Multiparametric immunotoxicity screening in mice during early drug development

Evaluation of potential adverse effects on the immune system should be incorporated into drug development prior to phase III clinical trials. In addition to standard toxicity results, T-dependent antibody response (TDAR) assays are widely used to evidence impaired immune function. The present study was aimed at validating a multiparametric screening approach in mice to investigate exaggerated pharmacologic or unintended immunosuppressive effects in early drug development. Male CD1 mice injected with a single IV dose of 2 mg KLH displayed a robust anti-KLH IgM response that peaked on day +5. Anti-KLH IgM response, standard haematology parameters, and thymus/spleen weight and histology were examined in mice treated once daily for 4 days with cyclophosphamide (CY; 5–20 mg/kg/day), cyclosporine (CS; 10–90 mg/kg/day), dexamethasone (DX; 5–20 mg/kg/day), prednisolone (PR; 3–30 mg/kg/day) or chlorpromazine (CZ; 10–30 mg/kg/day). CY and CS decreased anti-KLH IgM response at all dose levels. CY induced a marked decrease in WBC count and thymus/spleen weight with histological changes in both lymphoid organs. CS mainly decreased thymus weight (highest dose), which was associated with lymphoid depletion, without relevant effects on haematology parameters. Neither DX nor PR nor CZ induced significant changes in anti-KLH IgM response. DX and PR decreased lymphocyte counts and thymus/spleen weight, and induced histological changes in both lymphoid organs. CZ (higher doses) decreased lymphocyte count and thymus weight, and induced consistent histological changes in the thymus. This multiparametric study was able to detect 5 human drugs with variable immunosuppressive potency and thus may prove to be a useful early screening tool for predicting drug immunotoxicity.     

Fostamatinib, a Syk-kinase inhibitor, does not affect methotrexate pharmacokinetics in patients with rheumatoid arthritis

Fostamatinib (R788) is being investigated as an add-on therapy for the treatment of rheumatoid arthritis (RA) in patients with inadequate response to methotrexate (MTX). This study evaluated the potential pharmacokinetic interaction between R788 and MTX. Sixteen RA subjects on a stable weekly MTX regimen were enrolled and received MTX on days 1 and 8. Twelve subjects received 100 mg of R788 orally, and 4 subjects received a matching placebo twice daily from days 4 to 8 and once daily on days 3 and 9. Blood samples were collected on days 1 and 8 for MTX and 7-hydroxymethotrexate (7-OH-MTX), and days 3 and 9 for R788 and its active metabolite, R406. MTX and 7-OH-MTX pharmacokinetic parameters were similar on days 1 and 8. In the R788 group, the mean day 8 to day 1 ratios (90% confidence intervals) of maximum concentration and area under the plasma concentration-time curve estimates were 1.01 (0.85-1.20) and 1.12 (0.90-1.40) for MTX and 1.06 (0.82-1.35) and 1.06 (0.83-1.36) for 7-OH-MTX, respectively. Urinary excretion of MTX and 7-OH-MTX was also similar with or without R788, averaging 58% to 69% and 4% to 5% of the MTX dose, respectively. The data suggest that there is no clinically significant pharmacokinetic interaction of R788 and MTX in RA patients.     

Effect of prenatal administration of NSAIDs on the immune response in juvenile and adult rats

