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Administrative data

Key value for chemical safety assessment

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 the Escherichia coli strain WP2uvrA, performed according to OECD Guideline 471, it was concluded that T002907 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 T002907 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 T002907 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:
From 2005-10-14 to 2005-11-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D (dated May 19, 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): TIC2782 (T002907)
- Substance type: white solid
- Physical state: solid
- Analytical purity: 99.9%
- Lot/batch No.: 00467090 (= charge 05C0836)
- Expiration date of the lot/batch: 4 May 2006
- Storage condition of test material: room temperature
- Stability in solvent: not indicated by the sponsor
Target gene:
The histidine dependant strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
Solvent: Dimethylsulfoxide (DMSO)
Justification for choice of solvent: 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:
other: sodium azide 10 μg/plate in TA 1535 and TA 100
Remarks:
without metabolic activation
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, 4-NOPD 10 μg/plate in TA 98, 50 μg/plate in TA 1537
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methyl methane sulfonate, MMS, 4µL/plate in WP2 uvrA
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA, 2.5 μg/plate (TA 1535, TA 1537, TA 98, TA 100), 10 μg/plate (WP2 uvrA)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay
Experiment II: pre-incubation assay

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: at least 48 hours
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is 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 is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data was required according to the guideline
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:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with T002907at 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.TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a
clearing of the bacterial background lawn.
The pre-experiment was reported as main experiment I since the following criteria was met: evaluable plates (>0 colonies) at five concentrations or more in all strains used.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The laboratory´s historical control range was exceeded with metabolic activation in the untreated control of strain WP2 uvrA in experiment I, and in the untreated control of strain TA 100 without metabolic actvation in experiment II. These minor deviations are judged to be based on biological fluctuations in the number of colonies and have no impact on the outcome of the study.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-12 to 2016-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: M14LB5188
- Expiration date of the lot/batch: 2016-12-15 (retest date)
- Purity test date: 2015-08-14

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

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.
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, 156.3,313, 625, 1250, 1582 μg/mL without and with S9-mix;
Dose range finding test 24h: 0, 156.3, 313, 625, 1250, 1582 μg/mL without S9-mix;
Mutation experiment 1: 0, 20, 39, 78, 156, 313, 625, 1250, 1582 μg/mL without and with S9-mix;
Mutation experiment 2: 0, 20, 39, 78, 156, 313, 625, 1250, 1582 μg/mL without S9-mix;

Since the test item was poorly soluble in the exposure medium, the highest tested concentration in the dose range finding test was 1582 μg/mL exposure medium for the 3 hour and 24 hour treatment.
The highest tested concentration in the main mutation experiment was selected based on the toxicity of the test item.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in exposure medium. In DMSO, the test item was soluble at 158.2 mg/mL. The test item did not precipitate in the exposure medium up to and including the concentration of 1582 μg/ml (= 0.01 M). Based on these solubility findings, DMSO was selected as vehicle and 1582 μg/ml (= 0.01 M; the recommended top concentration in the guidelines) was selected as highest test item concentration in 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-diphenyltetrazoliumbromide (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).
Since the test item was poorly soluble in the exposure medium, the test item was dissolved in DMSO and the highest tested concentration was 1582 μg/mL exposure medium for the 3 hour and 24 hour treatment.
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:
no cytotoxicity, but tested up to precipitating concentrations
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 marked changes at the highest non-precipitating concentration of 1582 µg/mL (7.0 compared to 7.2)
- Effects of osmolality: No marked changes at the highest non-precipitating concentration of 1582 µg/mL (440 mOsm/kg compared to 452 mOsm/kg)
- Precipitation: No test item precipitation was observed up to the highest concentration tested of 1582 µg/mL in any of the experiments.

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 156.3 to 1582 μg/mL in the absence of S9-mixwith 3 hour treatment periods and in the presence of S9-mix with a 24-hour treatment period.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1582 μg/mL compared to the suspension growth of the solvent control.
Based on the results of the dose range finding test, 1582 µg/mL was selected as the highest test item concentration for the both first (3h) and second (24h) mutation experiment.

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 15 and 18 (3 hour treatment) and 72 (24 hour treatment)
Remarks on result:
other: Mutation experiment 1
Remarks:
(3 h treatment)

Mutation experiment 1:

Initially, a mutation experiment was performed with a 3 hour treatment period in the absence of S9-mix. Since no acceptable cell growth in the positive control group was obtained, this experiment was rejected and a repeat experiment was performed with the same dose range.

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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2005-11-02 to 2006-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Guideline adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4A: ”Mutagenicity – In vitro Mammalian Chromosome Aberration Test“, dated May 19, 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guideline S 2 A: ”Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests of Pharmaceuticals“ (CPMP/ICH/141/95), dated April 1996.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guideline S 2 B: ”Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals“ (CPMP/ICH/174/95), dated July 1997.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch No.: 00467090 (= charge 05C0836)
Agrgegate state at room temperature: Solid
Colour: White
Purity: 99.9 %
Stability in solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: May 04, 2006
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). 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 (the cells have a stable karyotype with a modal chromosome number of 22)
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Test on Toxicity:
Concentration range in the pre-test (with and without metabolic activation, 4h- and 24h exposures respectively): 12.3, 24.7, 49.4, 98.8, 197.5, 395.0, 790.0 and 1580.0 µg/mL

Main test:
Experiment I (without metabolic activation, 4h exposure, 14h- recovery): 49.4, 98.8, 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II (without metabolic activation, 18h exposure, no recovery): 49.4, 98.8, 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II* (without metabolic activation, 28h exposure, no recovery): 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II** (without metabolic activation, 28h exposure, no recovery): 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II (without metabolic activation, 28h exposure, no recovery): 197.5, 395.0 and 790.0, 1580.0 µg/mL

Experiment I (with metabolic activation, 4h exposure, 14h recovery): 49.4, 98.8, 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II* (with metabolic activation, 4h exposure, 24h recovery): 49.4, 98.8, 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II** (with metabolic activation, 4h exposure, 24h recovery): 49.4, 98.8, 197.5, 395.0 and 790.0, 1580.0 µg/mL
Experiment II (with metabolic activation, 4h exposure, 24h recovery): 49.4, 98.8, 197.5, 395.0 and 790.0, 1580.0 µg/mL

* Was repeated because the metaphases did not fulfil the criteria for evaluation
** Was repeated due to a technical error
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test item was dissolved in culture medium (minimum essential medium: MEM). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
300 - 400 μg/mL (2.4 – 3.2 mM) in nutrient medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
1.4 - 2.0 μg/mL (5.0 - 7.0 μM) in Saline (0.9 % [w/v])
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Pre-test: 4h and 24h
Without S9: Experiment I: 4 hours ; Experiment II: 18 hours and 28 hours
With S9 mix: Experiment I and II: 4 hours
- Expression time (cells in growth medium):
Experiment I: 14 hours (without and without S9 mix); Experiment II: 0 hours without S9 mix and 24 hours with S9 mix
After 4 hrs (4h- exposure period) the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
For the exposure period 18 and 28 hours, the culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
- Fixation time (start of exposure up to fixation or harvest of cells):
18 hours in Experiment I (with and without S9 mix) and 18 hours (without S9 mix) and 28 hours (with and without S9 mix) in Experiment II.
After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively).

SPINDLE INHIBITOR (cytogenetic assays): Colcemid was added (0.2 μg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later, 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.
STAIN (for cytogenetic assays): Giemsa (E. Merck, D-64293 Darmstadt)

NUMBER OF REPLICATIONS: duplicate

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, in the absence of S9 mix after 28 hrs exposure, where only 50 metaphase plates were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: reduced cell numbers (pre-test); mitotic index and number of polyploid cells (experiment I and II)

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
- Determination of endoreplication: investigated

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 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 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 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).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
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:
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4 - 3.8 %) as compared to the rates of the negative controls (1.2 - 4.1 %).
In both experiments, EMS (300 and 400 μg/mL, respectively) and CPA (1.4 and 2.0 μg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the pre-experiment, no relevant influence of the test item on the pH value was observed (negative control and at 1580 µg/mL pH 7.4)
- Effects of osmolality: In the pre-experiment, no relevant influence of the test item on osmolarity was observed (negative control 333 mOsm versus 347 mOsm at 1580 µg/mL)
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: In the pre-experiment, no precipitation of the test item was observed.
- Other confounding effects: no data

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 12.3 and 1580 μg/mL were applied. Clear toxic effects were observed after 4 hrs treatment with 1580 μg/mL in the absence of S9 mix. In contrast, in the presence of S9 mix after 4 hrs treatment and in the absence of S9 mix after 24 hrs continuous treatment no clear cytotoxicity was observed up to the highest concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: In both experiments, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.
The aberration rates of the cells after treatment with the test item (0.5 - 3.0 % aberrant cells, exclusive gaps) were within the range of the negative control values (0.5 - 3.0 % aberrant cells, exclusive gaps) and within the range of the historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In this study, neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest required concentrations of the test item.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative with metabolic activation
ambiguous without metabolic activation

In conclusion, it can be stated that under the experimental conditions reported, the test item TIC2782 (T002907) did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to the highest required concentration.
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 (Sokolowski, 2006), performed according to the OECD Guideline 471, T002907 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. 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:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 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 the test item 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.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, T002907 was considered to be non-mutagenic in the 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, T002907 was tested in the Salmonella typhimurium reverse mutation assay with three histidine-requiring strains TA98, TA100 and TA102 using the plate incorporation method at nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9). The dose range was 0.5 to 5000 μg/plate in the main experiment.

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.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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 (Kunz, 2006), performed according to the OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), T002907 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. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive controls in Experiment II, at 28 hrs exposure, without metabolic activation, where only 50 metaphase plates were scored.

The highest applied concentration in the pre-test on toxicity (1580 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.

In this study, in the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest required concentration.

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 relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

The test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, T002907 was considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to the highest required concentration.

 

In a K2 supporting study (Wright, 2004), performed according to a method similar to the OECD Guideline 473, T002907 was assessed for its potential to induce structural chromosomal aberrations in cultured human lymphocytes. Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material. A minimum of three concentration levels and the concurrent vehicle and positive controls were evaluated for chromosome aberrations for each exposure group. Except where there was the need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations, this would normally be the A culture. 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 and a 24-hour continuous exposure in the absence of S9.

The molecular weight of the test material was not provided and therefore the maximum dose level was 5000 μg/ml. The test material was found to be insoluble in dimethyl sulphoxide, minimal essential media (MEM) or acetone and therefore the selected vehicle was MEM because it provided the best suspension that was acceptable for dosing. There was no significant change in pH or osmolality at any of the dose levels used up to and including 5000 μg/ml. The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test. There was no toxicity in the pulse exposure groups although there was apparent slight inverted toxicity in the S9 group that was considered to be spurious. There was a dose-related toxic response in the 24 hours continuous exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 5000 μg/ml in the 4 (20)-hour exposure groups and at up to 3750 μg/ml in the 24-hour exposure group. A precipitate of the test material was observed at the end of the exposure period at 5000 μg/ml in the pulse exposure groups only. Therefore, the selection of the dose range for the chromosome aberration test included the maximum recommended dose level, 5000 μg/ml, as the upper dose level in the all exposure groups. The test material was did not induce any toxicity in the 4(20)-hour exposure groups. In the 24-hour exposure group without S9 the ideal level of toxicity of approximately 50% growth inhibition was achieved at 2500 μg/ml, therefore, in this case an acceptable level of toxicity was achieved. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The test material did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups. The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolising system after a 4(20)-hour exposure or after a continuous 24-hour exposure in the absence of metabolic activation. The test material was therefore 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 (2017) investigated the effect of T002907 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 DMSO.

In the first mutation experiment, the test item was tested up to concentrations of 1582 μg/ml (~ 0.01 M, recommended in the guidelines) in the absence and presence of S9-mix. The treatment period was 3 hours. No cytotoxicity was observed at this dose level in the absence and presence of S9-mix. No test item precipitation was observed up to the concentration of 1582 μg/ml.

In the second mutation experiment, the test item was tested up to concentrations of 1582 μg/ml in the absence of S9-mix. The treatment period was 24 hours. No cytotoxicity was observed at this dose level. No test item precipitation was observed up to the concentration of 1582 μg/ml.

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.

It was 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 with T002907 and the criteria of the CLP Regulation (EC) 1272/2008, T002907 should not be classified for mutagenicity.