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Genetic toxicity in vitro

Description of key information

 

Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100 TA1535, TA1537 and in Escherichia coli strain WP2uvrA, performed according to OECD Guideline 471, it was concluded that T002615 has no mutagenic properties towards any of the bacterial strains tested in the absence and in the presence of S9-mix under the test conditions described in the report.

 

In vitro chromosome aberration study in mammalian cells

In a well documented GLP study (K1) performed in accordance with the OECD Guideline 473, it was concluded that T002615 did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic and/or precipitating concentrations.

 

In vitro gene mutation study in mammalian cells

In a K1 mouse lymphoma assay, it was concluded that T002615 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-02-20 to 2003-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch No.: 00403487
Aggregate State at Room Temperature: solid
Colour: white
Purity: > 98 %
Stability in Solvent: not indicated by the sponsor
Storage: room temperature
Expiration Date: retest date - 2003-12
Target gene:
histidine locus ( S. Typhimurium strains) ; tryptophan locus (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see "Any other info. on material and methods incl. tables" section
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see "Any other info. on material and methods incl. tables" section
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/B-naphtoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment on toxicity: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Both plate incorporation test (experiment I) and the pre-incubation test (experiment II) (with and without S9 mix): 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation; TA 1535 and TA 100 at 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation; TA 1537 at 50 µg/plate and TA 98 at 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation; WP2 uvrA at 4 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation; 2.5 µg/plate( TA 1535, TA 1537, TA 98, TA 100), and 10 µg/plate (WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment I: in agar (plate incorporation test); experiment II: preincubation test

EXPERIMENTAL PERFORMANCE:
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μL: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL: Overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: 48 hours (experiment I and II)
- Selection time (if incubation with a selection agent): 48 hours ( simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine and tryptophan locus

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
- The test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent control, such an increase iss not considered biologically relevant.
Statistics:
No statistical evaluation of the data is required.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
E. coli WP2 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation: see detailed table in section "any other information on results"

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as part of experiment I since no toxic effect were observed and 5000 µg/plate were chosen as maximal concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical range of positive controls was exceeded in strains TA 1535 (exp.I) without metabolic activation and in strain 98 (exp.II) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested

The assay was performed in two independent experiments both with and without liver microsomal activation. Due to a technical failure, no bacterial growth was observed in strain WP2 uvrA in experiment I (plate incorporation test). An additional experiment was performed under identical conditions using strain WP2 uvrA. The results of this additional experiment are included in this report as experiment I.

Precipitation of the test item

strain  Experiment I  Experiment II 
without S9 mix  With S9 mix  without S9 mix  With S9 mix 
TA1535 1000-5000 1000-5000 2500, 5000 333, 2500, 5000
TA1537 1000-5000 1000-5000 1000-5000 1000-5000
TA98 1000-5000 1000-5000 2500, 5000 2500, 5000
TA100 5000 2500, 5000 2500, 5000 2500, 5000
WP2uvrA 1000-5000 1000-5000 2500, 5000 5000
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshfits in the genome of the strains used.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-01-29 to 2003-05-06
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:
Guideline Adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4A (dated 2000-05-19)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch No. 00403487
Aggregate state at Room Temperature: solid
Colour: white
Purity: >98%
Stability in solvent: Not indicated by sponsor
Storage: Room temperature
Expiration date: 2003-12 (restest date)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM supplemented with 10 % fetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Naphtoflavone induced rat liver S9 from male Wistar Hanlbm rats
Test concentrations with justification for top dose:
Range-finder test: 3.3, 6.6, 13.3, 26.6, 53.1, 106.3, 212.5, 425.0 μg/mL

Experiment I
4h exposure - 18h preparation time, without S9 mix: 1.3, 2.5, 5.0, 10.0, 20.0, 40.0 μg/mL (2.5, 5.0 and 10.0 μg/mL were selected for metaphase analysis)
4h exposure - 18h preparation time, with S9 mix: 1.3, 2.5, 5.0, 10.0, 20.0, 40.0 μg/mL (2.5, 5.0 and 10.0 μg/mL were selected for metaphase analysis)

Experiment II
18h exposure - 18h preparation time, without S9 mix: 0.6, 1.3, 2.5, 5.0, 10.0, 20.0 μg/mL (2.5, 10.0 and 20.0 μg/mL were selected for metaphase analysis)
28h exposure - 28h preparation time, without S9 mix: 2.5, 5.0, 10.0, 20.0 μg/mL (5.0 and 10.0 μg/mL were selected for metaphase analysis)
4h exposure - 28h preparation time, with S9 mix: 0.6, 1.3, 2.5, 5.0, 10.0, 20.0 μg/mL (2.5, 10.0 and 20.0 μg/mL were selected for metaphase analysis)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofurane
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

On the day of the experiment (immediately before treatment), the test item was suspended (pre-test) of dissolved (experiment I and II) in THF.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9-mix; 200 µg/mL (1.6 mM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix; 0.7-1.0 µg/mL (2.5-3.5 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h (experiment I); 4h (experiment II, with S9); 18h or 28h (experiment II, without S9)
- Expression time (cells in growth medium): 11.5h (4h exposure - experiment I); 21.5h (4h exposure -experiment II, with S9); none (18 h and 28h continuous exposure - experiment II)
- Fixation time (start of exposure up to fixation or harvest of cells): 18h (experiment I and II) or 28h (experiment II).

SPINDLE INHIBITOR: 15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 μg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts respectively). Per experiment two slides per group were prepared. After preparation the cells were stained.
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: triplicate (2 slides per culture)

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in experiment II at the 18h preparation interval without metabolic activation, where only 50 metaphases platees were scored. Only metaphases with characteristic chromosome numbers of 22 +/- 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) and/or reduced cell number

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype)
- Determination of endoreplication: investigated
- Other: Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data (0.0-4.0% aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data (0.0-4.0% aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory historical control data (0.0-8.5% polyploid cells)
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p<0.05)
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no influence of the test item on the pH value was observed (solvent control pH 7.3 versus pH 7.4)
- Effects of osmolality: no influence of the test item on the osmolality was observed (solent control 379 versus 379)
- Precipitation: at 6.6 µg/mL and above (Pre-test; 4h exposure; with and without S9); at 10 µg/mL and above (All experimental parts; with and without S9)

RANGE-FINDING/SCREENING STUDIES: In a range-finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 3.3 and 425 µg/mL were applied. No clear toxic effects were observed after 4 hrs and 24 hrs treatment up to the highest test item concentration in the absence and the presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The aberration rates of the cells after treatment with the test item (0.0-3.5% aberrant cells, excl gaps) were close to the range of the solvent control values (0.5-2.0% aberrant cells, exclusive gaps) and within the range of the laboratory historical control data: 0.0-4.0% aberrant cells, exclusive gaps.
In both experiments, EMS (200 µg/mL) and CPA (0.7 and 1.0 µg/mL, respectively) were used as positive controls and showed distinct increases inc ells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: It has to be considered that in the experimental parts in the presence of S9 mix the test item dissolved in THF interfered with the procedure for chromosome spreading. Although, no toxic effects were observed -neither reduced cell numbers nor reduced mitotic indices- cultures were not evaluable for cytogenetic damage. Thus, experiment I and II have been repeated once and the two highest applied concentrations in experiment I (repeat) were not evaluable for cytogenetic damage.
In this study, neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest tested concentrations of the test item.
Remarks on result:
other: all experiments

In both experiments, no biologically relevant increase in the rate of polyploid metaphase was found after treatment with the test item (1.2 -3.3%) as compared to the rates of the solvent controls (1.3 -4.1%).

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that under the experimental conditions reported, the test item T002615 did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-29 to 2016-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mouse lymphoma assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16CC0982
- Expiration date of the lot/batch: 2019-03-23
- Purity test date: 2016-03-23

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: Not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was suspended or dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test or until the test item had completely dissolved in the mutagenicity tests.

OTHER SPECIFICS: correction factor 1.00
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Cell cycle length, doubling time or proliferation index: not indicated
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10^6 cells/ml.
- Normal (negative control) cell cycle time: not indicated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50U/mL and 50μg/mL, respectively), 1mM sodium pyruvate and 2mM L-glutamin
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium)
Exposure medium 3h: basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium)
Exposure medium 24h: basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium)
Selective medium: basic medium supplemented with 20% (v/v) heat inactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat inactivated horse serum (R20-medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ßnaphthoflavone)
Test concentrations with justification for top dose:
Dose range finding test 3h: 0, 6.25, 12.5, 25, 50 and 100 μg/mL without and with S9-mix.
Dose range finding test 24h: 0, 6.25, 12.5, 25, 50 and 100 μg/mL without S9-mix.
Mutation experiment 1: 0, 0.2, 0.39, 0.78, 1.57, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL without and with S9-mix
Mutation experiment 2: 0, 0.20, 0.39, 0.78, 1.57, 3.13, 6.25, 12.5, 25 and 50 μg/mL without S9-mix.

Since the test item was soluble in DMSO at 46.98 mg/mL and precipitated directly at 5.9 mg/mL (=59 µg/mL) and above upon mixing with exposure medium, 100µg/mL was selected as the maximum final concentration for the dose range finding test.
The highest tested concentration in the main mutation experiment was selected based on the toxicity and the solubility of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In DMSO, the test item formed a suspension at 94 mg/mL and above and was soluble at 46.98 mg/mL. Upon mixing with exposure medium the test item precipitated directly at concentrations of 5.9 mg/mL (= 59 μg/mL) and above. After 3 hours, precipitation was also observed at 29 mg/mL. Based on these solubility findings, DMSO was selected as vehicle and 100 μg/mL was selected as the maximum final concentration for the dose range finding test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; 15 μg/mL (3h treatment), 5 μg/mL (24h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9; 7.5 μg/mL (3h treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hour treatment) were used.

DURATION
- Exposure duration: 3 h or 24 h
- Expression time (cells in growth medium): 48h (3h and 24h treatment)
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays): After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well.

NUMBER OF REPLICATIONS:
Determination of cloning efficiency: 2 x 96-well microtiter plates/concentration
Determination of mutation frequency: 5 x 96-well microtiter plates/concentration (solvent controls and treatment groups); 10 x 96-well microtiter plates/concentration (positive controls

NUMBER OF CELLS EVALUATED:
Determination of cloning efficiency (CEday2): One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
Determination of mutation frequency: 9.6 x 10^5 cells/concentration plated (5 x 96-well microtiter plates/concentration, each well containing 2000 cells in selective medium (solvent controls and treatment groups)); 9.6 x 10^5 cells/concentration plated (10 x 96-well microtiter plates/concentration), each well containing 1000 cells in selective medium (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth
Rationale for test conditions:
The highest concentration tested in the mutagenicity tests should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/ml or 0.01 M (whichever is the lowest).
Solubility limitations: No solubility test was performed in water/exposure medium based on the information provided by the sponsor. Since the test item was poorly soluble and precipitated upon mixing with exposure medium, the highest tested concentration was 100 μg/ml exposure medium.
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
No statistical analysis
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG 39% at 100µg/mL without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG 40% at 50 µg/mL and 27% at 25 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Water solubility: No solubility test was performed in water based on the information provided by the sponsor
- Precipitation:
Dose range finding test 3h: at 100µg/mL with and without S9-mix (start of treatment); at 25, 50 and 100 μg/mL with and without S9-mix (end of treatment)
Dose range finding test 24h: at 100µg/mL without S9-mix (start of treatment); at 25, 50 and 100 μg/mL without S9-mix (end of treatment)
Mutation experiment 1: at 25, 50 and 100 μg/mL with and without S9-mix
Mutation experiment 2: at 50 μg/mL wihtout S9-mix

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 6.25 to 100 μg/mL in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
In the absence of S9-mix, the relative suspension growth was 5% and 9% at the test item concentration of 100 µg/mL after 3h and 24h treatment, respectively. In the presence of S9-mix, no toxicity in the relative suspension growth was observed compared to the suspension growth of the solvent control; Based on these results, 100 µg/mL was selected as the top dose level for the first mutation experiment (3h treatment) and 50 µg/mL was selected as the top dose level for the second mutation experiment (24h treatment).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database

OTHER :
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 16 and 19 (3 hour treatment) and 55 and 82 (24 hour treatment)
Remarks on result:
other: mutation experiment 1
Remarks:
(3h treatment)
Conclusions:
Interpretation of results: negative with and without metabolic activation
In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

In a GLP compliant K1 bacterial reverse mutation assay (Poth, 2003), performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay), the test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains TA100, TA1535, TA98 and TA1537, and in the Escherichia coli assay with the tryptophan-requiring strain WP2uvrA for its potential to induce gene mutations in the plate incorporation assay (experiment 1) and the pre-incubation test (experiment 2). The test item was dissolved in DMSO. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with T002615 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In addition, appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a supporting K2 study (Bowles, 2004), performed according to a method similar to the OECD Guideline 471, the test item was tested in the Salmonella typhimurium reverse mutation assay with three histidine-requiring strains TA98, TA100 and TA102 using the plate incorporation method. The test item was dissolved in DMSO and was tested in the main experiment at nine dose levels, ranging from 0.5 to 5000 µg/plate, in triplicate in the presence and absence of a rat liver homogenate metabolising system (10% liver S9).

