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Diss Factsheets

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:
Between 14 April 1993 and 17 May 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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:
A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]
EC Number:
422-040-1
EC Name:
A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]
Cas Number:
426218-78-2
Molecular formula:
C16H26O
IUPAC Name:
1-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-2-oxabicyclo[2.2.2]octane; 3,6',6',6'a-tetramethyl-2',3'a,4',5',6',6'a-hexahydrospiro[cyclohexane-1,3'-cyclopenta[b]fura]-3-ene; 4,6',6',6'a-tetramethyl-2',3'a,4',5',6',6'a-hexahydrospiro[cyclohexane-1,3'-cyclopenta[b]fura]-3-ene; 5-methyl-1-(2,2,3-trimethylcyclopent-3-en-1-yl)-6-oxabicyclo[3.2.1]octane
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Fifteen healthy female nulliparous and non-pregnant albino guinea-pigs of the Dunkin/Hartley strain were obtained from D. Hall, Newchurch, Staffordshire, England.
The animals were in the weight range of 325 to 366 g on arrival and approximately six to seven weeks of age. All the guinea-pigs were acclimatised to the experimental environment for 12 days prior to allocation to the main study.
An additional six animals, from the same supplier, were used for the preliminary investigations
The animals on the main study were allocated without conscious bias to two groups as follows:
Group │Number of animals │ Animal numbers
Control animals │ 5 │ 1275 to 1279
Test animals │ 10 │ 1290 to 1299
The guinea-pigs were housed in groups of ten in suspended metal cages with wire mesh floors in Building R17 Room 13.
A vitamin C enriched guinea-pig diet FD 1 and drinking water were provided ad libitum. Hay was given weekly.
The batch of diet used for the study was not analysed for nutrients, possible contaminants or microorganisms.
However, other batches of diet are periodically tested, by the Supplier, and were found to be within the set limits.
Results of routine physical and chemical examination of drinking water at source, as conducted usually weekly by the supplier, are made available to Huntingdon Research Centre Ltd. as quarterly summaries.
Animal room temperature was maintained at approximately 21°C and relative humidity at 30 - 70% .
These environmental parameters were recorded daily. Air exchange was maintained at approximately 15 air changes per hour and lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period.
Each animal was identified by ear tattoo number. This number was unique within the HRC Industrial Toxicology Department throughout the duration of the study. Each cage was identified by a coloured label displaying the study schedul enumber, animal numbers and the initials of the Study Director and Home Office licensee.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D
Concentration / amount:
Intradermal injection: 7.5% (v/v) in Alembicol D
Topical application: as supplied
Day(s)/duration:
Topical application: 48 hours
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
As supplied and 50% v/v in Alembicol D
No. of animals per dose:
10 animals for the main test, 5 control animals
Details on study design:
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.

Selection of concentrations of test substance for the main study
Based on the results of the preliminary investigations, the following concentrations of the substance were selected:
Induction intradermal injection - 7. 5% v/v in Alembicol D
Induction topical application - as supplied
Topical challenge - as supplied and 50% v/v in Alembicol D

Main study
The procedure may be considered in two parts, Induction and Challenge.

Induction
Induction intradermal injections - test animals
A 40 x 60 mm 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 into a 2 x 4 cm area within the clipped area.
Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation (ph.Eur.) .
2. Test substance, 7.5% v/v in Alembicol D.
3. Test substance, 7.5% v/v in a 50:50 mixture of Freund's complete adjuvant and Alembicol D.
Induction topical application - test animals
The preliminary investigations indicated that the maximum practical concentration of the test substance for topical application (as supplied) did not produce skin irritation. Therefore, six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.2 mI per site of 10% w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch of What man No. 3 paper was saturated with approximately 0.4 ml of the substance, as supplied. The patch was placed on the skin <;If the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm").
This in turn was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") 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
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

Challenge
Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction application using the test substance, as supplied and 50% v/v in Alembicol D.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig.
A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of the substance, as supplied and applied to an anterior site on the flank. The test substance, 50% v/v in Alembicol D was applied in a similar manner 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
Clinical signs: All animals were observed daily for signs of ill health or toxicity.
Bodyweight: The bodyweight of each guinea-pig on the main study was recorded on Day 1 (day of intradermal injections) and on the last day observations were made of dermal responses to the challenge applications.
Dermal responses: The dermal reactions resul ting from intradermal injection and topical application on the preliminary study, and topical application at the challenge were assessed using the following numerical system.
Positive control substance(s):
yes

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
The substance as supplied
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
The substance as supplied
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in one animal
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
The substance as supplied
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in one animal
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
The substance as supplied
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
The substance as supplied
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
The substance as supplied
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Group:
positive control
Dose level:
As supplied
No. with + reactions:
29
Total no. in group:
30

Any other information on results 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. A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group. If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive. A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group. Clinical signs: No signs of ill health or toxicity were recorded. Body weight: Anticipated bodyweight increases were recorded for all guinea-pigs over the period of the study. Induction: Dermal reactions were seen following the induction applications. Intradermal injections Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving the test substance, 7. 5 % v/v in Alembicol D and very slight irritation was observed in control animals receiving Alembicol D. Topical application: Very slight erythema was observed in test animals following topical application with the test substance, as supplied. Very slight erythema was also seen in the control guinea-pigs. Challenge: No reactions were seen in any of the test animals or controls except that in one test animal dryness and sloughing of the epidermis was evident at 48 and 72 hours after the challenge application. The degree and duration of this reaction was not considered to represent evidence of skin sensitisation.

Applicant's summary and conclusion

Interpretation of results:
other: Not sensitising
Remarks:
in accordance with EU CLP (1272/2008 and its amendments)
Conclusions:
The substance is not a skin sensitiser in the Guinea Pig Maximisation Test (OECD TG 406).
Executive summary:

The substance was tested in a guinea pig maximisation test (OECD TG 406) using ten animals and five control animals. The concentrations were selected based on the results of a preliminary study. In the induction phase, the substance was tested both through intradermal injections at a concentration of 7.5% in Alembicol D as well as through topical application using the test substance as supplied. In the challenge phase, the substance was tested as supplied and at a concentration of 50% in Alembicol D. No signs of illness or toxicity were observed. Bodyweight increased as expected during the period of the study. In the induction phase, necrosis was observed in both test and control animals after intradermal injections of Freund's Complete Adjuvant. Slight irritation was seen in test animals after receiving the test substance, 7.5% v/v in Alembicol D and very slight irritation was seen in control animals after receiving Alembicol D without test substance. Topical application of the substance as supplied resulted in very slight erythema in test animals. Slight erythema was also observed in control animals. In the challenge phase, no reactions were observed in any of the test or control animals except for dryness and sloughing of the epidermis in one test animal 48 and 72 hours after the challenge phase. The degree and duration of this reaction was not considered to represent evidence of skin sensitisation. Based on the results obtained in this study, the substance is not sensitising to the skin.