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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Oct - 10 Nov 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996
Reference Type:
other: Amendment
Title:
Unnamed
Year:
1996
Report date:
1996
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 1992
Deviations:
yes
Remarks:
justification of choice of vehicle not provided, no justification for top dose intradermal induction, a higher number of animals is recommended
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same Regulation, the data was included to avoid unnecessary testing.

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin
EC Number:
601-147-9
Cas Number:
111988-49-9
Molecular formula:
C10H9ClN4S
IUPAC Name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Hsd Poc:DH, SPF-bred
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Marian Winkelmann GmbH Laboratory Animal Breeders, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 351 - 366 g
- Housing: in type IV Makrolon® cages in groups (5 animals per cage during acclimatisation and 2 - 3 animals per cage during the study) with low-dust wood shavings (Ssniff Spezialdiäten GmbH, Soest, Germany)
- Diet: "Altromin®3020 - Maintenance Diet for Guinea Pigs (Altromin, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1.5
- Humidity (%): 40 - 70
- Air changes (per hr): 12 -15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: 2% Cremophor EL®
Concentration / amount:
intradermal: 5.0%/0.1 mL
epicutaneous: 50%/0.5 mL
Day(s)/duration:
intradermal: single injection on Day 1, epicutaneous: starting on Day 8, exposure for 48 h
Adequacy of induction:
other: not tested up to irritant concentration causing mild irritation and no justification for selected doses (the highest dose tested did not cause skin irritation)
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 2% Cremophor EL®
Concentration / amount:
25%/0.5 mL
Day(s)/duration:
24 h
Adequacy of challenge:
other: not tested up to irritant concentration causing mild irritation and no justification for selected doses (some irritation was noted in some animals at 50%)
No. of animals per dose:
10 (treatment)/5 (control)
Details on study design:
RANGE FINDING TESTS
A. INDUCTION EXPOSURE
For intradermal induction: One guinea pig was given intradermal injections twice, in each case, with 0.1 mL of the following test item concentrations: 0 – 5%.
The injection sites were evaluated after 24 and 48 h. After 24 and 48 h, no reactions were observed after intradermal induction of the test formulations (0 - 5%).
For topical induction: Three concentrations (12%, 25% and 50%) and the vehicle were tested on four guinea pigs. The patches moistened with 0.5 mL of the test item formulations or the vehicle were applied to each animal under occlusive conditions for 24 h. At the end of the exposure period, the remaining test item was removed with physiological saline. No later than 21 h after removing the patches the treated areas were shorn in the zone of the challenge area. The skin reactions were evaluated 24 h and 48 h after patch removal. No skin reactions were recorded both 24 h and 48 h after topical application of the test formulations (0 - 50%).
B. CHALLENGE EXPOSURE
One week prior to the challenge, the challenge concentration was determined on 5 guinea pigs which were treated in the same manner as the control animals during the inductions. Four patches each loaded with 0.5 mL test item formulations (12%, 25% and 50%) or the vehicle were applied to each animal under occlusive conditions for 24 h. At the end of the exposure period, the remaining test item was removed with physiological saline. No later than 21 h after removing the patches the treated areas were shorn in the zone of the challenge area. The skin reactions were evaluated 48 and 72 h after start of the application. At 50% challenge, three animals showed skin reactions (Grade 1) after 48 h, two of them were reversible within the following 24 h. The other concentrations did not induce any reactions (0-25%). Based on these results, the following concentrations were selected for the main study: Intradermal induction: 5%, topical induction: 50% and challenge: 25%. For details on the results, please refer to the attached background material (Attachment 1).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) Freund's Complete Adjuvant (FCA)/sterile physicological saline solution
Injection 2: 5% test substance in 2% Cremophor EL®
Injection 3: 5% test substance in a 1:1 mixture of (v/v) FCA/2% Cremophor EL®
Epicutaneous: 50% test substance in 2% Cremophor EL®
- Control group:
Injection 1: a 1:1 mixture (v/v) Freund's Complete Adjuvant (FCA)/sterile physicological saline solution
Injection 2: 2% Cremophor EL®
Injection 3: 2% Cremophor EL® in a 1:1 mixture of (v/v) FCA/2% Cremophor EL®
Epicutaneous: 2% Cremophor EL®
- pre-treatment: One day before epicutaneous treatment, a 10% preparation of sodium lauryl sulfate in vaseline was applied to the skin
- Site: dorsal region (intradermal + epicutaneous)
- Frequency of applications: once
- Duration: intradermal induction was performed on Day 1, topical induction was performed on Day 8-10
- Concentrations: intradermal 5%, epicutaneous 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: three weeks after the intradermal induction
- Exposure period: 24 h
- Test groups: test substance and vehicle only
- Control group: test substance and vehicle only
- Site: left flank (test substance caudal, vehicle cranial)
- Concentrations: 25%
- Evaluation (hr after challenge): 24 h and 48 h after patch removal

