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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:
01.06. – 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)
Version / remarks:
Adopted 26th July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
EC Number:
304-149-5
EC Name:
Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
Cas Number:
94246-75-0
Molecular formula:
C23H11ClCuN7O11S3.3K
IUPAC Name:
tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
impurity 1
Chemical structure
Reference substance name:
Potassium chloride
EC Number:
231-211-8
EC Name:
Potassium chloride
Cas Number:
7447-40-7
Molecular formula:
ClK
IUPAC Name:
potassium chloride
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue 234 - Other name: Ostazin Blue H-5R- Lot/batch No.: 8002- Expiration date of the lot/batch: unlisted

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic- Number of animals:- Characteristics of donor animals (e.g. age, sex, weight): 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. - Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):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).- Time interval prior to initiating testing: The time interval between collection of the eyes and use of corneas in the BCOP was minimized, so the collected eyes were processed on the same day.All eyes used in the assay were from the same group of eyes collected on a specific day.- indication of any existing defects or lesions in ocular tissue samples:Only corneas from eyes free of defects including scratched, and neovascularisation were used.The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL - Amount(s) applied (volume or weight with unit): 750 μl of suspension2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.
Duration of treatment / exposure:
4 hrs
Duration of post- treatment incubation (in vitro):
1.5 hr
Number of animals or in vitro replicates:
The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.Number of corneas per group:Exposed group (test substance) - 3 corneas (No. 1, 4, 6) Positive control group (20% Imidazole) – 3 corneas (No. 12, 13, 14) Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 9)
Details on study design:
SELECTION AND PREPARATION OF CORNEASSelection criteria for eyes used in BCOP:Only corneas from eyes free of defects including scratched, and neovascularisation were used.The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Preparation: Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups. QUALITY CHECK OF THE ISOLATED CORNEASFrom 22 eyes the 3 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 6, 9, 12, 13 and 14), 7 eyes was superfluous and remaining 3 were used for the testing of another substance.NUMBER OF REPLICATESNumber of corneas per group:Exposed group (test substance) - 3 corneas (No. 1, 4, 6) Positive control group (20% Imidazole) – 3 corneas (No. 12, 13, 14) Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 9) NEGATIVE CONTROL USED0.9% NaClSOLVENT CONTROL USED (if applicable)0.9% NaClPOSITIVE CONTROL USED20% ImidazoleAPPLICATION DOSE AND EXPOSURE TIME750 µL of application form (2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution). 4 hrsTREATMENT METHOD: closed-chamber method POST-INCUBATION PERIOD: yes/no. If YES please specify durationREMOVAL OF TEST SUBSTANCE- Number of washing steps after exposure period: After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded. The test substance was removed from the anterior chamber with EMEM (containing phenol red) and sequentially was removed mechanically using a cotton swab (the test substance was coloured and had tendency to stick on the cornea surface) ; also the test substance was removed from the anterior chamber with EMEM (containing phenol red) once more. The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red). Rinsing was finalized after complete removal of the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.- POST-EXPOSURE INCUBATION: application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) METHODS FOR MEASURED ENDPOINTS: - Corneal opacity:measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale- Corneal permeability: 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. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.SCORING SYSTEM: In Vitro Irritancy Score (IVIS)IVIS = mean opacity value + (15 x mean permeability OD490 value)DECISION CRITERIA: IVISUN GHS≤ 3No Category> 3; ≤ 55No prediction can be made≥ 55Category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
9.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
other: no prediction can be made
Conclusions:
The In Vitro Irritancy Score (IVIS) for Reactive Blue 234 was 9.30. This value of IVIS is > 3 and simultaneously ≤ 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.
Executive summary:

The test substance, Reactive Blue 234, 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 26th July 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 Blue 234 was 9.30.

This value of IVIS is > 3 and simultaneously ≤ 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

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