Comparative gavage subchronic toxicity studies of o-chloroaniline and m-chloroaniline in F344 rats and B6C3F1 mice.

ortho-Chloroaniline (o-CA) andmeta-chloroaniline (m-CA) are chemical intermediates for pigment production in the textile industry. Comparative subchronic gavage studies were conducted to determine the effect of structure on toxicity.o-CA orm-CA was administered to 10 animals/sex/species in deionized water at dosages of 0, 10, 20, 40, 80, and 160 mg/kg for 13 weeks. Blood samples for clinical pathology were collected after 3 and 23 days in rats and at study termination (Day 93) in rats and mice. No mortalities occurred that could be directly attributed to treatment. Transient clinical signs of toxicity observed after dosing included cyanosis in rats and ataxia and tremors in mice. Methemoglobin formation was directly related to dosage (rats and mice) and duration of treatment (rats). At study termination, Heinz body formation in erythrocytes in association with decreased hemoglobin, hematocrit, and red blood cell count was a prominent treatment-related effect. Enlarged spleens (gross necropsy observation) and increased spleen weight were treatment effects of each chemical in both species. Microscopic lesions typical of increased red blood cell production were found in hematopoietic tissues (bone marrow, spleen, and liver), while lesions due to increased red cell destruction were found in these tissues and also the kidneys (rats). Microscopic changes were more frequently seen and severe, and involved more body organs, in rats than mice, and in m-CA-treated animals thano-CA-treated animals. Sex differences in lesion incidence/severity were not evident.

Hematological effects in F344 rats and B6C3F1 mice during the 13-week gavage toxicity study of methylene blue trihydrate.

Methylene blue trihydrate is used widely as a dye and therapeutic agent. Methylene blue was administered by gavage to 30 animals/sex/dose group in a 0.5% aqueous methylcellulose suspension at doses of 0, 25, 50, 100, and 200 mg/kg. Blood samples from 10 animals/sex/dose group were collected at the end of study weeks 1, 6, and 13. Methylene blue treatment resulted in methemoglobin formation and oxidative damage to red blood cells, leading to a regenerative anemia and a variety of tissue and biochemical changes secondary to erythrocyte injury. An early change was a dose-related increase in methemoglobin, where the response of rats and mice was similar in magnitude. Mice appeared to be more sensitive than rats to the formation of Heinz bodies and the development of anemia that was characterized by a decrease in hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was apparent in all treated mice and in the 100 mg/kg (males only) and 200 mg/kg rats at necropsy. There was a dose-related increase in absolute and relative spleen weight for both species. Microscopic examination revealed increased splenic hematopoiesis in all mice treatment groups and in rats at the 50 mg/kg dose level and above. Splenic congestion and bone marrow hyperplasia were also observed in these rat-dose groups. Mice at the higher doses showed hematopoiesis in the liver and accumulation of hemosiderin in Kupffer cells. These gross and microscopic findings are consistent with the development of hemolytic anemia. A dose-related increase in the reticulocyte count during study weeks 6 and 13 suggested a compensatory response to anemia.

Comparative absorption of lead from contaminated soil and lead salts by weanling Fischer 344 rats.

