Estr-4-ene-3,17-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Norandrostendione was not mutagenic with and without metabolic activation in a bacterial reverse mutation assay (Ames test) (Wollny, 1996). A micronucleus test in V79 cells with norandrostendion was positive without metabolic activation (Dorn, 2008).

In addition, the source substance norethisterone and/or other members of the category "steroidal progestins" did not induce gene mutations in these assays. Chromosome aberration tests yielded contradictory results for the source substance norethisterone and its ester derivates (norethisterone acetate).  As (1) norethisterone does not directly interact with DNA (2) clastogenic effects were not reproducible between different laboratories and typically occurred at high, unphysiological concentrations, available in vitro data do not indicate that the substances possess a relevant genotoxic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 1996

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:

adopted May 26, 1983

Deviations:

yes

Remarks:

Justification why the confirmation of the negative result is not considered necessary is not provided.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: Sodium azide, 4-Nitro-o-phenylenediamine, Methyl methane sulfonate; with metabolic activation: 2-Aminoanthracene

Details on test system and experimental conditions:

plate incorporation assay

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 102

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:

Slight precipitation of the test article occurred on the plates at 5000 µg/plate. Automatic colony counting was not affected.

Toxic effects, evident as a reduction in the number of revertants, occurred in all strains with metabolic activation at 2500 and 5000 µg/plate. In the absence of metabolic activation all strains except TA 98 showed these toxic effects at a concentration of 2500 µg/plate. At the maximal concentration of 5000 µg/plate toxicity of the test article is seemingly reduced in all strains except in TA 98 where strong toxic effects occurred.

Toxic effects, evident as a reduction in the number of revertants, occurred in all strains with metabolie activation at 2500 and 5000 µg/plate. In the absence of metabolie activation all strains except TA 98 showed these toxic effeets at a concentration of 2500 µg/plate. At the maximal concentration of 5000 µg/plate toxicity of the test article is seemingly reduced in all strains except in TA 98 where strong toxic effects occurred. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate control mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. The number of spontaneous revertants in the negative control of strain TA 98 is little lower than the historical range in 1994 (15 colonies versus a range of 17 - 45 colonies). This effect was judged as biologically irrelevant since small fluctuations of the number of spontaneous revertants are common in the bacterial assay system used and the number is within the historical range of negative controls in 1993.

Conclusions:

In this bacterial reverse mutation assay no evidence of mutagenic activity was seen up to the maximum recommended dose level of 5000 µg/plate in the presence and absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, norandrostendione was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The mutagenic potential of Norandrostendione was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471 using the plate incorporation test.

The assay was performed in one experiment with and without liver microsomal activation.

Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

10.0; 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate

Toxic effects, evident as a reduction in the number of revertants, occurred in all strains with and without metabolic activation at high concentrations.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate control mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Norandrostendione is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For the assessment of the genotoxic potential of Norandrostendione, a bacterial reverse mutation assay is available as well as a non-guideline micronucleus assay. Additional studies were conducted with the source substance Norethisterone and its esters Norethisterone acetate and Norethisterone enantate. A justification is attached to Iuclid section 13.

The mutagenic potential of Norandrostendione was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471 (Wollny, 1996). Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level in the presence and absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, Norandrostendione was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

The potency of Norandrostendione to induce micronuclei in V79 cells in vitro without metabolic activation was determined via CREST analysis (Dorn, 2008) before the corresponding OECD TG came into force (2010). Evidence of clastogenic activity was seen below the limit of cytotoxicity which was examined in a separate assay. Because the observed genotoxicity was neither due to apoptosis nor to production of reactive oxygen species the ability to induce micronuclei appears related to its lipophilicity. A non-specific chromosomal genotoxicity based on hydrophobic interactions appears likely.

For Norandrostendione (CAS No. 734-32-7) no further information on genotoxicity, e.g. HPRT or MNT in vivo is available. However, the German TRGS 905 (published by the German Federal Institute for Occupational Safety and Health; TRGS = Technische Richtlinien für Gefahrstoffe) already discusses the endpoint genotoxicity for Norethisterone several steroid hormones including Norandrostendione. While genotoxicity studies do not give a consistent picture throughout the substance class, evidence for a germ cell mutagenicity was not seen and therefore none of the 71 was proposed to classify accordingly.

This is in line with the available information on the source substance Norethisterone, for which several in vitro and in vivo studies on genotoxicity are available (including studies with its esters Norethisterone acetate and Norethisterone enantate). To fulfil formal data gaps a read across for Norandrostenedione is performed with available information on in vitro gene mutation in mammalian cells of the source Norethisterone.

 

 

Conclusion

Overall, the available information does not lead to the conclusion that a genotoxic potential is present for Norandrostenedione.  

 

Justification for classification or non-classification

Based on the study results with norandrostendion and the read-across substance androstendion a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.