N,2-dimethyl-N-phenylbutyramide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-04-18 to 1988-05-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Modified United States Federal Hazardous Substances Labelling Act Method (1973)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Home Office Guidelines on Eye Irritation/Corrosion Tests ('Draize' Eye Test) - February 1987.

Principles of method if other than guideline:

Modifications to standard test method:
1. Maximum of three animals per test group.
2. Treat one animal of Study Group A on Day 1.
3. Treat further animals of Study Groups A, B, C on Day 1 or Day 2 as indicated by the reactions of animals previously treated, with pretreatment of eyes with corneal anaesthetic as considered necessary.

In view of the degree of irritation produced by 0.01 mL of the material in this test, further testing at a dosage of 0.1 mL was precluded.

GLP compliance:

no

Remarks:

The study pre-dates the introduction of GLP

Test material

Test material information

Constituent 1

Reference substance name:

N,2-dimethyl-N-phenylbutyramide

EC Number:

282-817-4

EC Name:

N,2-dimethyl-N-phenylbutyramide

Cas Number:

84434-18-4

Molecular formula:

C12H17NO

IUPAC Name:

N,2-dimethyl-N-phenylbutanamide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Unknown

IUPAC Name:

Unknown impurities

Test material form:

liquid

Specific details on test material used for the study:

Test substance: Gardamide

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

New Zealand white rabbits, bought from a commercial supplier, are used. Each treatment group normally comprises of six rabbits at least 8 weeks old, weighing at least 1kg and having no spontaneous eye lesions. Fewer rabbits may be used to test a substance which may be an irritant to the eye, or in preliminary studies. In all cases the number of eye used is specified on the Protocol Summary.
The animals are housed individually in cages with wire mesh floors and have access to a certified pelleted commercial rabbit diet and tap water as libitum. Prior to treatment the corneal thickness of the eye to be treated is measured with a slit lamp as described by Burton (1972).

Test system

Vehicle:

other: unchanged (no vehicle) and Tween 80

Controls:

no

Amount / concentration applied:

Dose: 10μL
Concentrations: 10%, 50% and 100% (undiluted)
PREPARATION OF TEST SOLUTIONS
- 10 % solution: 1 g of test material was made up to 10 g with Tween 80.
- 50 % solution: 5 g of test material was made up to 10 g with Tween 80.
- 100 % solution: the test material was used undiluted.

Duration of treatment / exposure:

Duration of exposure not specified. Study report does not suggest that there was a rinsing step at any point.

Observation period (in vivo):

Animals are carefully observed during and immediately after treatment for evidence of discomfort or pain. If this is excessive or persistant the substance is immediately washed out with copious quantities of physiogical saline.
15 days. Observations are continued at intervals up to a maximum of 22 days after treatment or, if sooner, until the eyes are considered to be of normal appearance.

Number of animals or in vitro replicates:

Three - one animal was treated with each solution.

Details on study design:

The animal dosed with the neat test material was pre-treated with corneal anaesthetic.

REMOVAL OF TEST SUBSTANCE
Animals are observed carefully during and immediately after treatment for evidence of discomfort or pain. If this is excessive or persistent the substance is immediately washed out with copious quantities of physiological saline; in extreme cases the animal may be killed.

TOOL USED TO ASSESS SCORE: hand-slit lamp or torch. Prior to treatment the corneal thickness of the eye to be treated is measured with a slit lamp as described by Burton (1972).

SCORING SYSTEM: Animals showing no unusual responses to treatment are observed for macroscopic lesions and for corneal swelling as follows:

> Macroscopic Observations
The eyes are examined with the aid of a hand-held torch and/or a slit lamp. Conjunctival responses are assessed 15 minutes after treatment. Corneal conjunctival and iridial responses are assessed up to 15 days after treatment. The assessment is based on the following grades:

CORNEA
0 = Normal
½ = Any change from normal, i.e. slight dulling of corneal lustre
1 = Scattered/diffuse areas of opacity or loss of corneal epithelium, iris clearly visible
2 = Easily discernible greyish translucent areas, details of iris slightly obscured
3 = Grey/white areas, no details of iris visible, size of pupil barely discernible
4 = Complete corneal opacity, iris not discernible

IRIS
0 = Normal
1 = Any divergence from normal, i.e. congestion of iridial vessels, deepening of folds
2 = Severe congestion of iridial vessels or deepening of folds, loss of iridial reflex to light

AREA OF CORNEA OR IRIS AFFECTED
1 = Less than 1/4 area affected
2 = Between 1/4 and 1/2 area affected
3 = Between 1/2 and 3/4 area affected
4 = Between 3/4 and whole area affected

