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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
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
09 Nov - 22 Dec 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.”

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Freie und Hansestadt Hamburg, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Reference substance name:
Ashes (residues), coal
EC Number:
931-322-8
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
Ashes (residues), coal

Method

Target gene:
TK locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells used during experimental studies were maintained in RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and horse serum (10% by volume), hereinafter referred to as growth medium. Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone. Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10E-5 M thymidine, 1.2 x 10E-4 M hypoxanthine, 3.3 x 10E-5 M glycine and 7.2 x 10E-7 M methotrexate. Recovery medium is similar to cleansing medium, except that the methotrexate component is removed. Selection medium is growth medium that contains 3 µg/mL of 5-trifluoro-thymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat S9 fraction
Test concentrations with justification for top dose:
9.77, 19.5, 39.1, 78.13, 156.3, 312.5, 625, 1250, 2500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
other: The vehicle served as the negative control.
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 and 24 h without S9 mix; 3 h with S9 mix
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 11-12 days


SELECTION AGENT (mutation assays): 5-trifluoro-thymidine (TFT) at 3 µg/mL


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A dose-related or toxicity-related increase in mutant frequency should be observed.
If the mutant frequency obtained for a single dose at or near the highest testable toxicity is ca. 4 times the concurrent background mutant frequency or greater, the test item will be considered mutagenic in a single trial. Smaller increases will require confirmation by a repeat assay.
Sometimes correlation between toxicity and applied concentration is poor. The proportion of the applied test item effectively interacting with the cells to cause genetic alterations is not always repeatable or under control. Conversely, measurable changes in frequency of induced mutants may occur with concentration changes causing only small changes in observed toxicity. Therefore, either parameter, applied concentration or toxicity, can be used to establish whether the increase in mutant frequency is related to an increase in effective treatment.
Treatments inducing less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.
The ratio of small to large colonies will be calculated from the results of the determination of small to large colonies.
If the test item is positive, the small to large colony ratio for the test item will be compared with the corresponding ratios of the positive and negative controls. Based on this comparison the type of the mutagenic properties of the test item will be discussed.
A test item is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or in the case of relatively non-toxic items, a range or applied concentrations extending to the maximum of 5 mg/mL (or 0.01 M, or 5 µL/mL) or in the case of non-toxic, insoluble materials, a range of applied concentrations extending to at least twice the solubility limit in culture media.

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
Remarks:
Tested up to a concentration of 2500 µg/mL. The top concentration of 5000 µg/mL, as tested in the range-finding study, was a deep black coloured suspension and, thus, not analysable .
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiment. This study was performed both with and without S9 metabolic activation.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control.
A wide range of test item concentrations of 25, 100, 250, 1000, 2500 and 5000 µg Ash/mL was tested for cytotoxicity. After an exposure time of 3 hours at approx. 37 °C on a roller drum at 10 - 15 rpm, the cells were washed and resuspended in growth medium. The cells were then adjusted to 8 cells/mL and for each dose 0.2 mL was plated into 32 microtiter wells. The plates were incubated at 37 °C in a humidified incubator gassed with 5% CO2 in air for 3 days. Wells containing viable clones were identified under a microscope and counted.
The preliminary cytotoxicity information was then used to select dose levels for the mutation assay. Doses were selected using the following criteria: At least eight analysable concentrations were used. Where there would be cytotoxicity, these concentrations covered a range from the maximum to little or no toxicity; this means that the concentration levels were separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth. For relatively non-cytotoxic compounds the maximum concentration is 5 mg/mL, 5 µL/mL, or 0.01 M, whichever is the lowest. The top concentration of 5000 µg/mL, as tested in this range-finding study, was a deep black coloured suspension and, thus, not analysable .


COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of mutation frequencies of the negative controls ranged from 78.84 to 83.78 per 10E6 clonable cells in the experiments without metabolic activation, and from 79.93 to 105.51 per 10E6 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

Any other information on results incl. tables

The mutation frequencies of the cultures treated with Ash ranged from 61.08 to123.73 per 106clonable cells (3 hours exposure) and 80.56 to110.66 per 106clonable cells (24 hours exposure) in the experiments without metabolic activation and 56.77 to111.66 per 106clonable cells (3 hours exposure, first assay) and 68.53 to 97.16 per 106clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.71 to 2.00 for Ash treated cells and from 0.51 to 1.36 for the negative controls.

The positive controls Methylmethanesulfonate (10 and 15 µL/mL) and 3-Methyl­cholanthrene (2.5 and 4.0 µg/mL) caused pronounced increases in the mutation frequency ranging from 4061.79 to 7547.15 per 106clonable cells in the case of MMS and ranging from 3663.53 to 7380.73 per 106clonable cells in the case of 3-MC.

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.65 to 2.72 in the case of MMS.

According to the evaluation criteria for this assay, these findings indicate that a suspension of Ash, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, a suspension of Ash, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Ash did not exhibit clastogenic potential at the concentration range investigated.
According to the evaluation criteria for this assay, these findings indicate that a suspension of Ash, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.