5-[(4-chloro-2-nitrophenyl)azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Endpoint summary

Description of key information

Studies were carried out to evaluate skin irritation/corrosion and eye irritation potency of the test substance following OECD guidelines. The tests demonstrated that the test substance is non-irritant and non-corrosive to skin and non-irritant to eye.

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:

The experiment started: January 21, 1980; Experiment and study completion date: 04 February 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: The procedure used is described in the Proposed Guidelines of the United States Environmental Agency (EPA) § 163.81- 5 "Primary dermal irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

Name: FAT 36019/D
Purity: 50.2 %

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

The test was performed on 3 male and 3 female adult New Zealand White rabbits bred and raised on the premises weighing 2 to 3 kgs. They were housed individually in metal cages, numbered by ear tags, were kept at a constant room temperature of 22 ± 2 °C, at a relative humidity of 55 ± 10 % and on a 10 hours light cycle day. The animals received ad libitum standard rabbit food - NAFAG, No. 814, Gossau SG - and water. Prior to treatment they were adapted to our laboratories for a minimum of 4 days.

Type of coverage:

occlusive

Preparation of test site:

other: shaved and slightly scarified

Vehicle:

other: Propylene glycol+saline (70:30 parts), concentration 50%

Controls:

no

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

24 hours

Observation period:

7 days

Number of animals:

3 male and 3 female rabbits

Details on study design:

Before treatment the entire back and the flank of the rabbits were shaved with an electric clipper and immediately before treatment the shaven skin on one side was slightly scarified with the help of a "SchröpfSchnäpper", Aesculap, Switzerland. Gauze patches of 2.5 x 2.5 cm laden with 0.5 g of the test material were applied to the prepared abraded and intact skin. The patches were covered with an impermeable material and were fastened to the body of the rabbit with adhesive tape. The dressings were removed after a 24 hour application. The skin reaction was appraised upon removal and during an observation period of 7 days on the basis of the following evaluation scheme.

Score for skin irritation in rabbits
Erythema and eschar formation
- No erythema: 0
- Very slight erythema (barely perceptible): 1
- Well defined erythema: 2
- Moderate to severe erythema: 3
- Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4
Total possible erythema score 4

Edema formation
- No edema: 0
- Very slight edema (barely perceptible): 1
- Slight edema (edges of area well defined by definite raising): 2
- Moderate edema (raised approximately 1 mm): 3
- Severe edema (raised more than 1 mm and extending beyond area of exposure): 4
Total possible edema score: 4

Irritation parameter:

erythema score

Remarks:

Intact skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Remarks:

Abraded skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

Intact skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

Abraded skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Due to intensive staining by the test compound erythema was not assessable. For the purpose of calculating the primary irritation index erythema was assumed to be equal to edema based on the correlation existing (Marzulli and Maibach, Fol. Cosm. Tox. 13, 355, 1975). FAT 36091/D was found to cause a slight irritation when applied to intact and abraded rabbit skin.

Calculation of the primary skin irritation index

The scores read after 24 and 72 hours for erythema and edema for the intact as well as for the abraded skin were summed up and divided by 4. (FHSLA § 191.11)

	MEAN REACTION SCORE OF 6 RABBITS
	ERYTHEMA		EDEMA
Time after exposure	Intact skin	Abraded skin	Intact skin	Abraded skin
24 hours	0	1.3	0	1.3
72 hours	0	0.8	0	0.8
Total	0	2.1	0	2.1	4.2

primary irritation index = 4.2 / 4 = 1.1

Assessment of irritation

0 = none

>0 - 1 = minimal

>1 - 2 = slight

>2 - 4 = moderate

>4 - 6 = marked

>6 - 8 = extreme

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36091/D was found to cause a slight irritation when applied to intact and abraded rabbit skin.

Executive summary:

The skin irritation in albino rabbits after single application was performed to determine the irritant potency of the test item on the albino rabbit skin. The procedure used is described in the Proposed Guidelines of the United States Environmental Agency (EPA) 163.81-5 "Primary dermal irritation study", Federal Register Vol. 43, No. 163, August 22, 1978. The test was performed on 3 male and 3 female (New Zealand White rabbits), checked for normal skin conditions. Before treatment the flanks of the rabbits were shaved and immediately before treatment, the shaven skin on one side was slightly scarified. A gauze patch bearing 0.5 g of the test substance was applied to the prepared abraded and intact skin. The patches were covered by an impermeable material. Patches were held in place for 24 hours. Observations were made at 24, 48, 72 hours and 7 days, respectively. Due to intensive staining by the test compound erythema was not assessable. For the purpose of calculating the primary irritation index erythema was assumed to be equal to edema based on the correlation existing (Marzulli and Maibach, Fol. Cosm. Tox. 13, 355, 1975). The 24/48/72h mean erythema score and edema score was found to be 0 and 1 respectively. The effects were fully reversible within 7 days of observation period. Under the conditions of the present experiment FAT 36091/D was found to cause a minimal to slight irritation when applied to intact and abraded rabbit skin. The calculated primary irritation index was 1. FAT 36091/D was found to cause a minimal to slight irritation when applied to intact and abraded rabbit skin. In general, abration represents an elevated test condition that does normally not need to be taken into account for evaluation of the hazard assessment. Therefore, results on abraded skin are not taken into account.

