Grouping approaches based on structure alone are insufficient to conclude about toxicological properties—the example of monoamine-based chelates

Figures & data

Table 1. Monoamine based, aminocarboxylic acid chelating agents.

	Name	CAS No.	Molecular structure	MW
EDG-Na2	disodium 2-hydroxyethyl-iminodi(acetate)	135-37-5		221
NTA-Na3	trisodium 2,2′,2′′-nitrilotriacetate	5064-31-3		257
NTA-H3	2,2′,2′′-nitrilotriacetic acid	139-13-9		191
MGDA-Na3	alanine, N,N-bis(carboxy methyl)-, sodium salt (1:3)	164462-16-2		271
GLDA-Na4	tetrasodium (S)-2-[bis(carboxylatomethyl) amino]Pentanedioate	51981-21-6		351
GLDA-H4	2-[bis(carboxymethyl) amino]pentanedioic acid	58976-65-1		263

Table 2. Thermodynamic stability constants (log K values).

Metal ion	EDG	NTA	MGDA	GLDA	EDTA
Fe^3+	11.6	16.0	16.5	15.3	25.1
Cu^2+	11.8	13.0	13.9	13.1	18.8
Zn^2+	8.4	10.7	11.0	11.5	16.5
Fe^2+	6.8	8.9	8.1	8.7	14.3
Mn^2+	5.5	7.5	8.4	7.6	13.9
Ca^2+	4.7	6.4	7.0	5.9	10.7
Mg^2+	3.4	5.5	5.8	5.2	8.8

Values are based on Martell and Smith (2004), Begum et al. (2012a, b), and Trilon® Chelating Agents, BASF; Dissolvine® GL technical brochure, Nouryon, brochure-chelates-dissolvine-gl-global-en.pdf (Nouryon.com).

Table 3. Conditional complex building constants for EDG, NTA, MGDA, GLDA and EDTA.

Metal ion	Conditional stability constants for EDG depending on pH value
	pH 3	pH 4	pH 5	pH 6	pH 7	pH 8	pH 9	pH 10	pH 11
Cu^2+	6.0	7.1	8.1	9.1	10.0	10.6	10.3	9.3	7.7
Fe^3+	5.9	6.6	6.8	7.0	7.2	7.1	6.6	5.3	3.5
Zn^2+	2.6	3.7	4.7	5.7	6.7	7.6	7.4	6.2	5.3
Mn^2+	–	0.8	12.8	2.8	3.8	4.7	5.3	5.4	5.1
Ca^2+	–	0.0	1.0	2.0	3.0	3.9	4.5	4.7	4.7
Mg^2+	–	–	–	0.7	1.7	2.7	3.2	3.4	3.3
Metal ion	Conditional stability constants for NTA depending on pH value
	pH 3	pH 4	pH 5	pH 6	pH 7	pH 8	pH 9	pH 10	pH 11
Cu^2+	6.2	7.3	8.3	9.3	10.3	10.8	10.7	9.9	8.4
Fe^3+	8.5	8.5	8.2	8.0	7.4	6.9	6.6	5.8	4.4
Zn^2+	3.9	5.0	6.0	7.0	8.0	8.9	9.0	7.9	6.6
Mn^2+	0.7	1.8	2.8	3.8	4.8	5.8	6.7	7.2	7.0
Ca^2+	–	0.8	1.8	2.8	3.8	4.8	5.7	6.3	6.4
Mg^2+	–	–	0.8	1.8	2.8	3.8	4.8	5.3	5.4
Metal ion	Conditional stability constants for MGDA depending on pH value
	pH 3	pH 4	pH 5	pH 6	pH 7	pH 8	pH 9	pH 10	pH 11
Cu^2+	6.9	8.0	9.0	10.0	11.0	11.5	11.2	9.9	7.5
Fe^3+	8.9	8.7	7.9	6.7	5.1	3.1	1.0	–	–
Zn^2+	4.0	5.1	6.1	7.1	8.1	9.1	9.1	7.8	5.9
Mn^2+	1.4	2.5	3.6	4.6	5.6	6.5	7.5	8.1	8.0
Ca^2+	–	1.1	2.1	3.1	4.1	5.1	6.1	6.7	6.9
Mg^2+	–	–	1.0	2.0	3.0	4.0	4.9	5.6	5.7
Metal ion	Conditional stability constants for GLDA depending on pH value
	pH 3	pH 4	pH 5	pH 6	pH 7	pH 8	pH 9	pH 10	pH 11
Cu^2+	5.1	6.9	8.4	9.6	10.6	11.1	10.8	9.5	7.8
Fe^3+	6.7	7.2	7.3	7.2	6.6	5.8	5.3	4.2	2.4
Zn^2+	4.0	5.78	7.0	8.1	9.2	10.1	10.1	8.6	7.0
Mn^2+	–	1.1	2.9	4.2	5.2	6.2	7.1	7.5	7.5
Ca^2+	–	–	1.3	2.5	3.6	4.6	5.4	5.8	5.9
Mg^2+	–	–	0.5	1.8	2.8	3.8	4.7	5.1	5.1
Metal ion	Conditional stability constants for EDTA depending on pH value
	pH 3	pH 4	pH 5	pH 6	pH 7	pH 8	pH 9	pH 10	pH 11
Cu^2+	8.3	10.2	12.2	14.0	15.4	16.3	16.6	16.6	16.1
Fe^3+	13.9	14.7	14.8	14.6	14.1	13.7	13.6	14.0	14.3
Zn^2+	6.0	7.9	9.9	11.7	13.1	14.2	14.9	13.6	11.0
Mn^2+	3.6	5.5	7.4	9.2	10.6	11.7	12.6	13.4	13.4
Ca^2+	–	2.2	4.1	5.9	7.3	8.4	9.3	10.2	10.6
Mg^2+	–	–	2.2	4.0	5.4	6.5	7.4	8.3	8.6

