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Toxicological information

Skin sensitisation

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
read across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
In accordance with Annex VII of EC legislation 1907/2006 in-vivo testing studies apart from murine local lymph node assay (LLNA) meet the regulatory requirements if they were carried out or initiated before 11 October 2016. The described experimental test was performed according to the OECD 406 (GPMT method or Buehler test method) and meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4).

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:
- source: D. Hall, Newchurch, Staffordshire, England.
- weight: 296 to 341 g
- age: seven weeks
- acclimatization: thirteen days
- housing: groups of ten in suspended metal cages with wire mesh floors
- diet: vitamin C enriched guinea-pig diet FDl ad libitum
- water: drinking water ad libitum


Animal room temperature was maintained at approximately 21 °C and relative humidity at 30 - 70%. Air exchange was maintained at approximately 15 air changes per hour and lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal
Vehicle:
water
Concentration / amount:
Intradermal injection: 7.5 % w/v in distilled water
Topical application: 70 % w/v in distilled water
Challenge application: 70 and 35 % w/v in distilled water
Challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Intradermal injection: 7.5 % w/v in distilled water
Topical application: 70 % w/v in distilled water
Challenge application: 70 and 35 % w/v in distilled water
No. of animals per dose:
test group 10
control group 5
Details on study design:
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main smdy and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
Selection of concentrations of test substance for the main study. Based on the results of the preliminary investigations, the following concentrations of were selected:
Induction intradermal injection - 7.5% w/v in distilled water
A concentration of 7.5% elicited some irritation in preliminary investigations but did not adversely effect the animals.
Induction topical application - 70% w/v in distilled water
Topical challenge - 70 and 35% w/v in distilled water
From preliminary investigations 70% w/v in distilled water was the maximum practical concentration that could be dosed topically and did not give rise to irritating effects.

Main study
The procedure may be considered in two parts: Induction and Challenge.

Induction
Induction intradermal injections - test animals
A 40 X 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 2 x 4 cm area within the clipped area.
Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation
2. test item, 7.5% w/v in distilled water.
3. test item, 7.5% w/v in a 50 : 50 mixmre of Freund's complete adjuvant and distilled water.
Induction topical application - test animals
The preliminary investigations indicated that the maximum practical concentration of the test substance for topical application (70%) did not produce skin irritation. Therefore, six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.2 ml per site of 10% w/w sodium lauryl sulphate in petrolamm. Twenty-four hours later a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of the test substance, 70% w/v in distilled water. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm"). This in mm was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.
Induction - control animals
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction application using test article, 70 and 35% w/v in distilled water. Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml ofthe substance, 70% w/v in distilled water and implied to an anterior site on the flank. The test item, 35% w/v in distilled water was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".
Challenge controls:
The control and test animals were challenged topically two weeks after the topical induction application using the test article, 70 and 35% w/v in distilled water.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde

Results and discussion

Positive control results:
Intradermal injections
Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving the test substance, 7.5% w/v in distilled water and no irritation was observed in control animals receiving distilled water.

Topical application
Very slight erythema was observed in test animals following topical application with the test item, 70% w/v in distilled water. Very slight erythema was seen in the control guinea-pigs. Staining (yellow) of the dose site was observed on the dose site of all treated animals, this was
considered to be as a result of the physical namre of the test substance and not of toxicological importance.

CHALLENGE
There were no dermal reactions, other than staining of the dose site, seen in any of the test or control animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
70 and 35 %
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: 70 and 35 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
70 and 35 %
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: 70 and 35 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: third reading
Hours after challenge:
72
Group:
test group
Dose level:
70 and 35 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: third reading. . Hours after challenge: 72.0. Group: test group. Dose level: 70 and 35 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
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: 0 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
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: 0 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: third reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: other: third reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10 and 50%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 10 and 50%. No with. + reactions: 10.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study the test substance did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the test animals.
Executive summary:

Based on the results of a preliminary smdy and in compliance with the guideline , the following dose levels were selected:

Intradermal injection: 7.5% w/v in distilled water

Topical application: 70% w/v in distilled water

Challenge application: 70 and 35% w/v in distilled water

Ten test and five control guinea-pigs were used in this study.

In this study the test substance did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the test animals.