Teratogenic acitivity of the antiepileptic drugs phenobarbital, phenytoin, and primidone in mice

l=OXICOLOGYANDAPPLIEDPHARMACOLOGY~,

Teratogenic

271-282(1975)

Activity of the Antiepileptic Phenytoin, and Primidone

Drugs in Mice

Phenobarbital,

F. M. SULLIVAN AND P. R. MCELHATTON Department of Pharmacology, Guy’s Hospital Medical School, London SE1 9RT, England Received March 20,197s; accepted May 16,1975

Teratogenic Activity of the Antiepileptic Drugs Phenobarbital, Phenytoin, and Primidone in Mice. SULLIVAN, F. M. AND MCELHATTON, P. R. (1975). Toxicol. Appl. Pharmacol. 34, 271-282.The teratogenic activity of phenobarbital, phenytoin, and primidone was studied in mice derived from theICI pathogenfree strain. The drugswereadministeredin the diet or by gastric intubation either from days G16 or 12-16of pregnancy(day of vaginal plug is day 1). This covered the period during which the mice are susceptibleto agentsinducing cleft palate. The motherswere killed 1 day before term and the fetuseswereexamined.Cleft palateswererarely seenin the controls, the incidencebeing only 3 out of 1103(0.3%) fetuses.The incidenceof cleft palatein micetreatedwith phenobarbital50 or 150mg/kg in the diet was0.6 and3.9%, with phenytoin 40and 120mg/kgby intubation, or 250mg/kg in the diet was0,ll .l and 10.2%, and with primidone 100to 250mg/kg by intubation or 500-2500mg/kg in the diet rangedup to 15%. Since changesin blood folate concentrations following the use of anticonvulsantsmight be relatedto the teratogeniceffectsobserved,an attempt wasmadeto antagonizethe effectsby the simultaneousadministrationof folinic acid. Folinic acid had no teratogenicaction of its own, and had no effect on the incidenceof cleft palate inducedby phenobarbitalin the diet, or by phenytoin by gastric intubation. However, it did significantly potentiate the teratogeniceffectsof phenytoin administeredin the diet (p -C0.002) but significantly reduced the teratogenic effect of primidone given in the diet (p < 0.034). There have been a number of epidemiological surveys in various parts of the world reporting that there is an increasein the incidence of congenital malformations in the children of women with epilepsy (Elshove and Van Eck, 1971; Speidel and Meadow, 1972; Fedrick, 1973; Monson et al., 1973). It is difficult in such a situation to determine whether the increasedmalformation rate is due to the diseaseor to the drugs usedin its treatment, but in all of the surveys where it hasbeenmeasured,the incidence of malformations is higher in epileptics receiving drug treatment during the relevant pregnancy, than in epileptics receiving no treatment (Janz and Fuchs, 1964; Speidel and Meadow, 1972; Monson et a[., 1973). The evidence certainly seemsfairly strong that the antiepileptic drugs are teratogenic and this receives support from studies carried out on these drugs in animals. Massey (1966) first demonstrated that phenytoin (diphenylhydantoin) was teratogenie in mice and this has been confirmed for mice and rats (Elshove, 1969; Harbison and Becker, 1969, 1972). The most common defects found in rodents treated with Copyright 0 1975 by Academic Press, Inc. 271 All rights of reproduction Printed in Great Britain

in any fotm reserved.

