Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 July to August 18, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1993
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An appropriate guinea pig maximisation test was already avaiable, which would not justify conducting an additional LLNA due to animal welfare.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Remarks:
Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, 4414 Füllinsdorf/Switzerland
- Age at the beginning of acclimatization period: 5 - 7 weeks
- Body Weight at beginning of Acclimatization period: Control and Test Group 294 - 412 g; Pretest 352 - 396 g
- Housing: Individually in Makrol on type-3 cages with autoclaved standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 342, Batch nos. 63/94 (at delivery of the animals to 03-AUG-1994) and 64/94 (from 04-AUG-1994 to termination of test) guinea pig
breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 22 - 27
- Humidity (%): between 58 - 78 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light (approx. 100
Lux) /12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: - petrolatum; -mixture of Freund's Complete Adjuvant and physiological saline.
Concentration / amount:
5 %
Day(s)/duration:
From day 1 to day 7
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
50 %
Day(s)/duration:
From day 8 to day 21
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
50 %
Day(s)/duration:
From day 22
No. of animals per dose:
20 in the test group
10 in the control group
2 in the intracutaneous pretest
4 in the epicutaneous pretest
Details on study design:
RANGE FINDING TESTS:
The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main
study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge
application.
The procedure employed for these investigations was as follows:
INTRADERMAL INJECTIONS:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in petrolatum. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 5% test article dilution was selected.
EPIDERMAL APPLICATIONS:
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper ( 2 x 2 cm) were saturated with the test article at A = 50% (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 25%, C = 15% and D = 10% of the test article in vaselinum album and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize.
The allocation of the different test dilutions to the sites (A,B,C,D) on the animals was alternated in order to minimize site to site variation in
responsiveness. For the epidermal induction and challenge procedure the test article at 50% in vaselinum album was selected.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections / performed on test day 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5% with petrolatum.
3) The test article diluted to 5% by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Petrolatum.
3) 1:1 (w/w) mixture of petrolatum in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epi dermal applications / performed on test day 8
On test day 7 and 23 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and the test area was pretreated with 10 % Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10% concentration of SLS enhances sensitization by provoking a mild inflammatory reaction.
On test day 8 a 2 x 4 cm patch of filter paper was saturated with the test article (50% in vaselinum album) and placed over the injection sites of the test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea-pigs of the control group were treated as described above with vaselinum album only.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draizé.


B. CHALLENGE EXPOSURE
CHALLENGE / performed on test day 22
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. The test and control guinea-pigs were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig just prior to the application. Two patches ( 2 x 2 cm) of filter paper were saturated with the non-irritating concentration of 50% in vaselinum album (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application. The dressing were left in place for 24 hours. 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOL

Results and discussion

Positive control results:
In this study 60% of the animals were positive after treatment with a nonirritant test substance concentration of 25% in mineral oil.
The response of at least 30% positive animals is considered positive "R43" following the "Commission Directive 93/21/EEC, April 27, 1993 adapting to
technical progress for the 18th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to
the classification, packaging and labelling of dangerous substances".
Therefore the test article 2-MERCAPT0BENZ0THIAZ0L should be considered to be a sensitizer when described under the test conditions.
According to the rating of allergenicity by Magnusson and Kligmann (1969) the test article 2-MERCAPT0BENZOTHIAZOL is considered to be a moderate sensitizer
(grade III) when described under the test conditions.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
5
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
1
Total no. in group:
20

Any other information on results incl. tables

SKIN EFFECTS AFTER INTRADERMAL INDUCTION PERFORMED ON TEST DAY 1

CONTROL GROUP

Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 6, and then became necrotic from test day 7 to 11 followed by encrustation from test day 12 to 17 and exfoliation of encrustation up to the termination of

test.

Injection site 2 (petrolatum)

The area around the injection site was oedematous and erythematous from test day 2 to 6, and then became necrotic from test day 7 to 11 followed by encrustation from test day 12 to 15 and exfoliation of encrustation up to the termination of test.

Injection site 3 (1:1 (w/w) mixture of petrolatum in a 1:1 (v/v) mixture of FCA and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 6, and then became necrotic from test day 7 to 11 followed by encrustation from test day 12 to 19 and exfoliation of encrustation up to the termination of test.

As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

TEST GROUP

Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 6 and 2 to 5, and then became necrotic from test day 7 to 11 followed by encrustation from test day 12 to 17 and exfoliation of encrustation up to the termination of test.

Injection site 2 (5% dilution of test article in petrolatum)

The area around the injection site was oedematous and erythematous from test day 2 to 5 and black discolored on test day 2, and then became necrotic from test day 6 to 11 followed by encrustation from test day 12 to 17 and exfoliation of encrustation up to the termination of test.

Injection site 3 (5% dilution of test article in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 2 with the 5% dilution of test article in petrolatum.

As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION PERFORMED ON TEST DAY 8

CONTROL GROUP:

No erythematous or oedematous reaction was observed in the animals treated with vaselinum album only.

TEST GROUP:

No erythematous or oedematous reaction was observed in the animals treated with the test article at 50% in vaselinum album. The article stained the skin slightly black; however no influence on the reading of the skin reactions.

Slight black discolouration was noted from test day 10 to 20. All the animals were pretreated with a 10% SLS in paraffinum perliquidum.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL GROUP

No positive reactions were observed in the animals either when treated with vaselinum album alone or when treated with the test article at 50% in vaselinum album. Slight black discolouration was noted from test day 23 (after removal of the dressing) to 24.

TEST GROUP

No positive reactions were observed in the animals treated with vaseiinum album. Four animals were observed with very slight erythematous reactions at the 24 -hour reading when treated with the test article at 50% in vaseiinum album. At the 48-hour reading one animal still showed the same reaction. Slight black discolouration was noted from test day 23 (after removal of the dressing) to 24.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

Three animals of the epidermal pretest lost weight during the acclimatization period. The animals had recovered during the treatment period.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (skin sensitising) based on CLP criteria
Conclusions:
Skin sensitizer
Executive summary:

Method

To assess the allergenic potential of the test item in albino guinea pigs the Maximization-Test of B. Magnusson and A.M.

Kligman (1969) was used. Ten males were used as control group and 20 males were used as test group. The highest non-irritating test article concentration used for challenge application was 50% in vaselinum album.

Observations

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

At the challenge 4 guinea pigs out of 20 exhibited very slight erythema at 24 hours, one of which persisted to 48 hours.

Conclusion

Skin sensitizer