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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11 May 2010 to 5 July 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with an appropriate test guideline with no or minor deviations. Study read-across from shale oils heavy fraction.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
68308-34-9
Cas Number:
68308-34-9
IUPAC Name:
68308-34-9
Constituent 2
Reference substance name:
Shale oils
EC Number:
269-646-0
EC Name:
Shale oils
IUPAC Name:
269-646-0
Constituent 3
Reference substance name:
Shale oil
IUPAC Name:
Shale oil

Method

Target gene:
HPRT locus of V79 cells
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79 cells (Lot No. 05F013), originally derived from male Chinese hamster lung tissue by Ford and Yerganian in 1958, were used. The cells used in the study were obtained from ECACC (European Collection of Cell Cultures), England.
Additional strain / cell type characteristics:
other: HPRT deficient
Metabolic activation:
with and without
Metabolic activation system:
20-methylcholanthrene induced rate liver S9
Test concentrations with justification for top dose:
Five concentrations of the substance (1.625, 3.25, 6.5, 13, 26 µg/ml and 1.25, 2.5, 5, 10, 20 µg/ml) were evaluated in tests without metabolic activation system and five concentrations (1.25, 2.5, 5, 10, 20 µg/ml) in tests with S9, both in two independent experiments.
Vehicle / solvent:
Tested compound was dissolved in dimethylsulfoxide (DMSO) (2 mg/ml stock solution), further dilutions were made using the chosen vehicle and than diluted 1:100 into the serum free culture medium to the desired concentrations. Final DMSO concentration in the medium never exceeded 1% and the control group was exposed to an equivalent concentration of this solvent. All solutions of tested compound were made up freshly before use.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO in medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without metabolic activation: ethylmethanesulfonate; with metabolic activation: 7,12-dimethyl-benz(a)anthracene
Details on test system and experimental conditions:
MATERIALS AND METHODS

MATERIAL

SHALE OILS, HEAVY
Tested compound was dissolved in dimethylsulfoxide (DMSO) (2 mg/ml stock solution), further dilutions were made using the chosen vehicle and then diluted 1:100 into the serum free culture medium to the desired concentrations. Final DMSO concentration in the medium never exceeded 1%
and the control group was exposed to an equivalent concentration of this solvent. All solutions of tested compound were made up freshly before
use.
Concentration range was selected according to preliminary cytotoxicity assay (PE) (SOP 1). The used concentration range was from 26 µg/ml to 1.625 µg/ml for first experiment and 20 µg/ml to 1.25 µg/ml for second experiment without metabolic activation and with metabolic activation using 3h treatment. Five different concentrations were evaluated in each experiment.
Positive and negative controls
In each experiment following positive and negative controls were used:
a) negative control – cells were treated with medium containing 1% DMSO without test compounds -for determination of spontaneous mutations.

For the verification of cell line mutability were used positive controls:
b) positive control for experiments without metabolic activation:
ethylmethanesulfonate (EMS) - 0.4 mg/ml was dissolved in DMSO and diluted with culture medium immediately prior to use
c) positive control for experiments with microsomal fraction S9:
7,12-dimethyl-benz(a)anthracene (DMBA) - 0.003 mg/ml
The compound was dissolved in DMSO and diluted with culture medium immediately prior to use.
Handling with positives control according to the laboratories SOP.

Metabolic activation
All standard in vitro assays were performed in the absence and presence of a rat liver exogenous metabolic activation system (S9 mix). The post-mitochondrial fraction (S9) was derived from livers of adult Sprague-Dawley male rats (ANLAB, Czech republic), weighing approximately 200 g. The animals were pre-treated with the agent 20-methylcholanthrene (MCH), administered i.p. at 80 mg/kg 5 days prior to killing.
The S9 fraction (batch MCH18032008, protein content 41.3 mg/ml, Preparation of S9 fraction – Protocol No. 1/2008) was prepared according to (SOP 3) and stored in liquid nitrogen (-196°C) (SOP 16). The S9 mix was prepared with following composition and was added to the culture medium at a final concentration of 10%: 3ml of S9 fraction; 1 ml of 40 mM NADP; 1 ml of 50 mM glucose-6-phosphate; 1 ml of 330 mM KCl; 1 ml of 50 mM MgCl2; 2 ml of 20 mM HEPES buffer; 1 ml of deionised H2O (total volume 10.0 ml) (SOP 1).

