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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Research publication. Well documented meets generally accepted scientific principles, acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells.
Author:
Dunkel VC, San RHC, Seifried HE, Whittaker P
Year:
1999
Bibliographic source:
DOI 10.1002/(SICI)1098-2280(1999)33:1<28::AID-EM4>3.0.CO;2-S PMID 10037321 Environ Mol Mutagenesis 33(1):28-41.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Principles of method if other than guideline:
Method: other: Clive et al/1975 and 1979
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
not specified
Details on test material:
Ferrous Sulfate
The substance tested was the heptahydrate (CAS number 7782-63-0), but this does not affect the chemical species available to the test organisms.
Purity: 99%
Source: Sigma

Method

Target gene:
thymidine kinase (TK) gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
without S9: 309-1030 µg Fe/mL (i.e. mg Fe/L)
with S9: 0.206-1.236 µg Fe/mL (i.e. mg Fe/L)
Vehicle / solvent:
Vehicle used: Distilled water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
destilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
destilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no data
- 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): 12-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (final concentration 3ug/ml) added to cloning mediumfor mutant selection

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1E+06 for mutant selection, 200/plate for mutant count, colony size range 0.2-1.1 mm, counted with an Artek automated colony counter

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Doses of test compound selected for mutagenicity assay were within the range yielding approximately 0-90% cytotoxicity
Evaluation criteria:
Doubling of mutant frequency over  concurrent solvent treated control value together with dose relationship.
Statistics:
Colonies larger than 0.1 mm diameter were counted with an Artek automated colony counter. Colony size was also determined.

Results and discussion

Test results
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: L5178Y TK+/- 3.7.C mouse lymphoma cells

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of 2 plates) 

Concentration

µg Fe/ml

Mutant frequency*

Growth**

Concentration

µg Fe/ml

Mutant frequency*

Growth**

-S9

-S9

+S9

+S9

0*

25

100

0

27

100

20.1

25

84.5

0.804

53

61.5

50.25

29

72.5

1.005

67

54.5

100.5

32

51.5

1.206

70

41.5

150.75

46

27.5

1.508

88

5.0

201.0

80

10.5

Positive control

430

46

Positive control

171

52

* per 1E+06 survivors

** as % of control


PRECIPITATION CONCENTRATION: None reported

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In the mouse lymphoma TK+/- assay ferrous sulphate FeSO4 x 7H2O showed a weak positive response in the absence of metabolic activation at cytotoxic concentrations and a dose-related increase in mutant frequency in the presence of metabolic activation, with marked increase of cytotoxicity.
Executive summary:

A L5178Y TK+/- mouse lymphoma cell assay was conducted with FeSO4 x 7H2O. The cytotoxicity of the test substance was determined with and without metabolic activation (rat liver S9 mix) prior to the mutagenicity test in order to determine the appropriate testing concentrations. The mutagenicity assay was performed in duplicates. As indication of a positive effect doubling of the mutant frequency was used. The mouse lymphoma cells showed with and without metablic activation a weak increase in the number of induced mutants at cytotoxic concentrations.

FeSO4 x 7H2O is negative for mutagenicity in absence and presence of metabolic activation under the conditions of this test system.