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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test): positive [Reimann, 2004]

Gene mutation (bacterial reverse mutation assay / Ames test): positive [Li, 2020]

Gene mutation (bacterial reverse mutation assay/QSAR prediction with Leadscope and DEREK): negative [Wichard, 2021]

In vitro mammalian micronucleus test; OECD 487: negative [Ziegler, 2021]

Link to relevant study records

Reference

Reference 1

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

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

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.

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'

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

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

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'

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

 

 

 

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).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
DEREK Nexus 6.1
2. MODEL (incl. version number)
DEREK Nexus 6.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
n/a
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.6.1. Genetic toxicity in vitro
- Unambiguous algorithm: logic of argumentation. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features) inactive
- Defined domain of applicability: The scopes of the structure-activity relationships describing the mutagenicity endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for mutagenicity it can be considered to be within the applicability domain. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features. The applicability domain of each alert is defined by the alert developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For non-alerting compounds, users should determine the applicability of negative predictions by evaluating the information supplied by Derek (i.e. the presence or absence of misclassified and/or unclassified features).
- Appropriate measures of goodness-of-fit and robustness and predictivity: n/a
- Mechanistic interpretation: All alerts describing structure-activity relationships for the mutagenicity endpoint have a mechanistic basis wherever possible.
Mechanistic information is detailed in the comments associated with an alert and can include information on both the mechanism of action and biological target. The mechanistic basis of the model was developed a priori by examining the toxicological and mechanistic evidence before developing the structure-activity relationship.

5. APPLICABILITY DOMAIN

- Descriptor domain:
[1]Markush structures encoding activating and deactivating features (known as patterns in the Derek Nexus knowledge base)
[2]count of non-hydrogen atoms
[3]ClogP
[4]2D structural fragments
There is an a priori assumption that patterns and associated reasoning will be used to model toxicity within Derek Nexus. Further, experts identified that misclassified and unclassified features were useful descriptors for determining the reliability of negative predictions for non-alerting compounds.
- Similarity with analogues in the training set: Non-proprietary elements of the training set are available through the references, and illustrated by the examples, within Derek Nexus. The illustrative examples are not available, due to the proprietary nature of Derek Nexus.

6. ADEQUACY OF THE RESULT
Based on the common structure of the substance and the absence of any strucutral alert, the result is considered reliable.

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Version / remarks:

Version 3.1 July 2016

Principles of method if other than guideline:

- Software tool(s) used including version: DEREK Nexus 6.1
- Model(s) used: DEREK Nexus 6.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Species / strain / cell type:

bacteria, other: Predictions are made for the domain of bacteria and can be broken down into species (e.g. Salmonella typhimurium and Escherichia coli)

Additional strain / cell type characteristics:

other: The prediction is based on results from all tester strains recommended by the OECD Test Guideline

Evaluation criteria:

Two types of models were used to predict the mutagenic potential of the test item.
The DEREK Nexus model was used as a rule-based model which is based on the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. The second model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. If experimental data are available the prediction of the statistical model may be overruled.

Key result

Species / strain:

bacteria, other: not applicable for in silico study

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

The test item showed no alerts for mutagenicity. Therefore Delta-5-Norandrostendione was considered to be non-mutagenic and was assigned to mutagenic impurity class 5.

Cytotoxicity / choice of top concentrations:

other: not applicable for in silico study

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Based on the predictions performed with the statistical QSAR model Leadscope Applier and the rule-based model DEREK Nexus Delta-5-Norandrostendione is not mutagenic in a bacterial reverse mutation assay.

Executive summary:

In a QSAR prediction using DEREK Nexus v6.1 the potential of Delta-5-Norandrostendione to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

 

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Furthermore, the query structure does not contain an unclassified feature and is consequently not predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are available clearly reporting a negative result.

 

Based on these results Delta-5-Norandrostendione is considered non-mutagenic as predicted by DEREK Nexus.

 

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Leadscope model applier (v3.0.2)

2. MODEL (incl. version number)
Leadscope model applier (v3.0.2)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 3962-66-1; Chemical name: Delta-5-Norandrostendion; SMILES: C[C@]12CC[C@H]3[C@H]([C@@H]1CCC2=O)CCC4=C3CCC(=O)C4

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: QMRF 4.10. Mutagenicity OECD 471 Bacterial Reverse Mutation Test

- Unambiguous algorithm:
A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities (white paper) http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
The logic for matching alerts is detailed in "A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities" (white paper): http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf

- Defined domain of applicability: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).

