

Under the test conditions, test item is a strong skin sensitiser.

A human repeated insult patch test in human has been performed with the substance at 0.5% in ethanol/diethyl phthalate (1:3) (HRL, 2015). Under the conditions employed in this study, there was no evidence of sensitisation to the test material.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Under the REACH regulation there is no legal standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitisation. In addition, no validated or widely recognised in vitro or in vivo test methods specific to respiratory sensitisation are available yet. No human or animal data are available on the source or on the target substances to address respiratory sensitisation. No alert for respiratory sensitisation were found by the OECD QSAR Toolbox.

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. 1A, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, since EC3 was < 2% in a LLNA.

No direct scientific data are available on the substance to address respiratory sensitisation.