Ferrate(1-), [[N,N'-1,2-ethanediylbis[N-[[2-(hydroxy-.kappa.O)phenyl]methyl]glycinato-.kappa.N,.kappa.O]](4-)]-, sodium (1:1), (OC-6-13)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance Fe (Na)HBED did not produce a significant increase of mutation frequency in strains Salmonella typhimurium TA 100, TA 98, TA 97, TA 1535, TA 102 up to the dose 1 mg/plate. The test item Fe(III)HBEDNa analyzed in terms of micronucleus test in vitro (OECD 487) did not show genotoxic effects on the target test system and the result of the study is considered to be negative. The structurally related substance Fe(Na)EDDHA was examined in in vitro Mammalian Chromosome Aberration Test and in in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay). The tests were performed with and without metabolic activation. The substance tested up to cytotoxic concentrations did not induce structural chromosome aberrations in Chinese Hamster ovary cells and was therefore not considered clastogenic in the tested system. Fe(Na)EDDHA showed no mutagenic effect in a Mouse Lymphoma assay. Supporting studies conducted with the chelating agent have the same negative outcomes. Therefore, the target substance Fe(Na)HBED is considered non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2015 - January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study acc. to GLP

Qualifier:

according to guideline

Guideline:

other: OECD 487

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripherial bood human lymphocytes

Details on mammalian cell type (if applicable):

Blood from healthy, young women, (18-35 years of age), non-smoking.
Blood was taken from two healthy non-smoking, healthy female volunteers, who were not taking any medication.

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction S9 used for metabolic activation was prepared from livers of adult male Sprague-Dawley rats (Charles River (Velaz), Czech Republic) pre-treated with the agent 20-methylcholanthrene. S9-MIX conc. was 20%.

Test concentrations with justification for top dose:

1100 µg/mL , 550 µg/mL , 275 µg/mL , 137.5 µg/mL , 68.75 µg/mL

Vehicle / solvent:

sterile purified water

Untreated negative controls:

yes

Remarks:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: colchicine

Remarks:

Inthe absence of S9 mix as positive control was used mitimycinn C, in the presence of S9 mix colcicine was used

Details on test system and experimental conditions:

Principle of the method
Peripheral blood lymphocytes are exposed to the test item both with and without an exogenous source of metabolic activation. Concurrent solvent/vehicle and positive controls are included in all tests.
After exposure to the test item, lymphocytes are grown for a period sufficient to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Harvested and stained interphase cells are analysed for the presence of micronuclei. Micronuclei should only be scored in those cells that have completed nuclear division following exposure to the test item. It is important to demonstrate that cell proliferation has occurred in both the control and treated cultures, and the extent of test item-induced cytotoxicity or cytostasis should be assessed in the cultures (or in parallel cultures) that are scored for micronuclei (1, 3).

Treatment of cell cultures

3-hour treatment in the absence and presence of S9 mix
Lymphocyte cultures were incubated for approximately 44 hours following stimulation with PHA, before addition of the test item. The test item was prepared in the vehicle and dilutions were made for both sets of cultures. Two sets of duplicate cultures were used for each treatment level, solvent and positive control cultures. The test item was dosed at 2% v/v. S 9 homogenate was present in appropriate cultures at a final concentration of 2% v/v. All cultures were identified using unique number/colour code. All cultures were incubated at 371ºC in a humidified incubator gassed with 5% v/v CO2 in air for 3 hours.
The cells were centrifuged and the medium was replaced with fresh medium. The cultures were incubated for a further 17 hours in the absence of the test item.Cytochalasin B, formulated in DMSO, was added directly to all cultures (0.1 mL/culture) to give a final concentration of 6 µg/mL per culture. The cultures were incubated for a further 27-28 hours until the scheduled harvest time.

20-hour treatment in the absence of S9 mix
Human lymphocyte culture were set up as previously described. A 20-hour continuous treatment at 37°C was used in the absence of the S9 mix. The test item was added to cultures at 2% v/v Cytochalasin B ( final conc. of 6 µg/mL) was added to all culture. The cells were harvested at - 92 hours.
Changens in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in chromosome aberrations (7), (8). Osmolality and pH measurements on post treatment incubation medium were taken in the Range-Finder test.

Harvesting and fixation
The cells were harvested by centrifugation at 800 rpm for 8 minutes. The supernatant was carefully removed and cell pellet re-suspended and treated with a 5 mL (0.075M KCl) at 37°C, cultures were incubated for 5 minutes to cause swelling. Cultures were agitated, 5 mL of ice-cold fixative (3:1 v/v methanol: glacial acetic acid) was added slowly onto the culture surface. The fixative was changed by centrifugation (at 800 rpm for 8 minutes) and resuspension. The procedure was repeated several times until the cell pellets are clean.

