Androsta-1,4-diene-3,17-dione

Endpoint summary

Description of key information

Impairment of male fertility is the leading health effect for repeated administration of androstadiendion based on subchronic (3 months) oral toxicity studies in rats and mice with the read-across substance androstendion (NTP, 2010). The lowest NOAEL from these studies is 5 mg/kg bw/day for rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (supervised by NTP)

Qualifier:

according to guideline

Guideline:

other: NTP laboratory health and safety guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Cited from report: "The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public review."

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: F344/N
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation (mean): males 101 g, females 91 g
- Housing: in groups of five per cage (solid bottom polycarbonate cages)
- Diet and Water: ad libitum
- Acclimation period: 12 to 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 22 +/- 1.6 (72 +/- 3 °F)
- Humidity (%): 50 +/- 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

Administration volume: 5 mL/kg

VEHICLE: 0.5 % aqueous methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed three times during the 3-month study; animal room samples were also analysed. The analytical method used was HPLC.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

once daily, 5 days/week

Remarks:

Doses / Concentrations:
1, 5, 10, 20, and 50 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10; additionally 10 animals per sex and dose were treated for clinical pathology studies.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: For dose selection results from a previously conducted 2-week study were used. In this study groups of five male and five female rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5 % aqueous methylcellulose solution by gavage, 5 days per week for 12 days. All rats survived to the end of the study, and the mean body weights of dosed groups were similar to those of the vehicle control groups. The development of cytoplasmic vacuoles within centrilobular hepatocytes in male rats was the only treatment-related effect observed.

- Rationale for selecting satellite groups: Additional 10 animals per sex and dose were treated for 23 days for clinical pathology studies.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: observed twice daily; clinical findings were recorded weekly for core study animals.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Animals were weighed initially, on day 2 (female mice), day 3 (males), day 4 (female rats), weekly, and at the end of the studies.

CLINICAL PATHOLOGY:
Blood was collected from the retroorbital sinus of clinical pathology study rats on days 4 and 24 and from core study rats at the end of the studies for hematology and clinical chemistry.
- Hematology: automated and manual hematocrit; hemoglobin concentration; erythrocyte, nucleated erythrocytes, reticulocyte, and platelet counts; mean cell
volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials
- Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile acids

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, necropsies were performed on core study animals
Organs weighed were heart, right kidney, liver, lung, right testis, thymus, and uterus.

HISTOPATHOLOGY: Yes, complete histopathology was performed on vehicle control and 50 mg/kg core study rats
In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the adrenal gland, heart, liver, mammary gland, ovary, prostate gland, and thyroid gland of rats were examined in the remaining dosed groups.

SPERM MOTILITY AND VAGINAL CYTOLOGY: At the end of the studies, sperm samples were collected from male animals in the 0, 10, 20, and 50 mg/kg groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females administered 0, 10, 20, or 50 mg/kg for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.

Statistics:

Survival Analysis: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox¿s (1972) method for testing two groups for equality and Tarone¿s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
Calculation of Incidences were performed: Incidences of neoplasms or nonneoplastic lesions as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences: Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided.
Analysis of Continuous Variables: Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. 1) Parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). 2) Nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).

Details on results:

CLINICAL SIGNS AND MORTALITY
All rats survived to the end of the study.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights of the 20 mg/kg female group was significantly greater than those of the vehicle control group and there was significant increased weight gain in the 1, 20, and 50 mg/kg female groups.

CLINICAL PATHOLOGY
There were no changes in hematology or clinical chemistry variables that were considered attributable to androstenedione administration.

ORGAN WEIGHTS
Female thymus weights were significantly increased in the 20 and 50 mg/kg groups, which may be related to the increase in mean body weight.

GROSS PATHOLOGY
No lesions were observed through gross observation that could be attributed to the administration of androstenedione.

SPERM MOTILITY AND VAGINAL CYTOLOGY
The numbers of sperm per mg cauda epididymis in the 10, 20, and 50 mg/kg male groups and the total number of sperm per cauda epididymis in 50 mg/kg males were significantly less than those of the vehicle controls.

