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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 18, 2014 to September 05, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 487 and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 487
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine
EC Number:
292-053-3
EC Name:
2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine
Cas Number:
90530-15-7
Molecular formula:
C10 H22 N2 . C3 H3 N1
IUPAC Name:
2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine
Test material form:
other: liquid

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction)
Test concentrations with justification for top dose:
Experiments without S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were as follows:
31, 63, 125, 250, 375, 500 and 1,000 μg/mL for the 3 h treatment,
15.6, 31.3, 62.5, 125, 250, 375 and 500 μg/mL for the 24 h treatment.

Experiments with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were as follows:
31, 63, 125, 250, 375, 500 and 1,000 μg/mL for the first experiment,
62.5, 125, 250, 500, 750, 1,000 and 2,000 μg/mL in the second experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
yes
Remarks:
water for injection
Negative solvent / vehicle controls:
yes
Remarks:
water for injection
True negative controls:
not specified
Positive controls:
yes
Remarks:
Mitomycin C and Colchicine (Without S9 mix), and Cyclophosphamide (with S9 mix)
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Details on test system and experimental conditions:
L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 h.
L5178Y TK+/- cells were obtained from LGC standards (Teddington UK). The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

Culture conditions: Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and sodium pyruvate (200 μg/mL). This medium was supplemented by heat-inactivated horse serum at 10% (v/v).
Evaluation criteria:
The biological relevance of the results was considered first.
Evaluation of a positive response: a test substance was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.
Evaluation of a negative response: a test substance was considered negative if none of the criteria for a positive response were met.
Statistics:
For each experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the χ2 test, unless treated culture data were lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance.

Results and discussion

Test results
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

- No precipitate was observed in the culture medium at the end of the treatment period in any of the experiments.

- No significant increase in the frequency of micronucleated cells was noted in either experiments.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not induce any chromosome damage or damage to the cell division apparatus in cultured L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.
Executive summary:

A study was conducted to evaluate potential of the test substance to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-, as per OECD Guideline 487, in compliance with GLP. Based on the results of a preliminary experiment, the main assay was conducted in the form of two experiments. Experiment 1 consisted of concentrations of 31, 63, 125, 250, 375, 500 and 1,000 μg/mL for 3 h treatment and 24 h recovery without S9 mix, and concentrations of 31, 63, 125, 250, 375, 500 and 1,000 μg/mL for the 3 h treatment and 24 h recovery with S9 mix. Experiment 2 consisted of concentrations of 15.6, 31.3, 62.5, 125, 250, 375 and 500 μg/mL for the 24 h treatment and 20 h recovery, without S9 mix and concentrations of 62.5, 125, 250, 500, 750, 1,000 and 2,000 μg/mL for the 3 h treatment and 24 h recovery with S-9 mix. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (i.e., population doubling) of cells which is based on the starting cell count and the final cell count at the time of harvesting. For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test substance, for the vehicle and the positive controls, in 1,000 mononucleated cells per culture (total of 2,000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.No precipitate was observed in the culture medium at the end of the treatment periods in any of the experiments. No significant increase in the frequency of micronucleated cells was noted in either experiments. All acceptance criteria were met. The study was therefore considered to be valid. The test substance did not induce any chromosome damage or damage to the cell division apparatus in cultured L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system (Sarlang S, 2015).