In order to investigate the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the development of rat immunity, indomethacin (IND; 0.25, 0.5, or 1.0 mg/kg/day), acetyl salicylic acid (ASA; 90, 180, or 360 mg/kg/day), or diclofenac sodium salt (DSS; 0.5, 1.0, or 2.0 mg/kg/day) suspended in 0.5% methylcellulose aqueous solution, was orally administered once daily to five pregnant Sprague-Dawley (IGS) rats per group on days 18-21 of gestation. After parturition, the serum IgM and IgG levels, the spleen weight, and the number of spleen cells were measured in 3- and 8-week-old pups. Afterwards, immunophenotyping analysis of splenocytes or peripheral blood lymphocytes and T-dependent antibody response were performed. The number of spleen cells in 3-week-olds increased when 1.0 mg/kg of IND and 180 mg/kg of ASA were administered. Immunophenotyping analysis using flow cytometry (FCM) indicated that the proportion and number of CD45RA(+) cells increased, and the proportion of CD3(-) NKR-P1A(+) cells decreased in males when dosed with IND at 1.0 mg/kg or ASA at 180 mg/kg. The serum anti-KLH IgG antibody titer decreased in the males of the IND 1.0 mg/kg dosing group, the serum levels of anti-KLH IgM, total IgM, and IgG were not changed at all. These changes disappeared in 8-week-old pups. There were no effects on any of the parameters in the 3- and 8-week-olds of the DSS treatment group. These results suggest that IND or ASA administration to dams during late gestation either causes a change in the lymphocyte subsets, or that they suppress the T-dependent antibody response in juvenile males. Both of these changes eventually recover to intact levels later on during development. These results will contribute to the development of a technique for the assessment of developmental immunotoxicity and generate data on the effect of prenatal administration of NSAIDs on the developmental immune system in pups.     

The immunosuppressive effects of 10mg/kg cyclophosphamide in Wistar rats

The purpose of this study is to identify the immunosuppressive effects of cyclophosphamide (a known immunosuppressant) in male Wistar rats which were administered orally for 30 days repeatedly, and to find a proper dose of cyclophosphamide at which the immunosuppressive effects is identified. The dose of cyclophosphamide is 2, 5, 10mg/kg bw, respectively. The results showed that 10mg/kg cyclophosphamide treatment induced decreases in body weight, body weight gain, relative weight of spleen and thymus, antibody plaque-forming cells, delayed-type hypersensitivity reaction, natural killer cell activity, lipopolysaccharide-induced B-cell proliferation, and Concanavalin A-induced T-cell proliferation. The histopathology observation on spleen and thymus revealed as decreased cellularity. But, no significant clinical symptoms of toxicities and stress were observed and no rats died. These results indicated that the immunosuppressive effects of 10mg/kg cyclophosphamide dosed for 30 days orally were identified in male Wistar rats. Based on the results, it is considered that the dose of 10mg/kg of cyclophosphamide orally for 30 days in male Wistar rats could be used as the dose of positive control group for chemicals immunotoxicity assessment tests. This study also proved that the testing procedures and the experimental techniques established in our laboratory for immunotoxicity tests in male Wistar rats are reliable and feasible.     

汞对小鼠免疫功能和某些酶的影响

采用免疫毒理学方法,研究了汞对NIH纯系小鼠免疫功能和某些酶的影响。HgCl_2和CH_3HgCl染毒的各剂量组脾重量、腹腔巨噬细胞吞噬功能、外周血液T淋巴细胞计数、中性粒细胞均低于对按组。CH_3HgCl染毒的3个剂量组和HgCl_2中、高剂量组WBC总数低于对照组。CH_3HgCl、HgCl_2染毒的各剂量组动物肝、肾细胞中酶活性有改变。     

Pharmacokinetics of fostamatinib,a spleen tyrosine kinase (SYK) inhibitor,in healthy human subjects following single and multiple oral dosing in three phase I studies

Aim

Fostamatinib (R788) is an orally dosed prodrug designed to deliver the active metabolite R940406 (R406), a spleen tyrosine kinase (SYK) inhibitor, for the treatment of rheumatoid arthritis. The objectives were to evaluate the human pharmacokinetic properties of fostamatinib and R406.

Method

Three clinical studies were conducted in healthy subjects: (A) A single ascending dose study for R406 with doses ranging from 80–600 mg, (B) a single- and multiple-dose study of fostamatinib in aqueous suspension, with single doses ranging from 80–400 mg and multiple doses at 160 mg twice daily and (C) a study comparing suspension and tablet of fostamatinib, with the latter tested in both fed and fasted states.

Results

These studies demonstrated that when administered as a solution, R406 was rapidly absorbed. Increases in exposure were observed with doses up to 400 mg. A terminal half-life of 12–21 h was observed. Similar R406 exposure could be achieved with fostamatinib suspension and steady-state was achieved after 3–4 days following twice daily administration. Fostamatinib tablet and suspension exhibited similar R406 exposure. Upon co-administration with food, a delay in peak time and lower peak concentrations of R406 were observed but at the same time the overall exposure did not change.

