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EC number: 416-530-4 | CAS number: 178949-82-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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.
- 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) - 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.
- 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.
- 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.
Referenceopen allclose all
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 |
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
- 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.
Reference
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.
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