A comparison of the teratogenic activity of the antiepileptic drugs carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, and primidone in mice

TOXICOLOGYANDAPPLIED

PHARMACOL~GY~~,

365-378(1977)

A Comparison of the Teratogenic Activity of the AntiepiIeptic Drugs Carbamazepine, Clonazepam, Ethosuximide, Phenobarbital, Phenytoin, and Primidone in Mice F. M. SULLIVAN AND PATRICIA R. MCELHATTON Department of Pharmacology, Guy’s Hospital Medical School, London SE1 9RT, England Received November 5,1976; accepted December 29,1976

A Comparison of the Teratogenic Activity of the Antiepileptic Drugs Carbamazepine, Clonazepam, Ethosuximide, Phenobarbital, Phenytoin, and Primidone in Mice. SULLIVAN, F. M., AND MCELHATTON P. R. (1977). Toxicol. Appl. Pharmacol. 40, 365-378. The teratogenic activity of six commonly used antiepileptic drugs, carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, and primidone, was studied at three dose levels (3, 9, and 18 times the human therapeutic dose) in CD1 mice. The drugs, suspended in 1% carboxymethylcellulose, were administered by gastric intubation on Days 6-16 of pregnancy, which covers the period from implantation to the end of structural development. The dams were killed 1 day before term and the fetuses were examined. Full-length cleft palates were observed in all treated groups with the exception of the clonazepamtreated group. This defect was not observed in any of the control groups. Other defects such as exencephaly, enlarged cerebral ventricles, exomphalos, open eyes, undescended testes, abnormal palatal bones other than cleft palate, and fused ribs were seen less frequently. If these results are assessedby ranking the drugs in order of teratogenicity, assessedon either a pooled fetus or a pooled litter basis, the drugs fall into three categories: Phenytoin produced the highest incidence of teratogenic effects; carbamazepine, phenobarbital, and primidone produced a lower but still significant incidence of defects; and clonazepam and ethosuximide were the least teratogenic. Nevertheless, there was at least a two-fold increasein defects in these latter two treatment groups compared with the controls. In the last 10 years much evidence has accumulated associating the birth of congenitally malformed children to epileptic women with anticonvulsant medication (Janz and Fuchs, 1964; South, 1972; Speidel and Meadow, 1972; Lowe, 1973; Monson et al., 1973). As most epileptic patients are on muhiple drug therapy, it is difficult from the epidemiological reports published to determine which drug or combination of drugs (for it is important in such a situation that drug-drug interactions are considered) is responsible for the defects observed. Results reported in some of the earlier clinical articles seem to indicate that diphenylhydantoin (phenytoin) was the most likely teratogen, but more recent surveys have implicated almost the whole range of antiepileptic drugs (Fedrick, 1973; Hill 1973; Monson et al., 1973; Starreveld-Zimmerman et al., 1973; Annegers et al., 1974; Speidel and Meadow, 1974). In most of the animal copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

13

365 ISSN 0041-008X

366

SULLIVAN

AND

MCELHATTON

studies to date, phenytoin has been tested and found to be teratogenic (Massey, 1966: Elshove, 1969; Harbison and Becker, 1969 and 1972; Schardein et al., 1973; Sullivan and McElhatton, 1975). Poswillo (1972) examined the effects of paramethadione and trimethadione in six monkeys (Mucaca irus) and found no structural or behavioral abnormalities in offspring, although two of the animals did resorb or abort. More extensive primate studies by Wilson (1973) using both phenytoin and trimethadione suggested that both drugs produced a low incidence of teratogenicity. The work of the present authors also indicates that primidone and possibly phenobarbital are teratogens in the mouse (McElhatton and Sullivan, 1975; Sullivan and McElhatton, 1975; McElhatton et al., 1976). The teratogenicity of phenobarbital is less well established. Cahen (1964), in a review on the evaluation of teratogenic drugs, stated that barbiturates had no teratogenic capabilities, and this has become a generally accepted point of view, though in fact, there is experimental evidence against it. An increase in the incidence of fetal deaths and malformations has been reported in the rat (McCall et al., 1967) following maternal treatment with barbiturates. Walker and Patterson (1974) reported a high incidence of cleft palate in mice after exposure to phenobarbital in utero. However, the doses used caused sedation and as there was no current control group, these results are difficult to assesswith confidence. Apart from these studies, there is very little published work on the effects of other anticonvulsant drugs in animals. At the present time, there is no logical basis for preference for any particular drug regimen for pregnant epileptic women. Although most clinical studies have implicated phenytoin, this may be because it is the most commonly used drug, and there is little clear evidence that the other drugs are safer. Because of the lack of published evidence on the effects of the newer antiepileptic drugs in animals, we thought it would be worthwhile to compare all of the major antiepileptic drugs in one animal model system to see if any clear order of teratogenicity would emerge. It was decided to compare carbamazepine,’ clonazepam,2 ethosuximide,3 phenobarbital,4 phenytoin,* primidone,6 and sodium valproate.? The last mentioned drug, however, could not be included since the manufacturer stated that they had already found it to be teratogenic in animal studies, and had indicated that it should not be used during pregnancy. All of the other six major anticonvulsants were, therefore, tested individually in the mouse in one series of experiments to compare their teratogenic potential. METHODS