Conclusion

Fostamatinib demonstrates rapid and extensive conversion to R406, an inhibitor of SYK. Solid dosage forms of fostamatinib overcome the challenge of low aqueous solubility of R406. The PK profile of R406 could potentially allow once daily or twice daily oral administration of fostamatinib.     

Acetone in drinking water does not modulate humoral immunity in mice as measured by the antibody, plaque-forming cell assay

It has been reported that the repeated topical, nonoccluded application of acetone may modulate antibody production in mice, thus producing humoral immunosuppression. However, the evaporative loss expected following nonoccluded dermal application of acetone makes the systemic effect seem unlikely. This study was designed to investigate the immunotoxicity potential of acetone in mice following a more direct systemic route of dosing via drinking water for 28 days. CD-1 male mice consumed average daily acetone doses of 121, 621 or 1144 mg/kg/day. The antibody, plaque-forming cell (AFC) assay was performed to measure the T cell-dependent, anti-sheep red blood cell immunoglobulin M (IgM) response, and hematology and thymus weights were evaluated to provide additional insight into the potential effects to the immune system. Body weights, white blood cell (WBC), numbers, red blood cell (RBC) counts, and hemoglobin and hematocrit levels showed no treatment-related effects at any dose of acetone. Eosinophil percentages were variable but also showed no dose-related trends. Spleen and thymus weights were not statistically different from controls and there were no effects on spleen cellularity or AFC response as a result of acetone administration. The AFC responses ranged from 1088 to 1401 AFCs/10(6) splenocytes and were not statistically different from controls (1277 AFCs/10(6) cells). Mice treated with cyclophosphamide (20 mg/kg) on days 25 to 28 demonstrated a 94% reduction in AFC/10(6) cells. Thus, the direct systemic administration of acetone did not produce evidence for immunotoxicity in CD-1 mice and the no observed adverse effect level (NOAEL) in this study was determined to be 1144 mg/kg/day.     

Alterations in gene expression and testosterone synthesis in the testes of male rats exposed to perfluorododecanoic acid.

Perfluorododecanoic acid (PFDoA, C12), a synthetic perfluorinated chemical containing 12 carbons, has broad industrial applications and has been detected in sera from humans and other animals; however, few reports have addressed the effects of PFDoA exposure on male reproduction. In the present study, the effects of PFDoA exposure on testes ultrastructure, testosterone levels, and steroidogenic gene expression were investigated. Male rats were orally dosed for 14 days with 1, 5, or 10 mg PFDoA/kg/day or with vehicle. Absolute testis weight was diminished at the highest dose while relative testes weight was markedly increased at doses of 5 and 10 mg/kg/day. Total serum cholesterol levels were significantly increased at the highest dose. While luteinizing hormone was significantly decreased at the highest dose, testosterone was markedly decreased at doses of 5 and 10 mg PFDoA/kg/day. Serum levels of follicle-stimulating hormone were not significantly affected by PFDoA, and estradiol levels were markedly decreased only at 5 mg/kg/day. Leydig cells, Sertoli cells, and spermatogenic cells from rats that received 5 or 10 mg PFDoA/kg/day, exhibited apoptotic features including dense irregular nuclei, condensed chromatin, ill-defined nuclear membranes, and abnormal mitochondria. PFDoA exposure resulted in significant declines in mRNA expression of several genes involved in cholesterol transport and steroid biosynthesis at doses of 5 and 10 mg PFDoA/kg/day, while the gene expression of luteinizing hormone receptor and aromatase was not significantly changed. Our results demonstrate that PFDoA affects the reproduction function of male rats via alterations in steroidogenesis genes, testosterone levels, and testes ultrastructure.     

