Registration Dossier
Registration Dossier
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EC number: 200-699-4 | CAS number: 68-96-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation (HPRT in vitro) [according to OECD 476]: negative (Wollny, 2016)
Clastogenic/Aneugenic effect (MNT in vitro) [according to OECD 487]: positive (Chang, 2016)
Gene mutation (Bacterial reverse mutation assay / Ames test) [according to OECD 471]: negative with hydroxyprogesterone acetate (Wollny, 1995)
supporting data: DNA damage and/or repair study (UDS in vitro) [according to OECD 482]: negative (Fautz, 1994)
Link to relevant study records
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Feb to Jun 1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Version / remarks:
- (1986) This guideline was deleted in 2014.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
- Target gene:
- not applicable
- Species / strain / cell type:
- primary culture, other: hepatocytes of female rats
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 1, 5, 10, 15, and 20 µg/mL
According to the pre-experiment for toxicity the concentration range was selected to yield concentration-related toxic effects. - Vehicle / solvent:
- DMSO
(The solvent did not exceed 1 % in the culture medium.) - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- Remarks:
- Final concentration of positive control: 2.23 µg/mL
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 (1 used for UDS, 2 for determination of cytotoxicity)
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E05/ml
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 18 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- cytotoxicity evidenced by altered cell morphology and/or reduced number of adherent cells. In addition, the capability of the cel1s to incorporate a vital dye was determined by the neutral red absorption assay
METHODS FOR MEASUREMENTS OF GENOTOXICIY
see "Any other information on materials and methods incl. tables" - Species / strain:
- primary culture, other: hepatocytes of female rats
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Executive summary:
An in vitro genotoxicity test according to OECD TG 482 (Unscheduled DNS Synthesis, DNA damage and repair assay) was conducted on primary rat hepatocytes. Two independent experiments were performed, using identical procedures. The freshly isolated hepatocytes were exposed to the test article for 18 hours in the presence of 3HTdR (methyl-3H-thymidine). Uptake of radioactivity was determined by autoradiography. For each concentration, including controls, 100 cells were evaluated. The concentrations chosen for both experiments were 1.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, and 60 µg/mL, based on a pre-experiment for toxicity. To establish a concentration response five of these concentrations were evaluated: 1.0, 5.0, 10.0, 15.0, 20.0 µg/mL.
In both main experiments treatments of the cells with concentrations higher than 15.0 µg/mL revealed substantially reduced neutral red uptake due to toxicity. Precipitation of the test article could be observed starting with 20 µg/mL.
No reproducible concentration-dependent increase in the number of nuclear and net grain counts were observed up to the highest concentrations evaluated in the two independent experiments. An appropriate reference Mutagen (2-AAF, 2.23 µg/mL) showed, reproducibly, a distinct increase in the number of nuclear and net counts. Thus, it can be stated that the substance did not induce DNA-damage leading to increased repair synthesis under the conditions used in this assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Jan - Feb 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium) with supplements; for the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from the liver of phenobarbital/ß-naphthoflavone induced rats
- Test concentrations with justification for top dose:
- Pre-experiment: 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL (-/+ S9 mix)
Main experiment: 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL (-/+ S9 mix) - Vehicle / solvent:
- Dimethylsulfoxid (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. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- EMS was used without and DMBA with metabolic activation
- Details on test system and experimental conditions:
- - DOSE SELECTION: According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye and microscopically. Furthermore, test item induced changes in the osmolarity will influence dose selection. The highest applied concentration in the pre-test on toxicity (200 μg/mL) was based on the solubility of the test item in aqueous media. No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was determined up to the highest concentration with and without metabolic activation. The test medium was checked for precipitation at the end of treatment (4 hours) prior to removal to the test item. Precipitation occurred at 100.0 μg/mL and above after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the main experiment was set according to data generated in the preexperiment. The individual concentrations were spaced by a factor of 2.0. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations. (The cultures at the highest concentration [100 and 200 µg/ml in main experiment] with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations).
