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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:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study following a method equivalent to a recognised guideline; minor deviation - rechallenge conducted 24h after first challenge; to account for the movement of occlusive dressing.
Remarks:
Expert assessment indicates this is not considered a significant deviation: may increase the response rate due to irritation-mediated effects resulting from a lack of rest period between dose applications. Results in a predisposition towards positive dermal responses in rechallenge considering vehicle and method of application.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Rechallenge conducted 24h after first challenge; to account for the movement of occlusive dressing. Not considered a significant deviation: may increase the response rate due to irritation-mediated effects from lack of rest period between dose application
Principles of method if other than guideline:
Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman (1970) in "Allergic Contact Dermatitis in the Guinea-Pig: Identification of contact allergens"; C.C. Thomas, USA.
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected June 1990 ; signature: October 1990
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was conducted prior to 11 October 2016 as stated under Commission Regulation (EU) 2017/706 and is used by read-across adaptation under Regulation (EC) 1907/2006: Annex XI: section 1.5 read-across.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
432-350-9
EC Name:
-
Cas Number:
81752-87-6
Molecular formula:
C11H18O2
IUPAC Name:
methyl 2,2-dimethyl-6-methylidenecyclohexanecarboxylate
Test material form:
other: liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Housing: group housed (groups of 10) in stainless steel cages.
- Diet (e.g. ad libitum): FD1, SQC ad libitum; certificate of analysis documented
- Water (e.g. ad libitum): tap water ad libitum; certificate of analysis documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22°C; air conditioned
- Humidity (%): 52 - 66 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: 24 July 1991 To: 26 August 1991

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: 'light liquid paraffin' (intradermal injection) and ethanol for topical induction booster and/or challenge
Concentration / amount:
Preliminary irritation testing: intradermal: 100% (undiluted),50, 25, 5 and 1% v/v; topical: 100% (undiluted), 50, 25 and 12.5% v/v in ethanol
25% in light paraffin was chosen for intradermal induction (highest acceptable localised response concentration; non-irritating)
100% was found to be highest non-irritant topical induction. 50% in ethanol was selected for challenge concentrations

Main study conditions:
Induction:
- Intradermal: 25% test material in light liquid paraffin
- Topical: 100% test material (undiluted) in ethanol
- Challenge: 50% in ethanol
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: 'light liquid paraffin' (intradermal injection) and ethanol for topical induction booster and/or challenge
Concentration / amount:
Preliminary irritation testing: intradermal: 100% (undiluted),50, 25, 5 and 1% v/v; topical: 100% (undiluted), 50, 25 and 12.5% v/v in ethanol
25% in light paraffin was chosen for intradermal induction (highest acceptable localised response concentration; non-irritating)
100% was found to be highest non-irritant topical induction. 50% in ethanol was selected for challenge concentrations

Main study conditions:
Induction:
- Intradermal: 25% test material in light liquid paraffin
- Topical: 100% test material (undiluted) in ethanol
- Challenge: 50% in ethanol
No. of animals per dose:
Test group: 20; Control group: 10
Details on study design:
RANGE FINDING TESTS:
Preliminary Investigations: An intradermal injection concentration ranging study was performed to determine a suitable concentration of the test item for the intradermal induction stage of the main study. 0.1 ml aliquots of undiluted test article and 50%, 25%, 10%, 5%, and 1% v/v concentrations of the test article in light liquid paraffin were injected intradermally into the flanks of one guinea pig. Observations made daily for 6 days and the response at each injection site noted. A 25% v/v concentration test article was chosen for the main intradermal injection stage, this being the highest concentration caused an acceptable localised response during the observation period. In the topical range finding study the maximum non-irritating concentration and the minimum irritant concentration of the test item was determined with an ethanol vehicle using organisms previously treated with Freund's Complete Adjuvant. Four concentrations of the test item were used. These were undiluted test article and 50%, 25% and 12.5% v/v concentrations of the supplied test article in ethanol.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal induction: intradermal injections of 0.1ml pairs of injections of the following were made: 50% aqueous Complete Freund's Adjuvant alone, 25% v/v test item in light liquid paraffin and undiluted test item with equal volume of FCA in distilled water and with final test item concentration 25% v/v were injected into the clipped area dorsal area of each of 20 guinea pigs. The control group similarly received 50% v/v FCA in distilled water. Light liquid paraffin vehicle only. Then light liquid paraffin with equal volume of FCA in distilled water.

