Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-02-13 until 2002-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD test under GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: not stated in the report
- Weight at study initiation: 300-500 g
- Housing: The animals were barrier maintained (semi-barrier) in an air conditioned
room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Adequate

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
cotton seed oil
Concentration / amount:
For the intradermal injection (induction - first stage) the test item was applied at a concentration of 1% (diluted in Cotton Seed Oil).
For the topical application (induction - second stage) the test item was applied at a 100% concentration.
For the topical application (challenge) the test item was applied at a 100% concentration.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
For the intradermal injection (induction - first stage) the test item was applied at a concentration of 1% (diluted in Cotton Seed Oil).
For the topical application (induction - second stage) the test item was applied at a 100% concentration.
For the topical application (challenge) the test item was applied at a 100% concentration.
No. of animals per dose:
Test group: 10
Negative control group: 5
Details on study design:
RANGE FINDING TESTS:
For the justification of dose levels a preliminary test was performed. Three animals were topically treated with 25% / 50% and 100% concentration of
the test item. No signs of irritation and systemic toxicity were recorded for any of the concentrations.
Therefore the 100% concentration was chosen for the topical induction and for the challenge, respectively.
Two animals were intradermally treated with 1% / 2.5% and 5% concentration of the test item. Signs of erythema grade 4 were recorded 24, 48 and 72 h after application of the 5% concentration as well as 2.5% concentration. Signs of erythema grade 1 were recorded 24, 48 and 72 h after application of the 1% concentration.
Therefore the concentration of 1 % was chosen for the intradermal induction


MAIN STUDY
A. INDUCTION EXPOSURE
First Stage, Intradermal InJection

Three pairs of intradermal injections of 0.1 ml volume were given in the shoulder region which was cleared of hair by clipping so that one of each
pair lies on each side of the midline.

Test group: Day 0
Injection 1: Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
Injection 2: Prepared Test item
Injection 3: Prepared Test item at a concentration of 50% (VN) in Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline

Control group : Day 0
Injection 1: Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
Injection 2: Vehicle
Injection 3: Vehicle at a concentration of 50% (V/V) in Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
Injections 1 and 2 were given close to each other and nearest to the head, while 3 is given toward the caudal part of the test area.

Second Stage, Topical Application
Test and Control Group: Day 6
Approximately twenty-four hours before the topical induction application the test area was closely clipped.
Test Group: Day 7
A patch was loaded with 0.5 ml of the test item, applied to the test area and held in contact by an occlusive dressing for 48 hours.
Control Group: Day 7
A patch was loaded with 0.5 ml of the vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours.


B. CHALLENGE EXPOSURE
The flanks of treated and control animals were cleared of hair by closely clipping and the use of a depilation creme.

Test and Control Group: Day 20
A patch loaded with 0.5 ml of the prepared test item was applied to the left flank of the animals and, a patch loaded with 0.5 ml vehicle to the right flank
(intraspecific control), respectively. The patches were held in contact by an occlusive dressing for 24 hours. At the end of the challenge skin was cleaned
with the aid ofmoistened gauze patches.

Observation
Approximately 21 hours after removing the patch the challenge area was cleaned and cleared of hair. Approximately 24 hours after removing of the
patch the skin reaction was observed and recorded according to the grades shown below. Two more observations were recorded 48 and 72 hours after
patch removal.

Positive control substance(s):
no
Remarks:
but historical positive controls for the laboratory reported.

Results and discussion

Positive control results:
Positive control substance: Mercaptobenzothiazole, Purity> 98%, CAS No. 149-30-4, Lot 117H3487, Sigma Chemicals Co.
Concentrations:
2% induction I phase
25% induction 11 phase
15% challenge
The sensitization rate after application of the positive control substance Mercaptobenzothiazole (15% in Vaseline) was 60 %, confirming the reliability of the test system.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100 %
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 %. No with. + reactions: 7.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100 %
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 7.0. Total no. in groups: 10.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
100 %
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading:

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Considering the reported data of this sensitization test it can be stated that the test item caused skin reactions identified as sensitization.
Executive summary:

During the induction phase the guinea pigs (10 test group, 5 control group all females, strain: Hsd Poc: DH) were intradermally injected with 1% test item (diluted in Cotton Seed Oil) and topically treated with 100% test item. After a latency of 2 weeks - to allow a potential reaction of the immune system - the animals were challenged with 100% test item on the flank. The grade of skin reactions was compared to control animals, which were treated with Cotton Seed Oil during the induction stage, and during challenge phase with 100% test item, respectively.

The sensitization rate after application of the test item was 70 %.

Under the test conditions described below the test item showed clear sensitizing properties. According to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive

93/21 EEC) labelling is necessary as the sensitization rate was more than 30%.