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Dimethadione-induced fetotoxicity in rats
Toxicology, 9 (1978) 155--164
© Elsevier/North-Holland Scientific Publishers, Ltd.
DIMETHADIONE-INDUCED FETOTOXICITY IN RATS
H.S. BUTTAR, I. DUPUIS and K.S. KHERA Drug Research Laboratories, Health Protection Branch, Health and Welfare Canada, Ottawa K I A OL2 (Canada)
(Received May 10th, 1977) (Revision received August 3rd, 1977) (Accepted August 24th, 1977)
SUMMMARY The fetotoxic potential of dimethadione was studied in rats given single daily oral dosages of 0, 54, 216, 433 or 541 mg/kg on days 1--21 or 6--15 o f gestation. No maternal toxicity was observed following treatment on days 6--15. When administered from days 1 to 21 only the highest dose (541 mg/kg) produced a significant reduction in maternal body weight gain. Dimethadione caused a dose-related decrease in fetal weight and an increased incidence of umbilical hernia, ecchymoses and subcutaneous edema. There were also increased incidences of non-specific skeleton defects which consisted of unilateral or bilateral wavy ribs, additional ribs (14th rib, uni- and bilateral), retarded ossification of calvaria and a wide variety of sternal defects. Specific defects were bent radius and ulna, and bent tibia and fibula which increased with increasing dosages of dimethadione. Fetal mortality and incidence of skeletal anomalies were higher when the treatment was given on days 1--21 of gestation than on days 6--15 of pregnancy.
INTRODUCTION Our previous studies showed t h a t trimethadione, a suspected h u m a n teratogen, is fetotoxic in rats at maternally tolerated doses [1]. In the rat as in man, trimethadione is rapidly metabolized by N
155
H C.,zo..Co
[o]
..
TRIMETHADIONE
H3C'>r-O',~O + HCHO DIMETHADIONE
Fig. 1. Microsomal oxidation of trimethadione to dimethadione.
reported following oxazolidinedione medication [3], no information is available regarding fetotoxicity of dimethadione in mammals. The present study describes the fetotoxic effects of dimethadione in rats treated over different periods of gestation. MATERIAL AND METHODS Nulliparous female Wistar rats (175--220 g) obtained from Woodlyn Farms, Guelph, Ont., were used in this study. The animals were acclimatized to the laboratory environment for 1 week before mating, and were fed on Master Laboratory cubes and tap water ad libitum. Two females were paired overnight with one proven fertile male in breeding cages. Next morning, copulation was ascertained by finding sperms in vaginal smears or vaginal plugs, and this was counted as day 1 of pregnancy. The mated females were weighed and randomly assigned to experimental groups. Two experimental procedures were used. In experiment I, freshly prepared aqueous solutions o f dimethadione ( A b b o t t Laboratories, North Chicago, Ill.) were administered b y garage in single daily dosages of 54, 216, 433 or 541 mg/kg of b o d y weight on days 6--15 of gestation. In experiment II, rats were dosed once daily during days 1--21 of pregnancy. Control rats were given a volume of distilled water equal to the maximum volume administered to a test animal (1.25 ml/100 g). Pregnant rats were weighted daily to permit correction of dose with changing weight and to assess maternal toxicity. On day 22 of pregnancy, the dams were killed b y CO= inhalation. The fetuses were removed by cesarean section, weighed and examined for viability and external malformations, and maternal viscera including uteri were examined for pathological changes. Early resorptions and fetuses, dying late in development, were recorded. One-half to two-thirds of the live fetuses from each litter were examined for skeletal anomalies after staining with Alizarin Red-S. The remaining fetuses were fixed in Bouin's fluid and were inspected for gross visceral defects. Statistical evaluation Statistical evaluations were made with Student's t-test for unpaired data. The difference between means was considered significant when P was < 0.05. All results are reported as means with standard deviations.
156
RESULTS
Maternal toxicity No maternal death attributable to dimethadione treatment occurred during the study. Maternal body weight at term was reduced, in 433 and 541 mg/kg groups treated on days 6--15 of gestation. This reduction was attributed to small litter size and decreased litter weight since post-cesarean body weights without uterine contents were similar to those of control group (Fig. 2A).
® [ ] CONTROL GROUP(N=I8) DIMETHADIONE (Mg/Kg/DAY) [ ] 541 (N=20) [ ] 2 1 6 (N=20)
[ ~ 4 3 3 (N=22)
1
54 (N=Ig)
400 i~ ~ 3 5 0 ' ~+1
300
,~ £
25o
~E 2OO 175
I
6
12
9
15
22 BC
® [ ] CONTROL GROUP(N= 14) DIMETHADIONE (Mg/Kg/DAY) [ ] 541 (N:IO) [ ] 216 (N=IO)
22 AC
[ ] 433 (N=I3) •
54 ( N = I 2 )
400 I,"ILU U")
350 300
200
,751 i I',llll
I
T T
T
•
i" 6
9
12
'I
15
DAYS OF GESTATION
21
22 BC
22 AC
Fig. 2. M a t e r n a l body weights (on the last day, BC = b e f o r e , and AC = a f t e r cesarean s e c t i o n ) f o r groups o f rats treated during days (A) 6 - - 1 5 and (B) 1--21 o f gestation with daily oral d o s e s o f d i m e t h a d i o n e . Each bar represents the m e a n -+ S.D. o f animals s h o w n in p a r e n t h e s e s . * P < 0.05, and ** P < 0.001 w h e n compared with control values.
