Benzyl 4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxopyridin-3-yl)azo]benzoate

Endpoint summary

Description of key information

Not irritating to rabbit skin

Not irritating to rabbit 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:

12 August to 15 August, 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12 to 16 weeks old
- Weight at study initiation: 2.28 to 3.28 kg
- Housing: individually in suspended metal cages
- Diet: ad libitum; Rabbit Diet, Preston Farmers Limited, New Leake, Boston, Lincolnshire, UK
- Water: ad libitum; mains drinking water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 21 °C
- Humidity: 65 - 68 %
- Air changes: 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12 controlled by a time switch

Type of coverage:

occlusive

Preparation of test site:

clipped

Remarks:

fur of the dorsal/flank areas was closely clipped using veterinary clippers approximately 24 hours before the test

Vehicle:

water

Remarks:

slightly moistened with 0.5 ml distilled water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

- Amount applied: 0.5 g in 0.5 ml distilled water
- Negative control: untreated surrounding skin

Duration of treatment / exposure:

4 hours

Observation period:

72 hours after removal of substance

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: dorsal area
- Type of coverage if used: 2.5 × 2.5 cm gauze patch, secured with surgical adhesive tape (Blenderm; ca. 25 × 40 mm); then the trunk of the animal was wrapped in elasticated corset (Turbigrip)

REMOVAL OF TEST SUBSTANCE
- Removal: gentle swabbing with cotton wool soaked in distilled water
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
- 1, 24, 48 and 72 hours after removal of patch/test item

SCORING SYSTEM:
- Method of calculation: Draize, J. H. (1959) "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics", as follows:

Erythema and Eschar Formation:
0 - No erythema
1 - Very slight erythema (barely perceptible)
2 - Well-defined erythema
3 - Moderate to severe erythema
4 - Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Oedema Formation:
0 - No oedema
1 - Very slight oedema (barely perceptible)
2 - Slight oedema (edges of area well-defined by definite raising)
3 - Moderate oedema (raised approximately 1 mm)
4 - Severe oedema (raised more than 1 mm and extending beyond the area of exposure)

Irritation parameter:

erythema score

Basis:

animal: #1 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

edema score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

Very slight erythema was noted in all three animals at 1 hour after removal of the substance. Only one animal still showed slight erythema at 24 hours after exposure, which was reversible within 48 hours after exposure.
No oedema was observed.

Other effects:

Faint yellow coloured staining of the treatment sites was noted in all three animals which lasted until the end of the study period.

Table 1: Individual daily and individual mean scores for dermal irritation/corrosion.

Skin reaction	Reading (hours)	Individual scores
		#1	#2	#3
Erythema/eschar formation	1	1s	1s	1s
	24	0s	1s	0s
	48	0s	0s	0s
	72	0s	0s	0s
	total (24, 48, 72 h)	0	1	0
	mean (24, 48, 72 h)	0.0	0.3	0.0
Oedema formation	1	0	0	0
	24	0	0	0
	48	0	0	0
	72	0	0	0
	total (24, 48, 72 h)	0	0	0
	mean (24, 48, 72 h)	0.0	0.0	0.0

s = faint yellow-coloured staining over the test site not affecting evaluation of erythema

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

The test item was very slightly irritating to rabbit skin until 24 hours; skin was stained slightly yellow locally.

Executive summary:

The potential skin irritation/corrosion of the test item was evaluated in an experimental study according to a method based on the OECD guideline 404 (1981). The dorsal fur of three New Zealand White rabbits was clipped free of hair and 0.5 mg of test item moistened with 0.5 ml of distilled water was applied under a gauze. After four hours, the substance was removed with cotton and water, and observations of erythema and oedema were recorded at 1, 24, 48 and 72 hours after exposure.

Very slight erythema was noted in all three animals 1 hour after exposure. Only one animal still showed slight erythema 24 hours after exposure, which was reversible within 48 hours. No oedema was observed. Faint yellow coloured staining of the treatment sites was noted in all three animals which lasted until the end of the study period. The mean (24, 48 and 72 h) irritation scores for (i) erythema and (ii) oedema were subsequently calculated to be (i) 0 in two animals and 0.33 in one animal, and (ii) 0 in all animals.

