Adduction of Hemoglobin and Albumin in Vivo by Metabolites of Trichloroethylene, Trichloroacetate, and Dichloroacetate in Rats and Mice

Adducts to macromolecules from trichloroethylene formed by invivo and in vitro metabolism have been reported by many investigators.We examined the in vivo adduction of the blood proteins hemoglobin(Hb) and albumin in rats and mice dosed orally with [¹⁴C]trichloroethylene([¹⁴C]TRI) to explore the development of a protein adduct biomarkerof TRI exposure. We also examined the adduction of these twoproteins from doses of [¹⁴C]trichloroacetate (TCA) and [¹⁴C]dichloroacetate(DCA), two metabolites of TRI. Association of label with albuminpeaked at 4–8 hr in the rat (2480 nmol eq TRI/mg protein)and 2–4 hr in the mouse (1580 nmol eq TRI/mg protein).The decay was exponential with a half-life consistent with thatof rat or mouse albumin (approx 24 hr). The time course of labelwith Hb was characterized by an early plateau at 8 hr in rat(28 nmol eq TRI/ mg protein), 4 hr in mouse (7 nmol eq TRI/mgprotein), and followed by a slow steady increase, peaking at120 hr (54 nmol eq TRI/mg protein, rat; 38 nmol eq TRI/mg protein,mouse). This apparent binding was linear with dose in the rat,but was convex in the mouse albumin (mouse Hb label was belowdetection at low dose). We also found that a portion of theirreversibly associated label, referred to by previous investigatorsas "binding," could be accounted for as metabolic incorporationof label into glycine and serine. The fraction accounted forby metabolic incorporation was constant in albumin (approximately), while in Hb, this portion was time dependent, approximately30% at the early sampling time, 75% at the late time, implyingthe observed late increase could be accounted for by metabolicincorporation. TCA and DCA also formed Hb and albumin adducts.Portions of this binding was also due to metabolic incorporation.The pattern of the binding from TCA in albumin was differentfrom that of TRI, implying a route to adduct from TRI whichdoes not proceed through TCA.     

The Effect of Chloral Hydrate and its Metabolites,Trichloroethanol and Trichloroacetic Acid,on Bilirubin-Albumin Binding

Abstract: Chloral hydrate is used as a sedative in infants requiring ventilatory support. The metabolites, trichloroethanol and trichloroacetic acid, accumulate in the serum and are protein bound. The possibility that these chemicals might compete with bilirubin for albumin binding was tested using the peroxidase method and a dialysis rate method. Chloral hydrate and trichloroethanol had no effect on bilirubin-albumin binding. Trichloroacetic acid affects bilirubin-albumin binding but to a degree that would be dangerous only in infants with an unusual accumulation of this metabolite.     

Subchronic Studies of Doxylamine in B6C3F1 Mice

Subchronic Studies of Doxylamine in B6C3F, Mice. JACKSON, C.D. AND BLACKWELL, B.-N. (1988). Fundam. Appl. Toxicol. 10, 254-261.Doxylamine succinate, a histamine (HI) antagonist (antihistamine),was administered as an admixture in the feed to male and femaleB6C3F, mice for 14 or 90 days. Dose levels of 0,100,250, 500,1000, and 2000 ppm doxylamine were administered to males andfemales in the 14-day study while dose levels of 0, 80, 162,325, 750, and 1500 ppm were administered to both sexes in the90-day study. Little toxicity was seen in the 14-day study.Final body weights in the highest dose group were reduced 4.0and 7.3% in males and females, respectively. Treatment-relatedhistopathological changes in the 14-day study were limited toa very low incidence of hepatic necrosis in both sexes. Therewas little toxicity observed in the 90-day study and no cleardose response relative to weight gain was observed. Histologically,the liver was the only organ affected by doxylamine administration.The liver lesions consisted of hepatic cell cytomegaly and/orkaryomegaly which varied from mild to severe and a possibledose-related hepatic necrosis.     

