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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
An Ames test, an MN test and an MLA are available for the structurally related substance EDTA-MnNa2; several genotoxicity studies are available for other EDTA-compounds.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
10 Feb 2015 - 19 Aug 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The study was also in accordance with the recently issued OECD guideline 490: "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" issued on 28 July 2015
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The assay with mouse lymphoma (L5178Y) cells detects forward mutations at the thymidine kinase (TK) locus on chromosome 11. The TK mutation test detects base pair mutations, frame shift mutations, small and large deletions, and rearrangements of the relevant chromosome.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y cells (L5178Y tk +/- 3.7.2C line)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate
Test concentrations with justification for top dose:
13- 3891 μg/ml (the latter figure is equal to 10 mM), the maximum required concentration according to the OECD guideline 476, both in the absence and presence of S9-mix
Vehicle / solvent:
RPMI 1640 (culture medium)
Details on test system and experimental conditions:
The test substance EDTA-MnNa2 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Four experiments were conducted. In the first and second experiment, 13 single cultures were treated for 4 and 24 hours in the presence and absence of S9-mix, respectively. In the third experiment, 13 single cultures were treated for 24 hours in the absence of S9-mix, whereas in the fourth experiment, 13 single cultures were treated for 4 hours in the absence of S9-mix. The test substance was dissolved in culture medium (RPMI 1640).

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and culture medium (RPMI 1640) served as negative control in all experiments.

The L5178Y cells were grown in culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I), supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% v/v for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
The cells were cultured in a humidified incubator at ca. 37°C in air containing ca. 5% CO2. Five to seven days prior to treatment, the cells were generated from a frozen stock culture by seeding them in sterile, screw-capped tissue culture flasks (about 10,000,000 cells per flask: area ± 75 cm²) containing 50 ml culture medium (with 10% horse serum). Fresh cultures of L5178Y cells were harvested from a number of culture flasks and suspended in culture medium (with 10% horse serum) and the number of cells was counted. For the cytotoxicity and gene mutation tests ca. 3,000,000 and 5,000,000 L5178Y cells were used per culture in the absence and presence of S9-mix, respectively. On the day of exposure of the cells, the growth rate (doubling time should be 9-14h) and viability (>90 %; by trypan blue exclusion) of the cells were checked.

Four experiments were carried out with 13 different concentrations of the test substance and appropriate negative (RPMI 1640) and positive controls (MMS and MCA). A preliminary test to assess the toxicity of the test substance to the cells was not performed. In all experiments, just before use, the test substance was dissolved in culture medium (RPMI 1640) at a concentration of 38.91 mg/ml, based on a purity of 91.0%. Clear solutions were obtained. From these stock solutions serial dilutions in culture medium were prepared and 1.0 ml of each solution was added to a final volume of 10 ml culture medium.

In the assay without metabolic activation, the cells were exposed to the test substance according to the following procedure: 1.0 ml test substance solution or negative control and 4.0 ml culture medium without serum were added to ca. 3,000,000 L5178Y cells or 5,000,000 L5178Y cells (for 24h or 4h, respectively) in 5 ml culture medium (with 10 % horse serum) to a final volume of 10 ml. Two cultures treated with culture medium without serum were used as negative controls. One single culture treated with 100 μl stock solution of MMS (added to ca. 3,000,000 L5178Y cells in 5 ml culture medium (with 10% horse serum) together with 4.9 ml culture medium) was used as positive control substance at a final concentration of 0.1 mmol/l. Single cultures were used for each concentration of the test substance. The cells were exposed for 24h (experiment 1-3) and 4h (experiment 4) at ca. 37°C and ca. 5 % CO2 in a humidified incubator.

In the assay with metabolic activation, the cells were exposed to the test substance according to the following procedure; 1.0 ml test substance solution or negative control and 3.0 mL culture medium without serum was added to 1 ml 20% (v/v) S9-mix (§4.3) and 5 ml culture medium (with 10% horse serum) containing ca. 5,000,000 L5178Y cells to a final volume of 10 ml. Two cultures treated with culture medium without serum were used as negative controls. One single culture treated with 100 μl stock solution of MCA (added to 1 ml 20% (v/v) S9-mix ((§4.3) and 5 ml culture medium (with 10% horse serum) containing ca. 5,000,000 L5178Y cells) was used as positive control substance at a final concentration of 10 μg/ml. Single cultures were used for each concentration of the test substance. The cells were exposed for 4h at ca. 37°C and ca. 5 % CO2 in a humidified incubator.

