Reaction products of diazotized 2-amino-4-(methylsulfonyl)-phenol with 2-(3-Chlorophenyl)-2,4-dihydro-5-methyl-3H-pyrazol-3-one and Methyl-7-hydroxy-1-naphthylcarbamate – and chromium (III) 2:1, salted by ammonium, sodium and acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well-characterized material

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemieals No. 437, September 07,2009 (BCOP)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized 2-amino-4-(methylsulfonyl)-phenol with 2-(3-Chlorophenyl)-2,4-dihydro-5-methyl-3H-pyrazol-3-one and Methyl-7-hydroxy-1-naphthylcarbamate – and chromium (III) 2:1, salted by ammonium, sodium and acid

EC Number:

936-609-1

Molecular formula:

C40H32CrN4O12S2-R+] . [C36H28ClCrN7O10S2-R+] . [C34H26Cl2CrN8O8S2-R+]; R+ = NH4+, Na+ or H+

IUPAC Name:

Reaction products of diazotized 2-amino-4-(methylsulfonyl)-phenol with 2-(3-Chlorophenyl)-2,4-dihydro-5-methyl-3H-pyrazol-3-one and Methyl-7-hydroxy-1-naphthylcarbamate – and chromium (III) 2:1, salted by ammonium, sodium and acid

Details on test material:

Expiry date: 02 September 2012
purity: > 80% (see certificate of analysis)
Physical state: solid

Test animals / tissue source

Species:

other: Bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly
slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

Test system

Vehicle:

water

Controls:

yes, concurrent vehicle

Amount / concentration applied:

20% (w/v) suspension

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

none

Number of animals or in vitro replicates:

6 corneas per group

Details on study design:

Corneas free of defects (opacity, scratches, pigmentation etc.) are dissected with a 2 to 3 mm rim of sclera. Isolated corneas are mounted in specially designed cornea I holders that consists of anterior and posterior chambers. 80th chambers are filled to excess with prewarmed Eagles's MEM (without phenol red) and then equilibrated in a ver1ical position at about 32°C far at least 1 hour. After the equilibration period the medium in both chambers is replaced with fresh pre-warmed medium and initial cornea I opacity readings are taken for each cornea with an opacitometer.

Before application the medium in the anterior chamber is removed using a syringe. 0.750 mL of the 20% (w/v) test-substance preparation is applied directly to the epithelial surface of the cornea using a pipette (open chamber method). Control tissues are concurrently applied into the anterior chamber with 0.750 mL of highly deionized water (negative control, NC) or with 0.750 mL of 20% (w/v) solution of Imidazole in
highly de-ionized water (positive control, PC) using a pipette. The corneas will be incubated in a horizontal position at about 32°C for approximately 4 hours. The test substance, NC and PC is then be removed from the anterior chamber using a syringe and the epithelium is washed at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red). Both chambers are then refilled with fresh Eagle's MEM (without phenol red). Before measurement, each cornea is observed visually and observations are recorded.
Final corneal opacity readings are taken for each cornea with an opacitometer.

For determination of permeability the medium in the anterior chamber is replaced by 1 mL sodium fluorescein solution (5 mg/mL) and incubated for 90 ± 5 min in a horizontal position at about 32 °C.
The amount of sodium fluorescein that permeates through the corneas into the posterior chamber is measured spectrophotometrically. Three aliquots per cornea are transferred 10 a 96-well microtiter plale and the optical density (OD 490 nm) is determined.

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS

Basis:

mean

Time point:

other: 4h

Score:

33

Reversibility:

other: not relevant for BCOP assay

Remarks on result:

other: not "highly irritating". Two of six corneas were not scorable because of strong staining by the test item.

Applicant's summary and conclusion

Interpretation of results:

other: not higly irritating

Remarks:

Criteria used for interpretation of results: EU