Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

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:

01 July 2013 - 22 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

EC Number:

691-061-8

Cas Number:

1001320-38-2

Molecular formula:

C10H19NO2S2

IUPAC Name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

Test material form:

solid

Remarks:

yellow grease-like solid

Specific details on test material used for the study:

Molecular formula: C10H19NO2S2
Name: n-butoxycarbonyl n-butyl dithiocarbamate (active ingredient)
CAS Number: 1001320-38-2

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test: TA100 and WP2uvrA
Without and with S9-mix: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2: all strains
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 3: WP2uvrA
Without S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble and stable in DMSO and DMSO has been accepted and approved by authorities and international guidelines.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR 191 // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Not performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1537, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

but tested up to precipitating concentrations of 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at 1000 μg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Dose range finding test: Precipitation of S-10903 on the plates was not observed at the start or at the end of the incubation period in both tester strain TA100 and WP2uvrA.
First mutation experiment: At the end of the incubation period, precipitation of S-10903 on the plates was observed at all three tester strains TA98, TA1535 and TA5137 at concentrations of 3330 and 5000 μg/plate. Except in tester strain TA1535 (absence of S9-mix), where precipitation of S-10903 on the plates was already observed at 1000 μg/plate.
Second mutation experiment: precipitation of S-10903 on the plates was observed at the end of the incubation period at test substance concentrations of 1000 μg/plate and above in all tested strains.

RANGE-FINDING/SCREENING STUDIES:
- A slight to moderate reduction of the bacterial background lawn was observed at tested concentrations of 1000 μg/plate and above in tester strain TA100 in the absence of S9-mix.
- A moderate reduction in the number of revertant colonies was observed at test substance concentrations of 3330 and 5000 μg/plate both in the absence and presence of S9-mix in tester strain TA100 only. In the tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mean plate count of the positive control was for several strains not within the laboratory historical range: First experiment TA1535 (absence of S9), Second experiment: TA1535 and WP2uvrA (absence of S9) and TA100 (presence of S9-mix).
Evaluation: The values were above the limit of the range. These values were more than 3 times greater than the concurrent solvent control values, therefore this deviation in the mean plate count of the positive controls had no effect on the results of the study. The study integrity was not adversely affected by the deviation.
- In all other strains, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
TA1535 and TA1537: without S9: 1000 µg/plate and above
TA1537: with S9: 3330 and 5000 µg/plate
TA98 and TA1535 with S9: no toxicity was observed
Experiment 2:
TA1535, TA1537 and TA100: without S9: 1000 µg/plate and above
TA98, WP2uvrA and TA100, TA1535, TA1537 with S9: no toxicity was observed
- No mutagenicity was observed up to and including the top dose of 5000 µg/plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with and without S9-mix.

Executive summary:

The genetic toxicity of the substance was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and Escherichia coli WP2uvrA strain, in accordance with OECD 471 guideline and GLP principles.

All bacterial strains showed negative responses up to the highest tested concentration of 5000 μg/plate, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments. In several strains cytotoxicity and precipitation was observed.

Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with and without S9-mix.