Androst-4-ene-3,17-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test, GLP, OECD TG471): negative with and without metabolic activation [Schering AG, report no. AN15, 1996-07-23]

Gene mutation (mammalian cell gene mutation test / HPRT, GLP, OECD TG 476): negative with and without metabolic activation [Harlan Cytotest Cell Research GmbH, report no. 1554600, 2013 -09 -13]

Results of genotoxicity studies published within scope of NTP (NIH, US) assessment [NTP Technical Report 560, NIH Publication No. 10-5901, September 2010]:

Gene mutation (bacterial reverse mutation assay / Ames test; S. typhimurium TA 97, TA 98, TA 100, TA 1535): negative with and without S9

Gene mutation (bacterial reverse mutation assay / Ames test; S. typhimurium TA 100 and TA 98, E. coli WP2): negative with and without S9

Chromosome aberration in rats (3-day exposure, mammalian erythrocyte micronucleus test, bone marrow erythrocytes): negative

Chromosome aberration in mice (3-month exposure, mammalian erythrocyte micronucleus test, peripheral blood erythrocytes): negative in males, equivocal in females

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. to 24 Nov. 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

December 29, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of Aroclor 1254 induced male rats

Test concentrations with justification for top dose:

up to 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (NaN3; for TA1535 and TA100), 4-nitro-o-phenylene-diamine (4-NOPD; for TA1537 and TA98), methylmethanesulfonate (MMS; for TA102), 2-aminoanthracene (2-AA, for all strains)

Remarks:

The positive controls NaN3, 4-NOPD, and MMS were used without S9 mix; the positive control 2-AA with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in triplicates; plates were incubated at 37 °C for approx. 48 hours; the frequency of revertant colonies were assessed using the Autocount (Biosys GmbH, Karben, Germany).

DETERMINATION OF CYTOTOXICITY
- Method: The background growth of the bacteria was judged visually on a light bench. Normal range of spontaneous reversion rates: TA1535 10-29, TA1537 5-28, TA98 15-57, TA100 77-189, TA102 121-293.

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at least one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biological relevant positive response at any one of the test points is considered nonmutagenic in this system.
A significant response is described as folIows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 and TA 102 or thrice on TA 1535 and TA 1537. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not .

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(no cytotoxicity with metabolic activation, but tested up to limit concentration)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(no cytotoxicity with metabolic activation, but tested up to limit concentration)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation.
Appropriate control mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants, occurred in all test groups in the presence or absence of metabolic activation except in strains TA 100 and TA 102 with metabolic activation. These toxic effects started to occur in strain TA 1537 at concentrations of 1000 µg/plate and above with and without metabolic activation and in strain 1535 at 1000 µg/plate in the absence and at 2500 µg/plate in the presence of S9 mix. Toxic effects were restricted to the highest concentration of the test article in strains TA 98 with and without S9 mix and TA 100 and TA 102 in the absence of metabolic activation.
The background growth of the bacteria was reduced slightly at concentrations of 1000 µg/plate and severely at 2500 and 5000 µg/plate in strains TA 1535 and TA 1537.

Conclusions:

The substance did not show a mutagenic potential in a bacterial reverse mutation assay according to OECD TG 471 (Ames test in S. typhimurium TA98, TA1537, TA100, TA102, TA1535) when tested up to the highest recommended dose level of 5.0 mg/plate in the absence or presence of metabolic activation. Appropriate control mutagens showed the expected increases in induced revertant colonies.

Executive summary:

The mutagenic potential of Androstendione was evaluated in a Salmonella/microsome test with the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of S9 mix according to OECD TG 471.

The assay was performed in one experiment with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.

Toxic effects, evident as a reduction in the number of revertants, occurred in all test groups with and without metabolic activation except in strains TA 100 and TA 102 with metabolic activation.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate control mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Androstendione is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22. May to 22. Aug. 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM with supplements; for the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of phenobarbital/ß-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Experiment I: treatment time 4 hours, with and without S9 mix 7.5, 15.0, 30.0, 60.0, 120.0, and 240.0 µg/mL
Experiment II: treatment time 4 hours, with S9 mix 7.5, 15.0, 30.0, 60.0, 120.0, 180.0, and 240.0 µg/mL; treatment time 24 hours, without S9 mix 1.89, 3.75, 7.5, 15.0, 30.0, 60.0, 90.0, and 120.0 µg/mL.

Vehicle / solvent:

DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulfonate (EMS; 1.2 mM), 7,12-dimethylbenzanthracene (DMBA; 4.3 µM)

Remarks:

EMS was used without and DMBA with metabolic activation

Details on test system and experimental conditions:

PRE-TEST: Test item concentrations ranged between 15.0 ¿g/mL and 1920 ¿g/mL in the pre-experiment. As result no relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hour treatment. A transient reduction of the cloning efficiency to 44.1 % at 480 ¿g/mL without metabolic activation was judged as precipitation artefact rather than true cytotoxicity as the cloning efficiency was back to normal at higher concentrations. Following 24 hours treatment without metabolic activation cytotoxic effects occurred at 120 ¿g/mL and above.

