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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scietific principles.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells
Dunkel VC, San RHC, Seifried HE and Whittaker P.
Bibliographic source:
Environmental and Molecular Mutagenesis, 33: 28-41.

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
It is not customary to refer to GLP in publications in peer-reviewed scientific journals
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
electrolytic iron powder
electrolytic iron powder
Constituent 2
Reference substance name:
carbonyl iron
carbonyl iron
Details on test material:
- Name of test material (as cited in study report): electrolytic iron
- Physical state: solid (particles with an average size of 16 µm)
- Analytical purity: 99%
- Other: the source of electrolytic iron was SCM Metal Products

Name of test material (as cited in study report): carbonyl iron (purchased from ISP Technologies Inc.)
- Physical state: solid (uniform spheres; particles with an average size of 3 µm)
- Analytical purity: 99%
-Impurities: traces of carbon, oxygen and nitrogen
- Other: carbonyl iron is an extremely pure form of iron. It is produced after treatment of Fe with CO, that first results into iron pentacarbolyl. Thereafter, the latter is decomposed, yielding CO and pure iron powder.


Target gene:
no data
Species / strain
Species / strain / cell type:
other: S. typhimurium TA97a, TA98, TA 100, TA102, TA1535, TA1537 & TA1538 obtained from Dr. Bruce Ames, University of California, Barkeley, CA.
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 homogenates, male Sprague-Dawley rats and Syrian golden hamsters, that have been injected with Aroclor 1254 at 500 mg/kg bw.
Test concentrations with justification for top dose:
five doses up to and including 10.000 µg/ plate, with and without metabolic activation (see details in the text field below).
Vehicle / solvent:
no data
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Positive controls without metabolic activation:2-nitrofluorene (1 µg/plate) for TA98, sodium azide (1µg/plate) for TA100, cumene hydroperoxide (75 µg/plate) for TA102.
Positive controls with metabolic activation: 2-aminoanthracene (1 µg/plate) for TA98 and TA100, and sterigmatocystin (10 µg/plate) for TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation assay for initial mutagenicity testing (as originally described by Ames et al., 1975); subsequent experiments with the preincubation method (according to Yahagi et al., 1977).

- Preincubation period: 20 min (for preincubation method)
- Exposure duration: 48h (for both methods)

Evaluation criteria:
A test article should induce at least a doubling in the mean number of revertants per plate of at least one tester strain, so as to be considered as positive. The increase in the mean of revertants per plate should be accompanied by dose response to increasing concentrations of each substance. If a dose-response was observed but with a less than 3-fold increase on TA1537 or TA1538, the response was confirmed in a repeat experiment.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA97a, TA98, TA 100, TA102, TA1535, TA1537 & TA1538
Metabolic activation:
with and without
for both test substances
Cytotoxicity / choice of top concentrations:
not specified
for both test substances
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

None of the two substances gave a mutagenic response at all six test strains examined.

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative for both substances

Both Fe powders gave negative response in the Ames test performed.
Executive summary:

No positive mutagenic and cytotoxic response was observed in the Amest test with five doses up to and including 10.000 µg Fe/ plate of electrolytic and carbonyl Fe, with and without metabolic activation, when tested in the strains TA97a, TA98, TA 100, TA102, TA1535, TA1537 & TA1538 of Salmonella typhimurim.

Abstract from original publication:

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in Salmonella typhimurium and L5178Y mouse Iymphoma cells. Except for the weak response obtained with ferrous fumarate, none of the compounds induced a mutagenic response in Salmonella. In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe+3) to ferrous (Fe+2). Responses with the elemental forms of Fe were di­vergent. Electrolytic Fe with a relatively larger particle size and irregular shape was negative. The smaller-sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and with­out S9. With ferric chloride (FeCI3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9. With the Fe+2compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9. The lowest effective doses (LED) for induction of mutagenicity were identified for these compounds and an LED ratio calculated. The LED ratio ranges from 1 for FeSO4 to 30 for carbonyl Fe, which are similar to oral LD50 values obtained in animal studies.

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