[Reproductive and developmental toxicity studies of tazobactam/piperacillin or tazobactam (2)--Teratological study in rats with intravenously administration].

Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor and piperacillin (PIPC) is an antibiotics which is used in clinical field widely. The combination of TAZ and PIPC (TAZ/PIPC), which is combined with TAZ and PIPC at rate of 1:4, has been developed because of PIPC is unstable to various beta-lactamases. Teratogenic potential were studied in rats given daily intravenous doses of TAZ/PIPC (625, 1250, 2500 or 3750 mg/kg/day) or TAZ (125, 500 or 3000 mg/kg/day). TAZ/PIPC or TAZ were given from day 7 to day 17 of pregnancy. Total daily doses were administered in two equally divided doses. The study includes postnatal evaluation of the growth and development and reproductive performance of the F1 generation. Maternal deaths occurred in all groups given TAZ/PIPC. The incidence (range of 3 to 6 animals/group) was not dose dependent. Maternal body weight was decreased in rats receiving 3000 mg/kg of TAZ and food consumption was reduced in all drug-treated groups. Slight decreases in fetal body weights were observed at some doses that caused maternal body-weight or food-consumption decreases (2500 or 3750 mg/kg of TAZ/PIPC, 3000 mg/kg of TAZ). But these depressions of fetal body weights were not significant from control data. There were no fetal malformations or variations attributable to the test articles. Postnatal growth and development, behavior and reproductive performance of the F1 generation were not affected by the administration of TAZ/PIPC or TAZ. In conclusion, TAZ/PIPC or TAZ was not teratogenic in the rats. It is seemed that non-observed effect dose levels (NOELs) of TAZ/PIPC and TAZ for dams is less than 625 and 125 mg/kg/day in general toxicity respectively, however, NOELs of TAZ/PIPC is 3750 mg/kg/day or more and that of TAZ is 300 mg/kg/day or more for their offspring under the condition of this study.

[Reproductive and developmental toxicity studies of tazobactam/piperacillin or tazobactam(1)--Fertility and general reproduction study in rats with intraperitoneal administration].

Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor and piperacillin (PIPC) is an antibiotics which is used in clinical field widely. The combination of TAZ and PIPC (TAZ/PIPC), which is combined with TAZ and PIPC at rate of 1:4, has been developed because of PIPC is unstable to various beta-lactamases. Fertility and general reproductive performance were studied in rats given daily intraperitoneal doses of TAZ/PIPC (200, 800 or 1600 mg/kg/day) or TAZ (40, 160 or 640 mg/kg/day). TAZ/PIPC or TAZ were given during premating period (70 days in males and 15 days in females), the pairing period (in males and females) and the gestation and lactation periods (in females). Total daily doses were administered in two equally divided doses. The study includes evaluation of the F1 generation and the F2 generation through weaning. In the TAZ/PIPC, maternal toxicity (decreased food consumption) was observed at 200 mg/kg and above dosage groups. At maternotoxic doses of 800 and 1600 mg/kg groups, increased resorptions, decreased live litter size, and increased fetal variations (reversible changes in ribs) were observed. Reversible delays in ossification of caudal vertebrae were also observed at 1600 mg/kg group. In the TAZ, maternal toxicities were observed at 160 mg/kg group (decreased food consumption) and 640 mg/kg group (decreased body weight gain and food consumption). Furthermore, necropsy (raised and/or colored areas present in the cecum) revealed slight increases at 40 mg/kg and above dosage groups. Slight decreases in implantations and resultant slight decreases in live litter size, reversible delays in renal development, and increased stillbirths were observed at 640 mg/kg group. Postnatal growth and development, behavior and reproductive performance of the F1 generation were not affected by the administration of TAZ/PIPC or TAZ. There were no effects on any of the fetal or pup parameters evaluated in the F2 generation. In conclusion, mating behavior and fertility were not affected by TAZ/PIPC or TAZ in this study. TAZ/PIPC or TAZ caused adverse change in reproductive performance of the F0 generation only at doses that caused maternal toxicity. The F1 and F2 generation were not affected.(ABSTRACT TRUNCATED AT 400 WORDS)

Behavioral evaluations in developmental toxicity testing: MARTA survey results.

During 1991, the Middle Atlantic Reproduction and Teratology Association (MARTA) conducted a survey of laboratories performing behavioral evaluations as part of GLP developmental toxicity studies. This survey was conducted to determine the extent to which an "industry standard" had evolved for behavioral test batteries. The most commonly used developmental parameters were eye opening, pinna unfolding, and sexual maturation (physical landmarks); surface righting, pupil constriction, and nonautomated acoustic startle (reflexive landmarks). Locomotor activity was used by 80% of the laboratories. The majority (76%) of laboratories conducted at least one learning and/or retention evaluation; nearly one-fourth of the laboratories routinely performed two. The most common learning tests were watermaze (primarily a simple two-choice discrimination task) and passive avoidance. Automated startle paradigms (habituation, prepulse modification, and/or startle elicitation) were evaluated by 28% of the laboratories. This survey showed a remarkable similarity in methodology across laboratories and a progressive increase in the number of GLP studies that included behavioral assessments. The results indicate that behavioral tests have become a common component of developmental toxicity assessments of pharmaceuticals.