CD1 mice obtained from Charles River and kept under conventional animal house conditions for several weeks prior to use were used in these experiments. Groups of five mature females were caged overnight with one male and the morning of finding the vaginal plug was designated Day 1 of pregnancy. The pregnant mice were treated on Days 6-16 of pregnancy which covers the time from implantation to the end of structural 1 Tegretol, Ciba-Geigy. 3 Rivotril, Roche. 3 Zarontin, Parke-Davis. 4 Phenobarbitone Sodium, B.D.H. Laboratory 5 Epanutin, Parke-Davis. 6 Mysoline I.C.I. Pharmaceuticals. 7 Epilim, Reckitt and Colman.

Reagents.

TERATOLOGY

OF ANTIEPILEPTIC

367

DRUGS

development. Each of the drugs was suspended in 1% carboxymethylcellulose (1% CMC) and administered once daily by gastric intubation. The number of mice used at each dose level is shown in the relevant tables. All mice were killed on Day 19, the fetuses were removed, and the position, number of live fetuses, resorptions, and fullterm dead fetuseswere recorded. Each fetus was weighed and examined macroscopically, fixed in 70% alcohol and subsequently carefully dissected to look for internal defects using a magnifier or dissecting microscope as necessary. Finally, all the fetuses were stained with alizarin to examine for skeletal defects. Statistical analyses were carried out by the Fisher Exact probability test or the Student t test where appropriate. The mean percentage litter effect was calculated by taking the percentage of fetuses per litter having major defects and averaging these percentages for the total number of litters in each treatment group (Staples and Haseman, 1974). TABLE

1

DOSES OF THE SIX ANTIEPILEPTIC DRUGS ADMINISTERED ORALLY TO MICE AND THE TYPICAL HUMAN THERAPEUTIC DOSES ON WHICH THIS WAS BASED

Drug Carbamazepine Clonazepam Ethosuximide Phenobarbital Phenytoin Primidone

Typical human therapeutic dose mg/kg (range)

3 x TD“

mg/kg

mdkg

mg/kg

13.3 (1.5-23.0) 0.1 (0.05-O. 1)

40.0 0.3 60.0 10.0 15.0 30.0

120.0 0.9 180.0 30.0 45.0 90.0

240.0 1.8 360.0 60.0 90.0 180.0

20.0 (15.0-25.0) 3.3 (1 .o-5.0) 5.0 (3.0-10.0)

10.0 (lO.cWlO.0)

9xTD

18 x TD

ETD = therapeutic dose (human). It was difficult to choose a dose range for this experiment because there was such a wide variation in the animal ED50 (0.1-50 mg/kg, PO) and LD50 (280-4000 mg/kg, po) values amongst the six drugs chosen. As it was the aim of this study to compare the teratogenic effects of the drugs, it was decided to choose doses which were some multiple of the human therapeutic dose. This again proved difficult because of the wide (300-fold) range of therapeutic doses, e.g., 0.05-0.1 mg/kg for clonazepam to 15-25 mg/kg for ethosuximide. From previous work (Sullivan and McElhatton, 1975) it was known that in pregnant mice the maximum tolerated doses of phenytoin and primidone were 120 mg/kg and 200 mg/kg, respectively. These are equivalent approximately to 20 times the human therapeutic doses. Taking various other available factors for each of the six drugs into account, such as the pharmacokinetics in the mouse and the ratio of the doses effective in animals compared with the doses effective in man, it was decided to use dose levels of three and 18 times the human therapeutic dose of each drug in the first experiment and nine times the therapeutic dose in the second experiment. Preliminary 5-day toxicity tests in nonpregnant mice at the highest dose levels suggested that these doses would be satisfactory and the actual doses used are shown in Table 1.