272

SULLIVAN

AND

MCELHATTON

phenytoin during pregnancy are cleft lip and/or palate, hydrocephalus, hydronephrosis, and skeletal abnormalities including shortening of the long bones and ectrodactyly. The teratogenicity of phenobarbital in animals is much less well established. McCall et al. (1963) reported that in rats, phenobarbital induced skeletal defects and a high neonatal mortality. The same authors reported a similar high neonatal mortality and cardiac defects in rabbits (McCall et al., 1967). Several other workers, however, have found only a high embryolethality without malformations. The combination of phenobarbital and phenytoin has been shown in mice to be less teratogenic than the latter drug given alone. This was associated with reduced plasma concentrations of phenytoin. We are not aware of any published data on the teratogenicity of primidone in animals. The present experiments were designed to compare the effects of phenytoin, phenobarbital, and primidone in inducing cleft palate in mice, and to see whether the results would be affected by the concurrent administration of folinic acid. METHODS Albino mice derived from the ICI pathogen free strain and maintained under conventional animal house conditions at Guy’s Hospital Medical School were used for these experiments. Groups of five mature females were caged overnight with one male and the morning of finding the vaginal plug was called day 1 of pregnancy. The pregnant mice were normally treated either on days 6-16 of pregnancy or 12-16 of pregnancy which covers the period of palatal development and closure which occurs on day 16 in these mice. The drug was administered either mixed with powdered diet [FFG(M)] on the basis of 5 g diet per 40 g mouse, or directly by gastric intubation once each day. The number of mice used in each experiment is shown on the relevant tables. All mice were killed on day 19, the fetuses removed, and the position and number of live fetuses, resorptions, and full-term dead fetuses recorded. The fetuses were weighed andexamined macroscopically, fixed in 70% alcohol, and subsequently carefully dissected to look for internal defects and, finally, all fetuses were stained with alizarin to examine for skeletal defects. The drugs studied were phenobarbital sodium,’ 50 and 150 mg/kg in the diet, phenytoin sodium2 (diphenylhydantoin), 250 mg/kg in the diet, 40 and 120 mg/kg by gastric intubation, primidone3 500-2500 mg/kg in the diet and 100-250 mg/kg by gastric intubation, and folinic acid4 3.75 mg/kg subcutaneously. There are always problems in choosing appropriate drug dose levels for animal experiments, but we chose a range of doses such that the lowest dose produced little or no toxicity in the mother, whereas the highest dose was chosen to produce some maternal toxicity but low fetal mortality. Primidone which is insoluble in water, was suspended in an aqueous solution of 1% sodium carboxymethylcellulose and ultrasonicated to obtain a fine milky stable suspension. This suspension was kept in the dark and showed no loss of activity on analysis after 3 months. Statistical analysis was carried out by the Fisher Exact Probability test, two-tailed. 1 Phenobarbitone sodium, B.D.H. Laboratory z Epanutin, Parke Davis. 3 Mysoline, 1X2.1. 4 Calcium Leucovorin, Lederle.

Reagents.

TERATOLOGY

OF THREE

ANTIEPILEPTIC

DRUGS

213

RESULTS Phenobarbital

Mice were treated with 50 mg/kg or 150 mg/kg of phenobarbital mixed in the diet daily from days 6 to 16 of pregnancy. The controls were given powdered diet only and the results are shown in Table 1. There were no cleft palates in the control group but one fetus was exencephalic. One of 171 (0.6%) fetuses from the 50-mg/kg phenobarbital group had a cleft palate, rising to 6 of 155 foetuses (3.9%) in the 150-mg/kg group. There was no overall increase in resorptions though two mice in the 150-mg/kg group resorbed completely. There was no effect of treatment on either fetal body weight or maternal body weight. Phenytoin

Mice were treated with 40 mg/kg or 120 mg/kg phenytoin daily by gastric intubation from days 12 to 16 of pregnancy or with 250 mg/kg given in the diet over the same period. Two control groups were used, one on powdered diet only and the other intubated daily with distilled water. The results are shown in Table 2. There was a clear increase in the incidence of cleft palate rising to 7 of 63 fetuses (11.1 ‘A) in the 120-mg/kg group as a single oral dose and 19 of 186 fetuses (10.2%) in the dietary-treated mice, with half of the litters being affected in each case. There appeared to be a reduction in fetal body weight in the treated groups but this may be related only to the slightly increased litter size in two of these groups, since it is well known that litter size is negatively correlated with fetal weight. Thus phenytoin may be regarded as being teratogenic in these mice without being embryotoxic as judged by lack of increase in resorptions or fetal death. No other significant abnormalities were observed in the fetuses. There was no evidence of toxicity in the mothers treated with 40 or 120 mg/kg but there was failure of weight gain during the treatment period with 250 mg/kg in the diet. Primidone

