Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD 406 / Buehler Test): sensitising

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:
10 January 2012 to 21 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The study was conducted prior to the current requirement in Regulation (EC) 1907/2006 to perform an LLNA study (OECD 429) as the preferred in vivo skin sensitisation study.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdPoc: DH, SPF
- Source: Harlan Laboratories B.V. (Kreuzelweg 53, 5961 NM, The Netherlands)
- Age at study initiation: 6 weeks
- Weight at study initiation: 364.4-390.4 g (control group), 345.8-401.1 g (test group), 366.0-392.9 g (pretest group)
- Housing: in groups of up to 10 animals in stainless steel cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co KG, 73494 Rosenberg, Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst, Switzerland).
- Diet: Teklad Global Guinea pig diet 2040C (batch no. 63/11; Provimi Kliba AG, 4303 Kaiseraugst, Switzerland), ad libitum. A haystick 4642 (batch no. 45/11; Provimi Kliba AG, 4303 Kaiseraugst, Switzerland) was provided for environmental enrichment.
- Water: community tap-water from Itingen in water bottles, ad libitum
- Acclimation period: 1 day for pretest animals, 1 week for control and test animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (Music was played during the daytime light period.)

IN-LIFE DATES: from 11 January 2012 to 21 February 2012
Route:
epicutaneous, occlusive
Vehicle:
other: Diglyme
Concentration / amount:
100%
Route:
epicutaneous, occlusive
Vehicle:
other: Diglyme
Concentration / amount:
50 and 100%
No. of animals per dose:
- Pretest: 3 animals
- Main study (control group): 10 animals
- Main study (test group): 20 animals
Details on study design:
RANGE FINDING TESTS:
The pretest was performed during the acclimatisation of the main study animals. The test item concentrations used were selected during the preliminary solubility testing. The test item was formulated in the vehicle (25, 50, 75%) or was applied undiluted. As no skin reactions were observed 24±2 h after patch removal for any of the applied concentrations, the 100% concentration was chosen for the induction period, and 100% and 50% concentration was chosen for the challenge exposure of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6±0.25 h
- Test groups: undiluted test substance, applied by using a Hill Top Chamber
- Control group: naive control group (empty Hill Top Chambers were applied without vehicle or test item)
- Site: left shoulder
- Frequency of applications: 1 application per week
- Duration: 3 weeks
- Concentrations: 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 29
- Exposure period: 6±0.25 h
- Test groups: undiluted test substance, test substance in vehicle, vehicle only
- Control group: undiluted test substance, test substance in vehicle, vehicle only
- Site: left flank (vehicle only), right flank (100% test substance), right shoulder (50% test substance in vehicle)
- Concentrations: 100%, 50%, and vehicle only
- Evaluation (hr after challenge): 24±2 and 48±2
Challenge controls:
No challenge control group was included into the test.
Positive control substance(s):
yes
Remarks:
The sensitivity and reliability of the experimental method was assessed at least twice a year by use of positive control items such as alpha-hexylcinnamaldehyde or 2-mercapto-benzothiazole.
Positive control results:
The sensitivity and reliability of the experimental method was assessed at least twice a year by use of positive control items such as alpha-hexylcinnamaldehyde or 2-mercapto-benzothiazole which are recommended by the Commission Regulation (EC) No 440/2008, B.6 and OECD 406 and are known to have moderate skin sensitization properties in the guinea pig. The results from the most recent positive control study (Harlan Laboratories Study D24520, performed from 09-Jun-2011 to 29-Jul-2011) confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >15% of the test animals. Harlan Laboratories Study D51946, will be performed in March 2012 using alpha-hexylcinnamaldehyde as skin sensitizer.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 100%; Challenge: 0%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% ; Challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction 100%; Challenge: 50%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; Challenge: 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 100%; Challenge: 100%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
One animal with a skin reaction grade 1 (discrete or patchy erythema) was noted.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 100%; Challenge: 0%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; Challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 100%; Challenge: 50%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Two animals with skin reactions grade 1 (discrete or patchy erythema) were noted.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%, Challenge: 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 100%; Challenge: 100%
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
Five animals with skin reactions grade 1 (discrete or patchy erythema)and one animals with skin reactions grade 2 (moderate and confluent erythema) were noted.
Group:
positive control
Remarks on result:
positive indication of skin sensitisation
Viability/mortality: All animals survived the scheduled observation period. Clinical signs: No clinical signs were recorded in any animal. Skin reactions in the pretest: The undiluted test item (100%) and test item concentrations of 75%, 50% and 25% (w/w) did not produce any skin reactions, therefore the concentration 100% (w/w) was selected for the induction and two concentrations of 100% (w/w) and 50% (w/w) in diglyme were selected for the challenge. Skin reactions in the induction phase: No skin reactions were observed in the control group during the induction phase. In the test group, discrete or patchy erythema was observed in one animal after treatment with the test item at a concentration of 100% (w/w). Skin reactions in the challenge phase: No skin reactions were observed in the control group after challenge with the test item at concentrations of 100% (w/w) and 50% (w/w) in diglyme. After challenge with 100% (w/w) and 50% (w/w) in diglyme one animal showed discrete or patchy erythema at the 24-hour reading. Five animals showed discrete or patchy erythema and one animal showed moderate and confluent erythema at the 48-hour reading after the challenge with 100% (w/w) of test item. Two animals of the test group showed discrete or patchy erythema at the 48-hour reading after the challenge with 50% (w/w) test item in diglyme. Incidence indices of 30% and 10% and severity indices of 0.2 and 0.075 for the test group were calculated after challenge with 100% (w/w) and 50% (w/w) test item with diglyme, respectively. Body weights: The body weight of the animals was within the range commonly recorded for animals of this strain and age. Pathology/necropsy: No unscheduled deaths occurred, hence no necropsy was performed.
Interpretation of results:
other: Category 1B based on CLP criteria (EU criteria according to Regulation (EC) No. 1272/2008)
Conclusions:
The test item was tested for skin sensitising properties according to the OECD TG 406 (Buehler Test), and in compliance with GLP. The test material was applied undiluted in 3 epicutaneous induction exposures and in a single challenge exposure at 50% and 100% concentrations. No skin reactions were observed in any of the control animals after the challenge with 100% test item or with 50% test item in vehicle. One animal of the test group showed discrete or patchy erythema at the 24 h reading after the challenge with 100% test item or with 50% test item in vehicle. Five animals showed discrete or patchy erythema and one animal showed moderate and confluent erythema at the 48 h reading after the challenge with 100% of test item. Two animals of the test group showed discrete or patchy erythema at the 48 h reading after the challenge with 50% test item in vehicle. Based on these findings in a non-adjuvant sensitisation test in guinea pigs the test item has to be classified as a skin sensitiser (Cat 1B) according to EC/1272/2008.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Buehler Test:

In the available key study (Harlan, 2012d) the test item was tested for skin sensitising properties according to the OECD TG 406 and in compliance with GLP. Twenty male Albino Dunkin Hartley guinea pigs of the test group were treated topically with 100% test item once a week for a 3-week induction phase. Two weeks after the last induction, the animals were challenged with 100% and 50% test item in diglyme (vehicle). The ten animals of the control group were not treated with test item or vehicle during the induction. The animals were challenged with 100% and 50% test item in diglyme. No skin reactions were observed in any of the control animals.

Discrete or patchy erythema were observed in one animal of the test group at the third induction with 100% of test item. No skin reactions were observed in any of the control animals after the challenge with 100% test item or with 50% test item in diglyme. One animal of the test group showed discrete or patchy erythema at the 24 h reading after the challenge with 100% test item or with 50% test item in diglyme. Five animals showed discrete or patchy erythema and one animal showed moderate and confluent erythema at the 48 h reading after the challenge with 100% of test item. Two animals of the test group showed discrete or patchy erythema at the 48 h reading after the challenge with 50% test item in diglyme. Hence, as the test material produced skin reactions in 30% of the test animals after the challenge in a non-adjuvant sensitisation test in guinea pigs, the test item is considered to be a skin sensitiser.




Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on these data, the test item has to be classified as a skin sensitiser (Cat 1B) according to EC/1272/2008.