Figure 1. Metal-complexes of NTA (left panel) and MGDA (right panel). Due to steric hindrance the additional methyl group in MGDA is expected to stick out enlarging the overall three-dimensional size of the metal complex.

Table 4. Solubility of Ca-bound chelating agents.

	In water at 37 °C, pH = 7 (measured at Nouryon)	In water at 37 °C, pH = 7 (measured at BASF for confirmation)
EDG-Ca	∼0.1%
NTA-CaNa	∼0.4%	∼0.63%
MGDA-CaNa	∼13%	∼17.5%
GLDA-CaNa2	∼13%

Solubility of EDTA-CaNa2 is ∼28% (measured at 20 °C) (https://echa.europa.eu/registration-dossier/-/registered-dossier/5354/4/9).

Table 5. Repeat dose toxicity data and data on genotoxicity of EDG, NTA, MGDA and GLDA.

	Repeat dose toxicity	Genotoxicity	Chronic toxicity/carcinogenicity
EDG	Rats (0, 4, 41, 205 mg/kg bw EDG-Na3, 90-day): reversible kidney findings at 205 mg/kg; NOEL 41 mg/kg bw (Anonymous 1987)	Non-mutagenic in OECD 471 (Anonymous 1986a), in OECD 476 (Anonymous 2012), and in OECD 474 (Anonymous 1986b)	Not available
NTA	Several repeat dose toxicity studies (subacute, subchronic and chronic) are available (Nixon 1971; Nixon et al. 1972, Budny and Arnold 1973; NCI 1977; Anderson and Kanerva 1978; 1979; Merski 1982; Myers et al. 1982; BASF 1997a, 1998a, 2003). The organ system mainly affected by NTA treatment was the urinary tract. An overall NOAEL for non-neoplastic findings of 92 mg/kg bw (cytotoxicity) was established in a two-year carcinogenicity study (NCI 1977)	Results derived from ten in vitro and two in vivo genotoxicity tests showed that NTA does not appear to exert genotoxic effects (https://echa.europa.eu/de/registration-dossier/-/registered-dossier/14301/7/7/1)	Rats (0, 9, 92, 921 mg/kg bw NTA-Na3): primary urinary tract neoplasms at 921 mg/kg bw, one bladder papilloma at 92 mg/kg bw, transitional cell hyperplasia in renal pelvis, ureter and urinary bladder in all dose groups; NOAEL carcinogenicity 92 mg/kg bw (NCI 1977) Rats (0, 355, 526, 724, 1053 mg/kg bw NTA-Na3): increase in tumors of the urinary system at all dose levels; LOAEL carcinogenicity 355 mg/kg bw (NCI 1977) Mice (0, 752, 1504 mg/kg bw NTA-Na3): increase in tumors of the urinary system; LOAEL carcinogenicity 752 mg/kg bw Mice (0, 169, 338 mg/kg bw NTA-Na3.H2O/kg): no tumors, LOAEL non-neoplastic renal findings 169 mg/kg bw (NCI, 1977) Rats (0, 100 mg/kg bw NTA-NA3): increase of renal tumors, renal tubular hyperplasia; LOAEL carcinogenicity 100 mg/kg bw (Goyer et al. 1981) Rats (0, 15, 97, 322 mg/kg bw NTA-Na3): increase in urinary zinc level; NOAEL non-neoplastic findings 15 mg/kg bw (Nixon et al. 1972) Rats (0, 1167 mg/kg bw NTA-Na3 in males, and 0, 1039 mg/kg bw in females): kidney tumors in males and non-neoplastic changes in kidney and ureter in both sexes; LOAEL carcinogenicity 1167 mg/kg bw (BASF 2006)
MGDA	Rats (0, 170, 874, 1325, 1774 mg/kg bw MGDA-Na3 in males, and 0, 204, 1056, 1588, 2097 mg/kg bw in females, 90-day): non-neoplastic renal findings at the two highest doses; NOAEL non-neoplastic findings 874/1056 mg/kg bw (BASF 1998b)	Non-mutagenic in OECD 471 (BASF 1995a), in OECD 476 (BASF 2001), in OECD 473 (BASF 1995b), and in OECD 474 (BASF 1997c)	Rats (0, 54, 262, 1132 mg/kg bw MGDA-Na3 in males, and 0, 66, 334, 1317 mg/kg bw in females): no increase in tumors, non-neoplastic renal findings at high dose; NOAEL non-neoplastic findings 262/334 mg/kg bw (BASF 2006)
GLDA	Rats (0, 100, 300, 1000 mg/kg bw GLDA-Na4, 90-day): higher kidney weight at 1000 mg/kg bw but no histopathological renal findings; NOAEL 300 mg/kg bw (Anonymous 2007)	Non-mutagenic in OECD 471 (Anonymous 1994), in OECD 476 (Anonymous 1995a), in OECD 473 (Anonymous 1995b), and in OECD 474 (Anonymous 1995c)	Not available