The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG). The relative initial cell yield is the ratio of the number of cells after treatment to that of the negative control and is a measure for growth during treatment. The RSG is a measure for the cumulative growth rate of the cells 24h and 48h after treatment compared to untreated control cultures. The RTG is the product of the relative initial cell yield, the RSG and the relative colony-forming ability (‘cloning efficiency’) of the cells 48h after treatment compared with negative control cultures, and is a measure for cytotoxicity that occurs in all phases of the assay.

The frequency of TFT-resistant mutants and the cloning efficiency of the cells were determined two days after starting the test. The number of cells was counted and the cloning efficiency of the cells was determined as described in §4.4.5. To determine the frequency of TFT-resistant mutants, the cell suspensions were diluted to a density of 10,000 cells per ml in culture medium (with 20% horse serum) containing 4 μg TFT per ml. Portions (200 μl) of each dilution were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5% CO2 in a humidified incubator.
Evaluation criteria:
See below
Statistics:
No statistical analysis was performed (see below)
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
See below

In this study, four experiments were performed to evaluate the mutagenicity of EDTA-MnNa2. In the first and second experiment, cells were exposed to 13 single concentrations for 4h and 24h in the presence and absence of S9-mix. Due to a technical error in the first experiment this experiment was discontinued. The results of the first experiment are not discussed in this study report, but individual data are shown in Appendix 2.

In the second experiment, 9 concentrations each were evaluated for mutagenicity in both the absence and the presence of S9-mix.

In the third experiment, cells were exposed to 13 single concentrations for 24h in the absence of S9-mix. In this experiment, 9 concentrations were evaluated for mutagenicity.

In the fourth experiment, cells were exposed to 13 single concentrations for 4h in the absence of S9-mix. In this experiment, 8 concentrations were evaluated for mutagenicity.

The test substance was dissolved in culture medium (RPMI 1640).

The summarized results are presented in the tables attached. The results are summarized below.

Positive and negative controls

MMS and MCA were used as positive control substances in the absence and in the presence of S9-mix, respectively; culture medium (RPMI 1640) served as negative control in all cases. In all experiments the negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in MF compared to the negative control. Therefore, the study was considered valid.

Cytotoxicity

In both he absence and presence of S9 -mix, the test substance was toxic to the cells.

Mutagenicity

In the second experiment in the absence of S9-mix, after 24h treatment, at the highest five concentrations (3891, 2724, 1907, 1335 and 934 μg/ml), an increase in the mean MF by more than 88 or more than 126 mutants per 1,000,000 clonable cells was observed (ΔMF: 98, 153, 96, 104 and 152 for 3891, 2724, 1907, 1335 and 934 μg/ml, respectively). The increase in MF was only observed at concentrations causing almost 90% cytotoxicity or more (RTG ≤12%). In addition, a clear dose response relationship could not be established. As the observed cytotoxicity and increase in MF was unexpected based on data of the Ames test (V8405/14) and in vitro micronucleus test (V8417/06), the current experiment was repeated to investigate the reproducibility and biological relevance of the results. In the third experiment in the absence of S9-mix, after 24h treatment, at the highest two concentrations evaluated (1335 and 934 μg/ml), an increase in the mean MF by more than 88, but less than 126 mutants per 1,000,000 clonable cells was observed (ΔMF: 108 and 96 for 1335 and 934 μg/ml, respectively). These increases in MF were only observed at doses causing more than 90% cytotoxicity (RTG <10%). Therefore, these increases were considered to be not biologically relevant. At concentrations up to 90% cytotoxicity no increase in MF was observed.

Hence, although the cytotoxicity appears to be reproducible between experiment 2 and 3, the increase in MF observed at cytotoxicity levels required by the OECD guideline (RTG between 10% and 20%) in experiment 2 could not be reproduced in experiment 3. The observed increase in mutation frequency is likely to be more related to the high concentrations tested and accompanying extreme cytotoxicity caused by chelation of essential metals rather than an indication for mutagenicity. This phenomenon has been described by Heimbach et al. (2000).

In the fourth experiment in the absence of S9-mix, after 4h treatment no increase in the MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level. These results further support the conclusion from experiment 2 and 3, i.e. that the observed increases in mutation frequency are not indicative for mutagenicity.

