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Description of key information

Skin sensitisation: In vivo, Buehler Test, guinea pig ( Dunkin-Hartley) female (OECD 406, GLP): 60% (12/20) sensitisation rate and classified as a sensitiser to guinea pig skin

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-06-12 - 1995-07-13 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented study conducted under GLP according to OECD guideline 406 on the registered substance itself without deviations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Section 4, No. 406, "Skin Sensitisation", OECD 1992
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
EC directive B.6. Skin Sensitization, 1.6.2 Biihlertest. Appendix to the directive 92/69/EEC of the Commission of 31 July 1992 on the seventeenth adaptation of the directive 67/548/EEC of the council for adaptation of the legal and administration regulations for classification, packaging and labelling of hazardous substances to technical progress.
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
No new study has been conducted; data from a existing GLP-guideline study (1995) has been used as this study is totally sufficient to cover this endpoint.
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Pirbright White, Hsd/Win:DH [SPF]
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bred by: Harlan Winkelmann GmbH, Versuchstierzucht, Gartenstrafte 27, 33176 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Healthy young adults
- Weight at study initiation: Less than 500 g
- Housing: Conventional, maximum 5 animals/type IV Makrolon cage, Bedding: Softwood fibre type HW 300/500 W, supplied by JELU-Werk, Ludwigsmuhle, 73494 Rosenberg, Germany. The manufacturer provides regular reports on tests for contaminants.
- Diet (e.g. ad libitum): Ssniff G 4 complete feed for guinea pigs ad libitum supplied by Ssniff, Spezialfutter GmbH, 59494 Soest, Germany. The manufacturer carries out regular analyses of the feed.
- Water (e.g. ad libitum): Drinking water ad libitum, supplied by Gelsenwasser, Wasserwerk, 45721 Haltern, Germany. Samples of the tapwater are analysed in-house each quarter.
- Acclimation period: At least 5 days.
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 C
- Humidity (%): 30-70 % (slight deviations in the period from 1995-07-07 to 1995-07-13 were caused by extreme external climatic conditions, but there were no adverse effects on the validity of the test from this)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark rhythm (artificial light)
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
MEH 56
Concentration / amount:
100 % (w/w)
Day(s)/duration:
6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50 % (w/w)
Day(s)/duration:
6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
3 animals (preliminary test)
20 animals in test group, 10 animals in control group (main test)
Details on study design:
RANGE FINDING TESTS:
Aims of the preliminary test
The preliminary test was intended to establish a concentration for the induction phases which was well tolerated systemically by the animals and, in the case of irritant substances, causes slight to moderate dermal irritation in most animals. It was also intended to establish for the challenge treatment a maximum concentration not causing skin irritation in unsensitized animals.

Preliminary test procedure
Dissolving test:
According to the GLP information sheet, the test substance is insoluble in water and undergoes slow hydrolytic decomposition. No dissolving test with deionized water was therefore carried out. A dissolving test using an oily vehicle involved adding 80, 50 and 10 % test substance to MEH 56 corn oil.

Dermal administration:
2-3 hours before the administration, the fur was removed mechanically from the left and right flanks of 3 animals. The test substance was placed in the appropriate concentrations (5, 25 and 50 % in MEH 56 corn oil and 100 %) on gauze patches with an area of 2 * 2 cm and these were put on the sheared area of the animals' flanks. The amount administered was 0.3 cm^ per patch. Each gauze patch was covered with an occlusive plaster and fixed with a bandage for 6 hours. Each animal received two gauze patches on each flank.
After removal of the patches, the areas of administration were wiped off with MEH 56 corn oil on cottonwool. The dermal reactions were assessed 30 and 54 hours after the start of administration.