157
0o
days 6--15 of gestation
days 1--21 of gestation
I
II
0 54 216 433 541
0 54 216 433 541
Dose (mg[kg/day)
14 12 10 13 10
18 19 20 22 20
No. of dams p r e g n a n t at term
12.0 11.7 12.5 10.2
12.2 12.1 11.9 11.0 9.4 -+ 2 . 7 -+ 2.3 + 2.3 ± 3.9 0
-+ 1.4 -+ 3 . 3 -+ 3 . 5 ± 2.7 ± 3.0 b
N o . o f live fetuses (mean/litter -+ S . D . )
1.2 0 0 3.3 100
0.5 0 3.0 4.1 8.6
% Fetal death No. dead × 100 total fetuses
62.4 55.5 54.5 35.2
63.8 62.7 56.6 43.1 31.1
+ ± ± ±
13.4 11.6 12.9 12.0 b
-+ 8 . 2 ± 17.6 ± 16.2 ± 13.2 b ± 12.1 b
Litter weight (g, m e a n -+ S . D . )
IN R A T S D O S E D O N D A Y S 6 - - 1 5 O R 1 - - 2 1 O F G E S T A T I O N
ap< 0.05 b p < 0.001 when compared with control values.
Duration of dosing
OF DIMETHADIONE
Exp. no.
FETAL EFFECTS
TABLE I
-+ S.D.)
5.2 4.8 4.3 3.2
_+ 0 . 4 -+ 0 . 3 a -+ 0.3 a -+ 0 . 8 b
5 . 2 -+ 0 . 4 5.2-+ 0 . 4 4.9 ± 0.4 a 3.8 ± 0.5 b 3 . 3 -+ 0 . 4 b
mean
Fetal weight
(g,
~n ¢D
1.9 12.3 2.6 1.3 1.9 0 0 0 0.7 0.6 0.6
0.7 0 0
1.4
0 4.7 0 0.7 0 0 0 0
0
0 0
8/19
4]18 0 0 0 0
32/155 20.6
54
9/148 6.1
a A m a l f o r m e d litter c o n t a i n e d at least o n e m a l f o r m e d fetus.
Uni- a n d bilateral 1 4 t h rib Wavy ribs, uni- and bilateral F u s e d ribs, uni- and bilateral Missing s t e r n e b r a e Non-aligned s t e r n e b r a e S h o r t , k i n k y or missing tail B e n t radius a n d u l n a B e n t tibia a n d fibula R e t a r d e d ossification of: Sternum Skull S u t u r e defects o f skull b o n e s
Skeletal defects (%)
Subcutaneous edema Umbilical h e r n i a Runts Ecchymoses
Visceral defects (%)
No. of m a l f o r m e d fetuses Total examined M a l f o r m e d fetuses (%) No. o f m a l f o r m e d litters a Total examined
0
Dimethadione (mg/kg/day)
6.8 6.2 6.8
51.6 16.2 1.0 11.2 2.5 1.2 0 0
0 0 0 1.0
16]20
112/161 69.6
216
96.6 34.5 0 49.1 62.9 8.6 39.7 35.3 44.8 55.2 70.7
99.4 30.8 0 40.9 30.8 6.3 14.5 9.4 6.9 33.3 26.4
77.6 10.2 51.0 8.2
20]20 22/22 15.1 11.0 11.0 5.6
116/116 100
541
157/159 98.7
433
I N C I D E N C E OF A N O M A L I E S O B S E R V E D IN R A T S T R E A T E D WITH D I M E T H A D I O N E ON D A Y S 6 - - 1 5 O F G E S T A T I O N
T A B L E II
O
b-A
OBSERVED
0 30.0 0 0 0 0 0 0 0 0
0.9 6.1 1.7 0 0 0 0 0 0 0
0 0 0 2.9
9/12
3/14 0 0 0 0
26/81 32.1
54
8/115 7
0
0 14.1 15.6
56.3 46.9 9.4 1.6 1.6 0 0
3.0 0 3.0 3.0
10]10
53/64 82.8
216
WITH DIMETHADIONE
Dimethadione (mg/kg/day)
IN R A T S T R E A T E D
a A m a l f o r m e d litter c o n t a i n e d at least o n e m a l f o r m e d fetus.
Uni- a n d bilateral 1 4 t h rib Wavy ribs, uni- a n d bilateral Missing s t e r n e b r a e Non-aligned s t e r n e b r a e Short, k i n k y or missing tail B e n t radius and u l n a B e n t t i b i a a n d fibula R e t a r d e d ossification of: Sternum Skull S u t u r e defects o f skull b o n e s
Skeletal defects (%)
Subcutaneous edema Umbilical h e r n i a Runts Ecchymoses
Visceral defects (%)
No. of m a l f o r m e d fetuses Total e x a m i n e d M a l f o r m e d fetuses (%) No. o f m a l f o r m e d litters a Total examined
INCIDENCE OF ANOMALIES
T A B L E III
39.7 71.4 77.8
95.2 31.8 49.2 44.4 9.5 27.0 19.1
10.7 10.7 32.1 0
13/13
63/63 100
433
O N D A Y S 1--21 O F G E S T A T I O N
D a m s a d m i n i s t e r e d 541 m g / k g d i m e t h a d i o n e on d a y s 1--21 o f p r e g n a n c y a p p e a r e d ataxic and sedated at term and s h o w e d a m a r k e d r e d u c t i o n in b o d y w e i g h t gain (Fig. 2B). Vaginal bleeding was seen o n d a y s 1 7 - - 2 1 o f g e s t a t i o n in 10% and 30% o f d a m s d o s e d with 4 3 3 and 541 m g / k g , respectively, f o l l o w i n g t r e a t m e n t on d a y s 1 - - 2 1 o f p r e g n a n c y .
