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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 S9 (San and Wyman, 1993). In a GLP study conducted according to OECD Guideline 471 (available at the time), trisodium EDDS (in water) exhibited no mutagenic potential when tested at up to 5 mg/plate in an Ames test using five strains of S. typhimurium, with and without S9 (Jones et al. 1989). The related material, EDDS acid, also failed to show any mutagenic activity in a guideline study in five strains of S. typhimurium, with and without S9 (Thompson, 2002).

In a GLP study, trisodium EDDS showed no evidence of mutagenic potential at the tk locus in mouse lymphoma L5178Y tk+/- cells in an in vitro mammalian cell mutation assay when tested at up to about 5 mg/mL, with and without S9 (Bigger and Clarke, 1993).

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS induced statistically significant increases in chromosome numbers and structural aberrations (although the latter effect was not considered biologically relevant) in cultured Chinese hamster ovary cells after a 42-h exposure in the absence of S9. No evidence of numerical or structural chromosome aberrations were seen after 6-h or 18-h exposures without S9, or after a 6-h incubation in the presence of S9. Overall, trisodium EDDS is considered to cause chromosome aberrations in this in vitro mammalian cytogenicity assay (Putman and Curry, 1994).

Overall it can be concluded that trisodium EDDS is not genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

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

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

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)).

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)

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)

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)

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 supplemented with 10% fetal bovine serum, L-glutamine and antibiotics (penicillin and streptomycin)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0, 79, 157, 313, 625, 1250, 2500 and 5000 µg/mL without S9 (6 and 18 h exposure without S9; 6 h exposure with S9)
0, 5, 10, 20, 40, 79, 157, 313, 625 and 1250 µg/mL without S9 (42 h exposure)

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N¿-nitro-N-nitrosoguanidine

Remarks:

2 ug/mL, without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

30 ug/mL, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Exposure period (with and without metabolic activation): 6 h (with a 12 h recovery period)
Exposure period (without metabolic activation): 18 and 42 h

Fixation time: 18 or 42 h (from start of exposure to harvesting of the cells)

SPINDLE INHIBITOR (cytogenetic assays): colcemid added to the cultures 2 h before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate flasks prepared

NUMBER OF CELLS EVALUATED: 200 per dose level (100/flask)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell viability determined by trypan blue exclusion

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:

Gaps were noted, but not included in the total percentage of cells with aberrations or in the frequency of structural aberrations per cell. In the negative controls no cell must have more than two aberrations and the overall frequency must not be greater than 6%. A statistically significant frequency must be evident in the positive controls.

Statistics:

Fischer's exact test was used to compare the percent aberrant cells at each treatment level to that of the controls. The Cochran-Armitage trend test was used to determine dose responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/mL (study 1); 1250 µg/mL (study 2)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

313 µg/mL (study 2)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

numerical aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
none


RANGE-FINDING/SCREENING STUDIES: to determine cytotoxicity at 6, 18 and 42 h exposures without S9 and 6 h exposure with S9.


COMPARISON WITH HISTORICAL CONTROL DATA: yes (details not given)

There was a statistically significant increase in numerical aberrations (p < 0.025) at 20 and 40 ug/mL in study 1 and 2, respectively, after 42 h exposure. Although a statistically significant increase (p < 0.025) was also seen in structural aberrations in study 2 at 40 ug/mL at this exposure time, the percentage increase was within the acceptable range of historical control values, and therefore was considered by the investigators not to be biologically relevant.

Summary of cytogenetic analysis after 42 h exposure without metabolic activation

Treatment	No. cells scored	% Aberrant cells		Structural aberrations per cell
		Numerical	Structural
Study 1
Untreated	200	1.0	3.5	0.035 ± 0.184
Water (vehicle)	200	2.0	2.5	0.025 ± 0.157
Trisodium EDDS  5 ug/mL	200	1.0	1.0	0.010 ± 0.100
10 ug/mL	200	0.5	4.0	0.045 ± 0.231
20 ug/mL	200	27.5	5.5	0.070 ± 0.325
MNNG (2 ug/mL)	200	2.5	18.5	0.385 ± 1.193
Study 2
Untreated	200	0.5	1.0	0.020 ± 0.199
Water (vehicle)	200	0.5	0.0	0.000 ± 0.000
Trisodium EDDS 10 ug/mL	200	0.0	0.5	0.000 ± 0.000
20 ug/mL	200	6.5	0.0	0.000 ± 0.000
40 ug/mL	200	13.5	6.0	0.090 ± 0.416
MNNG (2 ug/mL)	200	1.0	42.0	2.370 ± 3.708