Determination of the challenge concentration
In the 4th week of the test, 3 additional guinea pigs which had not been treated up to this time were used for redetermination of the maximum non-irritant concentration for the challenge treatment.
This additional concentration testing was carried out because it was suspected that the sensitivity of the skin changes as the weight of the animals increases. This ensured that the challenge concentration was determined on animals which had approximately the same weights as the 30 animals in the challenge phase. The concentrations administered were the same as in the preliminary test (5, 25, 50 % in MEH 56 corn oil and 100 %). The test conditions were unchanged.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 epicutaneous occlusive
- Exposure period: 6 h
- Test groups: 1
- Control group: 1 (vehicle)
- Site: left flank
- Frequency of applications: 7 days
- Duration: 28 days
- Concentrations: 100 % (w/w)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28
- Exposure period: 6 h
- Test groups: 1 (front part of body: vehicle, rear part of body: 50 % test substance)
- Control group: 1 (front part of body: vehicle, rear part of body: 50 % test substance)
- Site: right flank
- Concentrations: 50 % (w/w)
- Evaluation (hr after challenge): 30 and 54 hours after the start of admisnistration
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole (2-MCBT)
Positive control results:
The sensitivity of the Dunkin Hartley, Pirbright White (DHPW) guinea pig strain was checked from 22.05. to 22.06.1995 in an investigation with the number HS-95/MCBT-1 using 2-mercaptobenzothiazole (2-MCBT) as well-known standard allergen. This standard allergen is a solid, and the sensitivity test was carried out by the Buhler method.
In order to test the sensitivity of the guinea pig strain to 2-MCBT, a group of 10 test animals and a group of 5 control animals were used for the main test. Any reaction, especially erythema and oedema, was assessed 24, 48 and 72 hours after the challenge treatment.The highest concentration which could be administered satisfactorily, 50%, and the 5, 10 and 30% standard allergen preparations in petrolatum, which were administered in the preliminary test, caused no primary dermal irritation 30 and 54 hours after administration. As a consequence, the 50% standard allergen concentration was fixed for the induction phases.
During the test, there were no systemic effects or impairments of body weight gain related to the test substance either in the test animals or in the controls.
The dermal treatments during induction phases I, II and III caused no dermal irritation either in the test animals or in the controls 30 hours after administration.
The challenge treatment led to a positive skin reaction in the form of erythema and oedema in the area of administration on the right rear flank of 4 of the 10 test animals 30 hours after administration. The skin irritation had resolved in one animal 54 hours after administration. 6 test animals and the 5 controls showed no signs of dermal irritation either 30 or 54 hours after administration. Administration of the vehicle to the right front flank led to no signs of dermal irritation in the test and control animals.
On the basis of these results (40% of the test animals showed a positive skin reaction), the guinea pig strain is assessed as sensitive under the test conditions described.
Reading:
1st reading
Hours after challenge:
30
Group:
test chemical
Dose level:
50 % concentration of test substance
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
6 test animals were found to have very slight erythema and oedema, and 3 test animals were found to have well-defined erythema and very slight oedema, on the posterior right flank
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
54
Group:
test chemical
Dose level:
50 % concentration of test substance
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
7 test animals were found to have very slight erythema and oedema on the posterior region of the flank
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
30
Group:
negative control
Dose level:
50 % concentration of test substance
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of dermal irritation were found on the control animals throughout the observation periods
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
54
Group:
negative control
Dose level:
50 % concentration of test substance
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of dermal irritation were found on the control animals throughout the observation periods
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
30
Group:
positive control
Dose level:
50% standard allergen concentration
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
4 of the 10 test animals showed a positive skin reaction in the form of erythema and oedema in the area of administration on the right rear flank
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
54
Group:
positive control
Dose level:
50% standard allergen concentration
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
The skin reaction had resolved in one animal 54h after administration.
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The study was performed according to the OECD TG 406 without deviations and therefore considered to be of high quality (reliability Klimisch 2). The validity criteria of the test system are fulfilled. The test material produced a 60 % (12/20) sensitisation rate and was classified as a sensitiser to guinea pig skin.
According to EU labelling regulations the test material was classified as skin sens. Cat. 1B. According to Commission Regulation (EU) No 286/2011 of 10 March 2011, Table 3.4.4, Animal test results for sub-category 1B, a substance must be classified as skin sensitizer Cat. 1B, if in a Buehler assay ≥ 15 % of the animals are responding at > 20 % topical induction dose. Topical induction dose was 100%, and 60% responded, so a subcategorisation into Skin Sens. Cat. 1B is indicated.
Executive summary:

A study was performed to assess the contact sensitisation potential of the test material in the albino guinea pig. The study was performed in compliance with the OECD Guidelines for Testing of Chemicals No. 406 "Skin Sensitisation (adopted 17 July 1992) and Method B6 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC). Twenty test and ten control animals were used for the main study.

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

-Topical Induction dose: 100% w/w in MEH 56 corn oil

-Topical Challenge dose: 50% w/w in MEH 56 corn oil

The test material produced a 60% (12/20) sensitisation rate and was classified as sensitiser to guinea pig skin. The test material was also classified as a sensitiser according to EU labelling regulations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to Regulation (EU) 286/2011, a substance must be classified as skin sensitizer Cat. 1B, if more than 15% of the animals show positive responses after >20% topical induction dose. In the present study, 9 resp. 7 out of 20 animals showed a positive response with 100% topical induction dose. Hence, the substance must be classified as skin sens. Cat. 1B.