Terminology of developmental abnormalities in common laboratory mammals (version 1).

This paper presents the first version of an internationally-developed glossary of terms for structural developmental abnormalities in common laboratory animals. The glossary is put forward by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology, and represents considerable progress toward harmonization of terminology in this area. The purpose of this effort is to provide a common vocabulary that will reduce confusion and ambiguity in the description of developmental effects, particularly in submissions to regulatory agencies worldwide. The glossary contains a primary term or phrase, a definition of the abnormality, and notes, where appropriate. Selected synonyms or related terms, which reflect a similar or closely related concept, are noted. Nonpreferred terms are indicated where their usage may be incorrect. Modifying terms used repeatedly in the glossary (e.g., absent, branched) are listed and defined separately, instead of repeating their definitions for each observation. Syndrome names are generally excluded from the glossary, but are listed separately in an appendix. The glossary is organized into broad sections for external, visceral, and skeletal observations, then subdivided into regions, structures, or organs in a general overall head to tail sequence. Numbering is sequential, and not in any regional or hierarchical order. Uses and misuses of the glossary are discussed. Comments, questions, suggestions, and additions from practitioners in the field of developmental toxicology are welcomed on the organization of the glossary as well as on the specific terms and definitions. Updates of the glossary are planned based on the comments received.

Use of cyclophosphamide as a positive control in dominant lethal and micronucleus assays.

Analyses of dominant lethal (DL) mutations and micronuclei (MN) are 2 important and widely used genotoxicity assays to measure drug-induced chromosome damage in germ cells and somatic cells, respectively. Cyclophosphamide (CP) has been widely used as a positive control in the single-dose mouse MN assay; however, its utility as a positive control for the DL assay has not been fully studied. In the present study, CP was tested in both assays under similar experimental conditions and MN seen in somatic tissue (bone marrow) were correlated with DL mutations seen in germinal tissue. In a dose-range finding study, groups of 5 male mice were dosed i.p. daily for 5 days at 0, 30 or 40 mg/kg CP and bone marrow was harvested 24 h later for MN assay. CP induced a dose-related increase (7- and 11-fold over control at 30 and 40 mg/kg) in micronucleated polychromatic erythrocytes (MNPCEs) and decreased %PCEs (to 60% and 54% of controls at 30 and 40 mg/kg, respectively). Based on this, a definitive DL and MN study was conducted using separate groups of 30 male mice at 0 and 40 mg/kg CP with a daily times 5 dosing regimen. For the MN assay, bone marrow was collected 24 h after the last dose from 5 animals and evaluated for MNPCEs and %PCEs. For the DL assay, each male was caged with 2 untreated females per week for 8 weeks to cover the postmeiotic germ cell stages. On day 17 after the initiation of breeding, the females were evaluated for the number of implantation sites and live, dead and resorbed implants. The results indicated that CP induced about a 17-fold increase in MNPCEs and a 46% decrease in PCEs in relation to controls. In the DL assay, CP produced a slight (13%) but statistically significant reduction in fertility index at week 7 of mating. Also, the total number of implants was significantly lower during weeks 1, 2, 3, 6 and 7 and the numbers of dead implants and postimplantation loss (PIL) were increased for weeks 1, 2 and 3 (55%, 71% and 34% PIL, respectively) over controls. These data clearly show that CP produced clastogenicity and some toxicity in both somatic tissue and germinal tissue. It was concluded that a dose of 40 mg/kg CP can be used as a positive control compound in the DL assay and in the multiple-dose marrow MN assay.

Developmental neurotoxicity of CI-943: a novel antipsychotic.