A 44-day dosed feed study was performed to compare the bioavailability of lead from contaminated soil versus two lead salts and the effect of soil on gastrointestinal absorption of ingested lead. Male Fischer rats (approximately 4 weeks of age) received lead, 17, 42, or 127 ppm, in the form of lead acetate, lead sulfide, lead-contaminated soil, or combinations thereof in the diet for 7, 15, or 44 days. Control soil was added to the diets of some animals to determine how it might alter lead bioavailability. Blood Delta-aminolevulinic acid dehydratase (Delta-ALAD) and blood, bone, kidney, and liver lead were determined in groups of animals at each time-point. Blood Delta-ALAD was inhibited in a dose-dependent manner and to the greatest degree in the lead acetate and lead acetate/control soil groups, followed by the lead sulfide and lead-contaminated soil groups. Bone and tissue lead levels increased in a dose-dependent manner and were greatest in animals receiving lead acetate and significantly less in animals receiving lead sulfide and lead-contaminated soil. Blood lead levels were generally greatest by 7 days and stabilized at lower levels thereafter. Bone lead concentration-time patterns did not demonstrate the biphasic change seen with tissues and continued to increase in most treatment groups through the course of the study. The presence of soil in the diet clearly attenuated the absorption of lead acetate, but had little effect on the absorption of lead sulfide. Results of these studies confirm previous observations that lead absorption is highly dependent on the form of lead ingested and the matrix in which it is ingested. More important, these studies demonstrate that lead in soil may be significantly less available than estimated by current default assumptions and that the presence of soil may decrease the availability of lead from lead salts on which the default assumptions are based. Results presented here also demonstrate that the weanling rat may represent an appropriate model that could be used to obtain relatively rapid and economical estimates of the availability of lead in complex matrices such as soil.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Carcinogenicity studies of oxazepam in mice.

Oxazepam is a benzodiazepine widely used as a sedative-hypnotic and antianxiety drug. In chronic studies, groups of 60 male and 60 female Swiss-Webster (SW) or B6C3F1 mice received oxazepam in feed at concentrations of 0,2500, or 5000 ppm. Additional groups of 60 male and female B6C3F1 mice received 125 ppm in feed to allow for study of mice with serum concentrations of oxazepam similar to those achieved in humans taking a therapeutic dose. At 57 weeks, treatment-related mortality of exposed SW mice caused the study to be terminated. Enhanced systemic amyloidosis contributing to heart failure was considered the principal cause of death. Hepatocellular adenomas and carcinomas were increased in exposed SW mice. Survival of B6C3F1 mice receiving 2500 and 5000 ppm oxazepam was also lower than that of controls. Early deaths were due to increased incidences of hepatoblastoma and hepatocellular carcinoma, and nearly all mice receiving 2500 or 5000 ppm developed hepatocellular neoplasia. An increase in follicular cell hyperplasia of the thyroid gland occurred in all exposed groups of B6C3F1 mice, and thyroid gland follicular cell adenoma was increased in exposed females. Further studies of the capacity of oxazepam to induce liver cell mitogenesis and an evaluation of the frequency of activated H- and K-ras oncogenes in the liver tumors of B6C3F1 mice has shown that many of the neoplastic and nonneoplastic responses of mice to oxazepam resemble those observed with phenobarbital.

Comparative carcinogenicity of polybrominated biphenyls with or without perinatal exposure in rats and mice.

Chronic toxicity and carcinogenicity studies of a polybrominated biphenyl mixture (PBB) were conducted in F344/N rats and B6C3F1 mice of each sex. The major objective of the study was to determine if exposure to PBB during the perinatal period, in addition to conventional exposure of animals for 2 years, enhances the sensitivity of the bioassay to identify the carcinogenic potential of this chemical. The studies were designed to determine the toxic and carcinogenic effects of dietary PBB in rats and mice receiving (i) perinatal exposure up to 8 weeks of age followed by control diet for 2 years, (ii) exposure for 2 years beginning at the age of 8 weeks, and (iii) combined perinatal/adult exposure to PBB (perinatal exposure to 8 weeks of age followed by adult exposure for 2 years). During the perinatal period, rats were exposed to PBB at dose levels ranging from 1 to 10 ppm and adult exposure concentrations ranged from 3 to 30 ppm in the diet. In the mice, the dose levels ranged from 3 to 30 ppm in both perinatal and adult exposure portions of the chronic studies. A total of eight dose groups (including controls) were used with 60 animals in each group. Liver was the major target organ of PBB toxicity. Perinatal exposure alone (through dietary administration of 10 ppm PBB to the dams) had no effect on the incidences of neoplasms in female F344/N rats, but in male rats, perinatal exposure was associated with a marginally increased incidence of hepatocellular adenomas that may have been related to chemical administration. In male and female B6C3F1 mice, perinatal exposure to 30 ppm PBB resulted in significantly increased incidences of hepatocellular neoplasms. In adult-only dietary exposure studies, PBB was carcinogenic in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of hepatocellular neoplasms. Combined perinatal and adult dietary exposure to PBB confirmed the findings of the adult-only exposures for the increased incidences of hepatocellular neoplasms in rats and mice. In male rats, there were no enhancing effects of combined perinatal and adult exposure. However, perinatal exposure enhanced the susceptibility of female rats receiving adult exposure of 10 or 30 ppm to the induction of liver neoplasms. For male and female rats, a combined analysis of the incidences of leukemia in the adult-only, perinatal-only, and combined perinatal and adult exposure groups revealed an apparent association between increasing incidences of mononuclear cell leukemia and exposure to PBB.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative carcinogenicity of 5,5-diphenylhydantoin with or without perinatal exposure in rats and mice.

