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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity was assessed in an Ames test (1977), an in vitro chromosome aberration test according to OECD 473 (2012) and a mouse lymphoma test according to OECD 476 (2012).
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-09-20 - 2012-12-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 476)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: the growth of the cells in RPMI 1640 prevents the creation of high backgrounds
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Wistar rats induced with phenobarbital and b-naphthoflavone
Test concentrations with justification for top dose:
Two preliminary tests were performed before the following two experiments
Experiment I
with metabolic activation: 0.05, 0.10, 0.20, 0.50, 0.75, 1.00, 1.25, 1.50 and 1.75 mM
without metabolic activation: 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 0.70 and 0.90 mM

Experiment II
with metabolic activation: 0.15, 0.30, 0.60, 0.90, 1.20, 1.40, 1.60 and 1.80 mM
without metabolic activation: 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 and 0.60 mM
Vehicle / solvent:
- Vehicle used: RPMI cell culture medium (RPMI +5% HS)
- Justification for choice of solvent/vehicle: based on solubility results
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
DURATION
short term experiment (Exp. I): 10^7 cells were suspended in 11 ml RPMI
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days, at 37 °C in 5% CO2/95% humidified air
The cell density was adjusted to 3*10^5 cells/ml in a culture volume of 20 ml

long term experiment (Expe. II): 5*10^6 cells suspended in 25 ml RPMI
- Exposure duration: 24 h
3*10^5 cells were suspended in 14 ml complete culture medium- expression time: 2 days, at 37 °C in 5% CO2/95% humidified air
The cell density was adjusted to 3*10^5 cells/ml in a culture volume of 14 ml

SELECTION AGENT (mutation assays): cells were seeded in 4 96-well plates (2000 cells/well) in 200 µl selective medium with TFT. Incubation for 14 days.
Selective medium- RPMI 1640 medium supplemented with:
20 % horse serum (HS)
100 U/100 μg/mL penicillin/streptomycin
1 mM sodium pyruvate
2 mM L-glutamine
25 mM HEPES
2.5 μg/mL amphotericin B
5 μg/mL TFT

NUMBER OF REPLICATIONS: 2

DETERMINATION OF GENOTOXICITY
Mutant frequency=[ -ln[NW/TW (selective medium)]/-ln[NW/TW (non-selective medium)]]*800
Relative Total Growth (RTG)= [Relative Suspension Growth (RSG)*Relative Cloning Efficiency (RCE)]/100
Colony sizing was performed for the highest concentrations and for the controls in order to detect any clastogenic potential of the test material.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
1.6 cells/well transferred in two 96-well plate and incubated for 6 days. Thereafter, the cloning efficiency was determined.

Evaluation criteria:
The test item is considered mutagenic if following criteria are met :
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.
Statistics:
non-parametric Mann-Whitney test
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below for details
Vehicle controls validity:
valid
Untreated negative controls validity:
other: same as vehicle control
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- pH value : within the physiological range
- Precipitation: yes, in pre-experiment I and experiment I with metabolic activation at concentrations of 0.2 mM and higher, while without S9 mix at concentrations of 0.5 mM and higher. In experiment II with S9 mix at concentrations of 0.3 mM and higher, and without S9 mix at concentrations of 0.2 mM and higher.
- Other confounding effects:

CYTOTOXICITY
Exp. I- with metabolic activation: RTG 22.3% and 4.8% at concentrations of 1.5 and 1.75 mM, respectively; without metabolic activation: RTG 15.4% at 0.9 mM
Exp II- with metabolic activation: RTG 23.2% AT 1.8 mM; without metabolic activation: RTG 16.1% at 0.6 mM

All validity criteria were met. The mutant frequencies induced by the test material did not show any biological relevance and the GEF was not exceeded in any of the dose groups. The statistically significant increase in mutant frequencies when compared to solvent controls seen in some cases was not dose-related. Colony sizing showed no clastogenic effects with and without metabolic activation Colony sizing showed no clastogenic effects with and without metabolic activation (increased occurence of small colonies< 40%).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this test PEROXAN BCC does not induce mutations in mouse lymphoma L5178Y cells. No clastogenic effects could be detected based on the colony sizing.
Executive summary:

In a mammalian cell gene mutation assay, L5178Ycells cultured in vitro were exposed to PEROXAN BCC (Bis(4-tert-butylcyclohexyl)peroxydicarbonate) at the following concentrations, based on range finding tests:

 

Experiment I

with metabolic activation: 0.05, 0.10, 0.20, 0.50, 0.75, 1.00, 1.25, 1.50 and 1.75 mM

without metabolic activation: 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 0.70 and 0.90 mM

 

Experiment II

with metabolic activation: 0.15, 0.30, 0.60, 0.90, 1.20, 1.40, 1.60 and 1.80 mM

without metabolic activation: 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 and 0.60 mM

 

S9 mix from male Wistar rats induced with phenobarbital and b-naphthoflavone was used for the metabolic activation. PEROXAN BCC was testedup to cytotoxic and insoluble concentrations.  The positive controlsdidinduce the appropriate response. The results of the experiment show that PEROXAN BCC is not mutagenic in this test.

 

This study is classified as acceptable.This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test item did not induce mutations in bacterial and mammalian cells and is not cytogenic in cultured human lymphocytes.


Justification for selection of genetic toxicity endpoint
GLP Guideline study (key study); no contradicting results with respect to mutagenicity.

Justification for classification or non-classification

No genotoxic effect was observed in an appropriate in vitro testing battery.