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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When the study was performed, the LLNA did not yet exist.

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Yellow powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: suspended in distilled water

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 6 - 7 weeks (at arrival)
- Weight at study initiation: 296 -341 g (at arrival)
- Housing: 10 animal per cage
- Diet: vitamin C enriched guinea-pig diet FDl ad libitum; Hay was given weekly.
- Water: drinking water ad libitum
- Acclimation period: 13 days
- Microbiological status when known: not specified
- Method of randomisation in assigning animals to test and control groups: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21°C
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: not specified

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Intradermal injection: 7.5 % w/v in distilled water
Topical application: 70 % w/v in distilled water
Challenge application: 70 and 35 % w/v in distilled water
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
70 and 35% w/v in distilled water
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
test group 10
control group 5
Details on study design:
RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main smdy and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
Based on the results of the preliminary investigations, the following concentrations of were selected:
- Induction intradermal injection - 7.5% w/v in distilled water (A concentration of 7.5% elicited some irritation in preliminary investigations but did not adversely effect the animals.)
- Induction topical application - 70% w/v in distilled water
- Topical challenge - 70 and 35% w/v in distilled water (In preliminary investigations 70% w/v in distilled water was the maximum practical concentration that could be dosed topically and did not give rise to irritating effects.)

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: one intradermal and one topical application (7 days after the injections)
- Exposure period: 48 h (topical application)
- Test groups: yes
- Control group: yes
- Site: dorsal skin on the scapular region of the guinea-pig
- Frequency of applications: single applications
- Concentrations:
intradermal injections: Three pairs of intradermal injections were made:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.).
2. Test substance, 7.5% w/v in distilled water.
3. Test substance, 7.5% w/v in a 50 : 50 mixture of Freund's complete adjuvant and distilled water.

topical application:
Six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.2 ml per site of 10% w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of the test substance, 70% w/v in
distilled water. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This in turn was firmly secured by elastic adhesive bandage wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.

During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: two weeks after the topical induction
- Exposure period: 24 h
- Test groups: yes
- Control group: yes
- Site: left flank of each guinea-pig
- Concentrations: A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of test substance, 70% w/v in distilled water and implied to an anterior site
on the flank. Test substance, 35% w/v in distilled water was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".
- Evaluation (hr after challenge): The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.
Challenge controls:
The control and test animals were challenged topically two weeks after the topical induction application using the test article, 70 and 35% w/v in distilled water.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used was checked periodically at the testing facility with hexyl cinnamic aldehyde, a known sensitiser.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
70 and 35 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
70 and 35 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
other: third reading
Hours after challenge:
72
Group:
test chemical
Dose level:
70 and 35 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Reading:
other: third reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study the test substance did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the test animals.
Executive summary:

A study was performed to assess the skin sensitisation potential of the test substance using theguinea-pig. The method followed was that described in EEC Methods for the determination of toxicity. Annex of Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Mediod B.6. Acute toxicity (skin sensitisation).

Based on the results of a preliminary smdy and in compliance with the guideline, the following dose levels were selected:

Intradermal injection: 7.5% w/v in distilled water

Topical application: 70% w/v in distilled water

Challenge application: 70 and 35% w/v in distilled water

Ten test and five control guinea-pigs were used in this study. For induction of animals, three pairs of intradermal injection were made. 6d later, topical induction in the same area was perfomed after treatment with sodium lauryl sulphate in petrolatum. Control and test animals were challenged topically two weeks after the topical induction application using the test substance, 70 and 35% w/v in distilled water.

There were no skin reactions or signs of toxicity recorded, other than staining of the dose site, seen in any of the test animals.