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)-

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:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

283-044-5

EC Name:

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

Cas Number:

84539-55-9

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

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

Details on test material:

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

Method

Target gene:

Not applicable

Species / strain

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%

Controls

Controls 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

Controls 2

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.