Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial reverse mutation assay

In a K2 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100 and TA102, performed according to a method similar to OECD Guideline 471, it was concluded that T001159 has no mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of S9-mix under the test conditions described in the report, up to the maximum test concentration of 5000 μg/plate. An expert statement was also added to justify the fact that no further testing is necessary.

 

- Chromosome aberration study

In a K1 in vitro chromosome aberration study in human lymphocytes, performed according to the OECD Guideline 473, T001159 was considered to be non-clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.

 

- In vitro gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T001159 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 cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-18 to 2016-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian chrommosome aberration test
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15LB5093
- Expiration date of the lot/batch: 2016-11-30 (retest date)
- Purity test date: 2016-03-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: In DMSO, the test item was dissolved at concentrations of 65 mg/ml and below but formed a suspension at concentrations of 160 mg/ml and above.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock solutions used for the dose range finding study and first cytogenetic assays were treated with ultrasonic waves until the test item had completely dissolved or to obtain a homogeneous suspension.

OTHER SPECIFICS: correction factor: 1.00
Target gene:
not applicable
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age):
Dose range finding study: age 30, AGT = 12.9 h
First cytogenetic assay: age 33, AGT = 12.7 h
Cytogenetic assay 1A: age 30, AGT = 13.8 h
Second cytogenetic assay: age 35, AGT = 12.9 h
Cytogenetic assay 2A: age 33, AGT = 14.2 h
Cytogenetic assay 2B: age 26, AGT = 12.8 h
Cytogenetic assay 2C: age 30, AGT = 12.9 h
AGT = Average Generation Time of the cells
- Suitability of cells: Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).
- Sex, age and number of blood donors if applicable: see in "Source of cells"
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: Immediately after blood collection lymphocyte cultures were started. 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). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.

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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone induced rat liver homogenate (S9-mix)
Test concentrations with justification for top dose:
Dose range finding test (3h exposure): 52, 164, 512, 1600 and 2000 µg/mL with and without 1.8% S9-mix
Dose range finding test (24h/48h continuous exposure): 52, 164, 512, 1600 and 2000 µg/mL without S9-mix

Cytogenetic assay 1 (3 h exposure time, 24 h fixation time): 50, 150, 500, 600, 700, 800, 900 and 1000 µg/mL without S9-mix; 145, 579, 772, 965, 1061, 1254 and 1447 µg/mL with 1.8% S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 1A (3 h exposure time, 24 h fixation time): 150, 500, 525, 550, 575, 600 and 650 µg/mL without S9-mix; 150, 500, 525, 550, 575, 600, 625 and 650 µg/mL with 1.8% S9-mix
The following dose levels were selected for scoring of chromosome aberrations, with and without S9-mix: 150, 575 and 650 µg/ml culture medium

Cytogenetic assay 2 (24 h continuous exposure, 24 h fixation time): 10, 25, 50, 75, 100, 125 and 150 µg/mL without S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 2A (24 h continuous exposure, 24 h fixation time): 1, 5, 10, 15, 20, 30, 40, 50 and 75 µg/mL without S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 2B (24 h continuous exposure, 24 h fixation time): 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL without S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 2C (24 h continuous exposure, 24 h fixation time): 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 µg/mL without S9-mix;
The following doses were selected for scoring of chromosome aberrations: 40, 160 and 180 µg/ml culture medium.

Since the test item precipitated in culture medium at 160 mg/mL (= 1600 µg/mL) and above in the solubility test, 2000 µg/mL was selected as maximum concentration for the dose range finding test.
The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in culture medium and ethanol. In DMSO, the test item was soluble up to the concentration of 65 mg/mL, and after treatment with ultrasonic waves up to the concentration of 160 mg/mL. Based on the solubility findings, DMSO was selected as vehicle.
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix; 0.5 and 0.75 µg/mL (3h), 0.2 and 0.3 µg/mL (24h)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix; 10 µg/mL (3h)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Cytogenetic assay 1A: 3h; Cytogenetic assay 2C: 24h continuous exposure
- Expression time (cells in growth medium): Cytogenetic assay 1A: 20-22h; Cytogenetic assay 2C: 0h.
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

SPINDLE INHIBITOR (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck):acetic acid (Merck) fixative (3:1 v/v).

STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8

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.