Fetal effects Table I s h o w s the fetal effects of d i m e t h a d i o n e in rats d o s e d on d a y s 6 - - 1 5 or 1--21 o f gestation. In c o m p a r i s o n with the c o n t r o l , the incidence o f fetal d e a t h was high and the m e a n fetal weight low at or above 216 m g / k g
NORMAL
A
B C
Fig. 3. Photographs of 22-day-old fetuses of rats given 433 mg/kg]day dimethadione on days 6--15 of pregnancy. Top left shows a normal fetus; (A) tailless fetus;(B) fetus with umbilical hernia; (C) edematous fetuses. 161
dosages given from days 6 to 15 of pregnancy. The mean values for live fetuses, litter weight and fetal weight were significantly reduced (P < 0.001) following treatment with 541 mg/kg/day. This dose given on days 1--21 of gestation was maternally toxic and caused 100% fetal mortality and an increased incidence of deciduomas as opposed to 8.6% fetal death following treatment on days 6--15 of gestation. The mean fetal weight at 54 mg/kg dose was significantly reduced following dosing on days 1--21 of gestation b u t n o t on days 6--15 of gestation. A
B N
I
2
3
Fig. 4. Skeleton defects in 22-day-old fetuses of rats given 433 mg/kg/day dimethadione on days 6--15 of gestation. In each photograph, N indicates skeleton of a normal fetus. (A) deformed forelimb (1--3, short humerus, bent radius and ulna); (B) deformed hindlimb (1--3, bent tibia and fibula); (C) bilateral 14th rib (arrows); (D) wavy ribs (arrow); (E) anomalous sternebrae (1--7, retardedly ossified, non-aligned and missing sternebrae); (F) suture defects (arrow) and retarded ossification of skull bones.
162
The incidences and t ype s of visceral and skeletal malformations observed in fetuses are presented in Tables II and III. The incidence of m a l f o r m e d fetuses was d o s e - d e p e n d e n t with 100% occurrence at 433 mg/kg. Umbilical hernia was a striking visceral anomaly at 433 or 541 mg/kg doses. Its freq u e n c y at the highest dose, regardless of the durat i on of t r e a t m e n t , was 11%. Also, a high incidence of runting, subcutaneous edema and ecchymoses at the cervical and shoulder regions was observed. F r e q u e n c y of runts at 433 mg/kg was 3 times greater ( 3 2 . 1 % vs. 11%) following t r e a t m e n t during days 1--21 than 6--15 o f gestation. There was a dose-related increased f r e q u e n c y of rib defects including wavy and ex tr a ribs ( 14t h rib, uni- and bilateral). Bilateral 14th rib occurred in all litters from the 433 mg/kg or higher dosage groups. Sternal defects (absent, retardedly ossified or non-aligned sternebrae) were m ore frequent in treated rats than in the c o n t r o l s . T h e incidence of short, kinky or missing tails was also dosed-related. Dimethadione caused an increased occurrence of retarded ossification of skull bones (parietal, interparietal, supraoccipital and frontal), and all the fetuses examined from litters exposed to 433 mg/kg during 6--15 or 1--21 days of gestation had defective calvaria. The numbers of litters with one or more littermates, showing bent radii and ulnae as well as bent tibiae and fibulae, were increased with increasing doses of di m et hadi one and usually anomalous fore and hindlimb bones were seen in the same fetus. Incidences of retarted ossification of sternal and skull bones and bending of fore and hindlimb bones were higher when 433 mg/kg di m et hadi one was administered on days 1--21 of gestation than on days 6--15 (compare T abl esIIand III). Dimethadione-induced visceral and skeletal defects are illustrated in Fig. 3 and 4. DISCUSSION T r i m e t h a d i o n e ingestion during pregnancy has been incriminated for a wide variety of birth defects including umbilical hernia in children [ 4 - - 6 ] . Both tr im et hadi one and dimethadione have been shown to be teratogenic in the chick e m b r y o , the principal anomaly p r o d u c e d by di m et hadi one being celosomia [ 7] . Fetal malformations, in the present study, resulted from maternal oral dosing of di m et hadi one and were d e p e n d e n t on dose and duration of treatment. A significantly increased incidence of malformations was observed at all dosages examined, but was p r o n o u n c e d at 433 and 541 mg/kg and more so when these doses were administered on days 1--21 o f gestation. These two doses did also elicit varying degrees of maternal t o x i c i t y and fetal growth retardation irrespective of t r e a t m e n t duration. The only dosage that had no adverse effect on mean fetal weight was 54 mg/kg given on days 6--15 of gestation. Reduced fetal weight and anomalous d e v e l o p m e n t appeared to be closely associated within a test dose group. Administration o f equimolar doses of t ri m et hadi one (480 mg/kg/day) {unpublished) and its metabolite, d i m e t h a d i o n e {433/mg/kg/day), on days 6--15 of gestation produced very similar types of malformations. However, the incidences of various anomalies were higher following t r e a t m e n t with d i m e t h a d i o n e than with trimethadione; suggesting that the metabolite is a mo r e p o t e n t teratogen than the parent drug. 163
