Registration Dossier

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 and TA1535, TA1537 and Escherichia coli strain WP2uvrA, performed according to a method similar to OECD Guideline 471, it was concluded that T003063 has no mutagenic properties towards the S. typhimurium and E. coli 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 an in vitro micronucleus assay in cultured peripheral human lymphocytes performed according to OECD Guideline 487, it was concluded that T003063 was not clastogenic or aneugenic in human lymphocytes in the absence and in the presence of S9 -mix under the experimental conditions described in the report.

In vitro gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay performed according to OECD Guideline 490, it was concluded that T003063 is not mutagenic in the mouse lymphoma L5178Y test system in the absence and presence of metabolic activation under the experimental conditions described in the report.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-26 to 2010-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Well documented study report, GLP compliant, and according to Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals. No deviations from the study protocol were recorded.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Mitsubishi Tanabe Pharma Corporation, 00541426
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
- Purity: 100%
- Physical description: Pale yellow-white crystalline powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not indicated
- Stability under test conditions: Stable at room temperature (under the airtight condition)
- Solubility and stability of the test substance in the solvent/vehicle: in DMSO: 50 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

OTHER SPECIFICS: The test substance was handled under the yellow light condition and stored under the light-shielded condition, because information about stability of the test substance under the light condition was not provided from sponsor.
Target gene:
histidine locus (histidine-dependent S. typhimurium strains); tryptophan locus (tryptophan-dependent E.coli strain)
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:
Phenobarbital/5,6-benzoflavone induced rat liver (S9) from male Sprague-Dawley rats
Test concentrations with justification for top dose:
Dose-finding assay: 0, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate (maximum dose recommended by the guideline)
Main assay: 156, 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: According to the information provided by the sponsor, the solubility in DMSO is 50 mg/mL or more, and the test substance is stable in DMSO. DMSO was selected as the solvent for the test substance, because the preliminary test on solvent selection in the test facility revealed that the test substance was soluble at 5% (w/v) in DMSO and the solution was stable. DMSO was dehydrated using Molecular Sieves 4A just before use.
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, all strains, 0.5 µg/plate (TA98), 1.0 µg/plate (TA100), 2.0 µg/plate (TA1535, TA1537), 10.0 µg/plate (WP2 uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Without S9-mix, 0.01 µg/plate (TA100, WP2 uvrA), 0.1 µg/plate (TA98)
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, 0.5 µg/plate (TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride
Remarks:
Without S9-mix, 1.0 µg/plate (TA1537)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation method
The 0.5 mL of 0.1 M Na-phosphate buffer with pH 7.4 (or 0.5 mL of S9 Mix in case of metabolic activation assay) was added to 0.1 mL of the test substance solution, and then 0.1 mL each of bacterial culture was added. The mixture was incubated at 37ºC for 20 minutes with shaking (“pre-incubation”). The 2.0 mL of top agar was added to the mixture, and the resultant mixture was overlaid onto a minimum glucose agar plate. The agar solution [0.6% agar (Bacto-Agar DIFCO), 0.5% NaCl] supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin solution for S. typhimurium or with 0.5 mM L-tryptophan solution for E. coli at a volume ratio of 10:1 was used as a top agar.
The plates were incubated at 37ºC for 48 hours, and stored at 4ºC until colony counting if the incubation finished on a holiday. After the incubation, the growth inhibition of bacterial strain and the precipitation were examined, and the revertant colonies were counted.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine (S. typhimurium); tryptophan (E. coli)

NUMBER OF REPLICATIONS: duplicate at each dose level; triplicate for negative control

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition (dose-related)

COLONY COUNTING:
Revertant colonies within a circle with about 80 mm diameter on a plate with 86 mm diameter (inner diameter: 84 mm) were counted with an automatic colony counter. The count data was corrected based on the area for uncounted colonies by using a personal computer. The revertant colonies at 1250 μg/plate or more under the conditions of S9 (±) could not be counted by the automatic colony counter due to the test substance precipitation; therefore, the colonies were counted manually. By manual counting,all number of revertant colonies were counted without correction.