- CULTURE MEDIUM: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- SEEDING: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×107 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- TREATMENT: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G". Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium. Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below. Three or four days after first sub-cultivation approximately 2.0×10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II). The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope. - Rationale for test conditions:
- Experiences from testing facility
- Evaluation criteria:
- Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
Evaluation of results:
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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
- Conclusions:
- negative
- Executive summary:
Hydroxyprogesterone was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 200 µg/mL with and without metabolic activation. The maximum test item concentration of the pre-experiment and the main experiment (200 μg/mL) was limited by the solubility of the test item in aqueous media. No substantial and reproducible dose dependent increase of the mutation frequency above was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance was considered to be non-mutagenic in this HPRT assay.
- 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
- 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
- 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. - 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.
- 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
- 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
- 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.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- 2-aminoanthracene only positive control for metabolic activation; but the metabolic activity of S9 mix preperation was checked with benzo(a)pyrene.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine gene locus
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced male rat liver S9 mix
- Test concentrations with justification for top dose:
- 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate
highest dose according to OECD Guideline 471 - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Remarks:
- untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: without metabolic activation: 4-Nitro-o-phenylenediamine, dissolved in DMSO, conc. 10 µg/plate; with metabolic activation: 2-Aminoanthracene, dissolved in DMSO, conc.: 2.5 µg/plate (10 µg/plate in TA 102)
- Details on test system and experimental conditions:
- plate incorporation assay
For each strain and dose level, including controls three plates were used as a minimum.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl: Test solution at each dose level, solvent (negative control) or control mutagen solution (positive control)
500 µl: S9 mix (metabolic activation) or S9 mix substitution buffer (without metabolic activation)
100 µl: Bacteria suspension
2000 µl: Overlay agar
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
Data recording:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, Biosys GmbH, 51184 Karben)
The background growth of the bacteria was judged visually on a light bench. - Evaluation criteria:
- Generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates
Evaluation of results:
A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at lest one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biological relevant positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous rate in strains TA 100 and TA 102 or thrice TA 1535, TA 1537, and TA 98.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Conclusions:
- negative
- Executive summary:
The mutagenic potential of Hydroxyprogesterone acetate was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level in the presence and absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, the test substance was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Justification for analogue approach:
As no bacterial reverse mutation assay of hydroxyprogesterone is available, a read-across to hydroxyprogesterone acetate (CAS 302-23-8) was performed. Usually esters are hydrolysed in the presence of water or in organisms due to enzymatic degradation by esterases. As a result, the respective alcohol derivative (hydroxyprogesterone (CAS 68-96-2)) and carboxylic acid are generated. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- 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:
- valid
- Positive controls validity:
- valid
- Conclusions:
- negative
- Executive summary:
No bacterial reverse mutation assay with the target substance Hydroxyprogesterone. Results of a study conducted with Hydroxyprogesterone acetate are regarded as representative as most likely ester cleavage occurs in vivo after administration.
The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level in the presence and absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, the test substance was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Based on read-across, this result is also valid for the target substance Hydroxyprogesterone.
Referenceopen allclose all
The following concentrations were chosen for both main experiments: 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, and 60 µg/mL.
To establish a concentration response effect five concentrations were selected for evaluation of the nuclear and cytoplasm grains: 1.0, 5.0, 10.0, 15.0, and 20.0 µg/mL.
In both main experiments treatments of the cells with concentrations higher than 15.0 µg/mL revealed substantially reduced neutral red uptake due to toxicity. Precipitation of the test article could be observed starting with 20 µg/mL. In the two independent experiments, after treatment with the test article, no reproducible concentration dependent increase in the number of nuclear and net grain counts was observed up to the highest concentration evaluated. Therefore, the net grain values obtained after treatment with the test article were consistently negative. In addition, no substantial shift to higher values was obtained in the percentage distribution of the nuclear grain counts. An appropriate reference mutagen showed, reproducibly, a distinct increase in nuclear and net grain counts.
