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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3700 AV Zeist, The Netherlands
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Celitement
- Chemical name: Calcium hydrosilicate
- Physical state: White powder
- pH-value: 10.5
- Expiration date of the lot/batch: 12/2015
- Stability under test conditions: Hydration reaction with water
- Storage condition of test material: Room temperature protected from light, in original container in evacuated desiccator

Method

Target gene:
thymidine kinase locus on chromosome 11
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rats (S9-mix).
Test concentrations with justification for top dose:
First test: 24h absence s9 mix and 4h with s9 mix: 300, 150, 75.2, 37.5, 18.8 µg/m2
Second test: 24h absence s9 mix: 300, 168, 101, 60.4, 30.4, 15.2 µg/m2
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (RPMI 1640)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Remarks:
In absence S9-mix: methylmethanesulfonate; In presence S9-mix: 3-methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:In the first test duplicate cultures were exposed for 24 hours in the absence of S9-mix and 4 hours in the presence of S9-mix to the test substance. In the second test, duplicate cultures were exposed for 24 hours in the absence of S9-mix. To avoid formation of a solid layer of the particles, the cell cultures were suspended three times during the 24 hours exposure.
- Selection time (if incubation with a selection agent): 10 - 14 days

SELECTION AGENT: trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).

DOSE RANGE FINDING
In this test, single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of the test substance ranging from 75.2 to 1200 μg/cm2. In addition, a single culture was exposed to culture medium (RPMI 1640). The highest concentration tested was based on the pH changes as determined in the solubility test and observations in an in vitro micronucleus test.
Evaluation criteria:
Following criteria were used to validate data obtained in the gene mutation assay:
-Average cloning efficiency of negative controls should not be <60% or >140%.
-Average suspension growth of negative controls should be between 8 and 32
-Average mutant frequency of negative controls should fall within the range of 40-300 TFT resistant mutants per 1,000,000 clonable cells.
-Mutant frequency of positive controls should be >400 TFT-resistant mutants per 1,000,000 clonable cells, and should be at least twice that of the corresponding negative control.
-Unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20%, or one data point between 1 and 10% and another between 20 and 30%.

A response was considered to be positive if induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) wars more than 126 mutants per 1,000,000 clonable cells. A response was considered to be equivocal if induced mutant frequency was more than 88 mutants (but not more than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity and with no evidence of mutagenicity at RTG > 10%, was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed. The test substance was considered not to be mutagenic if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test single cultures were treated for 24 hours in the absence of S9-mix with 5 concentrations the test substance ranging from 75.2 to 1200 μg/cm2. After 4 and 24 hours aliquots were taken to assess the cell yield. At the highest three concentrations (1200, 600 and 300 μg/cm2) the cells were completely covered by a semi solid layer of the test substance. In addition, at all concentrations a dose related sedimentation of the test substance was observed. The culture medium had changed from orange (normal pH value) to pink or slightly pink (indication for increased pH value) at the highest four concentrations. After 4 hours treatment at a concentration of 1200 μg/cm2 the cells were heavily affected (shrivelled), possibly due to suffocation under the layer of the test substance. The cell yield of the lower concentrations fluctuated between 82 and 104%. After 24 hours, at 1200 μg/cm2 the cells were still heavily affected (shrivelled) and the cell yield of the lower concentrations fluctuated between 83 and 123%. Based on the results and observations (presence of a semi solid layer of the test substance and significantly increased pH value) in the dose range finding study, the highest dose used in the main test was 300 μg/ml in the absence and presence of S9-mix.