368

SULLIVAN

AND

MCELHATTON

Experiment I

In the first pregnancy experiment two dose levels of each drug were used, each group consisting of at least 16 mice. Because of the large number of animals involved, the experiments had to be divided into two parts. The experiment was divided according to drug rather than dose because it was felt that in order to try to establish a dose-effect relationship both doses of drug needed to be tested at the same time, rather than testing all the high doses together and then all the low doses. The other possibility was to test each of the drugs at the same time in a small number of animals and repeat this procedure several times until there was a reasonable number of animals per treatment group. However, it was felt that the situation might arise when only one or two pregnant animals per group were being tested at a time, and thus there would not be an adequate number to serve as a control. Therefore, in the first part of the experiment ethosuximide (60 and 360 mg/kg), clonazepam (0.3 and 1.8 mg/kg), and phenytoin (15 and 90 mg/kg) were tested ; in the second part, primidone (30 and 180 mg/kg), phenobarbital (10 and 60 mg/kg) and carbamazepine (40 and 240 mg/kg) were used. In each part an untreated group and a CMC-treated group were included. Experiment 2

In the second experiment a single dose level of all of the drugs was tested at the same time, the dose used being nine times the human therapeutic dose, together with two control groups. RESULTS Pregnancy Study at Two Dose Levels (Experiment 1)

Although this study was carried out in two consecutive parts (for reasons stated above), the whole dosing schedule was completed within a 6-week period, and the results of this study will be discussed as a whole. Eflects on the Dams

There were no adverse effects on the dams in the primidone, phenobarbital, ethosuximide, or carbamazepine groups. In the clonazepam groups, the mice were rather quiet 1 hr after dosage but subsequently recovered; this seemed to be a dose-related effect. In the high-dose phenytoin group (90 mg/kg), there was some maternal toxicity in that three mice became ataxic and several dams were aggressive and had ruffled coats after about six doses. One mouse in the phenytoin (90 mg/kg) group died on Day 17 and all of the fetuses in this dam were found to have full-length cleft palates (not included in the tables of malformations). Each of the groups, with the exception of phenytoin (90 mg/kg), gained weight steadily during pregnancy in a similar manner to the controls. The high-dose phenytoin group gained weight, but significantly less rapidly during the treatment period (p < O.OOl), and the drug seemed to exert an antifertility effect in that there was a very low pregnancy rate (54 %) compared with the other groups (69-100 %) and more animals had to be mated to ensure a sufficient number of pregnant animals at term.

(360)

Ethosuximide

(53.8)

Tz

p+ae

(75.0)

16

(100.0) 12

16

12<+41

(100.0)

iii

(87.5) 16

16

Number of plugs (%) 16(+3 26 (76.9) 11’+3’ 16 (87.5) 11 16 (68.8) 14 9.1

(11.5) 17

102

127

105

147

127

(17.1)

(9.3) 21

(9.5) 13

(13.0) 11

8.5

10.6

8.8

9.2

8.4

(22.2) 12

(11.8) 22

8.3

(6.9) 26

91

92

12.6

15

Average number of fetuses per litter

201

Total number of dead and resorbed Number of fetuses live fetuses (“4

-

kO.02

kO.08 0.96

kO.01 1.3

kO.01 1.3

i-o.01 1.2

+0.01 1.2

kO.01 1.2

i-O.04 1.3

1.3

Mean fetal weight (g) +_SE

(11 .O)

11

0

(0.7) 0

1

0

0

0

FLCP” (“4 0

(3.0)

3

(1.6)

(3.8) 2

4

(2.0)

3

(1.6)

(4.3) 2

4

0

ECV” (Oh 0

0

0

0

0

(0.8)

1

0

0

(0.5)

1

Club foot (“4

110 (11.0) lk (1.0) 23“ (23.0)

13f (13.0)

0

0

Id (0.7)

0

0

0

Other major defects tn, (“XII 0

rl FLCP = full-length cleft palate. * ECV = enlarged cerebral ventricles (possibly a microform of hydrocephalus). c Figures in parentheses indicate dams that delivered spontaneously on Day 19 and so had to be excluded. d Separated basisphenoid bones. ’ One dam died on Day 17 and one delivered spontaneously on Day 19. f Undescended testes. h Open eye and partially fused palate. a Abnormal palatal bones.

