2-chloro-N,N-dimethyl-3-oxobutyramide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

2-chloro-N,N-dimethyl-3-oxobutyramide

EC Number:

227-372-9

EC Name:

2-chloro-N,N-dimethyl-3-oxobutyramide

Cas Number:

5810-11-7

Molecular formula:

C6H10ClNO2

IUPAC Name:

2-chloro-N,N-dimethyl-3-oxobutanamide

Constituent 2

Reference substance name:

Butanamide, 2-chloro-N,N-dimethyl-3-oxo

IUPAC Name:

Butanamide, 2-chloro-N,N-dimethyl-3-oxo

Details on test material:

Name:DIMETHYL-2-CHLORACETOACETAMID
Batch No.: not indicated
CAS No.: 005810-11-7
Aggregate State at RT: liquid
Colour: brown
Analysis: not indicated by the sponsor
Stability: Pure: stable
Storage: 20 0 C, light protected the test article was stored at 40 C

Method

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:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment 1: TA98 and TA 100: 33.3; 100.0; 333.3; 1000.0; 2500.0; 5000.0 µg/plate
Experiment 1: TA 1535, TA 1537, TA 102: 10.0; 33.3; 100.0; 333.3; 666.6; 1000.0 µg/plate
Experiment 2, all strains: 10.0; 33.3; 100.0; 333.3; 666.6; 1000.0 µg/plate

Vehicle / solvent:

water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA1535, TA 100, without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

for TA98, TA 1537, without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for TA102, without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

all strains with metabolic activation

Details on test system and experimental conditions:

Pre-Experiment for Toxicity
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. Dose Selection
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
Due to toxic effects, the maximum concentration was 5000.0 flg/plate (strains TA 98 and TA 100) in the pre-experiment and in experiment I and 1000.0 flg/plate (strains TA 1535, TA 1537, and TA 102) in experiment I and in experiment II (all strains). The con-centration range included two logarithmic decades. In this study six adequately spaced concentrations were tested. Two independent experiments were performed. As the re-sults of the pre-experiment were in accordance with the criteria, these data are reported as a part of the main experiment
According to the dose selection criteria the test article was tested at the following
concentrations:
Exp. I; strains TA 98 and TA 100:33.3; 100.0; 333.3; 1000.0; 2500.0; 5000.0 µg/plate
Exp. I; strains TA 1535, TA 1537, TA 102; Exp: II; all strains: 10.0; 33.3; 100.0; 333.3; 666.6; 1000.0 µg/plate
Experimental Performance
For each strain and dose level, including the controls three plates were used as a mini-mum. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µI Test solution at each dose level, solvent (negative control) or reference mutagen
solution (positive control),
500 µI S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test
without metabolic activation),
100 µI Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µl Overlay agar
In the pre-incubation assay 100 fll test solution, 500 fll S9 mix I S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes.
After pre-incubation 2.a ml overlay agar (45° C) was added to each tube. The mmixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
A test article is considered mutagenic if the number of reversions is at least twice the
spontaneous reversionrate in strains TA 100 and TA 102 or thrice on TA 1535, TA 1537,
and TA 98 (4).
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Results and discussion

Test results

Test results 1

Species / strain:

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

Test results 2

Species / strain:

S. typhimurium TA 102

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test article Dimetbyl-2-ch1oroacetoacetamide (97.3% pure) was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 according to OECD guideline 471.
The assay was performed in two independent experiments both with and without liver micro-somal activation. Each concentration and the controls, were tested in triplicate. The test article was tested at the following concentrations:
Exp.I; strainsTA98 andTA 100: 33.3; 100.0;333.3;1000.0;2500.0;5000.0µg/plate
Exp.l; strainsTA 1535,TA 1537,TA 102 and Exp:II; all strains: 10.0; 33.3; 100.0; 333.3; 666.6; 1000.0 µg/plate
Distinct toxic effects, evidenced by a reduction in the number of revertants, occurred in all strains in experiment I. Strain TA 1535 showed toxic effects at 1000.0 µg/plate without S9 mix, strain TA 1537 from 666.6 up to 1000.0 µg/plate with and without S9 mix, strain TA 98 from 1000.0 up to 5000.0 µg/plate without S9 mix, strain TA 100 from 25000 up tom5000.0 µg/plate with and without S9 mix and strain TA 102 from 666.6 up to 1000.0m µg/plate without S9 mix. In experiment II, toxic effects occurred in strain TA 102 at 1000.0m µg/plate with and without S9 mix.
The plates incubated with the test article showed normal background growth up to the highest investigated dose with and without S9 mix in all strains used. No substantial increases in rever-tant colony numbers of any of the five tester strains were observed following treatment with Di-metbyl-2-ch1oroacetoacetamide at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with in-creasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a dis-tinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the ex-perimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test article Dimetbyl-2-ch1oroacetoacetamide (97.3% pure) was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 according to OECD guideline 471.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test article was tested at the following concentrations:

Exp.I; strainsTA98 andTA 100: 33.3; 100.0;333.3;1000.0;2500.0;5000.0µg/plate

Exp.l; strainsTA 1535,TA 1537,TA 102 and Exp:II; all strains: 10.0; 33.3; 100.0; 333.3; 666.6; 1000.0 µg/plate

Distinct toxic effects, evidenced by a reduction in the number of revertants, occurred in all strains in experiment I. Strain TA 1535 showed toxic effects at 1000.0 µg/plate without S9 mix, strain TA 1537 from 666.6 up to 1000.0 µg/plate with and without S9 mix, strain TA 98 from 1000.0 up to 5000.0 µg/plate without S9 mix, strain TA 100 from 25000 up tom5000.0 µg/plate with and without S9 mix and strain TA 102 from 666.6 up to 1000.0m µg/plate without S9 mix. In experiment II, toxic effects occurred in strain TA 102 at 1000.0m µg/plate with and without S9 mix.

The plates incubated with the test article showed normal background growth up to the highest investigated dose with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with Dimetbyl-2-ch1oroacetoacetamide at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.