1-benzhydrylpiperazine

Endpoint summary

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 the OECD Guideline 471, it was concluded that T000750 is not mutagenic in the Salmonella typhimurium reverse mutation assay in the absence and presence of metabolic activation.

- Chromosome aberration study

In a K1 in vitro chromosome aberration study in human lymphocytes, performed according to the OECD Guideline 473,it was concluded that T000750 is not clastogenic in the human lymphocytes in vitro,in the absence and presence of metabolic activation.

- Gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, performed according to the OECD 490 Guideline, it was concluded that T000750 is not mutagenic in the mouse lymphoma L5178Y test system in the absence and presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-10 to 2016-10-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chrommosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15BB1451
- Expiration date of the lot/batch: 30 January 2021 (expiry date)
- Purity test date: 2015-05-21

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock solution of 200 mg/ml was treated with ultrasonic waves until the test item had completely dissolved.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in DMSO

OTHER SPECIFICS: correction factor 1.00

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Remarks:

primary culture

Details on mammalian cell type (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 35, AGT = 12.9 h
Dose range finding study 2: age 33, AGT = 14.2 h
First cytogenetic assay: age 21, AGT = 13.0 h
Second cytogenetic assay: age 27, AGT = 13.5 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 / cell type 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): 0, 62.5, 125, 250, 500, 1000, 2000 with and without S9-mix;
Dose range finding test 2 (3h): 0, 62.5, 125, 250, 500, 1000, 2000 with S9-mix;
Dose range finding test (24h): 0, 62.5, 125, 250, 500, 1000, 2000 without S9-mix;
Cytogenetic assay 1 (3h): 0, 50, 125, 150, 175, 200, 225, 250 µg/mL with and without S9-mix;
Cytogenetic assay 2 (24h): 0, 15, 30, 45, 60, 75, 90, 105, 120 µg/mL without S9-mix;

The test item precipitated in culture medium at the concentration of 100 mg/ml (=1000 μg/ml) and above. Based on these solubility findings, 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 / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item showed a turbid suspension in culture medium. In DMSO, the test item was dissolved. The stock solution of 200 mg/ml was treated with ultrasonic waves until the test item had completely dissolved. The test item precipitated in culture medium at the concentration of 100 mg/ml (=1000 μg/ml) and above. Based on these solubility findings, DMSO was selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix; at 0.5 and 0.75 µg/mL (3h exposure period), at 0.2 and 0.3 µg/mL (24h exposure 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 10 µg/mL (3h exposure period)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
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: acetic acid 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 (with a maximum deviation of 5%). 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
One hundred and fifty metaphase chromosome spreads per culture were examined 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. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed.

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 was used for statistical analysis of the data.

Species / strain:

lymphocytes: human

Remarks:

Cytogenetic assay 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Appropriate toxicity was reached at 200 μg/mL in the absence of S9-mix (mitotic index of 46% compared to concurrent vehicle control) and at 225 μg/mL in the presence of S9-mix (mitotic index of 39% compared to concurrent vehicle control)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Remarks:

Cytogenetic assay 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Appropriate toxicity was reached at 75 μg/mL (mitotic index of 25% compared to concurrent vehicle control)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / Effects of osmolality:
No marked changes in pH and osmolality were observed in culture medium containing the highest, non-precipitating test item concentration of 500 μg/ml (8.422 and 427 mOsm/kg, respectively) compared to the concurrent vehicle control (8.068 and 446 mOsm/kg, respectively).
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation:
Dose range finding test (3h): at 1000 μg/ml and above
Dose range finding test 2 (3h): at 1000 μg/ml and above
Dose range finding test (24h): at 500 μg/ml and above
Cytogenetic assay 1 (3h): No test item precipitation observed up to the highest tested concentration
Cytogenetic assay 2 (24h): No test item precipitation observed 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 str uctural 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. The test item was tested in the absence and in the presence of 1.8% (v/v) S9-mix. 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 48± 2 h and thereafter exposed to selected doses of the test item for 3 h and 24 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A vehicle control was included at each exposure time.
For the 3 hour exposure time, blood cultures were treated with 62.5, 125, 250, 500, 1000 and 2000 μg test item/ml culture medium with and without S9-mix.
For the 24 hour exposure time, blood cultures were treated with 62.5, 125, 250, 500, 1000 and 2000 μg test item/ml culture medium without S9-mix.
A complete cell lysis was observed at 250 μg/ml and above for the 3-hour exposure period without S9-mix, and at 500 μg/ml and above for the 3-hour exposure period with S9-mix and the 24-hour exposure period without S9-mix.
Based on the results of the dose range finding test, the following dose levels were selected for the first cytogenetic assay: 50, 125, 150, 175, 200, 225 and 250 μg/ml culture medium with and without S9-mix.
Based on the results of the dose range finding test, the following dose levels were selected for the second cytogenetic assay: 15, 30, 45, 60, 75, 90, 105 and 120 μg/ml culture medium.

