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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two structurally related substances Fe(3K)EDDHSA and Fe(Na)EDDHA were examined in Bacterial Reverse Mutation Assays and in in vitro Mammalian Chromosome Aberration Test. Fe(Na)EDDHA was tested also in in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay). The tests were performed with and without metabolic activation. The substances did not induce gene mutations by frameshift or base-pair substitution in the examined strains in the Ames test. Fe(Na)EDDHA tested up to cytotoxic concentrations did not induce structural chromosome aberrations in Chinese Hamster ovary cells and was therefore not considered as clastogenic in the tested system. Fe(3K)EDDHSA did not induce any clear evidence of chromosome aberrations in cultured human lymphocytes. Finally, Fe(Na)EDDHA showed no mutagenic effect in a Mouse Lymphoma assay. Therefore, the target substance Fe(3Na)EDDHSA is considered as non genotoxic.

Link to relevant study records

Reference

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

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

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 / 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.

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)

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.

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

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

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'.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Salmonella and E.Coli tests in vitro (AmesTests):

Fe(3K)EDDHSA (EC 462-490-6) was tested in the Bacterial Reverse Mutation Test in five strains of Salmonella typhimurium (TA 1535, TA 1537, TA98, TA 100 and TA 102) (OECD Guideline 471) (CIT, 2000e; Report No. 20510 MMO). The test substance Fe(3K)EDDHSA was dissolved in distilled water. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. In a preliminary toxicity test the test substance did not induce cytotoxicity up to the highest dose level of 5000 µg/plate with and without a metabolic activation system. The test substance was then tested in two independent experiments according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Each strain was exposed to five dose-levels of the test substance: 312.5, 625, 1250, 2500 and 5000 µg/plate (three plates/dose-level), with and without metabolic activation. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.
Except for a slight thinning of the bacterial lawn noted in the TA 1535 strain in the second experiment at dose-levels > 2500 µg/plate, no noteworthy toxicity was observed towards all the strains used, both with and without S9 mix. The test substance did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. The number of revertants for the vehicle and positive controls fulfilled the acceptance criteria. The study was therefore considered as valid. Under the experimental conditions, the test substance Fe(3K)EDDHSA does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to induce mutagenic effect in histidine-requiring strains of Salmonella mutagenic (TA 98, TA 100, TA 102, TA 1535, TA 1537) and in a tryptophan-requiring strain of Escherichia coli (WP2 uvr A) (OECD Guideline 471) (CIBA-GEIGY, 1994b; Report No. 931146a). The test compound was dissolved in bidistilled water and tested at five concentrations ranging from 312.5 to 5000 µg/plate with and without metabolic activation.

Suitable positive controls were used for each strain. All experiments were repeated in order to confirm the results.

The results revealed no increased incidence of mutants by the test item with and without metabolic activation. Therefore, it was concluded that the test compound did not show mutagenic activity in S. typhimurium and E. coli. The positive controls induced mutagenic activity. In conclusion, Fe(Na)EDDHA provoked no mutagenic activity in this test system.

In-vitro Mammalian Chromosome Aberation Test

Fe(3K)EDDHSA (EC 462-490-6) was tested for its potential to induce chromosome aberrations in cultured human lymphocytes (OECD Guideline 473) (CIT, 2001b; Report No. 20677 MHL ). The test substance was tested in two independent experiments, both with and without a metabolic activation. 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: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL; and for the second experiment: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL) and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity, indicated by reduction of mitotic index (MI). In the 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. In the second experiment,cells were exposed continuously to the test or control substances (without S9 mix) and for 3 hours and then rinsed (with S9 mix), respectively. 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. In both experiments, 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.
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.Except for a moderate toxicity induced at the highest dose-levels without S9 mix in the second experiment, no noteworthy toxicity was induced.
The metaphase analysis was performed at the dose levels of 2500, 3750 and 5000 µg/mL in the first experiment and at the1250, 2500 and 5000 µg/mL for the 20-hour harvest time in the second experiment, and at the 5000 µg/mL for the 44-hour harvest time in the second experiment both with and without S9 mix. 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 fulfilled the acceptance criteria. The study was therefore considered valid. In conclusion, the test substance Fe(3K)EDDHSA did not induce any clear evidence of chromosome aberrations in cultured human lymphocytes.

The test compound Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke clastogenic effects in Chinese hamster ovary cells (CCL61) in vitro (OECD TG 473) (CIBA-GEIGY, 1994c; Report No. 931147). The compound was dissolved in DMSO and tested without metabolic activation at concentrations of 0, 7.81,15.63 and 31.25 µg/mL for 18 and 42 hours. With metabolic activation (liver S9 fraction from Aroclor 1254 induced rat liver) concentrations of 0, 31.25, 62.5 and 125 µg/mL were applied for 3 hours followed by 15 hours recovery or 3 hours followed by 39 hours recovery. Higher concentrations could not be reached due to solubility limitations.

Three independent experiments of each with and without metabolic activation were performed. Two replicate culture per concentration and 200 cells per concentration were evaluated.

The results showed in both experiments with and without metabolic activation no increased number of metaphases with chromosomal aberrations. In contrast, the positive controls (Mitomycin 0.2 µg/mL and Cyclophosphamide 20 µg/mL) induced clastogenic effects. In conclusion, the test substance provoked no clastogenic activity in this test in vitro.

In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):

Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke mutations at the tk locus in L5178Y mouse lymphoma cells in vitro (OECD Guideline 476) (CIBA-GEIGY, 1994a; Report No. 931146b). The test compound was dissolved in DMSO. The range finding experiments showed that 1000 µg/mL was the highest concentration which could be used. Higher concentrations (greater than 100 mg/mL) produced precipitates in the vehicle.

In the presence of metabolic activation (liver S9 -fraction from Aroclor 1254 treated rats) the two highest concentrations revealed cytotoxicity. In absence of metabolic activation no toxicity was noted.

For the mutagenicity experiment, concentrations ranging from 0 to 125 µg/mL with metabolic activation and from 0 to 1000 µg/mL without metabolic activation were used. In the confirmatory experiment with metabolic activation concentrations ranging from 0 to 250 µg/mL were applied. The same concentrations (0 to 1000 µg/mL) were used in the confirmatory experiment without metabolic activation.

Corresponding positive controls (N-Nitrosodimethylamine, with metabolic activation and Ethylmethansulfonate without metabolic activation) were included. The mouse lymphoma cells were treated for 4 hours. After two days expression time, mutations at the tk locus were selected by resistance to 5-trifluorothymidine. Two types of colonies were selected, large colonies (base-pair substitutions and deletions) and small colonies (chromosome aberrations). The results showed no increase incidence of mutations at the tk locus of mouse lymphoma L5178Y cells in presence or absence of metabolic activation. Positive controls showed mutagenic activity. In conclusion, Fe(Na)EDDHA was not mutagenic in this test system in vitro.

Justification for selection of genetic toxicity endpoint
Key study conducted with the nearest analogue Fe(3K)EDDHSA (EC 462-490-6) in mammalian cells

Justification for classification or non-classification

Based on results of the three different in vitro genetic toxicity studies, performed with the nearest analogues Fe(3K)EDDHSA and Fe(Na)EDDHA, the target substance Fe(3Na)EDDHSA does not need to be classified and labelled as genotoxic according to Regulation 1272/2008/EC (CLP).