Chronic toxicity and carcinogenicity studies of 5,5-diphenylhydantoin (DPH), were conducted in F344/N rats and B6C3F1 mice of each sex. The major objective of the study was to determine if incorporating exposure to DPH during the perinatal period, in addition to conventional exposure of animals for 2 years, enhances the sensitivity of the bioassay to identify the carcinogenic potential of chemical. The studies were designed to determine the toxic and carcinogenic effects of dietary DPH in rats and mice receiving; (1) the perinatal administration including exposure of maternal animals prior to breeding, through gestation, lactation, weaning, and continued dietary exposure of offspring to the age of 8 weeks followed by control diet for 2 years, (2) exposure for 2 years beginning at the age of 8 weeks, and (3) of combined perinatal/adult exposure to DPH (perinatal exposure to 8 weeks of age followed by the adult exposure for 2 years). During the perinatal period, rats were exposed to DPH at dose levels ranging from 63 to 630 ppm and adult exposure concentrations ranged from 240 to 2400 ppm in diet. In the mice, the perinatal exposure ranged from 21 to 210 ppm in both males and females. In the adult exposure portion of the mouse studies, the dietary levels ranged from 30 to 300 ppm in males and 60 to 600 ppm in females. A total of eight dose groups (including controls) were used with 60 animals in each group. The only effect of perinatal exposure alone on tumor rate was a marginal increase in the incidence of hepatocellular neoplasms in female mice. The adult exposure to DPH significantly increased the incidence of hepatocellular neoplasms in female mice. There were also marginal increases in the incidence of liver tumors in male rats exposed to high DPH dietary concentrations during the adult-only regimen. Combined perinatal and adult dietary exposure to 5,5-diphenylhydantoin confirmed the findings for the increased incidences of hepatocellular neoplasms in male rats and female mice, although combined exposure did not enhance these effects. However, in male mice, perinatal and adult exposure resulted in an increase in the incidence of hepatocellular neoplasms that was not seen when dietary exposure was limited to the adult period only.

Comparative carcinogenicity of ethylene thiourea with or without perinatal exposure in rats and mice.

