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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-06-17 - 1996-08-04 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization", adopted by the Council on July 17,1992 (reported Paris, April 29,1993).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 96/54/EEC, B.6. "Acute Toxicity Skin Sensitization", July 30, 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
No new study has been conducted; data from an existing GLP-guideline study (1996) has been used as this study is totally sufficient to cover this endpoint. This study was conducted prior to the acceptance of alternative methods or REACH.

Test material

Constituent 1
Test material form:
solid: particulate/powder

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
- Females
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 358 - 443 g
- Housing: singly or in pairs in solid-floor polypropylene cages furnished with woodflakes. The animal room was maintained at a temperature of 20 to 23 °C and relative humidity of 52 to 70%. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.
- Diet: Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study.
- Acclimation period: After an acclimatisation period of at least five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen.
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 °C
- Humidity (%): 52 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Freund's Complete Adjuvant plus distilled water in the ratio 1:1
Concentration / amount:
5 %, 0.1 ml (intradermal)
50 % (epicutaneous)

Day(s)/duration:
topical induction was made 1 week (day 7) after intradermal induction (day 1)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
5 %, 0.1 ml (intradermal)
50 % (epicutaneous)
Day(s)/duration:
topical induction was made 1 week (day 7) after intradermal induction (day 1)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal and epicutaneous
Vehicle:
other: 5% w/v emulsion of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Concentration / amount:
5 %, 0.1 ml (intradermal)
50 % (epicutaneous)
Day(s)/duration:
topical induction was made 1 week (day 7) after intradermal induction (day 1)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
50 %
Day(s)/duration:
on day 21 for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
25 %
Day(s)/duration:
on day 21 for 24 hours
Adequacy of challenge:
other: To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 25% w/w in arachis oil BP was similarly applied to a skin site on the left shorn flank
No. of animals per dose:
10 (test group)
5 (control group)
Details on study design:
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:
1) Selection of Concentration for Intradermal Induction
Two concentrations of test material were investigated (1 % and 5% w/v in arachis oil BP). A total of two guinea pigs were used, each guinea pig receiving four 0.1 ml injections of only one 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 according to the Draize scale shown in the table below. The degree of oedema was not evaluated. 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.
2) Selection of Concentration for Topical Induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant 17 days earlier) were treated with four preparations of the test material (50%, 25%, 10% and 5% w/w in 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.
3) Selection of Concentration for Topical Challenge
Four preparations of the test material (50%, 25%, 10% and 5% w/w in 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 of the 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
Two main phases were involved in the main study; (a) an induction of a response and (b) a challenge of that response.
1) Induction
Induction of the 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:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 5% w/v suspension of the test material in arachis oil BP
c) a 5% w/v emulsion of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. injection site b) was evaluated according to the scale shown in the table below.
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 test material formulation. A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), loaded with the test material formulation (50% w/w in 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 (BLENDERM: approximate size 50 mm x 30 mm) covered with an overlapping length of aluminium foil. The patch and foil were further secured with 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.
The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches using the scale shown in Appendix X.
Any other reactions were also recorded.
Induction of the Control Animals: Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) arachis oil BP
c) a 50% w/v formulation of arachis oil BP in Freund's Complete Adjuvant/distilled water 1:1
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.

2) Challenge
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 a thick, even layer of test material at the maximum non-irritant concentration (50% w/w in arachis oil BP) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 25% w/w 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 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 challenge sites were swabbed with cotton wool soaked in distilled water 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.

3) Evaluation of Skin Reactions
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified using the scale shown in the table below.
Any other reactions were also recorded.

EVALUATION OF SKIN REACTIONS*
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar formation
(injuries in depth) 4
Oedema Formation
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending
beyond the area of exposure) 4
*Draize J.H., (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington D.C. p.31

Interpretation of Results
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:
Percentage of animals sensitised Classification of 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
The data obtained may be used to classify the test material according to Commission Directive 93/21/EEC adapting Council Directive 67/548/EEC on the classification, packaging and labelling of dangerous substances.
The test material will be classified as sensitising and assigned the symbol "Xi", the indication of danger 'irritant' and the risk phrase R 43 "MAY CAUSE SENSITISATION BY SKIN CONTACT" if 30% of the test animals show a sensitisation response.
Challenge controls:
25 % test item in the negative control animals
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Reading:
other: not applicable
Group:
positive control
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material produced a 0% (0/10) sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations. No risk phrase is required.
Executive summary:

In order to assess the cutaneous allergenic potential of the test material, the Maximization Test was performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6, under GLP.

The intradermal induction of sensitization in the test group was performed in the shoulder region with a 5 % dilution of the test item in arachis oil BP, 5% emulsion of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water and Freund's Complete Adjuvant plus distilled water in the ratio 1:1. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 50 % in arachis oil one week after the intradermal induction. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 50% and 25 % in arachis oil BP under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

 No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

Very slight erythema was noted at the intradermal induction sites of two control group animals at the 24-hour observation. No skin reactions were noted at the intradermal induction sites of control group animals at the 48-hour observation. None of the control and the test animals showed skin reactions after the challenge treatment with the test material.

Conclusion: Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC and Regulation 1272/2008, the test material does not have to be classified and labelled as a skin sensitizer.