METHOD OF APPLICATION: in medium
Approximately 1.5×10exp6 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 ¿l/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
Three or four days after treatment 1.5×10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory¿s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Water solubility: The maximum concentration of the pre-experiments (1920 ¿g/mL) was based on the solubility properties of the test item in DMSO and aqueous medium.
- Precipitation: No precipitation of the test item was observed up to the maximum analyzable concentration in both main experiments. In the pre-experiments precipitation occurred at 120 ¿g/mL and above after 4 hours treatment with and without metabolic activation, and at 480 ¿g/mL and above following 24 hours treatment without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects indicated by a relative cloning efficiency I or relative cell density below 50 % of the corresponding solvent control were noted at 120 ¿g/mL in the first main experiment without metabolic activation. In the presence of metabolic activation no cytotoxicity was observed at the maximum analyzable concentration of 120 ¿g/mL. At the next higher concentration of 240 ¿g/mL exceedingly severe cytotoxic effects precluded analysis of any data on mutagenicity. In the second experiment cytotoxicity as described above occurred at 180 ¿g/mL with and at 120 ¿g/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency I or relative cell density was covered with and without metabolic activation.

Conclusions:

negative

Executive summary:

Androstendion was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the pre-experiments (1920 μg/mL) was based on the solubility properties of the test item in DMSO and aqueous medium. The dose range of experiment I was limited by precipitation observed in the pre-experiment. The dose range of experiment II without metabolic activation was based on cytotoxic effects noted in the pre-experiment. The dose range of experiment II with metabolic activation was adjusted to cytotoxicity and precipitation observed in experiment I.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Androstendion is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

other: NTP laboratory health and safety guidelines

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

Specific details on test material used for the study:

no data in NTP TR 560

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

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

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).

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.

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

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

other: NTP laboratory health and safety guidelines

Principles of method if other than guideline:

A detailed discussion of this assay is presented by MacGregor et al., Fundam. Appl. Toxicol. 14, 1990, 513-522.

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Androstendione
- Lot/batch No.: H408
- Purity: > 98 %
- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 35 days (sealed amber glass bottles; 5 °C).

Species:

mouse

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
no further data in NTP TR 560

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Duration of treatment / exposure:

3 months

Frequency of treatment:

once daily

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
1 to 50 mg/kg bw
Basis:
actual ingested
exposure: 3 month

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

At the end of the 3-month toxicity study (see chapter repeated dose toxicity for further results), peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population was determined as a measure of bone marrow 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 results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs 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.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Sex:

female

Genotoxicity:

ambiguous

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Following 3 months of androstenedione administration (1 to 50 mg/kg) by gavage, no increase in the frequency of micronucleated NCEs was seen in peripheral blood samples from male B6C3F1 mice. In female mice, a small increase in the frequency of micronucleated NCEs was observed at the highest dose tested (50 mg/kg); although not significantly elevated above the vehicle control (P=0.0142), this increase resulted in a significant trend (P=0.001), and the test in female mice was judged to be equivocal.
No significant changes in the percentages of PCEs among total erythrocytes were seen in mice, suggesting no androstenedione-associated toxicity in the bone marrow.

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Gavage Administration of Androstenedione for 3 Months (a)

Compound	Dose (mg/kg)	Number of Male Rats with Erythrocytes Scored	Micronucleated PCEs/ 1,000 NCEs (b)	Pairwise p value (c)	PCE (b) (%)
Male
Methylcellulose (d)	0	5	2.60 ± 0.51		2.860 ± 0.19
Androstenedione	1	5	2.90 ± 0.62	0.3427	2.500 ± 0.37
	5	5	2.80 ± 0.30	0.3926	3.020 ± 0.18
	10	5	2.70 ± 0.54	0.4453	3.020 ± 0.20
	20	5	2.40 ± 0.46	0.6115	2.920 ± 0.18
	50	5	2.10 ± 0.10	0.7674	2.540 ± 0.29
			P=0.880 (e)
Female
Methylcellulose	0	5	1.60 ± 0.43		2.820 ± 0.24
Androstenedione	1	5	1.30 ± 0.20	0.7114	2.860 ± 0.28
	5	5	1.40 ± 0.29	0.6426	3.300 ± 0.29
	10	5	2.00 ± 0.57	0.2523	3.200 ± 0.18
	20	5	1.30 ± 0.12	0.7114	3.260 ± 0.27
	50	5	3.10 ± 0.40	0.0142	2.640 ± 0.14
			P=0.001

 

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990).

PCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte.

b Mean ± standard error

c Pairwise comparison with the vehicle control group; significant at P#0.005 (ILS, 1990)

d Vehicle control

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test, significant at P</=0.025 (ILS, 1990)   

Conclusions:

equivocal

Executive summary:

The genetic toxicity of androstenedione was assessed by testing the ability of the substance to induce increases in the frequency of micronucleated erythrocytes in mouse peripheral blood.

At the end of the 3-month toxicity study in male and female B6C3F1 mice, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population was determined as a measure of bone marrow toxicity.

Following 3 months of androstenedione administration (1 to 50 mg/kg) by gavage, no increase in the

frequency of micronucleated NCEs was seen in peripheral blood samples from male B6C3F1 mice. In

female mice, a small increase in the frequency of micronucleated NCEs was observed at the highest dose tested (50 mg/kg); although not significantly elevated above the vehicle control (P=0.0142), this increase resulted in a significant trend (P=0.001), and the test in female mice was judged to be equivocal.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

The mutagenic potential of Androstendion was evaluated in a Salmonella/microsome test with the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of S9 mix according to OECD TG 471.

The assay was performed in one experiment with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.

Toxic effects, evident as a reduction in the number of revertants, occurred in all test groups with and without metabolic activation except in strains TA 100 and TA 102 with metabolic activation.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate control mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Androstendion is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay (Reimann, 1996).

 

Androstendion was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the pre-experiments (1920 μg/mL) was based on the solubility properties of the test item in DMSO and aqueous medium. The dose range of experiment I was limited by precipitation observed in the pre-experiment. The dose range of experiment II without metabolic activation was based on cytotoxic effects noted in the pre-experiment. The dose range of experiment II with metabolic activation was adjusted to cytotoxicity and precipitation observed in experiment I.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Androstendion is considered to be non-mutagenic in this HPRT assay (Wollny, 2013).

 

Supporting data are reported in the NTP assessment [NTP Technical Report 560, NIH Publication No. 10-5901, September 2010]:

Androstenedione was not mutagenic in a bacterial mutation assay conducted with and without exogenous metabolic activation and at concentrations up to 10000 µg/plate for each tester strain (S. typhimurium TA 97, TA 98, TA 100, and TA 1535).

The assay was conducted as part of a NTP testing program and has to be seen as a whole together with a second indepently performed bacterial mutation assay. Therefore, only 4 relevant tester strains were used here, for results on E.coli WP2 see other endpoint study record (Ames test - part two, NTP, 2010).

Androstenedione was not mutagenic in a bacterial mutation assay conducted with and without exogenous metabolic activation and at concentrations up to 3500 µg/plate for S. typhimurium TA 100, up to 7500 µg/plate for S. typhimurium TA 98, and up to 10000 µg/plate for E. coli WP2.

The assay was conducted as part of a NTP testing program and has to be seen as a whole together with a second indepently performed bacterial mutation assay. Therefore, only 3 relevant tester strains were used here, for results on S. typhimurium TA 97, TA 98, TA 100, and TA 1535 see other endpoint study record (Ames test - part one, NTP, 2010).

In vivo studies

The genetic toxicity of androstenedione was assessed by testing the ability of the substance to induce mutations micronucleated erythrocytes in rat bone marrow and increases in the frequency of micronucleated erythrocytes in mouse peripheral blood.

 

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.

 

At the end of the 3-month toxicity study in male and female B6C3F1 mice, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population was determined as a measure of bone marrow toxicity.

Following 3 months of androstenedione administration (1 to 50 mg/kg) by gavage, no increase in the

frequency of micronucleated NCEs was seen in peripheral blood samples from male B6C3F1 mice. In

female mice, a small increase in the frequency of micronucleated NCEs was observed at the highest dose tested (50 mg/kg); although not significantly elevated above the vehicle control (P=0.0142), this increase resulted in a significant trend (P=0.001), and the test in female mice was judged to be equivocal.

 

No significant changes in the percentages of PCEs among total erythrocytes were seen in either the rats or mice, suggesting no androstenedione-associated toxicity in the bone marrow.

Justification for classification or non-classification

Overall, no classification required for genetic toxicity according to Regulation (EC) 1272/2008.

This is in accordance with German legislation for classification of androgenic steroids (See argumentation for the assessment of steroid hormones, Technical Rule for Hazardous substances 905; elaborated by the German Committee on Hazardous Substances (AGS) and published by the German Federal Ministry of Labour and Social Affairs, version of 2008/2005/1999, only available in German, http://www.baua.de/de/Themen-von-A-Z/Gefahrstoffe/TRGS/Begruendungen-905-906.html).