cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

Endpoint summary

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 (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA), performed according to the OECD Guideline 471, it was concluded that T001202 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay,in the absence and presence of metabolic activation.

-       Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T001202 was considered to be non-clastogenic to human lymphocytes in vitro,in the absence and presence of metabolic activation

-      Gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T001202 is not mutagenic in the mouse lymphoma L5178Y test system in the absence and presence of metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-23 to 2016-06-09

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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I14KB4717
- Expiration date of the lot/batch: 2016-11-25 (retest date)
- Purity test date: 2015-03-04

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
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

OTHER SPECIFICS: A correction factor of 1.09 for the purity/composition of the test item was applied in this study.

Target gene:

The histidine locus in S. typhimurium strains (TA98, TA100, TA1535 and TA1537) and the tryptophan locus in E. coli strains (WP2uvrA)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor 1254 induced rat liver metabolic activation system)

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA100 and WP2uvrA) (top dose selected based on the solubility findings)
Mutation experiment I: 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA98, TA1535 and TA1537) (top dose selected based on the dose-range finding test results)
Mutation experiment II: 154, 275, 492, 878, 1568 and 2800 μg/plate with and without 10% S9-mix (TA1535, TA1537, TA98 and TA100); 492, 878, 1568, 2800 and 5000 μg/plate with and without 10% S9-mix (WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water and ethanol at 50 mg/ml, the test item formed suspensions in both vehicles. In DMSO, the test item was soluble at 50 mg/ml after treatment with ultrasonic waves. Based on these solubility findings, DMSO was selected as vehicle.

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; 5 μg/plate (TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without metabolic activation; 2.5 μg/plate (TA1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation; 10 μg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; 650 μg/plate (TA100)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation; 10 μg/plate (WP2uvrA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix; 2.5μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2μg/plate (TA100 with 10% S9-mix), 15μg/plate (WP2uvrA with 5 and 10% S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn; increase in the size of the microcolonies; reduction of the revertant colonies

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.
Any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest dose levels. See details below

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: The test item was observed to be insoluble in water
- Precipitation: DRF test: at 1600 and 5000 µg/plate at the start of the incubation period, at 5000 µg/plate at the end of the incubation period; experiment I: at 5000 µg/plate at the start and at the end of the incubation period; experiment II: at 2800 and 5000 at the start of the incubation period, at 2800 and 5000 µg/plate at the end of the incubation period (WP2uvrA) and at 5000 µg/plate at the end of the incubation period (TA98, TA100, TA1535 and TA1537).

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Based on the results, the results of the dose range finding test are reported as a part of the mutation experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges
- Negative (solvent/vehicle) historical control data: The vehicle control values were within the laboratory historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
- Other observations when applicable:
Mutation experiment 1: Cytotoxicity was observed in the tester strains TA100, TA1535, TA1537 and TA98 in the absence and presence of S9-mix at the highest dose level(s) tested. Microcolonies were also observed in the absence and presence of S9-mix in the tester strain TA1535 at 5000 μg/plate. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
Mutation experiment 2: Cytotoxicity was observed in the tester strains TA100, TA1535, TA1537 and TA98 in the absence and presence of S9-mix at the highest dose level(s) tested. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Mutation experiment 2

Toxicity: In strain TA1537 (absence of S9-mix; experiment II), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 492 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-11 to 2016-11-22

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: I14KB4717
- Expiration date of the lot/batch: 2016-11-25 (retest date)
- Purity test date: 2015-03-04

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock formulation was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test (3h) or until the test item had completely dissolved in mutation experiment 2. In the dose range finding test (24h) and mutation experiment 1, the stock solution was already dissolved after a vortexing step only.

OTHER SPECIFICS: correction factor is 1.09

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, 125, 250, 500, 1000 μg/mL without and with S9-mix.
Dose range finding test (24h): 0, 31.3, 62.5, 125, 250, 500 μg/mL without S9-mix.
Mutation experiment 1: 0, 3.1, 6.13, 12.5, 25, 50, 100, 200, 300, 400 and 500 μg/mL without and with S9-mix
Mutation experiment 2: 0, 3.13, 6.25, 12.5, 25, 50, 65, 80, 100, 115, 130 and 150 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 1000 and 500 μg/mL exposure medium for the 3 hour and 24 hour treatment, respectively.
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 formed a suspension at the concentration of 100 mg/mL and above and was soluble at 50 mg/mL. Upon mixing with exposure medium the test item precipitated directly at the concentration of 100 mg/mL (= 1000 μg/mL) and above. After 3 hours, precipitation was also observed at the concentration of 50 mg/mL (= 500 μg/mL). Based on these solubility findings, 1000 and 500 μg/mL were selected as the maximum final concentration for the dose range finding test for the 3 and 24 hour treatment, respectively.

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: 3h or 24h
- 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:

Since the test item was poorly soluble in the exposure medium, the highest tested concentration for dose range finding test was 1000 and 500 μg/mL exposure medium for the 3 hour and 24 hour treatment, respectively.
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).

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: not measured
- Effects of osmolality: not measured
- Precipitation: Dose range finding test 3h: at 500 and1000 μg/ml with and without S9-mix
Dose range finding test 24h: at 500 μg/ml with and without S9-mix
Mutation experiment 1: at 300, 400 and 500 μg/ml
Mutation experiment 2: at 150 μg/ml

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 125 to 1000 μg/ml in the absence and in the presence of S9-mix with a 3-hour treatment period. No cell survival was observed at the test item concentration of 1000 µg/mL after a 3h treatment, and hardly any cell survival was observed at the test item concentration of 250 µg/mL and above after a 24h treatment. Based on the results of the dose range finding test, 500 and 150 µg/mL were selected as the highest test item concentration for the first mutation experiment (3h) and second mutation experiment (24h), respectively.

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 19 and 21 (3 hour treatment) and 109 and 114 (24 hour treatment)

Remarks on result:

other: mutation experiment 1

Remarks:

3 h 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 the report.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-12-01 (date test substance was received) to 2004-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: 1) No data on mitogenic stimulation, the metaphase-arresting substance (or time of addition) or identity of vehicle of test substance were provided. 2) Only 100 metaphase spreads scored per concentration.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

2 % rat liver homogenate metabolizing system (S9)

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 17.34, 34.67, 69.34, 138.69, 277.38, 554.75, 1109.5, 2219 and 4438 µg/mL
- Group 1 (4(20)-hour without S9): 0, 138.69, 277.38, 554.75, 832.13, 1109.5 and 2219 µg/mL (0, 277.38, 554.75, 832.13 and 1109.5 µg/mL were selected for metaphase analysis.)
- Group 2 (4(20)-hour with S9): 0, 34.69, 69.34, 138.69, 277.38, 416.07 and 554.75 µg/mL (0, 138.69, 277.38 and 554.75 µg/mL were selected for metaphase analysis.)
- Group 3 (24-hour without S9): 0, 8.67, 17.34, 34.69, 69.34, 104.02 and 138.69 µg/mL (0, 34.69, 69.34 and 138.69 µg/mL were selected for metaphase analysis.)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

: no data on identity

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C at 0.4 µg/mL for the 4h exposure and 0.2 µg/mL for the 24 h exposure

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

: no data on identity

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide at 10 µg/mL

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: no data

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: Groups 1 and 2: 20 hours was stated as the expression period in the study report. However, the study report failed to specify the time of addition of a spindle inhibitor.
Group 3: 0 hours
- Selection time: not applicable
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data

NUMBER OF REPLICATIONS:
Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material.

NUMBER OF CELLS EVALUATED:
100 metaphases per analyzed culture. 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.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

Statistics were performed. However, no data was provided on the tests used.

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: n the preliminary test, there was a precipitate of test substance observed at and above 554.75 µg/mL in all exposure groups.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
There was a precipitate of test material observed at and above 554.75 μg/ml in all exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were observed at up to 2219 μg/ml and 1109.5 μg/ml in the 4(20) hours exposure groups without and with-metabolic activation respectively. In the 24 hours continuous exposure group the maximum dose level with scorable metaphases was 138.69 μg/ml. The data show dose-related test material-induced toxicity with a steep toxicity curve in all exposure groups. Therefore, the selection of the dose range for the chromosome aberration test was based on toxicity and intermediate dose levels were included so as to achieve a dose level showing the ideal of 50% mitotic inhibition.

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range (historical data control range). The positive control substances induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

The test material did not induce statistically significant increases in the frequency of cells with aberrations at any dose level in any of the three exposure conditions within the limitations of the study design.

The test material did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A microscopic assessment of the slides showed that adequate numbers of metaphase cells for analysis were present at up to 1109.5 μg/ml in the 4(20)-hour exposure group without metabolic activation, 554.75 μg/ml in the presence of metabolic activation and at up to 138.69 μg/ml in the 24-hour continuous exposure group. Approximately 50% toxicity was achieved at 554.75 μg/ml in the 4(20) hours exposure with S9 and at 138.69 μg/ml in the 24 hours exposure. In the 4(20) hours exposure without S9 there was an approximate 60% mitotic inhibition at 1109.5 μg/ml, therefore acceptable levels of toxicity were achieved in all cases.
Haemolysis was observed at and above 1109.5 μg/ml in the pulse exposure groups only that was indicative of toxicity, there was no haemolysis seen in the 24 hours exposure group. Dose selection for metaphase analysis was based on toxicity in all exposure groups.

