Cylindrospermopsin toxicity in mice following a 90-d oral exposure.

Cylindrospermopsin (CYN) is a toxin associated with numerous species of freshwater cyanobacteria throughout the world. It is postulated to have caused an episode of serious illnesses in Australia through treated drinking water, as well as lethal effects in livestock exposed to water from farm ponds. Toxicity included effects indicative of both hepatic and renal dysfunction. In humans, symptoms progressed from initial hepatomegaly, vomiting, and malaise to acidosis and hypokalemia, bloody diarrhea, and hyperemia in mucous membranes. Laboratory animal studies predominantly involved the intraperitoneal (i.p.) route of administration and confirmed this pattern of toxicity with changes in liver enzyme activities and histopathology consistent with hepatic injury and adverse renal effects. The aim of this study was designed to assess subchronic oral exposure (90 d) of purified CYN from 75 to 300 µg/kg/d in mouse. At the end of the dosing period, examinations of animals noted (1) elevated organ to body weight ratios of liver and kidney at all dose levels, (2) treatment-related increases in serum alanine aminotransferase (ALT) activity, (3) decreased blood urea nitrogen (BUN) and cholesterol concentrations in males, and (4) elevated monocyte counts in both genders. Histopathological alterations included hepatocellular hypertrophy and cord disruption in the liver, as well as renal cellular hypertrophy, tubule dilation, and cortical tubule lesions that were more prominent in males. A series of genes were differentially expressed including Bax (apoptosis), Rpl6 (tissue regeneration), Fabp4 (fatty acid metabolism), and Proc (blood coagulation). Males were more sensitive to many renal end points suggestive of toxicity. At the end of exposure, toxicity was noted at all dose levels, and the 75 µg/kg group exhibited significant effects in liver and kidney/body weight ratios, reduced BUN, increased serum monocytes, and multiple signs of histopathology indicating that a no-observed-adverse-effect level could not be determined for any dose level.

The course of toxicity in the pregnant mouse after exposure to the cyanobacterial toxin cylindrospermopsin: clinical effects, serum chemistries, hematology, and histopathology.

Cylindrospermopsin (CYN) is a toxin produced by a variety of fresh-water cyanobacterial species worldwide and induces significant adverse effects in both livestock and humans. This study investigated the course of CYN-induced toxicity in pregnant mice exposed daily during either the period of major organogenesis (gestation days [GD] 8-12) or fetal growth (GD13-17). Endpoints include clinical signs of toxicity, serum analyses to evaluate hepatic and renal function, histopathology of liver and kidney, and hematology. Study animals were administered 50 µg/kg CYN once daily by ip route and euthanized 24 h after 1, 2, 3, 4, or 5 consecutive doses, or 6 or 13 d after the dosing period. The course of the CYN-induced effects was determined at all euthanasia times for the endpoints just outlined. Results indicated that CYN is a toxin, producing lethality in dams during the early part of gestation, significant weight loss, and bleeding in the gastrointestinal tract, tail tip, and peri-orbital tissues. Effects also included alterations in serum markers for liver function, histopathological changes in liver and kidney tissues, electrolyte abnormalities, leukocytosis, and posttreatment thrombocytopenia and reticulocytosis. The onset of symptoms was rapid, producing reductions in weight gain in GD8-12 animals, bleeding in the vaginal area in GD13-17 animals, and significant increases in sorbitol dehydrogenase (SDH) in both groups after a single dose. Although the GD8-12 dams displayed a 50% lethality, in GD13-17 animals only a single death occurred. Alterations seen in hepatic and renal function or histopathology do not appear to be of sufficient severity to produce death. Evidence indicates that bleeding may play a critical role in the onset of symptoms and eventually, in the observed lethality.

N,N-dimethyl-p-toluidine, a component in dental materials, causes hematologic toxic and carcinogenic responses in rodent model systems.

Because of the potential for exposure to N,N-dimethyl-p-toluidine (DMPT) in medical devices and the lack of toxicity and carcinogenicity information available in the literature, the National Toxicology Program conducted toxicity and carcinogenicity studies of DMPT in male and female F344/N rats and B6C3F1/N mice. In these studies, a treatment-related macrocytic regenerative anemia characterized by increased levels of methemoglobin and Heinz body formation developed within a few weeks of DMPT exposure in rats and mice. DMPT induced nasal cavity, splenic, and liver toxicity in rats and mice at 3 months and 2 years. DMPT carcinogenic effects were seen in the liver of male and female rats and mice, the nasal cavity of male and female rats, and the lung and forestomach of female mice. In rodents, DMPT is distributed to many of the sites where toxic and carcinogenic effects occurred. DMPT-induced oxidative damage at these target sites may be one mechanism for the treatment-related lesions. Methemoglobinemia, as seen in these DMPT studies, is caused by oxidation of the heme moiety, and this end point served as an early alert for other target organ toxicities and carcinogenic responses that followed with longer term exposure.

