Sodium hydrogen m-sulphonatobenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Sodium 3-sulfobenzoate was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

The test item was used as a solution in dimethylsulfoxide (DMSO).

It is concluded that the test item Sodium 3-sulfobenzoate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

The test item Sodium 3-sulfobenzoate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Three treatment series were included in the study. A short-term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism.

The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lenghts, was used.

A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at approximately 31 hours.

On the basis of the results obtained in a preliminary solubility trial, solutions of the test item were prepared in DMSO.

Dose levels of 2000, 1330, 889, 593, 395, 263, 176 and 117 and 78.0 µg/mL were used for the three-hour treatment in the absence and presence of S9 metabolism. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 52.0 µg/mL was also included.

The experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

For all treatment series, since no cytotoxicity of the test item calculated by the cytokinesis block proliferation index (CBPI) was observed, the dose levels of 2000, 1330 and 889 µg/mL, were selected for the scoring of micronuclei.

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control was observed at any dose level.

The incidences of cells bearing micronuclei were all within the distribution range of historical control data of the laboratory.

A concentration related increase of cells bearing micronuclei was observed for the short treatment series in the absence of S9 metabolism. This result was probably due to a very low incidence of micronucleated cells observed in the negative control cultures, hence the increase was considered to be of no biological significance.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that Sodium 3-sulfobenzoate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

The test item Sodium 3-sulfobenzoate was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. A Main Assay was performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO).

A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 2000 μg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL. No relevant toxicity was observed at any dose level in the absence or presence of S9 metabolic activation. Neither test item precipitation, nor opacity of the treatment mixture were noted at any concentration tested.

A Main Assay for mutation to 6-thioguanine resistance was performed. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 8 days to allow phenotypic expression of induced mutation. On the basis of the results obtained in the preliminary toxicity test, the following dose levels were selected: 2000, 1000, 500, 250 and 125 μg/mL.

No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism.

Negative and positive control treatments were included in each mutation experiment (Main Assay) in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system.

It is concluded that Sodium 3-sulfobenzoate does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity Sodium 3-sulfobenzoate
Alternative names 3-Sulpho Benzoic Acid Mono Sodium Salt
SBA (3-Sodiosulfobenzoic Acid)
Sodium hydrogen m-sulphonatobenzoate
Label name 3-Sodiosulfobenzoic Acid
Batch no. 170103
Expiry date 14 February 2019
Storage conditions Room temperature
RTC number 15432

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: deficient in a DNA excision repair system (uvrB mutation)

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA DNA repair deficiency

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue homogenate

Test concentrations with justification for top dose:

A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, was used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. A clear solution without any visible precipitation was obtained at 100mg/mL following few minutes of vortexing. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

Toxicity test
The test item Sodium 3-sulfobenzoate was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate.
No precipitation of the test item was observed at the end of the incubation period at any concentration. Neither toxic effects, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.

Main Assay
Two Main Assays were performed. Individual plate counts for these tests and the mean and standard error of the mean for each test point. On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000 μg/plate (the upper limit to testing indicated in the Study Protocol) and at four lower dose levels spaced by a factor of two: 2500, 1250, 625 and 313 μg/plate.

Vehicle / solvent:

Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. A clear solution without any visible precipitation was obtained at 100mg/mL following few minutes of vortexing. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Tester strain Absence of S9 Presence of S9
TA1535 sodium azide 2-aminoanthracene
TA100 sodium azide 2-aminoanthracene
TA1537 9-amino-acridine 2-aminoanthracene
TA98 2-nitrofluorene 2-aminoanthracene
WP2 uvrA methylmethanesulphonate 2-aminoanthracene

Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study.
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface
of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system. Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement No Growth on Minimal plates+Biotin.
Growth on Minimal plates+Biotin+Histidine.
Tryptophan requirement No Growth on Minimal agar plates.
Growth on Minimal plates+Tryptophan.
uvrA, uvrB Sensitivity to UV irradiation.
rfa Sensitivity to Crystal Violet.
pKM101 Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

Media
The following growth media were used:

Nutrient Broth
Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.

Nutrient Agar
Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.

Minimal Agar
Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-BonnerMedium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.

Top Agar
"Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).

