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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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).

Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well perfomed and reported GLP study
Qualifier:
according to
Guideline:
other: OECD 487
Principles of method if other than guideline:
OECD guideline 487 for the testing of chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit); adopted 22 July 2010.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Chromosome aberration
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Blood samples were obtained by venapuncture from young healthy, non-smoking individuals (30 and 32 years old) with no known recent exposures to genotoxic chemicals or radiation.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver (S9 mix)
Test concentrations with justification for top dose:
first test: 3977, 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 µg/ml
second test: 3977, 3000, 2500, 2000, 1500, 1000, 750, 500, 250, 125 and 62.5 µg/ml
Vehicle / solvent:
The test substance could be dissolved in culture medium up to the maximum required stock concentration of 39.77 mg/ml (100 mmol/l), based on a purity of 92.7%.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
and Vinblastine sulphate
Details on test system and experimental conditions:
In the presence of phytoheamagglutinine (PHA-L), aliquots of 0.5 ml of whole blood in 4.5 ml culture medium, were incubated for 48 hours at 37ºC in humidified air containing 5% CO2. The incubation was carried out in sterile screw-capped (loose) centrifuge tubes. After the 48-hour incubation period, the cells were exposed to different concentrations of the test substance, in both the presence and absence of the S9-mix. Culture medium was used as a solvent for the test substance. In the presence of S9-mix, a clastogenic compound (Cyclophosphamide), which requires metabolic activation, was used as positive control. In the absence of S9-mix, a direct acting clastogenic compound (Mitomycin C) and an aneugenic compound (Vinblastine sulphate) were used as positive control substances. Culture medium was used as a solvent for all positive control substances. In all instances duplicate cultures were used.
See further at remarks:

First test (with S9-mix)
At time point 48 hours after initiation of the cultures, the cells were harvested by low speed centrifugation and resuspended in freshly prepared tissue culture medium without foetal calf serum and PHA-L. A volume of 0.5 ml of each of the serial dilutions of the test substance or 50 µl of the positive control substance Cyclophosphamide was added to the tissue culture medium in individual culture tubes. To all cultures, 0.5 ml of S9-mix (see paragraph 4.6) was added. The total volume in each culture was 5 ml. After the 4 hours treatment period, the culture medium with the test substances and S9-mix was removed. The cells were washed twice with phosphate-buffered saline (pH 7.4) and subsequently supplied with 5 ml freshly prepared culture medium enriched with foetal calf serum (20%), PHA-L and cytochalasin B (6 μg/ml; final concentration). The cells were incubated for an additional 20 hours at 37°C in humidified air containing 5% CO2 and harvested 72 hours after initiation of the cultures (second cell-cycle).
Evaluation criteria:
The frequencies of binucleated cells with micronuclei were used for the evaluation of micronuclei induction. The CBPI was calculated for treated (selected doses) and control cultures as a measure of cell cycle delay. The test concentrations selected covered the appropriate range of cytotoxicity from maximum to little or no cytotoxicity. The standard maximum cytotoxicity level for the selection of the test substance concentration was 55 ± 5%.

The study was considered valid if the selected clastogenic and aneugenic positive controls gave a statistically significant increase in the number of binucleated cells containing micronuclei and the negative controls were within the historical data performed at the test facility.

A response was considered positive if a statistically significant concentration-related or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points.

A response was considered equivocal if the percentage of binucleated cells containing micronuclei was statistically marginal higher than that of the negative control (0.05
A test substance was considered negative if it produces neither a statistically significant concentration-related nor a reproducible statistically significant increase in the number of binucleated cells containing micronuclei, at any of the test points.

See also at remarks.
Statistics:
The frequencies of micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using Chi-square test (one-sided). The results were considered statistically significant when the p-value of the Chi-square test was less than 0.05 (P<0.05).
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity observed in the first in vitro micronucleus test:
In the pulse treatment group with metabolic activation, the highest three dose levels (3977, 2000 and 1000 µg/ml) showed 45%, 35% and 18% cytotoxicity, respectively. At the lower dose levels (7.8 to 500 µg/ml) the cytotoxicity fluctuated ( between 6% and 13%), when compared to the concurrent negative control. In the pulse treatment group without metabolic activation (S9-mix), the highest dose level (3977 µg/ml) showed 41% cytotoxicity, when compared to the concurrent negative control. None of the lower dose levels (7.8 to 2000 µg/ml) were cytotoxic to the cells. The positive control substances Cyclophosphamide (20 µg/ml), Mitomycin C (0.4 µg/ml) and Vinblastine sulphate (0.025 µg/ml) showed 41%, 52% and 18% cytotoxicity, respectively.

Cytotoxicity observed in the second in vitro micronucleus test:
In the continuous treatment group without metabolic activation, the highest four dose levels ( 3977, 3000, 2500 and 1500 µg/ml) were severely cytotoxic to the cells (only dead cells observed). At the lower dose levels (1000 and 750 µg/ml) some binucleated cells were detected. The dose level of 500 µg/ml was strong cytotoxic (77%) to the cells. The dose levels 250 and 125 µg/ml showed 55% and 35% cytotoxicity, respectively. At the lowest dose level (62.5 µg/ml ) the test substance was not cytotoxic to the cells when compared to the concurrent negative control. The positive control substances Mitomycin C (0.4 µg/ml) and Vinblastine sulphate (0.025 µg/ml) showed 31% and 37% cytotoxicity, respectively.

Remarks on result:
other: other: 4 h treatment
Remarks:
Migrated from field 'Test system'.
See tables attached.

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.

Conclusions:
Interpretation of results (migrated information):
other: positive (aneugenic activity), negative (no clastogenic activity)

From the results obtained in two independent in vitro micronucleus test it is concluded that the test substance EDTA-CuNa2 induced a dose dependent statistically significant increase in the number of binucleated cells containing micronuclei in the continuous treatment (20 h), when compared to the negative control, under the conditions used in this study.
The proportion of the large and small micronuclei induced by the test substance was not statistically different from the response of the aneugen Vinblastine sulphate at acceptable toxicity levels. The observed similar proportion of large and small micronuclei is considered to be an indication for aneugenic effects, under the conditions used in this study.
Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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.

Justification for classification or non-classification

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.