Oestr-5(10)-ene-3,17-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept - Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

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:

Oestr-5(10)-ene-3,17-dione

EC Number:

223-564-1

EC Name:

Oestr-5(10)-ene-3,17-dione

Cas Number:

3962-66-1

Molecular formula:

C18H24O2

IUPAC Name:

(3aS,3bR,9bS,11aS)-11a-methyl-1H,2H,3H,3aH,3bH,4H,5H,6H,7H,8H,9H,9bH,10H,11H,11aH-cyclopenta[a]phenanthrene-1,7-dione

Constituent 2

Reference substance name:

5(10)-Estrene-3,17-dione

IUPAC Name:

5(10)-Estrene-3,17-dione

Constituent 3

Reference substance name:

Delta-5-Norandrostendion

IUPAC Name:

Delta-5-Norandrostendion

Test material form:

not specified

Details on test material:

Purity not specified

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells were obtained from Merck KGaA, Darmstadt.
- Suitability of cells: The high proliferation rate (doubling time of V79 cells in stock cultures: approximately 12 hours, determined on 03 May 2021) were appropriate for the use of this cell line.
- Normal cell cycle time (negative control): 12 h

For cell lines:
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C and 5 % CO 2 in plastic flasks. Seeding was performed with about 1E+05 – 5 E+05 cells per flask. As culture medium ingredients with the following components was used: MEM (Earl´s with GlutaMAX and 25 mM HEPES); Pen/Strep: 1%; FBS: 10%
- Cell cycle length, doubling time or proliferation index : 12 h doubling time
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22 ± 2.
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: rat S9-mix
- source of S9: Liver homogenates (S9: 9000 x g fraction) were isolated in house (GLP-Prüfeinrichtung Early Development Bayer, Genetic Toxicology Wuppertal) from the livers of Aroclor 1254-induced male Sprague-Dawley rats. The used S9 fraction was derived from preparation dated 26 Nov 2019, color code green (protein content 23.8 mg/mL).
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium: For use, frozen aliquots of the S9 fraction were slowly thawed and mixed with a cofactor solution (2+3 parts). The S9 mix contained 40 % S9 fraction to result in a final concentration of 2 % S9 in cultures and was kept in refrigerator and used on the same day.

Test concentrations with justification for top dose:

Experiment 1:
-S9-mix; 4h: 0; 2; 6; 18; 50; 100; 300; 600 µg/mL
+ S9-mix; 4h: 0; 2; 6; 18; 50; 100; 300; 600 µg/mL
-S9-mix; 24h: 0; 2; 6; 18; 50; 100; 300; 600 µg/mL
Experiment 2:
-S9-mix; 4h: 0; 6; 12; 18; 24; 30; 36; 42; 48 µg/mL
+ S9-mix; 4h: 0; 30; 40; 50; 60; 70; 80; 90; 100 µg/mL
-S9-mix; 24h: 0; 2; 6; 9; 12; 15; 18; 24; 30 µg/mL
Experiment 3:
-S9-mix; 4h: 0; 6; 10; 12; 14; 16; 18; 22; 28 µg/mL
-S9-mix; 24h: 0; 2; 6; 10; 12; 14; 16; 20; 30 µg/mL

Due to the test item's toxicity, the first experiment did not meet the required concentration
range according to the guideline. The same was true for the setting 4 hours treatment without S9 mix of the second experiment. Also, as the laboratory’s internal historical control range of the solvent control (24 hours treatment without S9 mix) was not met, this setting was excluded from assessment and repeated in a third experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: In this solvent the test item was soluble at least up to 600 mg/mL. The solubility test showed that demineralized water was not suitable as solubility was lower than 5 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO at 1% (v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration six replicates/ 6 wells per concentration
- Number of independent experiments: Three experiments; due to to the test item's toxicity, the first experiment did not meet the required concentration range according to the guideline. The same was true for the setting 4 hours treatment without S9 mix of the second experiment. Also, as the laboratory’s internal historical control range of the solvent control (24 hours treatment without S9 mix) was not met, this setting was excluded from assessment and repeated in a third experiment.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2500 cells per well
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: over night
- Exposure duration/duration of treatment: 4h and 24h
- Harvest time after the end of treatment (sampling/recovery times): 24 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Approximately 24 hours after the start of treatment cells were harvested and then stained with EMA (Dye A). In addition, RNase and counting beads were added according to the pertinent instruction manual (version no. 171207) of the Micronucleus Analysis Kit (Litron).
Thereafter, cells were lysed and simultaneously the nuclei were stained with SYTOX green
(Dye B). After this, samples were submitted to flow cytometric analysis.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Micronuclei, Hypodiploid Nuclei and Apoptotic/Necrotic Nuclei
The percentage of micronuclei per nucleated events (%MN), indicative of clastogenic effects,
or hypodiploid nuclei per nucleated events (%HD), indicative of aneugenic effects, was determined. In parallel, the proportion of nuclei stemming from apoptotic or necrotic cells was detected (%A/N).
- %A/N = (A/N / Total Events) x100
- %MN = (MN / Nucleated) x100
- %HD = (HD / Nucleated) x100

