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Administrative data

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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(FREY-TOX GmbH, Herzberg / Elster, Germany)
Type of study:
Buehler test
Species:
guinea pig
Strain:
other: SPF albino guinea pigs of the stock Crl:HA
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Weight at study initiation: 287 - 365 g
- Housing: two or three animals in each macrolone cage
- Diet: Altromin 3123 (Altromin, Lage, Lippe, Germany); ad libitum
- Water: vitamin C enriched domestic quality water that was acidified to pH 2.5 with hydrochloric acid; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C +/- 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
(PEG E400)
Concentration / amount:
1st induction: 2.5%, 2nd induction: 1%, 3rd induction: 1%, challenge: 0.5%
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
(PEG E400)
Concentration / amount:
1st induction: 2.5%, 2nd induction: 1%, 3rd induction: 1%, challenge: 0.5%
No. of animals per dose:
control group: 10; test group: 20
Details on study design:
RANGE FINDING TESTS:
During a first preliminary investigation, two female animals were initially treated with four concentrations of the test item of 25%, 50%, 75% and 100%. The response of the guinea pigs to this treatment was unexpectedly intense indicating a moderate to severe reduction of the general state of health and severe skin reactions. Immediately after termination of 6-hour exposure, an intense heat and redness of skin was observed on the treated body areas. Posture, mimic, vocal utterance and behaviour of the animals as well as occurence of tremor and hypothermia revealed distinct signs of pain of the animals. After cleaning and drying of the treated skin areas, the animals were wrapped in warm towels and their cage was temorarily put into a very warm environment. Under these conditions, the general state of health of the animals improved and only a slight impairment was still discernible after 6 hours. After that, a modified procedure concerning further preliminary investigations and a reduction of the test concentration were decided. During a second and third preliminary investigation, only one concentration of the test item (5% or 10% (w/w)) was tested in two female animals each. These animals still showed disrete to intense erythema, but no comparable systemic reactions. Therefore, a fourth preliminary investigation was performed with the 1% and 2.5% (w/w) test item and two male animals. Based on the results, a concentration of 2.5% was initially selected for the induction and a concentration of 1% for the challenge application. However and due to the moderate skin reactions in the test group during the induction phase, it was decided to perform the 2nd and 3rd induction only with 1% (w/w) test item and the challenge treatement with 0.5% (w/w).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 3 x 6 h
- Test groups: day 0: A patch of multilayered gauze (2.5 x 2.5 cm) coated occlusively with impermeable tape (Blenderm) on one side was saturated with 0.5 mL of 2.5% (w/w) test item and the patch was placed on the skin of all test group animals. The fixation of this occlusive dressing was made by wrapping the trunk of the animals with Gothaplast tape. The dressings were removed after 6 h and residues of the test item were washed off from the skin initially with a watery 50% solution of PEG E 400 and subsequently with pure water. The procedure was repeated on days 7 and 14, but with a lower concetration (1%). The test sites were assessed for skin irritation 24 h after each patch application.
- Control group: treated like test groups, but with the vehicle PEG E 400 only
- Site: left flank region
- Frequency of applications: 3 applications (day 0, day 7, day 14)
- Duration: 6 h
- Concentrations: 1st induction: 2.5%, 2nd induction: 1%, 3rd induction: 1%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28 (4 weeks after the 1st induction)
- Exposure period: 6 h
- Test groups: A patch of multilayered gauze (2.5 x 2.5 cm) coated occlusively with impermeable tape (Blenderm) on one side was saturated with 0.5 mL of 0.5% (w/w) test item and the patch was placed on the skin of the anterior part of the right flank. In the same way an occlusively coated patch of multilayered gauze (2.5 x 2.5 cm) was saturated with 0.5 mL of the vehicle PEG E 400 and placed psterior on the right flank. The fixation of this occlusive dressing was made by wrapping the trunk of the animals with Gothaplast tape. The dressings were removed after 6 h and residues of the test item were washed off from the skin initially with a watery 50% solution of PEG E 400 and subsequently with pure water.
- Control group: treated like test groups, but with the vehicle PEG E 400 only
- Site: right flank region
- Concentrations: 0.5%
- Evaluation (hr after challenge): 24 and 48 h after the removal of the patch (about 6 h before the 24 h reading, the sites were clipped and shaved in order to facilitate the evaluation)

