Estradiol

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Studies for a Genotoxic Potential of Some Endogenous and Exogenous Sex Steroids

Author:

Rainer Lang and Roland Reimann

Year:

1993

Bibliographic source:

Environmental and Molecular Mutagenesis 21 :272-304 (1993)

Materials and methods

Test guideline

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

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

Method

Target gene:

His locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 5

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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'

Any other 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

Applicant's summary and conclusion

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.