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Diss Factsheets

Administrative data

Description of key information

Skin sensitisation


Buehler test, in vivo OECD 406, GLP: negative

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:
08 MAR 2021 - 27 OCT 2021
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
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
Although the murine Local Lymph Node Assay (LLNA) is currently the preferred assay for chemicals under EU REACH regulation, guinea pig assays are equally acceptable under other regulations.
The guinea pig has been found informative for predictive sensitisation tests and is used as animal model since several decades. The Buehler test is able to detect substances with moderate to strong sensitisation potential, including those with relatively weak sensitisation potential.
Species:
guinea pig
Strain:
Hartley
Remarks:
healthy Crl: HA albino guinea pigs
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 6–7 weeks old
- Weight at study initiation: 337–430 g
- Housing: Semi barrier in an air-conditioned room, animals were kept in groups in Terluran cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to autoclaved hay and to Altromin 3122 maintenance diet for guinea pigs, rich in crude fibre
- Water (e.g. ad libitum): Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least 5 days)
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
Route:
epicutaneous, occlusive
Vehicle:
other: undiluted, moistened with cottonseed oil
Concentration / amount:
Undiluted (100 %). A gauze patch was loaded with 0.1 g of the prepared test substance, which was the maximum technically applicable amount.
Day(s)/duration:
gauze patch was applied for 6 hours and repeated at weekly intervals
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: undiluted, moistened with cottonseed oil
Remarks:
undiluted, moistened with cottonseed oil
Concentration / amount:
Undiluted (100 %). A gauze patch was loaded with 0.1 g of the prepared test substance, which was the maximum technically applicable amount.
Day(s)/duration:
gauze patch was applied for 6 hours
Adequacy of challenge:
other: expected to be the highest non-irritant concentration, please refer to any other information on results for further information.
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
The test substance was applied at a concentration of 50%
Day(s)/duration:
gauze patch was applied for 6 hours
Adequacy of challenge:
other: The concentration was lowered to 50% in order to eliminate irritant reactions, in line with the technical report no. 78 by the ECETOC. It was considered still to be high enough to evoke potential allergic reactions.
No. of animals per dose:
10 animals in control group and 20 animals in the test group
Details on study design:
RANGE FINDING TESTS:
For the justification of dose levels a preliminary test was performed. The concentration for each induction exposure should be the highest to cause mild irritation and the concentration used for the challenge exposure will be the highest non-irritating dose.
A gauze patch was loaded with 0.1 g of the prepared test substance, which was the maximum technically applicable amount. The patch was applied to the test area of approximately 2.5 x 2.5 cm on the closed-clipped flank of the animals. Thus, an adequate dermal exposure of the test substance was ensured.
2 animals were treated topically with concentrations of 100 % (undiluted test item) for 6 hours.
The test item was moistened with cottonseed oil before application.
Based on the results of this preliminary test the following concentrations were chosen for the main test:
a concentration of 100 % for the dermal inductions
a concentration of 100 % for the challenge application

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: once a week for 6 hours
- Test groups: Test group and negative control group
- Site: The left flank was cleared of hair (close-clipped) prior to the first application and again prior to further applications. The patch was applied to the test area of approximately 2.5 x 2.5 cm and was held in contact with the help of an occlusive dressing.
- Frequency of applications: weekly
- Duration: 6 hours
- Concentrations: Test group: 100 %. A gauze patch was loaded with 0.1 g of the test substance moistened with cottonseed oil and applied to the test area; negative control group: 0%. A gauze patch moistened with cottonseed oil was applied to the test area.

B. CHALLENGE EXPOSURE
- No. of exposures: 1x challenge and 1x rechallenge
- Day(s) of challenge: 14 days after the last induction application (challenge), 14 days after challenge (rechallenge)
- Exposure period: once for 6 hours
- Test groups: Test group and original negative control group (challenge and rechallenge)
- Site: Both flanks were cleared of hair (close-clipped) prior to the challenge/rechallenge application.
Challenge: Test group and control animals: Test item application: right flank; intraspecific control: left flank
Rechallenge: Test group and control animals: Test item application: left flank; intraspecific control right flank
- Concentrations:
Challenge: 100%. 0.1 g of the test substance moistened with cottonseed oil was loaded on a patch and applied to the test area on the right flank. A patch only moistened with cottonseed oil was applied on the left flank at an untreated site (intraspecific control).
Rechallenge: Test substance was applied at a concentration of 50%, suspended with cottonseed oil to the test area on the left flank. A patch moistened with cottonseed oil was applied on the right flank (intraspecific control).
- Evaluation (hr after challenge or rechallenge): 24 and 48 hours
- Observations: approximately 24 and 48 hours after removing the patch the skin reaction was observed and recorded