T h e risk o f s p o n t a n e o u s a b o r t i o n s [4] and h e m o r r h a g i c disease in newb o r n i n f a n t s is increased b y m a t e n a l a n t i c o n v u l s a n t t h e r a p y [ 8 - - 1 0 ] . T h e s u b c u t a n e o u s f e t a l h e m o r r h a g e s seen in the p r e s e n t s t u d y m a y have b e e n r e l a t e d to t h e m a t e r n a l d i m e t h a d i o n e t r e a t m e n t . While the e x a c t cause o f vaginal b l e e d i n g t h a t o c c u r r e d late in g e s t a t i o n f o l l o w i n g d i m e t h a d i o n e d o s i n g r e m a i n s u n k n o w n , it c o u l d p o s s i b l y have b e e n d u e to a b o r t i o n or rupture of placental membranes. A l t h o u g h the a n t i c o n v u l s a n t e f f e c t s o f d i m e t h a d i o n e h a v e b e e n d e m o n s t r a t e d in h u m a n s [11] and in e x p e r i m e n t a l animals [ 1 2 ] , it is p r e s e n t l y n o t an a p p r o v e d d r u g in Canada. It is o f t o x i c o l o g i c a l interest b e c a u s e it is t h e principal, a n d p e r h a p s t h e o n l y , m e t a b o l i t e o f an e x t e n s i v e l y used antie p i l e p t i c drug, t r i m e t h a d i o n e , When t r i m e t h a d i o n e is a d m i n i s t e r e d chronically, t h e d e m e t h y l a t e d p r o d u c t a c c u m u l a t e s in t h e b o d y in a m o u n t s f a r g r e a t e r t h a n the a m o u n t o f p a r e n t d r u g and a b o u t 85% o f t h e t o t a l therap e u t i c e f f e c t is c o n t r i b u t e d b y d i m e t h a d i o n e a l o n e [ 1 3 ] . O u r u n p u b l i s h e d results suggest t h a t as in m a n , t r i m e t h a d i o n e is r a p i d l y m e t a b o l i z e d b y t h e r a t and t h e N - d e m e t h y l a t e d p r o d u c t , d i m e t h a d i o n e , a c c u m u l a t e s in t h e fetus. F o l l o w i n g l o n g - t e r m t r i m e t h a d i o n e dosing, s e r u m levels o f d i m e t h a d i o n e w e r e f o u n d t o increase [ 2 , 1 3 ] and a f e t o t o x i c e f f e c t o f d i m e t h a d i o n e at m a t e r n a l l y t o l e r a t e d doses is d e m o n s t r a t e d in t h e p r e s e n t study. Since it is d i f f i c u l t to e x t r a p o l a t e h u m a n e f f e c t s f r o m t h e r a t s t u d y , it w o u l d be u n w i s e t o s p e c u l a t e as to w h e t h e r d i m e t h a d i o n e or t r i m e t h a d i o n e is t h e real c a u s e o f d e v e l o p m e n t a n o m a l i e s in c h i l d r e n f r o m t r i m e t h a d i o n e - t r e a t e d mothers. In s h o r t , the p r e s e n t and p r e v i o u s investigations in e x p e r i m e n t a l animals e m p h a s i z e t h e c a u t i o n a l r e a d y c o n t a i n e d in the P h y s i c i a n s ' D e s k R e f e r e n c e [14] that care should be taken in the administration of o x a z o l i d i n e d i o n e d r u g s to e p i l e p t i c w o m e n o f child-bearing p o t e n t i a l . REFERENCES 1 H.S. Buttar, I. Dupuis and K.S. Khera, Toxicol. Appl. Pharmacol., 37 (1976) 126. 2 C.D. Withrow and D.M. Woodbury, Trimethadione and other oxazolidinediones, absorption distribution and excretion, in D.M. Woodbury, J.K. Penry and R.P. Schmidt (Eds.), Antiepileptic Drugs, Raven Press, New York, 1972, p. 389. 3 B.B. Gallagher, Trimethadione and other oxazolidinediones, toxicity, in D.M. Woodbury, J.K. Penry and R.P. Schmidt (Eds.), Antiepileptic Drugs, Raven Press, New York, 1972, p. 409. 4 J. German, A. Kowal and K.H. Ehlers, Teratology, 3 (1970) 349. 5 M.M. Nichols, J. Pediat., 82 (1973) 885. 6 E.H. Zackai, W.J. Mellman, B. Neiderer and J.W. Hanson, J. Pediat., 87 (1975) 280. 7 A.B. Rifkind, Toxicol. Appl. Pharmacol., 30 (1974) 452. 8 M.M. Stevenson and E.F. Gilbert, J. Pedlar., 77 (1970) 516. 9 D.O. Margolin and N.M. Kantor, Clin. Pediat., 11 (1972) 59. 10 W.A. Bleyer and A.L. Skinner, J. Am. Med. Assoc., 235 (1976) 626. 11 H.R. Chamberlin, W.J. Waddell and T.C. Butler, Neurology, (Minnecap.) 15 (1965) 449. 12 C.D. Withrow, R.J. Stout, L.J. Barton, W.S. Beacham and D.M. Woodbury, J. Pharmacol. Exp. Ther., 161 (1968) 335. 13 H.H. Frey and R. Schulz, Acta Pharmacol. Toxicol., 28 (1970) 477. 14 Physicians' Desk Reference, 3rd ed., Charles E. Baker, Jr., New Jersey, 1976, p. 550. 164
© Elsevier/North-Holland Scientific Publishers, Ltd.