Relative Increase in Nuclei Count (RINC)
Additionally, the number of nuclei originating from viable cells was related to an internal
standard (Cell Sorting Set-up Beads) as a measure of relative increase in nuclei count. For this relative increase in nuclei count, nuclei in cultures of up to 30 parallel wells were counted at the start of treatment time to determine start values.
The percentage was calculated as follows:
Mean (Nuclei/Beads) well 1-n Test Item - Mean (Nuclei/Beads) well 1-n Start
%RINC = -------------------------------------------------------------------------------------------------------------------------- x 100
Mean (Nuclei/Beads) well 1-n SC- Mean (Nuclei/Beads) well 1-n Start



METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
Determination of Relative Cytotoxicity
Relative cytotoxic effects of the test item were assessed using the relative increase in nuclei
count (RINC) in the presence and absence of S9 mix. The results of the solvent controls were
set 100 % and compared to the test substance treated cultures. A change of the RINC relative
to the corresponding solvent control was calculated as follows:
Relative Cytotoxicity % = 100% - RINC %

Rationale for test conditions:

as specified in the OECD test guideline

Evaluation criteria:

Assessment Criteria
Providing that all acceptability criteria were fulfilled, the test item was considered to be positive if:
- the test item induced a micronucleus frequency in one of the test item concentrations that is two-fold higher compared to the micronucleus frequency of concurrent solvent control
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data

Statistics:

Please refer to 'Any other information on materials and methods incl. tables'

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No biologically relevant increases of numbers of hypodiploid nuclei were detected after 4 h and 24 h treatment.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic effects were observed from 6 µg/mL on after 4 h and 24 h treatment -S9 mix, excessive cytotoxicity started at 18 µg/mL(4h) and 14 µg/mL (24h). With S9 mix, cytotoxic effects were observed from 70 µg/mL on, excessively increased at 90 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No biologically relevant increases of numbers of apoptotic/necrotic nuclei were detected after 4 h adn 24 h treatment.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic effects were observed from 6 µg/mL on after 4 h and 24 h treatment -S9 mix, excessive cytotoxicity started at 18 µg/mL(4h) and 14 µg/mL (24h). With S9 mix, cytotoxic effects were observed from 70 µg/mL on, excessively increased at 90 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no increase of micronuclei frequency in the absence or presence of S9 after 4 h and 24 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic effects were observed from 6 µg/mL on after 4 h and 24 h treatment -S9 mix, excessive cytotoxicity started at 18 µg/mL(4h) and 14 µg/mL (24h). With S9 mix, cytotoxic effects were observed from 70 µg/mL on, excessively increased at 90 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Concentrations of the test item of up to 600 µg/mL did not change the pH in the medium (phenol red-containing medium).
- Data on osmolality: The osmolality in the medium was not changed by concentrations of up to 600 µg/mL test item (highest concentration tested), analyzed with an Osmometer (Gonotec).
- Precipitation and time of the determination: No

RANGE-FINDING/SCREENING STUDIES (if applicable): Generally, the test item was dissolved in a suitable solvent according to the solubility test. For the test item, DMSO was selected as solvent. In this solvent the test item was soluble at least up to 600 mg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes, please refer to 'Any other information on results incl. tables'

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: n/a


Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: please refer to 'Any other information on results incl. tables'


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data): please refer to 'Any other information on results incl. tables'

Any other information on results incl. tables

Historical Controls

9000 – 18000 nuclei per study on flow cytometer MACSQuant 10 or Accuri C6 were evaluated.

