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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:
08 Nov - 02 Dec 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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 topical induction and challenge, no use of SLS to induce skin irritation, temperature of animal housing slightly divergent
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales Des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An in vivo skin sensitisation study was performed after 1 June 2008 for a non-EU regulatory scheme. In accordance with Regulation (EC) No. 1907/2006, the data were included to avoid unnecessary testing.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
433-460-1
EC Name:
-
Cas Number:
210880-92-5
Molecular formula:
C6H8ClN5O2S
IUPAC Name:
(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Animal Breeders, Kißlegg, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 355 - 423 g
- Housing: in groups of two to five per cage (Noryl cages)
- Diet: PROVIMI KLIBA 3420 - Maintenance Diet for Guinea Pigs, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 08 Nov 2011 To: 02 Dec 2011

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
intradermal: 5.0%/0.1 mL (= 20 mg test item/animal)
epicutaneous: 50%/0.5 mL (= 250 mg test item/animal)
Day(s)/duration:
intradermal: single injection on Day 1, epicutaneous: starting on Day 8, exposure for 48 h
Adequacy of induction:
other: After the first induction the animals in the control group and the test item group showed strong effects up to encrustation at the injection sites
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
50%/0.5 mL (= 250 mg test item/animal)
Day(s)/duration:
24 h
Adequacy of challenge:
not specified
No. of animals per dose:
10 (controls), 20 (in test group)
Details on study design:
RANGE FINDING TESTS
A. INDUCTION EXPOSURE
For intradermal induction: One guinea pig was given intradermal injections two, in each case, with 0.1 mL of the following test item concentrations: 0%, 1%, 2.5% and 5%. The injection sites were evaluated after 24 and 48 h.
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 48 h and 72 h after the start of the application. No skin reactions were recorded both 48 h and 72 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 2 guinea pigs in the main study 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. No skin reactions were recorded both 48 h and 72 h after application of the test formulations (0-50%). Based on these results, the following concentrations were selected for the main study: Intradermal induction: 5%, topical induction: 50% and challenge: 50%. For details on the results, please refer to the attached background material.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- 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 polyethylene glycol 400
Injection 3: 5% test substance in a 1:1 mixture of (v/v) FCA/polyethylene glycol 400
Epicutaneous: 50% test substance in polyethylene glycol 400
- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/sterile physicological saline solution
Injection 2: polyethylene glycol 400
Injection 3: polyethylene glycol 400 in a 1:1 mixture (v/v) FCA
Epicutaneous: polyethylene glycol 400
- Site: dorsal region (intradermal + epicutaneous)
- Frequency of applications: once (each)
- 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 (challenge)
- Day(s) of challenge: challenge was performed three weeks after intradermal induction on Day 22
- Exposure period: 24 h
- Test groups: test substance and vehicle only
- Control group: test substance and vehicle only
- Site: right flank (test substance caudal, vehicle cranial)
- Concentrations: 50%
- Evaluation (hr after challenge): 48 and 72 h

OTHER:
The animals were observed for clinical signs at least once daily throughout the entire study period. Body weights were recorded on Day1 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 female guinea pigs using alpha hexyl cinnamic aldehyde formulated in polyethylen glycol 400.

Results and discussion

Positive control results:
The following concentrations were used in regards to the positive control alpha hexyl cinnamic aldehyde (HCA) in a reliability test:
intradermal induction: 5%
topical induction: 25%
first challenge: 12%
second challenge: 6%

After the first challenge 100% of the test item animals and 60% of the control animals exhibited dermal reactions in the challenge treatment and after the second challenge 60% of the test item animals. There was no reddening of the skin to be observed in control group animals.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% (intradermal) 0% (epicutaneous), challenge: 50% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5% (intradermal) 50% (epicutaneous), challenge: 50% (epicutaneous)
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0% (intradermal) 0% (epicutaneous), challenge: 50% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
5% (intradermal) 50% (epicutaneous), challenge: 50% (epicutaneous)
No. with + reactions:
0
Total no. in group:
20
Key result
Group:
positive control
Dose level:
5% (intradermal), 25% (topical induction), first challenge: 12%, second challenge: 6%
Remarks on result:
other: reliability study: 60% of the HCA-treated animals exhibited dermal reactions after the second challenge

Any other information on results incl. tables

General examination:

Appearance and behaviour of the test item group were not 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 than that of the control group animals.

After the intradermal induction, the animals in the control group showed after 48 hours:
• red wheal
• white wheal with red surrounding

The animals in the test item group showed after 48 hours:
• red wheal
• white wheal with red surrounding
• encrustation

At Day 8, encrustation was recorded at the injection sites in the control group and wheals and encrustation in the test item group.

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 to OECD TG 406, was conducted under GLP and is considered valid and reliable. 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.