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Description of key information

Tested in a study according to OECD test guideline no. 406 and GLP conditions the test item was not a skin sensitiser.

In accordance with Column 2 of REACH Annex VII No. 8.3.1 in vitro or in chemico tests for skin sensitisation do not need to be conducted because adequate data from an in vivo skin sensitisation study are available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 JUL 2002 to 11 SEP 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17.07.1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30.7.1996
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
August 1998
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
According to Annex VII, Column 2 from Regulation (EC) No 1907/2006 (including amendments up to Commission regulation (EU) 2016/1688 of 20 September 2016) in vivo skin sensitisation studies that were carried out or initiated before 11 October 2016, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4) shall be considered appropriate to address standard information requirement for the endpoint skin sensitization. This applies for the guinea pig maximization test presented here.
Species:
guinea pig
Strain:
other: Mol:DH (Moellegaard)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:M & B A/S, P.O.box 1079, DK-8680 Ry, Denmark
- Weight at study initiation: 284 - 350 g
- Housing: In transparent macrolon cages (type V) on soft wood granulate in an air-conditioned room, 5 animals per cage
- Diet: ssniffe RIM-H, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at leat 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
water
Concentration / amount:
0.48 % test substance (42%) in deionized water corresponding to 0.2 % active ingredient
Day(s)/duration:
single application
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
4 % test substance (42 %) in deionized water corresponding to 1.68 % active ingredient
Day(s)/duration:
48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
4 % test substance (42 %) in deionized water corresponding to 1.68 % active ingredient
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in test group: 10
Number of animals in control group: 5

Pretest:
Determination of the tolerance of the intradermal injections: 2 animals
Determination of the primary non-irritating concentration: 3 animals
Details on study design:
RANGE FINDING TESTS:
DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION:
In a dermal-occlusive test three animals each received 4 intradermal injections of a 50 % Freund's Complete Adjuvant emulsion (4 x 0.1 mL) into the dorsal area, since Freund's Complete Adjuvant may lower the threshold of primary irritation. Thereafter, the test substance was administered to the right or left flank of the guinea pigs. The hair on the flanks of the animals was removed mechanically. 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema.

DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS:
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 2 guinea pigs (0.1, 0.48 and 2.4 % test substance). The injection sites were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders. 24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Three pairs of intradermal injections and an epidermal application on test day 8
- Exposure period: 3 weeks (1 week after the inreadermal injections epidermal applicationwas done. Two weeks after that, challenge was performed)

- Exposure group:
Two intradermal injections/site (3 sites, 0.2 ml/site) were made within the shaved dorsal area of 2 x 4 cm as follows:
1) 1:1 (v/v) mixture of Freud´s Complete Adjuvant and deionized water
2) Test item at 0.48 % (corresponding to 0.2 % active ingedient) in deionized water
3) Test item at 0.48 % (corresponding to 0.2 % active ingedient) in a 50 % Freund's Complete Adjuvant emulsion


-Control group:
Two intradermal injections/site (3 sites, 0.2 mL/site) were made within the shaved dorsal area of 2 x 4 cm as follows:
1) 50 % Freund's Complete Adjuvant emulsion in water
2) deionized water
3) 50 % Freund's Complete Adjuvant emulsion in water mixed with an equal volume of water.

- Site: dorsal region
- Duration: single intradermal injection, occlusive dressing from epidermal application was left for 48 h

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Site: 5 x 5 cm on the left flank
- Concentrations: 4 % test substance (42 %) in deionized water corresponding to 1.68 % active ingredient in deionized water
- Evaluation: 24 and 48 h after removal of the patches, reaction sited were assessed for erythema and oedema.
- Control animals were treated with the test item as well.

Reading and Scoring:
The scoring system was performed by visual scoring of erythema, oedema and other clinical changes of skin conditions. They were assessed using the following Magnusson and Kligman gradng scale:
0 = no visible change
1 = discrete or patchy erytheman
2 = moderate and confluent erythema
3 = intense erythema and swelling
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (tested in a separate experiment)
Positive control results:
90% (24 h and 48 h reading) of treated animals (9/10) showed a positive reaction during the observation period.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1.68 % active ingredient
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1.68 % active ingredient
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1.68 % active ingredient
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1.68 % active ingredient
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation

Intradermal induction treatment

Intradermal injections with Freund's Adjuvant (with and without test substance) caused well-defined erythema and edema as well as sporadically encrustations. The administration sites treated with the test substance in deionized water showed well-defined erythema and slight edema. Intradermal injections of the vehicle alone exhibited no signs of irritation.

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at day 7.

Dermal induction treatment

After the removal of the patches at day 10, well-defined erythema and edema, indurated and of the patches at day 10, well-defined erythema and edema, indurated and encrusted skin as well as sporadically necrosis were observed at the sites previously treated with Freund's Adjuvant. The administration sites treated with the test substance alone showed slight erythema and edema. The administration sites treated with the vehicle alone showed no signs of irritation.

Dermal challenge treatment

No skin reactions were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage.

The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexylcinnamaldehyde for the maximization test.

Interpretation of results:
GHS criteria not met
Conclusions:
Tested in this study according to OECD test guideline no. 406 and GLP conditions the test item was not a skin sensitiser.
Executive summary:

Testing for sensitising properties of the test item was performed in female guinea pigs according to the adjuvant sensitisation test by Magnusson and Kligman (Guinea pig Maximisation Test) according to OECD TG 406 and under GLP conditions. Pretests determined the maximal intradermal and epicutaneous concentrations causing mild to moderate skin irritation while beeig systemically tolerated.

Intradermal induction was performed using 0.2 % active ingredient in deionized water or in a mixture of Freund's Adjuvant and water (1:1 (v/v)). Epidermal induction was carried out with 1.48 % active ingredient in deionized water.

Using 1.68 % active ingredient in water for the challenge treatment resulted in a 0 % incidence of animals with skin reactions at the 24 and 48 hour reading in both the test group as well as the control group.

The positive control from the test lab showed distinctive positive results under the conditions applied in the lab (up to 90 % of animals with positive skin reactions weras the control animals showed no skin reactions).

Under the conditions of the test, the test item was not a skin sensitiser.

Endpoint:
skin sensitisation: in vitro
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Testing for sensitising properties of the test item was performed in female guinea pigs according to the adjuvant sensitisation test by Magnusson and Kligman (Guinea pig Maximisation Test) according to OECD TG 406 and under GLP conditions. Intradermal induction was performed using 0.2 % active ingredient in deionized water or in a mixture of Freund's Adjuvant and water (1:1 (v/v)). Epidermal induction was carried out with 1.48 % active ingredient in deionized water.

Using 1.68 % active ingredient in water for the challenge treatment resulted in a 0 % incidence of animals with skin reactions at the 24 and 48 hour reading in both the test group as well as the control group.

The positive control from the test lab showed distinctive positive results under the conditions applied in the lab (up to 90 % of animals with positive skin reactions whereas the control animals showed no skin reactions).

Under the conditions of the test, the test item was not a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

As no sensitising response was observed in a reliable guinea pig study, no classification is required according to Regulation (EC) No. 1272/2008 (CLP).