Androst-4-ene-3,17-dione

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (supervised by NTP); published data without detailed documentation

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2010

Report date:

2010

Reference 2

Reference Type:

publication

Title:

Toxicity and carcinogenicity of androstendione in F344/N rats and B6C3F1 mice

Author:

Blystone CR et.al.

Year:

2011

Bibliographic source:

Food and Chemical Toxicology 49, 2116-2124

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: NTP laboratory health and safety guidelines

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Principles of method if other than guideline:

The study was performed according to standard three-exposure protocol as described by Shelby et al., Environ. Mol. Mutagen. 21, 1993, 160-179.

Cited from report: "These are the basic guidelines for arriving at an overall assay result for assays performed by the National Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical procedures for data analysis have been described in the preceding protocols. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation. Results obtained in the absence of activation are not combined with results obtained in the presence of activation; each testing condition is evaluated separately." The summary table in the Abstract of the Technical Report presents a result that represents a scientific judgement of the overall evidence for activity of the chemical in an assay.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Androst-4-ene-3,17-dione

EC Number:

200-554-5

EC Name:

Androst-4-ene-3,17-dione

Cas Number:

63-05-8

Molecular formula:

C19H26O2

IUPAC Name:

androst-4-ene-3,17-dione

Specific details on test material used for the study:

no data in NTP TR 560

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: F344/N
no further data in NTP TR 560

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

Preliminary range-finding studies were performed. Factors affecting dose selection included chemical solubility and toxicity and the extent of cell cycle delay induced by androstenedione exposure.

Duration of treatment / exposure:

3 days; The animals were killed 24 hours after the third dosing and bone marrow samples prepared.

Frequency of treatment:

three times at 24-hour interval

Post exposure period:

24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
312.5 and 625 mg/kg bw
Basis:
actual ingested
exposure: 3 days

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control animals received injections of cyclophosphamide (15 or 25 mg/kg).

Examinations

Tissues and cell types examined:

The animals were killed 24 hours after the third dosing, and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained. 2000 polychromatic erythrocytes (PCEs; reticulocytes) were scored for the frequency of micronucleated cells in each of up to five animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of toxicity.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials. Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Statistics:

The frequency of micronucleated cells among PCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group (Integrated Laboratory Systems (ILS), Micronucleus Data Management and Statistical Analysis Software, Version 1.4. ILS, Inc., P.O. Box 13501, Research Triangle Park, NC 277071990, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In vivo, no significant increases in the frequencies of micronucleated PCEs (reticulocytes) were observed in bone marrow of male F344/N rats administered androstenedione (312.5 or 625 mg/kg) by gavage once daily for 3 days. No significant changes in the percentages of PCEs among total erythrocytes were seen in the rats, suggesting no androstenedione-associated toxicity in the bone marrow.

Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated
with Androstenedione by Gavage (a)
Compound Dose
(mg/kg) Number of Male Rats
with Erythrocytes Scored Micronucleated PCEs/
1,000 PCEs (b) Pairwise p value (c) PCE (b) (%)
Corn oil (d) 0 3 0.33 ± 0.17 5.200 ± 0.15
Androstenedione 312.5 5 0.40 ± 0.10 0.4165 5.120 ± 0.34
625 5 0.50 ± 0.39 0.3128 4.420 ± 0.60
P=0.304 (e)
Cyclophosphamide (f) 15 5 24.00 ± 2.47 0.0000 1.180 ± 0.17
25 4 19.17 ± 2.37 0.0000 0.700 ± 0.08

a Study was performed at ILS, Inc. The detailed protocol is presented by Shelby et al. (1993).
PCE=polychromatic erythrocyte
b Mean ± standard error
c Pairwise comparison with the vehicle control; dosed group values are significant at P#0.013; positive control values are significant at Pd Vehicle control
e Significance of micronucleated PCEs/1,000 PCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990).
f Positive control

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The genetic toxicity of androstenedione was assessed by testing the ability of the substance to induce mutations micronucleated erythrocytes in rat bone.

Male F344/N rats received androstenedion dissolved in corn oil by gavage three times at 24-hour intervals; vehicle control animals received corn oil only. The positive control animals received injections of cyclophosphamide (15 or 25 mg/kg). The animals were killed 24 hours after the third dosing, and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried

smears were fixed and stained; 2,000 polychromatic erythrocytes (PCEs; reticulocytes) were scored for the frequency of micronucleated cells in each of up to five animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of toxicity.

No significant increases in the frequencies of micronucleated PCEs (reticulocytes) were observed in bone marrow of male F344/N rats administered androstenedione (312.5 or 625 mg/kg) by gavage once daily for 3 days.

No significant changes in the percentages of PCEs among total erythrocytes were seen in this study, suggesting no androstenedione-associated toxicity in the bone marrow.