Target cells (test system)
V79 cells (Lot No. 05F013), originally derived from male Chinese hamster lung tissue by Ford and Yerganian in 1958, were used. The cells used in our experiments were obtained from ECACC (European Collection of Cell Cultures), England.

Cell growth and maintenance
Chinese hamster lung V79 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L glucose, with L-glutamine supplemented with10% v/v fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 µg/ml).
All cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. For subculture (twice-three times a week) cells were detached by brief treatment with trypsin (1:250; 0.025%)/EDTA solution and counted in suspension by Bürker’s chambers (SOP 4).

Chemicals
DMEM (1x) with 4,5 g/L glucose and L-glutamine Lot 9MB248 (BioWhittaker), Fetal bovine serum (FBS) Lot 9SB025 (BioWhittaker), Phosphate Buffered Salts (PBS) Lot 28104117 (Flow Laboratories), Penicillin G Lot 0811003 (Biotika), Streptomycin Lot 7D002526 (AppliChem), Trypsin 1:250 cell culture tested Lot 088K7025 (Sigma), Dimethylsulfoxide (DMSO) purum Lot 1104114 (Fluka), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) cell culture tested Lot 066K0721 (Sigma), Sodium carbonate (Na2CO3) (min. 99.0%) Lot 095K0043 (Sigma), 6-thioguanine (TG) (min. 98%) Lot 036K0173 (Sigma), D-glucose 6-phosphate sodium salt (G-6-P) (102% enzymat., 1% water) Lot 028K3783 (Sigma), ß-nicotineamideadeninedinucleotide phosphate sodium salt (NADP) (98%) Lot 077K7000 (Sigma), Kalium chloride (KCl) p.a. Lot 05894/02 (Mikrochem), Magnesium chloride (MgCl2.6H2O) cell culture tested Lot 037K02351 (Sigma), ethylmethanesulfonate (EMS) Lot 125K1797 (Sigma), 7,12-dimethyl-benz(a)anthracene DMBA (95%) Lot 079K1683 (Sigma), Hepes (min. 99.5%) Lot 30H5627 (Sigma), Methylene blue (certified, 87%) Lot 039H4378 (Sigma), liquid nitrogen (Duslo), deionized water (hameln rds a.s.)

Instruments
Digital pH meter Sentron Titan (SOP 5), laboratory balance H110 (Sartorius) (SOP 6), incubator – 37°C, humidified atmosphere, 5%/95% CO2 in air (Heraeus) (SOP 7), aseptic unit (SOP 8), centrifuge Labofuge 400R (Heraeus) (SOP 9), biological incubator BT 120 (SOP 10), laminar box HS 12 (SOP11), inverse phase contrast microscope (OPTIKA microscopes, Meopta), laboratory burner, fridge (Calex) SOP(12), freezer (Heraeus), shaker (Heidolph)

Disposable material
Sterile: cell culture Petri dishes – diameter 90, 60 mm, tissue culture flasks, cryotubes, centrifuge tubes, test tubes, laboratory glassware, single use syringe filter Millipore – 0,22 µm
Non sterile: cotton wool, automatic pipettes, Bürker`s chambers

TEST PERFORMANCE

Protocol
Tests were performed in compliance with OECD 476 (1) according to (2) and (SOP 1).
Cytotoxicity tests (PE) were performed according to (SOP 2).

