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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restriction (limited documentation)
Justification for type of information:
Read across is based on a structurally similar material. An analogue justification is attached in section 13 of dataset.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
uninduced and arochlor induced liver S9 mix of male Fischer 344 rats, B6C3F1 mice, and Syrian hamsters
Test concentrations with justification for top dose:
10; 33; 100; 333; 1000; 3333; 10000 µg/plate
Vehicle / solvent:
dest. water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: 2-Nitrofluorene; n-Methyl-N`-nitro-N-nitrosoguanidine; +S9: 2-aminoanthracene; 2-2(furyl)-3-(5-nitro-2-furyl)acrylamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

NUMBER OF REPLICATIONS:
3


Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Justification for type of information:
Read across is based on a structurally similar material. An analogue justification is attached in section 13 of dataset.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix
Test concentrations with justification for top dose:
50; 75; 100 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S 9: Mitomycin C; + S9: Cyclophosphamide
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster Ovary (CHO)
Remarks:
Migrated from field 'Test system'.

Table 1: Results of the Chromosome Aberrations Test for Na3EDTA.
Study Result: Negative
Activation Trial Trial Call
No Activation 1 Negative
Induced Rat Liver S9 2 Negative
Trial #:1   Activation: No Activation   Date: 10/17/1984   Harvest Time: 13.5 hrs   Trial Call: Negative  
Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
Abs. Per With Abs. Per With Abs. Per With Abs. Per With
Cell Abs. Cell Abs. Cell Abs. Cell Abs.
Abs: Aberrations
Vehicle Control: Dimethylsulfoxide 10          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Positive Control: Mitomycin C 0.25       100 29 0.29 26 24 0.24 22 5 0.05 5 0 0 0
1          50 31 0.62 46 24 0.48 44 7 0.14 14 0 0 0
Test Chemical: Ethylenediamine tetraacetate, trisodium salt (EDTA)  25          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
50          100 2 0.02 2 1 0.01 1 1 0.01 1 0 0 0
75          100 5 0.05 5 3 0.03 3 2 0.02 2 0 0 0
100          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Trend: 1.106 1.331 0.333
Probability: 0.134 0.092 0.37
Trial #:2   Activation: Induced Rat Liver S9   Date: 10/31/1984   Harvest Time: 14.0 hrs   Trial Call: Negative  
Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
Abs. Per With Abs. Per With Abs. Per With Abs. Per With
Cell Abs. Cell Abs. Cell Abs. Cell Abs.
Abs: Aberrations
Positive Control: Cyclophosphamide 15          100 55 0.55 40 29 0.29 22 26 0.26 22 0 0 0
Vehicle Control: Dimethylsulfoxide 10          100 3 0.03 3 2 0.02 2 1 0.01 1 0 0 0
Test Chemical: Ethylenediamine tetraacetate, trisodium salt (EDTA)  25          100 1 0.01 1 1 0.01 1 0 0 0 0 0 0
50          100 4 0.04 4 2 0.02 2 1 0.01 1 1 0.01 1
75          100 4 0.04 4 2 0.02 2 2 0.02 2 0 0 0
100          100 3 0.03 3 1 0.01 1 2 0.02 2 0 0 0
Trend: 0.686 -0.156 1.164
Probability: 0.247 0.562 0.122
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Justification for type of information:
Read across is based on a structurally similar material. An analogue justification is attached in section 13 of dataset.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats
Test concentrations with justification for top dose:
3000, 4000, 5000 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: methyl methanesulfonate; + S9 mix: methylcholanthrene
Species / strain:
mouse lymphoma L5178Y cells
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
Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells
Remarks:
Migrated from field 'Test system'.

