4-[4-(diethylamino)benzylidene]-5-ethoxy-2-phenyl-2,4-dihydro-3H-pyrazol-3-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2003 to 25 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Identification: HSY-2701
Description: Red powder
Batch: 03A-20
Purity: > 99.9 %
Test substance storage: At room temperature protected from light
Stability under storage conditions: Stable
Expiry date: 07 August 2003 (6 months from the date of receipt).

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: At least six weeks of age
- Weight at study initiation: At least 1 kg
- Housing: Individually labelled cages with perforated floors (Scanbur, Denmark, Dimension 56 x 44 x 37.5 cm).
- Diet (e.g. ad libitum):Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany), approximately 100 g per day.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 24.9 mg (23.8-26.6 mg) of the test substance at a volume of approximately 0.1 mL.

Duration of treatment / exposure:

Indefinite, no washing was performed

Observation period (in vivo):

Reported up to 72 hours as all animals had recovered by 48 hours.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
No washing reported

SCORING SYSTEM:
Immediately after 24 hours observation, a solution of 2 % fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quatitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage was estimated as a percentage of the total corneal area.

TOOL USED TO ASSESS SCORE: fluorescein

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 2

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 6

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 7

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 8

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 9

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 10

Irritation parameter:

conjunctivae score

Remarks:

Mean redness score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Results 11

Irritation parameter:

conjunctivae score

Remarks:

Mean redness score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Results 12

Irritation parameter:

conjunctivae score

Remarks:

Mean redness score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

The results were evaluated according to the EC criteria for classification and labelling of dangerous substance and preparations (Council directive 67/548/EEC and all adaptations to technical progress and ammendments of this Directive published in the Official Journal of the European Communities).
Instillation of approximately 25 mg of the test substance (approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which was seen as redness, chemosis and discharge. The irritation had completely resolved within 48 hours in all animals. OVer the three observation times used to calculate mean scores for EC classification purposes (24, 48, 72 hours) only grade 1 redness at 24 hours was observed.
No irridial irritation or corneal opacity was observed, and treatment of the eyes with 2 % fluorescein, 24 hours after test susbtance instillation revealed no corneal epithelial damage in any of the animals.
There was no evidence of of ocular corrosion.

Other effects:

Remnants of the test substance were present in the eyes of all animals on day 1. Yellow/orange staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Any other information on results incl. tables

Table 1: Individual eye irritation scores

Time after dosing	Cornea			Iris	Conjunctivae			Comments
	Opacity	Area	Fluor area (%)		Redness	Chemosis	Discharge
Animal No. 221
1 hour	0	0	0	0	2	0	1	e,b
24 hours	0	0		0	1	0	0	e
48 hours	0	0		0	0	0	0	e
72 hours	0	0		0	0	0	0	e
Animal No. 258
1 hour	0	0	0	0	1	1	1	e,b
24 hours	0	0		0	1	0	0	e
48 hours	0	0		0	0	0	0	e
72 hours	0	0		0	0	0	0	e
Animal No. 269
1 hour	0	0	0	0	2	0	1	e,b
24 hours	0	0		0	1	0	0	e
48 hours	0	0		0	0	0	0	e
72 hours	0	0		0	0	0	0	e
Fluor area (%): green staining (percentage of total corneal area) after fluorescein treatment b: Remnants of the test substance in the eye e: Yellow/orange staining of the head and paws by the test substance.

Table 2: Mean value irritation scores

Animal #	Mean 24-72 hours
	Corneal opacity	Iris	Conjuntivae
			Redness	Chemosis
221	0	0	0.3	0
258	0	0	0.3	0
269	0	0	0.3	0

Table 3: Animal specifications

Animal No.	Sex	Age at start (weeks)	Body weights (g)
			Prior to application	At termination
221	Male	7-9	2313	2441
258	Male	8-10	1991	2055
269	Male	8-10	1901	2059

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results, HSY-2701 is not considered an irritant to the rabbit eye.

Executive summary:

In a GLP study conducted in accordance with the OECD guideline 405, HSY-2701 was administered into the eye of three New Zealand white rabbits and observed for signs of irritation. Mild irritation of the conjunctivae occured shortly after instillation, however this had fully recovered by 48 hours. HSY-2701 was considered not irritating.