Estradiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in Ames test (OECD TG 471) with S. typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 when tested with five concentrations up to 2500 µg/plate in the presence and absence of metabolic activation (Lang & Reimann, 1993)

Study report: OECD 482 (UDS test): negative [Fautz, 1993]

Study report: OECD 482 (UDS test): negative [Dean, 1992]

Publications: HPRT Test and SCE_MNT_Chrom.Aberration: no effects with biological relevance/negative [Drevon, 1981 & Banduhn & Obe, 1985]

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

Remarks:

HPRT, SCE, MN, Chrom. Ab.

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

HPRT, SCE, MN and Chromosome aberrations

GLP compliance:

not specified

Conclusions:

negative for gene mtutaions in HPRT-like assay, negative for chromosome aberration and sister chromatid exchanges, positive for C-mitotic effects, polyploidies and micronuclei in human peripheral lymphocytes

Executive summary:

Estradiol 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.

Estradiol was highly toxic to V79 Chinese hamster cells when tested in the absence of hepatocytes. At concentrations of 50 µM a more than 90% decrease in viability was observed as compared with control cultures. The cytotoxicity somehow declined at higher concentrations to around 90% decrease. In the presence of hepatocytes from either male or female rats, a drastic reduction was observed in the toxicity of estradiol 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, estradiol did not show an increase in ouabain resistant mutants in the absence of a significant toxic effect, whereas the indirect mutagen DMN strongly increased the number of mutant colonies. However, at 75 µM Estradiol exposure in cultures with male rat liver cells a mutation frequency of 1.6 ± 1.6 in 8-azaguanine resistant mutants was noted. Since this was the only increase in mutation frequency, i.e. no dose dependency was observed this finding is considered accidental. Furthermore a slight increase of mutation frequency in 8-azaguanine  and quabain resistant mutants was observed when liver cells from Aroclor 1254 pretreated rats were used, this increase is also not considered to be of biological relevance (Drevon, 1981).

 

Estradiol was tested in one test system, namely, human peripheral lymphoctes in vitro with regard to the induction of chromosomal alterations (structural chromosomal aberrations and SCEs) and numerical chromosome changes (C-mitoses, polyploidies). Estradiol induced C-mitoses, polyploidies and micronuclei. The micronuclei are interpreted to result from the induction of numerical chromosome changes rather than from structural chromosomal aberrations. Cells were exposed to Estradiol in EtOH at concentrations of 1E-08, 1E-07, 1E-06, 1E-05 M for 24h and Estradiol in DMSO at concentrations of 1E-04 M for 24h, 1E-04 for 1h, 5E-05 for 6h and 1E-04 for 6 h for the determination of chromosomal aberrations in the presence and absence of metabolic activation. For sister chromatid exchanges the substance concentrations for exposure were 1E-08, 1E-07, 1E-06, 1E-05 in EtOH for 48 h, and 1E-05, 1E-04 M for 1h and 5E-05, 1E-04 for 6h and 1E-05, 1E-04 M for 24 h in the presence and absence of metabolic activation. Mitotic indices were determined after exposure with 1E-04 M Estradiol for 4h and the frequencies of polyploid cells were determined after 32h, 44h, 56h, and 72h of exposure to 5E-05 or 1E-4 M Estradiol. Also, after 30h and 50h exposure the frequencies were determined. The micronuclei were determined after 40h exposure to 5E-06, 1E-05, 5E-05 and 1E-04M Estradiol.

Estradiol does not induce chromosomal aberrations with or without metabolic activation or sister chromatid exchanges but increases the number of micronuclei, induces polyploidy and has clear C-mitotic effects similar to colcemid. Thus, the micronuclei are considered to be the result of failures in the distribution of chromosomes in partially destroyed spindles.

 

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Dec. 1991 to Jun. 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

23 October 1986

Deviations:

not specified

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

unspecified

Species / strain / cell type:

hepatocytes: male rats, primary cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Male Wistar rats purchased from Charles River UK Ltd, Margate
- Suitability of cells: The advantage of using hepatocytes over other cell types is that hepatocytes tend to be non-dividing which reduces the incorporation of radiolabel due to semi-conservative .DNA synthesis. Furthermore, these cells contain the enzymes necessary for the metabolic activation of many compounds without the need for exogenous activation systems

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The separated hepatocytes were gently washed through 150 um nylon mesh with Williams E medium-Complete (WE-C). The pellets resulting from subsequent centrifugation were resuspended in approximately 20 mL WE-C. The culture was then diluted with WE-C to provide 9.2 E+04 (Experiment 1) or 1.5 E+05 (Experiment 2) viable cells/mL. For treatment: Medium was removed from the cells and, following washing with 2 mL WE-I, replaced with 2 mL WE-I containing 10 uCi/ml [3H] thymidine and a 1:100 dilution of test compound, positive or negative control solutions. Cultures were then incubated overnight (18 hours 40 minutes to 20 hours 21
minutes) at 37°C in 5% CO2 in air (v/v).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Preliminary solubility assessments showed that Estradiol was soluble in DMSO in excess of 100 mg/mL. In culture medium, precipitation was seen at concentrations as low as 21.13 µg/mL. Dilutions were made up to 100.00 µg/mL in Experiment I and up to 20.00 µg/mL in Experiment II

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Preliminary tests

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate for viability, Sextuplicate cultures were used for test compound and quintuplicate cultures were used for each positive control treatment for Experiment 1. For Experiment 2 sextuplicate cultures were used for test compound and
positive control treatments. Ten negative (DMSO) controls were used.
- Number of independent experiments: Two independent Experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The culture was then diluted with WE-C to provide 9.2 E+04 (Experiment 1) or 1.5 E+05 (Experiment 2) viable cells/mL.
- Test substance added in medium



METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Trypan-blue staining

Evaluation criteria:

The following criteria were used for cell counting (Grain counting):
1) only cells with normal morphology were analysed
2) isolated nuclei with no surrounding cytoplasm were not analysed
3) cells with unusual staining artefacts were not analysed
4) heavily labelled cells in S-phase were not analysed
5) all other normal cells, up to 50 per slide were analysed
6) with the exception of the positive controls, slides were scored blind (coded).