Phenytoin (90)

Phenytoin (15)

(60)

Ethosuximide

Clonazepam (1.8)

Clonazepam (0.3)

I%CMC

Treatment (mg/W Untreated controls

Number pregnant

TABLE 2 THE EFFECTS OF CLONAZEPAM. ETHOSUXIMIDE. AND PHENYTOIN GIVEN TO MICE BY GASTRIC INTUBATION ON DAYS 6-l 6 OF PREGNANCY

(60)

Phenobarbital

AND

PRIMIDONE

10.9

(10.7) 18

175

12.6

(9.8) 18

145 (12.7)

21

10.4

10.3

(13.5) 18

165

(8.2)

9.8

(15.0) 23

147

202

10.9

25

142

(9.3)

11.7

(11.3) 21

2

kO.05 1.2 (0.01)

0

(0.6)

1

(0.11) 1.3

kO.01 1.4

0

0

+_O.Ol 1.3

0 0

kO.06 1.3

INTUBATION

FLCP” (“%I 0

kO.05 1.3

+0.01 1.3

1.2

12.0

fetal

BY GASTRIC

weight (g) &SE

Mean

TO MICE

Average number of fetuses per litter

GIVEN

175

Total number of dead and resorbed Number of fetuses fetuses (“A 313 40

PHENOBARBITAL

(0.5) 2

1

(2.4)

(0.7) 4

(1.4) 0

2

(0.6)

1

0

(3.0) 2

6

(0.6)

1

(0.7)

1

(1.4)

(2.3) 2

(1.1) 4

(0.3) 2

1

(“Xl

0

Club foot

(“4

14 (0.7)

0

1’ (0.6)

h (%N ___-.

Other major defects

6-16 PREGNANCY

ECV”

ON DAYS

(1.4) (1.4) (1.4) a FLCP = full length cleft palate. b ECV = enlarged cerebral ventricles (possibly a microform of hydrocephalus). c Figures in parentheses indicate dams that did not maintain their pregnancy to term. d Full-term dead fetus with cleft palate. e Exencephaly and open eyes. f Exomphalos and fused ribs. B Open eye.

Primidone (180)

Primidone (30)

(10)

Phenobarbital

Carbamazepine (240)

(75.0) 15 3 (78.9) 16 E-i (84.2) 13’W ai3 (73.7) 15 i5 (78.9) 16 ii? (84.2) 16 19 (84.2) 14 is (73.7)

z

26’“‘”

Number of plugs (73

Number pregnant

OF CARBAMAZEPINE,

Carbamazepine (40)

f%CMC

Treatment h/W Untreated Controls

THE EFFECTS

TABLE 3

2

TERATOLOGY

OF ANTIEPILEPTIC

371

DRUGS

Effects on the Fetuses The results are shown in Tables 2 and 3. There were no significant differences in litter size or resorption rates in any of the treated groups compared with the range seen in the controls. There was some variation in fetal weight but this was only significant for fetuses in the high-dose phenytoin (90 mg/kg) group which were significantly (p 6 0.001) lighter in weight than in the controls. One of the most common major defects observed was full-length cleft palate which occurred in the ethosuximide, primidone, phenobarbital, and phenytoin groups, and with the exception of the ethosuximide group, the incidence of the defect was doserelated although, of course, the significance of a single affected fetus in a group is TABLE 4 INCIDENCE

OF MAJOR PERCENTAGE

DEFECTS LITTER

Treatment Untreated controls Untreated controls l%CMC l%CMC Clonazepam Clonazepam Carbamazepine Carbamazepine Ethosuximide Ethosuximide Primidone Primidone Phenobarbital Phenobarbital Phenytoin Phenytoin

IN MICE CALCULATED BASIS FOR EXPERIMENT

Dose (m/W (Part 1) (Part 2) (Part 1) (Part 2) 0.3 1.8

ON A MEAN 1

Mean percentage litter effect 0.62 0.27

0

180 10

1.02 2.8 2.3 2.5 2.7 3.5 4.2 3.4 4.8 1.9

60

4.9

15

1.4 41.96

40 240 60 360 30

90

doubtful. No cleft palates were seen in any of the controI groups or in those exposed to clonazepam or carbamazepine. Enlarged cerebral ventricles, which may be a microform of hydrocephalus were detected in all of the drug-treated groups but not in any of of the control groups. Again this was a dose-related effect with the exception of the clonazepam group. The significance of a subjective assessment of increased cerebral ventricle size is difficult to determine, but it is unlikely to be due simply to retarded development since the fetuses showing this defect showed no other signs of retarded development as assessedby weight and degree of ossification in the skeleton, especially of the phalanges. Other major defects such as open eyes, exencephaly, exomphalos, fused ribs, separated basisphenoid bones, abnormally shaped palatal bones (not cleft palate), and undescended testes were seen occasionally in some of the drug-treated groups. These data were also assessedas a mean percentage litter effect for those major defects and the details are shown in Table 4.

