4-tert-butylcyclohexyl acetate

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

trans-4-tert-butylcyclohexyl acetate

EC Number:

217-598-6

EC Name:

trans-4-tert-butylcyclohexyl acetate

Cas Number:

1900-69-2

Molecular formula:

C12H22O2

IUPAC Name:

trans-4-tert-butylcyclohexyl acetate

Constituent 2

Reference substance name:

cis-4-tert-butylcyclohexyl acetate

EC Number:

233-881-7

EC Name:

cis-4-tert-butylcyclohexyl acetate

Cas Number:

10411-92-4

Molecular formula:

C12H22O2

IUPAC Name:

cis-4-tert-butylcyclohexyl acetate

Test material form:

liquid

Details on test material:

Description: Clear colorless liquid

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

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.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

81

Test group / Remarks:

%

Results 2

Key result

Parameter:

SI

Value:

1.39

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.98

Test group / Remarks:

50%

Results 4

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.

Applicant's summary and conclusion

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.