Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria 

A K1 GLP-compliant bacterial reverse mutation assay was performed according to OECD Guideline 471 and EU Method B.13/14 in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli strain WP2 uvrA (Verspeek-Rip, 2016). T000625 was concluded to be non-mutagenic in the presence or absence of metabolic activation.

Chromosome aberration test: 

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to OECD Guideline 473, T000625 did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after 4(20) hour exposures or 24 hour continuous exposure (Wright, 2004). The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Gene mutation in mammalian cells: 

In a K1 mouse lymphoma assay, performed according to the OECD Guideline 490, it was concluded that T000625 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report (Verspeek-Rip, 2017).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 meta phase spreads scored per concentration.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Description : White crystalline solid
Purity : 100%
Label : T 625 CODE NO. 002844
Date received : 2003-12-22
Storage conditions : Room temperature in the dark
Species / strain / cell type:
other: human lymphocytes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver homogenate metabolising system (S9)
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 11.72, 23.44, 46.88, 93.75, 187.5, 375, 750, 1500 and 3000 μg/mL
The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test:
- Group 1 (4-hour without S9 with 20 hour expression time): 0, 11.72, 23.44, 46.88, 93.75, 140.63, and 187.5 μg/mL (0, 46.88, 93.75 and 140.63 μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 23.44, 46.88, 93.75, 140.63, 187.5, and 281.25 μg/mL (0, 46.88, 93.75 and 140.63 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 5.86, 11.72, 23.44, 46.88, 70.32, and 93.75 μg/mL (0, 23.44, 46.88 and 70.32 μg/mL were selected for metaphase analysis)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; at 0.4 μg/mL for the 4(20) hour exposure and 0.2 μg/mL for the 24 hour continuous exposure (Groups 1 and 3 respectively)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with activation; at 10 µg/mL for the 4(20) hour exposure (Group 2)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
24-hour exposure without S9-mix, 0.2 µg/mL MMC
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: 20 hours (Groups 1 and 2) was provided in the study report as the expression period. However, no information was given on the time of addition of a spindle inhibitor ; 0 hours (Group 3)
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: 2 (only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations unless there was a need to clarify an equivocal response)

NUMBER OF CELLS EVALUATED: 100 cells per evaluated culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes



Rationale for test conditions:
No data
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increase in aberrations frequency a dose response relationship was generally required and appropriate statistical tests may have been applied in order to record a positive response.
Statistics:
No data, but statistical analysis was performed.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the dose range-finding experiment a precipitate of test material was observed at and above 93.75 μg/ml

RANGE-FINDING/SCREENING STUDIES: Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 750 μg/ml in the pulse exposure groups and 46.88 μg/ml in the continuous exposure group. In the 4(20) hours pulse exposure groups the data show dose-related test material-induced toxicity in both exposure groups with an approximate 50% growth inhibition at 93.75 and 187.5 μg/ml in the without and with-S9 exposure groups, respectively. In 24 hour exposure group there was a steep toxicity curve and no reduction in MI was observed at 46.88 μg/ml but no metaphases at 93.75 μg/ml. Therefore, the selection of the dose range for the chromosome aberration test was based on toxicity.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY AND INDUCTION OF CHROMOSOME ABERRATIONS: A microscopic assessment of the slides showed that adequate scorable metaphase cells were present at up to 140.63 μg/ml in the 4(20)-hour exposure group without S9 and at up to the maximum dose level tested, 281.25 μg/ml, in the with-S9 group. In the 24-hour continuous exposure group metaphases were present at up to 93.75 μg/ml. The test material induced the ideal level of toxicity of approximately 50% mitotic inhibition at 140.63 μg/ml, in both of the 4-hour pulse exposure groups. In the 24-hour exposure group without S9 there was a steep toxicity curve with approximately 50% mitotic inhibition seen at 46.88 μg/ml. Dose selection for metaphase analysis was based on toxicity for all exposure groups.
Remarks on result:
other: other: Group 1 and 2 (4 hour exposure groups)
Conclusions:
Interpretation of results:
negative

