Methyl 3-aminocrotonate

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:

22 October 1999 - 29 November 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Expiry date: 21 May 2000
Batch: 624

Specific details on test material used for the study:

Stored in the dark at ambient temperature.

Method

Target gene:

The purpose of this study was to establish the potential of LZ937 to induce gene mutations in Salmonella typhimurium and Eschericha coli.
The specific damage caused by a mutagen may not be detectable using a particular strain of bacteria. This is because the DNA site coding
for a feature selected in the test system may not be mutated by the type of agent under examination. It is necessary, therefore, to use a variety
of bacterial strains in order to test for a broad range of chemical mutagens. At the present time, available data suggest that the use of the 5 strains
used in this project permits the detection of a wide spectrum of mutagens.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

Mutations in the histidine operon, his G 46.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

Mutations in the histidine operon, his C 3076

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Mutations in the histidine operon, his D 3052.

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Mutations in the histidine operon, his G 46.

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain contains an ochre mutation in the trpE locus and can be mutated to tryptophan independence either by a base-pair reversion of an A-T base-pair in the trpE locus or, more likely, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromosome.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 enzymes (the supernatant of the post-mitochondial 9000 g fraction) were prepared from the livers of adult, male Fischer rats, as described by Ames eta/ (1975).

Test concentrations with justification for top dose:

17 ug/plate, 50 ug/plate, 167 ug/plate, 500 ug/plate, 1667 ug/plate and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide

Controls

Untreated negative controls:

no

Remarks:

Concurrent solvent control only

Negative solvent / vehicle controls:

yes

True negative controls:

no

Remarks:

Concurrent solvent control only

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (2-AAN)

Details on test system and experimental conditions:

Test Solution:
Dimethylsulphoxide (DMSO) was used to dissolve and dilute LZ937.
Vehicle Control:
Dimethylsulphoxide (DMSO) was used as the vehicle control.
Inducer:
The inducer was the polychlorinated biphenyl mixture, Aroclor 1254.
Agar Plates:
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames test were prepared in-house using purified agar.
Animals:
The animals used to prepare the activation system were male Fischer 344 rats.
Preparation of Bacteria:
Samples of each strain were grown by culturing for 16 h at 3]DC in nutrient broth (25 g Oxoid Nutrient Broth No.2 per litre). These cultures were kept for up to
2 days at +4 oc to allow relevant checks to be performed but fresh cultures were used for the experiments.
Preparation of the Assay Plates:
Diluted agar {0.6% Difco Bacto-agar, 0.6% NaCJ) was sterilised by autoclaving. L-histidine and biotin solutions, and L-tryptophan solutions were sterilised by
filtration. For use with the S. typhimurium strains, sterile 1.0 mM L-histidine.HCI/1.0 mM biotin solution was added, 5 ml per 1 00 ml of soft agar.
For E. coli WP2uvrA, 1.0 ml of 1.35 mM L-tryptophan was added per 100 ml agar. The agars {with additions) were thoroughly mixed prior to use and
maintained in a water bath at 45°C.

Evaluation criteria:

Evaluation:
----------
A test was considered acceptable if for each strain:
i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
ii) at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 5-35.
iii) on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean
vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for
assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for s. typhimurium strains TA 1535, TA 1537, and TA 98 and E.coli at least a doubling of the mean concurrent vehicle control values at some
concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the
vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required
before a significant mutagenic response was identified.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally,
(2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.

Statistics:

no data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

First mutation assay

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

first mutation assay

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:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

first mutation assay

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:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

first mutation assay

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:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

first mutation assay

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:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

second mutation assay

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

second mutation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Species / strain:

E. coli WP2 uvr A

Remarks:

Confirmatory test 1 and 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity Test:
------------
No toxicity to the bacteria was observed. No precipitation of the test material occurred.

Any other information on results incl. tables

Quality Control:

All strains were sensitive to crystal violet, whereas only the plasmid-containing strains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity

to u.v. light emitted over a period of 5-10 s from a lamp set at 254 nm. Increased sensitivity to u.v. light was demonstrated. These results are consistent with the known properties

of these bacteria.

Vehicle Control Groups:

The vehicle control values were generally within the normal ranges experienced in this laboratory and reported in the literature with these strains of S. typhimurium and E. coli,

(Ames eta/, 1975; Gatehouse eta/, 1994).

Positive Control Groups:

The results obtained in the positive control groups were within the normal ranges expected for each bacterial strain and activation condition.

Applicant's summary and conclusion

Conclusions:

It was concluded that LZ937 was very weakly mutagenic with Escherichia coli only (in the presence of S9 mix) and not the Salmonella typhimurium strains
when tested in dimethylsulphoxide up to a predetermined maximum limit.

Executive summary:

A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) was carried out in year 1999. Tested up to 5000 ug/plate without and with metabolic activation with five tester strains (TA 1535; TA 1537; TA 98; TA 100 and E coli WP2uvrA).

In the first mutation assay, no mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition.

In the second mutation assay, mutagenic activity was observed with Escherichia coli only and in the presence of S9 mix from 500 microg per plate, after incubation for

20 min in the pre-incubation method of treatment. No response was observed with E. coli in the absence of S9 mix or with the Salmonella typhimurium strains

in the presence or absence of S9 mix.

No precipitation of the test material was observed.

It was concluded that LZ937 was very weakly mutagenic with Escherichia coli only (in the presence of 59 mix only) and not the Salmonella typhimurium strains,

when tested in dimethylsulphoxide up to a predetermined maximum limit.