Lithium 2-aminobenzothiazole-6-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Range finding test: From 18-20 December, 2018.
Assay 1: From 16-18 January, 2019.
Assay 2: From 22-24 January, 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted according to OECD guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No. 440/2008, dated May 30th, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lithium 2-aminobenzothiazole-6-sulphonate

EC Number:

265-367-3

EC Name:

Lithium 2-aminobenzothiazole-6-sulphonate

Cas Number:

65072-36-8

Molecular formula:

C7H6N2O3S2.Li

IUPAC Name:

lithium 2-amino-1,3-benzothiazole-6-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Bulk Density (g/ml): 0.39
- Particle size distribution (μm): 19.6
- Water solubility: 20%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: IR2510181
- Expiration date of the lot/batch: 25 October 2020
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 °C, ≤70% relative humidity)
- Stability under test conditions: Analytical determination of 2-ABT lithium sulphonate stability was not performed because of the character and the short period of study
- Solubility and stability of the test substance in the solvent/vehicle: 2-ABT lithium sulphonate was soluble at a concentration of 100 mg/mL using distilled water; the certificate of analysis states that 2-ABT has "20% solubility in water"
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: A preliminary compatibility test was carried out using 50 µL of stock solution (100 mg/mL 2-ABT lithium sulphonate in distilled water), 2 mL of top agar solution and 500 µL of phosphate buffer. This mixture was deemed to be appropriate for the mutagenicity tests.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Range finding test: A stock solution of 100 mg/mL 2-ABT lithium sulphonate was prepared using distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10.
Assays 1 and 2: A stock solution of 100 mg/mL 2-ABT lithium sulphonate was prepared using distlled water. This was diluted in several steps to obtain the appropriate test concentrations.
- Preliminary purification step: Not specified
- Final dilution of a dissolved solid:
Stock solution: 100 mg/mL 2-ABT lithium sulphonate in distilled water

FORM AS APPLIED IN THE TEST
- A 50 µL solution of 2-ABT lithium sulphonate in distilled water

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

Assay 1 and Assay 2

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Remarks:

Range finding test

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:
Male Wistar rats

- method of preparation of S9 mix:
Rat livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions. The complete S9 mix was prepared with 100 mL rat liver homogenate (S9), 500 mL ice cold 0.2 M sodium phosphate buffer (pH 7.4) and 400 mL salt solution

- concentration or volume of S9 mix and S9 in the final culture medium:
500 µL of the S9 mix was present in the final culture medium, this contained 10% (v/v) of S9

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The enzymatic activity of the S9 batch was characterised using two mutagens, 2-aminoanthracene and benzo(a)pyrene, that require metabolic activation by microsomal enzymes.
The sterility of the S9 preparation was confirmed. The mean protein concentrations of the
S9 fractions used were 26.7 and 28.1 g/L

Test concentrations with justification for top dose:

Range finding test: 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Assays 1 and 2: 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate both with and without metabolic activation. The top dose was picked based on the results of the range finding test and the solubility findings

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water

- Justification for choice of solvent/vehicle: The solubility of 2-ABT lithium sulphonate was examined using distilled water, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). Partial dissolution was observed at a concentration of 100 mg/mL using DMSO and DMF, but 2-ABT lithium sulphonate was soluble at this concentration using distilled water.

- Justification for percentage of solvent in the final culture medium: Not specified

Controls

Untreated negative controls:

yes

Remarks:

Top agar and phosphate buffer control Top agar and S9 mix control

Negative solvent / vehicle controls:

yes

Remarks:

In the study dimethyl sulfoxide (DMSO) and distilled water were used as solvent control groups depending on the solubility of the test item and the solubility of strain specific positive control chemicals.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (NPD) 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Three (Range finding test and Assays 1 and 2)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk:

Range finding test and Assay 1: Standard plate incorporation procedure - Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). 50 µL of 2-ABT lithium sulphonate solution (or solvent or reference control), 100 µL of test strain and 500 µL of phosphate buffer (non-activated conditions) or S9 mix (activated conditions) were added to each test tube, then mixed and poured onto the surface of minimal glucose agar plates. After preparation, the plates were incubated at 37 °C for 48 hours.

