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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A registration of estrone was already submitted earlier and is public available on the ECHA website. Chapter 7, which is still valid from today's perspective, was amended to fulfill the current information requirements. Consequently the migrated data (IUCLID 5 to IUCLID 6) was kept unchanged and only modified if there was a need for further information and/or to pass the technical completeness check (TCC).

Estradiol and its metabolite estrone are essential endogeneous hormones. Estradiol-17ß is the most active naturally occuring estrogenic hormone. Estradiol and estrone are widely used for oral contraception and in post-menopausal hormonal therapy. Estradiol and estrone are generally negative in the ICH ( International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) core battery of tests according to the the literature review published by Joosten (Toxicology Letters 151, 113 -134) in 2004. A different picture with more positive tests appears when other endpoints and less commonly used cell types are used. Several examples of estrogens with conflicting results (positive, negative, variable results) are described.

Estrone and its surrogat estrone methylether are not mutagenic in gene mutation assays (bacteria and mammalian cells). Estrone can cause chromosomal aberrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
CHO cells used in the current investigation were routinely grown in 75cm² Corning plastic flasks in 10 ml of McCoy’s 5a medium supplemented with 10% fetal calf serum and antibiotics consisting of penicillin-G, (100 U/ml) and streptomycin, (100 µg/ml). The cultures were incubated in an atmosphere of 5% CO2 in air. To test the effect of steroid hormones, cultures were set up 24 h prior to the treatment by seeding approx. 1.6 x 10 to 6 cells per flask. Treatments were given by exposing the cultures to test compounds dissolved in DMSO and suspended in the culture medium for 12 h. The control cultures contained 0.5% of DMSO and were incubated under identical conditions. The procedure for the preparation of chromosomes to score for the abnormalities
is described in Experientia, 38 (1982) 845-846.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
The test material was procured from Sigma, St. Louis, MO.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO cells were obtained from Environmental Health Research and Testing, Lexington, KY. The cells were not used at a passage level of more than 15 after cloning, and were thawed routinely from liquid nitrogen storage and maintained by transferring twice a week.
Metabolic activation:
not specified
Test concentrations with justification for top dose:
10 to minus five M; 5 x 10 to minus five M; 10 to minus 4 M
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not examined
Conclusions:
positive
Executive summary:

The effect of steroid hormones on the chromosomes of cultured Chinese hamster ovary (CHO) cells was studied. It was noticed that estradiol-17fl, estrone, estriol and ethynyl estradiol were effective in producing various types of chromosome aberrations. The percentage of these abnormalities increased with increasing concentrations of steroids used.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Primary liver-cell-mediated mutagenesis assays were performed according to the method previously described by Langenbach et al. (Proc. Natl. Acad. Sci. 75, 2864-2867 (1978)). V79 Chinese hamster cells were plated in 25cm² flasks at a concentration of 3.0 X 1000000 in minimal essential medium (MEM) containing 10% fetal calf serum (FCS). After 24 h, the medium was removed, and freshly isolated liver cells were seeded at a concentration of 2x 10000000 viable cells/flask on top of the V79 Chinese hamster cells. Cells were left to stand in Williams’ E medium with 10% FCS at 37°C for 3 h to allow for attachment this medium was then replaced with medium containing the chemicals to be tested, After 48 h, the cells were washed twice with Earle’s BSS, Ca, Mg -free, and incubated for 2 additional h in MEM + 10% FCS. The V79 cells were then trypsinized and replated for the determination of the cytotoxicity and resistance to ouabain and 8 -azaguanine.
In one experiment,
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
The test material was purchased from Sigma Chemical Co (St. Louis, MO, USA)
Target gene:
resistance to ouabain and 8-azaguanine
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
originally obtained from dr. E. Hubermann (Oak Ridge National Laboratory, Oak Ridge, TN, USA)
Metabolic activation:
with and without
Metabolic activation system:
freshly isolated liver cells obtained from 6-8 week old male or female BDIV rats; in one experiment rats were given an i.p. injection of Aroclor 1254 (500 mg/kg) in olive oil 5 days before perfusion.
Test concentrations with justification for top dose:
0, 50, 75, 100 µM (in the absence or presence of liver cells)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Details on test system and experimental conditions:
The V79 cells were plated at a concentration of 2 X 100000 cells/60-mm dish for the determination of resistance to 8-azaguanine (AZA’) and I000000 cells/60-mm dish for resistance to ouabain (GUA’). After 72 h of expression time, the drugs, 8-azaguanine and ouabain, were added to give final concentrations of 20 µg/ml and 1 mM, respectively. The medium was changed 3 days later and replaced with Eagle’s MEM containing non-essential amino acids, dialyzed FCS and 8-azaguanine; for ouabain, the medium containing non-dialyzed FCS and the selective drug was changed 4 days later. The cultures were fixed and the colonies stained with Giemsa at 10 days for AZA’ and at 12 days for OWA’ mutants. The mutation frequency (resulting from the mean of
colonies observed in the 8 dishes) was calculated per 1000000 survivors.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
negative
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)
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: 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:
up to 5.0 mg/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
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 102 and 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Estrone methyether did not show a mutagenic potential in the bacterial reverse mutation assay.
Executive summary:
Estrone methyether did not show a mutagenic potential in a bacterial reverse mutation assay (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).
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
No genotoxicity studies were conducted with estrone; read across approach with results of a study with estron-methylether (ZK 5512).Estrone methylether is a steroidal hormone with a weak estrogenic activity. Pharmacological studies have yielded results which show that, despite the weak binding to the estrogen receptor in vitro, a full estrogenic effect can be expected following subcutaneous administration of 1 mg/kg estrone methylether in vivo in rats. It thus can be assumed that the 3-methylether of the estrone in the mammalien organism is transformed into the estrogenically active natural hormone estrone. Therefore, it is possible to use knowledge of estrone methylether for the assessment of estrone:
Reason / purpose for cross-reference:
read-across source
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 102 and 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Estrone methyether did not show a mutagenic potential in the bacterial reverse mutation assay.
Executive summary:
Estrone methyether did not show a mutagenic potential in a bacterial reverse mutation assay (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).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Estrone is an endogenous sex hormone and an approved drug since several decades. The active ingredient, synthetic estrone, is similar both chemically and biologically to endogenously produced human estrone. Estrone belongs to the category “steroidal estrogens” and is one of the pharmacologically less-active metabolites of 17β-estradiol which is the most the most potent of the naturally occurring estrogens (17ß-estradiol, estrone, estriol).The toxic effects of steroidal estrogens like estradiol <-> estrone are an exaggeration of the normal pharmacological effects and result in an increase of female characteristics. Estradiol and estrone are widely used for oral contraception and in post-menopausal hormonal therapy for many years. Therefore a wide base of clinical experience is available pointing out various types of adverse effects.