Slide preparation
Cells were pelleted and resuspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of suspension were gently spread onto multiple clean, dry microscope slides. After the slides had dried the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in purified water. Four slides were prepared per culture.


Selection of doses for micronucleus analysis
Slides from the main study experiments were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a 500 cells per culture. From these data the replication index (RI) was determined using the following formula:

((No. binucleated cells) + (2 × No. multinucleated cells )) ÷ (Total No. of cells)T
RI = × 100
((No. binucleated cells) + (2 × No. multinucleated cells )) ÷ (Total No. of cells)C

T = test item treatment culture
C = vehicle control culture

This indicates a value relative to the control. Expressed as a percentage cytotoxicity, the value is: 100 – RI = % cytotoxicity
If the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve 55±5% cytotoxicity using the recommended cytotoxicity parameters (reduction in RI to 45±5 % of the concurrent negative control). Care should be taken in interpreting positive results only found in the higher end of this 55±5% cytotoxicity range.
Five analyzable test concentrations were evaluated during main tests.

Slide analysis
One thousand binucleate cells from each culture (2000 per dose level) was analysed for micronuclei. Observations were recorded on raw data sheets.

Evaluation criteria:

Binucleate cells were only be included in the analysis if all of the following criteria are met:
1. the cytoplasm has remained essencially intact, and
2. the daughter nuclei are approximately aqual size
A micronucleus werel only be recorded if it meets for following criteria:
1. the micronucleus should have the same staining characteristic and a similar morphology to
the main nuclei, and
2. any micronucleus present is separate in the cytoplasm or only just touching a main nucleus,
and
3. micronuclei should be smooth edged and smaller than approximately one third the diameter
of the main nuclei.

Statistics:

The micronucleus frequencies observed in test item at defined concentrations were compared to those in negative control.
To determine whether the frequencies observed are statistically significant or not the procedure of chi-squared test was applied.

Species / strain:

lymphocytes: peripheral human blood lymphocytes

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

Additional information on results:

Selection of the test item concentrations for Range-Finder Test
Test highest test item concentration based on solubility in water (55 g/L) was 1100 g/mL. The use of stock solution of higher concentration (110 g/L) was not possible due its insolubility/ precipitate formation.

Range-Finder Test
The experiment was primarily designed for determination of cytotoxicity of the test item. On the other hand, the results also provided some initial mutagenic patterns of chosen test item concentrations (Tab.1a, Fig. 1) but these were not included in the final test interpretations.
Human lymphocyte culture were prepared as described in 12.2.2. The approx. 44h- cultures were treated with test item at 12 concentrations (from 0.55 to 1100 g/L) at 37°C in the absence of the S9 mix. The test item was added to cultures at 2% v/v, Cytochalasin B ( final conc. of 6 µg/mL) was added to all culture. The cells were harvested at 92 hours.
The results of potential cytotoxic effects testing based on the percentage of cytotoxicity are illustrated in the Tab. 1b and Fig. 2. Cytotoxicity data are reported only for the 5 highest concentrations of 1100, 550, 275, 137.5 and 68.75 g/L. It can be stated, that the test item did not show any cytotoxic effect at chosen concentrations because the cytotoxicity was below 55 ± 5 %.
Concerning the mitotic activity (cell proliferation) in the test item culture, it did not differ in comparison with mitotic activity in the solvent control (individual data not reported).
No marked changes of pH of treatment media and osmolality values were observed in the Range-Finder experiments at concentrations up to 1100 g/mL tested as compared to the concurrent vehicle controls (individual data not reported).
Based on Range-Finder test the concentrations 1100, 550, 275, 137.5 and 68.75 were selected for the Main test.

Remarks on result:

other: strain/cell type: peripheral human blood lymphocytes

Remarks:

Migrated from field 'Test system'.

 Main Test

The main test was performed at five concentrationsfollowing short (3 h) treatmentwith and without metabolic activationand extended (20 h) treatment without metabolic activation.

One thousand binucleate cells from each culture (2000 per dose level) was analysed for micronuclei under each experimental conditions.

The results are summarised in Tables 2a-b, 3a-b, 4a-b and Figures 3-4.

In negative control and test item treated cultures were not found binucleated cells with more than one nicronucleus.

The average number of micronuclei at 1000 binucleated lymphocytes and the frequency of micronuclei occurrence given separately for each treated and control cultures is specified in the Tables 1a, 2a, 3a and 4a.

