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Administrative data

Description of key information

Under the present test conditions, the test substance was not skin sensitising in a Buehler test according to OECD 406 (LPT, 2016).

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:
2016-06-09 to 2016-08-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
published in the Official Journal of the European Union L142, dated May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
As the test item was found to be irritating to the mouse ear skin in the preliminary experiment, the (main) LLNA in mice was stopped prematurely.


Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: Dunkin-Hartley
- Sex: male
- Breeder: Charles River Laboratories, France
- Supplier: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age: 29 days
- Weight at study initiation: 301  - 365 g, positive control group: 273 - 337 g
- Housing: max 5 animals per cage
- Diet (e.g. ad libitum): Commercial diet ssniff7 Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days
- Controls: 10 animals; treatment: vehicle

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3°C
- Humidity (%): 55 +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 hour light/dark rhythm
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
6 hour occlusive patch (left side), 0.5 ml undiluted; then removal of  residual test material
Day(s)/duration:
day 1/8/15 for 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
two 6 hour occlusive patches (right side), one with 0.5 ml test  substance (75%)  and one with vehicle,  subsequent removal of residual test material
Day(s)/duration:
day 29 for 6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 animals control group
20 animals test material
20 animals positive control
Details on study design:
RANGE FINDING TESTS:
A preliminary test was performed with 3 animals employing a range of concentrations of test item (as a 10, 25, 50 or 75% solutions in sesame oil or undiluted).
The flank regions of the animals were cleared of hair (closely-clipped) one day before application.
The test patch system, a square cotton pad of approximately 4 - 6 cm2, was fully loaded with 0.5 mL of the test item. The test patch system was applied to the test area and was held in contact with the skin by an occlusive patch and a suitable dressing for 6 hours. The test patch system was occlusive. The patch and the occlusive dressing were secured with adhesive strips (exposure time: 6 hours each).
For the main study for each induction stage, the concentration of test item was, if possible, adjusted to the highest level that could be well tolerated systemically and which, for irritant substances, produces mild to moderate irritation in the majority of test animals. The challenge concentration should be the maximum which produces no evidence of skin irritation in non-sensitised animals. No skin reactions were observed up to the concentration of 75%, the undiluted test item revealed a discrete or patchy erythema immediately and 24 hours after patch removal in this preliminary test.
Hence, the undiluted test item was chosen for the induction stages and a 75% concentration for the challenge of the main study.

ADMINISTRATION/EXPOSURE 

- Induction - Topical application
Day 1 - Treated Group
One day before application the left side of the flank region of the animals were cleared of hair (closely-clipped).
The test patch system, a square cotton pad of approximately 4 - 6 cm2 was fully loaded with 0.5 mL test item, undiluted.
The test patch system was applied to the test area and was held in contact with the skin by an occlusive patch and a dressing for 6 hours. The test patch system was occlusive.
The patch and the occlusive dressing were secured with adhesive strips, exposure time: 6 hours each.

Day 1 - Control Group
One day before application the left side of the flank region of the animals were cleared of hair (closely-clipped).
The control animals were treated in a similar manner to that used for the treated group, but received sesame oil instead of test item.
The test patch system was applied to the test area and was held in contact with the skin by an occlusive patch and a suitable dressing for 6 hours.

Days 8 and 15 - treated and control groups
The same application as on day 1 was carried out on the same test area (cleared of hair one day before application) on the same flank region on day 8 and again on day 15.

- Challenge
Day 29 - Treated and Control Groups:   
One day before application the untreated right flank of treated and control animals was cleared of hair (closely-clipped).
Two 6 hour occlusive patches (right side), one with 0.5 ml test  substance (75%) and one with vehicle, subsequent removal of residual test material.
Skin observations and scoring: 24, 48 and 72 hours after removing of the patch the skin reaction was observed and recorded (Days 30, 31 and 32)
- Concentrations used for challenge: 75 %

- Positive control:   alpha-hexylcinnamaldehyde (CAS No. 101-86-0) (not concurrent)  
The animals of the positive control group were treated with 0.5 mL of undiluted a-hexyl cinnamaldehyde (HCA, 85%) on test days 1, 8 and 15 in the same manner as the animals of the negative control and the test item groups.
For the challenge on test day 29, HCA was used undiluted, too. The exposure time was 6 hours.
The sensitivity of the strain of guinea pigs employed is checked at regular intervals of approximately 6 months.