Test concentrations in main experiment: 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL (-/+ S9 mix)
Precipitation occurred at 100.0 mg/mL at the beginning and at the end of treatment in the presence and absence of metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration.
The 95% confidence interval was slightly exceeded at 100 μg/mL in the first culture with metabolic activation (35.0 versus an upper limit of 28.7 mutant colonies/106 cells). This isolated increase was judged as irrelevant as it was not reproduced and there was no dose dependent increase as indicated by a lacking statistical significance. The mean value of both parallel cultures (35.0 and 16.9 equal to a mean of 26 colonies/10^6 cells) remained well within the 95% confidence interval.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In the main experiment with and without S9 mix the range of the solvent controls was from 12.1 up to 26.1 mutants per 10^6 cells; the range of the groups treated with the test item was from 9.1 up to 35.0 mutants per 10^6 cells.
EMS (450 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
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
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* 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
No toxic effects, evidenced by a reduction in the number of revertants, occured in the test groups with and without metabolic activation.
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rats with increasing concentrations in the range below the generally acknowledged border of biological relevance.
No toxic effects, evidenced by a reduction in the number of revertants, occured in the test groups with and without metabolic activation.
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rats with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
MNT in vivo: negative (Barraclough 2017)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March - July 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other:
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted 26 September 2014
- GLP compliance:
- yes
- Type of assay:
- other: micronucleus test in vivo
- Species:
- rat
- Strain:
- other: Crl:WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 10 -11 weeks (males), 9 - 10 weeks (females)
- Weight at start of dosing: 280.4 - 409.2 g (males), 186.4 - 242.4 g (females)
- Fasting period before study: not applicable
- Housing: housed in groups (up to four animals/cage by sex [both sexes pre-pairing and males post-pairing] or with one female and one male [pairing]), individually (mated females), or with their litter (lactation)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: up to 20 days
DETAILS OF FOOD AND WATER QUALITY:
No contaminants were present in diet and water at levels which might have interfered with achieving the objective of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): within 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: 11 April - 9 June 2016 - Route of administration:
- oral: gavage
- Vehicle:
- 0.1% Myrj S-50-PA, 1.0% Klucel LF add 100% with 0.9% physiological saline solution
- Details on exposure:
- Formulations were prepared weekly.
The test item formulations were formulated as a suspension in 0.1% Myrj S-50-PA, 1.0% Klucel LF add 100% with 0.9% physiological saline solution. The formulations were stored at room temperature (15 to 25°C) in a sealed container, protected from light. - Duration of treatment / exposure:
- Males: 42 days
Females: up to 64 days - Frequency of treatment:
- once daily
- Post exposure period:
- 24 hours
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Low dose
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Intermediate dose
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- High dose
- No. of animals per sex per dose:
- males: 5
females: 8 (control group), 4 (low and intermediate dose), 5 (high dose) - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- The positive control animals were dosed with cyclophosphamide (20 mg/kg).
- Tissues and cell types examined:
- bone marrow smears
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION: Bone marrow was flushed from the marrow cavity with foetal bovine serum. The collected samples were then filtered through cellulose columns. Once filtered, the bone marrow cells were pelleted by centrifugation and the
supernatant aspirated and discarded. Additional serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (by centrifugation) and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried prior fixing for 10 minutes in absolute methanol. Once fixed the slides were rinsed several times in distilled water and air-dried. Two slides per animal were stored at <-10°C with desiccant for subsequent staining. On the day of staining the slides were fixed and washed again (as described above) and immediately stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Stained slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. The dried, unstained slides were initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature.