DIMETHADIONE-INDUCED FETOTOXICITY IN RATS
H.S. BUTTAR, I. DUPUIS and K.S. KHERA Drug Research Laboratories, Health Protection Branch, Health and Welfare Canada, Ottawa K I A OL2 (Canada)
(Received May 10th, 1977) (Revision received August 3rd, 1977) (Accepted August 24th, 1977)
SUMMMARY The fetotoxic potential of dimethadione was studied in rats given single daily oral dosages of 0, 54, 216, 433 or 541 mg/kg on days 1--21 or 6--15 o f gestation. No maternal toxicity was observed following treatment on days 6--15. When administered from days 1 to 21 only the highest dose (541 mg/kg) produced a significant reduction in maternal body weight gain. Dimethadione caused a dose-related decrease in fetal weight and an increased incidence of umbilical hernia, ecchymoses and subcutaneous edema. There were also increased incidences of non-specific skeleton defects which consisted of unilateral or bilateral wavy ribs, additional ribs (14th rib, uni- and bilateral), retarded ossification of calvaria and a wide variety of sternal defects. Specific defects were bent radius and ulna, and bent tibia and fibula which increased with increasing dosages of dimethadione. Fetal mortality and incidence of skeletal anomalies were higher when the treatment was given on days 1--21 of gestation than on days 6--15 of pregnancy.
INTRODUCTION Our previous studies showed t h a t trimethadione, a suspected h u m a n teratogen, is fetotoxic in rats at maternally tolerated doses [1]. In the rat as in man, trimethadione is rapidly metabolized by N
155
H C.,zo..Co
[o]
..
TRIMETHADIONE
H3C'>r-O',~O + HCHO DIMETHADIONE
Fig. 1. Microsomal oxidation of trimethadione to dimethadione.
reported following oxazolidinedione medication [3], no information is available regarding fetotoxicity of dimethadione in mammals. The present study describes the fetotoxic effects of dimethadione in rats treated over different periods of gestation. MATERIAL AND METHODS Nulliparous female Wistar rats (175--220 g) obtained from Woodlyn Farms, Guelph, Ont., were used in this study. The animals were acclimatized to the laboratory environment for 1 week before mating, and were fed on Master Laboratory cubes and tap water ad libitum. Two females were paired overnight with one proven fertile male in breeding cages. Next morning, copulation was ascertained by finding sperms in vaginal smears or vaginal plugs, and this was counted as day 1 of pregnancy. The mated females were weighed and randomly assigned to experimental groups. Two experimental procedures were used. In experiment I, freshly prepared aqueous solutions o f dimethadione ( A b b o t t Laboratories, North Chicago, Ill.) were administered b y garage in single daily dosages of 54, 216, 433 or 541 mg/kg of b o d y weight on days 6--15 of gestation. In experiment II, rats were dosed once daily during days 1--21 of pregnancy. Control rats were given a volume of distilled water equal to the maximum volume administered to a test animal (1.25 ml/100 g). Pregnant rats were weighted daily to permit correction of dose with changing weight and to assess maternal toxicity. On day 22 of pregnancy, the dams were killed b y CO= inhalation. The fetuses were removed by cesarean section, weighed and examined for viability and external malformations, and maternal viscera including uteri were examined for pathological changes. Early resorptions and fetuses, dying late in development, were recorded. One-half to two-thirds of the live fetuses from each litter were examined for skeletal anomalies after staining with Alizarin Red-S. The remaining fetuses were fixed in Bouin's fluid and were inspected for gross visceral defects. Statistical evaluation Statistical evaluations were made with Student's t-test for unpaired data. The difference between means was considered significant when P was < 0.05. All results are reported as means with standard deviations.
156
RESULTS
Maternal toxicity No maternal death attributable to dimethadione treatment occurred during the study. Maternal body weight at term was reduced, in 433 and 541 mg/kg groups treated on days 6--15 of gestation. This reduction was attributed to small litter size and decreased litter weight since post-cesarean body weights without uterine contents were similar to those of control group (Fig. 2A).
® [ ] CONTROL GROUP(N=I8) DIMETHADIONE (Mg/Kg/DAY) [ ] 541 (N=20) [ ] 2 1 6 (N=20)
[ ~ 4 3 3 (N=22)
1
54 (N=Ig)
400 i~ ~ 3 5 0 ' ~+1
300
,~ £
25o
~E 2OO 175
I
6
12
9
15
22 BC
® [ ] CONTROL GROUP(N= 14) DIMETHADIONE (Mg/Kg/DAY) [ ] 541 (N:IO) [ ] 216 (N=IO)
22 AC
[ ] 433 (N=I3) •
54 ( N = I 2 )
400 I,"ILU U")
350 300
200
,751 i I',llll
I
T T
T
•
i" 6
9
12
'I
15
DAYS OF GESTATION
21
22 BC
22 AC
Fig. 2. M a t e r n a l body weights (on the last day, BC = b e f o r e , and AC = a f t e r cesarean s e c t i o n ) f o r groups o f rats treated during days (A) 6 - - 1 5 and (B) 1--21 o f gestation with daily oral d o s e s o f d i m e t h a d i o n e . Each bar represents the m e a n -+ S.D. o f animals s h o w n in p a r e n t h e s e s . * P < 0.05, and ** P < 0.001 w h e n compared with control values.