372

SULLIVAN

AND

MCELHATTON

Minor anomalies such as submucosal cleft palate, abnormal sternebrae, extra ribs, and retarded ossification of the hands and feet were observed in all groups, but on the whole, with the exception of phenytoin, these anomalies were not dose- or drug-related. It was only in the phenytoin-treated groups that there were marked differences in ossification when compared with the controls and the other anticonvulsant-treated groups. In the phenytoin (90 mg/kg)-treated group there was severely retarded ossification of the digits in the hands (42.6 %) and feet (62.3 “/,) compared with the untreated controls (4.6 and 29.5 %) and the CMC-treated controls (1.1 and 25.6 %). Single Dose Study (Experiment 2) In the first experiment, which had to be carried out in two parts, it was shown that each of the drugs did produce teratogenic effects. In order to try to rank these drugs in some order of teratogenicity, it was decided to carry out a single dose experiment testing all six drugs at the same time. In order to avoid the maternal toxicity observed with the high dose of phenytoin in the first experiment, an intermediate dose equal to nine times the human therapeutic dose was used for all of the drugs for the second experiment. Efects on the Dams There were no signs of toxicity asjudged by a lack of ataxia or respiratory depression; however, similar relaxant effects were seen in the clonazepam group as previously described. There were no significant adverse effects on maternal weight gain during the treatment period. There were no adverse effects on fertility; the pregnancy rate ranged from 74 to 90 % in the treated groups compared with 83 to 84 % in the controls. Efects on the Fetuses These results are summarized in Table 5. There were no significant differences in litter size or fetal weights in the anticonvulsant-treated groups compared with either control group. In the primidone-treated group there was an increase in fetal resorptions (19.4 %) compared with control values (8. l-l 1.8 %) or those from the other treated groups (7.3-12.7 %), and this was accounted for mainly by an increase in advanced resorptions. Two dams in the phenytoin group which had resorbed completely were excluded from the analysis as the hemorrhagic condition of the uterus prevented exact quantitation of the number of sites. Again, the major defects observed consisted of full-length cleft palate in the primidone carbamazepine, phenobarbital, and phenytoin-treated groups but not in those treated with ethosuximide or clonazepam, or in the controls. Enlarged cerebral ventricles were seen in some fetuses in all of the treated groups but not in the controls, which confirms the findings of the previous experiment. Cerebral hemorrhages were also observed in some of the fetuses exposed to carbamazepine, phenobarbital, phenytoin, and primidone. Exencephaly was observed in one fetus in each of the clonazepam- and phenobarbital-treated groups. In the carbamazepine group there was one grossly malformed fetus with multiple defects including phocomelia, full-length cleft palate, kyphosis, an unequal number of ribs, enlarged atria, and a hole in the right atrium. In the phenytointreated group 10 (5.6%) of the fetuses from 5 litters had open eyes; this defect was not seen in any of the other treated groups (with the exception of the grossly malformed

15 19 (73.9)

Primidone (90)

’ FLCP = full-length cleft palate. d One fetus with abnormal palatal bones, phocomelia, e Hemorrhage around lateral ventricles. ’ Separated basisphenoid bones. ’ Open eyes.

154

178

153 3

(1.7)

&O.Ol 1.2 kO.01

kO.01

1.2

+0.15

(0.6)

1

(5.1)

0

+0.01 1.3

1.1

(0.7) 0

1*

0

FLCP” (%I 0

*0.01 1.2

+0.01 1.2

+0.01 1.3

1.3

Mean fetal weight (g) +SE

(3.9)

6

(4.0)

(1.1)

2

(2.6)

4

(2.0)

3

(6.0)

9

0

ECVb (“4 0

0

(0.6)

(2.2)

(0.7) 4

(1.4) 1

(1.3) 2

2

(2.0)

4

(1.2)

Club foot (“A 2

MICEBY

space.

1’ (0.6) 1” (0.6) lg (0.6) 1” (0.6) 2’ (1.1) 3’ (1.7) 101 (5.6) 1” (0.6) 3’ (1.9)

0

2e (1.3) If (0.7)

ld (0.7)

0

Other major defects b, ( “%I1 4’ (2.4)

GASTRICINTU-

b ECV = enlarged cerebral ventricles. ’ Wolffian ducts still present. unequal number of ribs, open eyes, kyphosis, enlarged atria, and hole in right atrium. f Exencephaly. B Hemorrhage in the subarachnoid 1 Figures in parentheses dams that had resorbed completely (see text). k Abnormal palatal bones.