OTHER:
- For all plates, the growth inhibition of bacterial strain was observed with a stereoscopic microscope (40-fold magnification) and the precipitation was observed by unaided eyes.
- For each dose, the mean number of revertant colonies on two or more plates was calculated and rounded off to an integral number.
Evaluation criteria:
When the number of revertant colonies on the test substance treatment plate is increased 2-fold or more compared to the negative control value, and the increase is dose-dependent and reproducible between the dose-finding assay and the main assay, the test substance is judged to be positive for mutagenicity. Otherwise, the test substance is judged as negative. When the test substance is judged to be positive, the activity of mutagenicity is calculated.
Statistics:
No statistical analysis is performed for data analysis.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: Information about solubility and stability of the test substance in water is not provided.
- Precipitation: In the dose-finding assay, white precipitates of the test substance were observed at 20 μg/plate or more (without S9) and at 78 μg/plate or more (with S9); In the main assay, white precipitates of the test substance were observed at 156 μg/plate or more (with and without S9).

RANGE-FINDING/SCREENING STUDIES:
In the dose-finding assay, 5000 μg/plate was set as the maximum dose, which is recommended by the guideline, and a total of 7 doses were set with a common ratio of 4. Based on the results of the dose-finding assay, the highest dose in the main assay was set at 5000 μg/plate, and a total of 6 doses were set with a common ratio of 2, including at lease one dose producing precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the dose-finding assay and the main assay, the negative control values were within the acceptable range (mean± 2.5SD) specified based on the historical data of the test facility. The positive controls induced revertant colonies more than 2-fold of the negative control value in each strain. No bacterial growth due to contamination was observed in the sterility tests. These results indicated the assays were performed properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No growth inhibition of bacteria by the test substance was observed in any of the two experiments.
Remarks on result:
other: Dose-finding assay

Validity of assays

In the dose-finding assay and the main assay, the negative control values were within the acceptable range (mean±2.5SD) specified based on the historical data of the test facility. The positive controls induced revertant colonies more than 2-fold of the negative control value in each strain. No bacterial growth due to contamination was observed in the sterility tests. These results indicated the

assays were performed properly.

Maximum mutagenic activity

The maximum mutagenic activity was 5.64×10 rev./mg (at 5000 μg/plate in TA100 without S9 in the dose-finding assay).

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

Based on the results, it was concluded that T003063 was negative for mutagenicity under the test conditions of this study.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-16 to 2017-04-03
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: M14KB4863
- Expiration date of the lot/batch: 2017-11-10
- Purity test date: 2015-01-19

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 available


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 (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies)
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium)
Exposure medium 3h: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium)
Exposure medium 24h: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium)
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 µg/ml trifluorothymidine (TFT) (Sigma)
Non-selective medium consisted of 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 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:
phenobarbital and ß-naphthoflavone induced rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Dose range finding test 3h: 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/mL without and with S9-mix.
Dose range finding test 24h: 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/mL without S9-mix.
Mutation experiment 1: With and without S9-mix: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 μg/mL (Experiment 1 without S9-mix rejected since the values of the absolute cloning efficiency in both solvent controls in the absence of S9-mix were not within the range of the acceptability criteria and several treatment groups showed unexpected low absolute cloning efficiency values)
Mutation experiment 1A: Without S9-mix: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 μg/mL.
Mutation experiment 2: Without S9-mix: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75 and 100 μg/mL (repeated since, due to a technical error, no acceptable cell counts in the solvent controls were obtained)

Since the test item was poorly soluble in the exposure medium, THF was selected as vehicle based on a solubility test in Charles River Laboratories Study No. 513621, and 50 µg/mL was selected as the maximum final concentration for the dose range finding test.
The highest tested concentration in the main mutation assay was selected based on the toxicity of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: The test item was dissolved in tetrahydrofuran (THF) based on the solubility finding in Charles River Laboratories Study No. 513621. Based on this solubility test, THF was selected as vehicle and 50 μg/ml 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; 29.4 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-mix; 7.5 μg/mL (3h treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 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:
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).
The highest concentrations were determined by the solubility of the test item in the culture medium since the test item was not severely toxic and difficult to dissolve in aqueous solutions.
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
Remarks:
No severe toxicity was observed up to and including the highest tested dose level. The relative total growth was not decreased compared to the relative total growth of the solvent control group up to and including the highest precipitating dose level
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:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
No severe toxicity was observed up to and including the highest tested dose level. The relative total growth of the highest test item concentration of 50 μg/mL was 51% compared to the relative 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: not measured
- Effects of osmolality: not measured
- Precipitation:
Dose range finding test 3h: at 25 and 50 µg/mL at the start of the treatment; no precipitation at the end of treatment
Dose range finding test 24h: at 25 and 50 µg/mL at the start of the treatment; no precipitation at the end of treatment
Mutation experiment 1: at 50 μg/mL and above
Mutation experiment 2: at 50 μg/mL and above

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 1.56 to 50 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
No toxicity in the suspension growth was observed up to and including the highest test item concentration of 50 μg/ml compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix. The relative suspension growth was 63% at the test item concentration of 50 μg/mL compared to the relative suspension growth of the solvent control. Based on the results of the dose range finding test, 200 and 100 µg/mL were selected as the highest test item concentration for the first mutation experiment (3h) and the second mutation experiment (24h), respectively.

