L-Aspartic acid, N,N'-1,2-ethanediylbis-, sodium salt (1:3)

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:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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:

-

EC Number:

416-530-4

EC Name:

-

Cas Number:

178949-82-1

Molecular formula:

C10H13N2Na3O8

IUPAC Name:

trisodium 2-({2-[(1-carboxy-2-carboxylatoethyl)amino]ethyl}amino)butanedioate

Details on test material:

- Name of test material (as cited in study report): trisodium EDDS (under code)
- Substance type: technical product
- Physical state: clear, colourless liquid
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: 1 January 1998
- Stability under test conditions: no data
- Storage condition of test material: room temperature, protected from light

Method

Target gene:

Histidine, reversion to histidine independence (S. typhimurium strains); tryptophan, reversion to tryptophan independence (E. coli strains)

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA98, TA100, TA1535, TA1537, TA1538, Escherichia coli: WP2 (pKM101) and WP2 uvrA (pKM101)

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9, derived from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 and 5000 ug/plate

Vehicle / solvent:

Solvent: water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 only. At 1.0 ug/plate (TA1535, TA1537, TA1538, TA98, TA100); 10 ug/plate (WP2 uvrA (pKM101)); 10 and 30 ug/plate (WP2 (pKM101)).

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 only. At 1.0 ug/plate (TA1538 and TA98)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 only. At 1.0 ug/plate (TA1535, TA100)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 only. At 75 ug/plate (TA1537)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 only. At 1000 ug/plate (WP2 (pKM101), WP2 uvrA (pKM101))

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (used to confirm negative results in plate incorporation assay)

DURATION
- Preincubation period: 20 min
- Exposure duration: incubated for 48 h in agar

SELECTION AGENT (mutation assays): medium deficient in histidine (S. typhimurium) or tryptophan (E. coli) to select for mutants not requiring these amino acids for growth

NUMBER OF REPLICATIONS: preincubation tubes and plates prepared in triplicate

NUMBER OF CELLS EVALUATED: 10(8)

DETERMINATION OF CYTOTOXICITY
- Method: background lawn of non-revertant colonies, reduction in the lawn indicates toxicity

Evaluation criteria:

Dose-related increase in revertants for as least one tester strain with a minimum of two increasing concentrations of the test substance. TA1535, TA1537 and TA1538 were considered positive if the mutant colonies at the peak of the dose-response were equal to, or greater than, three times the mean vehicle control value. For the plasmid-bearing strains, the test was considered positive if the increase was at least double that of the vehicle control value

Statistics:

not applicable

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

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 applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli, other: WP2 (pKM101)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: none

RANGE-FINDING/SCREENING STUDIES: testing at up to 5000 ug/plate with all of the bacterial strains showed no evidence of cytotoxicity or precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA: data on acceptable ranges for spontaneous mutants for each of the tester strains is given in the report

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative

In a GLP study conducted according to a similar protocol to OECD Guideline 471, trisodium EDDS (in water) showed no mutagenic potential in a bacterial mutagenicity assay (Ames test) when tested at up to 5 mg/plate in five strains of S. typhimurium and two strains of E. coli, both in the presence and absence of a rat liver metabolic activation fraction.

Executive summary:

In a GLP study conducted according to the published methods of McCann and Ames (1976) and Maron and Ames (1983), trisodium EDDS (in water) was assessed for its ability to induce mutations in a bacterial assay for mutagenicity (the Ames test). This protocol is similar to that described in OECD Guideline 471.

Using Salmonela typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and Escherichia coli strains WP2 (pKM101) and WP2 uvrA (pKM101), trisodium EDDS was tested at a concentration range of 33-5000 ug/plate in the standard plate incorporation assay and by the preincubation method, both with and without addition of a rat liver metabolic activation fraction (S9). The concentrations used were determined by a range-finding study which showed no cytotoxicity or precipitation of the test substance at 5 mg/plate.

No increase in the mutation frequency was evident in any of the tester strains, both with and without S9, when compared to the vehicle controls.

In conclusion, trisodium EDDS showed no mutagenic potential in a bacterial mutagenicity assay (Ames test) when tested at up to 5 mg/plate in five strains of S. typhimurium and two strains of E. coli, both in the presence and absence of S9.