Metalloelastase (MMP-12) induced inflammatory response in mice airways: effects of dexamethasone, rolipram and marimastat

Direct instillation of a recombinant human form of MMP-12 (rhMMP-12) in mice airways elicited an early inflammatory response characterized by neutrophil influx, cytokine release and gelatinase activation followed by a delayed response, mainly characterized by macrophage recruitment. As this experimental model of lung inflammation partially mimics some features of chronic obstructive pulmonary disease (COPD), we have investigated the effects of treatment by anti-inflammatory compounds, dexamethasone and rolipram and a non-specific matrix metalloproteinase (MMP) inhibitor, marimastat. The compounds were administrated orally, 1 h before rhMMP-12 instillation (8 x 10(-3) U/mouse). Total and differential cell counts were evaluated in the bronchoalveolar lavage fluids. Cytokines and MMP-9 were quantified in bronchoalveolar lavage fluids and in lung homogenate supernatants. Marimastat (100 mg/kg), dexamethasone (10 mg/kg) and rolipram (0.1 and 0.3 mg/kg) were able to decrease significantly neutrophil recruitment at 4 and 24 h after rhMMP-12 instillation, but only marimastat (30 and 100 mg/kg) was effective at decreasing the macrophage recruitment occurring at day 7. Marimastat (100 mg/kg), dexamethasone (10 mg/kg) and rolipram (0.3 mg/kg) reduced significantly IL-6, KC/CXCL1, MIP-1alpha/CCL3 and MMP-9 levels in bronchoalveolar lavage fluid. Similar results were obtained in lung homogenates except with rolipram. Dexamethasone and rolipram were able to inhibit the early inflammatory response but were ineffective to limit the macrophage influx. In contrast, marimastat was able to reduce early and late response. These data indicate that MMP-12 instillation in mice could highlight some of the inflammatory response seen in COPD and could be used for the pharmacological evaluation of new anti-inflammatory mechanisms of action.     

Liu-Shen-Wan, a traditional Chinese medicine, improves survival in sepsis induced by cecal ligation and puncture via reducing TNF-alpha levels, MDA content and enhancing macrophage phagocytosis

Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. Here, we investigated putative protective effects of Liu-Shen-Wan (LSW), a well-known Chinese formula used in treating infectious diseases, against polymicrobial sepsis induced by cecal ligation and puncture (CLP). The oral administration of LSW, at the first dose of 60 mg/kg and then 30 mg/kg every 12 h, significantly improved the survival of CLP mice during a 4-day observation period. The effects of LSW on the inflammatory response (circulating tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) levels and malondialdehyde (MDA) content-an index of lipid peroxidation), infectious degree (peritoneal bacteria counts), and innate immunity function (leukocyte counts, macrophage phagocytosis and neutrophil respiratory burst) were further examined in rats. We demonstrated that treatment of LSW significantly decreased elevated levels of circulating TNF-alpha at 4 h and further reduced plasma MDA levels at 24 h after CLP, at first doses of 15 and 30 mg/kg and then 7.5 and 15 mg/kg every 12 h. Moreover, LSW markedly enhanced clearance of intraperitoneal bacteria associated with the increasing count of peritoneal leukocytes and enhancing phagocytic activity of macrophages partly impaired at 24 h after CLP. In contrast, LSW lightly reduced IL-1 levels at 4 h and failed to improve deactivated respiratory burst activity of neutrophils at 24 h after CLP. Thus, LSW exerts protective effects against sepsis induced by CLP, mainly by reducing plasma TNF-alpha and MDA levels and enhancing peritoneal macrophage phagocytosis, suggesting that it is a potential agent in the prevention and treatment of sepsis.     

The effect of Rhazya stricta Decne, a traditional medicinal plant, on spontaneous and drug-induced alterations in activity of rats.

The effect of acute and chronic treatment of rats with a lyophilized extract of the leaves of the medicinal plant Rhazya stricta on total and ambulatory activity was studied. Given acutely at single oral doses of 1, 2, 4, and 8 g/kg, the extract produced dose-dependent decreases in total activity and ambulatory activity. Diazepam (20 mg/kg, orally) produced a decrease in rat activity comparable to that produced by a dose of 1 g/kg of the extract. When given daily at an oral dose of 2 g/kg for 21 consecutive days, the extract produced, on the last day of treatment, significant decrease in activity amounting to about 30% of control activity levels. Subcutaneous (SC) treatment of rats with caffeine (7.5, 15, and 30 mg/kg), dose-dependently and significantly increased total activity and ambulatory activity. These effects were dose-dependently attenuated when the extract was given concomitantly with caffeine at oral doses of 1, 2, and 4 mg/kg. Treatment of rats with zoxazolamine alone (10, 20, or 40 mg/kg, SC) or R. Stricta (1 and 4 g/kg orally) alone significantly decreased total and ambulatory activities. Concomitant treatment with zoxazolamine and R. Stricta decreased the rats activity to a greater degree than with either treatment given alone.     