Absence of toxic effects in F344/N rats and B6C3F1 mice following subchronic administration of chromium picolinate monohydrate.

Chromium picolinate monohydrate (CPM) is a synthetic compound heavily marketed to consumers in the United States for use as a dietary supplement for muscle building and weight loss. The National Toxicology Program (NTP) tested the toxicity of this compound based on the potential for widespread consumer exposure and lack of information about its toxicity. Groups of 10 male and 10 female F344/N rats and B6C3F(1) mice were exposed to 0, 80, 240, 2000, 10,000, or 50,000 ppm CPM in feed for 13 weeks. CPM administration produced no effect on body weight gain or survival of rats or mice. Organ weights and organ/body weight ratios in exposed animals were generally unaffected by CPM. No compound-related changes in hematology and clinical chemistry parameters were observed. There were no histopathological lesions attributed to CPM in rats or mice.

Toxicology and carcinogenesis studies of microencapsulated citral in rats and mice.

Citral, a widely used natural ingredient, is added to foods and cosmetics as a flavoring and fragrance agent. Male and female F344/N rats and B6C3F1 mice were exposed to microencapsulated citral in the feed for 14 weeks or two years. All studies included untreated and vehicle control groups. In the 14-week studies, rats and mice were given diets containing 3,900, 7,800, 15,600, or 31,300 ppm citral. In rats, food consumption was reduced in the two highest dose groups. In mice an apparent increase in food consumption was observed, but was due to mice scattering the feed. Body weights of all treated animals were less than controls. All rats and four male mice were killed moribund in the high dose groups. In rats, forestomach and kidney lesions were observed. At the higher doses, lesions observed in the bone marrow, testes, and thymus in rats and in the ovary in mice were considered related to inanition and resultant moribundity. In the two-year studies, rats were exposed to 1,000, 2,000, or 4,000 ppm citral. Body weights were reduced in the 4,000 ppm rats. Mice were exposed to 500, 1,000, or 2,000 ppm citral. Body weights in the 1,000 and 2,000 ppm groups were reduced. No neoplasms were attributed to citral in rats or mice. Malignant lymphoma occurred with a positive trend and was significantly greater than controls in female mice in the 2,000 ppm group. However, the incidences were within the NTP historical control range and could not be clearly related to citral administration.

Subchronic sodium chlorate exposure in drinking water results in a concentration-dependent increase in rat thyroid follicular cell hyperplasia.

Chlorine dioxide (ClO2) is an effective drinking water disinfectant, but sodium chlorate (NaClO3) has been identified as a potentially harmful disinfection by-product. Studies were performed to describe the development of thyroid lesions in animals exposed to NaClO3 in the drinking water. Male and female F344 rats and B6C3F1 mice were exposed to 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/L NaClO3 for 21 days. Additional male F344 rats were exposed to 0, 0.001. 0.01. 0.1, 1.0. or 2.0 g/L NaClO3 for 90 days. Female F344 rats were exposed to 0, 0.5, 1.0, 2.0, 4.0, or 6.0 g/L of NaClO3 for 105 days. Thyroid tissues were processed by routine methods for light microscopic examination, and follicular cell hyperplasia was diagnosed using a novel method. Thyroid hormone levels were altered significantly after 4 and 21 days. NaClO, treatment induced a concentration-dependent increase in the incidence and severity of thyroid follicular cell hyperplasia. Male rats are more sensitive to the effects of NaClO3 treatment than females. Follicular cell hyperplasia was not present in male or female B6C3F1 mice. These data can be used to estimate the human health risk that would be associated with using ClO2, rather than chlorine, to disinfect drinking water.

14-Week toxicity and cell proliferation of methyleugenol administered by gavage to F344 rats and B6C3F1 mice.

