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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:
07 December 1990 - 14 February 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
The methodology used in this study was equivalent to the current guideline requirements for an OECD 406 study. The study was conducted in accordance with GLP and all relevant modern validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not applicable
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
not applicable
Principles of method if other than guideline:
The procedures used were modified from those described by Buehler, E.V., Arch. Dermatol., 91, 171, 1965 and updated by Buehler, E.V. and Ritz, H.L., Current Concepts in Cutaneous Toxicology, pp 25-40, 1980. This test was one of the accepted methods for skin sensitisation listed in document L251m an EEC Commission Directive (84/449/EEC) of 25 April 1984.
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The study was conducted prior to the LLNA method becoming the default approach for skin sensitisation under EU chemical control legislation. At the time of conducting this study, the Buehler method was considered the most appropriate test to assess skin sensitisation potential.

Test material

Constituent 1
Reference substance name:
Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal
EC Number:
944-855-6
Cas Number:
199445-85-7
Molecular formula:
Not applicable (reaction mass of stereochemical isomers)
IUPAC Name:
Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal
Test material form:
liquid
Details on test material:
Test Item Storage: +4 °C in the dark
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator (4ºC) - Dark
- Stability under test conditions: Stable.
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

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
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Microbiological status of animals, when known: Not reported
- Age at study initiation: Not reported
- Weight at study initiation: 300 - 350 g
- Housing: Stainless stell cage 85 x 57 x 25 cm with mesh floors
- Diet (e.g. ad libitum): FDI SQC guinea pig diet suppled ad libitum
- Water (e.g. ad libitum): Tap water supplied ad libitum
- Acclimation period: 5 days (prelim test) 10 days (main test)
- Indication of any skin lesions: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13 - 23 ºC
- Humidity (%): 30 - 60 % RH
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12:12
- IN-LIFE DATES: Not reported

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
25 % (v/v) / 0.5 mL
Day(s)/duration:
Days 0, 8 and 15 / 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
25% (v/v) on left flank & 12.5% on right flank / 0.5 mL
Day(s)/duration:
Day 29 / 6 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Ethanol
Concentration / amount:
1 % (v/v) on right flank & 0.2 % (v/v) on left flank / 0.5 mL
Day(s)/duration:
36 / 6 hours
Adequacy of challenge:
not specified
No. of animals per dose:
Preliminary test: 4 individuals
Main test: 20 individuals (10 test and 10 control)
Details on study design:
RANGE FINDING TESTS: 4 guinea pigs were exposed to 100, 50, 25 and 12.5 % v/v concentrations of the test substance (prepared in ethanol). An aliquot (0.5 mL) of the test solution was applied by way of a 2 cm patch secured to the animal at one of four sites on the back of the animal. Patches were removed after 6 h contact. Animals were examined after 24 and 48 h under a standard light to comply with the requirements of BS 950 Part 1. Presence of erythema and/or oedema was recorded using a point scoring system (Table 1).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 15 days
- Test groups: 10 individuals
- Control group: 10 individuals
- Site: Shoulder region
- Frequency of applications: Application once a week for 3 weeks
- Duration: 6 h
- Concentrations: 25 % v/v

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (per concentration)
- Day(s) of challenge: 29
- Exposure period: 6 h
- Test groups: 10 individuals
- Control group: 10 individuals
- Site: Flank
- Concentrations: 12.5, 6.25, 1.0, 0.2 % v/v
- Evaluation (hr after challenge): 24 and 48 h

OTHER:
Positive control substance(s):
no

Results and discussion

Positive control results:
No positive control substance

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
12.5 % v/v
No. with + reactions:
10
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:
12.5 v/v
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
6.25 % v/v
No. with + reactions:
10
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:
6.25 % v/v
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1.0 % v/v
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1.0 % v/v
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.2 v/v
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.2 % v/v
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
12.5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
12.5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
6.25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
6.25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
other:
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

Table 2       Main Test Skin Reaction Scores

 Animal

                      Test item Concentration - hours after challenge

 