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:

Start of experiment: November 7, 1989; End of experiment: November 14, 1989; Study completion date: 14 November 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

.

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Test article: FAT 36019/F
Batch No.: EN 162190.96
Storage conditions: room temperature
Validity: October 1994
Safety precautions: gloves and face masks
Test material received: October 26, 1999

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

The albino rabbit is the recommended species for Acute Eye Irritation/Corrosion studies.
- Animal strain: New Zealand white rabbits (Chbb:NZW)
- Breeder: Dr. K. Thomae GMBH Chemisch-pharmazeutische Fabrik D-7950 Biberach/Riss
- Acclimatisation period: at least 5 days-

Group size and husbandry:
The test was performed on 3 female rabbits, checked for normal eye conditions, weighing between 2630 to 2860 g (approx. 9-13 weeks old) . The animals were housed individually in metal cages, identified by ear tattoo, kept at a constant room temperature of 20+3 °C, at a relative humidity of 30-70 % and on a 12 hours light cycle day. The rabbits received ad libitum standard rabbit pellet - Nafag No. 814, Gossau, Switzerland - and fresh water.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml (42 mg)

Duration of treatment / exposure:

one single application

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

0.1 ml (42 mg) of FAT 36091/F was placed into the conjunctival sac of the left eye of each animal, after gently pulling away the lower lid from the eyeball. The lids were then held together for about one second in order to prevent loss of the test article. The animals were checked daily for systemic symptoms and mortality. The ocular reactions were evaluated 1, 24, 48, and 72 hours after the instillation of FAT 36091/F. A slit-lamp was used to facilitate the evaluation.

SCORING SYSTEM:
CORNEA
- Opacity: degree of density (area most dense taken for reading).
- No ulceration or opacity 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible 1
- Easily discernible translucent area, details of iris slightly obscured 2
- Nacrous area, no details of iris visible, size of pupil barely discernible 3
- Opaque cornea, iris not discernible through the opacity 4

IRIS
- Normal 0
- Markedly deepended rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive) 1
- No reaction to light, haemorrhage, gross destruction ( any or all of these ) 2

CONJUNCTIVAE
Redness: (refers to palpebral and bulbar conjunctivae, cornea and iris)
- Blood vessels normal 0
- Some blood vessels definitely hyperaemic (injected) 1
- Diffuse, crimson colour, individual vessels not easily discernible 2
- Diffusy beefy red 3

CHEMOSIS
lids and/or nictating membranes
- No swelling 0
- Any swelling above normal (includes nictating membranes ) 1
- Obvious swelling with partial eversion of lids 2
- Swelling with lids about half closed 3
- Swelling with lids more than, half closed 4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0.78

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Other effects:

No abnormal increase in body weight was noted.

Individual eye scores

	CORNEA		IRIS		CONJUNCTIVA
Animal number 310/F					Redness	Chemosis
	ce	te	ce	te	ce/te	ce/te
After 1 hour	0	0	0	0	0/2	0/1
After 24 hours	0	0	0	0	0/1	0/0
After 48 hours	0	0	0	0	0/1	0/0
After 72 hours	0	0	0	0	0/1	0/0
Mean 24 -72 hours	0	0	0	0	0/1	0/0
After 7 days	0	0	0	0	0/0	0/0

	CORNEA		IRIS		CONJUNCTIVA
Animal number 304/F					Redness	Chemosis
	ce	te	ce	te	ce/te	ce/te
After 1 hour	0	1	0	0	0/2	0/2
After 24 hours	0	0	0	0	0/1	0/0
After 48 hours	0	0	0	0	0/0	0/0
After 72 hours	0	0	0	0	0/0	0/0
Mean 24 -72 hours	0	0	0	0	0/0.33	0/0
After 7 days	0	0	0	0	0/0	0/0

	CORNEA		IRIS		CONJUNCTIVA
Animal number 318/F					Redness	Chemosis
	ce	te	ce	te	ce/te	ce/te
After 1 hour	0	0	0	0	0/2	0/2
After 24 hours	0	0	0	0	0/1	0/0
After 48 hours	0	0	0	0	0/1	0/0
After 72 hours	0	0	0	0	0/1	0/0
Mean 24 -72 hours	0	0	0	0	0/1	0/0
After 7 days	0	0	0	0	0/0	0/0

ce=control eye

te= test eye

M=Male

F=Female

Body weights (q)

Animal N°	310/F	404/F	318/F
At start of test	2860	2630	2670
After 3 days	2870	2690	2840
After 7 days (end)	2970	2760	2980

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions employed FAT 36091/F induced irritation of the cornea and conjunctiva when instilled into the conjunctival sac of albino rabbits.

