Trisodium hydrogen ethylenediaminetetraacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (equivalent or similar to OECD 471): negative

Gene mutation in mammalian cells (equivalent or similar to OCDE 476): negative

Cytogenicity/chromosome aberration in mammalian cells (equivalent or similar to OECD 473): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

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)

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

Key result

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

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

Conclusions:

Under the conditions of the test, no mutagenicity could be detected in any bacterial strain used with and without metabolic activation.

Reference 2

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:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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

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

Conclusions:

The test item was tested for its potential to induce clastogenicity in cultured CHO cells according to OECD 473. Experiments were performed with and without metabolic activation at concentrations of up to 100 µg/mL. Based on the results of the conducted study the test item did not induce chromosome abberations in CHO cells.

Reference 3

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:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

NTP-Standard Protocol

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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:

1000, 2000, 3000, 4000 and 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)

Conclusions:

The test item was tested for its potential to induce reverse mutations in mouse lymphoma L5178Y cells. Cells were treated with the test material with and without metabolic activation at concentrations of up to 5000 µg/mL. Based on the results of the conducted study the test item did not exhibit mutagenic properties in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test in somatic cells, mouse (OECD 474): negative

Read-across from disodium dihydrogen EDTA (CAS 139-33-3)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

The only adverse effect observed was piloerrection.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

source: CAS 139-33-3, BASF SE, 2000, MN

Conclusions:

The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their genotoxic potential. An in vivo mammalian erythrocyte micronucleus test (according to OECD guideline 474) was performed in male mice with the source substance disodium dihydrogen EDTA (CAS 139-33-3). No increase in frequency of micronucleated polychromatic erythrocytes was found after two oral gavage doses of 500, 1000 and 2000 mg/kg bw (24 h interdosing interval) and a bone marrow sampling at 24 hours after the second treatment from each of 5 male animals as compared to vehicle and positive control (cyclophosphamide and vincristine sulphate) animals. Therefore, no in vivo genotoxic potential is expected for target substance trisodium hydrogen EDTA (CAS 150-38-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro genetic toxicity

Trisodium hydrogen EDTA 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 trisodium hydrogen EDTA on Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 with and without metabolic activation.

Trisodium hydrogen EDTA was tested for its potential to induce clastogenicity in cultured CHO cells. Experiments were performed with and without metabolic activation at concentrations of up to 100 µg/mL (NTP, 1984). The test item did not induce chromosome abberations in CHO cells.

In a mouse lymphoma assay with trisodium hydrogen EDTA, 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). Supporting data also indicate no mutagenicity of trisodium hydrogen EDTA in a mouse lymphoma assay at concentrations of up to 5000 µg/mL with and without metabolic activation (McGregor, 1984). 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 trisodium hydrogen EDTA for 48 h without metabolic activation (Matthews, 1993).

In vivo genetic toxicity

Justification for read-across

There are no reliable experimental data available regarding the in vivo genetic toxicity epeated dose toxicity of trisodium hydrogen EDTA (CAS 150-38-9). However, an in vivo micronucleus study with the source substance disodium dihydrogen EDTA is available. Thus, read-across from the appropriate source substance, disodium dihydrogen EDTA (CAS 139-33-3), is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. (Bio)transformation to common compounds are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

The source substance disodium dihydrogen EDTA (CAS 139-33-3) was tested in an in vivo mammalian cytogenicty/erythrocyte micronucleus study, performed according to OECD 474 and GLP (BASF SE, 2000). No micronuclei were induced in polychromatic erythrocytes of NMRI mice after repeated oral administration (two administrations with a 24-hour interval) of 500, 1000 and 2000 mg/kg bw. 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.

Justification for classification or non-classification

Based on the available data, there is no indication that trisodium hydrogen EDTA has any mutagenic or clastogenic potential in vitro and in vivo. The available data on genetic toxicity are therefore conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008.