Reaction products of 2-(chloromethyl)oxirane and glycerol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

similar to OECD Guideline 415 (One-Generation Reproduction Toxicity Study)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Effects of Epichlorohydrin on Male and Female Reproduction in Long-Evans Rats

Author:

Toth, G., P., Zenick, H., Smith, M.K.

Year:

1989

Bibliographic source:

Fundamental and Applied Toxicology: 13. 16-25 (1989)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-chloro-2,3-epoxypropane

EC Number:

203-439-8

EC Name:

1-chloro-2,3-epoxypropane

Cas Number:

106-89-8

Molecular formula:

C3H5ClO

IUPAC Name:

2-(chloromethyl)oxirane

Constituent 2

Reference substance name:

3-chloro- 1 ,2 propane oxide

IUPAC Name:

3-chloro- 1 ,2 propane oxide

Details on test material:

- Name of test material (as cited in study report): Epichlorohydrin (3-chloro- 1 ,2 propane oxide)
- Analytical purity: 99%

Test animals

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI).
- Age at study initiation: males (80-90 days old) and females (70-80 days old)
- Weight at study initiation: no data
- Fasting period before study: no
- Housing:Males were singly housed in stainless-steel hanging cages. Females were housed three to a cage until pregnant, when they were transferred to individual shoebox cages.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Purina Lab Chow 5001 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 2 to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 10/14 with lights on at 20:00 h (males) and 12/12 with light commencing at 6:00 h (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were made fresh weekly and stored in the refrigerator. Chemical analyses showed that
these solutions were stable for this period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test material is well soluble in corn oil.
- Concentration in vehicle: Concentrations were adjusted so that animals received 1 mL solution per kilogram of body weight.
- Amount of vehicle (if gavage): 1 mL

Details on mating procedure:

- M/F ratio per cage: 1/3
- Length of cohabitation: In the mating trials designed to evaluate pre-and postimplantation loss, treated males were cohabited overnight (0 and 50 mg/kg bw only); In the trials to evaluate female fertility, an untreated male was placed in each female cage to cohabit overnight.
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 1] of pregnancy;
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no.
During the third week of treatemnt, males were allowed 5 days to mate, with a different proestrous female introduced each day until each male had copulated successfully with one female.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually in shoebox cages.
- Any other deviations from standard protocol: see below in "Any other information on materials and methods, incl. tables"

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: 21 days;
Females: 2 weeks prior to mating, 1 week during mating and during the gestation period until delivery.

Frequency of treatment:

once daily/5 days a week

Details on study schedule:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

females

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Remarks:

females

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day

Remarks:

females

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day

Remarks:

males

Doses / concentrations 5

Dose / conc.:

25 mg/kg bw/day

Remarks:

males

Doses / concentrations 6

Dose / conc.:

12.5 mg/kg bw/day

Remarks:

males

No. of animals per sex per dose:

Males: 20
Females: no data

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary studies.
Males:
Epichlorohydrin given for 15 days resulted in severe toxicity in male rats (weight loss, nephrosis, testicular edema, epididymal granulomas, decreased testicular and epididymal sperm counts). Treatment levels for the expanded study were, therefore, set at 0, 12.5, 25, and 50 mg/kg body wt (n = 20/group).
Females:
Epichlorohydrin was lethal, causing death in 10/10 animals within 48 hr of initiation of treatment. Administering epichlorohydrin for 15 days at levels of 50 and 100 mg/kg caused dose-dependent increases in liver, kidney, adrenal, and spleen organ-to-body weight ratios. In the 100 mg/kg group, 5/10 animals were so moribund as to warrant their sacrifice after 7-9 days treatment. The remaining 5 animals did not show weight loss or other overt toxic signs, but on sacrifice after 15 days treatment they displayed pale and enlarged kidneys. Since half of the females treated with 100 mg/kg/day survived, this was selected as the high dose, while 25 and 50 mg/kg/day were chosen as the low and middoses, respectively.

- Rationale for animal assignment (if not random): the animals were assigned to treatment groups such that mean body weights were the same across groups.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

The animals were weighed on alternate days (except pregnant females which were weighed daily).

