2-cyano-N-methylacetamide

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

Study period:

2000-11-07 - 2001-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-cyano-N-methylacetamide

EC Number:

228-705-0

EC Name:

2-cyano-N-methylacetamide

Cas Number:

6330-25-2

Molecular formula:

C4H6N2O

IUPAC Name:

2-cyano-N-methylacetamide

Details on test material:

- Name of test material: Methylcyanacetamid in Lsg. B. 100 %
- Storage condition of test material: at room temperature

Method

Target gene:

histidine (his-) and tryptophan (trp-)

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:

Mammalian liver post-mitochondrial fraction (S-9) was prepared from 5 male Sprague-Dawley rats with a single intraperitoneal injection of Aroclor 1254 according to Ames et.al.

Test concentrations with justification for top dose:

Standard plate test (SPT): 125 µg - 31,250 µg/plate
Preincubation test (PIT): 125 µg - 31,250 µg/plate

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: Water was chosen due to the good solubility of the test substance

Controls

Untreated negative controls:

yes

Remarks:

Sterility control (soft agar, S9-mix, buffer, vehicle/test substance, but without test strains)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

different for with and without metabolic activation test strains

Positive control substance:

other: With S-9 mix: 2-aminoanthracene (2-AA) / Without S-9 mix: TA 1535, TA 100: N-methyl-N´-nitro-N-nitrosoguanidine (MNNG); TA 98: 4-nitro-o-phenylendiamine (NOPD); TA 1537: 9-aminoacridine (AAC); E.coli WP2 uvrA: 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

SALMONELLA TYPHIMURIUM
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et.al.
Test tubes containing 2 mL portions of soft agar (overlay agar), which consists of 100 mL agar (0 .6 % agar + 0 .6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants : 0.5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle,
ca. 0.1 mL fresh bacterial culture,
0.5 mL S-9 mix (in tests with metabolic activation) or 0 .5 mL phosphate buffer (in tests without metabolic activation ).
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar :
980 mL aqua dest.,
20 mL Vogel-Bonner E medium,
15 g Difco bacto agar,
20 g D-glucose, monohydrate.

ESCHERICHIA COLI
The experimental procedure is based on the method of Ames et.al.
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 mL agar (0 .6 % agar + 0 .6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants : 0.5 mM tryptophan) are kept in a water bath at 45 °C, and the remaining components are added in the following order :
0.1 mL test solution or vehicle,
0.1 mL fresh bacterial culture,
0.5 mL S-9 mix (in tests with metabolic activation ) or 0 .5 mL phosphate buffer (in tests without metabolic activation).
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
Composition of the minimal agar :
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M .H .L. and Muriel, W .J ., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar),
100 mL solution A (saline solution),
8 mL solution C (glucose solution) IIA,
10 mL solution D (casein solution).

DURATION
- Exposure duration: the plates incubated at 37 °C in the dark for at 48 - 72 h

Evaluation criteria:

ACCEPTANCE CRITERIA
Generally, the experiment is to be considered valid if the following criteria are met :
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data .
- The titer of viable bacteria was > 10^9/mL.

EVALUATION CRITERIA
- The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tested strain either without S-9 mix or after adding a metabolizing system.
- A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tested strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No details are available.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed.
SOLUBILITY:
No test substance precipitation was found.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

All results are given as attached background material

Table 1: Results of Salmonella typhimurium TA 1535

Testdate:	07.NOV.2000
Method:	Standard plate test
Volume:	100 µL/plate
Strain:	TA 1535
Vehicle:	Water
S9-MIX	WITHOUT S-9 MIX				WITH S-9 MIX (1:9)
Dose/Plate	REV	M	SD	FAC	REV	M	SD	FAC	TITRE
Water	18	21	3	1.0	15	17	2	1.0	28
	21				19				24
	23				17				19
125 µg	20	20	2	1.0	14	14	4	0.8
	18				18
	22				11
625 µg	15	14	1	0.7	19	15	3	0.9
	15				14
	13				13
3125 µg	11	14	3	0.7	13	15	2	0.9
	16				14
	14				17
15625 µg	14	16	3	0.8	12	11	2	0.6	28
	14				12				22
	19				9				16
31250 µg	12	14	5	0.7	10	9	2	0.5	27
	20				11				20
	11				7				28
MNNG - 5.0 µg	539	598	51	28.9
	630
	625
2-AA - 2.5 µg					119	134	15	7.9
					148
					136
(REV = revertants/plate, M = mean, SD = standard deviation, FAC = factor)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study is considered as valid OECD Guideline 471 study without deviations and was performed under certificated Good Laboratory Praxis (GLP) and therefore considered to be of the highest quality (reliability Klimisch 1). The test material did not induce an increase in the frequency of revertant colonies in any of the bacterial strains, therefore the test material can be considered as non-mutagenic under these test conditions.

Executive summary:

The test substance 2 -cyano-N-methylacetamide was investigated according to OECD TG471 for its potential to cause gene mutation in Salmonella typhimurium strains (TA98, TA100, TA1537, TA1535 and TA1538) and Escherichia coli WP2 uvrA. The Standard plate test (SPT) as well as the Preincubation test (PIT) were performed with test concentrations of 125 µg - 31,250 µg/plate. Due to the good water solubility of the substance, aqua dest. was used as vehicle. The test was performed with and without a metabolic activation system. For this purpose, the mammalian liver post-mitochondrial fraction (S-9) was prepared (Aroclor induced rat liver S-9 mix). Results: No toxic effects were reported. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain as well as in the E.coli strain in comparison with the corresponding controls. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material caused neither base-pair substitutions, nor frameshift mutations. Therefore the test results revealed no indication of gene mutagenic activity.