Acetamide, N-(2-amino-4,5,6,7-tetrahydro-6-benzothiazolyl)-, hydrobromide (1:1)

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-Methyl-4,5,6,7-tetrahydro-benzothiazol-6-yl)-acetamide hydrobromide

EC Number:

600-588-4

Cas Number:

104617-50-7

Molecular formula:

C9 H13 O N3 S x HBr

IUPAC Name:

N-(2-Methyl-4,5,6,7-tetrahydro-benzothiazol-6-yl)-acetamide hydrobromide

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the previous Ames test, the bromide salt, 2-amino-6-acetylamino-4,5,6,7- tetrahydrobentothiazol HBr (SND 919 Thiazol HBr) was dissolved in distilled water [i]. However, in a non-GLP solubility test, the base, SND 919 Thiazol was not soluble in water at 2500 µg/100 µL but was soluble in DMSO at 2500 µg/100 µL. However, no precipitation was observed when 2500 and 5000 µg/100 µL SND 919 Thiazol was added to soft agar.
Therefore, a suspension of 5000 µg/100 µL/plate SND 919 Thiazol dissolved in DMSO was used as top concentration in this study.

Vehicle / solvent:

Dimethylsulfoxide was used as solvent and negative control

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Details on test system and experimental conditions:

0.1 mL of the vehicle, SND 919 Thiazol or positive control solution, 0.1 mL of a bacterial shaking culture (6 h, exponential phase) and 0.5 mL phosphate buffer or S9 mix were added to 2 mL soft agar. After vortexing, these mixtures were overlaid immediately onto minimal medium plates in triplicate (n = 6 for the negative controls).

The test was performed in the presence and absence of rat liver microsomal enzymes (S9 mix). The plate test method was used since the previous Ames study with
SND 919 Thiazol Br was also performed using this method.

Rationale for test conditions:

The assay was considered valid since all tester strains exhibit a characteristic number of spontaneous revertants per plate as reported in the literature and/or close to our in house historical negative control data ranges (non-GLP).

The addition of the metabolic activation system did not alter the number of spontaneous revertants per plate and therefore these numbers were combined.

In addition, the assay is considered valid since the reference mutagens induced a distinct increase in the number of revertants reflecting also the activity of the metabolizing system. The number of revertants of the respective positive controls were calculated from data obtained in our laboratory from April 2011 to February 2014 for the S. typhimurium strains and from March 2013 to February 2014 for the E.coli strain

Evaluation criteria:

The assessment and interpretation of the results follows the OECD Guideline No. 471. In addition, the historical negative control data ranges experienced in our laboratory were also considered though for orientating purposes only.

Statistics:

Revertant his+ colonies were counted after incubation at 37°C for 2 days using the Sorcerer colony counter and the values were captured with the Ames Study Manager. For all replicate platings, the mean number of revertants per test concentration was calculated automatically by the computerized system. The condition of the background bacterial lawn (residual growth on minimum histidine) was evaluated macroscopically for evidence of bacteriotoxicity induced by SND 919 Thiazol. If extreme thinning or complete lack of the microcolony lawn compared to the negative, vehicle control plates was observed, no revertants were counted.
Evidence of precipitates in the agar overlay was recorded.

Results and discussion

Test results

Test results 1

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:

not specified

Positive controls validity:

valid

Test results 2

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:

not specified

Positive controls validity:

valid

Test results 3

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:

not specified

Positive controls validity:

valid

Test results 4

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:

not specified

Positive controls validity:

valid

Test results 5

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:

not specified

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

SND 919 Thiazol caused neither base-pair substitutions nor frameshift mutations in different S. typhimurium strains and one E. coli strain in the absence and presence of metabolic activiation system when tested up to the recommended concentration. Based on these results ist was concluded, that SND 919 Thiazol is "Ames negative".