Offspring from Sprague-Dawley rats administered 0, 10, 25, or 75 mg/kg/day CI-943 in the diet prior to mating and throughout gestation and lactation (fertility study) or during the last week of gestation and throughout lactation (perinatal/postnatal study) were evaluated for developmental neurotoxicity using a screen of behavioral tests designed to evaluate rotorod performance, motor activity, acoustic startle responding, and learning and memory via a two-way shuttle avoidance paradigm. Treatment-related effects were evident for each behavioral parameter; they occurred at parentally toxic and nontoxic doses and in the absence of detrimental effects on offspring growth and development. Behavioral effects were in general more robust and occurred at lower doses in the perinatal-postnatal study than in the fertility study. Vertical movement was the most sensitive motor activity parameter in each study; decreases of the greatest magnitude occurred during the first minute of testing, and in males more often than in females. Acoustic startle responding and learning and memory were diminished in each study; these effects were in general concomitant with diminished motor activity, although the pattern of response differed for each study. These results indicate that behavior of offspring from parents administered CI-943 was altered regardless of the developmental stage of exposure, although the pattern of response was dependent on exposure regimen.

Developmental toxicity studies in mice, rats, and rabbits with the anticonvulsant gabapentin.

The developmental toxicity of the anticonvulsant agent gabapentin was evaluated in mice, rats, and rabbits treated by gavage throughout organogenesis. Mice received 500, 1000, or 3000 mg/kg on gestation days (GD) 6-15 and rats and rabbits received 60, 300, or 1500 mg/kg on GD 6-15 (rats) or 6-18 (rabbits). Additional groups received an equivalent volume of the vehicle, 0.8% methylcellulose, or remained untreated. All dams were observed daily for clinical signs of toxicity. In mice, body weights and food consumption were recorded on GD 0, 6, 12, 15, and 18 while in rats and rabbits these parameters were evaluated daily. Near term (mouse, GD 18; rat, GD 20; and rabbit, GD 29) each female was euthanatized, necropsies were performed, and litter and fetal data were collected. Live fetuses were examined for external, visceral, and skeletal variations and malformations. No adverse maternal or fetal effects were observed in mice or rats given doses up to 1500 or 3000 mg/kg, respectively. No treatment-related maternal or fetal effects were apparent in rabbits given 60 or 300 mg/kg. At 1500 mg/kg, one rabbit died, four others aborted, and reduced food consumption and body weight gain were observed. No other reproductive, litter, or fetal parameters were affected, except that the incidence of visceral variations in rat fetuses was slightly but statistically significantly increased at 1500 mg/kg due to a slight increase in the incidence of dilated renal pelvis. This finding was not considered biologically significant because this degree of variability has been seen in this strain of rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of sustained low-level muscarinic agonism in rats.

Sustained, low level muscarinic activity was induced in rats by feeding the muscarinic agonist and experimental drug candidate CI-969 at 50, 100 and 200 mg/kg body weight/day for 4 wk. Except for urine staining, clinical signs typical of acute high-dose exposure to muscarinic agonists were not observed. A dose-related suppression of body weight gain approached 60% at the high dose, but no significant effects on haematology or clinical chemical parameters were observed after 4 wk of exposure. Corneal opacities with histopathological features including neovascularization, acanthosis and stromal proliferation were observed in a dose-related fashion in both sexes at 100 and 200 mg/kg/day. Hypertrophy of the Harderian and lacrimal glands also occurred, probably as an adaptive response to sustained muscarinic activity. Lacrimal gland concentrations of the muscarinic agonist were in the range of pmol/mg tissue and therefore significant direct exposure of cornea to the compound through the tears was discounted. The presence of corneal muscarinic receptors was investigated to determine whether opacities could be related to direct, receptor-mediated events in the cornea; however, no specific binding of the muscarinic receptor radioligand [3H]quinuclidylbenzilate was detected. Because muscarinic agonist-induced opacities can be inhibited by scopolamine, the apparent lack of muscarinic receptors in the cornea indicates that the opacities are not a direct effect, but are instead secondary to muscarinic events at another site. To our knowledge, this is the first report of corneal opacities induced by a muscarinic agonist.

Food restriction during organogenesis in rabbits: effects on reproduction and the offspring.

