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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:
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 guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD “GUIDANCE DOCUMENT ON THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; Series No. 160, 25. Oct. 2011
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium hydrogen m-sulphonatobenzoate
EC Number:
241-602-5
EC Name:
Sodium hydrogen m-sulphonatobenzoate
Cas Number:
17625-03-5
Molecular formula:
C7H6O5S.Na
IUPAC Name:
sodium 3-sulfobenzoate
Test material form:
solid: particulate/powder
Details on test material:
White crystalline powder.
Specific details on test material used for the study:
Test Item
Designation in Test Facility: 17042002G
Date of Receipt: 20. Apr. 2017
Condition at Receipt: Room temperature, in proper conditions

Specification
Batch no. 170103
Appearance White crystalline powder
Composition Sodium 3-Sulfobenzoate
Purity > 99.0% (Titration with NaOH, Glass indicating electrode, Calomel reference electrode)
Homogeneity totally homogeneous
Expiry date Jan. 2019
Storage Room Temperature (20 ± 5°C), keep away from humidity
CAS No. 17625-03-5
EINECS-No. 241-602-5
Chemical Class not stated
Volatility not applicable
pH-value weakly acidic
Stability H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: >1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Safety Precautions
For handling, information given in the MSDS will be observed.

Storage
The test item will be stored in the test facility in a closed vessel at room temperature (20±5°C).

Preparation
The test item is a solid substance. It was tested directly.

Test animals / tissue source

Species:
other: Fresh bovine corneas
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
The BCOP test method uses isolated corneas from the eyes of cattle which are freshly slaughtered in the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany. The cattle were between 12 and 60 months old.
The eyes are removed as soon as possible after death of the cattle and immersed in Hanks’ Balanced Salt Solution in a suitable cooled container. Then, they are transported to the laboratory within max. 4 hours where they are immediately used for the test.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
Tissue 1 amount 342.7 mg
Tissue 2 amount 347.2 mg
Tissue 3 amount 359.2 mg
Duration of treatment / exposure:
Open Chamber Method
Exposure time on the corneas is 4 hours ± 5 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers are filled with cMEM without phenol red, and the final illuminance value of each cornea is recorded at once.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red is removed from the front chamber, and 1 mL sodium fluorescein solution is added to the front chamber for the detection of permeability of the corneas.
For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL is used.
The chambers are then closed again and incubated for 90 ± 5 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber is thoroughly mixed. Then, its optical density at 492 ± 2 nm is measured with the spectrophotometer.
Observation period (in vivo):
-
Duration of post- treatment incubation (in vitro):
The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.
Number of animals or in vitro replicates:
For each treatment group (negative control solution, test item and positive control solution), three replicates were used.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
After having carefully cleaned and sterilised the corneal holders, they are kept in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After arrival of the corneas, they are examined and only corneas which are free from defects are used.

NUMBER OF REPLICATES
For each treatment group (negative control solution, test item and positive control solution), three replicates were used.

NEGATIVE CONTROL USED
HBSS- solution: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10)

POSITIVE CONTROL USED
Imidazole C3H4N2, CAS-No. 288-32-4, dissolved in HBSS solution, 20% solution (200 g/L)

APPLICATION DOSE AND EXPOSURE TIME
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
Tissue 1 amount 342.7 mg
Tissue 2 amount 347.2 mg
Tissue 3 amount 359.2 mg
The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

TREATMENT METHOD: Open Chamber Method

Calculation of Opacity
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea.
The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

Calculation of Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

Calculation of IVIS (In Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)

The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Tissue 1
Value:
303.38
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Tissue 2
Value:
307.55
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Tissue 3
Value:
416.57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean IVIS
Value:
342.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
mean score 0.94
Positive controls validity:
valid
Remarks:
mean score 117.12

Any other information on results incl. tables

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Table. Illuminance Values

Parameter

Negative Control

Test Item

Positive Control

(I) Measured values before exposure

1020

997

985

972

985

999

953

973

1013

(I) Measured values after exposure

989

966

975

122

121

92

336

282

373

 

The values in the following tables present the calculated opacity values, according to evaluation:

Table. Opacity Values Negative Control

Parameter

Negative Control

Opacity before exposure

1.81

2.76

3.28

Opacity after exposure

3.11

4.12

3.72

Opacity Difference

1.29

1.35

0.44

Mean Opacity Difference

1.03

 

Table. Opacity Values Test Item and Positive Control

Parameter

Test Item

Positive Control

Opacity before exposure

3.85

3.28

2.68

4.71

3.81

2.10

Opacity
after exposure

305.31

308.16

417.72

85.75

109.72

73.33

Opacity
Difference

301.46

304.88

415.04

81.04

105.91

71.24

Opacity
Difference

corrected

300.43

303.85

414.01

80.01

104.89

70.21

Mean Opacity Difference

339.43

85.03

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Table. Optical density at 492 nm of Blank

Parameter

cMEM without phenol red

1. Measurement

0.069

2. Measurement

0.070

3. Measurement

0.074

Mean

0.071

 

Table. Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter

Negative Control

Test Item

Positive Control

1. Measurement

0.059

0.077

0.053

0.259

0.311

0.237

0.477

0.428

0.575

2. Measurement

0.058

0.084

0.054

0.263

0.312

0.235

0.470

0.429

0.580

3. Measurement

0.064

0.086

0.054

0.264

0.314

0.236

0.504

0.427

0.589

 

1. Measurement - blank

-0.0120

0.0060

-0.0180

0.1880

0.2400

0.1660

0.4060

0.3570

0.5040

2. Measurement - blank

-0.0130

0.0130

-0.0170

0.1920

0.2410

0.1640

0.3990

0.3580

0.5090

3. Measurement - blank

-0.0070

0.0150

-0.0170

0.1930

0.2430

0.1650

0.4330

0.3560

0.5180

 

Mean of each replicate

-0.0107

0.0113

-0.0173

0.1910

0.2413

0.1650

0.4127

0.3570

0.5103

Mean of the three

replicates

-0.0056

--

--

Corrected

--

--

--

0.1966

0.2469

0.1706

2.0689

1.7906

2.5572

 

* Note: All values for the positive control were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

 

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table. IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS

1.13

0.94

73.28%

1.52

0.18

Test Item
sodium 3-sulfobenzoate

303.38

342.50

18.74%

307.55

416.57

Positive Control
20% imidazole

solution

111.04

117.12

10.87%

131.74

108.57

Note: the high relative standard deviation of the IVIS of negative control is due to mathematical reasons, as the respective means are very small.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item sodium 3-sulfobenzoate induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 342.50.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.
Executive summary:

Evaluation of sodium 3-sulfobenzoate in the Bovine Corneal Opacity and Permeability (BCOP) Test Method has been performed following OECD Guideline 437 and EU Method 8.47. The study was performed to assess corneal damage potential of sodium 3-sulfobenzoate by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item sodium 3-sulfobenzoate was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

The test item was tested pure. Under the conditions of this test, the test item sodium 3-sulfobenzoate induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 342.50.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I. The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria.

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