(19.4)

10.3

11.1

(8.9) 14

37

9.0

15

(8.1)

10.6

(12.7) 13

(8.3)

(30)

(180)

148

10.0

22

17’+1” zz (86.4)

10.7

(11.8) 18

(8.1)

10.3

Average number of fetuses per litter

22

Phenytoin (45)

Phenobarbitone

Ethosuximide

Clonazepam (0.9)

Carbamazepine

151

203

164

(7.3)

(120)

number of plugs (“%I 16 is (84.2)

Total number of dead and resorbed Number of fetuses live fetuses (“4

(82.6) 15 i5 (78.9) 14 i5 (73.7) 17 i5 (89.5) 16 19 (84.2)

l%CMC

Treatment (m/W Untreated controls

Number pregnant

‘TABLE 5 THE EFFECTSOF CARBAMAZEPINE,CLONAZEPAM,ETHOSUXIMIDE,PHENOBARBITALPHENYTOIN,ANDPRIMIDONEGIVENTO BATIONON DAYS~-16 OFPREGNANCY

Y w

9 ii $j s r/~

$ g P

s p 8 4 %

374

SULLIVAN

AND

MCELHATTON

fetus exposed to carbamazepine). Other defects such as abnormal palatal bones (not cleft palate), separated basisphenoid bones, and undescended testes were seen occasionally as in the previous experiment. The incidence of major defects expressed as a mean percentage litter effect are shown in Table 6. Skeletal variants occurring in the drug-treated groups all fell within the normal range of control values, except that fetuses exposed to phenytoin had marked retardation in ossification of the hands and feet. TABLE 6 INCIDENCE CULATED

OF MAJOR DEFECTS IN MICE ON A MEAN PERCENTAGE LITTER FOR EXPERIMENT 2

CALBASIS

Treatment

Mean percentage litter effect

Untreated controls l%CMC Ethosuximide Clonazepam Primidone Phenobarbital Carbamazepine Phenytoin

3.4 1.3 3.2 3.9 6.5 8.9 9.1 18.0

If, for the overall assessment of the results, the numbers of fetuses or litters with major malformations are pooled for each drug regardless of dose, then the incidences of defects are shown in Table 7. Since the incidences in the untreated and CMC controls were the same, these have been pooled. DISCUSSION The aim of this study was to try to establish whether or not six of the commonly used antiepileptic drugs (carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, and primidone) would produce teratogenic effects in the mouse, and if so, whether or not they could be placed in some order of teratogenicity. Because this study had to be divided up into parts, as previously described, the animals were probably subjected to minor differences in environmental influences, as is reflected in the variation in control values obtained in each part of the study. Since the middle dose formed a separate experiment and there were clear differences between the control groups, the data obtained from the two experiments cannot be directly assessed on a dose-effect basis. However, within each part of the experiment the results indicate quite clearly that the drugs can produce teratogenic effects, the incidences of which are higher in the drugtreated groups than in the corresponding CMC-treated or untreated control groups. The teratogenic effects obtained in each part of the experiment can be assessed on a drug-related basis, if the data from each treatment group, regardless of dose, are pooled. Therefore, if the number of fetuses with major defects is added together for each of the

2.7

3.2

4.7

4.7 4.8

20.9

Pooled controls

Clonazepam

Ethosuximide

Carbamazepine

Phenobarbital Primidone

Phenytoin

AND

WITH

MAJOR

$5)

(ii) (5%)

$3)

$5)

(3%)

(lii7)

Number of fetuses with major defects (total number of fetuses)

LITTERS

IN MICE;

0.3 x lo-=

0.00009

o.ooo1

0.0002

0.03

0.12

-

P'

DEFECTS

7

’ All drug groups compared with pooled controls; Fisher Exact probability test, 2-tailed.

1.3

Treatment

OF FETUSES

Percentage of fetuses with major defects

INCIDENCE

TABLE RESULTS

56.1

29.8 37.8

31.8

26.7

23.1

12.6

Percentage of litters with major defects

POOLED

ALL DOSELEVELS

& (E,

ii

(&

(3;)

$3)

Number of litters with major defects (total number of litters)

FROM

0.4 x 10-6

0.02 0.001

0.01

0.07

0.2

-

P"

: E Q 0-2

ti 2 g i= %! =!