To assess the effects of markedly restricted food intake versus ad libitum feeding or a slightly restricted feeding regimen during the period of organogenesis we fed groups of 16-18 pregnant rabbits Purina Certified High Fiber Chow ad libitum, 150 g/day, 75 g/day, or 15 g/day on Gestation Days 6 to 18 inclusive. Prior to and after organogenesis the animals were provided food ad libitum (ad lib). Clinical observations, body weights, and food and water consumption were recorded daily. On Gestation Day 30 each doe was euthanatized and necropsied, and maternal and fetal data were collected. Each fetus was examined for external, visceral, and skeletal variations and malformations. Ossification parameters were also evaluated. Statistical analyses were conducted in two ways, first comparing the restricted groups to the ad lib group and second comparing the 15 and 75 g/day groups to the 150 g/day group. During Days 6-18, the 15 and 75 g/day groups had significantly decreased weight gain (actual weight loss), compared to the groups fed 150 g/day or ad lib. Water consumption was also significantly decreased in the 15 g/day group during this period, compared to the ad lib group. When food was provided ad lib on Days 19-30 to the restricted groups, weight gain was significantly higher in the 15 and 75 g/day groups than the group previously given 150 g/day and the ad lib group. There were no differences in water consumption during that period. Abortion occurred in three 15 g/day animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental studies on reproduction with the lipid-regulating agent gemfibrozil.

Gemfibrozil, a new lipid-regulating agent, was evaluated in rats and rabbits for effects on various phases of the reproduction process. In teratology studies groups of pregnant rats and rabbits received gemfibrozil at doses up to 200 mg/kg during organogenesis (rat, Days 6-15; rabbit, days 6-18). For peri- and postnatal studies, groups of pregnant rats were given 92 or 331 mg/kg from Day 15 of gestation through weaning. In fertility studies groups of sexually mature male rats were given 93 or 326 mg/kg of gemfibrozil for 61 days and females were given 94 or 318 mg/kg for 15 days prior to mating within treatment groups. Drug administration continued in females through gestation and weaning of the F1 offspring. In subsequent fertility experiments, treated male rats were mated with untreated females and treated females were cohabitated with untreated males. Gemfibrozil did not elicit a teratogenic response in either rats or rabbits up to doses that resulted in maternal toxicity. Reduced pup weights during the neonatal and weaning periods in the female fertility study as well as in the perinatal-postnatal study were the only apparent drug effect. Treatment of female rats prior to mating had no significant effects on general reproductive parameters. Male rats given doses of about 300 mg/kg/day showed inconsistent and equivocal lower rates of fertility relative to the concurrent controls. No adverse effects were seen in the reproductive performance of offspring of gemfibrozil-treated male rats.

Teratology studies of ametantrone acetate in rats and rabbits.

Ametantrone acetate is an antineoplastic drug chemically described as 1,4-bis [[2-[(2-hydroxyethyl)-amino] ethyl]amino] -9,10-anthracenedione diacetate salt. The drug has activity against leukemia and solid tumors in animal models. The purpose of this study was to investigate the teratogenic potential in pregnant rats and rabbits when administered during the critical period of organogenesis. Daily doses of 1.5, 3.0, and 6.0 mk/kg were administered IP to pregnant rats on days 6 through 15 of gestation, and 0.2, 0.4, and 0.8 mg/kg to rabbits on days 6 through 18. Dose-related weight loss occurred in both species during treatment as well as in the entire gestation period. Maternal and fetal parameters were evaluated upon uterotomies in rats on gestation day 20 and rabbits on day 28. In both species, there was dose-related blue discoloration of abdominal viscerae and of skin at injection sites. In rats, fetal malformations and developmental variations were comparable between treated and control fetuses. However, the incidence of fetal malformations was increased in rabbits given 0.4 and 0.8 mg/kg but not at 0.2 mg/kg. Based on these data, ametantrone was considered teratogenic at dose levels of 0.4 mg/kg and above in rabbits.

