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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.-02.06.2016
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

Constituent 1
Chemical structure
Reference substance name:
Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate
EC Number:
277-616-3
EC Name:
Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate
Cas Number:
73816-74-7
Molecular formula:
C28H24ClN7O10S3.3Na
IUPAC Name:
trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate
impurity 1
Chemical structure
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 Red 45:1
- Physical state: red solid powder
- Analytical purity: 85% (w/w)
- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 15% (w/w)
- Lot/batch No.: 7010
- 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
Republic
Eyes 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.

Duration of treatment / exposure:
4 h
Duration of post- treatment incubation (in vitro):
1.5h
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 quanti
tatively 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 epit
helium on the outer cornea surface through the endothelium on the inner cornea surface) measured in
directly 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 RESULTS
Mean 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 empiricallyderived
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

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
28.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
31.51
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Optical dnesity value
Value:
0.212
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made on test substance potential to cause eye irritation or serious eye damage.
Conclusions:
The In Vitro Irritancy Score (IVIS) for Reactive Red 45:1 was 31.51. 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 Red 45:1,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 26thJuly 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 Red 45:1 was 31.51. 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.

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