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Diss Factsheets

Toxicological information

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 22nd to August 14th, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The reliability of the source read-across study was established to be R2: guideline study acceptable for assessment
Justification for type of information:
Justification for read-across is detailed at section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test already available on the target substance. No further tests were conducted.

Test material

Constituent 1
Reference substance name:
Parent substance #1
IUPAC Name:
Parent substance #1

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: bred and reared at the Haffkine Biopharmaceuticals, Mumbai.
- Age at study initiation: 11-18 weeks
- Weight at study initiation: 289 to 404 g
- Diet : 'Amrut' brand extruded pelleted Guinea-Pig feed manufactured by Pranav Agro Industries Ltd., Pune, was provided ad libitum. This diet was supplemented by a daily dose of Vitamin C administered as oral drops.
- Water: potable water, passed through Aqua guard water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized glass bottles with stainless steel sipper tubes, throughout the acclimation and study period.
- Acclimation period: at least five days under experimental room conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: 10-15 per hrs
- Photoperiod:12 hrs dark/12 hrs light.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
Induction 1 - 5 % w/v
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction 2 - 25 % w/v
Day(s)/duration:
day 6
Challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25 % w/v
Day(s)/duration:
day 20
No. of animals per dose:
Dose range finding test: 8 animals
Definitive study: 30 animals
Details on study design:
INDUCTION
- Type of induction: intradermal injection and epicutaneous, occlusive.
- No. of exposures: the test animals were initially exposed to the test article by an intradermal injection on day 0, followed by topical application (induction exposures) on day 6.
- Applications: three pairs of intradermal injections of 0.1 ml volume were given in the shoulder region. Topical application was maintained over a period of 48 hours; after contact period the test patch was removed and skin was wiped with physiological saline swab and dried.
- Duration: rest period of 14 days (induction period).
- Preparation of the treated area: approximately 24 hours before treatment, the hair on the shoulders (back) of the guinea pigs was closely clipped with an electric clipper exposing an area of approximately 40 cm2 (6 x 7 cm).
- Intradermal injection area: the intradermal injection area was cleared of hair, so that one of each pair lay on each side of the midline.
- Topical application patch: the test patch, a filter paper (2 cm x 4 cm), was loaded with test article or the vehicle and was applied to the clipped skin area on the back of the animals. The test patch was covered with an aluminum foil and was secured in position with an adhesive tape (Johnsonplast, USP).

CHALLENGE EXPOSURE
- Type challenge: epicutaneous, occlusive.
- Day(s) of challenge: the animals were exposed to a challenge exposure on day 20.
- Applications: topical application was maintained over a period of 24 hours; after contact period the test patch was removed and skin was wiped with physiological saline and dried.
- Preparation of the treated area: the flanks of treated and control animals were cleared of hair approximately 24 hours before treatment. The test patch, a filter paper (2 cm x 4 cm) was loaded with test article (25 % w/v) and was applied to the clipped skin area on the left flank of the animals. The test patch was covered with an aluminum foil and was secured in position with an adhesive tape (Johnsonplast, USP).


RECHALLENGE EXPOSURE
- Type challenge: epicutaneous, occlusive.
- Application: in absence of any ambiguity in the results obtained in the first challenge, a second challenge (i.e. a rechallenge) was not carried out.
Positive control substance(s):
yes
Remarks:
1-chloro-2,4 Dinitrochlorobenzene

Results and discussion

Positive control results:
A positive response was observed in 60 % of the test animals, classifying the control article as a moderate sensitizer.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 % w/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no incidence of signs of systemic toxicity in any of the animals throughout the study period.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 % w/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no incidence of signs of systemic toxicity in any of the animals throughout the study period.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no incidence of signs of systemic toxicity in any of the animals throughout the study period.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no incidence of signs of systemic toxicity in any of the animals throughout the study period.

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation
Conclusions:
Based upon the findings of this Guinea Pig maximization Test (GPMT), the test article is classified as non-sensitiser to guinea pig as per the Magnusson and Kligman criteria of classification.
Executive summary:

Method

The assessment of the allergic contact sensitisation potential of the target substance was performed as per the Guinea Pig Maximization Test (GPMT), in compliance with the OECD Guidelines for Testing of Chemicals, Section 4, No. 406 - Skin sensitisation, adopted on 17 July 1992. The Guinea Pig maximization Test (GPMT) of Magnusson and Kligman, which uses Freund’s complete adjuvant (FCA), was employed as recommended by the OECD test guideline.

As an attempt to induce sensitisation, the test animals were initially exposed to 5 % (w/v, aqueous) suspension of test item by intradermal injections made on either side of shoulder (back) area on day 0. This was followed by topical application of a 25 % suspension (w/v, aqueous) on their back on day 6 with an exposure period of 48 h.

Because the test article was not found to be a topical skin irritant in guinea pigs during pilot study, in order to create a local irritation, the test area was painted with 0.5 ml of 10 % sodium lauryl sulphate in vaseline, about 24 hours before the topical induction application.Thereafter, following a rest period of 14 days (induction period), during which an immune response was likely to develop, the animals were exposed to a challenge exposure on day 20 of the study, by topical application of test item (25 % suspension, w/v, aqueous) on their left flank.All topical applications made were occlusive in nature and the periods of exposure were 48 h for induction and 24 h for the purpose of challenge.

 

Obserations

Skin reaction was recorded at 48 and 72 hours after removal of the inductionpatches, and at 24 and 48 hours after removal of the challenge patches. The extent and degree of skin reaction to the challenge exposure in the test animals was compared with that demonstrated by vehicle control animals which underwent sham treatment during induction and received the challenge exposure. The class of the sensitisation potential of test article was assigned according to the frequency (in percentage) of animals giving a positive response in the test group and graded as per the classification of Magnusson, B. and Kligman, A. (1970). The guinea pigs were examined for survival and abnormal clinical signs daily during the study, and their body weights were recorded at start and termination of the experiment. In this study, all guinea-pigs survived through the duration of the study. Clinical observations made periodically during the study revealed that the test item did not induce any signs of systemic toxicity in the exposed animals. There was no adverse effect of the treatment on the body weight gain by the exposed guinea pigs. As evident at 24 h and 48 h after the intradermal injections made for the purpose of ‘induction’, and as per the requirement of the study protocol, the test article, induced very slight to well defined erythema at the injections sites in the treated guinea pigs. As evident at 24 h and 48 h after the topical induction patch removal, the target substance induced a mild irritation at these skin sites, pre-treated with sodium lauryl sulphate.

 

Results

Following, challenge application on day 20, there was no evidence of any acute dermal response at the site of application in the form of erythema and/or swelling in all animals from the control and the treatment groups on observation day 22 and 23.In absence of any positive skin response indicative of sensitisation in treated animals following the challenge, the test article, is considered as non-sensitiser to guinea pig.