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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:
2001-11-08 to 2001-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996-09-30
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2001-08-22
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The conduct of this study was prior to the establishment of the LLNA method (OECD 429).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: light beige powder, nearly odourless

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored dry

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstraße 27, D-33178 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: younger than 1 year
- Weight at study initiation: Treatment group: 249 - 286 g and control group: 260 - 290 g
- Housing: housed individually in Makrolon cages (780 cm²)
- Diet: Raiffeisen Ringfutter, Trockenfutter 52 P, Raiffeisen Kraftfuttermischwerk Kehl
- Water: ad libitum, tap water (with addition of 20 mg / L ascorbic acid and 100 mg citric acid per 100 ml water)
- Acclimation period: 5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 23 °C
- Relative humidity: 30 - 70 °C
- Photoperiod (hrs dark / hrs light): 12:12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Concentration / amount:
0.1 ml of Freund's Adjuvant (FCA) in aqua ad incietabilia (1:1)
Day(s)/duration:
Day 0
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
0.1 ml 5 % test substance
Day(s)/duration:
Day 0
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
intradermal
Vehicle:
other: aqua ad iniectabilia
Concentration / amount:
0.1 ml 5 % test substance in Freund's Adjuvant (FCA)
Day(s)/duration:
Day 0
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Vehicle:
other: aqua ad iniectabilia
Concentration / amount:
not specified
Day(s)/duration:
Day 7
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
50 % test substance (w/w)
Day(s)/duration:
Day 21 (treatment duration: 24 hours)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (test substance) + 5 (controls)
Details on study design:
RANGE FINDING TESTS:
On the basis of two preliminary investigations (each with 3 guinea pigs) the doses for the main study were chosen.
The intradermal irritancy of the test article was investigated in order to find the minimal irritant test article concentration for the intradermal induction using intradermal injections as in the main study. The following test item concentrations were tested as 0,1 ml intradermal injection: 1 %, 3 % and 5 % (w/w) test substance in aqua ad iniectabilia. Observation time points for oedema or erythema were 24, 48 and 72 hours post-exposure.

The topical irritancy of the test article was investigated in order to find the minimal irritant test article concentration for the dermal induction and the maximum non-irritating test article concentration for the challenge application using procedures similar to the main study. The following test item concentrations were tested: 20 %, 33,3 % and 50 % test substance in aqua ad iniectabilia. A filter paper (2x4 cm²) was moistened with the test material and occlusively fixed for 24 hours on the guinea pig's clipped back. Observation time point for oedema or erythema was 24 hours post-exposure.

Results:
intradermal injection: no skin reaction was observed, the 5 % test concentration was determined for intradermal induction in the main study.
topical application: no skin reaction was observed, the test concentration was determined as challenge concentration in the main study.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and dermal application)
- Site: area of dorsal skin 4 x 6 cm in the scapular region was clipped free of pair with an electric clipper.
- Frequency of applications: three pairs of intradermal injections were given once and 6 days later the same site was treated with 0.5 ml sodium lauryl sulphate (10% in vasaline) to provoke a mild inflammatory reaction. 24 hours later the test item was topically applied to the skin by using filter paper (moistened with the test item) and a occlusive bandage.
- Concentrations
Test animals:
Intradermal:
i) 0.1 mL FCA diluted 1:1 with aqua ad iniectabilia
ii) 0.1 mL test article in sesame oil (final concentration: 5 % (w/w))
iii) 0.1 mL test article in FCA mixed with aqua ad iniectabilia in the ratio 1:1 (final concentration: 5 % (w/w))
Topical application:
not specified

Control animals:
Intradermal injection:
i) 0.1 mL FCA diluted 1:1 with aqua ad iniectabilia
ii) 0.1 mL sesame oil
iii) 0.1 mL sesame oil and FCA with aqua ad iniectabilia (1:1) in the ratio 1:1 (w/w)
Topical application:
sesame oil


B. CHALLENGE EXPOSURE
- No. of exposures: 1 (topical application three weeks after the intradermal induction)
- Exposure period: 24 hours
- Site: both flanks clipped (area: 3x5 cm²), right flank: sesame oil; left flank: 50 % of the test item in sesame oil
- Concentrations:
test animals: 50 % test item in aqua ad iniectabilia ((1:1) (w/w))
controls: sesame oil
- Evaluation (hr after challenge): 48 and 72 hours

Challenge controls:
5 guinea pigs were used as control animals.
Challenge dose: right flank: sesame oil; left flank: 50 % of the test item in sesame oil
Positive control substance(s):
yes

Results and discussion

Positive control results:
The postive control (benzocaine) has induced in 6 of 10 guinea pigs in the treatment group slight or moderate, diffuse redness 48 hours post-exposure.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 % (w/w) test substance in sesame oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50 % (w/w) test substance in sesame oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % (w/w) test substance in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50 % (w/w) test substance in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
100 % benzocaine in sesame oil
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
100 % benzocaine in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none

Any other information on results incl. tables

Body weight gain was observed in all tested animals to the end of the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the Regulation (EC) 1272/2008 and subsequent adaptions, the substance is not sensitising to the skin.