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Description of key information

A LLNA is available on 3,3,5-trimethylcyclohexyl acrylate to evaluate the skin sensitising potential.

Based on this study, 3,3,5-trimethylcyclohexyl acrylate is considered to be a skin sensitizer with a EC1.4 of 5.14%.

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:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
yes
Remarks:
no radioactivity used
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Elevage Janvier Labs
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9-11 week old
- Weight at study initiation: 18-25 g
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%):30-70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25, 50 and 100%
No. of animals per dose:
4 females / dose
Details on study design:
Preliminary study : As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at 100% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from Day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on Day 1, Day 3 and on Day 6. The body weight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.

Main study : The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days ( Days 1, 2 and 3). The test item formulation was administerred using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

Clinical observations:
All animals were observed daily on Days 1, 2, 3, 4, 5 and 6. The bodyweight pf each mouse was recorded on day 1 (prior to dosing) and Day 6 (prior to termination).
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses. On day 1 and day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item) was noted.

Determination of the EC1.4 value : The EC1.4 value (theoritical concentration resulting in a SI value of 1.4) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold.
For cell count indices such cut-off values are much lower: 1.4 times increase of stimulation index. This in understandable by the facts that cell count indices have : lower individual variance compared to 3H-thymidine incorporation, and lower maximum stimulation indices compared to radioactive labelling.

Interpretation of the results (according to CLP): If at least one concentration of the test item results in a SI > or = 3, the test item is considered to be sensitizer.
Positive control substance(s):
other: no
Statistics:
no
Positive control results:
n/a
Key result
Parameter:
EC3
Value:
5.14
Parameter:
SI
Value:
1.38
Test group / Remarks:
5%
Parameter:
SI
Value:
2.09
Test group / Remarks:
10%
Parameter:
SI
Value:
3.01
Test group / Remarks:
25%
Parameter:
SI
Value:
3.91
Test group / Remarks:
50%
Parameter:
SI
Value:
5.4
Test group / Remarks:
100%
Cellular proliferation data / Observations:
PRELIMINARY SCREENING TEST
No mortality and no signs of systemic toxicity were noted.
No cutaneous reactions were recorded at the concentration of 100%. Therefore this concentration was chosen as the highest concentration for the main study.

MAIN TEST
No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.
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.

A stimulation index (SI) cof more than 1.4 was recorded with the four higher tested concentrations.
The SI calculated by pooled approach was 1.38, 2.09, 3.01, 3.91 and 5.40 for the treated groups at 5%, 10%, 25%, 50% and 100% respectively.
The EC1.4 determined by linear regression was 5.14%

Dryness of the skin was noted in one animal treated at 100% on D5. No other cutaneous reaction was noted during the study.
No significant increase in ear thickness and in ear weight was noted in animals treated at 5%, 10%, 25%, 50% and 100%. The test item has to be considered as non-irritant at these concentrations.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, in view of these results, under these experimental conditions, the test item 3,3,5 -trimethylcyclohexyl acrylate has to be classified as a sensitizer.
Executive summary:

The test was performed to assess the skin sensitisaition potential of the test item 3,3,5 -trimethylcyclohexyl acrylate in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

Five groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 µL (25 µL per ear) of the test item undiluted (100%) and diluted in acetone/olive oil (4:1, v/v) (AOO) at concentrations of 50%, 25%, 10% and 5%. A further group of 4 animals was treated with acetone/olive oil (4:1, v/v) (AOO).

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according to the OECD test guideline no.429 (2010).

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No significant increases in ear thickness and in ear weight was noted in animals treated at 5%, 10%, 25%, 50% and 100%. The test item has to be considered as non-irritant at these concentrations.

The stimulation index (SI) calculated by pooled approach was 1.38, 2.09, 3.01, 3.91 and 5.40 for the treated groups at 5%, 10%, 25%, 50% and 100%, respectively. The EC1.4 determined by linear regression was 5.14%.

In conclusion, in view of these results, under these experimental conditions, the test item 3,3,5 -trimethylcyclohexyl acrylate has to be classified as a sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test was performed to assess the skin sensitisaition potential of the test item 3,3,5 -trimethylcyclohexyl acrylate in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

Five groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 µL (25 µL per ear) of the test item undiluted (100%) and diluted in acetone/olive oil (4:1, v/v) (AOO) at concentrations of 50%, 25%, 10% and 5%. A further group of 4 animals was treated with acetone/olive oil (4:1, v/v) (AOO).

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according to the OECD test guideline no.429 (2010).

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No significant increases in ear thickness and in ear weight was noted in animals treated at 5%, 10%, 25%, 50% and 100%. The test item has to be considered as non-irritant at these concentrations.

The stimulation index (SI) calculated by pooled approach was 1.38, 2.09, 3.01, 3.91 and 5.40 for the treated groups at 5%, 10%, 25%, 50% and 100%, respectively. The EC1.4 determined by linear regression was 5.14%.

In conclusion, in view of these results, under these experimental conditions, the test item 3,3,5 -trimethylcyclohexyl acrylate has to be classified as a sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, 3,3,5 -trimethylcyclohexyl acrylate should be classified as skin sensitizer (Skin Sens.1B, H317) according to the Regulation EC n°1272/2008.

Justification: Positive results in the LLNA and EC1.4 > 2%