Conclusions:

Interpretation of results:
negative without metabolic activation after 6 or 18 h exposure
negative with metabolic activation after a 6 h exposure
positive without metabolic activation after 42 h exposure

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS induced a statisically significant increase in chromosome numbers and structural aberrations (although the latter effect was not considered biologically relevant) in cultured Chinese hamster ovary cells after a 42-h exposure in the absence of S9. No increases in numerical or structural chromosome aberrations were seen after 6 h or 18-h exposures without S9, or after a 6-h incubation in the presence of S9.

Executive summary:

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS was assessed for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells.

CHO cells were incubated with seven or more dose levels of the test sustance for either 6, 18 or 42 h without S9, or for 6 h with S9. The concentration ranges were determined on the basis of cytotoxicity studies using the same exposure times; 5 mg/mL was selected as the highest dose for the 6 and 18 h exposures and 1.25 mg/mL for the 42 h exposure. After a total incubation period of either 18 or 42 h, cells were fixed and stained and the three highest doses with 200 scorable metaphases were selected for evaluation of chromosome aberrations.

A statistically significant increase in numerical aberrations was evident after an exposure of 42 h without S9. Although a statistically significant increase was also seen in structural aberrations at 40 ug/mL at this exposure time, the percentage increase was within the acceptable range of historical control values, and therefore was considered by the investigators not to be biologically relevant. No significant increase in either numerical or structural aberrations was detected after 6 or 18 h without S9, or after 6 h with S9.

Overall, trisodium EDDS caused chromosome aberrations in an in vitro mammalian cytogenicity assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium for leukemic cells in mice with 0.1% Pluronics, supplemented with 10% horse serum and L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from a mixture of Aroclor 1242- and 1254-induced rat liver

Test concentrations with justification for top dose:

0, 2514, 2765, 3017, 3268, 3519, 3771, 4022, 4273, 4525 and 4776 µg/mL with metabolic activation (study 1 only)
0, 2765, 3017, 3268, 3519, 3771, 4022, 4273, 4524, 4776 and 5028 µg/mL without metabolic activation (studies 1 and 2), and with metabolic activation (study 2 only).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

(culture medium used as the solvent)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: 0.25 and 0.5 uL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

culture medium used as the solvent

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation Migrated to IUCLID6: 2.5 and 5.0 uL/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-16 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: plated in triplicate, duplicate tests carried out

NUMBER OF CELLS EVALUATED: 3 X 10(5) for tk-/- mutants; 60 cells for cloning efficiency

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The mutant frequency was calculated by dividing the average number of colonies in the selective plates by the average number of colonies in the non-selective plates and multiplying by 200 to give the number of mutants per 10(6) clonable cells. The test substance was determined to be positive if a dose-related increase was evident with more than one dose having a cloning efficiency of 10% or above and exhibiting a mutant frequency of two-fold or more above that of the spontaneous frequency. Or, detection of a reproducible two-fold increase for at least one test concentration.

The mutant frequencies of the positive controls must be at least three times that of the appropriate negative controls. The spontaneous mutant frequency must be no higher than 150 mutants per 10(6) surviving cells.

Statistics:

not applicable, negative response

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In both repeats there were no increases in mutant frequencies observed at any dose level of the test substance, with or without metabolic activation.

Conclusions:

Interpretation of results: Negative

In a GLP study, trisodium EDDS showed no evidence of mutagenic potential at the tk locus in mouse lymphoma L5178Y tk+/- cells in an in vitro mammalian cell mutation assay when tested at up to about 5 mg/mL, with and without metabolic activation.

