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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 24, 1988 to June 20, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Experiment 1:
Doses: 0, 20, 100, 500, 2500 and 5000 ug/plate in DMS0 - 3 test plates per dose or per control

Experiment 2:
Doses: 0, 4, 20, 100, 500 and 2500 ug/plate in DMS0 - 3 test plates per dose or per control

Vehicle / solvent:
Comlete solubility of the test substance in DMSO.
Untreated negative controls:
yes
Remarks:
vehicle: DMSO
Positive controls:
yes
Positive control substance:
other: 10 ug 2-aminoanthracene in DMSO (with S9), 5 ug N-methyl-N -nitro-N-nitrosoguanidine (MNNG) (without S-9))
Details on test system and experimental conditions:
S-9 mix:
The S-9 mix is prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S-9 -fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are:

MgCl2 8mM
KCl 33 mM
glucose-6--phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 100 mM. The phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4:1.

Bacteria:

The indicator organisms TA 1535, TA 1537, TA 98 and TA 100 selected for this test are derivatives of Salmonella typhimurium. All strains have a defective excision repair System (uvr9), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.

The strains TA 1535 and TA 100 are derived from histidineprototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 96 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the .1 mutant his C 3076 in the case of TA 1537 and the .2 type his D 3052 in the case of TA 96.

The strains TA 96 and 1A 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Preincubation test
The experimental procedure is based on the method described by Yahagi et al. and Matsushime et al. 0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37 °C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
960 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Oifco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Standard plate test
The experimental procedure is based on the method of Ames et al. Test tubes containing 2 ml portions of soft agar which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml 5-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates
Titer determination
Ttiter is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, 0.1 mL of the overnight cultures is diluted to 10 –E06 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino-acid solution (5 mM histidine * 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial Suspension (dilution: 10-E06)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-8onner agar plates within approx. 30 seconds. After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

Test design:
Experiment 1:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 ug/plate in DMS0
Type of test, Standard plate test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

Experiment 2:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 4, 20, 100, 500 and 2500 ug/plate in DMS0
Type of test, preincubation test with and without S-9 mix
Number of plates: 3 test plates per dose or per control





Evaluation criteria:
Evaluation criteria
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the study conditions, the test substance is not mutagenic in the Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 at the dose range of: 20 µg - 5000 µg/plate (SPT) and at the dose range of: 4 µg - 2500 µg/plate (PIT) in the absence and presence of metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 in Bacterial Reverse Mutation Test. The test substance was examined using four strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, and TA 98). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). DMSO was used as a vehicle. Toxic effects were monitored at doses: 20 µg - 5000 µg/plate in the Standard plate test (SPT) and 4 µg - 2500 µg/plate in the Preincubation test (PIT). In both experiments 3 test plates per dose or per control were investigated. Bacteriotoxic effect were observed at doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT). An increase in the number of his revertants was not observed both in the SPT and in the PIT either without S-9 mix or after the addition of a metabolizing system. Under the study conditions, the test substance is not mutagenic in the Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 at the dose range of: 20 µg - 5000 µg/plate (SPT) and at the dose range of: 4 µg - 2500 µg/plate (PIT) in the absence and presence of metabolic activation (Gelbke, 1988).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 22, 2016 to November 08, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
L5178Y TK+/-
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose formulation preparation
The test substance was dissolved in DMSO at concentrations of:
. 200 mg/mL for the preliminary cytotoxicity test - highest recommended dose-level,
. 80 mg/mL for the first experiment - criteria specified in the international guidelines,
. 20 mg/mL for the second experiment.

Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine: 0.5 μg/mL (without S9 mix, Aneugen)
Details on test system and experimental conditions:
Cells
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 hours.
L5178Y TK+/- cells were obtained from ATCC (USA), by the intermediate of Biovalley (Marne-La-Vallée, France). 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).

Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.
The S9 fraction was preserved in sterile tubes at -80 °C, until use. The S9 mix was prepared at 4 °C immediately before use and maintained at this temperature until added to culture medium.