OTHER:
The animals were weighed prior to the administration, after the challenge (48 hours after removing the patch) and at the end of the observation period (rechallenge: 48 hours after removing the patch).
Challenge controls:
After challenge, weak skin reactions were noted in test and control animals. A lower concentration was used in the rechallenge in order to clarify the nature of the skin reactions.
The rechallenge was performed using the test item at a concentration of 50%, in line with the technical report no. 78 by the ECETOC. The concentration was lowered to 50% in order to eliminate irritant reactions and was considered still to be high enough to evoke potential allergic reactions.
The rechallenge was performed with same negative control group.
After rechallenge, no positive reactions were observed in any of the test or control animals. Therefore, the effects observed after challenge were considered as irritant in their nature. In contrast, positive rechallenge data, regardless of the used concentration, would have rendered the study positive.
Positive control substance(s):
yes
Positive control results:
The recent reliability check was performed during the period from 22 Feb to 25 Mar 2021. The sensitisation rate after application of the positive-control substance 2-mercaptobenzothiazole (50 % (in vaseline) inductions I, II and III and 25 % (in vaseline) challenge) was 70 %, confirming the reliability of the test system.
20 animals used for positive-control group.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
test substance was applied at a concentration of 50%, suspended with cottonseed oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
test substance was applied at a concentration of 50%, suspended with cottonseed oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
test substance was applied at a concentration of 50%, suspended with cottonseed oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
test substance was applied at a concentration of 50%, suspended with cottonseed oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % undiluted (0.1 g of the test substance moistened with cottonseed oil)
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
other: erythema grade 1 in 6/20 test animals at the test site
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 % undiluted (0.1 g of the test substance moistened with cottonseed oil)
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
other: erythema grade 1 in 8/20 test animals at the test site
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 % undiluted (0.1 g of the test substance moistened with cottonseed oil)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
other: erythema grade 1 in 2/10 negative-control animals
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 % undiluted (0.1 g of the test substance moistened with cottonseed oil)
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.
Remarks on result:
other: erythema grade 1 in 4/10 negative-control animals
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25 % in vaseline
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
not specified
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 % in vaseline
No. with + reactions:
14
Total no. in group:
20
Clinical observations:
not specified
Remarks on result:
positive indication of skin sensitisation

Table a. Summary: Frequency of Positive Skin Reactions after Challenge in Comparison to Control Animals

































































 E0E1E2E3O0O1O2O3Skin Reaction Positive* Animals (%)
T24146----20------30
T48128----20------40
C2482----10------20
C4864----10------40

E= erythema; O = oedema; T = test group; C = control group; 0 – 3 = grade; 24/48 = 24/48 hours after end of challenge treatment; * = skin reactions were not considered to be allergenic


Table b. Summary: Frequency of Sensitisation after Rechallenge in Comparison to Control Animals

































































 E0E1E2E3O0O1O2O3Sensitised Animals (%)
T2420------20------0
T4820------20------0
C2410------10------0
C4810------10------0

E= erythema; O = oedema; T = test group; C = control group; 0 – 3 = grade; 24/48 = 24/48 hours after end of rechallenge treatment


Table c. Frequency of Sensitisation in Positive-Control Animals




































































Group / Hours after Challenge TreatmentNumber of Sensitised AnimalsPersentage of Sensitised Animals
E0E1E2E3O0O1O2O3
T24712102000065
T48611302000070
C2410000100000
C4810000100000

T = test group (positive control); C = negative control group; 24/48 = 24/48 hours after end of rechallenge treatment; E0/1/2/3 = erythema grade 0/1/2/3; O0/1/2/3 = oedema grade 0/1/2/3

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Under the conditions of the present study the test item Carbonic acid bis-[1-(benzothiazol-2-ylsufanyl)-2,2,6,6-tetramethyl-piperidin-4-yl] ester was considered not to cause reactions identified as sensitisation.
Executive summary:

The study was performed in order to assess the potential of the test item to induce delayed contact hypersensitivity when applied topically to albino guinea pigs, according to OECD 406 (Buehler test) under GLP compliance. 