The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after 4(20) hour exposures or 24 hour continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-05 to 2017-02-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mouse lymphoma assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16AB0081
- Expiration date of the lot/batch: 2018-01-11
- Purity test date: 2016-02-18

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

OTHER SPECIFICS: correction factor 1.00
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Cell cycle length, doubling time or proliferation index: not indicated
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10^6 cells/ml.
- 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 containing penicillin/streptomycin (50U/mL and 50μg/mL, respectively), 1mM sodium pyruvate and 2mM L-glutamin
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium)
Exposure medium 3h: basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5- medium)
Exposure medium 24h: basic medium supplemented with 10% (v/v) heat inactivated horse serum ( R10-medium)
Selective medium: basic medium supplemented with 20% (v/v) heat inactivated horse serum (R20- medium) and 5 μg/ml trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat inactivated horse serum (R20-medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ßnaphthoflavone)
Test concentrations with justification for top dose:
Dose range finding test (3h): 0, 7.8, 15.6, 31.3, 62.5, 125 μg/mL without and with S9-mix.
Dose range finding test (24h): 0, 7.8, 15.6, 31.3, 62.5, 125 μg/mL without S9-mix.
Mutation experiment 1 (3h) (rejected: suspension growth over two days was below the acceptability criteria of 8): 0, 0.32, 0.63, 1.25, 5, 10, 20, 30, 40, 50 and 60 μg/mL without S9-mix; 0, 1.25, 5, 10, 20, 30, 45, 62.5, 80, 100 and 125 μg/mL with S9-mix;
Mutation experiment 1A (3h): 0, 0.63, 1.25, 5, 10, 20, 30, 35, 40, 45, 50, 55 and 60 μg/mL without S9-mix (rejected. no appropriate survival was observed at the positive control group); 0, 1.25, 5, 10, 20, 30, 45, 60, 80, 100 and 125 μg/mL with S9-mix (rejected: no appropriate levels of toxicity were obtained for the determination of the mutation frequency);
Mutation experiment 1B (3h): 0, 1.25, 2.5, 5, 10, 20, 40, 50, 60, 70, 80, 90 and 125 μg/mL without S9-mix; 0, 1.25, 5, 10, 20, 30, 45, 60, 80, 100 and 125 μg/mL with S9-mix;
Mutation experiment 2 (24h): 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 85, 100 and 125 μg/mL without S9-mix

Since the test item was poorly soluble in the exposure medium (precipitated directly at the concentration of 6.25 mg/ml (= 62.5 μg/ml) and above, and at 12.5 mg/mL (= 125 µg/mL) and above after 3h), the highest tested concentration was 125 μg/ml exposure medium in the dose range finding test.
The highest tested concentration in the main mutation experiment was selected based on the toxicity of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was dissolved in dimethyl sulfoxide (DMSO) based on the solubility finding in Charles River Laboratories Study No. 513695.
The test item was soluble in DMSO at 50 mg/mL. Upon mixing with exposure medium the test item precipitated directly at the concentration of 6.25 mg/mL (= 62.5 μg/mL) and above. After 3 hours, precipitation was only observed at 12.5 mg/mL (= 125 μg/mL) and above. Based on these solubility findings, 125 μg/mL was selected as the maximum final concentration for the dose range finding test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; 15 μg/mL (3h treatment), 5 μg/mL (24h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9; 7.5 μg/mL (3h treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): - Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used.

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 48h (3h and 24h treatment)
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays): After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well.

NUMBER OF REPLICATIONS:
Determination of cloning efficiency: 2 x 96-well microtiter plates/concentration
Determination of mutation frequency: 5 x 96-well microtiter plates/concentration (solvent controls and treatment groups); 10 x 96-well microtiter plates/concentration (positive controls)


