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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Jan - 26 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministère de l'Économie et des Finances, vry-sur-Seine Cedex, France
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Japan Health Science Foundation
- Suitability of cells: recommended test system in international guidelines
- Number of passages if applicable: 24 and 39
- Methods for maintenance in cell culture: Cell cultures were maintained in suspension cell culture flasks at 37 °C and 5% CO2.

MEDIA USED
- Type and identity of media including CO2 concentration: The culture medium used was the R10 medium: Roswell Park Memorial Institute medium (RPMI 1640) + 10% horse serum, heat inactivated (v/v),
50 IU/mL penicillin, 50 µg/mL streptomycin + 1 mM sodium pyruvate + 0.05% pluronic acid.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I
4 h treatment (with and without metabolic activation): 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 and 500 µg/mL

Experiment I Repetition
4 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL

Experiment II
24 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL

Test item concentrations were initially selected based on the solubility in the solvent ethanol. In cases where the test item is cytotoxic, the highest concentration should aim to reach a maximum cytotoxicity (cytostasis) of 55 ± 5%. In the first experiment (4 h) without metabolic activation, only two of the eight tested concentrations met the acceptability criteria, therefore the test was not valid. Cytotoxicity (cytostasis) of more than 71% was observed at concentrations of ≥ 15.6 µg/mL, but only when tested without metabolic activation. Therefore the first experiment (without metabolic activation) was repeated at lower concentrations.
Vehicle / solvent:
- Vehicle used: ethanol (1.0% (v/v))
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine (-S9), 0.1 µg/mL in ethanol (4 h exposure) and 0.01 µg/mL in ethanol (24 h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E6 cells/tube in 8 mL cultivation medium

DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20-24 h; 24 h treatment: 44-48 h

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in all experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, the cell cultures were homogenised, centrifuged and underwent hypotonic treatment. Afterwards, the cells were fixed with methanol-acetic acid (3:1 ratio) and spread on glas slides for microscopic analysis.

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cytostasis
- Any supplementary information relevant to cytotoxicity: please refer to “Any other informations on materials and methods”
Evaluation criteria:
A test substance was considered positive (clastogenic or aneugenic) in the micronucleolus test if:
- At least one of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared to the concurrent negative control
- A dose effect was observed in at least one experimental condition when evaluated with an appropriate trend test
- The results were outside the data distribution range of historic negative controls

A test substance was considered negative (not clastogenic or aneugenic) in the micronucleus test if:
- None of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared with the concurrent negative control
- There was no concentration-related increase in the frequency of micronucleated cells when evaluated with an appropriate trend test
- All results were inside the distribution of the historical negative control data
Statistics:
Chi square test (p < 0.05)

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA: please refer to Table 2 under “Any other informations on results incl. tables”

Any other information on results incl. tables

Table 1: Experimental results of the micronucleus test

Mononuclear cells number Micronucleated cells number P-value Interpretation
Experiment I: 4 h + S9 mix
Vehicle control 994 6
995 5
CP 1.5 µg/mL 979 21 1.84E-08 significant
964 36
250 µg/mL 996 4 4.90E-01 non significant
996 4
125 µg/mL 995 5 8.27E-01 non significant
995 5
62.5 µg/mL 997 3 4.90E-01 non significant
995 5
31.25 µg/mL 993 7 8.27E-01 non significant
997 3
Negative control 999 1
995 5
Experiment I (Repetition): 4 h - S9 mix
Vehicle control 995 5
997 3
MMC 0.2 µg/mL 814 186 1.50E-94 significant
781 219
Col 0.1 µg/mL 879 121 1.61E-43 non significant
917 83
250 µg/mL 996 4 1.00E+00 non significant
996 4
125 µg/mL 998 2 7.96E-01 non significant
995 5
62.5 µg/mL 996 4 1.00E+00 non significant
996 4
31.25 µg/mL 998 2 7.96E-01 non significant
995 5
Negative control 997 3
995 5
Experiment II: 24 h - S9 mix
Vehicle control 994 6
996 4
MMC 0.02 µg/mL 916 84 3.34E-28 significant
940 60
Col 0.01 µg/mL 961 39 1.23E-11 non significant
969 31
250 µg/mL 997 3 4.66E-01 non significant
996 4
125 µg/mL 998 2 4.66E-01 non significant
995 5
62.5 µg/mL 997 3 3.16E-01 non significant
997 3
31.25 µg/mL 998 2 6.37E-01 non significant
994 6
Negative control 995 5
997 3
CP: Cyclophosphamide; MMC: Mitomycin C; Col: Colchicine

Table 2: Historical control data

Without S9 With S9
4 h treatment (n=30) 24 h treatment (n=29) 4 h treatment (n=30)
Vehicle* Mitomycin Colchicine Vehicle* Mitomycin Colchicine Vehicle* Cyclophosphamide
Concentration (µg/mL) 0.2 0.1 0.02 0.01 1.5
Minimum 5 261 91 5 93 43 5 58
Maximum 13 489 326 14 228 231 15 233
Mean 8 368 180 8 135 76 9 128
Standard deviation 2 70 55 2 28 42 3 40
Vehicle*: culture medium or ethanol; data were generated in 2015-2018

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in mouse lymphoma L5178 cells with and without metabolic activation.