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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro (mutagenic effects - bacterial). OECD 471. Bacterial reverse mutation assay. Negative. Reliability = 1. In Vitro (mutagenic effects - mammalian). OECD 473. Mammalian chromosome aberration test. Negative. Reliability = 1. In Vitro (mutagenic effects - mammalian). OECD 476. Mouse lymphoma gene mutation assay. Negative. Reliability = 1. In Vivo (mutagenic effects - mammalian). OECD 474: Mammalian erythrocyte micronucleus assay. Negative. Reliability = 1.

Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as a key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at study conduct.
Qualifier:
according to
Guideline:
other: EC Commission, Directive 2000/32/EC, Annex 4A-B10, No. L 136
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at study conduct.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were obtained from non-smoking donor(s) less than 50 years old without previous chemotherapy or radiotherapy; and without recent (within the last 6 months) viral disease or X-ray exposure.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
A correction factor based on the percent active ingredient was used for preparation of the dosing solution.
Preliminary Toxicity Assay: 10, 50, 100, 250, 500, 1000, 1500,2810, 5000 μg/mL
Chromosome Assay: 100, 250, 500, 1000, 2810, 5000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility of the test substance and compatibility with the target cells.
Untreated negative controls:
yes
Remarks:
dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide (DMSO)
Positive controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
-S9 treatments
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
+S9 treatments
Details on test system and experimental conditions:
METHOD OF APPLICATION:
For the chromosome aberration assay, 0.5 mL heparinized blood was inoculated into centrifuge tubes containing complete medium supplemented with PHA-M. The tubes were incubated at 37±2°C in a humidified atmosphere of 5±2% CO2 in air for approximately 48 hours. At the time of test substance treatment the culture tubes were centrifuged and the culture medium was discarded and replaced with RPMI 1640 treatment medium (with or without 10% S9 mixture), such that addition of the test substance volume (100 μL) resulted in a total volume of 10 mL. In order to obtain the limit dose of 5000 μg/mL, 178 μL of the neat 281 mg/mL (% weight of active ingredient) test substance mixture was added to 10 mL of treatment media.

Sets of duplicate cultures were administered an aliquot of the test substance (6 concentrations were applied), the vehicle control, and 2 positive control substance concentrations for each test condition. The cells were exposed for 4 or 22 hours (non-activated test conditions) and 4 hours (activated test conditions) at 37±2°C in a humidified atmosphere of 5±2% CO2 in air. After completion of the 4-hour exposure periods only, the cells were collected by centrifugation, and the treatment medium replaced with complete RPMI 1640 culture medium and incubated until cell harvest. The incubations were conducted at 37 ± 2°C in a humidified atmosphere of 5 ± 2% CO2 in air.

Cells were harvested approximately 19 hours after treatment initiation by adding Colcemid® to the cultures at a 0.1 μg/mL final concentration in the culture media. Approximately 22 hours after treatment initiation the cells were collected by centrifugation and the medium removed. The cells were treated with 0.075M KCl hypotonic buffer, fixed in methanol: glacial acetic acid (3:1 v/v), and stored frozen. To prepare slides, the cells were collected by centrifugation and resuspended in fixative. Slides were prepared by applying an aliquot of the fixed cells onto clean microscope slides and air-drying them. The slides were stained by Giemsa and permanently mounted.

In the preliminary toxicity assay, the cells were exposed to solvent alone and to nine concentrations of the test substance for 4 hours in both the presence and absence of S9 activation, and for 22 hours continuously in the absence of S9 activation. The maximum dose tested was 5000 μg/mL. Precipitation was observed in the treatment media at 2810 μg/mL for all 3 test conditions. Observed changes in osmolality did not exceed a 20% increase over the vehicle control and were, therefore, not considered significant. Substantial toxicity (50% reduction in mitotic index relative to the vehicle control) was observed at 500, 1000 and 1500 μg/mL in the 4 hour non-activated condition only. The mitotic index was increased in the 22 hour non-activated test condition in the test substance treated cultures compared to the negative control. Based on the findings from the preliminary toxicity assay, the highest concentration chosen for the chromosome aberration assay was 5000 μg/mL for all 3 treatment conditions.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent trials (tox and main)

NUMBER OF CELLS EVALUATED: Cells were scored for the 500, 1000, 2810 μg/mL (non-activated and activated) 4-hour treatment, 22-hour harvest; and for the 1000, 2810, 5000 μg/mL (non-activated) 22-hour continuous treatment. The mitotic index was recorded as the percentage of cells in mitosis per 1000 cells counted per concentration level (500 from each duplicate culture). Slides were scored in a blind manner. Metaphase cells were selected for scoring based on good chromosome morphology and staining characteristics. Only metaphase cells with 46 centromeres were analysed. Two hundred metaphases per concentration level (100 from each duplicate culture), when available, were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphases evaluated per duplicate flask was less if 10 or more aberrant cells were observed among the first 25 cells scored.