OTHER:
The animals were observed for clinical signs at least once daily throughout the entire study period. Body weights were recorded on Day 1 before the first induction and at the end of the study.
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
The GPMT was checked for reliability in a separate study with 10 male guinea pigs using 2-mercaptobenzothiazole in 2% Cremophor EL®. The following substance concentrations were used: Intradermal induction: 2.5%, topical induction: 40%, challenge: 40%.

Results and discussion

Positive control results:
The following concentrations were used in the positive control 2-mercaptobenzothiazole reliability test:
intradermal induction: 2.5%
topical induction: 40%
challenge: 40%
48 h after the challenge, 55% of the test item animals and 0% of the control animals exhibited dermal reactions in the challenge treatment and after 72 h 44% of the test item animals showed clear dermal reactions. There was no reddening of the skin to be observed in control group animals. Summarized results can be found in Attachment 3.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5% (intradermal) 50% (epicutaneous), challenge: 25% (epicutaneous)
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
skin irritation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5% (intradermal) 50% (epicutaneous), challenge: 25% (epicutaneous)
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
skin irritation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0% (intradermal) 0% (epicutaneous), challenge: 25% (epicutaneous)
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% (intradermal) 0% (epicutaneous), challenge: 25% (epicutaneous)
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
2.5% (intradermal) 40% (epicutaneous), challenge: 40% (epicutaneous)
No. with + reactions:
5
Total no. in group:
9
Clinical observations:
skin irritation, 1 animal died on Day 4 and was not used for evaluation. On Day 6 and 7, 1 animal showed lacrimation and turbidity of the right eye, turbidity of the right eye continued until the end of the study
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
2.5% (intradermal) 40% (epicutaneous), challenge: 40% (epicutaneous)
No. with + reactions:
4
Total no. in group:
9
Clinical observations:
skin irritation, 1 animal died on Day 4 and was not used for evaluation. On Day 6 and 7, 1 animal showed lacrimation and turbidity of the right eye, turbidity of the right eye continued until the end of the study

Any other information on results incl. tables

The appearance and behaviour of the animals in the test item group were not significantly different from the control group.
At the end of the study, the mean body weight of the treatment group animals was in the same range as that of the control group animals.

 

Summarized results can be found in Attachment 2. 

 

The following results were obtained for further groups:

test chemical: 5% (intradermal), 50% (epicutaneous), challenge: 0% (epicutaneous): 0/10 animals showed reactions) after both 24 and 48 h

Vehicle: 0% (intradermal), 0% (epicutaneous), challenge: 0% (epicutaneous): 0/5 animals showed reactions) after both 24 and 48 h

Positive control: 2.5% (intradermal), 40% (epicutaneous), challenge: 0% (epicutaneous): 0/9 animals showed reactions) after both 24 and 48 h

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 12 72/2008.
Conclusions:
The study is in accordance with OECD guideline 406, was conducted under GLP and is considered valid and reliable. The challenge treatment with the 25% test substance formulation led to skin reactions
(grade 1) in 1/10 (10%) of the test group animals. There was no skin reaction in the control group animals. Under the conditions chosen, the test substance did not exhibit a skin sensitisation potential. According to criteria of the CLP Regulation (EU) No. 1272/2008, no classification of the test item in regards to skin sensitisation is required.