Androst-4-ene-3,17-dione

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

NTP protocol followed – dose range finding for 2-year study (no testing of sensory reactivity/motor activity, not all mandatory endpoint with regard to ED investigated (no thyroid hormones)

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

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:

- 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).

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

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

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.

Examinations

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).

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

All rats survived to the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes in hematology variables that were considered attributable to androstenedione administration.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes in clinical chemistry variables that were considered attributable to androstenedione administration.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 pathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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.

Effect levels

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).

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

cauda epididymis

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Survival and body Weights of Rats in the 3-Month Gavage Study of androstenedione

	dose (mg/kg)	Survival a	Mean Body Weight b (g) Final Weight			Final Weight Relative to Controls (%)
			Initial	Final	Change
Male	0	10/10	101 ± 2	333 ± 7	232 ± 6
	1	10/10	101 ± 2	340 ± 6	239 ± 6	102
	5	10/10	101 ± 1	344 ± 4	243 ± 4	103
	10	10/10	102 ± 2	343 ± 5	241 ± 4	103
	20	10/10	102 ± 1	343 ± 3	241 ± 3	103
	20	10/10	101 ± 2	340 ± 5	239 ± 4	102
Female	0	10/10	92 ± 1	189 ± 2	97 ± 3
	1	10/10	89 ± 1	200 ± 4*	111 ± 4*	106
	5	10/10	92 ± 1	198 ± 2*	107 ± 2	105
	10	10/10	93 ± 1	198 ± 2*	106 ± 2	105
	20	10/10	90 ± 2	206 ± 3**	116 ± 3**	109
	20	10/10	89 ± 1	199 ± 4**	111 ± 4**	105

 

* Significantly different (P#0.05) from the vehicle control group by Williams’ test

** P#0.01

a Number of animals surviving at 2 weeks/number initially in group

b Weights and weight changes are given as mean ± standard error.

 

 

Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Gavage Study

of Androstenedione

	Vehicle Control	10 mg/kg	20 mg/kg	50 mg/kg
N	10	10	10	10
Weights (g)
Necropsy body wt	333 ± 8	343 ± 5	343 ± 3	340 ± 5
L. Cauda epididymis	0.1954 ± 0.0057	0.2082 ± 0.0044	0.2050 ± 0.0034	0.2083 ± 0.0048
L. Epididymis	0.4562 ± 0.0119	0.4610 ± 0.0075	0.4540 ± 0.0082	0.4303 ± 0.0110
L. Testis	1.5171 ± 0.0277	1.5272 ± 0.0259	1.5135 ± 0.0175	1.5053 ± 0.0220
Spermatid measurement
Spermatid heads (1E03 /mg testis)	124.86 ± 3.27	127.11 ± 4.83b	131.80 ± 4.53	126.49 ± 3.56
Spermatid heads (1E06 /testis)	169.38 ± 5.53		178.00 ± 6.85	169.38 ± 5.91
Epididymal spermatozoal measurements
Sperm (1E03 /mg cauda epididymis)	575 ± 27	475 ± 28*	463 ± 27*	407 ± 21**
Sperm (1E06 /cauda epididymis)	112 ± 5	98 ± 5	95 ± 6	85 ± 5**
Sperm motility (%)	75.7 ± 2.2	76.0 ± 2.1	73.8 ± 2.4	74.7 ± 1.5

* Significantly different (P</=0.05) from the vehicle control group by Shirley’s test

** (P</=0.01)

a Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett’s test (body and tissue weights) or Dunn’s test (spermatid measurements).

b n = 9

Estrous Cycle Characterization for Female Rats in the 3-Month Gavage Study of Androstenedione a

	Vehicle Control	10 mg/kg	20 mg/kg	50 mg/kg
Number weighed at necropsy	10	10	10	10
Necropsy body wt	189 ± 2	198 ± 2	206 ± 3**	199 ± 4
Proportion of regular cycling females b	10/10	10/10	9/10 c	7/10 d
Estrous cycle length (days)	4.8 ± 0.2	5.0 ± 0.1	4.9 ± 0.1	5.2 ± 0.2
Estrous stages (% of cycle)
Diestrus	60.0	60.0	60.2	61.1
Proestrus	10.0	16.7	13.0	7.4
Estrus	29.2	22.5	21.3	22.2
Metestrus	0.8	0.8	5.6	7.4
Uncertain diagnoses	0.0	0.0	0.0	1.9

 

** Significantly different (P#0.01) from the vehicle control group by Dunnett’s test

a Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunn’s test (estrous cycle length). By multivariate analysis of variance, dosed females do not differ significantly from the vehicle control females in the relative length of time spent in the estrous stages.

b Number of females with a regular cycle/number of females cycling

c Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.

d Estrous cycle was longer than 12 days or unclear in 3 of 10 animals.

Applicant's summary and conclusion

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.