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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 January 2014 to 05 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with OECD GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The humidity fell below the protocol range several times, to a minimum of 39% for a maximum of 3 hours. The study integrity was not adversely affected by the deviation.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 30531
- Substance type: Mono-constituent
- Physical state: Liquid
- Analytical purity: 98.6%
- Purity test date: 10 January 2012
- Lot/batch No.: 41260127919/Lot 2
- Storage condition of test material: At room temperature in the dark

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: Human blood peripheral lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated fetal calf serum, L-glutamine (2 mM), pnicillin/streptomycin (50 U/mL), and heparin (30 U/mL).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 mix
Test concentrations with justification for top dose:
First Cytogenetic Assay: With and without S9-mix: 25, 100, 200, 220, 240, 260, 280, 300 ug/mL
Second Cytogenetic Assay: With and without S9-mix: 25, 75, 100, 150, 200, 250, 300 ug/mL
Cytogenetic Assay 2A: Without S9-mix: 25, 100, 200, 220, 240, 260, 280, 300 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Mitymycin C. With metabolic activation: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: First assay: 3 hours in the presence and absence of S9 mix, Second assay: 24 and 48 hours in the absence of S9 mix and 3 hours in the presence of S9 mix.
- Expression time (cells in growth medium): First Assay: 51 hours, Second assay: 51-96 hours
- Fixation time (start of exposure up to fixation or harvest of cells): First assay: 24 hours, Second assay: 24 and 48 hours

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 ug/mL medium)
STAIN (for cytogenetic assays): Giemsa solution in Sorensenbuffer pH 6.8

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: Mototic index/dose selection scoring of the cytogenetic assay: At least 1000 cells. Analysis of slides for chromosome aberrations: One hundred metaphase chromosome spreads per culture were examined.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if: it induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) in crease in the number of cell with chromosome aberrations. The test article was also considered positive if it induced a statistically significant, biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship. A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Species / strain:
mammalian cell line, other: Human blood peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
The test article was not clastogenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cell lysis was observed at concentrations above 100 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals both produced statistically significant increase in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cell lysis occurred in cells exposed to concentrations greater than 100 ug/mL.
Remarks on result:
other: strain/cell type: Human blood peripheral lymphocytes
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of the study, the test article is not clastogenic in human lymphocytes in the presence or absence of metabolic activation.
Executive summary:

The potential of the test article (Clear colorless liquid, Purity 98.6%, Batch 41260127919/Lot 2) to induce chromosome aberrations was evaluated in human blood peripheral lymphocytes in the presence and absence of metabolic activation (S9 phenobarbitol and beta-naphthoflavone induced rat livers). The study was performed under GLP (OECD, 1997) conditions. The test method was based on OECD Guideline no. 473 (1997), and EC No. 440/2008, Part B (2008). The test material was dissolved in dimethyl sulfoxide (DMSO). The final concentration of the solvent in the culture medium was 1.0% (v/v). A negative control (DMSO) and positive controls were also prepared (without metabolic activation: Mitomycin C, with metabolic activation: Cyclophosphamide) and tested in parallel with the test article. A dose range finding test was conducted in the presence and absence of metabolic activation beyond the limit of solubility to obtain adequate toxicity data. In the first cytogenetic assay the test article was tested up to 300 µg/ml with and without metabolic activation for a 3 hour exposure (24 hour fixation time). In the second cytogenetic assay the test article was tested up to a concentration of 300 µg/ml without metabolic activation for a 24 hour exposure (24 hour fixation time) and a 48 hour exposure(48 hour fixation time), and with metabolic activation for a 3 hour exposure (24 hour fixation time). Precipitation of the test article occurred at the 1000 µg/ml dose levels. In the absence of S9-mix (24 hour exposure time) no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 200 ug/mL not enough cytotoxicity was observed, whereas at 250 ug/mL too much toxicity was observed. As a result, dose levels of 200, 220, and 240 ug/mL were retested in that range. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed relative to the controls in the absence or presence of S9-mix. Based on the results of the study, the test article is not clastogenic in human lymphocytes in the presence or absence of metabolic activation.