Androst-4-ene-3,17-dione

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Androstendione is an endogenous intermediate in steroid hormone synthesis and as such an endogenous precursor of testosterone and estrone, which can be metabolized to estradiol. Exposure during pregnancy may lead to signs of masculinization in the sex organs of a female child. If taken by nursing women androstendione may reach into the mother's milk and thus impair the development of the infant.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

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

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

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 females

Control animals:

yes, concurrent vehicle

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.

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: 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.

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).

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

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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

viability

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

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

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.

Reference 2

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

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 (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 throughout gestation. Caesarean sections were performed on GD 20.

- Parameters analysed / observed: serum androstenedione, estradiol, testosterone, progesterone, LH and FSH, 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:

yes

Specific details on test material used for the study:

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

Species:

rat

Strain:

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

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 (49 days old; 150–175 g), male (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 5002M (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

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%).

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: approximately 15 h/day, 5 days/week for up to 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 (n = 3) were necropsied.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Female rats received androstenedione in corn oil via intragastric intubations for
two weeks prior to mating, during the mating period and during GD 0–19.
The male rats were not treated and were only used as sires therefore the effect of androstenedione on male reproductive function was not tested in this study.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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.

(The control group (double dose control or ‘‘00’’ control) for the animals receiving androstenedione at a concentration of 60 mg/kg body weight received 0.6 ml corn oil alone per 100 g body weight.)

Control animals:

yes, concurrent vehicle

Details on study design:

Experimental animals received androstenedione in corn oil at 5.0, 10.0, 30.0 or 60 mg/kg of body weight. The dosages utilized in the present study were derived based on an androstenedione exposure of approximately 400 mg androstenedione taken per day (100 mg taken 4 times/day) by an individual weighing approximately 70 kg. The animals utilized in the present study were exposed to androstenedione at approximately 1, 2, 6 or 10 times this dose.

Oestrous cyclicity (parental animals):

The estrous cycles of randomly selected female rats (n = 20 rats/group) from the single and double dose control groups as well as the 30 and 60 mg/kg androstenedione treatment groups were monitored daily during the two week
pre-mating exposure period.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

Each viable fetus was removed from the uterus and examined individually
for any gross external morphological effects.
Records were kept as to its uterine position, sex, weight, crown-rump length, and anogenital distance, as well as any externally visible abnormalities.
A subset of fetuuses were examined for skeletal anomalies or 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.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

The mean number of estrous cycles observed during the 14 day pre-treatment in the 60 mg/kg androstenedione treatment group (2.85 ± 0.15) was significantly different from the double dose control group (3.45 ± 0.15). (Table 2) Similar effects were not observed when the single dose control group was compared with the 30 mg/kg dose group.

Reproductive performance:

no effects observed

Description (incidence and severity):

The time to mating was not affected as most animals mated within the first
week of co-habitation. Effects were not observed in the numbers of pregnant females, corpora lutea, implants, implantation efficiency, early and late deaths.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Effects were not observed in the numbers of viable fetuses, the total number of fetuses, number of viable fetuses/litter, anogenital distance, sex distribution, fetal body weight or crown-rump length data (Table 4).

The average number of soft-tissue variations per litter was similar in both control animals and the androstenedione treated animals except for a statistically significant increase in the number of fetuses with a moderately enlarged renal pelvis in the 30 mg/kg dose group (Table 5).
The increase appears to be random because of a lack of effect at 60 mg/kg. Exposure to androstenedione at the doses utilized in the present study did not affect fetal ossification.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: At the doses given, androstenedione had no specific effect on the development of individual bones or soft tissues.

Reproductive effects observed:

yes

Lowest effective dose / conc.:

60 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:

not specified

Relevant for humans:

yes

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.

 

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Androstendione is an endogenous intermediate in steroid hormone synthesis and as such an endogenous precursor of testosterone and estrone, which can be metabolized to estradiol. Exposure during pregnancy may lead to signs of masculinization in the sex organs of a female child. If taken by nursing women androstendione may reach into the mother's milk and thus impair the development of the infant.

For an assessment of steroid hormones see also the argumentation for steroid hormones related to the Technical Rule for Hazardous Substances 905, which was 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). In this argumentation 73 steroid hormones or precursors of steroid hormones were each allocated to one of seven hormone classes based on their predominant pharmacological activity (i.e. androgenic, mild androgenic, anabolic, estrogenic, gestagenic, mild gestagenic or glucocorticoide), and recommendations for their classification were elaborated.