Teratogenesis of calcium valproate in rabbits.

The calcium salt of valproic acid (Valontin) has been proposed for use in the treatment of absence, myoclonic, and tonic clonic seizures of the primarily generalized type. The present study was conducted to determine the teratogenic potential of calcium valproate in rabbits. Groups of 20 Dutch-belted rabbits were given oral doses of 50, 150, or 350 mg/kg on days 6-18 of gestation. A reference group was given 350 mg/kg sodium valproate and control groups were untreated or given vehicle alone. Animals were observed daily and body weights were recorded on gestation days 0, 6, 13, 18, and 30. Litter and fetal parameters were evaluated following uterotomies on day 30. No drug-related clinical signs or deaths occurred. Postimplantation loss and the incidence of malformed vertebrae and ribs, rudimentary or absent pollices, and extra vertebrae and ribs were increased at 350 mg/kg with both calcium and sodium salts of valproic acid. At the 150-mg/kg dose level, calcium valproate markedly increased the incidence of supernumerary ribs. No teratogenic or embryotoxic effects were seen with calcium valproate at 50 mg/kg. These data indicate that the sodium and calcium salts of valproic acid exhibit teratogenic potential in rabbits.

Teratology study in rats with amsacrine, an antineoplastic agent.

Amsacrine, an acridinylamino derivative used in the treatment of refractory leukemias, was evaluated for its teratogenic potential in pregnant rats. The compound was given by intraperitoneal (ip) administration on Days 6 to 9 of gestation to groups of 20 female CD rats at levels of 0.5, 1.0, and 2.0 mg/kg. Appropriate vehicle and untreated controls were included. Dams given 2.0 mg/kg lost weight during and after the treatment period. Food consumption was comparable to controls at all dose levels except for the high dose group in the post-treatment period. Decreased litter size, increased postimplantation loss, and reduced fetal weights occurred with doses of 2.0 mg/kg. Significantly reduced fetal body weight and increased incidence of stunting were the only adverse findings at 0.5 and 1.0 mg/kg, respectively. Two fetuses at 2.0 mg/kg, one at 1.0 mg/kg, one at 0.5 mg/kg, and two vehicle control fetuses had gross abnormalities. Fetotoxicity, manifested by inhibition of osteogenesis and minor skeletal abnormalities, occurred with doses of 0.5 mg/kg or more. The results indicate that amsacrine was embryolethal to rats at doses of 2.0 mg/kg and embryotoxic at lower dose levels. Teratogenicity was not evident at doses which did not affect fetal survival.

Studies on reproduction in rats with pirmenol, an antiarrhythmic agent.

Fertility and perinatal-postnatal studies were performed in CD rats given pirmenol, an antiarrhythmic agent, at dosages of 0, 25, 50, and 100 mg/kg. The drug was administered orally as diet admixtures in all studies. In the male fertility study, mature male rats were treated for 61 days prior to mating with virgin, untreated female rats. In the female fertility study, mature virgin female rats were treated for 15 days prior to mating with untreated partners with treatment continuing throughout mating, pregnancy, parturition, and weaning of the litters. In both studies, one-half of the dams in each group were killed on Day 21 of pregnancy and the remaining dams were allowed to deliver and wean their offspring and postnatal development was monitored. At weaning, two males and two females were arbitrarily selected from each litter, allowed to mature on unmedicated diet, and then mated within treatment groups to produce the F2 generation. In the perinatal-postnatal study, pregnant females were treated continuously from Day 15 of pregnancy until weaning of the litters on Day 21 postbirth. No adverse effects on fertility, general reproductive parameters, or offspring survival and development were evident at doses employed in these studies.

Preclinical toxicology studies with the lipid-regulating agent gemcadiol.