HISTOPATHOLOGY
No treatment-related histological lesions were observed in males or females.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Steroid hormone-related effect on male fertility in the next higher dose group based on sperm investigations (The numbers of sperm per mg cauda epididymis were significantly decreased).

Critical effects observed:

not specified

Executive summary:

Cited from NTP report:

"Groups of 10 male and 10 female core study rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5 % aqueous methylcellulose solution by gavage, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats received the same doses for 23 days. All rats survived to the end of the study. The mean body weights of the 20 mg/kg female group was significantly greater than those of the vehicle control group and there was significant increased weight gain in the 1, 20, and 50 mg/kg female groups. Female thymus weights were significantly increased in the 20 and 50 mg/kg groups, which may be related to the increase in mean body weight. The numbers of sperm per mg cauda epididymis in the 10, 20, and 50 mg/kg male groups and the total number of sperm per cauda epididymis in 50 mg/kg males were significantly less than those of the vehicle controls. No treatment-related histological lesions were observed in males or females."

No NOAEL was concluded in the report, as the study served only for the purpose of dose selection for a 2 -year carcinogenicity study. As effects on male fertility revealed the most sensitive health effect in the study a NOAEL of 5 mg/kg is concluded as point of departure for the delineation of a DNEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is GLP compliant and is of high quality (Klimisch score=1)

System:

male reproductive system

Organ:

testes

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For androstadiendion (CAS No. 897-06-3) no repeated dose toxicity data are available. Therefore, repeated dose toxicity data of androstendion (CAS No. 63-05 -8) were used. A search for structure-analogue substances using the QSAR Toolbox 3.3.5 recommended androstendion as one out of 11 category substances for a read-across approach (for details see QSAR OECD Toolbox Report on Androstadiendion attached in chapter 7, Endpoint Summary: Toxicological information).

The once daily oral administration of androstendion to male and female rats over 4 weeks at doses of 0, 15, 50 and 150 mg/kg led for females to dose-dependent effects such as increased body weight, atrophy of uterus, cervix, pituitary gland and adrenals, as well as increased numbers of erythrocytes and increased hemoglobin. For males alterations in thymus were reported. These effects were regarded to represent endocrine effects typical for a steroid hormone (androstendion is an intermediate in endogenous steroid synthesis and as such an endogenous precursor of testosterone and estrone, which can be metabolized to estradiol). Regarding these effects a NOAEL could not be established in the study.

An increase in liver weights together with increased liver transaminases was additionally observed at 150 mg/kg in both sexes and was considered to indicate beginning liver toxicity (Treher, 1996).

Moreover, results of two 90 -day repeated oral toxicity studies on rats and mice were published within the scope of National Toxicology Program (NIH, US) assessment [NTP, 2010]:

 

In one of these studies groups of 10 male and 10 female rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedion/kg body weight in a 0.5 % aqueous methylcellulose solution by gavage, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats received the same doses for 23 days. All rats survived to the end of the study. The mean body weights of the 20 mg/kg female group was significantly greater than those of the vehicle control group and there was significant increased weight gain in the 1, 20, and 50 mg/kg female groups. Female thymus weights were significantly increased in the 20 and 50 mg/kg groups, which may be related to the increase in mean body weight. The numbers of sperm per mg cauda epididymis in the 10, 20, and 50 mg/kg male groups and the total number of sperm per cauda epididymis in 50 mg/kg males were significantly less than those of the vehicle controls. No treatment-related histological lesions were observed in males or females. No NOAEL was concluded in the report, as the study served only for the purpose of dose selection for a 2 -year carcinogenicity study. As the effect on male fertility revealed the most sensitive health effect a NOAEL of 5 mg/kg is concluded from the study. In a second study groups of 10 male and 10 female mice were administered 0, 1, 5, 10, 20, or 50 mg androstenedion/kg body weight in a 0.5 % aqueous methyl cellulose solution by gavage, 5 days per week for 14 weeks. Except for one 10 mg/kg female that died early due to a dosing accident, all mice survived to the end of the study. The mean body weights of dosed groups were similar to those of the vehicle control groups. The number of spermatids per mg testis and the total number of spermatids per testis in 20 mg/kg males were significantly greater than those of the vehicle controls. Sperm motility in 50 mg/kg males was significantly lower than that in the vehicle controls. The incidences of x-zone atrophy of the adrenal cortex, an androgen-sensitive endpoint, were significantly increased in females administered 5 mg/kg or greater. There were also significant decreases in the incidences of x-zone cytoplasmic vacuolization in 20 and 50 mg/kg females. The incidences of bone marrow hyperplasia were significantly increased in 5 and 50 mg/kg males. Again, the leading health effect is concluded to be the effect on male fertility starting at 50 mg/kg (= LOAEL). Concerning the incidences of x-zone atrophy this is not regarded to be of relevance for humans, as the x-zone of the adrenal gland is located at the junction of the cortex and medulla, and is unique to the mouse.