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150 metaphase chromosome spreads per culture were examind by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Rationale for test conditions:
The highest concentration examined for chromosome aberrations should be cultures that produce an inhibition of the mitotic index of 55 ± 5 %, whereas the mitotic index of the lowest concentration should have little or no cytotoxicity (approximately the same as vehicle control). Also cultures treated with an intermediate concentration should be examined.
For poorly soluble test items, the highest concentration analysed should be the lowest insoluble concentration (determined at the end of treatment) irrespective of toxicity. The extent of precipitation may not interfere with the scoring of chromosome aberrations.
If no precipitate or limiting toxicity is observed, the highest concentration examined should correspond to 2 mg/ml or 0.01 M, whichever is the lowest.
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) The increase is dose related 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) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s 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:
Graphpad Prism version 4.03 and ToxRat Professional v 3.2.1 were used for statistical analysis of the data.
Since the Fisher’s exact test shows that there are statistically significant differences between one or more of the test item groups and the concurrent 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:
Cytogenetic Assay 1A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
appropriate toxicity was reached at 650 µg/mL (mitotic index of 44%)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
lymphocytes: human
Remarks:
Cytogenetic Assay 2C
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Appropriate toxicity was reached at 180 µg/mL (mitotic index of 39%)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH and osmolality were observed in culture medium containing the highest, non-precipitating test item concentration of 512 μg/ml (pH 7.827) compared to the concurrent vehicle control (pH 7.866)
- Effects of osmolality: No marked changes in osmolality were observed in culture medium containing the highest, non-precipitating test item concentration of 512 μg/ml (424 mOsm/kg) compared to the concurrent vehicle control (432 mOsm/kg)
- Water solubility: no data
- Precipitation:
Dose range finding test (3h and 24h exposure): at 1600 µg/mL and above
Cytogenetic Assay 1A: No test item precipitation up to the highest tested concentration.
Cytogenetic Assay 2C: No test item precipitation up to the highest tested concentration.
- Definition of acceptable cells for analysis: Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with structural aberrations and the number of structural aberrations were recorded. Gaps were also recorded and reported separately, but not included in the total aberration frequency.

RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Lymphocytes were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3h, 24h and 48h in the absence of S9-mix or for 3h in the presence of 1.8% (v/v) S9-mix. For the 3 hour exposure time, blood cultures were treated with 52, 164, 512, 1600 and 2000 μg test item/ml culture medium with and without S9-mix. For the 24 and 48 hour exposure time, blood cultures were treated with 52, 164, 512, 1600 and 2000 μg test item/ml culture medium without S9-mix. A complete cell lysis was observed at 1600 μg/ml and above for all the exposed groups. Based on the results of the dose range finding test, the following dose levels were selected for the Cytogenetic Assay 1A: 50, 150, 500, 600, 700, 800, 900 and 1000 μg/ml culture medium without S9-mix and 145, 579, 772, 965, 1061, 1254 and 1447 μg/ml culture medium with S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. The number of cells with chromosome aberrations 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 cells with chromosome aberrations found in the vehicle control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the vehicle control cultures was within the 95% control limits of the distribution of the historical vehicle control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

Cytogenetic Assay 1A:

In the absence of S9-mix, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest concentration only, both when gaps were included and excluded. The increase was considered not biologically relevant since the sum of the number of cells with chromosome aberrations in the duplicate cultures was within the historical vehicle control data range.

In the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Cytogenetic Assay 2C:

At the 24 h continuous exposure time, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest concentration only, both when gaps were included and excluded. The increase was considered not biologically relevant since the increase could not be confirmed by 2 independent scorers.

It was noted that the test item increased the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix. This may indicate that the test item has the potential to disturb mitotic processes and cell cycle progression.

Conclusions:
It is concluded that this test is valid and that the test item is not clastogenic in human lymphocytes 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:
2005-03-30 to 2005-05-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 3 bacterial strains used, no repeat experiment and limited details on methodology were provided.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
Description: Pale beige powder
Batch number: 00456784 RT001159G3BA11
Purity: ca. 100 %
Date received: 2005-03-18
Storage conditions: Room temperature in the dark
Target gene:
histidine locus
Species / strain:
other: Salmonella typhimurium TA100, TA98 and TA102
Details on mammalian cell lines (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, rat liver homogenate metabolizing system (10% in standard co-factors)
Test concentrations with justification for top dose:
0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9: 5 μg/plate for TA98
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1-8-Dihydroxyanthroquinone
Remarks:
With S9: 10 μg/plate for TA102
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: 1 μg/plate for TA100
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9: 0.2 μg/plate for TA98
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9: 0.5 μg/plate for TA102
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9: 3 μg/plate for TA100
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix of phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of the test material both with and without S9-mix.