Methyleugenol, a food flavor and fragrance agent, was tested for toxicity in male and female F344/N rats and B6C3F1 mice. Groups of 10 males and 10 females per sex per species were administered 0, 10, 30, 100, 300 or 1000 mg methyleugenol/kg body weight in 0.5% aqueous methylcellulose by gavage, 5 days per week for 14 weeks. Additional groups of rats and mice of each sex were dosed similarly and used for hematology and clinical chemistry studies. Groups of 10 male and 10 female rats and mice received the vehicle by gavage on the same dosing schedule and served as vehicle controls. For serum gastrin, gastric pH and cell proliferation studies groups of 10 female rats were given 0, 37, 75 or 150 mg/kg, once daily 5 days per week for 30 or 90 days or 300 or 1000 mg/kg for 30 days; male mice were given 0, 9, 18.5, 37, 75, 150 or 300 mg/kg for 30 or 90 days. For the gastrin, pH and cell proliferation studies, groups of 10 female rats and 10 male mice were given the vehicle for 30 or 90 days and served as controls. Methyleugenol administration to rats induced erythrocyte microcytosis and thrombocytosis in male and female rats. It also caused an increase in serum alanine aminotransferase and sorbitol dehydrogenase activities and bile acid concentration, suggesting hepatocellular injury, cholestasis or altered hepatic function. Additionally, methyleugenol induced hypoproteinemia and hypoalbuminemia, evidenced by decreased total protein and albumin concentrations in both male and female rats, suggesting in inefficiency of dietary protein utilization due to methyleugenol-induced toxic effects on the liver and glandular stomach of rats and mice. The increase in gastrin and gastric pH of rats and mice given methyleugenol suggests that gastrin feedback was impaired and resulted in conditions not conducive to protein digestion. In rats, methyleugenol caused an increase in the incidences of hepatocyte cytologic alteration, cytomegaly, Kupffer cell pigmentation, mixed foci of cellular alteration and bile duct hyperplasia of the liver and atrophy and chronic inflammation of the mucosa of the glandular stomach. In mice, it caused an increase in the incidence of cytologic alteration, necrosis, bile duct hyperplasia and subacute inflammation of the liver and atrophy, degeneration, necrosis, edema, mitotic alteration, and cystic glands of the fundic region of the glandular stomach. The increased incidences of adrenal gland cortical hypertrophy and/or cytoplasmic alteration in the submandibular salivary glands, adrenal glands, testis and uterus of rats were considered secondary to the chemical-related effects observed in the liver and glandular stomach. Based on mortality, body weight gain, clinical chemistry and gross and microscopic evaluation of tissues of rats and mice, the no-observed-effect level (NOEL) of methyleugenol for both species was estimated at 10 mg/kg.

Toxicology and carcinogenesis studies of p,p'-dichlorodiphenyl sulfone in rats and mice.

p,p'-Dichlorodiphenyl sulfone (DDS) is used as a starting material in the production of polysulfones and polyethersufones, a family of thermoplastics. DDS was studied because of its high production volume and use. In toxicology studies, 10 Fischer 344 rats and 10 B6C3F1 mice/sex/group were fed diets containing 0, 30, 100, 300, 1,000 or 3,000 ppm DDS for 14 weeks. All animals survived until the end of the studies. Mean body weights of groups exposed to 300 ppm or greater were significantly decreased. Liver and kidney in rats and liver in mice were the major target organs of DDS toxicity. Dose-related increases in liver weights and incidences of centrilobular hepatocyte hypertrophy were observed in DDS-exposed groups. Nephropathy was seen in male and female rats only at and above 300 ppm. Neurotoxicity evaluations were negative in DDS-treated animals. Clinical chemistry and hematology parameters were minimally affected. In the 2-year toxicity and carcinogenicity studies, 50 rats and 50 mice/sex/group were fed diets containing 0, 10 (male rats), 30, 100, or 300 ppm DDS for 104 to 105 weeks. Survival of exposed groups was not affected. There were no clinical signs of toxicity related to DDS exposure. Final mean body weights were 2-17% lower in DDS-treated groups. Liver was the only target organ of DDS-induced toxicity. The incidence of centrilobular hepatocyte hypertrophy in mice and rats, and the incidence of bile duct hyperplasia and centrilobular degeneration in female rats was significantly greater than in controls. A no-observed-adverse-effect level (NOAEL) of 30 ppm DDS in the diet (1.5 mg/kg body weight) was established for rats. DDS was not carcinogenic in these studies.

The effect of short intermittent light exposures on the melatonin circadian rhythm and NMU-induced breast cancer in female F344/N rats.