Two groups of experiments were carried out using primidone. In the first, mice were given 500, 1250,2000, or 2500 mg/kg of primidone mixed in with the diet on days 6-16 of pregnancy. In the second experiment, mice were given a single daily dose of 100,150,200, or 250 mg/kg by gastric intubation on days 12-16 of pregnancy. Corresponding control groups were given either powdered diet alone or intubated with a 1% carboxymethylcellulose solution which had been used to suspend the primidone. The results are shown in Tables 3 and 4. When given in the diet, primidone produced a doserelated increase in the incidence of cleft palate as judged by the percentage of fetuses affected though only one or two litters were involved in each case. No other drug-related significant malformations were observed. The primidone was toxic to the mothers at the two highest dose levels as shown by ataxia in many of the animals and probably also by the low pregnancy rates as shown in Table 3, since resorption occurring on day 6 may not be detected. With single daily oral administration very much lower dose levels had to be used since preliminary toxicity experiments showed that following administration by gastric intubation for 4 consecutive days to groups of nonpregnant mice the LD50 was approximately 280 mg/kg. A range of doses from 100 to 250 mg/kg was

,::;

cl:)

(E)

155

171

210

No. live fetuses

GIVEN

o Exencephaly. * Cutaneous haemorrhage. c Exomphalos. d Excluding two mice which resorbed completely days 11-12. eDislocated hind limbs.

Phenobarbital (150 mg/kg)

Phenobarbital (50 mg/kg)

Control (powdered diet only)

Drug

OF PHENOBARBITAL

No. pregnant mice at term (no. with vaginal plugs)

EFFECTS

TABLE TO

1

10.3

9.5

9.5

Mean no. live fetuses per litter

IN THE DIET

DAYS

(81.58)

(X)

,:.“,

Total no. dead and resorbed fetuses (%)

MICE ON OF PREGNANCY

(6!7)

(5f6)

0

No. litters with palatal defects (%I

6-16

(3p9)

(0!6)

0

Total no. fetuses with palatal defects (%)

lb 1’ 1’

1”

Total no. fetuses with other abnormalities

E 3 8

fi4 8m

2 F: 7 $

OF PHENYTOIN

’ Dislocated hind limbs. ’ Syndactyly. c Cutaneous haemorrhage. d Exencephaly.

Phenytoin (120 mg/kg, po)

Phenytoin (40 mg/kg, PO)

Control (water)

Phenytoin (250 mg/kg, diet)

No. live fetuses

No. pregnant mice at term (no. with vaginal plugs)

63

34

98

186

181

IN THE DIET

EITHER

ADMINISTERED

Control (powdered diet alone)

Drug

EFFECTS

TABLE

2

10.5

8.5

9.8

11.6

9.1

Mean no. live fetuses per litter

OR BY GASTRIC

(K)

&)

(&

(496)

Total no. dead and resorbed fetuses (%)

INTWATION

ON DAYS

(5i.O)

(li.0) 0

(5i.O)

0

No. litters with palatal defects (%I

TO MICE

(1:I)

(l!O) 0

$2)

0

2” 1’ I*

;:

2”

1”

1”

Total no. fetuses with other abnormalities

PREGNANCY

Total no. fetuses with palatal defects (%)

12-l 6 OF

3

Phw)

No. pregnant mice at term (no. with vaginal

33

19

214

188

210

No. live fetuses

8.3

6.3

8.2

9.9

9.5

live fetuses per litter

Mean no.

(lo:*)

(3!!4)

(l?O)

:sIb)

(Y9)

Total no. deadand resorbed fetuses(%)

* Exencephaly. ’ Polydactyly. * Dislocated limbs. e Two pregnant mice died during treatment--excluded. * Cutaneous haemorrhages.

’ As experiments werecarriedout at the sametimethis isthe samecontrol groupas in Table 1.

