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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Substance was tested for in vitro mutagenicity in a bacterial mutation (Ames) test, for the potential for clastogenicity/aneugenicity in an in vitro micronucleus assay and for the mutagenic activity in a mammalian cell gene mutation test (HPRT).
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD 487: In Vitro Mammalian cell Micronucleus Test (adopted 26 September 2014)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No. 440/2008, Part B, Guideline B.49 In Vitro Mammalian cell Micronucleus Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Species / strain:
other: Peripheral Human Lymphocytes
Details on mammalian cell lines (if applicable):
- Type and identity of media: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine, penicillin/streptomycin, and heparin.
- Properly maintained: yes
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Mitomycin C, Colchicine (without activation) and cyclophopsphamide (with activation)
Test concentrations with justification for top dose:
Dose Range finding test: The highest test concentration was 2000 μg/ml.

First Cytogenetic assay: 52, 164, 512, 1600 and 2000 μg/ml (with and without activation)

Second Cytogenetic assay: 100, 500, 700, 900, 1100, 1300, 1500 and 1700 μg/ml (without activation).
Vehicle:
The test substance was dissolved in dimethyl sulfoxide (DMSO) of spectroscopic quality.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and conditions:
Cell source:
Cells were obtained from healthy human volunteers (non-smoking males (aged 18 - 35)). The average generation time (AGT) of the cells and the age of the donor was determined to be in the range 26 to 32 and 12.6 to 12.8 hours for age and AGT respectively for all experimental phases of study.

Culture medium:
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM) ,penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.

Metabolic activation:
The metabolic activation system used was rat S9 homogenate prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test substance preparation:
The test substance was dissolved in DMSO to create a stock solution. This was treated with ultrasonic waves until completely dissolved. Stocks were used within 2.5 hours after preparation.

Environmental conditions:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.7 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations occured when opening the incubator door, which are not considered significant.

First cytogenetic assay:
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test substance for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasin B (5 μg/ml) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay.

Second Cytogenetic assay:
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of test substance with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Preparation of slides:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol: acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated coverslipper.
Evaluation criteria:
A test substance is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei, compared with the concurrent negative or solvent control as appropriate.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if
a) None of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei, compared with the concurrent negative or solvent control as appropriate.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:
Species / strain:
other: Peripheral Human Lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
An upper concentration of 2000 μg/ml showed no precipitation in the culture medium.

RANGE-FINDING/SCREENING STUDIES:

The pH and osmolarity of a concentration of 2000 μg/ml were 7.32 and 444 mOsm/kg respectively (compared to 7.36 and 456 mOsm/kg in the solvent control). The pH and osmolarity of a concentration of 2928 μg/ml (0.01 M) were 7.74 and 419 mOsm/kg respectively (compared to 7.95 and 443 mOsm/kg in the solvent control).
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

As such, the test substance is not considered to be clastogenic or aneugenic in human lymphocytes under the experimental conditions described.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In all three in vitro genotoxicity tests conducted, the test substance provided a negative response both in the presence and absence of metabolic activation indicating a lack of genotoxicity under the experimental conditions described in the experimental reports.

Justification for classification or non-classification

It was concluded that the test material was not mutagenic based on the conditions employed in the bacterial reverse mutation test, micronucleus test and gene cell mutation test and would not be classified based on the negative results obtained.