Diiron tris(sulphate)

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 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

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

Test material information

Constituent 1

Reference substance name:

7782-63-0

Cas Number:

7782-63-0

IUPAC Name:

7782-63-0

Constituent 2

Reference substance name:

ferrous sulfate (FeSO4 x 7 H2O)

IUPAC Name:

ferrous sulfate (FeSO4 x 7 H2O)

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

Controls

Controls 1

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

Controls 2

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.