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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2009-01-16 to 2009-03-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
Please see attached file.
Cross-reference
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October 2015 - January 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study acc. to GLP
Justification for type of information:
Plese see attached file.
Reason / purpose for cross-reference:
read-across source
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
sodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]ferrat(1-)
EC Number:
700-327-5
Cas Number:
1061328-86-6
Molecular formula:
C20H20FeN2NaO6
IUPAC Name:
sodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]ferrat(1-)
Details on test material:
- Name of test material (as cited in study report): Fe (III) HBED
- Substance type: chelate
- Physical state: microgranulate
- Analytical purity: 8.6% of Fe3+
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
Data concerning identity, purity and stability of test item are responsibility of Sponsor.
- Purity test date: 2008-05-26
- Lot/batch No.: 215/P/Fe-HBED
- Expiration date of the lot/batch: not reported
- Stability under test conditions: Life time of the test article is 36 months from manufacturing date (15.05.2008)
- Storage condition of test material: at temp. from -10°C to +30°C
- Other:

Method

Target gene:
his-
Species / strain
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%)
Controlsopen allclose all
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

Results and discussion

Test resultsopen allclose all
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'.

Applicant's summary and conclusion

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.