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, except in the first experiment without S9-mix, in which the mutation frequency of the solvent control culture was above the upper control limit (166 per 10^6 survivors versus 95% upper control limit of 135 per 10^6 survivors). However, the observed mutation frequency of the solvent control culture was still within the range of the acceptability criteria of this assay.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with THF was between 7 and 14 (3 hour treatment) and 60 and 61 (24 hour treatment).
Remarks on result:
other: 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 this report.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-12 to 2016-06-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.49 (In vitro mammalian cell micronucleus test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: M14KB4863
- Expiration date of the lot/batch: 2017-11-10 (retest date)
- Purity test date: 2015-01-19

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No specific handling conditions required
- Solubility and stability of the test substance in the solvent/vehicle: Not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not available
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Remarks:
cultured peripheral human lymphocytes
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 35
- 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: 14.2h (age 33; dose-range finding test and first cytogenetic assay); 12.9h (age 35; 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-inactivated (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, 0.55, 1.7, 5.4, 17 and 52 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, 1.7, 5.4 and 17 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, 2, 5, 20, 30, 40, 50, 60 µg/mL without S9-mix (24h treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble at 320 mg/ml in culture medium, dimethyl sulfoxide, ethanol and acetone. In THF, the test item was dissolved at 320 mg/mL, resulting in a clear yellow solution after treatment with ultrasonic waves.
The test item precipitated in culture medium at the concentration of 3.4 mg/mL (= 17 μg/mL) and above. Based on these solubility findings, THF 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: Colchcine
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 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.
Reference: Fenech M. (1996) The cytokinesis-block micronucleus technique. In: GP Pfeifer, editor: Technologies for detection of DNA damage and mutations. New York: Plenum Press, 25 - 36.

%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).
Since the Fisher exact test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.
Species / strain:
lymphocytes: human
Remarks:
dose-range finding test; first cytogenetic assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no appropriate levels of cytotoxicity up to 52 µg/mL (dose-range finding test) or 17µg/mL (first cytogenetic assay)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Remarks:
second cytogenetic assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no appropriate levels of cytotoxicity up to 60 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not specified
- Precipitation: at 17 µg/mL and above (first cytogenetic assay; dose range finding test); at 20 µg/mL and above (second cytogenetic assay)

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was 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, 0.55, 1.7, 5.4, 17 and 52 µg/mL. The test item precipitated at concentrations of 17 μg/mL and upwards, and no cytotoxicity was observed. Therefore, 17 µg/mL was chosen as top dose for the first cytogenetic assay;

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
dose range finding test / first cytogenetic assay: Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei after a 3 hour treatment period
second cytogenetic assay: The test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the concentrations of 5 and 20 μg test item/ml culture medium. Also a statistically significant dose-related trend in the induction of micronuclei was observed. However since the number of micronuclei was within the 95% control limits of the historical control data these increases were not considered
is biologically relevant

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); no appropriate levels of cytotoxicity were observed
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 report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

A key study K1 (Matsumoto K, 2010) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). T003063 was tested in the Salmonella typhimurium reverse mutation assay with five histidine-requiring strains TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA.

The test item was dissolved in dimethyl sulfoxide.

In the dose-finding assay, the test item was tested at a concentration range of 1.2 to 5000 μg/plate, and a total of 7 doses were tested in the absence and presence of S9 -mix in all strains.

Based on the results of the dose-finding test, the test item was tested in the main mutation assay at a concentration range of 156 to 5000 µg/plate in the absence and presence of S9 -mix in in all strains.

In both the dose-finding and the main assay, the test substance did not increase the number of revertant colonies 2-fold or more compared to the negative control value both in the presence and absence of metabolic activation in any of the strains. The growth inhibition of bacteria by the test substance was not observed. The white precipitates of the test substance were observed at 20 μg/plate or more under the conditions of S9 (−) and at 78 μg/plate or more under the conditions of S9 (+).