METHOD OF ANALYSIS: Scoring was carried out using fluorescence microscopy at an appropriate magnification. A sample of slides from the vehicle control animals and the positive control slides were checked for quality and/or response prior to analysis. All slides (including the positive control slides) were allocated a random code and analysed by an individual not connected with the dosing phase of the study. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Then at least 4000 PCE/animal (2000 PCE per positive control slide) were examined for the presence of MN (micronucleus).
The following criteria were applied during slide assessment:
1. Cells were to be of normal cell morphology
2. Areas where erythrocytes overlap were to be ignored
3. A MN was to be round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN which were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.
Slide analysis was performed by a competent analyst trained in the applicable Covance Laboratories standard operating procedures.
DATA EVALUATION: After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. %MN PCE for each animal and the group mean %MN PCE (±standard deviation).
The numbers of MN PCE in the vehicle control animals were compared with the laboratory's standard micronucleus historical control data to determine whether the assay was acceptable. - Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. At points that were significant the group mean MN PCE value was outside the 95% historical standard micronucleus vehicle control range
3. At the same dose levels the distribution of MN PCE in the majority of animals exceeded the laboratory’s historical vehicle control data
4. A dose-response trend in the proportion of MN PCE (where more than two dose levels were analysed) was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels. - Statistics:
- For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation. For female data the numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. For males the heterogeneity chi-squared calculation showed a significant difference and therefore the male data were evaluated using Wilcoxon Rank Sum test. The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The data confirm that:
1. The vehicle control MN PCE data were comparable with the laboratory’s standard micronucleus historical vehicle control ranges
2. The positive control chemical (CPA) induced a clear increase in the frequency of MN PCE that was comparable with the laboratory’s historical positive control data
3. Adequate numbers of cells and doses were analysed.
The assay data were therefore considered valid. - Conclusions:
- negative
- Executive summary:
The potential for Hydroxyprogesterone to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow was evaluated in male and female rats following daily oral (gavage) administration for at least 42 days. Bone marrow was sampled 1 day after the final dose administration (Day 43 for male rats or for females either Day 14 post-partum or Day 26 after mating for females that did not litter).
The vehicle control data for %PCE and MN PCE were comparable with the laboratory’s standard micronucleus historical vehicle control data. The positive control slides exhibited a clear increase in MN PCE over the concurrent vehicle control. The assay was therefore accepted as valid.
Male and female rats treated with Hydroxyprogesterone at all doses had group mean %PCE that were similar to the concurrent vehicle control group and which were comparable to the laboratory’s standard micronucleus historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.
Male and female rats treated with Hydroxyprogesterone at all doses had MN PCE frequencies that were similar to the concurrent vehicle control group and which were considered consistent with the laboratory's standard micronucleus historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle controls.
It is concluded that Hydroxyprogesterone did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female rats treated up to 1000 mg/kg/day, under the experimental conditions employed.
Reference
Hydroxyprogesterone: Summary of Micronucleus Data:
Heterogeneity | Statistics a) | |||||||||
Group/Sex/Treatment (mg/kg/day) | PCE Scored | Number of MN PCE | %PCE | MN / 4000 PCE | %MN PCE | SD | Chi² | Significance | P-value | Significance |
1M / Vehicle (0) | 20000 | 62 | 48.16 | 12.40 | 0.31 | 0.09 | 4.63 | NS | - | - |
2M / Hydroxyprogesterone (100) | 20000 | 62 | 50.64 | 12.40 | 0.31 | 0.14 | 9.97 | P≤0.05 | 0.5238 | NS |
3M / Hydroxyprogesterone (300) | 20000 | 30 | 47.68 | 6.00 | 0.15 | 0.06 | 4.34 | NS | 0.9802 | NS |
4M / Hydroxyprogesterone (1000) | 20000 | 31 | 50.16 | 6.20 | 0.16 | 0.05 | 2.39 | NS | 0.9881 | NS |
1F / Vehicle (0) | 32000 | 67 | 51.63 | 8.38 | 0.21 | 0.10 | 12.67 | NS | - | - |
2F / Hydroxyprogesterone (100) | 16000 | 27 | 49.90 | 6.75 | 0.17 | 0.07 | 3.38 | NS | 0.70 | NS |
3F / Hydroxyprogesterone (300) | 16000 | 24 | 46.75 | 6.00 | 0.15 | 0.09 | 7.01 | NS | 1.69 | NS |
4F / Hydroxyprogesterone (1000) | 20000 | 37 | 51.96 | 7.40 | 0.19 | 0.08 | 5.58 | NS | 0.25 | NS |
Positive control slides | 4000 | 199 | 51.80 | 199.0 | 4.98 | 0.46 | - | - | - | - |
Linear Trend tests:
Males: P-value: 0.9945 NS
Females:P-value (z) -0.367 NS
a Wilcoxon Rank Sum Test for male data, Chi-squared 2 x 2 Contingency for female data
M Male
F Female
MN Micronucleus
NS Not significant
PCE Polychromatic erythrocyte
SD Standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
17-Hydroxyprogesterone is a naturally occurring metabolite in the human body, thus at low doses a genotoxic effect is not assumed.
