Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

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.

An additional poorly documented Ames test of Na2EDTA using bacteria strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 yielded also negative results up to concentrations of 446 µg/plate with and without metabolic activation (DeFlora 1981).

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). In a third mouse lymphoma assay which was done only without metabolic activation, 2-fold to 6-fold increases of mutant frequencies were induced by EDTA free acid at very high concentrations of 7400, 8800 µg/ml and a treatment time of 4 hours; relative total growths at these concentrations were 57% and 16% respectively. The pH, measured in a parallel experiment where EDTA was dissolved in culture medium (pH of 7.2), was reduced to 5.8 at 8800 µg/ml and to 6.1 at 5900 µg/ml, when measured directly after preparation of the solution. Whether the mutagenic activity was due to pH effects or due to the high tested concentrations remains to be unclear (Wangenheim and Bolcsfoldi, 1988).

Several alkaline elution assays with mouse lymphoma cells using EDTA free acid have been performed in vitro. EDTA induced DNA single strand breaks in very high concentrations (8800, 11800, 14800 µg/ml). Slight cytotoxicity was reported but no details given; the assay was conducted without a metabolic activation system only (Garberg et al., 1988). However, in concentrations up to 8766 µg/ml EDTA induced no effects with and without S-9 mix in alkaline elution assays with V79 cells (Swenberg et al, 1976; Swenberg, 1981).

A sister chromatid exchange assay with Na3EDTA in CHO cells with and without metabolic activation resulted in negative findings. Cells were incubated with up to 100 µg/ml of the test substance for 26 h (NTP, 1984). Another SCE Assay reported an increase of SCE by 1.5 fold after incubation to 292 µg/ml edetic acid for 18 - 20 h. However, no detailed information on the conductance of the test is available (Fukada, 1987). Additionally, a dose dependant increase of unscheduled DNA synthesis in SHE cells was reported after incubation with up to 87.7 µg/ml edetic acid. However no details are given (Fukada, 1987).

Cell transformation assays with edetic acid as well as Na2 and Na3 salts of EDTA were negative. One test was performed with SHE cells up to concentration of 87.7 µg/ml edetic acid (Fukada, 1987). Another 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).

A non-standard micronucleus test using Tradescantia Paludosa Sax found no increase after incubation with up to 372 µg/ml Na3EDTA (Ma, 1984).

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, 1,000 and 2,000 mg/kg bw Na2EDTA. As clinical sign only piloerection was observed after the second administration of 2,000 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 were not given. Only males (3 per 24-hour group; 4 per 48-hour group) were used (Russo and Levis, 1992).

One positive micronucleus assay in male mice (strain:CFT; n = 4) was reported after low oral doses of Na2EDTA (15 and 20 mg/kg bw). A dose-dependent increase in the incidence of micronucleated polychromatic erythrocytes was reported for the only sampling time of 24 hours. The highest micronucleus frequency was 1.43% (20 mg/kg) as compared to 0.35% in the vehicle control. Neither clinical signs nor lethal effects were described. The PCE/NCE ratio was not affected by EDTA treatment. (Muralidhara and Narasimhamurthy; 1991). The study result does not appear to be plausible, since it contrasts to negative results obtained with much higher doses. Only very mutagenic substances would show such effects at 15 mg/kg bw.

Compared with the negative results of the micronucleus tests after oral (BASF, 2000) and intraperitoneal administration (Russo and Levis, 1992), the positive result reported by Muralidhara and Narasimharnurthy (1991) seems to be not reliable. The positive effect after oral administration of low doses, such as 15 and 20 mg/kg bodyweight, is not plausible. The genral conclusion is that EDTA does not induce micronuclei in bone marrow cells.

Additionally tests for aneuploidy and sister chromatid exchange have been performed in bone marrow cells of BALB/c mice using Na2EDTA. Single i.p. administration of 93 and 186 mg/kg bw did not induce aneuploidy in bone marrow cells or induce sister chromatid exchanges (Zordan, 1990).

Additonally 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 analysed 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. In addition, Na2EDTA does not induce chromosomal aberrations in the spermatogonial phase, the most suitable germ cell population to detect chromosomal aberrations. (Russo and Levis, 1992).

The remaining studies on rodent germ cells had a negative outcome. 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.

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).

In a dominant lethal assay Na2EDTA did not induce dominant lethal mutations when mice (strain:CFT) were administered orally 10 mg/kg bw/day day for 5 consecutive days (Muralidhara and Narasimhamurthy, 1991).

Additional tests have been performed with Drosophila melanogaster. A somatic mutation and recombination test (SMART) was negative using Na2EDTA. In this test lavae were treated with 2.75; 3.72 9.3 mg/ml for 24 h (Zordan 1990). Two other drosophila cells produced positive results: An assay for nondisjunction and loss of the sex chromosomes EDTA free acid (700 ppm in substrate) was positive with respect to the endpoint chromosome loss (Ramel and Magnusson, 1979). Zordan et al. (1990) investigated the genetic effect of Na2EDTA by using the FIX test for heritable aneuploidy after treatment of adult female Drosophila melanogaster. Na2EDTA was assayed at 2.79 mg/ml and 9.3 mg/ml. Genetic effects were observed for both exposure levels.

A test for chromosomal aberration in grasshoppers with edetic acid was positive. Grasshoppers were injected with 0.29 g/l solution. After 30 - 31 h single-break chromosome aberration of anaphase 1 cells were determined (Saha 1974).

Short description of key information:
No genotoxicity studies with tetraammonium EDTA or any other ammonium EDTA salts are available. Therefore data from EDTA sodium salts and EDTA free acid have 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. Only one mouse lymphoma assay using edetic acid was positive. However, it was not clear whether this effect was due to the test substance or the pH change. Several other in vitro tests have been performed, and in general EDTA was not genotoxic in vitro.
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 lethals. A positive result was obtained in a micronucleus test with spermatids after ip application, indicating that aneugenic effects may be induced in specific phases of spermatogenesis (late spermacytogenesis). The effect was linked to the use of an extremely high dose in the LD50 range. Since the induction of aneuploidy is based on a threshold mode of action, the potential for induction of aneuploidy will not be expressed at low doses. Furthermore, the effects may be indirect, resulting from the lower intracellular bioavailability of essential elements. On balance, EDTA and its sodium salts may show a low aneuploidogenic potential at extremely high doses.
On the basis of the various negative findings and the assumption of a threshold mode of action for aneugens, it can be concluded that EDTA and its sodium salts are not mutagenic for humans.
Besides, several other ammonium salts are not genotoxic (SIDS Ammonia Category) and it can be concluded that the a ammonium salts of EDTA are also not mutagenic for humans.

Endpoint Conclusion:

Justification for classification or non-classification

Based on the results obtained in the toxicity studies and taking into account the provisions laid down in Council Directive 67/548/EEC and CLP, a classification has not to be done with respect to genetic toxicity.