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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:
From September 2 to October 3, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study completion (2003), the LLNA OECD test method was not adopted.

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(5,5-dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one
EC Number:
260-486-7
EC Name:
1-(5,5-dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one
Cas Number:
56973-85-4
Molecular formula:
C13H20O
IUPAC Name:
1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one
Constituent 2
Chemical structure
Reference substance name:
1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one
Cas Number:
56973-84-3
Molecular formula:
C13H20O
IUPAC Name:
1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one
Test material form:
liquid
Remarks:
clear liquid
Details on test material:
- Name of test material (as cited in study report): 030822-4D
- Solubility: Soluble in methanol, ethanol, and acetone. Solubility in corn oil or mineral oil unknown. Soluble in mineral oil (determined by PSL).

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Remarks:
albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Laboratories, Chelmsford, MA
- Weight at study initiation: 324-377 g (test and sham groups: males)
- Housing: Animals were group housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet: Pelleted Purina Guinea Pig Chow #5025
- Water: Filtered tap water supplied by an automatic water dispensing system, ad libitum
- Acclimation period: 4 days (Test and Sham groups)

ENVIRONMENTAL CONDITIONS
- Temperature: 20-22 °C
- Humidity: 34-56 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: not reported

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: mineral oil
Concentration / amount:
5 % w/w
Day(s)/duration:
Day 1
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:
100 % w/w
Day(s)/duration:
Day 8 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
75 % w/w
Day(s)/duration:
Day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
5 and 10 animals for sham and treatment group, respectively
Details on study design:
PRELIMINARY TEST:
- Intradermal injection ranging study: Two animals were used to determine the minimally irritating concentration (MIC) of test substance via itradermal injection. The fur was removed by clipping the dorsal area and flanks of each animal. This area was divided into six test sites on each animal. Each guinea pig received six ID injections; three concentrations of test material (0.1 mL each) at 1, 3 and 5 % w/w in mineral oil and the same three concentrations in an emulsion of CFA. 24 hours after the injections, each site was evaluated for local reactions (erythema). Based on these findings, the MIC selected for the ID injections of the main study was a 5% w/w mixture in mineral oil.
- Topical irritancy ranging study: Two animals were used to determine the minimally irritancy concentration (MIC). The previously clipped dorsal area of each guinea pig was divided into two sites. The test substance was applied neat and also diluted with a mineral oil to yield a w/w concentration of 75 %. Each concentration was applied to one of the to sites using a 2 cm x 4 cm, 2-ply gauze patch. The patch was covered with plastic wrap and secured in place with non-allergenic Durapore adhesive tape to avoid dislocation of the patch and to minimize loss of the test substance. After the 48-hour exposure period, the patches were removed and the test sites were gently cleansed of any residual test substance. One hour after removal, readings were made of local reactions (erythema).
Four animals were used to determine the Highest Non-irritating Concentration (HNIC). The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites on each animal. The test substance was applied neat and also diluted with mineral oil to yield a w/w concentration of 75 %, 50% and 25%. Each concentration was applied to a test site using an occlusive 25 mm Hill Top Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 24-hours of exposure, the chambers were removed and the test sites were gently cleansed of any residual test subtance. Twenty-four hours after patch removal, each site was evaluated for local reactions (erythema).
Based on these findings, the MIC selected for topical application of the main study was 100% with a pretreatment of SLS. The HNIC established and used for challenge was a 75% w/w mixture in mineral oil.


MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Test groups: Intradermally injected with 3 injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant and water; test article (5% w/w) in mineral oil; test article at a final concentration of 5% w/w in FCA/water (1:1), on Day 1.
- Control group: Intradermally injected with 3 injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant and water;undiluted vehicle (mineral oil); vehicle (mineral oil) in FCA/water (1:1), on Day 1.
- Exposure period: 6 days
- Site: dorsal area (anterior shoulder)
- Evaluation: 24 h after administration of the intradermal injection

Six days after administration of the intradermal injections, the injection sites were clipped free of fur. As the results of the topical range finding study indicated that undiluted test article was non-irritant, 10 % sodium lauryl sulphate in light liquid paraffin was applied to the test and control animals in an attempt to produce local irritation.