g

;;1 F 8 6 2

376

SULLIVAN

AND

MCELHATTON

drugs, it is possible to divide up the drugs into three main categories of toxicity as shown in Table 7. Phenytoin is in a category of its own because it produces the highest incidence of fetal defects (20.9 %). In fact, there is a 4- to 7-fold increase in defects in the phenytoin groups compared with the other five drugs and about a 16-fold increase in malformations when compared with the controls (1.3 %). Primidone (4.8 %), phenobarbital (4.7 %), and carbamazepine (4.7%) fall into the next category. These three drugs, although they produce a lower incidence of defects than phenytoin, still have an incidence about 3.5 times higher than observed in the controls and this difference is very highly significant. In the third category are clonazepam (2.7 %) and ethosuximide (3.2 %) which produced the lowest incidence of fetal malformations. Although this is still between 2 to 2.5 times higher than the incidence of “spontaneous” defects in the controls, it is of doubtful statistical significance. When these results are assessedon a pooled litter basis (mean percentage litter effect), a similar pattern emerges, i.e., phenytoin (20.1), then phenobarbital (5.3), primidone (4.9), and carbamazepine(4.9) followed by ethosuximide (3.5) andclonazepam (3.0). The control values are 0.9 and (absolute controls) 1.2. There is no agreed satisfactory method for the statistical analysis of the percentage litter effect results, so the proportions of affected litters with each drug were analysed statistically in Table 7 using the Fisher Exact probability test. Again, the drugs fall into three categories with the clonazepam and ethosuximide groups being of doubtful significance. On the whole, the drugs did not produce toxic effects in the dams as judged by lack of ataxia or respiratory depression and by an adequate weight gain throughout pregnancy. The highest dose of phenytoin (90 mg/kg) did produce some toxic effects in the dams viz., ataxia, ruffled coat, irritability, and reduced weight gain, and probably an antifertility effect also asjudged by the low pregnancy rate in this group, but a high incidence of malformations was still obtained with phenytoin at the 45-mg/kg dose which produced very little sign of toxicity in the dams. In general, there were no adverse effects on fetal weight (except in the phenytointreated groups) or fetal resorptions and death, which suggests that the drugs are teratogenic rather than embryolethal. Full-length cleft palate was observed in each part of the study mainly in the phenytoin, primidone, and phenobarbital groups and less frequently in the ethosuximide and carbamazepine groups. It is very interesting that no full-length clefts were observed in any of the clonazepam-treated groups. Enlarged cerebral ventricles (possibly a microform of hydrocephalus) have also been observed by Harbison and Becker (1969) in mice treated with phenytoin. In the “two dose” experiment the incidence of this defect seemed to be dose-related in all except the clonazepam groups. There are no published reports on the teratogenicity of clonazepam. Other major defects such as exencephaly (phenobarbital, 10 and 30 mg/kg; and clonazepam, 0.9 mg/kg), exomphalos plus fused ribs (phenobarbital, 60 mg/kg) and phocomelia plus multiple defects (carbamazepine, 240 mg/kg) were observed occasionally, but the significance of such single fetuses with defects is difficult to assess. Defects such as open eyes, abnormally shaped palatal bones other than cleft palate, and undescended testes were seen mainly in the phenytoin groups (30 and 60 mg/kg) but not in the lowest (15 mg/kg) dose group. Skeletal anomalies were distributed throughout the

TERATOLOGYOF ANTIEPILEPTICDRUGS

377

groups, marked differences occurring only in the phenytoin groups (30 and 60 m/k). In summary, the results of this comparative study, when judged on either a pooledper-fetus basis or a pooled-per-litter basis, indicate clearly that four of the six drugs tested are teratogenic, but that the degree of teratogenicity is variable. Phenytoin produced the highest incidence of major defects in the fetuses together with an overall retardation in fetal growth and possibly an antifertility effect at high doses. Primidone, phenobarbital, and carbamazepine produced a lower but still significant incidence of fetal malformations also. Ethosuximide and clonazepam produced the lowest incidence of fetal malformations in this study and the difference from controls was not statistically significant. However, the overall incidence of defects with both of these drugs is more than twice that of the controls, and certain defects such as enlarged cerebral ventricles were not seen in the controls. It is, therefore, quite possible that the use of larger numbers of litters would show a significant increase in malformations over the controls. It is interesting that with the exception of clonazepam each of the drugs which have produced teratogenic effects in CD1 mice have also been implicated singly or in combination in the reported clinical data (Speidel and Meadow, 1972 and 1974; Fedrick, 1973; Millar and Nevin, 1973; Annegers et al., 1974). The main defect in the mouse, i.e., cleft palate, is also one of the main defects reported to occur in humans. As these results indicate that some of the anticonvulsant drugs are safer than others in the mouse, it is possible that this may be true for man also. As epileptic patients require anticonvulsant drugs for long periods of time and are usually on multiple drug therapy, it would be worthwhile to try to determine from epidemiological surveys which drug or combination of drugs is the safest for women of childbearing age. REFERENCES ANNEGERS, J. F., ELVEBACK,L. R., HAUSER,W. A., AND KURLAND, L. T. (1974). Do anti-

convulsants have a teratogenic effect? Arch. Neural. 31,364373. CAHEN,R. L. (1964). Evaluation of the teratogenicity of drug. Clin. Pharmacol.