For reproductive toxicity classification warranted as Repr. 1A (H360F/D: May damage fertility or the unborn child) according to Regulation (EC) 1272/2008. Additionally, classification is warranted for effects on or via lactation H362 according to Regulation (EC) 1272/2008 (May cause harm to breast-fed children).

 

A 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.

 

In a second study 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.

 

Further data on reproductive toxicity/fertility are cited in RTECS database (April 2013):

 

The subcutaneous administrations of androstendione to male rats over 25 days prior to mating result in no further specified effects on spermatogenesis (including genetic material, sperm morphology, motility, and count) testes, epididymis and sperm duct; TDLo: 10500 µg/kg (25D male) [Acta Medica Turcica. (Dr. Ayhan Okcuoglu, Cocuk Hastalikari Klinigi, c/o Ankara Univ., Tip Facultesi, Cebeci, Ankara, Turkey) V.1-10/11, 1948-58: New series: V.1- 1964- v. 8, p. 68, 1971 (AMTUA3)]

 

The subcutaneous administrations of androstendione to female rats over 14 days prior to mating result in maternal effects on ovaries, fallopian tubes, uterus, cervix and vagina and not further specified effects on fertility; TDLo: 7 mg/kg (14D pre) [Contraception. (Geron-X, Inc., POB 1108, Los Altos, CA 94022) V.1- 1970- v. 5, p. 489, 1972 (CCPTAY)]

 

The administration of androstendione to female rats via an implantat over16 days prior to mating results in menstrual cycle changes or disorders and not further specified effects on fertility ; TDLo: 5056 µg/kg (16D pre) [Journal of Reproduction and Fertility. (Biochemical Soc. Book Depot, POB 32, Commerce Way, Colchester, Essex CO2 8HP, UK) V.1- 1960- v. 56, p. 675, 1979 (JRPFA4)]

 

Effects on developmental toxicity

Description of key information

Androstendione is an endogenous intermediate in steroid hormone synthesis and as such an endogenous precursor of testosterone and estrone, which can be metabolized to estradiol. Exposure during pregnancy may lead to signs of masculinization in the sex organs of a female child. If taken by nursing women androstendione may reach into the mother's milk and thus impair the development of the infant.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

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

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

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

endocrine findings

Abnormalities:

no effects observed

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

 

 

 

 

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.

Reference 2

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

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

Specific details on test material used for the study:

obtained from Steraloids, Newport, RI
>99% pure

Species:

rat

Strain:

other: CD-CRL: CD-BR

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

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%).

Analytical verification of doses or concentrations:

yes

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.

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 females

Control animals:

yes, concurrent vehicle

Maternal examinations:

estrous cycle monitored during the 14-day pre-mating dosing period

Ovaries and uterine content:

- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

serum estradiol, estrone, testosterone and androstenedione levels

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Anogenital distance

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.

Details on maternal toxic effects:

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


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.

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).

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: reproductive function (oestrous cycle)

Abnormalities:

no effects observed

Reduction in number of live offspring:

effects 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).

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

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.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

no effects observed

Developmental effects observed:

no

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

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Androstendione is an endogenous intermediate in steroid hormone synthesis and as such an endogenous precursor of testosterone and estrone, which can be metabolized to estradiol. Exposure during pregnancy may lead to signs of masculinization in the sex organs of a female child. If taken by nursing women androstendione may reach into the mother's milk and thus impair the development of the infant.

For an assessment of steroid hormones see also the argumentation for steroid hormones related to the Technical Rule for Hazardous Substances 905, which was 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). In this argumentation 73 steroid hormones or precursors of steroid hormones were each allocated to one of seven hormone classes based on their predominant pharmacological activity (i.e. androgenic, mild androgenic, anabolic, estrogenic, gestagenic, mild gestagenic or glucocorticoide), and recommendations for their classification were elaborated.

 

A 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.

 

In a second study 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.