The composition of S9 mix was as follows (in volumes):
Glucose-6-phosphate (180 mg/mL): 1
NADP (25 mg/mL): 1
KCl (150 mM): 1
S9 fraction (final concentration in S9 mix: 40 % (v/v); batch No. 3556, protein concentration: 42.5 mg/mL): 2
In the assay with metabolic activation, the culture medium was supplemented with 5% of this S9 mix so that the final concentration of S9 in the treatment medium was 2 %.

EXPERIMENTAL DESIGN

Preliminary cytotoxicity test
- To assess the cytotoxicity of the test substance, six dose-levels (one culture/dose-level) were tested:4, 40, 200, 400, 1000 and 2000 μg/mL, as follows:
without S9 mix: 3 h treatment + 24 h recovery; 24 h treatment + 0 h recovery
with S9 mix: 3 h treatment + 24 h recovery.
- The treatments of this preliminary test were performed as for the main experiments.
- Assessment of cytotoxicity was performed by evaluation of Population Doubling.
- After the final cell counting, the cells were not harvested and no slides were prepared.

Main cytogenetic experiments
In the main study seven dose-levels of the test substance were tested in duplicate (two cultures/dose-level), both with and without metabolic activation.

Experiments without S9 mix
With a treatment volume of 1 % (v/v) in culture medium, the dose-levels selected for the treatments were as follows:
25, 50, 100, 200, 300, 400 and 800 μg/mL for the 3 h treatment + 24 h recovery,
3.13, 6.25, 12.5, 25, 50, 100 and 200 μg/mL for the 24 h treatment + 0 h recovery in the first experiment,
12.5, 25, 50, 100, 125, 150, 175 and 200 μg/mL for the 24 h treatment + 0 h recovery in the second experiment.
No precipitate was observed in the culture medium at the end of the treatment periods.

Experiment with S9 mix: 3 h treatment + 24 h recovery
With a treatment volume of 1% (v/v) in culture medium, the dose-levels selected for the treatment were 25, 50, 100, 200, 300, 400 and 800 μg/mL.
No precipitate was observed in the culture medium at the end of the treatment period.

Treatment:
On the day of treatment, cells were counted and suspended in order to reach approximately 3 x 10+E05 cells/mL (final concentration = N0) in the final treatment medium (culture medium containing 5 % inactivated horse serum). Cells were exposed in 24-well plates to the test or control substances, with or without S9 mix, at +37 °C in a humidified atmosphere of 5 % CO2/95 % air. At the end of the treatment period, the cells were washed twice. Cells were suspended in culture medium containing 10 % inactivated horse serum and the plates were incubated for the recovery period, at +37 °C in a humidified atmosphere of 5 % CO2/95 % air. At the end of the recovery period (if any), the cells were counted to determine the final count at the time of harvesting (N) in order to assess the cytotoxicity by the evaluation of PD.

Assessment of cytotoxicity
For each culture, the Population Doubling (PD) was calculated and used relative to that of the vehicle control. The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.
PD = [log (N/N0)]/log 2
Mean PD as % of control = (Mean PD treated * 100 %) / Mean PD vehicle control
The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100 %).
Decrease in PD (%) = 100 - Mean PD as % of control

Cell harvesting and slides preparation
After the final cell counting, the cells were washed with culture medium containing 10 % inactivated horse serum and 1 % pluronic acid. The cells were suspended in 49.5 % culture medium containing 10 % inactivated horse serum, 50 % PBS and 0.5 % pluronic acid, before being fixed. Following the fixation, the cells were kept at 4° C for at least an overnight period. Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), four dose-levels of the test substance-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5 % Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.

Analysis of the slides
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 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Appropriate test substance dose-levels for scoring of micronuclei were selected mainly on the basis of the achieved reduction of PD and on the presence of precipitate. Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995).



Evaluation criteria:
The biological relevance of the results was always taken into account when evaluating results.
Evaluation of a positive response: a test substance is 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 demonstrated by a statistically significant trend test,
. 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 is considered clearly negative if none of the criteria for a positive response was met.
Statistics:
For each condition of the cytogenetic 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 are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose-levels. This statistical analysis was performed using SAS Enterprise Guide software.
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
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOTOXICITY TEST

- A precipitate was observed in the culture medium at the end of the treatment period, at the dose-level of 2000 μg/mL.
- Following the 3-hour treatments with and without S9 mix, a marked to severe cytotoxicity was induced at dose-levels ≥ 400 μg/mL as shown by a 55 to 100 % decrease in the PD.
- Following the 24-hour treatment (without S9 mix), a severe cytotoxicity was observed at dose-levels ≥ 200 μg/mL, as shown by a 100 % decrease in the PD.