Primidone(2500mg/kg)

Primidone(2000mg/kg)

Primidone(1250mg/kg)

Primidone(500mg/kg)

Control” (powdereddiet alone)

Drug

TABLE

(2i.O)

(3i.3)

&)

0

No. litters with palatal defects (%I

(A)

$3)

(&)

(:.6)

0

fetuses with palatal defects(%)

Total no.

EFFECTS OF PRIMIDONE GIVEN IN THE DIET TO MICE ON DAYS 6-16 OF PREGNANCY Total no.

1” lb 1* 1” lb

1’

lb

fetuseswith other abnormalities

(150 mg/kg)

(200 mg/kg)

(250 mg/kg)

Primidone

Primidone

Primidone

GIVEN

(1;)

PlwF)

No. pregnant mice at term (no. with vaginal

OF PRIMIDONE

11.7

140 12.9

12.5

125

10.0

Mean no. live fetuses per litter

11.5

90

4

INTUBATION

TABLE

150

90

No. live fetuses

BY GASTRIC

u CMC, carboxymethyl cellulose 1% used as suspending agent. * Dislocated hind limbs. c One full-term dead fetus had cleft palate. d h< 0.01 compared with control. e Club foot. f Cutaneous haemorrhage. 9 One mother had her litter early on day 19-excluded. “p < 0.025 compared with control. I Kinked tail. 1 Three mothers died days 14-15-excluded.

(100 mg/kg)

(1% CMC)

Primidone

Controls

Drug

EFFECTS

(&)

(282.6)

(3343)

(4040)

(1:4)

(1:s)

(222)

(1:;)

:8!i)

(I’op”,,

(111)

Total no. fetuses with palatal defects (%)

12-16 OF PREGNANCY

No. litters with palatal defects (%I

ON DAYS

Total no. dead and resorbed fetuses (%)

TO MICE

2b

Total no. fetuses with other abnormalities

3 F 8 6

278

SUUIVAN

AND

MCELHATTON

therefore used in these experiments, and there was no sign of toxicity or ataxia in the groups receiving 100, 150, or 200 mg/kg but two mice became ataxic and three died during treatment with 250 mg/kg. A significant increase in total palatal defects as judged by number of fetuses affected was seen in all except the highest dose group. With 100 mg/kg this consisted mainly of submucosal cleft palates (12 of the 16 fetuses) but at the higher doses of 150 and 200 mg/kg there was also an increase in the incidence of full-length cleft palates (8/l 1 and 6/l 5, respectively). Histological examination of the submucosal cleft palates showed failure of the mesoderm to meet in the midline so that the palate was bridged only by a layer of epithelium. No other drug-related malformations were observed except for an increase in cutaneous haemorrhages. Combined Efects of Drugs with Folinic Acid Since it is known that anticonvulsant drugs reduce blood folic acid concentrations (Reynolds, 1973a) attempts were made to see if the teratogenic effects could be antagonized by concurrent administration of folic acid. Since it is usually easier to reverse folic acid antagonists with folinic acid rather than folic acid, this compound was used. The anticonvulsant drugs, phenobarbital, phenytoin, and primidone were all administered in the diet on days 12-16 of pregnancy and the folinic acid was injected subcutaneously once daily during the same period in a standard dose of 3.75 mg/kg. In addition, in one experiment, phenytoin was administered once daily by gastric intubation along with folinic acid as above. The results are shown in Table 5. The incidence of cleft palate in the phenobarbitaltreated groups was not affected by folinic acid administration, though the three affected fetuses in the phenobarbital group alone had submucosal cleft palates and in the combined phenobarbital and folinic acid group, they had full-length cleft palates. In the groups treated with primidone, folinic acid produced a slight but just significant (p = 0.034) reduction in the incidence of cleft palate from 17 of 156 foetuses (10.9%) to 4 of 117 foetuses (3.4 %). When phenytoin was given in the diet the incidence of cleft palate was increased by concurrent administration of folinic acid from 2 of 88 fetuses (2.3 %) to 8 of 39 fetuses (20.5%) (p = 0.002). When phenytoin was given by stomach tube the folinic acid did not affect the incidence of cleft palate. DISCUSSION