 

Reliability of the accurate detection of the tests item with known clastogenic activities was confirmed. Obtained results demonstrated very high statistically significant results for all the positive controls in comparison with the untreated controls.

Clastogen (-S9 mix): Mitomycin, Colchicine (p<0.001)

Clastogen (+S9 mix): cyclophosphamide monohydrate, (p<0.001)

 

The test item concentrations used in the main test showed nostatistically significant increase in incidence of micronuclei in cultured human lymphocytes in comparison with the solvent control in the variant without metabolic activation and as well as with metabolic activation (P-values ranges between 0.315 - 1).

The test results with all positive controls, which significantly increased the incidence of micronuclei in cultured human lymphocytes compared with the solvent control, demonstrated the functionality of the test system. 

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that test item Fe(III)HBEDNa administered for 3 hours at concentrations up to 1100 g/L, in both the absence and presence of S9 MIX, did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes in this in vitro test system under the experimental conditions described.
The extended (20 h) treatment with Fe(III)HBEDNa without metabolic activation did not result in
an increase in the induction of micronuclei in cultured human lymphocytes in this in vitro test system under the experimental conditions described.
Therefor the test item Fe(III)HBEDNa analyzed in terms of micronucleus test in vitro did not show genotoxicc effects on the target test system and the result of the study is considered to be negative.

Executive summary:

The purpose of the study was to evaluate the potential genotoxic effect of the test item Fe(III)HBEDNa in the in vitro micronucleus test conducted according to the OECD Guideline 487. 

In the Range-Finder Test aimed to find potential cytotoxic effects of the test item, the test item at the highest test dose (limited by solubility) - 1100mg/mL did not reduce the cell proliferation.

 

In the main test 3 different treatments (3h treatment with and without metabolic activation and 20 h treatment without metabolic activation) were tested. Under each test condition five test item concentrations (conc. range 1100- 68.75mg/mL)were evaluated. Nostatistically significant increase of the incidence of micronuclei in comparison with the solvent control in all the test variants were determined.  (P-values ranges between 0.315 and 1.000).

No difference was observed in mitotic – cell proliferation activity for the test item concentrationsin comparison with mitotic activity of the solvent control. 

 

Functionality of the test system was demonstrated bythe test results obtained with all positive controls where the incidence of micronuclei compared with untreated negative or solvent control was significantly increased.

 

In conclusion, the test item Fe(III)HBEDNa analyzed in terms of micronucleus testin vitrodid not showgenotoxic effects on the target test system and the result of the study is considered to benegative.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2009-01-16 to 2009-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 98, TA 97, TA 102 and TA 1535

Additional strain / cell type characteristics:

other: his-, rfa mutation, uvrB mutation, the resistance to ampicilin (R factor) (were confirmed according to Maron and Ames, 1983)

Metabolic activation:

with and without

Metabolic activation system:

Methylcholanthrene or Aroclor 1254 induced post-mitochondrial S9 fraction

Test concentrations with justification for top dose:

Range-finding test: 0.001 - 5.0 mg/plate
Main test: 1.0 to 0.0001 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (in experiments with preincubation cone, up to 1%)

Untreated negative controls:

yes

Remarks:

solvent/vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Migrated to IUCLID6: TA 1535, TA 100 - 0.0015, 0.002 mg/plate, solvent: aqua

Untreated negative controls:

yes

Remarks:

solvent/vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Migrated to IUCLID6: TA 98 - 0.003 mg/plate, solvent DMSO

Untreated negative controls:

yes

Remarks:

solvent/vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Migrated to IUCLID6: TA 97 - 0.075 mg/plate, solvent DMSO

Untreated negative controls:

yes

Remarks:

solvent/vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Migrated to IUCLID6: TA 102 - 0.0005 mg/plate, solvent aqua

Untreated negative controls:

yes

Remarks:

solvent/vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-acetamidofluorene (2AAF): TA 98, TA 100 - 0.100 mg/plate, solvent DMSO

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation:
The main test consisted of two plate incorporation experiments (without any modification: with and without metabolic activation) and one preincubation test (without metabolic activation)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours




OTHER:

Evaluation criteria:

Evaluation criteria
Positive results: concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation, MF>2. Student's t-test was used for evaluation of statistical significance of mutation frequency increasing against solvent control value.

Statistics:

Student's test

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:

The highest doses 5.0 and 2.5 mg/plate reduced the number of spontaneous revertants of most of tested strains (please refer to table 1 (attached)).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The Fe (III) HBED produce neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according these results is considered non mutagenic in this system.

