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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (cofactor-supplemented liver fraction of phenobarbital/5,6-benzoflavone-induced rats)
Test concentrations with justification for top dose:
Experiment 1: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate
Experiment 2: 0; 50; 150; 500; 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: A solution suitable for use was obtained in ethanol at a concentration of 50 mg/mL (to provide a final concentration of 5000 μg/plate, the maximum guideline recommended test substance concentration).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used in tests without metabolic activation for strains TA100 and TA1535 at 2 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Used in tests without metabolic activation for strain TA1537 at 50 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Used in tests without metabolic activation for strain TA98 at 2 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Used in tests without metabolic activation for strain WP2 uvrA (pKM101) at 2 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Used in tests with metabolic activation for strains TA100 and TA1535 at 5 µg/plate; for strain WP2 uvrA (pKM101) at 10 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Used in tests with metabolic activation for strains TA98 and TA1537 at 5 µg/plate.
Details on test system and experimental conditions:
Two independent standard plate incorporation assays were performed with and without metabolic activation (S9 mix). Test procedures varied in the proportion of S9 content in the S9 mix (10% v/v and 20% v/v in Experiment 1 and 2, respectively).

3 Pleates per treatment were incubated at approx. 37 °C for ca. 72 h. The appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter.

Cytotoxiciy was defined as a substantial reduction in mean revertant colony counts and/or by a sparse or absent background bacterial lawn.

Evaluation criteria:
The test material is considered to exhibit mutagenic activity in this assay if a reproducible increase in revertant colony numbers of at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship occurs.

A test substance is considered non-mutagenic in this test if exposure to the test material does not produce a reproducible increase in revertant colony numbers.
Statistics:
The data were not statistically analysed. The study result was unequivocal.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No evidence of toxicity was obtained.
At a test material concentration of 5000 µg/plate, precipitate was observed in all treatments.
All sterility control plates were colony free. Hence the absence of microbial contamination of the S9 mix, buffer and test material formulation was confirmed. Viability counts met the acceptance criteria. See tables on results and viability attached as background material

Conclusions:
no mutagenicity observed in this Ames test
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human (blood collected aseptically from two healthy, non-smoking adult donors and pooled, cultured in whole blood culture)
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male Sprague-Dawley derived rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction (5% v/v in final medium)
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST
3-hour treatment in the absence and presence of S9 mix, and 21-hour continuous treatment in the absence of S9 mix:
30.2, 50.4, 84, 140, 233.3, 388.8, 648, 1080, 1800 and 3000 µg/mL (the maximum concentration was chosen based on a fluctuation in osmolality of the medium greater than 50 mOsm/kg at 5000 µg/mL).

MAIN TEST
3-hour treatment in the absence of S9 mix:
Exposure concentrations: 200, 275*, 350, 425, 500*, 575*, 650 and 725 µg/mL

3-hour treatment in the presence of S9 mix:
Exposure concentrations: 50, 100*, 200*, 300*, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 and 3000 µg/mL

21-hour continuous treatment in the absence of S9 mix:
Exposure concentrations: 50, 100*, 150, 200*, 250*, 300, 350 and 400 µg/mL

* assessed for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the test material was soluble in Ethanol at 500 mg/mL. On dosing a 500 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 5000 µg/mL, precipitate (visible by eye) was observed, when compared to the vehicle control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
used in tests without metabolic activation (-S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
used in tests with metabolic activation (+S9 mix)
Details on test system and experimental conditions:
CELL DIVISION STIMULANT:
Phytohaemagglutinin (PHA)

METHOD OF APPLICATION: in cell culture medium

DURATION
- Exposure duration: 3-hour treatment with and without metabolic activation; 21-hour continuous treatment without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells):
21 hours in each of both experiments (after the 3 h treatment the cells were cultivated with fresh media for a further18 h)

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® (0.1 µg/mL culture medium, added 2 hours before harvest)

STAIN (for cytogenetic assays):
10% Giemsa (pH 6.8)

NUMBER OF REPLICATIONS:
Duplicate cultures per treatment

NUMBER OF CELLS EVALUATED:
100 metaphases per culture. This number was reduced in cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy and endoreplication: Polyploid and endoreduplicated cells (i.e. the ploidy status) were recorded as a percentage of the 100 metaphases analysed per slide.

OTHER:
Microscopic examination of the metaphases included the recording of the following parameters:
- Number of aberrant cells (including and excluding gaps)
- Types of aberrations (Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap)
- Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes

Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed.
Evaluation criteria:
An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
-Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) at one or more test concentration.
-The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
Statistics:
The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) [SAS INSTITUTE (2002) SAS OnlineDoc® Version Nine. SAS Institute Inc., Cary, NC, USA].

The number of aberrant metaphase cells in each test substance concentration group was compared with the vehicle control value using the one-tailed Fisher exact test [Fisher, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York].

A Cochran-Armitage test for trend [Armitage, P. (1955) Tests for linear trends in proportions and frequencies. Biometrics, 11, 375-386.] was applied to the control and all test substance groups. If this was significant at the 1% level, the test was reiterated excluding the highest concentration group - this process continued until the trend test was no longer significant.

