Registration Dossier

Administrative data

Description of key information

Key study:

A skin sensitization study was performed in accordance to the standardized guidelines OECD 406, and EU Test Method B.6., under GLP conditions.

 

Two phases were involved in the main study; an induction of a response by intradermal injection and topical application and a topical challenge of that response.

 

Based on the on the results of sightings tests, the concentrations of test material for the induction and challenge phases were selected as follows: intradermal induction: 5% v/v in arachis oil BP; topical induction: undiluted test material; topical challenge: undiluted test material and 75% v/v in arachis oil BP.

 

Under the conditions of the test, the test material produced a 0% (0/10) sensitization rate and was classified as a non skin sensitizer

(SafePharm Laboratories, 2002).

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:
28 March 2002 to 10 December 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid guinea pig maximisation test (GPMT) was performed according to guideline and GLP - no further data is necessary.
Specific details on test material used for the study:
- Purity: Not specified
- Description: Brown viscous liquid
- Storage: Room temperature in the dark
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton on Trent, Staffordshire, UK
- Age at study initiation: Eight to twelve weeks old
- Weight at study initiation: 300 to 450 g
- Housing: Singly or in pairs, in solid-floor polypropylene cages furnished with woodflakes
- Diet and water: Free access to mains tap water and food (Certified Guniea Pig Diet (Code 5026) supplied by IPS Product Supplies Limited, Wellingborough, Northants, UK)
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Humidity: 30 to 70%
- Air changes: Fifteen changes per hour
- Photoperiod: Twelve hours continuous light (06:00 to 19:00) and twelve hours darkness
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
0.1 mL
Day(s)/duration:
7 days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
25% v/v
Day(s)/duration:
48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
75% and 100% v/v
Day(s)/duration:
48 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control Group: 5
Test Group: 10
Details on study design:
RANGE FINDING TESTS: Test material concentration to be used at each stage of the main study was determined by sighting tests in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:

Selection of concentration for intradermal induction:
Intradermal injections (0.1 mL/injection site) were made on the clipped shoulder of one guinea pig, at a concentration of 5% v/v in arachis oil BP. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours, and 7 days after injection. Any evidence of systemic toxicity was also recorded. The concentration caused only mild to moderate skin irritation, was well tolerated systemically, and was selected for the intradermal induction stage of the main study.

Selection of concentration for topical induction:
Two guinea pigs (injected intradermally with Freund’s Complete Adjuvant eight days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50%, and 25% v/v in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. Erythema and oedema was evaluated approximately 1, 24, and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

Selection of concentration for topical challenge:
The undiluted test material and three preparations of the test material (75%, 50%, and 25% v/v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 48 hours. Erythema and oedema was evaluated approximately 1, 24, and 48 hours after dressing removal. 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
A. INDUCTION EXPOSURE
Shortly before treatment in Day 0, the hair was removed from 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 into a 20 mm x 40 mm area. The injections were:
- Freund’s complete Adjuvant plus distilled water in the ratio 1.1
- A 5% v/v formulation of the test material in arachis oil BP
- A 5% v/v formulation of the test material in a 1:1 preparation of Freund’s Complete Adjuvant plus arachis oil BP

Approximately 24 and 48 hours after intradermal injection, the degree of erythema at the test material injection sites was evaluated.

On day 7, the same area on the shoulder region was clipped again and treated with a topical application of the undiluted test material. A filter paper patch (WHATMAN No. 4: approximate size 40 mm x 20 mm), loaded with the undiluted test material was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

The degree of erythema and oedema was quantified at one and twenty-four hours, following removal of the patches using a scale.

Any other reactions were also recorded.

B. CHALLENGE EXPOSURE
Shortly before treatment on day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal was clipped free of hair with veterinary clippers.

A square filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm), loaded with the undiluted test material was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. The test material at a concentration of 75% v/v in arachis oil BP was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with elastic adhesive bandage around the torso of each animal.

After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. 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.

Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified using a scale.

Any other reactions were also recorded.
Challenge controls:
Five animals were used in the challenge control group. The topical induction procedure was identical to that used for the test animals, except that the test material was omitted.
Positive control substance(s):
yes
Remarks:
Historical positive control data for 2-Mercaptobenzothiazole and α-Hexylcinnamaldehyde was used
Positive control results:
The historical data showed induction of a positive response positive control substances were applied.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% v/v in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% v/v in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100% v/v in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100% v/v in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0% v/v in arachis oil BP
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% v/v in arachis oil BP
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
2-Mercaptobenzothiazole 50 and 25% in acetone:PEG 400 (70:30)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Yes
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
2-Mercaptobenzothiazole 50% and 25% in acetone:PEG 400 (70:30)
No. with + reactions:
9
Total no. in group:
9
Clinical observations:
Yes
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
alpha-Hexylcinnamaldehyde 100% and 75% in arachis oil BP
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Yes
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
alpha-Hexylcinnamaldehyde 100 and 75% in arachis oil BP
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Yes
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
alpha-Hexylcinnamaldehyde 100% and 75% in arachis oil BP
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Yes
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
2-Mercaptobenzothiazole 50% and 25% in acetone:PEG 400 (70:30)
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Yes
Remarks on result:
positive indication of skin sensitisation

Skin reactions observed after intradermal injection:

Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of test group animals.

 

Discrete or patchy erythema was noted at the intradermal induction sites of control group animals.

 

Skin reactions observed after topical induction:

Light brown coloured staining and residual test material were noted at the topical induction sites of all test group animals during the study. This did not affect evaluation of skin reactions.

 

Discrete or patchy to moderate and confluent erythema, with or without very slight oedema, was noted at the topical induction sites of test group animals.

 

Discrete or patchy erythema was noted at the topical induction sites of one control group animal.

 

Skin reactions observed after topical challenge:

Light brown coloured staining was noted at the topical induction sites of all test and control group animals during the study. This did not affect evaluation of skin reactions.

Undiluted as Supplied

No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48 -hour observations. Redness was noted around the edge of the challenege sites of nine test group animal and all control group animals at the 24 -hour observation. This was considered to be a possible reaction between the test material and the blenderm tape securing the challenge patches.

75% v/v in Arachis Oil BP

No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48 -hour observations. Redness was noted around the edge of the challenge sites of seven test group animals and all control group animals at the 24 -hour observation. This was considered to be a possible reaction between the test material and the blenderm tape securing the challenge patches.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test material has been determined as a non-sensitizer to the skin.
Executive summary:

A skin sensitization study was performed in accordance to the standardized guidelines OECD 406, and EU Test Method B.6., under GLP conditions.

 

Two phases were involved in the main study; an induction of a response by intradermal injection and topical application and a topical challenge of that response.

 

Based on the on the results of sightings tests, the concentrations of test material for the induction and challenge phases were selected as follows: intradermal induction: 5% v/v in arachis oil BP; topical induction: undiluted test material; topical challenge: undiluted test material and 75% v/v in arachis oil BP.

 

Under the conditions of the test, the test material produced a 0% (0/10) sensitization rate and was classified as a non skin sensitizer.

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

Justification for classification or non-classification