A further study with repeated subcutaneous administration is available for androstendion. In this study the once daily subcutaneous injection of the substance to male and female rats over 4 weeks at doses of 0, 5, 15 and 50 mg/kg led to dose-dependent effects such as decrease of thymus and testes weights, increase of organ weights such as prostate, seminal vesicles or uterus and related histological alterations. These effects can be attributed to the endocrine action of the test compound with partial androgenic, estrogenic and progestagenic action. Additionally signs of general toxicity like decreased body weight gain, decreased food- and increased water consumption together with the occurrence of protein in the urine and increased kidney weight were observed at higher doses. Due to the onset of some of the described effects already at the low dose, a NOAEL could not be established (Steger-Hartmann, 2000).

Further study results for androstendion are cited in RTECS database (April 2013):

The once daily oral administration of androstendion over 14 days results in endocrine effects on menstrual cycle; TDLo: 14 mg/kg/14D-I [Food and Chemical Toxicology. (Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, NY 10523) V.20- 1982- v. 42, p. 917, 2004 (FCTOD7)]

The daily application of androstendion to rats over 34 days results in endocrine effects not further specified; TDLo: 34 mg/kg/34D-I [Food and Chemical Toxicology. (Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, NY 10523) V.20- 1982- v. 42, p. 917, 2004 (FCTOD7)]

The daily oral administration to rats over 33 days leads to not specified changes in the liver and enzyme inhibition, induction, or change in blood or tissue levels: Phosphatases; TDLo: 1980 mg/kg/33D-I [Food and Chemical Toxicology. (Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, NY 10523) V.20- 1982- v. 44, p. 579, 2006 (FCTOD7)]

Androstendion applied once daily orally over 33 days results in changes in serum composition (e.g., TP, bilirubin, cholesterol) and metabolism (intermediary): Lipids, including transport; TDLo: 165 mg/kg/33D-I [Food and Chemical Toxicology. (Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, NY 10523) V.20- 1982- v. 44, p. 579, 2006 (FCTOD7)]

The daily intramuscular administration to mice over 4 days results in maternal effects in uterus, cervix, vagina ; TDLo: 200 mg/kg/4D-I [Journal of Pharmacology and Experimental Therapeutics. (Williams and Wilkins Co., 428 E. Preston St., Baltimore, MD 21202) V.1- 1909/10- v. 297, p. 1099, 2001 (JPETAB)]

A lot of of human data is available from the use of androstendion to achieve a pharmacological effect (e.g. hormone replacement therapy, over-the-counter sports supplementation). As these data does not give relevant information in the context of REACH they were not included in the IUCLID.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the lowest NOAEL related to the leading health effect of the substance (effect on fertility) is selected.

Justification for classification or non-classification

No classification required for repeated dose toxicity according to Regulation (EC) No. 1272/2008 (CLP), as the leading health effect after repeated exposure to the substance is reproductive toxicity, and the substance is classified accordingly (see chapter "Toxicity to reproduction").