Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-21.04.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate

EC Number:

274-418-9

EC Name:

Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate

Cas Number:

70210-21-8

Molecular formula:

C26H20ClN7O10S3.3Na

IUPAC Name:

trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reactive Orange 5- Physical state: solid, powder- Analytical purity: 90% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Lot/batch No.: 8001- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

TEST MATERIAL: Bovine eyes- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech RepublicEyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. Only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: 750 µl of suspension- The test substance was tested as suspension prepared from test substance at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution. Positive control substance: Name: ImidazoleLot no: WXBC1234VExpiration: 01/2024Supplier: Sigma-AldrichNegative control substance: Name: 0.9% NaClLot: 15IM422A1 Expiration: 11/2018Supplier: B. Braun Melsungen AG, Germany

Duration of treatment / exposure:

4h

Duration of post- treatment incubation (in vitro):

1,5 h

Number of animals or in vitro replicates:

Exposed group (test substance) - 3 corneas (No. 9, 10, 12) Positive control group (20% Imidazole) – 3 corneas (No. 13, 14, 15) Negative control group (0.9% NaCl) – 3 corneas (No. 6, 8, 11)

Details on study design:

TEST PROCEDURE- Selection of corneas, mounting in holders- incubation with EMEM 1 hour (32 ± 1°C)- removed EMEM, measurement of baseline opacity- treatment by positive and negative control substances and test substance (incubation 4 hour)- washing epithelium, measurement of opacity after application -application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) - measurement of absorbance (490 nm).Endpoints measured:Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale. Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values.EVALUATION OF RESULTSMean opacity:Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.Mean permeability:Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean opacity is calculated. IVIS calculation:Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:IVIS = mean opacity value + (15 x mean permeability OD490 value)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

28.17

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: no predicton can be made

Conclusions:

The In Vitro Irritancy Score (IVIS) for Reactive Orange 5 was 28.17 but this result could be affected by higher opacity values (colouring of corneas after removing the test substance). On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed.No prediction can be made on test substance potential to cause eye irritation or serious eye damage.

Executive summary:

The test substance,Reactive Orange 5,was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 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, Adopted 26^thJuly 2013

 

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured.The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

 

The In Vitro Irritancy Score (IVIS) for Reactive Orange 5was 28.17 but this result could be affected by higher opacity values (colouring of corneas after removing the test substance).

On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed.

No prediction can be made on test substance potential to cause eye irritation or serious eye damage.