B. INDUCTION EXPOSURE: TOPICAL
- No. of exposures: One, on Day 8
- Exposure period: 48 h
- Test groups: 2-ply gauze patch (4 cm x 2 cm) with fivee-tenths of a milliliter of undiluted test material applied topically via occlusive dressing.
- Control group: 2-ply gauze patch (4 cm x 2 cm) with 0.5mL of vehicle (mineral oil) applied topically via occlusive dressing.
- Site: on the dorsal midline, between the injection sites
- Frequency of applications: Single application
- Evaluation: 24 h after removal of the topical induction patches in both test and control groups

C. CHALLENGE EXPOSURE: TOPICAL
- No. of exposures: One
- Day of challenge: Day 22
- Exposure period: 24 h
- test material at concentration of 75% w/w (HNIC) in mineral oil was applied to the right flank of all test and control animals using an occlusive 25 mm Hill Top Chamber.
- Evaluation (h after removal of challenge patch): 24 and 48 h

OTHER:
- Body weight of each animal was recorded at the start (Day 1) and end of the study (Day 25).
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde, technical (HCA)

Results and discussion

Positive control results:
alpha-Hexylcinnamaldehyde is a sensitizer under the conditions of this study.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75% w/w in mineral oil
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75% w/w in mineral oil
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
mineral oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
mineral oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Topical induction phase:

- Test animals (test substance, 100%): moderate to severe erythema (2 -3) was noted at all test sites one hour after removal of the topical induciton patch.

- Sham animals (mineral oil, 100%): very faint to severe erythema (0.5 -3) was noted for all sham control sites one hour after removal oh the topical induction patch.

- Historical positive control animals (HCA technical, applied as received): very faint to moderate erythema (0.5 -2) was noted at all test sites following the topical induction phase.

- Historical positive Vehicle control animals (mineral oil, 100%): very faint erythema (0.5) was noted at two of five vehicle control sites following the topical induction phase.

Challenge phase:

- Test animals (75% w/w mixture of the test substance in mineral oil): Eight of ten test animals exhibited signes of a sensitization response (faint erythema [1]) 24 hours following challenge patch removal. Similar indications were observed at seven sites through 48 hours. Very faint erythema (0.5) was noted at the other test sites following challenge.

- Sham animals (75% w/w mixture of the test substance in mineral oil): Very faint erythema (0.5) was noted at two of five sham sites 24 hours following the challenge patch removal. Irritation persisted at the two sites through 48 hours.

- Historical positive control animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Eight of ten test animals exhibited signs of a sensitization response (faint to severe erythema [1 -3]) 24 hours following the challenge patch removal. Similar indications persisted at seven sites through 48 hours. Very faint erythema (0.5) was noted for two sites at the 24-hour and three sites at the 48-hour interval.

- Historical positive Vehicle control animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Very faint erythema (0.5) was noted at three of five vehicle control sites 24 hours after challenge patch removal. All animals were free of irritation by 48 hours.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, the test material is classified in Category 1B according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, 10 Hartley guinea pigs (male/female) were induced with three pairs of intradermal injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant solution; 5 % concentration of the test article in mineral oil; 5 % concentration of the test article in FCA solution, on Day 1, on three different sites on dorsal area between the shoulder. Control group of 5 aimals was intradermally induced with 0.1 mL of 50 % solution of Freund’s Complete Adjuvant; mineral oil; mineral oil in FCA solution, on Day 1.

On Day 8, the topical induction phase was conducted. Prior to the topical induction, sites were re-clipped free of fur and the shoulder area of each est group animal was pre-treated with SLS in order to enhance sensitization by provoking a mild inflammatory reaction. The injection sites was topically induced with undiluted test article by occluded 2-ply gauze patch for 48 h in test group, whereas control group was applied with vehicle (mineral oil) alone.

On Day 22, a challenge dose of the test article (75 % w/w mixture in mineral oil) was applied to a naive site on each test and sham animal. The test concentrations for the main study were determined from a sighting study.

 

Following challenge with test article at 75% w/w in mineral oil, eight of ten test animals responded positively at the 24h observation and seven of ten at the 48h observation, resulting in a worst-case response incidence of 80%. None of the sham animals responded positively to challenge with test material. A known sensitizer, HCA, which induced skin sensitization indicating the validity of the study.

 

Under the test conditions, the test material is classified in Category 1B according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.