Androst-4-ene-3,17-dione

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Effects of androstenedione on in utero development in rats

Author:

Sprando RL, et al.

Year:

2004

Bibliographic source:

Food and Chemical Toxicology 42 (2004) 917–924

Materials and methods

Principles of method if other than guideline:

- Principle of test: Thirty-day old rats received corn oil alone or androstenedione (in corn oil) at one of four concentrations (0, 1.0, 5.0, 10.0 or 30.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and throughout gestation

- Parameters analysed / observed: Feed and fluid consumption, Body weight, estrous cycle, number of corpora lutea, the number of implantation sites, and the number and position of resorption sites and fetuses (dead or alive), gravid uterus weight;
fetuses: sex, weight, crown-rump length, anogenital distance, external, skelatal, visceral abnormalities

GLP compliance:

not specified

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:

SOURCE OF TEST MATERIAL
- Source: Steraloids, Newport, RI
- Purity: >99%

Test animals

Species:

rat

Strain:

other: CD-CRL: CD-BR

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Wilmington, MA)
- Females nulliparous and non-pregnant: yes
- female (n=60; 49 days old; 150–175 g), male (n=30; 56 days old; 175–200 g)
- Housing:
acclimation period: singly
mating period: two female rats co-habitated with a single male
- Diet (e.g. ad libitum): Purina Rodent Chow 5002 (Purina Mills, Inc., Richmond,
IN), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approx. 1 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64–79 F
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were stable for >35 days as determined by HPLC analysis. All solutions of androstenedione were prepared and used according to the results of the stability tests. The dosing solutions were not utilized until HPLC analysis confirmed the solutions were within the prescribed concentrations (±10%).

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: 5 times per week for 3 weeks or until mating occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
The females that did not mate at the end of the week were re-mated with a different
randomly selected male. At the end of the three week mating period, females that did not mate were necropsied.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

females: 2 weeks prior to mating, during the mating period (up to 3 weeks) and days 0–20 of gestation
male rats were not treated and were only used as sires

Frequency of treatment:

daily

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.:

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

No. of animals per sex per dose:

12 females

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

serum estradiol, estrone, testosterone and androstenedione levels

Oestrous cyclicity (parental animals):

monitored during the 14-day pre-mating dosing period

Postmortem examinations (parental animals):

number of corpora lutea, number of implantation sites, number and position of resorption sites and fetuses (dead or alive), gravid uterus weight

Postmortem examinations (offspring):

uterine position, sex, weight, crown-rump length, and anogenital distance,
abnormalities (external, skeletal, soft-tissue)

Statistics:

For the parameters feed and fluid consumption, number of corpora lutea, implants, alive, males and females alive and estrous cyclicity data an Analysis of
Variance (ANOVA) followed by a protected least significance difference (LSD) test (one-tail, if ANOVA p<0.05) was used to compare the control with each
of the treated groups. For the parameters implant efficiency and percent early, late, and total (early+late) resorptions, the data were transformed using a Freeman-Tukey Arc-Sine Transformation followed by an ANOVA and a protected LSD test as discussed above.
An Analysis of Covariance (ANCOVA) followed by a protected LSD (two-tail, if the ANCOVA p<0.05) test was used to compare the control with each treated group for the parameters adult organ weights, and mean body weight gain and gravid uterine weight of pregnant females. The ANCOVA adjusted the body weight gain and the gravid weight by the Day 0 dam weight, and the organ weight by the final dam weight.
For both the fetal weight and crown rump measurements a Nested ANOVA followed by a protected LSD test (p<0.05) was used to compare the control and
treated groups.
A Fisher’s Exact Test was used to compare the treated groups with the control for the incidence of specific softtissue, sternebral, and skeletal variations in fetuses, and clinical signs in female rats and Day 20 fetuses. The of the treated groups for the number of litters with 1+, 2+, or 3+ soft-tissue, sternebral, and skeletal variations.
For the average number of fetuses per litter with 1+, 2+ or 3+ soft-tissue, sternebral, and skeletal variations the data were first transformed by a Freeman-Tukey Arc-Sine transformation. The transformed data were then analyzed using an ANOVA followed by a protected LSD test as explained above. A Nested ANOVA followed by a protected LSD test was used to compare the control and treated groups for the average number of ossified vertebrae in fetuses.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Dose-related increases in serum androstenedione levels were observed in all treated groups, and statistically significant increases were seen in the 5.0, 10.0 and 30.0 mg/kg dose groups. Dose related statistically significant increases in serum estrone levels were observed in all treated groups in comparison to the controls. A dose-related increase in serum estradiol levels was observed in all treated groups, and the increase was significant in the 10.0 and 30.0 mg/kg dose groups.
Serum testosterone levels were elevated in the 10 and 30 mg groups and significantly elevated in the 30.0 mg/kg dose group

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in the number of androstenedione treated animals having a regular estrous cycle was observed in the 10.0 and 30.0 mg/kg dose groups during the pre-mating period (Table 3). Only one animal from the 10.0 mg/kg dose group had a highly irregular cycle and at least one animal from each androstenedione dose group was acyclic.