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:

17 August to 21 August, 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1981

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: David Percival Limited, Moston, Sandbach, Cheshire, UK and Nottingham University, School of Agriculture, Sutton Bonington, Leicestershire, UK
- Age at study initiation: 12 to 16 weeks old
- Weight at study initiation: 2.72 to 3.08 kg
- Housing: individually in suspended metal cages
- Diet: ad libitum; Rabbit Diet, Preston Farmers Limited, New Leake, Boston, Lincolnshire, UK
- Water: ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18-21 °C
- Humidity: 65-68 %
- Air changes: ca. 15 per hr
- Photoperiod (hrs dark / hrs light): 12/12 controlled by a time switch

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 ml finely ground test item

Duration of treatment / exposure:

ongoing (not removed after application)

Observation period (in vivo):

As no ocular irritation was observed at the 72 h reading, the study was terminated at 72 hours

Number of animals or in vitro replicates:

3

Details on study design:

APPLICATION
- Preparation: a volume of 0.1 ml of unchanged test material (found to weigh ca. 42 mg) was ground into a fine powder using a mortar and pestle, and measured by gently compacting the required volume into an adapted syringe
- Application: placed into the right eye of each rabbit by gently pulling the lower lid away from the eyeball to form a cup into which the test item was dropped; the upper and lower lids were then held together for ca. 1 second to prevent loss of test item
- Observations: 1, 24, 48 and 72 hours after application

REMOVAL OF TEST SUBSTANCE
- Removal: no removal or washing was performed

TOOL USED TO ASSESS SCORE: light source of a standard ophthalmoscope (Keeler)

SCORING SYSTEM:
- Method of calculation: Draize, J. H. (1959) "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics", as follows:

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

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

Conjunctival redness: refers to palpebral and bulbar conjunctiva excluding cornea and iris
0 - Blood vessels normal
1 - Some blood vessels definitely hyperaemic (injected) above normal
2 – Diffuse, crimson colour, individual vessels not easily discernible
3 – Diffuse beefy red*

Chemosis: lids and/or nictitating membranes
0 – No swelling
1 – Any swelling above normal (includes nictitating membrane)
2 – Obvious swelling with partial eversion of lids
3 – Swelling with lids about half closed
4 – Swelling with lids about half closed to completely closed

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

- No corneal opacity was noted in the study period.
- Iridial inflammation was observed only in one animal and only at 1 hour after application.
- Grade 2 conjunctival redness was observed in all three animals at one hour after application, which persisted at 24 h (grade 1) and was reversible within 48 hours after application.
- Grade 1-2 chemosis was observed in all three animals at one hour after application, which persisted at 24 h in just one animal (grade 1), and was reversible within 48 hours after application.

Individual and mean scores for cornea, iris & conjunctivae.

Rabbit no.	Time after treatment (h)	Corneal opacity	Iritial inflammation	Conjunctival redness	Conjunctival chemosis
#1	1	0	0	2	2
	24	0	0	1	1
	48	0	0	0	0
	72	0	0	0	0
	total (24, 48, 72 h)	0	0	1	1
	mean (24, 48, 72 h)	0	0	0.333333	0.333333
#2	1	0	0	2	1
	24	0	0	1	0
	48	0	0	0	0
	72	0	0	0	0
	total (24, 48, 72 h)	0	0	1	0
	mean (24, 48, 72 h)	0	0	0.333333	0
#3	1	0	1	2	2
	24	0	0	1	0
	48	0	0	0	0
	72	0	0	0	0
	total (24, 48, 72 h)	0	0	1	0
	mean (24, 48, 72 h)	0	0	0.333333	0

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

The test item induced slight conjunctival redness and chemosis until 24 hours after application.

Executive summary:

The eye irritation/eye damage potential of the test item was evaluated in an experimental study according to a method based on the OECD guideline 405 (1981). 0.1 ml of finely ground test item (weighing ca. 42 mg) was placed into the right conjunctival sac of three New Zealand White rabbits using an adapted syringe (no rinsing/removal performed). The eye was then monitored for corneal opacity, iridic inflammation, conjunctival redness and chemosis (using the Draize irritation scale) at 1, 24, 48 and 72 hours after application using a standard ophthalmoscope. The left eye may be considered an untreated control.

As no signs of irritation or damage were evident at 72 hours after application, the study was terminated. No corneal opacity was noted in the study period, and iridial inflammation was observed only in one animal and only at 1 hour after application. Grade 2 conjunctival redness was observed in all three animals at one hour after application, which persisted at 24 h (grade 1) and was reversible within 48 hours after application. Grade 1 -2 chemosis was observed in all three animals at one hour after application, which persisted at 24 h in just one animal (grade 1), and was reversible within 48 hours after application. The mean (24, 48 and 72 h) irritation scores for (i) corneal opacity, (ii) iridial inflammation, (iii) conjunctival redness and (iv) chemosis were subsequently calculated to be (i) 0 in all animals, (ii) 0 in all animals, (iii) 0.33 in all animals, and (iv) 0.33 in one animal and 0 in two animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The potential skin irritation/corrosion of the test item was evaluated in three experimental studies according to the OECD guideline 404 (1981), or an equivalent method. All three studies were patch tests which monitored the mean (24, 48 and 72 h) erythema and oedema scores according to the Draize scale. The key study was chosen based on the purity of the test sample and the test procedure, e.g. guideline available and GLP.

