potassium {[(2Z)-4-ethoxy-4-oxobut-2-en-2-yl]amino}acetate

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:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The Study was conducted in compliance with GLP and according to the OECD guideline 471 (bacetrial reverse mutation assay)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

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

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:

32085-79-3

EC Number:

608-700-3

Cas Number:

32085-79-3

IUPAC Name:

32085-79-3

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

TA 98: hisD3052, rfa, uvrB
TA 100: hisG46, rfa, uvrB
TA 102: hisG428 (pAQ1), rfa
TA 1535: hisG46, rfa, uvrB
TA 1537: hisC3076, rfa, uvrB

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat)

Test concentrations with justification for top dose:

25-5000 µg/plate

Vehicle / solvent:

Water for the test item and the positive controls Sodium Azide and Mitomycin C as well as DMSO (dimethyl solfoxide) for the positive controls 2-Nitrofluorene, 9-Aminoacridine, 2-Aminofluorene and 2-Aminoanthracene.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Water or DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive control substance:

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st main experiment: in agar (plate incorporation):
ln a sterile tube,
0.1 ml of the appropriately diluted test material
(or 0.1 ml of the solvent)
(or 0.1 ml [0.05 ml for TA 1535] of the strain specific positive control item)
(or 0.05 ml of the positive control items for proving metabolic activation)
0.5 ml phosphate buffer (or 0.5 ml 89 mix in the experiment with metabolic activation) 2 ml of molten trace histidine supplemented top agar at approx. 45°C 0.1 ml of the bacterial overnight culture were mixed.
Mixing was done in triplicate, for each bacterial strain and for each concentration of the test material. The mixture was then poured onto the surface
of minimal agar plates. These plates were incubated at 37° for 72 hours and then the number of revertant colonies was counted.
2nd main experiment: preincubation
ln a sterile tube a 0.1 ml aliquot of each one of the bacterial overnight cultures was mixed with a 0.5 ml volume of 89 mix (for tests with metabolic activation) or phosphate-buffer (for tests without metabolic activation). Then either 50 !JI of the positive controls 2-Aminofluorene and
2-Aminoanthracene (+S9), 100 µI of the solvent, or 100 µI of the test item solution were added. The tubes were incubated at 30°C for 30 min with gentle agitation. At the end of the incubation period, 2 ml of molten trace histidine supplemented top agar was added to each
tube, mixed briefly and poured onto minimal agar plates. These plates were incubated at 37°C for 72 hours and then the number of revertant
colonies was counted.

DURATION
- 1st main experiment: 72 hours incubation (37°C)
- 2nd main experiment: 30 min preincubation (30°C), 72 hours incubation (37°C)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
determination of background lawn:

Evaluation criteria:

For a test item to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test item. A test item that does not meet these criteria was called non-mutagenic in bacteria. Single increases in revertants frequencies, which are not dose-related and not reproducible in two independent experiments were considered non-relevant.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

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 with metabolic activation
negative without metabolic activation

Gly-Dane-salt did not induce a mutagenic effect in S. typhimurium with and without metabolic activation. lt is therefore not considered to be a bacterial mutagen.

Executive summary:

Gly-Dane-salt did not induce a mutagenic effect in S. typhimurium with and without metabolic activation. lt is therefore not considered to be a bacterial mutagen.