Androst-4-ene-3,17-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

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:

Unnamed

Year:

2005

Materials and methods

Principles of method if other than guideline:

- Principle of test:
Thirty-day old female rats received corn oil or androstenedione (in corn oil) at one of four concentrations (5.0, 10.0, 30.0 or 60.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and until gestation day (GD) 19. Caesarean sections were performed on GD 20.

- Parameters analysed / observed:
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;
viable fetuses examined individually for any gross external morphological effects, sex, weight, crown-rump length, and anogenital distance, any externally visible abnormalities, skeletal anomalies, soft-tissue anomalies;
serum hormones levels: Androstenedione, Estradiol, LH, FSHTestosterone, Progesterone

GLP compliance:

yes

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:

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

Test animals

Species:

rat

Strain:

other: CD-CRL:CD-BR, VAF+

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Wilmington, MA)
female (n = 250; 49 days old; 150–175 g)
male (n = 91; 56 days old; 175–200 g)
- Housing:
females: singly during the pre-mating dosing period and from GD 0 until GD 20
- Diet (e.g. ad libitum): Purina Rodent Chow 5002M (Purina Mills, Inc., Richmond, IN), ad libitum
males: singly, except during mating period
mating period: two female rats + one male rat
- 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 only utilized after HPLC analysis confirmed that the solutions were within the prescribed concentrations (±10%).

VEHICLE
- Amount of vehicle (if gavage):
0.3 ml corn oil /100 g bw: control, 5.0, 10.0 or 30.0 mg/kg bw/d dose groups
0.6 mL/100 g bw: double dose control or ‘‘00’’ control, 60 mg/kg bw/d dose group

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2
- Length of cohabitation: 15 h/day, 5 days/week for up to 3 weeks or until mating occurred
-females that did not mate at the end of the week were re-mated with a different randomly selected
- At the end of the three-week mating period, females that did not mate (n = 3) were necropsied.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

- males: not exposed
- females: 2 weeks prior to mating, during the mating period (up to 3 weeks) and from GD 0 through GD 19

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

The distribution of the animals among 0 control, 00 control, 5, 10, 30 and 60 mg/kg dose groups were 39, 29, 39, 29, 33 and 40, respectively.

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

Estrous cyclicity was evaluated in 20 randomly selected animals from the 30 mg/kg and 60 mg/kg treatment groups monitored daily during the two week pre-mating exposure period

Ovaries and uterine content:

- corpora lutea, number of implantation sites, and number and position of resorption sites and fetuses (dead or alive)
- gravid uterus was removed in toto and weighed

Blood sampling:

Blood was collected from the inferior vena cava of all pregnant females, serum hormone (Androstenedione, Estradiol, LH, FSH, Testosterone, Progesterone) and gonadotrophin levels were determined by radioimmunoassay

Fetal examinations:

gross external morphological effects
anogenital distance
skeletal anomalies
soft-tissue anomalies

Statistics:

A p value of 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 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 soft-tissue variations in fetuses, and clinical external signs in female rats and day 20 fetuses.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

endocrine findings

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no specific effect on fetal parameters

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Serum endocrine parameters (mean ± SEM) from day 20 pregnant rats a

	Dose levels (mg/kg)
	0 (19)	00 (21)	5.0 (25)	10.0 (27)	30.0 (24)	60.0 (27)
Androstenedione b	1.67 ± 0.14	1.65 ± 0.16	1.53 ± 0.10	1.64 ± 0.11	1.75 ± 0.10	1.67 ± 0.11
Estradiol c	61.68 ± 3.51	50.60 ± 3.96	56.09 ± 4.30	54.29 ± 3.59	62.61 ± 4.51	57.08 ± 3.24
LH d,b	1.06 ± 0.07	1.12 ± 0.04	1.28 ± 0.10	1.30 ± 0.15	1.08 ± 0.06	1.07 ± 0.07
FSH b	5.92 ± 0.38	6.13 ± 0.31	6.53 ± 0.32	6.26 ± 0.27	6.53 ± 0.37	5.81 ± 0.26
Testosterone b	0.47 ± 0.06	0.59 ± 0.08	0.48 ± 0.06	0.48 ± 0.04	0.52 ± 0.06	0.46 ± 0.05
Progesterone b	16.68 ± 1.99	16.15 ± 2.50	15.57 ± 1.78	15.53 ± 1.80	17.04 ± 1.79	16.82 ± 1.34

( ) = number of litters.

a If the p value is not given then p > 0.10.

b Data expressed in ng/ml.

c Data expressed in pg/ml.

d Data transformed using a log transformation.

