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Description of key information

Not sensitising in a Buehler assay

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Predates requirement of the LLNA and in vitro methods
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Guideline study under GLP
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.4100 (Skin Sensitisation)
Principles of method if other than guideline:
Similar to OECD 406, Buehler protocol
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
This in vivo study was undertaken for compliance with the U.S Federal Hazardous Substances Act, and predates the EU requirement to use the LLNA and in vitro methods in lieu of guinea pig studies. The repitition of an in vivo study in mice is not indicated, based on Article 25 of Regulation EC No. 1907/2006.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
28 young adult Hartley Guinea Pigs, 14 from each sex, were used in the study.
Animals were purchased from a registered commercial breeding laboratory (Elm Hill Breeding Labs, Chelmsford, MA)
The animals weighed between 250-400 grams (21-43 days old) at the start of the study
Quarantined for 6-9 days.

Animals were group housed in stainless steel cages.
Hardwood chips were used as a contact bedding within the cages.
Animal rooms were maintained at 68 ±3 °F, with a relative humidity of 30-70%
Minimum of 10-13 air exchanges per hour
12 hour light/dark cycle using full spectrum fluorescent lights.

Animals were supplied with a diet of commercial guinea pig ration (Agway Prolab, Agway, Waverly, NY) and municipal tap water ad libitum

Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
USP grade
Concentration / amount:
25%
Day(s)/duration:
6 hours/day, once weekly for 3 consecutive weeks
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
USP grade
Concentration / amount:
25%
Day(s)/duration:
24 hours, in week 5, a 2 week rest after induction
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
4
Details on study design:
A preliminary irritation study was performed with four previously unexposed animals before the start of the challenge phase. Four different concentration of 100%, 50%, 25%, and 10% were applied to the skin for 6 hours. Severe irritataion with necrosis was noted in the 100% group within 48 h; lesser degrees of irritation were found at 50% and 25%. 25% was chosen as the maximally tolerated dose for the sensitisation study.

In the main study, the test substance was applied once per week for 3 consecutive weeks on one side of the animal. The positive control substance was applied in the same manner. Induction scoring was performed 24 hours after the test substance application.
The day preceding the challenge, a 4x3 cm virgin skin site was shaved on the backs of the experimental and control animals. During week 05, the challenge test was performed on freshly clipped skin sites in the same way as the 6 hour closed patch test of the induction phase. The skin was exposed to the test substance for 24 hours. One virgin site was prepared per animal.
At 24 hours after application of the challenge dose, the area of the challenge was marked and the whole back shaved. Two hours after shaving, the test site was examined for erythema and edema.

At the end of the study, the animals were euthanised with CO2.
Positive control substance(s):
yes
Remarks:
DCNB, 0.05-0.1% in acetone
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
no skin reactions or toxicity
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
no skin reactions or toxicity
Remarks on result:
other: valid negative control
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.05% DCNB
No. with + reactions:
4
Total no. in group:
4
Clinical observations:
24 h score 2.25; 48 h score 2.75
Remarks on result:
positive indication of skin sensitisation
Remarks:
valid positive control

Challenge with the test substance following 3 weeks of induction caused no erythema or edema in any of the 10 treated animals, either at 24 or 48 hours after elicitation. No reactions were noted in the negative control group, and 4/4 animals reacted in the positive control group. The score for the positive control at 24 h was 2.25, and at 48 h, was 2.75. Under the conditions of this study the test substance is not considered a skin sensitizer.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this guideline Buehler study in guinea pigs, the test substance is not considered a skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a guideline study under GLP, the test substance was found to be non-sensitising under occlusive patch conditions of a Buehler assay. The criteria for classification under Regulation EC No. 1272/2008 are not met.