We investigated the effects of altered endogenous nighttime melatonin concentrations on mammary tumor production in an N-nitroso-N-methylurea (NMU)-induced breast cancer model in female Fischer 344 (F344)/N rats. Experiments were designed 1) to evaluate whether short-duration intermittent exposures to light at night would affect the nocturnal rise of melatonin, resulting in a decrease in nighttime serum melatonin concentrations, 2) to evaluate whether any suppression of nighttime serum melatonin concentrations could be maintained for a period of weeks, and 3) to determine the effects of suppressed serum melatonin concentrations on the incidence and progression of NMU-induced breast cancer. In vivo studies were used to assess serum melatonin concentrations after 1 day and 2 and 10 weeks of nightly administration of short-duration intermittent light exposure at night and incidence of NMU-induced tumors. Five 1-minute exposures to incandescent light every 2 hours after the start of the dark phase of the light: dark cycle decreased the magnitude of the nocturnal rise of serum melatonin concentrations in rats by approximately 65%. After 2 weeks of nightly intermittent light exposures, an average decrease of the peak nighttime serum melatonin concentrations of approximately 35% occurred. The amelioration continued and, at 10 weeks, peak nighttime serum melatonin concentrations were still decreased, by approximately 25%. Because peak endogenous nighttime serum melatonin values could be moderately suppressed for at least 10 weeks, a 26-week NMU mammary tumor study was conducted. Serum melatonin concentrations and incidence, multiplicity, and weight of NMU-induced mammary tumors were assessed. A group of pinealectomized (Px) animals was also included in the tumor study. No effect on the development of mammary tumors in an NMU-induced tumor model in rats occurred when endogenous nighttime serum melatonin concentrations were moderately suppressed by short-duration intermittent light exposures at night. At necropsy, there were no alterations in mammary tumor incidence (28/40 NMU controls, 28/40 NMU + light, 31/40 NMU + Px), multiplicity (2.18 tumors/tumor-bearing NMU control, 1.89 NMU + light, 2.39 NMU + Px), or average tumor weight (1.20 g NMU control, 1.19 g NMU + light, 0.74 g NMU + Px). Tumor burden had no effect on the serum melatonin cycle. At 26 weeks, however, animals exposed to intermittent light at night exhibited approximately 3-fold higher serum melatonin concentrations as compared with controls. Additionally, rats that had been pinealectomized at 4 weeks of age had serum melatonin concentrations that were markedly higher than the expected baseline concentrations for pinealectomized rats (<15 pg/ml), suggesting the reestablishment of a melatonin cycle. This finding was unexpected and suggests that melatonin can be produced by an organ or tissue other than the pineal gland.

Disseminated thrombosis and bone infarction in female rats following inhalation exposure to 2-butoxyethanol.

Groups of 10 male and 10 female F344/N rats were exposed to 0, 31, 62.5, 125, 250, and 500 ppm of 2-butoxyethanol (BE) by inhalation, 6 hr/day, 5 days/wk, for 13 wk. Four moribund female rats from the 500 ppm group were sacrificed during the first 4 days of exposure, and 1 moribund female from the same group was sacrificed during week 5. Dark irregular mottling and/or loss of the distal tail were noted in sacrificed moribund rats. Similar gross lesions were noted in the terminally sacrificed females exposed to 500 ppm BE. Histologic changes noted in the day 4 sacrificed moribund rats included disseminated thrombosis involving the coccygeal vertebrae, cardiac atrium, lungs, liver, pulp of the incisor teeth, and the submucosa of the anterior section of the nasal cavity. Alterations noted in coccygeal vertebrae from the 500 ppm sacrificed moribund rats included ischemic necrosis and/or degeneration of bone marrow cells, bone-lining cells, osteocytes (within cortical and trabecular bone), and chondrocytes (both articular and growth plate), changes that are consistent with an infarction process. The moribund female rat that was sacrificed during week 5 and those female rats treated with 500 ppm and sacrificed following 13 wk of treatment lacked thrombi, but they had coccygeal vertebral changes consistent with prior infarction and transient or complete bone growth arrest. No bone lesions or thrombi were noted in the male rats treated with the same doses of BE. In conclusion, exposure to 500 ppm BE vapors caused acute disseminated thrombosis and bone infarction in female rats. Possible pathogenic mechanisms are discussed.

Toxicity of 3,3',4,4'-tetrachloroazoxybenzene in rats and mice.

The toxicity of 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice. In addition to histopathology, evaluations included clinical chemistry, hematology, thyroid hormone analyses, and effects on sperm morphology and estrous cycle length. Groups of 10 rats and 10 mice of each sex were exposed to TCAOB at dose levels of 0, 0.1, 1, 3, 10, or 30 mg/kg 5 days a week for 13 weeks. In the rat studies, the major effects included death in the 30 mg TCAOB/kg dose group; at lower exposure levels, a decrease in body weight gain, a decrease in thymus weight, an increase in liver weight, an increase in hematopoietic cell proliferation in the spleen and liver, a responsive anemia, a decrease in platelet counts, a chronic active inflammation of the vasculature in the lung, an increase in cardiomyopathy, hyperplasia of the forestomach, and a marked decrease in circulating thyroxine concentrations were observed. In male rats a decrease in sperm motility in the epididymides was observed. In addition, in female rats an increase in lung, spleen, kidney, and heart weights and nephropathy was observed. Furthermore, the estrous cycle length was increased. In the mouse studies, the major effects for males and females included a decrease in thymus weights, an increase in liver and kidney weights, centrilobular hypertrophy in the liver, hematopoietic cell proliferation, hyperplasia of the forestomach, and dilatation of hair follicles. The spectrum of effects in both rats and mice after exposure to TCAOB indicates that dioxin-like effects occur in addition to effects that have not been observed with dioxin-like compounds. No no-observed-adverse-effect level was reached in male or female rats or mice.