The present study has shown that phenytoin, primidone, and phenobarbital are teratogenic when judged on a per fetus basis in the strain of mouse used, the most common abnormalities being either full-length cleft palate or submucosal clefts. Other defects such as exomphalos, exencephaly, fetal haemorrhages, skeletal abnormalities, and minor defects such as open eye and kinked tails were also observed. There were no significant differences in the numbers of full-term dead fetuses or fetal resorptions in any of the groups treated with anticonvulsants when compared with the control groups. When judged on a per litter basis, the number of litters at each dose level is, in general, too small to allow valid statistical analysis. However, if the results from all of the litters treated with any one of the drugs are pooled, irrespective of dose, then

OF CONCURRENT

-

3.75

-

-

3.75

-

3.75

-

-

3.75

15

Phenobarbital(150mg/kg, diet)

Phenobarbital(150 mg/kg, diet)

None (diet alone)

Phenytoin (250mg/kg, diet)

Phenytoin (250mg/kg, diet)

Water (0.5 ml SC)plus Water (0.1 ml po) Phenytoin (120mg/kg, PO)plus Water (0.5 ml sc) Phenytoin (120mg/kg, po)

None (diet alone)

Primidone(1250mg/kg, diet)

Primidone (1250mg/kg, diet)

None (folinic acid alone)

LIThreemicehadlittersprematurely-excluded.

-

-

Folinic acid (SC)

ADMINISTRATION

None (diet alone)

Treatment Anticonvulsant

EFFECTS

TABLE

5

(it; 12 (18)

,:;

(E)

(1%

(1:)

(174,

(1:)

$1

(1:)

(E,

(2)

$)

No. pregnant females (no. with vaginal plugs)

117

117

156

152

65

62

62

39

88

94

141

154

210

Total no. live fetuses

OF FOLINIC ACID SUBCUTANEOUSLY ON DAYS 12-l 6 OF PREGNANCY

9.8

11.7

11.1

10.9

10.8

10.3

8.9

6.5

9.8

10.4

10.8

11.8

9.5

Mean no. live fetuses per litter

WITH

THE

(453) 11 (8.6)

(:16)

(1;Yl)

(22974)

(2T5)

(868)

(if,

(lY8)

(788)

(lY9)

(473)

(Y!9)

Total no. deadand resorbed fetuses(%)

TO MICE

(2020) 0

(649.3)

$0) 0

(664.6)

(332.3) 0

(2l.2)

(lltl)

(lZ4)

(233.0)

0

Litter (%)

(344) 0

(X9)

(2%) 0

(i.0)

(2i.5) 0

(22.3,

(Ll)

(23.2)

039)

0

Fetuses(%)

Total no. palatal defects

ANTICONVULSANT DRUGS

?I \o

z 5

l

P

E 5 8

3

;

