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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 19, 1993 to August 26, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test was conducted on a similar substance. The complete justification for the read across is attached at section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
(2 concentrations were applied on the same animal during first and rechallenge, skin sensitization study with the positive control was conducted within 7 months (instead of 6 months))
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
(2 concentrations were applied on the same animal during first and rechallenge, skin sensitization study with the positive control was conducted within 7 months (instead of 6 months))
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Füllinsdorf/Switzerland
- Age at study initiation: Preliminary study- 6 to 8 weeks; Main study- 7 to 9 weeks
- Weight at study initiation: Preliminary study- 316 to 432 g; Main study- 301 to 426 g
- Housing: Individually in Makrol on type-3 cages (size: 22x37x15 cm) with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 342, Batch nos. 77/93 (from acclimatization start to July 20, 1993) and 78/93 (from July 21, 1993 to termination) guinea pig breeding/maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water: Community tap water from Fullinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water.

ENVIRONMENTAL CONDITIONS
Air-conditioned with 10-15 air changes/h and continuously monitored environment with a temperature of 22±3°C, a relative humidity between 40-70 %, 12 h artificial fluorescent light (approx. 100 lux) /12 h dark, music during the light period.

IN-LIFE DATES: From July 19, 1993 to August 26, 1993

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: Acetone and FCA were used for the intradermal applications and vaselinum album for the epidermal applications.
Concentration / amount:
Test group:
1) A 1:1 (w/w) mixture of Freund's complete adjuvant and physiological saline.
2) The test article, diluted to 5 % with acetone.
3) The test article diluted to 5 % Freund's complete adjuvant.
Control Group:
1) A 1:1 (w/w) mixture of Freund's complete adjuvant and physiological saline.
2) Acetone.
3) A 1:1 (w/w) mixture of acetone and Freund's complete adjuvant.

Epi dermal applications:
Test group:
25 % in vaselinum album.
Control Group:
vaselinum album only.
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone and FCA were used for the intradermal applications and vaselinum album for the epidermal applications.
Concentration / amount:
10 % (applied on the posterior left flank)
15 % (applied on the anterior left flank)
vehicle only (vaselinum album, applied to the right flank)
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone and FCA were used for the intradermal applications and vaselinum album for the epidermal applications.
Concentration / amount:
Test group:
10 % (applied on the posterior right flank)
15 % (applied on the anterior right flank)
vehicle only (vaselinum album, applied to the left flank)
No. of animals per dose:
Preliminary study:
Intradermal injections group: 2 female animals
Topical applications group: 4 female animals

Main study:
Control group: 10 female animals
Test group: 20 female treated animals
Details on study design:
RANGE FINDING TESTS:
Intradermal injections: Intradermal injections (0.1 mL/site) were made into the clipped flank of two animals at concentrations of 5, 3 and 1% of the test substance in acetone. The resulting dermal reactions were assessed 24 h later. For intradermal induction application a 5% test substance dilution was selected.
Epidermal applications: Patches of filter paper (2 cm x 2 cm, held in place with occlusive dressing) were saturated with test substance at 25, 15, 10 and 5% in vaselinum album and applied to the clipped and shaved flanks of 4 animals. The dressings were removed after an exposure period of 24 h and the depilated reaction sites were assessed 24 and 48 h after removal of the bandage for skin reactions using Draize scoring system.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Topical induction: 48 h
- Test group: Intradermal injection: On Day 1, 3 pairs (top, middle and down) of 5% test substance in acetone were administered by intradermal injection in the clipped scapular region. Topical induction: On Day 8, a filter paper (2 cm x 4 cm; held in place with occlusive dressing) was fully-loaded with 25% test substance in vaselinum album), and then applied to the clipped and shaved scapular area (approx. 6 cm x 8 cm), over the intradermal injection sites for 48 h. The induction phase was followed by a 14 d rest period.
- Control group: Animals were treated as described above with the omission of test substance (acetone and vaselinum album only for intradermal and topical inductions respectively).
- Site: Scapular region
- Frequency of applications: Once intradermal, once topical
- Evaluations: Reaction sites were assessed for erythema and oedema 24 and 48 h after removal of the dressing using Draize scoring system.

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: First challenge: Day 22; Second challenge: Day 36
- Exposure period: 24 h
- First challenge: Test and control groups: On Day 22, three patches of filter paper ( 2 cm x 2 cm, held in place with occlusive dressing) were saturated with the 10 % (applied on the posterior left flank), 15 % (applied on the anterior left flank) of the test substance and the vehicle only (vaselinum album, applied to the right flank), using the same method as for the epidermal application. The dressings were removed after approx. 24 h.
- Second challenge: On Day 36, animals in the test group were treated in the same manner as that described for the first challenge except the applications were made to the opposite flanks of the animals. The control animals were treated with the vehicle alone on the left flank.
- Evaluation: Reaction sites were assessed for erythema and oedema 24 and 48 h after removal of the dressing using Draize scoring system.

