Effect of Administration of Thiopental Sodium to Pregnant Mice Upon The
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Okajimas Fol. anat. jap., 43: 219-226, 1967 Effect of administration of thiopental sodium to pregnant mice upon the development of their offspring By Takashi Tanimura, Yasuo Owaki and Hideo Nishimura Department of Anatomy, Faculty of Medicine, Kyoto University Introduction In recent years, investigations have been carried out on the possible teratogenicity of various drugs on mammalian offspring. It was shown in our laboratory that some psychoactive drugs such as glutethimide (T a k a n o et al., '63) and thiamylal sodium (T a n i- m u r a, '65) manifested teratogenicity upon mouse offspring at fairly high dosages. The embryotoxic effect of barbiturates on human beings has not been established (B a k e r, '61 G i r o u d et al., '62). Since the observations on humans concerning that area are limited and yield only fragmentary information, it seems that thorough investigations on animals in this line are needed. Considering that the results of some animal experiments so far are rather inconsistent, the present study was attempted on mice with thiopental sodium, one of the ultra-short acting barbiturates, which is most widely used as an intravenous anesthetic during various human operations including induced abortions. Materials and Methods The animals used in this experiment are ICR-JCL mice supplied by the Central Laboratories for Experimental Animals in Tokyo. They were fed compressed pellets for mice and rats manufactured by Funabashi Farm (Funabashi City, Chiba Prefecture). Water was available ad libitum by bottle. The mice were kept in an air- conditioned room at 20° to 24°C. Nonparous females of 13 to 18 weeks of age were mated all night (16 hours). The mice with a copulatory vaginal plug were 219 220T. Tanimura,Y. Owakiand H. Nishimura isolated the next morning and that day was designated as zero day of gestation. On day 11, those females with evident signs of pregnancy on their vaginal smear were taken and injected intra- peritoneally with an aqueous solution of thiopental sodium (Ravonal, Tanabe Seiyaku Co., Osaka) at the dosage of 25 to 150 mg/kg. The same volume, 10 ml/kg was used for injection of each dose. Control mice were injected similarly with 10 ml/kg of distilled water. The body weight and daily food intake of the pregnant mice were measured from day 10 up to day 18. All the mice were autopsied on day 18. Implantation sites and the number of live and dead fetuses were counted on pregnant cases. Live fetuses were weighed and examined for external gross ma!- formations including those of the oral cavity. Then, they were fixed with 95% alcohol and the cleared specimens dyed with alizarin red S were prepared by Da w so n's method ('27) for examining skeletal defects and states of ossification. Results Pharmacological effects found in the mice, which received thio- pental sodium on day 11 are summarized in Table 1. All the animals injected at 150 mg/kg fell into deeper narcosis and died usually within two hours. In the group treated at 75 mg/kg, all showed deep sleep for several hours. In the group injected at 50 mg/kg, most of the animals showed a moderate narcotic state from 1 to 3 hours, which is similar to the human therapeutic level, but some failed to sleep and righting reflex was still positive. Maternal body weight on day 12 in the groups treated at 75 or Table 1. Pharmacologicaleffect of a single intraperitonealinjection of thiopentalsodium to pregnant mice on day 11 Effect of administration of thiopental sodium to pregnant mice221 100 mg/kg was slightly decreased compared with that on the previous day (Fig. 1). Food intake in those groups was severely lowered during the 24 hours after injection, but it returned to the control level after three days (Fig. 2). The groups treated at the lower dosages showed almost no loss of body weight nor reduction of food intake. Main findings on the fetuses are summarized in Table 2. In two mice in the group treated at 100 mg/kg, no sign of implan- tation was detected at autopsy, though they were confirmed pregnant on day 11. Thus, they are regarded as having miscarriages. Increase of fetal mortality was shown in the groups treated at 75 or 100 mg/kg. Average body weight in the groups treated at and over 50 mg/kg was significantly lower than that in the control. No externally malformed fetuses were noted in any of the treated as well as the control group. The findings on skeletal abnormalities of the cleared specimens are listed in Table 3. A few cases with fused ribs were found in 222 T. Tanimura, Y. Owaki and H. Nishimura Effect