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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-Nov-1996 to 02-Jan-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD test guideline 406)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted on 17-Jul-1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4100 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When this GPMT was performed, the LLNA did not yet exist as an OECD testing guideline.
Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd, Wölferstrasse 4, 4414 Füllinsdorf, Switzerland
- Age at study initiation: 5-7 weeks old
- Weight at study initiation: 337-369 g
- Housing: individually, in Makrolon type-3 cages
- Diet: pelleted standard guinea pig diet (Nafag Ecosan 845 25W4; Nafag, Nähr- und Futtermittel AG, 9202 Gossau, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: none for pretest animals, 1 week for main test animals

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10-15 air changes/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: 25-Nov-1996 to 02-Jan-1997
Route:
intradermal and epicutaneous
Vehicle:
other: bidistilled water with 1 drop of Tween 80
Concentration / amount:
Intradermal induction exposure: 5 %
Epicutaneous induction exposure: 25 %
Epicutaneous challenge exposure: 15 %
Route:
epicutaneous, occlusive
Vehicle:
other: bidistilled water with 1 drop of Tween 80
Concentration / amount:
Intradermal induction exposure: 5 %
Epicutaneous induction exposure: 25 %
Epicutaneous challenge exposure: 15 %
No. of animals per dose:
10 test animals, 5 control animals
Details on study design:
RANGE FINDING TESTS:
A pretest was performed to identify appropriate test item concentrations for induction and challenge in the main study.
- Intradermal injections: Two pairs of intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made simultaneously into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig with the test article at concentrations of 5, 3 and 1 % and the dermal reactions assessed 24 hours later.
- epidermal applications (after intradermal application with FCA/physiological saline): Two pairs of intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made simultaneously into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper ( 2 x 2 cm) were saturated with the test article at 50%, 25%, 15% and 10% and applied to the clipped and shaved flanks for 24 hours. 21 hours after removal of the dressing the application site was depilated. The reaction sites were assessed 24 and 48 hours after removal of the bandage.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 exposures, intradermal and epicutaneous. Three pairs of intradermal injections (0.1 ml/site) were given (1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline, test article in the vehicle, test article in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline). On test day 8 the test site was treated epicutaneously with a filter paper saturated with the test article.
- Exposure period: single application (intradermal exposure), 48 hours (epicutaneous exposure)
- Test groups: 1 group of 10 animals
- Control group: 1 group of 5 animals (same treatment except that the test item was omitted)
- Site: scapular region (intradermal exposure), test item placed over injection sites (epicutaneous exposure)
- Frequency of applications: 1 intradermal application, 1 epicutaneous application
- Concentrations: 5 % (intradermal exposure), 25 % (epicutaneous exposure)
- Other: all animals were treated epicutaneously with sodium lauryl sulfate (10 % in paraffinum perliquidum) on day 7 as no primary irritation was observed in the pretest

B. CHALLENGE EXPOSURE
- No. of exposures: 1 epicutaneous exposure
- Day of challenge: day 22
- Exposure period: 24 hours
- Test group: 10 animals
- Control group: 5 animals
- Site and concentration: left flank (15 % test item), right flank (vehicle) in test and control group animals
- Evaluation: approximately 24 and 48 hours after removal of the dressing

The observed skin reactions were scored on the basis of the Draize score (1959). Rating was performed according to the Magnusson and Kligman grading scale.
Challenge controls:
Control animals were treated similar to test animals during challenge (note: during the induction phase the test item was omitted in the control animals).
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole and alpha-hexylcinnamaldehyde, separate experiments
Positive control results:
In the 2-MERCAPTOBENZOTHIAZOLE positive control assay, 90 % (at the 24-hour reading) and 100 % (at the 48-hour reading) of the animals were observed with positive skin reactions. In the ALPHAHEXYLCINNAMALDEHYDE positive control assay, 65 % (at the 24-hour reading) and 60 % (at the 48-hour reading) of the animals were observed with positive skin reactions.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no sign of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no sign of systemic toxicity.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no signs of systemic toxicity.

SKIN EFFECTS AFTER INTRADERMAL INDUCTION - performed on test day 1

A normal development of the local symptoms was observed in the animals of the control and test group after the intradermal injections.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION - performed on test day 8

CONTROL GROUP: No erythematous or oedematous reaction was observed in the animals treated with bi-distilled water containing one drop of tween 80 only.

TEST GROUP: No erythematous or oedematous reaction was observed in the animals treated with the test article at 25% in bi-distilled water containing one drop of tween 80.

SKIN EFFECTS AFTER THE CHALLENGE - performed on test day 22

CONTROL AND TEST GROUP: No positive reactions were observed in the animals neither when treated with bidistilled

water containing one drop of tween 80 alone nor when treated with the test article at 15% in bi-distilled water with one drop of tween 80. A slight beige discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

One animal of the epidermal pretest group lost weight during its test article treatment period. The body weight of the other animals was within the range of physiological variability known for this strain and age.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this study, the test article is considered to be a non-sensitizer when used under the described test conditions.
Executive summary:

In order to assess the cutaneous allergenic potential of the test material, a GLP-compliant Maximization-Test in accordance with OECD Guideline No. 406 and Directive 92/69 EEC B.6 was carried out on 15 (10 test and 5 control) male albino guinea pigs. Induction was a two stage operation. First, three pairs of intradermal injections (0.1 ml/site) were given into the scapular region (1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline, 5% test article in the vehicle, 5% test article in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline). On test day 8, the epicutaneous induction of sensitization was conducted under occlusion with the test article at 25% in the vehicle (bi-distilled water with one drop of tween 80). Two weeks after the epicutaneous induction application the challenge was conducted by epicutaneous application of the test article at 15% in the vehicle under occlusive dressing. The animals of the control group were induced with bi-distilled water with one drop of tween 80 and FCA/ physiological saline and challenged similarly as those of the test group. Cutaneous reactions, i.e erythema and eschar, as well as oedema formation 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 and no deaths occurred. In this study 0% of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 15%. No skin reactions were observed in the control group. Therefore, the test article applied at a concentration of 15% in bi-distilled water with one drop of tween 80 is considered to be a non-sensitizer when used under the described test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test substance was investigated for its sensitizing potential in a GLP-compliant Maximization test according to OECD guideline 406 (RCC, 1997). Himalayan guinea pigs received intradermal induction treatments with 5% test material in bi-distilled water with one drop of tween 80 and one epicutaneous induction treatment with 25% test material in the vehicle. For intradermal induction, three pairs of injections (adjuvant/saline; test item in vehicle; test item in vehicle plus adjuvant/saline) were given. Epidermal challenge was performed by occlusive application for 24 h two weeks later (15% test article in the vehicle). Control animals were treated with the vehicle during induction and with vehicle and test material during challenge. No skin reactions were observed either for control or test group animals and therefore no animal was sensitized by the test article under the experimental conditions employed. The sensitivity of the strain was controlled separately with 2 -mercaptobenzothiazole and alpha-hexylcinnamaldehyde. In conclusion, the test substance is not considered to be a skin sensitizer in albino guinea pigs and does not require classification.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No local reactions were observed in a guinea pig maximization test (OECD 406) after an intradermal induction with 5%. Therefore, the substance does not require classification as a skin sensitizer under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.