Ther. 5,

480-514. ELSHOVE, J. (1969). Cleft palate in the offspring of female mice treated with phenytoin. Lancet ii, 1074. FEDRICK,J. (1973). Epilepsy and pregnancy: A report from the Oxford Record Linkage Study. Brit. Med. J. 2,442-448.

HARBISON,R. D., AND BECKER,B. A. (1969). Relation of dosage and time of administration of diphenylhydantoin to its teratogenic effect in mice. Teratology 2, 305-311. HARBISON,R. D., AND BECKER,B. A. (1972). Diphenylhydantoin teratogenicity in rats. Toxicol. Appl. Pharmacol.

22,193-200.

HILL, R. M. (1973). Drugs ingested by pregnant women. Clin. Pharmacol. Ther. 14, 654-6.59. JANZ,D., AND FUCHS,U. (1964) .Sind Antiepileptische Medikamente wlhrend der Schwangershaft schadlich? Deutsche Med. Wschr. 89, 241-243. LOWE, C. R. (1973). Congenital malformations among infants born to epileptic women. Lancet i, 9-10, MCCOLL, J. D., GLOBUS,M., ANDROBINSON, S. (1963). Drug induced skeletal malformations in the rat. Experientia 19, 183-184. MCCOLL, J. D., ROBINSON, S., ANDGLOBUS,M. (1967). Effects of some therapeutic agents on the rabbit foetus. Toxicol. Appl. Pharmacol. 10,244252. MCELHATTON,P. R., AND SULLIVAN,F. M. (1975). Teratogenic effects of primidone in mice. Brit. J. Pharmacol. 54, 267-268P.

378

SULLIVAN

AND

MCELHATTON

MCELHATTON, P. R., SULLIVAN, F. M., AND TOSELAND, P. A. (1976). The teratogenic activity and metabolism of primidone in the mouse. Epilepsia, in press. MASSEY, K. M. (1966). Teratogenic effects of diphenylhydantoin sodium. J. Oral Ther. Pharmacol. 2,380-385. MILLAR, J. H. D., AND NEVIN, N. C. (1973). Congenital malformations and anticonvulsant drugs. Lancet i, 328. MONSON, R. R., ROSENBERG, L., HARTZ, S. C., SHAPIRO, S., HEINONEN, 0. P., AND SLONE, D. (1973). Diphenylhydantoin and selected congenital malformations. New Engl. J. Med. 289,

1049-1052. PERSAUD, T. V. N. (1965). Tierexperimentelle untersuchungen fur Frage der Teratogenen wirkung von Barbituraten. Acta Biol. Med. Ger. 4, 89-90. POSWILLO, D. E. (1972). Tridione and Paradione as suspectedteratogens. Ann. Roy. Coll. Surg. Engl. 50,367-370. SCHARDEIN, J. L., DRESNER, A. J., HENTZ, D. L., PETRERE, J. A., FITZGERALD, J. E., AND KURTZ, S. M. (1973). The modifying effect of folinic acid on the diphenylhydantoin induced teratogenicity in mice. Toxicol. Appl. Pharmacol. 24, 150-158. SOUTH, J. (1972). Teratogenic effects of anticonvulsants. Lancet ii, 1154. SPEIDEL, B. D., AND MEADOW, S. R. (1972). Maternal epilepsy and abnormalities of the foetus and newborn. Lancef ii, 839-843. SPEIDEL, B. D., AND MEADOW, S. R. (1974). Epilepsy, anticonvulsants and congenital malformations. Drug 8, 354-365. STAPLES, R. E., AND HASEMAN, J. K. (1974). Selection of appropriate experimental units in teratology. Teratology 9,259-260. STARREVELD-ZIMMERMAN, A. A. E., VAN DER KOLK, W. J., MEINARDI, H., AND ELSHOVE, J. (1973). Are anticonvulsants teratogenic? Lancef ii, 48-49. SULLIVAN, F. M., AND MCELHA~ON, P. R. (1975). Teratogenic activity of the antiepileptic drugs phenobarbital, phenytoin, and primidone in mice. Toxicol. Appl. Pharmacol. 34,271282. WALKER, B. E., AND PATTERSON, A. (1974). Induction of cleft palate in mice by tranquillisers and barbiturates. Teratology 10, 159-l 63. WILSON, J. G. (1973). Teratologic causation in man and its evaluation in non-human primates. In Birth Defects (A. G. Motulsky and W. Lenz, eds.), pp. 191-203. Excerpta Medica,

Amsterdam.