Developmental toxicity study of Aquacoat ECD ethylcellulose aqueous dispersion administered orally to rats.

A developmental rat toxicity study of Aquacoat((R)) ECD was performed as part of a program to evaluate the safety of the product. Groups of 25 presumed-pregnant Charles River Sprague-Dawley CD rats received doses of 0, 903, 2709 and 4515mg/kg/day (dry weight basis) of Aquacoat ECD administered undiluted once daily via oral gavage on days 6-15 of gestation. All surviving dams underwent caesarean sectioning on day 20 of gestation. Foetuses were weighed, sacrificed and subject to external, visceral and skeletal evaluations. No test material-related maternal deaths occurred; one high-dose female died on day 14 due to gavage error. The only treatment-related clinical sign noted among dams receiving 2709mg/kg/day and greater was pale faeces which was attributed to the presence of the test material in the faeces. No statistically significant differences were noted among the measured maternal parameters. Foetal sex ratios and body weights were similar in all groups. The results of external and visceral foetal evaluations revealed no treatment-related alterations. The only statistically significant findings noted during the skeletal evaluation were increased litter incidences of incompletely ossified or wavy ribs noted among foetuses receiving 4515mg/kg/day, and a significant increase in the litter incidence of thickened ribs at doses of 2709 and 4515mg/kg/day. Given the nature of these findings and the lack of effects on any other parameter measured in this study, they were not considered adverse effects of treatment. Under the conditions of this study, the maternal and foetal no-observed-adverse-effect level (NOAEL) is in excess of 4515mg/mg/day.

Subchronic oral toxicity study of Aquacoat ECD ethylcellulose aqueous dispersion in the rat.

Groups of 20 male and 20 female Sprague-Dawley rats were administered undiluted Aquacoat ECD ethylcellulose aqueous dispersion by oral gavage at doses of 903, 2709 or 4515 mg/kg body weight/day (dry weight basis) for 90 days. Control animals received water at the same dosage volume as the high-dose group. Body weights and food consumption were recorded weekly. Blood was collected prior to study termination for haematology and clinical chemistry measurements. Survivors underwent complete necropsies on days 91 94. Selected organs were weighed and histologically examined. The only treatment-related clinical sign observed was pale faeces which was noted among males and females receiving 2709 and 4515 mg/kg/day Aquacoat ECD. No statistically significant differences in body weights, body weight gains, food consumption and organ weights were noted among males and females when compared with controls. No treatment-related effects in haematology parameters were noted. Significantly decreased total protein and globulin levels and increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in male rats receiving 2709 and 4515 mg Aquacoat ECD/kg/day were considered to be treatment related. No gross or microscopic lesions were attributed to Aquacoat ECD treatment. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for female rats is in excess of 4515 mg/kg/day: the NOAEL for male rats is 903 mg/kg/day.

A dietary oncogenicity study of tributyl phosphate in the CD-1 mouse.

Tri-n-butyl phosphate (TBP), an industrial chemical, was administered in the diet at concentrations of 0, 150, 1000 or 3500 ppm to groups of 50 male and 50 female CD-1 mice for 18 months. Survival, clinical signs and hematology parameters were unaffected by treatment at any concentration. Initial weight losses and significant decreases in body weight gain occurred in males and females receiving the high dose (3500 ppm) of TBP in diet. A significant dose-related increase in absolute and relative liver weights was seen in male and female mice which received the two highest dietary concentrations of TBP (1000 and 3500 ppm). The incidence of hepatocellular adenomas was significantly increased in male mice treated with 3500 ppm TBP in diet. No other tumors were associated with TBP administration in this study. The NOEL for chronic toxicity was 150 ppm, or 28.9 mg/kg/day for females and 24.1 mg/kg/day for males. Although rats treated chronically with TBP have exhibited urinary bladder hyperplasia and urinary bladder papillomas and transitional cell carcinomas, no urinary bladder alterations attributed to TBP administration occurred in this study.

Respiratory sensitization to konjac flour in guinea pigs.

