4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Feb - 10 Mar 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 July 1992

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The test was performed in 1995 before the alternative OECD guidelines (LLNA and in vitro test methods) were set into force.

Test material

Test material information

1

Reference substance name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

EC Number:

605-140-1

Cas Number:

158237-07-1

Molecular formula:

C16H20ClN5O2

IUPAC Name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Hsd/Win:DH

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes (females only used in range-finding study to determine induction concentration)
- Microbiological status of animals, when known: SPF-bred, Healthy animals exhibiting no clinical signs of illness were used for the study. one animal showd symptoms like emaciation, sunken flanks and labored breathing and temporary breathing sounds.
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 259 - 382 g
- Housing: in groups of five during the adaptation period and groups of two or three per cage throughout the study period type IV makrolon cages
- Diet: Altromin®3020 - Maintenance Diet for Guinea Pigs supplied by Altromin Gmb, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

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: sterile physiological saline solution containing 2% Cremophor EL® (v/v)

Concentration / amount:

intradermal induction: 5%
epicutaneous induction: 40% (maximum achievable concentration)

Day(s)/duration:

48 h (epicutaneous)

Adequacy of induction:

other: induction doses based on dose range-finding test; after intradermal injection, concentrations of 2.5% - 5% induced local skin reactions; topical application up to 40% did not induce local reactions, thus, the skin was pre-treated with 10% SDS

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: physiological saline solution containing with 2% Cremophor EL® (v/v)

Concentration / amount:

40% and 25% (w/v), 0.5 mL

Day(s)/duration:

24 h

Adequacy of challenge:

other: the dose for challenge was selected based on the results of the dose range-finding test; concentrations up to 40% (maximum achievable concentration) did not induce local reactions; thus, the highest achievable concentration was selected

No. of animals per dose:

20 (in test groups), 10 (controls)

Details on study design:

RANGE FINDING TESTS:
Dose-range finding study for intradermal induction:
One guinea pig was given intradermal injections twice, in each case, with 0.1 mL of the following test substance concentrations: 0%; 1%; 2.5%; 5%
The injection sites were evaluated after 24 and 48 h:
Scoring after 24 h: 0%: no reaction, 2.5% - 5%: reddish wheal
Scoring after 48 h: 0% - 1%: no reaction, 2.5% - 5%: wheal

Dose range-finding study for topical induction:
In each case, 4 concentrations were tested on 4 guinea pigs. 4 patches each saturated with 0.5 mL of test substance formulation were applied to each animal under occlusive conditions for 24 h. At the end of the exposure period, the remaining test substance was removed with physiological saline solution and 21 h later the treated areas were shorn. The skin reactions were evaluated 48 h and 72 h after the start of the application.

Dose range-finding study for challenge:
One week prior to the challenge, the challenge concentrations were determined on 5 guinea pigs that were treated in the same manner as the control animals during the inductions. 4 patches each saturated with 0.5 mL test substance formulation were applied to each animal under occlusive conditions for 24 h. At the end of the exposure period, the remaining test substance was removed with physiological saline solution and 21 h later the treated areas were shorn. The skin reactions were evaluated 48 h and 72 h after the start of the application.

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: 1:1 mixture complete Freund's adjuvant (Difco) and physiological NaCl solution
Injection 2: 5% test substance in physiological NaCl solution containing 2% v/v Cremophor El®
Injection 3: 5% test substance in a 1:1 mixture physiological NaCl solution containing 2% v/v Cremophor El® and complete Freund's adjuvant (Difco)
Epicutaneous: 0.5 mL test substance 40%
- Control group:
Intradermal (3 pairs of injections):
Injection 1: 1:1 mixture complete Freund's adjuvant (Difco) and physiological NaCl solution
Injection 2: physiological NaCl solution containing 2% v/v Cremophor El®
Injection 3: physiological NaCl solution containing 2% v/v Cremophor El® and complete Freund's adjuvant (Difco)
Epicutaneous: 0.5 mL physiological NaCl solution containing 2% v/v Cremophor El®
- Site: cranial, medial and caudal bilateral
- Frequency of applications: every 7 days, topical induction was performed one week after the intradermal induction
- Duration: Days 0-8
- Concentrations: intradermal 5%, epicutaneous 40%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 3 weeks after intradermal induction
- Exposure period: 24 h
- Test groups: 40% and 25% test substance and vehicle
- Control group: vehicle and test substance
- Site: dorsal region and the flanks
- Concentrations: 40% and 25%
- Evaluation (hr after challenge): 48 and 72 h

Challenge controls:

The control group is actually a challenge control

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Results and discussion

Positive control results:

Challenge with 2-mercaptobenzothiazole formulated with physiological NaCl at 40% and 25%, exhibited in 80% and 65% of the test animals dermal alterations after the first challenge. After the 2nd challenge both with 12% and 6% test substance formulations 80% and 75% of the test animals developed dermal signs. There was no reddening of the skin to be observed on control group animals. In another test series performed with female animals, 70% of the treated animals showed positive reactions after the 1st challenge. Thus, sensitivity as well as reliability of the experimental technique is confirmed in both studies.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

induction: 5% intradermal, 40% epicutaneous, challenge: 40% and 25%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

One animal showed symptoms like emaciation, sunken flanks and beside on day 5 labored breathing and temporary breathing sounds. From day 15 up to the end of the study it showed only labored breathing.

Results 2

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenge: 40% and 25%

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction: 5% intradermal, 40% epicutaneous, challenge: 40% and 25%

No. with + reactions:

0

Total no. in group:

20

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

challenge: 40% and 25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

After the 2nd induction on Day 10, encrustations at the treatment areas were observed in 3 animals, which lasted latest until Day 22.

Results 5

Reading:

1st reading

Group:

positive control

Dose level:

induction: 2,5% intradermal, 40% epicutaneous, challenge with 25 and 40%

Clinical observations:

in respective control animals, no reddening was observed

Remarks on result:

positive indication of skin sensitisation

Remarks:

test performed in males (for further information, please refer to "Positive control results")

Results 6

Reading:

1st reading

Group:

positive control

Dose level:

induction: 2,5% intradermal, 40% epicutaneous, challenge with 25 and 40%

Clinical observations:

in respective control group, no reddening was observed

Remarks on result:

positive indication of skin sensitisation

Remarks:

test performed in females (for further information, please refer to "Positive control results")

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

Challenge with up to 40% test substance formulation (maximum achievable concentration) induced skin redness in only 1 animal at the first reading time point. As no skin redness was observed at the later time point and in none of the remaining animals, the test substance is not considered to exhibit skin sensitising properties.