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

Endpoint summary

Description of key information

Skin: not irritant

Eye: not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 27 to 30, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Species: Albino Rabbit, New Zealand White, (SPF-Quality).
- Original Source: Broekman Institute, Someren, The Netherlands.
- Selection of animals: healthy animals, not previously used, were selected prior to treatment. Both flanks of each animal were free of interfering lesions, irritation or defects.
- Total number of animals: 3 male rabbits.
- Allocation and age at start of treatment: animals 1248, 1249 & 1254; approx.12 weeks.
- Identification: ear tag.
- Acclimatisation: at least five days after veterinary examination.

HUSBANDRY
- Room No.: animal 1254 was housed in room 11 during acclimatisation, but was moved to room 10 at day-1 (one day before treatment). Animals 1248 and 1249 were housed in room 10 during acclimatisation. All animals were housed in room 10 during the observation period of the study.
- Conditions: standard laboratory conditions. Air-conditioned with 15 air changes per hour and controlled environment with optimal conditions considered as being a temperature of 21°C and a relative humidity of 55%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day. Music during the light period.
- Accommodation: individually in labelled cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
- Diet: standard laboratory rabbit diet (LKK-20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands) approx.100 gram per day.
- Water: free access to tap-water diluted with decalcified water.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TREATMENT
Approximately 24 hours before treatment, the dorsal fur was shaved with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm2). Prior to test substance administration, the animals were physically examined and the shaved area of skin to be treated was normal. On test day 1, 0.5 gram of the test substance was applied to the intact skin of the shaved area on one flank, using a Scotchpak-non-woven patch (2x3 cm, 3M, St.Paul, U.S.A.) mounted on Micropore tape (3M, St. Paul, U.S.A.). The contralateral flank was similarly prepared (but without test substance) to act as a procedural control. The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, St. Paul, U.S.A.). Four hours after the application, the dressing was removed and the remaining test substance removed using a tissue moistened with tap-water and subsequently a dry tissue. The skin areas concerned of the animals were re-shaved at least 3 hours before the observations, when considered necessary to facilitate scoring.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Other effects:

COLOURATION:
Red staining of the treated skin by the test substance was observed during the observation period. The staining did not interfere with the scoring of the erythema.

BODY WEIGHTS:
The body weights and variability in body weight gain of the animals were considered to be normal.

TOXICITY SYMPTOMS/MORTALITY
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Not irritant

Executive summary:

Method:

The Skin irritation was determined in an irritation test on rabbits according to the OECD Guideline 404 and the EU Method B.4.

 

Observations:

The test substance was applied onto shaved skin of three male albino rabbits using semi-occlusive dressings for 4 hours, followed by four observations at approximately 1, 24, 48 and 72 hours days after removal of the dressings and remaining test substance. Under the conditions of this study, no skin irritation was caused by the test substance and no corrosive effect occurred on the skin in any of the three rabbits. In the area of application, red staining of the treated skin was observed, which did not interfere with the scoring of erythema. No signs of systemic intoxication were observed during the study period. The test substance resulted in a primary irritation index of 0.0 (non-irritating) when applied to the intact rabbit skin.

 

Result:

Not irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 24 to 27, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Species: Albino rabbit, New Zealand White (SPF-Quality).
- Original Source: Broekman Institute, Someren, The Netherlands.
- Selection of animals: healthy animals, not previously used, were selected prior to treatment. Both eyes of each animal were free of interfering lesions, irritation or defects.
- Total number of animals: 3 male rabbits.
- Allocation and age at start of treatment: animals 1298, 1302 and 1303; approx. 14 weeks.
- Identification: ear tag.
- Acclimatisation: at least five days under laboratory conditions after veterinary examination.

HUSBANDRY
- Conditions: standard laboratory conditions. Air-conditioned with 15 air changes per hour and controlled environment with optimal conditions considered as being a temperature of 21°C and a relative humidity of 55%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day. Music during the light period.
- Accommodation: individually in labelled cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). 0.1 et standard laboratory rabbit diet (LKK-20, pellet diameter 4 mm, Hope Farms, Woerden, The Netherlands) approx. 100 g per day.
- Water: free access to tap-water diluted with decalcified water.

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

100 mg per animal

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

TREATMENT
On day 1, prior to test substance administration, the animals were physically examined and the eyes were found to be normal. The test substance (100 ± 1 mg per animal) was instilled in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control. Immediately after the 24 hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

IRRITATION
Instillation of the test substance into one eye of each of the three male albino rabbits affected the iris in two animals and the conjunctivae in all three animals. Slight dulling of normal lustre (corneal opacity grade 0) was observed in animal 1302 on day 1 only. Treatment of the eyes with 2% fluorescein, 24 hours after test substance instillation revealed no corneal epithelial damage in any of the animals. The iridic irritation had resolved within 24 hours in animals 1298 and 1302.
The irritation of the conjunctivae had resolved within 72 hours after instillation in all animals.
CORROSION
There was no evidence of ocular corrosion.