MAIN CYTOGENETIC EXPERIMENTS

Since the test substance was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

EXPERIMENTS WITHOUT S9 mix:

Cytotoxicity:

- Following the 3-hour treatment, a marked to severe cytotoxicity was induced at dose-levels ≥400 μg/mL, as shown by a 66 to 100 % decrease in the PD.
- Following the 24-hour treatment of the first experiment, a severe cytotoxicity was induced at 200 μg/mL as shown by a 85% decrease in the PD.
- Following the 24-hour treatment of the second experiment, a moderate to severe cytotoxicity was induced at dose-levels ≥50 μg/mL, as shown by a 38 to 100 % decrease in the PD

Micronucleus analysis:

The dose-levels selected for micronucleus analysis were as follows:
100, 200 and 300 μg/mL for the 3-hour treatment, the latter inducing a 4 % decrease in the PD but higher dose-levels being considered as too cytotoxic,
25, 50 and 100 μg/mL for the 24-hour treatment in the first experiment, the latter inducing no decrease in the PD, but the higher dose-level being considered as too cytotoxic,
25, 50 and 100 μg/mL for the 24-hour treatment in the second experiment, the latter inducing a 57 % decrease in the PD.

- No statistically significant or dose-related increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges.
- The recommended level of cytotoxicity was not reached after the 3-hour treatment. However, considering the narrow dose-levels spacing used in this experiment, the available results were considered as suitable to allow a reliable interpretation.
- In the absence of S9 mix, the overall results were considered to meet the criteria for a negative response.

EXPERMIMENT WITH S9 mix:

Cytotoxicity

- A marked to severe cytotoxicity was induced at dose-levels ≥400 μg/mL, as shown by a 64 to 100 % decrease in the PD.

Micronucleus analysis

The dose-levels selected for micronucleus analysis were 100, 200 and 300 μg/mL, the latter inducing only a 19 % decrease in the PD, but higher dose-levels being considered as too cytotoxic.

- No increase in the frequency of micronucleated cells was noted relative to the vehicle control. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed.
- The recommended level of cytotoxicity was not reached, however, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation.
- In the presence of S9 mix, the overall results were considered to meet the criteria of a negative response.


-

Following the 24-hour treatment of the first experiment, none of the selected dose-levels induced the recommended level of cytotoxicity and since there was only a 2-fold spacing between dose-levels in this experiment, a second experiment was undertaken under the same experimental conditions, i.e. a 24-hour treatment + 0-hour recovery, without metabolic activation, but using a more narrow range of dose-levels.

Conclusions:
Under the study conditions, the test substance did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

A study was conducted to determine the potential of the test substance, to induce an increase in the frequency of micronucleated cells in the mouse lymphoma cell line L5178Y TK+/-, according to OECD Guideline 487, in compliance with GLP. The test substance was dissolved in DMSO and it consisted of 1 % (v/v) volume in culture medium. No precipitate was observed in the culture medium at the end of the experimental periods. First to assess the cytotoxicity of the test substance, six dose-levels (one culture/dose-level) were tested both with and without metabolic activation system S9 mix up to dose level of 2000 μg/mL in preliminary test.

In the main study seven dose-levels of the test substance were tested in duplicate (two cultures/dose-level), both with and without metabolic activationfor 3 hours and 24 hours of recovery period. Following test substance concentrations were evaluated: 25, 50, 100, 200, 300, 400 and 800 μg/mL. Additionally two tests were conducted without S9 metabolic system with 24 hours treatment and no recovery period. This treatment covered a range of concentration of the test substance from 3 to 200 μg/mL and was subsequently narrowed from 12.5 to 200 μg/mL. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from four dose-levels of the test substance-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5 % Giemsa. Slides from vehicle and positive controls cultures were also prepared. 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 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture. Since the test substance was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity.