Executive summary:

Fe(III)HBED was tested in Bacterial Reverse Mutation Assay (Ames Test) according to OECD TG 471 and in compliance with GLP rules (Bednarikova, 2009). Five strains of Salmonella typhimurium: TA100, TA98, TA97, TA1535 and TA102 were used for evaluation of mutagenic activity of the test substance. The substance Fe(III)HBED was tested in range finding assay up to a maximum dose 5.0 mg/plate selected according to the guideline as the highest tested dose. Since the highest doses 5.0 and 2.5mg/plate reduced the number of spontaneous revertants most of tested strains, the concentrations of test compound in following experiments ranged between 0.0001and 1.0 mg/plate. The main test consisted of two standard plate incorporation assays (each with and without metabolic activation) and one preincubation assay in the absence of external metabolic activation. In the standard plate assay, two external metabolic activation systems were used: S9 fraction prepared from Sprague-Dawley rat after induction with 20-methylcholanthrene and S9 fraction prepared from Sprague-Dawley rat after induction with Aroclor1254. Adequate strain specific positive controls were included in the experiments. DMSO served as solvent/negative control. The positive mutagens yielded a significant increase in the number of revertants as compared to solvent controls, therewith demonstrating the sensitivity of the assay. Fe (III) HBED neither produce a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according to these results is considered non-mutagenic in this test system.

Reference 3

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:

1993-10-15 to 1994-02-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

The underlying hypothesis for the read-across is that Fe(Na)HBED and Fe(Na)EDDHA have the same mode of action based on their ability to chelate, remove or add iron to body causing perturbation of body’s iron balance leading, possibly, to iron deficiency anaemia (IDA) effects. The target and the source substances are six-dentate chelates which ligands (here called also chelators) have the same functional groups (= donor groups: carboxylic, amine and phenolic, each double), that bind iron (central metal atom).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

The typical purity of the marketed target substance Fe(Na)HBED is in the range of 78-88 % (w/w) whereby the typical concentration of the main component sodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]ferrat(1-) is 81 % and water 5-9 % (average 7 %). Sodium chloride (19 %) is specified as impurity. As mentioned above, sodium chloride will not affect validity of the read-across statement, since the percentages of sodium and chloride ions (both are macro elements) are negligible to cause toxicity effects in living organisms.

In contrast, Fe(Na)EDDHA is UVCB substance, containing except the structures of complexes also considerable amounts of polycondensation products as well as by-products remaining after the synthesis reaction:

Product [%]
Fe[o,o]EDDHANa 34.2
Fe[o,p]EDDHANa 4.4
Fe[p,p]EDDHANa 2.3
Fe polycondensate Na 13.3
NaCl 24.7
NaNO3 13.4
KCl 1.3
Moisture content 5.9
Sum 99.5

Molecular weight of Fe-polycondensate chelate of 678-680 g/mol was determined by HPLC-MS analyses (plase see RA). The substances with such a high molecular weight have difficulties to pass cell membrane in the gut according to ECHA guidance on Toxicokinetics (Chapter R.7C, section R. 7.12; 2014). They can be transported by pinocytosis or per sorption, but, if the polycondensates are very hydrophilic (negative log Kow, similar to the main components), the absorption is likely to be limited. Therefore, no extensive absorption into systemic circulation is expected for polycondensates. Their affinity to iron is not determined experimentally, but if absorption into systemic circulation is negligible, no remarkable concern can be attributed to the polycondensates as potential sequesters of iron from the body.

The amount of sodium, and chloride ions from NaCl, KCl and Na ions from NaNO3 are comparable to the amounts in the Fe(Na)HBED. Thus, no considerable differences in the toxicological activity of the target and the source substance related to these ions can be expected. Nitrate, however, is present only in the source substance. Its impact on the toxicological activity is not expected to be considerable, since only a small amount of nitrate originates from the source substances which are negligible with regard to the toxicity profile.
Water content is also similar by the target and the source substances.
According to published literature, commercial EDDHA consists of mixture of positional OH isomers: orto-orto (o-o), orto-para (o-p) and para-para (p-p) (Lucena, 2003). EDDHA o-o and o-p can form stable iron chelates while “p-p-EDDHA was completely unable to form iron cheltes” (Lucena, 2003). Since the source substances contain low amount of p-p isomers, they are not toxicologically relevant. No differences in the binding capacity were reported between o-o and o-p isomers (Lucena, 2003, Yunta et al., 2003b). The iron binding capacity of the chelator EDDHA is 36.89 (mean of meso and racemic forms). The stability constant of Fe(Na)HBED (39.01) (Ma et al., 1994) is however higher than those of its analogues. The higher Fe(III) affinity of Fe(Na)HBED relative to that of Fe(Na)EDDHA is due to a more favourable steric orientation of donor groups (Ma and Martell, 1993).
Based on this information, the presence of secondary components of the UVCB source substance i.e. polycondensates, geometrical isomers (o-o, o-p and p-p) as well as nitrates, that are different from the monoconstituent target substance Fe(Na)HBED, is considered not to influence the toxicological activity of the main components o-o Fe(Na)EDDHA.


