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Toxicological information

Genetic toxicity: in vitro

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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:
Between September and December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Draft OECD Guideline 487, December 2007
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Purity: 92.3%
Batch no: CFC 9380
Expiry date: 31 August 2012

Method

Target gene:
Formation of small membrane-bound DNA fragments such as micronuclei (chromosome aberrations); this test may detect both clastogenic and aneugenic chemicals.
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: 0.5 ml whole blood from young healthy male non-smoking individuals in RPMI-1640 with Glutamax-1, foetal calf serum, penicillin, streptomycin, phytohaemagglutinin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
with and without S9 mix: 0, 125, 250, 500, 1000, 2000 and 3891 µg/ml (4 h treatment & 20 h recovery)
without S9 mix: 0, 1000, 1500, 2000, 2500, 3000 and 3891 µg/ml (20 h treatment & 28 h recovery)
3891 µg/ml corresponds to 10 mmol/L
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (supplemented RPMI 1640)
- Justification for choice of solvent/vehicle: good water solubility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide (with S9-mix; 4 h treatment), mitomycine C and vinblastine sulphate (without S9-mix; 4 h and 20 h treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: cultures were set up within 1 h after blood withdrawal, incubated for 48 h
- Exposure duration: 4 h (with and without S9-mix), 20 h (without S9-mix)
- Expression time (cells in growth medium): 48 h preincubation + 4 h exposure + 20 h recovery = 72 h in total, and
48 h preincubation + 20 h exposure + 28 h recovery = 96 h in total
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): in case of 4-h exposures, cells were harvested and slides were fixed 20 h later; in case of 20 h exposure, cells were harvested and slides were fixed 28 h later

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): acridin-orange

NUMBER OF REPLICATIONS: 3 sides per concentration (1 for CBP index (cytotoxicity), and 2 for micronucleus formation)

NUMBER OF CELLS EVALUATED: 500 per slide, thus 1000 per culture

DETERMINATION OF CYTOTOXICITY
- Method: by using CBP index = (1x no. of mononucleates + 2x no. of binucleates + 3x no. of multinucleates) / total number of cells
Replication index (%) = (CBPI ^ T(mean) -1 / CPBI ^ C(mean) -1) x 100
Cytotoxicity (%) = 100 - replication index
with T(mean) = mean of 2 test cultures and C(mean) = mean of 2 control cultures

OTHER EXAMINATIONS: none
Evaluation criteria:
The study was considered valid because the selected clastogenic and aneugenic positive controls gave a statistically significantly increase in the number of binucleated cells containing micronuclei and the negative controls were within the historical data presented by test facility and in the literature (E. Lorge et al, 2006).
A response was considered to be positive if a statistically significant concentration-related or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points.
A response was considered to be equivocal if the percentage of binucleated cells containing micronuclei was statistically marginal higher than that of the negative control (<= 0.05A test substance was considered to be negative if it produces neither a statistically significant concentration-related or reproducible statistically significant increase in the number of binucleated cells containing micronuclei, at any of the test points (p>0.1).
The statistical method was used as an aid in evaluating the test results but was not the only determining factor for a positive response. Both statistical methods and biological relevance of the test results were considered together in the evaluation.
Statistics:
The frequencies of micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using Fisher's exact probability test (one-sided). The results were considered statistically significant when the p-value of the Fisher's exact probability test was <= 0.05.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes, only at the highest levels tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (measured)
- Effects of osmolality: no (measured)
- Evaporation from medium: no (test substance is not volatile)
- Water solubility: no (good water solubility)
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: not done

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is conluded that EDTA-MnNa2 under the conditions of this test was neither clastogenic nor aneugenic to cultured human lymphocytes.
Executive summary:

The test substance EDTA-MnNa2 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. Two separate in vitro micronucleus tests were conducted for which blood was obtained from two different donors. In the first test, in the presence and absence of S9 -mix, the treatment/recovery time was 4/20 hours (pulse treatment method). In the second test, concentration spacing was modified and the treatment/recovery times were 20/28 hours (continuous treatment) in the absence of S9-mix. Dose levels, ranging from 7.8 to 3891 µg/ml, were tested. In all instances, the maximum final concentration in the culture medium was 10 mmol/l. A purity of 92.3% was taken into account while preparing the dosing solutions. Culture medium was used as solvent for the test substance. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). 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 the presence and absence of S9-mix, the highest dose level (3891 µg/ml) was cytoxic to the cells based on the CBPI. Analysis of micronuclei formation was carried out in the cultures of three dose levels (1000, 2000 and 3891 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive controls. In the first test, the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei.

In the second test, the two highest dose levels (3000 and 3891 pg/ml) were cytotoxic to the cells based on the CBPI. Analysis of micronuclei formation was carried out in the cultures of three dose levels (2500, 3000 and 3891 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive controls. In the second test, the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei.

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 the study. From the results obtained in two in vitro micronucleus tests it is concluded that, under the conditions used in this study, the test substance EDTA-MnNa2 was neither clastogenic nor aneugenic to cultured human lymphocytes.