Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000

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
Justification for non-LLNA method:
The study was conducted prior to the validation of the LLNA method and the finalisation of the OECD testing guideline (429).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

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, UK
- Age at study initiation: approx 8 - 12 weeks
- Weight at study initiation: 305 - 372 g
- Housing: Singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Free access to food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK)
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23°C
- Humidity (%): 30 - 70%
- Air changes (per hr): approx 15 / hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Main study:
Induction phase:
intradermal injection:
Test Item:
1% (w/w) in dried arachis oil BP
Freud's Complete Adjuvant plus distilled water in the ratio (1:1)
1% w/w in a mixture of Freud's Complete Adjuvant plus dried arachis oil BP (1:1)
Control
Dried arachis oil BP
Freud's Complete Adjuvant plus distilled water in the ratio (1:1)
50% w/w formulation of dried arachis oil BP in a1:1 mixture of Freud's Complete Adjuvant/dried arachis oil BP

Topical induction:
50%* w/w in dried arachis oil BP

Challenge phase: topical application, 50%* and 25% w/w in dried arachis oil BP



* Maximum attainable concentration suitable for topical application
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Main study:
Induction phase:
intradermal injection:
Test Item:
1% (w/w) in dried arachis oil BP
Freud's Complete Adjuvant plus distilled water in the ratio (1:1)
1% w/w in a mixture of Freud's Complete Adjuvant plus dried arachis oil BP (1:1)
Control
Dried arachis oil BP
Freud's Complete Adjuvant plus distilled water in the ratio (1:1)
50% w/w formulation of dried arachis oil BP in a1:1 mixture of Freud's Complete Adjuvant/dried arachis oil BP

Topical induction:
50%* w/w in dried arachis oil BP

Challenge phase: topical application, 50%* and 25% w/w in dried arachis oil BP



* Maximum attainable concentration suitable for topical application
No. of animals per dose:
10 animals in test item group
5 animals in control group
Details on study design:
PRELIMINARY IRRITATION STUDY:
Selection of concentration for intradermal induction
Two test substance concentrations were investigated (1% and 5% w/w in dried arachis oil BP). The 5% concentration was found impossible to dose and therefore one guina pig was used. One guinea pig received four 0.1ml injections of the concentration of test material. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation and which was well tolerated systemically was selected for the intradermal induction stage of the main study.

Selection of concentration for topical induction
Two guinea pigs (intradermally injected with Freud's Complete Adjuvant nine days earlier) were treated with four preparations of the test material (50%, 25%, 10% and 5% w/w in dried arachis oil BP) Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of 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
Four preparations of the test material (50%, 25%, 10% and 5% w/w in dried arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals ofthe main study, up to Day 14. The degree of 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
Dose levels were selected based on the results of the preliminary irritation study
A. INDUCTION EXPOSURE - treated animals
Intradermal induction
Day 0; The hair was removed from the shoulder region of each animal. A row of three injections (0.1ml) was made on each side of the midline as follows:
A)Freunds' Complete Adjuvant with distilled water in the ratio 1:1
B) a 1% w/w formulation of the test material in dried arachis oil BP
C) a 1% w/w formulation of the test material in a 1:1 preparation of Freunds Complete Adjuvant plus dried arachis oil BP

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

Topical induction
On Day 7 the same area on the shoulder region used previously for intradermal injections was clipped and treated with a topical application of the test material formulation. A filter paper patch loaded with the test material formulation (50% w/w in dried arachis oil BP) as a thick, even layer 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. This occlusive dressing was kept in place for 48 hours.
The degree of erythema and oedema was quantified 1 and 24 hours following removal of the patches. Any other reactions were also recorded.


Induction - Control animals
Intradermal injections were administered using an identical procedure to that used for the test animlas except that the injections were:
A)Freunds' Complete Adjuvant with distilled water in the ratio 1:1
B) Dried arachis oil BP
C) 50% w/w formulation of dried arachis oil BP in a 1:1 mixture of Freunds Complete Adjuvant /dried arachis oil BP

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

The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals


B. CHALLENGE EXPOSURE - all animals
Day 21 an area on both flanks of each animal was clipped free of hair. A filter paper patch loaded with a thick even layer of the test material at the maximum non irritant concentration (50% w/w in dried arachis oil BP) was was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure the maximum non iritant concentration was used at challenge, the test material at a concentration of 25% w/w in dried arachis oil BP was applied to a skin site on the left shorn flank, The patches were occluded with an overlapping lenghth of aluminium foil and secured with a strip of elastic adhesive bandage.
After 24 hours, the dressing was removed and the challenge sites swabbed with cotton wool soaked in diethyl ether to remove residual material.
Prior to the 24 hour observation the flanks were clipped to remove regrown hair.


