Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 January 2017 - 03 January 2017

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)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

EC Number:

227-497-9

EC Name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Cas Number:

5858-81-1

Molecular formula:

C18H12N2Na2O6S

IUPAC Name:

disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Test material form:

solid: particulate/powder

Details on test material:

Name as cited in the report: D&C Red 6
Appearance: red powder
Storage conditions: at room temperature

Specific details on test material used for the study:

Correction for purity: no
The test item was tested for solubility in physiological saline. Since no workable suspension of the test item in physiological saline could be obtained, it was used as delivered by the sponsor and added pure on top of the corneas.

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 335.6 to 349.6 mg (complete coverage of the cornea)

NEGATIVE CONTROL:
- Amount applied: 750 µL

POSITIVE CONTROL:
- Amount applied: 750 µL

Duration of treatment / exposure:

240 +/- 10 minutes

Duration of post- treatment incubation (in vitro):

90 +/- 5 minutes in sodium fluroescein

Number of animals or in vitro replicates:

3

Details on study design:

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed and applied directly on the corneas in such a way that the cornea was completely covered (317.8 to 354.9 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red.
- POST-EXPOSURE INCUBATION: 90 ±5 minutes 32 ± 1 °C in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

ACCEPTABILITY CRITERIA:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Single run/mean of 3 replicates

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS: 1.5 - 3.4

Positive controls validity:

valid

Remarks:

IVIS: 111.9 - 180.9

Other effects / acceptance of results:

The corneas treated with D&C Red 6 showed opacity values of -0.4, 0.3 and 0.7 and OD values of 0.010, 0.110 and 0.101 (corrected for negative control). IVIS scores were 0.9, 1.3 and 1.8 (n=3). The corneas were clear after the 240 minutes of treatment.

OTHER EFFECTS:
- The corneas treated with the positive control item were turbid after the 240 minutes of treatment.
- No pH effect of the test item was observed on the rinsing medium.
- The IVIS of one of the negative controls was >3. However, since the opacity, permeability and IVIS of this cornea were within the historical control data range and the test item gives a clear negative result this does not affect the study result.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (e.g. 1.5-3.4).
- Acceptance criteria met for positive control: yes, results were within historical range (e.g. 111.9-180.9).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, it is concluded that D&C Red 6 does not need to be classified under GHS (IVIS ranging from 0.9 to 1.8 after 240 minutes of treatment).

Executive summary:

The eye hazard potential of D&C Red 6 was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test). Three corneas from young cattle were exposed to 335.6 to 349.6 mg (complete coverage) of the test item, next to a negative control group of physiological saline (n=3) and a positive control of 20% (w/v) Imidazole (n=3). Duration of treatment was approximately 240 minutes. Results of the negative and positive controls were within the historical data. Therefore, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. D&C Red 6 did not induce ocular irritation, resulting in a mean in vitro irritancy score of 1.3 after 240 minutes of treatment. Based on this results (IVIS < 3), no classification for eye irritation or serious eye damage is required for D&C Red 6 according to GHS criteria.