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Genetic toxicity in vitro

Description of key information

Concerning Na4EDTA no genotoxicity studies are available, therefore data from other EDTA sodium salts and EDTA free acid have been considered. Natrium salts of EDTA were tested negative in several Ames tests. Natrium salts of EDTA were tested negative in several mouse lymphoma assays. Several other in vitro tests have been performed, and in general EDTA was not genotoxic in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
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:
Non-induced and Aroclor 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:
2-nitrofluorene
other: - S9 mix: 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


Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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
Key result
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
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
other: Mouse lymphoma Assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from 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
Key result
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

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)
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
other: Chromosomal aberration Assay
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:
cyclophosphamide
mitomycin C
Key result
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

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
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Concerning Na4EDTA no genotoxicity studies are available, therefore data from other EDTA sodium salts and EDTA free acid have been considered. In vivo, somatic cells in mice (bone marrow cells) showed negative results with respect to the endpoints micronuclei, aneuploidy and sister chromatid exchanges. In germ line cells negative results were obtained for induction of structural chromosomal aberrations in spermatogonia, for induction of aneuploidy in primary and secondary spermatocytes, and also for induction of dominant lethal effects. This result was also confirmed by the independent evaluation of the MAK Commission for the Investigation of Health Hazards of Chemical Compounds in the work area (MAK, 46; 2009).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology
Type of assay:
other: Erythrocyte Micronucleus Assay
Specific details on test material used for the study:
- Purity test date: 31.Oct 1996
- Batch No.: 31-9412
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle water over a period of 4 hours has been verified analytically
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Weight at study initiation: Mean 29 g
- Housing: Makrolon cages type Mill, in groups of 5
- Diet: Kliba, standardized pelleted feed, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70% .
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water
Details on exposure:
- The low dose group was given 500 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 2.5 g/100 mL.
- The intermediate dose group was given 1000 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 5.0 g/100 mL.
- The top dose group was given 2000 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 10.0 g/100 mL.
Duration of treatment / exposure:
48 h (animals were treated twice at 24 h intervals)
Frequency of treatment:
twice
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
20 mg of cyclophosphamide (CPP)/kg body weight or 0.15 mg of vincristine sulphate (VCR)/kg body weight, both, dissolved in purified water, were administered to male animals once, orally each in a volume of 10 mL/kg body weight.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
METHOD OF ANALYSIS:
2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters have been evaluated:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter )
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed .
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range .

A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
- The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
As clinical signs only piloerrection were observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- After two administrations of the highest dose of 2000 mg/kg body weight, 0.4% of polychromatic erythrocytes containing micronuclei were found after 24 hours.
- In the two lower dose groups, rates of micronuclei of about 0.6% (1000 mg/kg group) and 0.7% (500 mg/kg group) were detected.
- With 12.7% the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight.
- With 81.7% the positive control vincristine for induction of spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i .e. 10.5%.
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups

Thus, the test substance Trilon BD did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control value and was within the historical control range. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three dose groups of Trilon BD.

No inhibition of erythropoiesis, induced by the treatment of mice with Trilon BD was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.
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 non-induced and Aroclor-induced liver S9 Mix from male Fischer 344 rats, B6C3F1 mice or Syrian hamsters. The substance was tested up to concentrations of 10000 µg/plate (Dunkel 1985).

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 when 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 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 concentrations up to 770 µg/mL Na3EDTA without metabolic activation for 48 h (Matthews, 1993).

In vivo

Several in vivo tests for genotoxicity on somatic cells have been performed. The key study which was performed according to OECD 474 guideline and GLP concluded that no micronuclei were induced in polychromatic erythrocytes of NMRI mice after repeated oral administration (twice with a 24-hour interval between administrations) of 500, 1000 and 2000 mg/kg bw Na2EDTA. As clinical sign only piloerection was observed after the second administration of 2000 mg/kg. No lethal effects or cytotoxicity (PCE/NCE ratio) were induced. Only males (5 per group) were used because no distinct symptomatic differences between males and females were noticed in a pre-test (BASF 2000).

In another well-conducted in vivo micronucleus assay Na2EDTA was negative in bone marrow cells of mice (strain: BALB/c). Mice were treated with a single intraperitoneal dose of 186 mg/kg bodyweight; the sampling times were 24 hours and 48 hours after treatment. The tested dose was near to the LD50 value. No cytotoxic effects (PCE/NCE) were induced; information about clinical signs or lethal effects was not given. Only males (3 per 24-hour group; 4 per 48-hour group) were used (Russo and Levis, 1992).

Additionally in vivo mutagenicity tests have been performed on rodent germ cells. Na2EDTA induced micronuclei in germ cells at the late stages of spermacytogenesis of mice (strain: BALB/c) after intraperitoneal administration of a very high dose of 186 mg/kg bw in the range of the i.p. LD50 value. The frequency of micronuclei was analyzed in Golgi phase and Cap phase, representing the two earliest phases of spermatids development. The sampling times were 24 hours and 48 hours after administration. Na2EDTA induced micronuclei in Golgi phase spermatids (0.30% and 0.38% micronucleated spermatids at 24 hours and 48 hours sampling as compared to 0.08% in controls); in Cap phase spermatids negative results were obtained. Toxicity data were not given. Aneuploidy is discussed as most probable origin of micronuclei produced by NA2EDTA in secondary spermatocytes because the substance generally induced micronuclei of larger size in comparison with other substances. Micronuclei induction in germ cells was evaluated after after i.p. application in this study. Due to the very alkaline character of the test item and its application very close to the gonads the route of application chosen in this test is not suitable and results obtained are considered to be equivocal In addition, Na2EDTA does not induce chromosomal aberrations in the spermatogonial phase, the most suitable germ cell population to detect chromosomal aberrations. An in vivo chromosomal aberration assay with mouse spermatogonia (strain: BALB/c) led to a negative result after a single i.p. administration of 186 mg/kg bw Na2EDTA. The sampling time was 24 hours sampling time after administration (Russo and Levis, 1992).

Zordan et al. (1990) investigated aneugenic properties of Na2EDTA in primary and secondary spermatocytes of mouse (strain: BALB/c). After single i.p. administration of 93 and 186 mg/kg bw no increases in aneuploid spermatocytes were observed. The sampling was 6 hours and 5 days after administration; higher doses resulted in lethality.



Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.