3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

The chelates Fe(Na)HBED and Fe(Na)EDDHA are not mutagenic. All available studies are negative.
Please refer to section 13 of the IUCLID file where the read-across statement is attached.


4. DATA MATRIX
Data matrix on the source substance used in this robust study summary is described in greater details in the read-across statement attached to the section 13 of the IUCLID file.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line CCL 61 (Chinese hamster ovary cells, CHO) was maintained in culture medium consisting of Nutrient Mixture F-12 supplemented with 10 % fetal calf serum + Penicillin/Streptomycin 100 units/mL ug/mL in 75 cm2 tissueculture (plastic) flaks. The cultures were incubated at 37 degree C in humidified atmosphere containing 5% CO2. The cell culture were periodically checked for mycoplysma contamination.

Genome stability of the cell line:
The cell line CHO CCL 61 has been used for cytogenetic studies for several years. The stability of the genome of these cells can be assessed on the basis of the regular (cytogenetic) analysis of control cultures in the course of the cytogenetic studies. It is judged to be adequate for the particular purpose of cytogenetic studies.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver post mitochondrial fraction S9

Test concentrations with justification for top dose:

Experiments without metabolic activation:
- 18 hours treatment time:
original experiment: 7.81, 15.63 and 31.25 µg/mL
confirmatory experiment: 7.81, 15.63 and 31.25 µg/mL
- 42 hours treatment time: 7.81, 15.63 and 31.25 µg/mL

Experiments with metabolic activation:
- 3 hours treatment followed by 15 hours recovery period:
original experiment: 31.25, 62.5 and 125 µg/mL
confirmatory experiment: 31.25, 62.5 and 125 µg/mL
- 3 hours treatment followed by 39 hours recovery, period: 31.25, 62.5 and 125 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The final concentration of the vehicle was 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 hours before treatment
- Exposure duration: without metabolic activation: 18 hours and 42 hours; with metabolic activation: 3 hours (followed by 15 and 39 hours recovery period)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemide 0.4 µg/mL for 2 hours (2 hours before cell harvest)


NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
The cytotoxicity test was performed as an integral part of the mutagenicity test.
- Method: mitotic index

Evaluation criteria:

Criteria for a positive response
The test substance is generally considered to be active in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significant from the respective value ofthe negative control.
- A concentration-related response should be demonstrable.

Criteria for a negative response
The test substance is generally considered to be inactive in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in all treatment groups is less than or equal to 6.0 (based on historical negative control range) and does not differ statistically significant from the respective value ofthe negative control.

Statistics:

In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using a chi-squared test. The non significant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly a chi-square test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased, resulting in a rather save judgement ofthe observed effects.
The tests were performed based upon the presence of any specific aberration.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: cytotoxicity test was performed , results were not indicated

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 ug/ml (without metabolic activation) or 125 ug/ml (with metabolic activation) could not be scored.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity test / Selection of concentrations

The selection of the highest scorable concentrations could not primarily be based on cytotoxicity data. Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 µg/mL (without metabolic activation) or 125 µg/mL (with metabolic activation) could not be scored. An inhibition in mitotic activity by 62.3% could be observed in the original experiment performed with metabolic activation (3h/15h) at the highest scorable concentration of 125 µg/mL. In the respective confirmatory experiment cytotoxicity was noted at the non-scorable concentration of 250 µg/mL only. In the absence of metabolic activation toxicity appeared only after 42 hours treatment at the highest concentration of 1000 µg/mL (non-scorable).

Original mutagenicity study

In the experiment performed without metabolic activation (experiment 1; 18 hours treatment), 2.0% of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 7.81 µg/mL, 15.63 µg/mL and 31.25 µg/mL 1.0%, 2.0% and 1.5% of cells with specific chromosomal aberrations were found.