Oestrous cyclicity (parental animals):

Estrous cyclicity and copulatory behaviour were examined.

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility and sperm morphology.

Litter observations:

Litters were sexed and weighed on Postnatal Day 1 (day of delivery). Pups were weighed weekly thereafter. At Day 4, litters were culled to 8 pups (approximately 4 male and 4 female). At postnatal Day 21, dams were sacrificed and litters reduced to 1 male and 1 female. Weanlings were weighed weekly until sacrifice at Postnatal Day 42. No further data.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals: on Day 21; On day 23 (an additional group of males which was used for the measurement of cauda epididymal spem motion.
- Maternal animals: All surviving animals: One group of females was sacrificed on Day 15 of pregnancy and corpora lutea, implantations,
and resorptions were counted. Other females were sacrificed on Postnatal Day 21.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: no data

HISTOPATHOLOGY / ORGAN WEIGHTS
Males:
At sacrifice, the following organs were excised and weighed: liver, kidney, adrenals, spleen. heart, testes, accessory organs (prostate and seminal vesicles), vas deferens, and epididymis.

Postmortem examinations (offspring):

No data

Statistics:

Body weights for all animals were analyzed using a repeated-measures analysis of variance (BMDP, Los Angeles. CA). Fertility and pregnancy rates were analyzed by X2 methods (Snedecor and Cochran, 1980). All other data were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison procedure (SAS, Gary, NC), with the following parameters being transformed before analysis: mount latency period and ejaculation latency period (logarithmic): numbers of mounts and intromissions (square root); and percentage normal sperm morphology; and percentage motile sperm (arcsin). CellSoft-generated sperm motion parameters were analyzed using t tests (control vs treated groups) with a modified Bonferroni correction (Simes, 1986). Dose dependence was determined by linear regression for trend.

Reproductive indices:

In females which were sacrificed on Day 15 of pregnancy, corpora lutea, implantations, and resorptions were counted.

Offspring viability indices:

No data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

all females in the 100 mg/kg group died or were moribund.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males in the 50 mg/kg bw group exhibited a 7% decline in weight gain compared to controls. Females: no effects.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males in the 50 mg/kg bw group exhibited a 7% decline in weight gain compared to controls. Females: no effects.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Description (incidence and severity):

Test substance intake: not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

In the group of males, used for sperm motion analysis, cauda epididymal sperm counts were slightly but significantly decreased (50 mg/kg bw)

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Within the first week of treatment, all females in the 100 mg/kg group died or were so moribund as to warrant sacrifice. Animals in the remaining groups appeared healthy and in good condition.

Males: All animals appeared healthy and active throughout the experiment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males in the 50 mg/kg bw group exhibited a 7% decline in weight gain compared to controls. This difference was not significant (Table 1).
Females: Mean body weights across groups showed no differences during pregestational exposure nor during pregnancy (data not shown).


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The fertility rate and litter outcome data are shown in Table 6. Although mating success was somewhat low across all groups (since proestrous females were not chosen for mating trials), no differences were seen in the number of females mating or in number of successful pregnancies. Litter size and mean pup birth weight were not significantly different. One dam in the 25 mg/kg group neglected her pups, which all died by Postnatal Day 4. This accounts for the slightly decreased percentage survival seen in the 25 mg/kg group. Male and female pup weights, measured weekly, did not significantly differ during lactation or in the 3 weeks postweaning.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no significant differences in cauda epididymal sperm count, testicular spermatid count, or epididymal sperm morphology in treated animals (Table 3). In the second group of males used for sperm motion analyses, testis and epididymis organ-to-body weight ratios were unchanged. However, cauda epididymal spem counts were slightly, but significantly decreased in the 50 mg/kg bw group (p<0.05) (Table 4). Table 5 shows shows the effects of ECH treatment on cauda epididymal sperm motion parameters as measured with the Cell-Soft computer-assisted sperm motion analysis
system. Consistent with the analyses of ejaculated sperm, there was no change in the percentage of motile cells in the cauda epididymides of animals from any dose group. Mean curvilinear velocity and straight-line velocity of cauda epididymal sperm were reduced across all groups in a dose-dependent fashion (p = 0.0001). Table 5 also shows theeffect of ECH treatment of male rats on specific motion endpoints related to sperm swimming pattern: linearity, amplitude of lateral head displacement, flagellar beat frequency, and number of cells displaying circular motion. Sperm track linearity was reduced only at the 50 mg/kg dose level. However, a dose-dependent trend was evident (p = 0.000 1). The amplitude of lateral head displacement
(ALH) was decreased and the beat/cross frequency was increased in all dose groups in a dose-dependent manner (p < 0.0006). No effect was seen on the percentage of sperm swimming in a circular motion.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance was unimpaired by ECH treatment, as were copulatory plug weight, ejaculated sperm count, percentage motil sperm, and sperm morphology (Table 2). In the mating trials designed to evaluate pre- and postimplantation loss, all control and 50 mg/kg - males mated, as verified by a copulatory plug or vaginal sperm. However, none of the females that mated with ECH males was pregnant, compared to a 90% pregnancy rate in controls (data not shown).

