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Genetic toxicity in vitro

Description of key information

No adverse effects were observed under the conditions of this study.

Link to relevant study records
in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February 2014 to 17 September 2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21 July 1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (induced using Aroclor 1254)
Test concentrations with justification for top dose:
- Cytotoxicity Test: 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL in the absence and presence (2% v/v final concentration S9) of metabolic activation
- Mutagenicity Test (2 independent experiments in the absence and presence (2% v/v final concentration S9) of metabolic activation: Trial 1: 312.5, 625, 1250, 2500 and 5000 μg/ml, Trial 2 (confirmation test): 500, 1000, 2000 4000 and 5000 μg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Epofloc L-1R was soluble in sterile distilled water at 500 mg/mL.
Untreated negative controls:
Sterile Distilled Water
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Ethyl Methanesulfonate (0.4 µL/mL final concentration in the absence of metabolic activation); Benzo(a)pyrene (6 µL/mL final concentration in the presence of metabolic activation).
Details on test system and experimental conditions:

- Preincubation period: approximately 24 hours prior to treatment
- Exposure duration: Mutagenicity Test Trial I: 3 hours 45 minutes; Trial II: 3 hours 45 minutes, both in the absence and presence (2% v/v final concentration S9) of metabolic activation
- Expression time (cells in growth medium): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days post treatment

SELECTION AGENT (mutation assays): 2-amino-6-mercaptopurine (6-thioguanine) at a final concentration of 5 µg/ml.

NUMBER OF REPLICATIONS: 2 per treatment concentration, positive and negative control.

NUMBER OF CELLS EVALUATED: 10000000 cells/culture for each treatment and negative and positive controls.

- Method: Other: assessed by calculating the percent relative cloning efficiency following treatment.

- Determination of polyploidy: No
- Determination of endoreplication: No
Evaluation criteria:
Epofloc L-1R was considered positive if the following criteria were met:
- A concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test item caused a three-fold increase (Nestmann, E.R. et al., 1991) in the number of 6-thioguanine resistant colonies relative to concurrent negative control with statistically significant increases outside the laboratory historical negative control range.
- A net increase in mutant colonies of treated cultures above the concurrent control in at least two of the concentrations tested. Negative results were confirmed by a repeat test (short duration, 3 hour 45 mins), using same experimental conditions considering the toxicity pattern achieved in the trial I mutagenicity experiment.

The analytical acceptance criteria for the concentration of the test item in the vehicle was ± 20% deviation from nominal value and CV% < 10%.
Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on Epofloc-L-1R treatment groups against negative control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
No cytotoxicity of biological relevance at the limit concentration.
Vehicle controls validity:
not examined
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
- No significant change in pH (± 1 pH unit) or osmolality (≥ 50 mOsm/kg H2O) was observed in culture medium at any tested concentration up to 5000 μg Epofloc-L-1R/ml in the absence or presence (2% v/v final concentration S9) of metabolic activation.
- Water solubility: Soluble in sterile distilled water at 500 mg/mL.
- Precipitation: None observed in culture medium up to 5000 μg Epofloc-L-1R/mL in either the absence or presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: No significant reduction/toxicity in cell count was observed at the end of treatment in cultures treated at 5000 μg/mL both in the absence (relative cloning efficiency 86.82%) and presence (relative cloning efficiency 90.58%) of metabolic activation (2% v/v final volume S9) at 5000 μg/mL, the guideline limit dose. This was therefore the highest concentration tested in the absence and presence of metabolic activation for the main study.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

The absolute cloning efficiency of the CHO-K1 cell line (negative control) was above 60% in both the trials. The spontaneous mutation level (negative control) was within the acceptable limit [less than 20 per 106 clonable cells (Li, A.P. et al., 1987)] in both the trials, validating the acceptability of the test system. The increased mutant frequency in positive controls in trial I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Interpretation of results (migrated information): negative

It is concluded that Epofloc-L-1R does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the present experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
The OECD 476; in vitro mammalian cell gene mutation test, is a very recent study undertaken to a recognised guideline and to GLP. It is also the most definitive of the three in vitro studies.

Justification for classification or non-classification

No adverse effects were observed under the conditions of any of the in vitro genetic studies, therefore this substance will not be classified for genetic toxicity.

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