Toxicity of 3,3',4,4'-tetrachloroazobenzene in rats and mice.

The toxicity of 3,3',4,4'-tetrachloroazobenzene (TCAB) was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice. In addition to histopathology, evaluations included clinical chemistry, hematology, thyroid hormone analyses, and reproductive parameters. Groups of 10 rats and 10 mice of each sex were exposed to TCAB at dose levels of 0, 0.1, 1, 3, 10, or 30 mg/kg for 5 days a week for 13 weeks. In the rat studies, the major effects for both males and females included a 10% decrease in terminal body weight at 30 mg/kg/day, an increase in hematopoietic cell proliferation in the spleen at 10 and 30 mg/kg/day, and a responsive anemia at 10 and 30 mg/kg/day. A 15 to 30% decrease in platelet counts and a 20 to 40% decrease in thymus weights was observed at 10 and 30 mg/kg/day. An increase in liver weight up to 15% was found at 3 mg/kg/day and higher doses in males and at 10 and 30 mg/kg/day in females, respectively. An increase in spleen weights up to 15% was observed at 10 and 30 mg/kg/day in males and at 30 mg/kg/day in females. A marked decrease in circulating total thyroxine (TT4) was found in both males and females at all dose levels tested. TT4 could hardly be detected at 10 and 30 mg TCAB/kg/day. In addition, hyperplasia of the forestomach was increased at 3 mg/kg/day and higher doses in males and at 30 mg/kg/day in females. In the mouse studies, an increase in liver and spleen weight was observed up to approximately 25% in both males and females at 10 and 30 mg/kg/day. Hyperplasia of the forestomach was observed at 1 mg/kg/day and higher doses in both males and females. In males, a 30% decrease in thymus weights at 30 mg/kg/day and a 60% decrease in epididymal sperm density at 3 and 30 mg/kg/day was observed. Also in males, centrilobular hypertrophy of hepatocytes and an increase in hematopoietic cell proliferation in the spleen was observed at 3 mg/kg/day and higher doses. Based on the current study and information in the literature, TCAB has dioxin-like properties. Comparison of the effects of TCAB in the present study and in the literature to those with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) indicates that TCAB is from two to six orders of magnitude less potent than TCDD depending on the end point.

Melatonin does not inhibit estradiol-stimulated proliferation in MCF-7 and BG-1 cells.

Melatonin, an indolic pineal hormone, is produced primarily at night in mammals and is important in controlling biological rhythms. Previous research suggested that melatonin can attenuate proliferation in the estrogen-responsive MCF-7 breast cancer cell line. We tested whether these anti-proliferative effects may have physiological consequences upon two estrogen-responsive cell lines, MCF-7 (a breast cancer cell line) and BG-1 (an ovarian adenocarcinoma cell line). Melatonin (10(-9)-10(-5) M) attenuated proliferation of MCF-7 and BG-1 cells by >20% in the absence of estrogen. However, 17beta-estradiol exposure negated the ability of melatonin to inhibit proliferation. To substantiate this finding, cells were estrogen starved followed by multiple treatments with estradiol and melatonin. Melatonin did not inhibit estradiol-stimulated proliferation under this protocol. Estradiol increased MCF-7 and BG-1 cell cycle transition from G1 to S phase, however, melatonin did not inhibit this transition nor did it down-regulate estradiol-induced pS2 mRNA levels measured by northern blotting, further indicating that melatonin was unable to attenuate estradiol-induced proliferation and gene expression. We also examined the effects of melatonin on estradiol-induced proliferation in MCF-7 cell xenografts in athymic nude mice. Melatonin at a dose 28 times greater than 17beta-estradiol did not inhibit estradiol-induced proliferation in vivo. Furthermore, pinealectomy did not increase proliferation. Therefore, we conclude that melatonin does not directly inhibit estradiol-induced proliferation.

Nutritional status modulates rat liver cytochrome P450 arachidonic acid metabolism.