Topical Application: Seven days after intradermal injections, the topical induction phase was conducted. Prior to the topical induction, the injection sites were clipped free of hair. 0.4 ml of the test item undiluted were attached at the injection sites using an occlusive dressing. The control groups were similarly treated. The dressing was left in place for 48 hours.

B. CHALLENGE EXPOSURE
14 days after the induction period, the fur was clipped and the test item undiluted (0.1 ml) was applied to a 2 x 2 cm Whatman No, 3 MM paper in a similar fashion to that used for topical induction application. The right flank was treated with 50% v/v in ethanol similarly with occlusive dressing for 24 hours. On removing the dressings, 24 hours later, it was noted that on 11 animals the patches used for application of the undiluted test article had become detached from the intended application sites. At this stage it was decided to proceed with the assessment of all application sites, at 24 and 48 hour intervals, after removal of the dressings and then perform a second challenge.
A second challenge was performed 24 hours after completion of the first challenge. Approximately 3 hours after application patches were again observed being dislodged from beneath the occlusive dressings. In an attempt to remedy this all patches and dressings were removed and fresh patches applied and secured with additional lengths of adhesive bandage. These patches remained in position for the required 24 hours. After removal of the patches and dressings the application sites were assessed in the same manner as the first challenge.
The scoring system for responses was fully described within the study report.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
other: rechallenge 1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
other: rechallenge 1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 20.0.
Reading:
other: rechallenge 2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
other: rechallenge 2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

All organisms gained body weight during the course of the study. There were no reports of any clinical signs. All responses were limited to score 1 during rechallenge only and the response rate was 10% in the 50%v/v rechallenge only. No responses were seen in the 100% v/v (undiluted) challenge or rechallenge.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study used, the test material is not considered to be a contact sensitizer.
Executive summary:

The study was performed according to a method equivalent to guideline OECD TG 406 consistent with Magnusson-Kligman maximisation test to assess the skin sensitisation potential of the test substance. The study was conducted using a preliminary irritation screen, a two-stage induction phase and a challenge phase with a rechallenge. Preliminary irritation testing was used to define the doses in the induction phases of the study and the challenge phase of the study. The first stage of the induction phase involved intradermal injections of 0.1ml pairs of injections of: 50% aqueous Complete Freund's Adjuvant alone, 25% v/v test item in light liquid paraffin and undiluted test item with equal volume of FCA in distilled water and with final test item concentration 25% v/v were injected into the clipped area dorsal area of each of 20 guinea pigs. The control group similarly received 50% v/v FCA in distilled water. Light liquid paraffin vehicle only. Then light liquid paraffin with equal volume of FCA in distilled water. After 7 days the second stage of the induction phase entailed the topical application of 0.4 ml of the test item undiluted attached at the injection sites using an occlusive dressing. The control groups were similarly treated. The dressing was left in place for 48 hours. 14 days after the induction period, a challenge dose of the undiluted test substance selected for application to the left flank on each test animal; and 50% v/v in ethanol applied to the right flank using the same procedure as in the topical induction. The site was inspected at 24 and 48 hours and due to dislodging of the application patches beneath the occlusive dressing a rechallenge was initiated 24hours after the first challenge. Again 3 hours into the second challenge dislodging occurred and so fresh application patches applied. Following the first challenge application no response was observed at either challenge dose. The overall sensitisation rate was 0%. No responses were exhibited in any member of the test group (undiluted, 100% v/v) or control group. Following the second challenge using 50%v/v concentration in the test group two responses were exhibited (scored 1). No responses were seen in the control group. The overall sensitisation rate was 10%. Since the overall response rate both in the challenge and rechallenge was < 30 %, under the conditions of this study the test substance is not a contact skin sensitizer.