Estr-5(10)-en-17-one, 3,3-dimethoxy-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3,3-Dimethoxyestr-5(10)-en-17-one

EC Number:

606-278-5

Cas Number:

19257-34-2

Molecular formula:

C20 H30 O3

IUPAC Name:

3,3-Dimethoxyestr-5(10)-en-17-one

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: As recommended by the OECD test guideline. The high proliferation rate were appropriate for the use of this cell line.
- Normal cell cycle time (negative control): doubling time of V79 cells in stock cultures: approximately 12 hours, determined on 03 May 2021

For cell lines:
- Absence of Mycoplasma contamination: Yes, cells were routinely checked for mycoplasma contamination
- 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 1 x E+05 – 5 x E+05 cells per flask.
- Cell cycle length, doubling time or proliferation index : doubling time 12 h
- Modal number of chromosomes: 22 ± 2
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM (Earle’s with GlutaMAX and 25mM HEPES); Pen/Strep: 1 %; FBS: 10 %, 5% CO2, 37°C.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Sprague Dawley rats
- method of preparation of S9 mix: 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).
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:

For the test item, DMSO was selected as solvent. In this solvent the test item was soluble at
least up to 133.33 mg/mL. In the solubility test precipitation in the medium was observed at 666.7 µg/mL and above. Thus, 666.7 µg/mL was chosen for top dose.

Vehicle / solvent:

DMSO

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

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration sextuplicate (6 wells per concentration were measured)
- Number of independent experiments: 3:
- 4 hours treatment, 20 hours recovery, without S9 mix
- 4 hours treatment, 20 hours recovery, with S9 mix
- 24 hours treatment, without S9 mix

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h and 24 h
- Harvest time after the end of treatment (sampling/recovery times): Approximately 24 hours after the start of treatment cells were harvested and then stained with EMA (Dye A).


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: 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 %

METHODS FOR MEASUREMENTS OF GENOTOXICITY

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

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

Rationale for test conditions:

As recommended by the OECD test guideline

Evaluation 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:

without

Genotoxicity:

not determined

Remarks:

4h treatment. Due to an induction of the micronucleus frequency in the presence of S9 mix, micronuclei in V79 cells treated with the test item in the absence of S9 mix were neither further evaluated nor statistically analyzed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 24.7 µg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

starting at 74.1 µg/mL after 4h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 74.1 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:

STUDY RESULTS
- Concurrent vehicle negative and positive control data: see 'any other information on results incl. tables'

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o When cytokinesis block is not used: For relative cytotoxicity, additional nuclei were counted at the start of treatment. The start value was determined by calculating the nuclei beads ratio from cultures of up to 30 parallel wells resulting in a mean value of 0.1. This value was allocated for RINC which was the base of the relative cytotoxicity.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see 'any other information on results incl. tables'
- Negative (solvent/vehicle) historical control data: see 'any other information on results incl. tables'

Any other information on results incl. tables

Frequency of Micronuclei
With S9 mix, the test item showed a statistically significant increase in the number of micronuclei at a concentration of 74.1 μg/mL. The induction of the micronucleus frequency was less than two-fold, but the increase was considered biologically relevant since it was outside the range of the historical solvent control.
Moreover, a concentration-related trend in the micronucleus frequency across the increasing concentration levels of the test item was found following 4 hours treatment with S9 mix.
Thus, overall, the micronucleus test showed a relevant increase in the micronucleus frequency in V79 cells treated with the test item in the presence of S9 mix (4 hours treatment). Due to an induction of the micronucleus frequency in the presence of S9 mix, micronuclei in V79 cells treated with the test item in the absence of S9 mix were neither further evaluated nor statistically analyzed.

Frequency of Apoptotic/Necrotic Nuclei
No biologically relevant increases in the numbers of apoptotic/necrotic nuclei were detected after 4 hours treatment. After the 24 h treatment period an increase in the number of apoptotic/necrotic nuclei (16 % A/N) was detected at a test item concentration of 74.1 µg/mL. This concentration was excluded from analysis due to excessive cytotoxicity.

Frequency of Hypodiploid Nuclei
No biologically relevant increases of numbers of hypodiploid nuclei were detected after 4 hours treatment. The same was true for a treatment period of 24 hours.

