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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
in vivo
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 92/69 EEC B6
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Guinea pig maximisation test was available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Age at study initiation: 5-8 weeks
- Weight at study initiation: control and test group: 341-489 g. Pretest 317-435 g
- Housing: individually
- Diet: pelleted standard Kliba 342, Batch no 68/95 guinea pig breeding/maintenance diet, ad libitum
- Water: community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/l) via the drinking water.
- Acclimation period: one week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21-23 °C
- Humidity: 48-60 %
- Air changes: 10-15 per hr
- Photoperiod: 12 hour light, 12 hour dark cyle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
Test:
FCA : phys. saline 1:1
5 % bi-distilled water
5 % FCA : phys. saline 1:1
Control:
FCA : phys.saline 1:1
bi-distilled water
FCA : phys. sal. 1:1/bi-distilled water
Day(s)/duration:
day 1
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
- 25 % bi-distilled water
Day(s)/duration:
day 8
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
15 % bi-distilled water
Day(s)/duration:
day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 10 males
Test group: 20 males
Intradermal pretest: 2 males
Epidermal pretest: 4 males
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: three pairs of intradermal injections
- Exposure period: 7 days
- Test groups:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2) test article, diluted to 5 % with bi-distilled water
3) test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Control group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2) bi-distilled water
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Site: dorsal skin from the scapular region (approximaterly 6 × 8 cm) was clipped free of hair.
- Frequency of applications: 1 week
- Duration: 48 h


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 - 48 h
- Site: hair was clipped and shaved from a 5 × 5 cm area on the left and right flank of each guinea-pig just prior to the application.
- Concentrations: 2 patches ( 2 × 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15 % (left flank) and the vehicle only (bi-distilled water, applied to the right flank) using the same method as for the epidermal application.
- Duration: 24 h
- Evaluation: 24 and 48 h after removal of the dressing
Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester and 2- mercaptobenyothiazol

Results and discussion

Positive control results:
For validation of sensitivity, a known sensitizer (2-mercaptobenzothiazol) was selected as a positive control.
In this study, 95 % and 90 % of animals of test group were observed with positive skin reactions at the 24- and 48-hour reading respectively after treatment with a non-irritant test substance concentration of 5 % in peanut oil. No skin reactions were observed in the control group. The results obtained with test article at 10 % in peanut oil were not taken into consideration since both control group were observed with erythematous reactions.
A known mild sensitizer was also selected as a positive control (4-aminobenzoic acid ethyl ester). In this study 30 % and 35 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 30 % in mineral oil. No skin reactions were observed in the control group.
The response of at least 30 % positive animals is considered positive.
Test article 2-mercaptobenzothiazol at concentration of 5 % in peanut oil is considered an extreme sensitiser.
Test article 4-aminobenzoic acid ethyl ester at concentration of 30 % in mineral oil is considered a moderate sensitiser.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythem and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

After epidermal induction, due to orange-red discoloration produced by test substance, a possible erythema reaction could not be determined.

Viability / mortality / macroscopic findings: as there were no deaths during the treatment period, no necropsies were performed.

Clical signs, systemic: no symptoms of systemic toxicity were observed in the animals.

Body weight: 2 out of 10 animals of the control group (nos. 325, 330) and 1 out of 20 animals of the test group (no. 346) incidentally lost weight during the acclimatisation period. No weight loss was observed during treatment period.

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
Not skin sensitiser.
Executive summary:

Method

A guinea pig maximisation test was carried out to assess the allergenic potential of the substance. Ten males were used as control group and 20 males were used as test group.

Before the main test, a pretest was performed in order to identify a maximally tolerated concentration of test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of test article, by the topical route of administration, was identified for the challenge application.

The pretest was performed by intradermal (0.1 ml/site, at concentrations of 1, 3 and 5 % of test article in bi-distilled water) and epidermal route (5, 10, 15 and 25 % of test material in bi-distilled water).

Based on pretest findings:

- application a 5 % test substance dilution in bi-distilled water was selected for intradermal induction

- 25 % test substance in bi-distilled water was selected for the induction application and 15 % test substance in bi-distilled water was selected for the challenge application.

Results

None of the animals of the test group showed positive skin reactions responding to a 5 % intradermal induction dose. Therefore, test substance was considered as not skin sensitising.