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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:
Experiments were carried out between 1991-06-20 and 1992-05-14.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Only 4 strains of S. typhimurium tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Principles of method if other than guideline:
There was the following deviation from the requirements of the above mentioned principles:
- Analytical investigation (stability of the test substance in DMSO and aqua dest) was not carried out.

The Ames-standard plate test (SPT) was carried out according to:
- Ames BN et al (1973). Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci USA 70: 2281-2285; and
- Ames BN et al (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res 31: 347-364.

The Ames-preincubation test (PIT) was carried out according to:
- Yahagi T et al (1977). Mutagenicities of N-nitrosoamines in Salmonella. Mutat Res 48: 121-130; and
- Matsushima T et al (1980). Factors modulating mutagenicity in microbial tests. In: Short-Term Test Systems for Detecting Carcinogens. Edited by Norpoth KH and Garner RC. Springer Verlag Berlin, Heidelberg, New York.

The Liquid suspension assay (LSA) was carried out according to:
- Rannug U et al (1976). The mutagenicity of chloroethylene oxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid, conceivable metabolites of vinyl chloride. Chem Biol Interaction 12: 251-263; and
- Lutz D et al (1982). Structure-mutagenicity relationship in alpha,beta-unsaturated carbonylic compounds and their corresponding allylic alcohols. Mutat Res 93: 305-315
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(E)-3-formylbut-2-enyl acetate
EC Number:
247-825-4
EC Name:
(E)-3-formylbut-2-enyl acetate
Cas Number:
26586-02-7
Molecular formula:
C7H10O3
IUPAC Name:
(2E)-3-methyl-4-oxobut-2-en-1-yl acetate
Details on test material:
- Name of test material (as cited in study report): C5-Acetat
- Physical state: colourless liquid
- Analytical purity: 98.8%
- Impurities (identity and concentrations): no data
- Lot/batch No.: no data; date of manufacturing: 1991-04-05
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: the stability of the test substance throughout the study period was verified analytically by reanalysis
- Storage condition of test material: room temperature (N2 conditions)

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9
Test concentrations with justification for top dose:
4 - 5000 µg/plate (SPT)
4 - 1000 µg/plate (PIT)
4 - 1250 µg/plate (LSA)
For details, see freetext.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, N-methy-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine, 9-aminoacridine chloride monohydrate, crotonaldehyde, methylvinyl ketone
Remarks:
Positive control substances depended on the tester strain and test conditions.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension
Five independent experiments were carried out; see freetext.

NUMBER OF REPLICATIONS: 3 plates per dose and control

DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth, decrease in the number of his+ revertants
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfil the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
>= 500 µg/plate; see additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 µg/plate; see additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 µg/plate; see additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance was completely soluble; no precipitation was noted in any experiment under any test condition.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect was observed at doses >= 1500 µg/plate (SPT; all tester strains), at 1000 µg/plate (PIT; TA 1537 and TA 98) and at doses >= 500 µg/plate (LSA; TA98).
Remarks on result:
other: other: all strains (SPT with and without S-9; PIT with and without S-9; LSA with S-9);strains TA 1535 and TA1537 (LSA without S-9)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No increase in the number of revertants was observed for:

·        SPT without S-9; all tester strains

·        SPT with S-9; all tester strains

·        PIT without S-9; all tester strains

·        PIT with S-9; all tester strains

·        LSA without S-9; tester strains TA 1535 and TA 1537

·        LSA with S-9; all tester strains.

Controversial results were observed in the LSA without S-9 for TA 98 (slightly enhanced figures at 500 - 1000 µg/plate; factor 1.8-2.0; this could not be confirmed by a second experiment).

Positive reaction was observed in the LSA without S-9 for TA100 from about 250 µg/plate (factor 1.8) onward with an increase in the number of mutant colonies by a factor of 8.1 at 1250 µg/plate.

Positive controls gave the expected results.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: negative in the standard Ames Test( Standard Plate Test and in the Preincubation Test); positive in the modified Ames Test (Liquid Suspension Assay)

According to the authors, the test substance is mutagenic in a modified Ames test (liquid suspension assay) under the experimental conditions chosen.