Medical reports linking asthma and occupational exposure to airborne powder produced during the manufacture of konjac flour have been reported in the literature. This study was conducted to investigate whether exposure to food grade konjac flour, which is the end product of the manufacturing process, could produce respiratory hypersensitivity using an animal bioassay developed by Karol et al. (Karol, Y., Ioset, H.H., Riley, E.J., Alarie, Y.C., 1978. Am. Ind. Hyg. Assoc. J. 39, 546-556). Groups of guinea pigs were randomly assigned to a negative control, a konjac flour exposure group, or a positive control group exposed to trimellitic anhydride (TMA). The study design included five consecutive days of inhalation induction exposures followed by three inhalation challenge exposures on days 19, 26 and 40. Positive control guinea pigs were exposed to 98 mg/m3 TMA during the induction exposures and 57-67 mg/m3 TMA during the challenge exposures. The mean (+/-S.D.) konjac flour concentration during the induction exposures was 111+/-8.3 mg/m3 and the exposure concentrations of konjac flour during the challenge exposures ranged from 50 to 68 mg/m3. The criteria used to define respiratory tract sensitization (an increase in respiratory rate of 36% and a change in the respiratory waveform) were met by 25% of the animals at each challenge in the konjac flour group. In addition, a few animals responded with slightly lower increases in respiratory frequency and a change in waveform suggestive of a slight pulmonary hypersensitivity response. Guinea pigs that responded during the first challenge also responded during subsequent challenges. No changes in respiratory rate or waveform were noted in animals assigned to the negative control group when challenged with konjac flour. The results of this study indicate that respiratory hypersensitivity to food grade konjac flour can be induced in guinea pigs following repeated inhalation exposure. Therefore, proper engineering controls or personal protection equipment should be utilized to prevent respiratory sensitization in those who may be occupationally exposed to food grade konjac flour.

Qualitative investigation of uptake of fine particle size microcrystalline cellulose following oral administration in rats.

A subchronic toxicity study was conducted to evaluate the potential toxicological effects associated with intestinal translocation of a special fine particle size (median particle size 6 microns) microcrystalline cellulose (MCC). Four groups of Sprague-Dawley rats (20/sex/group) received either 0 (control), 500, 2500 or 5000 mg/kg/day MCC (25% w/v in tap water) daily by oral gavage for 90 d. At study termination, organs and tissues from high-dose and control animals, including multiple sections of intestine with gut-associated lymphoid tissue, were processed for light microscopy with subsequent examination under polarised light for the presence of birefringent MCC particles. None were observed in any tissue examined. No toxicologically significant effects or lesions were found in any other parameter or organ evaluated. The 'no observed adverse effect level' (NOAEL) for toxicological effects was greater than 5000 mg/kg/day MCC, which was the highest dosage tested. These results further verify the safety of commercial MCC products for use in food and pharmaceutical applications.

Basic animal research.

Recent research with animal models of the fetal alcohol syndrome has resulted in advances in the areas of prenatal and postnatal growth retardation, facial dysmorphology, immune system deficiencies, endocrine dysfunction, and central nervous system alterations, including behavior. The rat is the most common species used in these studies, although similar experimental changes have been produced in a limited number of studies in other species. The specific deleterious effects of ethanol on the fetus are dose-related and dependent on the stage of gestation, as with other fetotoxic agents. However, the precise timing and dose-effect curves have yet to be established for most types of ethanol-induced damage. Various exposure regimens have been employed, including inhalation of ethanol vapor and intraperitoneal injection, as well as oral exposure by gavage, diet, or drinking water. In the early postnatal period, following gestational exposure, altered function is most marked, with recovery in some measurements with postnatal maturation. The present literature clearly defines developmental damage from gestational exposure to ethanol as well as areas for additional research.

Acute response of the fetal telencephalon to short-term maternal exposure to ethanol in the rat.

Pregnant rats were exposed to either ethanol (total dose 18 g/kg) on gestational days 14 and 15 or whole-body ionizing radiation (0.5 Gy) on gestational day 15. On gestational day 16, 24 h following the last dose of ethanol or exposure to ionizing radiation, the developing cerebral cortex of the fetus was examined histologically. Ionizing radiation caused extensive cell death within the fetal cerebral cortex whereas ethanol caused more subtle morphological changes such as cortical thinning and petechial intraventricular hemorrhages. These findings suggest that ethanol, unlike ionizing radiation, acts by some mechanism other than cell death to cause cortical thinning and cortical malformations. The pathogenesis of ethanol-induced cortical dysgenesis may include fetal hypoxia and inhibition of neuroblast proliferation within the developing cerebral cortex.

Morphometric analysis of developing rat cerebral cortex following acute prenatal ethanol exposure.

