L-Aspartic acid, N,N'-1,2-ethanediylbis-

Endpoint summary

Additional information

In a GLP study conducted according to OECD Guideline 471, EDDS acid was studied for mutagenic potential in a bacterial reverse mutation assay (Ames test). The test substance, at concentrations of 0, 50, 150, 500, 1500 or 5000 ug/plate, was tested with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in a plate incorporation assay, with and without a rat liver metabolic activation system (S9). Three plates were prepared for each test concentration and the numbers of revertant colonies were counted after a 48-h incubation period. Vehicle and positive control plates were similarly prepared. The study was carried out in duplicate. EDDS acid did not induce a significant increase in mutant frequency at any dose level and showed no cytotoxicity. The spontaneous frequencies were within the historical range for each bacterial strain. The positive controls showed the expected increase in mutant numbers, confirming the satisfactory performance of the assay. In conclusion, EDDS acid showed no evidence of mutagenic potential in a bacterial mutagenicity assay (Ames test) when tested at up to 5 mg/plate in five strains of S. typhimurium, both in the presence and absence of S9 (Thompson, 2002).

In a GLP study conducted according to a similar protocol to OECD Guideline 471, trisodium EDDS (in water) showed no mutagenic potential in a bacterial mutagenicity assay (Ames test) when tested at up to 5 mg/plate in five strains of S. typhimurium and two strains of E. coli, both in the presence and absence of S9 (San and Wyman, 1993). In a guideline study to GLP, trisodium EDDS (in water) exhibited no mutagenic potential when tested at up to 5 mg/plate in a bacterial mutagenicity assay using five strains of S. typhimurium, with and without S9 (Jones et al. 1989).

In a GLP study, conducted according to the published methods of Clive and Spector (1975) and Clive et al. (1979), trisodium EDDS was assessed for its ability to induce mutation at the tk locus in an in vitro mammalian cell mutation assay. The protocol is similar to that described in OECD Guideline 476. Mouse lymphoma L5178Y tk+/- cells were incubated with concentrations of up to 5028 ug/mL of the test substance, with and without S9. The assay was carried out on two separate occasions. None of the treated cultures, with or without S9, exhibited an increase in mutant frequency that was at least twice that of the untreated culture medium. The positive controls induced a considerable increase in mutants showing that the assay was performing correctly. In conclusion, trisodium EDDS showed no evidence of mutagenic potential at the tk locus in mouse lymphoma L5178Y tk+/- cells in an in vitro mammalian cell mutation assay when tested at up to about 5 mg/mL, with and without S9 (Bigger and Clarke, 1993).

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS was assessed for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells. CHO cells were incubated with seven or more dose levels of the test sustance for either 6, 18 or 42 h without S9, or for 6 h with S9. The concentration ranges were determined on the basis of cytotoxicity studies using the same exposure times; 5 mg/mL was selected as the highest dose for the 6 and 18-h exposures and 1.25 mg/mL for the 42-h exposure. After a total incubation period of either 18 or 42 h, cells were fixed and stained and the three highest doses with 200 scorable metaphases were selected for evaluation of chromosome aberrations. A statistically significant increase in numerical aberrations was evident after an exposure of 42 h without S9. Although a statistically significant increase was also seen in structural aberrations at 40 ug/mL at this exposure time, the percentage increase was within the acceptable range of historical control values, and therefore was considered by the investigators not to be biologically relevant. No significant increase in either numerical or structural aberrations was detected after 6 or 18 h without S9, or after 6 h with S9. Overall, trisodium EDDS was concluded to be positive in this in vitro mammalian cytogenicity assay (Putman and Curry, 1994).

In a GLP study, equivalent to OECD Guideline 475, trisodium EDDS was assessed for its ability to induce chromosome aberrations in the bone marrow cells of Sprague-Dawley rats following single oral administration. Twenty animals of each sex were given a single dose of 0 or 2000 mg/kg bw and 15/sex were administered 200 or 670 mg/kg bw by gavage (in water) two hours after receiving subcutaneous implantation of bromodeoxyuridine tablets (to allow differentiation of first- and second-division metaphases). Colchicine was given 2 h before harvesting to arrest the cells in metaphase. Cells were harvested (from 5 animals/dose/sex) at 10, 16, 28 or (for the control and top dose groups only) 40 h after exposure. Bone marrow cells were flushed from the femurs and after fixation and staining, 100 metaphases per animal were scored for both numerical and structural aberrations. The test substance produced no treatment-related structural or numerical chromosomal aberrations at any tested dose or harvest time (Putman, 1994).

Good quality studies, involving single gavage administration of radiolabelled trisodium EDDS at about 2 g/kg bw to male (Powers, 1993a) and female (Powers, 1993b) rats, demonstrate that the bone marrow is exposed to EDDS and/or its metabolites following oral dosing under conditions similar to those employed in the in vivo cytogenetic study.

[Data on trisodium EDDS is considered relevant to use for understanding the potential genotoxic activity of EDDS acid, and is acceptable for using as read-across information.]

Therefore, overall, it can be concluded that EDDS acid and its trisodium salt are not genotoxic.

Short description of key information:
In a GLP study conducted according to OECD Guideline 471, EDDS acid showed no evidence of mutagenic potential when tested at up to 5 mg/plate in a bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102, both in the presence and absence of a rat liver metabolic activation fraction (S9) (Thompson, 2002). On the related material, trisodium EDDS also showed no mutagenic activity in Ames tests when tested at up to 5 mg/plate in five strains of S. typhimurium and two strains of E. coli, with and without S9 (Jones et al. 1989; San and Wyman, 1993).

In a GLP study, trisodium EDDS showed no evidence of mutagenic potential at the tk locus in mouse lymphoma L5178Y tk+/- cells in an in vitro mammalian cell mutation assay when tested at up to about 5 mg/mL, with and without S9 (Bigger and Clarke, 1993).

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS induced statistically significant increases in chromosome numbers and structural aberrations (although the latter effect was not considered biologically relevant) in cultured Chinese hamster ovary cells after a 42-h exposure in the absence of S9. No evidence of numerical or structural chromosome aberrations were seen after 6-h or 18-h exposures without S9, or after a 6-h incubation in the presence of S9. Overall, trisodium EDDS was considered to cause chromosome aberrations in this in vitro mammalian cytogenicity assay (Putman and Curry, 1994).

However, in a in vivo GLP study equivalent to OECD Guideline 475, trisodium EDDS showed no evidence of induction of numerical or structural chromosome aberrations when administered as a single oral dose at up to 2000 mg/kg bw in a bone marrow cytogenetic assay in male and female rats (Putman, 1994).

Overall, it can be concluded that EDDS acid and its trisodium salt are not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to EU CLP and DSD regulations, EDDS acid and its trisodium salt would not be classified as genotoxic based on the available data described.