Potassium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']ferrate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

EDTA-FeNa tested up to 10,000 µg/plate Fe in the plate incorporation and pre-incubation Ames test dit not result in an increased number of revertant colonies in strains S. typhimurium strains TA 98, 97a., 100, 102, 1537, 1538. Another test showed absence of genotoxicity using E coli strains (WP2 Mutoxitest). The mutagenic activity of EDTA-FeNa and EDTA-Na2 was tested in a mammalian gene mutation assay with L5178Y mouse lymphoma cells. The authors of this study concluded that EDTA-FeNa is positive in the mouse lymphoma mutagenicity study. However, doubling of the number of mutants was only observed at test concentrations that cause significant cytotoxicity. Not taking into account the cytotoxic concentrations does not give a dose related increase in the mutant frequency. EDTA-Na2 is much less cytotoxic and is negative in this assay. An in vitro micronucleus test in human lympocytes did not result in an increased number of micronuclei following exposure for 4 h (with and without S9 mix), but it did following exposure for 20 h (without S9 mix).

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: several

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well written overview; however, limited details were available

Qualifier:

no guideline available

Principles of method if other than guideline:

Overview of tests is given

GLP compliance:

not specified

Type of assay:

other: several

Conclusions:

Interpretation of results (migrated information):
negative

The absence of mutagenicity in the Ames test for EDTA-FeNa but positive results in the mouse lymphoma test suggests that mouse lymphoma cells may be particularly sensitive to incorporation of excessive quantities of iron salts in the tissue culture growth medium. The significance of mutations produced by iron compounds added at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms. This finding with iron EDTA is not unique and it is of minor importance in the overall assessment of its safety.

Executive summary:

The authors of this overview concluded that:

"A review of the literature by Heindorff et al. (1983) indicated that EDTA inhibited DNA synthesis and repair and produced a low degree of chromosomal damage and gene mutations in vitro. FDA scientists (Lerneret al., 1986) concluded that the observed events were probably spurious indications of genotoxic potential caused by the chelation of cations that are important as enzymatic cofactors involved in DNA synthesis in the cell. Taylor and Jones (1972) reported that transient inhibition of DNA synthesis was observed in rat kidneys following intraperitoneal injection of various calcium, sodium or manganese chelated forms of EDTA, but not by zinc salts of EDTA. These authors speculated that the lack of effects by the zinc EDTA salt provided evidence that zinc is required for the initiation or continuation of DNA synthesis and that the other EDTA salts probably

caused a depletion of the required zinc ions in the kidney tissues. The importance of zinc in tissues undergoing rapid growth and cell division was also noted in the developmental studies of Kimmel (1975) and Kimmel and Sloan (1975). Na3EDTA was not mutagenic in in vitro assays with Salmonella typhimurium (strains TA98, TA100, TA1535, TA1537, TA1538) or with Escherichia coli WP uvrA (Dunkel et al., 1985). The L5178Y mouse

lymphoma cell forward mutation assay (McGregor et al., 1988), either with or without a rat liver S9 metabolic activation system, showed no mutagenic activity. A recent publication by Dunkelet al. (1999; see enclosed robust summary) reported that of 11 forms of iron salts tested in the

Ames Salmonella assay with or without S9 metabolic activation, only ferrous fumarate produced slight increases above control levels and iron EDTA (NaFeEDTA) and other salts were not mutagenic. In the L5178Y mouse lymphoma assay, in contrast to negative findings by McGregor et al.(1988), positive increases in mutants were obtained with most of the iron compounds. Iron EDTA produced a concentration-related, but only a marginally positive, increase of 2.5 -2.7 -fold over control values at the highest dose tested in the presence and absence of a rat liver S9 metabolic activation system, respectively. At the highest concentrations that were considered mutagenic, increases in mutant incidence above control values for common iron compounds were approximately 3.2 -fold for ferrous sulfate either with (+) or without (-) S9; 2.8 (+) and 2.0 (-) for ferric chloride; and 2.5 (+) and 1.3 (-) for ferric phosphate. Ferrocene complex was not mutagenic with S9 but produced 7.2 -fold increases above control values without S9. The absence of mutagenicity in the Ames test for the same compounds suggests that mouse lymphoma cells may be particularly sensitive to incorporation of excessive quantities of iron salts in the tissue culture growth medium. The significance of mutations produced by iron compounds added at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms. As the finding with iron EDTA is typical of the other irons salts tested, and was not the highest response observed, it most likely reflects the sensitivity of the L5187Y cells to abnormal iron concentrations. Thus, the finding with iron EDTA is not unique and it is of minor importance in the overall assessment of its safety. The overall findings indicate that EDTA-metal complexes lack significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function."

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

EDTA-FeNa was negative in the Ames test, in the WP2 Mutoxitest and in the in vitro micronucleus test using a treatment period of 4 h (with and without S9 -mix). In a mouse lymphoma assay with EDTA-FeNa, increases in mutants were observed but only at high cytotoxic concentrations. Not taking the cytotoxic concentrations into account, no increase in mutant incidences above control values were seen. The absence of mutagenicity in the Ames test for the same compound suggests that mouse lymphoma cells may be particularly sensitive to incorporation of excessive quantities of iron salts in the tissue culture growth medium. In the in vitro micronucleus test using a treatment period of 20 h (continuous treatment without S9 -mix), EDTA-FeNa was positive at levels >= 500 µg/mL, inducing aneugenic but no clastogenic effects. This long treatment period together with the high concentrations of chelant may have resulted in exchange and substantial binding of essential elements such as zinc. Heimbach et al (2000; see robust summary) concluded that the lack of effects by the Zn-EDTA salt in contrast to effects induced by Ca-, Na- and Mn-salts of EDTA, provided evidence that zinc is required for the initiation or continuation of DNA synthesis and maintaining cell function. As such, the significance of mutations produced by EDTA-FeNa at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms. EDTA-MnNa2 was negative in an MLA.

Therefore, the overall findings indicate that EDTA-FeNa lacks significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function. The same is expected for EDTA-FeK (see also read across document in section 13).

Justification for selection of genetic toxicity endpoint

This study gives an overview of the read across substance EDTA-FeNa (see also read across document in section 13).

Justification for classification or non-classification

EDTA-FeNa gave negative results in three in vitro mutagenicity studies, viz. the Ames test, the WP2 Mutoxitest, and the micronuclueus test following exposure for 4 h (with and without S9 mix) but gave positive results (aneugenicity) following exposure for 20 h (without S9 -mix). The latter was most probably explained by induction of Zn deficiency. The ambiguous results in the mouse lymphoma test were ascribed to cytotoxicity; moreover, EDTA-MnNa2 was negative in the MLA. Overall, based on both in vitro and in vivo studies, it was concluded that classification for genotoxicity is not warranted, also not for EDTA-FeK.