Morphologic alterations of fetal rat cerebral cortex were quantified by morphometric analysis following acute ethanol exposure on Gestational Day 14, a critical period of development of cerebral cortex. Pregnant rats were intubated with a total dose of 9 g/kg of ethanol on Gestational Day 14. Maternal blood ethanol levels ranged from 51 to 202 mg% during the period of ethanol exposure. Fetal brains were examined on Gestational Day 15, 24 h after the last dose of ethanol. The acute morphologic changes associated with ethanol exposure include enlargement of subventricular zone nuclei, cortical swelling, and dilation of pial blood vessels over the affected cortex. In some fetuses, cortical swelling was accompanied by the protrusion into the lateral ventricles of cytoplasmic blebs of ventricular zone cells. It is concluded that maternal ethanol exposure during a critical period of brain development produces measurable morphologic changes in fetal rat cerebral cortex within 24 h after ethanol exposure.

Cerebral cortical morphology and behavior in rats following acute prenatal ethanol exposure.

Cerebral cortical morphology and early motor development were evaluated in offspring from ethanol-exposed mothers, pairfed control mothers and ad libitum control rats. Ethanol-exposed rats received a total dose of 18 g/kg of ethanol by intubation on gestational Days 14 and 15, a critical period of development of the cerebral cortex. Pairfed control mothers received isocaloric sucrose on gestational Days 14 and 15 and were pairfed to ethanol-exposed animals from gestational Day 12 through gestational Day 20. Ethanol-exposed offspring weighed significantly less than control offspring from postnatal Day 7 through postnatal Day 28. Ethanol-exposed offspring also showed significant delays in reflex suspension (time an animal maintained its grip on a crossbar) and continuous corridor (number of turns in 5 min). The thickness of the cerebral cortex of ethanol-exposed offspring was significantly different from ad libitum and pairfed control offspring on postnatal Day 1. However, only Layer V and total cortical thickness were affected in ethanol-exposed offspring on postnatal Day 28. The results of this study indicate that ethanol exposure during a critical period of development causes alterations in central nervous system development and developmental delays.

Prenatal brain malformations following acute ethanol exposure in the rat.

Morphology of the cerebral cortex was studied in fetuses on gestational Day 21 following oral administration of several doses of ethanol (for total doses of 10, 15, or 18 g/kg) to pregnant rats on gestational Days 14 and 15, a critical period for the development of the cerebral cortex. All doses of ethanol were associated with a reduction in maternal weight gain, fetal body weight, and placental weight. Only the high dose of ethanol (total dose 18 g/kg) caused significant fetal cortical thinning. Acute exposure of pregnant rats to ethanol produced dose-dependent malformations of the cerebral cortex and hippocampus in fetuses. On gestational Day 21, the 18 g/kg group contained fetuses with severely disorganized cortical architecture, heterotopias of the cerebral cortex, pia and choroid plexus, and status verrucosus deformis. Fetuses from the 10 g/kg group had less severe malformations, such as disorganization of layers of cortical neurons and dentate granule cells while fetuses from the 15 g/kg group had a mixture of severe and minor malformations. This study demonstrates that acute ethanol exposure during a critical period of development in rats can result in brain malformations similar to those reported in human fetuses and neonates from alcoholic mothers.

3-Hydroxylation of salicylamide in mice.

Salicylamide is an important model compound for use in investigations concerning drug disposition. In this study the metabolic fate of salicylamide at high doses was evaluated in male mice using HPLC methodology. The concentrations of salicylamide and its metabolites were determined in urine and in blood at various times after the administration of 2 or 4 mmol kg-1 salicylamide. Salicylamide, gentisamide, and their glucuronide and sulfate conjugates were detected. 2,3-Dihydroxybenzamide, the 3-hydroxy metabolite of salicylamide, as well as its glucuronide and sulfate conjugates, were identified and quantitated for the first time by HPLC. 2,3-Dihydroxybenzamide had previously been detected only as a minor metabolite of salicylamide by paper chromatography. However, in the present study, 18% of the salicylamide metabolites appearing in urine after either dosage of salicylamide were 3-hydroxylation products. When a previously published HPLC method for salicylamide analysis was used, 2,3-dihydroxybenzamide glucuronide coeluted with salicylamide glucuronide. The possible formation of 3-hydroxy metabolites must be evaluated in any study of drug metabolism using salicylamide as a model compound.