Critical developmental periods for effects on male rat genitalia induced by finasteride, a 5 alpha-reductase inhibitor.

The conversion of testosterone to 5 alpha-dihydrotestosterone by the enzyme 5 alpha-reductase is inhibited by finasteride. In a study in which maternal dosing with finasteride commenced on Gestational Day 15 and terminated on Postpartum Day 21, there were 13 and 27% decreases in anogenital distance of male pups on Postnatal Day 1 at 0.03 and 3 mg/kg/day, respectively. These decreases were largely reversed by Postnatal Day 22 even though treatment of the dams continued. Treatment at 3 mg/kg/day also resulted in hypospadias with cleft prepuce and a 5-day delay in the separation of the prepuce from the glans penis in those animals without hypospadias. A second study in which 20 mg/kg/day finasteride was administered on successive 2-day periods during late gestation in rats demonstrated that the period of Gestational Days 16 to 17 was the most sensitive (critical period) for finasteride-induced hypospadias, cleft prepuce, decreased anogenital distance, reduced prostate weight, and nipple formation in F1 male offspring. This critical period is just prior to the appearance on Day 18 of gestation of a midline mesenchymal plate between the urogenital sinus and the rectum in normal male fetuses. This midline plate does not appear in finasteride-exposed fetuses destined to have hypospadias as demonstrated in a previous study. Based on these observations, we hypothesize that finasteride causes hypospadias by preventing the formation of the medial mesenchymal plate which is necessary for assisting the movement of the urogenital sinus from the base to the tip of the genital tubercle.

Developmental variation of the diaphragm and liver in Fischer 344 rats.

The nature and frequency of a developmental variation of the diaphragm and liver in Fischer 344 rats are described. Totals of 20, 98 and 55 (25 for caesarean-sectioning and 30 for natural delivery) mated female rats were used for Experiments 1, 2, and 3, respectively. Each rat was intubated (gavage) with either an aqueous suspension of 0.2% METHOCEL, 0.25% methyl cellulose, or distilled water as a single daily dose from days 6 through 15 (inclusive) of gestation. On the 20th day of gestation, a caesarean-section was performed, and the uterine contents of each rat were examined. A gross necropsy was performed on the pups of 30 mated female rats on day 21 postpartum. The visceral examinations conducted on these fetuses and pups included an evaluation of a developmental variation in the diaphragm and liver. The variation consisted of a thin fibrous central tendon of the diaphragm with an area of liver (0.5-3 mm diameter) that protruded within the thin central tendon of the diaphragm. The incidence (mean % of fetuses affected per litter) of the diaphragm/liver developmental variation was 9% and 11% for METHOCEL- and water-treated groups, respectively. A thin central tendon was present in the diaphragm of all fetuses of methyl cellulose-treated dams; these fetuses did not have a raised area of the liver present within the diaphragm's central tendon. However, in a few weaned pups of the Fischer 344 rats in this study, liver protruded within the central tendon of the diaphragm.(ABSTRACT TRUNCATED AT 250 WORDS)

Currently used alternatives to the Chernoff-Kavlock short-term in vivo reproductive toxicity assay.

The Chernoff-Kavlock assay was initially designed to identify developmental toxins within a relatively short time frame, using a mammalian system. Although it has been used for initial prioritization of multiple agents studied concurrently or for dosage-range evaluations, it has not gained wide usage in a commercial setting. As proposed, use of the Chernoff-Kavlock assay in an industrial setting is relatively inefficient, in terms of animal usage and data produced, when compared with available alternative study designs. Only a single dosage level was studied, and the data obtained from the assay do not provide sufficient information to meet the minimum requirements of safety evaluations submitted for regulatory review. For these reasons, other methods of prioritization are generally used. Alternate methods used for initial screening and prioritization at our laboratory are: 1) the hydra developmental toxicity assay; and 2) a dosage-range study in rats. When the results of several pilot and definitive developmental toxicity assays performed in rats were compared, it appeared that the described pilot study served the dual functions of predicting and prioritizing the developmental hazard of the test agent and providing a dosage-range study that identified the appropriate dosages to be tested in the definitive study.

Detection of prenatal effects on learning as a function of differential criteria.