Additional information

A registration of estrone was already submitted earlier and is public available on the ECHA website. Chapter 7, which is still valid from today's perspective, was amended to fulfill the current information requirements. Consequently the migrated data (IUCLID 5 to IUCLID 6) was kept unchanged and only modified if there was a need for further information and/or to pass the technical completeness check (TCC).

No genotoxicity studies were conducted with estrone (ZK 5019); read across approach with results of a study with estron-methylether (ZK 5512). Estrone methylether is a steroidal hormone with a weak estrogenic activity. Pharmacological studies have yielded results which show that, despite the weak binding to the estrogen receptor in vitro, a full estrogenic effect can be expected following subcutaneous administration of 1 mg/kg estrone methylether in vivo in rats. It can thus to be assumed that the 3-methylether of the estrone in the mammalien organism is transformed into the estrogenically active natural hormone estrone (analogous to the transformation of mestranol into ethinylestradiol). Therefore, it is possible to use knowledge of estrone methylether for the assessment of estrone:

ZK 5512 did not show a mutagenic potential in a bacterial reverse mutation assay (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). [Schering AG, Report No. AL92; 1996-06-05]

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)]




Justification for classification or non-classification

Various in vitro and in vivo test systems for investigation of genotoxic properties of estradiol and estrone have led to contradictive results. Different publications show mutagenic effects in vivo and in vitro. However, different estrogens are tested in standard genotoxicity tests and did not show any genotoxic potential. Therefore, there is no sufficient evidences available to classify the substance estrone as genotoxic.

Classification according to Regulation (EC) No. 1272/2008 (CLP) would not appear to be appropiate.

The non-classification is in accordance with German legislation for classification of estrogenic steroid hormones. The German Committee on Hazardous Substances (AGS) recommended for estrogenic steroid hormones classification as:

Toxicity to reproduction - Fertility: Category 1A

Toxicity to reproduction - Development: Category 2

Carcinogenicity: Category 2

See Technical Rule for Hazardous Substances 905; elaborated by the German Committee on Hazardous Substances (AGS) and published by the German Federal Ministry of Labour and Social Affairs, version: 19.04.2016, only available in German,URLhttp://www.baua.de/de/Themen-von-A-Z/Gefahrstoffe/TRGS/Begruendungen- 905-906.html.

The associated documentation and justification for grouping steroid hormones and their classification was published in 09/1999. Estrone is mentioned in attachment 2 on page 16.