Immunotoxicity of 2,4-Diaminotoluene in Female B6C3F1 Mice

ABSTRACT

2,4-Diaminotoluene (DAT) has been demonstrated to be a potent carcinogen. The present studies were carried out to determine the toxic and immunotoxic potential of DAT. Mice exposed to DAT at 25-100 mg/kg per day for 14 days by gavage showed a 42% increase in liver weight and a slight decrease in spleen weight. Histopathologic evaluation of selected organs showed the liver to be the major target with morphological changes which were dose dependent. The high dose (100 mg/kg) was associated with moderate centrilobular necrosis. No abnormal structure was noted in the spleen, lungs, thymus, kidney or mesenteric lymph nodes. The liver toxicity was associated with an elevation in alanine aminotransferase activity. The only change noted in selected hematologic parameters was a 64% increase in peripheral blood leukocytes. Mice exposed to DAT showed a decreased IgM and IgG response to sheep erythrocytes. The decrease was not a function of a decreased number of B cells because the number of B cells increased dose dependently. Proliferative capacity of immunocompetent cells was not impaired by exposure to DAT as measured by the response to several mitogens. The delayed hypersensitivity response to keyhole limpet hemocyanin in mice exposed to DAT was increased. Natural killer cell activity was decreased dose dependently and may represent a spleen cell pool shift because the number of B cells increased in the presence of a decreasing spleen size. Serum C3 was suppressed at the high dose of DAT. Phagocytosis by splenic macrophages, but not peritoneal macrophages, was inhibited by DAT exposure. DAT exposure for 14 days decreased host resistance to the bacteria, Streptococcus pneumoniae and Listeria monocytogenes, while host resistance to the pulmonary tumor model, B16F10, and the PYB6 fibrosarcoma was unaffected by DAT exposure. These data indicate that DAT is hepatotoxic and perturbs the differentiation and maturation of leukocytes.     

Comparison of Styrene Hepatotoxicity in B6C3F1 and Swiss Mice

Inhalation exposure to styrene at concentrations that causemetabolic saturation results in significantly greater hepatotoxicityin B6C3F1 mice than in Swiss mice; females of both strains aremore susceptible than males. These studies were conducted toinvestigate the mouse strain and gender differences in susceptibilityto hepatotoxicity caused by repeated exposure to styrene atconcentrations that do not cause metabolic saturation. Maleand female B6C3F1 and Swiss mice (8 weeks old) were exposedto 0, 150, or 200 ppm styrene for 6 hr/day, 5 days/week, forup to 2 weeks. Changes in body and liver weights, serum alanineaminotransferase (ALT) and sorbitol dehydrogenase (SDH) levels,liver histopathology, and total liver glutathione (GSH) wereevaluated after 2, 3, 5, and 10 exposures (six mice/sex/strain/timepoint/concentration). Blood levels of styrene and styrene-7,8-oxide(SO) were measured in mice exposed to 200 ppm styrene for 2,3, or 5 days (six mice/sex/strain/time point/concentration).Serum ALT and SDH levels were significantly elevated only infemale B6C3F1 mice after 3 exposures to 200 ppm styrene; enzymelevels had returned to control levels when measured after 5and 10 exposures. Degeneration and coagulative necrosis of centrilobularhepatocytes were observed in female B6C3F1 mice exposed 2, 3,and 5 days to 150 or 200 ppm styrene; incidences of these lesionswere greater in the 200 ppm than in the 150 ppm dose group.After 10 days of exposure to 150 or 200 ppm styrene, hepatocellularlesions had resolved, although a residual chronic inflammationwas present in livers of most female B6C3F1 mice. Degenerationof centrilobular hepatocytes was observed in one male B6C3F1mouse after 3 exposures to 200 ppm, and no significant lesionswere observed in livers of exposed Swiss mice. Significant dose-relateddecreases in hepatic GSH were observed in both sexes of bothstrains throughout the 2-week exposure. In general, hepaticGSH depletion was greatest in female B6C3F1 mice. Exposure to200 ppm caused 60–70% GSH depletion in female B6C3F1 miceat each time point. GSH depletion generally decreased in B6C3F1mice and increased in Swiss mice with continued exposure to150 ppm styrene. With continued exposure to 200 ppm, GSH depletiongenerally decreased in all mice. Blood styrene and SO levelsincreased in all groups with the number of exposures. Styrenelevels were significantly higher in B6C3F1 mice than in Swissmice; however, within each strain gender differences were notsignificant. These data suggest that the transient hepatotoxicityin female B6C3F1 mice was related to greater hepatic GSH depletionand/or slower GSH regeneration in these animals.     

