2-[(2-methyl-1-oxoallyl)oxy]ethyl acetoacetate

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:

16/November/1990 - 11/December/1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted in accordance with international guidelines.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: light yellow liquid

Details on test material:

Name: P5306 (Methacrylsäure-2-Acetoacetoxyethylester)
Batch received: 15/November/1990
Storage: in the dark at ambient temperature

Method

Target gene:

The purpose of this study was to establish the potential of P5306 to induce gene mutations in the bacterium Salmonella typhimurium, causing
the histidine-dependent strains to revert to the wild-type phenotype. The particular damage induced to DNA by a mutagen may cause reversions
to occur which are observable in certain strains of Salmonella typhimurium, but not in others. 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):

not applicable.

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):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat Arochlor 1254 induced microsomal fraction (S9-mix)

Test concentrations with justification for top dose:

15 ug/plate, 50 ug/plate, 150 ug/plate, 500 ug/plate, 1500 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

other: 2-aminoanthracene (2-AAN)

Details on test system and experimental conditions:

Test Solution:
------------
P5306 and the positive control substances, except sodium azide, were dissolved in dimethylsulphoxide. Sodium azide was dissolved in sterile,
ultra-pure water.

Inducer:
--------
The polychlorinated biphenyl mixture, Aroclor 1254, was obtained from Monsanto (UK) Limited, Newport, Wales.

Agar Plates:
-----------
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames test were obtained from Becton Dickinson Limited, Scotland.

Animals:
--------
Male Fischer 344 rats were obtained from Charles River (UK).

Preparation of Bacteria:
--------------------
Samples of each strain were grown by culturing for 16 h at 37°C in nutrient broth (25 g Oxoid Nutrient Broth NO.2). These cultures were kept for up
to 2 days at +4°C, used within that period, then discarded.

Preparation of the Metabolic Activation System:
------------------------------------------
ANIMALS
Male Fischer 344 rats (average weight 256 g) were injected once intraperitoneally with Aroclor 1254 (diluted in corn oil to a concentration of 200 mg.ml-1) at a dosage of 500 mg.kg-1. They were allowed drinking water continuously, but food was withheld for 16 h before they were killed in an
atmosphere of carbon dioxide 5 days after injection.

Preparation of the Assay Plates:
----------------------------
Diluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was autoclaved and, just before use, 5 ml of sterile 1.0 mM L-histidine.HC1, 1.0 mM biotin solution
was added to each 100 ml of soft agar and thoroughly mixed. This molten agar, maintained in a water bath at 45°C, was dispensed in 2 ml volumes
into small sterile tubes to which were added in order:
0.5 ml S9 mix or 0.05 M phosphate buffer, pH 7.4
0.1 ml bacteria (ca 2 x 109 cells.ml-1)
0.1 ml solvent or test solution
The tube contents, which were continually cooling, were mixed and then poured in minimal medium plates obtained from Becton Dickinson
limited, Scotland. These plates contained 25 ml of 1.7% Gibco Bacteriological agar in Bonner Medium E (2) with 2% glucose.
When the soft agar had set,· the plates were inverted and incubated at IRI 750756 14 37°C for 2 days whereupon colonies were counted using a Biotran III automated counter (New Brunswick Incorporated, NJ, USA) at maximum sensitivity ie colonies of 0.1 mm or more in diameter counted. The
plates were also examined for precipitates and, microscopically, for microcolony growth.

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, TA 1538 and TA 98, 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 I.S-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

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 1538

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium 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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity Test:
------------
There was no toxicity to the bacterial strain TA 100 in either the presence or absence of S9 mix at any concentration tested.
There was no precipitation of the test substance.

Any other information on results incl. tables

Quality Control:

All strains of S. typhimurium 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 10 s from a CAMAG u.v. 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.

Positive Control Groups:

The positive control values were generally within the range expected for each bacterial strain and activation system.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No mutagenic activity was observed in either the presence or absence of S9 mix. There was no toxicity to the bacteria in any of the experiments performed. There was no precipitation of the test substance. It was concluded that the test item was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide up to the predetermined maximum limit of 5000 µg per plate.

Executive summary:

A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) was carried in year 1990. Tested up to 5000 ug/plate without and with metabolic activation the test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA 1535; TA 1537; TA 1538; TA 98 and TA 100). The positive control values were generally within the range expected for each bacterial strain and activation system. There was no toxicity to the bacteria in any of the experiments performed. There was no precipitation of the test substance. It was concluded that the test item was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide up to the predetermined maximum limit of 5000 µg per plate.