- Appropriate measures of goodness-of-fit and robustness and predictivity: Chemicals/descriptor ratio: 241 alerts for 11,528 reference chemicals (ratio = 48); Alerts are run within the Leadscope model applier that provides the capability to specify one or more compounds (using SMILES, Mol files, SD files, or copying from the clipboard), select and run the alerts, assess the applicability domain, and view the results including an explanation for any prediction (such as a full description of any matched alerts). The performance was assessed using the Hansen dataset comprised of 3,700 chemicals (47% positive).
Concordance = 83%, Sensitivity = 92%, Specificity = 70%, Positive
Predictivity = 81%, Negative Predictivity = 86% , coverage = 95% were
obtained.

- Mechanistic interpretation: Accompanying any positive prediction, any alert(s) that match the test compounds are described including a description of the mechanistic basis from the literature reference that cites the alert.

5. APPLICABILITY DOMAIN

- Descriptor domain: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).

- Structural domain: Leadscope Predictive Data Miner is a software program for systematic sub‐structural analysis of a chemical using predefined structural features stored in a template library, training set‐dependent generated structural features (scaffolds) and calculated molecular descriptors. The feature library contains approximately 27,000 pre‐defined structural features and the structural features chosen for the library are motivated by those typically found in small molecules: aromatics, heterocycles, spacer groups, simple substituents. Leadscope allows for the generation of training set‐dependent structural features (scaffold generation), and these features can be added to the pre‐defined structural features from the library and be included in the descriptor selection process.

- Mechanistic domain: The global model identifies structural features and molecular descriptors which in the model development was found to be statistically significant associated with effect. Many predictions may indicate modes of action that are obvious for persons with expert knowledge for the endpoint

- Similarity with analogues in the training set: The original data set from Kazius et al. (2005) consisted of 4337 molecular structures with corresponding Ames test data.
The structural similarity of the test compound with respect to the training set compounds was analysed and quantified in terms of Tanimoto distance, which provides a quantitative measure of structural relatedness between the test compound and each training set compound. The 25 training set compounds found to be mostly similar to the test compound.

6. ADEQUACY OF THE RESULT
As can be seen from Annex A and B of the QPRF the result is considered adequate due to the presence of almost all structural features of the parent compound which can also be found in the training/validation dataset. Furthermore the prediction substantiate the experimental result for the substance of interest.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version: Leadscope model applier (v3.0.2)
- Model(s) used: Leadscope model applier (v3.0.2)
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: Combination of results from the S. typhimurium histidine reversion gene mutation test using tester strains TA97, TA97a, TA1537, TA98, TA100, TA1535, TA102, E.coli (any variant)

Additional strain / cell type characteristics:

other: The QSAR prediction is based on results from all tester strains recommended by the OECD Test guideline

Evaluation criteria:

The model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. Only descrete organic compounds can be predicted. The model searches for structural fragments and combines them with eight molecular descriptors. Thus, a probability of either a negative or positive result is calculated. If experimental data are available the prediction of the statistical model may be overruled.

Key result

Species / strain:

other: not applicable for in silico study

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

The test item showed no alerts for mutagenicity. Therefore the test item was considered to be non-mutagenic.

Cytotoxicity / choice of top concentrations:

other: not applicable for in silico study

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Based on the predictions performed with the statistical QSAR model Leadscope Applier Delta-5-Norandrostndione is not mutagenic in a bacterial reverse mutation assay.

Executive summary:

In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of  Delta-5-Norandrostendione to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 28 training compounds were identified in the model training set being structurally similar to the test compound.

 

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope. Furthermore, the query structure does not contain an unclassified feature and is consequently predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are also available reporting a negative result.

 

Based on these results Delta-5-Norandrostendione is considered non-mutagenic as predicted by Leadscope.

 

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30/03/2020 to 14/05/2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

only plate incorporation test

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-auxotrophic Salmonella typhimurium
including TA97a, TA98, TA100, TA102, and TA1535.

Species / strain / cell type:

S. typhimurium TA 97a

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- S9 liver mix
- concentration or volume of S9 mix and S9 in the final culture medium:
Preparation of S9 mixture (S9 mix)-related solutions:
(a) Salt solution [1.65 mol/L potassium chloride (KCl) + 0.4 mol/L magnesium chloride (MgCl2 )]
Potassium chloride (KCl) 6.15 g
Magnesium chloride (MgCl2 x 6 H2O) 4.07 g
Ultra-pure water diluted to 50 mL
(b) 0.2 mol/L phosphate buffer solution (pH 7.4)
Disodium hydrogen phosphate (Na2 x HPO4) 12.496 g
Sodium dihydrogen phosphate (NaH2PO4 x 2 H2O) 1.872 g
Ultra-pure water added to 500 mL
(c) 0.025 mol/L co-enzyme-II (oxidation model) solution
Oxidation model co-enzyme-II (NADP) 1.9685 g
Sterile, ultra-pure water added to 100 mL
(d) 0.05 mol/L glucose-6-disodium phosphate salt solution
Glucose-6-disodium phosphate salt solution (G-6-P-Na2) 1.5205 g
Sterile, ultra-pure water added to 100 mL