Benzene-Induced Hematotoxicity and Bone Marrow Compensation in B6C3F1 Mice

Long-term inhalation exposure of benzene has been shown to causehematotoxicity and an increased incidence of acute myelogenousleukemia in humans. The progression of benzene-induced hematotoxicityand the features of the toxicity that may play a major rolein the leukemogenesis are not known. We report the hematologicalconsequences of benzene inhalation in B6C3F1 mice exposed to1, 5, 10, 100, and 200 ppm benzene for 6 hr/day, 5 days/weekfor 1, 2, 4, or 8 weeks and a recovery group. There were nosignificant effects on hematopoietic parameters from exposureto 10 ppm benzene or less. Exposure of mice to 100 and 200 ppmbenzene reduced the number of total bone marrow cells, progenitorcells, differentiating hematopoietic cells, and most blood parameters.Replication of primitive progenitor cells in the bone marrowwas increased during the exposure period as a compensation forthe cytotoxicity induced by 100 and 200 ppm benzene. In miceexposed to 200 ppm benzene, the primitive progeni or cells maintainedan increased percentage of cells in S-phase through 25 daysof recovery compared with controls. The increased replicationof primitive progenitor cells in concert with the reported genotoxicityinduced by benzene provides the components necessary for producingan increased incidence of lymphoma in mice. Furthermore, wepropose this mode of action as a biologically plausible mechanismfor benzene-induced leukemia in humans exposed to high concentrationsof benzene.     

Subchronic Toxicity of Triethylenetetramine Dihydrochloride in B6C3F1 Mice and F344 Rats

Triethylenetetramine dihydrochloride (trien-2HCl; CAS No. 38260-01-04),a chelating agent used to treat Wilson's disease patients whoare intolerant of the drug of choice, was tested for subchronictoxicity in B6C3F1 mice and F344 rats. Mice and rats receivedtrien-2HCl in the drinking water at concentrations of 0, 120,600, or 3000 ppm for up to 92 days. Twenty mice and 18 ratsof each sex were assigned to each dose group fed either a cereal-based(NIH-31) or a purified (AIN-76A) diet, both containing nutritionallyadequate levels of copper. An additional control group of ratsand mice received a Cu-deficient AIN-76A diet. This low copperdiet resulted in Cu-deficiency symptoms, such as anemia, liverperiportal cytomegaly, pancreatic atrophy and multifocal necrosis,spleen hematopoietic cell proliferation, and increased heartweight, together with undetectable levels of plasma copper inrats but not in mice. Trien-2HCl lowered plasma copper levelssomewhat (at 600 and 3000 ppm) in rats fed the AIN-76A diet,but did not induce the usual signs of copper deficiency. Trien-2HClcaused an increased frequency of uterine dilatation at 3000ppm in rats fed AIN-76A diet that was not noted in females fedthe Cu-deficient diet. Trien-2HCl toxicity occurred only inmice in the highest dose group fed an AIN-76A diet. Increasedfrequencies of inflammation of the lung interstitium and liverperiportal fatty infiltration were seen in both sexes, and hematopoieticcell proliferation was seen in the spleen of males. Kidney andbody weights were reduced in males as was the incidence of renalcytoplasmic vacuolization. There were no signs of copper deficiencyin mice exposed to trien-2HCl. The only effect of trien-2HClin animals fed the NIH-31 diet was a reduced liver copper levelin both rat sexes, noted at 3000 ppm.     