As the test material did not demonstrate any evidence of causing a biologically relevant increase in the frequency of structural chromosome aberrations under any treatment condition, no D20 values for structural aberrations are presented.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: (assessed by eye at the end of treatment)
absence of S9 mix
3-h treatment: 575 µg/mL and above
24-h treatment: 200 µg/mL and above
presence of S9 mix
3-h treatment: no notable culture medium changes were observed

RANGE-FINDING/SCREENING STUDIES:
- 3-hour exposure in the absence and presence of S9 mix:
Precipitate (observed by eye) was observed at concentrations of 648 µg/mL and greater. Exposure to 1080 µg/mL (-S9 mix) and 233.3 µg/mL (+S9 mix) resulted in reduction in the mitotic index to 36% and 48% respectively.
- Continuous exposure for 21 hours in the absence of S9 mix:
Precipitate (observed by eye) was observed at concentrations of 140 µg/mL and greater. Exposure to 233.3 µg/mL resulted in reduction in the mitotic index to 59%.

COMPARISON WITH HISTORICAL CONTROL DATA:
All values were within the historical control range, when taken at the 99% confidence limit.

ADDITIONAL INFORMATION ON CYTOTOXICITY (MAIN STUDY):
- 3-hour exposure in the absence and presence of S9 mix:
Reduction in the mitotic index to 52% and 55% at 575 µg/mL (-S9 mix) and 300 µg/mL (+S9 mix) respectively
- Continuous exposure for 21 hours in the absence of S9 mix:
Reduction in the mitotic index to 51% at 250 µg/mL

See summary table attached as background material.

The test material did not cause biological significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration in the absence or presence of metabolic activation by S9 mix, when compared to the vehicle control.

 

See table summary HML attached as background material.

Conclusions:
no mutagenicity observed in this chromosome abberration test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase gene (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
basic medium: RPMI 1640, buffered with sodium bicarbonate (2 mg/mL) and supplemented with L-glutamine (2.0 mM) and gentamicin (50 µg/mL);
cell culture medium: basic medium, supplemented with surfactant (Synperonic F68, 0.1% v/v), sodium pyruvate (1.0 mM) and heat-inactivated donor horse serum (HiDHS, 10% v/v);
cloning efficiency plating: equal volumes of basic medium (supplemented with surfactant (Synperonic F68, 0.02% v/v), sodium pyruvate (1.0 mM) and HiDHS (30% v/v)) and cell culture medium
selective medium: cell culture medium containing trifluorothymidine (TFT, 4 µg/mL).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male Sprague Dawley derived rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TESTING (suspension growth relative to that of vehicle controls)
Test concentrations at 3 h exposure with (+S9) and without (-S9) metabolic activation and at 24 h exposure without metabolic activation (-S9):
4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL (the maximum concentration was chosen based on a fluctuation in osmolality of the medium greater than 50 mOsm/kg at 5000 µg/mL).

MUTATION TESTS
3 h exposure (with and without S9):
Exposure concentrations: 150*, 175*, 200*, 225*, 250, 275, 300, 325, and 350 μg/mL

24 h exposure (-S9):
Exposure concentrations: 50*, 150, 175*, 200*, 225*, 250*, 275, and 300 μg/mL

* assessed for determinationof the mutant phenotype
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (final volume added to the cultures was 1% v/v)
- Justification for choice of solvent/vehicle: the test material was soluble at 500 mg/mL in ethanol. A solution of 500 mg/mL, dosed at 1% in medium, showed precipitate in the culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
used in tests without metabolic activation (-S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
used in tests with metabolic activation (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours with and without metabolic activation; 24 hours without metabolic activation
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration 4 µg/mL

NUMBER OF REPLICATIONS:
- Exposure phase: two cultures for each concentration of test material and positive control; four cultures for vehicle controls.
- Assessment of cloning efficiency: two 96-well plates per culture
- Assessment of mutant potential: two 96-well plates per culture

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- colony size distribution in vehicle and positive controls
Evaluation criteria:
The mutation test result was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF = 126 x 10^–6; Moore et al. 2006. Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis. 47, 1-5).

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1.
Statistics were not reported since the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was not exceeded and no significant positive linear trend (p<0.05) was seen.
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
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: (assessed by eye at the end of treatment)
3-h treatments: 175 µg/mL
24-h treatment: 150 µg/mL and above

RANGE-FINDING/SCREENING STUDIES: Precipitate (observed by eye at the end of treatment) was observed at concentrations of 156.25 µg/mL and greater in both the absence and presence of S9 mix, following a 3-hour exposure. Exposure at concentrations from 4.88 to 2500 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in relative suspension growth (RSG) values from 122 to 0% and from 124 to 0% respectively.
Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at concentrations of 625 µg/mL and greater. Exposure to concentrations from 4.88 to 2500 µg/mL resulted in RSG values from 109 to 0%.

See tables on toxicity and mutations attached as background material

The results obtained in response to the exposure of cell cultures to the test material in the absence or presence of metabolic activation by S9 mix did not demonstrate mutagenic potential: there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.

See table on MLA results attached as background material.

 

Conclusions:
no mutagenicity observed in this MLA test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the negative results attained in the three in vitro genotoxicity tests the test material is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].