Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

EC Number:

812-037-7

Cas Number:

1793011-72-9

Molecular formula:

Unknown for all components

IUPAC Name:

Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes were collected at the slaughterhouse (Vitelco, Den Bosch, The Netherlands).
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in a cooler with physiological saline and the cornea were isolated within 1 hour after arrival.
- Time interval prior to initiating testing: Eyes were used as soon as possible after slaughter.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: Not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 315 to 360.4 mg per cornea.

VEHICLE
- Amount(s) applied (volume or weight with unit): Physiological saline used as a negative control.

Duration of treatment / exposure:

Incubated for 240 +/- 10 minutes at 32°C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minute incubation period with sodium fluorescein.

Number of animals or in vitro replicates:

3 corneas per group (test, negative and positive controls)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

NUMBER OF REPLICATES :
Three replicates per group

NEGATIVE CONTROL USED:
Physiological saline was used as the negative control.

POSITIVE CONTROL USED :
20% (w/v) Imidazole [CAS Number 288-32-4] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME : Various weights of the substance as such were added to the corneas and incubated for 4 hours.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: Yes: 90 minute incubation period with sodium fluorescein.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Not specified, although not all test substance could be removed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. A volume of 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as per the test guideline used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

>= 63 - <= 147

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
The test substance induced the opacity and increased the permeability. This resulted in a mean in vitro irritancy (IVIS) score of 99 after 240 minutes of treatment. However, since the test substance is a black powder and could not be completely removed after rinsing this likely increased the opacity values.

Although the substance induced an IVIS ≥ 55, it cannot be concluded that it induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report. Since the black colored test item forms spots on the cornea which cannot be complete removed, the outcome of the present study is inconclusive.

As the test substance could not be adequately removed from the corneas, it is likely that the inconclusive nature of this test would be replicated if other validated in vitro techniques were used based on a similar testing method (i.e. application of the test substance directly to animal corneas).

DEMONSTRATION OF TECHNICAL PROFICIENCY: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 130 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Although the test substance induced an IVIS score >/= 55, it cannot be concluded that the substance induces serious eye damage. Since the black coloured test item forms spots on the cornea which cannot be completely removed, the outcome of the present study is inconclusive.