S9 tissue homogenate
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 3770
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL):
S9 tissue fraction 1.0 mL
NADP (100 mM) 0.4 mL
G-6-P (100 mM) 0.5 mL
KCl (330 mM) 1.0 mL
MgCl2 (100 mM) 0.8 mL
Phosphate buffer (pH 7.4, 200 mM) 5.0 mL
Distilled Water 1.3mL

Rationale for test conditions:

A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, was used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates from the preliminary toxicity test were held at 4°C for approximately 24 hours before scoring, while plates from Main Assays were immediately scored by counting the number of revertant colonies on each plate.

Statistics:

Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility
Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. A clear solution without any visible precipitation was obtained at 100mg/mL following few minutes of vortexing. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

Toxicity test
The test item Sodium 3-sulfobenzoate was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate.
No precipitation of the test item was observed at the end of the incubation period at any concentration.
Neither toxic effects, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.

Main Assay
Two Main Assays were performed. On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000 μg/plate (the upper limit to testing indicated in the Study Protocol) and at four lower dose levels spaced by a factor of two: 2500, 1250, 625 and 313 μg/plate. No toxicity was observed at any concentration with any tester strain/activation condition combinations.
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments and using the same dose levels ofMain assay I.
Neither toxic effects, nor increases in the revertant colonies were noted with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
No precipitation of the test item was observed at the end of the incubation period in the absence or presence of S9 metabolic activation, in any experiment.

Acceptance criteria
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each tester strain. No plates were lost through contamination or cracking. The study was
accepted as valid.
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

It is concluded that the test item Sodium 3-sulfobenzoate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Sodium 3-sulfobenzoate was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

The test item was used as a solution in dimethylsulfoxide (DMSO).

It is concluded that the test item Sodium 3-sulfobenzoate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

The test item: Sodium 3-sulfobenzoate
Alternative names: 3-Sulpho Benzoic Acid Mono Sodium Salt
SBA (3-Sodiosulfobenzoic Acid)
Sodium hydrogen m-sulphonatobenzoate
Label name: 3-Sodiosulfobenzoic Acid

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese hamster V79 cells were obtained from Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK. This cell line, V79 4(H) can be traced back directly to the original V79 isolate prepared by Ford and Yerganian in 1958. The karyotype, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for generation time and the absence of mycoplasmal contamination.
Permanent stocks of the V79 cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Cultures of the cells are grown in EMEM medium supplemented with 10% Foetal Calf Serum (EMEM Complete). All incubations are at 37°C in a 5% carbon dioxide atmosphere (100% humidity).

Additional strain / cell type characteristics:

other: HPRT-deficient cells

Cytokinesis block (if used):

Not used.

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue homogenate
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 3773
The production and quality control certificate can be found in Addendum 2 of this report.

The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL):
S9 tissue fraction 2.0mL
NADP (0.1 M) 0.4mL
G-6-P (0.1 M) 0.5mL
KCl (0.33 M) 1.0mL
MgCl2 (0.1M) 0.5mL
Phosphate Buffer (0.2 M) 5.6mL
Total 10.0mL

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 2000 μg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL.

On the basis of the results obtained in the preliminary toxicity test, the following dose levels were selected:
2000, 1000, 500, 250 and 125 μg/mL.

Vehicle / solvent:

The solvent used in this study was DMSO (Honeywell, batch no: H012S).

Solutions of ethylmethanesulphonate (Labelled as: EthylMethane Sulfonate; Sigma, batch no.: BCBN1209V) were prepared in ethanol (Sigma, batch no.: BCBK5059V) and served as positive control in the absence of S9 metabolism. Solutions of 7,12-dimethylbenzanthracene (labelled as 7,12-dimethylbenz(a)anthracene; Sigma, batch no.: SLBF3276V) were prepared in DMSO (Fluka, batch no.: STBF8595V) and served as positive control in the presence of S9 metabolism.

Solubility test
Solubility of the test item was evaluated in the previous study (i.e. Study No. A2768, named "In vitro Micronucleus Test in Human Lymphocytes") where the test item was found to be soluble in DMSO at the concentration of 400 mg/mL. Since DMSO is known to be cytotoxic in the selected assay system at a concentration greater than 1% (volume fraction), this solvent has to be present at a constant volume of 1% (v/v) in the negative controls and in all test item concentrations. Based on this result, the test item was assayed at the maximum dose level of 2000 μg/mL, the upper limit to testing indicated in the Study Protocol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Solutions of ethylmethanesulphonate were prepared in ethanol and served as positive control in the absence of S9 metabolism. Solutions of 7,12-dimethylbenzanthracene were prepared in DMSO and served as positive control in the presence of S9 metabolism.