Chronic Toxicity and Carcinogenicity Studies of 1-Methylnaphthalene in B6C3F1 Mice

The carcinogenic potential of 1-methylnaphthalene (1-MN), acompound which exists widely in the environment, was investigatedin B6C3F1 mice. Groups of 50 male and 50 female mice were givendiets containing 0, 0.075, or 0.15% 1-MN for 81 weeks. Bothtreatment groups developed pulmonary alveolar proteinosis athigh incidence, with 46.0 and 34.7% of females and 46.0 and38.0% of males, respectively, being affected. Total lipid andphospholipid levels in sera and monocytes in peripheral bloodwere also significantly increased in 1-MN-treated female andmale mice in contrast with control values. The incidences ofbronchiolar/alveolar adenomas in the lungs of male mice givenboth 0.075 or 0.15% 1-MN were 26.0 and 24.0%, respectively,in both cases significantly increased in contrast with the 4.1%observed for control males. However, neither dose dependencenor significant difference in the incidences of bronchiolar/alveolarcarcinomas between 1-MN-treated and control male mice was observed.The incidences of other tumors also were similar in both 1-MN-treatedand control groups. The results of the present experiment thussuggested a possible weak carcinogenic potential of 1-MN tothe lung of male but not female B6C3F1 mice.     

The Chronic Hepatotoxic, Tumor-Promoting, and Carcinogenic Effects of Acetaminophen in Male B6C3F1 Mice

The Chronic Hepatotoxic, Tumor-Promoting, and Carcinogenic Effectsof Acetaminophen in Male B6C3F₁ Mice. HAGIWARA, A., AND WARD,J.M. (1986). Fundam. Appl. Toxicol. 7, 376-386. Acetaminophen(ACT), the most commonly used analgesic and antipyretic in theUnited States, was previously demonstrated to be a hepatocarcinogenin one mouse study but not in rats. In order to help elucidatethe potential mechanisms of carcinogenesis by this nongen-otoxicchemical and its relationship to hepatotoxicity, ACT was fedto groups of 60-120 male B6C3F₁ mice at dietary concentrationsof 5000 or 10,000 ppm from 6 weeks of age for periods of upto 70 weeks to study the hepatotoxic effects of ACT. To testfor potential liver tumor-promoting effects of ACT, N-nitrosodiethylamine(DEN) was injected intraperitoneally at 40 mg/kg into additionalgroups of 30-60 male B6C3F₁ mice at 4 weeks of age. Two weekslater some mice received ACT at dietary concentrations of 5000or 10,000 ppm. Mice were sacrificed either at 24 weeks afterDEN injection or after 22 or 70 weeks of ACT exposure. The liverswere weighed and prepared for qualitative and quantitative histologicalevaluation of focal hepatocel-lular proliferative lesions (FHPL)including microscopic hyperplastic foci and neoplasms by automatedimage analysis. At 24 weeks the incidence and number of FHPLper square centimeter were significantly increased only in DEN-treatedmice receiving 10,000 ppm ACT. Chronic hepatotoxicity was mildat this time. At 72 weeks ACT alone had no effect on the incidenceor number of naturally occurring liver tumors despite severechronic hepatotoxicity and suppression of body weight gain inmice receiving 10,000 ppm and only mild toxicity at 5000 ppm.There were histological findings suggesting that the chronichepatotoxicity had, in part, a vascular pathogenesis. This studyprovided evidence against the hypothesis that chronic hepatotoxicity,in and of itself, results in an increased incidence of naturallyoccurring liver tumors in mice.     

