Dipotassium [[N,N'-ethylenebis[N-(carboxylatomethyl)glycinato]](4-)-N,N',O,O',ON,ON']zincate(2-)

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:

October-November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']manganate(2-)

EC Number:

239-407-5

EC Name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']manganate(2-)

Cas Number:

15375-84-5

Molecular formula:

C10H12N2O8MnNa2

IUPAC Name:

Disodium; 2-[2-(bis(carboxylatomethyl)amino)ethyl-(carboxylatomethyl)amino]acetate; manganese(+2) cation

Details on test material:

Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Purity: 92.3%
Batch no: CFC 9380
Expiry date: 31 August 2012

Method

Target gene:

Point mutations in one or more DNA base pairs

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 62, 185, 556, 1667, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffered saline
- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: The following positive controls were used in the absence of S9-mix: sodium azide (TA1535 and TA100), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98), N-ethyl-N-nitrosourea (WP 2 uvrA). The following positive controls were used in the presence of S9-mix:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10-16 h
- Exposure duration: 48-72 h
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth compared to the vehicle control

OTHER EXAMINATIONS: none

Evaluation criteria:

Positive in case if the mean number of revertant colonies is concentration-related increased or if a reproducible 2-fold or more increase is observed compared to the negative control. The test is considered valid if the mean colony counts of the vehicle control are within acceptable ranges, if the results of the positive control meets the criteria for a positive response and if no more than 5% of the plates are lost through contamination or other unforeseen events.

Statistics:

Not needed.

Results and discussion

Test results

Test results 1

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:

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:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: no problem
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: not done

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test substance was not toxic to any strain

Applicant's summary and conclusion

Conclusions:

The results obtained in this study using tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA, in both the absence and presence of S9-mix, indicated that EDTA-MnNa2 is not mutagenic.

Executive summary:

The test substance, EDTA-MnNa2, was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella

typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a

liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance was dissolved in phosphate buffered saline (PBS). One

bacterial reverse mutation test was performed. All strains were used, in the absence and presence of S9-mix, with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (PBS) and positive controls were run simultaneously with the test substance. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected

increase in the mean number of revertant colonies. The test was considered valid. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In both the absence and presence of S9-mix in all strains, EDTA-MnNa2 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that EDTA-MnNa2 is not mutagenic under the conditions employed in this study.