Chronic toxicity and carcinogenicity studies of ethylene thiourea (ETU), 97% pure, were conducted in F344/N rats and B6C3F1 mice of each sex. The major objective of the study was to determine if incorporation of perinatal exposure, in addition to the conventional exposure of young adult animals for 2 years, enhances the sensitivity of the bioassay in identification of the carcinogenic potential of chemicals when compared to the conventional exposure of animals to a chemical for 2 years, usually beginning at the age of 6-8 weeks. The studies were designed to determine (1) the toxic and carcinogenic effects of dietary ETU in rats and mice receiving perinatal exposure up to 8 weeks of age followed by control diet for 2 years, (2) the effects of ETU in rats and mice receiving exposure for 2 years beginning at the age of 8 weeks, and (3) the effects of combined perinatal/adult exposure to ETU (perinatal exposure to 8 weeks of age followed by the adult exposure for 2 years). During the perinatal period, rats were exposed to dietary ETU concentrations ranging from 9 to 90 ppm and adult exposure concentrations ranged from 25 to 250 ppm. In the mice, the perinatal exposure concentrations of ETU in the diet ranged from 33 to 330 ppm, and in the adults the concentrations were 100 to 1000 ppm. A total of eight exposure groups (including controls) were used with 60 animals in each group. Ten animals from each group were killed at Month 9 of the study for interim evaluation. The thyroid gland in rats and mice and the liver in mice were identified as target organs of ETU toxicity at the 9-month interim evaluation. The perinatal only exposure to ETU was not carcinogenic in rats or mice, while adult or perinatal/adult combination exposures to ETU were carcinogenic both in rats and in mice. The thyroid gland was the major site of ETU carcinogenicity both in rats and in mice. The liver and pituitary glands were other major sites of ETU carcinogenicity in mice. The carcinogenic effects of ETU were generally similar by adult and perinatal/adult combination protocols except that the incidences of thyroid tumors were slightly higher in the rats receiving the perinatal/adult combination of ETU exposure in the diet.

Duration and intensity of behavioral change after sublethal exposure to soman in rats.

The behavioral effects produced by acute exposure to sublethal doses of pinacolyl methylphosphonofluoridate (soman) were examined in the Sprague Dawley rat. Two hours after exposure to soman (100-150 micrograms/kg IM), dose-related decreases in spontaneous motor activity (SMA), fore- and hindlimb grip strength, thermal sensitivity, and rectal temperature were observed. In addition, acoustic startle response amplitude decreased, while response latency increased. Soman also depressed the percentage of conditioned avoidance and escape responses and increased response latency. In both the 103 and 116 micrograms/kg dose groups, effects on hindlimb grip strength persisted up to 14 days after exposure, while effects on hot plate response lasted for 7 days. A biphasic change in motor activity was seen in the 103 and 116 mg/kg soman groups: Initial SMA depression during the first 24 hours after exposure was followed by SMA increases which persisted up to 21 days. Animals that showed delayed hyperactivity often exhibited seizures and increased excitability when handled. The results of these studies demonstrate that sublethal doses of soman can cause marked and often long-lasting changes in behavior in the rat.

The pharmacological activity of the fatty acid conjugate 11-palmitoyloxy-delta 9-tetrahydrocannabinol.

A long-retained cannabinoid metabolite has been detected in rat tissue after intravenous administration of delta 9-tetrahydrocannabinol (THC) and has been identified as a fatty acid conjugate of psychoactive 11-hydroxy-delta 9-tetrahydrocannabinol (11-OH-delta 9-THC) and palmitic acid. The objective of these studies was to determine if this compound, 11-palmitoyloxy-delta 9-tetrahydrocannabinol (11-palm-delta 9-THC) is pharmacologically active. Intravenously injected 11-palm-delta 9-THC decreased thermal sensitivity and induced catalepsy in rats, responses similar to those produced by 11-OH-delta 9-THC, but less pronounced and more delayed. To further characterize the response, animals were intracisternally injected with 11-OH-delta 9-THC or 11-palm-delta 9-THC. Catalepsy and decreased thermal sensitivity were seen in the 11-OH-delta 9-THC and 11-palm-delta 9-THC groups, and again, 11-OH-delta 9-THC appeared to be the more potent of the two cannabinoids. In contrast to the intravenous study, 11-palm-delta 9-THC-induced effects were seen soon after treatment and appeared to be fully developed by the first test time (15 min). The intracisternal results suggest that 11-palm-delta 9-THC itself is active; however, since it is known that the fatty acid conjugate is hydrolyzed in vivo to 11-OH-delta 9-THC in the rat, the possibility remains that the effects of 11-palm-delta 9-THC are due to metabolic conversion to 11-OH-delta 9-THC.