Immunotoxicological Profile of Morphine Sulfate in B6C3F1 Female Mice

This study was undertaken to investigate a number of immuneparameters which may be compromised with exposure to morphinesulfate. Mice were implanted subcutaneously with 8-, 25-, or75-mg morphine sulfate pellets. Placebo pellets of identicalmakeup to the 75-mg morphine pellet (without morphine of course)were used as a control. Twenty-four hours after implantationof a 75-mg morphine pellet, blood levels reached a peak of 1610ng/ml. Corticosterone increased in parallel with morphine andreached a peak level of 966 ng/ml 24 hr after implantation.The dose response of morphine to increase corticosterone, however,was fiat. The weight of the lymphoid organs, spleen and thymus,and the liver were significantly reduced in the morphine-treatedgroups. Morphine treatment was associated with an increase inserum albumin, SGPT, BUN, and alkaline phosphatase indicativeof hepatic damage. In contrast to increased serum proteins,the C3 component of complement was reduced in a dose-dependentmanner. Leukocyte number in the peripheral blood was significantlyreduced, while erythro-cyte number and hematocrit were bothincreased. The number of B cells and T cells was decreased inmorphine-treated animals. However, the percentage of T cellsrelative to B cells was increased. The primary IgM antibodyresponse to the T-depen-dent antigen, sheep red blood cells,was decreased. Natural killer cell activity was reduced in responseto morphine, as was the phagocytic capacity of Kupffer cells.Host-resistance models of Listeria monocytogenes or Streptococcuspneumoniae showed an increased resistance following administrationof morphine. This increased host resistance, however, was notdue to an increase in antimicrobial action of sera obtainedfrom mice treated with morphine. The majority of morphine'seffects on the immune system exhibited a flat dose response,suggesting that these effects may be mediated secondarily throughcorticosterone.     

Chronic Toxicity Carcinogenicity Studies of Triethanolamine in B6C3F1 Mice

The chronic toxicity and carcinogenic potential of triethanolaminewas examined in B6C3F1 mice. Triethanolamine, dissolved in distilledwater at levels of 0 (control), 1, and 2%, was given to groupsof 50 males and 50 females ad libitum in drinking water for82 weeks. Neoplasms developed in all groups, including the controlgroup, but no dose-related increase of the incidence of anytumor was observed in treated groups of both sexes. There wereno adverse effects as regards survival of the mice, organ weights,and specific incidence of neoplasms in the treated, comparedto the control group. This chronic toxicity test provides noevidence of carcinogenic potential of triethanolamine in B6C3F1mice.     

Chronic Toxicity and Carcinogenicity of Methylmercury Chloride in B6C3F1 Mice

Chronic Toxicity and Carcinogenicity of Methylmercury Chloridein B6C3F1 Mice. MRRSU-MORI, K., HIRANO, M., UEDA, H., MAITA,K., AND SHIRASU, Y. (1990). Fundam. Appl. Toxicol. 14, 179–190.A 2-year feeding study of methylmercury chloride (MMC: 0, 0.4,2, or 10 ppm) was conducted in B6C3F1 mice (60 mice of eachsex/group) to compare chronic toxicity and carcinogenicity resultswith those for ICR mice from our previous study in which malesof the 10-ppm group showed an increased incidence of renal tumorswithout any abnormal in-life parameters. In B6C3F1 mice of the10-ppm group, neurotoxic signs characterized by posterior paralysiswere observed in 33 males after 59 weeks and in 3 females after80 weeks. In males, a marked increase in mortality and a remarkabledecrease in body weight gain were observed after 60 weeks. Toxicencephalopathy consisting of neuronal necrosis of the brainand toxic peripheral sensory neuropathy were induced in bothsexes in this group. Chronic nephropathy, testicular atrophy,and glandular stomach ulcer increased in incidence in the males;chronic nephropathy also increased in incidence in females.In proliferative lesions, there were significant increases inthe incidence of renal adenoma and/or carcinoma (16/60) andtubular cell hyperplasia (14/ 60) in males of the 10-ppm group,as compared to the control group. The incidence of chronic nephropathyalso increased in males of the 2-ppm group. The results of thisstudy indicate that the susceptibility of B6C3F1 mice to renaltoxicity and renal carcinogenicity is comparable to that ofICR mice, and B6C3F1 mice are more sensitive to the chronicneurotoxic effects of MMC than are ICR mice.     