The 500 μL phosphate buffer solution in the non-metabolic activation test was replaced with 500 μL S9-mix

Test concentrations with justification for top dose:

The pre-test used DMSO as the solvent and a total of six doses were established, respectively, 5000, 3000, 2000, 1000, 300, and 60 μg/plate. Under not metabolically activated conditions, the plate incorporation test was carried out on the test bacterial strains TA97a, TA98, TA100, TA102, and TA1535, two plates per group, and a simultaneous solvent control was established.
The results of the pre-test indicated that, under not metabolically activated conditions, prior to the start of the test and after the end of culturing, large amounts of precipitation were present at dose 5000 μg/plate and the count was affected. At dose 3000 μg/plate large amounts of precipitation were present prior to the start of culturing while after culturing ended there was no precipitation. At dose 2000 μg/plate a small amount of precipitation was present prior to culturing while after culturing ended there was no precipitation. For the other dose groups there was no precipitation present either prior to culturing or after culturing ended. At dose 5000 μg/plate, revertant bacteria count, deaths, and background could not be counted or were clearly lower than the solvent control group. At dose 3000 μg/plate, revertant bacteria count and background could not be counted or were clearly lower than in the solvent control group.

Based on these results the treatment concentrations were 0, 37, 111, 333, 1000, and 3000 µg/plate.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Based on the results of the above-described pre-test, DMSO was selected as the solvent.

- Justification for percentage of solvent in the final culture medium: In the first test 0.1797 g of test sample was weighed and in the repeat test 0.1799 g of test sample was weighed. They were dissolved in 6 mL DMSO. Under sterile conditions at the prepared maximum dose solution 30 mg/L, gradient dilution was performed using DMSO, to prepare test sample solutions at the other doses. The concentrations were 10, 3.33, 1.11, and 0.37 mg/mL. All test samples were freshly prepared on the day of exposure.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; effects on count number, i.e. counting not possible or a strong reduction of counts


METHODS FOR MEASUREMENTS OF GENOTOXICIY
At least one bacterial strain among the bacteria strains was tested. When it was a metabolically activated or not metabolically activated system, the revertant bacteria count in the test sample group was compared to the solvent control group and exceeded a certain numerical range (i.e. for the test bacteria TA1535 test sample group, the revertant bacteria count was equal to or greater than three times the mean of the solvent control group while, in the other test bacteria test sample groups, revertant bacteria counts were equal to or greater than two times the solvent control group). In addition, a dose-response relationship existed among all doses, or at a certain test point (bacterial strain or dose) the increase in revertant bacteria count was reproducible, and the test results could be judged positive. Otherwise, the test results were negative.

Rationale for test conditions:

As recommended by the guideline

Evaluation criteria:

At least one bacterial strain among the bacteria strains was tested. When it was a metabolically activated or not metabolically activated system, the revertant bacteria count in the test sample group was compared to the solvent control group and exceeded a certain numerical range (i.e. for the test bacteria TA1535 test sample group, the revertant bacteria count was equal to or greater than three times the mean of the solvent control group while, in the other test bacteria test sample groups, revertant bacteria counts were equal to or greater than two times the solvent control group). In addition, a dose-response relationship existed among all doses, or at a certain test point (bacterial strain or dose) the increase in revertant bacteria count was reproducible, and the test results could be judged positive. Otherwise, the test results were negative.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 3000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 3000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

observed at 3000 µg/plate, slightly higher than in vehicle control

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

slightly higher than in the vehicle contorl at 333 µg/plate, clearly higher (twofold or more) at 3000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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
- Precipitation and time of the determination: Whether metabolically activated or not metabolically activated, at the 3000 μg/plate dose, large amounts of precipitation were macroscopically observed prior to starting the culture and after ending the culture but there were no signs of precipitation in the other dose groups.