In the experiments without S9 mix following the 3-hour treatment, a marked to severe cytotoxicity was induced at dose-levels ≥400 μg/mL. Following the 24-hour treatment of the first experiment, a severe cytotoxicity was induced at 200 μg/mL. Following the 24-hour treatment of the second experiment, a moderate to severe cytotoxicity was induced at dose-levels ≥50 μg/mL. No statistically significant or dose-related increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges. The recommended level of cytotoxicity was not reached after the 3-hour treatment. However, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation. In the absence of S9 mix, the overall results were considered to meet the criteria for a negative response.

In the experiment with S9 mix a marked to severe cytotoxicity was induced at dose-levels ≥400 μg/mL. No increase in the frequency of micronucleated cells was noted relative to the vehicle control. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. The recommended level of cytotoxicity was not reached, however, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation. In the presence of S9 mix, the overall results were considered to meet the criteria of a negative response.

Under the study conditions, the test substance did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system (Sire, 2017).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 01, 2017 to October 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Treatment concentrations for the mutation assays of the main tests were selected based on a preliminary toxicity test.
Assay 1
5-hour treatment in the presence of S9-mix:
1500, 1000, 333.3, 111.1, 37.037 and 12.35 μg/mL
5-hour treatment in the absence of S9-mix:
1500, 1250, 1000, 750, 500, 250 and 125 μg/mL

Assay 2
5-hour treatment in the presence of S9-mix:
1500, 1000, 333.3, 111.1, 37.037 and 12.35 μg/mL
24-hour treatment in the absence of S9-mix:
800, 700, 600, 500, 250, 125, 62.5 and 31.25 μg/mL

Assay 3
24-hour treatment in the absence of S9-mix:
2000, 1500, 1250, 1000, 750, 500, 250 and 125 μg/mL
Vehicle / solvent:
The test substance was soluble in Dimethyl sulfoxide.The formulation of 200 mg/mL concentration was used in the preliminary experiment and 150 mg/mL was used in the main assays. DMSO is also compatible with the survival of the mammalian cells and the metabolic activation system. The test substance was weighed into a calibrated volumetric flask (no correction for purity of the test substance was applied). Approximately 80 % of required DMSO) volume was added and the formulation was stirred until homogeneity was reached, then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared to obtain dosing solutions. The stock solution and DMSO were filtered sterile using a 0.22 μm syringe filter (Supplier: Millipore) before the preparation of the dosing formulations in each case.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
INDICATOR CELLS
CHO K1: Sub-line (K1) of Chinese hamster ovary cell line CHO
ATCC No.: CCL-61
Lot No.: 58244452
Supplier: American Type Culture Collection (Manassas,Virginia, United States)

The CHO cell line was originally derived from the ovary of a female Chinese hamster. The CHO K1 is a sub-line of CHO cell line. Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium.
Cells were stored as frozen stocks in a liquid nitrogen tank. Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative. For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium “culture medium” and incubated at 37°C (± 0.5°C) in a humidified atmosphere (5± 0.3 % CO2 in air). When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.
Rationale for test conditions:
PRELIMINARY TEST:
Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. The highest test concentration in the preliminary test was 2000 μg/mL. In the preliminary experiment, a 5-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed.
Evaluation criteria:
The assay was considered valid if all the following criteria are met:
1. The mutant frequency in the negative (vehicle) control cultures was in accordance with the historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test substance concentrations in duplicate cultures were presented.
Statistics:
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
The mutation frequencies were statistically analyzed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd.) The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1500 and 1000 μg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1500 μg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1500 μg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Assay I, in the presence of S9-mix (5-hour treatment):

- Marked cytotoxicity of the test substance was observed at the concentrations of 1500 and 1000 μg/mL (relative survival of 2.2 %, 40.0 % and 2.0 %, 30.0 % after treatment and on the survival plates, respectively).
- Statistically significant increase (at p<0.01 level) was observed in this experiment at 1000 μg/mL concentration, but the observed value was within the historical control range. Furthermore, the observed mutant frequency (15.2-E6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-E6). No clear dose response to the treatment was observed (by trend analysis).