Based on these results, it was concluded that the test substance was negative for mutagenicity under the conditions of this study.

A supporting study (K2, Sokolowski A, 2009) was performed according to the OECD Guideline 471 ( Bacterial Reverse Mutation Assay). T003063 was tested in the Bacterial reverse mutation assay in triplicate using three histidine-requiring Salmonella typhimurium strains TA98, TA100 and TA102 in the presence and absence of S9 -mix.

The test item wasdissolved in dimethyl sulfoxide.

In the dose-range finding test/Experiment I, the test item was tested at a concentration range of 3 to 5000 μg/plate in the absence and presence of S9 -mix in all strains. Based on the results of the dose-finding test, the test item was tested in Experiment II at a concentration range of 33 to 5000 µg/plate in the absence and presence of S9 -mix in in all strains.

Precipitation was observed in all tester strains at concentrations of 1000 µg/plate and above. Minor toxic effects were observed only in strain TA98. In none of the two mutation experiments did any of the S. typhimurium strains reveal any increase in mutation rate, either in the presence or absence of S9 -mix.

Based on these results, it was concluded that the test substance was negative for mutagenicity under the conditions of this study.

 

In vitro micronucleus assay

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 with a 3 hour exposure period and in the absence of S9-mix with a 3 and 24 hour exposure period, according to the OECD Guideline 487 (In vitro Mammalian cell micronucleus test) (key study, K1).

The test item was dissolved in tetrahydrofuran at a concentration of 320 mg/ml.

In the first cytogenetic assay, the test item was tested up to 17 μg/ml for a 3 hour exposure period with a 27 hour harvest time in the absence and presence of S9-mix. No appropriate levels of cytotoxicity were observed up to test item concentrations of 17 μg/ml compared to the solvent control. The test item precipitated in the culture medium at this dose level. All dose levels were selected for scoring of micronuclei in the first cytogenetic assay.

In the second cytogenetic assay, the test item was tested up to 20 μg/ml for a 24 hour exposure period with a 24 hour harvest time in the absence of S9-mix. No appropriate levels of cytotoxicity were observed up to test item concentrations of 60 μg/ml compared to the solvent control. The test item precipitated in the culture medium at the concentrations of 20 µg/ml and above. The following dose levels were selected for the scoring of micronuclei: 2, 5 and 20 µg test item/ml culture medium.

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.

The test item was not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in the report.

A supporting study (K2, Bohnenberg S., 2009) was performed according to a draft proposal for the OECD Guideline 487 (In vitro Mammalian cell micronucleus test). The test item was dissolved in tetrahydrofuran.

In the first cytogenetic assay, the test item was tested up to 2569 μg/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 at 80.3 µg/mL and above in the absence of S9 -mix and at 160.6 µg/mL in the presence of S9 -mix.

In the second cytogenetic assay, the test item was tested up to 2569 μg/mL for a 24 hour exposure period with a 24 hour harvest time in the absence of S9-mix and up to 642.3 µg/mL for a 24 hour exposure period with a 24 hour harvest time in the absence of S9 -mix. The test item precipitated at 80.3 µg/mL and above in the absence of S9 -mix and at 160.6 µg/mL in the presence of S9 -mix.

The test item was not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in the report.

 

In vitro gene mutation study in mammalian cells

Verspeek-Rip C (2017) 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 9-mix 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 tetrahydrofuran at a concentration of 40 mg/ml.

In the first mutation experiment, the test item was tested up to concentrations of 100 μg/ml in the absence and presence of S9-mix. The treatment period was 3 hours.

No toxicity was observed up to the concentration 100 μg/ml in the absence and presence of S9-mix. In the absence and presence of S9-mix, the relative total growth was not decreased compared to the relative total growth of the solvent control group up to and including the highest precipitating dose level. The test item precipitated in the culture medium concentrations of 50 μg/ml and above.

In the second mutation experiment, the test item was tested up to concentrations of 50 μg/ml in the absence of S9-mix. The treatment period was 24 hours. The Relative Total Growth was 51%. The test item precipitated in the culture medium at the concentration of 50 μg/ml.

In the absence of S9-mix, the test item did not induce 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 this report.

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T003063 and the criteria of the CLP Regulation (EC) 1272/2008, T003063 should not be classified for mutagenicity.