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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An LLNA is not performed because other reliable information is available.

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl rel-acetate
Molecular formula:
C12H22O2
IUPAC Name:
(1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl rel-acetate
Constituent 2
Chemical structure
Reference substance name:
(1R,2R)‐2,6,6‐trimethylcycloheptyl rel‐acetate
Molecular formula:
C12H22O2
IUPAC Name:
(1R,2R)‐2,6,6‐trimethylcycloheptyl rel‐acetate
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Sex: male
- Age at study initiation: 4 – 6 weeks
- Weight on arrival: 400 ± 50 g
- Housing: The guinea-pigs were housed in suspended cages with wire mesh floors
- Diet: A vitamin C enriched guinea-pig diet FD1 was provided ad libitum. Hay was given once weekly.
- Water: tap water was given ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21
- Humidity: not controlled but was recorded daily using a wet/dry bulb thermometer
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: March 1981

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
Injection: 0.1 mL
Treatment: adjuvant/water for injection 1:1 (v/v)
Control: adjuvant/water 1:1 (v/v)
Day(s)/duration:
Day 0: First injection of the three pairs of intradermal injections
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
Injection: 0.1 mL
Treatment: 10 % test substance in liquid paraffin
Control: 10 % test substance in paraffin oil
Day(s)/duration:
Day 0: Second injection of the three pairs of intradermal injections
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
Injection: 0.1 mL
Treatment: 10 % test substance in an adjuvant/liquid paraffin oil mixture 1:1 (v/v)
Day(s)/duration:
Day 0: Third injection of the three pairs of intradermal injections
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Patch: 2x4 cm; occlusive dressing
Treatment: 100% test substance
Day(s)/duration:
Day 8: occlusive dressing for 48 hours
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Patch: 2x2 cm; occlusive dressing
Both treatment and control: 20% v/v test substance in liquid paraffin (anterior site on flank), 10% v/v liquid paraffin (posterior site on flank)
Day(s)/duration:
Day 15: occlusive dressing for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
15 (test animals) / 15 (control animals)
Details on study design:
TREATMENT PROCEDURE
PRELIMINARY INVESTIGATIONS
The intradermal and topical irritancy of a range of dilutions of the test substance in liquid paraffin was investigated to identify (a) irritant test substance concentrations suitable for the induction phase of the main study and (b) non-irritant concentrations by the topical route of administration for the challenge phase.

The following concentrations of the test substance were selected:
Induction:
- Intradermal injection: 10% v/v in liquid paraffin.
- Topical application: test substance, as supplied.
Challenge
- 20% and 10% v/v in liquid paraffin

MAIN STUDY
The procedure may be considered in two parts, Induction and Challenge.

INDUCTION EXPOSURE
Induction intradermal injections - test animals
A 4 x 6 cm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made simultaneously into this area. Injectables were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for injection.
2. Test substance, 10% v/v in liquid paraffin.
3. Test substance, 10% v/v in a 50 : 50 mixture of Freund's complete adjuvant and liquid paraffin.

Induction topical application - test animals
One week after the injections, the same 4 x 6 cm interscapular area was clipped and shaved free of hair. A 2 x 4 cm patch of Whatman No. 3 MM paper was saturated with test substance, as supplied. The patch was placed on the skin and covered by a length of impermeable plastic adhesive tape (5 cm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage ("Elastoplast" 5 cm width ) wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.

Induction - control animals
Control animals: During the induction period the control animals were treated similarly to the test animals with the exception that the test compound was omitted from the intradermal injections and topical application.

CHALLENGE EXPOSURE
Challenge - control and test animals
The animals were challenged topically two weeks after the induction period using the test substance, 20 % and 10 % v/v in liquid paraffin. The guinea-pigs of the control group were similarly challenged at this time.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 2 x 2 cm Whatman No. 3 MM paper was saturated with approximately 0.2 ml of the test substance, 20 % v/v in liquid paraffin and applied to an anterior site on the flank. The test substance, 10% v/v in liquid paraffin was applied in a similar fashion to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".


OBSERVATIONS
Reading challenge reactions
The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches. Reactions were scored according to the arbitrary scale given in Table 1 of "Any other information materials and methods incl. tables".

Interpretation of results
Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
10 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
10 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
20 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
20 % v/v test substance in liquid paraffin
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

ANTERIOR SITE, EXPOSED TO THE TEST SUBSTANCE, 20 % V/V IN LIQUID PARAFFIN.

Test animals

One guinea-pig died prior to the challenge application. Death was not considered to be related to treatment with the test substance. Slight to well-defined dermal reactions were seen throughout the observation period in four guinea-pigs, and another one at the 48 and 72 hours readings. The skin reactions were localised (restricted to a small area of the challenge site) on most occasions. No dermal reactions were recorded in the remaining nine animals.

 

Control animals

One guinea-pig was in poor condition and was killed prior to the topical induction application. Slight or well-defined dermal reactions were seen at the 24 hours reading in eight animals. The skin reactions, which were localised on most occasions, persisted at the 48 hours reading in one guinea-pig and throughout the observation period in four other guinea-pigs. No erythema or oedema was recorded in the remaining six animals.

POSTERIOR SITE, EXPOSED TO THE TEST SUBSTANCE, 10 % V/V IN LIQUID PARAFFIN

Test animals

Slight, localised erythema was observed at the 24 hours reading in three guinea-pigs numbers and persisted at the 48 hours reading in guinea-pig one of them. A further two guinea-pigs developed slight, localised erythema at the 48 hours reading only. No erythema or oedema was seen in the remaining nine animals.

Control animals

Slight or well-defined, localised erythema was seen at the 24 hours reading in four animals. The skin reaction persisted at the 48 hours reading in one guinea-pig and throughout the observation period in two other guinea-pigs. No dermal reactions were recorded in the remaining ten animals.

Applicant's summary and conclusion

Interpretation of results:
other: not a skin sensitiser
Remarks:
in accordance with EU CLP (EC 1272/2008 and its amendments)
Conclusions:
The substance is not a skin sensitiser in the GPMT test (similar to OECD TG 406).
Executive summary:

A Guinea pig maximisation test was performed similar to OECD TG 406. Fifteen test and fifteen control animals were used. Induction consisted of two stages, intradermal injection followed one week later by a 48 hour occluded patch application. Based on the results of a preliminary study dose levels selected were as follows. A total of 6 intradermal injections were administered: 2 injections of 0.1 mL of 50 % Freund's Complete Adjuvant, 2 injections of 0.1 mL of a 10 % solution of the test material in liquid paraffin and 2 injections of 0.1 mL of a 10 % suspension of the test material in Freund's Complete Adjuvant and liquid paraffin (1:1). The topical induction concentration was 100 %. In the challenge phase, the substance was applied topically at 10 and 20 % (v/v) in liquid paraffin. Erythema and oedema were scored 24, 48 and 72 hours after the challenge. The sensitising potential of the substance was classified according to the grading of Magnusson and Kligman. None of the animals had a positive reaction to the test substance. The substance is, therefore, not a skin sensitiser.