Sodium [[α,α'-(ethylenediimino)bis[2-hydroxybenzene-1-acetato]](4-)]ferrate(1-)

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:

experimental study

Adequacy of study:

key study

Study period:

1993-11-09 to 1994-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: Black red, fine grained and free-flowing solid

Details on test material:

- Test material: CGA 65047 SG 100, (A-5787 A); identical to FeNaEDDHA
- Chemical name/type: Iron(III)-complexes
- Common/Trade name: Sequestrene 138 Fe 100 SG
- Physical state: granules
- Analytical purity: 100% (UVCB)
- Stability under test conditions: stable

Method

Target gene:

tk locus (5-trifluoro-thymidine resistance)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Four types of RPMI 1640 were prepared: RM0, RM5, RM10 and RM20
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 rat liver post-mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

The cell cultures were evaluated at the following concentrations:
Experiment I:
without S9 mix: 3.91; 15.63; 62.50; 250; and 1000 µg/mL
with S9 mix: 0.49; 1.95; 7.81; 31.25; and 125 µg/mL
Experiment II:
without S9 mix: 3.91; 15.63; 62.50; 250; and 1000 µg/mL
with S9 mix: 0.98; 3.91; 15.63; 62.50; and 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The highest concentration of the test substance was determined in a preliminary solubilisation test to be 100.0 mg/mL soluble in DMSO, which resulted in a homogeneous suspension after sonication during 10 to 20 minutes. Higher concentration produced non tolerable precipitates in the vehicle. The final concentration of DMSO in the culture medium was 1%.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: overnight
- Exposure duration: 4h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 1-2 weeks: "At the end of the expression period cultures were plated for viability and 5-trifluorothymidine (TFT) restistance. A sample of the post-expression culture was diluted to give 100 mL at 1 x 10E4 cells/mL ('mutation cultures').TFT was added to the 'mutation cultures' to give a final concentration of 4 µg/mL. Each TFT treated culture was dispensed at 200 µL per well into four 96-well microtitre plates (2000 cells per well). These plates were incubated for 1-2 weeks to allow cell growth.
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) was added to the "mutation cultures" to give a final concentration of 4 µg/mL.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 5.0x10E6 L5178Y cells were placed in growth medium (RM10) for the determination of mutation rate and toxicity, respectively.

DETERMINATION OF CYTOTOXICITY
A range-finding cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiments. The concentrations applied ranged from 0.49 to 1000.0 mg/mL separated by 2-fold intervals. 1000.0 mg/mL represent the highest concentration which could be applied in the experiment.

Evaluation criteria:

The test substance was considered to be mutagentic if:
- the assay was valid
- the mutant frequency at one or more concentrations was statistically significant greater than that of the negative control
- there was a statistically significant dose-relationship as indicated by the linear trend analysis
- the effects observed were reproducible

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. These test required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity

In the cytoxicity range-finder experiment, 12 concentrations of the test substance were tested. In the part with metabolic activation, the concentrations ranged from 0.49 to 1000.0 µg/mL separated by 2 fold intervals. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. In the part with metabolic activation, down to the concentration of 31.25 µg/mL, a growth inhibiting effect greater than 50.0% in comparison to the negative control could be seen. No relevant cytotoxicity could be detected after treatment with the test chemical in the part without metabolic activation.

Accordingly, five concentrations were selected for the original mutagenicity experiment. In the part with metabolic activation the following concentrations were selected: 0.49, 1.95, 7.81, 31.25 and 125.0 µg/mL. At the top concentration of 125.0 µg/mL the mean zero hour survival value was 49.33%. The relative total growth at the end ofthe expression period revealed a mean value of 31.40%. In the part without metabolic activation the concentrations appplied were: 3.91, 15.63, 62.50, 250.0 and 1000.0 µg/mL. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. The highest concentration showed a mean relative survival of 62.01%. The mean relative total growth value was 80.31%. All concentrations were selected to determine viability and TFT-resistance two days after treatment.

In the confirmatory mutagenicity experiment, in the part with metabolic activation, the concentration range was increased. The concentrations applied were: 0.98, 3.91, 15.63, 62.50 and 250.0 µg/mL. In the part without metabolic activation the same concentration range as in the original experiment was selected. The mean relative survival value obtained in the part with metabolic activation was 47.45% at the highest concentration. The mean relative total growth value obtained after the two days expression period was 42.05%. The zero hour survival value in the part without metabolic activation was 81.90%. The relative total growth determined after the expression revealed a value of 75.74%. All concentrations were selected to determine viability and TFT-resistance 2 days after treatment.

