Tetrasodium ethylenediaminetetraacetate

Endpoint summary

Key value for chemical safety assessment

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

Reference

Reference 1

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

Reference 2

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)

Reference 3

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.