RANGE-FINDING/SCREENING STUDIES (if applicable): See: 'Test concentration with jusitification for top dose'

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


Ames test:
- Signs of toxicity: yes, see: 'Details on test system and experimental conditions'
- Mean number of revertant colonies per plate and standard deviation: see '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)
not reported

First test (-S9)
		-S9
Dose µg/plate	Statistical results	TA97a	TA98	TA100	TA102	TA1535
Solvent control	Mean ± SD ratio	185.33±9.45 1.00	18.67±3.51 1.00	89.00±8.19 1.00	323.00±14.93 1.00	7.67±3.21 1.00
3000	Mean ± SD ratio	936.33±125.91 5.05	121.33±5.86 6.50	139.00±22.27 1.56	151.33±20.65 0.47	1.67±0.58 0.22
1000	Mean ± SD ratio	731.00±25.94 3.94	75.00±6.00 4.02	119.00±4.58 1.34	236.33±7.51 0.73	10.00±1.00 1.30
333	Mean ± SD ratio	347.67±91.77 1.88	26.00±2.00 1.39	101.67±0.58 1.14	255.00±28.79 0.79	6.00±3.46 0.78
111	Mean ± SD ratio	186.33±14.64 1.01	18.33±3.06 0.98	72.67±4.16 0.82	290.67±5.13 0.90	6.00±3.61 0.78
37	Mean ± SD ratio	175.33±7.23 0.95	18.00±1.00 0.96	83.00+2.65 0.93	295.00±11.79 0.91
positive control	Mean ± SD ratio	4097.33±32.58 22.11	1094.67±88.12 58.64	638.33±55.08 7.17	2418.00±35.37 7.49	313.00±35.38 40.83
First test (+S9)
		+S9
Dose µg/plate	Statistical results	TA97a	TA98	TA100	TA102	TA1535
Solvent control	Mean ± SD ratio	221.00±4.58 1.00	35.00±7.00 1.00	92.00±9.85 1.00	322.67±10.41 1.00	9.33±1.53 1.00
3000	Mean ± SD ratio	866.33±32.65 3.92	130.67±16.77 3.73	85.67±5.03 0.93	126.33±7.02 0.39	2.33±1.53 0.25
1000	Mean ± SD ratio	195.00±2.00 0.88	26.00±4.36 0.74	70.33±4.73 0.76	231.67±31.56 0.72	7.00±1.73 0.75
333	Mean ± SD ratio	209.33±22.23 0.95	26.33±2.08 0.75	91.67±4.04 1.00	253.33±10.02 0.79	7.00±1.73 0.75
111	Mean ± SD ratio	237.33±21.78 1.07	28.00±6.56 0.80	78.67±1.53 0.86	281.67±10.41 0.87	8.00±3.46 0.86
37	Mean ± SD ratio	245.33±19.60 1.11	34.33±4.73 0.98	87.00±7.94 0.95	295.33±5.51 0.92	9.00±2.00 0.96
positive control	Mean ± SD ratio	856.33±12.22 3.87	920.00±16.00 26.29	628.33±29.94 6.83	996.00±23.07 3.09	106.00±8.00 11.36
Repeat test (-S9)
		-S9
Dose µg/plate	Statistical results	TA97a	TA98	TA100	TA102	TA1535
Solvent control	Mean ± SD ratio	203.33±17.21 1.00	35.67±3.06 1.00	105.00±15.13 1.00	305.67±24.70 1.00	11.33±5.03 1.00
3000	Mean ± SD ratio	1019.33±176.16 5.01	142.67±5.86 4.00	195.67±30.55 1.86	127.33±13.58 0.42	0.33±0.58 0.03
1000	Mean ± SD ratio	492.67±21.57 2.42	87.67±14.22 2.46	174.67±9.02 1.66	221.33±16.80 0.72	22.33±2.89 1.97
333	Mean ± SD ratio	387.00±18.33 1.90	54.33±2.31 1.52	121.33±8.74 1.16	324.33±23.86 1.06	9.33±2.08 0.82
111	Mean ± SD ratio	226.33±10.02 1.11	39.33±3.06 1.10	103.33±17.21 0.98	256.33±16.26 0.84	12.00±3.61 1.06
37	Mean ± SD ratio	206.67±6.43 1.02	42.33±2.08 1.19	97.33±6.03 0.93	338.33±55.95 1.11	11.00±2.65 0.97
positive control	Mean ± SD ratio	3952.00±44.54 19.44	989.33±86.57 27.74	913.33±65.43 8.70	3240.00±62.48 10.60	360.33±24.19 31.79
Repeat test (+S9)
		+S9
Dose µg/plate	Statistical results	TA97a	TA98	TA100	TA102	TA1535
Solvent control	Mean ± SD ratio	233.67±10.60 1.00	31.67±4.16  1.00	105.67±7.09 1.00	318.00±10.15 1.00	13.67±3.21 1.00
3000	Mean ± SD ratio	1134.33±127.45 4.85	126.33±4.93 3.99	102.33±3.06 0.97	114.33±7.77 0.36	6.33±3.21 0.46
1000	Mean ± SD ratio	343.00±45.08 1.47	27.67±7.77  0.87	98.67±10.26 0.93	228.33±5.51 0.72	9.67±3.06 0.71
333	Mean ± SD ratio	319.00±35.68 1.37	30.00±6.56  0.95	105.00±7.21 0.99	254.67±7.51 0.80	10.33±2.52 0.76
111	Mean ± SD ratio	219.00±45.83 0.94	27.00±7.00  0.85	104.33±8.74 0.99	296.33. ±43.41 0.93	12.67±3.21 0.93
37	Mean ± SD ratio	279.33±11.06 1.20	29.00±3.61  0.92	101.67±12.50 0.96	293.67±15.95 0.92	13.00±3.61 0.95
positive control	Mean ± SD ratio	865.33±16.17 3.70	1258.00±193.40 39.73	784.00±13.11 7.42	1127.33±105.19 3.55	104.67±13.61 7.66