Treatment
Plating efficiency PE. Cells were treated with SHALE OILS, HEAVY for 3 h in medium without serum. Then the cells were washed twice with serum free medium. After this the cells from each sample were trypsinized, diluted and plated in five Petri dishes (diameter 9 cm) at amounts of 3x10^2 cells/dish. Seven days later the colonies had grown, they were stained and the numbers of viable cells were determined.
Estimation of mutant frequency. V79 cells (1x10^6) were seeded in Petri dishes (diameter 6 cm) containing 5 ml MEM with FBS and cultivated for 24 h. The medium was removed and the cells were treated with SHALE OILS, HEAVY for 3 h in medium without serum, in absence or presence of an external metabolising enzyme system (S9 mix from rat liver). After treatment, cells were washed with FBS free medium and trypsinized. The cells were plated into five Petri dishes (diameter 9 cm), at amounts of 3x10^2 cells/dish for determination of cytotoxicity (PE 1) and in two Petri dishes (diameter 9 cm) for mutant freqeuency. After the expression period of 7 days (two subcultures), cells were trypsinized and sampled for resistance to TG. Cells (2x10^5) were seeded in 10 Petri dishes (diameter 9 cm) containing 10 ml of MEM with 10% FBS and 10 µg TG to select for HPRT mutants. In order to determine cell viability (PE 2) at the time of mutant selection, 300 cells each were seeded in 9-cm Petri dishes containing 8 ml MEM with 10 % FBS but without TG. Surviving colonies were counted 7 days after plating and frequency of mutation to thioguanine resistance determined.

Staining method
The colonies of viable cells were stained with 1 % methylene blue in deionized water;
0.2 ml of 1% methylene blue was added into Petri dishes with medium for 20 min.

Analysis of samples
After air drying, colonies were counted and the plating efficiency, i.e. number of colonies per number of seeded cells, and mutation frequency, i.e. number of colonies per number of seeded cells times the plating efficiency, were calculated.

Treatment of results
Data included cytotoxicity and viability determination, colony counts and mutant frequencies for the treated and control cultures. Plating efficiency (PE) was calculated as percent colonies in treated dishes relative to negative controls. Mutant frequency from 10^5 cells was expressed as number of mutant cells per number of surviving cells. All data were summarised in tabular form in protocols (SOP 1, 2).



Evaluation criteria:
Evaluation criteria
Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. Negative results indicate that under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.

There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. Biological relevance of the results is considered first.
A substance is classified as mutagenic if it induces a concentration-related increase in the number of mutants with at least three concentrations or a reproducible unequivocal increase in the number of mutants after treatment with at least one concentration. In the latter case, the induced number of mutants should be at least 3-fold as compared to the actual spontaneous mutation frequency of the solvent group.
Statistics:
Statistical analysis
Multiple sample comparison of treated and untreated cell sets was processed applying Kruskal-Wallis test. The P-value outcoming from the test
was considered to draw the relevant conclusion about statistical significance. Significance level of 0.01 was taken into account.
Multiple sample comparison was followed by two sample test applying Mann-Whitney W test to compare the medians of the two samples. The statistical significance between the medians was indicated for P-values smaller than 0.01.
All individual values of frequencies are presented together with summary statistics involving count, average, standard deviation, coefficient of variation, minimum, maximum, range and standard skewness.
Electronic Database of raw data was formed following double entry principle.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS AND DISCUSSION

EVALUATION OF CYTOTOXIC EFFECTS

Preliminary cytotoxicity test was undertaken to define the dose range for V79/HPRT gene mutation test. The evaluation of the cytotoxic effect of SHALE OILS, HEAVY on V79 cells was based on their colony forming ability after treatment with test product due to the fact that only viable cells are able to grow, proliferate and form colonies in medium (plating efficiency). SHALE OILS, HEAVY was tested in PE test in concentration range up to maximum dose 500 µg/ml. The lethal effect was manifested at concentrations of 500, 100 and 50 µg/ml. The cytotoxic effect was observed at concentration of 25 µg/ml (22.45 %). In this experiment test article at the lower concentrations of 10 and 5 µg/ml didn’t produce reduction of the colony forming ability (PE) (> 84.08 %) . For this experimental condition IC50-value (concentration of test article at which colony formation was reduced to 50% of the control value) of 13.80 µg/ml was calculated.
Based on these results, the concentrations ranging from 1.625 to 26 µg/ml were chosen for in vitro mammalian cell gene mutation tests with and without metabolic activation.