Mutagenicity

In the presence and absence of metabolic activation, in the original and confirmatory experiments, reproducible, statistically significant and dose-related relevant increases in mutant frequencies were not observed. In the original experiment, in the part with metabolic activation, the highest concentration of 125.0 µg/mL was excluded from statistical analysis due excessive heterogeneity in the duplicate survival plates. In the part without metabolic activation the concentration of 15.36 µg/mL was excluded from statistical anlysis due to heterogeneity in the survival plates. Nevertheless, the mutant frequency values found at these concentrations are lying within the normal range and do not influence the validity of the study in any respect.

Large and small colonies

For the negative and positive controls the number of wells containing small colonies and the number of wells containing large colonies were scored. With metabolic activation the mean proportion of small colonies in the negative controls ranged from 40.0 to 44.4%, whereas with the positive controls (DMN) an increased proportion of 55.4 to 59.5% was observed. Without metabolic activation the proportion of small colonies in the negative controls ranged from 67.3 to78.0%, whereas with the positive controls (EMS) a proportion of 42.0 to 53.3% was observed.

Small and large colony numbers are not reported for CGA 65 047 SG 100, (A-5787 A) as the data did not fulfil the criteria for a positive response.

Table1 : Summary of the mutagenicity test (Experiment with metabolic activation)

Treatment	Zero hour survival %	Relative total growth (RTG) %
Negative control DMN 2 µL/mL	100.00 78.35	100.00 48.88
CGA 65 047 SG 100  (A-5787 A):
125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL	49.33 103.76 93.79 85.79 75.22	31.40 86.83 64.80 63.13 60.61
Treatment	Mutant frequency X 10E-6	Significance (P)
Negative control DMN 2 µL/mL	24.58 387.77	#
CGA 65 047 SG 100.  (A-5787 A):
125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL	54.08 27.73 25.17 36.01 27.83	§ NS NS NS NS
Test for linear trend: NS

Table2 : Summary of the mutagenicity test (Experiment without metabolic activation)

Treatment	Zero hour survival %	Relative total growth (RTG) %
Negative control EMS 1 µL/mL	100.00 1.82	100.00 2.54
CGA 65 047 SG 100 (A-5787 A):
1000.00 µg/mL 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL 3.91 µg/mL	62.01 74.51 100.13 134.44 144.04	80.31 109.86 96.75 92.87 115.83
Treatment	Mutant frequency x 10E-6	Significance (P)
Negative control EMS 1 µL/mL	33.76 841.91	#
CGA 65 047 SG 100 (A-5787 A):
1000.00 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL  3.91 µg/mL	38.61 22.40 35.37 40.38 39.89	NS NS NS § NS
Test for linear trend		NS

 § excluded from experiment;

NS not significant.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the result of two indipendently performed experiments and under the given experimental conditions, it is concluded that FeNaEDDHA and its metabolites did not show any mutagenic activity at the tk locus of mouse lymphoma L5178Y cells in the presence and absence of metabolic activation.

Executive summary:

FeNaEDDHA was tested for its ability to induce mutations at the tk locus (5-trifluoro-thymidine resistance) in L5178Y mouse lymphoma cells. The study consisted of a preliminary cytotoxicity range-finder and two independent experiments, each performed with and without metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9 fraction).

In a first range-finder experiment the concentrations applied ranged from 0.49 to 1000.0 µg/mL separated by 2-fold intervals. Based on the results of the range finding study, 5 concentrations were chosen for the first mutagenicity experiment, separated by 4-fold intervals and ranging from 0.49 to 125.0 µg/mL in the part with and from 3.91 to 1000.0 µg/mL in the part without metabolic activation. At the highest concentrations the zero hour survival was 49.33% and 62.01% in the presence and absence of metabolic activation. In the confirmatory experiment, in the part with metabolic activation, the concentration range was increased ranging from 0.98 to 250.0 µg/mL. Without metabolic activation the same concentration range was used. With metabolic activation the relative survival at the highest concentration was 47.45%, without metabolic activation the value found was 81.90%. Negative (vehicle) and positive control treatments were included in each experiment with and without metabolic activation. The mutant frequencies of the negative controls were within normal ranges and the positive controls N-Nitrosodimethylamine (DMN, with metabolic activation) and Ethylmethansulfonate (EMS, without metabolic activation) produced statistically significant increases of mutant frequency. In the part performed with and without metabolic activation, no relevant increases in mutant frequency were observed after treatment with FeNaEDDHA.