For a full evaluation of the ability of 17-Hydroxyprogesteron (17-OHP) to induce possible types of genetic damage the available data for 17-OHP on gene mutation in mammalian cells in vitro (hypoxanthine-guanine phosphoribosyl transferase; HPRT assay), chromosomal damage (Micronucleus test; MNT in vitro and in vivo, Unscheduled DNA Synthesis; UDS in vitro) as well as read-across data of OHPA on Bacterial Reverse Mutation (Ames test in vitro) were taken into account.
In vitro tests
Ames test
No bacterial reverse mutation assay with the target substance Hydroxyprogesterone. Results of a study conducted with Hydroxyprogesterone acetate are regarded as representative as most likely ester cleavage occurs in vivo after administration. A read-across approach from hydroxyprogesterone acetate to hydroxyprogesterone is justified as described in the document "Justification for a read-across between 17-Hydroxyprogesterone acetate (CAS 302-23-8, EC 206-119-6) as source and 17-Hydroxyprogesterone (CAS 68-96-2, EC 200-699-4) as target".
The mutagenic potential of Hydroxyprogesterone acetate was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level in the presence and absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, the test substance was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay. (Wollny, 1995)
Gene mutation assay in mammalian cells
Hydroxyprogesterone was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 200 µg/mL with and without metabolic activation. The maximum test item concentration of the pre-experiment and the main experiment (200 μg/mL) was limited by the solubility of the test item in aqueous media. No substantial and reproducible dose dependent increase of the mutation frequency above was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance was considered to be non-mutagenic in this HPRT assay. (Wollny, 2016)
Micronucleus test
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 occurred in the highest concentration without observation of cytotoxicity, 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. (Chang, 2016)
Unscheduled DNA Synthesis
An in vitro genotoxicity test according to OECD TG 482 (Unscheduled DNA Synthesis, DNA damage and repair assay) was conducted on primary rat hepatocytes. Two independent experiments were performed, using identical procedures. The freshly isolated hepatocytes were exposed to the test article for 18 hours in the presence of 3HTdR (methyl-3H-thymidine). Uptake of radioactivity was determined by autoradiography. For each concentration, including controls, 100 cells were evaluated. The concentrations chosen for both experiments were 1.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, and 60 µg/mL, based on a pre-experiment for toxicity. To establish a concentration response five of these concentrations were evaluated: 1.0, 5.0, 10.0, 15.0, 20.0 µg/mL.
In both main experiments treatments of the cells with concentrations higher than 15.0 µg/mL revealed substantially reduced neutral red uptake due to toxicity. Precipitation of the test article could be observed starting with 20 µg/mL.