Cytochrome P450 1B1 is required for 7,12-dimethylbenz(a)-anthracene (DMBA) induced spleen cell immunotoxicity.

7,12-Dimethylbenz(a)anthracene (DMBA) is a potent carcinogen that induces immunosuppression of both humoral and cell-mediated immunity in mice and other species. Previous studies have shown that CYP1B1 is required for bone marrow toxicity produced by DMBA in mice. Therefore, the purpose of these studies was to determine whether CYP1B1 was required for spleen cell immunotoxicity. Female C57BL/6N wild-type (WT) and CYP1B1 knockout (-/-) mice were treated with 0, 17, 50, or 150 mg/kg (cumulative dose) DMBA in corn oil by oral gavage once a day for five days. Several immunotoxicological assays were used to assess the effects of DMBA on systemic immunity. These included the in vitro T-dependent antibody response to sheep red blood cells (SRBC) measured using a direct plaque forming cell (PFC) assay, T- and B-cell mitogenesis induced by Con A and LPS, and nonspecific cell-mediated immunity was evaluated using an NK cytotoxicity assay. In addition, lymphocyte subpopulations were measured by flow cytometry using specific cell surface markers. Following five days of DMBA treatment, the body weights and spleen cell surface markers of the WT and CYP1B1 (-/-) mice showed no significant changes. A decrease in NK activity was found at the 50 mg/kg DMBA dose in WT mice, but not in the CYP1B1 (-/-) mice. Interestingly, at the 150 mg/kg dose of DMBA, CYP1B1 null mice had decreased NK activity, whereas WT mice did not. The SRBC PFC response demonstrated that the IgM antibody response was suppressed by DMBA in WT mice in a dose-dependent manner (significant at 50 and 150 mg/kg). However, there were no changes in the SRBC PFC responses in any DMBA test group in the CYP1B1 (-/-) mice. Similarly, while DMBA suppressed B- and T-cell mitogenesis at the 50 and 150 mg/kg dose levels in C57BL/6N WT mice, no effect was seen in CYP1B1 (-/-) mice. Thus, CYP1B1 appears to be critical for the immunosuppression of DMBA in mice, suggesting a role for bioreactive metabolites in the spleen cell immunotoxicity produced by DMBA.     

Evaluation of neurobehavioral abnormalities and immunotoxicity in response to oral imidacloprid exposure in domestic chickens (Gallus gallus domesticus)

ABSTRACT

Domestic chickens (Gallus gallus domesticus) were exposed to imidacloprid by gavage once daily for 7 consecutive days at 0, 0.03, 0.34, 3.42, 10.25, and 15.5 mg/kg/day (n = 20 per group; 5 6-week-old males, 5 6-week-old females, 5 9-week-old males, and 5 9-week-old females). The severity and duration of neurobehavioral abnormalities were recorded. Components of the innate and adaptive immune system were assessed with 7 standard functional assays. Temporary neurobehavioral abnormalities were observed in a dose-dependent manner, including muscle tremors, ataxia, and depressed mentation. Based upon mean clinical severity scores, the no observed adverse effect level (NOAEL) was 3.42 mg/kg/day, and the lowest observed adverse effect level (LOAEL) was 10.25 mg/kg/day. The effective dose value for the presence of any neurobehavioral abnormalities in 50% of the test group (ED₅₀) was 4.62 ± 0.98 mg/kg/day. The ED₅₀ for an adjusted score that included both severity and duration of neurobehavioral abnormalities was 11.24 ± 9.33 mg/kg/day. These ED₅₀ values are equivalent to a 1 kg bird ingesting 29 or 70 imidacloprid treated soybean seeds respectively. Immunotoxicity was not documented, possible causes include the assays were insensitive, relevant immune functions were not examined, or imidacloprid is not immunotoxic at this dosing schedule in this species. Neurobehavioral abnormalities were a more sensitive indicator of the sublethal effects of imidacloprid than immunotoxicity.     

