Registration Dossier

Administrative data

Description of key information

The test substance is not considered sensitizing to the skin

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:
12 Dec 2016 to 13 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 17, 1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March, 2003
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
The Buehler test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD 406.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Test iem No.: 15/0531-1
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
HsdDhl: DH, SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo CRS (Switzerland) Limited
- Female animals should be nulliparous and non-pregnant.
- Weight at study initiation: 275 g – 325 g
- Housing: Stainless steel wire mesh cages with grating with shallow cage body; floor area: 4225 cm2; 5 animals per cage
- Enrichment: Wooden gnawing blocks (Type KNH E-041); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria
- Bedding: H 15005-29; Ssniff, Spezialdiäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany)
- Diet: FD1 SQC; SDS Special Diets Services, 67122 Altrip, Germany, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: Approx. 10
- Photoperiod: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
deionized
Concentration / amount:
5% / 0.5 mL
Day(s)/duration:
Day 0 / 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
deionized
Concentration / amount:
5% / 0.5 mL
Day(s)/duration:
Day 7 / 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5% / 0.5 mL
Day(s)/duration:
Day 14 / 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
water
Remarks:
deionized
Concentration / amount:
2% / 0.5 mL
Day(s)/duration:
Day 28 / 6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
- Control group: 10
- Test group: 20
Details on study design:
EXPERIMENTAL PROCEDURE
- Route of application: epicutaneous occlusive
- Occlusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance preparations were applied to the skin of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Clipping of the fur: At least 15 hours before each test substance application at the appropriate application sites. If necessary, additionally at least 3 hours before evaluation of the skin reactions.
- Time of day of application: In the morning
- Reason for the selection of the route of application: Since a skin contact with the test substance cannot be excluded, the test substance was examined for possibly sensitizing properties to the skin. For a comprehensive assessment of the sensitizing potential, 3 epicutaneous inductions were carried out in accordance with the provisions of the BUEHLER Test prior to the challenge application.
- Clinical observations: Clinical signs for each animal were recorded several times on the day of administration and at least once during each workday thereafter.
- Illumination used for reading: Daylight tubes "Lumilux" (L 58W/860 PLUS ECO 25X1, Osram, Germany)
- Body weight determination: Individual body weights on day 0 and on the last day of observation.
- Mortality: A check for any dead or moribund animal was made at least once each workday.

RANGE FINDING TEST 1
- Amount applied: 0.5 mL of the test substance preparation was applied to each animal.
- Oclcusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance preparations were applied to the skin of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Duration of exposure: 6 hours
- Application site: Right and left flank, anterior and posterior
- Application frequency: One application
- Number of test animals: 3
- Readings: 1, 24, 48 and 72 h after removal of the patch
- Concentrations: 80, 50, 25 and 10 %

RANGE FINDING TEST 2
- Amount applied: 0.5 mL of the test substance preparation was applied to each animal.
- Oclcusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance preparations were applied to the skin of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Duration of exposure: 6 hours
- Application site: Right and left flank, anterior and posterior
- Application frequency: One application
- Number of test animals: 3
- Readings: 1, 24 and 48h after removal of the patch
- Concentrations: 5, 2.5, 1.0 and 0.5 %

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Occlusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance preparation were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Application site: Intact flank; test item preparation: left flank anterior
- Application frequency: Three times on weekly intervals; days 0, 7 and 14 on the same application area
- Duration: 6 hours per application
- Concentration: 5% (w/w)
- Readings: 24h after removal of the patch

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Occlusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance preparation were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Application site: Intact flank; test item preparation: right flank posterior
- Day(s) of challenge: The challenge was carried out 14 days after the third induction.
- Duration of exposure: 6 hours
- Concentrations: 2% (w/w)
- Readings: 24 and 48 h after the removal of the patch
Challenge controls:
The control group was treated analogously to the test group. As deionized water was used as vehicle, no vehicle was applied as vehicle control on the animals since water was not expected to show any influence on the test result.
Positive control substance(s):
yes
Remarks:
A separate study is performed twice a year in the laboratory. The positive controls with Alpha-Hexylcinnamaldehyde, techn. 85% showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.
Positive control results:
The last positive control test with the reference material Alpha-Hexylcinnamaldehyde, technical grade, 85 % was performed from July until August 2016. The sensitization potential of the positive reference item has been sufficiently shown by the response of 13 of 20 guinea pigs (65 %) of the test group with positive skin reactions (erythema with grade 1 and 2).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2% (w/w)
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2% (w/w)
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a BUEHLER test study performed according to OECD TG 406 under GLP conditions, the skin sensitizing potential of the test substance was assessed on the skin of guinea pigs (Bioassay, 2017). Based on two preliminary range-finding tests, 20 animals were exposed (epicutaneous, occlusive) to 5% for the induction phase and 2% for the challenge of the test substance prepared dionized water. The control group consisted of 10 animals. In the test group 12 animals showed discrete erythema (grade 1) after the first induction, while 14 animals showed discrete or moderate erythema (grade 1 or 2) after the second induction. 20 animals showed discrete or moderate erythema after the third induction. After the challenge 1 animal in total showed discrete erythema (grade 1). Based on these results, it was concluded that the test substance does not have a sensitizing effect on the skin of the guinea pig in the BUEHLER Test under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information classification for skin sensitisation is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.