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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July, 2016
Deviations:
no
Remarks:
No deviations ocurred that adversely impacted the integrity or interpretability of the results.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
C4 sulfonamido methacrylate
IUPAC Name:
C4 sulfonamido methacrylate
Details on test material:
- Name of test material (as cited in study report): 2-Propenoic acid, 2-Methyl-, 2-[Methyl[(Nonafluorobutyl)Sulfonyl]Amino]Ethyl ester, FZ-9262
- Physical state: white to grey waxy solid
- Analytical purity: 99.5%
- Storage condition of test material: at room temperature in the dark
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 40010
- Expiration date of the lot/batch: 24 May, 2020
- Purity test date: 24 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 C
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Test article solubility was evaluated in the vehicle (DMSO) and it was deemed that it was soluble up to the highest concentration to be dosed in the study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dissolved in DMSO.
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: 200 mg/mL (dilutions were performed to reach the appropriate dose levels).
- Final preparation of a solid: Dissolved in DMSO

Method

Species / strain
Species / strain / cell type:
lymphocytes: Cultured human lymphocytes.
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: A 29 and a 35 year old subject (non-smoking)
- Suitability of cells: Recommended in OECD 473
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: 2 donors, 29 and 35 years old
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The media for culting the human peripheral blood lymphocytes consisted of RPMI 1640 medium (with HEPES and Glutamax), supplemented with heat-inactivated fetal calf serum (20%), penicillin (100 U/mL medium), streptomycin (100 ug/mL medium) and phytohaemagglutinin (2.4 ug/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data
Metabolic activation:
with and without
Metabolic activation system:
S9-mix consisting of liver homogenate fraction (S9).
Test concentrations with justification for top dose:
4 hour exposure with and without S9: 0 (solvent control), 50, 100, 200, 300, 400, 500 ug/mL
24 hour exposure without metabolic activation: 0 (solvent control(, 6.25, 12.5, 25, 50, 100, 150, 200, 300, 400, 500 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test article solubility and test system compatibility
Controls
Untreated negative controls:
no
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:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): 2% Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Three slides were prepared from each selected culture. The slides were stained in a 2% solution of Giemsa, rinsed in water, air-dried and mounted with a coverslip. The slides were coded by a qualified person not involved in scoring the slides, to enable “blind” scoring.

NUMBER OF CELLS EVALUATED: A number of 1000 stimulated lymphocytes (500 per slide) were examined in each culture to determine the percentage of cells in mitosis (mitotic index). Based on the results of the mitotic index scoring and the observations made with respect to the number and quality of the metaphases, at least three concentrations of the test substance together with the solvent and positive controls were selected for chromosomal aberration analysis. For each treatment group, 300 well-spread metaphases per concentration (150 metaphases per culture and 75 metaphases per slide), each containing 46 centromeres, were analyzed by microscopic examination for chromatid-type aberrations (gaps, breaks, fragments, interchanges), chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 per dose.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Rationale for test conditions:
According to OECD 473
Evaluation criteria:
The number of metaphases containing one or more aberrations of the test substance treated groups were compared with those of the concurrent solvent controls using Fisher's exact test (one-sided). The difference was considered statistically significant when the p-value of the Fisher’s exact test was less than 0.05.
The response was considered positive if all of the following criteria are met:
- at least one of the test concentrations exhibits a statistically significant increase
compared to the concurrent negative control.
- the increase is dose-related in at least in one experimental condition when
evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical solvent control data.
A response was considered negative if all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase
compared to the concurrent negative control.
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data.
A test substance was considered equivocal if the response was neither positive or negative even after further investigation.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Cultured human peripheral lymphocytes.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the test article is not clastogenic to cultured human lymphocytes.
Executive summary:

The test substance was examined for its potential to induce structural chromosomal aberrations in cultured human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix). The study was conducted according to OECD 473 and was conducted in compliance with OECD GLP regulations. Dimethylsulfoxide (DMSO) was used as the solvent. Dose levels ranging from 3.13 to 500 μg/ml were tested. The maximum final concentration (500 μg/ml) was based on the solubility of the test substance in the culture medium. The purity of the test substance (99.3%) was taken into account while preparing the dosing solutions. In all instances, duplicate cultures were used. The mitotic index was used as measurement for cytotoxicity. In the first chromosomal aberration test, in both the absence and presence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment). In the second test, the treatment/harvesting times were 24/24 hours (continuous treatment). Solvent and positive controls were run in parallel. In the first test, in the pulse treatment groups both with and without S9-mix, the highest test substance concentration induced a moderate cytotoxicity to the cells. At the higher concentrations, cytotoxicity showed some fluctuation and did not exceed the maximal cytotoxicity of 55±5% as stated in the OECD guideline 473. In both pulse treatment groups, four test substance concentrations (with S9-mix: 50, 200, 400 and 500 μg/ml; without S9-mix: 50, 100, 300 and 500 μg/ml), together with the cultures of the solvent and the positive control (Cyclophosphamide) were analyzed. In the second test, a dose-related increase in cytotoxicity was observed. It should be noted that, at the four highest tests substance concentrations (200 – 500 μg/m) the observed cytotoxicity (as measured by mitotic index) was comparable between the concentrations. Four test substance concentrations (12.5, 50, 150 and 500 μg/ml), together with the cultures of the solvent and the positive control (Mitomycin C) were analyzed. In both chromosomal aberration tests, the numbers of cells with structural aberrations observed in the solvent control (1% DMSO) cultures were within the historical range. Treatment with the positive controls Cyclophosphamide and Mitomycin C resulted in statistically significant increases in the numbers of metaphases containing one or more chromosomal aberrations, when compared to the numbers observed in the cultures treated with the solvent. This demonstrates the validity of the study. In both chromosomal aberration tests, the test substance did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment conditions analyzed, when compared to the number of aberrant cells observed in the solvent control cultures. Based on the results of the study, the test article is not clastogenic to cultured human lymphocytes.

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