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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:
experimental study
Adequacy of study:
key study
Study period:
2010-08-11 to 2010-09-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The present study was performed according to the respective OECD guideline and EU method

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests For Pharmaceuticals, 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
no
Principles of method if other than guideline:
no aplicable
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Charcoal
EC Number:
240-383-3
EC Name:
Charcoal
Cas Number:
16291-96-6
IUPAC Name:
Charcoal
Details on test material:
CAS no.: 16291-96-6
Batch no.: 29004
Purity: C-Fix: 80.5 %; dose calculation not adjusted to purity
Storage conditions: At room temperature, moisture protected
Expiry date: December 2030

An extract of the test item was prepared using the following procedure: 250 g of the test item was extracted with 1 L acetone/n-hexane 50:50 (v:v) at 50°C in the ultrasonic bath for 1 h. After cooling to room temperature the extract was filtered. This procedure was repeated; thereafter the sample was mixed mechanically with 250 mL of the abovementioned solvent mixture. After a filtration step, the volume of the extract was reduced in several steps to approximately 150 mL using a rotary evaporator at 50°C. The extract was quantitatively filled up to 250 mL in a volumetric flask. Accordingly, the nominal concentration of the extract was 1,000 mg/mL.
The extract, a pale yellowish-brown liquid, was then stored in the refridgerator at 2-8°C.

Method

Target gene:
In this study in vitro mouse lymphoma assay (MLA), employing the in L5178Y cells carrying a single gene mutation in the tk (thymidine kinase) are employed. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-), producing the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products. In the presence of a toxic pyrimidine analogue 5-trifluorothymidine (TFT), the enzyme will incorporate this analogue into the cells.
Cells deficient in thymidine kinase (TK) due to the mutation tk +/- → tk -/- are resistant to the cytotoxic effects of TFT. Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TFT (the mutant cells have non-functional pyrimidine salvage pathway), whereas normal cells, which contain thymidine kinase, are not.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Supplier: American Type Culture Collection (ATCC) (Manassas,Virginia)
Product no.: CRL-9518
Batch no.: 3794355
Date of arrival: 2009-02-10

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Date of arrival: February 10, 2009.
Cells are stored as frozen stocks (Master Copies: MC) in liquid nitrogen. Each batch of frozen cells is purged of TK-/--mutants (producing Master Products) and checked for the absence of mycoplasma.

For the experiments, 4-4 vials (Assay 1 - Assay 2) were thawed rapidly, cells were diluted in RPMI 10 medium and incubated at 37  0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Well growing cells, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver homogenate obtained from rats induced with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
128; 320; 800; 2,000 and 5,000 µg/mL
Vehicle / solvent:
acetone/n-hexane 50:50 (v:v)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
concentration: of 0.2 µg/mL for the 3-h treatment and 0.1 µg/mL for the 24-h treatment; for tests without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in RPMI 5 Medium; final concentration: 5 µg/mL; for tests with metabolic activation
Details on test system and experimental conditions:
The study included a preliminary solubility test, a preliminary range finding test (toxicity test) and 2 main mutation assays (assay 1 and 2).
Assay 2 was performed because of the negative results obtained in assay 1.

Preliminary Solubility and Toxicity Tests

A preliminary solubility test (as a part of the toxicity test) was performed for investigation of the test item extract behavior in the MLA test system.
The examined concentration ranges were should cover the nominal concentration range of 51.2, 128, 320, 800, 2,000, and 5,000 µg/mL).
In the toxicity test, the treatment procedure was the same as described below for the main mutation assays, however single cultures were used and positive controls were not included.
The treated cells were washed following treatment with RPMI 10 Medium and re-suspended in 10 mL RPMI 10 culture medium.
Cell concentrations were adjusted to 8 cells/mL and, for each dose, 0.2 mL was plated into each well of a 96-well microtiter plate. The microtiter plates were incubated at 37±1°C in a humidified incubator gassed with 5% (v/v) CO2 in air for 11-13 d.
Wells containing viable clones were identified by eye and counted. At the end of this test the harmonized relative survival of each culture was determined.
The obtained cell growth data were used to choose the dose levels for the main assays. Five concentrations were selected for the main mutation experiments.

Main Mutation Assays

Treatment of the Cells

Main Mutation Assay 1

A 3-h treatment was performed in the presence and absence of S9 Mix.
For the 3-h treatment incubation, 1 x 10^7 cells were placed in a series of sterile flasks (culturing surface about 75 cm^2). The treatment medium (RPMI 5) contained a reduced serum level of 5 % (v/v).
A suitable volume of vehicle, test compound, or positive control solution, and 1.0 mL of S9 Mix or of 150 mM KCl was added to a final volume of 20 mL per culture containing 1 x 10^7 cells.
Duplicate cultures were used for each treatment.
Solubility and behaviour of the test item extract in the cultures was assessed by the unaided eye at the beginning and end of treatment.
After the 3-h incubation at 37±1°C (approximately 5% CO2 in air) with gentle shaking, the cell cultures were centrifuged at 1200 rpm for 5 min, washed with 10 mL RPMI 10 Medium, and suspended in 10 mL RPMI 10/tube. The cell densities were adjusted to a concentration of 2x105/mL. Cells were transferred to flasks for growth through the expression period and diluted to be plated for survival.

Main Mutation Assay 2

A 24-h treatment was performed without metabolic activation, and a 3-h treatment with addition of metabolic activation.
The procedure for the 3-h treatment was the same as described above.
For the 24-h treatment, 4 x 10^6 cells were placed in each of a series of sterile flasks (culturing surface 25 cm2). The treatment medium (RPMI 5) contained a reduced serum level of 5% (v/v).
A suitable volume of vehicle, test compound or positive control solution was added to a final volume of 20 mL per culture containing 4 x 10^6 cells.
Duplicate cultures were used for each treatment.
Solubility and behaviour of the test item extract in the cultures was assessed by the unaided eye at the beginning and end of treatment.
After the 24-h incubation at 37±1°C (approximately 5% CO2 in air) the treated cultures were adjusted on the same way as it was performed at the 3-h treatment.

