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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.11.-04.12.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dipotassium propanedioate
EC Number:
820-064-0
Cas Number:
13095-67-5
Molecular formula:
C3H2K2O4
IUPAC Name:
Dipotassium propanedioate
Specific details on test material used for the study:
purity > 95%

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
- the test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
- since no relevant toxic effects were observed, 5000 μg/plate were chosen as maximal concentration.
- concentrations in the experiment I and II: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
aqua dest.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
aqua dest.
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD; 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
aqua dest.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) in Experiment I and pre-incubation in
Experiment II.

DURATION
- Exposure duration: 48h at 37°C

NUMBER OF REPLICATIONS: 3

ACCEPTANCE CRITERIA:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100 and TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data range of the test facility
- corresponding background growth on solvent control, negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analyzable
Rationale for test conditions:
these are according to the OECD guideline 471
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control

A test item producing neither a dose related increase in the number of revertants nor a reproducible
biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- no precipitation of the test item was observed in any tester strain used in experiment I and II (with
and without metabolic activation)
- no toxic effects of the test item were noted in any of the five tester strains used up to the highest
dose group evaluated (with and without metabolic activation) in experiment I and II
Remarks on result:
other: not mutagenic

Applicant's summary and conclusion

Conclusions:
Dipotassium malonate is considered to be non-mutagenic in this bacterial reverse mutation assay
Executive summary:

In the current study the potential of the test item dipotassium malonate to induce gene mutations according to the plate

incorporation test (experiment I) and the pre-incubation test (experiment II) was assessed according to OECD TG 471 and in compliance to GLP.

The plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

31.6, 100, 316, 1000, 2500 and 5000 μg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with dipotassium malonate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, dipotassium malonate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, dipotassium malonate is considered to be non-mutagenic in this bacterial reverse mutation assay.