Cardiovascular defects in rat embryos cultured on serum from rats chronically exposed to phenytoin.

Headfold-stage rat embryos were cultured for 48 hours on serum from rats chronically exposed to phenytoin for periods as long as from conception until 11 months of age. Serum from phenytoin-exposed rats caused approximately 50% of the cultured rat embryos to develop cardiovascular defects as compared to 12% for controls. These morphological abnormalities included hemorrhaging of blood vessels within the embryo, pericardial edema, and absence of yolk sac circulation. Neither serum glucose nor phosphate levels nor serum osmolality were appreciably affected by phenytoin treatment. However, serum protein concentration was reduced in rats exposed to phenytoin as compared to controls. An absence of the serum protein hemopexin was associated with the reduction in serum protein levels but did not appear to be responsible for the observed cardiovascular defects.

Subchronic toxicity of all-trans-retinoic acid and retinylidene dimedone in Sprague-Dawley rats.

Sprague-Dawley rats received daily oral gavage doses of either 2-retinylidene-5,5-dimethyl-1,3-cyclohexanedione (retinylidene dimedone; 14, 50, 150, or 330 mg/kg) or all-trans-retinoic acid (1, 4, 14, or 50 mg/kg) for 13 weeks. Rats given 50 mg/kg of all-trans-retinoic acid developed numerous long-bone fractures and became moribund during the third week of the study. Those receiving lower dosages survived until scheduled termination, but the 14 mg/kg group showed clear signs of retinoid intoxication including growth depression, anemia, serum alkaline phosphatase elevation, bone fracture, and testicular degeneration. Exposure to retinylidene dimedone did not result in any treatment-related deaths, growth depression, or histopathologic lesions, even at the highest dose, 300 mg/kg. Animals given this dosage exhibited mild anemia, equivocal evidence of bone fracture, but no increase in alkaline phosphatase activity. Retinylidene dimedone appears to be considerably less toxic than all-trans-retinoic acid.

Effects of single and repeated exposures to abate on rat behavior and cholinesterase activity.

Rats were injected i.p. with the organophosphate insecticide ABATE and tested over the next 16 days. Animals given 1000 mg/kg showed impaired performance of a previously conditioned avoidance response 6 days after injection but not 2, 8, 10, or 16 days after injection. No behavioral changes were observed in animals given 316 or 562 mg/kg. A subsequent experiment showed that the avoidance impairment in animals given 1000 mg/kg was accompanied by significant erythrocyte, plasma, and brain cholinesterase activity inhibition and decreased spontaneous motor activity. If administration of the same ABATE dose was distributed over 6 days (167 mg/kg/day), cholinesterase and motor activity depression was still evident but conditioned avoidance performance was unimpaired. The results were interpreted as differential behavioral adaption to repeated injections of ABATE.

Behavioral and biochemical effects of the carbamate insecticide, MOBAM.

Decreases in rat plasma, erythrocyte and brain cholinesterase levels after intraperitoneal injection of 1 to 5 mg/kg of 4-benzothienyl-N-methylcarbamate (MOBAM) were compared with decrements in both spontaneous motor activity and conditioned avoidance performance produced by this compound. Significant effects were observed with all five measured phenomena at dosages producing no obvious clinical signs. In albino rats, a dosage of 2 mg/kg significantly depressed plasma and erythrocyte cholinesterase activity, and decreased motor activity 15 min after injection but only higher dosages (3 and 5 mg/kg) significantly depressed brain cholinesterase activity and avoidance performance. In Long-Evans rats, both brain cholinesterase activity and avoidance performance were significantly reduced by the lower (2 mg/kg) dosage. The avoidance impairments observed after 3 mg/kg could be prevented by prior injection with atropine sulfate. It is suggested that both central and peripheral cholinesterase changes are important in determining the nature of the behavioral effects observed after exposure to this compound.