Executive summary:

In a GLP study, conducted according to the published methods of Clive & Spector (1975) and Clive et al. (1979), trisodium EDDS was assessed for its ability to induce mutation at the tk locus in an in vitro mammalian cell mutation assay. The protocol is similar to that described in OECD Guideline 476.

Mouse lymphoma L5178Y tk+/- cells were incubated with concentrations of up to 5028 ug/mL of the test substance, with and without a rat liver metabolic activating fraction (S9), placed in untreated medium for 2 days to allow expression of the tk-/- mutation before incubating for 10-12 days in the presence of trifluorothymidine to allow selection of the mutant colonies. The cloning efficiency was determined by incubation in the absence of the selective agent. The assay was carried out on two separate occasions.

None of the treated cultures, with or without S9, exhibited an increase in mutant frequency that was at least twice that of the untreated culture medium. The positive controls induced a considerable increase in mutants showing that the assay was performing correctly.

In conclusion, trisodium EDDS showed no evidence of mutagenic potential at the tk locus in mouse lymphoma L5178Y tk+/- cells in an in vitro mammalian cell mutation assay when tested at up to about 5 mg/mL, with and without S9.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 April - 6 May 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no strain to detect oxidative mutagens or cross-linking agents

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

other: TA1535, TA1537, TA1538, TA98, TA100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

- Solvent: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; 0.5 ug/plate with TA1538 and TA98, 1 ug/plate with TA100, 2 ug/plate with TA1535 and TA1537

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation Migrated to IUCLID6: 80 ug/plate with TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation Migrated to IUCLID6: 3 ug/plate with TA100, 5 ug/plate with TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation Migrated to IUCLID6: 1 ug/plate with TA98, 2 ug/plate with TA1538

Key result

Species / strain:

other: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: Negative

In a guideline study to GLP, trisodium EDDS (in water) exhibited no mutagenic potential when tested at up to 5 mg/plate in a bacterial mutagenicity assay using five strains of S. typhimurium, with and without rat liver metabolic activation.

Executive summary:

In a GLP study conducted according to OECD Guideline 471 (available at the time), trisodium EDDS was assessed for its ability to induce mutation in an assay for bacterial mutagenicity (Ames test).

In two independent assays, the test substance (in water) was used at concentrations of up to 5 mg/plate in a plate incorporation assay with Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, with and without a rat liver metabolic activation fraction (S9). (These strains do not have the ability to detect oxidising mutagens or cross-linking agents.) The plates were scored for revertant colonies after 3 days incubation.

No evidence of mutagenic activity was apparent with any strain, with or without S9. The positive control substances gave the expected increases in mutation frequency, confirming the sensitivity of the assay.

In conclusion, trisodium EDDS exhibited no mutagenic potential when tested at up to 5 mg/plate in an in vitro assay for bacterial mutagenicity (Ames test) using five strains of S. typhimurium, with and without S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a GLP study equivalent to OECD Guideline 475, trisodium EDDS showed no evidence of induction of numerical or structural chromosome aberrations when administered as a single oral dose at up to 2000 mg/kg bw in a bone marrow cytogenetic assay in male and female rats (Putman, 1994).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 March - 24 June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Frederick, Maryland, USA
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males: 284-312 g; females: 191-220 g
- Assigned to test groups randomly: yes, under following basis: distributed according to body weight
- Fasting period before study: no data
- Housing: plastic cages on woodchip bedding
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): "controlled environment"
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: prepared immediately before use

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

10, 16, 28 h for all test groups and vehicle control; also at 40 h for the vehicle control and 2000 mg/kg bw groups only

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

670 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/dose/bone collection time (i.e. 15 animals/sex/dose at 200 and 670 mg/kg bw and 20/sex/dose at 0 and 2000 mg/kg bw)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (structural aberrations); vinblastine sulphate (numerical aberrations)
- Justification for choice of positive control(s): cyclophosphamide is included in the list of recommended substances
- Route of administration: oral gavage
- Doses / concentrations: cyclophosphamide, 20 mg/kg bw; vinblastine sulphate, 6 mg/kg bw

Tissues and cell types examined:

Femur bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on the acute oral toxicity in rats; as the test material was nontoxic, the top dose was set at 2 g/kg bw and the mid and low doses were set at 1/3 and 1/10 of this.


TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): rats were dosed with a volume of 10 mL/kg bw about 2 h after subcutaneous implantation of bromodeoxyuridine tablets (to allow differentiation of first- and second-division metaphases). Colchicine was given by intraperitoneal injection about 2 h before sacrifice.


DETAILS OF SLIDE PREPARATION: bone marrow cells were flushed out of the femurs, washed and fixed in methanol:acetic acid (3:1) and stored overnight at 2-6oC. The resuspended cells were dropped onto slides, air-dried, and stained by fluorescence plus Giemsa stain.


METHOD OF ANALYSIS: slides were randomly coded. A minimum of 100 first-division metaphases per animal containing 42 ± 1 centromeres were scored for numerical and structural aberrations and endoreduplication in the 10 and 16 h exposure groups. Where possible 100 second-division metaphases per animal were scored for total chromosome numbers and endoreduplication in the 28 and 40 h exposure groups.

Evaluation criteria:

A positive response was interpreted as a statistically significant dose-related increase in aberrations. Gaps were reported in the data but were not included in the total percentage of cells with aberrations, or in the average number of aberrations per cell. The percentage of cells in the vehicle controls having aberrations should not exceed 4%.

Statistics:

Fisher's exact test was used to compare the incidence of numerical or structural aberrations in the treated and vehicle control groups. Evidence of a dose-response was analysed using the Cochran-Armitage trend test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY in male rats
- Dose range: 2000 mg/kg bw
- Solubility: soluble in water
- Clinical signs of toxicity in test animals: no data
- Evidence of cytotoxicity in tissue analyzed: no, cell cycle times similar to controls
- Rationale for exposure: performed to determine appropriate harvest times
- Harvest times: 24 h


RESULTS OF RANGE-FINDING STUDY in Chinese hamster ovary cells
- Dose range: 20 and 40 ug/mL, with and without metabolic activation
- Solubility: soluble
- Evidence of cytotoxicity: cell cycle delay was noted in the 40 ug/mL dose level only without S9
- Rationale for exposure: performed to assist in selection of metaphase collection times in the in vivo assay
- Harvest times: 24 h


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: no structural aberrations detected in treated or vehicle control groups.
- Appropriateness of dose levels and route: appropriate doses based on acute toxicity data
- Statistical evaluation: no statistically significant increase in structural aberrations (p>0.025) or numerical aberrations (p>0.025) at any tested dose or harvest time
- Clinical signs of toxicity in test animals: no adverse effect on body weight gain was evident. In the high dose group, 15/20 males and 1/19 females had diarrhoea; one female at the high dose died during the study from undetermined causes.

Conclusions:

Interpretation of results: Negative

In a GLP study equivalent to OECD Guideline 475, trisodium EDDS showed no evidence of induction of numerical or structural chromosome aberrations when administered as a single oral dose at up to 2000 mg/kg bw in a bone marrow cytogenetic assay in male and female rats.

Executive summary:

In a GLP study equivalent to OECD Guideline 475, trisodium EDDS was assessed for its ability to induce chromosome aberrations in the bone marrow cells of Sprague-Dawley rats following single oral administration.

Twenty animals of each sex were given a single dose of 0 or 2000 mg/kg bw and 15/sex were administered 200 or 670 mg/kg bw by gavage (in water). Cells were harvested (from 5 animals/dose/sex) at 10, 16, 28 or (for the control and top dose groups only) 40 h after exposure. Bone marrow cells were flushed from the femurs and after fixation and staining, 100 metaphases per animal were scored for both numerical and structural aberrations.

The test substance produced no treatment-related increases in structural or numerical chromosomal aberrations at any tested dose or harvest time.

In conclusion, trisodium EDDS showed no evidence of induction of numerical or structural chromosome aberrations when administered as a single oral dose at up to 2000 mg/kg bw in a bone marrow cytogenetic assay in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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 TA 1535, TA 1537, TA 1538, TA 98 and TA 100 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 the 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 (San and Wyman, 1993).