Gemcadiol is a medium-length diol moiety with lipid-regulating properties in animals and man. The compound was not toxic when single doses were administered to rodents with the lethal dose greater than 7000 mg/kg in rats and mice. Rats treated for 13 or 52 weeks with 30 to 300 mg/kg had reversible food intake suppression and weight gain inhibition, decreased blood cholesterol, slight anemia, and generally dose-related but reversible decreases in glucose, and increases in alkaline phosphatase and blood urea nitrogen. Liver weights were increased, and there was accompanying hypertrophy and increased cytoplasmic eosinophilia of hepatocytes with associated peroxisome proliferation. Rats treated for 52 weeks also had mild renal tubular dilatation. Dogs given 25 to 300 mg/kg of gemcadiol for up to 52 weeks tolerated the compound better than rats. Effects related to compound administration were elevated serum alanine aminotransferase activity in female animals only, and microscopic cytoplasmic vacuolation and hyaline body formation in both sexes. Monkeys given 25 to 300 mg/kg gemcadiol for 13 weeks had slightly decreased serum cholesterol and slightly increased serum creatine phosphokinase. Teratology studies in rats or rabbits indicated no teratogenic response. Gemcadiol affects principally the liver, and the hepatic alterations seen in rats and dogs may reflect compensatory manifestations of altered metabolism related to the lipid-regulating activity of the compound.

Studies on reproduction in rats with meclofenamate sodium, a nonsteroidal anti-inflammatory agent.

Reproduction and teratology studies were performed in rats given meclofenamate sodium, a nonsteroidal anti-inflammatory agent. Dosages of 0, 3, 6, and 9 mg/kg were administered orally as dietary admixtures in the Fertility and Perinatal-Postnatal studies. In the Teratology study, dosages of 10, 12, 15, and 20 mg/kg were administered by intragastric intubation. In the Male-Fertility study no adverse effects on fertility or litter and offspring parameters were observed in two generations. In the Female-Fertility and Perinatal-Postnatal studies, maternal toxicity (death associated with intestinal ulceration and adhesions) was particularly evident during lactation. Prolonged gestation periods, decreased weanling weights, and increased weanling mortality were evident at dosages of 6 and 9 mg/kg. Increased postimplantation loss occurred at 6 and 9 mg/kg in the Term Sacrifice subgroup of the Female-Fertility study. Fertility rates were unaffected and all other litter and offspring parameters of the F1 and F2 generations appeared normal. In the Teratology study no adverse effects on embryonic or fetal development were evident at maternally toxic dosages up to 20 mg/kg.

Teratology study with the synthetic prostaglandin ONO-802 given intravaginally to rabbits.

ONO-802, a synthetic E1 prostaglandin, was administered intravaginally via pessaries to Dutch belted rabbits at doses of 250, 62.5, and 12.5 micrograms/kg on days 6 through 18 of gestation. Rabbits in a vehicle control group were treated with pessaries that did not contain ONO-802 during the same period. Another group of animals remained untreated throughout gestation. Necropsies were performed on rabbits found dead and on those killed on gestation day 30. Body weight, food and water consumption, and clinical signs were monitored during the experiment. Major organs were weighed when the dams were necropsied on gestation day 30, and litter and fetal data were collected. Abortion and maternal deaths occurred in drug-treated groups. Body weight gains and food and water consumption were adversely affected by treatment particularly at the 250 and 12.5 micrograms/kg dose levels. Wastage (postimplantation loss) was significantly increased among treated groups (all dose levels), while other litter and fetal parameters were unaffected. ONO-802 was not teratogenic at maternal and embryotoxic dose levels.

Two-phase teratology study with the synthetic prostaglandin ONO-802 given intravaginally to rats.