Conclusions:

In the present test conducted according to OECD guideline 471 (1997) S. typhimurium Strains TA97a, TA98, TA100, TA102, and TA1535 were exposed to 0, 37, 111, 333, 1000, 3000 µg/plate Delta-5-Norandrostendion, in the presence and absence of metabolic activation, in a plate incorporation assay. In the tester strains TA97a, TA98 and TA100 the test item induced an induction of revertant colonies of twofold or more at 3000 µg/plate with and without metabolic acitvation. In the tester strains TA102 and TA1535 no increase at this concentration could be observed due to high toxicity of the substance. Based on these results the substance is considered mutagenic under the conditions of the test.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 97a, TA98, TA 100, TA 1535, and TA 102 of S. typhimurium were exposed to Delta-5-Norandrostendione in DMSO at concentrations of 0, 37, 111, 333, 1000, and 3000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

The test item was tested to the concentration of 3000 µg/plate because higher concentration showed precipitation. TA97a, TA98 and TA100 showed increased reversion to prototrophy at the highest tested concentration 3000 µg/plate, either in the absence or presence of S9 mix. The other tester strains showed no increased reversion to prototrophy at all concentrations with or without metabolic activation except for the highest concentration where a strong reduction of counts was observed (i.e.: cytotoxicity). The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Evaluation of the data does indicate that the test substance is a mutagen in the Ames-Test. Three of the five tester strains (all strains of Salmonella typhimurium) showed increased reversion to prototrophy with the test substance at the doses tested in the absence and presence of S9 mix. Only TA102 and TA1535 showed no increased reversion to prototrophy.

 

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from March to April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

only plate incorporation procedure was conducted

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus; tryptophan gene locus

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Sprague-Dawley rat liver S9 mix

Test concentrations with justification for top dose:

0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 mg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

ethylmethanesulphonate

other: anthracen-2-amine, 4-Nitro-o-phenylenediamine, N-Methyl-N¿-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

- Exposure duration: ca. 72 hours (S. typhimurium) and ca. 48 hours (E. coli)
- all plates were prepared in triplicates

Evaluation criteria:

A positive response was considered if the number of revertants of the compound groups compared to the number of revertants of the
negative group was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.

Statistics:

The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 0.25 mg onwards

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

from 0.05 mg onwards

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

from 0.5 mg onwards

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

from 0.25 mg onwards

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

from 0.5 onwards

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

between 0.05 and 0.25 mg

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 2.5 mg on

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitates in the agar were found starting at 2.5 mg/plate onwards.
Growth inhibition of the background lawn was observed at 2.5 and 5.0 mg/plate.

All S. typhimurium tester strains used, i.e. TA1535, TA100, TA1537, TA1538 and TA98, showed increased reversion to prototrophy in assays with the test substance at the doses tested between 0.05 and 5.0 mg/plate in the absence of S9 mix. In the experiments with S9 mix only the S. typhimurium strains TA1535 and TA98 showed an increased reversion to prototrophy. However, with E. coli no mutagenic effect occurred without and with metabolie activation after incubation with the test substance.

Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.