The following criteria were used for cell counting (S-phase analysis):
1) only cells with normal morphology were analysed
2) isolated nuclei with no surrounding cytoplasm were not analysed
3) cells with unusual staining artefacts were not analysed
4) all heavily labelled S-phase cells in a total of 1000 cells
per slide where possible were counted
5) slides were scored blind (coded).

UDS analysis
The following were calculated for each slide and for each treatment:
1) the population average NG and the standard deviation (SD)
2) the percent of cells responding or in repair and the SD (ie 2 SNG)
3) the population average NG and the SD for the sub-population of cells in repair
4) the population average cytoplasmic and nuclear grain count.

Evaluation criteria
UDS analysis
A test chemical at a particular dose level would be considered clearly positive in this study if:
1) the test chemical yielded 5 NG or above, and at least 20% of cells were found to be in repair (i.e. NG 25)
2) a dose related increase was seen in both NG and the percentage of cells in repair
3) any induction. of UDS was reproduced in the independent experiment.

S-phase analysis
A test chemical at a particular dose level would be considered clearly positive in this test system if the percent of cells in S-phase was significantly greater than the vehicle control and
exceeded 5% of the population.

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

top concentration was also limited by solubility

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item precipitates at 20.00 µg/mL

No biologically relevant increase in number of S-phase cells could be observed.

Mean viability of triplicate hepatocyte cultures (Experiment I)

Treatment (µg/mL)	Percent viability (mean)	Percent of control
DMSO	55	100
0.00128	NS	NS
0.0064	NS	NS
0.032	NS	NS
0.16	NS	NS
0.8	NS	NS
4 P	50	91
20 P	T	-
100 P	T	-

Mean viability of triplicate hepatocyte cultures (Experiment II)

Treatment (µg/mL)	Percent viability (mean)	Percent of control
DMSO	51	100
0.03906	NS	NS
0.07813	NS	NS
0.1563	NS	NS
0.3125	NS	NS
0.625	NS	NS
1.25	NS	NS
2.5	NS	NS
5	58	115
10 P	33	65
20 P	6	13

NS = not scored, P = precipitate, T = toxic

Net grain count values for each treatment from Experiment I

Dose (µg/mL)	Net nuclear grain count (NG)		Net grain count of cells in repair		Percent of cells in repair (NG ≥ 5)
	mean	SD	mean	SD	mean	SD
0 DMSO	-1.7	2.2	8.2	5.0	1.6	1.7
0.0064	-1.1	0.3	6.0	0.0	0.7	1.2
0.032	-0.5	0.4	7.6	2.5	2.0	2.0
0.16	-1.1	1.0	0	-	-	-
0.8	-2.7	2.4	0	-	-	-
4	-1.1	0.7	0	-	-	-
5 2-AAF	11.3	3.2	12.7	2.4	84.0	12.0

 

Net grain count values for each treatment from Experiment II

Dose (µg/mL)	Net nuclear grain count (NG)		Net grain count of cells in repair		Percent of cells in repair (NG ≥ 5)
	mean	SD	mean	SD	mean	SD
0 DMSO	-2.3	1.0	0	-	-	-
0.625	-4.9	1.7	0	-	-	-
1.25	-2.8	1.3	6.0	0.0	0.7	1.2
2.5	-2.8	0.7	6.7	0.0	1.3	2.3
5	-2.1	0.2	0	-	-	-
10	-0.9	1.8	5.3	0.3	3.3	4.2
2.5 2-AAF	6.7	2.2	9.3	1.3	63.3	18.6

 

Percentage of cells in S-phase for each treatment in Experiment I

Dose (µg/mL)	Percent cells in S-phase
	mean	SD
DMSO	3.3	0.4
0.00128	NS	NS
0.0064	0.9	0.6
0.032	1.0	0.7
0.16	1.6	1.2
0.8	1.8	0.8
4 P	1.2	0.7
20 P	NS	NS
100 P	NS	NS

NS = not scored

P = precipitate

 

Percentage of cells in S-phase for each treatment in Experiment II

Dose (µg/mL)	Percent cells in S-phase
	mean	SD
DMSO	1.5	0.6
0.03906	NS	NS
0.07813	NS	NS
0.1563	NS	NS
0.3125	NS	NS
0.625	0.6	0.5
1.25	0.7	0.6
2.5	0.8	0.2
5	0.5	0.5
10 P	0.2	0.3
20 P	NS	NS

NS = not scored

P: precipitate

 

 

Conclusions:

Primary rat hepatocytes from male Wistar rats were incubated with estradiol. No increased unscheduled DNA synthesis were observed upt ot the cytotoxic dose.

Executive summary:

In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures from male Wistar rats were exposed to Estradiol in DMSO and at concentrations of 0, 0.00128, 0.0064, 0.032, 0.16, 0.8, 4, 20, and 100 µg/mL in Experiment I and at concentrations of 0, 0.03906, 0.07813, 0.1563, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 µg/mL in Experiment II for 18 h.

Estradiol was tested up to cytotoxic concentrations appearing at 10.00 and 20.00 µg/mL, respectively. There was no increase of unscheduled DNA synthesis at any non-toxic or toxic concentration. The positive control (2-AAF, 2.5 and 5 µg/mL, respectively) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1993-02-11 to 1993-07-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

23 October 1086

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

unspecified

Species / strain / cell type:

hepatocytes: female rats

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Hepatocytes isolated from female Sprague Dawley rats, 6-9 weeks of age, 177-235g, animals were purchased from Charles River Wiga GmbH, D-Sulzfeld, F.R.G.
- Suitability of cells: The population of freshly isolated hepatocytes has a small proportion of dividing cells. Their isolation can be done by standard procedures thus rendering a very standardizable test substrate with a low semi-conservative DNA replication.
Hepatocytes\are capable to metabolise actively many promutagens to their active forms within the target cells. They also retain normal detoxification pathways and may therefore be more realistic cellular test units than cells from most continuous cell lines.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The washed hepatocytes were centrifuged and transferred into Williams medium E (WME, Gibco/BRL, D-7514 Eggenstein, F.R.G.) supplemented with:
Hepes 2.38 mg/mL
Glutamin 0.29 mg/mL
Penicillin 100 units/mL
Insulin 0.50 µg/mL
Streptomycin 0.10 mg/mL
Fetal Calf Serum (FCS) 100 µL/mL