EXAMINATIONS
Skin observations and scoring
Days 30, 31 and 32: 24, 48 and 72 hours after removing of the patch the skin reaction was observed and recorded.
In addition, the skin condition was evaluated immediately and 18 hours after the end of induction exposures (6 and 24 hours after start of treatment).
- Grading system: Magnusson and Kligman, see below

OTHER OBSERVATIONS
Mortality: daily during the observation period
Clinical signs: daily during the observation period
Body weight: before the test commences and at study termination
Records were maintained on all additional and standard observations

Challenge controls:
sesame oil
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (CAS No. 101-86-0) (not concurrent)   Bühler test with 20 test and 10 control animals   Occlusive epicutaneous induction, undiluted   Occlusive epicutaneous challenge with 50 % in corn oil
Positive control results:
The positive control a-hexyl cinnamaldehyde solution revealed sensitising properties in all 20 animals employed.
Key result
Reading:
1st reading
Hours after challenge:
30
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Key result
Reading:
2nd reading
Hours after challenge:
54
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Key result
Reading:
other: 3nd reading
Hours after challenge:
78
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Key result
Reading:
1st reading
Hours after challenge:
30
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Key result
Reading:
2nd reading
Hours after challenge:
54
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Key result
Reading:
other: 3nd reading
Hours after challenge:
78
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no

RESULTS OF PILOT STUDY: 

Four concentrations of test item dissolved in sesame oil (10, 25, 50 and 75%) and the undiluted test item (100%) were tested by dermal application to guinea pigs.

No skin reactions were observed up to the concentration of 75%, the undiluted test item revealed a discrete or patchy erythema immediately and 24 hours after patch removal in this preliminary test.


RESULTS OF TEST

Sesame oil served as negative control.

Test item revealed discrete or patchy erythema (grade 1) or moderate and confluent erythema (grade 2) in all 3 induction stages.

A 75% concentration of the test item in sesame oil chosen for the challenge did not cause any skin reactions.

The positive control a-hexyl cinnamaldehyde revealed pronounced sensitising properties.

No deaths or clinical signs were observed.

The body weight gain of the animals was not influenced.

Conclusions:
Under the present test conditions, test item revealed no sensitising properties in guinea pigs in a skin sensitisation test according to E. V. BUEHLER.
Executive summary:

The purpose of this study was to determine the sensitising potential of test item in a skin sensitisation test in guinea pigs according to E. V. BUEH­LER.  (Remark: As the test item was found to be irritating to the mouse ear skin in the preliminary experiment, the (main) LLNA in mice was stopped prematurely.)

The test item was used as supplied (100%) for the 3 induction stages and as a 75% concentration in sesame oil (v/v) for the challenge.

Sesame oil served as negative control.

The test item revealed discrete or patchy erythema (grade 1) or moderate and confluent erythema (grade 2) in all 3 induction stages.

A 75% concentration of the test item in sesame oil chosen for the challenge did not cause any skin reactions.

The positive control a-hexyl cinnamaldehyde solution revealed sensitising properties in all 20 animals employed.

 

Conclusion

Under the present test conditions, test item revealed no sensitising properties in guinea pigs in a skin sensitisation test according to E. V. BUEHLER.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

As the test item was found to be irritating to the mouse ear skin in the preliminary experiment, the LLNA in mice was stopped prematurely.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the skin sensitization test according to OECD 406 (Buehler method) in guinea pigs and according to the EC Regulation 1272/2008 and subsequent regulations on classification, labelling and packaging of substances and mixtures, the test substances has no skin sensitizing properties and hence it does not require to be classified.