Based on the results of the preliminary test, for the first, second, third induction and challenge, the test item was used as provided (powder), i.e. at a 100% concentration. The test item was moistened with cottonseed oil before application.


At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.


Induction I, II and III (6-hour exposure, occlusive):


Immediately and 24 hours after removing the patch neither erythema nor oedema was observed in any animal at any time of observation.


Challenge Exposure (6-hour exposure, occlusive):
24 hours after removing the patch: erythema grade 1 in 2/10 control and 6/20 test animals
48 hours after removing the patch: erythema grade 1 in 4/10 control and 8/20 test animals
Neither control nor test animal showed oedema at any observation time.
There was evidence of positive skin reactions in the challenge in test as well as in control animals.


In order to clarify the results of the challenge, a rechallenge (second challenge) using the same negative control group was performed with the test item at the concentration of 50%.


Rechallenge Exposure (6-hour exposure, occlusive):
24 hours after removing the patch: neither erythema nor oedema was observed in any animal at any time of observation
48 hours after removing the patch: neither erythema nor oedema was observed in any animal at any time of observation


There was no evidence of sensitisation at the rechallenge and the percentage of sensitised animals was 0%.


After challenge, weak skin reactions were noted in test and control animals. A lower concentration was used in the rechallenge in order to clarify the nature of the skin reactions.
After rechallenge, no positive reactions were observed in any of the test or control animals. Therefore, the effects observed after challenge were considered as irritant in their nature. In contrast, positive rechallenge data, regardless of the used concentration, would have rendered the study positive.


Therefore, under the conditions of the present study the test item Carbonic acid bis-[1-(benzothiazol-2-ylsufanyl)-2,2,6,6-tetramethyl-piperidin-4-yl] ester was considered not to cause reactions identified as sensitisation.

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

The study was performed in order to assess the potential of the test item to induce delayed contact hypersensitivity when applied topically to albino guinea pigs, according to OECD 406 (Buehler test) under GLP compliance. 


Based on the results of the preliminary test, for the first, second, third induction and challenge, the test item was used as provided (powder), i.e. at a 100% concentration. The test item was moistened with cottonseed oil before application.


At the clinical observation the animals did not show any visible clinical symptoms and no mortality occurred.


Induction I, II and III (6-hour exposure, occlusive):


Immediately and 24 hours after removing the patch neither erythema nor oedema was observed in any animal at any time of observation.


Challenge Exposure (6-hour exposure, occlusive):
24 hours after removing the patch: erythema grade 1 in 2/10 control and 6/20 test animals
48 hours after removing the patch: erythema grade 1 in 4/10 control and 8/20 test animals
Neither control nor test animal showed oedema at any observation time.
There was evidence of positive skin reactions in the challenge in test as well as in control animals.


In order to clarify the results of the challenge, a rechallenge (second challenge) using the same negative control group was performed with the test item at the concentration of 50%.


Rechallenge Exposure (6-hour exposure, occlusive):
24 hours after removing the patch: neither erythema nor oedema was observed in any animal at any time of observation
48 hours after removing the patch: neither erythema nor oedema was observed in any animal at any time of observation


There was no evidence of sensitisation at the rechallenge and the percentage of sensitised animals was 0%.


After challenge, weak skin reactions were noted in test and control animals. A lower concentration was used in the rechallenge in order to clarify the nature of the skin reactions.
After rechallenge, no positive reactions were observed in any of the test or control animals. Therefore, the effects observed after challenge were considered as irritant in their nature. In contrast, positive rechallenge data, regardless of the used concentration, would have rendered the study positive.


Therefore, under the conditions of the present study the test item Carbonic acid bis-[1-(benzothiazol-2-ylsufanyl)-2,2,6,6-tetramethyl-piperidin-4-yl] ester was considered not to cause reactions identified as sensitisation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the result of the in vivo skin sensitisation study following OECD Guideline 406 under GLP compliance, the registered substance is not classified in accordance with Regulation (EC) No. 1272/2008.