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn in TA98, with S9-mix only, at and above 1500 μg/plate. No toxicity was observed to the remaining tester strains at any dose level of the test material. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A powdery precipitate was observed at and above 500 μg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of the test.

In vitro chromosome aberration study in mammalian cells

In a K1 study (Schulz, 2003), performed according to the OECD Guidelie 473 (In Vitro Mammalian Chromosome Aberration Test), the test item was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up.

A preliminary toxicity test was performed with a concentration range of 3.3 to 425 µg/plate. Precipitation was observed at 10 µg/mL and above in the absence and presence of S9 -mix, and no clear toxic effects were observed. Based on these results, the concentration ranges of 1.3 to 40 µg/mL (experiment 1; 4h exposure + 18h preparation time), 0.6 to 20 µg/mL (experiment 2; 4h exposure + 28h preparation time, and 18h exposure + 18h preparation time) and 2.5 to 20 µg/mL (experiment 2; 28h exposure + 28h preparation time) were selected, i.e. concentrations exhibiting clear test item precipitation as recommended in the OECD Guideline 473.

Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in experiment 2, without metabolic activation at the 18 hrs preparation interval, where only 50 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (425 µg/ml; approx. 1 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.

No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Furthermore, the highest concentrations scored for chromosomal aberrations were far in the range where test item precipitates occurred.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

In addition, the positive controls EMS and CPA were used in both experiments and showed distinct increases in cells with structural chromosome aberrations. The solvent control values were within the range of the historical control data.

In conclusion, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells in vitro. Therefore, the test item was considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations.

 

In a non-GLP K2 supporting study (Wright, 2004), performed according to a method similar to OECD Guidelie 473 (In Vitro Mammalian Chromosome Aberration Test), the test item was assessed for its potential to induce structural chromosome aberrations in human lymphocytes.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material. Three exposure groups were used in the study. These were: a 4-hour exposure in the absence of metabolic activation (S9) with a 20 -hour expression period; a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period; a 24-hour continuous exposure in the absence of S9.

The dose range for the toxicity test was 14.69 to 3760μg/ml, based on a 10mM maximum dose level. There was a precipitate of test material observed at and above 470μg/ml in all exposure groups. Some apparent toxicity was observed in the exposure groups without metabolic activation (S9) and a slight reduction in mitotic index was seen in the exposure group with metabolic activation (S9). This was considered to be related to the precipitate of the test material obscuring the metaphase cells and not to test material toxicity. Therefore, the selection of the dose range for the chromosome aberration test was based on the lowest precipitating dose level of 470μg/ml.

For all three exposure groups in the main experiment, the dose range was 14.69 to 470 µg/mL. The test material did not induce mitotic inhibition at any dose level in any exposure group. Dose selection for metaphase analysis was based on the lowest precipitating dose level, 470 μg/ml.

The test material did not induce statistically significant increases in the frequency of cells with aberrations at any dose level in either the pulse exposure groups (4 -hour exposure) or the continuous exposure group (24 -hour exposure).

In addition, all of the vehicle control cultures had frequenscies of cells with chromosome aberrations within the expected range, and the positive control materials induced statistically significant increases in the frequence of cells with aberrations.

In conclusion, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation study in mammalian cells

In a K1 study, Verspeek-Rip C (2016) investigated the effect of the test item on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, according to the OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene). The test was performed in the presence of S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone) with a 3 hour treatment period and in the absence of S9-mix with a 3 and 24-hour treatment period. The test item was dissolved in dimethyl sulfoxide at a concentration of 47 mg/ml.

In the first mutation experiment, the test item was tested up to concentrations of 25 μg/ml in the absence and presence of S9-mix. The treatment period was 3 hours. Relative total growth (RTG) was 39 and 74% in the absence and presence of S9 mix, respectively. The test item precipitated in the culture medium at the concentration of 25 μg/ml.

In the second mutation experiment, the test item was tested up to concentrations of50 μg/ml in the absence of S9-mix. The treatment period was 24 hours. The RTG was 40% at the highest dose level tested, whereas at one dose level below the RTG was 27%. The test item precipitated in the culture medium at the concentration of 50 μg/ml. In the absence of S9-mix, the test item did not induce asignificant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

In addition, the positive control chemicals produced significant increases in the mutation freaquency within the 95% control limits of the distribution of the historical positive control database. The mutation frequency found in the solvent control cultures was considered valid for the evaluation of the mutagenic activity of the test item.

It was therefore concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.  

 

 

 

 

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests and according to the criteria of the CLP Regulation (EC) 1272/2008, T002615 should not be classified for mutagenicity.