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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:
24 February 2015 to 25 April 2015
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.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
LLNA is considered prone to false positive results with this type of test substance so a maximisation method was chosen instead.
Test material information:
Composition 1
Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Species and strain: Guinea pigs (LAL/HA/BR)
- Age at study initiation: Young adult, ~ 6 weeks at onset the treatment
- Weight at study initiation: 313 to 360 g
- Housing: 5 animals / cage (to allow socialisation)
- Diet: ad libitum
- Water: ad libitum (tap water, containing 50 mg/100 mL ascorbic acid)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.1 to 22.8 °C
- Humidity: < 24 to 40 % (relative)
- Air changes: 15 to 20 per hour
- Photoperiod: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)

Route:
intradermal and epicutaneous
Vehicle:
other: Methylcellulose solution
Concentration / amount:
- 1% (w/v) for intra-dermal injections
- 75% (w/v) for topical sensitisation treatment
- 50% (w/v) for challenge dose
- 25% (w/v) for safeguard dose
- 1% methylcellulose for control dose
Route:
epicutaneous, occlusive
Vehicle:
other: Methylcellulose solution
Concentration / amount:
- 1% (w/v) for intra-dermal injections
- 75% (w/v) for topical sensitisation treatment
- 50% (w/v) for challenge dose
- 25% (w/v) for safeguard dose
- 1% methylcellulose for control dose
No. of animals per dose:
- Test group: 10 animals
- Control group: 5 animals
Details on study design:
-PREPARATION OF ANIMALS: A day prior to the test, the hair was removed from the right and left surface of the animals (approximateky 5 x 5 cm). The hair removal was performed carefully to ensure animals are closely shaven.

RANGE FINDING TEST
A series of test material concentrations was tested to identify the primary irritation following intra-dermal injection and dermal application: 0.5, 1, 2.5 and 5 % (w/v) concentrations were used for intra-dermal injection and 10, 25, 50 and 75 % (w/v) for dermal application. Local effects were examined and scored 1, 24, 48 and 72 hours after the treatment or after patch removal. Skin effects were scored for erythema and oedema, any other observations of changes to the skin was recorded.
For the intra-dermal application, 0.1 mL per concentration was injected intra-dermally into the hair free skin of the flanks. One concentration was injected on the right side and another concentration on the left side of the animals. Each concentration was injected in duplicate. Two animals were used per concentration.
It was found that concentrations of 2.5 and 5 % (w/v) concentrations produced necrosis 48 hours after the treatment; therefore these concentrations are not acceptable for the main study. The concentrations of 0.5 and 1 % (w/v) caused no reaction (scores 0-0 for erythema and oedema) in the skin of guinea pigs.
For the dermal application, approximately 0.5 mL or a 2.5 x 2.5 cm area per concentration was applied onto the clipped and shaved skin of the animals. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main study.
One concentration was used on the right side and another concentration on left side of the animals. Two animals were used per concentration. Time of exposure was 48 hours.
It was found that approximately 0.5 mL of the test material formulations at concentrations of 75 and 50 % (w/v) caused moderate to severe erythema or well defined erythema (score 3 and 2, respectively) 1 hour after the patch removal. The skin irritation was persistent at the 75 % (w/v) formulation (score 1 or 2). No further skin irritation was observed in case of 50 % (w/v) concentration 24, 48 and 72 hours after the patch removal. Concentrations of 25 and 10 % (w/v) produced no reaction (scores 0-0) on the skin of guinea pigs.
On the basis of results of the Preliminary Dose Range Finding Study, the 1 % (w/v) concentration was used for intra-dermal treatment and 75 % (w/v) formulation was used for dermal induction treatment.
Control animals were treated with 1 % methylcellulose.
For the challenge exposure, the 50 % (w/v) concentration was used as a challenge dose and at concentration of 25 % (w/v) as a safeguard dose.