Remarks on result:

other: Groups 1, 2 and 3

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after 4(20)-hours pulse exposures or 24-hours continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

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

A key study (K1, Verspeek-Rip CM, 2016) was performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay).T001202 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA).

A correction factor of 1.09 was used to correct for the purity/composition of the test item.The test item was dissolved in dimethyl sulfoxide at a concentration of 50 mg/mL.

In a dose range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the strains TA100 and WP2uvrA. Results of the dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction in the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix at the highest dose level(s) tested.

In a second mutation assay, the test item was tested at a concentration range of 154 to 2800 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at a range of 492 to 5000 μg/plate in WP2uvrA, in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 2800 μg/plate and upwards, except in the presence of S9-mix in the Salmonella typhimurium strains, where no precipitation was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction in the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence and presence of S9-mix at the highest dose level(s) tested. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation in any of the experiments.

The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

A K2 study (De Meester, 1987) was used as supporting evidence. This GLP screening test was only performed on two tester strains: Salmonella typhimurium TA 98 and TA 100. Based on the lack of increase of the reversion rates, it can be concluded that the test substance has no mutagenic properties towards Salmonella typhimurium TA98 and TA100 in the absence and in the presence of rat metabolic activation system.

In vitro Chromosome aberration test:

Wright (K2, 2004) performed an in vitro chromosome aberration study in human lymphocytes according to a method equivalent/similar to OECD Guideline 473.

The test item, dissolved in vehicle, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and the presence of metabolic activation by S9 mix.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test item, ranging from 138.69 to 2219 µg/mL without metabolic activation and from 34.69 to 554.75 µg/mL with metabolic activation in the first experiment (4 hour exposure period followed by a 20 hour recovery period), and from 8.67 to 138.69 µg/mL in the second experiment (24 hour exposure period). 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 presence chromosome aberrations.

A microscopic assessment of the slides showed that adequate numbers of metaphase cells for analysis were present at up to 1109.5 μg/mL in the 4(20)-hour exposure group without metabolic activation, at up to 554.75 μg/mL in the presence of metabolic activation and at up to 138.69 μg/mL in the 24-hour continuous exposure group. Approximately 50% toxicity was achieved at 554.75 μg/mL in the 4(20) hour exposure with S9 and at 138.69 μg/mL in the 24 hours exposure. In the 4(20) hour exposure without S9 there was an approximate 60% mitotic inhibition at 1109.5 μg/mL, therefore acceptable levels of toxicity were achieved in all cases. Haemolysis was observed at and above 1109.5 μg/mL in the pulse exposure groups which was indicative of toxicity. No haemolysis has been observed in the 24 hour exposure group. Dose selection for metaphase analysis was based on toxicity in all exposure groups. 100 metaphases were scored per concentration for the chromosome aberrations.

The test item did not induce statistically significant increases in the frequency of cells with aberrations in any of the exposure groups.

The test item did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

T001202 was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation study in mammalian cells:

Verspeek - Rip (2016) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (key study, K1).

The test item was assessed for its potential to induce forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3- and 24-hour treatment period.
A correction factor of 1.09 was used to correct for the purity/composition of the test item. At concentrations of 100 mg/mL and higher the test item was suspended in dimethyl sulfoxide. At concentrations of 50 mg/mL and lower the test item was dissolved in dimethyl sulfoxide.

 

In the first mutation experiment,the mutation frequency induced at the thymidine-kinase locus by the test item was determined up toconcentrations of 300 µg/ml in the absence and presence of S9-mix. The treatment period was 3 hours. The relative total growth (RTG) was 33 and 39% in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at the concentration of 300 µg/ml.        
In the second mutation experiment,the mutation frequency induced at the thymidine-kinase locus by the test item was determined up toconcentrations of 130 µg/ml in the absence of S9-mix. The treatment period was 24 hours. The relative total growth (RTG) was 11% at the concentration of 130 µg/ml. No precipitation of the test item in the culture medium was observed up to the concentration of 130 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 is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Based on the results of positive control chemicals the test conditions were considered adequate and it was concluded that the metabolic activation system (S9 -mix) functioned properly. In addition, the acceptability criteria for the negative control substance were met and the mutation frequency was situated within the 95% control limits of the distribution of the historical solvent control database, except in the first experiment without S9-mix. However, the observed mutation frequency of the solvent control culture was still within the range of the acceptability criteria of this assay.

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T001202 and the criteria of the CLP Regulation (EC) 1272/2008, T001202 should not be classified for mutagenicity.