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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 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

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium hydrogen m-sulphonatobenzoate
EC Number:
241-602-5
EC Name:
Sodium hydrogen m-sulphonatobenzoate
Cas Number:
17625-03-5
Molecular formula:
C7H6O5S.Na
IUPAC Name:
sodium 3-sulfobenzoate
Test material form:
solid: particulate/powder
Details on test material:
White crystalline powder.
Specific details on test material used for the study:
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

Method

Species / strainopen allclose all
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.

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