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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-24 - 2018-06-18 (Experimental phase: 2018-05-15 - 2018-05-29)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 473, "In Vitro Mammalian Chromosome Aberration Test", adopted 29th July, 2016.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Council Regulation (EC) No 440/2008, B.10. Mutagenicity – IN VITRO MAMMALIAN CHROMOSOME ABRERRATION TEST, COMMISSION REGULATION (EU) 2017/735 adopted 14 February 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
additive
Test material form:
liquid
Details on test material:
- State of aggregation: clear liquid

Method

Target gene:
n/a
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: purchased from ECACC (European Collection of Cells Cultures)
- Suitability of cells: 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 V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Methods for maintenance in cell culture if applicable: The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. 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 a humidified atmosphere in an incubator, set at 5% CO2.
- Modal number of chromosomes: diploid, 2n=22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were 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%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S9
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 62.5, 125, 250 and 500 μg/mL test item
with S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 125, 250, 500 and 750 μg/mL test item
Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at least for the highest test concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DME (Dulbecco’s Modified Eagle’s) medium
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database. The test item was dissolved in a concentration of 4 mg/mL DME (Dulbecco’s Modified Eagle’s) medium (stock solution) at the first step. A clear solution of Hexanal was obtained in DME medium up to a concentration of 4 mg/mL. There was no precipitation in the medium at any concentration tested.
Controls
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DME (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:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: n/a
- Exposure duration: 3h (±S9) or 20h (-S9)
- Expression time (cells in growth medium): Cell counts were conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix)
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): 40h or 31h

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS:
Duplicate cultures were used at each test item concentration and the negative control cultures as well as the positive controls for treatment without and with S9 mix.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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 free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5% Giemsa for subsequent scoring of chromosome aberration frequencies.
Analysis of Metaphases 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, 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.

NUMBER OF CELLS EVALUATED: 300 well-spread metaphase cells (150 metaphases/slide) per incubation

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well-spread metaphase cells (150 metaphases/slide) per incubation

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)
Rationale for test conditions:
as indicated in the guideline
Evaluation criteria:
Evaluation of Result
Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– 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, in any of the experimental conditions examined:
– 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, the test item is considered clearly negative if, in all experimental conditions examined:
– 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,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance are considered together.
There is no requirement for verification of a clearly positive or negative response.
Statistics:
For statistical analysis the 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. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment dose using the adequate regression analysis by Microsoft Excel software.

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Remarks:
solvent controls
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / Effects of osmolality: There were no relevant changes in pH or osmolality after treatment with the test item.
- Evaporation from medium: none known
- Water solubility / Precipitation: No precipitation of the test item was observed at any of the applied concentrations

RANGE-FINDING/SCREENING STUDIES:
Solubility and Dose Selection
A clear solution of Hexanal was obtained DME (Dulbecco’s Modified Eagle’s) medium up to a concentration of 4 mg/mL. There was no precipitation in the medium at any concentration tested.
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.
Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 62.5, 125, 250 and 500 μg/mL test item
with S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 125, 250, 500 and 750 μg/mL test item
All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
3h/20h treatment/sampling time without S9-mix
incl. Gaps excl. Gaps
Mean 40.50 31.70
SD 3.51 3.88
Range 35-50 26-39
Lower CI 33.22 23.64
Upper CI 47.78 39.75
n 23 23

3h/20h treatment/sampling time with S9-mix
incl. Gaps excl. Gaps
Mean 46.07 39.43
SD 2.39 2.65
Range 39-51 34-46
Lower CI 41.11 33.95
Upper CI 51.02 44.92
n 23 23

- Negative (solvent/vehicle) historical control data: 3h/20h treatment/sampling time without S9-mix
incl. Gaps excl. Gaps
Mean 6.26 2.85
SD 0.75 0.65
Range 4-8 2-5
Lower CI 4.70 1.59
Upper CI 7.82 4.11
n 23 23

3h/20h treatment/sampling time with S9-mix
incl. Gaps excl. Gaps
Mean 6.39 3.02
SD 0.83 0.64
Range 5-9 2-5
Lower CI 4.66 1.69
Upper CI 8.12 4.35
n 23 23

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

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

Applicant's summary and conclusion

Conclusions:
The study was performed according to the OECD TG 473 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). Hexanal did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.
Executive summary:

The test item Hexanal, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix), in accordance with the current OECD Guideline 473, under GLP:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 62.5, 125, 250 and 500μg/mL test item

with S9 mix: 125, 250, 500 and 750μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 125, 250 and 500μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix:125, 250 and 500μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix:125, 250, 500 and 750μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at least for the highest test concentration.

No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation.

In the experiment A in the absence of metabolic activation one value at the dose of 500 μg/mL (5 aberrant cells excluding gaps/150 cells) was within the historical control range (2-5 aberrant cells without gaps/150cells) but slightly above the 95% control limits of the historical control data (upper limit approximately 4,11 aberrant cells excluding gaps/150 cells).

However, no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups. Therefore, the finding was considered as accidential and not being biologically relevant.

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

The number of aberrations found in the solvent controls was in the range of the 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.

In conclusion, Hexanal did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Thus, the test item is considered as being non-clastogenic in this system.