In the experiment performed with metabolic activation (experiment 2; 3 hours treatment/15 hours recovery), 1.0% of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 31.25 µg/mL, 62.5 µg/mL and 125 µg/mL the respective values were 2.5%, 2.0% and 5.0%. The value obtained with the highest concentration of the second experiment showed a statistically significant difference when compared with the respective negative control. The value however is below the critical limit required for a positive response and it is within the historical range for negative controls. Furthermore no increase at all was observed in the respective confirmatory experiment. The slight increase in the frequency of aberrant metaphases is therefore considered to be spontaneous in origin.

Flow cytometry

The influence of the test substance on the cell cycle of CHO cells was tested at the concentrations selected for chromosome analysis. The DNA distribution was determined by flow cytometry and compared with the profile of the respective control culture. In the absence and in the presence of metabolic activation a shift in the DNA distribution profile could not be detected. Therefore, no evidence of a cell cycle disturbance by the test substance was obtained in the CHO cells.

Analytical results

The test material in suspension was analysed by UV/VIS-spectroscopy to confirm the intended concentrations to be used in the mutagenicity tests and the stability of the test substance in the vehicle used. The concentration values found were 120.6 and 124.1% of the calculated concentrations, thus indicating a sufficient stability of the test substance in the vehicle.

TABLE1 MITOTIC INDEX VALUES / CYTOTOXICITY
Original study, experiment 1        18h treatment without metabolic activation
Cells scored			Mitosis	M.I. %	Frequency % of control
Solvent control		2000	263	13.15	100
CGA65047SG100 (A-5787A)
1000 µg/mL		2000	250	12.50	95.06
500 µg/mL		2000	234	11.70	88.97
250 µg/mL		2000	268	13.40	101.90
125 µg/mL		2000	273	13.65	103.80
62.5 µg/mL		2000	282	14.10	107.22
31.25 µg/mL		2000	290	14.50	110.27
15.63 µg/mL		2000	300	15.00	114.07
7.81 µg/mL		2000	310	15.50	117.87
Original study, experiment 2        3h treatment with metabolic activation/ 15h recovery
Cells scored			Mitosis	M.I. %	Frequency % of control
Solvent control		2000	220	11.00	100.00
CGA65047SG100 (A-5787A)
1000 µg/mL		2000	114	5.70	51.82
500 µg/mL		2000	70	3.50	31.82
250 µg/mL		2000	62	3.10	28.18
125 µg/mL		2000	83	4.15	37.73
62.5 µg/mL		2000	218	10.90	99.09
31.25 µg/mL		2000	224	11.20	101.82
15.63 µg/mL		2000	238	11.90	108.18
7.81 µg/mL		a)

a) When three subsequent concentrations with a frequency of 70% mitosis or more in relation to the solvent control are found, the evaluation of the lower concentrations is omitted.

TABLE 2 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 1
18 h treatment without metabolic activation
Treatment	total no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exc	cs del	cs exc	mab	pol	end
Solvent control	200	2.0	1	2		1		1	7
CGA 65047 SG 100 (CA-5787 A)
7.81 µg/mL	200	1.0	3			1	1		3
15.63 µg/mL	200	2.0	2	1		2	1		3
31.25 µg/mL	200	1.5	3			2	1		6
positive control (Mito-C, 0.2 µg/mL	50 )	24.0***	5	6	6				2
TABLE 3 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 2
3 h treatment with metabolic activation / 15 h recovery
Treatment	total no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exc	cs del	cs exc	mab	pol	end
Solvent control	200	1.0	6			2			8
CGA 65047 SG 100 (CA-5787 A}
31.25 µg/mL	200	2.5	6	2		2	1		3	1
62.5 µg/mL	200	2.0	13	1		3			9
125 µg/mL	200	5.0*	10	6	3		1		5	1
positive control (CPA, 20 µg/mL)	100	17.0***	2	6	9	3			3

	Legend to Tables2 -3
ctdel	Chromatid deletions (including deletions, breaks, fragments)
ct exc	Chromatid exchanges (including triradials, quadriradials, endfusions and acentric rings)
csdel	Chromosome deletions (including deletions, breaks, fragments)
cs exc	Chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings)
mab	Multiple aberrations: metaphases containing more than10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps)
gaps	Chromatid and chromosome type gaps
pol	Polyploid metaphases (>30 centromers)
end	Endoreduplications
CPA	Cyclophosphamide
Mito-C	Mitomycin-C
*)	Statistical significance:0.05 =P> 0.01
	Statistical significance:0.01 =P> 0.001
	Statistical significance: P< 0.001
#)	%cells with aberrations excluding gaps and numerical alterations(pol,end)

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FeNaEDDHA.

