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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:
10 October, 2017 - 09 February, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not applicable
Remarks:
No analysis was carried out to determine the homogeneity, concentration and stability of the test formulation. it was formulated within é hours of it being applied to the test system. It is assumed that it was stable for this duration.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55503625
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 100%
- Physical state / Apparence : Pale yellow powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

- Solubility and stability of the test substance in the solvent/vehicle:


TREATMENT OF TEST MATERIAL PRIOR TO TESTING


- Final dilution of a dissolved solid, stock liquid or gel: The test item is soluble in DMS O


OTHER SPECIFICS:

Method

Target gene:
Chromosome aberrations
Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Peripheral circulation of a non-smoking volonteer who had been previously screened for suitability
- Suitability of cells: done
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: 18-35 years old, 1 male and 2 females
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Celles were grown in Eagle's minimal essential medium with HEPES buffer (MEM),
supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10ù foetal bovine serum (FBS) at approximately 37°C with 5% CO2 in humidified air.
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
The dose level used in the Main Experiment were selected using data from the Preliminary Toxicity test where the results indicated that the maximum
concentration should be limited on both precipitate and toxicity, depending on the exposure group.

Group Final concentration of the test item DNAN (µg/mL)
4(20)-hour without S9 0, 40, 80, 160, 320, 560, 640, 960
4(20)-hour with S9 0, 40, 80, 160, 320, 560, 640, 960
24-hour without S9 0, 10, 20, 60, 80, 120, 240


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not souble in aqueous media
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 48 hours incubation
- Exposure duration: 4-hours exposure in the presence of an induce rat liver homogenate metaolizing system (S9), 4 hours exposure in the absence of metabolic activation (S9) and a 24-hour exposure in the absence of metabolic activation.
- Expression time (cells in growth medium): 20-hour expression period

- Fixation time (start of exposure up to fixation or harvest of cells):



SPINDLE INHIBITOR (cytogenetic assays): Mitosis was arrested by addition of demecolcine 2,5 hours before the required harvest time



NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: the cells (from the KCL suspension) were fixed into fresh methanl/glacial
acetic acid. The fixative was changed at least 3 times and the cells stored at approx. 4°C to ensure complete fixation prior to slide preparation.
The lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and re-suspension in a small aount of fixative. Several
drops of this suspension were dropped onto clean, wet microscope slide and left to air dry. Each sllide was permanently labeled with the appropriate
identification data.

NUMBER OF CELLS EVALUATED: 2000 lymphocyte cell nuclei

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate.



DETERMINATION OF CYTOTOXICITY
- Method: precipitation and mitotic index. A total of 2000 lymphocyte cell nuclei were counted and the number of the cells in metaphase recorded and expressed as the mitotic index ans as a percentage of the vehicle control value.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


- OTHER:
Rationale for test conditions:
The following criteria were used to determine a valid assay :
- The frequency of cells with structural chromosome aberrations in the vehicle control cultures was within the laboratory historical control data range
- All the positive control chemicals induced a positive response and demonstrated the validity of the experiment and the integrity of the S9-mix
- The stuyd was performed using all 3 exposure conditions using a top concentration which meets the requirements of the current testing guideline
- The required number of cells and concentrations were analyzed
Evaluation criteria:
A test item can be considered to be clearly negative if, any of the experimental conditions examined :
- The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data
- No toxicology or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis
- There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if :
- The number of cells with strucutral chromosome aberrations is outside the range of the laboratory historical control data
- At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the conccurent negative control
- The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using a Fisher's Exact Test (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells aberrations excludings gaps is less than 0.05 when compared to
its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproductible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results
Key result
Species / strain:
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:
not applicable
Positive controls validity:
valid
Additional information on results:
As the criteria required to valid the assay are provided, the assay was considered as valid.
The test item did not induce any statistically significant increases the the frequency of cells with aberrations either in the absence or presence of metabolic activation at any dose level in any of the exposure groups.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure group.
Remarks on result:
other: Valid results

Applicant's summary and conclusion

Conclusions:
The test item is considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

A Chromosome aberration test in Human Lymphocytes in vitro, Bowles A., 2018, has been performed according to the 473 OECD guideline.

Duplicate culture of human lymphocytes, treated with the test item were evaluated for chormosome aberrations at 3 dose levels, together with vehicle and positive controls, at 3 exposure conditions : 4 hours exposure in the presence of an induce rat liver homogenate metabolizing system (S9), 4 hours expposure in the absence of metabolic activation (S9) both with a 20-hour expression period and a 24 -hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity test where the results indicated that the maximum concentration should be limited on both precipitate and toxicity, depending on the exposure group (0, 40, 80, 160, 320, 560, 640, 960 µg/mL : concentrations tested for the 4 -hour exposure either with and without S9 and 0, 10, 20, 40, 60, 80 120, 240 µg/mL: concentrations used for the 24 -hour exposure without S9).

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberration either in the presence or absence of metabolic activation at any dose level in any of the exposure groups.

The test item, DNAN was considered to be non-clastogenic to human lymphocytes in vitro.