MAIN STUDY: INTRA-DERMAL INDUCTION EXPOSURE
The day before the treatment, an area of 5 x 5 cm² on the scapular region of animals was clipped free of hair and shaved.
> Test group: A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
-2 injections with 0.1 mL of Freund's Complete Adjuvant mixed with saline (1:1) (v/v)
- 2 injections with 0.10 mL of the test material in 1 % methylcellulose at 1 % (w/v) concentration
- 2 injections with 0.1 mL of test material in 1 % (w/v), formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and saline

> Control group: The control animals were treated similarly as the test group; however, the vehicle without the test material was used for injections as follows:
- 2 injections with 0.1 mL mix of Freund's Complete Adjuvant and saline (1:1) (v/v)
- 2 injections with 0.1 mL of 1 % methylcellulose
- 2 injections with 0.1 mL of 50 % formulation of 1 % methylcellulose in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and saline

MAIN STUDY:
DERMAL INDUCTION EXPOSURE
The same inter-scapular region which received the intradermal injections, were used for dermal induction exposure.
Since the test material was skin irritant in the Preliminary Dose Range Finding Study, the test area was not painted with sodium dodecyl sulphate.
Seven days after the intra-dermal injections, the same hair-free scapular area (approximately 6 x 8 cm) was treated. A 2.5 x 2.5 cm sterile gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test material formulation at 75 % (w/v) concentration (the highest dose caused mild-to-moderate irritation in the preliminary dose range finding study) and placed over the injection sites. The control group was treated with 0.5 mL 1 % methylcellulose.
The gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The treated areas were covered for 48 hours with a fully occlusive foil (Closed Patch Test). After the patch removal any remaining test material was removed with a wet gauze swab.
Following the dermal induction treatment, the animals were left untreated for 14 days prior to challenge applications.

CHALLENGE EXPOSURE
Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose. Approximately twenty four hours before the treatment, the hair was removed from an area of approximately 6 x 8 cm on the left and right flank of each animal. A 5 x 5 cm patch of sterile gauze patch was saturated with the test material at 50 % (w/v) concentration and applied to the left flank of all animals (both the test and the control). The right shaved flank area of all animals was treated with a 50 % dilution of the maximum dermal challenge dose (25 % (w/v)).
The volume of formulated test material was approximately 0.5 mL. The time of the exposure was 24 hours. After the patch removal any remaining test material was removed with a wet gauze swab.
Challenge controls:
Approximately 24 hours before treatment, the hair was removed from an area approximately 6 x 8 cm on the left and right flank of each animal. A 5 x 5 cm patch of sterile gauze patch was saturated with the test material at 50% (w/v) concentration and applied to the left flank of all animals (both test and control). The right shaved flank area of all animals was treated with a 50% dilution of the maximum dermal challenge dose (25% (w/v)). The volume of formulated test material was approximately 0.5 mL.
Method of administration: The time of the exposure was 24 hours under an occlusive patch. After the patch removal, any remaining test immaterial was removed with a wet gauze swab.
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: Control animals
Hours after challenge:
24
Group:
negative control
Dose level:
25% (w/v)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: Control animals
Hours after challenge:
48
Group:
negative control
Dose level:
25% (w/v)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
no indication of skin sensitisation

Table 1: Dermal Response Scores for Guinea Pigs Challenged with the Reference Item (2-Mercaptobenzothiazole)

Test Animal Number Sex: Male

Scores of Dermal Reaction

Control Animal Number Sex: Male

Scores of Dermal Reaction

24 hours

48 hours

24 hours

48 hours

after the patch removal

after the patch removal

99

1

1

102

0

0

100

1

1

104

0

0

101

0

0

108

0

0

105

1

1

111

0

0

106

1

1

112

0

0

107

1

1

 -

 -

109

1

1

 -

 -

 -

110

1

1

 -

 -

 -

113

1

0

 -

 -

 -

114

1

1

 -

 -

 -

Mean of scores

0.90

0.80

Mean of scores

0.00

0.00

Number of positive / number of tested

9/10

8/10

Number of positive / number of tested

0/5

0/5 

Scoring of Skin Sensitisation

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Following challenge with the concentrations of the test material at 50 and 25 % (w/v), there were no skin reactions in the test or control animals.
The net response value represented an incidence rate of 0 % after a challenge exposure at both 50 and 25 % (w/v) test material.
In conclusion, under the conditions of the present assay the test material was shown to have no sensitisation potential and classified as a non-sensitizer, according to current EU-regulations.
Executive summary:

A skin sensitisation study was performed in the guinea pig according to the Magnusson-Kligman method, using a maximisation method with Freund's Complete Adjuvant to evaluate the sensitisation potential of test material. The study was performed according to OECD Guideline No. 406 and in compliance with GLP guidelines. In accordance with the criteria set forth for assessing data quality by Klimisch et al. (1997) the study was awarded a reliability score of 1.