 

 

Table 2: Estrous cycle measurements during 14-day pre-mating period (day, mean ± SEM)

	Dose levels (mg/kg)
	0 (20)	00 (20)	30.0 (20)	60.0 (20)
Regular cycle (4–5 days)	2.05 ± 0.17	2.20 ± 0.17	2.05 ± 0.17	1.65 ± 0.18
Irregular cycle (2,3,6–10 days)	0.25 ± 0.12	0.25 ± 0.14	0.10 ± 0.07	0.30 ± 0.15
Highly irregular cycle (11–13 days)	0.00 ± 0.00	0.00 ± 0.00	0.05 ± 0.05	0.00 ± 0.00
Acyclic (14 days)	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Number of estrous stages observed	3.30 ± 0.13	3.45 ± 0.15	3.20 ± 0.14	2.85 ± 0.15*

( ) = number of animals per group. *Indicates statistically significant difference from the ‘‘00’’ control group; p </= 0.05.

 

 

Table 3: Mean feed and fluid consumption and body weight gain during pre-mating (days 0–14) and gestation (days 0–20) a

	Dose levels (mg/kg)
	0	00	5.0	10.0	30.0	60.0
Feed consumption (g ± SEM)
0–14 b	229.3 ± 2.7	207.4 ± 2.3	231.7 ± 3.0	225.0 ± 2.9	229.2 ± 2.7	211.9 ± 1.7
0–20 c	399.2 ± 6.0	344.7 ± 6.2	396.9 ± 6.2	397.3 ± 7.0	402.6 ± 5.8	358.6 ± 4.3
Fluid consumption (ml ± SEM)
0–14 b	387.8 ± 9.7	376.0 ± 10.6	375.0 ± 10.1	356.5 ± 10.9	390.8 ± 8.1	363.7 ± 7.0
0–20 c	727.7 ± 16.8	650.9 ± 24.0	698.5 ± 17.4	711.3 ± 24.6	732.3 ± 18.8	655.3 ± 14.2
Mean body weight gain (g ± SEM)
0–14 b, d	34.4 ± 1.7	31.8 ± 1.4	34.1 ± 1.5	34.5 ± 1.7	37.5 ± 1.6	38.6 ± 1.3
0–20 c, d	138.4 ± 4.6	132.0 ± 7.1	133.6 ± 4.3	134.5 ± 4.7	134.2 ± 5.2	131.9 ± 4.7

a If p value not given p > 0.10.

b n = 47, 38, 39, 30, 41 and 57 animals for the 0, 00, 5.0, 10.0, 30.0 and 60 mg/kg dose groups, respectively.

c n = 39, 29, 39, 29, 33 and 40 animals for the 0, 00, 5.0, 10.0, 30.0 and 60 mg/kg dose groups, respectively.

d Total weight gain minus gravid uterine weight.

 

 

