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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Feb 2011 - 26 Feb 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The LLNA method was not available yet by the time the study was conducted.
Species:
guinea pig
Strain:
other: Kwl:Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kiwa Laboratory Animals Co., Ltd, Japan
- Age at study initiation: 6 weeks
- Weight at study initiation: 318-398 g
- Housing: stainless-steel wire-mesh cages (W 266 x D 266 x H 200 mm: Riko Denki Co., Ltd.). The animals were housed in groups of 2 or individually during the study
- Diet: pelleted diet RC4 (Oriential Yeast Co., Ltd., Lot No.; 101116) was provided ad libitum
- Water: tap water (Fujimi Water Union) was provided ad libitum via an automatic water supply system
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 40-76
- Air changes (per hr): 11-14
- Photoperiod (hrs dark / hrs light): 12/12

Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
First induction (intradermal): 0.5%
Second induction (epicutaneous): 100%
Challenge: 25 and 10%
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
First induction (intradermal): 0.5%
Second induction (epicutaneous): 100%
Challenge: 25 and 10%
No. of animals per dose:
5 (controls), 10 (in test group)
Details on study design:
RANGE FINDING TESTS:
Preliminary test 1 (for determination of the concentration for intradermal induction): Two guinea pigs were injected intradermally with 0.1 mL (midline of the shaved shoulder region) of the test substance at concentrations of 10, 5, 2.5, 1, 0.5 and 0.25% (six sites, three of each on the left and right side of the midline of the shaved shoulder region).
Preliminary test 2 (for determination of the concentrations for topical induction and challenge): For topical applications, 0.1 mL of the test substance at concentrations of 100, 50, 25, 10, 5 and 2.5% were applied to four further animals. The animals were wrapped with occlusive dressing for 24 hours.
The skin reactions were then evaluated at 24 and 48 hours after intradermal injection and 24 and 48 hours after patch removal according to the evaluation standard of skin reaction by Magnusson and Kligman.
The following concentrations were chosen for the main study based on the results of this preliminary study: The concentrations for the intradermal induction was selected as 0.5% as this was the highest concentration that did not produce necrosis at the intradermal injection sites. The concentrations for the epicutaneous induction was selected at 100%, as it was the highest concentration that produced slight skin reactions at the topical application sites. For the challenge induction, the maximum no-irritation concentration of 25% and approximately its ½ of 10% were selected.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of 0.1 mL injections):
Injection 1: a 1:1 mixture of FCA/water
Injection 2: 0.5% of test substance
Injection 3: a 1:1 mixture of 1% test susbtance/FCA

Epicutaneous: 0.2 mL of 100% test substance

- Control group:
Intradermal (3 pairs of 0.1 mL injections):
Injection 1: a 1:1 mixture of FCA/water
Injection 2: water
Injection 3: a 1:1 mixture of FCA/water
- Site: shoulder region
- Frequency of applications: every 7 days
- Duration: 0-8 days
- Concentrations: 0.5% intradermal and 100% epicutaneous

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: day 20
- Exposure period: 24 h
- Test and control groups: 0.1 mL of the 25 and 10% test substance
- Site: flank
- Concentrations: 25 and 10%
- Evaluation (hr after challenge): 24 and 48
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
1-Chloro-2,4-dinitrobenzene (DNCB)
Positive control results:
The positive control substance (0.1% DNCB in acetone) induced positive reactions in 5/5 animals (100%) at 24 and 48 hours after challenge patch removal. On the other hand, the sensitisation rates for the acetone challenge sites were determined to be 0% at 24 and 48 hours after challenge patch removal.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction intradermal: 0; challenge; 25%
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: induction intradermal: 0; challenge; 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction intradermal: 0%; challenge: 10%
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: induction intradermal: 0%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction intradermal: 0.5%; challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: induction intradermal: 0.5%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction intradermal: 0.5%; challenge: 10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: induction intradermal: 0.5%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
induction intradermal: 0.1%; challenge: 0.1%
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: induction intradermal: 0.1%; challenge: 0.1%. No with. + reactions: 5.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction intradermal: 0%; challenge: 25%
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: induction intradermal: 0%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction intradermal: 0; challenge: 10%
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: induction intradermal: 0; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction intradermal: 0.5%; challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: induction intradermal: 0.5%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction intradermal: 0.5%; challenge: 10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: induction intradermal: 0.5%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
induction intradermal: 0.1%; challenge: 0.1%
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: induction intradermal: 0.1%; challenge: 0.1%. No with. + reactions: 5.0. Total no. in groups: 5.0.

Table 3. Mean skin reaction scores

Test group

Substance for induction

Substance for challenge

Score of skin reactions

No. of animals

No. of animals with positive reactions

Sensitisation rate (%)

Intradermal (concentration)

Topical (concentration)

(concentration)

24 ha)

48 hb)

Test substance sensitisation group

Test substance in water (0.5%)

Test substance (100%)

Test substance in water (25%)

0

10

10

0/10

0

1

0

0

2

0

0

3

0

0

Mean score

0

0

Test substance in water (10%)

0

10

10

0/10

0

1

0

0

2

0

0

3

0

0

Mean score

0

0

Water

0

10

10

0/10

0

1

0

0

2

0

0

3

0

0

Mean score

0

0

Control group

Water

Water

Test substance in water (25%)

0

5

5

0/5

0

1

0

0

2

0

0

3

0

0

Mean score

0

0

Test substance in water (10%)

0

5

5

0/5

0

1

0

0

2

0

0

3

0

0

Mean score

0

0

Water

0

5

5

0/5

0

1

0

0

2

0

0

3

0

0

Mean score

0

0

Positive control substance sensitisation group

0.1% DNCB in olive oil 

0.1% DNCB in olive oil 

0.1% DNCB in acetone

0

0

0

5/5

100

1

0

0

2

0

0

3

5 (3)c)

5 (3)c)

Mean score

3

3

0/5

0

 

Acetone

0

5

5

1

0

0

2

0

0

3

0

0

Mean score

0

0

a) Hours after removal of the patch for challenge

b) The number of animals with individual score of 1 or above

c) The number of animals revealed escher formation

Interpretation of results:
not sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation

The skin sensitising potential of the test item was evaluated in a Guinea Pig Maximization Test (GPMT) according OECD TG 406 and in compliance with GLP (Nomura, 2011). 10 female Hartley guinea pigs were used for this study. Test substance concentrations selected for the main study were based on the results of a preliminary study. During the induction phase the animals were intradermally injected with 0.5% test item (diluted in water) and then seven days later topically treated with 100% test material. After a latency of 14 days (to allow a potential reaction with the immune system) the animals were challenged with 25 and 10% test material on the flank. The sensitisation rate after application of the test item was 0%.The positive control substance (0.1% DNCB in acetone) induced positive reactions in 5/5 animals (100%) at 24 and 48 hours after challenge patch removal. On the other hand, the sensitisation rates for the acetone challenge sites were determined to be 0% at 24 and 48 hours after challenge patch removal.The grade of skin reactions was compared to those of the 5 female control animals, which were treated with water during the topical induction phase and, during the challenge phase, with the test item, respectively. Under the test conditions described, the test item showed no sensitising effects. No other signs of clinical toxicity were reported. Based on the results, the test item was considered not sensitising under the conditions of the test.

Migrated from Short description of key information:

Skin sensitising (OECD TG 406/GPMT): not sensitising

Justification for selection of skin sensitisation endpoint:

There is only one study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification