D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetrakis(2,2-dimethylpropanoate), (1S)-

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 the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and in the Escherichia coli strain WP2uvrA, performed according to a method similar to OECD Guideline 471, it was concluded that T003422 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 micronucleus assay

In a K1 in vitro micronucleus assay in cultured peripheral human lymphocytes, performed according to OECD Guideline 487, it was concluded that T003422 was not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in the test.

 

In vitro gene mutation study in mammalian cells

In a K1 in vitro mouse lymphoma assay, performed according to the OECD Guideline 490, it was concluded that T003422 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:

2016-05-30 to 2016-06-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP study report in accordance with OECD guideline 471 and EU Method B. 13/14. No deviations were recorded.

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: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity test date: 2015-09-22

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock solutions were treated with ultrasonic waves to obtain homogenous suspensions or until the test item had completely dissolved

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)

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: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in absence and presence of S9
Mutation experiment I: 0, 17, 52, 164, 512 and 1600 μg/plate in absence and presence of S9
Mutation experiment II: 0, 154, 275, 492, 878, 1568 and 2800 μg/plate in absence and presence of S9

Justification for top dose: at 1600 µg/plate the test item exhibited limited solubility and was therefore used as the highest concentration of the test item in the subsequent mutation assay

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: in Test Facility Study no. 513615 (Micronucleus test study with test item T003422), the test item was suspended at 51.2 mg/mL in DMSO. 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 S9-mix; 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 S9-mix; 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 S9-mix; 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 S9-mix; 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 S9-mix; 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 ± 4h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium strains); Tryptophan (E. coli strains)

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

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of 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.
In addition to the criteria stated below, 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.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in number of revertants at 1600 µg/plate (mutation experiment I)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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
- Precipitation:
Dose range finding test: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards. In strain WP2uvrA, precipitation at the end of the incubation period was already observed at 164 μg/plate.
First mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 and 1600 μg/plate and at 1600 μg/plate at the end of the incubation period in all tester strains.
Second mutation experiment: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 878 μg/plate and upwards, except in tester strain TA100 in the absence of S9-mix, where precipitation was only observed at 1568 and 2800 μg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: In the 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 tester strains TA100 and WP2uvrA. The dose range finding test results are reported as a part of mutation experiment I.

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: cytotoxicity (decrease in number of revertant colonies) was only observed in tester strain TA1537 at 1600 µg/plate (top dose) in the first mutation experiment in the presence and absence of S9

Mutation experiment I:

Toxicity: In strain TA98 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose of 17 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. This reduction is more likely caused by incidental fluctuations in the number of revertant colonies.

Mutation experiment II:

Toxicity: In strain TA100 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed at 878 and 2800 μg/plate. However, since no dose-relationship was observed and the reductions in the mean number of revertant colonies were only minor when compared against relevant historical control data, these reductions are caused by incidental fluctuations in the number of revertant colonies and are considered as not biologically relevant.

In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the presence of S9-mix at the mid-dose level of 878 μg/plate. However, since the increase was not two-fold (a maximum of 1.5-fold was reached), this increase was not considered to be relevant.

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with 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:

2017-01-03 to 2017-01-30

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: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not available
- 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


FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: correction factor 1

Target gene:

thymidine kinase (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).
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was 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 (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5- medium); For 24 hour exposure: 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) (Sigma).
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R 20-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 x 10^-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 treatment): 31.25, 62.5, 125, 250 and 500 μg/mL with and without S9-mix;
Dose range finding test (24h treatment): 31.25, 62.5, 125, 250 and 500 μg/mL without S9-mix;
Mutagenicity assay I (3h treatment): 0.99, 1.97, 3.93, 7.85, 15.7, 31.3, 62.5, 125 and 250 μg/mL with and without S9-mix;
Mutagenicity assay II (24h treatment): 0.99, 1.97, 3.93, 7.85, 15.7, 31.3, 62.5, 125 and 250 μg/mL without S9-mix;

Top dose justification: Based on the findings of the solubility test, DMSO was selected as vehicle and 500 µg/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 of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in exposure medium and ethanol. In DMSO, the test item formed a slight, translucent formulation at the concentration of 1.7 mg/ml. Upon mixing with exposure medium the test item precipitated directly at the concentration of 5.2 mg/ml (= 52 μg/ml) and above. After 3 hours, precipitation was observed at concentrations of 16.4 mg/ml (= 164 μg/ml) and above. Based on these solubility findings, DMSO was selected as vehicle and 500 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-mix; at 29.4 µg/mL (3h treatment) and at 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-mix; at 7.5 µg/mL

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)

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 48 (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 inclubation, 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.
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: 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 (RSG)

Rationale for test conditions:

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

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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: In the presence of S9-mix, the relative total growth of the highest test item concentration was 46% compared to the total growth of the concurrent solvent controls

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 data
- Effects of osmolality: no data
- Water solubility: no data
- Precipitation:
Dose range finding test (3h): The test item precipitated in the exposure medium at 125, 250 and 500 μg/mL
Dose range finding test (24h): The test item precipitated in the exposure medium at 125, 250 and 500 μg/mL
Mutation experiment 1: The test item precipitated in the exposure medium at the two highest concentrations tested (125 and 250 μg/mL).
Mutation experiment 2: The test item precipitated in the exposure medium at the highest concentration tested (250 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 31.25 to 500 μg/mL n 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. After 3 hours of treatment, 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 500 μg/mL compared to the suspension growth of the solvent control. After 24 hours of treatment, no severe toxicity in the relative suspension growth was observed up to test item concentrations of 500 μg/ml compared to the solvent control.
Based on the results of the dose range finding test, 250 µg/mL was selected as the highest test item concentration for the first (3h) and second (24h) mutation experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: 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 8 and 15 (3 hour treatment) and 36 (24 hour treatment)

Evaluation of test item precipitation (first mutation experiment)

To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 250 μg/ml was not selected for mutation frequency measurement.

Evaluation of mutagenicity (first experiment)

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Evaluation of mutagenicity (second experiment)

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-12 to 2016-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not available
- 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy adult, non-smoking volunteers
- Suitability of cells: stimulated human lymphocytes were used because they are sensitive indicators of clastogenic and aneugenic activity of a broad range of chemicals
- Sex, age and number of blood donors if applicable: ages 33 and 30
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively).
- Methods for maintenance in cell culture if applicable: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: AGT: 12.7h (age 33; dose-range finding test and first cytogenetic assay); 13.8h (age 30; second cytogenetic assay)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-in activated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
exposure medium: Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasin B (Sigma) was added to the cells simultaneously with the test item at the 24 hour exposure time.
- Properly maintained: yes (immediately after blood collection, lymphocyte cultures were started)
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital-/ß-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Dose-range finding test: 0, 5.4, 17, 52, 164, 512 μg/mL without S9-mix (24h treatment); the highest tested concentration was determined by the solubility of the test item in the culture medium
First cytogenetic assay: 0, 17, 52, 164 μg/mL with and without S9-mix (3h treatment); the highest test concentration was determined based on the solubility of the test item in the culture medium and on the result of the dose-range finding test
Second cytogenetic assay: 0, 5.4, 17, 52, 164 μg/mL without S9-mix (24h treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble at 500 mg/ml in culture medium (the test item floated on the medium) and ethanol (the test item sank to the bottom). In DMSO, the test item was suspended at 500 mg/ml. The test item formed a white viscous suspension after treatment with ultrasonic waves and was selected as appropriate. The test item precipitated in culture medium at the concentration of 16.4 mg/ml (= 164 μg/mL) and above. 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:

mitomycin C

Remarks:

without S9; at 0.25 and 0.38 µg/mL (3h treatment); at 0.15 and 0.23 µg/mL (24h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

without S9; at 0.1 µg/mL (3h treatment); at 0.05 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; at 15 and 17.5 µg/mL (3h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasin B (Sigma) was added to the cells simultaneously with the test item at the 24 hour exposure time. A vehicle control was included at each exposure time.
After 3 hours of exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with cytochalasin B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.

DURATION
- Exposure duration: 3h (first cytogenetic assay) or 24h (dose range finding test; second cytogenetic assay)
- Expression time (cells in growth medium): 24h (first cytogenetic assay) or 0h (dose range finding test; second cytogenetic assay)
- Fixation time (start of exposure up to fixation or harvest of cells): 27h (3h treatment) or 24h (24h treatment)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (positive control chemical in the first and second cytogenetic assay without S9-mix)

STAIN (for cytogenetic assays): 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. The slides were marked with the Test Facility Study number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene / pertex and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED: At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: CPBI index (cytokinesis-block proliferation index)
- Any supplementary information relevant to cytotoxicity: Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI). Three analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded.

%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}
t = test item or control treatment culture
c = vehicle control culture
and
CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / Total number of cells

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) The increase is dose-related in at least one experimental condition when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical vehicle control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the historical vehicle control data range.

Statistics:

Fisher exact test, one-sided, p < 0.05; Excel 2013 and ToxRat Professional v 3.2.1 were used for statistical analysis of the data.

Species / strain:

lymphocytes: human

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: not indicated
- Precipitation: at 164 and 512 µg/mL (dose range finding test); at 164 µg/mL (cytogenetic assay 1); at 52 and 164 µg/mL (cytogenetic assay 2)

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data were obtained in a dose range finding test. The highest tested concentration was determined by the solubility of the test item in the culture medium. The tested dose levels were 0, 5.4, 17, 52, 164, 512 μg/mL. The test item precipitated at concentrations of 164 μg/mL and upwards, and no cytotoxicity was observed. Therefore, 164 μg/mL was chosen as top dose for the first cytogenetic assay;

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Prior to the mitosis (during or after exposure of the test item) the chemical cytochalasin B was added to the cultures. Cytochalasin B arrests the formation of actin filaments. Consequently, the cell is not able to divide, but nuclear division still continues. In this way, cytochalasin B allows discrimination between cells that have undergone nuclear division (binucleated) and cells that have not (mononucleated).

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: at least 1000 (+- 5% deviation maximum)
- Indication whether binucleate or mononucleate where appropriate: yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. Furthermore, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- Negative (solvent/vehicle) historical control data: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical vehicle control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CPBI index (cytokinesis-block proliferation index)
- Other observations when applicable: No appropriate levels of cytotoxicity were observed up to test item concentration of 512 μg/ml (dose-range finding test) or 164 μg/ml (cytogenetic assy 1 and 2) compared to the solvent control.

Conclusions:

It is concluded that this test is valid and that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Bacterial reverse mutation assay

In a K1 key study (Verspeek R, 2016), performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay), T003422 was evaluated for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains (TA98, TA100, TA1535 and TA1537) resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain (WP2uvrA), resulting in a tryptophan-independent strain. The test was performed in two independent experiments in the presence and absence of S9-mix The test item was dissolved in dimethyl sulfoxide.

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 tester strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 512μg/plate and upwards in the tester strain TA100 and at 164μg/plate and upwards in the tester strain WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 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 1600μg/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 level of 1600μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA1537 in the absence and presence of S9-mix at the highest tested concentration. In a second mutation assay, the test item was tested at a concentration range of 154 to 2800μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.The test item precipitated on the plates at dose levels of 878μg/plate and upwards in the absence and presence of S9-mix in all tester strains, except in strain TA100, where precipitation in the absence of S9-mix was only observed at 1568 and 2800μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

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.

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.

 

In a K2 supporting study (Procaccino, 2012), performed according to a method similar to the OECD Guideline 471, T003422 was evaluated for its ability to induce reverse mutations at the histidine locus in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537), and at the tryptophan locus of Escherichia coli strain WP2uvrA, in the presence and absence of a metabolic activation system (S9).The test item was dissolved in dimethyl sulfoxide.

In a plate incorporation assay all five tester strains were tested at the dose range of 1 to 5000 µg/plate with and without rat S9 in triplicate. No indications of toxicity, apparent by reduced bacterial colony counts and/or reduced or absent bacterial lawns, were observed. Precipitation was observed in top agar at >= 500 µg/plate.

Precipitation was also observed on plates at 500μg/plate and obscured colony counting at >= 1000μg/plate. Revertant frequencies for all doses of T003422, in all tester strains with and without S9, approximated or were less than those observed in the concurrent vehicle control cultures.

All positive and vehicle control values were within acceptable ranges, as were tester strain characterization results.

In conclusion, the test item was negative in the in vitro bacterial/microbial activation reverse mutation assay under the conditions, and according to the criteria, of the test protocol.

In vitro micronucleus assay

In a K1 key study, performed according the OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test), Eurlings (2016) investigated the effect of the test item on the induction of micronuclei formed in cultured peripheral human lymphocytes in the presence of S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ßnaphthoflavone) with a 3-hour exposure period and in the absence of S9-mix with a 3 and 24 hour exposure period. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.

The test item was suspended in DMSO at a concentration of 500 mg/ml. In the first cytogenetic assay, the test item was tested up to 164μg/ml for a 3 hour exposure period with a 27 hour harvest time in the absence and presence of S9-mix. The test item precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 52μg/ml for a 24 hour exposure period with a 24 hour harvest time in the absence of S9-mix. The test item precipitated in the culture medium at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mono- and binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, T003422 was found not to be clastogenic or aneugenic in human lymphocytes under the experimental conditions described in the test.

 

 

In vitro gene mutation study in mammalian cells

In a K1 key study, performed according to the OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene), Verspeek-Rip C. (2017) investigated the effects of T003422 on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. 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. DMSO was selected as vehicle.

In the first mutation experiment, the test item was tested up to concentrations of 125μg/mL in the absence and presence of S9-mix. The treatment period was 3 hours. No toxicity was observed up to the concentration 125μg/mL in the absence of S9-mix.The relative total growth (RTG) was 46% in the presence of S9-mix. The test item precipitated in the culture medium at the concentration of 125μg/mL.

In the second mutation experiment, the test item was tested up to concentrations of 250μg/mL in the absence of S9-mix. The treatment period was 24 hours. No toxicity was observed at this dose level. The test item precipitated in the culture medium at the concentration of 250μg/mL.

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. 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.

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.

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Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T003422 and the criteria of the CLP Regulation (EC) 1272/2008, T003422 should not be classified for mutagenicity.