Executive summary:

FeNaEDDHA was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in vitro with and without extrinsic metabolic activation (S9). The test compound FeNaEDDHA was dissolved in DMSO and tested at each of the following conditions:

Experiments without metabolic activation:

- 18 hours treatment time:

original experiment: 7.81, 15.63 and 31.25 µg/mL

confirmatory experiment: 7.81, 15.63 and 31.25 µg/mL

- 42 hours treatment time: 7.81, 15.63 and 31.25 µg/mL

Final concentrations higher than 31.25 ug/mL of culture medium could not be scored due to solubility limitations. Mitomycin C (0.2 ug/mL) was used as a positive control in the 18 hours experiments.

Experiments with metabolic activation:

- 3 hours treatment followed by 15 hours recovery period:

original experiment: 31.25, 62.5 and 125 µg/mL

confirmatory experiment: 31.25, 62.5 and 125 µg/mL

- 3 hours treatment followed by 39 hours recovery, period: 31.25, 62.5 and 125 µg/mL

Final concentrations higher than 125 ug/mL of culture medium could not be scored due to solubility limitations. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/15 hours experiments.

In addition, DNA distribution of cultures treated under the above described conditions (18 hours only) was determined by flow cytometry. These measurements allow to analyse the influence of the test substance on the cell cycle of CHO cells.

In both the experiments performed without and with metabolic activation no significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed.

Flow cytometry experiments did not reveal any evidence for cell cycle disturbing activities of FeNaEDDHA either in the absence or in the presence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

1993-11-09 to 1994-03-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

The underlying hypothesis for the read-across is that Fe(Na)HBED and Fe(Na)EDDHA have the same mode of action based on their ability to chelate, remove or add iron to body causing perturbation of body’s iron balance leading, possibly, to iron deficiency anaemia (IDA) effects. The target and the source substances are six-dentate chelates which ligands (here called also chelators) have the same functional groups (= donor groups: carboxylic, amine and phenolic, each double), that bind iron (central metal atom).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

The typical purity of the marketed target substance Fe(Na)HBED is in the range of 78-88 % (w/w) whereby the typical concentration of the main component sodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]ferrat(1-) is 81 % and water 5-9 % (average 7 %). Sodium chloride (19 %) is specified as impurity. As mentioned above, sodium chloride will not affect validity of the read-across statement, since the percentages of sodium and chloride ions (both are macro elements) are negligible to cause toxicity effects in living organisms.

In contrast, Fe(Na)EDDHA is UVCB substance, containing except the structures of complexes also considerable amounts of polycondensation products as well as by-products remaining after the synthesis reaction:

Product [%]
Fe[o,o]EDDHANa 34.2
Fe[o,p]EDDHANa 4.4
Fe[p,p]EDDHANa 2.3
Fe polycondensate Na 13.3
NaCl 24.7
NaNO3 13.4
KCl 1.3
Moisture content 5.9
Sum 99.5

Molecular weight of Fe-polycondensate chelate of 678-680 g/mol was determined by HPLC-MS analyses (plase see RA). The substances with such a high molecular weight have difficulties to pass cell membrane in the gut according to ECHA guidance on Toxicokinetics (Chapter R.7C, section R. 7.12; 2014). They can be transported by pinocytosis or per sorption, but, if the polycondensates are very hydrophilic (negative log Kow, similar to the main components), the absorption is likely to be limited. Therefore, no extensive absorption into systemic circulation is expected for polycondensates. Their affinity to iron is not determined experimentally, but if absorption into systemic circulation is negligible, no remarkable concern can be attributed to the polycondensates as potential sequesters of iron from the body.

The amount of sodium, and chloride ions from NaCl, KCl and Na ions from NaNO3 are comparable to the amounts in the Fe(Na)HBED. Thus, no considerable differences in the toxicological activity of the target and the source substance related to these ions can be expected. Nitrate, however, is present only in the source substance. Its impact on the toxicological activity is not expected to be considerable, since only a small amount of nitrate originates from the source substances which are negligible with regard to the toxicity profile.
Water content is also similar by the target and the source substances.
According to published literature, commercial EDDHA consists of mixture of positional OH isomers: orto-orto (o-o), orto-para (o-p) and para-para (p-p) (Lucena, 2003). EDDHA o-o and o-p can form stable iron chelates while “p-p-EDDHA was completely unable to form iron cheltes” (Lucena, 2003). Since the source substances contain low amount of p-p isomers, they are not toxicologically relevant. No differences in the binding capacity were reported between o-o and o-p isomers (Lucena, 2003, Yunta et al., 2003b). The iron binding capacity of the chelator EDDHA is 36.89 (mean of meso and racemic forms). The stability constant of Fe(Na)HBED (39.01) (Ma et al., 1994) is however higher than those of its analogues. The higher Fe(III) affinity of Fe(Na)HBED relative to that of Fe(Na)EDDHA is due to a more favourable steric orientation of donor groups (Ma and Martell, 1993).
Based on this information, the presence of secondary components of the UVCB source substance i.e. polycondensates, geometrical isomers (o-o, o-p and p-p) as well as nitrates, that are different from the monoconstituent target substance Fe(Na)HBED, is considered not to influence the toxicological activity of the main components o-o Fe(Na)EDDHA.


