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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:
2002-04-25 to 2002-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17 July 1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
Safepharm Laboratories Limited, U.K.
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid guinea pig maximization study is available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 300 to 450 g
- Housing: singly or in pairs in solid-floor polypropylene cages finnished with woodflakes.
- Diet: ad libitum; Certified Guinea Pig Diet (Code 5026) supplied by PMI Nutrition International, Nottingham, UK and IPS Product Supplies Limited, Northants, UK
- Water: e.g. ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
1 %
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
day 8 / 48 h
Challenge
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
25 and 50 % (v/v)
Day(s)/duration:
day 21 / 24 h
No. of animals per dose:
10 test animals
5 control animals
Details on study design:
RANGE FINDING TESTS: intradermal induction: 1 and 5% (w/v) in arachis oil
epidermal applications (induction and challenge): 25, 50% and 75% (v/v) in arachis oil and 100% (undiluted)
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests'. In a first pre-test the test substance was intradermally injected in various concentrations and the highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage. In a second pre-test, the highest concentration producing only mild to moderate dermal irritation after topical application in two animals intradermally injected with Freund's Complete Adjuvant fifteen days earlier was selected for the topical induction stage. Finally, in a third pre-test the highest non-irritant concentration of the test material after topical application and one lower concentration were selected for the topical challenge stage.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 pairs of intradermal injections and one epidermal induction
- Test group:
Three pairs of intradermal applications (0.1 mL per injection): 1:1 mixture of adjuvant and distilled water, a 1% v/v formulation of the test compound in arachis oil, and a 1% v/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water. Epicutaneous application: 1 week after intradermal application, with undiluted test compound for 48 h under occlusive condition, patch 2x4 cm.
- Control group: Treated with adjuvant and the vehicle during induction period.
- Site: shoulder
- Concentrations: intradermal injection: 1%, epidermal application: 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 21 (2 weeks after the epicutaneous induction application)
- Exposure period: 24 hours
- Test and control groups: test compound in arachis oil BP, patch 2x2 cm
- Site: left and right flank clipped free of hair with veterinary clippers
- Concentrations: 25 and 50% v/v
- Evaluation (hr after challenge): 24, 48 and 72 hours after challenge

OTHER:
After 24 hours challenge exposure, the sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Erythema score 1 (4 animals) and 2 (5 animals).
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Erythema scores of 1 (2 animals) and 2 (2 animals) observed. Severe desquamation, preventing the evaluation of erythema and oedema in 6/10 animals.
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Erythema score of 3 in 1/10 animals; severe desquamation, preventing the evaluation of erythema and oedema in 9/10 test group animals.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Erythema score 1 (3 animals) and 2 (6 animals) and edeam score 1 (2 animals).
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Erythema score 1 (3 animals) and 2 (1 animal). Severe desquamation, preventing the evaluation of erythema and oedema in 5/10 animals.
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Erythema score of 3 in one animal. Severe desquamation, preventing the evaluation of erythema and oedema in 8/10 animals.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5

Any other information on results incl. tables

50% v/v in Arachis Oil BP:

Positive skin responses were noted at the topical challenge sites of all test group animals. Discrete or patchy to moderate and confluent erythema was noted at the topical challenge sites of nine test group animals at the 24-hour observation and in four test group animals at the 48-hour observation. Intense erythema and swelling was noted at the topical challenge site of one test group animal at the 72-hour observation. Severe desquamation, which prevented evaluation of erythema and oedema, was noted at the topical challenge sites of six test group animals at the 48-hour observation and in nine test group animals at the 72-hour observation. Desquamation was noted at the topical challenge sites of four test group animals at the 48-hour observation and in one test group animal at the 72-hour observation. The reaction extended beyond the topical challenge site of one test group animal at the 48 and 72-hour observations. No skin reactions were noted at the challenge sites of the control group animals at the 24, 48 or 72-hour observations.

25% v/v in Arachis Oil BP:

Positive skin responses were noted at the topical challenge sites of all test group animals. Discrete or patchy to moderate and confluent erythema was noted at the topical challenge sites of nine test group animals at the 24-hour observation and in four test group animals at the 48-hour observation. Very slight oedema was also noted at the topical challenge sites of two test group animals at the 24-hour observation. Intense erythema and swelling was noted at the topical challenge site of one test group animal at the 72-hour observation. Severe desquamation, which prevented evaluation of erythema and oedema, was noted at the topical challenge sites of five test group animals at the 48-hour observation and in eight test group animals at the 72-hour observation. Desquamation was noted at the topical challenge sites of five test group animals at the 48-hour observation and in two test group animals at the 72-hour observation. The reaction extended beyond the topical challenge site of one test group animal at the 48 and 72-hour observations. No skin reactions were noted at the challenge sites of the control group animals at the 24, 48 or 72-hour observations.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the test, the test material produced a 100% (10/10) sensitisation rate and was classified as an extreme sensitiser to guinea pig skin.
Executive summary:

In a GLP compliant guinea pig maximization test according to OECD guideline 406, the contact sensitization potential of the test material was assessed. Ten test and five control animals were used for the study. Two phases were involved in the main study; an induction of a response by intradermal injection and topical application and a topical challenge of that response. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:

Intradermal Induction 1% v/v in arachis oil BP.

Topical Induction undiluted as supplied.

Topical Challenge : 50% and 25% v/v in arachis oil BP.

Under the conditions of the test, the test material produced a 100% (10/10) sensitization rate and was classified as strong sensitizer to guinea pig skin.