Historical Controls 2018-2020, 4 Hours Treatment, 24 Hours Harvest Time
Solvent or substance	S9 Mix	Conc.	No. of Studies	Micronuclei in %
				Mean	SD	Min	Max
Water		1% v/v	11	1.1	0.6	0.4	2.5
DMSO		1% v/v	150	1.0	0.4	0.4	2.0
Mitomycin C		0.1 µg/mL	168	15.8	3.7	7.0	27.4
Water		1% v/v	11	1.4	0.5	0.7	2.4
DMSO		1% v/v	162	1.2	0.4	0.5	2.2
CP		2 µg/mL	173	16.0	4.3	5.9	29.2
Historical Controls 2018 - 2020, 24 Hours Treatment, 24 Hours Harvest Time
Water		1% v/v	12	1.2	0.8	0.4	2.9
DMSO		1% v/v	158	1.2	0.5	0.4	2.5
Vinblastine		0.0018 µg/mL	177	18.1	5.6	8.5	42.6

 

Summary of the Results (4 Hours Treatment –S9 Mix)
	Conc. µg/mL	% A/N	% MN	% HD	% Rel. Cytotoxicity	Precipitation (P)
Solvent Control		1.1	1.0	0.1		no
Positive Control MMC	0.1	4.1	14.5*	0.4	41.6 a	no
Test Item	6	0.9	1.1	0.0	20.4 a	no
	10	0.8	0.9	0.1	28.5 a	no
	12	0.7	0.8	0.0	24.6 a	no
	14	0.8	1.0	0.0	38.2 a	no
	16	0.8	0.8	0.1	52.7 a	no
	18	1.1	0.7	0.0	74.4  b	no
	22	1.1	0.9	0.0	83.1  b	no
	28	1.5	1.1	0.0	93.8  b	no
a relevant cytotoxicity
b concentration excluded from statistical analysis due to excessive cytotoxicity (above limit = 55 ± 5%)
* statistically significant increase of micronucleated events (P = < 0.05)
Summary of the Results (4 Hours Treatment +S9 Mix)
	Conc. µg/mL	% A/N	% MN	% HD	% Rel. Cytotoxicity	Precipitation (P)
Solvent Control		1.2	1.0	0.1		no
Positive Control CP	2	4.0	9.1*	0.1	17.5	no
Test Item	30	1.0	0.7	0.1	2.0	no
	40	0.9	0.8	0.1	11.1	no
	50	1.1	1.1	0.2	18.9	no
	60	1.1	1.4* §	0.2	11.1	no
	70	0.7	0.9	0.1	34.5 a	no
	80	0.8	1.0	0.1	54.2 a	no
	90	1.2	1.0	0.1	84.0  b	no
	100	1.5	1.2	0.1	91.9  b	no
a relevant cytotoxicity
b concentration excluded from statistical analysis due to excessive cytotoxicity (above limit = 55 ± 5%)
* statistically significant increase of micronucleated events (P = < 0.05)
§ statistical significance not considered biologically relevant
Summary of the Results (24 Hours Treatment –S9 Mix)
	Conc. µg/mL	% A/N	% MN	% HD	% Rel. Cytotoxicity	Precipitation (P)
Solvent Control		0.6	1.2	0.1		no
Positive Control VSS	0.0018	6.3	13.5*	2.0#	42.8 a	no
Test Item	2	0.6	0.8	0.0	8.4	no
	6	0.7	0.9	0.1	28.1 a	no
	10	0.5	0.7	0.0	36.9 a	no
	12	0.7	0.8	0.1	50.4 a	no
	14	1.0	0.8	0.1	68.6  b	no
	16	1.3	0.8	0.1	85.9  b	no
	20	1.7	0.8	0.0	93.5  b	no
	30	2.5	1.0	0.0	102.0  b	no
a relevant cytotoxicity
b concentration excluded from statistical analysis due to excessive cytotoxicity (above limit = 55 ± 5%)
* statistically significant increase of micronucleated events (P = < 0.05)
# biologically relevant increase of hypodiploid events

 

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported the test item did
not induce chromosome breakage (structural chromosomal aberrations) or misdistribution of
chromosomes leading to micronucleus formation stemming from V79 cells in vitro neither in
the absence nor in the presence of metabolic activation.

Executive summary:

Delta-5-Norandrostendion was tested up to cytotoxic concentrations (i.e., 6 µg/mL (24 h) and 4 h without S9 mix and 70 µg/mL (4h, with S9 mix). For the test item, no biologically relevant or/and statistically significant increases of numbers of micronuclei were detected after 4 hours and 24 hours treatment without metabolic activation. In agreement with the assessment criteria the statistically significant increased micronucleus frequency observed at 60 µg/mL after treatment with the test item for 4 hours in the presence of S9 mix, was considered to be of no biological relevance, since it was in the range of the historical controls (see Historical Controls) and not concentration correlated.

Also, no concentration-related trend in the micronucleus frequency across the increasing concentration levels of the test item was found in any of the settings.

The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background. The acceptance criteria were fulfilled.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline 487 for in vitro mammalian cell micronucleus data.

Based on the described results Delta-5-Norandrostentdion is considered non-genotoxic in the micronucleus test in vitro, when tested up to cytotoxic concentrations in Chinese hamster lung fibroblasts (V79).