Assay I, in the absence of S9-mix (5-hour treatment):

- Marked cytotoxicity of the test substance was observed (the highest concentration of 1500 μg/mL showed a relative survival of 0.5 % and 34 % after treatment and on the survival plates, respectively).
- Slight, statistically significant increase (at p<0.05 level) was observed in this experiment at the 1250 μg/mL concentration, but the observed value was within the historical control range. Furthermore, the observed mutant frequency (13.4-E6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-E6). No clear dose response to the treatment was observed (by trend analysis).

Assay II, in the presence of S9-mix (5-hour treatment):

- Excessive cytotoxicity was observed at 1500 μg/mL concentration with no surviving cells detected after the treatment. An evaluation of the mutant frequency was made using data of the concentration of 1000 μg/mL (relative survival of 39.1 % and 26 % after treatment and on the survival plates, respectively) and the next four concentrations (a total of five concentrations).
- Statistically significant reproducible increase (at p<0.01 level) was observed in this experiment at 1000 μg/mL concentrations and slight statistically significant increase (at p<0.05 level) was observed in this experiment at 333.3 μg/mL concentrations, but the observed values were within the historical control range. Furthermore, the observed mutant frequencies (17.9 and 15.0 -E6) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20-E6). An apparent dose response to the treatment was observed (by trend analysis).

In Assay II, in the absence of S9-mix (24-hour treatment):
- No marked cytotoxicity of the test substance was observed (the highest concentration of 800 μg/mL showed a relative survival of 31.2 % and 77 % after treatment and on the survival plates, respectively
- No statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (by trend analysis).

Based on the results for the 24 hours treatment of Assay II, the analysable doses did not meet the recommendations of the test guidelines. Therefore, an additional experiment was performed for validity of the study using modified experimental conditions to give more information about the cytotoxic range and to complete the data.

In Assay III, in the absence of S9-mix (24-hour treatment):
- Excessive cytotoxicity was observed at 2000, 1500 and 1250 μg/mL concentration with no surviving cells detected after the treatment. An evaluation was made using data of the concentrations of 1000 and 750 μg/mL (relative survival of 36.4 %, 45.8 % and 4.0 %, 44 % after treatment and on the survival plates, respectively) and the next three concentrations (a total of five concentrations).
- No statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (by trend analysis).

At the end of the treatment period, insolubility (precipitate) was detected in the final treatment medium for the following assays: assay I at 1500 μg/mL, with and without metabolic activation, assays I and II at 1000 μg/mL, with metabolic activation, assay III at 2000, 1500 and 1250 μg/mL. The precipitation did not interfere with the reading of the results. There were no large changes in pH and osmolality after treatment in any cases.
Remarks on result:
other: assay I: 5 h treatment

An additional in vitro mammalian gene mutation test was performed in order to provide additional information for a Weight of Evidence approach. The conditions used in this Mouse Lymphoma Assay were the same: identical concentrations were used and exposure was for 5-hours in the presence of S9-mix. In conclusion, no mutagenic effect of the test substance was observed in the presence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Conclusions:
Under the study conditions, based on the weight of evidence approach, no mutagenic effect of the test substance was concluded either in the presence or absence of a metabolic activation system.
Executive summary:

A study was conducted to determine the potential of the test substance to induce mutations in CHO K1 Chinese hamster ovary cells according to In Vitro Mammalian Cell Gene Mutation Test (OECD Guideline 476) and EU Method B.17, in compliance with GLP. Dimethyl sulfoxide was used as the vehicle. Treatment concentrations for the mutation assays of the main tests were selected based on a preliminary toxicity test. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) in experiment I and II and for 24 hours without metabolic activation (-S9-mix) in experiment III. In experiment I nominal concentrations were within the range of 1500 - 12.35 μg/mL (+S9) and 1500 – 125 μg/mL (-S9). In experiment II: 1500 - 12.35 μg/mL (+S9) and 800 - 31.25 μg/mL (-S9). In experiment III: 2000 - 125 μg/mL. In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined. At the end of the treatment period, insolubility (precipitate) was detected in the final treatment medium for the following assays: assay I at 1500 μg/mL, with and without metabolic activation, assays I and II at 1000 μg/mL, with metabolic activation, assay III at 2000, 1500 and 1250 μg/mL. The precipitation did not interfere with the reading of the results. There were no large changes in pH and osmolality after treatment in any cases. Based on the statistical results for 5-hour treatments in the presence of S9-mix (statistically significant reproducible increase and dose response were observed) the test substance seems to be positive. However, regarding the spontaneous mutant frequency which is generally between 5 and 20-E6 based on the guideline, the observed mutant frequencies (Assay I: 15.2-E6 over the negative control 8.2-E6 and Assay II: 17.9 and 15.0-E6 over the negative control 7.7-E6) were within this expected range of the negative control. Therefore, the observed values were considered as having no clear biological relevance. In this case the test substance result is a borderline in in the presence of metabolic activation system under the conditions of this HPRT assay. This result is not declared as clearly positive, and is also not clearly negative; there are other assays in this study with trends or statistical differences that are not clearly negative. To further help in the interpretation of this inconclusive result, an additional in vitro mammalian gene mutation test was performed in order to provide additional information for a weight of evidence approach. The conditions used in this Mouse Lymphoma Assay were the same: identical concentrations were used and exposure was for 5 hours in the presence of S9-mix. In conclusion, no mutagenic effect of the test substance was observed in the presence of metabolic activation system under the conditions of Mouse Lymphoma Assay. The spontaneous mutation frequency of the negative (vehicle) and positive control were in accordance with the general historical control range in all assays. At least five evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the cytotoxic range in each test). The overall study was considered to be valid. Under the study conditions, based on the weight of evidence approach, no mutagenic effect of the test substance was concluded either in the presence or absence of a metabolic activation system (Kovács, 2018).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Under the conditions of the HPRT assay, the result is borderline in the presence of metabolic activation system.

Based on the statistical results for 5-hour treatments in the presence of S9-mix (statistically significant reproducible increase and dose response were observed) the test substance seems to be positive. However, regarding the spontaneous mutant frequency which is generally between 5 and 20-E6 based on the guideline, the observed mutant frequencies (Assay I: 15.2-E6 over the negative control 8.2-E6 and Assay II: 17.9 and 15.0-E6 over the negative control 7.7-E6) were within this expected range of the negative control. Therefore, the observed values were considered as having no clear biological relevance.

An additional in vitro mammalian gene mutation test was performed in order to provide additional information for a Weight of Evidence approach. The conditions used in this Mouse Lymphoma Assay were the same: identical concentrations were used and exposure was for 5-hours in the presence of S9-mix. In conclusion, no mutagenic effect of the test substance was observed in the presence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Additional information

Study 1:

A study was conducted to determine the potential of the test substance, to induce an increase in the frequency of micronucleated cells in the mouse lymphoma cell line L5178Y TK+/-, according to OECD Guideline 487, in compliance with GLP. The test substance was dissolved in DMSO and it consisted of 1 % (v/v) volume in culture medium.No precipitate was observed in the culture medium at the end of the experimental periods. First to assess the cytotoxicity of the test substance, six dose-levels (one culture/dose-level) were tested both with and without metabolic activation system S9 mix up to dose level of 2000 μg/mL in preliminary test.

In the main study seven dose-levels of the test substance were tested in duplicate (two cultures/dose-level), both with and without metabolic activationfor 3 hours and 24 hours of recovery period. Following test substance concentrations were evaluated: 25, 50, 100, 200, 300, 400 and 800 μg/mL. Additionally two tests were conducted without S9 metabolic system with 24 hours treatment and no recovery period. This treatment covered a range of concentration of the test substance from 3 to 200 μg/mL and was subsequently narrowed from 12.5 to 200 μg/mL. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from four dose-levels of the test substance-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5 % Giemsa. Slides from vehicle and positive controls cultures were also prepared. 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 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture. Since the test substance was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity.

In the experiments without S9 mix following the 3-hour treatment, a marked to severe cytotoxicity was induced at dose-levels ≥400 μg/mL. Following the 24-hour treatment of the first experiment, a severe cytotoxicity was induced at 200 μg/mL. Following the 24-hour treatment of the second experiment, a moderate to severe cytotoxicity was induced at dose-levels ≥50 μg/mL. No statistically significant or dose-related increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges. The recommended level of cytotoxicity was not reached after the 3-hour treatment. However, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation. In the absence of S9 mix, the overall results were considered to meet the criteria for a negative response.