Details on results (P0)

Mean corpora lutea counts from androstenedione treated animals were indistinguishable from control values (Table 4). A slight (non-statistically significant) reduction in the number of implants, the average percent of early plus late deaths, and the mean number of viable fetuses and the mean number of viable male fetuses was observed in the 30.0 mg/kg dose group (Table 4).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Remarks on result:

other: only females treated

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A slight (non-statistically significant) reduction in the mean number of viable fetuses and the mean number of viable male fetuses was observed in the 30.0 mg/kg dose group (Table 4).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Androstenedione exposure did not affect sternebral ossification, the incidence of fetal sternebal variations or the incidence of specific fetal skeletal variations.

A statistically significant increase in the number of fetuses with a moderately enlarged ureter at the kidney was observed in the 1.0 and 5.0 mg/kg dose groups, and slight non-statistically significant increases were observed in the 10.0 and 30.0 mg/kg dose groups (Table 5). The average number of soft-tissue variations per litter was similar in both control animals and the androstenedione treated animals (Table 5). Statistically significant differences in dam organ weights expressed per gram of body weight and dam organ weights expressed per gram of brain weight were not observed when values obtained for treated and control groups were compared.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

viability

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 1: Mean serum endocrine parameters (±SEM) from day 20 pregnant rats

Dose (mg/kg)	0	1.0	5.0	10.0	30.0
No. Dams	11	11	10	11	10
Androstenedione (ng/ml)	1.27±0.15	1.36±0.14	1.83±0.20**	2.19±0.25**	4.71±0.41****
Estradiol (pg/ml)	65.86±4.94	77.54±7.72	79.89±7.12	90.63±6.43*	128.34±10.86****
Estrone (pg/ml)	33.02±4.92	42.37±3.72*	55.97±6.24**	62.26±7.05****	110.79±13.82****
Testosterone (ng/ml	0.21±0.03	0.18±0.04	0.19±0.03	0.27±0.03	0.70±0.11****

 

Asterisks indicate significant difference from controls (* p</=0.05; ** p</=0.01; *** p</=0.001; **** p</=0.0001).

 

 

Table 2: Mean feed and fluid consumption and body weight during the pre-mating period

Days	Dose (mg/kg)
	0	1.0	5.0	10.0	30.0
Feed Consumption (g±SEM)
0–14a	209.9±3.9	205.2±4.3	201.6±4.1	214.2±6.0	219.2±5.4
0–20b	401.3±7.1	399.6±14.0	400.0±7.4	408.3±8.7	400.9±11.4
Fluid Consumption (ml±SEM)
0–14a	376.4±18.1	388.3±26.5	369.2±17.6	415.8±23.7	404.7±15.1
0–20b	642.5±25.9	684.3±34.7	644.4±24.0	727.7±30.4	656.2±19.1
Terminal Body Weight (g±SEM)
14a	225.4±3.7	223.2±3.5	222.7±3.1	230.6±5.1	236.6±4.5
20b	364.1±5.3	361.5±9.9	365.4±6.1	368.5±9.7	366.3±8.2
Mean Body Weight Gain (g±SEM)
0–14a	37.4±3.5	32.7±2.6	33.4±3.5	40.6±2.8	45.8±3.0
0–20b	136.2±2.0	139.3±7.2	138.8±4.7	134.2±6.8	125.7±6.2
Adjusted Weight Gain (g±SEM)
20b,c	56.4±3.3	58.2±4.0	59.7±4.0	57.8±3.5	57.1±7.2

a n=12 animals per group.

b n=11, 11, 10, 11, 10 animals per group for the 0.0, 1.0, 5.0, 10.0 and 30.0 mg/kg body weight dose groups.

c Total weight gain minus gravid uterine weight.

 

 

Table 3: Summary of estrous cycle measurements collected for 14 consecutive days prior to mating for female rats treated with androstenedione

Dose Level (mg/kg)	0	1.0	5.0	10.0	30.0
No. Dams	12	12	12	12	12
Regular Cycle (4-5 Days)	10	8	9	5*	5*
Irregular Cycle (3, 6–10 Days)	2	3	2	5	5
Highly Irregular Cycle (11–13 Days)	0	0	0	1	0
Acyclic (14 Days)	0	1	1	1	2
Total No. Estrous Cycles	42	34	34	31	28
Mean No. Estrous Cycles (Mean±SEM)	3.6±0.2	2.9±0.2*	2.9±0.2*	2.9±0.1**	2.4±0.2**

Asterisks indicate significant difference from controls (* p</=0.05; ** p</=0.01).