Results for each tester strain:

TA 1535:

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
DMSO	50 µL	17	14	7	7	10	3	1.0	1.0
		18			9
		6			13
Phosphate buffer	50 µL	16	16	2	18	13	6	1.1	1.3
		17			13
		14			7
Test item	0.05 mg	18	16	6	15	14	3	1.1	1.4
		20			10
		9			16
Test item	0.1 mg	17	17	3	11	10	1	1.2	1.0
		14			9
		20			10
Test item	0.25 mg	28	26	6	13	12	1	1.9	1.2
		31			11
		19			11
Test item	0.5 mg	35	31	4	12	14	2	2.3	1.5
		31			16
		28			15
Test item	1.0 mg	38	44	6	16	17	3	3.2	1.8
		46			15
		49			20
Test item	2.5 mg	43 mPB	48	6	24 PB	22	3	3.5	2.2
		55 mPB			22 mPB
		46 mPB			19 mPB
Test item	5.0 mg	31 mPB	33	5	20 mPB	13	7	2.4	1.3
		38 mPB			7 mPB
		29 mPB			12 mPB
Anthracen-2-amine	2.5 µg	15	13	2	122	126	12	1.0	13.0
		11			139
		14			117
Cyclophosphamide	400.0 µg	38	38	5	218	220	19	2.8	22.7
		33			239
		43			202
Sodium azide	5.0 µg	436	436	29	50	52	10	31.9	5.3
		407			43
		464			62

 

TA 100:

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
DMSO	50 µL	129	123	6	92	98	5	1.0	1.0
		118			100
		123			101
Phosphate buffer	50 µL	160	146	13	99	104	9	1.2	1.1
		143			114
		134			99
Test item	0.05 mg	129	151	20	96	103	12	1.2	1.1
		168			95
		156			117
Test item	0.1 mg	156	155	13	107	101	7	1.3	1.0
		142			104
		168			93
Test item	0.25 mg	232	208	21	90	101	12	1.7	1.0
		193			99
		198			114
Test item	0.5	295	302	6	99	101	15	2.5	1.0
		306			88
		306			117
Test item	1.0	391	395	24	94	104	10	3.2	1.1
		374			104
		421			113
Test item	2.5	89 mPB	92	18	97 mPB	85	11	0.7	0.9
		112 mPB			83 mPB
		76 mPB			76 mPB
Test item	5.0	101 mPB	104	9	88 mPB	85	7	0.8	0.9
		98 mPB			77 mPB
		114 mPB			90 mPB
Anthracen-2-amine	2.5 µg	108	158	45	1344	1409	87	1.3	14.4
		168			1507
		197			1375
Benzo[a]pyrene	2.5 µg	116	127	10	512	556	40	1.0	5.7
		129			589
		136			567
Sodium azide	5.0 µg	371	431	53	129	133	8	3.5	1.4
		467			128
		456			142

 

TA 1537:

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
DMSO	50 µL	12	9	3	15	14	1	1.0	1.0
		7			15
		8			13
Phosphate buffer	50 µL	9	9	3	15	13	2	1.0	0.9
		11			13
		6			11
Test item	0.05 mg	17	12	4	16	16	1	1.4	1.1
		9			17
		11			16
Test item	0.1 mg	12	14	2	11	11	1	1.5	0.7
		16			11
		13			10
Test item	0.25 mg	20	20	2	16	15	1	2.2	1.1
		22			14
		18			16
Test item	0.5 mg	30	29	2	11	14	2	3.2	1.0
		27			15
		30			15
Test item	1.0 mg	42	10	4	13	11	2	4.5	0.8
		36			10
		43			10
Test item	2.5 mg	77 PB	64	17	16 PB	14	3	7.1	1.0
		45 mPB			16 PB
		70 mPB			11 PB
Test item	5.0 mg	25 mPB	25	6	5 mPB	7	2	2.8	0.5
		31 mPB			7 mPB
		20 mPB			8 mPB
Anthracen-2-amine	2.5 µg	11	8	4	58	61	7	0.9	4.2
		10			55
		4			69
4-NPDA	10.0 µg	51	54	4	17	18	3	6.0	1.2
		53			15
		59			21

 

TA 1538:

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
DMSO	50 µL	15	13	3	26	25	7	1.0	1.0
		14			18
		9			32
Phosphate buffer	50 µL	10	12	4	39	32	6	0.9	1.3
		9			27
		17			30
Test item	0.05 mg	16	18	5	27	30	5	1.4	1.2
		23			27
		14			35
Test item	0.1 mg	25	18	6	18	22	3	1.4	0.9
		15			23
		14			24
Test item	0.25 mg	11	17	8	33	29	4	1.3	1.1
		14			28
		26			26
Test item	0.5 mg	29	28	2	24	25	4	2.2	1.0
		26			29
		30			22
Test item	1.0 mg	34	39	5	22	21	5	3.1	0.8
		43			15
		40			25
Test item	2.5 mg	46 mP	62	14	54 P	49	5	4.9	1.9
		67 mP			45 P
		73 mP			48 P
Test item	5.0 mg	47 mPB	51	19	38 mPB	29	9	4.0	1.1
		34 mPB			28 mPB
		72 mPB			21 mPB
Anthracen-2-amine	2.5 µg	12	11	2	668	644	25	0.8	25.4
		11			645
		9			619
2-Nitrofluorene	10.0 µg	1031	1098	61	311	320	8	86.6	12.6
		1151			326
		1111			322

