Disodium benzene-1,3-disulphonate

Endpoint summary

Description of key information

Skin Irritation:

The dermal irritation potential of target chemical was assessed in various in- vitro and in-vivo experimental studies.Based on the available studies,it can be concluded that the test chemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified''.

Eye Irritation:

The ocular irritation potential of target chemical disodium benzene-1,3-disulphonate (831-59-4) was assessed in in- vitro and in vivo experimental studies for test chemical and its functionally similar read across substances .Based on the available experimental studies,it can be concluded that the test chemical is likely to cause eye irritation and considered as irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2 (irritating to eyes)”.

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:

supporting study

Study period:

May 05, 2017 to July 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model (MatTek Corp., Ashland, MA).

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material:- Expiration date of the lot/batch:- Purity test date:RADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature or Fridge storage- Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test articles is tested as provided (neat).- Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data availableFORM AS APPLIED IN THE TEST: SolidOTHER SPECIFICS: No data available

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ 3-dimensional human tissues used in this study

Source strain:

other: Not applicable

Details on animal used as source of test system:

- Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.- Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.

Justification for test system used:

The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

The tissues were exposed to the test article neat (undiluted) on June 28, 2017 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours, 57 minute and 25 second MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette. Tissues were exposed to controls and the test articles for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS, positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b)Test ArticleFor solid test article, the tissues were moistened with 25 μL of ultrapure water to improve contact of the tissue surface with the test article. Approximately 25 mg of each test article was evenly applied to the apical surface of each tissue (n=3). All the tissues were placed into the ~37°C incubator with 5% CO2. The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature. 3.Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: TwiceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (OD) mean from all replicates for each plate (ODblank). Negative Controls (NC):·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·        The OD mean per NC tissue was calculated. ·        The mean OD for all tissues corresponds to 100% viability. ·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Positive Control (PC):·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.ODtvt= optical density of treated viable tissue incubated in MTT mediaODvt= optical density of viable tissues incubated in media alone- Evaluation of data The results of the assay was evaluated and compared to negative control.  Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mg - Concentration (if solution): neat (undiluted)VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate

Duration of treatment / exposure:

The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

For a total of an approximately 42 hour post-exposure incubation.

Number of replicates:

3 tissues will be used per test compound and control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

106.2

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of OD: 2.215; Non-Irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Interpretation of results:

other: not irritating

Conclusions:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The mean of OD for test chemical was determined to be 2.215.The standard deviation of viabilities for test chemical were calculated to be 4.49.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 106.2%. Thus, test chemical was considered to be not irritating to the human skin.

Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met and passed the acceptance of criteria.

The mean of OD for test chemical was determined to be 2.215. The standard deviation of viabilities for test chemical were calculated to be 4.49.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 106.2%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In different studies, the test chemical has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in- vitro and in-vivo experimental conducted in human and rabbits which have been summarized as below:

In an in vitro study,the dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data shows that the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control.The Mean % tissue viability compared to negative control (n=3) of Disodium benzene-1,3-disulphonate [CAS: 831-59-4] was determined to be 106.2 %.Hence, under the experimental test conditions it was concluded that Disodium benzene-1,3-disulphonate [CAS: 831-59-4] was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

In another in vitro study,the dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.The MTT data show the assay quality controls were met and passed the acceptance of criteria.The mean of OD for test chemical was determined to be 2.215. The standard deviation of viabilities for test chemical were calculated to be 4.49.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 106.2%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

A Skin irritation study was performed by Chin H. Tay et.al (International Journal of Toxicology ,January 2004 vol. 23 no. 1 pp.11-16) on New Zealand White rabbits to assess the irritation potential of disodium Benzene meta-disulfonate. The study was performed according to EPAOPPTS 870.2500, “Acute Dermal Irritation” Guidelines.

Two concentrations of disodium benzene meta-disulfonate were tested in separate groups of animals, 3 female and 3 male rabbits: 5000 and 2000 mg/L. The pH of the 5000 mg/L test substance ranged from 7.07 to 6.22 prior to dosing and after the 4-hour exposure. The pH of the 2000 mg/L test substances, measured prior to dosing and after the 4-hour exposure, ranged from 7.04 to 6.96.

