Magnesium, EDTA cobalt copper iron manganese zinc complexes

Genetic toxicity: in vitro

Administrative data

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:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed according to methods similar to OECD471, non-GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity of Iron Compounds in Salmonella typhimurium and L5 178Y Mouse Lymphoma Cells

Author:

Dunkel, V.C., San, R.H.C., Seifried, H.E., Whittaker, P.

Year:

1999

Bibliographic source:

Environmental and Molecular Mutagenesis 33:28-41

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Clive and Spector 1975

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium feredetate

EC Number:

239-802-2

EC Name:

Sodium feredetate

Cas Number:

15708-41-5

Molecular formula:

C10H12FeN2O8.Na

IUPAC Name:

iron(3+) sodium 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate

Details on test material:

Substance: Sodium iron (III) EDTA
CAS No.: 15708-41-5
Molecular formula: NaFeEDTA (Hamp-ene, 13% Fe)
Purity: 98%
Supplier: W.R. Grace & Co.

EDTA-Na2 was used as non-Fe contaning control compound

Substance: Disodium EDTA
CAS No.: 6381-92-6
Molecular formula: Na2EDTA 2H2O (Hamp-ene Na, pure)
Purity: 100%
Supplier: W.R. Grace Bc Co.

Method

Target gene:

thymidine kinase (TK) deficiency

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells wcre grown in Fischer's medium for leukemic cells of mice (Irvine Scientific, Irvine, CA) supplemented with 10% horse serum (Hyclone Laboratories, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

New cultures were initiated at approximately 3-month intervals rrom cells stored in liquid N2.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

EDTA-FeNa
- S9 mix
0, 1.3, 2.6, 162.5, 325.0 µg Fe/ml = 0, 9.80, 19.60, 1225.21, 2450.43 EDTA-FeNa
+ S9 mix
0, 0.026, 0.052, 1.625, 3.250 µg Fe/ml = 0, 0.20, 0.39, 12.25, 24.50
(mol weight Fe 55.847, mol weight EDTA-FeNa 3H2O 421.096)

EDTA-Na2
- S9 mix
0, 250, 500, 1000, 1500, 2000 µg/ml
+ S9 mix
0, 250, 500, 1000, 1500, 2000, 4000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: EDTA-FeNa: -S9 mix 4.7*10-6M EMS, +S9mix 9*10-5M MCA, EDTA-Na2: -S9 mix 0.25 µg/ml EMS, +S9mix 5.0 µg/ml 3-MCA

Remarks:

EDTA-Na2 was used as non-Fe contaning control compound

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 292-340 hours

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: colonies > 0.1 mm were counted.

DETERMINATION OF CYTOTOXICITY
- Method: growth rate

Evaluation criteria:

Doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence
of a dose-related increase.

Statistics:

Not applicable.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

EDTA-FeNa

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9 mix >2.6 µg Fe/ml, + S9 mix >0.052 µg Fe/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

EDTA-Na2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+S9 mix > 2000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: study was performed, results not reported.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous

The authors of this study concluded that EDTA-FeNa is positive in the mouse lymphoma mutagenicity study. However doubling of the number of mutants is only observed at test concentrations that cause significant cytotoxicity. Not taking into account the cytotoxic concentrations does not give a dose related increased in mutant frequency. OECD476: "Care should be taken to avoid conditions which would lead to results not reflecting intrinsic mutagenicity. Positive results which do not reflect intrinsic mutagenicity may arise from changes in pH, osmolality or high levels of cytotoxicity." Therefore we concluded that the results from this study are ambiguous.

Executive summary:

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 is only observed at test concentration that cause significant cytotoxicity. Not taking into account the cytotoxic concentrations does not give a dose related increased in mutant frequency. EDTA-Na2 is much less cytotoxic and is negative in this assay.