Based on the results of a preliminary test, ten test animals were subjected to sensitisation procedures in a two-stage process, named induction phase: i.e. an intra-dermal treatment and a 48h topical application (dermal treatment under an occlusive dressing). The test material was used at a concentration of 1 % (w/v) for intra-dermal injections and at a concentration of 75 % (w/v) for topical sensitisation treatment. Five control guinea pigs were simultaneously exposed to 1 % methylcellulose during the sensitisation phase.

Two weeks after the last induction exposure, a challenge dose (at a concentration of 50 % (w/v)) was administered on the left flank of all animals. The right flank area of the animals was treated with 50 % dilution of the maximum dermal challenge dose as a safeguard dose (25 % (w/v)). Challenge was performed by dermal application of the test material. Skin reactions were measured 24 and 48 h after patch removal.

No signs of systemic toxicity were observed in any animal. During the induction period well defined and very slight erythema (score 2 and 1) and/or very slight oedema (score 1) were observed in all test animals 1 hour after the dermal treatment (on Day 10) and very slight erythema (score 1) and/or very slight oedema (score 1) in 5/10 test animals 24 hours after the dermal treatment (on Day 11). No further local skin effects were observed in either the test group or in the control group.

Following challenge with the concentrations of 50 and 25 % (w/v) test material, there were no skin reactions in the test or control animals.

The net response value represented an incidence rate of 0 % after a challenge exposure at both 50 and 25 % (w/v) test material.

In conclusion, under the conditions of the present assay the test material (Batch No.: MB -140709) was shown to have no sensitisation potential and classified as a non-sensitizer, according to current EU-regulations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A skin sensitisation study was performed in the guinea pig according to the Magnusson-Kligman method, using a maximisation method with Freund's Complete Adjuvant to evaluate the sensitisation potential of test material. The study was performed according to OECD Guideline No. 406 and in compliance with GLP guidelines.

Based on the results of a preliminary test, ten test animals were subjected to sensitisation procedures in a two-stage process, named induction phase: i.e. an intra-dermal treatment and a 48h topical application (dermal treatment under an occlusive dressing). The test material was used at a concentration of 1 % (w/v) for intra-dermal injections and at a concentration of 75 % (w/v) for topical sensitisation treatment. Five control guinea pigs were simultaneously exposed to 1 % methylcellulose during the sensitisation phase.

Two weeks after the last induction exposure, a challenge dose (at a concentration of 50 % (w/v)) was administered on the left flank of all animals. The right flank area of the animals was treated with 50 % dilution of the maximum dermal challenge dose as a safeguard dose (25 % (w/v)). Challenge was performed by dermal application of the test material. Skin reactions were measured 24 and 48 h after patch removal.

Incidence Rate:

No signs of systemic toxicity were observed in any animal. During the induction period well defined and very slight erythema (score 2 and 1) and/or very slight oedema (score 1) were observed in all test animals 1 hour after the dermal treatment (on Day 10) and very slight erythema (score 1) and/or very slight oedema (score 1) in 5/10 test animals 24 hours after the dermal treatment (on Day 11). No further local skin effects were observed in either the test group or in the control group.

Following challenge with the concentrations of 50 and 25 % (w/v) test material, there were no skin reactions in the test or control animals.

The net response value represented an incidence rate of 0 % after a challenge exposure at both 50 and 25 % (w/v) test material Acid Blue 249.

In conclusion, under the conditions of the present assay the test material (Batch No.: MB -140709) was shown to have no sensitisation potential and classified as a non-sensitizer, according to current EU-regulations.


Migrated from Short description of key information:
Not-sensitising (guinea pig maximisation test), OECD 406, Török-Bathó (2015)

Justification for selection of skin sensitisation endpoint:
Only one study is available. The study was carried out in accordance with standardised guidelines under GLP conditions. In accordance with the criteria set forth for assessing data quality by Klimisch et al. (1997) the study was awarded a reliability score of 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin sensitisation.