The role of appropriate criteria for detecting prenatal effects on learning was evaluated in Crl:COBS CD (SD)BR rats using a two-choice spatial discrimination escape paradigm. Alcohol was the prenatal treatment, as it has been reported to produce learning deficits in rats. The offspring of dams which consumed either a lab chow diet or isocaloric liquid diets containing 0%, 17.5% or 35% ethanol-derived calories during pregnancy were reared by surrogate control dams and tested for watermaze learning at 20 days postparturition. Learning performance and the required number of trials to the selected criterion were interrelated: (1) four or five consecutive errorless trials were too difficult, as high error rates occurred in all groups, including the control; (2) two consecutive errorless trials, or the number of trials before the first errorless trial, were too simple, as low error rates occurred in all groups, including the high dosage group; and (3) three consecutive errorless trials revealed significant dosage-dependent learning decrements in the alcohol-exposed groups. The results indicate that selection of the appropriate criterion in learning paradigms is critical for valid testing of prenatal effects.

The effect of paternal alcohol consumption on fetal development in mice.

The purpose of this study was to evaluate the effect of chronic paternal alcohol consumption on fetal growth and development in C3H mice. Male mice were pair-fed isocaloric liquid diets containing either 30%, 20%, or 0% ethanol-derived calories, or given free access to lab chow. After four weeks of treatment, all males were allowed to mate with untreated females. No differences were found between the litters of alcohol-treated males and controls in terms of the number of implantation sites, prenatal mortality, fetal weight, sex ratio, or frequency of soft tissue malformations. The results suggest that paternal alcohol consumption does not grossly alter fetal growth and development in C3H mice.

Effects of acute alcohol exposure during selected days of gestation in C3H mice.

This investigation was designed to evaluate the teratogenic potential of a single alcohol exposure on one of nine specific days of gestation using a mouse model. C3H mice were intubated with alcohol on either Day 7, 8, 9, 10, 11, 12, 14, 16, or 18 of pregnancy in a dose of 0.0, 2.5, or 5.0 g/kg. On day 19 of gestation, the fetuses were removed by caesarian section, weighted, and fixed in Bouin's solution for subsequent free-hand sectioning. The results demonstrated that (1) acute prenatal alcohol exposure tended to decrease fetal body weight as the dose of alcohol increased, an effect that was most pronounced following exposure on Day 18 of gestation, and (2) neither the dose of alcohol nor the day of acute exposure significantly influenced implantations, resorptions, dead fetuses, or the incidence of fetal malformations across groups.

Alcohol sensitivity in female mice: effect of ovariectomy.

This study tested the hypothesis that decreased estrogen levels accomplished by removing the ovaries affect the response to acutely administered alcohol in female mice. Sensitivity to alcohol was measured in ovariectomized, sham-operated, and non-surgical control C3H/HEN mice. Each animal received an IP injection of alcohol (3.0 or 4.0 g/kg). Core temperature, fall time, sleep time, and waking blood alcohol levels were the dependent variables. For each of these measures, alcohol sensitivity was found to be a function of the dose of alcohol administered, but not the surgical condition. Additionally, the stage of estrus in control animals was not found to be related to alcohol sensitivity.

Department of Psychology, State University of New York at Albany.

The effects of prenatal alcohol on learning and retention of passive avoidance and discriminated shock escape were examined in offspring of rats who consumed isocaloric liquid diets containing either 35, 17.5 or 0% ethanol derived calories (EDC) or lab chow during pregnancy. Alcohol exposed progeny required more trials to reach criterion during passive avoidance acquisition and had shorter second trail latencies into the shock compartment than did controls. Both these measures were found to be direct functions of prenatal alcohol exposure. No differences between groups were evident during retention testing (1, 3, or 7 days later). During the 25 trial acquisition phase of T-maze escape, alcohol exposed progeny made more errors despite equivalent group performance by the end of training. During retention testing 24 hours later, these offspring again evidenced more errors regardless of whether or not the original contingencies were reversed. Both learning and retention deficits in the T-maze were directly related to the percent EDC consumed by the mother during pregnancy.

Decreased ethanol consumption as a function of pregnancy and lactation in C57BL mice.

The effects of pregnancy and lactation on alcohol consumption were examined in the C57BL mouse. Pregnant mice were given a two-bottle choice between water and 10% w/v alcohol solution from Day 5 of pregnancy, through two weeks of lactation, and an additional four weeks following the removal of nursing pups. Alcohol consumption was expressed in terms of g absolute ethanol per kg body weight and as an alcohol preference ratio (ml alcohol/ml total fluid). Alcohol consumption (g ethanol per kg body weight) fell below control values during pregnancy and lactation but increased to control levels during postlactation. Similarly, alcohol preference ratio was found to decline during pregnancy, and to further decrease during lactation, a trend that was reversed during postlactation. The results support the findings of decreased alcohol consumption during pregnancy in alcoholic as well as nonalcoholic women.

Nose-poking and head-dipping behaviors in rats prenatally exposed to alcohol.

