Registration Dossier

Administrative data

Description of key information

LLNA, 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 2012-09-04 to 2012-09-18
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 standards
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
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:
100%, 50% and 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Procedure: The mouse was treated by daily application of 25 µl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited
biological relevance to the endpoint of sensitisation.
- Results: No signs of systemic toxicity, visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

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 thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6 A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
- 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 will not be regarded as a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
All formulations will be used within two hours of preparation and will be assumed to be stable for this period. The concentration, homogeneity or stability of the formulations was 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 = 5.76, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
EC3
Value:
81
Test group / Remarks:
%
Key result
Parameter:
SI
Value:
1.39
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.98
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
3.61
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% : mean DPM / animal = 3864.68.
25%: mean DPM / animal = 5383.15.
50%: mean DPM / animal = 7649.61.
100%: mean DPM / animal = 13956.12.

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:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, the test item is classified as a skin sensitizer in the Local Lymph Node Assay Skin Sens. 1B (H317: May cause an allergic skin reaction) 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 the test item 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 a test concentration of 100%, this concentration of 100% 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 the test item as a solution in acetone/olive oil 4:1 at concentrations of 100%, 50% or 25% 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

3864.68

N/A

N/A

25%

5383.15

1.39

Negative

50%

7649.61

1.98

Negative

100%

13956.12

3.61

Positive

Since the data indicated a positive response, the EC3 value (concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation) was calculated and determined to be 81%.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 5.76, 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, the test item is classified as a skin sensitizer in the Local Lymph Node Assay Skin Sens. 1B (H317: May cause an allergic skin reaction) 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:
adverse effect observed (sensitising)
Additional information:

A FCAT was available on the substance (Roche, 1982). However it did not meet criteria of today's standard method (basis for the selection of induction and challenge concentrations was not reported, positive controls were not included). Therefore a new in vivo test was performed (Harlan, 2012, rel.1).

This new LLNA 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 a test concentration of 100%, this concentration of 100% was selected as the highest dose to be investigated in the main test. Three groups, each of five animals, were treated with 50μl (25μl per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 100%, 50% or 25% 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 of3H-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 are as follows: 1.39, 1.98 and 3.61 for the concentrations of 25%, 50% and 100%, respectively. Since the data indicated a positive response, the EC3 was calculated and determined to be 81%. The historical positive control,α-Hexylcinnamaldehyde, gave a SI of 5.76, 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, 4 -tert-butylcyclohexyl acetate is classified as a skin sensitizer in the Local Lymph Node Assay.

Two HRIPT conducted on 31 and 33 volunteers were available on 4 -tert-butylcyclohexyl acetate. Both showed no evidence of sensitization.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP)

Self classification:

Based on the available information additional self-classification is proposed Skin Sens. 1B (H317: May cause an allergic skin reaction) according to the CLP and the GHS.

No information is available regarding respiratory sensitisation.