NUMBER OF CELLS EVALUATED:
Determination of cloning efficiency (CEday2): One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
Determination of mutation frequency: 9.6 x 10^5 cells/concentration plated (5 x 96-well microtiter plates/concentration, each well containing 2000 cells in selective medium (solvent controls and treatment groups)); 9.6 x 10^5 cells/concentration plated (10 x 96-well microtiter plates/concentration), each well containing 1000 cells in selective medium (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Rationale for test conditions:
The highest concentration tested in the mutagenicity tests should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/ml or 0.01 M (whichever is the lowest).
Solubility limitations: A solubility test was performed in Charles River Laboratories Study No. 513695. Based on this solubility test, DMSO was selected as vehicle. Since the test item was poorly soluble and precipitated upon mixing with exposure medium, the highest tested concentration was 125 μg/ml exposure medium.
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:
70 to 125 μg/mL were too toxic for further testing and were not used for MF measurement (without S9);
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:
70 to 125 μg/mL were too toxic for further testing and were not used for MF measurement
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: not indicated
- Precipitation:
Dose range finding test 3h: at 125μg/mL with and without S9-mix
Dose range finding test 24h: no precipitation observed
Mutation experiment 1: at 80 μg/mL and above with and without S9-mix
Mutation experiment 2: at 125 μg/mL at the start of the incubation, no precipitation observed at the end of 24h treatment.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 7.8 to 125 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. In the absence of S9-mix, no cell survival was observed at 62.5 and 125 µg/mL (3h treatment) and the RTG was 3% at 125 µg/mL (24h treatment). In the presence of S9-mix, the RTG was 14% at 125 µg/mL compared to the RTG of the solvent control.
Based on the results of the dose range finding test, 125 µg/mL was selected as the highest test item concentration for the first (3h) and second (24h) mutation experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database, except in the first experiment in the absence of S9-mix in which the mutation frequency of one of the solvent control cultures was just below the acceptability criteria range of this assay. Since the mutation frequency was just below the lower limit of the range and the mutation frequency of the other solvent control culture was within the range, this deviation in the mutation frequency had no effect on the results of the study.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 13 and 15 (3 hour treatment) and 54 and 59 (24 hour treatment).
Remarks on result:
other: Mutation experiment 1
Remarks:
(3 h treatment)
Conclusions:
Interpretation of results: negative with and without metabolic activation
In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-30 to 2016-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16AB0081
- Expiration date of the lot/batch: 2018-01-11 (retest date)
- Purity test date: 2016-02-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions : Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated
Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose-range finding test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (top dose selected based on the solubility findings)
Mutation experiment I: 0, 52, 164, 512, 1600 and 5000 μg/plate (top dose selected based on the dose range finding test results)
Mutation experiment II: 0, 275, 492, 878, 1568, 2800 and 5000 μg/plate (top dose selected based on the dose range finding test and mutation experiment I results)



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml (the test item floated on the water). In DMSO, the test item was dissolved at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9; 5μg/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; 2.5μg/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; 10μg/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; 650μg/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; 10μg/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; 2.5 µg/plate (TA1535 with 5 and 10% S9, TA1537 with 5% S9), 5µg/plate (TA1537 with 10% S9), 1µg/plate (TA98 with 5 and 10% S9, TA100 with 5% S9), 2 µg/plate (TA100 with 10% S9), 15 µg/plate (WP2uvrA with 5 and 10% S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: ; in agar (plate incorporation)

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

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

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

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

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
Species / strain:
S. typhimurium, other: TA1535 and TA100
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:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity at 5000 µg/plate with and without S9 in mutation experiment I; no cytotoxicity observed in mutation experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed at 5000 µg/plate in mutation experiment I without S9; no cytotoxicity observed in mutation experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml (the test item floated on the water).
- Precipitation:
Dose range finding test: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period
First mutation experiment: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 and 5000 μg/plate.
Second mutation experiment:Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1568 μg/plate and upwards and at 2800 and 5000 μg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: in the dose-range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. The dose range finding test results are reported as a part of mutation experiment I.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
- Other observations when applicable: cytotoxicity (decrease in number of revertant colonies) was only observed in tester strains TA1537 (with and without S9) and TA98 (without S9) at 5000 μg/plate (top dose) in the first mutation experiment

Mutation experiment I:

Cytotoxicity: In strains TA1537 and TA98 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are caused by incidental fluctuations in the number of revertant colonies and are considered as not biologically relevant.