No reproducible concentration-dependent increase in the number of nuclear and net grain counts were observed up to the highest concentrations evaluated in the two independent experiments. An appropriate reference Mutagen (2-AAF, 2.23 µg/mL) showed, reproducibly, a distinct increase in the number of nuclear and net counts. Thus, it can be stated that the substance did not induce DNA-damage leading to increased repair synthesis under the conditions used in this assay. (Fautz, 1994)
In vivo tests
Micronucleus test
The potential for Hydroxyprogesterone to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow was evaluated in male and female rats following daily oral (gavage) administration for at least 42 days. Bone marrow was sampled 1 day after the final dose administration (Day 43 for male rats or for females either Day 14 post-partum or Day 26 after mating for females that did not litter).
The vehicle control data for %PCE and MN PCE were comparable with the laboratory’s standard micronucleus historical vehicle control data. The positive control slides exhibited a clear increase in MN PCE over the concurrent vehicle control. The assay was therefore accepted as valid.
Male and female rats treated with Hydroxyprogesterone at all doses had group mean %PCE that were similar to the concurrent vehicle control group and which were comparable to the laboratory’s standard micronucleus historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.
Male and female rats treated with Hydroxyprogesterone at all doses had MN PCE frequencies that were similar to the concurrent vehicle control group and which were considered consistent with the laboratory's standard micronucleus historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle controls.
It is concluded that Hydroxyprogesterone did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female rats treated up to 1000 mg/kg/day, under the experimental conditions employed. (Barraclough 2017)
The in vivo micronucleus test was included in the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test. While no adverse effects to the bone marrow were observed (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependant decrease), systemic availability was proven by substance-related effects in females of all dose groups in the OECD TG 422 part of the study, which consisted of elevated cholesterol levels, which may have been attributed to administration of Hydroxyprogesterone, and resulted in a down regulation of progesterone production. Microscopically, an increased incidence and/or severity of tubular basophilia and/or cortical scar of kidneys was present in test item-treated groups. Slightly elevated urea and creatinine, together with an increased incidence and severity of tubular basophila and/or scarring, observed for females administered 1000 mg/kg/day were indicative of renal injury, and as such, was considered adverse. According to EFSA (2017), systemic toxicity observed in repeated dose toxicity studies is an indicator of systemic bioavailability. Since the bone marrow is a well-perfused tissue, systemic bioavailability of a test substance can be considered as a line of evidence of bone marrow exposure. Therefore, the negative outcome of this in vivo micronucleus test is fully valid and disproves the initial positive finding in vitro.
In summary, there is no indication of Hydroxyprogesterone to induce gene mutation since the HPRT for 17-OHP was negative. An initial finding for a potential to induce chromosomal damage in a MNT in vitro assay was opposed by the negative in vivo proof (MNT in vivo). Based on read-across to OHPA an Ames-test is considered, which also yielded a negative result. Overall, 17-OHP gives no indication for a potential to induce genetic toxicity based on the available data.
As supportive information on genetic toxicity the Ames test result of the other ester of 17-OHP, namely OHPC, maybe be considered with no mutagenic potential found within an identical protocol as used for OHPA. Moreover, it should be noted that the German TRGS 9053 has already concluded on the endpoint genotoxicity for various gestagenic steroid hormones. There, for the group of gestagens no convincing indication for a relevant mutagenic potential is seen and consequently no classification for mutagenicity proposed.