Details on test system and experimental conditions:

Cytotoxicity assay
A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of the test item was used; cell cultures were treated using the same treatment conditions as the mutation assay, and the survival of the cells was subsequently determined.
Treatments were performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included. In order to evaluate baseline count, at the beginning of treatment two additional control cultures were included in the experimental scheme. These two cultures were trypsinized and cell counts were performed approximately at time 0. The baseline count was the average of the total number of cells from the two flasks. At the end of treatment, cell monolayers were washed with PBS, the cultures were trypsinised, counted, diluted and plated. After incubation for eight days, the colonies were stained with Giemsa solution and counted.

Mutation assay
Treatment of cell cultures
A main assay was performed including negative and positive controls, in the absence and presence of S9 metabolising system. The two treatment series were assayed in separate runs. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. For each run, two additional control cultures were included in the experimental scheme, in order to evaluate baseline count. Two days before the experiment, sufficient numbers of 175 cm2 flasks were inoculated with 5 million freshly trypsinised V79 cells from a common pool, in order to have at least 20 million of cells for treatment. At the treatment time, the growth medium was removed from the flasks and replaced with treatment medium; cultures were incubated at 37°C for three hours.

Determination of survival (Day 0)
At the end of treatment, the medium was removed and the cell monolayers were washed with PBS. The cultures were trypsinised, counted and an aliquot from each culture was diluted and plated to estimate the viability of the cells. Each cell suspension was re-plated (2 x 106 cells/ F175) in order to maintain the treated cell populations. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Subculturing (Day 2 and Day 5)
On Days 2 and 5, the cell populations were subcultured in order to maintain them in exponential growth. The cultures were trypsinised, counted and the number of cells taken forward was adjusted to give two million viable cells seeded in 225 cm2 flasks.

Determination of mutant frequency (Day 8)
At the expression time (Day 8), each culture was trypsinised, resuspended in complete medium and counted by microscope. After dilution, an estimated 1 x 105 cells were plated in each of twenty 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine (at 7.5 μg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Cloning Efficiency (CE).

Evaluation criteria:

Assessment of cytotoxicity and mutant frequency
Cytotoxicity was evaluated by relative survival (RS), i.e. cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, as compared with adjusted cloning efficiency in solvent controls (assigned a survival of 100%). The following formulas were applied:
CE = Number of colonies/Number of cells plated

Adjusted CE = CE x (Number of cells at the end of treatment / Number of cells at the beginning of treatment (baseline count))

Relative survival of treated cultures was calculated as follows:
RS = (Adjusted CE in treated culture / Adjusted CE in solvent control culture) x 100

Mutant frequency (MF) per million surviving cells was calculated using the following formula:
MF = (CEmutant/CEviable) x 10EXP6
where:
CEmutant = cloning efficiency of mutant colonies in selective medium
CEviable = cloning efficiency of colonies in non-selective medium
When no mutant colonies were observed at a given test point, the mutation frequency was calculated assuming one colony.

Statistics:

Statistical analysis
The individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y=(x+a)EXPb
where a = 0 and b = 0.275.
The mutant frequency in the solvent control and treated cultures was compared using the Dunnett’s test (one-tailed).
For each experimental point, the corrected sum of squares of transformed mutation frequencies was calculated. The error mean square (EMS) was calculated as the sum of SSy values divided by the sum of degrees of freedom.
For each experimental point the t value was calculated.
For each comparison of treatment with control, the calculated t value was compared with
tabulated critical values for the one tailed Dunnett’s test.

The results of the experiment were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation were examined. For each experiment, a two way analysis of variance was performed (without interaction) fitting to two factors:
– Replicate culture: to identify differences between the replicate cultures treated.
– Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility test
Solubility of the test item was evaluated in the previous study (i.e. Study No. A2768, named "In vitro Micronucleus Test in Human Lymphocytes") where the test item was found to be soluble in DMSO at the concentration of 400 mg/mL. Since DMSO is known to be cytotoxic in the selected assay system at a concentration greater than 1% (volume fraction), this solvent has to be present at a constant volume of 1% (v/v) in the negative controls and in all test item concentrations. Based on this result, the test item was assayed at the maximum dose level of 2000 μg/mL, the upper limit to testing indicated in the Study Protocol.

Cytotoxicity test
A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 2000 μg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL. No relevant toxicity
was noted at any concentrations tested in the absence or presence of S9 metabolism. Neither precipitation of the test item, nor opacity of the treatment mixture were observed at any concentration tested in the absence or presence of S9 metabolic activation.

