Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th November 11997 - 14th December 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed in compliance with the recommendations of the OECD Guidelines for the Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B6 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC). The test system was chosen because the guinea pig has been shown to be a suitable species for this type of study and is recommended in the test method. The strain used in these laboratories has been shown to produce satisfactory sensitisation responses using known positive sensitisers.

Test material

Reference
Name:
Unnamed
Test material form:
solid: bulk
Details on test material:
Buff-colored
Specific details on test material used for the study:
Sponsor's identification: UK-114,958
Batch number: 116044/D/16/X2/1
Date received: 28 October 1997
Description: buff solid
Storage conditions: 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:
The animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes. Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study.
For the main study the animal room was maintained at a temperature of 19 to 23"C and relative humidity of 39 to 61%. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
A row of three injections (0.1 ml each) was made on each side of the mid-line. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 1°4) w/v formulation of the test material in arachis oil BP
C) a 1% w/v formulation of the test material in a 1:1 preparation
of Freund's Complete Adjuvant plus distilled water.
Day(s)/duration:
Two series of injections over seven days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
The test material at the maximum non-irritant concentration (50% w/w in arachis oil BP) was applied.
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Twenty animals were given the test substance and ten were given the control.
Details on study design:
The method used for assessing the sensitising properties of the test material was based on the Guinea Pig Maximisation test of Magnusson B & Kligman A M, J. Invest. Dermatol. (1969) 52: 268 -276.

Selection of Concentrations for Main Study (Sighting Tests):
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:

Selection of Concentration for Intradermal Induction:
Four concentrations of test material were investigated (1%, 5°/0, 10 0/0 and 25% wiv in arachis oil BP). A total of four guinea pigs were used, each guinea pig receiving four 0.1 ml injections of only one concentration of test material. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and up to 7 days after injection according to the Draize scale shown in Appendix X. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.

Selection of Concentration for Topical Induction:
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant 15 days earlier) were treated with four preparations of the test material (5%, 10%, 25% and 50% w/w in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

Main Study:
A group of thirty guinea pigs was used for the main study, twenty test and ten control. The bodyweight of each animal was recorded at the start and end of the study. Two main phases were involved in the main study; (a) an induction of a response and (b) a challenge of that response.

Induction (Induction of the Test Animals):
Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 ml each) was made on each side of the mid-line. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 1°4) w/v formulation of the test material in arachis oil BP
C) a 1% w/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.

Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites were evaluated.

One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), loaded with the test material formulation (50% w/w in arachis oil BP) as a thick, even layer was applied to the prepared skin and held in place with a strip of surgical adhesive tape (BLENDERM: approximate size 50 mm x 30 mm) covered with an
overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours. Any other ractions were also recorded.

Induction of the Control Animals:
Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) arachis oil BP
c) 50% w/w formulation of arachis oil BP in Freund's Complete
Adjuvant/distilled water 1:1

Approximately 24 and 48 hours after intradermal injection the degree of erythema at the vehicle injection sites were evaluated. The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.
Challenge controls:
Selection of Concentration for Topical Challenge:
Four preparations of the test material (5%, 10%, 25% and 50% w/w in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.

A square filter paper patch (WHATMAN No.4: approximate size 20 mm x 20 mm), loaded with a thick, even layer of test material at the maximum non-irritant concentration (50% w/w in arachis oil BP) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 25% w/w in arachis oil BP was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The challenge 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. Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
Positive control substance(s):
yes

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%
Clinical observations:
+ve skin responses and incidents of very slight - slight oedema were noted at the challenge sites of all test group animals at the 24 and 48-hour observations. Also incidents of desquamation and an isolated incident of hardened dark brown/black scabbing.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
Clinical observations:
No adverse skin reactions were noted at the challenge sites of control group animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
Clinical observations:
+ve skin responses and incidents of very slight - slight oedema were noted at the challenge sites of all test group animals at the 24 and 48-hour observations. Also incidents of desquamation and an isolated incident of hardened dark brown/black scabbing.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50
Clinical observations:
No adverse skin reactions were noted at the challenge sites of control group animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
Clinical observations:
+ve skin responses and incidents of very slight - slight oedema were noted at the challenge sites of all test group animals at the 24 and 48-hour observations. Also incidents of desquamation and an isolated incident of hardened dark brown/black scabbing.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
Clinical observations:
No adverse skin reactions were noted at the challenge sites of control group animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25
Clinical observations:
+ve skin responses and incidents of very slight - slight oedema were noted at the challenge sites of all test group animals at the 24 and 48-hour observations. Also incidents of desquamation and an isolated incident of hardened dark brown/black scabbing.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
Clinical observations:
No adverse skin reactions were noted at the challenge sites of control group animals.
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test material, UK-114,958, produced a 100% (20/20) sensitisation rate and was classified as an EXTREME SENSITISER to guinea pig skin. The test material was also classified as a sensitiser according to EU labelling regulations.
Executive summary:

A study was performed to assess the contact sensitisation potential of the test material in the albino guinea pig. The study was performed in compliance with the OECD Guidelines for Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B6 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Twenty test and ten control animals were used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction: 1% w/v in arachis oil BP

Topical Induction: 50% w/w in arachis oil BP

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

The test material produced a 100% (20/20) sensitisation rate and was classified as an extreme sensitiser to guinea pig skin. The test material was also classified as a sensitiser according to EU labelling regulations.