Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Endpoint summary

Description of key information

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation" - Magnusson and Kligman Maximisation Test.

Twenty test and ten control animals were used for the main study.

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as

follows :

Intradermal Induction: 25% (w/v) in arachis o i l B.P.

Topical Induction : undiluted as supplied

Topical Challenge : 75% and 50% (v/v) in arachis oil B.P.

The test material produced a 10% (2/20) sensitisation rate.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Information provided in SNIF file. Study was conducted prior to the mandatory requirement to conduct LLNA over in vivo methods

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Specification
Thirty-eight female, albino Dunkin-Hartley guinea pigs were supplied by David Hall Limited, Burton-on-Trent, Staffordshire, U.K. At the start of the main study the animals weighed 316 -438g, and were approximately eight to twelve weeks old. After a minimum acclimatisation period of five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen.
Husbandry
The animals were housed in groups of up to three in solid-floor polypropylene cages furnished with softwood shavings. Free access to mains tap water and food (Guinea Pig FDl Diet, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.The animal room was maintained at a temperature of 18 - 21°C and relative humidity of 32 - 56%. On occasions the temperature was below the limit specified in the protocol (19°C). This was considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

25% (w/v) in arachis oil B.P.
25% (w/v) in a mixture of Freund's
Complete Adjuvant plus arachis oil B.P. (1:1)

Day(s)/duration:

Day 7: intradermal dose

Adequacy of induction:

other: The highest concentration that did not cause local necrosis, ulceration or systemic toxicity.

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

Undiluted as supplied

Day(s)/duration:

Day 0

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

75% (v/v) in arachis oil B.P.

Day(s)/duration:

Challenge dose on day 21

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

50% (v/v) in arachis oil B.P.

Day(s)/duration:

Challenge dose on day 21

Adequacy of challenge:

other: The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

No. of animals per dose:

Number of animals in test group: 20
Number of animals in negative control group: 10

Details on study design:

The method used for assessing the sensitising properties of the test material was based on the Guinea Pig Maximisation test of Magnusson B. & Kligman A.M., J. Invest. Dermatol. (1969) 52: 268 - 276.
Selection of Concentrations for Main Study (Sighting Tests)
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material . The procedures were as follows:
a) Selection of Concentration for Intradermal Induction
Four animals were intradermally injected with preparations of test material (1%, 5%, 10% or 25% w/v in arachis oil B.P.). The highest concentration that did not cause local necrosis, ulceration or systemic toxicity, was selected for the intradermal induction stage of the main study.
b) Selection of Concentration for Topical InductionTwo guinea pigs (intradermally injected with Freund's Complete Adjuvant ten days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in arachis oil B.P.).The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.Selection of Concentration for Topical Challenge
The undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in arachis oil B.P.) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.
Main Study
A group of thirty guinea pigs was used for the main study, twenty test and ten control. The bodyweight of each animal was recorded at the start and end of the study.
Two main procedures were involved in the maximisation test;
(a) an induction of a response and (b) a challenge of that response.a) InductionInduction of the Test Animals: Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm X 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line. The injections are reported above. One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the undiluted test material. The undiluted test material (0.2 - 0.3 ml) was applied on filter paper (WHATMAN No.4: approximate size 40 mm X 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm X 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm X 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.Erythematous reactions were quantified one and twenty-four hours following removal of the patches using the 0 - 3 scale: 0 - no reaction / l - scattered mild redness / 2 - moderate and diffuse redness / 3 - intense redness and swelling Induction of the Control Animals : Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were as reported above. The topical applications followed the same procedure as for the test animals except that nothing was applied to the filter paper.Skin reactions were quantified as for the test animals.
b) Challenge
Shortly before treatment on Day 21, an area, approximately 50 mm X 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.A quantity of 0.1 - 0.2 mL of the test material formulation (75% v/v in arachis oil B.P.) was applied to the shorn right flank of each animal on a square of filter paper (WHATMAN No.4: approximate size 20 mm X 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm X 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% (v/v) in arachis oil B.P. was also similarly applied to a separate skin site on the right shorn flank. The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm X 75 mm) wound in a double layer around the torso of each animal.After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. The vehicle sites were similarly swabbed. The position of the treatment sites was identified by using a black indelible marker-pen. Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
c) Evaluation of Skin Reactions
Approximately 24 and 48 hours after challenge dressing removal erythematous reactions were quantified using the four-point scale.

Challenge controls:

The vehicle alone was applied as a control.

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 %

No. with + reactions:

1

Total no. in group:

20

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

1

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75 %

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

75 %

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

75 %

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

a) Skin Reactions Observed After Topical Induction

Scattered mild redness was elicited by the test material.

b) Skin Reactions Observed After Topical Challenqe

75% (v/v) in Arachis Oil B.P.

A positive skin response (redness grade 1) was noted at the challenge site of one test group animal at the 24-hour observation.

No skin reactions were noted at the challenge sites of test group animals at the 48-hour observation and at the challenge sites of control group animals at the 24 and 48-hour observations.

50% (v/v) in Arachis Oil B.P.

A positive skin response (redness grade 1) was noted at the challenge site of one test group animal at the 24-hour observation.

No skin reactions were noted at the challenge sites of test group animals at the 48-hour observation and at the challenge sites of control group animals at the 24 and 48-hour observations.

Vehicle Control

No skin reactions were noted at the vehicle control sites of the test or control group animals at the 24 and 48-hour observations.

Bodyweight

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material, ET-344 SP, produced a 10% (2/20) sensitisation rate. The test material does not need to be classified according to the CLP criteria.

Executive summary:

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing o f Chemicals (1981) No. 406 "Skin Sensitisation" - Magnusson and Kligman Maximisation Test referenced as Method B6 in Commission Directive 84/449/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Twenty test and ten control animals were used for the main study.

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as

follows :

Intradermal Induction: 25% (w/v) in arachis o i l B.P.

Topical Induction : undiluted as supplied

Topical Challenge : 75% and 50% (v/v) in arachis oil B.P.

The test material produced a 10% (2/20) sensitisation rate.

The test mateial does not need to be classified according to the CLP criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the Guinea Pig Maximisation Test, the substance is not classified as a skin sensitiser in accordance with the CLP Regulation.