Detection of immunotoxicity of benzo[a]pyrene in a subacute toxicity study after oral exposure in rats.

In an extended OECD 407 study protocol, including immune parameters, male Riv:Tox Wistar SPF rats were treated for 35 days with benzo[a]pyrene (B[a]p) (3, 10, 30, or 90 mg/kg body weight) by gavage. Oral administration of B[a]p in rats resulted not only in general toxicity, as indicated by the effects on body weight, but also in immunotoxicity, as indicated by the effects on bone marrow, thymus, spleen, and lymph nodes. Oral B[a]p induced a dose-related decrease in thymus weight (at 10, 30, and 90-mg/kg). Lymph node weights (popliteal, mandibular, and mesenteric) were decreased in the 90-mg/kg rats only. Histologically, indications for cortical atrophy were noted in the thymuses of the 30- and 90-mg/kg dose groups, which was confirmed by morphometric analysis. Nucleated spleen and bone marrow cell counts were decreased in the 90-mg/kg group. Both the absolute number (90 mg/kg) and relative number (10, 30, and 90 mg/kg) of B cells in the spleen were decreased. Red blood cell (RBC) and white blood cell (WBC) counts were significantly decreased; for the WBC at 90 mg/kg, and for the RBC at 10, 30, and 90 mg/kg. The absolute number of lymphocytes and eosinophilic granulocytes was decreased in the 90-mg/kg group, while the absolute number of monocytes was increased in the 10- and 30-mg/kg dose groups. Serum immunoglobulin levels showed a decrease of IgM and IgA after treatment of the animals with 30 and 90 mg/kg, respectively. The highest dose of B[a]p treatment (90 mg/kg) resulted in a significant decrease of natural killer (NK)-cell activity in the spleen. Most toxic effects were only observed in the highest-dose group (90 mg/kg), but compared to the general toxicity, some parameters indicating immunotoxic effects were also affected at lower doses (10 and 30 mg/kg). In conclusion, immunotoxicity of B[a]p can be detected using parameters of the immune system such as described in the recently updated OECD 407 guideline. In the present study thymus weight changed and spleen B-cell populations were affected at a dose of 10 mg/kg, a level where no overt general toxicity was noted.     

Effects of diacerein on the quantity and phagocytic activity of thioglycollate-elicited mouse peritoneal macrophages

Diacerein (DAR: 1,8-diacetoxy-9,10-dioxo-dihydroanthracene-3-carboxylic acid) is an anthraquinone drug which displays anti-inflammatory effects in experimental animals and antirheumatic activity in humans. The drug was administered for orally for four consecutive days to mice injected intraperitoneally with thioglycollate. The following dose levels of DAR were used: 2.5, 5 and 10 mg/kg/day. At the end of the experimental period the macrophage content of peritoneal exudate was dose-dependent and significantly lower in DAR-treated mice compared with animals that were given saline orally. The macrophages isolated from the peritoneal exudate of mice that received DAR displayed a dose-dependent reduced phagocytosis. The effects of DAR were found to be similar to those of indometacin and dexamethasone, which were used as reference drugs. The ability of DAR to interfere with macrophage functioning may contribute to its overall therapeutic activity.     

MNT and Muta™Mouse studies to define the in vivo dose response relations of the genotoxicity of EMS and ENU