Additional strain / cell type characteristics:

not applicable

Test concentrations with justification for top dose:

1. Experiment: 0.5, 1.0, 5.0, 10.0 or 20.0 µg/mL
2. Experiment: 5.0, 10.0, 15.0, 20.0 or 25.0 µg/mL

To evaluate the toxicity of the test article a pre-experiment was performed with 10 concentrations. The experimental conditions in the pre-experiment were the same as described below for the repair assay except the omission of 3HTdR. Toxicity could be evidenced by altered cell morphology and/or reduced number of adherent cells. In addition, the capability of the cells to incorporate a vital dye was determined by the neutral red absorption assay. According to the results from the pre-experiments the concentrations to be applied in the repair assay were chosen. The highest concentration normally used should be 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity but not so great that incorporation of neutral red was completely suppressed.
According to the criteria mentioned above at least five adequate spaced concentrations were tested.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: On the day of the experiment, the test article was dissolved in DMSO (Merck, D-6100 Darmstadt). The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells. The solvent did not exceed 1 % in the culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.0 E+05 living cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 18 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: other: neutral red uptake

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER: Nuclear and net grain counts are taken into consideration together. Increased net grains which are based on enhanced nuclear grain counts are considered relevant. Increased net grain values which are mainly due to decreased cytoplasmic grains are considered non-relevant. The result of a particular test point is classified positive if the mean nuclear grain count is statistically significant increased over the corresponding control value and if the mean net grain value is higher than five per nucleus. A test article is classified positive if it induces either a significant concentration-related increase in radiolabel incorporation expressed as nuclear and net grains or, in the absence of a concentration response, a reproducible and statistically significant positive response for at least one of the test points. A test article producing neither a significant concentration-related increase in radiolabel incorporation expressed as nuclear and net grains nor a statistically significant and reproducible positive response at any one of the test points is considered non-effective in this system.

Evaluation criteria:

Nuclear and net grain counts are taken into consideration together. Increased net grains which are based on enhanced nuclear grain counts are considered relevant. Increased net grain values which are mainly due to decreased cytoplasmic grains are considered non-relevant. The result of a particular test point is classified positive if the mean nuclear grain count is statistically significant increased over the corresponding control value and if the mean net grain value is higher than five per nucleus. A test article is classified positive if it induces either a significant concentration-related increase in radiolabel incorporation expressed as nuclear and net grains or, in the absence of a concentration response, a reproducible and statistically significant positive response for at least one of the test points. A test article producing neither a significant concentration-related increase in radiolabel incorporation expressed as nuclear and net grains nor a statistically significant and reproducible positive response at any one of the test points is considered non-effective in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform, as the number of nuclear and net grain counts of the groups treated with the test article were not substantially
enhanced.

Key result

Species / strain:

hepatocytes: rat, female

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to cytotoxic and precipitating concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: before the experiment the pH of the cells was adjusted to 7.4 and the pH of the medium was adjusted to 7.6
- Precipitation and time of the determination: Concentrations higher than 10.00 µg/mL precipitated in the culture medium during the treatment period.
- Definition of acceptable cells for analysis: Increased net grains which are based on enhanced nuclear grain counts are considered relevant. Increased net grain values
which are mainly due to decreased cytoplasmic grains are considered non-relevant.

RANGE-FINDING/SCREENING STUDIES (if applicable): A pre-experiment was performed to determine the appropriate concentration range for the main experiments.
The following concentrations were tested:
0.10; 0.33; 0.67; 1.00; 3.33; 6.67; 10.00; 33.33; 66.67; and 100.00 µg/mL. In the pre-experiment treatment with concentrations higher.than 10.00 µg/mL yielded toxic effects evidenced by a reduction of neutral red uptake. Thus, The following concentrations were chosen for the main experiments:
Experiment I: -
0.50; 1.00; 5.00; 10.00; 20.00; 30.00; 40.00; and 50.00 µg/mL
Experiment II:
0.50; 1.00; 5.00; 10.00; 15.00; 20.00; 25.00; 30.00; 35.00; and 40.00 µg/mL

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes

Toxicity data of the pre-experiment:

Concentration (µg/mL)	Neutral red absorption (OD 540 nm)*	Percentage of viable cells relative to the control
Solvent control (1% DMSO)	0.891	100
Negative control (Medium)	0.902	101
0.10	0.961	108
0.33	0.772	87
0.67	0.764	86
1.00	0.849	95
3.33	0.937	105
6.67	0.518	58
10.00	0.723	81
33.33	0.371#	42
66.67	0.241#	27
100.00	0.185#	21

*= two cultures were treated similar to the cultures for autoradiography. After the treatment period the cultures were rinsed with PBS and incubated with neutral red solution (50 µg/mL) in order to allow viable cells to incorporate the vital dye. After 3 h the neutral red solution was discarded, the cultures rinsed, and the dye eluted with 50% ethanol supplemented with 1% acetic acid. The data represent the mean value of the two independent cultures.

#= the test article precipitated during the treatment period.

 

Toxicity data test item:

	Experiment I		Experiment II
Concentration (µg/mL)	Neutral red absorption (OD 540 nm)*	Percentage of viable cells relative to the control	Neutral red absorption (OD 540 nm)*	Percentage of viable cells relative to the control
Solvent	0.673	100	1.491	100
0.00	0.651	97	1.574	106
2.23 (2-AAF)	0.646	96	1.398	94
0.50	0.614	91	1.539	103
1.00	0.671	100	1.422	95
5.00	0.685	102	1.354	91
10.00	0.628	93	1.378	92
15.00	n.t.	n.t	1.236	83
20.00	0.198	29	0.958	64
25.00	n.t.	n.t.	0.652	44
30.00	0.149	22	0.513	34
35.00	n.t.	n.t.	0.459	31
40.00	0.128#	19	0.328	22
50.00	0.130#	19	n.t.	n.t.