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. 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.
- 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 1:

The test item increased the number of polyploid cells both in the absence and presence of S9-mix.

Cytogenetic assay 2:

The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Conclusions:

Both in the absence and 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 in two independent experiments.
It was noted that the test item increased the number of polyploid cells both in the absence and presence of S9-mix. This may indicate that the test item has the potential to disturb mitotic processes.
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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-24 to 2017-03-06

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: M15BB1451
- Expiration date of the lot/batch: 2021-01-30 (expiry date)
- Purity test date: 2015-05-21

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 indicated

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 / 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). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10^6 cells/ml.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium 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: 25, 50, 100, 150, 200, 400 μg/ml without and with S9-mix.
Dose range finding test 24h: 25, 50, 100, 150, 200, 400 μg/ml without S9-mix.
Mutation experiment 1: Without S9-mix: 6.25, 12.5, 25, 50, 70, 80, 90 and 100 μg/ml; With S9-mix: 12.5, 25, 50, 60, 70, 80, 90 and 100 μg/ml
Mutation experiment 2: 0.63, 1.25, 2.5, 5, 10, 20, 30, 35, 40, 45, 50, 60 and 70 μg/ml
Since in Charles River Laboratories Study No. 513663, the test item was cytotoxic at dose levels of 150 μg/ml and above, 400 μg/ml was selected as the maximum concentration for the dose range finding test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was dissolved in dimethyl sulfoxide based on the solubility finding in Charles River Laboratories Study No. 513663

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): 8 x 10^ 6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6cells (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-diphenyltetrazoliumbromide (MTT) to each well.

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

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

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Rationale for test conditions:

The highest concentration tested 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).
Since in the Charles River Laboratories Study No. 513663, the test item was cytotoxic at dose levels of 150 μg/ml and above, 400 μg/ml was selected as the maximum concentration for the dose range finding test.

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:

Cytotoxicity at 150 μg/mL and above, 400 μg/mL selected as maximum

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Precipitation: Dose range finding test 3h: 400 μg/mL with and without S9-mix
Dose range finding test 24h: at 400 μg/mL with and without S9-mix
Mutation experiment 1: at 130 μg/mL
Mutation experiment 2: at 70 μg/mL

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 25 to 400 μg/mL in the absence of S9-mix with 3 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.

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.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 14 and 16 (3 hour treatment) and 53 and 54 (24 hour treatment)

Remarks on result:

other: 3 h treatment

Conclusions:

Interpretation of results: negative with and without metabolic activation
In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-19 to 2006-05-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

3 strains used i.s.o. 5

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D dated May 19, 2000

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch Number: 0000008325
Molecular Weight: 252.36
Aggregate State at
Room Temperature: Solid
Colour: White to pale beige
Purity: 100 %
Stability in Solvent: Not indicated by the sponsor
Storage: Room temperature
Expiry Date: 2006-10-01

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Genotype: his D 3052; rfa-; uvrB-; R-factor

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: Genotype: his G 428; rfa-; uvrB+; R-factor

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Genotype: his G 46; rfa-; uvrB-; R-factor

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment / experiment I:
3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Experiment II:
10; 33; 100; 333; 1000 and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

strain TA100; without metabolic activation; 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

strain TA98; without metabolic activation; 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

strain TA102; without metabolic activation; 4 µL/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains; with metabolic activation; 2.5 µg/plate (10 µg/plate in TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: both preincubation (exp. II) and plate incorporation (exp. I) assays performed.

The pre-experiment was reported as main experiment I.

Experimental performance:
The following materials were mixed in a test tube and poured onto the minmal agar plates:
- 100 μL of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 μL of S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
- 100 μL of bacteria suspension (c.f. test system, pre-culture of the strains)
- 2000 μL overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 h at 37°C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (clearing of the bacterial background lawn) or reduction in the number of spontaneous revertants

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed for the strains tested: TA 98, TA 100 and TA 102).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in and independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data was not required.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

as from 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

as from 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

as from 333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation: No precipitation of the test item was observed in the overlay agar

