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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2000-09-04 to 2000-11-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): EDDHAS Fe 3K; EDDHAS Fe, sel de K
- Molecular formula (if other than submission substance): C18H14FeN2O12S2(3K)
- Molecular weight (if other than submission substance): 687.3
- Smiles notation (if other than submission substance): O=C(O)C(NCCNC(C(=O)O)c1cc(ccc1(O))S(=O)(=O)O)c2cc(ccc2(O))S(=O)(=O)O
- InChl (if other than submission substance): InChI=1S/C18H20N2O12S2/c21-13-3-1-9(33(27,28)29)7-11(13)15(17(23)24)19-5-6-20-16(18(25)26)12-8-10(34(30,31)32)2-4-14(12)22/h1-4,7-8,15-16,19-22H,5-6H2,(H,23,24)(H,25,26)(H,27,28,29)(H,30,31,32)
- Substance type: chelate
- Physical state: brownish/red powder
- Analytical purity: 55±2
- Purity test date: 2000-10-24
- Expiration date of the lot/batch: 2001-01-24
- Stability under test conditions: at least 6 months from receipt (date of receipt: 24 July 2000)
- Storage condition of test material: at room temperature and protected from humidity

Method

Target gene:

Not applicable
The in vitro chromosome aberration test is able to identify substances that cause structural chromosome aberrations in cultured mammalian cells.

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (containing 20% fetal calf serum)

Additional strain / cell type characteristics:

not applicable

Remarks:

stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours

Metabolic activation:

with and without

Metabolic activation system:

S9 mix consisiting of enzymatic systems induced by Aroclor 1254 in rat liver microsomal fraction (S9 fraction) and the cofactors necessary for their function

Test concentrations with justification for top dose:

With a treatment volume of 100 µL/5.5 mL culture medium, the dose-levels were as follows:
First experiment: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL (with and without S9 mix)
Second experiment: 156.25, 312.5, 625, 1250, 2500 and 5000 mg/mL (with and without S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: The test substance was freely soluble in the culture medium.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Migrated to IUCLID6: 3 µg/mL (3h of treatment) or 0.2 µg/mL (continuous treatment)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Migrated to IUCLID6: 50 µg/mL or 25 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in the first experiment (with and without S9 mix) and in the second experiment (with S9 mix); 20 hours and 44 hours in the second experiment (without S9 mix)
- Expression time (cells in growth medium): 17 hours in the first experiment (with and without S9 mix) and in the second experiment (with S9 mix); 41 hours in the second experiment (with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours in the first experiment (with and without S9 mix); 20 and 44 hours in the second experiment (with and without S9 mix).

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (10 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED:
200 metaphases/dose level (with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes (were recorded when present)
- Determination of endoreplication: yes (were recorded when present)

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the X2 test, in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

in the first experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second experiment, after 20-hour exposure to the test substance, up to 44% decrease in the mitotic index was noted, mainly at dose-levels > 1250 µg/mL. After 44-hour exposure, up to 51% decrease in the mitotic index was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No noteworthy toxicity was induced in both experiments

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the dose level of 5000 µg/mL, pH was about 7.6 (as for the vehicle control)
- Effects of osmolality: At the dose level of 5000 µg/mL, the osmolality was equal to 320 mOsm/kg H20 (292 for the vehicle control)
- Water solubility: The test substance was freely soluble in the culture medium.
- Precipitation: The final dose-level of 5000 µg/mL showed no precipitate in the culture medium.


RANGE-FINDING/SCREENING STUDIES: No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility.

COMPARISON WITH HISTORICAL CONTROL DATA: in the range

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosomal aberration analysis (experiments without S9 mix):

In the first experiment, after 3-hour exposure to the test substance, no significant increase in the frequency of cells with structural chromosomal aberrations was noted. After 20-hour exposure, a slight but a non significant increase (frequency of 3%) in the frequency of aberrant cells was noted at 5000 µg/mL. However, this increase was not considered as biologically relevant since it was neither dose-related nor reproducible between the two cultures. After 44-hour exposure, even though a slight increase in the frequency of aberrant cells was noted at 5000 µg/mL (3.5%), no statistically significant difference was obtained.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, the test substance EDDHAS Fe 3K did not induce any clear evidence of chromosome aberrations in cultured human lymphocytes.

Executive summary:

The objective of this study was to evaluate the potential of the test substance EDDHAS Fe 3K to induce chromosome aberrations in cultured human lymphocytes.

Methods

The test substance was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37°C in a humidified atmosphere of 5% CO2 /95% air, for 48 hours.

First experiment

Lymphocyte cultures were exposed to the test or control substances, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hour before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis.

Second experiment

- without S9 mix, cells were exposed continuously to the test or control substances,

- with S9 mix, cells were exposed to the test or control substances for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. One and a half hour before harvest, each culture was treated with a colcemid solution (10 pg/ml) to block cells at the metaphase-stage of mitosis.

For both experiments, after hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test substance EDDHAS Fe 3K was dissolved in culture medium.

The dose-levels of the positive controls were as follows:

- without S9 mix, mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

- with S9 mix, cyclophosphamide: 50 µg/mL or 25 µg/mL.

Results

The test substance was freely soluble in the culture medium. The final dose-level of 5000 mg/mL showed no precipitate in the culture medium and pH and osmolality values were equivalent to those of the vehicle control culture. With a treatment volume of 100 µL/5.5 mL culture medium, the dose-levels both with and without S9 mix were as follows:

- 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL: for the first experiment,

- 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL: for the second experiment.

Cytotoxicity:

Except for a moderate toxicity induced at the highest dose-levels without S9 mix in the second experiment, no noteworthy toxicity was induced.

Chromosomal aberration analysis:

The metaphase analysis was performed at the following dose-levels, both with and without S9 mix:

- 2500, 3750 and 5000 µg/mL: for the first experiment,

-1250, 2500 and 5000 µg/mL: for the 20-hour harvest time in the second experiment,

- 5000 µg/mL: for the 44-hour harvest time in the second experiment.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Conclusion

Under the experimental conditions, the test substance EDDHAS Fe 3K did not induce any clear evidence of chromosome aberrations in cultured human lymphocytes.