DURATION
- Exposure duration: 3 days

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
other: Salmonella typhimurium TA100, TA102, and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control substances used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated. The S9 mix used in both experiments was shown to be sterile. Results for the negative controls were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to the maximum recommended dose level of 5000 µg/plate.
Conclusions:
Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for mutagenic potential in the Ames Test using S. typhimurium strains TA98, TA100 and TA102 in the presence and absence of metabolic activation. The test substance was considered to be non-mutagenic under the conditions of the test in all tester strains either in the presence or absence of metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-17 to 2017-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15LB5093
- Expiration date of the lot/batch: 2017-03-07 (retest date)
- Purity test date: 2016-12-19

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was suspended or dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test or until the test item had completely dissolved in the mutagenicity tests.


OTHER SPECIFICS: correction factor 1.00
Target gene:
TK locus
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (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 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, 47, 94, 188, 375, 750, 1500 μg/ml without and with S9-mix.
Dose range finding test 24h: 0, 47, 94, 188, 375, 750, 1500 μg/ml without S9-mix.

Mutation experiment 1: 0, 10.9, 21.9, 43.8, 87.5, 175, 200, 225, 250, 275, 300, 325 and 350 μg/ml without and with S9-mix (rejected: no appropriate levels of toxicity were obtained for the determination of the mutation frequency)
Mutation experiment 1 A: 0, 50, 100, 200, 225, 250, 265, 280, 300, 315, 330 and 345 μg/ml with S9-mix
Mutation experiment 2: 0, 3.13, 6.25, 12.5, 25, 50, 100, 125, 150, 175, 200, 225 and 250 without S9-mix.

Since the test item was poorly soluble in the exposure medium, the highest tested concentration for the dose range finding test was 1500 μg/ml exposure medium.
The highest tested concentrations for the mutation experiments were selected based on the cytotoxicity observed in the dose range finding tests.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on these solubility findings, DMSO was selected as vehicle and 1500 μg/ml was selected as the maximum final concentration for the dose range finding test.
Negative controls:
yes
Solvent 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)
Negative controls:
yes
Solvent 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 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: 3 h or 24 h
- 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:
Solubility limitations: No solubility test was performed in water/exposure medium based on the information provided by the sponsor. Since the test item was poorly soluble and precipitated upon mixing with exposure medium, the highest tested concentration in the dose range finding tests was 1500 μg/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:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: 3 h treatment
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: 24 h treatment
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes in pH were observed in culture medium containing the highest, non-precipitating concentration of 512 µg/mL: 7.827 compared to the concurrent solvent control 7.866
- Effects of osmolality: no marked changes in osmolality were observed in culture medium containing the highest, non-precipitating concentration of 512 µg/mL: 0.424 Osm/kg compared to the concurrent solvent control 0.432 Osm/kg
- Water solubility: No solubility test was performed in water based on the information provided by the sponsor
- Precipitation: Dose range finding test 3h: at 1500 μg/mL with and without S9-mix
Dose range finding test 24h: at 1500 μg/mL with and without S9-mix
Mutation experiment 1A: No precipitation up to the highest concentration tests (345 µg/mL)
Mutation experiment 2A: at 250 μg/mL without S9-mix

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 47 to 1500 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.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1500 μg/mL compared to the suspension growth of the solvent control.

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 without S9-mix, in which the mutation frequency of the solvent control culture was just above the upper control limit (150 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 DMSO was between 9 and 15 (3 hour treatment) and 61 and 65 (24 hour treatment)
Conclusions:
Interpretation of results: negative with and without metabolic activation
In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.
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 (K2, Bowles, 2005) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). Salmonella typhimurium strains TA98, TA100 and TA102 were treated with solutions of the test item using the Ames plate incorporation method at 9 dose levels, in triplicate, both with and without the addition of 10% liver S9 in standard co-factors. The dose range was 0.5 to 5000 µg/plate in the main experiment.

The vehicle (dimethyl sulfoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control substances used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated. Results for the negative controls were considered to be acceptable.

The test substance caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to the maximum recommended dose level of 5000 µg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Expert statement

According to Annex VII, section 8.4, in vitro gene mutation study using bacteria (Ames test) is a standard information requirement at the present tonnage level. The registrant is aware of the version of the EU Test Method B.13/14/OECD TG 471 in force since 1997 which indicates that at least five strains of bacteria should be tested. The Ames test, currently included in the dossier, is performed following OECD 471 but included testing on three S. typhimurium strains (TA 98, TA 100, TA 102) only.