*= two cultures were treated similar to the cultures for autoradiography. After the treatment period the cultures were rinsed with PBS and incubated with neutral red solution (50 µg/mL) in order to allow viable cells to incorporate the vital dye. After 3 h the neutral red solution was discarded, the cultures rinsed, and the dye eluted with 50% ethanol supplemented with 1% acetic acid. The data represent the mean value of the two independent cultures.

n.t.= not tested

#= the test article precipitated during the treatment period.

Experiment I: Results

Treatment	Grains per nucleus	Grains per cytoplasm area	Net grains per nucleus
	Mean* Standard deviation	Mean* Standard deviation	Mean* Standard deviation
Negative control: Medium	11:65 ± 4.16	13:23 ± 3.83	-1:58 ± 3.76
Solvent control : DMSO	14:70 ± 4.37	16:23 ± 4.93	-1:53 ± 3.55
Positive control: 2-AAF ( 2.23 µg/mL)	23:65 ± 9.35	12:44 ± 3.32	11:21 ± 7.68
1. Concentration ( 0.50 µg/mL)	13:11 ± 5.28	16:01 ± 5.82	-2:90 ± 3.69
2. Concentration ( 1.00 µg/mL)	14:19 ± 4.37	14:56 ± 3.69	-0:37 ± 2.93
3. Concentration ( 5.00 µg/mL)	13:57 ± 4.47	14:57 ± 4.58	-1:00 ± 3.29
4. Concentration ( 10.00 µg/mL)	15:02 ± 4.62	17:78 ± 4.61	-2:76 ± 3.58
5. Concentration ( 20.00 µg/mL)#	7:96 ± 3.4	8:32 ± 3.47	-0:36 ± 2.34

* Mean of 100 cells

# Mean of 89 cells

 

Experiment II: Results

Treatment	Grains per nucleus	Grains per cytoplasm area	Net grains per nucleus
	Mean* Standard deviation	Mean* Standard deviation	Mean* Standard deviation
Negative control: Medium	15.32 ± 4.75	20.32 ± 5.33	-5.00 ± 4.32
Solvent control : DMSO	14.99 ± 5.30	22.30 ± 5.22	-7.31 ± 5.19
Positive control: 2-AAF ( 2.23 µg/mL)	60.04 ± 31.99	26.99 ± 7.81	37.05 ± 31.54
1. Concentration ( 5.00 µg/mL)	17.59 ± 6.44	24.85 ± 5.11	-7.26 ± 5.72
2. Concentration ( 10.00 µg/mL)	20.49 ± 6.43	26.64 ± 6.41	-6.15 ± 6.02
3. Concentration ( 15.00 µg/mL)	16.80 ± 4.60	21.26 ± 5.02	-4.46 ± 4.76
4. Concentration ( 20.00 µg/mL)	15.13 ± 5.00	17.17 ± 4.74	-2.04 ± 4.01
5. Concentration ( 25.00 µg/mL)#	11.67 ± 3.91	13.17 ± 3.60	-1.50 ± 2.88

* Mean of 100 cells

 

 

 

 

 

 

Conclusions:

Primary rat hepatocytes were incubated with estradiol. No increased unscheduled DNA synthesis were observed at cytotoxic dose. Estradiol is considered not genotoxic under the conditions of the test.

Executive summary:

In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures were exposed to Estradiol in DMSO at concentrations of 0, 0.5, 1.00, 5.00, 10.00, and 20.00µg/mL in Experiment I and at concentrations of 0, 5.00, 10.00, 15.00, 20.00, and 25.00 µg/mL in Experiment II for 18 h.

Estradiol was tested up to cytotoxic concentrations appearing at 10.00 and 15.00 µg/mL, respectively. There was no increase of unscheduled DNA synthesis at any non-toxic concentration. The positive control (2-AAF, 2.23 µg/mL) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data.

 

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

1983

Deviations:

yes

Remarks:

only one method: plate incorporation method, TA102 or E.coli WP2 not tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His 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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Sprague-Dawley rats
- method of preparation of S9 mix: pretreated with Aroclor 1254, commercially available
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S 9 mix were 8 mM MgCI,, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate, pH 7.4, and S 9 at a concentration of 0.1 ml (plate incorporation assay)

Test concentrations with justification for top dose:

0, 5, 25, 125, 500, 2500 µg/plate

Vehicle / solvent:

DMSO for the positive controls and Phosphate buffer for the treatment groups

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffer

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

N-dimethylnitrosamine

benzo(a)pyrene

cyclophosphamide

ethylmethanesulphonate

other:

Remarks:

For testing of Estradiol only anthracene-2-amine and 2-aminofluorene were used at concentrations of 2µg/plate each substance

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E-+06 cells/plate
- Test substance added in medium; in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

The plates were scored for the number of mutant colonies manually in the very early experiments and later with an automated colony counter (Biotran II, Model] C 11 1, New Brunswick Scientific Co., Edison, NJ, and Artek M 982B, Artek Systems Corp.. Farmingdale, NY). The arithmetic means of the number of mutant colonies of the three parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneous ones was higher than twofold. Also, a dose-dependent increase in the number of revertants was considered to indicate a mutagenic effect. A toxic effect of the substance on the background lawn of nonrevertant bacteria and precipitates in the agar were examined stereomicroscopically.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: at 2500 µg/plate after 72 h

Ames test:
- Signs of toxicity: no reduced background lawn observed
- Mean number of revertant colonies per plate and standard deviation: see 'Additional information on results incl. tables'

Mutant colonies per plate (mean of three plates ± SD)

