Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2014
Deviations:
yes
Remarks:
Ethyl methanesulfonate was used as the positive control.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro mammalian chromosome aberration test.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 31233510
CAS No.: 3598-16-1
Storage conditions: At room temperature

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h).
The cell stocks were kept in a freezer at -80 +/- 10 °C. Checking for mycoplasma infections was carried out. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in an incubator with a humidified atmosphere, set at 5 % CO2. The V79 cells for this study was grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rodent S9 mix
Test concentrations with justification for top dose:
Experiment A:
Without S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 20 h after start of exposure).
With S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 20 h after start of exposure).

Experiment B:
Without S9 mix: 0 - 500 - 1000 - 2000 µg/mL (20 h exposure; sampling 20 h after start of exposure)
With S9 mix: 0 - 500 - 1000 - 2000 µg/mL (20 h exposure; sampling 28 h after start of exposure)
With S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 28 h after start of exposure).

Selection of doses according to the results of the preliminary test and the maximum recommended concentration of 2000 μg/mL
Vehicle / solvent:
the test item solutions were prepared in the testing laboratory using DME (Dulbecco’s Modified Eagle’s) medium as solvent.
This vehicle is compatible with the survival of the V79cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dulbecco’s Modified Eagle’s medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Components of Media:
Name: DME (Dulbecco’s Modified Eagle’s) medium
Supplier: Sigma-Aldrich, Germany
Name: Fetal Bovine Serum
Supplier: Sigma-Aldrich, Germany
Name: Antibiotic-antimycotic
Supplier: Sigma-Aldrich, Germany

Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH, Germany; manufacturer: MOLTOX INC., USA. Certificate of Analysis was obtained from the supplier.

Pre-test for Cytotoxicity (Concentration selection)
The pre-experiments on solubility of the test item and the non GLP Pre-test for Cytotoxicity (Concentration selection) were performed.
In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay the cells were seeded into 92 x 17 mm dishes (for tissue cultures in TC sterile quality) at 5 x 105 cells each and were incubated for 24 hours in 10 mL of DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/mL) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. Cell counts were performed after 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). The cells were counted using a Bürker chamber. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. At harvest the cells were trypsinised, collected and cell counts were determined. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. The volume of culture medium was 5 mL/dish for each per group. The results obtained were used for dose selection of the test item used in the Chromosome Aberration Assays (Experiments A and B). In addition, the pH and osmolality was measured in this pre-test.

Chromosome Aberration Assays:
Experiment A: The test item was dissolved in DME (Dulbecco’s Modified Eagle’s) medium for the treatment (stock solution: 50 mg/mL). The appropriate amount of this stock solution was completed with DME (Dulbecco’s Modified Eagle’s) medium to reach the constant volume. The constant volume was diluted with medium to obtain the examination concentrations. Duplicate cultures were used at each concentration and the negative control cultures as well as the positive controls for treatment without and withS9 mix. 5 x 105 cells were set up at each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the test item. The exposure period was 3 hours. The exposure period was followed by washing the cells with DME (Dulbecco’s Modified Eagle’s) medium and then growth medium was added. Sampling was made at 20 hours after treatment start (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 105 cells were set up.
Experiment B:
In the cytogenetic Experiment B the exposure period without metabolic activation was 20 hours. The exposure period with metabolic activation was 3 hours.
Experiment B, as Experiment A, included a concurrent S9 non-activated and S9 activated positive and negative control. For each group 5 x 105 cells/dish cells were seeded. Sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

The pH value and osmolality of negative (solvent) control and test item treatment solutions (for every treatment concentrations) were measured in the Chromosome Aberration Assay.

Preparation of Chromosomes:
Cell cultures were treated with Colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.

Analysis of Methaphase Cells:
All slides were independently coded before microscopic analysis and scored blind. 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.
Electronic copy 1 of 1
Rationale for test conditions:
See above.
Evaluation criteria:
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test
Statistics:
For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

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:
valid
Positive controls validity:
valid
Additional information on results:
In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to the maximum recommended concentration without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start.
Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration were inside the distribution of the laboratory historical negative control data.
There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
There was no precipitation of the test item at any dose level tested.
No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.
The number of aberrations found in the solvent controls was compatible with the laboratory historical laboratory control data.
The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and Cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Applicant's summary and conclusion

Conclusions:
Sodium Phenoxyacetate, tested up to the maximum recommended concentration 2000 μg/mL, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item is not clastogenic in this system.
Executive summary:

A Chromosome Aberration Assay in V79 cells was performed. The test item was dissolved in Dulbecco’s Modified Eagle’s medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study. In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:

Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 500, 1000 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time without S9 mix: 500, 1000 and 2000 μg/mL test item

Experiment B with 20/28 h treatment/sampling time without S9 mix: 500, 1000 and 2000 μg/mL test item

Experiment B with 3/28 h treatment/sampling time with S9 mix: 500, 1000 and 2000 μg/mL test item

Results:

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration were inside the distribution of the laboratory historical negative control data.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The number of aberrations found in the solvent controls was compatible with the laboratory historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and Cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.