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males: liver, kidney, and epididymal organ-to-body weight ratios were significantly higher (p<0.05) in 50 mg/kg bw animals relative to controls, although all differences were marginal. In the second group of males used for sperm motion analyses, testis and epididymis organ-to-body weight ratios were unchanged. However, cauda epididymal spem counts were slightly, but significantly decreased in the 50 mg/kg bw group (p<0.05) (Table 4).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Females: Autopsy of females from the 100 mg/kg bw dose group that died showed hemorrhagic stomachs and kidneys with a very pale cortex and hemorrhagic medulla.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Impaired fertility (none of females mated with the treated males from 50 mg/kg bw dose group were pregnant)

Remarks on result:

not determinable

Remarks:

no NOAEL identified

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects in any parameter examined.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
Litter size and mean pup birth weight were not significantly different. One dam in the 25 mg/kg group neglected her pups, which all died by Postnatal Day 4. This accounts for the slightly decreased percentage survival seen in the 25 mg/kg group.

CLINICAL SIGNS (OFFSPRING)
No detailed description.

BODY WEIGHT (OFFSPRING)

Mean pup birth weight were not significantly different. Male and female pup weights, measured weekly, did not significantly differ during lactation or in the 3 weeks postweaning (Table 7).

SEXUAL MATURATION (OFFSPRING) Not reported in details

Effect levels (F1)

Remarks on result:

other: no information available

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

TABLE 1
Male Body Weight and Organ/ 100 g Final Body Weight Ratios following Treatment with Epichlorohydrin (Means ± SD)
		Dose (mg/kg po)
	0 ^a	12.5 ^a	25 ^a	50 ^a
	(« = 20)	(n= 20)	(n = 20)	(n= 20)
Initial body weight (g)	356 ±37.7	353 ±37.1	352 ± 28.6	355 ±40.3
Final body weight (g)	403 ±38.5	390 + 34.6	386 + 24.2	377 ± 42.0
Liver	3.580 ±0.282	3.654 + 0.362^b	3.722 ±0.377 ^b	3.876 ±0.224 ^b
Kidney	0.413 ±0.025	0.425 ±0.049 ^b	0.412 ±0.026	0.445 ±0.037 ^b
Adrenal	0.0134 ±0.0028	0.0148 ±0.0033	0.0148 ±0.0031	0.0143 ±0.0034
Spleen	0.174 ±0.032	0.165 ±0.021	0.167 ±0.022	0.175 ±0.029
Heart	0.298 ± 0.023	0.303 ± 0.024	0.303 ±0.017	0.314 ±0.020
Testis	0.409 ±0.043	0.406 ±0.042	0.420 ±0.042	0.423 ±0.045
Vas deferens	0.0263 ±0.0025	0.0266 ± 0.0039	0.0283 ±0.0031 ^b	0.0291 ±0.0044 ^b
Accessory organs	0.735 ±0.107	0.771 ±0.094	0.791 ±0.092	0.782 ±0.144
Epididymis	0.134±0.015	0.138 + 0.01^b	0.143 + 0.012 ^b	0.148 ±0.020 ^b
Note.Organ weights expressed as g/100 g body wt.   ^a,b Groups with different superscripts are significantly different(p <0.05).