Executive summary:

The eye irritation in New Zaeland White after single application was performed to determined the irritant potency of the test item on the rabbit eyes. This test was based on the OECD Guideline No. 405, adopted May 12, 1981, by the OECD council, and on Annex V, part B of Council Directive 79/831/EEC (Commission Directive 84/449/EEC of April 25, 1984; Official Journal L251, September 19, 1984). 0.1 ml (42 mg) of FAT 36091/F was placed into the conjunctival sac of the left eye of each animal, after gently pulling away the lower lid from the eyeball. The lids were then held together for about one second in order to prevent loss of the test article. The right eye remained untreated and served as a control. The animals were checked daily for systemic symptoms and mortality. Because reactions were observed within 72 hours after instillation of the test article, the observation period was extended to 7 days to determine the reversibility of the eye reactions. No effects were observed on corneal opacity, iris and chemosis. Mean 24/48/72h conjuctiva score was found to be 0.78. According to the EEC classification of the results obtained 24/48.72 hours after instillation FAT 36091/F can be classified as non-irritant to albino rabbit's eye. The eye reactions observed were reversible within 72 hours.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key study was performed to determine the potential of FAT 36091/E (Study number: 875189) to induce irritation/corrosion to albinos rabbits skin according to the OECD Guideline 404 (Acute Dermal Irritation / Corrosion). The test was performed on 3 rabbits. A gauze patch bearing 0.5 gr. of the test item was applied to the flank of each animals. Patched were held in place for 4 hours. During the experiment, no mortality occuried and no edema or erythema were observed. The primary index irritation was found to be 0. The test item, did not induce skin irritation when applied to the shaved albino rabbits skin. Three supporting studies were carried out with FAT 36091 on different batches (A and D) according to the Proposed Guidelines of the United States Environmental Agency (EPA) 163.81-5 "Primary dermal irritation study", Federal Register Vol. 43, No. 163, August 22, 1978.

- Batch D: two studies were performed to determine the skin irritation in the rabbit after a single application.

- In the study report number 801358, FAT 36091/D was found to be a minimal irritant with a primary irritation index of 0.9

- In the study report number 791713, FAT 36091/D was found to cause slight irritation when applied to intact and abraded skin. The primary skin irritation index was 1.1.

Skin reaction were fully reversible within 7 days as reported in the study number 791713 and fully reversible within 7 days for the intact skin in the study number 801358.

- Batch A: One more study, (study number 385/19), was performed to determine the skin irritation in the rabbit after a single application. The calculated primary irritation index was found to be 4.2 and under these the test item was found to cause marked irritation when applied to intact and extreme irritation when applied to abraded rabbit skin. One more supporting study was carried out with FAT 36091/A according to the the procedure used is described in the Proposed Guidelines of the United States Environmental Agency (EPA) 163.81-5 "Primary dermal irritation study", Federal Register Vol. 43, No. 163, August 22, 1978.

-Batch A (study number 801357): Due to intensive staining by the test compound, erythema was not assessable. For the purpose of calculationg the primary irritation index erythema was assumed to be equal to edema based on the correlation existing (Marzulli and Maibach, Fol. Cosm. Tox. 13, 355, 1975). The calculated primary irritation index was 1. The skin reactions were reversible within 7 days for the intact and abraded skin.

FAT 36091/A was found to cause minimal to slight irritaion when applied to intact and abraded rabbit skin but classified as non-irritant to the rabbit skin. Considering the above key and supporting studies FAT 36091 can be considered as non-irritant to skin of rabbits.

A key study was performed to determine the potential of FAT 36091/A to induce irritation to rabbit eye according to OECD guideline 405 and no GLP methodology. Because reaction were observed within 72 hours after instillation of the test article, the observation period was extended to 7 days determine the reversibility of the eye reactions. According to the EEC classification of the results obtained 24 to 72 hours after instillation, FAT 36091/F can be classified as non-irritant to albino rabbit. The eye reaction observed were reversible within 7 days. FAT 36091/F is to be considered as a non-irritant to the eye of rabbits. The two supporting studies were carried out with FAT 36091 (batch A and D) to determine the eye irritation according to non GLP guideline methods.

The primary irritation index, for batch A, was found to be 7.5 for unrinsed eyes and 7.1 for rinsed eyes. Rinsing the eye following instaillation has no significant effect. The primary irritation index, for batch D, was found to be 0.7 for unrinsed eyes and 0.0 for rinsed eyes.

FAT 36091/A is considered to be a slightly irritant to the rabbit eye mucosa. FAT 36091/D is considered to be a minimal irritant to the rabbit eye mucosa. Since the reactions in the supporting studies are fully reversible, it is considered as non-irritant to eye.

Therefore, considering the results from the key and supporting studies, FAT 36091 is considered as non-irritant on skin and eye.

Justification for selection of skin irritation / corrosion endpoint:
Most recent study available and the experiment was performed according to the OECD Guideline 404.

Justification for selection of eye irritation endpoint:
Most recent study available and the experiment was performed according to the OECD Guideline 405.

Justification for classification or non-classification

Based on the above mentioned results, the substance does not need to be classified for eye irritation. The degree of skin and eye irritation was insufficient for classification and labelling and thus no classification is proposed for skin irritation.

Therefore, FAT 36091 is classified as non-irritant for skin and eyes.