Table 4: Analysis of maternal and reproductive necropsy findings a

	Dose level (mg/kg)
	0 (39)	00 (29)	5.0 (39)	10.0 (29)	30.0 (33)	60.0 (40)
	No. (%) of pregnant females
	37 (95)	27 (93)	38 (97)	28 (97)	32 (97)	38 (95)
	Corpora lutea (Mean ± SEM)
	16.27 ± 0.29	16.19 ± 0.52	16.03 ± 0.46	16.86 ± 0.39	16.03 ± 0.41	16.13 ± 0.38
	Implants/litter (Mean ± SEM)
	15.27 ± 0.30	14.89 ± 0.70	14.03 ± 0.66	14.86 ± 0.58	14.44 ± 0.52	14.21 ± 0.57
	Implantation efficiency (%, Mean ± SEM)
	94.14 ± 1.34	90.47 ± 3.45	86.88 ± 3.09	88.12 ± 2.91	89.63 ± 2.56	87.63 ± 3.04
	Early + late deaths/litter (%, Mean ± SEM)
	3.96 ± 0.84	6.50 ± 3.69	2.55 ± 0.69	7.14 ± 2.10	9.53 ± 3.21	4.20 ± 0.89
	Viable fetuses/litter (Mean ± SEM)
	14.68 ± 0.32	14.44 ± 0.73	13.61 ± 0.62	13.79 ± 0.62	13.44 ± 0.61	13.61 ± 0.56
	Total number of fetuses (litters)
Male	276 (37)	195 (26)	273 (38)	176 (28)	221 (31)	264 (38)
Female	267 (37)	195 (26)	244 (38)	210 (28)	209 (31)	253 (37)
Viable fetuses/litter (Mean ± SEM)
Male	7.46 ± 0.32	7.22 ± 0.53	7.18 ± 0.43	6.29 ± 0.51	6.91 ± 0.44	4.50 ± 0.52
Female	7.22 ± 0.39	6.95 ± 0.39	6.42 ± 0.37	7.50 ± 0.49	6.53 ± 0.42	7.10 ± 0.62
Anogenital distance (Mean ± SEM)
Male	3.40 ± 0.02	3.40 ± 0.03	3.40 ± 0.02	3.34 ± 0.03	3.38 ± 0.02	3.45 ± 0.02
Female	1.68 ± 0.01	1.73 ± 0.02	1.69 ± 0.01	1.66 ± 0.02	1.74 ± 0.02	1.76 ± 0.01
Sex distribution (%)
Male	50.83	50	52.8	45.6	51.4	51.06
Female	49.17	50	47.2	44	48.6	48.94
Fetal body weight (g, Mean ± SEM)
Males	3.80 ± 0.02	3.70 ± 0.02	3.76 ± 0.02	3.75 ± 0.03	3.74 ± 0.03	3.73 ± 0.03
Females	3.63 ± 0.02	3.54 ± 0.02	3.56 ± 0.02	3.56 ± 0.03	3.62 ± 0.02	3.59 ± 0.02
Fetal crown-rump length (cm, Mean ± SEM)
Males	4.02 ± 0.01	3.99 ± 0.01	4.01 ± 0.01	4.01 ± 0.01	4.00 ± 0.01	3.99 ± 0.01
Females	3.94 ± 0.01	3.92 ± 0.01	3.91 ± 0.01	3.93 ± 0.01	3.94 ± 0.01	3.91 ± 0.01
No. runts (litters with runts)
Males	1 (1)	2 (2)	1 (1)	2 (2)	2 (2)	5 (5)
Females	3 (3)	0 (0)	2 (2)	2 (2)	0 (0)	4 (4)

a If the p value is not given then p > 0.10.

 

 

 

Table 5: Representative incidence of specific fetal soft-tissue variations *

Dose level (mg/kg)	0	0	5	10	30	60
No. fetuses (litters) examined	236 (35)	196 (26)	225 (36)	194 (28)	214 (31)	232 (37)
Hydroureter, severe	6 (5)	7 (5)	5 (5)	10 (6)	4 (4)	10 (7)
Hydroureter, moderate	0 (0)	24 (12)	0 (0)	0 (0)	0 (0)	13 (11)
Enlarged renal pelvis, moderate	6 (3)	8 (7)	14 (8)	8 (5)	12 (10)*	12 (9)
Enlarged ureter at kidney, severe	12 (7)	16 (8)	11 (7)	10 (6)	4 (4)	9 (7)
Enlarged ureter at kidney, moderate	16 (12)	15 (8)	23 (14)	20 (12)	17 (13)	18 (14)
Kidney, ectopic	1 (1)	0 (0)	1 (1)	1 (1)	1 (1)	3 (3)
Anophthalmia	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (1)

( ) = number of litters.

*p 6 0.05.