Challenge controls:
Induction - Control animals
Intradermal injections were administered using an identical procedure ti that used for the test animlas except that the injections were:
A)Freunds' Complete Adjuvant with distilled water in the ratio 1:1
B) Dried arachis oil BP
C) 50% w/w formulation of dried arachis oil BP in a 1:1 mixture of Freunds Complete Adjuvant /dried arachis oil BP

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

The topical applications folowed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals


B. CHALLENGE EXPOSURE - all animals
Day 21 an area on both flanks of each animal was clipped free of hair. A filter paper patch loaded with a thick even layer of the test material at the maximum non irritant concentration (50% w/w in dried arachis oil BP) was was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure the maximum non iritant concentration was used at challenge, the test material at a concentration of 25% w/w in dried arachis oil BP was applied to a skin site on the left shorn flank, The patches were occluded with an overlapping lenghth of aluminium foil and secured with a strip of elastic adhesive bandage.
After 24 hours, the dressing was removed and the challenge sites swabbed with cotton wool soaked in diethyl ether to remove residual material.
Prior to the 24 hour observation the flanks were clipped to remove regrown hair.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
1
Total no. in group:
9
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 9.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
1
Total no. in group:
9
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 9.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
1
Total no. in group:
9
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 9.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25
No. with + reactions:
1
Total no. in group:
9
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25. No with. + reactions: 1.0. Total no. in groups: 9.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 4.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 4.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 4.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 4.0.
Reading:
1st reading
Group:
positive control
Dose level:
Intradermal: 5 % in arachis oil BP; Topical: 50 % in acetone: PEG 400 (70:30); Challenge: 50 % and 25 % in acetone: PEG 400 (70:30)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
100 % incidence of sensitisation (10/10)
Remarks on result:
positive indication of skin sensitisation
Remarks:
Historical positive control data from 1999 (see attached results)

Any other information on results incl. tables

Please see attached background information for results.

PRELIMINARY IRRITATION STUDY

Based on the intradermal and topical sighting tests the following concentrations were selected for the main study

Intradermal induction : 1% w/w in dried arachis oil BP

Topical induction : 50%* w/w in dried arachis oil BP

Topical challenge : 50%* and 25% w/w in dried arachis oil BP

*Maximum attainable concentration suitable for topical application

MAIN STUDY

Induction Phase

Skin reactions after intradermal induction

Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of all test group animals at 24 and 48 hour observations

Discrete or patchy erythema was noted at the intradermal induction sites of four control group animals at 24 hour observations and all control group animals at the 48 hour observations

Skin reactions after topical induction

Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of all test group animals at the 1 hour observation. Discrete or patchy erythema was noted at the topical induction sites of six test group animals at the 24 hour observation. Bleeding from the intradermal injection sites was noted in four test group animals at the 1 hour observation.

Bleeding from the intradermal injection sites was noted in one control group animal at the 1 hour observation. No signs of erythema or oedema were noted at the treatment sites of control group animals at the 1 and 24 hour observations

Skin reactions after topical challenge

50% w/w in dried arachis oil BP

A positive skin response was noted at the challenge site of one test group animal at the 24 and 48 hour observations. Desquamation was also noted at the challenge site of this test group animal at the 48 hour observation.

No skin reactions were noted at the challenge sites of control group animals at the 24 or 48 hour observations.

25% w/w in dried arachis oil BP

A positive skin response was noted at the challenge site of one test group animal at the 24 and 48 hour observations. Desquamation was also noted at the challenge site of this test group animal at the 48 hour observation.

No skin reactions were noted at the challenge sites of control group animals at the 24 or 48 hour observations.

Mortality - One control group animal was found dead on Day 18. The cause of death was not determined but was thought not to be treatment related. One test group animal was killed in extremis on Day 2.

Clinical signs - There were no symptoms of systemic toxicity

Bodyweight - Bodyweights gain of the test group animals between day 0 and day 24 were comparable to those observed in the control animals.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

Introduction. 

A study was performed to assess the contact sensitisation potential of the test item in the albino guinea pig. The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No. 406 "Skin Sensitisation (adopted 17 July 1992) and Method B6 of Commission Directive 96/54/EC

Methods. 

Based on the results of sighting tests, the concentrations of test material selected for induction and challenge phases were

Intradermal indcution : 1% w/w in dried arachis oil BP

Topical induction : 50%* w/w in dried arachis oil BP

Topical challenge : 50%* and 25% w/w in dried arachis oil BP

*Maximum attainable concentration suitable for topical application

Results. 

The test material produced a 11% (1/9) sensitisation rate and was classified as a mild sensitiser to guinea pig skin.

Conclusion. 

The test item does not meet the criteria for classification according to the Classification, Labelling and Packaging Regulation (CLP) or the Globally Harmonised Classification System.