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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100 and TA1535, TA1537 and in Escherichia coli strain WP2 uvrA, performed according to OECD Guideline 471, it was concluded that T003421 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in the absence and in the presence of S9 -mix.

In vitro micronucleus assay

An in vitro micronucleus assay in cultured peripheral human lymphocytes performed according to OECD Guideline 487, it was concluded that T003421 was not clastogenic or aneugenic in human lymphocytes 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 T003421 is not mutagenic in the mouse lymphoma L5178Y test system in the absence and presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-29 to 2016-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: M15KB4494
- Expiration date of the lot/batch: 2016-11-13 (retest date)
- Purity test date: 2016-01-19

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 indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock formulations were treated with ultrasonic waves to obtain a homogenous suspension 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: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5%(v/v) S9-mix (top dose selected based on the solubility findings)
Mutation experiment I: 1.7, 5.4, 17, 52 and 164 μg/plate without S9-mix and 5.4, 17, 52, 164 and 512 μg/plate with 5%(v/v) S9-mix in TA1535, TA1537 and TA98 (top dose selected based on the dose range finding test results)
Mutation experiment II: 15, 27, 48, 86, 154 and 275 μg/plate without S9-mix and 48, 86, 154, 275, 492 and 878 μg/plate with 10%(v/v) S9-mix (top dose selected based on the dose range finding test and mutation experiment I results)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol.
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water or DMSO at 50 mg/ml. In ethanol the test item formed a white homogenous suspension at 50 mg/ml (sank slowly to the bottom). The test item was dissolved at 16 mg/ml in ethanol using ultrasonic waves. Based on these solubility findings, ethanol 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), 15 μg/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 ethanol and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

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

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

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

DETERMINATION OF CYTOTOXICITY
- Method: Reduction 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 above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:
Dose-range finding test (part of experiment I): at 164 μg/plate and upwards (start of incubation); at 52 μg/plate and upwards (TA100 without S9; end of incubation), at 164 μg/plate and upwards (TA100 and WP2uvrA with S9; end of incubation), and at 512 μg/plate and upwards (WP2uvrA with S9; end of incubation)
Mutation experiment I: at 512 µg/plate (start of incubation); at 164 and 512 µg/plate (end of incubation)
Mutation experiment II: at 275 µg/plate and upwards (start of incubation); at 154 µg/plate and upwards (without S9; end of incubation) and at 275 µg/plate and upwards (with S9; end of incubation)

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: strain-specific positive control values were within the laboratory historical control data ranges
- Negative (solvent/vehicle) historical control data: 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
- No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed under all conditions tested.

Mutation experiment II:

Toxicity:

In strain TA1537 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid-dose level of 27 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 an incidental fluctuation in the number of revertant colonies.

Mutagenicity:

In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix at the lowest dose of 15 μg/plate. However, since the increase was less than two-fold (a maximum of 1.1-fold was reached), this increase was not considered to be biologically relevant.

Conclusions:
Interpretation of results:
negative with and without metabolic activation
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2017-03-13
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: M16FB2273
- Expiration date of the lot/batch: 2017-06-14 (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 test item was suspended ethanol at a concentration of 40 mg/ml. At concentrations of 20 mg/ml and lower the test item was dissolved in ethanol. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test and first mutation experiment or until the test item had completely dissolved in the second mutation experiment.

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)
- 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): 6.25, 12.5, 25, 50, 100, 200 μg/mL without S9-mix; 5.8, 11.6, 23.1, 46.3, 92.3, 185.2 µg/mL with S9-mix;
Dose range finding test (24h treatment): 6.25, 12.5, 25, 50, 100, 200 μg/mL without S9-mix;
Mutagenicity assay I (3h treatment): 0.78, 1.57, 3.13, 6.25, 12.5, 25, 50, 100, 200 μg/mL with and without S9-mix;
Mutagenicity assay II (24h treatment): 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 μg/mL without S9-mix;

Top dose justification: Based on the solubility test, ethanol was selected as vehicle and 200 μg/mL 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) : ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed in Charles River Laboratories Study No. 513681: Based on this solubility test, ethanol was selected as vehicle and 200 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; at 15 µg/mL (3h treatment period), at 5 µg/mL (24h treatment period)
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 (3h treatment period)
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): 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):
0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

