2-(acetylamino)-N-benzyl-3-methoxypropanamide

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:

17 October 2016 - 03 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

N-benzyl-2-acetamido-3-methoxypropanamide

EC Number:

700-539-8

Cas Number:

175481-26-2

Molecular formula:

C13H18N2O3

IUPAC Name:

N-benzyl-2-acetamido-3-methoxypropanamide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder

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 Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with 5% (v/v) S9-mix): TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with 5% (v/v) S9-mix: 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2: all strains:
Without and with 10% (v/v) S9-mix: 492, 878, 1568, 2800 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance was dissolved in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (in DMSO) for all tester strains

Remarks:

with S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191: 2.5 μg/plate (in DMSO) for TA1537

Remarks:

without S9-mix

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

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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 follow up experiment.

Results and discussion

Test results

Test results 1

Key result

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The 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.

TA1535 TA1537 TA98
S9-mix - + - + - +
Range 78 - 1381 78 - 1058 55 – 1565 55 – 1112 410 – 2057 263 - 1907
Mean 785 228 653 387 1155 860
SD 167 105 290 143 370 323
n 1684 1662 1448 1536 1646 1686

TA100 WP2uvrA
S9-mix - + - +
Range 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean 892 1404 1263 342
SD 178 327 461 165
n 1650 1677 1370 1410

- The negative control values were within the laboratory historical control data ranges, except the response for TA1537 in the absence and presence of S9-mix, first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (2 revertant colonies) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered to be not affected.

TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 4 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 12 – 68 12 - 70
Mean 14 13 7 9 17 25 100 103 26 32
SD 6 5 3 4 5 7 16 18 7 8
n 1662 1677 1548 1547 1662 1703 1659 1691 1421 1424

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Applicant's summary and conclusion

Conclusions:

The mutagenic activity of SPM20200 was evaluated in accordance with OECD 471 (1997) and according to GLP principles. Based on the results of this study 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 metabolic activation.

Executive summary:

The mutagenic activity of SPM20200 was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The substance was tested up to and including 5000 µg/plate in all tester strains (as no cytotoxicity and/or precipitation of the test substance was observed). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). Adequate solvent and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. Based on the results of this study 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 metabolic activation.