Assay 2: Standard pre-incubation procedure (except S. typhimurium TA98 strain with metabolic activation where the plate incorporation procedure was used since no biologically relevant
increase in the number of revertant colonies was observed in Assay 1). Molten top agar was prepared and kept at 45 °C. Before the overlaying, 50 µL 2-ABT lithium sulphonate solution (or solvent or reference control), 100 µL of bacterial culture and 500 µL of S9 mix (activated conditions) or phosphate buffer (non-activated conditions) were added into appropriate tubes to provide direct contact between bacteria and the test item (in its solvent). The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 minutes at 37 ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The plates were then incubated at 37 ºC for 48 hours.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
Assay 2: 20 minutes
- Exposure duration/duration of treatment:
Range finding test, Assays 1 and 2: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Not specified
- Any supplementary information relevant to cytotoxicity: Not specified


METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel™ software.

Rationale for test conditions:

Partial dissolution was observed with a 100 mg/mL concentration of 2-ABT lithium sulphonate in DMSO and DMF, but the test substance was soluble at this concentration using distilled water. Distilled water was therefore selected as the appropriate solvent.
The doses selected for Assays 1 and 2 were picked based on the results of the range finding test and the solubility findings.

Evaluation criteria:

Criteria for a positive response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose
groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the
negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli
WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the
negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial
strains.

According to the OECD, EC and EPA guidelines, statistical method may be used as an aid in
evaluating the test results. However, statistical significance should not be the only
determining factor for a positive response.

Criteria for a negative response:
A test item was considered non-mutagenic if:
- it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

According to the OECD, EC and EPA guidelines, a statistical method may be used as an aid in
evaluating the test results. However, statistical significance should not be the only
determining factor for a positive response.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Remarks:

(Range finding test)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Clear dose-related effect. Mutation factor values at 1000, 2500 and 5000 µg/plate were > 2 (up to 20.84) and, therefore above the biologically relevant threshold.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Remarks:

(Assay 1)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Clear dose-related positive effect. Mutation factor values at 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate were > 2 (up to 25.60) and, therefore above the biologically relevant threshold.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Remarks:

(Assay 2)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Clear dose-related positive effect. Mutation factor values at 625, 1250, 2500, 3750 and 5000 µg/plate were > 2 (up to 27.60) and, therefore above the biologically relevant threshold. This result demonstrated that the positive effect was reproducible.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A

Remarks:

TA 98, TA 100: Result from Range finding test and Assays 1 and 2 TA 1535, TA 1537 and E. coli WP2 uvr A: Results from Assays 1 and 2

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

other: S. typhimurium TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A

Remarks:

TA 100: Result from Range finding test and Assays 1 and 2 TA 1535, TA 1537 and E. coli WP2 uvr A: Results from Assays 1 and 2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Sporadically lower or higher revertant counts, compared to the vehicle (solvent) control, were observed in the tested strains in Assays 1 and 2 at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range. Therefore, these results were considered to be due to the biological variability of the test system.

Applicant's summary and conclusion

Conclusions:

In a bacterial reverse mutation (Ames) assay, conducted according to OECD test guideline 471 and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was determined to be mutagenic in a single Salmonella typhimurium strain (TA98) in the presence, but not absence, of metabolic activation. No mutagenic activity was seen in the other four Salmonella strains, nor in E. coli strain WP2uvrA, with or without metabolic activation.

Executive summary:

In a bacterial reverse mutation (Ames) assay, conducted according to OECD guideline 471 and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was added (up to 5000 μg/plate) to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA in the presence and absence of metabolic activation (rat liver-derived S9).

In the range finding test (TA98 and TA100 only), a clear dose-related mutagenic response was identified in S. typhimurium TA98 in the presence of S9. This response was also observed in S. typhirmurium TA98 in Assay 1, whilst all other strains and TA98 without metabolic activation were negative for mutagenicity. In Assay 2, the mutagenic response in S. typhimurium TA98 with S9 was determined to be reproducible. The other strains and TA98 without S9 were confirmed to be negative for mutagenicity.

In this reliable in vitro study, lithium 2-aminobenzothiazole-6-sulphonate was determined to be mutagenic in a single Salmonella typhimurium strain (TA98) in the presence, but not absence, of metabolic activation. No mutagenic activity was seen in the other four Salmonella strains, nor in E. coli strain WP2uvrA, with or without metabolic activation.