TA1535

TA100

TA1537

TA1538

TA98

+S9

+S9

+S9

+S9

+S9

Test substance

Dose/ plate

-S9

Rat

Mouse

-S9

Rat

Mouse

-S9

Rat

Mouse

-S9

Rat

Mouse

-S9

Rat

Mouse

Direct plate incorporation assay

DMSO

34 ± 8

15 ± 3

10 ± 4

81 ± 16

117 ± 9

73 ± 13

8 ± 5

10 ± 3

8 ± 2

14 ± 3

26 ± 4

16 ± 4

31 ± 3

33 ± 8

21 ± 3

Phosphate buffer

41 ± 5

-

14 ± 4

90 ± 7

-

79 ± 11

7 ± 2

-

7 ± 1

12 ± 1

-

21 ± 2

27 ± 7

-

23 ± 3

Estradiol [µg]

5

31 ± 3

10 ± 2

9 ± 3

68 ± 10

101 ± 4

84 ± 16

7 ± 4

6 ± 2

8 ± 4

15 ± 4

17 ± 8

25 ± 4

25 ± 5

30 ± 5

28 ± 3

25

36 ± 18

14 ± 2

13 ± 4

73 ± 9

96 ± 14

73 ± 10

6 ± 2

5 ± 2

5 ±1

12 ±1

17 ±4

23 ±1

27 ± 5

28 ±12

23 ±3

125

51 ± 9

12 ± 1

12 ± 8

68 ± 5

99 ± 5

78 ± 5

8 ± 1

9 ± 2

10 ± 3

13 ± 1

21 ± 2

18 ± 3

22 ± 1

26 ± 2

28 ± 1

500

55 ± 7

13± 2

11 ± 3

72 ± 8

85 ± 9

70 ± 3

8 ± 1

7 ± 2

5 ±1

14 ±5

25 ± 3

24 ± 2

16 ± 3

29 ± 6

30 ± 6

2500

P 45 ± 12

P 11 ± 5

P 9 ± 2

P 68 ± 11

P 91 ± 3

P 83 ± 11

P 8 ± 1

P 5 ± 2

P 5 ± 2

P 19 ± 3

P 27 ± 2

P 18 ± 2

P 15 ± 2

P 34 ± 5

P 23 ± 4

2-AA [µg]

2

59 ± 4

92 ± 16

348 ± 15

-

631 ±136

-

-

-

-

781 ± 165

-

555 ± 110

-

2-AF [µg]

2

-

-

-

-

-

-

-

-

22 ± 3

-

1542 ± 94

-

-

-

10

-

-

-

88 ± 3

-

1533 ± 165

8 ± 2

281 ± 27

86 ± 5

-

-

-

-

-

> 3000

Conclusions:

In the present test Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA 1538 were exposed to Estradiol at concentations of 0, 5, 25, 125, 500, 2500 µg/plate in the plate incorporation assay similar to OCED Guideline 471(1983). Precipitates were observed at the highest concentration in each tester strain, no reduced background lawn was observed. The number of revertants did not increase at least twofold as compared with the respective negative control, thus, Estradiol is not considered a mutagen under the conditions of the test.

Executive summary:

In a reverse gene mutation assay in bacteria similar to OECD guideline 471 (1983), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to Estradiol in phosphate buffer in concentrations of 0 (control), 5, 25, 125, 500, and 2500 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix and mouse liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to precipitating concentrations. Cytotoxic effects were not noted in all tester strains. Precipitation was observed at the highest concentration of 2500 µg/plate.The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA1535, or TA1537 and TA1538) examined at dose levels up to 2500 µg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA1535, TA1537 and TA 1538 under the conditions employed (plate incorporation assay).

There was no evidence of induced mutant colonies over background.

 

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

other information

Study period:

7. Jul 1994 to 3. Aug 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well reported study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schering
- Age at study initiation: males ca. 7-8 weeks; females: ca. 8-10 weeks
- Weight at study initiation: males: 155 - 212 g; females: 145 - 189 g
- Assigned to test groups randomly: by Topsy
- Fasting period before study: not reported
- Housing: individually in Makrolon, type lll cages with embedding
- Diet (e.g. ad libitum): ad libitum, Altromin® R, pulverized
- Water (e.g. ad libitum):ad libitum, demineraiized, acidified water (pH about 2.5)
- Acclimation period: 7-13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 62-68
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 900 mg NaCl + 85 mg Myri 53 ad 100 mL bidest. water
- Concentration of test material in vehicle: 100 mg/100 mL
- Amount of vehicle (if gavage or dermal): 2 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the formulations were prepared for immediate use. The administrations were carried out within approximately 3.5 hours after preparation

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Post exposure period:

no

Dose / conc.:

2 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3/sex/group

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

DNA adduct analysis on liver samples.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals received daily intragastric administrations of the respective formuiations over a period of 14 days. They were sacrified 24 h after the iast administration and livers were removed and shock frozen in iiquid nitrogen immediately after sacrifice. The livers were stored frozen at ca. - 70 °C until DNA isolation and 32P-postlabelling.

METHOD OF ANALYSIS:
Rat liver was homogenized in extraction buffer (10 mM Tris-HCI, 100 mM EDTA, 0.5 % SDS,
pH 8.0) at a concentration of about 1g/mL with a Polytron. Aliquots of the homogenate
equivalent to ca. 18 mg wet tissue from 3 animals per sex and treatment were combined and
the volume was increased to 1 ml with extraction puffer. The combined Liver homogenate was incubated with 50 units RNase A plus 200 units RNase T1 for ca. 18 h at 37°C followed by an additionaL incubation with 8.0 units proteinase K at 37 °C for 1 h. The DNA was extracted with phenol and sevag (chloroform-isoamyl alcohol 24:1), precipitated with absolute ethanol after addition of 5 M NaCl and washed with 70 % ethanol. The precipitate was dissolved in 10 mM Tris-HCl, pH 8.0 and the DNA concentration was measured spectrophotometrically assuming an A260 of 1 equals 50 pg DNA/mL.
Hydrolysis of DNA and nuclease P1 enrichment
From each sample, 15 µg of the DNA dissolved in 10 mM Tris--HCI pH 8.0 were dried in vacuo, redissolved in 0.1 M Na-succinate and 50 mM CaCl2, pH 6.0 and digested with a mixture of 4.0 units micrococcus nuclease and ca. 0.02 units spleen phosphodiesterase. The samples were incubated at 37 °C for 3h to hydrolyse the DNA to individual deoxynucleoside 3'-monophosphates. In the following step, the samples were incubated with 15 units (3‘ AMP as substrate) nuclease P1 for 40 min at 37 °C in order to remove the 3‘-phosphate from
unmodified nucleotides. The reaction was stopped by adding 0.5 M Tris-base (final concentration of Tris-base was 65 mM). From each sample, 2 aliquots with volumes equivalent to 6 µg DNA were taken for DNA adduct analysis. The remaining DNA (3 µg) was discarded.
ln an additional experiment, rat liver DNA isolated from animals which were treated with EE2 or the vehicle control was digested and enriched with nuclease P1 under modified conditions.
Each sample containing 10 µg DNA was dried, redissolved and digested with 2.0 units
micrococcus nuclease and ca. 0.01 units spleen phosphodiesterase as described above. The
nuclease P1 enrichment was carried out with 9.6 units nuclease P1. The hydrolysate was
evaporated and the residue was dissolved in 8 µl water and taken for DNA adduct analysis.