Alterations in nutritional status affect hepatic cytochrome P450 levels. Since cytochromes P450 participate in the metabolism of arachidonic acid, we hypothesized that changes in liver P450 arachidonic acid metabolism occur during fasting and refeeding. Male Fisher 344 rats were either fed, fasted 48 hr (F48), fasted 48 hr and then refed 6 hr (F48/R6), or fasted 48 hr and then refed 24 hr (F48/R24). F48 rats had reduced body weight, increased plasma beta-hydroxybutyrate, and reduced plasma insulin compared with the other groups. Although there was no significant change in total liver P450 content, there was a significant 20%, 48%, and 24% reduction in total hepatic microsomal arachidonic acid metabolism in F48, F48/R6, and F48/R24 rats, respectively, compared with fed rats. Epoxygenase activity decreased by 28%, 51%, and 26% in F48, F48/R6, and F48/R24 rats, respectively. In contrast, omega-1 hydroxylase activity increased by 126% in F48 rats compared with fed rats. Immunoblotting revealed that levels of CYP2C11 protein were markedly reduced, whereas levels of CYP2E1 protein were markedly increased in the F48 and F48/R6 groups. In contrast, levels of CYP1A1, CYP1A2, CYP2B1, CYP2J3, CYP4A1, and CYP4A3 were unchanged with fasting/refeeding. Northern blots revealed that levels of CYP2C11 mRNAs were decreased, whereas CYP2E1 mRNAs were increased in F48 and F48/R6 rats. Recombinant CYP2C11 metabolized arachidonic acid primarily to epoxides with preference for the 14(S),15(R)-, 11(R), 12(S)-, and 8(S),9(R)- epoxyeicosatrienoic acid enantiomers. We conclude that (1) nutritional status affects hepatic microsomal arachidonic acid metabolism, (2) reduced epoxygenase activity in F48 and F48/R6 rats is accompanied by decreased levels of CYP2C11, (3) increased omega-1 hydroxylase activity is accompanied by augmented levels of CYP2E1, and (4) the effects of fasting on CYP2C11 and CYP2E1 expression occur at the pretranslational level.

Eight-week toxicity study of 60 Hz magnetic fields in F344 rats and B6C3F1 mice.

Toxicity studies were performed by exposing F344/N rats and B6C3F1 mice (10 animals per sex per species per group) to transient-free, linearly polarized 60 Hz magnetic fields for 8 weeks. Targeted magnetic fields strengths used were 0 gauss (G; sham control fields did not exceed 0.001 G), 0.02 G, 2 G, and 10 G. Exposure was whole-body and continuous for 18.5 hr per day, 7 days per week. An additional group of rats and mice was exposed intermittently (1 hr on/1 hr off) to 10 G fields for the same period of time. Endpoints evaluated included morbidity, mortality, gross pathology, histopathology, body/organ weights, clinical chemistry (rats only), and hematology (rats only). All mice and all male rats survived until the end of the study. One female rat (2-G exposure group) died during Week 7 of the study; the death was not attributed to magnetic field exposure. In both studies, the mean body weight gains of exposed animals were similar to those of the respective controls. There were no gross, histological, hematological, or biochemical lesions attributed to magnetic field exposure. Statistically significant increases in liver weight and liver to body weight ratio occurred in female rats of all exposure groups but only at the termination. These data suggest that, for the variables evaluated in these studies, an 8-week exposure to linear-polarized, transient-free 60 Hz magnetic fields at field intensities of up to 10 G is not associated with significant toxicity in F344/N rats and B6C3F1 mice. Furthermore, there was no toxicity observed in animals receiving intermittent (1 hr on/1 hr off) exposures to 10-G fields. A 2-year study in F344/N rats and B6C3F1 mice is nearing completion of the in-life phase without overt toxicity in any exposed group. It is premature, however, to make any prediction concerning the possible influence of exposure to 60 Hz magnetic fields on cancer rates.

Thirteen-week inhalation toxicity of 2- and 4-chloronitrobenzene in F344/N rats and B6C3F1 mice.