 

TA 98:

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
DMSO	50 µL	25	25	6	38	40	3	1.0	1.0
		30			38
		19			43
Phosphate buffer	50 µL	17	23	7	37	36	1	0.9	0.9
		30			37
		22			35
Test item	0.05 mg	68	55	13	44	42	2	2.2	1.1
		43			42
		55			41
Test item	0.1 mg	90	100	11	33	39	6	4.1	1.0
		99			39
		111			44
Test item	0.25 mg	201	208	29	42	42	7	8.4	1.1
		239			36
		183			49
Test item	0.5 mg	417	345	63	38	40	5	14.0	1.0
		316			36
		301			45
Test item	1.0 mg	780	761	67	49	56	7	30.8	1.4
		816			62
		687			57
Test item	2.5 mg	875 P	892	25	96 P	93	9	36.2	2.3
		921 P			99 P
		880 P			83 P
Test item	5.0 mg	835 PB	740	83	45 mPB	49	5	30.0	1.2
		700 PB			55 mPB
		684 PB			47 mPB
Anthracen-2-amine	2.5 µg	20	21	1	624	631	7	0.8	15.9
		21			630
		21			638
Benzo[a]pyrene	2.5 µg	32	23	8	224	256	45	0.9	6.4
		19			307
		18			236
2-Nitrofluorene	10.0 µg	857	832	24	293	296	37	33.7	7.5
		828			334
		810			260

 

E. coli WP2uvrA:

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
DMSO	50 µL	23	24	10	24	26	3	1.0	1.0
		15			25
		35			29
Phosphate buffer	50 µL	21	28	6	27	25	4	1.2	1.0
		33			21
		31			28
Test item	0.05 mg	20	24	6	20	26	7	1.0	1.0
		31			34
		21			23
Test item	0.1 mg	21	23	3	22	23	4	1.0	0.9
		27			20
		22			28
Test item	0.25 mg	19	23	8	29	25	6	0.9	0.9
		17			18
		32			27
Test item	0.5 mg	21	22	2	16	25	6	0.9	0.9
		22			26
		24			26
Test item	1.0 mg	19	15	5	27	23	4	0.6	0.9
		16			22
		10			19
Test item	2.5 mg	23 mPB	19	4	18 mPB	21	3	0.8	0.8
		16 mPB			24 mPB
		19 mPB			20 mPB
Test item	5.0 mg	19 mPB	19	3	24 mPB	22	2	0.8	0.9
		22 mPB			21 mPB
		16 mPB			22 mPB
Anthracen-2-amine	10.0 µg	36	37	5	182	178	5	1.5	6.8
		32			172
		42			180
EMS	5.0 µL	640	599	41	151	607	166	24.6	23.3
		600			507
		558			798
MNNG	5.0 µg	222	253	28	197	215	16	10.4	8.3
		274			226
		264			222

M=mean

P=Precipitation

m=manually scored

C=Contamination

B= Background lawn reduced

± SD=Standard Deviation

+S9= With S9 mix

-S9=Without S9 mix

Quotient= Mean revertants (test substance)/Mean revertants (Solvent)

 

 

 

 

 

Conclusions:

The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, 1538 and TA 1537 and E. Coli in the presence and absence of S9 mix according to OECD TG 471. Evaluation of the data does indicate that the test substance is a mutagen in the Ames-Test. Five of the six tester strains (all strains of Salmonella typhimurium) showed increased reversion to prototrophy with the test substance at the doses tested in the absence of S9 mix. Additionally an increased reversion to prototrophy occurred with the strains TA1535 and TA98 in the tests with metabolic activation. Only E. coli showed no increased reversion to prototrophy.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium and E.coli WP2uvrA were exposed to Delta-5-Norandrostendione in phosphate buffer at concentrations of 50, 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

