Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Genetic toxicity: in vitro

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)

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 (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

EC Number:

283-044-5

EC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Cas Number:

84539-55-9

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Details on test material:

- Test material: CGA 65047 SG 100, (A-5787 A); identical to FeNaEDDHA
- Chemical name/type: Iron(II)-complexes
- Common/Trade name: Sequestrene 138 Fe 100 SG
- Physical state: granules
- Analytical purity: 100% (UVCB)
- Lot/batch No.: P.201845
- Stability under test conditions: stable

Method

Target gene:

tk locus (5-trifluoro-thymidine resistance)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

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 ug/mL
with S9 mix: 0.49; 1.95; 7.81; 31.25; and 125 ug/mL
Experiment II:
without S9 mix: 3.91; 15.63; 62.50; 250; and 1000 ug/mL
with S9 mix: 0.98; 3.91; 15.63; 62.50; and 250 ug/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

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) was added to the "mutation cultures" to give a final concentration of 4 ug/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 ng/ml separated by 2-fold intervals. 1000.0 ng/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 ug/mL separated by 2 fold intervals. 1000.0 ug/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 ug/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 ug/mL. At the top concentration of 125.0 ug/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 ug/mL. 1000.0 ug/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 ug/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 ug/mL was exciuded from Statistical anaiysis due excessive heterogeneity in the duplicate survival plates. In the part without metabolic activation the concentration of 15.36 ug/mL was exciuded 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 ofthe study in any respect.

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 ug/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 ug/mL in the part with and from 3.91 to 1000.0 ug/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 ug/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.