In the experiment with S9 mix a marked to severe cytotoxicity was induced at dose-levels ≥400 μg/mL. No increase in the frequency of micronucleated cells was noted relative to the vehicle control. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. The recommended level of cytotoxicity was not reached, however, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation. In the presence of S9 mix, the overall results were considered to meet the criteria of a negative response.

Under the study conditions, the test substance did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system (Sire, 2017).

Study 2:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 in Bacterial Reverse Mutation Test. The test substance was examined using four strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, and TA 98). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). DMSO was used as a vehicle. Toxic effects were monitored at doses: 20 µg - 5000 µg/plate in the Standard plate test (SPT) and 4 µg - 2500 µg/plate in the Preincubation test (PIT). In both experiments 3 test plates per dose or per control were investigated. Bacteriotoxic effect were observed at doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT). An increase in the number of his revertants was not observed both in the SPT and in the PIT either without S-9 mix or after the addition of a metabolizing system. Under the study conditions, the test substance is not mutagenic in the Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 at the dose range of: 20 µg - 5000 µg/plate (SPT) and at the dose range of: 4 µg - 2500 µg/plate (PIT) in the absence and presence of metabolic activation (Gelbke, 1988).

Study 3:

A study was conducted to determine the potential of the test substance to induce mutations in CHO K1 Chinese hamster ovary cells according to In Vitro Mammalian Cell Gene Mutation Test (OECD Guideline 476) and EU Method B.17, in compliance with GLP. Dimethyl sulfoxide was used as the vehicle. Treatment concentrations for the mutation assays of the main tests were selected based on a preliminary toxicity test. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) in experiment I and II and for 24 hours without metabolic activation (-S9-mix) in experiment III. In experiment I nominal concentrations were within the range of 1500 - 12.35 μg/mL (+S9) and 1500 – 125 μg/mL (-S9). In experiment II: 1500 - 12.35 μg/mL (+S9) and 800 - 31.25 μg/mL (-S9). In experiment III: 2000 - 125 μg/mL. In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined. At the end of the treatment period, insolubility (precipitate) was detected in the final treatment medium for the following assays: assay I at 1500 μg/mL, with and without metabolic activation, assays I and II at 1000 μg/mL, with metabolic activation, assay III at 2000, 1500 and 1250 μg/mL. The precipitation did not interfere with the reading of the results. There were no large changes in pH and osmolality after treatment in any cases. Based on the statistical results for 5-hour treatments in the presence of S9-mix (statistically significant reproducible increase and dose response were observed) the test substance seems to be positive. However, regarding the spontaneous mutant frequency which is generally between 5 and 20-E6 based on the guideline, the observed mutant frequencies (Assay I: 15.2-E6 over the negative control 8.2-E6 and Assay II: 17.9 and 15.0-E6 over the negative control 7.7-E6) were within this expected range of the negative control. Therefore, the observed values were considered as having no clear biological relevance. In this case the test substance result is a borderline in in the presence of metabolic activation system under the conditions of this HPRT assay. This result is not declared as clearly positive, and is also not clearly negative; there are other assays in this study with trends or statistical differences that are not clearly negative. To further help in the interpretation of this inconclusive result, an additional in vitro mammalian gene mutation test was performed in order to provide additional information for a weight of evidence approach. The conditions used in this Mouse Lymphoma Assay were the same: identical concentrations were used and exposure was for 5 hours in the presence of S9-mix. In conclusion, no mutagenic effect of the test substance was observed in the presence of metabolic activation system under the conditions of Mouse Lymphoma Assay. The spontaneous mutation frequency of the negative (vehicle) and positive control were in accordance with the general historical control range in all assays. At least five evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the cytotoxic range in each test). The overall study was considered to be valid. Under the study conditions, based on the weight of evidence approach, no mutagenic effect of the test substance was concluded either in the presence or absence of a metabolic activation system (Kovács, 2018).

Justification for classification or non-classification

Based on the results of in vitro testing, the test substance does not require classification for mutagenicity according to EU CLP (EC 1272/2008) criteria).