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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:
January 1994 - February 1994
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
"Experimental Skin Sensitization in the Guinea Pig and Man", Buehler E. V. and Griffith F. in: Animals models in dermatology (ed. H.I. Maibach), pp. 56-66, Edinburgh, Churchill Livingstone, 1975.
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
An appropriate Buehler test is available which would not justify conducting an additional LLNA due to animal welfare. A Buehler test was selected, as no intradermal injection of an acceptable concentration of the test substance could be carried out in the
Guinea Pig Maximization Test.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-380-1
EC Name:
-
Cas Number:
133336-92-2
Molecular formula:
C29H28N4O2
IUPAC Name:
1-(4-methylphenyl)-3-{4-[(4-{[(4-methylphenyl)carbamoyl]amino}phenyl)methyl]phenyl}urea
Test material form:
solid: particulate/powder
Details on test material:
- Particle size distribution: 10%: < 23.94 µm
- Density: 1.32 g/cm3 (20°C)
- Test substance storage: at room temperature in the dark
- Stability under storage conditions: stable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
- Source: BRL Ltd., Basel, Switzerland.
- Age at study initiation: Young adult females (approx. 5-6 weeks old)
- Weight at study initiation: 250 - 395 g
- Housing: Group housing of 2 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system.
- 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). In addition, hay was provided once a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 January 1994 to 23 February 1994

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
0.5 ml 50% (w/w) test substance
Day(s)/duration:
6 h on days 1, 8 and 15
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
0.5 ml of 50% (w/w) test substance
Day(s)/duration:
Day 29 for 6 hours
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
0.05 ml of 10%, 25% and 50% (w/w) test substance
Day(s)/duration:
Day 36 for 6 hours
Adequacy of challenge:
not specified
No. of animals per dose:
control group: 10
experimental group: 20
Details on study design:
RANGE FINDING TESTS:
One animal: 50% (w/w) test substance concentration in propylene glycol (0.5 ml) was applied epidermally on a shaved flank. After 24 hours, residual test substance was removed using a tissue moistened with tap water and treated skin was assessed for erythema and oedema 24 and 48 hours later.
Four animals: Four concentrations of the test substance in propylene glycol (50%, 25%, 10% and 5% w/w, 0.05 ml each) were applied occlusively on a shaved flank, using Square chambers. After 6 hours, the residual test substance was removed using a tissue moistened with tap water and the treated sites were assessed for erythema and oedema 24 and 48 hours later.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h each
- Test group: TS
- Control group: vehicle
- Site: left flank
- Frequency of applications: every 7 days
- Duration: 1 - 15 days
- Concentration: same troughout (0.5 ml of the 50% (w/w) test substance)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Exposure period: 6 h
- Day(s) of challenge: day 29
- Test group: TS
- Control group: TS
- Site: right flank
- Concentration: same as induction
- Evaluation (hr after challenge): 24 h and 48 h

C. RE-CHALLENGE EXPOSURE
- No. of exposures: 1
- Exposure period: 6 h
- Day(s) of challenge: day 36
- Test group: TS
- Control group: TS
- Site: right flank
- Concentration: 0.05 ml of 10%, 25% and 50% (w/w) test substance
- Evaluation (hr after re-challenge): 24 h and 48 h

In addition to the skin reactions the following data were recorded:
Mortality/Viability/Toxicity: Twice daily.
Body weights: Prior to start and at termination of the study.
Challenge controls:
vehicle
Positive control substance(s):
yes
Remarks:
A positive control experiment with formaldehyde is performed every six months as a sensitivity check of the test system.

Results and discussion

Positive control results:
The six-month reliability check with formaldehyde indicates that the Himalayan strain of guinea pig is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
discrete or patchy erythema
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete or patchy erythema
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
discrete or patchy erythema
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
10%, 25% and 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10%, 25% and 50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
10%, 25% and 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10%, 25% and 50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1% Formaldehyde
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1% Formaldehyde
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Interpretation:

Positive skin reactions (grade 1 or more) were considered signs of sensitisation, provided that such reactions were not observed in the control group. The results for the experimental animals at the challenge application(s) were compared with the results for the control animals. A sensitisation rate (%) was calculated for each concentration as follows: the number of sensitised animals to one concentration in proportion to the total number of animals of the experimental group.

Results Pre-screen test:

No signs of systemic toxicity were observed during the preliminary study. However, body weight loss was noted in one of the five animals. The test substance concentrations used in the Main Study were based on the findings in the preliminary study. Since no or slight erythema was recorded, the maximum test substance concentration (50%) could be selected for the induction and the first challenge.

Other results - main study:

- Induction

Five experimental animals showed slight erythema and one of these animals also showed slight oedema after the 48 hours occluded epidermal induction exposure.

 

- Bodyweights

A difference between the average body weight gain of experimental and control animals was noted. No reason can be given to the cause of the difference. 

- Toxicity symptoms / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study during the study period.

Applicant's summary and conclusion

Interpretation of results:
other: Sensitising
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
In an skin sensitisation study (Buehler-test), performed according to OECD/EC test guidelines, KY-AF was considered to be a skin sensitiser.
Executive summary:

A skin sensitisation study (Buehler-test) was performed according to OECD/EC test guidelines and in accordance with GLP principles. Based on the results of a preliminary study, the test concentration was selected at 50% w/w. Five experimental animals showed slight erythema and one of these animals also showed slight oedema after the 6 h occluded epidermal induction exposure on day 15. After the first challenge (day 29) one control animal and 4 experimental animals showed discrete or patchy erythema. No animals showed a skin reaction after the second challenge exposure (day 36). No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. A difference between the average body weight gain of experimental and control animals was noted. No reason can be given to the cause of the difference. Taking into account the intensity of the responses and comparing these with the skin reactions seen in the control animals, no experimental animals showed a positive reaction in response to any of the concentrations tested. These results lead to a sensitisation rate of 0 per cent.

In the Risk assessment performed by the Dutch CA (RIVM, 10-08-2005) it was concluded that the substance is a skin sensitiser as in the first challenge a 20% sensitisation rate was found. Therefore the substance needs to be classified as skin sensitiser category 1B and shall be labelled with H317: May cause an allergic skin reaction.

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