Hydroxyprogesterone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyprogesterone

EC Number:

200-699-4

EC Name:

Hydroxyprogesterone

Cas Number:

68-96-2

Molecular formula:

C21H30O3

IUPAC Name:

17-hydroxypregn-4-ene-3,20-dione

Details on test material:

Purity: 99.8 %

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany
- Suitability of cells: used successfully for many years in in vitro experiments; high proliferation rate, and a reasonable plating effeiciency
- Cell cycle length, doubling time or proliferation index: doubling time approximately 13 hours
- Methods for maintenance in cell culture if applicable: stocks are stored in liquid nitrogen; before freezing each batch is screened for mycoplasm contamination and
checked for karyotype stability
- Modal number of chromosomes: 22 +/- 1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal essential medium (MEM) with different supplements; incubations were done at 37°C in a humidified atmosphere with 1.5% carbon dioxide.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

pre-test = Experiment I; concentrations in µg/mL: 0.65, 1.29, 2.58, 5.16, 10.33, 20.66, 41.31, 82.63, 165.3, 330.5 (with and without S9 mix)
Highest test item concentration was selected according to "ICH Guideline S2 (R1) on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use" and should be 500 µg/mL or 1 mM, whichever it the lowest. With regard to the molecular weight of the test item, 330.5 μg/mL of Hydroxyprogesteron (approx. 1 mM) was selected as the highest dose. Precipitation of the test item was observed at the end of treatment at 165.3 µg/mL and above.

Vehicle / solvent:

DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- Culture conditions: Thawed stock cultures were propagated at 37 °C in 80 cm^2 plastic flasks. About 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes containing microscopic slides. Into each chamber 1.0 x 10^5 – 1.5 x 10^5 cells were seeded. For RICC (relative increase in cell count) determination 1.5 x 10^3 cells per well were seeded in duplicates in a 24-well-plate.
All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

- TREATMENT: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
The cells were treated on the slides in the chambers with deionised water for 1 – 1.5 min at 37 °C. Afterwards the cells were fixed twice with a mixture of ethanol and glacial acetic acid (3+1 parts, respectively) containing 1.25 % formaldehyde. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

- Pre-experiment/Experiment I: A preliminary cell growth inhibition test (determination of proliferation index) was performed to determine the concentrations to be used in the main experiment. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

- Evaluation: Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle [COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.] The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. Per culture at least 1000 cells from clones with 2 - 8 cells were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells.
Cytotoxicity was assessed by the determination of the relative increase in cell counts (RICC).

RICC= [(Increase in number of cells in treated cultures (final - starting))/(Increase in number of cells in control cultures (final - starting))]*100

Cytotoxicity [%] = 100 – RICC

In addition, cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represent the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated (see formula below). Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee a sufficient cell proliferation during treatment and recovery.

PI = [(c1 x 1) + (c2 x 2) + (c4 x 3) + (c8 x 4)] / [c1 + c2 + c4 + c8]
PI = Proliferation index
cx = Number of clones with x cells (with x: 1, 2, 4, or 8)

Evaluation criteria:

Acceptability criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range.
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in details on test system and conditions were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− The criteria for the selection of top concentration are consistent with those described above.

Evaluation Criteria:
Clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation of the test item in the culture medium was observed at 165.3 μg/mL and above in the absence and presence of S9 mix at the end of treatment. No relevant influence on osmolarity or pH was observed. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, where precipitation occurred. In the absence of S9 mix, a dose dependent increase in the number of micronucleated cells was observed after treatment with the test item. The two highest evaluated concentrations (82.63 and 165.3 μg/mL) showed statistically significant increases (2.05 and 3.25 %) compared to the concurrent solvent control. The values of the four highest evaluated concentrations exceeded the 95 % control limit of the historical control data (0.0 – 1.63 % micronucleated cells). No mutagenicity was observed in presence of S9 mix. MMC (0.1 μg/mL) or CPA (20.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Table: Summary of results  Exp.  Preparation interval Test item concentration in µg/mL  Proliferation index   RICC in % Cytotoxicity in %  Micronucleated cells* in %                     Exposure period 4 hrs without S9 mix  1  24 hrs  Solvent control^1  2.77 100.00  0.00  1.25       Positive control^2 2.51  78.16  21.84  3.75^S        10.33  1.92 142.13  n.c.  1.45       20.66 2.84  86.58  13.42  1.80       41.31 2.61  111.99  n.c.  1.70       82.63 2.58  115.40  n.c.  2.05^S       165.3^P  2.74 135.75  n.c.  3.25^S                     Exposure period 4 hrs with S9 mix  1  24 hrs  Solvent control^1  2.42 100.00  0.00  1.50       Positive control^3  1.75 44.42  55.58  11.25^S      41.31 2.01  88.21  11.79  1.25       82.63 2.29  96.99  3.01  1.30       165.3^P  2.23 84.61  15.39  1.30

* The number of micronucleated cells was determined in a sample of 2000 cells

S Number of micronucleated cells statistically significantly higher than corresponding control values

P Precipitation was observed at the end of treatment

n.c. Not calculated as the RICC is equal or higher than the solvent control value

1 DMSO 0.5 % (v/v)

2 Mitomycin C 0.1 μg/mL

3 CPA 20.0 μg/mL

Applicant's summary and conclusion

Conclusions:

positive

Executive summary:

Hydroxyprogesterone was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro according to OECD Guideline 487 at concentrations of 0.65, 1.29, 2.58, 5.16, 10.33, 20.66, 41.31, 82.63, 165.3, 330.5 µg/mL with and without metabolic activation (S9 mix).

Precipitation occured in the highest concentration without observation of cytotoxicty, which were evaluated in the absence and presence of S9 mix.

In the absence of S9 mix, a dose dependent increase in the number of micronucleated cells was observed after treatment with the test item. No mutagenicity was observed in presence of S9 mix.

Appropriate mutagens were used as positive controls. In conclusion, it can be stated that under the experimental conditions reported, the test item induced micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells. Therefore, it is considered to be clastogenic in this in vitro micronucleus test, when tested up to precipitating concentrations.