Hydrolysis of DNA and butanol enrichment
Samples with 10 µg DNA were dried, redissolved and digested with a mixture of micrococcus
nuclease and spleen phosphodiesterase (2.0 units and ca. 0.01 units, respectively) as described before. From each sample, 2 aliquots with volumes equivalent to 5 µg DNA
were taken for DNA adduct analysis. The modified nucleotides were repeatedly extracted with water-saturated 1-butanol. To remove impurities of unmodified nucleotides, the butanol phase was re-extracted four times with butanol-saturated water and evaporated to dryness. The residue was then used for DNA adduct analysis.

Labelling after nuclease P1 enrichment
To the hydrolysed samples were individually added 8 µL labelling mixture containing 45 pCi [γ-32P]-ATP (ca. 3000 Ci/mmol) equivalent to ca. 15 µmol [γ-32P]-ATP/sample and 4 U T4-
polynukleotide kinase in kinase buffer. The final concentration of the kinase buffer was
31.3 mM bicine, 15.6 mM DTT, 15.6 mM spermidine, 3.9 mM MgCl2.
The samples were incubated at 37°C for 45 min followed by the addition of 8 µl apyrase
(40 mU) and a further incubation at 37°C for 30 min. The resulting mixture was subjected to
thin layer chromatography.

Labelling after butanol enrichment
To the hydrolysed samples were individually added 8 pi labelling mixture. The labelling mixture contained 100 pCi [γ-32P]-ATP (ca. 6700 Ci/mmol; [γ-32P]-ATP was evaporated to dryness and dissolved in water) equivalent to ca. 33 µmol [γ-32P]-ATP/sample and 4 U T4-
polynukleotide kinase in kinase buffer. The final concentration of the kinase buffer was
31.3 mM bicine, 15.6 mM DTT, 15.6 mM spermidine, 3.9 mM MgCl2.
The samples were incubated at 37 °C for 45 min followed by the addition of 8 µl apyrase
(40 mU) and a further incubation at 37 “C for 30 min. The resulting mixture was subjected to

thin layer chromatography.
Prior to adduct analysis, a rapid chromatographic test was performed to ensure that an excess of [γ-32P]-ATP was present during the labelling reaction and to monitor the efficiency of the enrichment procedures. For this purpose, 2 µl Eabelied nucleotides were diluted in 98 µl and 5 µl of this solution were applied to the origin of a polyethyleneimine (PEl) cellulose thin layer plate (10 x 20 cm). The plate was then developed in AF-buffer for a modified phosphate-buffer (AP-buffer) for 2-3 h. After drying, the distribution of radioactive spots was analysed using a Fujix Bioimaging analyzer BAS 2000. Only samples showing a clear excess
of [γ-32P]-ATP were further evaluated.
For adduct analysis, the remaining voiume was applied to the origin of a PEI-cellulose thin
layer plate to which a 10 x 20 cm Whatman filter paper which was folded in the middle (5 x
20 cm) was attached. Two samples were applied per sheet and the sheet was developed overnight (~18 h) in direction D1 with D1-buffer (Table 2). Under these conditions, bulky and lipophilic adducts remain at or close to the origin. The plates were cut along the dotted line as indicated and all were washed by placing them vertically into an acrylic rack which was then immersed in 2-3L of demineralized water for 2 x 5 min. After drying, the plates were developed in direction D3 and subsequently - after washing - in D4 for about 1 h each using high-salt, high urea solvents (D3-, D4-buffer). The plates were washed and dried as described, attached to a 6 x 10 cm Whatman filter paper and developed in direction D5 with D5-buffer for 3-4 h in order to remove the remaining impurities. In a second experiment, the buffers D3 and D4 were used with a higher concentration of urea.
The Whatman filter paper was removed and the plate was washed and dried. The
chromatograms were analysed using a Fujix Bio-imaging analyser BAS 2000 after an
exposure time of 1.5 h or, if necessary, of ca. 17 h.

DNA adduct quantification
DNA adduct levels were calculated as reiative adduct labelling (RAL)-value, which is the ratio
of the measured radioactivity of DNA adducts [nCi] to specific radioactivity [nCi/pmol] of [y-
32F]-ATP and analysed amount of DNA [pmol]
The plates were analysed together with PEi-plates containing 3 calibration spots, which were
obtained by pipetting 2, 4 and 6 pl of the diluted nucleotides (s. 2.5.5) - if necessary further
dilutions were made - on a PEl thin layer plate (2 x 5 cm). The radioactivity of the used dilution was calculated directly from the data provided by the supplier (Amersham). The analysis was performed using a Fujix Bio-imaging analyzer BAS 2000 which enables the visualization of the radioactivity as spots and the on-line determination of the amount of radioactivity as PSL-values. The radioactivity of the DNA adduct spots was calculated by comparison of the adduct PSL-values with those determined from the calibration spots. The specific activity at the day of analysis was calculated from the data provided by the supplier (Amersham).
Only drug-specific DNA adducts were quantified, which were qualitatively or quantitatively
distinguishable from control samples. A DNA addduct spot was only considered when the
RAL-value was at least two fold higher than the RAL-value determined in the corresponding
region of a control sample (common background spot). A DNA adduct spot was classified as
n.q. (e not quantifiable) when the spot differed only qualitatively from control samples. but
showed a RAL-value of less than twice the corresponding background region.
For the nuclease P1 procedure, each sample was run in duplicate. Therefore, each sample
was divided into two aliquots after incubation with the nuclease P1 which were further
analysed. In an additional experiment in which the DNA isolated from EE2 treated animals was used, the DNA samples were divided into two aliquots before hydrolysis of DNA and enrichment with nuclease P1.
Using the butanol enrichment procedure each sample was single determination.