OTHER: The animals were observed at least once a day for signs of illness and reactions to the treatment. The animals of the main study were weighed at the beginning of the study (day 0) and at the end of the study (day 33).
Positive control substance(s):
yes
Remarks:
(The last positive control test with the reference material alpha-Hexylcinnamaldehyde (tech., 85%) was performed from March 2013 until May 2013. 85% of the guinea pigs showed a positive response ( erythema grade 1 and 2).)
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible change.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No visible change..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible change.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No visible change..
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No visible change.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.5%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No visible change..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No visible change.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No visible change..

No signs of illness were observed in the animals of the main study. They had a normal body weight gain during the study period.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitization:

 

The dermal sensitizing potential of the test substance was investigated on the basis of GLP and OECD 406 (BASF SE, 65V0731/11A403, 2013; reliability score 1). The Buehler test was used and the main test was performed on 20 guinea pigs in the test group and 10 animals in the control group. The main study started with an induction phase including an occlusive topical application for 6 hours once a week for three consecutive weeks. The animals of the test group were induced with 2.5% of the test item in PEG E 400, whereas the animals of the control group were induced with PEG E 400. None of the control group animals showed signs of skin irritation during the induction phase. A discrete or patchy erythema (grade 1) was observed in 10 test group animals after the first induction as well as a moderate and confluent erythema (grade 2) in five test animals. Therefore and in accordance with the sponsor, a reduced concentration level of the test item of 1% was used for the application of the 2nd and 3rd induction, whereas the control group was induced again with the vehicle PEG E 400. Nine test group animals showed a discrete or patchy erythema (grade 1) after the second induction. A moderate and confluent erythema (grade 2) was also observed in two test group animals. After the third induction, four test group animals showed a moderate erythema and nine animals of the test group a slight erythema, whereas the animals of the control group were free of any reaction after both the second and third induction. The 6-hour challenge procedure of an occlusive topical application of the test item on the right flank of all animals followed four weeks after the first induction. The skin reactions were evaluated 24 hours after the challenge application. For the challenge, the test item was used in a concentration of 0.5%. None of the animals of the test group and control group responded with skin reactions to the challenge treatment with the test item and the vehicle. Under the experimental conditions described, the test substance did not supply evidences for delayed contact hypersensitivity.

 

Additionally, in-vitro studies were performed. The tests have undergone internal validation.

 

The first assay identifies the peptide binding properties in chemico (BASF SE, 64V0731/11A402, 2012; reliability score 1). Two incubations with model peptides containing either cysteine or lysine were performed. The substance did not deplete cysteine or lysine peptides during the 24h incubation period.

The second assay targets the ability to activate dentritic cells to release the surface marker CD86 (BASF SE, 65V0731/11A403, 2012; reliability score 1). Ethylene diamine was used as positive control. The test substance did not induce CD86 levels beyond the threshold level to identify sensitizing substances.

 

The outcome of the in-vitro assays confirmed the result of the in vivo sensitization study and therefore, the test substance would not be identified as a skin sensitizer.


Migrated from Short description of key information:
in vivo: Buehler-test - not sensitising (BASF SE, 32H0731/11X591, 2013);
in vitro: direct peptide binding assay - non protein binding (BASF SE, 64V0731/11A402, 2012); dentritic cell activation assay - no activation of dentritic cells (BASF SE, 65V0731/11A403, 2012);

Justification for selection of skin sensitisation endpoint:
Key study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC):

The available data is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin sensitization under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

 

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. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).