TABLE 2 Male Mating Behavior and Ejaculated Semen Parameters after 21 Days of Epichlorohydrin Treatment (Means ± SD)
	Dose (mg/kg po)
	0(n= 20)^a	12.5(n =20)^a	25(n =20)^a	50(n= 20)^a
Number of mounts	2.6 ±2.33	1.6 ± 1.54	2.2+1.56	2.9 + 2.02
Number of intromissions	15.8 ± 4.10 ^b	12.6 ±3.69	15.0 ± 3.49 ^b	14.8±3.90 ^b
Mount latency (sec)	7.8 ± 4.22	15.0 ±30.52	12.6 ± 12.36	10.3 ± 8.6
Ejaculation latency (sec)	280 ±81.2	267 ± 196.5	249 ± 140.4	247 ± 73.4
Plug weight (g)	0.12 ±0.022	0.12 ±0.022	0.12 ±0.020	0.12 ±0.017
Sperm count X 10^6	48 ± 13.3	53 ± 12.1	54 ±9.2	45± 15.1
% Normal morphology	98 + 2.0	98 ± 1.8	98 ± 1.6	98±0.1
% Motile	45± 1.1	37 ± 1.4	42 ±0.8	41 ± 1.0
^a,bGroups with different superscripts are significantly different(p <0.05).

TABLE 3 Sperm Parameters Obtained at Sacrifice (Means ± SD)"
	Dose (mg/kg po)
	0	12.5	25	50
Cauda epididymal sperm count (X10^6/g cauda)	645 ± 188.7  (n = 10)	698 ± 95.1 (n=19)	736 ± 113.4 (n = 20)	612±199.4 (n = 20)
Spermatid count (X10^6/g testis)	119 ± 21.8 (n = 20)	110 ± 15.9 (n = 19)	120 + 21.7 (n = 20)	120+ 12.7 (n = 20)
% Normal morphology	96.7 ± 0.02 (n = 10)	94.6 ± 0.07 (n = 16)	96.0 ± 0.05 (n = 18)	97.3 + 0.33 (n = 18)
'' No significant differences observed between groups on any of the sperm parameters.

TABLE 4. Male Body Weight, Organ-to-Body Weight Ratios, and Cauda Epididymal Sperm Counts  for Second Group of Males Used in Motion Analyses (Means ± SD)
		Dose (mg/kg po)
		0'' (n = 18)	12.5'' (n = 16)	25''(n = 18)	50 (n= 15)
Initial body weight (g)		434 + 16.8	428 ± 18.8	429 ± 14.9	432 ± 25.1''
Final body weight (g)		445 ± 17.4	444 ± 26.2	438 ± 21.1	426 ± 36.1''
Testis		0.38 ± 0.044"	0.38 ± 0.020	0.38 ± 0.023	0.40 ± 0.030''
Epididymis		0.13±0.013	0.13 ± 0.011	0.13 ± 0.010	0.14 + 0.015''
Sperm count (X10^6/g cauda)		574 ± 93.5	568 ± 93.8	648 ± 117.9	446 ± 115.8*
'',* Groups with different superscripts are significantly different(p <0.05).

TABLE 6 Fertility Rates and Litter Outcomes in Females Treated with Epichlorohydrin (Means ± SD)''
	Dose (mg/kg po)
	0		25	50
Fertility rate*	60		70	57
Litter size	14.4 ± 1.4 (n = 12)		13.5 ± 3.3 (n = 12)	13.6 ± 1.8  (n = 10)
% Survival at Day 4	97  (n = 12)		90 (n = 12)	99 (n = 10)
Birth weight	5.8 ± 0.48 (n = 12)		5.7 ± 0.46 (n = 12)	5.9 ±0.52 (n = 10)
Weaning weight (Day 21)	44.4 ± 3.28 (n=12)		46.1 ± 4.18 (n= 11)	44.6 ± 2.00 (n = 10)
" Groups did not differ significantly on parameters. * Fertility rate = (No. pregnant/No. mated) x 100.

