1-benzhydrylpiperazine

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

1-benzhydrylpiperazine

EC Number:

212-667-7

EC Name:

1-benzhydrylpiperazine

Cas Number:

841-77-0

Molecular formula:

C17H20N2

IUPAC Name:

1-(DIPHENYLMETHYL)PIPERAZINE

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-130806-AAA (T000750)
- Physical state: solid (powder)
- Appearance: white to slight beige powder

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

Method

Target gene:

not applicable

Species / strain

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.

Controls

Controls 1

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)

Controls 2

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.