Toxicity studies were performed by exposing F344/N rats and B6C3F1 mice to 2- and 4-chloronitrobenzene (CNB) by whole-body inhalation 6 hr/day, 5 days/week, for 13 weeks. Animals were evaluated for clinical chemistry (rats), hematology (rats), histopathology, and body/organ weights. Exposure concentrations were 0, 1.1, 2.3, 4.5, 9, and 18 ppm for 2-CNB and 0, 1.5, 3, 6, 12, and 24 ppm for 4-CNB. All rats in the 2-CNB study survived until the end of the study. Two male mice in the 18-ppm group in the 2-CNB study, however, died during Week 12; no deaths attributable to 4-CNB exposure occurred in rats or mice. In both studies, the mean body weight gains of exposed animals were similar to those of the respective controls. In rats, inhalation exposure to 2- or 4-CNB resulted in methemoglobinemia leading to a regenerative anemia and a variety of tissue changes secondary to the oxidative erythrocyte injury. In the 2-CNB study, methemoglobinemia resulted in a normocytic, normochromic, responsive anemia, whereas with 4-CNB, the methemoglobinemia was more severe and resulted in a macrocytic, hyperchromic, responsive anemia. Alterations of erythrocyte morphology were observed in both studies; changes included Heinz bodies, poikilocytes, and polychromasia. In rats, both isomers caused increases in serum activities of alanine aminotransferase and sorbitol dehydrogenase and increased bile acid concentrations. Microscopic liver changes included hemosiderin deposition in Kupffer cells (rats and mice exposed to 4-CNB), hepatocytomegaly (mice), and cytoplasmic basophilia (rats). Hepatocellular necrosis and chronic inflammation observed in mice were rather specific to the 2-CNB isomer, as only slight evidence of focal necrosis in the liver was observed in mice exposed to 4-CNB. Splenic lesions included hemosiderin accumulation capsular fibrosis, and increased hematopoietic cell proliferation. Increased bone marrow hemosiderin and hematopoietic cell proliferation and kidney tubule hemosiderin deposition were also observed. Other findings, attributed to chemical exposure but not to the hematotoxicity, were described. Lesions included hyaline droplet nephropathy and degeneration of the testis in male rats exposed to 4-CNB, inflammation of the harderian gland in rats exposed to 4-CNB, hyperplasia of the nasal cavity epithelium in rats exposed to 2-CNB, and hyperplasia of the forestomach epithelium in mice exposed to 4-CNB; these lesions have not been described previously in studies with these chemicals. Based on the exposure concentrations evaluated, A no-observed-adverse-effect level (NOAEL) for histopathological injury in mice was 4.5 ppm for 2-chloronitrobenzene and 6 ppm for 4-chloronitrobenzene; a NOAEL was not determined for rats.

Frequency and relationships of clinical chemistry and liver and kidney histopathology findings in 13-week toxicity studies in rats.

The relative sensitivities of eight commonly used clinical chemistry end points and histopathology to detect potential toxic effects in liver and kidney were evaluated for a series of 61 13-week rat toxicity studies conducted for the National Toxicology Program. The data consisted of 1-,2- to 3-, and 13 week clinical chemistry measurements and 13-week histopathological assessments of liver and kidney. Except for serum alkaline phosphatase, treatment-related alterations of individual clinical chemistry variables occurred in 20-48% of the studies, depending on the analyte, sampling time, and sex. Liver and kidney lesions were reported for 31% and 41% of the studies respectively. There was an association between treatment-related increases in alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities and histopathological changes in the liver. SDH activity had greater positive and negative predictive values than similar changes in ALT; by week 1 in females and weeks 2-3 in both sexes. SDH predicted morphological hepatic change at study termination with 75% or better accuracy. If increases in activities of both enzymes occurred simultaneously, however, terminal histopathological changes could be predicted, in both sexes, with 75% accuracy by week 1, increasing to 100% by weeks 2-3. There also was an association between treatment-related increases in urea nitrogen (UN) and creatinine (Cre) concentrations and morphological kidney change. Cre concentration had greater positive predictive values than similar changes in UN; by weeks 2-3 in males and week 13 in both sexes. Cre predicted morphological renal change at study termination with 56% or better accuracy. UN concentration was associated and predictive of morphological kidney change only in females at week 13. Depending on time point and sex, serum alkaline phosphatase activity increased in 5-22% of the studies. Increases in total bile acid concentration occurred in 33-48% of the studies. Because both tests are used as markers of cholestasis, this marked discrepancy was unexpected. Treatment-related decreases in alkaline phosphatase activity occurred, however, in 39-56% of the studies; serum alkaline phosphatase may be more useful as an indicator of decreased food intake (decreased activity) than of cholestasis (increased activity). In summary, treatment-related alterations of clinical chemistry and histopathology occurred frequently in this series of toxicity studies in rats. Changes in the chemistry end points also occurred frequently at interim time points, indicating that clinical chemistry evaluations can be useful for detecting potential treatment effects throughout a study. This observation is important, since histopathological evaluations are limited to animal termination and not useful for detecting transient responses or the onset of treatment-related effects.

Early changes in sex hormones are not evident in mice exposed to the uterine carcinogens chloroethane or bromoethane.