RANGE-FINDING/SCREENING STUDIES:
Toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation at the following concentrations (µg/plate) - Experiment I:
Strain TA 98: 1000-5000 µg/plate without S9 mix; 2500-5000 µg/plate with S9 mix
Strain TA 100: 1000-5000 µg/plate without S9 mix; 2500-5000 µg/plate with S9 mix
Strain TA 102: 1000-5000 µg/plate without S9 mix; 5000 µg/plate with S9 mix
Reduced background growth was observed with and without S9 at 2500 and 5000 µg/plate in experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation at the following concentrations (µg/plate) - Experiment II:
Strain TA 98: 1000, 2500 µg/plate with and without S9 mix
Strain TA 100: 1000, 2500 µg/plate with and without S9 mix
Strain TA 102: 333-2500 µg/plate without S9 mix; 1000, 2500 µg/plate with S9 mix

Remarks on result:

other: all strains/cell types tested

Conclusions:

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

Under the experimental conditions of this test, the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

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, Sokolowski A., 2006) was performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay). T000750 was tested in the Salmonella typhimurium reverse mutation assay with three histidine requiring strains (TA98, TA100 and TA102). The test item was dissolved in dimethyl sulfoxide.

In a dose range finding test, the test item was tested at a concentration range of 3 to 5000 µg/plate in the absence and presence of 15% liver S9 -mix in standard co-factors. Results of the dose range finding test were reported as the first mutation experiment. No precipitation of the test item was observed in the overlay agar. Cytotoxicity, evident as a reduction in the number of spontaneous revertants, was observed at 1000 µg/plate and upwards in the absence of S9 -mix and at 2500 and/or 5000 µg/plate in the presence of S9 -mix.

In the second mutation experiment, the test item was tested at a concentration range of 10 to 5000 µg/plate in the absence and presence of 15% liver S9 -mix in standard co-factors. No precipitation of the test item was observed in the overlay agar. Cytotoxicity, evident as a reduction in the number of spontaneous revertants, was observed at 1000 µg/plate and upwards, except in strain TA102 without S9 -mix, where it was observed at 333 µg/plate and upwards.

The test item did not induce a substantial increase in the revertant colony numbers in any of the three tester strains (TA98, TA100 and TA102) at any dose level, neither in the presence nor absence of 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.

Expert statement in addition of Ames test

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 assay

 A key study (K1, Eurlings I., 2017) was performed according to the OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).

The test item, dissolved in dimethyl sulfoxide, was assessed for its ability to induce chromosome aberrations in cultured peripheral human lymphocytes in the presence of 1.8% (v/v) S9 -mix.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test item ranging from 50 to 250 µg/mL with and without metabolic activation in the first experiment (3 hours exposure period) and from 15 to 120 µg/mL without metabolic activation in the second experiment (24 hours exposure period). At least three analysable concentrations were used for scoring of the cytogenetic assay.

In the first cytogenetic assay the test item was tested up to 200 and 225 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, respectively. Appropriate toxicity (minimal reduction of the mitotic index to 45 ± 5%) was reached at 200 µg/mL culture medium in the absence of S9 -mix, and 50, 150 and 200 µg/mL were selected for scoring of chromosome aberrations. In the presence of S9 -mix, appropriate toxicity was reached at 225 µg/mL culture medium, and 50, 150, 200 and 225 µg/mL were selected for scoring of chromosome aberrations.

In the second cytogenetic assay, the test item was tested up to 75 μg/mL for a 24 hour continuous exposure time 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 75 µg/mL, and 15, 30, 60 and 75 µg/mL were selected for scoring of chromosome aberrations.

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.

The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

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

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.

A K3 study (Kunz S., 2007), with an expert statement, was used as supporting evidence. In this study, performed according to the OECD Guideline 473, the toxicity level was not reached. Under the conditions of this test, no biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed, and no biologically relevant increases in the rate of polyploid metaphases were found after treatment with the test item as compared to the rates of the solvent control. It was concluded that the substance was not considered to be clastogenic with and without metabolic activation.

In vitro gene mutation study in mammalian cell

Verspeek-Rip (2017) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (key study, K1).

The test item, 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 (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.

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 7 and 6% at the concentration of 100 μg/mL in the absence and presence of S9-mix, respectively. No test item precipitation was observed up to the concentration of 100 μg/mL.

In the second mutation experiment, the test item was tested up to concentrations of 20 μg/mL in the absence of S9 -mix. The treatment period was 24 hours. The Relative Total Growth (RTG) was 14% at the concentration of 20 μg/ml. No precipitation was observed up to the concentration of 20 μg/mL.

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

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T000750 and the criteria of the CLP Regulation (EC) 1272/2008, T000750 should not be classified for mutagenicity.