Other effects:

COLOURATION:
Red staining of the outside of the eyelids, caused by the test substance, was observed in two animals on days 2 and 3.

TOXIC SYMPTOMS/MORTALITY:
No toxic symptoms were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Not irritant

Executive summary:

Method:

The eye irritation of the test substance was determined in an irritation test on rabbits according to OECD Guideline 405 and EU Method B.5.

 

Observations:

The test substancewas instilled into one eye of each of three male albino rabbits, followed by four observations at approximately 1, 24, 48 and 72 hours after test substance administration. Under the conditions of this study,the test substanceresulted in adverse effects on the iris in two animals and on the conjunctivae in all three animals. Slight dulling of normal lustre (corneal opacity grade 0) was observed in one animal on day 1. The iridic irritation had resolved within 24 hours and the irritation of the conjunctivae had resolved within 72 hours after instillation in the animals. Ocular corrosion was not observed in any of the rabbits. No signs of systemic intoxication were observed during the study period.

 

Result:

Not irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

The skin irritation was determined in an irritation test on rabbits according to the OECD Guideline 404 and the EU Method B.4.

The test substance was applied onto shaved skin of three male albino rabbits using semi-occlusive dressings for 4 hours, followed by four observations at approximately 1, 24, 48 and 72 hours days after removal of the dressings and remaining test substance. Under the conditions of this study, no skin irritation was caused by the test substance and no corrosive effect occurred on the skin in any of the three rabbits. In the area of application, red staining of the treated skin was observed, which did not interfere with the scoring of erythema. No signs of systemic intoxication were observed during the study period. The test substance resulted in a primary irritation index of 0.0 (non-irritating) when applied to the intact rabbit skin.

 

EYE IRRITATION

The eye irritation of the test substance was determined in an irritation test on rabbits according to OECD Guideline 405 and EU Method B.5.

The test substance was instilled into one eye of each of three male albino rabbits, followed by four observations at approximately 1, 24, 48 and 72 hours after test substance administration. Under the conditions of this study,the test substance resulted in adverse effects on the iris in two animals and on the conjunctivae in all three animals. Slight dulling of normal lustre (corneal opacity grade 0) was observed in one animal on day 1. The iridic irritation had resolved within 24 hours and the irritation of the conjunctivae had resolved within 72 hours after instillation in the animals. Ocular corrosion was not observed in any of the rabbits. No signs of systemic intoxication were observed during the study period.

 

These results are in line with the one obtained in the tests listed in the table attached below.

Justification for classification or non-classification

SKIN IRRITATION/CORROSION

A corrosive substance is a substance that produces destruction of skin tissue, namely, visible necrosis through the epidermis and into the dermis, in at least 1 tested animal after exposure up to 4 hours duration.

Three sub-categories are provided within the corrosive category 1:

Sub-category 1A = where responses are noted following up to 3 minutes exposure and up to 1 hour observation;

Sub-category 1B = where responses are described following exposure between 3 minutes and 1 hour and observations up to 14 days;

Sub-category 1C = where responses occur after exposures between 1 hour and 4 hours and observations up to 14 days.

To classify a substance as irritant, the major criterion is that at least 2 of 3 tested animals have a mean score of ≥ 2.3 - ≤ 4.0.

Category 2:

- Mean value of ≥ 2.3 - ≤ 4.0 for erythema/eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions; or

- Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

- In some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical eposure in a single animal but less than the criteria above.

The substance is not classified as skin irritant because in the performed test it doesn't meet the classification criteria of the CLP regulation n. 1272/2008.

 

EYE IRRITATION

Serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

Eye irritation means the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

Irreversible effects on the eye (Category 1):

If, when applied to the eye of an animal, a substance produces:

- At least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/or

- At least in 2 of 3 tested animals, a positive response of corneal opacity ≥ 3 and/or iritis > 1.5 calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight. In this context, persistent lesions are considered those which are not fully reversible within an observation period of normally 21 days. Substances are also classified in Category 1 if they fulfill the criteria of corneal opacity ≥ 3 or iritis > 1.5 detected in a Draize eye test with rabbits, recognising that such severe lesions usually do not reverse within a 21-day observation period.

The substance does not meet the requirements to be classified in Category 1.

Irritating to eyes (Category 2):

When applied to the eye of an animal, a substance produces:

- At least in 2 of 3 tested animals, a positive response of: corneal opacity ≥ 1 and/or iritis ≥ 1, and/or conjunctival redness ≥ 2 and/or conjunctival oedema (chemosis) ≥ 2.

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

The substance is not classified as eye irritant because in the performed test it doesn't meet the classification criteria of the CLP regulation n. 1272/2008.