The selection of these three strains is based on internal historical data gathered during more than 25 years of testing. This is not a specific observation but is widely accepted in pharmaceutical companies. In general, most compound screening strategies are based on this limited set of 2 strains (TA 98 and TA 100). Moreover, the introduction of plasmid pKM101 in strains TA1535 and TA1538 resulted in the corresponding isogenic strains TA100 and TA98. Plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone recombinational DNA repair pathway. Thus TA100 and TA98 are believed to be more sensitive than their plasmid-free counterparts. Therefore the number of compounds that is exclusively positive in TA1535 and/or TA1537 (the 2 strains that are not tested) is extremely small (less than 2.3% or 90 on 3083 compounds tested).  The relationship and historical aspects are clearly described in Mortelmans et al, Mutation Research 455 (2000). We therefore are convinced that the proposed 3 strains are sufficient for registration of intermediates in the production of active pharmaceutical ingredients. It should be noted however that, as TA 102 is also tested, the potential to detect certain specific types of mutagens has been elaborated. This mutation is also reverted by mutagens that cause oxidative damage. In addition, this DNA repair proficient strain TA 102 detects cross-linking agents such as bleomycin and mitomycin C. Therefore, no additional test is performed to evaluate five strains.  

 

Chromosome aberration:

A key study (K1, Eurlings, 2017) performed an in vitro chromosome aberration study in human lymphocytes according to OECD Guideline 473.

The test item, dissolved in dimethyl sulfoxide, was assessed for its potential to induce chromosomal aberrations in cultured peripheral human lymphocytes in the presence and absence of S9-mix.

 

In the first cytogenetic assay, the test item was tested up to 650 μg/mL for a 3 hour exposure period with a 24 hour fixation time in the absence and presence of 1.8% (v/v) S9-mix. Appropriate toxicity (minimal reduction of the mitotic index to 45 ± 5%) was reached at this dose level.

In the second cytogenetic assay, the test item was tested up to 180 μg/mL for a 24 hour continuous exposure period with a 24 hour fixation time in the absence of S9-mix. Appropriate toxicity (minimal reduction of the mitotic index to 45 ± 5%) was reached at this dose level.

The number of cells with chromosome aberrations found in the vehicle 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 cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations 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.

First cytogenetic assay:

In the absence of S9-mix, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest concentration only, both when gaps were included and excluded. Since the number of cells with chromosome aberrations was well within the historical control data range the increase was considered not biologically relevant.
In the presence of S9-mix, the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Second cytogenetic assay:

At the 24 h continuous exposure time, the test item induced a statistically significant increase in the number of cells with chromosome aberrations above the historical control data range at the highest concentration. Since this increase could not be confirmed by 2 independent scorers, this increase was considered biologically not relevant.

It was noted that the test item increased the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix. This may indicate that the test item has the potential to disturb mitotic processes and cell cycle progression.

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

 

The K2 supporting chromosome aberration study was performed by Wright (2005), according to a method similar to OECD 473. The test substance was evaluated for induction of chromosome aberrations in human lymphocytes and was tested with and without metabolic activation at concentrations up to 1250 µg/mL in a 4 -hour exposure period, and without metabolic activation up to 312.5 µg/mL for a 24 -hour continuous exposure period.
There was a statistically significant increase in the frequency of aberrant cells at 625 µg/mL after 4-hour treatment in the presence of a metabolic activation system, which was equal to the maximum value of the historical vehicle control range. Due to the steep toxicity curve the ideal 50% mitotic inhibition was not achieved at 625 µg/mL (22% mitotic inhibition) and no scorable metaphases were present at the subsequent higher dose level in the 4-hour treatment arm in the presence of a metabolic activation system. It is currently unknown whether the increase in the frequency of aberrant cells at 625 µg/mL, which was equal to the maximum value of the historical control range, is of biological relevance. Based on the results in the 4-hour treatment arm in the presence of a metabolic activation system, the study was considered inconclusive. Therefore, this study was then not used to conclude on the classification of the test substance and an additional chromosome aberration test was performed to cover this endpoint (chromosome aberration test of Eurlings, 2017).

 

In vitro gene mutation study in mammalian cell

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

The test item, dissolved in dimethyl sulfoxide, was assessed for its potential to induce forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3- and 24-hour treatment period.

In the first mutation experiment, the mutation frequency induced at the thymidine-kinase locus by the test item was determined up to concentrations of 300 µg/mL in the absence and presence of S9-mix. The treatment period was 3 hours. The Relative Total Growth (RTG) was 15 and 11% in the absence and presence of S9-mix, respectively. No test item precipitation was observed up to the concentration of 300 µg/mL.

In the second mutation experiment,the mutation frequency induced at the thymidine-kinase locus by the test item was determined up to concentrations of 150 µg/mL in the absence of S9-mix. The treatment period was 24 hours. The Relative Total Growth (RTG) was 6% at the concentration of 150 µg/mL. No precipitation was observed up to the concentration of 150 µg/mL.

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

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

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

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

 

 

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T001159 and the criteria of the CLP Regulation (EC) 1272/2008, T001159 should not be classified for mutagenicity.