 

 

Table 6A: Absolute organ weights for selected maternal organs (g, mean ± SEM)

	Dose level (mg/kg)
	0 (31)	00 (26)	5.0 (33)	10.0 (28)	30.0 (32)	60.0 (33)
Heart	0.913 ± 0.016	0.882 ± 0.014	0.909 ± 0.011	0.902 ± 0.015	0.892 ± 0.015	0.942 ± 0.034
Liver	14.531 ± 0.241	14.282 ± 0.238	13.987 ± 0.280	14.215 ± 0.288	14.237 ± 0.276	14.647 ± 0.243
Spleen	0.710 ± 0.065	0.609 ± 0.016	0.621 ± 0.023	0.647 ± 0.019	0.611 ± 0.014	0.604 ± 0.012
Adrenal
Rt.	0.035 ± 0.002	0.033 ± 0.001	0.035 ± 0.001	0.032 ± 0.001	0.032 ± 0.001	0.030 ± 0.001
Lft.	0.037 ± 0.002	0.033 ± 0.001	0.036 ± 0.001	0.033 ± 0.001	0.032 ± 0.001	0.030 ± 0.001
Both	0.072 ± 0.003	0.066 ± 0.002	0.071 ± 0.002	0.065 ± 0.002	0.064 + 0.002	0.060 ± 0.002
Ovary
Rt.	0.077 ± 0.003	0.072 ± 0.002	0.074 ± 0.002	0.070 ± 0.002	0.076 ± 0.003	0.074 ± 0.003
Lft.	0.072 + 0.002	0.071 ± 0.003	0.070 ± 0.0021	0.072 ± 0.002	0.071 ± 0.002	0.070 ± 0.003
Both	0.150 + 0.004	0.143 ± 0.003	0.144 ± 0.0042	0.142 ± 0.003	0.147 ± 0.004	0.144 ± 0.005
Brain	1.898 ± 0.014	1.879 ± 0.021	1.888 ± 0018	1.925 ± 0.016	1.910 ± 0.016	1.889 ± 0.016

( ) = number of organs examined.

 

 

Table 6B: Organ to brain weight ratios for selected maternal organs (g, mean ± SEM)

	Dose level (mg/kg)
	0 (31)	00 (26)	5.0 (33)	10.0 (28)	30.0 (32)	60.0 (33)
Heart	0.481 ± 0.008	0.470 ± 0.008	0.482 ± 0.006	0.469 ± 0.008	0.468 ± 0.008	0.498 ± 0.016
Liver	7.659 ± 0.144 (25)a	7.625 ± 0.155	7.458 ± 0.156	7.390 ± 0.150	7.463 ± 0.150 (26)a	7.776 ± 0.133 (27)a
Spleen	0.373 ± 0.032	0.325 ± 0.008	0.392 ± 0.012	0.337 ± 0.011	0.320 ± 0.006	0.320 ± 0.006
Adrenal
Rt.	0.018 ± 0.001	0.018 ± 0.001	0.018 ± 0.001	0.017 ± 0.001	0.017 ± 0.001	0.016 ± 0.001**
Lft.	0.019 ± 0.001	0.018 ± 0.001	0.019 ± 0.001	0.017 ± 0.001*	0.017 ± 0.001*	0.016 ± 0.001**
Both	0.037 ± 0.001	0.036 ± 0.001	0.037 ± 0.001	0.034 ± 0.001*	0.034 + 0.001*	0.032 ± 0.001**
Ovary
Rt.	0.041 ± 0.001	0.038 ± 0.001	0.039 ± 0.002	0.037 ± 0.001	0.040 ± 0.002	0.039 ± 0.002
Lft.	0.038 + 0.001	0.038 ± 0.002	0.037 ± 0.001	0.037 ± 0.001	0.0378 ± 0.001	0.037 ± 0.002
Both	0.079 + 0.002	0.076 ± 0.002	0.076 ± 0.002	0.074 ± 0.002	0.077 ± 0.002	0.076 ± 0.002

( ) = number of organs examined.

* Significantly different than the ‘‘0’’ control group, p </= 0.05.

** Significantly different than the ‘‘00’’ control group, p </= 0.05.

a Indicates that six livers were transferred to another protocol.

 

 

 

 

Applicant's summary and conclusion

Executive summary:

Thirty-day old female rats received corn oil or androstenedione (in corn oil) at one of four concentrations (5.0, 10.0, 30.0 or 60.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and until gestation day (GD) 19. Caesarean sections were performed on GD 20. No dose related changes were observed in serum androstenedione, estradiol, LH, FSH, testosterone or progesterone. A statistically significant decrease in estrous cycle length was observed in the 60.0 mg/kg dose group only. Feed and fluid consumption, mean body weight gain, organ weight and fetal parameters were not affected by androstenedione treatment. At the doses given, androstenedione had no specific effect on the development of individual bones or soft tissues.