of administration of thiopental sodium to pregnant mice 223 some groups, but the incidence in any treated group did not differ from that in the control. No modification in skeletal variations such as dyssymphysis of axis and cervical rib was observed in the treated group. Ossification of mid-phalanges in the forefoot was slightly inhibited in the group treated at 50 to 100 mg/kg, but the one treated at 25 mg/kg did not show any retardation of ossification as compared to the control. Table .3. Effect of a single intraperitoneal injection of thiopental sodium to pregnant mice on day 11 upon the skeletal development of their offspring Discussion It is presumed from our present data, that the effective narcotic dose on pregnant mice lies between 50 and 75 mg/kg, and this dose is about ten times higher than the human anesthetic dose (usually 6 mg/kg). The results show that this effective dose induces a slight growth supression and fetal lethality but no teratogenic effect on the external form and the skeleton. With respect to embryotoxicity , the dose effect relationship was presented. Lethal effects of barbi- turates on the offspring of experimental animals have been noted by several investigators (D ill e, '37 ; Beck e r et al ., '63 ; Se t a l et al., '64 ; T a n i m u r a, '65). P e r s a u d ('65) injected thiopental , hexobarbital or barbital sodium intraperitoneally to pregnant rats once at various gestational stages and found no teratogenic effect on the fetuses. McColl et al. ('63) fed Sprague-Dawley rats a diet containing phenobarbital (0.16%) from three days prior to 224T. Tanimura,Y. Owakiand H. Nishimura mating up to delivery and found that many skeletal abnormalities such as double vertebral centra were induced in their offspring. Oliver crona ('64) injected phenobarbital intraperitoneally to preg- nant NMRI mice at mid-term at 1 to 4 mg/kg and found no teratogenic effect on the fetuses. Setala et al. ('64) administered pentobarbital sodium to pregnant RA mice on days 1,2,3,4, and 1 to 15 at the dosage of 1.6 mg/mouse (approximately 50 to 60 mg/kg), which caused a transitory narcosis in the mothers, and found various types of malformations such as absence of limbs in the newborns. T a n m u r a ('65) injected Japanese colony bred stock dd mice intraperi- toneally with thiamylal sodium at a dosage of 60 mg/kg once on each of days 7 to 14. A slight teratogenicity was shown from the treatment on day 10 but not any other day. Administration of 60 mg/kg caused a moderate narcosis with duration of 1 to 3 hours in dd mice, which is similar to the effect induced by the treatment at 50 mg/kg in the present study with thiopental. The findings mentioned above indicate that a slight intraperitoneal injection of a barbiturate to pregnant rats or mice hardly induces malformation in the offspring. Placental transfer of thiopental sodium in pregnant mice was studied in our laboratory and it was shown that this compound passed through the placenta on day 11 of gestation to embryos in a few minutes and persisted in those cases for more than 60 minutes (T a n i m u r a et al., in press). Placental transfer thiopental near term was established by Flowers ('63) in rabbits and guinea pigs. Sever- al other barbiturates are known to pass through the placenta during the late fetal stage in humans as well as in animals. Studies in the above mentioned investigations coincide with ours. S etal a ('63) noted sodium pentobarbital was an extremely potent mitotic poison to the normal epidermis of RA mice. T a n m u r a (in press) observed the inhibitory effect of thiamylal sodium on mitotic activity of mouse corneal epithelium. Subsequently, it seems plausible to assume that the embryotoxic effect of thiopental sodium shown in the present experiment is due to the direct action of the drug on the embryonic organs,which results in inhibition of mitosis. But a possibility remains that the decrease of food intake of the mother mice contributed to the embryotoxicity to some extent, since T a k a n o et al. ('63) demonstrated that a limited food intake in mice on days 10 to 15 increased fetal mortality though did not induce malformations. The problem should be clarified by further investigations. Effect of administration of thiopental sodium to pregnant mice 225 Summary The effect of thiopental sodium, one of the ultra-short acting barbiturates, upon the embryonic development of ICR-JCL mice was investigated. They were intraperitoneally injected at various dosages of from 25 to 150 mg/kg on day 11 of gestation. A moderate narcotic effect was shown by the treatment at 50 to 75 mg/kg, while all the mice died from the injection at 150 mg/kg.