2 $ g

280

SULLIVAN

AND

MCELHATTON

comparison with the pooled controls is possible. The pooled results from all of the tables (excluding any animals treated also with folinic acid) show that 2 of 82 control litters had abnormal fetuses. This compares with 5 of 46 litters treated with phenol barbital (p = 0.114), 15 of 32 litters treated with phenytoin (p = 0.52 x lo-‘), and 26 of 108 litters treated with primidone (p = 0.19 x 10-3. Thus as judged on a litter basis, the effect of phenobarbital is not significant, but the teratogenic effects of phenytoin and primidone are very highly significant. The three anticonvulsants studied increased the incidence of full-length cleft palate when compared with the spontaneous incidence in this strain of mice. Cleft palate rarely occurs in the control mice, the incidence of cleft palate being only 3 out of 1103 fetuses. However, the incidence of cleft palate in mice treated with phenytoin was 2 to 21% depending on the dose. An increase in the incidence of cleft palate in rats and mice treated with phenytoin has also been reported by other workers (Massey-, 1966; Elshove, 1969; Harbison and Becker, 1969, 1972; Schardein et al., 1973; Marsh and Fraser, 1973). In the phenobarbital-treated groups up to 4% of the fetuses had cleft palate. There have been no other reports of phenobarbital producing cleft palate in animals, although embryolethal effects but not abnormalities have been reported to occur in NNRI mice treated with phenobarbital (Olivecrona, 1964). Other workers have reported skeletal abnormalities (McCall et al., 1963, 1967; Persaud, 1965), and cardiac defects (McCall et al., 1967) in offspring of rats and rabbits treated with phenobarbital. The incidence of cleft palate in the mice treated with primidone in the present experiments ranged up to 15 ‘A and when given in the diet was dose-related. When given by gastric intubation, an incidence of about 10% was seen with all dose levels and presumably a maximum effect is already reached with 100 mg/kg. The highest dose of 250 mg/kg was toxic for the pregnant dams and three died. It is interesting that despite this toxicity the resorption rate in this group was not elevated, suggesting a low level of fetotoxicity as opposed to teratogenicity. Skeletal abnormalities have been reported in the offspring of animals (McCall et al., 1963, 1967; Harbison and Becker, 1969) and in children (Fedrick, 1973; Hill, 1973; Loughnan et al., 1973) whose mothers had been treated with anticonvulsants during pregnancy. Similar skeletal malformations have been observed occasionally in this series of experiments with polydactyly and syndactyly occurring in the phenytoin-and primidone-treated groups. Fetuses with kinked tails were also observed in the primidone-treated groups. Folate, the metabolically active form of which is folinic acid, is an important cofactor for many metabolic processes such as nucleoprotein synthesis, mitosis, and for drug hydroxylation reactions including the metabolism of anticonvulsants. It has been observed that in some patients on anticonvulsant therapy, there is a reduction in serum folate with varying degrees of macrocytic anemia (Reynolds, 1973a, 1973b). As folate is required as a cofactor for the biotransformation of anticonvulsant drugs, a folate deficiency could lead to a decrease in the rate of inactivation of the drug, and thus lead to higher blood concentrations of anticonvulsant and perhaps an increase in toxicity. On the other hand, high serum folate concentrations could lead to rapid inactivation of the drug and a subsequent decrease in fit control. Some anticonvulsant drugs have structural similarities to the folate molecule and it has been suggested that they might competitively block the intestinal absorption of

TERATOLOGY OF THREE ANTIEPILEPTIC DRUGS

281

folate (Hoffbrand and Necheles, 1969) and inhibit folate conjugase. Furthermore, it has been demonstrated that folic acid and its derivatives may themselves have convulsant activity (Spector, 1972; Reynolds, 1973a), and it is possible therefore that folate may in some way contribute to the teratogenic effects observed, and that giving excess folate, may not be entirely harmless. There are many conflicting reports as to the effects of giving folate supplements concurrently with anticonvulsants in animals. In the present series of experiments the action of folate is by no means clearly defined. We have shown that folinic acid can have variable effects on the incidence of drug-induced malformations, in that it potentiated the teratogenic effects of phenytoin (250 mg/kg) administered in the diet, but had no effect on the malformation rate when phenytoin (120 mg/kg) was administered by gastric intubation. When folinic acid was given concurrently with phenobarbital (I 50 mg/kg in diet) it did not reduce the incidence of cleft palate from that produced by the drug alone whereas, there seemed to be some slight improvement when folinic acid was given with primidone (1250 mg/kg in diet). Other workers (Kernis et al., 1973; Marsh and Fraser, 1973) have shown that folic acid provided little or no protection against the production of fetal abnormalities induced by phenytoin. Marsh and Fraser (1973) also demonstrated that while folic acid had no effect, folinic acid reduced the incidence of phenytoin-induced cleft palate. Schardein et al. (1973), on the other hand, showed that with a constant dose of phenytoin and variable doses of folinic acid there was a potentiation of phenytoin-induced teratogenicity. In conclusion, it has been shown that in this strain of mouse, when judged on a per fetus basis, phenobarbital, phenytoin, and primidone are teratogenic, producing cleft palate as the most common defect. When assessed on a pooled litter basis only phenytoin and primidone were teratogenic. Because of variation in results between experiments, the significance of the interaction of folinic acid with primidone and phenytoin is difficult to assess with confidence. However, it cannot be assumed that concurrent administration of folinic acid to patients receiving anticonvulsants could only be beneficial.

