Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-06 to 2015-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
The Buehler test was conducted because the test substance is also subject to other registration processes outside EU, where a skin sensitisation study according to OECD guideline 406 is mandatory.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 003
- Expiration date of the lot/batch: January 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (RT), no direct sunlight, hygroscopic
- Stability under test conditions: stable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories GmbH, NL-5800 AN Venray
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 280 - 338 g
- Housing: groupwise (2 - 3 aniamls) in one macrolone cage (floor area 2280 cm²)
- Diet: ad libitum, pelleted diet "Altromin 3123" (Altromin, D-32791 Lage, Lippe, Germany)
- Water: ad libitum, vitamin C enriched domestic quality water
- Acclimation period: at least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Concentration / amount:
50 % (w/w) test item / 0.5 mL
Day(s)/duration:
Day 1, 7, 14 / 6 hours
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Concentration / amount:
50 % (w/w) test item / 0.5 mL
Day(s)/duration:
Day 28 / 6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 (test group); 10 (control group)
Details on study design:
RANGE FINDING TESTS: The skin irritant effect was observed during a range finding test. Three animals were exposed to 4 concentrations each on the left and rigt anterior and posterior flank. Animals were treated with 5, 10, 25 and 50 % solutions of the test item. Skin reactions were read after 24 and 48 hours. The follwing grading scale was used:

no visible change 0
discrete or patchy erythema 1
moderate and confluent erythema 2
intense erythema and swelling 3

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours
- Test groups: Patch of multi-layered gauze (2.5 x 2.5 cm) coated occlusive with impermeable tape (Blenderm ™) on one side was saturated with 0.5 mL of the 50 % (w/w) test item and the patch was placed on the skin of all the animals in the test group.
- Control group: was treated in the same manner, but with the vehicle aqueous 1 % (w/w) CMC only.
- Site: left flank region
- Frequency of applications: 3 times
- Duration: 7 days per treatment
- Concentrations: 50 % (w/w) in CMC

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 28
- Exposure period: 6 hours
- Test group: a patch of multi-layered gauze (2.5 x 2.5 cm) coated occlusive 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 occlusive coated patch of multi-layered gauze (2.5 x 2.5 cm) was saturated with 0.5 ml of the vehicle 1 % (w/w) CMC and placed on the posterior part of the right flank.
- Control group: treated like the test group.
- Site: right flank region
- Concentrations: 50 % (w/w) in CMC
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
The challenge control was treated similarly to the test group.
Positive control substance(s):
yes
Remarks:
a-Hexylcinnamaldehyde

Results and discussion

Positive control results:
The last positive control test with the reference material a-Hexylcinnamaldehyde, technical grade, 85 % was performed from December 2014 until January 2015. The sensitization potential of the positive reference item has been sufficiently shown by the response of 18 of 20 guinea pigs (90 %) of the test group with positive skin reactions (erythema with grade 1 and 2).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 % test substance
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 % test substance
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 % test substance
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % test substance
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

No skin irritation effects were observed during preliminary investigation with three animals at concentrations of 5, 10, 25 and 50 % test substance in CMC.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met