In the second experiment, in the presence of S9-mix after 4h treatment, no increase in the MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

Colony sizing

In the third experiment colony sizing was performed on all concentrations tested. The distribution of small and large colonies for all concentrations was not significantly different from the negative control, further supporting the conclusion from experiment 2 and 3, i.e. that the observed increases in mutation frequency are not indicative for mutagenicity.In the second experiment in the presence of S9-mix and in the fourth experiment colony sizing was not performed as no positive responses were observed in cultures exposed to the test substance.

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that under the conditions used in this study, the test substance EDTA-MnNa2 is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix).
Executive summary:

The test substance EDTA-MnNa2 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Four experiments were conducted. In the first and second experiment, 13 single cultures were treated for 4 and 24 hours in the presence and absence of S9 -mix, respectively. In the third experiment, 13 single cultures were treated for 24 hours in the absence of S9-mix, whereas in the fourth experiment, 13 single cultures were treated for 4 hours in the absence of S9-mix. The test substance was dissolved in culture medium (RPMI 1640).

The highest concentration of EDTA-MnNa2 evaluated for mutagenicity was 3891 μg/ml (equal to 10 mM), the maximum required concentration according to the OECD guideline 476, both in the absence and presence of S9-mix.

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and culture medium (RPMI 1640) served as negative control in all experiments. As all acceptance criteria were met, the study was considered valid.

Both in the absence and presence of S9-mix, the test substance was toxic to the cells, resulting in a reduction in initial cell yield and suspension growth. The relative total growth (RTG) at the highest concentration evaluated in the absence of S9-mix (3891 (24h), 1335 (24h) and 3543 (4h) μg/ml) was 1%, 7% and 60%, for experiment 2, 3 and 4, respectively. The relative total growth (RTG) at the highest concentration evaluated in the presence of S9-mix (3891 μg/ml) was 49%, for experiment 2.

In the second experiment in the absence of S9-mix, after 24h treatment, at the highest five concentrations (3891, 2724, 1907, 1335 and 934 μg/ml), an increase in the mean mutant frequency (MF) by more than 88 or more than 126 mutants per 1,000,000 clonable cells was observed. The increase in MF was only observed at concentrations causing almost 90% cytotoxicity or more (RTG ≤12%). In addition, a clear dose response relationship could not be established. The second experiment was repeated to investigate the reproducibility and biological relevance of the results. In the third experiment in the absence of S9-mix, after 24h treatment, at the highest two concentrations evaluated (1335 and 934 μg/ml), an increase in the mean MF by more than 88, but less than 126 mutants per 1,000,000 clonable cells was observed. These increases in MF were only observed at concentrations causing more than 90% cytotoxicity (RTG <10%). Therefore, these increases were considered to be not biologically relevant. At concentrations up to 90% cytotoxicity no increase in MF was observed. Hence, although the cytotoxicity appears to be reproducible between experiment 2 and 3, the increase in mutation frequency observed at acceptable cytotoxicity levels (as required by the OECD guideline 476: RTG between 10% and 20%) in experiment 2 could not be reproduced in experiment 3.

In the fourth experiment in the absence of S9-mix, after 4h treatment no increase in the MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level. These results further support the conclusion from experiment 2 and 3, i.e. that the observed increases in MF are not indicative for mutagenicity.

In the presence of S9-mix after 4h treatment (experiment 2), no increase in the MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

It is concluded that under the conditions used in this study, EDTA-MnNa2 is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells, in the presence of metabolic activation (S9-mix).

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

Additional information

Additional information from genetic toxicity in vitro:

Although no genotoxicity studies have been carried out with EDTA-Mn(NH4)2, several genotoxicity studies are available for EDTA-MnNa2 and other EDTA-compounds such as EDTA-Na2H2, EDTA-Na3H and several metal-chelates. None of these showed genotoxic activity (see also read across document in section 13).


Justification for selection of genetic toxicity endpoint
Several studies available for other (metal) EDTA chelates (see read across document in section 13).

Justification for classification or non-classification

Because both the Ames test and the in vitro micronucleus test with EDTA-MnNa2 were negative, and genotoxicity was not observed with other EDTA (metal) chelates, it was concluded that EDTA-Mn(NH4)2 is not mutagenic and that no classification is needed for this endpoint.