Estradiol

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Estradiol

EC Number:

200-023-8

EC Name:

Estradiol

Cas Number:

50-28-2

Molecular formula:

C18H24O2

IUPAC Name:

estra-1,3,5(10)-triene-3,17-diol

Test animals

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

Administration / exposure

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

Doses / concentrations

Dose / conc.:

2 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3/sex/group

Control animals:

yes, concurrent vehicle

Examinations

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.

Results and discussion

Test results

Test results 1

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.

Test results 2

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

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.