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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:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

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

Test material

Reference
Name:
Unnamed

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (28 years old) for Experiment I and from a male donor (22 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.

Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
Dose selection was performed according to the current OECD Guideline for the in vitro chromosome aberration test.

With regard to the solubility properties of the test item, 2500 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 9.5 to 2500 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 88.9 μg/mL and above in the absence of S9 mix and at 155 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation in the absence of S9 mix, this preliminary test was designated Experiment I. The experimental part with S9 mix was repeated in Experiment II, because the cytotoxicity criteria in the positive controls were not met.

Dose selection of Experiment II was affected by the occurrence of phase separation. 250 μg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix and short exposure in the presence of S9 mix.

Experiment 1 (4h exposure; without S9 mix; μg/mL): 9.5, 16.6, 29, 50.8, 88.9, 155, 272, 476, 833, 2500
Experiment 2 (22h exposure; without S9 mix; μg/mL): 9.8, 14.6, 21.9, 32.9, 49.4, 74.1, 111, 167, 250
Experiment 2 (4h exposure; with S9 mix; μg/mL): 32.9, 49.4, 74.1, 111, 167, 250
Vehicle / solvent:
ethanol (99.99% pure)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
ethylmethane sulfonate (absence of S9), Cyclophosphamide (presence of S9)
Positive control substance:
ethylmethanesulphonate
mitomycin C
Details on test system and experimental conditions:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the main experiment.
The pre-test was performed with 10 concentrations of the study substance separated by no more than a factor of √10 and solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each study substance concentration. The culture medium was replaced with serum-free medium containing the study substance. For the treatment with metabolic activation, 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. After washing, the cells were resuspended in complete culture medium (with 10 % FBS) and cultured until preparation of the cells.

Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each study substance concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the study substance. The culture medium was not changed until preparation of the cells.

Preparation of metaphases
Cultures were treated with the metaphase-arresting substance colcemid (final concentration: 0.2 μg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g), the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, and, after drying covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

Evaluation of the slides was performed according to the standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" using microscopes with 100 x oil immersion objectives.
Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate.
At least 150 well-spread metaphases were evaluated per culture for structural aberrations. Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established.
Evaluation criteria:
A study substance is classified as non-clastogenic if:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

A study substance is classified as clastogenic if all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.

In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the study substance response will be concluded to be equivocal.

An increase in the number of polyploid cells may indicate that the study substance has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the study substance has the potential to inhibit cell cycle progress. Therefore incidence of polyploid cells and cells with endoreduplicated chromosomes should be recorded separately.
Statistics:
The statistical significance is confirmed by the Fisher’s exact test (modified) (p < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics.

A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to assess a possible dose dependent increase of mutation frequencies. The number of chromosomal aberrations, obtained for the groups treated with the study substance were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Both, biological and statistical significance were considered together.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-clastogenic
Remarks:
The study substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The study substance was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the study substance did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, the study substanceis considered to be non-clastogenic in this chromosome aberration test, when tested up to phase separating or to the highest evaluable concentration.
Executive summary:

The study substance, dissolved in Ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments.


 


In each experimental group two parallel cultures were analyzed. Per culture at least 150 metaphases were evaluated for structural chromosomal aberrations.


The highest applied concentration in this study (2500 μg/mL of the study substance) was chosen with regard to the solubility properties of the study substance and with respect to the current OECD Guideline 473.


 


Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of the study substance phase separation in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section 3.5.1. The chosen treatment concentrations are reported in Table 1 and the results are summarized in Table 2.


 


In Experiment I in the absence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration (49.4 μg/mL). The next higher tested concentration (74.1 μg/mL), however, which was separated by a by a factor smaller than requested by the guideline (factor 1.5), was not evaluable for cytogenetic damage due to an insufficient number of adequate metaphases.


 


In Experiment I in the absence of S9 mix and in Experiment II in the presence of S9 mix neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the study substance.


 


In Experiment II in the absence of S9 mix, two values after continuous treatment with 21.9 and 49.4 μg/mL (2.2 and 2.3 % aberrant cells, excluding gaps) exceeded the 95% Control limit of the historical control data (0.0 – 1.9 aberrant cells, excluding gaps), but were within the min-max range of the historical control data (0.0 – 2.3% aberrant cells, excluding gaps). Since the values were not statistically significant and no dose-dependency, tested by trend test was observed, the findings can be regarded as biologically irrelevant.


 


No evidence of an increase in polyploid metaphases was noticed after treatment with the study substance as compared to the control cultures.


 


Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.