Studies for assessment of genetic toxicity for OHPA and 17-OHP
| Hydroxyprogesterone acetate OHPA | 17-Hydroxyprogesterone 17-OHP |
CAS No. | 302-23-8 | 68-96-2 |
ZK | 5189 | 5149 |
Ames (OECD TG 471) with & without S9-mix, plate incorporation method | · Report AL93, study no. TX95141 (Reimann/ Wollny, 1996) · GLP, guideline study · No deviations · Compound purity: 98.3% (HPLC) · Formulation: DMSO; final suspensions freshly prepared · Strains: TA1535, TA1537, TA 98, TA100, TA102 · Six concentrations up to 5 mg/plate tested · Appropriate controls used · Results: no increases in revertant colony numbers in any strain, no tendency of higher mutation rate with increasing concentrations · Reliability 1 | No data
RA from OHPA |
HPRT in vitro (OECD TG 476) with & without S9-mix | No data | · Report T102359-0, study no. 1727201 (Wollny, 2016) · GLP, guideline study; no deviations · Compound purity: 99.8%; stability tested (stable over 4 and 24 hours in DMSO (Envigo Study 41502470) · Formulation: DMSO; final suspensions freshly prepared · Cells: Chinese hamster V79 · Six concentrations up to 200µg/ml tested (limited by solubility; pre-experiment performed) · Appropriate controls used · Results: no gene mutation induced at the HPRT locus · Reliability 1 |
UDS in vitro (OECD TG 482 -> deleted 2014) | No data | · Report AC44, study no. 456400 (Fautz/ Reimann, 1994) · GLP, guideline study; no deviations · Compound purity: unknown but certificate of analysis 20447/93 approved December 14, 1993 · Formulation: dissolved in DMSO; stability unknown -> therefore freshly prepared · Cells: primary rat hepatocytes (female) · Five concentrations up to 20.0µg/ml tested (pre-experiment performed) · Appropriate control used · Results: no DNA-damage induced leading to increased repair synthesis · Reliability 1 |
MNT in vitro (OECD TG 487) with & without S9-mix | No data | · Report T102360-2, study no. 1727204 (Chang, 2016) · GLP, guideline study; no deviations · Compound purity: 99.84%; stability tested (stable over 4 and 24 hours in DMSO (Envigo Study 41502470) · Formulation: DMSO; final suspensions freshly prepared · Cells: Chinese hamster V79 · Ten concentrations up to 330.5µg/ml tested (pre-experiment performed, no cytotoxicity observed) · Appropriate controls used; mitomycin C (w/o S9) used in 10-fold lower concentration than usual · Results: dose dependent increase of micronucleated cells in the absence of S9 mix, no mutagenicity observed in with metabolic activity (S9) · Reliability 1 |
MNT in vivo (OECD 474) | No data | · Report T102532-3, study no. 8335930 (Barraclough, 2017) · GLP, OECD 474 guideline study (within a OECD 422); no deviations · Rats, treated daily p.o. for 42d · Compound purity: 100%; homogenicity & stability tested (stable up to 10d; Covance Study 8335929) · Formulation: 0.1% (w/v) Myrj S-50-PA and 1.0% (w/v) Klucel LF in 0.9% saline · Evaluation of micronuclei in erythrocytes (PCEs) of bone marrow sampled 1 day after the final dose · Results: 17-OHP did not induce micronuclei in PCEs of the bone marrow in animals administered up to 1000 mg/kg/day. · Reliability 1 |
In conclusion, assessment of reliability and adequacy of the source study Bacterial Reverse Mutagenicity test (Ames test) supports read-across from OHPA as source to 17-OHP as target resulting in no mutagenic potential. This is supported by negative findings of 17-OHP itself in in vitro mutagenicity tests in mammalian cells (HPRT and UDS). An initial positive finding in inducing chromosomal damage in the micronucleus test in vitro was disproved in a subsequent micronucleus analysis in vivo investigated in the combined repeated oral toxicity and reprotoxicity study in rats. All studies conducted with 17-OHP itself are scored to be of reliability 1 (Klimisch 1997).
Accordingly, 17-OHP will be not be categorized for genotoxicity.
Reference:
EFSA, 2017. Clarification of some aspects related to genotoxicity assessment (https://doi.org/10.2903/j.efsa.2017.5113)
Justification for classification or non-classification
Based on the available genotoxicity test battery in vitro and in vivo the test substance showed no indication to be mutagenic in vivo and therefore no classification is warranted for genotoxicity according to Regulation (EC) 1272/2008 (CLP).
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