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: 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) (see calculations in section "any other information on materials and methods incl. tables")

Rationale for test conditions:

Since the test item was poorly soluble in the exposure medium, the highest tested concentration for dose range finding test was 200μg/mL exposure medium.
The highest concentration tested in the mutagenicity tests should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/mL or 0.01 M (whichever is the lowest).
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
No statistical analysis
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG was 32% at the highest concentration in the absence of S9-mix; no significant decrease in RTG in the presence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG was 10% at 50µg/mL. The dose levels of 60 to 90 µg/mL were too toxic.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Water solubility: no data
- Precipitation:
Dose range finding test (3h): at 100 and 200 μg/mL without S9-mix and at 92.3 and 185.2 μg/mL with S9-mix
Dose range finding test (24h): at 50 μg/mL and above without S9-mix
Mutagenicity test 1 : at 100 and 200 μg/mL with and without S9-mix
Mutagenicity test 2 : at 40 μg/mL without S9-mix

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with test item concentration ranged of 6.25 to 200 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and at 5.8 to 185.2 μg/ml in the presence of S9-mix with a 3 hour treatment period. After a 3h treatment period, no significant toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 185.2 in the absence of S9-mix and 200 μg/mL in the presence of S9-mix compared to the suspension growth of the solvent control. After a 24h treatment period, the relative suspension growth was 9% at the test item concentration of 200 μg/mL compared to the relative suspension growth of the solvent control. Based on the results of the dose range finding test, 200 µg/mL and 90 µg/mL were selected as the highest test item concentrations for the first mutation experiment (3h treatment period) and the second mutation experiment (24h treatment period), 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.
- 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 ethanol was 14 and 15 (3 hour treatment) and 61 and 71 (24 hour treatment).
Remarks on result:
other: Mutation experiment 1
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:
2016-07-29 to 2016-09-19
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: M15KB4494
- Expiration date of the lot/batch: 2016-11-13 (retest date)
- Purity test date: 2016-01-19

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 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 to obtain a homogeneous suspension.

Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Remarks:
cultured peripheral 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 21, 28 and 24
- 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: 13.0h (age 21; dose-range finding test and first cytogenetic assay); 12.9h (age 28; cytogenetic assay 1A); 12.9h (age 24; 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, 9.8, 19.5, 39.1, 78.1, 156.3 and 312.5 µg/mL without S9 (24h treatment); the highest tested concentration was determined by the solubility of the test item in the culture medium
Cytogenetic assay 1: 0, 39.1, 78.1 and 156.3 µg/mL with and without S9 (3h treatment); based on the solubility of the test item and on the result of the dose-range finding test
Cytogenetic assay 1A: 0, 19.5, 39.1 and 78.1 µg/mL with and without S9 (3h treatment); due to precipitation at 78.1 µg/mL and above in cytogenetic assay 1, this experiment was repeated with lower test concentrations
Cytogenetic assay 2: 0, 5, 10, 20, 30, 50 and 75 µg/mL without S9 (24h treatment);
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in culture medium (test item floated on the solvent) and dimethyl sulfoxide (non-homogeneous suspension with big lumps) at concentrations of 125 mg/ml. In ethanol a homogeneous suspension was formed at a concentration of 125 mg/ml and clear solutions were obtained at a concentration of 15.63 mg/ml and below. The test item precipitated in the culture medium at 15.63 mg/ml (= 156.3 μg/ml) and above. Based on these solubility findings, ethanol 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 re-suspended 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 (cytogenetic assay 1/1A) or 24h (dose range finding test/cytogenetic assay 2)
- Expression time (cells in growth medium): 24h (cytogenetic assay 1/1A) or 0h (dose range find test/cytogenetic assay 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 27h (cytogenetic assay 1/1A) or 24h (dose range find test/cytogenetic assay 2)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (positive control chemical in the cytogenetic assays 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 2016 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 specified
- Precipitation:
Dose range finding test: at 39.1 µg/mL and above
Cytogenetic assay 1/1A: at 78.1 µg/mL and above
Cytogenetic assay 2: at 50 µg/mL and above

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, 9.8, 19.5, 39.1, 78.1, 156.3 and 312.5 µg/mL. The test item precipitated at concentrations of 39.1 μg/mL and upwards, and no cytotoxicity was observed. 156.3 μg/mL was chosen as top dose for cytogenetic assay 1.

