Registration Dossier

Administrative data

Description of key information

LLNA, Not sensitising (OECD 429, GLP, K, rel. 1)

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 2012-10-23 to 2012-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 429 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspected on 2012-07-10/ signed on 2012-11-30)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Individually housed, in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: : ad libitum (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK).
- Water: mains tap water ad libitum.
The diet and water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10%, 5% and 2.5%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: maximum attainable concentration = 10%
- Irritation: No signs of systemic toxicity, local irritation or irritation indicated by ≥25% increase in mean ear thickness noted at the maximum attainable concentration).
Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in acetone/olive oil 4:1.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: individual approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Additional investigation: measurement of ear thickness. The ear thickness of each ear was measured using an Oditest micrometer (Dyer, PA) immediately prior to and 1-hour post application of test item on Day 1 and 1-hour post application of the test item on Days 2 & 3. Additional measurements will be taken on Days 4, 5 & 6 (Day of lymph node harvest for the LLNA) to monitor for any changes in ear thickness. Group mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness of ≥ 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Criteria used to consider a positive response: The decision process with regard to identification of a positive response will include a SI of ≥ 3, together with consideration of dose-response, and where appropriate, statistical significance. Any test item failing to produce a SI of ≥ 3 at all test concentrations was not regarded as a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
All formulations were used within two hours of preparation and will be assumed to be stable for this period. The concentration and homogeneity of the formulations were not determined by analysis.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Positive control results:
A group of five animals was treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 6.29, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.46
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.09
Test group / Remarks:
10%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% : mean DPM / animal = 2918.93.
2.5%: mean DPM / animal = 2050.60.
5%: mean DPM / animal = 4271.47.
10%: mean DPM / animal = 3183.67

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated.

BODY WEIGHTS: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, ST 10 C 08 is not classified as a skin sensitiser according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of ST 10 C 08 in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity were noted at the maximum attainable concentration (10%), ST 10 C 08 concentration of 10% was selected as the highest dose to be investigated in the main test.

Three groups, each of five animals, were treated with 50 μl (25 μl per ear) of ST 10 C 08 as a solution in acetone/olive oil 4:1 at concentrations of 10%, 5% or 2.5% v/v for 3 consecutive days. A further group of five 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 3H-Methyl Thymidine.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 to 6.

The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration

Mean dpm/animal

Test / Control Ratio

Result

Vehicle

2918.93

N/A

N/A

2.5%

2050.60

0.70

Negative

5%

4271.47

1.46

Negative

10%

3183.67

1.09

Negative

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 6.29, when tested at 25 % v/v. The test system was therefore considered to be valid.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Under the test conditions, ST 10 C 08 is not classified as a skin sensitiser in the Local Lymph Node Assay according to 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:
no adverse effect observed (not sensitising)
Additional information:

A key study was identified (Harlan, 2013, rel.1). The study was conducted according to the OECD test guideline No 429 and in compliance with GLP. Following a preliminary screening test in which no clinical signs of toxicity were noted at the maximum attainable concentration (10%), ST 10 C 08 concentration of 10% was selected as the highest dose to be investigated in the main test. Three groups were treated with ST 10 C 08 as a solution in acetone/olive oil 4:1 at concentrations of 10%, 5% or 2.5% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

The irritant potential of ST 10 C 08 was assessed in parallel by measurement of ear thickness on days 1 to 6.

The Stimulation Index values were 0.70, 1.46 and 1.09 for the concentrations of 2.5%, 5% and 10%, respectivey.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 6.29, when tested at 25 % v/v. The test system was therefore considered to be valid.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Under the test conditions, ST 10 C 08 is not classified as a skin sensitiser in the Local Lymph Node Assay.

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 (CLP).

Self classification:

Based on the available data, no additional self-classification is proposed according to the CLP and the GHS.

No data was available for respiratory sensitisation. However, this substance is not a skin sensitizer, therefore according to Figure R.7.3 -2 of the Chapter R.7 (V 4.1 - October 2015) the chemical is not considered as a respiratory sensitizer.