The test item was tested limit concentration 5000 µg/plate All of the five tester strains showed increased reversion to prototrophy at different concentrations tested between 50 and 2500 µg/plate, either in the absence or presence of S9 mix. The E.coli WP2uvrA tester strain showed no increased reversion to prototrophy at all concentrations with or without metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed at the high concentrations tested (2500 and 5000 µg/plate) in the strains TA1535, TA100 and TA1537, from 5000 µg/plate onwards in strain TA98 with or without 59 mix. There were precipitates in the agar found starting from 2500 µg/plate onwards in all strains used in the tests without and with S9 mix.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Evaluation of the data does indicate that the test substance is a mutagen in the Ames-Test. Five of the six tester strains (all strains of Salmonella typhimurium) showed increased reversion to prototrophy with the test substance at the doses tested in the absence of S9 mix. Additionally, an increased reversion to prototrophy occurred with the strains TA1535 and TA98 in the tests with metabolic activation. Only E. coli showed no increased reversion to prototrophy.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium and E.coli WP2uvrA were exposed to Delta-5-Norandrostendione in phosphate buffer at concentrations of 50, 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

The test item was tested limit concentration 5000 µg/plate. All of the five tester strains showed increased reversion to prototrophy at different concentrations tested between 50 and 2500 µg/plate, either in the absence or presence of S9 mix. The E.coli WP2uvrA tester strain showed no increased reversion to prototrophy at all concentrations with or without metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed at the high concentrations tested (2500 and 5000 µg/plate) in the strains TA1535, TA100 and TA1537, from 5000 µg/plate onwards in strain TA98 with or without 59 mix. There were precipitates in the agar found starting from 2500 µg/plate onwards in all strains used in the tests without and with S9 mix.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Evaluation of the data does indicate that the test substance is a mutagen in the Ames-Test. Five of the six tester strains (all strains of Salmonella typhimurium) showed increased reversion to prototrophy with the test substance at the doses tested in the absence of S9 mix. Additionally, an increased reversion to prototrophy occurred with the strains TA1535 and TA98 in the tests with metabolic activation. Only E. coli showed no increased reversion to prototrophy.

 

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 97a, TA98, TA 100, TA 1535, and TA 102 of S. typhimurium were exposed to Delta-5-Norandrostendione in DMSO at concentrations of 0, 37, 111, 333, 1000, and 3000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

The test item was tested to the concentration of 3000 µg/plate because higher concentration showed precipitation. TA97a, TA98 and TA100 showed increased reversion to prototrophy at the highest tested concentration3000 µg/plate, either in the absence or presence of S9 mix. The other tester strains showed no increased reversion to prototrophy at all concentrations with or without metabolic activation except for the highest concentration where a strong reduction of counts was observed (i.e.: cytotoxicity). The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Evaluation of the data does indicate that the test substance is a mutagen in the Ames-Test. Three of the five tester strains (all strains of Salmonella typhimurium) showed increased reversion to prototrophy with the test substance at the doses tested in the absence and presence of S9 mix. Only TA102 and TA1535 showed no increased reversion to prototrophy.

 

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).

 

 

In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of  Delta-5-Norandrostendione to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 28 training compounds were identified in the model training set being structurally similar to the test compound.

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope. Furthermore, the query structure does not contain an unclassified feature and is consequently predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are also available reporting a negative result.

Based on these results Delta-5-Norandrostendione is considered non-mutagenic as predicted by Leadscope.

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

 

In a QSAR prediction using DEREK Nexus v6.1 the potential of Delta-5-Norandrostendione to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Furthermore, the query structure does not contain an unclassified feature and is consequently not predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are available clearly reporting a negative result.

Based on these results Delta-5-Norandrostendione is considered non-mutagenic as predicted by DEREK Nexus.

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

 

Justification for classification or non-classification

In summary, D-5-Norandrostendion exhibited genotoxic properties in bacteria (Ames) but not in mammalian cells (V79; MNT in vitro). Furthermore, an in silico Ames prediction was negative. According to the current REACH mutagenicity testing strategy following a positive result in a gene mutation test in bacteria and a negative micronucleus test in mammalian cells in vitro a subsequent in vivo testing shall be considered (e.g. a combined in vivo Comet Assay (OECD TG 489) and Micronucleus Test (OECD TG 474)). This in vivo test is, however, not considered to provide further useful information since a conclusion on carcinogenicity as the predominant resulting risk associated with human exposure to mutagenic substances is already drawn. D-5-Norandrostendion is metabolized to nandrolone (CAS 434-22-0) in vivo and therefore belongs to the class 2 of anabolic steroids which are categorized as non-mutagen, Carc. 2, RF1A and RD1B according to TRGS 905 (Federal Institute for Occupational Safety and Health, Germany, March 2020).

 

Based on these considerations a mutagenic potential is not concluded for D-5-Norandrostendion and a classification according to Regulation (EU) No. 1272/2008 (CLP) is not justified.