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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
points 8.1 and 8.2 of Annex VIII of REACH have been amended. Nevertheless, adequate information from existing in vivo studies can still be used to fulfil the information requirement at any tonnage level.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 14th of January to the 5th of February, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
appropriate guinea pig maximisation test is available which would not justify conducting an additional LLNA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Expiry date: 29 August 2002
Storage condition: at room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
As required by the Dutch Act on Animal Experimentation, the study protocol was reviewed and agreed by the Article 14-functionary and the EthicalCommittee of NOTOX.
Species: Dunkin Hartley strain, albino guinea pig (SPF-quality)
Source: Charles River Deutschland, Germany
Age and body weight: young adult animals (approx. 5 weeks old). Individual body weight did not exceed 500 grams.
Identification: ear tattoo
ENVIRONMENTAL CONDITIONS
Conditions: air-conditioned room with approximately 15 air changes per hour
Temperature: 21°C
Relative humidity of 50%.
Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity.
Lighting: 12 hours artificial fluorescent light and 12 hours dark per day.
Accomodation: group housing of 5 animals per labelled cage
Cage: with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
Acclimatisation period: at least 5 days before start of treatment under laboratory conditions.
Diet: free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands)
Certificates of analysis were examined and then retained in the NOTOX archives.
Water: free access to tap-water diluted with decalcified water
Certificates of quarterly analysis were examined and then retained in the NOTOX archives.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
0.2, 04, 50
Day(s)/duration:
2 days
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
50%
Day(s)/duration:
2 days
No. of animals per dose:
Experimental group: 10 females
Control group: 5 females
Nulliparous and non-pregnant
Details on study design:
RANGE FINDING TEST:
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting-and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and, if needed, further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during the main study, unless otherwise specified. The animals were between 4 and 9 weeks old, and as a consequence the body weights may exceed 500 grams. Body weights were determined prior to treatment.
Intradermal injections
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 ml/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment. Epidermal application: A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be appllied. Two different concentrations were applled (0.5 ml each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on medical tape, which were held in place with Micropore tape' and subsequently Coban elastic bandage. The animails receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.
Epidermal application
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were appllied (0.5 mi each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on medical tape, which were held in place with Micropore tape and subsequently Coban elastic bandage. The animails receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.

MAIN TEST
MAIN STUDY INDUCTION
Experimental animals
Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds‘Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 0.2% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds’Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
Day 3
The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 7
The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl sulfate (SDS, Boom, Meppel, The Netheriands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.
Day 8
The 10% SDS treated area between the injection sites was treated with 0.5 ml of a 50% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal exposure were assessed for irritation.
INDUCTION
Control animals The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.
Challenge controls:
Day 21 One fiank of all animais was ciipped and treated by epidermai application of a 50% test substance concentration and the vehicle (0.15 ml each), using Patch Test Piasters (Leukotest', Beiersdorf Medical, Aimere, The Netheriands). The patches were held in piace with Micropore tape and subsequentiy Coban elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residuai test substance and vehicle. The treated sites were assessed for chalienge reactions 24 and 48 hours after removai of the dressing.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Red/brown staining was observed at aii test substance treated skin sites 24 and 48 hours after challenge.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% test substance concentration and the vehicle 0.15 ml
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of irritation were observed
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

Based on the results, the test substance concentrations selected for the main study were a 0.2% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure. No signs of irritation were observed to the highest test substance concentration tested in the preliminary irritation study. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reactionA 50% test substance concentration was selected for the challenge phase.

MAIN STUDY

Induction study The reactions noted in the experimental animals after the epidermal induction.Challenge phase. No skin reactions were evident after the challenge exposure in the experimental and control animals. Red/brown staining was observed at the test substance treated skin sites, 24 and 48 hours after challenge exposure were considered to be enhanced by the SDS treatment. Toxicity/MortalityNo mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main studyBody WeightsBody weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Classification criteria according to the CLP Regulation 1272/2008 and its amendments
Conclusions:
There was no evidence that the substance had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in the challenge phase.
This result indicates a sensitisation rate of 0 per cent.
Executive summary:

The study was carried out based on the guidelines described in: EC Commission Directive 96/54/EC, Part B.6, "Skin Sensitisation" and OECD No. 406, "Skin Sensitisation“, and based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig Identification of Contact Allergens".

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, ten experimental animals were intradermally injected with a 0.2% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. No skin reactions were evident after the challenge exposure in the experimental and control animals.

Red/brown staining was observed at the test substance treated skin sites, 24 and 48 hours after challenge.

There was no evidence that the substance had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in the challenge phase. This result indicates a sensitisation rate of 0 per cent.