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EC number: 277-749-7 | CAS number: 74181-84-3
EDTA-CuNa2 tested in the Ames test (standard plate and preincubation test) did not result in an increased number of revertant colonies in strains S. typhimurium strains TA 98, 100, 1537, 1538. An in vitro micronucleus test with EDTA-CuNa2 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).
As it has been reported that aneugenic compounds do not show adverse effects up to a certain exposure level of the test substance, suggesting the existence of a threshold (Aardema et al., 1998; Bentley et al 2000; Elhajouji et al., 1995), a threshold level (or NOAEL) was calculated for EDTA-CuNa2by applying linear regression analysis to the dataset (r= 0.92). The highest concentration that would not produce a statistically significant increase in the number of cells containing micronuclei in the current in vitro study was calculated to be 56 µg/ml.
The test substance EDTA-CuNa2was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix) according to the OECD guideline 487. Two independentin vitromicronucleus tests were conducted for which blood was obtained from two different donors. Culture medium was used as solvent for the test substance. Dose levels ranging from 7.8 to 3977 µg/ml (3977 µg/ml = 10 mM) were tested as final concentrations in the culture medium. Concurrent negative and positive controls were run simultaneously. Duplicate cultures were used. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment).
In the second test,in thecontinuous treatment group without metabolic activation, the treatment/recovery time was 20/28 hours.
In both the first and the second test, the negative controls were within the historical data of the test facility. Treatment with the positive controls Cyclophosphamide, Mitomycin C and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the negative control. This demonstrates the validity of the study.
In the pulse treatment group with metabolic activation, a dose related cytotoxicity was observed and three dose levels (3977, 2000 and 500 µg/ml) of the test substance, together with the negative controls and positive controls, were analysed for micronucleus induction in binucleated lymphocytes.In the pulse treatment group without metabolic activation, only the highest dose level was cytotoxic to the cells.Three dose levels of the test substance (3977, 2000 and 1000 µg/ml), together with the negative controls and positive controls, were analysed for micronucleus induction in binucleated lymphocytes.
In both the pulse treatment groups, the test substance did not show a significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed, when compared to the numbers found in the concurrent negative controls.
In the second test, in the continuous treatment group without metabolic activation, a dose related cytotoxicity was observed. At the selected dose levels(250, 125 and 62.5 µg/ml)and concurrent negative control cultures, 4000 instead of 2000 cells cells wereanalysed for micronucleus induction in binucleated lymphocytes.The selected dose levels induced an appropriate level of cytotoxicity. For the positive control cultures 2000 cells were analysed. The test substance induced a dose dependent statistically significant increase in the number of binucleated cells containing micronuclei when compared to the number found in the concurrent negative control (culture medium).
In the second test, with respect to the size-classified micronucleus counting, treatment with the positive control Mitomycin C resulted in a statistically significant different response in the number of small and large micronuclei when compared to the aneugen Vinblastine Sulphate. The small/large micronuclei proportion for Vinblastine sulphate and Mitomycin C were 49%/51% and 70%/30%, respectively. This demonstrates the validity of the size-classified micronucleus counting.
From the results obtained in the first and secondin vitromicronucleus test it is concluded that, under the conditions used in this study, the test substanceEDTA-CuNa2induced a statistically significant increase in the number of binucleated cells containing micronuclei in the continuous treatment, when compared to the negative control.Based on the results of the size-classified micronucleus counting, the proportion of the large micronuclei and small micronuclei found at the three test substance dose levels (250, 125 and 62.5µg/ml) analysed was not statistically different from the response of Vinblastine Sulphate at acceptable toxicity levels. The observed similar proportions of large and small micronuclei compared to the aneugen Vinblastine Sulphate, is considered to be an indication for an aneugenic effect, under the conditions used in this study.
Additional information from genetic toxicity in vitro:
EDTA-CuNa2 was negative in the Ames test and in the in vitro micronucleus test using a treatment period of 4 h (with and without S9 -mix). In the in vitro micronucleus test using a treatment period of 20 h (continuous treatment without S9 -mix), EDTA-CuNa2 was positive at levels >= 62.5 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. Similar results were obtained with EDTA-FeNa and DTPA-FeNaH (see robust summaries and read across document; section 13). Heimbach et al (2000; see also 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-CuNa2 (and also EDTA-FeNa and DTPA-FeNaH) at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms.
Although no in vivo genotoxicity studies have been carried out with EDTA-CuNa2, EDTA-Cu(NH4)2 and EDTA-CuK2, several in vivo genotoxicity studies are available for other EDTA-compounds such as EDTA-Na2H2. No genotoxic activity was observed (see also read across document in section 13).
Therefore, the overall findings indicate that EDTA-CuK2 lacks significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function.
Justification for selection of genetic toxicity endpoint
Results of tests with EDTA-CuNa2 and other (metal) chelates were used for read across with EDTA-CuK2; see also read across document in section 13.
The test substance EDTA-CuNa2 gave negative results in two in vitro mutagenicity studies, viz. the Ames test and the micronuclueus test following exposure for 4 h (with and without S9 mix) but gave positive results (aneugenicity but not clastogenicity ) following exposure for 20 h (without S9 -mix). The latter was most probably explained by induction of Zn deficiency. Overall, it was concluded that classification for genotoxicity, also for EDTA-Cu(NH4)2 is not warranted.
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