157
0o
days 6--15 of gestation
days 1--21 of gestation
I
II
0 54 216 433 541
0 54 216 433 541
Dose (mg[kg/day)
14 12 10 13 10
18 19 20 22 20
No. of dams p r e g n a n t at term
12.0 11.7 12.5 10.2
12.2 12.1 11.9 11.0 9.4 -+ 2 . 7 -+ 2.3 + 2.3 ± 3.9 0
-+ 1.4 -+ 3 . 3 -+ 3 . 5 ± 2.7 ± 3.0 b
N o . o f live fetuses (mean/litter -+ S . D . )
1.2 0 0 3.3 100
0.5 0 3.0 4.1 8.6
% Fetal death No. dead × 100 total fetuses
62.4 55.5 54.5 35.2
63.8 62.7 56.6 43.1 31.1
+ ± ± ±
13.4 11.6 12.9 12.0 b
-+ 8 . 2 ± 17.6 ± 16.2 ± 13.2 b ± 12.1 b
Litter weight (g, m e a n -+ S . D . )
IN R A T S D O S E D O N D A Y S 6 - - 1 5 O R 1 - - 2 1 O F G E S T A T I O N
ap< 0.05 b p < 0.001 when compared with control values.
Duration of dosing
OF DIMETHADIONE
Exp. no.
FETAL EFFECTS
TABLE I
-+ S.D.)
5.2 4.8 4.3 3.2
_+ 0 . 4 -+ 0 . 3 a -+ 0.3 a -+ 0 . 8 b
5 . 2 -+ 0 . 4 5.2-+ 0 . 4 4.9 ± 0.4 a 3.8 ± 0.5 b 3 . 3 -+ 0 . 4 b
mean
Fetal weight
(g,
~n ¢D
1.9 12.3 2.6 1.3 1.9 0 0 0 0.7 0.6 0.6
0.7 0 0
1.4
0 4.7 0 0.7 0 0 0 0
0
0 0
8/19
4]18 0 0 0 0
32/155 20.6
54
9/148 6.1
a A m a l f o r m e d litter c o n t a i n e d at least o n e m a l f o r m e d fetus.
Uni- a n d bilateral 1 4 t h rib Wavy ribs, uni- and bilateral F u s e d ribs, uni- and bilateral Missing s t e r n e b r a e Non-aligned s t e r n e b r a e S h o r t , k i n k y or missing tail B e n t radius a n d u l n a B e n t tibia a n d fibula R e t a r d e d ossification of: Sternum Skull S u t u r e defects o f skull b o n e s
Skeletal defects (%)
Subcutaneous edema Umbilical h e r n i a Runts Ecchymoses
Visceral defects (%)
No. of m a l f o r m e d fetuses Total examined M a l f o r m e d fetuses (%) No. o f m a l f o r m e d litters a Total examined
0
Dimethadione (mg/kg/day)
6.8 6.2 6.8
51.6 16.2 1.0 11.2 2.5 1.2 0 0
0 0 0 1.0
16]20
112/161 69.6
216
96.6 34.5 0 49.1 62.9 8.6 39.7 35.3 44.8 55.2 70.7
99.4 30.8 0 40.9 30.8 6.3 14.5 9.4 6.9 33.3 26.4
77.6 10.2 51.0 8.2
20]20 22/22 15.1 11.0 11.0 5.6
116/116 100
541
157/159 98.7
433
I N C I D E N C E OF A N O M A L I E S O B S E R V E D IN R A T S T R E A T E D WITH D I M E T H A D I O N E ON D A Y S 6 - - 1 5 O F G E S T A T I O N
T A B L E II
O
b-A
OBSERVED
0 30.0 0 0 0 0 0 0 0 0
0.9 6.1 1.7 0 0 0 0 0 0 0
0 0 0 2.9
9/12
3/14 0 0 0 0
26/81 32.1
54
8/115 7
0
0 14.1 15.6
56.3 46.9 9.4 1.6 1.6 0 0
3.0 0 3.0 3.0
10]10
53/64 82.8
216
WITH DIMETHADIONE
Dimethadione (mg/kg/day)
IN R A T S T R E A T E D
a A m a l f o r m e d litter c o n t a i n e d at least o n e m a l f o r m e d fetus.
Uni- a n d bilateral 1 4 t h rib Wavy ribs, uni- a n d bilateral Missing s t e r n e b r a e Non-aligned s t e r n e b r a e Short, k i n k y or missing tail B e n t radius and u l n a B e n t t i b i a a n d fibula R e t a r d e d ossification of: Sternum Skull S u t u r e defects o f skull b o n e s
Skeletal defects (%)
Subcutaneous edema Umbilical h e r n i a Runts Ecchymoses
Visceral defects (%)
No. of m a l f o r m e d fetuses Total e x a m i n e d M a l f o r m e d fetuses (%) No. o f m a l f o r m e d litters a Total examined
INCIDENCE OF ANOMALIES
T A B L E III
39.7 71.4 77.8
95.2 31.8 49.2 44.4 9.5 27.0 19.1
10.7 10.7 32.1 0
13/13
63/63 100
433
O N D A Y S 1--21 O F G E S T A T I O N
D a m s a d m i n i s t e r e d 541 m g / k g d i m e t h a d i o n e on d a y s 1--21 o f p r e g n a n c y a p p e a r e d ataxic and sedated at term and s h o w e d a m a r k e d r e d u c t i o n in b o d y w e i g h t gain (Fig. 2B). Vaginal bleeding was seen o n d a y s 1 7 - - 2 1 o f g e s t a t i o n in 10% and 30% o f d a m s d o s e d with 4 3 3 and 541 m g / k g , respectively, f o l l o w i n g t r e a t m e n t on d a y s 1 - - 2 1 o f p r e g n a n c y .
