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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:
7 December 1992 to 7 January 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study pre-dates the availability of the LLNA guideline.

Test material

Constituent 1
Reference substance name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
EC Number:
309-912-6
EC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
Cas Number:
101357-15-7
Molecular formula:
This is a UVCB substance. See section 1.2 for individual components.
IUPAC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
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 - 368g
- Housing: groups of up to three in solid-floor polypropylene cages furnished with softwood shavings
- Diet and water: ad libitum throughout study
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21°C . On one occasion the temperature was below the limit specified in the protocol (19°C). This was considered not to have affected the purpose or integrity of the study.
- Humidity (%):31 - 66%
- Air changes (per hr): 15/hr
- Photoperiod: 12 hrs dark / 12 hrs light


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Sighting Test for Main Study
1) Intradermal Induction: 1%, 5%, 10% or 25% w/v test material in arachis oil B.P. in 4 animals. The highest concentration that did not cause local necrosis, ulceration or systemic toxicity was selected for the intradermal induction stage of the main study.
2) Topical Induction: Two guinea pigs (intradermally injected with Freund's Complete Adjuvant fourteen days earlier) were treated with three preparations of the test material (50%, 25% and 10% w/w in arachis oil B.P.). The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure was selected for the topical induction stage of the main study.
3) Topical Challenge: Two preparations of the test material (50% and 25% w/w in arachis oil B.P.) were applied occlusively to the flanks of two guinea pigs for a 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 15. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Main study:
1) Intradermal Induction: 25% w/v test material in arachis oil B.P.
2) Topical Induction: 50% w/w test material in arachis oil B.P.
3) Topical Challenge: 25% and 10% w/w test material in arachis oil B.P.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Sighting Test for Main Study
1) Intradermal Induction: 1%, 5%, 10% or 25% w/v test material in arachis oil B.P. in 4 animals. The highest concentration that did not cause local necrosis, ulceration or systemic toxicity was selected for the intradermal induction stage of the main study.
2) Topical Induction: Two guinea pigs (intradermally injected with Freund's Complete Adjuvant fourteen days earlier) were treated with three preparations of the test material (50%, 25% and 10% w/w in arachis oil B.P.). The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure was selected for the topical induction stage of the main study.
3) Topical Challenge: Two preparations of the test material (50% and 25% w/w in arachis oil B.P.) were applied occlusively to the flanks of two guinea pigs for a 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 15. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Main study:
1) Intradermal Induction: 25% w/v test material in arachis oil B.P.
2) Topical Induction: 50% w/w test material in arachis oil B.P.
3) Topical Challenge: 25% and 10% w/w test material in arachis oil B.P.
No. of animals per dose:
Sighting Test for Main Study:
Intradermal injection: 4
Topical Induction: 2
Topical Challenge: 2

Main Study:
Test animals: 20
Control animals: 10
Details on study design:
The bodyweight of each animal was recorded at the start and end of the study.
Two main procedures were involved in the maximisation test; (a) an induction of a response and (b) a challenge of that response.

a) 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:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
ii) a 25% (w/v) dilution of test material in arachis oil B.P.
iii) a 25% (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant plus arachis oil B.P.

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 (50% w/w in arachis oil B.P.). The test material formulation (0.2 - 0.3 ml) was applied on filter paper (WHATMAN No.4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by 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.
Erythematous reactions were quantified one and twenty-four hours following removal of the patches using the 0 - 3 scale shown below.

b) Challenge

Shortly before treatment on Day 21, an area, approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A quantity of 0.1 - 0.2 ml of the test material formulation (25% w/w in arachis oil B.P.) was applied to the shorn right flank of each animal on a square of filter paper (WHATMAN No.4: approximate size 20 mm x 20 mm) which was held in place by 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 10% (w/w) in arachis oil B.P. was also similarly applied to a separate skin site on the right shorn flank. The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by 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 vehicle sites were similarly swabbed. 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.

c) Evaluation of Skin Reactions

Approximately 24 and 48 hours after challenge dressing removal erythematous reactions were quantified using the four-point scale shown below:

0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling
Challenge controls:
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:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
ii) arachis oil B.P.
iii) Freund's Complete Adjuvant plus arachis oil B.P. in the ratio 1:1.
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.
Positive control substance(s):
yes
Remarks:
2,4-Dinitrochlorobenzene (DNCB)

Results and discussion

Positive control results:
The known contact sensitiser, 2,4-Dinitrochlorobenzene (DNCB), produced a 90% (9/10) sensitisation rate. This was considered to be a satisfactory
sensitisation response for this material under the conditions of the test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% (w/w) in Arachis Oil B.P.
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
redness grade 1
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% (w/w) in Arachis Oil B.P.. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: redness grade 1.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% (w/w) in Arachis Oil B.P
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
redness grade 1
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% (w/w) in Arachis Oil B.P. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: redness grade 1.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% (w/w) in Arachis Oil B.P.
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
redness grade 1
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% (w/w) in Arachis Oil B.P. . No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: redness grade 1.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% (w/w) in Arachis Oil B.P.
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
redness grade 1
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% (w/w) in Arachis Oil B.P.. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: redness grade 1.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% (w/w) in Arachis Oil B.P.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% (w/w) in Arachis Oil B.P.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% (w/w) in Arachis Oil B.P.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% (w/w) in Arachis Oil B.P.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10% (w/w) in Arachis Oil B.P.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10% (w/w) in Arachis Oil B.P.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% (w/w) in Arachis Oil B.P.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% (w/w) in Arachis Oil B.P.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1% (w/v) in arachis oil B.P.
No. with + reactions:
16
Total no. in group:
18
Clinical observations:
16/18 animals showed erythematous responses
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Main Study

a) Skin Reactions Observed After Topical Induction

Black-coloured staining caused by the test material was noted at the induction sites of all test group animals one and 24 hours after dressing removal. The staining prevented accurate evaluation of erythema.

b) Skin Reactions Observed After Topical Challenge

Slight black/grey-coloured staining caused by the test material was noted at the challenge sites of all test and control group animals at the 24 and 48-hour observations. The staining did not prevent accurate evaluation of erythema.

25% (w/w) in Arachis Oil B.P.

Positive skin responses (redness grade 1) were noted at the challenge sites of two test group animals at the 24-hour observation and persisted at the challenge site of one test group animals at the 48-hour observation. No skin reactions were noted at the challenge sites of control group animals at the 24 and 48-hour observations.

10% (w/w) in Arachis Oil B.P.

A positive skin response (redness grade 1) was noted at the challenge site of one test group animal at the 24 and 48-hour observations. No skin reactions were noted at the challenge sites of control group animals at the 24 and 48-hour observations.

Vehicle Control

No skin reactions were noted at the vehicle control sites of the test or control group animals at the 24 and 48-hour observations.

Bodyweight

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to EU criteria.
Conclusions:
The test material produced a 10% (2/20) sensitisation rate.
Executive summary:

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation' - Magnusson and Kligman Maximisation Test.

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: 25% (w/v) in arachis oil B.P.

Topical Induction: 50% (w/w) in arachis oil B.P.

Topical Challenge: 25% and 10% (w/w) in arachis oil B.P.

The test material produced a 10% (2/20) sensitisation rate.