Each dose of test compound (0.5 ml of each concentration for each dose cohort) was applied to a small area of skin (approximately 6cm2) and covered with four layers of gauze patch held in place with nonirritating tape. Since the compound was in liquid form, it was applied to the gauze before application to the skin. The patch was loosely held in contact with the skin using a semi-occlusive dressing for the exposure period of 4 hours. At the end of the 4-hour exposure period, the patches were removed and the skin wiped to remove any test substance still remaining. Animals were observed for signs of erythema and edema at 1, 24, 48, and 72 hours after patch removal. Observations of erythema or edema were scored according to the Draize Scale for Scoring Skin Reactions (USEPA 1998). This scale assesses irritation as follows: 0, no erythema or edema; 1, very slight erythema and/or edema (barely perceptible); 2, well-defined erythema and/or slight edema; 3, moderate to severe erythema or moderate edema, and 4, severe erythema and/or edema. Animals were weighed before exposure and at the end of the exposure period (day 3) and observed daily for the incidence of any clinical signs of toxicity (other than erythema and edema). Control skin showed no signs of irritation. At 5000 mg/L, very slight (one animal, Draize Score 1) and well defined erythema (two animals, Draize Score 2) were observed 1 hour after removal of the test substance. At the 24-hour observation point, only two animals demonstrated very slight irritation. By 48 hours, very slight irritation remained on only one of the two test animals. By 72 hours, no irritation was observed on any animal. No erythema or edema was observed in any test animals at 2000 mg/L.

Since the effects at 5000mg/l were fully reversible within 72 hours and not observed at 2000 mg/l, disodium Benzene meta-disulfonate can be considered not irritating to rabbit skin.

This result is also supported by the experimental study performed by Chin H. Tay et.al (International Journal of Toxicology, January 2004 vol. 23 no. 1 pp.11-16) on New Zealand White rabbits to assess the irritation potential of the functionally similar read across substance,  Sodium benzene sulfonate [CAS: 515-42-4]. Four doses were applied: 5000 mg/L, 2000 mg/L, 1000 mg/L, and 500 mg/l to 6 male and 6 female rabbits per dose group. The pH of the 1000 mg/L dosage was 7.01 and 6.98 prior to dosing and after the 4-hour exposure, respectively. For the 5000 mg/L dosage, the pH was 7.00 and 7.02 prior to dosing and after the 4-hour exposure, respectively.

Each dose of test compound (0.5 ml of each concentration for each dose cohort) was applied to a small area of skin (approximately 6cm2) and covered with four layers of gauze patch held in place with nonirritating tape. Since the compound was in liquid form, it was applied to the gauze before application to the skin. The patch was loosely held in contact with the skin using a semi-occlusive dressing for the exposure period of 4 hours.

At the end of the 4-hour exposure period, the patches were removed and the skin wiped to remove any test substance still remaining. Animals were observed for signs of erythema and edema at 1, 24, 48, and 72 hours after patch removal. Observations of erythema or edema were scored according to the Draize Scale for Scoring Skin Reactions (USEPA 1998). This scale assesses irritation as follows: 0, no erythema or edema; 1, very slight erythema and/or edema (barely perceptible); 2, well-defined erythema and/or slight edema; 3, moderate to severe erythema or moderate edema, and 4, severe erythema and/or edema. Animals were weighed before exposure and at the end of the exposure period (day 3) and observed daily for the incidence of any clinical signs of toxicity (other than erythema and edema). Control skin showed no signs of irritation.At a dose of 5000 mg/L, very slight erythema (Draize Score 1) was observed in all three test animals 1 hour after removal of the gauze containing the benzene sulfonate solution.At the 24-hour observation point, very slight irritation (Draize Score 1) was observed in one of the three test animals. At the 48- and 72 -hour observation points, the irritation was reversed, and no irritation was observed in any of the animals. Edema was not observed at any time. Control skin showed no signs of irritation. At 2000 mg/L, very slight erythema (Draize Score 1) was observed in two of the three test animals 1 hour after removal of the benzene sulfonate solution. The irritation was reversed by 24 hours. No erythema was observed in any test animals at this dosage at 24, 48 or 72 hours.

Since the effects at all dose levels were fully reversible within 72 hours, Sodium Benzene sulfonate can be considered not irritating to rabbit skin.