Hepatocarcinogenicity of Chioral Hydrate, 2-Chloroacetaldehyde, and Dichioroacetic Acid in the Male B6C3F1 Mouse

The chlorinated acetaldehydes, chloral hydrate (CH) and 2-chloroacetaldehyde(CAA), have been identified as chlorination by-products in finisheddrinking water supplies. Although both chemicals are genotoxic,their potential for carcinogenicity had not been adequatelyexplored. The studies reported here are chronic bioassays conductedwith male B6C3F1 mice exposed to levels of 1 g/liter CH and0.1 g/liter CAA via the drinking water for 104 weeks. Distilledwater (H₂O) served as the untreated control and dichloroaceticacid (DCA; 0.5 g/liter), another chlorine disinfection by-product,was included. The mean daily ingested doses were approximately166 mg/kg/day for CH, 17 mg/kg/day for CAA, and 93 mg/kg/dayfor DCA. Evaluations included mortality, body weight, organweights, gross pathology, and histopathology. The primary targetorgan was the liver as the organ weights and pathological changesin the other organs (spleen, kidneys, and testes) were comparablebetween the treated groups and the H₂O control group. Liverweights were increased for all three test chemicals at the terminaleuthanasia with the greatest increase seen in the CH and DCAgroups. Hepatocellular necrosis was induced by all three testchemicals, and it was also most prevalent and severe in theCH and DCA groups. A significant increase in the prevalenceof liver tumors was seen for all three chemicals. The strongestresponse was with DCA, in which 63% of the 104-week survivorshad hepatocellular carcinomas (carcinomas) and 42% possessedhepatocellular adenomas (adenomas) and the combined prevalencefor carcinomas plus adenoma was 75%. The corresponding prevalencerate for carcinomas, adenomas, and combined tumors were 46,29, and 71% 31, 8, and 38% and 10, 5, and 15% for CH, CAA, andH₂O, respectively. In addition to the tumors we evaluated theprevalence of a possible preneoplastic lesion, the hepatocellularhyperplastic nodule (nodules), a lesion which occurred in allthree treated groups but not in the H₂O group.     

The Effect of Methadone on the Immune Status of B6C3F1 Mice

Previously, morphine has been shown to elevate corticosteronevia the hypothalamic–pituitary–adrenal axis andto suppress the immune system. The present investigation soughtto determine if the µ-opiate receptor agonist methadoneincurred a similar immune suppression in B6C3F1 mice. Serummethadone and corticosterone levels peaked 1 hr following asingle subcutaneous injection of 20 mg/kg methadone HCl. Indeed,the rise in corticosterone levels paralleled that of methadone.After a single injection with 20 mg/kg methadone a pharmacokineticanalysis revealed a serum half-life of 2 hr. Following fiveinjections of methadone over a 24-hr period (every 6 hr), methadonelevels were elevated as would be expected; however, corticosteronelevels did not become elevated. This suggests that the abilityof methadone to elevate corticosterone becomes uncoupled followingrepeated dosing, indicative of either a tolerance or an increasedcatabolic mechanism. Moreover, dosing every 6 hr for 5 daysinduced an increase in the catabolism of methadone itself. Therefore,all assays were begun 1 hr after subcutaneous administrationof methadone HCl, a time at which both methadone and corticosteroneserum levels were elevated. The primary IgM antibody responseto sheep red blood cells (sRBC) was suppressed when splenocyteswere immunized in vitro. In contrast, animals immunized withsRBC and assayed for the primary IgM antibody response 4 dayslater were not suppressed. The activity of the resident macrophagesof the liver and spleen as measured by the uptake of ⁵¹C-sRBCwas suppressed in a dose-dependent manner. Previously, it hasbeen demonstrated that morphine suppresses hepatic and splenicphagocytic activity through an opiate receptor-mediated pathway that involves the release of corticosterone. It would appearthat methadone plays a similar role in the suppression of hepaticand splenic phagocytosis.     

