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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-Sep-2011/ 19-Jan-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP compliant (with GLP certificate)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Whole blood was collected from healthy volunteer donors. The volunteers were non-smokers and were not receiving any medication or radiation exposure prior to the time of sampling.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
male rat S9 liver induced by phenobarbital
Test concentrations with justification for top dose:
First experiment: Dose levels of 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/ml were used both in the absence and presence of S9 metabolism.
As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism with a treatment time of 24 hours and using the same harvest time (24 hours). A continuous treatment until harvest was used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle:Solubility of the test item
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin-C (without S9) and cyclophosphamide (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:3 hours +/-S9; 24 hours -S9
- Fixation time (start of exposure up to fixation or harvest of cells): The harvest time of 24 hours, corresponding to approximately 1.5 cell cycle, was used.

SPINDLE INHIBITOR (cytogenetic assays):Colcemid
STAIN (for cytogenetic assays):Giemsa
Three air-dried slides were prepared from each culture and stained in 3% Giemsa in tap water.

NUMBER OF REPLICATIONS: Two independent experiments for chromosomal damage were performed.
Two cultures were prepared at each test point.

NUMBER OF CELLS EVALUATED:100/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy
- Determination of endoreplication
Evaluation criteria:
In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled:
(i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control.
(ii) The increases are reproduced in both replicate cultures.
(iii) The increases must exceed historical controls. Any significant increase over the concurrent negative controls is therefore compared with the historical control.
The evaluation is based on the set of results which excludes gaps.
Statistics:
For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility:
A preliminary solubility trial was performed starting from the concentration of 472 mg/ml in DMSO. This concentration, when added to culture medium in the ratio of 1:100, gave a maximum dose level of 4720 µg/ml corresponding to 10.0 mM. Since this concentration is lower than 5000 µg/ml it was chosen as upper limit to be tested as indicated in the Study Protocol.
The test item was found to be soluble at the concentration of 15.0 mg/ml after approximately 10 minutes of vortex mixing.
An aliquot of this solution, added to supplemented RPMI 1640 medium (Dutch modification), in the ratio of 1:100, gave turbidity of the medium. Opacity of the medium was obtained by adding an aliquot at 7.50 mg/ml. A clear solution was obtained by adding an aliquot at 3.75 mg/ml. Based on solubility results, the concentration of 150 µg/ml was selected as the highest dose level to be used in the cytogenetic assay.
Additional solubility trials were performed by using sterile distilled water of injectable grade and ethanol where the test item was found not soluble.

- Osmolarity and pH: Following treatment with the test item, no remarkable variation of osmolality and pH was observed in the absence or presence of S9 metabolism, at any dose level, in any treatment series in any experiment.



Any other information on results incl. tables

Following treatment with the test item, no remarkable increase in the incidence of cells bearing aberrations, including or excluding gaps over the control value, was observed in any experiment.

For the first experiment, following the short treatment in the absence of S9 metabolism, one endoreduplicated cell was observed at the intermediate dose level. One endoreduplicated cell was also observed in the second experiment following the continuous treatment in the absence of S9 metabolism for one replicate culture from the low dose level selected for scoring and for one replicate culture from the solvent control. Since the incidence of cells bearing numerical changes was comparable to historical values observed in our laboratory, these observations were not considered biologically relevant.

Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps over the control values, was observed in both experiments in the absence and presence of S9 metabolism.

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Copper dibutyl dithiocarbamate does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions, in the absence or presence of S9.
Executive summary:

The test item Copper dibutyl dithiocarbamate was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Two independent experiments for chromosomal damage were performed, as limited by solubility.

For the first main experiment, the dose levels selected for treatment, as limited by solubility, were: 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/ml, both in the absence and presence of S9 metabolism.

In the first main experiment, the cells were treated for 3 hours both in the presence and absence of S9 metabolism. A harvest time of 24 hours, corresponding to approximately 1.5 cell cycles, was used.

As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism with a treatment time of 24 hours and using the same harvest time (24 hours). A continuous treatment until harvest was used.

Since no cytotoxicity was observed in the first main experiment, dose levels of 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586µg/ml were used.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Each experiment included appropriate negative and positive controls.Two cell cultures were prepared at each test point.

For both experiments, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Since the test item did not induce toxicity at any dose level, the highest treatment level was selected as the highest dose (150 µg/ml) for scoring. Two lower dose levels (37.5 and 75.0 µg/ml) were also selected for scoring.

One hundred metaphase spreads were scored for chromosomal aberrations from each culture.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level or any treatment time in the absence or presence of S9 metabolic activation.

Statistically significant increases in the incidence of cells bearing aberrations (both including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

It is concluded that Copper dibutyl dithiocarbamate does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.