 

 

Table 4: Analysis of maternal and reproductive autopsy findings

Dose Level (mg/kg)	0	1.0	5.0	10.0	30.0
No of pregnant females	11	11	10	11	10
Corpora Lutea (Mean±SEM)	14.45±0.41	14.91±0.44	15.40±0.72	14.91±0.49	14.60±0.54
Implants (Mean±SEM)	14.00±0.50	13.82±1.02	14.20±0.76	13.45±1.11	12.70±1.03
Implantation Efficiency	96.84±2.00	92.18±5.74	93.11±4.36	90.08±6.97	86.94±6.43
Av. Percent early / late deaths/litter (%, Mean±SEM)	4.43±1.73	2.92±1.57	5.17±2.25	3.60±1.37	7.90±3.03
Viable Fetuses (Mean±SEM)	13.36±0.51	13.36±0.97	13.50±0.86	12.91±1.05	11.60±0.97
Male	6.45±0.37	6.36±0.72	6.30±0.73	6.36±0.59	4.50±0.52
Female	6.91±0.65	7.00±0.74	7.20±0.55	6.55±0.68	7.10±0.62
Sex Distribution (%)
Male	48.30	47.62	46.67	49.30	38.79
Female	51.70	52.38	53.33	50.70	61.21
Fetal body weight (g, Mean±SEM)
Males	3.99±0.03	4.06±0.04	3.87±0.04	3.91±0.05	3.92±0.06
Females	3.81±0.03	3.83±0.03	3.71±0.03	3.73±0.04	3.63±0.06
Fetal crown-rump length (cm, Mean±SEM)
Males	4.1±0.01	4.2±0.02	4.1±0.01	4.1±0.02	4.1±0.02
Females	4.0±0.01	4.0±0.01	4.0±0.01	4.0±0.01	4.0±0.02
No. runts (litters with runts)
Males)	0(0)	0(0)	0(0)	2(1)	1(1)
Females	0(0)	0(0)	0(0)	1(1)	2(2)

 

 

 

 

 

 

Table 5: Incidence of specific fetal soft-tissue variations

Dose Level (mg/kg)	0	1.0	5.0	10.0	30.0
No. fetuses (litters) examined	76(11)	73(11)	69(10)	70(11)	57(10)
Hemorrhage, internal	1(1)	1(1)	-	-	-
Hydroureter, severe	1(1)	8(4)	4(4)	2(2)	4(3)
Hydroureter, moderate	5(4)	13(6)	15(8)	8(5)	11(7)
Enlarged renal pelvis, severe	-	-	-	-	-
Enlarged renal pelvis, moderate	1(1)	-	1(1)	-	1(1)
Enlarged ureter at kidney, severe	2(2)	7(4)	3(2)	3(2)	5(3)
Enlarged ureter at kidney, moderate	5(2)	15(7)*	17(9)**	9(6)	7(6)
Kidney, ectopic	3(3)	-	-	-	-
Cleft Palate	1(1)	-	-	-	-
Undescended Testicle	-	-	-	-	1(1)

Number of litters involved in parentheses. Asterisks indicate: * p</=0.05; ** p</=0.01

Applicant's summary and conclusion

Executive summary:

This study was conducted to characterize the effect of androstenedione on estrous cyclicity, mating behavior and fetal development.
Thirty-day old rats received corn oil alone or androstenedione (in corn oil) at one of four concentrations (0, 1.0, 5.0, 10.0 or 30.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and throughout gestation. Dose related increases in serum androstenedione, estradiol and estrone were observed in all androstenedione treated animals at gestation day 20. A statistically significant increase in serum testosterone concentration was observed in the 30 mg/kg dose group. Feed and fluid consumption were not affected by androstenedione treatment during the pre-mating or gestational periods, however a statistically significant decrease in the number of females with regular estrous cycles was observed in the 10.0 and 30.0 mg/kg dose groups. Exposure to androstenedione did not affect mean body weight gain during pre-mating or gestation. Slight not statistically significant reductions in the number of implants, number of viable fetuses and number of viable male fetuses were observed in the
30.0 mg/kg androstenedione group. Reductions were not observed in the number of corpora lutea. Fetal growth in terms of fetal weight, crown-rump length, anogenital distance and the number of external abnormalities was not affected by androstenedione exposure. At the doses given, androstenedione had no specific effect on the development of individual bones, including sternebrae.
Dose related effects of androstenedione were not observed on the development of soft tissues. A statistically significant increase in moderately enlarged ureter at the kidney was observed in both the 1.0 and 5.0 mg/kg dose groups. Organ weights (expressed per gram of body weight or per gram of brain weight) were not affected by androstenedione treatment.