Chloroethane and bromoethane have been shown to cause a marked uterine tumor response in B6C3F1 mice exposed for 2 years. These chemicals are nearly unique in this regard among the nearly 400 chemicals studied by the National Toxicology Program, and the reasons for this carcinogenic activity are unclear. The possible relationship of changes in blood concentrations of sex hormones to this response was evaluated by examining the estrous cycle of mice prior to and during a 21-day exposure to concentrations of the haloethanes which resulted in the tumorigenic response in the 2-year studies. Serum concentrations of estradiol and progesterone were determined at the termination of the exposures and compared to exposure group and stage of the estrous cycle. No consistent patterns of change were found in estrous cyclicity or in blood concentrations of sex hormones. Thus, the findings suggest that early changes in circulating sex hormones are not important contributing factors in the uterine neoplasia caused by these chemicals.

Toxicity and carcinogenicity of riddelliine following 13 weeks of treatment to rats and mice.

Toxicity studies of riddelliine, a member of a class of pyrrolizidine alkaloids, were conducted because riddelliine has been found to contaminate human food sources. Groups of male and female Fischer rats were administered riddelliine by gavage in phosphate buffer at doses up to 10 mg/kg, and B6C3F1 mice at doses up to 25 mg/kg, five times a week. The animals were necropsied after 13 weeks of treatment or after a 7 or 14 week recovery period. Body weight gains were inversely related to dose in both rats and mice. Body weight of the 1.0 and 3.3 mg/kg female rats and 10.0 and 25.0 mg/kg mice remained depressed during the 14 week recovery period. At 13 weeks, significant findings included dose-related hepatopathy and intravascular macrophage accumulation in rats and hepatocytomegaly in mice. During the 14 week recovery period these lesions persisted and hepatic foci of cellular alteration in male rats and bile duct proliferation in female rats and male and female mice increased in severity. In the 10 mg/kg group of female rats adenomas of the liver occurred in two of ten at 13 weeks and in one of five at the 14 week recovery period. In separate studies, the frequency of micronucleated erythrocytes in peripheral blood was increased in male mice administered a single dose (150 mg/kg) of riddelliine. Increases in unscheduled DNA and S-phase syntheses were detected in primary hepatocytes from rats and mice treated with riddelliine at doses up to 25.0 mg/kg for 5 or 30 days. In mating trials in rats and mice, pup weights from treated dams at birth and during suckling were lower than controls. Thus, riddelliine is genotoxic and carcinogenic and may cross the placenta and/or be found in milk, causing developmental toxicity in rodents.

Subchronic toxicity of cupric sulfate administered in drinking water and feed to rats and mice.

The effects of acute poisoning by cupric sulfate in a number of species are well known; however, the effects of chronic low-level ingestion of cupric sulfate are less well characterized. Because exposure of humans to cupric sulfate may occur through drinking water, food, soil, or ambient air, subchronic toxicity studies were conducted in male and female F344/N rats and B6C3F1 mice by the drinking water (2-week exposure) and dosed feed (2- and 13-week exposure) routes. Animals were evaluated for histopathology, clinical pathology, reproductive toxicity, and tissue metal accumulation, and target organs were examined by a variety of special stains and by electron microscopy to characterize the observed lesions. In drinking water, cupric sulfate concentrations of 300 to 100 ppm produced no ill effects, whereas concentrations of 3000 to 30,000 ppm were lethal to rats and mice within 2 weeks. In feed, cupric sulfate concentrations of 4000 to 16,000 ppm caused significant reductions in body weight gain in both species in the 2- and 13-week studies. Hyperplasia and hyperkeratosis of the limiting ridge of the forestomach were present in both species in the 2- and 13-week studies. Rats in the dosed feed studies had a dose-related increase in inflammation in the liver and changes in clinical chemistry parameters which were indicative of hepatocellular damage and cholestasis. Histologic changes in the kidneys of rats consisted of a dose-related increase in the number and size of eosinophilic protein droplets in the epithelial cytoplasm and the lumina of the proximal convoluted tubules. Droplets were larger and more numerous in males than in females. Urinalysis results were suggestive of renal tubular epithelial damage. Iron staining of spleens from treated animals indicated a marked depletion of iron stores in both male and female rats, but not in mice, while hematologic and clinical chemistry alterations in rats in the 13-week study, along with histologic changes in bone in the 2-week dosed feed study, were indicative of a microcytic anemia. Cupric sulfate produced no adverse effects on any of the reproductive parameters measured in rats or mice of either sex. These results indicate that cupric sulfate at high exposure levels is a hepatic and renal toxicant, as well as an inducer of anemia in rodents, with rats more sensitive than mice following subchronic exposure.