19-norpregn-4-en-20-yn-3-one, 17-hydroxy-7-methyl, (7alpha, 17alpha-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2018 - 25 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2(R1). Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human use. CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(8R,9S,10R,13S,14S,17R)-17-ethynyl-17-hydroxy-13-methyl-1,2,8,9,10,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one

EC Number:

608-630-3

Cas Number:

31528-46-8

Molecular formula:

C20H24O2

IUPAC Name:

(8R,9S,10R,13S,14S,17R)-17-ethynyl-17-hydroxy-13-methyl-1,2,8,9,10,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one

Test material form:

solid: particulate/powder

Details on test material:

Appearance: Off-white powder
Test item storage: At room temperature

Specific details on test material used for the study:

The test item is toxic to reproduction.

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose-range finding test, reported as part of the first experiment (TA100 and WP2uvrA):
Without and with S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item exhibited limited solubility.

First experiment (TA1535, TA1537 and TA98):
Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate.
The top dose was selected based on the results of the dose-range finding study.

Second experiment (all tester strains):
Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1000 μg/plate.
The top dose was selected based on the results of the first experiment.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: according to guidelines.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

For details on the positive controls, see table 1 in 'Additional information on materials and methods'

Details on test system and experimental conditions:

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3 (in both independent experiments)

EXPERIMENTAL DESIGN: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test results

Key result

Species / strain:

other: All tested strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Cytotoxicity: in both experiments, without and with S9, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

- Mutagenicity: in the first experiment, in tester strain WP2uvrA in the presence of S9-mix, the test item induced a 2.2-fold increase
in the number of revertant colonies compared to the solvent control. In the second experiment, in tester strain TA1537 in the absence of S9-mix, the test item induced a 3.0-fold increase in the number of revertant colonies compared to the solvent control. However, both these increases were only observed in one experiment. Furthermore, the increases were within the historical control data range. Therefore, the increases are considered to be not biologically relevant.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the dose range finding test, precipitation was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards. In the first experiment, precipitation was observed at the concentration of 1600 μg/plate at the start of the incubation period and at 512 μg/plate and above at the end of the incubation period.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: reported as the first experiment

HISTORICAL CONTROL DATA: see table 2 and 3 in 'Any other information on results'
- Results of the solvent control and of the positive control were within the historical data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Any other information on results incl. tables

Table 2 Historical data for the solvent control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 - 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 - 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Table 3 Historical data for the positive control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	-	+	-	+	+	-	+
Range	125 -1248	73 - 1206	55 – 1353	54 – 1051	365 - 1995	250 – 1977	439 - 1848	408 –2651	93 – 1951	111 - 1359
Mean	846	219	787	353	1406	887	901	1232	1094	437
SD	146	119	345	162	258	349	168	343	477	149
n	2348	2229	2003	2234	2200	2276	2335	2327	2021	2084

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Applicant's summary and conclusion

Conclusions:

The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that D6-Noreth is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.