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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:
28 October, 2015 to 29 April, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
None
Specific details on test material used for the study:
Test Substance
Identification: FAT 36038/J TE
Commercial Name: Terasil Violet BL Crude Moist
Batch/Lot No.: 1404301 (China)
Purity: 99.1% (provided by Sponsor)
Expiration Date: 13 August 2019
Description by BioReliance: Very dark blackish violet powder
Storage Conditions: Room temperature, protected from light
Receipt Date: 29 May 2015

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 liver microsomal fraction
Test concentrations with justification for top dose:
dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥ 200 μg/mL in the non-activated 20-hour exposure group
Vehicle / solvent:
Dimethyl formamide (DMF)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Experimental Design
The in vitro mammalian chromosome aberration assay was conducted using standard procedures (Galloway et al, 1994; Preston et al, 1981; Swierenga et al, 1991) by exposing Chinese hamster ovary (CHO) cells to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.

Preliminary Toxicity Test for Selection of Dose Levels
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose level, and the lowest precipitating dose level was measured. Dose levels for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control) or visible precipitate at the conclusion of the treatment period.

Chromosome Aberration Assays
Seven to nineteen dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was either based on cytotoxicity (cell growth inhibition relative to the vehicle control) or visible precipitate at the conclusion of the treatment period. Two or three additional dose levels were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assay)
The pH at the highest test substance concentration was measured prior to dosing using a pH meter or test strips. Treatment was carried out by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test substance dosing solution or vehicle alone. Untreated controls were re-fed with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions.
For the chromosomal aberration assay only, two hours prior to cell harvest, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts, and then Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL. Thus the treatment time for the precipitating dose levels was 18 hours instead of 20 hours.

Collection of Metaphase Cells (Preliminary Toxicity Test and Chromosome Aberration Assayd)
For the preliminary toxicity test and chromosome aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).

Scoring for Metaphase Chromosome Aberrations (Chromosome Aberration Assays)
The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture were reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 150 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level).
The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

Statistical Analysis
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Criteria for Determination of a Valid Test
Vehicle Controls
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).

Positive Controls
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).

Cell Proliferation
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.

Test Conditions
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 20-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Analyzable Concentrations
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored were reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.
Evaluation criteria:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95 % control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations.

The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None

Any other information on results incl. tables

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 200 μg/mL in the non-activated 4-hour exposure group, at dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥ 200 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at all dose levels in all three treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 250 μg/mL for the non-activated 4 and 20-hour exposure groups, and from 0.25 to 10 μg/mL for the S9-activated 4-hour exposure group.

In the initial chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at dose levels ≥ 3 μg/mL in the S9-activated 4-hour exposure group and at dose levels 100, 175, 200 and 250 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 50 μg/mL in the non-activated 4 and 20-hour exposure groups.

The dose levels selected for microscopic analysis were 10, 25, and 50 μg/mL for the non-activated 4 and 20-hour exposure groups; and 0.25, 0.5, 1, and 3 μg/mL for the S9-activated 4-hour exposure group.

No significant or dose-dependent increases in structural aberrations were observed in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, a statistically significant increase (6.3%) in structural aberrations was observed at 3 μg/mL (p ≤ 0.05; Fisher’s Exact test). In order to confirm dose-responsiveness, an additional dose level of 1 μg/mL was included in the microscopic evaluation. However, the Cochran-Armitage test was negative for a dose-response (p > 0.05). In addition, the statistically significant increase was within the historical control range of 0.0% to 9.5%; but outside the 95% historical control limit.

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the test substance treated groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL. Due to insufficient cell growth during baseline counts in the vehicle and untreated controls, the assay was repeated again in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL.

In the second repeat assay, 55 ± 5% cytotoxicity was observed at dose levels ≥ 5 μg/mL in the S9-activated 4-hour exposure group. The dose levels selected for microscopic analysis were 1, 2.5, and 5 μg/mL. No significant or dose-dependent increases in structural or numerical aberrations were observed in the S9-activated 4-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicated that the statistically significant increase observed in the initial assay at the cytotoxic dose was an isolated event which was not reproducible. Therefore, the test substance was considered to be negative for the induction of structural aberrations in all three exposure groups.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Executive summary:

This study was performed to evaluate the clastogenic potential of FAT 36038/J TE, which was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dimethyl formamide (DMF) was used as the vehicle. Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was observed at doses ≥200 μg/mL in the non-activated 4-hour exposure group, at dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥200 μg/mL in the non-activated 20-hour exposure group.

At the conclusion of the treatment period, visible precipitate was observed at all dose levels in all three treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 250 μg/mL for the non-activated 4 and 20-hour exposure groups, and from 0.25 to 10 μg/mL for the S9-activated 4-hour exposure group.

In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at dose levels ≥ 3 μg/mL in the S9-activated 4-hour exposure group and at dose levels 100, 175, 200 and 250 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥50 μg/mL in the non-activated 4 and 20-hour exposure groups. The dose levels selected for microscopic analysis were 10, 25, and 50 μg/mL for the non-activated 4 and 20-hour exposure groups; and 0.25, 0.5, 1, and 3 μg/mL for the S9-activated 4-hour exposure group. No significant or dose-dependent increases in structural aberrations were observed in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, a statistically significant increase (6.3 %) in structural aberrations was observed at 3 μg/mL (p ≤0.05; Fisher’s Exact test). In order to confirm dose-responsiveness, an additional dose level of 1 μg/mL was included in the microscopic evaluation. However, the Cochran-Armitage test was negative for a dose-response (p >0.05). In addition, the statistically significant increase was within the historical control range of 0.0 % to 9.5 %; but outside the 95 % historical control limit.

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the test substance treated groups (p >0.05; Fisher’s Exact and Cochran-Armitage tests). In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL. Due to insufficient cell growth during baseline counts in the vehicle and untreated controls, the assay was repeated again in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL. In the second repeat assay, 55 ± 5 % cytotoxicity was observed at dose levels ≥5 μg/mL in the S9-activated 4-hour exposure group. The dose levels selected for microscopic analysis were 1, 2.5, and 5 μg/mL. No significant or dose-dependent increases in structural or numerical aberrations were observed in the S9-activated 4-hour exposure group (p >0.05; Fisher’s Exact and Cochran-Armitage tests). These results indicated that the statistically significant increase observed in the initial assay at the cytotoxic dose was an isolated event which was not reproducible. Therefore, the test substance was considered to be negative for the induction of structural aberrations in all three exposure groups. All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met. Under the conditions of the assay described in this report, FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.