OBSERVATIONS:
- Viability/mortality: Daily
- Clinical signs (local/systemic): Daily
- Skin reactions: At the times specified during the induction and challenge periods, scored in each animal according to the following scale:
Erythema and eschar formation:
No erythema..................................................................................................................................................0
Very slight erythema (barely perceptible).................................................................................................1
Well-defined erythema.................................................................................................................................2
Moderate to severe erythema......................................................................................................................3
Severe erythema (beet redness) to slight eschar formation (injuries in depth)..................................4

Oedema formation:
No oedema......................................................................................................................................................0
Very Slight oedema (barely perceptible).....................................................................................................1
Slight oedema (edges of area well-defined by definite raising)..............................................................2
Moderate oedema (raised approximately 1 mm)............................................................................3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure)....................4

Any observations of edema or other lesions were recorded.
- Body weight: The body weight of each animal was recorded at the beginning of the acclimatization period, at Day one and at the termination of the test
- Necropsy: No necropsies performed except in one animal of the control group which was found dead on Day 18 of the treatment period.
Challenge controls:
First challenge: Test substance (left flanks) and vehicle (right flank)
Second challenge: Vehicle (left flank)
Positive control substance(s):
not required
Remarks:
(mercaptobenzothiazole tested in another study)

Results and discussion

Positive control results:
No data

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15% epidermal application (anterior left flank)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
very slight erythematous reaction, grade 1
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15% epidermal application (anterior left flank). No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: very slight erythematous reaction, grade 1.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% epidermal application (posterior left flank)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no positive reactions
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% epidermal application (posterior left flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no positive reactions.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15% epidermal application (anterior left flank)
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
very slight erythematous reaction, grade 1
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15% epidermal application (anterior left flank). No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: very slight erythematous reaction, grade 1.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10% epidermal application (posterior left flank)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no positive reactions
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% epidermal application (posterior left flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no positive reactions.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
15% epidermal application (anterior right flank)
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 3
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 15% epidermal application (anterior right flank). No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: erythematous reactions, grade 1 to 3.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
10% epidermal application (posterior right flank)
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 3
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 10% epidermal application (posterior right flank). No with. + reactions: 15.0. Total no. in groups: 20.0. Clinical observations: erythematous reactions, grade 1 to 3.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
15% epidermal application (anterior right flank)
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 3
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 15% epidermal application (anterior right flank). No with. + reactions: 17.0. Total no. in groups: 20.0. Clinical observations: erythematous reactions, grade 1 to 3.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
10% epidermal application (posterior right flank)
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 3
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 10% epidermal application (posterior right flank). No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: erythematous reactions, grade 1 to 3.

Any other information on results incl. tables

Cutaneous reactions:

- In the preliminary test: At the 24 h reading of intradermal injection: A very slight erythema (grade 1) was noted at the injection sites of 2/2 animals.

- At both 24 and 48 h readings of epidermal application: Very slight erythema (grade 1) was noted at 15% (1/4) and 25% (3/4) concentrations of test substance.

- In the control group: The area around the intradermal injection sites were oedematous and erythematous from Day 2 to 4, and became necrotic from Day 5 to 12. Encrustation was observed from Day 13 to 17 and exfoliation of encrustation from Day 18 to 39 (termination of test).

- After first challenge in the control group, at the 24 and 48 h readings, no positive reactions were observed in the animals, neither when treated with vehicle alone (right flank) nor when treated with the test substance at 10% (posterior left flank) and 15% (anterior left flank). Similarly, after rechallenge in the control group, at the 24 and 48 h readings, no positive reactions were observed in the animals, when treated with vehicle alone (left flank).

- In the test substance treated group: the area around the intradermal injection sites was oedematous and erythematous from Day 2 to 4 (no erythema in second pair of injections), necrotic from Day 5 to 12/13 . Encrustation was observed from Day 13/14 to 17 and exfoliation of encrustation from Day 18 to 39 (termination of test).

- The cutaneous reactions observed after rechallenge on the right flank (test substance-treated) of the test substance treated group were of higher incidence and severity than those recorded on the left flank (vehicle treated) of the control group. Therefore, these findings were considered to be attributed to delayed contact hypersensitivity.

- Mortality: No unscheduled deaths occurred except one death in control group.

- Clinical signs (systemic): No symptoms of systemic toxicity were observed in the animals.

- Body weight: The body weight gain of the animals was not affected adversely during the study.

- Necropsy: One control animal was found dead on Day 18 of the treatment period. At necropsy, lungs, dark-red discoloured, were observed.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1B (indication of skin sensitising potential) based on CLP criteria
Conclusions:
Under the study conditions, the test substance was considered to be sensitizing.
Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6 in compliance with GLP.

Based on the results of a preliminary study, 5 % and 25 % were selected as intradermal and topical induction doses, respectively. The highest non-irritating test substance concentration used for challenge application was 10 %. However, an additional test substance concentration of 15 % was applied in the challenge procedure because only one animal out of 4 showed a positive skin reaction during the epidermal pretest. A second challenge was performed two weeks after the first one. The treatment procedure was the same as that described for the first challenge except that the applications were made to the opposite flanks of the guinea pigs.

In the first challenge, 5 and 15 % of the animals showed erythematous reactions after the 24 and 48 h reading when treated with the test substance at 15 %. No reactions were noted at 10 %. In the second challenge, 80 % and 85 % of the animals showed erythematous reactions after the 24 and 48 h reading at 15 % and 75 % to 80 % of animals with the test substance at 10 %.

Under the study conditions, the test substance was considered to be sensitizing.