methyl (2Z)-2-(2-nitrobenzylidene)-3-oxobutanoate

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: see 'Remark'

Remarks:

This study followed the procedures indicated by the following internationally accepted gudelines and recommendations: - Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997 - Council Regulation (EC) No 440/2008 B 13/14 Mutagenicity: Reverse Mutation Test Using Bacteria dated May 30, 2008 - EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test" EPA 712-C-98-247, August 1998 - ICH Guideance S2 (R1): Guideance on Genotoxicity testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: CH Guideance S2 (R1): Guideance on Genotoxicity testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012

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:

methyl (2Z)-2-(2-nitrobenzylidene)-3-oxobutanoate

EC Number:

810-021-4

Cas Number:

111304-31-5

Molecular formula:

C12H11NO5

IUPAC Name:

methyl (2Z)-2-(2-nitrobenzylidene)-3-oxobutanoate

Test material form:

solid: crystalline

Details on test material:

ame: Niliden
Cas no.: 111304-31-5
Batch no.: ND-78
Date of analysis: October 2014
Re-test date: October 2015
Storage: In well-closed vial, up to 25°C
Description: a white to pale yellow crystalline substance
Purity by HPLC: 99,6%

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Test concentrations with justification for top dose:

Concentration tested (µg/plate): 500,158, 50, 15.8, 5, 1.58

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Control groups: Dimethyl sulfoxide (DMSO), Ultrapure water

True negative controls:

not specified

Positive controls:

yes

Remarks:

with and without metabolic activation

Positive control substance:

other: 4-nitro-1,2-phenylendiamine ; 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains arrived to the test facility in a form of the disc cultures. The origin of the tester strains: S. typhimurium TA 98 and TA 100.
In addition to histidine mutation ,each strain has additional mutations which enhance its sensitivity to mutagens.
The strains are stored in the Laboratory of Toxi Coop Zrt. in the form of lyophilized discs and at (-) 80 ±10 °C in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO is added as cryoprotective agent.

Evaluation criteria:

The test item is considered mutagenic if:
- a dose-related increase in the number of revertants occur and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
Criteria for a negative response:
A test item is considered non-mutagenic if it produces neither a a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No biologically relevant increases were observed in revertant colony numbers of nay of the examined strains following treatment with Niliden at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reference mutagen treatments ( positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in both tester strains.

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 item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Niliden has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.