CONJUNCTIVAE
- Redness (palpebral and bulbar conjunctivae and nictitating membrane)
0 = Vessels normal
1 = Some vessels definitely injected
2 = Diffuse, crimson red, individual vessels not easily discernable
3 = Diffuse, beefy red
- Chemosis
0 = No swelling
1 = Any swelling above normal
2 = Obvious swelling with partial eversion of eyelid
3 = Swelling with lid about half closed
4 = Swelling with lid more than half closed
- Discharge
0 = No discharge
1 = Any discharge different from normal
2 = Discharge, with moistening of lid and hairs adjacent to lid
3 = Discharge, with moistening of lid and considerable area around eye

PANNUS
- Severity
0 = No vessels growing into the cornea
1 = Vessels just onto the cornea
2 = Vessels quite clearly onto the cornea but extending less than half-way across the iris
3 = Vessels more than half-way across the iris
4 = Vessels extending across to the pupil
- Extent
1 = Four vessels or fewer
2 = Band of vessels extending up to 1/4 of corneal circumference
3 = Band of vessels extending up to 1/2 of corneal circumference
4 = Band of vessels extending beyond 1/2 of corneal circumference

CONTENT OF BLOOD VESSELS
1 = All vessels empty
2 = More than half (but not all) vessels empty
3 = More than half (but not all) vessels contain blood
4 = All vessels contain blood

CORNEAL SWELLING
Corneal thickness is measured with a slit lamp 24 hours after treatment and thereafter at daily intervals until the thickness has returned to its pre-treatment value. The percentage corneal swelling is calculated from:
Corneal swelling = [(corneal thickness on day of measurement / corneal thickness before treatment) - 1] x 100

Results and discussion

In vivo

Results

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Group A: 10%
The eye showed slight conjunctival erythema and oedema 15 minutes after treatment. The cornea was unaffected by treatment, The eye appeared normal 24 hours after treatment.

Group B: 50%
The animal showed signs of discomfort on treatment. The eye subsequently showed loss of epithelium from approximately half the area of the cornea, together with moderate corneal swelling. Slight conjunctival erythema was evident. The eye had healed to mormality by Day 10.

Group C: 100%
The rabbit was pretreated with corneal anaesthetic, and subsequently showed no sign of discomfort on treatment with test material. The eye subsequently showed total loss of corneal epithelium, together with severe corneal swelling and iritis. Conjunctivitis was slight. Healing was complete at Day 15.

Any other information on results incl. tables

Table 1: Summary of Results

Dosing Solution (%)	Number of rabbits with stated ocular lesion
	Positive conjunctival reaction	Iritis	Pannus	Maximum corneal opacity					Duration of opacity	Maximum corneal swelling (%)
				0.5	1	2	3	4	>1 on Day 8	Nil (0 - 10)	Slight (11 - 45)	Moderate (46 - 80)	Severe (81)
10	0	0	0	0	0	0	0	0	0	1	0	0	0
50	0	0	0	0	1	0	0	0	0	0	0	1	0
100	0	1	0	0	1	0	0	0	1	0	0	0	1

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of this study, administration of undiluted test material produced moderate/severe eye irritation.

Executive summary:

A test was conducted to investigate the irritant effects of the test material after a single application to the eye of new Zealand White rabbits. The method was based upon that prescribed for the purposes of the United States Federal Hazardous Substances Labelling Act (1973) and meets OECD Guideline 405 (1987). The study also meets Home Office Guidelines on Eye Irritation/Corrosion Tests ('Draize' Eye Test, 1987).

The test material was administered as a 10, 50 and 100 % solution to the eye of a single rabbit at each dose level. The 10 and 50 % solutions used Tween 80 as the vehicle.

The test substance was applied to the eyes by gently pulling the lover eyelid away from the eyeball and placing 0.01 mL in the sac so formed. Eyes were examined 24 hours after treatment and thereafter at specified daily intervals and graded for corneal, conjunctival and iridial damage. Corneal thickness was also measured and corneal swelling was recorded if present.

- Treatment with 10 % solution: The rabbit showed no sign of discomfort. The eye subsequently showed only slight, transient effects 15 minutes after treatment (conjunctival erythema and oedema). The cornea was unaffected. The eye appeared normal 24 hours after treatment.

- Treatment with 50 % solution: The rabbit showed signs of discomfort on treatment. The eye subsequently showed the loss of an extensive area of corneal epithelium (approximately half the area of the cornea), together with moderate corneal swelling. Slight conjunctival erythema was evident. The eye had healed to normality by day 10.

- Treatment with neat test material: The animal showed no sign of discomfort on test material administration, having been pre-treated with a corneal anaesthetic. The eye subsequently showed total loss of corneal epithelium with severe corneal swelling and iritis. Conjunctivitis was slight. The eye had healed to normality by day 15.

Under the conditions of this study, administration of undiluted test material produced moderate/severe eye irritation.