Mutagenicity: In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the presence of S9-mix at the mid-dose level of 164 μg/plate. However, since the increase was less than two-fold (a maximum of 1.3-fold was reached), this increase was not considered to be biologically relevant.

Mutation experiment II:

Cytotoxicity: In strains TA1537 and TA98 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are caused by incidental fluctuations in the number of revertant colonies and are considered as not biologically relevant.

Mutagenicity: In strain TA100, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the presence of S9-mix. However, since the increases were less than two-fold (a maximum of 1.8-fold was reached) and no dose-relationship was observed, these increases were not considered to be biologically relevant.

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

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No study is available. 

 

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

A key study bacterial reverse mutation assay (Verspeek-Rip C, 2016) was performed according to OECD Guideline 471 and EU Method B.13/14. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia. coli strain WP2 uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor 1254 induced rat liver metabolic activation system).

The dose range finding test, showed the test item precipitated at the top dose of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertant was observed.

During the first mutation assay, the concentrations ranging from 52 to 5000 μg/plate, in the absence and presence of 5% (v/v) S9-mix, were tested with TA1535, TA1537 and TA98.  T000625 precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Also, cytotoxicity, was observed in tester strains TA1537 in the absence and presence of S9-mix and TA98 in the absence of S9-mix at the highest dose level tested.

In a second mutation assay, the test item was tested at a concentration range of 275 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 2800 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertant 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).

In a supporting, K2 study performed similar to OECD guideline 471, the test substance failed to increase reversion rates in TA98 and TA100 in the presence and absence of metabolic activation up to a concentration of 1000 µg/plate, and therefore considered negative for mutagenic potential under the described test conditions of the Ames Salmonella/Microsomal Activation Assay (Vanparys, 1986).

Chromosome aberration 

A key study on genotoxicity - chromosomes aberration in mammalian cells (Wright N., 2004) was performed according to guidelines equivalent to OECD Guideline 473.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material. Three exposure groups were used in this study: 

- 4(20) hour exposure in absence of metabolic activation (S9)

- 4(20) hour exposure in presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 hour expression period. 

- 24 hour continuous exposure in absence of S9

A Preliminary Toxicity Test was set the concentration range for the chromosome aberration test at a dose range of  11.72 to 3000 μg/ml, based on a 10mM maximum dose level. 

The chromosome aberration test showed that The test material did not induce statistically significant increases in the frequency of cells with aberrations at any dose level in any of the three exposure conditions. A microscopic assessment of the slides showed that adequate scorable metaphase cells were present at up to 140.63 μg/ml in the 4(20)-hour exposure group without S9 and at up to the maximum dose level tested, 281.25 μg/ml, in the with-S9 group. In the 24-hour continuous exposure group metaphases were present at up to 93.75 μg/ml. The test material induced the ideal level of toxicity of approximately 50% mitotic inhibition at 140.63 μg/ml, in both of the 4-hour pulse exposure groups. In the 24-hour exposure group without S9 there was a steep toxicity curve with approximately 50% mitotic inhibition seen at 46.88 μg/ml. The test material did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups either. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Gene mutation in mammalian cells 

A key study on mammalian cells Gene mutation (Verspeek-Rip C, 2017) was performed according to OECD Guideline 490. 

In this study, T000625 was dissolved in dimethyl sulfoxide at a concentration of 12.5 mg/ml.

In the first mutation experiment, the test item was tested up to concentrations of 60 and 125 μg/ml in the absence and presence of S9-mix, respectively. The treatment period was 3 hours. The relative total growth (RTG) was 4% at the concentration of 60 μg/ml in the absence of S9 and 49% at the concentration of 125 μg/ml presence of S9-mix. The test item precipitated in the culture medium at concentrations of 80 μg/ml and above.

In the second mutation experiment, the test item was tested up to concentrations of 60 μg/ml in the absence of S9-mix. The treatment period was 24 hours. The relative total growth (RTG) was 13% at the concentration of 60 μg/ml. No precipitation was observed up to the concentration of 60 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. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

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