Registration Dossier

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
Study period:
Study was performed between 24 August 1992 and 3 October 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP compliant study. Read-across study therefore categorised as Klimisch 2.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: Mixture containing zinc 3,5-bis(α-methylbenzyl)salicylate
- Physical state: White opaque liquid
- Lot/batch No.: SB-1051
- Storage condition of test material: room temperature
- Other:
- Date received: 24 July 1992
- Container: white opaque plastic jar

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, U.K.
- Age at study initiation: approximately 8 to 12 weeks old
- Weight at study initiation: 310 - 413g
- Housing: The animals were housed in groups of up to three in solid-floor polypropylene cages furnished with softwood shavings.
- Diet (e.g. ad libitum): Free access food (Guinea Pig FD1 Diet, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap waterwas allowed throughout the study.
- Acclimation period: at least f five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 51 - 75
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.

On one occasion the humidity was above the limit specified in the protocol (70%). This was considered not to have affected the purpose or integrity of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
Distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:
- Intradermal Induction: 5% (w/v) in distilled water
- Topical Induction: undiluted as supplied
- Topical Challenge: 75% and 50% (v/v) in distilled water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
Distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:
- Intradermal Induction: 5% (w/v) in distilled water
- Topical Induction: undiluted as supplied
- Topical Challenge: 75% and 50% (v/v) in distilled water
No. of animals per dose:
20 animals for test substance
10 animals for control
Details on study design:
RANGE FINDING TESTS:
Selection of Concentrations for Main Study (Sighting Tests): The concentrations of test material to be used at each stage of the main study were determined by ‘sighting tests’ in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:
a) Selection of Concentration for Intradermal Induction: Four animals were intradermally injected with preparations of test material (1%, 5%, 10% or 25% w/v in distilled water). The highest concentration that did not cause local necrosis, ulceration or systemic toxicity, was selected for the intradermal induction stage of the main study.
b) Selection of Concentration for Topical Induction: Two guinea pigs (intradermally injected 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 distilled water). The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.
c) Selection of Concentration for Topical Challenge: The undiluted test material andthree preparations of the test material (75%, 50% and 25% v/v in distilled water) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. 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:
Test Animals: Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on 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. The injections were:
1) Freund’s Complete Adjuvant plus distilled water in the ratio 1:1.
2) a 5% (w/v) dilution of test material in distilled water.
3) a 5% (w/v) dilution of test material in a 1:1 preparation of Freund’s Complete Adjuvant plus distilled water.

One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the undiluted test material. The undiluted test material (0.2 - 0.3 ml) was applied on filter paper (WHATMAN No.4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal, This occlusive dressing was kept in place for 48 hours.

Erythematous reactions were quantified one and twenty-four hours following removal of the patches using the 0 - 3 scale shown below:
0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

Control Animals: Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
1) Freund’s Complete Adjuvant plus distilled water in the ratio 1:1.
2) distilled water.
3) Freund’s Complete Adjuvant plus distilled water in the ratio 1:1

The topical applications followed the same procedure as for the test animals except that nothing was applied to the filter paper. Skin reactions were quantified as for the test animals.

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

A quantity of 0.1 - 0.2 ml of the test material formulation (75% v/v in distilled water) was applied to the shorn right flank of each animal on a square of filter paper (WHATMAN No.4: approximate size 20 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that that maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% (v/v) in distilled water was also similarly applied to a separate skin site on the right shorn flank. The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours, the dressing was carefully cut using blunt- tipped scissors, removed and discarded. The treatment sites were decontaminated with distilled water and the position of the treatment sites identified by using a black indelible marker-pen. On Day 23 the treatment sites were clipped free of hair with veterinary clippers.

OTHER:
Evaluation of Skin Reactions: Approximately 24 and 48 hours after challenge dressing removal erythematous reactions were quantified using the four-point scale below:

0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

Evaluation of data: The percentage of test animals that showed a more severe reaction at the test material challenge site than the most severe reaction seen in the control animals, was compared with the following scale:

% of animals Classification of
sensitised: sensitisation potential:

0 non-sensitiser
> 0 - 8 weak sensitiser
> 8 - 28 mild sensitiser
> 28 - 64 moderate sensitiser
> 64 80 strong sensitiser
> 80 - 100 extreme sensitiser

A response in at least 30% of the animals is considered positive and the risk phrase R 43 “MAY CAUSE SENSITISATION BY SKIN CONTACT” is required.
Positive control substance(s):
yes
Remarks:
2,4 dinitro chlorobezene in arachis oil

Results and discussion

Positive control results:
produced an 89% sensitization rate.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% and 50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75% and 50%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% and 50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% and 50%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
9
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: 9.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
9
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: 9.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1% DNCB
No. with + reactions:
16
Total no. in group:
18
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 1% DNCB. No with. + reactions: 16.0. Total no. in groups: 18.0.

Any other information on results incl. tables

a) Skin Reactions Observed After Topical Induction (See Appendices IV and V of attached report for full results):

- One control animal was found dead on day 9. The cause of death was not determined but the absence of this animal was not considered to affect the purpose or integrity of the study.

- Scattered mild redness and moderate and diffuse redness were elicited by the test material. Residual test material was noted at the test sites of all test animals one and 24 hours after dressing removal.

 

b) Skin Reactions Observed After Topical Challenge (See Tables 1 and 2 of attached report for full results):

- No adverse reactions were noted at the test material and vehicle control sites of the test or control animals at the 24 and 48-hour observations.

 

c) See Appendices VI and VII of attached report for individual body weights and body weight gains of test animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material, a mixture containing zinc 3,5-bis(α-methylbenzyl)salicylate, produced a 0% (0/20) sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin.
The test material was also classified as a non-sensitiser according to EEC labelling regulations. No risk phrase is therefore required.
Executive summary:

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig.

Twenty test and ten control animals were used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

 

Intradermal Induction: 5% (w/v) in distilled water

Topical Induction: undiluted as supplied

Topical Challenge: 75% and 50% (v/v) in distilled water

 

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material is therefore not classified as a sensitiser according to the CLP Regulation.