Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: draft OECD guideline 487

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium feredetate

EC Number:

239-802-2

EC Name:

Sodium feredetate

Cas Number:

15708-41-5

Molecular formula:

C10H12N2O8FeNa

IUPAC Name:

Sodium; 2-[2-(bis(carboxylatomethyl)amino)ethyl-(carboxylatomethyl)amino]acetate; iron(+3) cation

Details on test material:

Name : EDTA-FeNa
Chemical name : Ethylenediaminetetraacetic acid, ferric sodium complex, trihydrate
Other name: Dissolvine ® E-Fe-13
Molecuar formula: C10H12 FeN2O8Na.3H2O
Molecular weight: 421.1 g/mol
Batch number: CFC 7539
Purity: 100.0 ± 0.3%
CAS number: 15708-41-5
Appearance : Yellow brown solid crystals
Storage conditions : Ambient temperature, protected from light
Date of receipt : 21 May 2010
Expiry date: 01 March 2013

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate

Test concentrations with justification for top dose:

First test (4h, with and without S9-mix): 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000, 4211 µg/mL
Second test (20 h, without S9-mix): 10, 50, 125, 250, 500, 1000, 1500, 2000, 2500, 3000, 4211 µg/ml

Vehicle / solvent:

culture medium

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Mitomycin C (clastogen) and Vinblastine sulphate (aneugen)

Remarks:

without S9-mix

Controls 2

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix Migrated to IUCLID6: (clastogen)

Details on test system and experimental conditions:

Blood samples for the first and second test were obtained by venapuncture from a young female (27 years old) and a young male (35 years old), respectively. Both blood donors were healthy non-smoking persons with no known recent exposures to genotoxic chemicals or radiation. The blood was collected in sterile, heparinized vacutainer tubes and gently mixed before use to prevent clotting. The cultures were set up within 1 hour after withdrawal of the blood.

Evaluation criteria:

See below

Statistics:

See below

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: 4 h treatment

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Test 1: Observed cytotoxicity

In the presence of metabolic activation, all dose levels analysed (4211, 2000, 1000, 500, 250 and 125 μg/ml) showed 38%, 24%, 17%, 28%, 12% and 11% toxicity, respectively. In the absence of metabolic activation, all dose levels analysed (4211, 2000, 1000, 500, 250 and 125 μg/ml) were slightly toxic to the cells and showed 13%, 13%, 18%, 5%, 9% and 13% toxicity, respectively. The positive control substances Cyclophosphamide (20 μg/ml), Mitomycin C (0.4 μg/ml) and Vinblastine sulphate (0.025 μg/ml) showed 64%, 55% and 9% toxicity, respectively.

Test 1: Micronuclei induced by the test substance

Three dose levels (1000, 2000 and 4211 µg/mL) and the controls were analysed for micronuclei. In the presence and absence of a metabolic activation system (S9-mix), pulse treatment (4 hours) with the test substance did not result in a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed, when compared to the numbers found in the concurrent control cultures.

Test 2: Observed cytotoxicity

As a result of continuous treatment with the test substance the two highest dose levels analysed (4211 and 3000 μg/ml) showed severe toxicity of 76% and 69%, respectively. The four next lower dose levels analysed (2500, 2000, 1500 and 1000 μg/ml) were clearly to slightly toxic to the cells and showed 57%, 41%, 31%, 23% and 6% toxicity, respectively. At the lowest dose levels analysed (250, 125, 50 and 10 μg/ml) no toxicity was observed. The positive control substances Mitomycin C (0.05 μg/ml) and Vinblastine sulphate (0.025 μg/ml) showed 25% and 19% toxicity, respectively.

Test 2: Micronuclei induced by the test substance

Four dose levels of the test substance (2500, 1500, 500 and 125 μg/ml), ranging from 55 ± 5% toxicity to no toxicity, together with the negative and positive controls, were analysed for micronuclei formation in binucleated lymphocytes. Continuous treatment with the test substance for 20 hours resulted in a statistically significant increase in the number of binucleated cells containing micronuclei at one dose level (2500 μg/ml; ***p<0.001) with high (55 ± 5%) but acceptable toxicity, at one moderate dose level (1500 μg/ml; ***p<0.001) with average toxicity and at one low dose level (500 μg/ml; *p<0.05) with slight toxicity, when compared to the numbers found in the concurrent control cultures. At the lowest dose level (125 μg/ml), the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei, when compared to the number found in the concurrent negative control (culture medium).

