Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001/05/07 - 2001/08/23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: vlaid guideline studies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan spotted
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wolferstrasse 4, CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 290-418 g
- Housing: Makrolon type-4 cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Induction: 100% (substance was applied undiluted)
Challenge: 50% diluted in water
Rechallenge: 25%, 50% diluted in water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Induction: 100% (substance was applied undiluted)
Challenge: 50% diluted in water
Rechallenge: 25%, 50% diluted in water
No. of animals per dose:
test group: 20 animals
control group: 10 animals
Details on study design:
Irritation Screening Test:

An irritation screening test was performed to determine the minimal irritating concentration used in the induction period and the highest non-irritating concentration used for the challenge. Four different concentrations (5%, 25%, 50%, 100% diluted with bi-distilled water) were used on each animal for a 6-hour exposure period. 4 guinea pigs were used. Application sites were assessed after 24 and 48 h.

The most representative concentration to stimulate a state of immune hypersensitivity was 100 % used in the induction phase and a concentration of 50% was used in the challenge as the highest non-irritating concentration.

Main study
Induction

The fur was clipped from the left shoulder of each test animal and the patches applied, over a period of 3 weeks. The animals were treated with the test substance applied undiluted. Each animal received one patch per week which remained in place for approximately 6 hours each. The repeated application was performed at the same site. The interval between exposure was one week. The control animals remained untreated. After the last induction exposure the test animals were left untreated for 2 weeks before the challenge. The skin responses were graded 24 hours after the patches had been removed. Any gross skin reactions were recorded without depilation.

Challenge- performed on test day 29

The animals previously exposed during the induction period (i.e. test group) as well as the previously untreated control animals were challenged two weeks after the last induction exposure using the test substance at 50 % in bi-distilled water. The fur was clipped from the left posterior quadrant of the side and back of the animals. The exposure period was 6 hours on a naive skin site. The responses were graded at 24 and 48 hours after the patches had been removed.

Second challenge

The test group was rechallenged 14 days following primary challenge. All animals in the test group were included in the rechallenge. The test substance was applied on the right cranial flank at 50 % in bi-distilled water and on the the right caudal flank at 25 % in bi-distilled water.

The grading method used for irritation screen, induction and challenge was identical. The scoring system was performed by visual assessment of erythema, oedema and other clinical changes in skin conditions. They were assessed as follows:

0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Grading of all animals was done by positioning each animal under true-light (Philips TLD 36W/84 or Osram 36W/31 830).
Challenge controls:
During induction the control animals remained antreated. At the challenge the controls were treated the same as the test group. Only the test group was challenged a second time.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control results:
Five out of 10 test animals were observed with discrete/patchy erythema at the 24-hour reading and moderate/confluent erythema was observed at the 48-hour reading in all ten test animals after the challenge treatment with the highest tested non-irritating concentration of 2-Mercaptobenzothiazole at 0.03 % in mineral oil. No skin reactions were observed in the control group.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
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: 20.0. Clinical observations: none.

Any other information on results incl. tables

In the first challenge application 12 out of 20 animals reacted with discrete erythema after 24 and 48 hours. the challenge was repeated with a purified sample in the sensitized animals. None of the animals reacted to the purified sample. The purification step is always applied to the commercial product.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
During challenge and re-challenge, distinct signs for sensitization were observed with the test material. THerefore, a second rechallenge was performed using a purified test material sample. With this sample no effects were observed. The commercial product is equivalent to the purified test sample.
Executive summary:

The test substance has been investigated for its sensitization potential in accordance with OECD guideline (Buehler protocol) under GLP conditions. For the induction phase undiluted material was used. During challenge 12 out of 20 animals reacted with distinct erythema. Therefore, a purified sample has been prepared for the re-challenge procedure. During re-challenge with the purified sample no skin reactions were observed after 24 and 48 hours in any animal.The registered substance is not considered to be sensitizing because the purification step is applied to all commercial material.

The read across justification is given in an extra rationale document.