Evaluation criteria:

DNA adduct spots were determined in comparison to common background spots.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: E2 generated one adduct spot only in male rat iiver.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Non-significant decreases in body weight gain were observed in female animals of group 8 (E2). Theses effects are suspected to be compound-related.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Body weight gain decreased in female rats. DNA adduct spots of estradiol were detected after administration in male rat liver cells.

DNA adducts levels of Estradiol in male and female rat liver

Steroid	Dose [mg/kg/d]		RAL-values* [add./E+09 nucleotides]/sex
			female	male
E2	2.0	adduct 1	-	1.81 ± 0.10
		∑		1.81 ± 0.10#

*= means ± SD of two determinations

# after 17h exposure

 

 

Conclusions:

After 14 days oral treatment with estradiol decreased body weight gain was observed in female rats and DNA adducts were detected in male rat liver cells. There is no correlation between DNA adduct formation and tumorigenic potential observed.

Executive summary:

In a test conducted to detect the formation of DNA adducts, performed similar to OECD guideline 486, female and male Wistar rats were exposed orally to Estradiol at 2 mg/kg bw/d for 14 days. Subsequently the animals were sacrificed and the occurrence of DNA adducts in liver homogenates was determined. During the administration there were no premature deaths and the animals were in good health with respect to general examination. There was only a slight decrease in body weight in females exposed to Estradiol. However, DNA adducts were only observed in male animals after 17 h. Thus, it is considered that the occurrence of DNA adducts is correlated with the sex-specific metabolism of steroids. No correlation was found between DNA adduct formation and tumorigenic potential. Based on the results obtained from the present study Estradiol is considered positive with regard to DNA adduct formation in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a reverse gene mutation assay in bacteria similar to OECD guideline 471 (1983), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to estradiol in phosphate buffer in concentrations of 0 (control), 5, 25, 125, 500, and 2500 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix and mouse liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to precipitating concentrations. Cytotoxic effects were not noted in all tester strains. Precipitation was observed at the highest concentration of 2500 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA1535, or TA1537 and TA1538) examined at dose levels up to 2500 µg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA1535, TA1537 and TA 1538 under the conditions employed (plate incorporation assay).

There was no evidence of induced mutant colonies over background.

In addition, an Ames test (OECD TG 471) with estradiol-valerate which is converted in vivo into estradiol showed negative results in the tester strain TA102. Based on these information and the already concluded risk on carcinogenicity for estrogens (mutag. Cat. 2) a repeat of the Ames test with estradiol including the tester strain S.typhimurium TA102 or E.coli WP2 was omitted. 

Under the conditions of the study, the test substance was negative for mutagenic potential.

Accordingly, estradiol is not classified (Reg(EC) No. 1272/2008) for genotoxicity.

 

 

In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures were exposed to Estradiol in DMSO at concentrations of 0, 0.5, 1.00, 5.00, 10.00, and 20.00µg/mL in Experiment I and at concentrations of 0, 5.00, 10.00, 15.00, 20.00, and 25.00 µg/mL in Experiment II for 18 h.

Estradiol was tested up to cytotoxic concentrations appearing at 10.00 and 15.00 µg/mL, respectively. There was no increase of unscheduled DNA synthesis at any non-toxic concentration. The positive control (2-AAF, 2.23 µg/mL) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data.

 

In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures from male Wistar rats were exposed to Estradiol in DMSO and at concentrations of 0, 0.00128, 0.0064, 0.032, 0.16, 0.8, 4, 20, and 100 µg/mL in Experiment I and at concentrations of 0, 0.03906, 0.07813, 0.1563, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 µg/mL in Experiment II for 18 h.

Estradiol was tested up to cytotoxic concentrations appearing at 10.00 and 20.00 µg/mL, respectively. There was no increase of unscheduled DNA synthesis at any non-toxic or toxic concentration. The positive control (2-AAF, 2.5 and 5 µg/mL, respectively) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data.

 

 

Estradiol 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.

Estradiol was highly toxic to V79 Chinese hamster cells when tested in the absence of hepatocytes. At concentrations of 50 µM a more than 90% decrease in viability was observed as compared with control cultures. The cytotoxicity somehow declined at higher concentrations to around 90% decrease. In the presence of hepatocytes from either male or female rats, a drastic reduction was observed in the toxicity of estradiol 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, estradiol did not show an increase in ouabain resistant mutants in the absence of a significant toxic effect, whereas the indirect mutagen DMN strongly increased the number of mutant colonies. However, at 75 µM Estradiol exposure in cultures with male rat liver cells a mutation frequency of 1.6 ± 1.6 in 8-azaguanine resistant mutants was noted. Since this was the only increase in mutation frequency, i.e. no dose dependency was observed this finding is considered accidental. Furthermore a slight increase of mutation frequency in 8-azaguanine  and quabain resistant mutants was observed when liver cells from Aroclor 1254 pretreated rats were used, this increase is also not considered to be of biological relevance (Drevon, 1981).