Test 2: Size-classified micronucleus counting

To discriminate aneugens from clastogens from in vitro micronucleus test positive compounds, size-classified micronucleus counting was performed on the slides of all dose levels of the test substance with statistically significant increases in the number of binucleated cells containing micronuclei (2500, 1500 and 500 μg/ml), together with the slides of the positive controls Mitomycin C and Vinblastine sulphate. The proportion of large micronuclei, found at the three test substance dose levels (2500, 1500 and 500 μg/ml) were 38%, 42% and 18%, respectively, which was greater than the border (10 ± 2%) between aneugens and clastogens 10 ± 2%. The test substance EDTA-FeNa clearly increased the proportion of large micronuclei at all dose levels analysed. The relatively small proportion (9%) of large micronuclei induced by the clastogen Mitomycin C and the relatively large proportion (34%) of large micronuclei induced by the aneugen Vinblastine sulphate, demonstrated the expected response of a clastogen and aneugen, respectively and were comparable with data presented in literature.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

The results of the first in vitro micronucleus test with EDTA-FeNa in human lymphocytes was negative following 4 h exposure with and without metabolic activation. The 2nd test (20- h treatment without metabolic activation) was positive. Based on the results of the size-classified micronucleus counting in the
second micronucleus test, EDTA-FeNa clearly increased the proportion of large micronuclei at three dose levels, at acceptable toxicity levels. This increased
proportion of large micronuclei is considered to be an indication for aneugenic effects, under the conditions used in this study.

Executive summary:

The test substance EDTA-FeNa was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix) with duplicate cultures. The micronucleus study consisted of two separate tests for which blood was obtained from two different donors. In the second test, size-classified micronucleus counting was additionally performed on the slides of three dose levels of the test substance and the positive controls Mitomycin C and Vinblastin sulphate, to discriminate aneugens from clastogens.

In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment time was 4 hours (pulse treatment) and the recovery time 20 hours. In the second test, in which metabolic activation was absent and concentration spacing was modified, the treatment time was 20 hours (continuous treatment) and the recovery time 28 hours. Dose levels of the test substance ranging from 7.8 to 4211 μg/ml were tested in the culture medium. In all instances, the maximum final concentration in the culture medium was 10 mmol/l. Culture medium was used as solvent for the test substance. Cytotoxicity was calculated from the Cytokinesis- Block Proliferation Index (CBPI). Based on cytotoxicity, at least three dose levels

were selected for micronuclei analysis. Cyclophosphamide, a clastogenic compound which requires metabolic activation, was used as positive control in the presence of S9-mix. A known clastogenic compound (Mitomycin C) and a known aneugenic compound (Vinblastine sulphate) were used as positive controls in the absence of S9-mix.

In the first test, in both pulse treatment groups, analysis of micronuclei formation was carried out in the cultures of three dose levels of the test substance (4211, 2000 and 1000 μg/ml), the cultures of the solvent control (culture medium) and the cultures of the positive controls. The test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the dose levels analysed, when compared to the numbers found in the concurrent negative control (culture medium).

In the second test, after continuous treatment with the test substance, analysis of micronuclei formation was carried out in the cultures of four dose levels (2500, 1500, 500 and 125 μg/ml) of the test substance, the cultures of the solvent control (culture medium) and the cultures of the positive controls. At the three highest dose levels analysed, the test substance induced a statistically significant increase (p<0.001 at 2500 μg/ml and 1500 μg/ml; p<0.05 at 500 μg/ml) in the number of binucleated cells containing micronuclei, when compared to the numbers found in the concurrent negative control (culture medium). At the lowest dose level analysed (125 μg/ml), the test substance did not induce a statistically significant

increase in the number of binucleated cells containing micronuclei, when compared to the number found in the concurrent negative control (culture

medium).

To discriminate aneugens from clastogens from in vitro micronucleus test positive compounds, size-classified micronucleus counting was performed on the slides of all dose levels with a statistically significant increase in the number of binucleated cells containing micronuclei (2500, 1500 and 500 μg/ml), together with those of the positive controls Mitomycin C and Vinblastine sulphate. The proportions of large micronuclei, found at the three test substance dose levels analyzed (2500, 1500 and 500 μg/ml) were 38%, 42% and 18%, respectively which were greater than the border (10 ± 2%) between aneugens and clastogens. The test substance EDTA-FeNa clearly increased the proportion of large micronuclei at all dose levels analysed. The relatively small proportion (9%) of large micronuclei induced by the clastogen Mitomycin C and the relatively large proportion (34%) of large micronuclei induced by the aneugenVinblastine sulphate demonstrated the expected response of a clastogen and aneugen, respectively and were comparable with data presented in literature.

In both the first and second test, with respect to the formation of micronuclei, the negative controls were comparable with the data presented in the literature and the historical data. Treatment with the positive controls Cyclophosphamide, Vinblastine sulphate and Mitomycin C resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the solvent control. This demonstrates the validity of both the first and second in vitro micronucleus test.

In the second test, with respect to the size-classified micronucleus counting, treatment with the positive controls Mitomycin C and Vinblastine sulphate resulted in an expected small and large proportion of large micronuclei, respectively. This demonstrates the validity of the size-classified micronucleus counting carried out in the second in vitro micronucleus test.

Based on the results obtained in two in vitro micronucleus tests, the test substance EDTA-FeNa induced a statistically significant increase in the number of binucleated cells containing micronuclei in the second test (continuous treatment), when compared to the negative control (culture medium), under the conditions used in this study. Based on the results of the size-classified micronucleus counting in the second test (continuous treatment), EDTA-FeNa clearly increased the proportion of large micronuclei at three dose levels, at acceptable toxicity levels. This increased proportion of large micronuclei is considered to be an indication for aneugenic effects, under the conditions used in this study.