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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.08 - 01.10.1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
not specified
Type of study:
Buehler test
Justification for non-LLNA method:
The Maximisation test was selected because it is regarded as the most sensitive and the preferred method with regard to testing for sensitisation potential.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: <500 g
- Housing: 5 animals in 1 metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands)
- 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).
- Water: Free access to tap-water, diluted with decalcified water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 45 - 55
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 7.00 a.m. to 7.00 p.m.
- Lighting: Fluorescent light, 4000°K, 120 Lux

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
0.1%
Challengeopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
0.1%
No. of animals per dose:
10
Details on study design:
PRELIMINARY STUDY
- Justification: to select the test concentrations to be used in the main study
- Test substance concentrations: 50, 20, 10, 5, 2, 1 and 0%.
- Method: identical to those during the main study
- Intradermal injections: 2 animals received 2 different concentrations in duplicate (0.1 ml/site) in the clipped scapular region; assessment for irritation 24 and 48 hours after treatment.
- Epidermal application: 2 different concentrations were applied per animal to the clipped flank, using Metallive patches on Medical tape, held in place with Micropore tape and subsequently Coban elastic bandage. 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 STUDY
INDUCTION
- 3 pairs of intradermal injections (0.1 mL/site): 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; undiluted test substance; 1:1 w/w mixture of the undiluted test substance and Freunds' Complete Adjuvant.
- Exposure period: 24 hours
- Site: clipped area: 2 x 3 cm

CHALLENGE
- No. of exposures: 1
- Exposure period: 24 hours
- Site: Metalline patches (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently Coban elastic bandage.
- Concentrations: 0,5 mL
- Evaluation (hr after challenge): 24 and 48 hours: Any signs of erythema and other lesions were recorded.

RE-CHALLENGE
- one week after the first challenge: to clarify the results in the first challenge. The contralateral flank of all animals was similarly treated.

OTHER:
- Randomization: by way of lottery drawing
- Controle group: 10 animals were treated as described for the experimental animals, except that the vehicle was administered.

OBSERVATIONS:
- Mortality: twice daily
- Body weights: Prior to start and at termination of the study.
- Irritation: Skin reactions were graded according to the following numerical scoring systems. Furthermore, a description of all other (local) effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.

GRADING IRRITATION REACTIONS
ERYTHEMA AND ESCHAR FORMATION
0: no erythema
1: slight erythema (barely perceptible)
2: well defined erythema
3: moderate to severe erythema
4: severe erythema (beet redness) to slight eschar formation (injuries in depth)

OEDEMA FORMATION

0: no oedema
1: very slight oedema (barely perceptible)
2: slight oedema (edges of area well defined by definite raising)
3: moderate oedema (raised approximately 1 mm)
4: severe oedema (raised more than 1 mm and extending beyond area of exposure)

GRADING CHALLENGE REACTIONS
0: no visible change
1: Discrete or patchy erythema
2: Moderate and confluent erythema
3: Moderate erythema and swelling
4: Intense erythema and swelling

At the end of the study all animals were killed by asphyxiation using an oxygen/carbon dioxide procedure.
Positive control substance(s):
not specified

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No skin reactions
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No skin reactions.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No skin reactions
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No skin reactions.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item is not sensitising under the conditions of the current study.
Executive summary:

The purpose of this study was to evaluate whether the test item induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure of the animals under the conditions described in this report.

The Maximisation test is selected because it is regarded as the most sensitive and the preferred method with regard to testing for sensitisation potential.

 

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.

 

The test substance concentrations selected for the main study were based on the results of a preliminary study.
 In the main study, 10 animals were intradermally injected with the undiluted test substance and epidermally exposed to the undiluted test substance. Five control animals were similarly treated, but with the vehicle only. Two weeks after the epidermal application all animals were challenged with the undiluted test substance and the vehicle. A second challenge was performed one week later with again the undiluted test substance and the vehicle.

 

In the first challenge
 a skin reaction of grade 1 (score 1 accounts for the left side area of the application site) was observed in 1 animal in response to the undiluted test substance concentration, 48 hours after exposure. No skin reactions were evident in the control animals.

 

In the second challenge, performed one week later, no skin reactions were evident after the challenge exposure in the experimental and control animals.

 

The skin reaction of grade 1, observed in response to the undiluted test substance in 1 animal in the first challenge phase, is equal to 10% of the animals responding. According to the criteria set out in Annex I (Table 3.4.4.) of the CLP Regulation N° 1272/2008 the substance does not have to be classified.