MAIN TEST RESULTS:
As all acceptance criteria were met, the test was considered valid. Although the average mutant frequency of the negative controls in test 2 was at the lower limit of the range, the test was still considered valid as the average mutant frequency found in test 2 was very similar to the average mutant frequency found in test 1. Dose levels and visual observations before and after treatment In the first test, the dose levels of the test substance ranged from 18.8 to 300 μg/cm2 in both the absence and presence of S9-mix. At the start of the treatment in both the absence and presence of S9-mix, sedimentation was observed at all concentrations tested and at the highest concentration (300 μg/cm2) the cells were completely covered by a semi solid layer of the test substance. In both the absence and presence of S9-mix, at the highest concentration (300 μg/cm2) the colour of the culture medium was slightly pink. At the end of the treatment period, at a concentration of 300 and 150 μg/cm2 in the absence of S9-mix, formation of a semi solid layer of the test substance and change of the medium colour (slightly pink) was observed. The cells were fixed under the layer of the test substance and could not be removed from the cell culture flasks. At the end of the treatment period, viability of the cells (as determined with trypan blue dye exclusion) of the highest concentration was about 80%. In the presence of S9-mix at the end of the treatment period, viability of the cells at the highest concentration (300 μg/cm2) was about 90%. In the second test, the cell cultures were resuspended three times during the 24h treatment (ca. 2-3 hours, ca. 4-6 hours and ca. 20-22 hours after start of exposure) to avoid formation of a solid layer of the particles. At the start of the treatment in the absence of S9-mix, turbidity and sedimentation was observed at all concentrations tested, in a dose related manner. The colour of the cultures was slight pink when compared to the negative control cultures. At the end of the treatment period, turbidity was observed in all cultures and the colour of the highest four cultures was pink to slightly pink in a dose related manner. At the end of the treatment period, at the highest concentration (300 μg/cm2) almost no viable cells were present. The viability of the cells (as determined with trypan blue dye exclusion) of the three lower concentrations (168, 101 and 60.4 μg/cm2) were about 75%, 95% and 96%, respectively.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first test in the absence of S9-mix, the initial cell yield and/or relative suspension growth (RSG) and/or relative total growth (RTG) were reduced by more than 10% at and above 18.8 μg/cm2. The three highest dose levels of the test substance evaluated for mutagenicity were 300, 150 and 75.2 μg/cm2; the RTG at these doses was 3%, 8% and 78% respectively. It was anticipated that the observed reduced cell growth was mainly caused by the fact that the cells could not be removed from the cell culture flasks and not due to cytotoxicity. To demonstrate this, the 24h treatment was repeated and cells were resuspended regularly during exposure. In the presence of S9-mix, the initial cell yield and/or relative suspension growth (RSG) and/or relative total growth (RTG) were not reduced when compared to the negative control.
In the second test in the absence of S9-mix (repeat of 24h treatment group), the initial cell yield and/or relative suspension growth (RSG) and/or relative total growth (RTG) were reduced by more than 10% at and above 60.4 μg/mL. The four highest dose levels of the test substance evaluated for mutagenicity were 300, 168, 101 and 60.4 μg/cm2; the RTG at these doses was 1%, 3%, 22 and 80% respectively. Apparently, the observed reduction in initial cell yield, relative suspension growth and RTG in test 1 was not only due to the fact that the cells could not be removed from the cell culture flasks, but also due to cytotoxicity.

MUTAGENICITY
In the first and second test, in the absence and presence of S9-mix, no increase in the mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

COLONY SIZING
Colony sizing was not performed as no positive responses were observed in cultures exposed to the test substance.
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

Under the test conditions OECD 476 and GLP the test substance is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix).
Executive summary:

According to OECD guideline 476 and GLP the test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Two tests were conducted. In the first test, 5 duplicate cultures were treated for 4 and 24 hours in the presence and absence of S9-mix, respectively. In the second test (repeat of the 24h treatment group) 6 duplicate cultures were treated in the absence of S9-mix. The test substance was suspended in culture medium (RPMI 1640). In both tests in the absence and presence of S9-mix, the highest concentration of the test substance tested and evaluated for mutagenicity was 300μg/cm2 (equal to 750μg/mL). This was considered the maximum feasible concentration and was based on the physiological conditions; formation of a semi solid layer of the test substance and marked increase of pH at the higher concentrations. Methyl methanesulphonate and 3-methylcholanthrene were used as positive control substances in the absence and in the presence of S9-mix, respectively; culture medium (RPMI 1640) served as a negative control in the test. As all acceptance criteria were met, the test was considered valid. In the presence of S9-mix the test substance was not cytotoxic to the cells at the maximum feasible concentration. In the absence of S9-mix the test substance was cytotoxic to the cells resulting in a reduction in initial cell yield and suspension growth. In the first and second test in the absence of S9-mix, the relative total growth at the highest concentration (300 μg/cm2) evaluated was 3% and 1%, respectively. In both the absence and presence of S9-mix no increase in mutant frequency by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any of the dose levels tested. It is concluded that under the conditions used in this study, the test substance is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells, in both the absence and presence of metabolic activation (S9-mix).