3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

The chelates Fe(Na)HBED and Fe(Na)EDDHA are not mutagenic. All available studies are negative.
Please refer to section 13 of the IUCLID file where the read-across statement is attached.


4. DATA MATRIX
Data matrix on the source substance used in this robust study summary is described in greater details in the read-across statement attached to the section 13 of the IUCLID file.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

tk locus (5-trifluoro-thymidine resistance)

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%.

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

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.

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'.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Bacterial Revers Mutation Assay (Ames Test)

Fe(Na)HBED was tested in Bacterial Reverse Mutation Assay (Ames Test) according to OECD TG 471 and in compliance with GLP rules (Bednarikova, 2009). Five strains of Salmonella typhimurium: TA100, TA98, TA97, TA1535 and TA102 were used for evaluation of mutagenic activity of the test substance. The substance Fe(Na)HBED was tested in range finding assay up to a maximum dose 5.0 mg/plate selected according to the guideline as the highest tested dose. Since the highest doses 5.0 and 2.5 mg/plate reduced the number of spontaneous revertants most of tested strains, the concentrations of test compound in following experiments ranged between 0.0001and 1.0 mg/plate. The main test consisted of two standard plate incorporation assays (each with and without metabolic activation) and one preincubation assay in the absence of external metabolic activation. In the standard plate assay, two external metabolic activation systems were used: S9 fraction prepared from Sprague-Dawley rat after induction with 20-methylcholanthrene and S9 fraction prepared from Sprague-Dawley rat after induction with Aroclor1254.Adequate strain specific positive controls were included in the experiments. DMSO served as solvent/negative control. The positive mutagens yielded a significant increase in the number of revertants as compared to solvent controls, therewith demonstrating the sensitivity of the assay. Fe(Na)HBED neither produce a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according to these results is considered non-mutagenic in this test system.

In-vitro Mammalian Cell Micronucleus Test (OECD 487)

The purpose of the study was to evaluate the potential genotoxic effect of the test item Fe(Na)HBEDNa in the in vitro micronucleus test conducted according to the OECD Guideline 487 (Beňo, 2016). 

In the Range-Finder Test aimed to find potential cytotoxic effects of the test item, the test item at the highest test dose (limited by solubility) - 1100mg/mL did not reduce the cell proliferation. In the main test 3 different treatments (3h treatment with and without metabolic activation and 20 h treatment without metabolic activation) were tested. Under each test condition five test item concentrations (conc. range 1100- 68.75mg/mL)wereevaluated. Nostatistically significant increase of the incidence of micronuclei in comparison with the solvent control in all the test variants were determined.  (P-values ranges between 0.315 and 1.000).

No difference was observed in mitotic – cell proliferation activity for the test item concentrationsin comparison with mitotic activity of the solvent control. 

Functionality of the test system was demonstrated bythe test results obtained with all positive controls where the incidence of micronuclei compared with untreated negative or solvent control was significantly increased.

In conclusion, the test item Fe(III)HBEDNa analyzed in terms of micronucleus test in vitro did not show genotoxic effects on the target test system and the result of the study is considered to be negative.

In-vitro Mammalian Chromosome Aberration Test

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, 1994; 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 TG 476) (CIBA-GEIGY, 1994; 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.

The results of the toxicity experiment which revealed cytotoxicity at the two highest concentrations with metabolic activation (Liver S9-fraction from Arochlor 1254 treated rats). 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. Conclusion: Fe(Na)EDDHA was not mutagenic in this test system in vitro.

Justification for selection of genetic toxicity endpoint
Guideline and GLP study detecting mutations in mammalian cells.

Justification for classification or non-classification

Based on results of Ames Test and In-vitro Mammalian Cell Micronucleus Test (OECD 487) performed with the target substance Fe(Na)HBED and two in vitro genetic toxicity studies, performed with the structurally related analogue Fe(Na)EDDHA, the target substance Fe(Na)HBED does not need to be classified and labelled as genotoxic according to Regulation 1272/2008/EC (CLP).