EVALUATION OF MUTATION FREQUENCY

The mutagenic effect of SHALE OILS, HEAVY was evaluated as resistance to TG. Resistance to the purine analogue TG is a widely used genetic marker in a number of mammalian mutagenesis systems. Mutants resistant to TG arise mainly by alterations of the gene encoding the salvage pathway enzyme hypoxanthine-guanine phosphoribosyl-transferase (HPRT), which normally enables cells to utilize exogenous guanine, hypoxanthine, or their analogues for nucleotide synthesis. Since a functional enzyme is not essential for DNA synthesis, TG mutants may arise through base pair substitution, frameshift mutation, or deletion of the HPRT gene.
According to the cytotoxicity of the test product (first-pilot experiment), the concentration range was adjusted.
Five concentrations of SHALE OILS, HEAVY (1.625, 3.25, 6.5, 13, 26 µg/ml and 1.25, 2.5, 5, 10, 20 µg/ml) were evaluated in tests without metabolic activation system and five concentrations (1.25, 2.5, 5, 10, 20 µg/ml) in tests with S9, both in two independent experiments.
In all experiments negative (solvent) control (1 % DMSO in medium) for determination of spontaneous mutations was used. For the verification of cell line mutability positive controls EMS (0.4 mg/ml) in tests without S9 and DMBA (0.003 mg/ml) in tests with S9 were used.

In the first experiment without external metabolic system (S9) statistically significant increase in mutant frequency wasn’t found.
In this experiment test article at the top concentration of 26 µg/ml produced reduction of the colony forming ability (PE1) (0.76 %). Recovery ability of the cells (in PE 2) at this concentration was 101.97 % .
On the base of the results from first experiment the concentration of 26 µg/ml wasn’t included.
in the second experiment and test article was evaluated in concentration range from 1.25 to 20 µg/ml.
In the second experiment without external metabolic system (S9), SHALE OILS, HEAVY did not induce statistically significant increase in mutation frequency in Chinese hamster lung cells at any concentration from range of 1.25 µg/ml – 20 µg/ml.
In this experiment test article at the top concentration of 20 µg/ml produced reduction of the colony forming ability (PE1) (21.31 %). Recovery ability of cells (in PE 2) at this concentration was 98.83 % .
Responsiveness of the test system was verified by exposing the cells to the direct–acting mutagen ethylmethanesulfonate (EMS). The well known genotoxic agents produced a high frequency of HPRT mutations (23.03 and 21.43 mutants/10^5 cells).
In first and second experiments with metabolic activation SHALE OILS, HEAVY did not induce statistically significant increase in mutation frequency in Chinese hamster lung cells at any concentration from range of 1.25 µg/ml – 20 µg/ml.
In these experiments test article at the top concentration of 20 µg/ml did not produce reduction of the colony forming ability (PE1) (100.0 %, 116.66 %).
Responsiveness of the test system as well as metabolic activity of S9-mix were verified by exposing the cells to the 7,12-dimethyl-benz(a)anthracene (DMBA). The positive control induced a high frequency of HPRT mutations (31.54 mutants/10^5 cells and 36.66 mutants/10^5 cells.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not induce a concentration-related increase in mutation frequency at the HPRT locus in V79 cells, both in the absence and presence of metabolic activation and under the test conditions the test product is not mutagenic in the cultured mammalian cells used.
Executive summary:

A GLP compliant in vitro mammalian cell gene mutation test has been conducted in accordance with OECD Guideline 476. The test substance did not induce a concentration-related increase in mutation frequency at the HPRT locus in V79 cells, both in the absence and presence of metabolic activation and under the test conditions the test product is not mutagenic in the cultured mammalian cells used.

Based on the rationale for read-across, it is considered acceptable to use this study to address the same endpoint for the light fraction of shale oil.