Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
October-November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study under GLP
Justification for type of information:
Members of the aminocarboxylic acid (ethylenediamine-based) chelants chemical category possess similar molecular structures that contain common functional groups. All members have a molecular structure with an ethylenediamine or a diethylenetriamine backbone, which has 4 or 5 acetic acid groups attached to the nitrogens. The diethylenetriamine struc¬tures contain five acetic acid groups (DTPA); the ethylenediamine structure has four acetic acid groups (EDTA).
Therefore, all category members have identical functional groups (DTPA and EDTA) and have an ethylene-diamine-backbone. It is the presence of multiple carboxylic acid groups on the amine that provides chelants with their unique metal ion chelating or sequestering properties. This common property is the important feature to consider in assessing the aquatic and mammalian toxicity of chelants and in justifying their consideration as a category.
The substantial body of evidence that chelants are not directly toxic to aquatic and mammalian organisms but exert their influence by affecting mineral balance, together with the fact that the backbone structures of the chelants in the category have qualitative similar affinities for metals supports the inclusion of these chelants in a category. Subtle differences in toxicity due to the presence of calcium, magnesium, manganese, ferric (or ferrous) iron, copper or zinc can be explained by their affinity towards these metals and their ability to supply or deny metals to organisms.
As tested substance EDTA-MnNa2 and target substance DTPA-MnNa3 have the same metal cation and the chelating agents (EDTA and DTPA) are very similar structure, the results obtained for EDTA-MnNa2 are also valid for DTPA-MnNa3.

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

Constituent 1
Chemical structure
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other:
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 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:
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
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:
Interpretation of results (migrated information):
negative

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.