Mutation assays
Experimental design
Based on the results obtained in the preliminary cytotoxicity assay, a Main Assay for mutation to 6-thioguanine resistance was performed using the following dose levels:
2000, 1000, 500, 250 and 125 μg/mL.

The mutant frequencies in the negative control cultures fell within the 95% control limits of the distribution of the laboratory’s historical control database. Treatment with the positive control items gave marked responses that were compatible with those generated in the historical control database and produced a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control, indicating the correct functioning of the test system. Adequate number of cells and concentrations was analysed. The study was accepted as valid.

Osmolality and pH
The pH values and osmolality of the post-treatment media were determined. The addition of the test item solution did not have any remarkable effect on the osmolality or pH of the treatment medium. Both parameters were within acceptable values since pH did not shift over 1 unit and osmolality did not increase over 100 mOsm/kg with respect to the negative control values (Scott et al., 1991).

Survival after treatment
In the absence of S9 metabolic activation, mild toxicity was observed at higher dose levels reducing relative survival (RS) to 46% of the concurrent negative control value at 2000 μg/mL.
This value was not coherent with the RS value obtained in the preliminary toxicity test. However, differences in cytotoxicity may be attributed to the variability of a biological system and to the use of only one replicate in the preliminary test. This consideration is also confirmed by the relevant guideline, where cytotoxicity for each culture is required in the main experiment even if a preliminary cytotoxicity test is performed.
Moreover, it should be noted that the RS value obtained was adequate for the evaluation of mutagenic effect.
No relevant toxic effects were observed in the treatment series in the presence of S9 metabolic activation at any concentration tested.

Mutation results
No statistically significant increase over the spontaneous mutation frequency was observed at any treatment level, in the absence or presence of S9 metabolic activation. All results were inside the distribution of the historical negative control data, both in the absence and presence of S9 metabolism.
Analysis of variance indicated that nor replicate culture neither dose level were significant factors in explaining the observed variation in the data, in the absence or presence of S9 metabolism.

Conclusions:

It is concluded that Sodium 3-sulfobenzoate does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Sodium 3-sulfobenzoate was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. A Main Assay was performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO).

A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 2000 μg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL. No relevant toxicity was observed at any dose level in the absence or presence of S9 metabolic activation. Neither test item precipitation, nor opacity of the treatment mixture were noted at any concentration tested.

A Main Assay for mutation to 6-thioguanine resistance was performed. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 8 days to allow phenotypic expression of induced mutation. On the basis of the results obtained in the preliminary toxicity test, the following dose levels were selected: 2000, 1000, 500, 250 and 125 μg/mL.

No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism.

Negative and positive control treatments were included in each mutation experiment (Main Assay) in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system.

It is concluded that Sodium 3-sulfobenzoate does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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
Expiry date 14 February 2019
Storage conditions Room temperature
RTC number 15432

Species / strain / cell type:

lymphocytes: Lymphocyte cultures from human peripheral blood

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

inhibitor of actin polymerisation cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction (Rat, Strain Sprague Dawley, Tissue Liver, Inducing Agents Phenobarbital – 5,6-Benzoflavone).

Test concentrations with justification for top dose:

Dose levels were selected on the basis of the solubility of the test item in the culture medium in agreement with the Study Protocol.
Dose levels of 2000, 1330, 889, 593, 395, 263, 176, 117 and 78.0 µg/mL were used for the three hour treatment in the absence and presence of S9 metabolism. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 52.0 µg/mL was also included.
Appropriate negative and positive control cultures were included in the experiments. Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only with Cyclophosphamide at the dose levels of 20.0 and 15.0 µg/mL. Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the dose levels of 80.0 and 40.0 ng/mL.

Vehicle / solvent:

Solutions of the test item, as received, were prepared immediately before use in DMSO on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 53 minutes from the initial formulation. All dose levels in this report are expressed to three significant figures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Solutions of Colchicine were prepared in sterile water and served as positive control in the absence of S9 metabolism. Solutions of Cyclophosphamide were prepared in sterile water and served as positive control in the presence of S9 metabolism.