In a GLP study conducted according to OECD Guideline 471 (available at the time), trisodium EDDS was assessed for its ability to induce mutation in an Ames test. In two independent assays, the test substance (in water) was used at concentrations of up to 5 mg/plate in a plate incorporation assay with S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without S9. (These strains do not have the ability to detect oxidising mutagens or cross-linking agents.) The plates were scored for revertant colonies after 3 days incubation. No evidence of mutagenic activity was apparent with any strain, with or without S9. The positive control substances gave the expected increases in mutation frequency, confirming the sensitivity of the assay (Jones et al. 1989).

In a GLP study conducted according to OECD Guideline 471, the related material EDDS acid showed no evidence of mutagenic potential when tested at up to 5 mg/plate in a bacterial reverse mutation assay using S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102, with and without S9 (Thompson, 2002).

In a GLP study, conducted according to the published methods of Clive and Spector (1975) and Clive et al. (1979), trisodium EDDS was assessed for its ability to induce mutation at the tk locus in an in vitro mammalian cell mutation assay. The protocol is similar to that described in OECD Guideline 476. Mouse lymphoma L5178Y tk+/- cells were incubated with concentrations of up to 5028 ug/mL of the test substance, with and without S9. The assay was carried out on two separate occasions. None of the treated cultures, with or without S9, exhibited an increase in mutant frequency that was at least twice that of the untreated culture medium. The positive controls induced a considerable increase in mutants showing that the assay was performing correctly. In conclusion, trisodium EDDS showed no evidence of mutagenic potential at the tk locus in mouse lymphoma L5178Y tk+/- cells in an in vitro mammalian cell mutation assay when tested at up to about 5 mg/mL, with and without S9 (Bigger and Clarke, 1993).

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS was assessed for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells. CHO cells were incubated with seven or more dose levels of the test sustance for either 6, 18 or 42 h without S9, or for 6 h with S9. The concentration ranges were determined on the basis of cytotoxicity studies using the same exposure times; 5 mg/mL was selected as the highest dose for the 6 and 18-h exposures and 1.25 mg/mL for the 42-h exposure. After a total incubation period of either 18 or 42 h, cells were fixed and stained and the three highest doses with 200 scorable metaphases were selected for evaluation of chromosome aberrations. A statistically significant increase in numerical aberrations was evident after an exposure of 42 h without S9. Although a statistically significant increase was also seen in structural aberrations at 40 ug/mL at this exposure time, the percentage increase was within the acceptable range of historical control values, and therefore was considered by the investigators not to be biologically relevant. No significant increase in either numerical or structural aberrations was detected after 6 or 18 h without S9, or after 6 h with S9. Overall, trisodium EDDS was concluded to be positive in this in vitro mammalian cytogenicity assay (Putman and Curry, 1994).

Good quality studies, involving single gavage administration of radiolabelled trisodium EDDS at about 2 g/kg bw to male (Powers, 1993a) and female (Powers, 1993b) rats, demonstrate that the bone marrow is exposed to EDDS and/or its metabolites following oral dosing under conditions similar to those employed in the in vivo cytogenetic study.

In a GLP study, equivalent to OECD Guideline 475, trisodium EDDS was assessed for its ability to induce chromosome aberrations in the bone marrow cells of Sprague-Dawley rats following single oral administration. Twenty animals of each sex were given a single dose of 0 or 2000 mg/kg bw and 15/sex were administered 200 or 670 mg/kg bw by gavage (in water) two hours after receiving subcutaneous implantation of bromodeoxyuridine tablets (to allow differentiation of first- and second-division metaphases). Colchicine was given 2 h before harvesting to arrest the cells in metaphase. Cells were harvested (from 5 animals/dose/sex) at 10, 16, 28 or (for the control and top dose groups only) 40 h after exposure. Bone marrow cells were flushed from the femurs and after fixation and staining, 100 metaphases per animal were scored for both numerical and structural aberrations. The test substance produced no treatment-related structural or numerical chromosomal aberrations at any tested dose or harvest time (Putman, 1994).

Therefore, overall, it can be concluded that trisodium EDDS is not genotoxic.

Justification for classification or non-classification

According to EU CLP regulation, trisodium EDDS would not be classified as mutagenic based on the available data described.