The above results are also supported by the experimental study performed by Chin H. Tay et.al (International Journal of Toxicology, January 2004 vol. 23 no. 1 pp.11-16) on New Zealand White rabbits to assess the irritation potential of the functionally similar read across substance, sodium 4-hydroxybenzenesulfonate [CAS: 825-90-1]. 2 doses were applied: 5000 mg/L, 2000 mg/L to 3 male and 3 female rabbits per dose group. Each dose of test compound (0.5 ml of each concentration for each dose cohort) was applied to a small area of skin (approximately 6cm2) and covered with four layers of gauze patch held in place with nonirritating tape. Since the compound was in liquid form, it was applied to the gauze before application to the skin. The patch was loosely held in contact with the skin using a semi-occlusive dressing for the exposure period of 4 hours. At the end of the 4-hour exposure period, the patches were removed and the skin wiped to remove any test substance still remaining. Animals were observed for signs of erythema and edema at 1, 24, 48, and 72 hours after patch removal. Observations of erythema or edema were scored according to the Draize Scale for Scoring Skin Reactions (USEPA 1998). This scale assesses irritation as follows: 0, no erythema or edema; 1, very slight erythema and/or edema (barely perceptible); 2, well-defined erythema and/or slight edema; 3, moderate to severe erythema or moderate edema, and 4, severe erythema and/or edema. Animals were weighed before exposure and at the end of the exposure period (day 3) and observed daily for the incidence of any clinical signs of toxicity (other than erythema and edema). Control skin showed no signs of irritation. At the 24- and 48-hour observation points, very slight irritation (Draize Score of 1) was seen in this animal. By 72 hours, the irritation had been reversed. In the other two test animals, very slight irritation (Draize Score of 1) was observed 1 hour after removal of the test substance. By 24 hours, the irritation was reversed in these two animals. No erythema or edema was observed in any test animals at 2000 mg/L.

Since the effects at 5000mg/l were fully reversible within 72 hours and not observed at 2000 mg/l, sodium 4-hydroxybenzenesulfonate can be considered not irritating to rabbit skin.

The above experimental study leads to a conclusion that Test chemical is indeed not irritating to skin. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “Not Classified”.

 

Eye Irritation:

In different studies, the test chemical disodium benzene-1,3-disulphonate (831-59-4) has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in- vitro data of target chemical and in-vivo data of functionally similar read across substances conducted in rabbits conducted which have been summarized as below:

In an in vitro study, the ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance was determined to be 9.1%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

In the another supporting in vivo study for functionally similar read across substance, a Draize Test was carried out for chemical Sodium 3-nitrobenzenesulphonate (CAS no: 127-68-4) to assess the eye irritation potential of six white Vienna rabbits. About 0.1ml of test chemical was instilled into the eye of each rabbits and the eyes were not washed out after 24 hours after substance application. Findings were graded as described in OECD test guideline 405 and were recorded after 24, 48, 78 hours, and at the end of the observation period (8 days). 24 h after application of the test substance, 2/6 animals showed opacicity of the cornea grade 1 (area 1/4-1/2), 1/6 animal showed circumcorneal injection, 6/6 animals showed redness of the conjunctivae grade 2, 5/6 showed swelling of the conjunctivae grade 1 and 6/6 showed secretion. 48 h after application of the test substance, 2/6 animals showed opacity of the cornea grade 1 (area 1/4-1/2), 1/6 animal showed circumcorneal injection, 5/6 animals showed redness of the conjunctivae grade 1-2, 4/6 showed swelling of the conjunctivae grade 1 and 4/6 showed secretion. 72 h after application of the test substance, 2/6 animals showed opacity of the cornea grade 1 (area 1/4-1/2), 1/6 animal showed circumcorneal injection, 5/6 animals showed redness of the conjunctivae grade 1-2, 4/6 showed swelling of the conjunctivae grade 1 and 3/6 showed secretion. Opacity of the cornea (grade 1, area 1/4-1/2) persisted in 2/6 animals while conjunctival redness grade 1 persisted in 3/3 animals until the end of the observation period. Other signs of toxicity were not noted. Thus from the observed findings, it is concluded that the chemical Sodium 3-nitrobenzenesulphonate (CAS no: 127-68-4) was irritating to the eyes of white Vienna rabbits.

Both the above studies were further supported by another in vivo study for functionally similar read across substance, the ocular irritation potential of the test chemical was assessed in rabbits. The test chemical was instilled into the eyes of rabbits and observed for signs of irritation (dose, duration not mentioned). Signs of irritation were observed whenthe test chemical was instilled into rabbit eyes. Hence, the test chemical can be considered to be irritating to eyes.

Based on the available data in vitro for the target as well as in vivo data for read across chemicals , disodium benzene-1,3-disulphonate (831-59-4) can be considered as irritating to eyes.Comparing the above annotations with the criteria of CLP regulation, test chemical can be classified under the category “Category 2 (irritating to eyes)”.

Justification for classification or non-classification

Available data for disodium benzene-1,3-disulfonate suggests that it is not likely to cause any irritation to skin and but irritant to eyes.

Disodium benzene-1,3-disulfonate can be classified under the category “Not Classified” for skin but for eye it is classified as “Category 2 (irritating to eyes)” as per CLP regulation.