Results and concurrent control data:

 

 

 

 

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
				Micronuclei in %
Solvent or Substance	S9 Mix	Conc.	Number of studies	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
Solvent control	0.0	0.8	1.1	0.1	-	no
Positive control MMC	0.1	2.8	18.2#	0.3	39.3 a	no
Test item	0.3	0.7	1.1	0.1	0.0	no
	0.91	0.8	1.2	0.1	0.0	no
	2.74	0.8	1.2	0.1	0.0	no
	8.23	0.7	1.0	0.1	0.0	no
	24.7	0.9	1.4	0.1	28.0 a	no
	74.1	2.9	2.6	0.1	96.3 b	no
	222.2	DIV/0!	DIV/0!	DIV/0!	DIV/0! c	no
	666.7	DIV/0!	DIV/0!	DIV/0!	DIV/0! c	yes
a relevant cytotoxicity b excessive cytotoxicity, (above limit = 55 ± 5%) c concentration excluded from flow cytometric assessment # biologically relevant increase
Summary of the Results (4 Hours Treatment +S9 Mix)
	Conc. µg/mL	% A/N	%MN	%HD	% rel. Cytotoxicity	Precipitation
Solvent control	0.0	0.7	1.3	0.1	-	no
Positive control CP	2	3.2	15.5*	0.1	34.5 a	no
Test item	0.3	0.5	1.3	0.1	13.0	no
	0.91	0.7	1.4	0.1	2.7	no
	2.74	0.8	1.4	0.1	0.0	no
	8.23	1.0	1.3	0.1	0.2	no
	24.7	0.7	1.6	0.1	9.2	no
	74.1	1.0	2.4*	0.1	45.2 a	no
	222.2	DIV/0!	DIV/0!	DIV/0!	DIV/0! c	no
	666.7	DIV/0!	DIV/0!	DIV/0!	DIV/0! c	yes
a relevant cytotoxicity c concentration excluded from flow cytometric assessment * statistically significant increase of micronucleated events (P = < 0.05)
Summary of the Results (24 Hours Treatment –S9 Mix)
	Conc. µg/mL	% A/N	%MN	%HD	% rel. Cytotoxicity	Precipitation
Solvent control	0.0	0.6	1.3	0.1	-	no
Positive control VSS	0.00018	2.9	20.8#	5.5#	65.5 b	no
Test item	0.3	0.4	1.2	0.1	0.9	no
	0.91	0.5	1.3	0.1	0.0	no
	2.74	0.5	1.3	0.1	0.0	no
	8.23	0.5	1.1	0.1	11.8	no
	24.7	1.3	1.0	0.1	79.0 b	no
	74.1	16.4	1.8	0.3	106.4 b	no
	222.2	DIV/0!	DIV/0!	DIV/0!	DIV/0! c	no
	666.7	DIV/0!	DIV/0!	DIV/0!	DIV/0! c	yes
b excessive cytotoxicity, (above limit = 55 ± 5%) c concentration excluded from flow cytometric assessment # biologically relevant increase

Applicant's summary and conclusion

Conclusions:

The present study was conducted according to OECD guideline 487 (2016). Chinese hamster lung fibroblasts (V79) were exposed to 0, 0.3, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, and 666.7 µg/mL Dimethoxyketal for 4h (with and without metabolic acitvation) and 24 h (without metabolic activation). The frequency of micronuclei, apoptotic/necrotic nuclei and of hypodiploid nuclei were determined. Under the experimental conditions reported the test item induced chromosome breakage (structural chromosomal aberrations) leading to micronucleus formation stemming from V79 cells in vitro in the presence of metabolic activation.

Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487 (2016), V79 cells cultured in vitro were exposed to Dimethoxyketal in DMSO at concentrations of  0, 0.3, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, and 666.7 µg/mL in the presence and absence of mammalian metabolic activation [rat S9 liver mix] for either 4 h (with and without metabolic activation) and 24 h (without metabolic activation).

Dimethoxyketal was tested up to cytotoxic concentrations (i.e., 24.7 µg/mL (24 h) and 74.1 µg/mL (4 h. With S9 mix, the test item showed a statistically significant increase in the number of micronuclei at a concentration of 74.1 μg/mL. The induction of the micronucleus frequency was less than two-fold, but the increase was considered biologically relevant since it was outside the range of the historical solvent control. The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background.

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 Dimethoxyketal is considered to increase micronuclei in Chinese hamster lung fibroblasts (V79) after metabolic activation.