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Key value for chemical safety assessment

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 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.

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).

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 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).

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, 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).

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 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.

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.

Short 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. (For read-across justification also refer to section 13). Na salts of EDTA were tested negative in several ames tests. Na 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.

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. 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 it can be concluded that EDTA and its sodium salts are not mutagenic for humans.
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. Lieferung, 2009).

Endpoint Conclusion: No adverse effect observed (negative)

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 genetic toxicity.