Reaction mass of ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;magnesium, ammonium;potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;magnesium and ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;magnesium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is a reaction mixture of Magnesium (Mg), EDTA, DTPA and HEEDTA. None of these substances have known mutagenic properties, based on literature, QSAR predictions and/or classification of individual components:

Mg: Magnesium and magnesium compounds (MgCl2, MgSO4) are not considered mutagenic (cfr. classification on ECHA dissemination website). The most reliable source for this is an in vitro gene mutation study (TK) in L5178Y mouse lymphoma cells.

EDTA, DTPA and HEEDTA are classified as non-mutagenic on the ECHA dissemination website. To support this, a published negative Ames test for EDTA and QSAR results for EDTA, DTPA and HEEDTA are provided that predict negative outcomes of the Ames test with sufficient reliability.

Because of the lack of mutagenic potential in Mg compounds, EDTA, DTPA and HEEDTA, it is concluded that the reaction mass of MgEDTA, MgDTPA and MgHEEDTA also should not be classified for genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
VEGA v1.4.4

2. MODEL (incl. version number)
Mutagenicity (Ames test) model (CONSENSUS) 1.0.2.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
DTPA = C(CN(CC(=O)O)CC(=O)O)N(CCN(CC(=O)O)CC(=O)O)CC(=O)OC(CN(CC(=O)O)CC(=O)O)N(CCN(CC(=O)O)CC(=O)O)CC(=O)O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
See attached QMRF

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
See attached QMRF

6. ADEQUACY OF THE RESULT
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
An in-vitro gene mutation study in bacteria is required for REACH dossiers in the 1-10 tonnage band. This QSAR provides a prediciton for this endpoint.

Guideline:

other: REACH Guidance on QSARs R.6

Specific details on test material used for the study:

SMILES: C(CN(CC(=O)O)CC(=O)O)N(CCN(CC(=O)O)CC(=O)O)CC(=O)O

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the CAESAR model for Mutagenicity (Ames test) v2.1.13, DTPA is non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
VEGA v1.4.4

2. MODEL (incl. version number)
Mutagenicity (Ames test) model (CONSENSUS) 1.0.2.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
HEEDTA = C(CN(CC(=O)O)CC(=O)O)N(CCO)CC(=O)O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
See attached QMRF

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
See attached QMRF

6. ADEQUACY OF THE RESULT
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
An in-vitro gene mutation study in bacteria is required for REACH dossiers in the 1-10 tonnage band. This QSAR provides a prediciton for this endpoint.

Guideline:

other: REACH Guidance on QSARs R.6

Specific details on test material used for the study:

SMILES: C(CN(CC(=O)O)CC(=O)O)N(CCO)CC(=O)O

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the CAESAR model for Mutagenicity (Ames test) v2.1.13, HEEDTA is non-mutagenic.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
VEGA v1.4.4

2. MODEL (incl. version number)
Mutagenicity (Ames test) model (CONSENSUS) 1.0.2.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
EDTA = C(CN(CC(=O)O)CC(=O)O)N(CC(=O)O)CC(=O)O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
See attached QMRF

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
See attached QMRF

6. ADEQUACY OF THE RESULT
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
An in-vitro gene mutation study in bacteria is required for REACH dossiers in the 1-10 tonnage band. This QSAR provides a prediciton for this endpoint.

Guideline:

other: REACH Guidance on QSARs R.6

Specific details on test material used for the study:

SMILES: C(CN(CC(=O)O)CC(=O)O)N(CC(=O)O)CC(=O)O

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the CAESAR model for Mutagenicity (Ames test) v2.1.13, EDTA is non-mutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Remarks:

Only abstract and tables are available in English

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Executive summary:

The mutagenicity potential of magnesium sulfate was re-assessed using the current procedure of the reverse mutation test with bacteria and chromosomal aberration test with mammalian cells (a Chinese hamster lung fibroblast cell line; CHL/IU) in culture. In the reverse mutation test with bacteria, Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA were use and the maximum dose level was set at 5000 micrograms/plate irrespective of the absence or presence of metabolic activation. Five dose levels (313-5000 micrograms/plate) were selected for all strains except for TA98 without metabolic activation and for TA1537 with metabolic activation, for which 6 dose levels (156-5000 micrograms/plate) were selected. Magnesium sulfate induced no increase in the number of colonies with reverse mutation in any of the strains irrespective of the absence of presence of metabolic activation in the dose-range-finding study or in the main study. In the chromosomal aberration test with mammalian cells, a Chinese hamster lung fibroblast cell line (CHL/IU) in culture was used and the maximum dose level was set at 5.0 mg/mL both in the direct and metabolic activation methods. Three dose levels (1.25-5.0 mg/mL) were selected. Magnesium sulfate induced no increase in the incidence of cells with chromosomal aberration or those with genome mutation (polyploidy) in any of the strains irrespective of the absence of presence of metabolic activation. Thus, it is concluded that magnesium sulfate does not have mutagenic potential under the presence experimental conditions.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Only abstract and tables are available in English

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Executive summary:

The mutagenicity potential of magnesium sulfate was re-assessed using the current procedure of the reverse mutation test with bacteria and chromosomal aberration test with mammalian cells (a Chinese hamster lung fibroblast cell line; CHL/IU) in culture. In the reverse mutation test with bacteria, Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA were use and the maximum dose level was set at 5000 micrograms/plate irrespective of the absence or presence of metabolic activation. Five dose levels (313-5000 micrograms/plate) were selected for all strains except for TA98 without metabolic activation and for TA1537 with metabolic activation, for which 6 dose levels (156-5000 micrograms/plate) were selected. Magnesium sulfate induced no increase in the number of colonies with reverse mutation in any of the strains irrespective of the absence of presence of metabolic activation in the dose-range-finding study or in the main study. In the chromosomal aberration test with mammalian cells, a Chinese hamster lung fibroblast cell line (CHL/IU) in culture was used and the maximum dose level was set at 5.0 mg/mL both in the direct and metabolic activation methods. Three dose levels (1.25-5.0 mg/mL) were selected. Magnesium sulfate induced no increase in the incidence of cells with chromosomal aberration or those with genome mutation (polyploidy) in any of the strains irrespective of the absence of presence of metabolic activation. Thus, it is concluded that magnesium sulfate does not have mutagenic potential under the presence experimental conditions.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The TK+/- 3.7.2 heterozygote of L5178Y mouse lymphoma cells was used throughout these studies (Clive et al., 1972). Media, serum, and reagents have been described by Clive and Spector (1975).
All cells were thawed from frozen stock and maintained in Fischer's medium for leukemic cells of mice (Gibco Laboratories, Grand Island, N.Y.) containing 10% heat-inactivated horse serum (Flow Laboratories, McLean, Va.), Pluronic F68 (Wyandotte, Wyandotte, Mich.), sodium pyruvate (Gibco), penicillin G (Eli Lilly, Indianapolis, Ind.), and streptomycin sulfate (Lilly).
Background spontaneous TK"'~ mutant frequencies were reduced weekly by 24-h treatment of the cells with medium containing thymidine (Gibco), hypoxanthene (Gibco), methotrexate (Sigma Chemical Co., St. Louis, Mo.), and glycine (Sigma).

Metabolic activation:

with and without

Metabolic activation system:

S9 (Aroclor-induced)

Test concentrations with justification for top dose:

22, 24, 26, 28, 30, 32, 36 mg/mL

Vehicle / solvent:

distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

ethylmethanesulphonate

Remarks:

The positive controls EMS and AAF were tested at 620 and 50 Mg/ml, respectively.

Details on test system and experimental conditions:

The test system was based on the procedure described by Clive et al. (1975, 1979) with modifications to the cloning procedure (Fig. 1). Metal compounds were diluted in sterile glass-distilled water, and 0.1 ml of each dilution was added to a 10-ml suspension containing 6 X 10^6 cells from a culture recently cleansed of TK-/- cells. When testing with activation, the 10-ml suspension included 4 ml of an appropriate dilution of S9 with cofactor mix. EMS and AAF were used as positive controls in the absence and presence of S9 metabolic activation, respectively, while the negative controls received the solvent, sterile deionized glass-distilled water. Cultures containing either test chemical, positive or negative controls were incubated for 4 h at 37°C. After exposure, the cells were washed twice, fresh medium was added, and the cultures were carried through a 2-d expression period. The cultures were counted after d 1 and readjusted to 3 X 10^5 cells per milliliter if necessary.

On day 2, a modified cloning procedure was followed. A sample from each culture was centrifuged and the cells resuspendedat 500,000 viable cells per milliliter in Fischer's medium. The concentrated cells were serially diluted and appropriate dilutions plated in triplicate in cloning medium with and without trifluorothymidine (TFT). Approximately 500,000 viable cells (as determined by exclusion of trypan blue) were plated on each of three selective medium plates containing 2 Mg/ml TFT (Sigma), and 100 cells were cloned on each of three nonselective plates for each test and control tube. Cell inocula were added directly into 100-mm tissue culture plates (Costar, Vineland, N.J.), followed by the addition of about 30 ml cloning medium. The plates were swirled gently to ensure even dispersal of the inocula, allowed to gel, and then incubated at 37°C for approximately 12 d before they were counted. A New Brunswick Scientific automatic colony counter was used to determine the number of colonies per plate. Total survival was determined by the method of Clive and Spector (1975) which combines growth in suspension culture and soft cloning efficiency data. The mutation frequency (MF) was calculated asthe number of mutants per 10^5 colony-forming cells.

Evaluation criteria:

Total survival was determined and the mutation frequency was calculated as the number of mutants per 10^5 colony-forming cells.

Statistics:

no

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at concentration of 28 mg/mL and above due to changes in osmotic pressure.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Test doses of MgCI2 evoked little or no enhancement of mutation compared to the solvent control. Only at 36 mg/ml was the response to MF greater than the negative control, and this was at 1% total survival.

Conclusions:

No evidence of mutagenicity of MgCl2 was found after an in vitro gene mutation study with L5178Y mouse lymphoma cells (TK locus).

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

uninduced and arochlor induced liver S9 mix of male Fischer 344 rats, B6C3F1 mice, and Syrian hamsters

Test concentrations with justification for top dose:

10, 33, 100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other:

Details on test system and experimental conditions:

All plates were prepared in triplicate, and concurrent positive and negative controls were run at all times.

Statistics:

not needed

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Based on the Ames test, EDTA has no mutagenic properties.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

The substance consists of Magnesium and the chelating agents EDTA, DTPA and HEDTA. Mg is an inorganic that is very common in nature and the human body and is typically not considered mutagenic. None of the chelating agents are classified as mutagenic on the ECHA dissemination website.

Because none of its constituents are considered mutagenic, the reaction mass of MgEDTA, MgDTPA and MGHEDTA is also not expected to have mutagenic properties and further tests are not required.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

The substance consists of Magnesium and the chelating agents EDTA, DTPA and HEDTA. Mg is an inorganic that is very common in nature and the human body and is typically not considered mutagenic. None of the chelating agents are classified as mutagenic on the ECHA dissemination website.

Because none of its constituents are considered mutagenic, the reaction mass of MgEDTA, MgDTPA and MGHEDTA is also not expected to have mutagenic properties and further tests are not required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)