Table 6. Levels of (metal)ions in urine compared to controls (biomarker study; HIHS 2013).

	NTA	MGDA	GLDA	EDTA
Sodium	2.6	2.7	2.4	2.8
Magnesium	0.3	0.7	0.8	0.6
Calcium	1.7	2.1	1.0	0.9
Zinc	7.5	7.5	2.4	5.0

Values are mean of x-fold change on days 7, 14, 21 and 28 compared to control; values in bold show statistically significant changes (p < 0.05) on one or more measurement days; n = 8–10 male rats/group.

Figure 2. Overview of urinary tract findings induced by EDG, NTA, MGDA, GLDA and EDTA. *Read-across to NTA as Ca-microcrystal formation is likely due to the very low solubility of EDG-Ca and because marked renal toxicity was noted in vivo.

Table 7. Levels of sodium, calcium and zinc in blood and urine of untreated rats.

	Blood	Reference	Urine	References
Sodium	140 mmol/L	90-day study GLDA (Anonymous 2007)	48 mmol/L	Biomarker study (HIHS 2013)
Calcium	3 mmol/L	90-day study GLDA (Anonymous 2007)	7.8 mg/dL (2 mmol/L)	Biomarker study (HIHS 2013)
Zinc	∼170 μg/dL (0.03 mmol/L) ∼22 μmol/L (0.02 mmol/L)	Jayalakshmi and Platel (2016) Walker and Kelleher (1978)	2 ppm (0.03 mmol/L) 0.2–0.24 μmol/L	Biomarker study (HIHS 2013) Walker and Kelleher (1978)

Table 8. Levels of kidney biomarkers in urine compared to controls (biomarker study; HIHS 2013).

	NTA	MGDA	GLDA	EDTA
Kim-1 (Day 7)	2.45**	1.70*	1.40	1.45
Kim-1 (Day 14)	1.64*	1.19	1.03	1.05
CAL (Day 28)	1.39	2.46*	1.45	1.16
CLU (Day 28)	2.42**	10.3*	1.02	0.96
OST (Day 28)	1.58	2.25*	1.01	1.10
EGF-R (Day 28)	0.90	1.78*	1.17	1.38
GLU (Day 7)	4.90**	3.12**	2.17*	1.19
GLU (Day 14)	7.33**	3.70**	2.74*	1.47
GLU (Day 21)	4.14**	3.82**	2.16*	0.93
GLU (Day 28)	3.95**	4.50*	1.80	1.63

Values are mean of x-fold change compared to control. Kim-1 = kidney injury molecule 1, CAL = calbandin, CLU = clusterin, OST = osteopontin, EGF-R = epidermal growth factor-rat; GLU = glutathion-S-transferase alpha; n = 8–10 male rats/group; *p < 0.05, **p < 0.01.

Table 9. Mean labeling indices (LI) for kidney cortical proximal tubular cells (biomarker study; HIHS 2013).

Treatment group	Mean LI (%)	SD	N
Vehicle control	2.06	0.58	10
NTA	3.26*	0.96	8
MGDA	2.99	0.98	9
GLDA	2.11	0.74	10
EDTA	1.92	0.83	9

*Significantly different from vehicle control (p < 0.05).

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