Sodium 3-[{4-[{5-cyano-2,6-bis[(3-methoxyalkyl)amino]alkylpyridin-3-yl}diazenyl]dialkylphenyl}diazenyl]benzenesulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February 2015 to 10 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Compliant to GLP and testing guidelines; coherence between data, results and conclusion.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assesment no. 160

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Use of an alternate testing framework for classification of eye irritation potential of EPA pesticide products - Office of Pesticide Programs, U.S. Environmental Protection Agency (31 May 2013).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test item: Acid Red EAY 9656

Test animals / tissue source

Species:

cattle

Strain:

not specified

Remarks:

bovine cornea

Details on test animals or tissues and environmental conditions:

Test system: isolated corneas from the eyes of freshly slaughtered bovine.
Age of animals: 6-12 months

Transport solution: Hanks balanced salt solution (HBSS) Modified supplemented with Penicillin sulphate and Streptomycin sulphate.
Transport conditions: in transport solution at approximately 4°C.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

The test item was spread on each epithelial surface of three idoneous bovine corneas as supplied (150 mg/cornea); 0.75 mL of physiological saline was then added on each cornea in order to better distribute the substance on the cornea surface (final concentration on cornea = 200 mg/mL, approximately 20% w/v)
Number of replicates: 3

Duration of treatment / exposure:

Corneas were exposed in horizontal position for 4 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.
There was no post-exposure period.

Details on study design:

Positive control item: corneas treated 20% (w/v) Imidazole (Sigma, batch no. 0001422794) in physiological saline (0.9% NaCl) (Baxter, batch no. 13D0406).
Negative control item: corneas treated with physiological saline (0.9% NaCl) (Baxter, batch no. 13D0406).
Volume of treatment: 0.75 mL
Number of replicates: 3

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

mean score of three corneae

Value:

22.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean permeability OD490 score

Value:

0.186

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

IVIS = mean opacity score + (15 x mean permeability OD490 score)

Value:

25.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean opacity detected with an opacitometer at the end of the test item exposure period was 22.7. This was confirmed at the macroscopic observation in which slight opacity of the treated corneas was noted. Plentiful residual test item, adhering on the surface of all treated corneas, was observed. Slight increases in cornea permeability were observed after treatment with the test item, with a mean permeability OD490 value of 0.1863.

The following results were obtained in the negative control:
– mean opacity value of the negative control = 0.00
– mean permeability value of the negative control = 0.0077

The following results were obtained in the positive control:
– IVISpositive control = 72.4

Based on the evaluation criteria the experiment was accepted as valid.

Any other information on results incl. tables

The following results were obtained:

– mean opacity value of the negative control = 0.00

– mean permeability value of the negative control = 0.0077

– IVISpositive control = 72.4

Based on the stated criteria (§ 4.6.2) the experiment was accepted as valid.

The calculated in vitro irritancy score for the test item was:

– IVIStest item = 25.5

indicating that the test item cannot be clearly classified.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

The test item Acid Red EAY 9656 cannot be clearly classified.

Executive summary:

The potential of the test item, Acid Red EAY 9656, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 26 July 2013), the Guidance Document OECD series on testing and assessment no. 160 and the method described in the document issued by U.S. EPA/OPP (31 May 2013). The test item was spread on each epithelial surface of three idoneous bovine corneas as supplied (150 mg/cornea); 0.75 mL of physiological saline was then added on each cornea in order to better distribute the substance on the cornea surface (final concentration on cornea = 200 mg/mL, approximately 20% w/v); an exposure period of 4 hours ± 10 minutes was used. The mean opacity detected with an opacitometer at the end of the test item exposure period was 22.7. This was confirmed at the macroscopic observation in which slight opacity of the treated corneas was noted. Plentiful residual test item, adhering on the surface of all treated corneas, was observed. After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 0.1863. The calculated in vitro irritancy score (IVIS) for the test item was 25.5. Positive and negative controls [a 20 % (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar conditions and gave the expected results. According to the OECD Guideline no. 437, the test item can not be clearly classified.