In three experiments pregnant female rats consumed liquid diets containing various amounts of the total calories in the form of ethanol. In the first study, offspring of these females were tested in a nose-poking paradigm. The frequency of this response was found to be a direct function of the level of alcohol exposure in utero. In a second study when nose poking produced the onset of a dim light, animals prenatally exposed to alcohol were again found to poke more often, and this effect was not attenuated by preweanling handling. Finally, the generality of these findings became evident when head dipping rather than nose poking was examined; head-dip frequency was higher in alcohol-exposed offspring, and this effect was independent of stimulus complexity. Additionally, offspring body weights and survival rates following this prenatal alcohol exposure are presented.

Lack of response inhibition in rats prenatally exposed to alcohol.

Offspring of mothers who consumed either 32, 19, 8, or 0% of their daily caloric intake in the form of ethanol during pregnancy were tested for passive avoidance. At 18 days of age, the number of trials to criterion and the within-group variability were direct functions of the amount of ethanol consumed by the mother during pregnancy. At 41--53 days of age, alcohol-treated pups still required more trials to criterion than controls and had faster speeds into the shock compartment on the first trial. When the progeny of mothers consuming either 35, 17, or 0% ethanol-derived calories during pregnancy were compared for conditioned taste aversion to a lithium chloride solution, a linear dose-response function was again evident. Animals in the alcohol-treated groups showed less suppression of drinking than controls. These investigations indicated that the effects of alcohol exposure in utero were manifested in behavioral outcomes involving response inhibition that were not correlated with the more familiar physical symptoms.

Hypnotic susceptibility to various depressants in rats selected for differential ethanol sensitivity.

MA rats, bred for greater motor impairment following subhypnotic doses of ethanol, were found to be more sensitive to the hypnotic effects of phenobarbital and chloral hydrate than were LA rats. In addition, the previously reported finding of a difference between the two lines of rats in duration of loss of righting reflex following a hypnotic dose of ethanol was replicated. The results are discussed in terms of a phenotypic difference in central nervous systems sensitivity to a range of sedative-hypnotics.

Response perseveration in rats exposed to alcohol prenatally.

Pregnant female rats consumed liquid diets containing either 35, 17, or 0% of the total calories as ethanol. Offspring of these females were tested for spontaneous alternation at 21 days of age and for reversal learning in a T-maze shock-escape paradigm at 20--21 days of age. In the spontaneous alternation test, rats exposed to alcohol prenatally took more trials than controls to enter the goal compartment opposite to that initially entered. In the T-maze escape study, alcohol-exposed offspring made more mistakes prior to criteron and more mistakes per trial than controls when the previously incorrect goal was made safe during reversal learning. In both studies linear dose-response functions were found. Furthermore, there was a significant tendency for the within-group variabitliy to increase as the level of prenatal exposure increased, perhaps indicating that the incidence as well as the severity of behavioral dysfunction was dose dependent. The results are interpreted in terms of a delay in the development of a central inhibitory system.

Differential tolerance to pentobarbital in rats bred for differences in alcohol sensitivity.

Two lines of rats bred for differences in motor impairment following alcohol treatment were also found to be differentially affected by sodium pentobarbital in three experiments. The most affected (MA) animals, bred for sensitivity to alcohol, showed a decrement in stabilimeter activity at doses of 8 mg and 16 mg pentobarbital per kg body weight. The least affected (LA) animals, bred for insensivity to alcohol, were affected only by the higher dose, at which the resulting impairment was still less than that of the MA group. This finding was partially replicated in a second study designed to test the possibility of an activating effect of pentobarbital on LA animals at 8 mg/kg. In a final study, MA animals were more likely to lose their righting reflex than LA animals at a dose of 18 mg/kg, and 'slept' longer following this dose. These results indicate that the differential sensitivity shown by these animals is not specific to alcohol, but can be generalized to another depressant.

Effects of initial tolerance on acquired tolerance to alcohol in two selectively bred rat strains.

The extent to which initial sensitivity to alcohol influences acquired tolerance was investigated in two selectively bred rat lines. These two lines have been selectively bred for differences in initial sensitivity to alcohol. The most affected (MA) line shows about a 90% reduction in motor activity following a 1.5 g/kg dose of alcohol compared with only a 40% reduction in least affected (LA) animals. In an initial investigation using stabilimeter activity, MA animals demonstrated increased tolerance following regular alcohol exposure, while LA animals showed little change from their initial levels of tolerance. In a second study following a similar intubation procedure, tolerance was assessed by sleeping time. Both MA and LA rats evidenced increased tolerance to alcohol, although MA animals did not approach the levels of tolerance shown by LA animals. In both studies the amount of initial tolerance was correlated with the degree of acquired tolerance. The potential for using these animals in the study of the underlying mechanisms of tolerance is discussed.