3-methoxyoestra-1,3,5(10)-trien-17-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in Ames test in S. typhimurium strains TA98, TA100, TA102, TA1535, TA1537) when tested up to the highest recommended dose level of 5.0 mg/plate in the absense or presense of extrinsic metabolic activation (liver S9 mix form Aroclor 1254 -treated rats) (Wolly, 1995)

 

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment, the test article was dissolved in the vehicle. The test article formed a suspension in the stem solution.

Target gene:

Histidine locus

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 102 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 microsomal fraction

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:

yes

Positive controls:

yes

Positive control substance:

other: - S9 Mix: Sodium azide: TA100, TA 1535; 4-NOPD: TA 1537, TA 98; MMS: TA 102; + S9 Mix: 2-AA all strains

Details on test system and experimental conditions:

plate incorporation assay

For each strain and dose level, including controls three plates were used as a minimum.

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl: Test solution at each dose level, solvent (negative control) or control mutagen solution (positive control)
500 µl: S9 mix (metabolic activation) or S9 mix substitution buffer (without metabolic activation)
100 µl: Bacteria suspension
2000 µl: Overlay agar

After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

Data recording:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, Biosys GmbH, 51184 Karben)

The background growth of the bacteria was judged visually on a light bench.

Evaluation criteria:

Generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates

Evaluation of results:

A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at lest one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biological relevant positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous rate in strains TA 100 and TA 102 or thrice TA 1535, TA 1537, and TA 98.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Minor toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at concentrations of 1000 µg/plate and above.

Conclusions:

negative
Estrone methyether did not show a mutagenic potential in the bacterial reverse mutation assay.

Executive summary:

The mutagenic potential of Estrone methylether 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.

The assay was performed in one experiment with and without Iiver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

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

Minor toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at concentrations of 1000 µg/plate and above.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with ZK 5512 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 article did not induce gene mutations by base-pair changes or frame-shifts in the genome of the strains used.

Therefore, the substance 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 study available

Additional information

The mutagenic potential of Estrone methylether 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.

The assay was performed in one experiment with and without Iiver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

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

Minor toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at concentrations of 1000 µg/plate and above.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with ZK 5512 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 article did not induce gene mutations by base-pair changes or frameshifts in the genome of the strains used.

Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay (Wolly, 1995).

 

Accordingly, estrone-3-methylether is not classified (Reg (EC) No. 1272/2008) for genotoxicity.

No bacterial genotoxicity screening (TG 471) was performed with estrone but literature data demonstrated estrone having a clastogenic potential in a sister-chromatid exchange assay in CHO cells (Kochar, 1988), and in a MNT and Comet assay in human breast cells (Yared, 2002). The potential to induce gene mutations by estrone was, however, found to be negative in an HPRT-like assay in V79 cells (Drevon, 1981).

 

The influence of estrone on the induction of sister-chromatid exchange (SCE) in cultured CHO cells was studied. Cells were exposed to the substance at concentrations of 1E-05 , 2.5E-05, 5E-05 and 7.5E-05 M for 48 h; after 24 h BrdU was added. Four hours prior to harvest, colcemid (0.1 µg/ml) was added to each flask. The cells were fixed and stained with Hoechst dye. SCE was scored by counting 25 metaphases per concentration and the experiments were replicated. Only chromosome spreads of 21 ± 2 chromosomes were scored. In this study, estrone produced more than 2-fold increase in SCE frequency over the controls (Kochar, 1988).

 

Estrone was tested in an in vitro gene mutation assay in V79 cells (primary liver cell mediated mutagenesis assay). The cells were co-cultured with freshly isolated liver cells obtained from 6-8-week-old male or female BDIV rats following collagenase perfusion (in one experiment the rats were treated with Aroclor 1254 5 days before the perfusion). The cells were exposed to the test item for 48 h at concentrations of 50, 75, and 100 µM, and 75 µM in co-culture with Aroclor-induced primary liver cells. For the mutagenicity assays, resistance to 8-azaguanine and ouabain was determined.

Estrone was highly toxic to V79 Chinese hamster cells when tested in the absence of hepatocytes. At concentrations of 50 µM or above, a 90% or greater decrease in viability was observed as compared with control cultures. In the presence of hepatocytes from either male or female rats, a drastic reduction was observed in the toxicity of estrone to V79 cells. This could be attributed to the binding of the tested hormones and of their metabolites to the liver cell’s receptor or to detoxication processes occurring in the liver cells. Under the experimental conditions described in this publication, at concentrations ranging from 50 to 100 µM, estrone did not show an increase in 8-azaguanine or ouabain resistant mutants in the absence of a significant toxic effect, whereas the indirect mutagen DMN strongly increased the number of mutant colonies (Drevon, 1981).

 

The potential of Estrone to induce chromosomal damage was examined in a cytokinesis block micronucleus (CBMN) assay. MCF-7 cells (human mammary carcinoma cell line) cells were treated for either 24 or 120 h with estrone at concentrations of 1E-10, 1E-08, 1E-06, 1E-04 M in DMSO (maximum concentration 1% v/v). Micronuclei in 500 binucleate MCF-7 cells from a minimum of three experiments were scored. Estrone induced dose-related increases in micronucleus-forming activity following both 24 and 120 h treatment. Up to 5- and 2-fold increases in micronucleus formation were observed following 24 and 120 h treatment, respectively. A feature of estrone induced micronucleus-forming activity was the dose-related induction of multiple micronuclei in binucleate MCF-7 cells (Yared, 2002).

