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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:
October - November 2002.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA method was used as this study was performed prior to the publication and implementation of the OECD LLNA Test Guideline.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: off-white powder
Date received: 14 August 2002
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:
Twenty male albino Dunkin Hartley guinea pigs were supplied. After an acclimation period of at least five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black inedible marker pen. At the start of the main study the animals were in the weight range of 300 to 450g, and were eight to twelve weeks old.
The animals were housed singly or in pairs in solid-floor polypropylene cages furnished with wood flakes. Free access to mains tap water and food was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 deg C and 30 to 70% respectively. The rate of air exchange was at least 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)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
5% w/w formulation of the test material
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
5% w/w formulation of the test material
No. of animals per dose:
Ten test, five control.
Details on study design:
RANGE FINDING 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:
Intradermal injections (0.1 ml/injection site) were made on the clipped shoulder of one guinea pig, at a concentration of 5% w/w w in arachis oil BP. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours and 7 days after injection. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The concentration caused only mild to moderate skin irritation, was well tolerated systemically, and was selected for the intradermal induction stage of the main study.
- Selection of concentration for Topical Induction
Two guinea pigs (intradermally injected with Freud's Complete Adjuvant sixteen days earlier) were treated with four preparations of the test material (50%, 25%, 10% and 5% 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.
- Selection of concentration for Topical Challenge
Four preparations of the test material (50%, 25%, 10% and 5% 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 was selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
Shortly before treatment on Day 0 the hair was removed from an area approximately 40mm x 60mm 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 into a 20mm x 40mm area. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ration 1:1
b) a 5% w/w formulation of the test material in arachis oil BP
c) a 5% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant distilled water
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (i.e. injection site b) was evaluated.
On 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 loaded with the test material formulation (50% w/w in arachis oil BP) was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.
The degree of erythema and oedema was quantified one and twenty four hours following removal of the patches.

- Control animals
The intradermal induction was performed using an identical procedure to that used for the test animals except that the test material was omitted from the intradermal injections. Injection b) was therefore the vehicle alone, injection c) was a 50% formulation of the vehicle in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water. Similarly, the topical induction procedure was idential to that used for the test animals except that the test material was omitted.

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area of approximately 50mm x 70mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A square filter paper patch loaded with the test material formulation at the maximum non-irritant concentration (25% 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 tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10% 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 would in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully 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 inedible marker pen.
Prior to the 24 hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
very slight Erythema observations
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: very slight Erythema observations.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no Oedema observations
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no Oedema observations.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
very slight Erythema observations
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: very slight Erythema observations.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no Oedema observations
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no Oedema observations.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
ZMB2 produced a 50% (5/10) sensitisation rate and is considered a sensitiser to guinea pig skin under the conditions of the test.