Fetal effects Table I s h o w s the fetal effects of d i m e t h a d i o n e in rats d o s e d on d a y s 6 - - 1 5 or 1--21 o f gestation. In c o m p a r i s o n with the c o n t r o l , the incidence o f fetal d e a t h was high and the m e a n fetal weight low at or above 216 m g / k g
NORMAL
A
B C
Fig. 3. Photographs of 22-day-old fetuses of rats given 433 mg/kg]day dimethadione on days 6--15 of pregnancy. Top left shows a normal fetus; (A) tailless fetus;(B) fetus with umbilical hernia; (C) edematous fetuses. 161
dosages given from days 6 to 15 of pregnancy. The mean values for live fetuses, litter weight and fetal weight were significantly reduced (P < 0.001) following treatment with 541 mg/kg/day. This dose given on days 1--21 of gestation was maternally toxic and caused 100% fetal mortality and an increased incidence of deciduomas as opposed to 8.6% fetal death following treatment on days 6--15 of gestation. The mean fetal weight at 54 mg/kg dose was significantly reduced following dosing on days 1--21 of gestation b u t n o t on days 6--15 of gestation. A
B N
I
2
3
Fig. 4. Skeleton defects in 22-day-old fetuses of rats given 433 mg/kg/day dimethadione on days 6--15 of gestation. In each photograph, N indicates skeleton of a normal fetus. (A) deformed forelimb (1--3, short humerus, bent radius and ulna); (B) deformed hindlimb (1--3, bent tibia and fibula); (C) bilateral 14th rib (arrows); (D) wavy ribs (arrow); (E) anomalous sternebrae (1--7, retardedly ossified, non-aligned and missing sternebrae); (F) suture defects (arrow) and retarded ossification of skull bones.
162
The incidences and t ype s of visceral and skeletal malformations observed in fetuses are presented in Tables II and III. The incidence of m a l f o r m e d fetuses was d o s e - d e p e n d e n t with 100% occurrence at 433 mg/kg. Umbilical hernia was a striking visceral anomaly at 433 or 541 mg/kg doses. Its freq u e n c y at the highest dose, regardless of the durat i on of t r e a t m e n t , was 11%. Also, a high incidence of runting, subcutaneous edema and ecchymoses at the cervical and shoulder regions was observed. F r e q u e n c y of runts at 433 mg/kg was 3 times greater ( 3 2 . 1 % vs. 11%) following t r e a t m e n t during days 1--21 than 6--15 o f gestation. There was a dose-related increased f r e q u e n c y of rib defects including wavy and ex tr a ribs ( 14t h rib, uni- and bilateral). Bilateral 14th rib occurred in all litters from the 433 mg/kg or higher dosage groups. Sternal defects (absent, retardedly ossified or non-aligned sternebrae) were m ore frequent in treated rats than in the c o n t r o l s . T h e incidence of short, kinky or missing tails was also dosed-related. Dimethadione caused an increased occurrence of retarded ossification of skull bones (parietal, interparietal, supraoccipital and frontal), and all the fetuses examined from litters exposed to 433 mg/kg during 6--15 or 1--21 days of gestation had defective calvaria. The numbers of litters with one or more littermates, showing bent radii and ulnae as well as bent tibiae and fibulae, were increased with increasing doses of di m et hadi one and usually anomalous fore and hindlimb bones were seen in the same fetus. Incidences of retarted ossification of sternal and skull bones and bending of fore and hindlimb bones were higher when 433 mg/kg di m et hadi one was administered on days 1--21 of gestation than on days 6--15 (compare T abl esIIand III). Dimethadione-induced visceral and skeletal defects are illustrated in Fig. 3 and 4. DISCUSSION T r i m e t h a d i o n e ingestion during pregnancy has been incriminated for a wide variety of birth defects including umbilical hernia in children [ 4 - - 6 ] . Both tr im et hadi one and dimethadione have been shown to be teratogenic in the chick e m b r y o , the principal anomaly p r o d u c e d by di m et hadi one being celosomia [ 7] . Fetal malformations, in the present study, resulted from maternal oral dosing of di m et hadi one and were d e p e n d e n t on dose and duration of treatment. A significantly increased incidence of malformations was observed at all dosages examined, but was p r o n o u n c e d at 433 and 541 mg/kg and more so when these doses were administered on days 1--21 o f gestation. These two doses did also elicit varying degrees of maternal t o x i c i t y and fetal growth retardation irrespective of t r e a t m e n t duration. The only dosage that had no adverse effect on mean fetal weight was 54 mg/kg given on days 6--15 of gestation. Reduced fetal weight and anomalous d e v e l o p m e n t appeared to be closely associated within a test dose group. Administration o f equimolar doses of t ri m et hadi one (480 mg/kg/day) {unpublished) and its metabolite, d i m e t h a d i o n e {433/mg/kg/day), on days 6--15 of gestation produced very similar types of malformations. However, the incidences of various anomalies were higher following t r e a t m e n t with d i m e t h a d i o n e than with trimethadione; suggesting that the metabolite is a mo r e p o t e n t teratogen than the parent drug. 163
