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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:
1998-12-27 to 1998-11-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

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:
At the time this study was conducted, an in-vivo maximization test was an acceptable test for determining skin sensitization.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: At room temperature protected from light
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared in arachis oil BP/Freund's Complete Adjuvant to the desired concentration based on the type of test

FORM AS APPLIED IN THE TEST (if different from that of starting material)
lntradermal Induction: 5% w/v in arachis oil BP and 5% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1 :1)
Topical Induction: 50% w/w in arachis oil BP
Topical Challenge: 10% and 5% w/w in arachis oil BP

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 (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known:
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 361-437 g
- Housing: Singly or in paris in in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): ad libitum (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK)
- Water (e.g. ad libitum): ad libtium tap water
- Acclimation period: >5 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-21
- Humidity (%): 43-54
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
50% w/w
Day(s)/duration:
2
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
5% w/v
Day(s)/duration:
9
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
other: Freund's Complete Adjuvant plus distilled water (1:1)
Concentration / amount:
5% w/v
Day(s)/duration:
9
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:
10% w/w
Day(s)/duration:
1 d
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
5 w/v %
Day(s)/duration:
1 d
No. of animals per dose:
10 animals for the test concentration
5 animals for the 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:
Selection of Concentration for lntradermal Induction
Three concentrations of test material were investigated (1 %, 5% and 10% w/v in arachis oil BP). A total of three 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 Appendix X. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that could be intradermally injected and 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 Freund's Complete Adjuvant ten 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.

Selection of Concentrations 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
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 9 days
- Test groups: 1
- Control group: 1
- Site: Shoulder region
- Frequency of applications: Once on day 0 intradermally, and once on day 7 topically
- Duration: 9 days for the intradermal, and 2 days for topical
- Concentrations: a) Freund's Complete Adjuvant plus distilled water in the ratio 1: 1 (vehicle control)
b) a 5% w/v formulation of the test material in arachis oil BP
c) a 5% w/v formulation of the test material in a 1: 1 preparation of Freund's Complete Adjuvant plus distilled water.

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 1
- Exposure period: 1 day
- Test groups: 1
- Control group: 1
- Site: Shorn right flank and shorn left flank of each animal
- Concentrations: 10% w/w in arachis oil for the right shorn flank, and 5% w/w in arachis oil for the left shorn flank.
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
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
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the vehicle injection sites (ie injection site b) was evaluated according to the scale shown in Appendix X. 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.
Positive control substance(s):
no

Results and discussion

Positive control results:
No concurrent positive control was used.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% w/w
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animal 9 was found dead on day 9, Cause of animal death not considered substance related. No impact to overall study results
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 % w/w
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animal 9 was found dead on day 9. Cause of animal death not considered substance related. No impact to overall study results
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10 % w/w
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animal 9 was found dead on day 9. Cause of animal death not considered substance related. No impact to overall study results
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
5% w/w
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animal 9 was found dead on day 9. Cause of animal death not considered substance related. No impact to overall study results
Remarks on result:
no indication of skin sensitisation
Reading:
other: No positive control group used
Group:
positive control
Remarks on result:
not measured/tested
Reading:
other: No negitive control goups used
Group:
negative control
Remarks on result:
not measured/tested

Any other information on results incl. tables

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 23 were comparable to those observed in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
This study validly determined that the test substance does not meet the criteria to be classified as a skin sensitizer according to GHS criteria.