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Description of key information

Skin irritation/corrosion

The potential of the test item Sodium 3-sulfobenzoate to be irritant to the skin was investigated through an in vitro skin irritation study (according to OECD Guideline 439), using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.

The blank, negative and positive controls gave acceptable results and the study was accepted as valid. The mean cell viability of the test item treated tissues, after the blank subtraction, was 102%. Based on the results obtained, the test item Sodium 3-sulfobenzoate is classified as non-irritant to the skin (UN GHS No Category).

Eye irritation/damage

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 B.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.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted on 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identity Sodium 3-sulfobenzoate
Alternative names 3-Sulpho Benzoic AcidMono Sodium Salt
SBA (3-Sodiosulfobenzoic Acid)
Sodium hydrogen m-sulphonatobenzoate
Label name 3-Sodiosulfobenzoic Acid
Batch no. 170103
Retest date 14 February 2019
Storage conditions Room temperature
RTC number 15432
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
The test system EPISKINTM is commercially available from SkinEthic Laboratories.

Characteristic of the test system
The SkinEthic reconstructed human tissue model EPISKINTM consists of an airlifted, living, multi-layered tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionality equivalent to human tissue in vivo.
Normal human keratinocytes are used to reconstruct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker chemicals.

Functional conditions
The barrier function should be demonstrated. The containment properties of the RhE model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The RhE model supplier should ensure that each batch of the RhE model used meets defined production release criteria. A certificate will be supplied for each batch of the test system.

Preparation and storage of the test system
Immediately after arrival, the plate will be opened under a sterile airflow and the sterile filter paper removed. Each insert containing the epidermal tissue will be carefully taken out. Any remaining agarose that adheres to the outer sides of the insert will be rapidly removed by gentle blotting on the sterile filter paper. Inserts will be quickly placed in a 12-well plate (supplied) in which each well has previously been filled with 2 mL/well SkinEthic. Maintenance Medium (at room temperature) making sure that no air bubbles are formed underneath the inserts.
Culture dishes will be placed in the incubator at 37°C, 5% CO2 and saturated humidity. Testing can initiate after at least two hours of incubation.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
In the Main Assay, alive tissues were treated with the test item, positive and negative controls. The treatment scheme was the following:
Negative control (D-PBS) amount per well 20 µL (3 replicates)
Positive control (5% (w/v) SDS) amount per well 20 µL (3 replicates)
Test Item (sodium 3-sulfobenzoate) amount per well 20 mg (3 replicates).
Duration of treatment / exposure:
Exposure period
An exposure time of 15±0.5 minutes was allowed in a ventilated cabinet at room temperature.

Washing
At the end of the exposure, each tissue was rinsed with approximately 25mL of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2mL/well of maintenance medium.
Duration of post-treatment incubation (if applicable):
Post-exposure period
A 42 ± 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 1 (A1)
Value:
93.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
110.4 % tissue viability
Positive controls validity:
valid
Remarks:
3.1 % tissue viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 2 (A2)
Value:
118
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
89.8 % tissue viability
Positive controls validity:
valid
Remarks:
4.6 % tissue viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 3 (A3)
Value:
96.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
99.8 % tissue viability
Positive controls validity:
valid
Remarks:
4.5 % tissue viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
102
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100 % tissue viability (mean)
Positive controls validity:
valid
Remarks:
4 % tissue viability (mean)

Preliminary test

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. An opaque yellow colour was noted in the MTT solution at the end of the incubation period. This no relevant colour change indicated that the test item could not direct interact with MTT.

In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed, indicating that the test item has no potential interfering ability.

Based on the results obtained, no additional control was added in the Main Assay.

Main Assay

A Main Assay was performed. The mean Optical Density of Blank Controls was 0.038, lower than the maximum acceptable value (0.1). The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

Positive control results indicated an appropriate cell death with an acceptable relative cell viability (4% of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 0.8). Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 102%, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 13.5 lower than 18).

Interpretation of results:
GHS criteria not met
Conclusions:
The blank, negative and positive controls gave acceptable results and the study was accepted as valid. The mean cell viability of the test item treated tissues, after the blank subtraction, was 102%. Based on the results obtained, the test item Sodium 3-sulfobenzoate is classified as non-irritant to the skin (UN GHS No Category).
Executive summary:

The potential of the test item Sodium 3-sulfobenzoate to be irritant to the skin was investigated through an in vitro skin irritation study (according to OECD Guideline 439), using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.

The blank, negative and positive controls gave acceptable results and the study was accepted as valid. The mean cell viability of the test item treated tissues, after the blank subtraction, was 102%. Based on the results obtained, the test item Sodium 3-sulfobenzoate is classified as non-irritant to the skin (UN GHS No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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)
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.
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.
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)]
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

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to CLP Regulation the substance is classified as Eye Damage 1 H318 since an IVIS score of 342.5 (then >55, limit value for substances that induces eye damage acording to OECD 437) has been determined in a Guideline test according OECD 437.

As a result of a Guideline test according to OECD 439, the substance is not classified for skin irritation/corrosion.

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