Butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo-, tetradecyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 September 2003 to 3 October 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

-

EC Number:

412-600-3

EC Name:

-

Cas Number:

152827-98-0

Molecular formula:

C32H48ClN5O3

IUPAC Name:

tetradecyl 3-[(4-{6-tert-butyl-7-chloro-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl}phenyl)carbamoyl]propanoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo-, tetradecyl ester
- HAEL Number: 2003-0090
- KAN: 632021-1
- CIN: 10084241
- SRID or Lot No.: DEV84241
- Physical state and appearance: brown powder
- Storage conditions of test material: at room temperature
- Purity determination: 96.5 weight percent
- Stability: stable

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: The rabbits were young adults (at least three months old).
- Weight at study initiation: Not reported
- Housing: Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The rabbits were singly housed in suspended, stainless-steel mesh cages. Cages and racks were washed once a week. Absorbent paper, used to collect excreta, was changed every other day.
- Diet: Certified High Fiber Rabbit Diet was available ad libitum.
- Water: Water was available ad libitum through an automatic watering system. The source of the water was the local public water system.
- Acclimation period: The animals were isolated upon arrival and allowed to acclimate for a period of 5 days. Animals were judged to be healthy prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.3 to 23.0 °C
- Humidity: 46.3 to 56.6 %
- Photoperiod: A photoperiod of 12 hours of light was maintained.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: No control substance was used. The untreated eye (left) of each animal served as a control for the test.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 grams

Duration of treatment / exposure:

N/A

Observation period (in vivo):

Eyes were observed immediately after instillation of the test substance and 1, 24, 48, and 72 hours thereafter.

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eyes of three animals were immediately washed with running distilled water; the eyes of the other three animals were not irrigated.
- Time after start of exposure: Immediately

SCORING SYSTEM
- The eyes were examined and grades of ocular reactions recorded for each animal at all observation periods. Ocular lesions were graded and scored as described below:

Grading Of Ocular Lesions (as described in OECD 405):

Cornea:-
0 = No ulceration or opacity
1 = Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible
2 = Easily discernible translucent area, details of iris slightly obscured
3 = Nacreous area, no details of iris visible, size of pupil barely discernible
4 = Opaque cornea, iris not discernible through the opacity

Area of Corneal Opacity:-
1 = One-quarter or less, but not zero
2 = Greater than one-quarter up to one-half
3 = Greater than one-half up to three-quarters
4 = Greater than three quarters up to the whole area

Iris:-
0 = Normal
1 = Markedly deepened folds, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combinations of any thereof, iris still reacting to light(sluggish reaction is positive)
2 = No reaction to light, hemorrhage, gross destruction (any or all of these)

Conjunctivae:-
Redness: refers to palpebral and bulbar conjunctivae excluding cornea and iris
0 = Blood vessels normal
1 = Some blood vessels definitely injected
2 = Diffuse, crimson red, individual vessels not easily discernible
3 = Diffuse beefy red

Chemosis: lids and/or nictitating membranes
0 = No swelling
1 = Any swelling above normal (including nictitating membranes)
2 = Obvious swelling with partial eversion of lids
3 = Swelling with lids about half closed
4 = Swelling with lids more than half closed

TOOL USED TO ASSESS SCORE
- Observations included indications of immediate sensory irritation and estimations of chemosis and redness of the adnexal structures. Also evaluated were effects on the iris, the presence of corneal opacity, andor discharge from the eye. Eyes were treated with a 2 % ophthalmic solution of fluorescein at 24 hours and observed for staining.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects seen

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects seen

Results 3

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

other: no effects seen

Results 4

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects seen

Irritant / corrosive response data:

In the in vivo eye irritation study, signs of irritation consisted of redness (grade 1) for all unwashed eyes one hour after administration of the test substance. At the 24-hour examinations, all three unwashed eyes appeared clinically normal. Immediate irrigation of the eyes was slightly palliative. Signs of irritation for the three washed eyes were limited to redness (grade 1) for two of three washed eyes at the one-hour examinations. At the 24-hour examination, all three washed eyes appeared clinically normal. Staining was not evident in unwashed or washed eyes when tested with fluorescein dye.

Other effects:

No other lesions were noted for the unwashed or washed eyes during the 72-hour observation period. Staining was not evident for unwashed or washed eyes when tested with fluorescein dye 24 hours after administration of the test substance.
No toxic effects were noted during the study.

Any other information on results incl. tables

Observations for Irritation

The treated eye of each animal was examined for signs of irritation at 1, 24, 48, and 72 hours after instillation of the test substance. Observations of corneal opacity, iris effects, redness, and chemosis are presented in Table 1.

Table 1: Ocular Irritation

Animal Number	Irrigated	Corneal Opacity, Iris effects, Redness and Chemosis*
		1 hour	24 hours	48 hours	72 hours
751	No	0,0,1,0	0,0,0,0	0,0,0,0	0,0,0,0
752	No	0,0,1,0	0,0,0,0	0,0,0,0	0,0,0,0
753	No	0,0,1,0	0,0,0,0	0,0,0,0	0,0,0,0
754	Yes	0,0,1,0	0,0,0,0	0,0,0,0	0,0,0,0
755	Yes	0,0,0,0	0,0,0,0	0,0,0,0	0,0,0,0
756	Yes	0,0,1,0	0,0,0,0	0,0,0,0	0,0,0,0

*Graded as described in OECD GuideIine 405 (Annex V., Test B.5) (Grading for Ocular Lesions)

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of the test, the substance was determined to be a non-irritant to the eye.

Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 405 and EU Method B.5, the eye irritation of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was investigated in a total of six rabbits. A single dose of 0.1 gram of the test substance was administered into the conjunctival sac of one eye of each animal. Immediately after administration, the treated eyes of three rabbits were washed with distilled water. The remaining eyes were not irrigated. Ocular lesions were graded according to OECD Guideline 405 (Annex V., Test B.5) (Grades for Ocular Lesions).

For the unwashed eyes, signs of irritation consisted of redness (grade 1) for all unwashed eyes one hour after administration of the test substance. At the 24-hour examinations, all three unwashed eyes appeared clinically normal. Staining was not evident in unwashed eyes when fluorescein dye was applied. Immediate irrigation of the eyes was slightly palliative. Signs of irritation for the three washed eyes were limited to redness (grade 1) for two of three washed eyes at the one-hour examinations. At the 24-hour examination, all three washed eyes appeared clinically normal. Staining was not evident in washed eyes when tested with fluorescein dye. Based on these results, the test substance is not classified for eye irritation in line with current EU requirements.