Although there are a multitude of in vitro and in vivo studies on the genotoxic activity of EMS, no lifetime carcinogenicity studies, repeat dose mutation data or exposure analysis are available to serve as a solid basis for risk assessment for human exposure cases. The present studies were undertaken to investigate whether a threshold for mutagenic and clastogenic activity in vivo could be established, using the bone marrow micronucleus (MNT) and Muta™Mouse test systems, in the hope to provide reassurance to the patients that their accidental exposure to EMS at doses up to 0.055 mg/kg did not carry a toxicological risk. Dose levels ranging from 1.25 to 260 mg/kg/day were applied orally for up to 28 days. As a reference we included ENU at doses of 1.1–22 mg/kg/day. Our studies showed that daily doses of up to 25 mg/kg/day (bone marrow, GI tract) and 50 mg/kg/day (liver) did not induce mutations in the lacZ gene in the three organs tested. Doses up to 80 mg/kg/day (7-day dosing regime) did not induce micronuclei in mouse bone marrow. The genotoxic activity of EMS became apparent only at higher dose levels. Dose fractionation of EMS (28 times 12.5 mg/kg versus a single high dose 350 mg/kg) provided further evidence for the thresholded dose response of EMS and showed that no cumulation of gene mutations below a threshold was occurring. In contrast, for ENU no threshold was apparent and dose fractionation indicated full additivity of individual dose effects.     

Immunotoxicity of bis(tri-n-butyltin) oxide in the rat

Previous studies in the rat have shown that bis(tri-n-butyltin) oxide (TBTO), used as a biocide, was immunotoxic at dose levels that did not affect other organs. In order to determine a no-effect level, weanling rats were treated for at least 28 consecutive days with TBTO at 0, 0.5, 2, 5, or 50 mg/kg of diet. Studies on clinical chemistry, hematology, pathology, and immune function, that is, plaque-forming cell (PFC) assay, delayed-type hypersensitivity (DTH) response, and the splenic clearance of Listeria monocytogenes, were performed at the end of treatment. No treatment-related effects were noted on clinical chemistry and hematology parameters and on PFC and DTH response, whereas thymic atrophy and impaired clearance of L. monocytogenes were noted only at a dietary concentration of 50 mg/kg. These results confirm the thymus as a target organ of TBTO immunotoxicity. Under the conditions of these experiments the dietary concentration of 5 mg/kg, equivalent to a dose of 0.5 mg/kg body weight, represents a no observed effect level (NOEL) for immunotoxicity in the Sprague-Dawley rat.     

Reproduction study of rokitamycin. Teratological study in rabbits

A teratological study was performed on rokitamycin (TMS-19-Q), a new macrolide antibiotic, using rabbits. TMS-19-Q, at dose levels of 100, 300 and 600 mg/kg, was orally administered from the day 6 to the day 18 of gestation to dams. Diarrhea and decrease in body weight gain and food consumption were observed in dams at or above 300 mg/kg. Decrease in numbers of live fetuses was observed at dose levels of 300 and 600 mg/kg but there was no teratogenicity at any dose levels. The maximum non-toxic dose level for TMS-19-Q in this study was 100 mg/kg.     

Toxicologic, reproductive and teratologic studies of colestipol hydrochloride, a new bile acid sequestrant

Colestipol hydrochloride is a high molecular weight, essentially insoluble, polyethylenepolyamine polymer with 1-chloro-2,3-epoxypropane hydrochloride, developed as a serum cholesterol-lowering agent. This article presents the results of toxicity studies done to determine the safety of colestipol HCl before testing in man. The LD50 in mice and rats ip was >4000 mg/kg; orally in the rat it was >1000 mg/kg. In short-term, high-dose studies, the compound did not affect the rat at a dose of 4000 mg/kg/day for 6 days in the feed, or the rabbit at a dose of 4000 mg/kg/day for 2 weeks by gastric intubation. In dogs 13–14 months old, 3000 mg/kg/day in the diet had no adverse effects during a 30-day period, but in dogs 6–8 months old given 4000 mg/kg/day in a diet of canned horsemeat for 11 days, 3 of 4 developed a possibly significant decrease in serum inorganic phosphorus. Levels of 500, 1000 or 2000 mg/kg/day caused no drug-related abnormalities in dogs for 1 year in the diet, nor in rats at the same levels in the diet for 18 months.In reproduction studies, F₀ and F₁ rats given levels of 500 or 1000 mg/kg/day in the diet did not develop any drug-related abnormalities. Males had received the drug from the age of 40 to 100 days, and females were dosed from 14 days before breeding at 100 days through weaning of the litters at 21 days postparturition. No drug-related malformations developed in rats or rabbits born to females treated with 300 or 1000 mg/kg/day from days 6 to 15 in rats and to day 18 in rabbits.