Immunotoxicity of the Semiconductor Gallium Arsenide in Female B6C3F1 Mice

The effects of gallium arsenide (GaAs) exposure on immunocompetenceof B6C3F1 female mice were investigated. GaAs was administeredas a single intratracheal instillation at doses of 50, 100,and 200 mg/kg. Fourteen days after exposure, various cellularand humoral immune parameters were assessed. GaAs exposure increasedspleen cellularity in a dose-dependent manner. However, thepercentages of Thy 1.2 positive and 1g positive cells were decreasedand that of F4/80 positive cells was increased dose dependency.The IgM and IgG antibody-forming cell response of the spleento the T-dependent antigen sheep erythrocytes was reduced by66 and 48%, respectively, at 200 mg/kg. Levels of the serumcomplement protein, C3, were increased by as much as 16% withno significant change in CH50 levels. The mitogenic responseof splenic T cells to Con A and PHA was unaffected by GaAs,but that of B cells to LPS was increased by 52%. The delayedhypersensitivity response to keyhole limpet hemocyanin and mixedlymphocyte response were significantly reduced in a dose-dependentmanner by GaAs exposure. Natural killer cell activity againstthe YAC-1 mouse lymphoma was enhanced in treated mice. Analysisof peritoneal exudate cells (PEC) revealed a dose-dependentdecrease in number and a shift in the composition of PECs. Thepercentage of PEC monocytes increased from 53% of the populationto 81%, while the lymphocytes decreased from 46 to 20%. Theadherent PEC population demonstrated decreased phagocytosisof covaspheres and increased phagocytosis of chicken erythrocytes(CRBC). GaAs exposure had no effect on host resistance to Plasmodiumyoelii or Streptococcus pneumoniae, but dose dependency increasedresistance of the mouse to Listeria monocytogenes Treated micedemonstrated a significantly decreased resistance to the B16F10melanoma with a sevenfold increase in tumor burden at 200 mg/kg.GaAs affects both humoral and cellular immune parameters inmice and impairs the ability of the immune system to protectagainst B16F10 tumor challenge.     

Prechronic Toxicity Studies on 2-Ethylhexanol in F334 Rats and B6C3F1 Mice

Data on the subchronic toxicity of 2-ethylhexanol (2EH) wererequired to establish the dose vehicle and dose levels for oncogenicitystudies. In preliminary studies 2EH was given subacutely (11days) to male and female Fischer 344 rats and B6C3F1 mice asan aqueous emulsion by oral gavage (0, 100, 330, 1000, and 1500mg/kg/day). Clinical observations were made, body weights, foodconsumption, clinical chemistries, hematologies, and selectedorgan weights were measured, and gross and micropathologieswere performed. Target organs were the central nervous system,liver, forestomach, spleen, thymus, and kidney in rats and thecentral nervous system, liver, and forestomach in mice. 2EHwas then administered by oral gavage to male and female F344rats and B6C3F1 mice as an aqueous emulsion (0, 25, 125, 250,and 500 mg/kg/day) for 13 weeks. At 500 mg/kg/day in the ratthere was reduced body weight gain (6% male, 7% female), increasedrelative liver (29% male, 15% female), kidney (16% male, 6%female), stomach (11% male, 16% female), and testes (6%) weights,and moderate gross and microscopic changes in the liver andforestomach. There were no behavioral effects or effects onthe spleen or thymus. A no-effect level for target organ effectsin the rat was 125 mg of 2EH/kg/day. At 500 mg of 2EH/kg/dayin the mouse the only effects were increased relative stomachweights in males (13%) and a low incidence of gross and microscopicfindings in the forestomach (male and female) and liver (female).A no-effect level for target organ effects in the mouse was125 mg of 2EH/kg/day. 2EH was a peroxisome proliferator in therat but not in the mouse at subchronic dose levels of 500 mg/kg/day.Dose levels in oncogenicity studies were set at 50 mg/kg/dayfor the absence of treatment-related effects in rats and mice,and 500 and 750 mg/kg/day, respectively, in rats and mice ashigh doses producing minimal toxicity without altering the lifespan.     

Chronic Toxicity and Carcinogenicity Studies of 2-Methylnaphthalene in B6C3F1 Mice

The toxicity and carcinogenic potential of 2-methylnaphthalene(2-MN) were examined in B6C3F₁ mice. Groups of 50 male and 50female mice were given diets containing 0, 0.075, and 0.15%2-MN for 81 weeks. Both 0.075 and 0.15% 2-MN caused pulmonaryalveolar proteinosis at high incidence: 55.1 and 45.8% in femalesand 42.9 and 46.9% in males, respectively. The incidences oftotal lung tumors, including bronchiolar/alveolar adenomas andcarcinomas, were 20.4 and 12.2% in male mice given 0.075 and0.15% 2-MN, respectively, the former value being significantlyincreased compared with the 4.1% in control males. However,in the respective incidences of the adenomas and carcinomas,neither in-tergroup differences nor dose dependencies were observed.The incidences of other tumors did not differ between mice treatedwith 2-MN and the controls. The results indicated that 2-MNinduces pulmonary alveolar proteinosis but does not possessunequivocal carcinogenic potential in B6C3F₁ mice.     