REFERENCES ELSHOVE, J. (1969).Cleft palatein the offspring of femalemice treated with phenytoin. Lancef ii, 1074.

ELSHOVE, J. ANDVAN ECK, J. H. M. (1971).Congenitalmalformations,particularly cleft lip with or without cleft palate in children of epileptic mothers. Nederlands Tijdschrift voor Geneeskunde 115, 1371. FEDRICK,J. (1973). Epilepsy and pregnancy: A report from the Oxford Record Linkage Study. Brit. Med. J. 2, 442-448. HARBISON, R. D., ANDBECKER, B. A. (1969).Relation of dosageand time of administration of diphenylhydantoin to its teratogeniceffect in mice. Teratology 2, 305-3 11. HARBISON, R. D., AND BECKER,B. A. (1972). Diphenylhydantoin teratogenicity in rats. Toxicol. Appl. Pharmacol. 22, 193-200. HILL, R. M. (1973).Drugs ingestedby pregnantwomen. Clin. Pharmacol. Ther. 14,654659. HOFFBRAND, A. V., ANDNECHELES, T. F. (1968).Mechanismof folate deficiencyin patientsreceiving phenytoin. Lancet ii, 528-530. JANZ, VON D., AND FUCHS,U. (1964). Sind Antiepileptische Medikamente wahrend der Schwangershaftschadlich?Deutsche Med. Wschr. 89, 241-243. 10

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KERNIS, M. M., PASHAYAN,H. M., AND PRUZANSKY, S. (1973). Dilantin induced teratogenicity and folic acid deficiency. TeratoIogy 7, Al 9-20. LOUGHNAN, P. M., GOLD, H., AND VANCE,J. C. (1973). Phenytoin teratogenicity in man. Lancet, i, 7&72. MARSH,L., ANDFRASER,F. C. (1973).Studieson dilantin-inducedcleft palate in mice. Teratology, 7, A23. MASSEY,K. M. (1966).Teratogeniceffectsof diphenyihydantoin sodium.J. Oral Ther. Pharmacol.2,380-385. MCCOLL,J. D., GLOBUS, M., ANDROBINSON, S. (1963).Drug inducedskeletalmalformations in the rat. Experientia 19, 183-184. MCCOLL,J. D., ROBINSON, S., ANDGLOBUS, M. (1967).Effects of sometherapeuticagentson the rabbit fetus. Toxicol. Appl. Pharmacol.10,244-252. MONSON,R. R., ROSENBERG, L., HARTZ,S. C., SHAPIRO, S., HEINONEN, 0. P., ANDSLONE, D. (1973). Diphenylhydantoin and selectedcongenital malformations.N. Engl. J. Med. 289, 1049-1052. OLIVECRONA, H. (1964).Embryo destroyingdefectsof injected phenobarbital in the mouse. Acta Anat. 58,217-221. PERSAUD, T. V. N. (1965). Tierexperimentelleuntersuchungenfur Frage der Teratogenen wirkung von Barbituraten. Acta Biol. Med. Ger. 4,89-90. REYNOLDS, E. H. (1973a).Anticonvulsants, folic acid and epilepsy.Lancet i, 1376-1378. REYNOLDS, E. H. (1973b).Anticonvulsant drugs, folic acid and vitamin B,, metabolism,and schizophrenia-likepsychosesin epilepsy.Epilepsia(Amst.) 14, 462. 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 inducedteratogenicity in mice. Toxic01Appl. Pharmacol.24, 150-l 58. SPECTOR, R. G. (1972). Influence of folic acid on excitable tissues.Nature New Biol. 240, 247-249. SPEIDEL, B. D., ANDMEADOW,S. R. (1972).Maternal epilepsyand abnormalitiesof the foetus and newborn.Lancet ii, 839-843.