Plating for Survival

Following adjustment of the cultures to 2 x 10^5 cells/mL, samples from these cultures were diluted to 8 cells/mL as follows:
Initial Cell Concentration: 2 x 10^5/mL (A)
1. Dilution Step: 0.1 mL of Cell Suspension (A) + 9.9 mL of RPMI 10
Intermediate Cell Concentration: 2 x 10^3/mL (B)
2. Dilution Step: 0.2 mL of Cell Suspension (B) + 49.8 mL of RPMI 20
Final Cell Concentration: 8/mL

Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microtiter plates (192 wells) resulting in average of 1.6 cells per well. The microtiter plates were incubated at 37±1°C containing approximately 5% (v/v) CO2 in air for 12-13 d. Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period

The cultures were maintained in flasks for 2 d, during this time the tk-/- mutation expressed. During the expression period, subculturing was performed daily with the aim of not exceeding 1 x 10^6 cells per mL and, where possible and retaining a total of at least 5 x 10^6 cells/flask. For this purpose cell densities were adjusted to a concentration of 2 x 10^5/mL and transferred to flasks for further growth.
From observations on recovery and growth of the cultures during the expression period, six dose levels plus negative and positive controls were plated for viability and 5-trifluorothymidine (TFT) resistance.

Plating for Viability

At the end of the expression period, the cell density in the cultures was determined and adjusted to nominally 1x10^4/mL with RPMI 20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL as follows:

Initial Cell Concentration: 1 x 10^4/mL (A)
1. Dilution Step: 0.5 mL of Cell Suspension (A) + 9.5 mL of RPMI 10
Intermediate Cell Concentration: 5 x 10^2/mL (B)
2. Dilution Step: 0.8 mL of Cell Suspension (B) + 49.2 mL of RPMI 20
Final Cell Concentration: 8/mL

Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microtiter plates (192 wells) resulting in an average of 1.6 cells per well. The microtiter plates were incubated at 37±1°C containing approximately 5% (v/v) CO2 in air for 12-13 d. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 5-trifluorothymidine (TFT) Resistance

At the end of the expression period, the cell concentrations were adjusted to 1 x 10^4/mL.
The TFT (300 µg/mL) was diluted 100-fold in the cell suspensions to give a final concentration of 3 µg/mL according to the followings: 89.1 mL of cell suspension + 0.9 mL of TFT.
Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four 96-well microtiter plates (384 wells) resulting in average 2x10^3 cells per well. The microtiter plates were incubated at 37±1°C containing approximately 5% (v/v) CO2 in air for two weeks and wells containing clones were identified as above and counted. The evaluation completed on the 13th-14th day. In addition, scoring of large and small colonies was performed as additional information.
Evaluation criteria:
Assay acceptance criteria

The assay was considered valid if all of the following criteria were met:
1. The MF of the negative (vehicle) control cultures fell within the normal range (above 50-170 mutants per 10^6 viable cells).
2. The positive control chemicals induced a statistically significant increase in MF.
3. The PE of the negative controls was within the range of 65% to 120% on day 3.
4. At least 4 test concentrations were present, with the highest concentration producing 80-90% toxicity, precipitation, or being 5 mg/mL or the highest practical concentration.

Mutagenicity criteria

The test item was considered to be mutagenic in this assay if all the following criteria were met:
1. The assay was valid;
2. Statistically significant (p < 0.05) increases in MF were observed in treated cultures compared to the corresponding vehicle control values at one or more concentrations;
3. The increases were reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There was a significant dose-relationship as indicated by the adequate trend analysis;
5. The MF at the test concentration showing the largest increase was at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.
Statistics:
The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The positive control data were compared with the respective vehicle control or untreated control data with 2 Sample t-Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
not applicable
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH ans osmolality:
In assay 1, the pH and osmolality values of the treatments (in absence and also in presence of S9 mix) were in the same range and did not show any dose-dependent relationship. In assay 2, osmolality and pH were values for the 24-h treatment were in the same range as those observed for the 3-h treatments in assay 1. In assay 2, osmolality and pH values for the 3-h treatment were not measured.

- Water solubility and precipitation:
In both assays, micro-drops (as colloid chemical phenomenon) appeared just after the treatment at the concentration of 5,000 µg/mL (with and without S9 mix). In assay 1, precipitate or micro-drops were not observed after the 3-h treatment (with and without S9 Mix). In assay 2, precipitate appeared after the 24-h treatment at the concentrations of 2,000 and 5,000 µg/mL (with S9 mix). The precipitate did not disturb the evaluation of the assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous MF values of the negative (vehicle) control cultures were in line with the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the whole examined concentration range (of 128-5,000 µg/mL) based on harmonised RS%. In assay 1 in the presence of S9 mix, the relative total growth (RTG) values were lower (51-67% of the RTG of the vehicle control) than the RTG values of the vehicle control. However, no dose-relationship was observed and these lower values were not evaluated as a test item caused effect, but as the biological variability of the applied test system.
Remarks on result:
other: other: Assay 1
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of this study, the extract of the test item charcoal (Probe 2: C-Fix=80.5%) did not induce gene mutations in the cultured mammalian cells (L5178Y TK+/- cells, neither in the presence nor in the absence of metabolic activation by rat S9 mix.
Accordingly, the test item was concluded to be non-mutagenic under the conditions of the present study.