TABLE 7
Body Weights of Male and Female Pups Postweaning (Means ± SD)''
	Dose (mg/kg po)
	0	25	50
Day 28
Male	78 ±5.12	80 ± 6.32	78 ± 5.66
	(n = 11)	(n = 11)	(n = 10)
Female	69 ± 3.59	72 ±6.41	71 ±3.72
	(n = 12)	(n = 11)	(n = 10)
Day 35
Male	129 ±8.85	132 ±9.75	124 ± 12.84
	(n = 11)	(n = 11)	(n =10)
Female	109 ±3.88	111 ± 8.29	110 ±7.48
	(n = 12)	(n = 11)	(n = 10)
Day 42
Male	185 + 10.84	188 ± 14.39	178 ± 12.74
	(n = 11)	(n = 11)	(n = 10)
Female	139 ± 5.18	141 ± 12.60	138 ± 10.40
	(n = 12)	(n = 11)	(n = 10)
" Groups did not differ significantly on parameters.

Applicant's summary and conclusion

Conclusions:

Epichlorohydrin reduced sperm motion parameters at a dose as low as 12.5 mg/kg/day and total infertility at 50 mg/kg/day fertility in exposed male rats for 21 days. No reproductive or developmental parameters were affected in femal rats.

Executive summary:

Effects of Epichlorohydrin on Male and Female Reproduction in Long-Evans Rats was studied (Toth et al., 1989). The animals were treated with epichlorohydrin (ECH) by oral gavage (males: 12.5, 25, and 50 mg/kg/day; females: 25, 50, and 100 mg/kg/day) for 21 and 14 days, respectively, prior to mating trials with untreated animals. Treated females were further dosed until delivery. Fertility was assayed in the high-dose males only and was found to be totally impaired. None of the females that mated with ECH males was pregnant, compared to a 90% pregnancy rate in controls. However, treated males showed normal copulatory behavior. Sperm morphology and percentage motile sperm were not statistically different from control values in both ejaculated and cauda epididymal samples from ECH-treated animals. The number of sperm in ejaculates was normal while cauda epididymal sperm count was slightly decreased in males at the 50 mg ECH/kg dose level. The decreased cauda epididymal sperm count is unlikely to be responsible for the observed infertility. However, it may in part be a result of the effects of ECH on the vigor and swimming pattern of cauda epididymal sperm. Mean curvilinear velocity, straight-line velocity, and amplitude of lateral head displacement of cauda epididymal sperm were significantly reduced by ECH at 12.5 mg/kg/day and above. Sperm track linearity was also reduced, but only at 50 mg/kg/day. Beat/cross frequency of sperm was significantly increased at 12.5 mg/kg/day and above. All of the above sperm motion parameters showed dose-dependent trends. At necropsy, liver, kidney, and epididymal organ-to-body weight ratios were significantly higher in 50 mg/kg males (p<0.05) relative to controls, although all differences were marginal. Liver and kidneys are known targets for epichlorohydrin toxicity. Increased epididymis-to-body weight ratios are possibly a result of inflammation and swelling from spermatocoeles and sperm granulomas.In the second group of males used for sperm motion analyses, testis and epididymis organ-to-body weight ratios were unchanged.

Within the first week of treatment, all females in the 100 mg/kg group died or were so moribund as to warrant sacrifice. Autopsy showed hemorrhagic stomachs and kidneys with a very pale cortex and haemorrhagic medulla. Animals in the remaining groups appeared healthy and in good condition. Mean body weights across groups showed no differences during pregestational exposure nor during pregnancy. No measured parameters of female reproduction were changed relative to controls. No differences were seen in the number of females mating or in number of successful pregnancies. Litter size and mean pup birth weight were not significantly different. One dam in the 25 mg/kg group neglected her pups, which all died by Postnatal Day 4. This accounts for the slightly decreased percentage survival seen in the 25 mg/kg group. Male and female pup weights, measured weekly, did not significantly differ during lactation or in the 3 weeks postweaning.

In conclusion, no evidence for ECH-induced female reproductive or developmental toxicity was observed at doses up to 100 mg/kg/day. However, male rats showed reductions in sperm motion parameters at a dose as low as 12.5 mg/kg/day and total infertility at 50 mg/kg/day.