The first study was a 4-hour exposure to intact skin only, followed by washing/removal of the substace. In the second and third study, both an abraded and an intact flank were tested and the exposure time was 24 hours, after which the dressing was removed but the substance was not removed/washed. The vehicle used in the second study was 50 % propylene glycol + saline (70:30 parts), whereas the test item was simply moistened with water in the other studies.

The first and second studies both demonstrated very slight irritation based on the mean irritation scores as well as yellow local staining, whereas the third study did not demonstrate any erythema, oedema or staining.

Signis of irritation were always reversible within 48 hours.

Eye

The eye irritation/eye damage potential of the test item was evaluated in three experimental studies according to the OECD guideline 405 (1981), or an equivalent method. In all three studies, unchanged test item (42 mg, 0.1 g or 57 mg) was applied topically to the conjunctival sac of rabbits and the mean (24, 48 and 72 h) irritation scores were recorded according to the Draize scale.

The key study was chosen based on the purity of the test sample and the test procedure, e.g. guideline available and GLP.

The eyes were not rinsed in the first or third study; three out of six animals had their treatment eye rinsed 30 seconds after application in the second study. No signs of irritation were recorded in the second study; mild irritation based on mean irritation scores was observed in the first and third study. However, all parameters were completely reversible within 48 and 72 hours, respectively.

Justification for classification or non-classification

Skin Irritation/Corrosion Criteria

According to Annex I: 3.2.2.1.1 of the CLP Regulation (EC 1272/2008) for skin corrosion and irritation, a substance is classified as a Category 1: Skin Corrosive if it produces irreversible damage to the skin; namely, visible necrosis through the epidermis and into the dermis, in at least 1 test animal following the application of a test substance for up to 4 hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, discolouration due to blanching of the skin after 14 days, complete areas of alopecia, and scars. Skin corrosives can be further divided into 3 sub-categories:

- Sub-Category 1A: corrosive responses in at least one animal following exposure ≤ 3 min during an observation period ≤ 1 h

- Sub-Category 1B: corrosive responses in at least one animal following exposure > 3 min and ≤ 1 h and observations ≤ 14 days

- Sub-Category 1C: corrosive responses in at least one animal after exposures > 1 h and ≤ 4 h and observations ≤ 14 days

A substance is classified as a Category 2: Skin Irritant if:

(i) it produces reversible damage (mean score of ≥ 2.3 and ≤ 4.0 for erythema and/or oedema at 24, 48 and 72 hours after removal) following the application of a test substance for ≤ 4 hours in at least 2 of 3 tested animals; or

(ii) it produces inflammation that persists to the end of an observation period of 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

(iii) very definite positive effects related to chemical exposure are apparent in a single animal but it does not meet the criteria above.

In available studies, the mean irritation scores for the test item do not satisfy the criteria for either Category 1: Skin Corrosive or Category 2: Skin Irritant.

Eye Irritation/Corrosion Criteria

According to Annex I: 3.3.2.1.1 and Annex I: 3.3.2.1.2 of the CLP Regulation (EC 1272/2008) for serious eye damage and eye irritation, a substance is classified as Category 1: Serious Eye Damage if it produces:

(i) effects on the cornea, iris or conjunctiva which are not reversible within 21 days in at least one animal, and/or

(ii) a positive response of corneal opacity (mean score ≥ 3) or iritis (mean score > 1.5) in at least 2 of 3 animals following grading at 24, 48 and 72 hours after application of the test material to the anterior surface of the eye.

A substance is classified as a Category 2: Eye Irritant if:

- it produces a positive response of corneal capaity (mean score 1), iritis (mean score 1), conjunctival redness (mean score 2) and/or chemosis (mean score 2) in at least 2 of 3 animals following grading at 24, 48 and 72 hours after application of the test material to the anterior surface of the eye which is fully reversed within 21 days.

In available studies, the mean irritation scores of the test item did not meet the criteria for classification in either Category 1: Serious Eye Damage or Category 2: Eye Irritant.