The synthetic prostaglandin ONO-802 was administered intravaginally to Sprague Dawley rats at doses of 1.0, 0.5, and 0.125 mg/kg on days 6 through 15 of gestation. A vehicle control group was treated with pessaries that did not contain the drug while another group remained untreated. Body weight, food, water consumption, and clinical signs were monitored during the experiment. In Phase One, 20 pregnant animals from each group were sacrificed at term, major organs were weighted, and litter and fetal data were collected. In Phase Two ten dams per group were allowed to deliver their litters, and the offspring were evaluated for survival, growth, developmental signs, and physiological function. Selected F1 offspring were retained to assess learning and emotional behavior or reproductive capacity. Administration of either 0.5 or 1.0 mg/kg of ONO-802 resulted in a slight reduction in food consumption and body weight gain. Water consumption was increased both during and after the dosing period for the mid and high dose dams. Significantly increased weights for the heart, lungs, liver, adrenals, and ovaries and decreased weights for the thymus gland were noted at term sacrifice of the 1.0 mg/kg dams, whereas the 0.5 mg/kg group had increased weights of the adrenals and ovaries only. Litter parameters were unaffected by treatment. Weights of the female fetuses of the 1.0 and 0.5 mg/kg groups were significantly reduced when compared to controls. There were no significant drug-related abnormalities among the F1 offspring and no evidence that treatment of the F0 dams affected the development, behavior, or reproductive performance of the F1 offspring. Thus, ONO-802 was not teratogenic when given to rats by the intravaginal route.

Teratogenesis of calcium valproate in rats.

Studies were conducted to determine the teratogenic potential of the calcium salt of valproic acid in rats when given orally at doses of 600, 150, and 50 mg/kg on days 6--15 of gestation. The sodium salt of valproic acid was used as a reference agent at a dose level of 600 mg/kg. The administration of 600 mg/kg/day of either calcium or sodium valproate resulted in transient, severe sedation in the dams. Four dams receiving 600 mg/kg of either salt died during the experiment, with deaths occurring between day 7 and 11 of gestation. Food consumption and body weight gain were significantly reduced during the dosing period with both salts at dose levels of 600 mg/kg. Embryotoxicity at the high doses (600 mg/kg) with either salt was manifested by increases in fetal resorption, reduced body weights, and significantly increased incidence of supernumerary ribs and bifid vertebral centra among the surviving fetuses. A teratogenic effect was evident at 600 mg/kg with either salt of valproic acid. Seven of 16 fetuses from dams given the calcium salt were abnormal. Findings included one with omphalocele and six others with skeletal malformations. Eleven of 24 fetuses from dams given the sodium salt were abnormal: three littermates had bilateral ectrodactyly of the rear feet and malformed vertebral centra and eight others had skeletal malformations. No teratogenic effect was evident among the fetuses from dams given 150 mg/kg calcium salt. Embryotoxicity was demonstrated by a significant increase in the incidence of supernumerary ribs. No adverse effect was observed among the fetuses from dams given 50 mg/kg of the calcium salt.

Cannibalistic traits observed in rats treated with a teratogen.

Rat offspring malformed as a result of maternal exposure to a teratogen were cannibalized preferentially to apparently normal offspring. Lack of viability also appeared to be a factor in the cannibalistic tendency, since both normal and malformed dead pups were consumed more frequently than viable pups of either category. Of the 29 cannibalized pups observed, 86% were cannibalized in the 1st 24 h. The cause of this behavior is not known with certainty but maternal hormone imbalance may play an important role.

The effect of vidarabine on the development of the offspring of rats, rabbits, and monkeys.

The effect of vidarabine, a new antiviral agent, on the offspring of rats, rabbits, and monkeys was studied by varying routes of administration during several periods of gestation. Vidarabine demonstrated a dose-related teratogenic effect in rats when given parenterally at doses of 30 mg/kg and greater. The drug was also teratogenic in the rabbit at dosages of 5 mg/kg and greater by the parenteral route or when applied topically in 10% concentration to 5 or 10% of the body surface area. The pattern of malformation was similar in the two species, and consisted of multiple, severe abnormalities of the head, trunk, and limbs. The drug had no demonstrable teratogenic effect in a limited study in the rhesus monkey; nor were there adverse effects on the offspring when it was applied intravaginally to pregnant rats in the perinatal period.