Cyclohexanone, 2-methyl-4-(1,1,2-trimethylpropyl)-, cis-

Endpoint summary

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:

29 February - 6 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to EU method and in compliance with Good Laboratory Practice.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

The animals were female (CBA/J@Rj) strain mice from Elevage Janvier, Le Genest-Saint-Isle, France. 8 weeks old animals, nulliparous and nonpregnant, were selected having body weights in the range of 21.1-23.8 g.
A controlled environment was maintained in the room with optimal conditions of approximately 15/h air changes, a temperature of 22±3ºC, a relative humidity of 30-70% and a 12 hour artificial fluorescent light/12 hour dark cycle per day (light cycle 7.00-19.00h).
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
Drinking water (tap) and food were supplied freely.
After an acclimatisation period of 5 days, animals were randomly selected and marked on the tail.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Three dose groups of four animals per group were treated for three consecutive days with the test item. The concentrations of the dose groups were 100% (undiluted), 50% (v/v), 25% (v/v) and 0% (negative control).

No. of animals per dose:

4 mice per dose group.

Details on study design:

RANGE FINDING TESTS:
A preliminary screening test was performed treating one mouse daily by application of 25 μL of the test item at 100% to the dorsal surface of each ear for three consecutive days. The preliminary test was a screening by daily clinical observation, body weigth evolution measured on Day 1 (prior to dosing) and Day 6, ear thickness measurement on Day 1, 3 and 6, the weight of the lymph nodes and the cell count.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Criteria used to consider a positive response: If the EC1.4 for any of the treated groups ≤100%, the test item is classified as a sensitiser. EC1.4 is the theoretical concentration resulting in a SI value of 1.4.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the test compound (and negative control) was daily applied to the entire dorsal surface of each ear of each mouse using the tip of a micro pipette for three consecutive days. On day 6, the mice were killed with an intraperitoneal injection of sodium pentobarbital. The draining Auricular lymph node were excised and a single cell suspension was prepared by pooling the lymph node cells of four mice through mechanical tissue disaggregation in 4ml of PBS containing 0.5% BSA into a well of a multi-well 6.
10µl of the cell suspension was diluted in physiological saline solution (NaCl 0.9%). The lymphocytes were counted using a Beckman Coulter Z2 cell counter. The lower and upper selected sizes were 5µm and 15µm resp.

The weight of the animals were recorded prior to exposure to the test compound and on day 6 prior to the intraperitoneal injection. The thickness of the right ear of the animals were measured using a micrometer prior to exposure on day 1, prior to the third exposure on day 3 and after the intraperitoneal injection. Punch biopsies of 8mm in diameter of the apical area of both ears were prepared and weighed on day 6 in order to assess the irritation potential. Any irritation reaction (erythema and oedema) was recorded in parallel and any other observation (dryness, presence of residual test item…) was noted.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The EC1.4 is determined by linear interpolation of the points in the stimulation index dose-response curve immediately above and below the 1.4 threshold.

Positive control results:

A study was performed to assess the sensitivity of the strain mouse with the known sensitiser (positive control) α-hexylcinnamaldehyde (95% pure, CAS 101-86-0). Three groups, each of four animals were treated with 50µl (25µl/ear) at concentrations of 5%, 10% and 25% as a solution in acetone/olive oil (4:1 v/v). A negative control group was included. The EC1.4 value of the positive control was 8.73%.

Key result

Parameter:

SI

Value:

2.86

Test group / Remarks:

25% test substance

Key result

Parameter:

SI

Value:

2.61

Test group / Remarks:

50% test substance

Key result

Parameter:

SI

Value:

3.21

Test group / Remarks:

100% test substance

Preliminary test

No mortality and no significant clinical abnormalities or increase in ear thickness was noted in the animal treated at 100%. The body weight on day 1 and 6 were 19.3 and 20.4 g resp. Ear weight on day 6 was 30.5 mg. The weight of the lymph nodes was 9.4 mg and the cell count 10.73·10⁶/ml. The test item is not considered as excessively irritant at the three concentrations. The concentration of 100% has therefore been choosen as the highest concentration for the main test.

Main test

No mortality and no signs of systematic toxicity were noted in test and control animals.

On day 6, a slight dryness to dryness was noted on the treatment site of all animals treated at 50% (4/4) and 100% (4/4).

Body weight evolution of the test animals between day 1-6 were comparable to the control group.

The EC1.4 can not be determined in this study since no interpolation is possible.

No significant increase in ear thickness was noted in animals treated at 25% and 50% (see Table II). Increase in ear thickness (+17.6%) and in ear weight (+47.1%) were noted on day 6 in animals dosed at 100%. Therefore, the test item must be considered as excessively irritant at this concentration.

Table I Cell count, stimulation index and calculation of EC1.4.

Group	concentration	Cell count (x10^6 cells/ml)	Stimulation Index	Result	EC1.4
1	0%	20.33	n.a.	n.a.	n.a
2	25%	58.13	2.86	Positive	n.a.
3	50%	53.14	2.61	Positive
4	100%	65.25	3.21	Positive

n.a.: not applicable

Table II Ear thickness & weight increases and the assessement of excessive local irritation.

Group	concentration	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessiveirritation
1	0%	8.7	n.a.	no
2	25%	4.1	3.1	no
3	50%	3.7	9.0	no
4	100%	17.6	47.1	yes

n.a.: not applicable

Interpretation of results:

sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The stimulation index is in excess of 1.4 at 25%, 50% and 100% and therefore test substance should be regarded as a skin sensitiser.

Executive summary:

The skin sensitisation potential of the test substance Iriswood was assessed according to OECD method 429, in the CBA/J strain mouse following topical application to the dorsal ear surface.

Three groups of four animals were treated for three consecutive days (day 1,2 and 3)with 50µl (25µ per ear) with the test compound in acetone/olive oil (4:1 v/v) at concentrations (v/v) of 100% (undiluted), 50%, 25% and 0% (negative control).

On day 6, the proliferation of the lymphocytes in the draining Auricular lymph nodes was determined by cell counting.

No mortality and no signs of systematic toxicity were found in all the animals at all concentrations. A slight dryness to dryness was noted on the treatment site of all animals treated at 50% and 100% on day 6. No significant thickness in ear thickness and ear weight were observed at 25% and 50%, but increased ear thickness and ear weight were noted on day 6 in animals dosed at 100%. The test substance was therefore considered as excessively irritant at 100%.

The stimulation index (SI) calculated by pooled approach was 2.86, 2.61 and 3.21 for the treated groups with test compound concentrations of 25%, 50% and 100%. The EC1.4 could not be determined by linear interpolation. Since the SI>1.4 at concentrations of 25%, 50% and 100%, the test substance is classified as a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:

Skin Sensitisation, Local Lymph Node Assay (EU Method B.42): Sensitising category 1.

Justification for selection of skin sensitisation endpoint:

Study performed on the substance according to EU method and in compliance with Good Laboratory Practice.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available