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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
FREY-TOX GmbH, Osteroda 38, 04916 Hertzberg (Elster), Germany
Type of study:
Buehler test
Justification for non-LLNA method:
The Bühler assay was regarded as the most suitable test for this substance because free fatty acids could lead to false positive results in a LLNA.
Species:
guinea pig
Strain:
other: SPF albino guinea pigs of the stock Crl:HA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, D-88353 Kisslegg
- Weight at study initiation: between 297-353 grams
- Housing: two or three guinea pigs per macrolone cage (floor area 2280 cm2). These cages contained Lignocel-Granulat or Lignocel-Fasern (Altromin D-32791 Lage, Lippe) bedding material. Hay bricks called "Raufutter Rolls" (from the company Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland) were used as enrichment. The cages were cleaned and the bedding changed 3 times a week. The bedding material was regularly analysed.
- Diet: Pelleted diet (Altromin 3123) ad libitum, from Altromin D-32791 Lage, Lippe. Analyses of the diet is performed regularly.
- Water: free access to vitamin C enriched domestic quality water that was acidified to pH 2.5 with hydrochloric acid in order to prevent microbiological growth.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Humidity (%): at least 30% and preferably not exceeding 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness. Light was on from 6 a.m. to 6 p.m.

Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG E 400
Concentration / amount:
- Induction exposure: 75% (w/w) (solution)
- Challenge exposure: 50% (w/w) (solution)
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG E 400
Concentration / amount:
- Induction exposure: 75% (w/w) (solution)
- Challenge exposure: 50% (w/w) (solution)
No. of animals per dose:
- Control: 10
- Test item: 20
Details on study design:
RANGE FINDING TESTS:
During two preliminary investigations with two animals each, the skin irritant effect of the test item in different levels of concentrations was examined
in order to choose suitable test concentrations for the topical applications during the induction and challenge phase. The procedures used in the preliminary investigations were identical to those used in the main study. Based on the results of the preliminary investigations and in agreement with the sponsor, a concentration of 75% (w/w) (solution) was selected for the induction and a concentration of 50% (w/w) (solution) for the challenge application.

MAIN STUDY
A. INDUCTION EXPOSURE
On day -1, an area of skin on the left flank region measuring approx. 4x6 cm was clipped free of hair with an electric clipper and an electric razor. On the first experimental day (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 the 75% (w/w) test item and the patch was placed on the skin of all the animals in the test group. The fixation of this occlusive dressing was made by wrapping the trunk of the animals with Gothaplast tape (5 cm width). The dressings were removed after 6 hours 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. The test sites were assessed for skin irritation 24 hours after each patch application. The guinea pigs in the control group were treated in the same manner, but with the vehicle PEG E 400 only.

B. CHALLENGE EXPOSURE
Four weeks after the first induction (day 28), all animals of the test and control group were subjected to a challenge. On day 27, an area (approx. 4x6 cm) of skin on the animals right flank region was clipped free of hair with an electric clipper and subsequently with an electric razor. On day 28, a patch of multilayered gauze (2.5 x 2.5 cm) coated with occlusively with impermeable tape (Blenderm) on one side was saturated with 0.5 mL of the 50% (w/w) test item. 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 posterior on the right flank. The fixation of these occlusive dressings was made by wrapping the trunk of the animals with Gothaplast tape (5 cm width). The dressings were removed after 6 hours and the treated skin areas were washed initially with a watery 50% solution of PEG E 400 and subsequently with pure water.

OBSERVATIONS:
- Reading skin reactions: Each challenge site was examined 24 and 48 hours after the removal of the patch and the skin reactions were evaluated according to the Magnusson/Kligman grading scale. About six hours before the 24 hour reading, the sites were clipped and shaved in order to facilitate the evaluation.
- Health and Bodyweight: The animals were observed at least once a day for signs of illness and reactions to the treatment. The animals in the main study were weighted at the beginning of the study (day 0) and at the end of the study (day 31).

SCORING SCALE (Magnusson/Kligman):
- no visible change: 0
- discrete or patchy erythema: 1
- moderate and confluent erythema: 2
- intense erythema and swelling: 3

VERIFICATION OF THE TEST PROCEDURE
The sensitivity and reliability of the experimental technique and the test systems used were verified. The last positive control test with the reference material α-hexylcinnamaldehyde tech., 85% was performed from September 2012 until October 2012. The sensitization potential of the positive reference item has been shown by the response of 13 of 20 guinea pigs (65%) of the test group with positive skin reactions.
Challenge controls:
The control animals were treated as described above.
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde
Positive control results:
The last positive control test with the reference material α-hexylcinnamaldehyde tech., 85% was performed from September 2012 until October 2012. The sensitization potential of the positive reference item has been shown by the response of 13 of 20 guinea pigs (65%) of the test group with positive skin reactions. Positive responses were observed in six animals only after 24 hrs and in two animals only after 48 hrs.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
test item 50% (w/w)
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: test item 50% (w/w) . No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
test item 50 % (w/w)
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: test item 50 % (w/w). No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
vehilcle (PEG E 400)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: vehilcle (PEG E 400). No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
vehicle (PEG E 400)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: vehicle (PEG E 400). No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
test item 50% (w/w)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: test item 50% (w/w) . No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
test item 50% (w/w)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: test item 50% (w/w) . No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle (PEG E 400)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle (PEG E 400). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle (PEG E 400)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle (PEG E 400). No with. + reactions: 0.0. Total no. in groups: 10.0.
Group:
positive control
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
For verification of the test procedure, the last positive control test with a-Hexylcinnamaldehyde was performed from September 2013 until October 2012.

None of the animals showed signs of illness. The animals 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:

In a GLP compliant sensitivity study (Buehler test), performed according to OECD guideline 406, Crl:Ha female guinea pigs were treated with the test substance (FREY-TOX 2013). During two preliminary investigations with two animals each, the skin irritant effect of the test item in different levels of concentrations was examined. Based on the results of the preliminary investigations, a concentration of 75% (w/w) (solution) was selected for the induction and a concentration of 50% (w/w) (solution) for the challenge application. The animals were topically induced to the test substance on day 0 using an occlusive patch for 6 hours after which the patch was removed. This procedure was repeated on days 7 and 14. The control animals were treated in the same manner but with the vehicle PEG E 400 only. During the challenge phase the test animals were topically exposed on the anterior right flank to the test substance and on the posterior right flank to the vehicle. The control animals were treated the same way during the challenge phase. During the challenge phase, formation of erythema was not observed on the two test fields of the animals of the control group treated with the 50% (w/w) test item and the vehicle PEG E 400. In the test group, a discrete or patchy erythema (grade 1) was observed in 1 of 20 animals (5%) on the test field treated with 50 % (w/w) test item during the challenge after 24 hrs and 48 hrs. All remaining 19 animals were free of any skin reactions after 24 hrs and 48 hrs. The test fields treated with the vehicle PEG E 400 were free of any reactions in all animals of the group. The low frequency of the shown reactions (5%) supplied no evidences for a skin sensitizing effect of the test item.


Migrated from Short description of key information:
Soybean oil, epoxidized, reaction products with methanol was not skin sensitizing in the Buehler test with female guinea pigs.

Justification for classification or non-classification

Based on the findings in the skin sensitisation study (Buehler test), the test substance does not have to be classified according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008