3,3'-[[4-[(2,6-dichloro-4-nitrophenyl)azo]phenyl]imino]bispropiononitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From June 02 to July 05, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Study predates LLNA method

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]bis[propiononitrile]

EC Number:

223-765-4

EC Name:

3,3'-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]bis[propiononitrile]

Cas Number:

4058-30-4

Molecular formula:

C18H15ClN6O2

IUPAC Name:

3,3'-({4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}imino)dipropanenitrile

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wolferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Number of animals for main study / Irritation screen: 30 female / 4 female animals (nulliparous and non-pregnant). Challenge:- 20 test animals; - 10 control animals. Irritation Screen: - 4 animals
- Age at delivery/ acclimatization start: 4 - 6 weeks
- Body weight at delivery/ acclimatization start: Test and control animals 301 - 400 g
- Identification: By unique cage number and corresponding individual number of animals.
- Randomization: Randomly selected at time of delivery.
- Acclimatization: One week for the control and test group under test conditions after health examination. However, contrary to the test group the control group remained untreated during the 3 induction weeks. One day for the animals used in the irritation screen for induction and challenge. Only animals without any visible signs of illness were used for the study.
- Husbandry: The animals were accommodated under standard laboratory conditions. The animal room was air-conditioned with 10-15 air changes per hour and the air was continuously monitored with a range for room temperature of 22 ±3 °C and for relative humidity between 40-70 % (values above 70 %
during cleaning process possible). The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Fluorescent "Gold" lamps (Silvania Gold F40T1260) were present in the room used for the study. Music was played during the light period.
- Accommodation: Individually in Makroion type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Nafag Ecosan 845 25W4, batch no. 23/99, guinea pig breeding / maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum. Results of analyses for contaminants are archived at RCC Itingen.
Water: Community tap water from Füllinsdorf, ad libitum. Once weekly from day 7 of the acclimatization to the end of the study additional supply of ascorbic acid (approx. 1 g/1) was provided via the drinking water. The water analysis was performed at RCC Ltd, Environmental Chemistry & Pharmanalytics Division.Results of bacteriological, chemical and contaminant analyses are archived at RCC Itingen.

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50 % / 0.5 mL

Day(s)/duration:

Days 1, 8, 15 for 6 hours each

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50 %

Day(s)/duration:

Day 29 for 6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Challenge:
- 20 test animals
- 10 control animals
Irritation Screen:
- 4 animals

Details on study design:

IRRITATION SCREEN FOR INDUCTION AND CHALLENGE - PERFORMED DURING THE ACCLIMATIZATION PERIOD
For patch placements, the format described below was used on 4 guinea pigs. Four different concentrations were used on each animal for a 6-hour exposure period. The test article concentration of 50 % in bi-distilled water was considered to be the most qualified to assure an optimum technical application procedure.
The allocation of the different test dilutions to the sites (A, B, C, D) on the four animals was alternated in order to minimize site-to-site variation in responsivenessThe application sites were assessed for erythema and oedema 24 and approximately 48 hours after removal of the patches.
The highest test article concentration of 50 % in bi-distilled water was considered to be the most representative concentration to stimulate a state of immune hypersensitivity in the induction phase and the highest non-irritating concentration to be used for the challenge.

INDUCTION
The concentration of the test article required for the induction was agreed between the Study Director and responsible Technical Coordinator after the irritation screen had been completed. The fur was clipped from the left shoulder of each test animal and the patches applied, over a period of 3 weeks. Each animal received one patch per week which remained in place for approximately 6 hours each. The repeated application was performed at the same site. The interval between exposure was one week. After the last induction exposure the animals were left untreated for 2 weeks before the challenge.
The skin responses were graded approximately 24 hours after the patches have been removed. Any gross skin reactions were recorded without depilation.

CHALLENGE - PERFORMED ON TEST DAY 29
The animals previously exposed during the induction period (i.e. test group) as well as the previously untreated control animals were challenged two weeks after the last induction exposure using the dose as prescribed. The fur was clipped from the left posterior quadrant of the side and back of the animals. Patch sites for challenge are indicated below. The exposure period was 6 hours on a naive skin site.
The responses were graded at 24 and 48 hours after the patches have been removed.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:

90 % of the animals of the test group were observed with significant skin reactions (grade of 0 and ± are considered to be non-significant responses, whereas those of 1 and greater are considered to be significant) after challenge performed with the highest nonirritating concentration of ALPHA-HEXYLCINNAMALDEHYDE at 10 % in PEG 400.
No skin reactions were observed in the control group treated in the same conditions during the challenge phase.
Therefore, the test article ALPHA-HEXYLCINNAMALDEHYDE applied at a concentration of 10 % in PEG 400 is considered to be a sensitizer when used under the described test conditions.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

8

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 %

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Results 5

Reading:

2nd reading

Hours after challenge:

24

Group:

positive control

Dose level:

10%

No. with + reactions:

18

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10%

No. with + reactions:

13

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

There were no deaths during the course of the study, hence no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

SKIN EFFECT IN THE INDUCTION

As the test article at 50 % in bi-distilled water stained the skin violet, it was not possible to determine whether erythema was present or not. However, no oedema was observed.

SKIN EFFECT IN THE CHALLENGE Discrete/patchy erythema was observed in eight (at the 24-hour reading) and four (at the 48- hour reading) out of 20 test animals after treatment with the test article at 50 % in bi-distilled water. No skin reactions were observed in the animals of the control group treated with the same test article, vehicle and concentration.

BODY WEIGHTS

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Sensitising

Executive summary:

Method

The skin sensitisation of the test substance has been evaluated using a Buheler test, according to the OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization", adopted by the Council on July 17, 1992 (reported Paris, April 29,1993) and the Directive 96/54/EEC, B.6. "Skin Sensitization", July 30, 1996.

Observations

Twenty female animals of the test group were treated topically with the test substance at 50 % in bi-distilled water once a week for a 3 week induction phase. Two weeks after the final induction application the animals were challenged with the same test article concentration of 50 % in bi-distilled water as used for induction. The ten animals of the control group were not treated during the induction but were treated once at challenge with the test substance at 50 % in bi-distilled water.

In this study 40 % of the animals of the test group were observed with skin reactions after challenge treatment performed with the highest non-irritating concentration of the test substance at 50 % in bi-distilled water. No skin reactions were observed in the control group treated in the same conditions during the challenge phase.

Conclusion

The test substance , under these experimental conditions, is considered as a sensitizer.