 

The potential of Estrone to induce DNA single-strand breaks (SSBs) was examined in an alkaline single cell-gel electrophoresis 'Comet' assay. MCF-7 cells (human mammary carcinoma cell line) cells or Human mammary epithelial cells isolated from freshly expressed breast milk at concentrations of 1E-10, 1E-09, 1E-08, 1E-06, 1E-04, 1E-02 M presence and absence of the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C). At least 5 experiments were performed in duplicates. The cells were incubated with the test item for 2 h. In MCF-7 cells as well as primary culture of breast milk celIs estrone induced clear dose-related increases in CTL in this Comet assay (Yared, 2002).

Studies in various laboratory animal species have demonstrated, that estradiol (E2), its metabolite estrone (E1) and their catechol metabolites, in particular 4-hydroxy E2/E1, are carcinogenic in the kidney, uterus and mammary gland. Two pathways in the initiation, promotion and progression of cancer were identified (Yager, 2015; Cavaliere, 2014; Fleck, 2012; Matthews, 2010):

 

- One pathway involves the binding and activation of estrogen receptors (ERs) which stimulate the transcription of genes involved in cell proliferation. The increased cell replication may lead to an increase in the chances of spontaneous mutations via error-prone DNA repair

 

- The second pathway involves the oxidative metabolism of E2/E1 to catechols (2-OH and 4-OH catechols) and then reactive quinones that can contribute to oxidative DNA damage and form specific, mutagenic depurinating adducts with adenine and guanine which then in turn can serve as biomarkers for the occurrence of these processes.

 

Cavalieri et al. (2014) showed that the quinone metabolites of estrone cause DNA damage by mutagenic depurinating adducts with adenine and guanine.

 

The 2-hydroxylation pathway results in formation of ROS and formation of a low level of depurinating adducts. In contrast, the 4-hydroxylation pathway and the resulting 4-E1 quinone cause the formation of much greater levels of depurinating adenine and guanine adducts (Yager, 2015).

 

In humans, a link between female reproductive history and increased risk of developing cancer in the breast and endometrium has been established: Women exposed to estrogen, through early menarche, late menopause, and/or prolonged hormone replacement therapy have a higher risk of developing certain types of hormone-dependent cancers. (Matthews, 2010)

 

Studies for assessment of genetic toxicity of estrone and estrone-3-methylether

	Estrone	Estrone-3-methylether
CAS No.	53-16-7	1624-62-0
ZK	5019	5512
Ames (OECD TG 471) with & without S9-mix	No data	·         Report AL92, study no. TX95143 (Reimann/ Goerke, 1996) ·         GLP, guideline study; plate incorporation method ·         No deviations ·         Batch 11427106; purity: 95.6% (HPLC) ·         Formulation: DMSO; final suspensions freshly prepared ·         Strains: TA1535, TA1537, TA 98, TA100, TA102 ·         Seven doses up to 5 mg/plate tested ·         Appropriate controls used ·         Results: no increases in revertant colony numbers in any strain, no tendency of higher mutation rate with increasing concentrations ·         Reliability 1

 

Additionally, positive results of genotoxicity studies with estrone are cited in RTECS database (Aug 2011):

DNA adduct were found at 870 nmol/kg after oral application of estrone to rats [Chemico-Biological Interactions. (Elsevier Scientific Pub. Ireland Ltd., POB 85, Limerick, Ireland) V.1- 1969- v. 23, p. 13, 1978 (CBINA8)]

 After intraperitoneal application in rats mutation was found in cytogenetic analysis at a dose of 10 mg/kg [Current Science. (Current Science Assoc., Sadashivanagar P.O., Bangalore 560 080, India) V.1- 1932- v. 50, p. 425, 1981 (CUSCAM)]

Sister chromatid exchange was reported in hamster ovary cells at 10 umol/L [Experientia. (Birkhaeuser Verlag, POB 133, CH-4010 Basel, Switzerland) V.1- 1945- v. 44, p. 62, 1988 (EXPEAM)]

 In hamster lung cells a specific locus test was positive at 100 nmol/L [Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1964- v. 550, p. 109, 2004 (MUREAV)]

Saccharomyces cerivisiae (microorganism) mutation test was positive at 31.6 umol/L/6H [Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1964- v. 676, p. 113, 2009 (MUREAV)]

In hamster ovary cells cytogenetic analysis was found positive at 50 umol/L [Toxicology Letters. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1977- v. 29, p. 201, 1985 (TOLED5)]

 

References:

Cavalieri E, Rogan E. The molecular etiology and prevention of estrogen-initiated cancers: Ockham's Razor: Pluralitas non est ponenda sine necessitate. Plurality should not be posited without necessity. Molecular aspects of medicine, (2014 Apr) Vol. 36, pp. 1-55.

 

Drevon C. et al., Mutagenicity assays of estrogenic hormones in mammalian cells. Mutation Research, 89 (1981) 83-90

 

Fleck SC et al. Catechol metabolites of zeranol and 17β-estradiol: A comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase. Toxicology Letters (2012), 210(1), 9-14

 

Matthews, J. Estrogens as endogenous toxins. Endogenous Toxins (2010), Volume 1, 227-248.  Editor(s): O'Brien, Peter; Bruce, W. Robert. Publisher: Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany

 

Yager JD. Mechanisms of estrogen carcinogenesis: The role of E2/E1-quinone metabolites suggests new approaches to preventive intervention--A review. Steroids, (2015 Jul) Vol. 99, No. Pt A, pp. 56-60. 

Justification for classification or non-classification

Based on the available genotoxicity test in vitro (bacterial reverse muration assay) the test substance showed no indication to be mutagenic and therefore no classification is warranted for genotoxicity according to Regulation (EC) 1272/2008 (CLP).