OTHER: Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials, triradials and complex rearrangements. Chromosome-type aberrations included chromosome breaks and exchange figures. Pulverized chromosome(s) and cells, and severely damaged cells (i.e., cells with ≥ 10 aberrations per cell) were recorded and included in the analyses. The XY coordinates for the microscope stage were recorded for cells with structural aberrations. The percentage of polyploid and endoreduplicated cells was evaluated per 100 cells.
Evaluation criteria:
Data were evaluated using scientific judgment. Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose-responsive trend. However, values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant.

The following conditions were used as a guide to determine a positive response:
• A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response.
• The observed increased frequencies were accompanied by a concentration-related increase.
• A statistically significant increase was observed at the highest dose only.

The following condition was used as a guide to determine an equivocal response:
• Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.

The test substance was judged negative if the following condition was met:
• There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC FACTORS
- Effects of pH: The pH of the highest concentration of test substance in treatment medium was approximately 7.92.
- Effects of osmolality: The osmolality of the non-activated test system was 437 in the vehicle compared with 326 at 2810 μg/mL and 353 at 5000 μg/mL. The osmolality of the S9-activated test system was 456 in the vehicle compared with 344 at 2810 μg/mL and 375 at 5000 μg/mL. The observed changes in osmolality did not exceed a 20% increase over the vehicle control and were, therefore, not considered significant.
- Evaporation from medium: No data
- Solubility: A solubility determination was conducted to determine the maximum soluble concentration of a workable suspension up to a maximum of 50 mg/mL for aqueous vehicles and 500 mg/mL for organic vehicles. The vehicle of choice for this study was DMSO, which permitted preparation of
the highest workable/soluble stock concentration. Under the conditions of this test system, the final concentration of DMSO in the treatment medium did not exceed 1% of the treatment medium.
- Precipitation: Precipitation was observed in the treatment media at 2810 μg/mL for all 3 test conditions.

RANGE-FINDING/SCREENING STUDIES:
Substantial toxicity (50% reduction in mitotic index relative to the vehicle control) was observed at 500, 1000 and 1500 μg/mL in the 4 hour non-activated condition only. The mitotic index was increased in the 22 hour non-activated test condition in the test substance treated cultures compared to the negative
control.

COMPARISON WITH HISTORICAL CONTROL DATA: The percent aberrant cells in the test substance-treated groups were within the historical solvent control range.

ADDITIONAL INFORMATION:
The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not increased relative to the solvent control at any dose level (p>0.05, Fisher’s Exact test).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). The test substance was concluded under the conditions of this assay to be negative for the induction of structural or numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes.
Executive summary:

The test substance was evaluated for clastogenic (chromosome-damaging) activity in human peripheral blood lymphocytes in vitro following 4 -hour treatments with and without metabolic (S9) activation and a 22-hour treatment without S9. All cells were harvested 22 hours after treatment initiation. The maximum concentration tested was 5000 μg/mL. Dilutions of the test substance were prepared in dimethyl sulfoxide (DMSO) as this vehicle was determined to be the solvent of choice based on the solubility of the test substance and compatibility with the target cells. A correction factor based on the percent active ingredient was used for preparation of the dosing solution. In the preliminary toxicity assay substantial toxicity (50% reduction in mitotic index relative to the vehicle control) was observed at 500, 1000 and 1500 μg/mL in the 4 hour non-activated condition only. Precipitation was observed in the treatment media at 2810 μg/mL for all 3 test conditions. Changes in osmolality did not exceed a 20% increase over the vehicle control and were, therefore, not considered significant. In the chromosome aberration assay, precipitation was observed in the treatment media at 2810 μg/mL for all 3 test conditions. Substantial toxicity was not observed; however, a 55.9% increase in the mitotic index was observed at 5000 μg/mL in the 22-hour non-activated test condition. In the chromosome aberration assay, the percentage of cells with structural or numerical aberrations in the test substance-treated groups was not increased relative to the solvent control at any dose level (p < 0.05, Fisher’s Exact test). Based on the findings of this study, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test substance did not produce mutagenicity or clastogenicity when evaluated in vitro  in bacterial or mammalian cell cultures or in vivo in animals.


Justification for selection of genetic toxicity endpoint
Multiple OECD guideline, GLP studies have been identified as key for this endpoint. In addition the study selected above, a bacterial reverse mutation assay, a mouse lymphoma gene mutation assay, and an in vivo mouse micronucleus assay are pertinent to the hazard conclusion for this endpoint.

Justification for classification or non-classification

The test substance did not produce mutagenicity or clastogenicity when evaluated in vitro  in bacterial or mammalian cell cultures or in vivo in animals. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.