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 in the first cytogentic assay. 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.
- 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, except for the number of mononucleated cells with micronuclei in one of the solvent controls at the 3 hour exposure period in the presence of S9-mix. Since the number of mononucleated cells with micronuclei (4 cells) was slightly above the upper 95% control limit (3.04 cells), the number of binucleated cells with micronuclei was within the 95% control limits and the number of mono- and binucleated cells with micronuclei in the other solvent control was within the 95% control limits, this observation was considered to have no effect on the study results.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CPBI index (cytokinesis-block proliferation index); no appropriate levels of cytotoxicity were observed

In cytogenetic assay 1, the test item precipitated in the culture medium at the concentrations of 78.1 μg/ml and above, in particular 2 out of the 3 concentrations showed precipitate.

Therefore the experiment was repeated with concentrations of 19.5, 39.1 and 78.1 μg test item/ml culture medium with and without S9-mix (cytogenetic assay 1A with a 3 hour exposure to the test item).

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 of this test.
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 (Verspeek R, 2016) was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay). T003421 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA.

The test item was suspended in ethanol at a concentration of 50 mg/ml. At concentrations of 16 mg/ml and lower the test item was dissolved in ethanol.

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 was tested up to or beyond a precipitating dose level. 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.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay in the tester strains TA1535, TA1537 and TA98 at the concentration ranges of 1.7 to 164 μg/plate and 5.4 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix, respectively. The test item precipitated on the plates at the highest tested concentrations. 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.

In a second mutation assay, the test item was tested at the concentration ranges of 15 to 275 μg/plate and 48 to 878 μg/plate in the absence and presence of 10% (v/v) S9-mix, respectively, in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 154 μg/plate and upwards and at 275 μg/plate and upwards in the absence and presence of S9-mix, respectively. 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 biologically relevant and/or 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-mix in any of the experiments. It was concluded that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

A supporting in vitro gene mutation study in bacteria (K2, Preston, 2012) was performed according to OECD Guideline 471. T003421 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA, in the presence and absence of S9 metabolic activation. The following dose levels were tested: 1, 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 μg/plate. The plate incorporation test was used. All doses were evaluated in triplicate plates. Indications of toxicity (reduced bacterial colony counts, sparse lawns (reduced lawns), or absent bacterial lawns) were not observed. Precipitation was observed in top agar at > 250 μg/plate. Precipitation also obscured colony counting at > 500 μg/plate. Revertant frequencies for all doses of T003421, 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.

It was concluded that T003421 was negative in the in vitro bacterial reverse mutation assay under the conditions, and according to the criteria, of the test protocol.

In vitro micronucleus assay

Eurlings (2016) 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 experiment was performed according to the OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test).

The test item was soluble in ethanol at a concentration of 15.6 mg/ml but formed a suspension at concentrations of 31.3 mg/ml and above.

In the first cytogenetic assay, the test item was tested up to 78.1 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 in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 50 μg/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 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.

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 the report.

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 mononucleated 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9 - mix) functioned properly.

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 Test using the Thymidine Kinase Gene), Verspeek-Rip C (2017) investigated the effects of JNJ-42808389-AAA (T003421) 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.

The test item was suspended in ethanol at a concentration of 40 mg/ml. At concentrations of 20 mg/ml and lower the test item was dissolved in ethanol.

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. The relative total growth (RTG) was 32% at the concentration of 100 μg/ml in the absence of S9-mix. No significant toxicity was observed up to the concentration 100 μg/ml in the presence of S9-mix. The test item precipitated in the culture medium at the concentration of 100 μg/ml.

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 (RTG) was 10% at the concentration of 50 μg/ml. The test item precipitated in the culture medium at the concentrations of 40 and 50 μg/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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

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

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T003421 and the criteria of the CLP Regulation (EC) 1272/2008, T003421 should not be classified for mutagenicity.