T h e risk o f s p o n t a n e o u s a b o r t i o n s [4] and h e m o r r h a g i c disease in newb o r n i n f a n t s is increased b y m a t e n a l a n t i c o n v u l s a n t t h e r a p y [ 8 - - 1 0 ] . T h e s u b c u t a n e o u s f e t a l h e m o r r h a g e s seen in the p r e s e n t s t u d y m a y have b e e n r e l a t e d to t h e m a t e r n a l d i m e t h a d i o n e t r e a t m e n t . While the e x a c t cause o f vaginal b l e e d i n g t h a t o c c u r r e d late in g e s t a t i o n f o l l o w i n g d i m e t h a d i o n e d o s i n g r e m a i n s u n k n o w n , it c o u l d p o s s i b l y have b e e n d u e to a b o r t i o n or rupture of placental membranes. A l t h o u g h the a n t i c o n v u l s a n t e f f e c t s o f d i m e t h a d i o n e h a v e b e e n d e m o n s t r a t e d in h u m a n s [11] and in e x p e r i m e n t a l animals [ 1 2 ] , it is p r e s e n t l y n o t an a p p r o v e d d r u g in Canada. It is o f t o x i c o l o g i c a l interest b e c a u s e it is t h e principal, a n d p e r h a p s t h e o n l y , m e t a b o l i t e o f an e x t e n s i v e l y used antie p i l e p t i c drug, t r i m e t h a d i o n e , When t r i m e t h a d i o n e is a d m i n i s t e r e d chronically, t h e d e m e t h y l a t e d p r o d u c t a c c u m u l a t e s in t h e b o d y in a m o u n t s f a r g r e a t e r t h a n the a m o u n t o f p a r e n t d r u g and a b o u t 85% o f t h e t o t a l therap e u t i c e f f e c t is c o n t r i b u t e d b y d i m e t h a d i o n e a l o n e [ 1 3 ] . O u r u n p u b l i s h e d results suggest t h a t as in m a n , t r i m e t h a d i o n e is r a p i d l y m e t a b o l i z e d b y t h e r a t and t h e N - d e m e t h y l a t e d p r o d u c t , d i m e t h a d i o n e , a c c u m u l a t e s in t h e fetus. F o l l o w i n g l o n g - t e r m t r i m e t h a d i o n e dosing, s e r u m levels o f d i m e t h a d i o n e w e r e f o u n d t o increase [ 2 , 1 3 ] and a f e t o t o x i c e f f e c t o f d i m e t h a d i o n e at m a t e r n a l l y t o l e r a t e d doses is d e m o n s t r a t e d in t h e p r e s e n t study. Since it is d i f f i c u l t to e x t r a p o l a t e h u m a n e f f e c t s f r o m t h e r a t s t u d y , it w o u l d be u n w i s e t o s p e c u l a t e as to w h e t h e r d i m e t h a d i o n e or t r i m e t h a d i o n e is t h e real c a u s e o f d e v e l o p m e n t a n o m a l i e s in c h i l d r e n f r o m t r i m e t h a d i o n e - t r e a t e d mothers. In s h o r t , the p r e s e n t and p r e v i o u s investigations in e x p e r i m e n t a l animals e m p h a s i z e t h e c a u t i o n a l r e a d y c o n t a i n e d in the P h y s i c i a n s ' D e s k R e f e r e n c e [14] that care should be taken in the administration of o x a z o l i d i n e d i o n e d r u g s to e p i l e p t i c w o m e n o f child-bearing p o t e n t i a l . REFERENCES 1 H.S. Buttar, I. Dupuis and K.S. Khera, Toxicol. Appl. Pharmacol., 37 (1976) 126. 2 C.D. Withrow and D.M. Woodbury, Trimethadione and other oxazolidinediones, absorption distribution and excretion, in D.M. Woodbury, J.K. Penry and R.P. Schmidt (Eds.), Antiepileptic Drugs, Raven Press, New York, 1972, p. 389. 3 B.B. Gallagher, Trimethadione and other oxazolidinediones, toxicity, in D.M. Woodbury, J.K. Penry and R.P. Schmidt (Eds.), Antiepileptic Drugs, Raven Press, New York, 1972, p. 409. 4 J. German, A. Kowal and K.H. Ehlers, Teratology, 3 (1970) 349. 5 M.M. Nichols, J. Pediat., 82 (1973) 885. 6 E.H. Zackai, W.J. Mellman, B. Neiderer and J.W. Hanson, J. Pediat., 87 (1975) 280. 7 A.B. Rifkind, Toxicol. Appl. Pharmacol., 30 (1974) 452. 8 M.M. Stevenson and E.F. Gilbert, J. Pedlar., 77 (1970) 516. 9 D.O. Margolin and N.M. Kantor, Clin. Pediat., 11 (1972) 59. 10 W.A. Bleyer and A.L. Skinner, J. Am. Med. Assoc., 235 (1976) 626. 11 H.R. Chamberlin, W.J. Waddell and T.C. Butler, Neurology, (Minnecap.) 15 (1965) 449. 12 C.D. Withrow, R.J. Stout, L.J. Barton, W.S. Beacham and D.M. Woodbury, J. Pharmacol. Exp. Ther., 161 (1968) 335. 13 H.H. Frey and R. Schulz, Acta Pharmacol. Toxicol., 28 (1970) 477. 14 Physicians' Desk Reference, 3rd ed., Charles E. Baker, Jr., New Jersey, 1976, p. 550. 164