Tables: Results of the mouse lymphoma test with Na3EDTA

Nonactivation Trial: 1 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: H2O 0          64 87 113 59 69
63 112 132 70
65 108 133 68
64# 92 148.5 78
Test Chemical: 60          65 107 177 91 83
74 109 165 74
70          58 110 97 56 68
67 113 162 81
80          67 100 131 65 59
63# 116 99 52
90          58 86 119 69 72
63 93 140 75
100          71 111 142 67 73
66 101 159 80
Positive Control: MMS 15          49 42 293 198 181*
53 45 261 164
Trial Notes:
Nonactivation Trial: 2 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          78 103 24 10 18
93 91 50 18
103 104 57 18
84 102 63 25
Test Chemical: 1000          80 42 61 25 23
76 51 45 20
2000          87 53 52 20 21
88 52 61 23
3000          79 38 47 20 21
77 50 50 22
4000          93 32 80 29 27
65 30 49 25
5000          79 23 49 21 22
76 29 54 24
Positive Control: MMS 15          52 25 146 93 93*
38 18 107 93
Trial Notes:
Nonactivation Trial: 3 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          69 94 88 43 38
62 109 48 26
58 87 79 46
76 110 84 37
Test Chemical: 1000          61 59 95 52 57
55 60 101 61
2000          68 73 111 55 49
61 61 77 42
3000          62 64 110 59 60
52 50 95 61
4000          61 45 74 40 42
61 51 81 45
5000          50 34 68 45 46
55 37 77 47
Positive Control: MMS 15          23 24 163 235 217*
26 23 156 200
EMS 250          41 61 427 347 325*
52 59 474 302
Trial Notes:
Induced S9 Trial: 1 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          108 93 101 31 36
102 109 98 32
109 101 119 36
97 97 130 45
Test Chemical: 1000          105 74 137 43 47
100 78 152 51
2000          82 58 101 41 42
97 67 126 43
3000          99 56 159 54 44
109 62 113 35
4000          99 55 106 36 38
85 45 101 40
5000          77 36 124 54 55
85 36 145 57
Positive Control: MCA 2.5        72 44 615 286 305*
70 40 680 323
Trial Notes:
Induced S9 Trial: 2 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          60 96 36 20 20
64 110 35 18
56 92 48 29
66 103 25 13
Test Chemical: 1000          52 61 41 26 21
63 75 31 16
2000          67 63 56 28 27
62 61 48 26
3000          61 40 41 22 31
71 48 85 40
4000          66 41 56 28 29
57 35 51 30
5000          70 34 65 31 28
59 36 45 25
Positive Control: MCA 2.5        34 19 231 229 232*
34 17 237 236
Trial Notes:
Footnotes:
Asterisks(*) indicate significant responses.
r = rejected value due to quality control criteria
# = reduced sample size because of the loss of one culture dish due to contamination
MMS = methyl methanesulfonate
MCA = methylcholanthrene
DMSO = dimethylsulfoxide (solvent)
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro:

Na3EDTA was negative in a reverse gene mutation assay using bacteria Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 as well as E. Coli WP2uvrA without and with uninduced and arochlor induced liver S9 from male Fischer 344 rats, B6C3F1 mice or Syrian hamsters. The substance was tested up to concentrations of 10000 µg/plate (Dunkel 1985).

Similar results were obtained by Zeiger (1988), who tested up to 10000 µg/plate Na3EDTA on Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 with and without Arochlor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers S9 mix.

Several mammalian cell line gene mutations assays are available. In a mouse lymphoma assay with Na3EDTA the mutant frequency was not increased at concentrations of 3000, 4000, 5000 µg/ml and a treatment time of 4 h. The assay was conducted with and without metabolic activation and no cytotoxicity was detected (NTP, 1984).

Another mouse lymphoma assay with Na2EDTA was also negative at concentrations of 250, 500, 1000, 1500 and 2000 µg/ml with and without metabolic activation. However, 2000 µg/ml Na2EDTA reduced the relative growth to 65.5% compared to the control in cells without metabolic activation. This was not the case if cells were treated with S9 mix (Whittaker, 2001).

Additionally, in a chromosome aberration test Na3EDTA was tested negative (NTP, 1984).

Cell transformation assays with Na2 and Na3 salts of EDTA were negative. One test was performed on SHE cells with exposure of up to 100 µg/ml for 24 h or 150 µg/ml Na2EDTA for up to 7 days without metabolic activation (LeBoef, 1996).

Additionally a cell transformation assay using BALB/c-3T3 cells was performed without metabolic activation. Cells were exposed to up to up to 770 µg/ml Na3EDTA for 48 h without metabolic activation (Matthews, 1993).


Short description of key information:
Concerning free edetic acid only few studies are available, therefore data from EDTA sodium salts have also been considered. Na salts of EDTA were tested negative in several ames tests. Na salts of EDTA were tested negative in several mouse lymphoma assays.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results obtained in the toxicity studies and taking into account the provisions laid down in Regulation EC 1272/2008, classification is not fulfilled with respect to genetic toxicity.