Styrene Inhalation Toxicity Studies in Mice: I. Hepatotoxicity in B6C3F1 Mice

Studies were conducted to evaluate the toxic effects of short-termrepeated styrene inhalation in B6C3F1 mice. Male and femalemice were exposed to 0, 125, 250, or 500 ppm styrene, 6 hr/day,for up to 14 days. Styrene toxicity was characterized by severecentrilobular hepatic necrosis and deaths after one exposureto 500 ppm or two exposures to 250 ppm. Mortality and hepatotoxicitywere not increased by additional exposures, and in survivingmice, regeneration and repair of initial hepatic injury occurredin spite of continued exposure for 14 days. A marked sex differencewas observed, with male mice significantly more susceptibleto styrene toxicity than females. A nonlinear dose responsewas observed where mortality in male and female mice was greaterin the 250 ppm dose group than that in the 500 ppm dose group.Severe congestion and necrosis of the liver was present in moribundmice; hepatic congestion and serum alanine aminotransferaseand sorbitol dehydrogenase were significantly greater in moribundanimals.     

Disposition and Pharmacokinetics of Isopropanol in F-344 Rats and B6C3F1 Mice

The absorption, metabolism, disposition, and excretion of isopropanol(IPA) were studied in male and female rats and mice. Animalswere exposed by iv (300 mg/kg) and inhalation (500 and 5000ppm for 6 hr) routes; additionally, IPA was given by gavageto rats only in single and multiple 300 and 3000 mg/ kg doses.In the rat approximately 81–89% of the administered dosewas exhaled (as acetone, CO₂, and unmetabolized IPA); approximately76% of the dose in mice was exhaled after iv bolus but 92% wasexhaled following inhalation. Approximately 3–8% of theadministered dose was excreted in urine as IPA, acetone, anda metabolite tentatively identified as isopropyl glucuronicacid. Small amounts of radiolabel were found in feces and inthe carcass. There were no major differences in the rates orroutes of excretion observed either between sexes or betweenroutes of administration. Additionally, repeated exposure hadno effect on excretion. However, both the route of administrationand the exposure or dose level influenced the form in whichmaterial was exhaled. Following exposure to 5000 ppm, a greaterpercentage of unmetabolized IPA was recovered in the expiredair than following exposure to 500 ppm, implying saturationof metabolism.     

Metabolism and Disposition of Phenolphthalein in Male and Female F344 Rats and B6C3F1 Mice

A recent 2-year carcinogenicity/toxicology study determinedthat phenolphthalein (PHTH) is a multisite carcinogen in bothmice and rats at all doses evaluated. In response to this findingthe metabolism and disposition of PHTH has been evaluated inboth F344 rats and B6C3F1 mice at a single oral dose of 800mg/kg. This dose fell within the range previously found to becarcinogenic in rats and mice. Studies were also performed using1 and 50 mg/kg doses. At 800 mg/kg recovery of [¹⁴C]PHTH after72 h was near 100% in females but closer to 75% in males. Radioactivitywas primarily recovered in the feces in rats (>90%), whilemice excreted 30–40% of administered activity in the urine.There was no significant retention of radioactivity in tissuesby 72 h and no significant accumulation of radioactivity inany tissue at any time point. Covalent binding to protein intarget tissues, bone marrow and ovary, was at or less than thepmol/mg protein range. The major metabolite was PHTH glucuronide.Three minor metabolites were detected. A sulfate conjugate andand a hydroxylated metabolite were identified by comparisonof retention times and ¹H NMR and/or mass spectra with syntheticstandards. A diglucuronide conjugate was tentatively identified.Biliary elimination was extensive in rats (35% of dose within6 h); the only product detected in bile was phenolphthaleinglucuronide.