 

Additional data on developmental toxicity are cited in RTECS database (April 2013):

 

The daily subcutaneous administration of androstendione to hamsters on day 1-4 of pregnancy results in pre-implantation mortality (e.g., reduction in number of implants per female; total number of implants per corpora lutea); TDLo: 35 mg/kg (1-4D preg) [Endocrinology (Baltimore). (Williams and Wilkins Co., 428 E. Preston St., Baltimore, MD 21203) V.1- 1917- v. 80, p. 1152, 1967 (ENDOAO)]

 

The daily subcutaneous administration of androstendione to hamsters on day 1 -4 of pregnency results in maternal effects in ovaries, and fallopian tubes not further specified; TDLo: 17500 ug/kg (1-4D preg) [Endocrinology (Baltimore). (Williams and Wilkins Co., 428 E. Preston St., Baltimore, MD 21203) V.1- 1917- v. 80, p. 1152, 1967 (ENDOAO)]

 

Androstendione is administered subcutaneous to hamsters on day 1-4 of pregnancy. This leads in unspecified maternal effects of uterus, cervix and vagina; TDLo: 70 mg/kg (1-4D preg) [Endocrinology (Baltimore). (Williams and Wilkins Co., 428 E. Preston St., Baltimore, MD 21203) V.1- 1917- v. 80, p. 1152, 1967 (ENDOAO)]

 

Adrostendion is administered subcutaneous to rats on day 2-5 of pregnancy leading to maternal effects in uterus, cervix and vagina as well as pre-implantation mortality (e.g., reduction in number of implants per female; total number of implants per corpora lutea) and further unspecified effects on fertility; TDLo: 80 mg/kg (2-5D preg) [Endocrinology (Baltimore). (Williams and Wilkins Co., 428 E. Preston St., Baltimore, MD 21203) V.1- 1917- v. 81, p. 1091, 1967 (ENDOAO)]

 

The daily oral application of androstendion to rats two weeks prior to mating and 0-20 day of pregnancy results in specific developmental abnormalities of the urogenital system; TDLo: 280 mg/kg (2W pre/0-20D preg) [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 adminsitration of androstendione to female rats 14 day pre-mating and until day 19 after conception led to specific developmental abnormalities (central nervous system); TDLo: 1980 mg/kg (14D pre/-19D preg) [Toxicology and Industrial Health. (Princeton Scientific Pub. Co., POB 2155, Princeton, NJ 08540) V.1 -1985- v. 23,p. 65, 2007 (TIHEEC)]

 

Intramuscular application of androstendione to rats on day 14-21 of pregnancy results in specific developmental abnormalities on skin and skin appendages as well as effects on newborn: Live birth index (similar to fetuses per litter, except measured after birth) and delayed effects; TDLo: 80 mg/kg (14-21D preg) [Journal of Comparative and Physiological Psychology. (Washington, DC) V.40-96, 1947-82. v. 92, p. 13, 1978 (JCPPAV)]

 

Intramuscular administration of androstendione to rats on day 14-21 day of pregnancy leads to specific developmental abnormalities of the urogenital system and effects on newborn (e.g., reduced weight gain); TDLo: 40 mg/kg (14-21D preg [Journal of Comparative and Physiological Psychology. (Washington, DC) V.40-96, 1947-82. v. 92, p. 13, 1978 (JCPPAV)]

 

Androstendione administered subcutaneously to rats on day 5 of pregnancy results in pre-implantation mortality (e.g., reduction in number of implants per female; total number of implants per corpora lutea); TDLo: 20 mg/kg (5D preg) [Journal of Endocrinology. (Biochemical Soc. Book Depot, POB 32, Commerce Way, Colchester, Essex CO2 8HP, UK) V.1- 1939- v. 18, p. 271, 1959 (JOENAK)]

 

Subcutaneous application of androstendione to rats on day 8 of pregnancy results in post-implantation mortality (e.g., dead and/or resorbed implants per total number of implants); TDLo: 20 mg/kg (8D preg) [Journal of Endocrinology. (Biochemical Soc. Book Depot, POB 32, Commerce Way, Colchester, Essex CO2 8HP, UK) V.1- 1939- v. 18, p. 271, 1959 (JOENAK)]

Justification for classification or non-classification

For reproductive toxicity classification warranted as Repr. 1A (H360F/D: May damage fertility or the unborn child) according to Regulation (EC) 1272/2008. Additionally, classification is warranted for effects on or via lactation H362 according to Regulation (EC) 1272/2008 (May cause harm to breast-fed children).

 

The classification is in accordance with German legislation for classification of androgenic steroids. The German Committee on Hazardous Substances (AGS) recommended for androgenic steroids classification as Repr. Cat. 1 for effects on fertility and Repr. Cat. 2 for developmental toxicity (each according to criteria of Directive 67/458/EEC). (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).