 12.5 % v/v - 24 h

 12.5 % v/v- 48 h

 6.25 % v/v- 24 h

6.25 % v/v- 48 h 

1.0 % v/v- 24 h 

1.0 % v/v- 48 h 

0.2 % v/v- 24 h 

0.2 % v/v- 48 h 

 1

2 

2 

1 

1 

0 

0 

0 

0 

 2

0.5 

0.5 

0.5 

0.5 

0 

0 

0 

0 

 3

3 

2 

2 

1 

0 

0 

0 

0 

 4

1 

0.5 

1 

1 

0 

0 

0 

0 

 5

1 

0 

1 

1 

0 

0 

0 

0 

 6

2 

2 

1 

0.5 

0 

0 

0 

0 

 7

2 

2 

2 

2 

0 

0 

0 

0 

 8

1 

1 

1 

1 

0 

0 

0 

0 

 9

2 

1 

1 

0.5 

0 

0 

0 

0 

 10

2 

2 

1 

1 

0 

0 

0 

0 

Table 3       Bodyweights of Test Animals

 Animal

       Bodyweight (g)

 

 Day -1

Day 31 

Day 38 

 1

446 

689 

705 

 2

416 

690 

753 

 3

430 

677 

719 

 4

454 

766 

833 

 5

400 

610 

632 

 6

478 

701 

774 

 7

455 

683 

741 

 8

407 

651 

725 

 9

472 

594 

791 

 10

426 

659 

702 

 Mean

438 

672 

738 

 S.D.

26.8 

48.3 

55.3

Table 3       Bodyweights of Control Animals

 Animal

       Bodyweight (g)

 

 Day -1

Day 31 

Day 38 

 1

401 

536 

572 

 2

455 

744 

794 

 3

429 

755 

821 

 4

401 

577 

635 

 5

404 

649 

700 

 6

429 

727 

788 

 7

405 

634 

680 

 8

420 

615 

641 

 9

461 

757 

824 

 10

431 

680 

745 

 Mean

424 

667 

720 

 S.D.

21.8 

74.1 

87.6 

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance produced a delayed dermal hypersensitiviy in guinea pig using test substance concentrations ≥ 6.25 % v/v. The test demonstrated the failure to elicit a response using test substance concetrations of ≤ 1.0 % v/v.
Executive summary:

The skin sensitisation potential of the test substance was assessed in guinea pig using the Beuhler method similar to EU Method B.6 and GLP.

In a preliminary test, 4 guinea pigs were exposed to 100, 50, 25 and 12.5 % v/v concentrations of the test substance (prepared in ethanol).  An aliquot (0.5 mL) of the test solution was applied by way of a 2 cm patch secured to the animal at one of four sites on the back of the animal.  Patches were removed after 6 h contact.  Animals were examined after 24 and 48 h under a standard light to comply with the requirements of BS 950 Part 1. Presence of erythema and/or oedema was recorded using a point scoring system. A test concentration of 25 % v/v was selected for application in the main test as it was the lowest concentration tested that elicited a response.

Twenty individuals within the weight range 400 - 478 g were selcted for the main test, 10 of which were allocated as control animals and the remaining 10 as test animals. On the first day of the test a 0.5 mL aliquot of the test solution (25 % v.v, prepared in ethanol) was applied to the shoulder region of each of the test animals by way of a 2 x 2 cm patch. The patch was occluded using surgical tape and covered using and elastic bandage. The dressings were removed after 6 h contact time. The dosing process was repeated weekly for a total of 3 weeks (Day 1, 8 and 15) during which time the control animals were left untreated. The challenge test was conducted on Day 29, after a two week rest period. Test solutions prepared at 12.5 % v/v and 6.25 % v/v (prepared in ethanol) were applied, as previously described, to the left and right flanks of each test animal. The control animals were left untreated. The contact patches were removed after 6 h contact time. Animals were examined visually 24 h and 48 h after test item application for erthema or oedema. A second challenge test was conducted 5 days after completion of the first challenge test by applying test solutions of concentration 1.0 % v/v and 0.2 % v/v to virgin sites on the flanks of each of the test animals. The procedures applied were the same as those employed for the first challenge test. During visual assessment animals were scored on two indices, incidence and severity.

During the observation period nine test animals exhibited responses following the challenge application of 12.5 and 6.25 % v/v solution that equated to a reaction score of 1 or greater. No response was observed in the control animals or test animals when treated at 1.0 and 0.2 % v/v.

It was concluded that the test substance produced a delayed dermal hypersensitivity in the guinea pig at concentrations ≥ 6.25 % v/v.