2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 JUN 2012 to 15 JUN 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 405) and according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

EC Number:

229-419-9

EC Name:

2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Cas Number:

6528-34-3

Molecular formula:

C18H18N4O6

IUPAC Name:

2-[(4-methoxy-2-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxobutanamide

Test material form:

solid: bulk

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.31 to 2.75 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930 Teklad Global Certified Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

A volume of 0.1 ml of the test item, which was found to weigh approximately 35 mg

Duration of treatment / exposure:

single application / no washing

Observation period (in vivo):

Examinations were done approximately 1 hour and 24, 48 and 72 hours following treatment

Number of animals or in vitro replicates:

3 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. )

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A volume of 0.1 ml of the test item, which was found to weigh approximately 35 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made.
After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. In order to minimise pain on application of the test item, one drop of local anaesthetic (Tetracaine hydrochloride 0.5%, Bausch & Lomb, Surrey, UK) was instilled into both eyes of these animals 1 to 2 minutes before treatment. Due to a technician error the initial pain reaction was not recorded for the third animal. This deviation was considered not to affect the purpose or integrity of the study.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Results 3

Irritation parameter:

other: redness

Basis:

animal: 72094 Male

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 4

Irritation parameter:

other: redness

Basis:

animal: 72123 Male

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Results 5

Irritation parameter:

other: redness

Basis:

animal: 72125 Male

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 6

Irritation parameter:

chemosis score

Basis:

animal: 72094 and 72123 Male

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 7

Irritation parameter:

chemosis score

Basis:

animal: 72123 Male

Time point:

other: Mean score at 24, 48 and 72 hours

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Ocular Reactions
Individual and mean scores for eye irritation are given in Table 1 and Table 2.
Orange coloured staining of the fur was noted around all treated eyes throughout the study.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in two treated eyes at the 24 Hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 48 Hour observation.
One treated eye appeared normal at the 48 Hour observation and two treated eyes appeared normal at the 72 Hour observation.
All signs of irritation were fully reversible within 72 Hours.
No corrosive effects were noted during the study.

Other effects:

Clinical Observations
No clinical signs of toxicity were noted during the study.

Bodyweight
Individual bodyweights and bodyweight changes are given in Table 2.
All animals showed expected gains in bodyweight during the study.

Any other information on results incl. tables

Table 2              Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
72094Male	2.75	2.81	0.06
72123 Male	2.41	2.47	0.06
72125 Male	2.31	2.35	0.04

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

According to the classification criteria of Regulation (EC) No 1272/2008 the test material is not irritating to eyes under these test conditions.

Executive summary:

Introduction

The primary eye irritation potential of the test material was investigated according to a method compatible with OECD test guideline no. 405 and Method B5.

Method

The test item was applied by instillation of 0.1 ml into the right eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation. 

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iritis, redness and chemosis of the conjunctivae.

Results

The individual mean scores for corneal opacity and iritis were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.67, 0.33 and 1.00 for reddening and 0.00, 0.00 and 0.33 for chemosis.

The instillation of test substance into the eye resulted in minimal to moderate irritation, early‑onset and transient ocular changes, such as iridial inflammation and reddening of the conjunctivae, ocular discharge and chemosis. These effects were reversible and were no longer evident 72 hours after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea of any animals at any of the examinations. No corrosion was observed at any of the measuring intervals. No clinical signs of toxicity were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Conclusion

Based upon the referred classification criteria (Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008), the test item does not have to be classified with respect to eye irritation.