 

Estradiol was tested in one test system, namely, human peripheral lymphoctes in vitro with regard to the induction of chromosomal alterations (structural chromosomal aberrations and SCEs) and numerical chromosome changes (C-mitoses, polyploidies). Estradiol induced C-mitoses, polyploidies and micronuclei. The micronuclei are interpreted to result from the induction of numerical chromosome changes rather than from structural chromosomal aberrations. Cells were exposed to Estradiol in EtOH at concentrations of 1E-08, 1E-07, 1E-06, 1E-05 M for 24h and Estradiol in DMSO at concentrations of 1E-04 M for 24h, 1E-04 for 1h, 5E-05 for 6h and 1E-04 for 6 h for the determination of chromosomal aberrations in the presence and absence of metabolic activation. For sister chromatid exchanges the substance concentrations for exposure were 1E-08, 1E-07, 1E-06, 1E-05 in EtOH for 48 h, and 1E-05, 1E-04 M for 1h and 5E-05, 1E-04 for 6h and 1E-05, 1E-04 M for 24 h in the presence and absence of metabolic activation. Mitotic indices were determined after exposure with 1E-04 M Estradiol for 4h and the frequencies of polyploid cells were determined after 32h, 44h, 56h, and 72h of exposure to 5E-05 or 1E-4 M Estradiol. Also, after 30h and 50h exposure the frequencies were determined. The micronuclei were determined after 40h exposure to 5E-06, 1E-05, 5E-05 and 1E-04M Estradiol.

Estradiol does not induce chromosomal aberrations with or without metabolic activation or sister chromatid exchanges but increases the number of micronuclei, induces polyploidy and has clear C-mitotic effects similar to colcemid. Thus, the micronuclei are considered to be the result of failures in the distribution of chromosomes in partially destroyed spindles.

 

In a test conducted to detect the formation of DNA adducts, performed similar to OECD guideline 486, female and male Wistar rats were exposed orally to Estradiol at 2 mg/kg bw/d for 14 days. Subsequently the animals were sacrificed and the occurrence of DNA adducts in liver homogenates was determined. During the administration there were no premature deaths and the animals were in good health with respect to general examination. There was only a slight decrease in body weight in females exposed to Estradiol. However, DNA adducts were only observed in male animals after 17 h. Thus, it is considered that the occurrence of DNA adducts is correlated with the sex-specific metabolism of steroids. No correlation was found between DNA adduct formation and tumorigenic potential. Based on the results obtained from the present study Estradiol is considered positive with regard to DNA adduct formation in mammalian cells.

 

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 estradiol

	Estradiol
CAS-No	50-28-2
ZK	5018
Ames (OECD TG 471) with & without S9-mix	·        Lang & Reimann, 1993 (estradiol table XII, p 289) ·        DMSO for the positive controls and Phosphate buffer for the treatment groups, freshly prepared ·        Non-GLP, guideline study with deviations (Strains: TA98, TA100, TA1535, TA1537, TA1538), no 2nd independent experiment with preincubation method, differences in positive controls ·        0, 5, 25, 125, 500, 2500 µg/plate ·        Result: not mutagen ·        Reliability 2
UDS in vitro OECD TG 482	·        GLP, guideline study OECD TG 482 (version 1986); meanwhile deleted ·        Result: negative Reliability 1
UDS in vitro	·        Report A090, Study TX 91.258, Hazleton Microtest (Dean, 1992) ·        Non-GLP, equivalent to (deleted) OECD TG 482 ·        Result: negative Reliability 2
UDS in vivo OECD TG 482	·        Report AG18, Study TX 94.188 (Zügner, 1995) ·        GLP, equivalent to OECD TG 486 ·        Result: positive Reliability 1
HPRT in vitro; with & without S9-mix	·        Drevon C. et al., Mutation Research, 89 (1981) 83-90 ·        Non-guideline study; scientifically well documented ·        Compound purchased from Sigma Aldrich; purity unknown ·        Formulation: DMSO; final suspensions freshly prepared ·        Cells: Chinese hamster V79 ·        Four concentrations tested w/o and one with metabolic activation (liver cells from male & female rats) ·        Dimethylnitrosamine used as positive control ·        Results: no gene mutation induced at the HPRT locus ·        Reliability 2
Chromosomal damage MNT, chrom. abb., SCE	·        Banduhn & Obe, Mut Res 156 (1985) ·        Non-guideline study; scientifically well documented ·        Estradiol provided by Schering; purity unknown ·        Formulation: DMSO; final suspensions freshly prepared ·        Cells: human PBMCs ·        Results: no chrom.abb. or SCE but MN induction ·        Reliability 2

 

Summary:

17ß-estradiol has been shown to be non-mutagenic in the Ames Salmonella assay (Lang and Reimann R, 1993). Ashby J et al. (1997) have recently reviewed controversial results as regards the potential of 17ß-estradiol to induce chromosome aberrations in human blood Iymphocytes in vitro and to induce micronuclei in vivo: Dhillon and Dhillon (1997) reported that 17ß-estradiol induces micronuclei in the bone marrow of mice after 0.1 to 10 mg/kg i.p. and chromosomal aberrations in human Iymphocytes in vitro. Banduhn and Obe (1985) had reported earlier that 17ß-estradiol is able to induce micronuclei, but not structural chromosome aberrations, in cultured human Iymphocytes. A large proportion of the micronuclei contained centromeric heterochromatin, consistent with an effect by the compound on metaphase spindle tubulin, rather than on DNA. These investigators also observed, and essentially discounted, a weak SCE response for 17ß-estradiol in the human Iymphocyte assay. Among these several data sets, the mouse bone marrow micronucleus assay results are probably the most significant in terms of potential human hazard (TinweIl H and Ashby J, 1994). However, Ashby J et al. (1997) were not able to reproduce the previously published positive findings in mice even when using 15 times high er doses of 17ß-estradiol (up to 150 mg/kg i.p.). Furthermore, a limited micronucleus assay conducted in sexually immature female rats indicated that 17ß-estradiol was inactive at a dose level that elicited a potent estrogenic response in the uterus of the treated animals (20 µg/kg i.p.). Thus, Ashby et al. and the reviewers of their paper (Shelby MD et al., 1997) came to the conclusion that until and unless the positive results reported by Dhillon and Dhillon (1997) are confirmed, it cannot be concluded that 17ß-estradiol induces chromosomal damage in bone marrow cells of rodents.

Therefore, according to the present state of knowledge, no relevant mutagenic risk of 17ß-estradiol for humans is assumed.

 

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

Due to the results of the key study no classification is required according to Regulation (EC) 1272/2008/EC (CLP).