Details on test system and experimental conditions:

One Main Experiment was performed including solvent and positive controls. Two cultures were prepared at each test point.
Lymphocyte cultures were treated fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.
For the short treatment, the composition of the treatment media was as follows:
Treatment medium in the presence of S9 metabolism
Test item solution 0.05mL
S9 mix 1.00mL
Culture medium (without PHA) 3.95mL

Treatment medium in the absence of S9 metabolism
Test item or control solution 0.05mL
Culture medium (without PHA) 4.95mL

For the continuous treatment, due to the concurrent addition of Cytochalasin B dissolved in DMSO, it was necessary to reduce the volume of test item solutions (25 µL/tube instead of 50 µL/tube), to maintain the final concentration of organic solvents to 1%.
The composition of the treatment medium was as follows:
Test item or control solution 0.025mL
Culture medium (without PHA) 4.975mL
For the short term exposure, the treatment media were added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.
For the continuous treatment, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting at 31 hours.

Experimental design
Dose levels were selected on the basis of the solubility of the test item in the culture medium in agreement with the Study Protocol.
Dose levels of 2000, 1330, 889, 593, 395, 263, 176, 117 and 78.0 µg/mL were used for the three hour treatment in the absence and presence of S9 metabolism. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 52.0 µg/mL was also included.
Appropriate negative and positive control cultures were included in the experiments. Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only with Cyclophosphamide at the dose levels of 20.0 and 15.0 µg/mL. Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the dose levels of 80.0 and 40.0 ng/mL.

Harvesting and slide preparation
The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was
then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this waywere dropped onto clean, wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture.
The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Evaluation criteria:

The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI = (mononucleated + 2 x binucleated + 3 x multinucleated) / total number of cells counted

where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells. CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed.
Since no cytotoxicity occurred, scoring was terminated after scoring the highest five dose levels in agreement with the Study Protocol.

The percentage cytotoxicity was evaluated according to the following formula:
%Cytotoxicity = 100 - 100 (CBPIt-1/CBPIc-1)

where:
t = test item treated culture
c = solvent control culture

Scoring of micronuclei
The highest dose level for genotoxicity assessment should be selected as a dose which produces a substantial cytotoxicity (approximately 55±5%) compared with the negative control. If the test item does not induce relevant toxicity at any concentration, then the highest treatment level is selected as the highest dose level for scoring.
Two lower dose levels are also selected for the scoring of micronuclei. For the three selected doses, for the solvent and positive controls (Mitomycin-C and Cyclohosphamide), 1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.
The criteria for identifying micronuclei were as follows:
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin.

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Species / strain:

lymphocytes: Lymphocyte cultures from human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Assay results

Following treatment with the test item, no remarkable increase in the incidence of micronucleated cells over the concurrent negative control value was observed in the absence or presence of S9 metabolism.

Marked increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

Analysis of results

Results show that the incidence of micronucleated cells of the negative controls was within the distribution range of our historical control values.

Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

The study was accepted as valid.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent control was observed at any dose level. The incidences of cells bearing micronuclei were all within the distribution range of historical control data of the laboratory.

A concentration related increase of cells bearing micronuclei was only observed for the short treatment series in the absence of S9 metabolism. This result was probably due to a very low incidence of micronucleated cells observed in the negative control cultures, hence the increase was considered to be of no biological significance.

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in human lymphocytes after in vitro treatment.

Conclusions:

On the basis of the above results, it is concluded that sodium 3-sulfobenzoate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item Sodium 3-sulfobenzoate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Three treatment series were included in the study. A short term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism.

The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lenghts, was used.

A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at approximately 31 hours.

On the basis of the results obtained in a preliminary solubility trial, solutions of the test item were prepared in DMSO.

Dose levels of 2000, 1330, 889, 593, 395, 263, 176 and 117 and 78.0 µg/mL were used for the three hour treatment in the absence and presence of S9 metabolism. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 52.0 µg/mL was also included.

The experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

For all treatment series, since no cytotoxicity of the test item calculated by the cytokinesis block proliferation index (CBPI) was observed, the dose levels of 2000, 1330 and 889 µg/mL, were selected for the scoring of micronuclei.

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control was observed at any dose level.

The incidences of cells bearing micronuclei were all within the distribution range of historical control data of the laboratory.

A concentration related increase of cells bearing micronuclei was observed for the short treatment series in the absence of S9 metabolism. This result was probably due to a very low incidence of micronucleated cells observed in the negative control cultures, hence the increase was considered to be of no biological significance.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that Sodium 3-sulfobenzoate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to CLP Regulation the substance is not classified for genetic toxicity since results obtained from three assay (i.e. Bacterial reverse mutation, Micronucleus study, and Chinese Hamster V79 Cells) were conclusive but not sufficient for the classification of the substance.