Distribution of emamectin benzoate in Atlantic salmon (Salmo salar L.).

The aims of this study were to investigate the content of emamectin in blood, mucus and muscle following field administration of the recommended dose, and correlation with sea lice infection on the same fish (elimination study). The tissue distribution of tritiated emamectin benzoate after a single oral dose in Atlantic salmon was also investigated by means of whole-body autoradiography and scintillation counting (distribution study). In the elimination study, concentrations of emamectin benzoate reached maximum levels of 128, 105 and 68 ng/g (p.p.b.) for blood, mucus and muscle respectively, on day 7, the last day of administration. From day 7, the concentration in the blood declined until concentration was less than the limit of detection on day 77. The concentration was higher in mucus compared with plasma (P < 0.05) except on days 7 and 21. The concentration of emamectin benzoate decreased gradually from the end of treatment (day 7) to day 70 with half-lives of 9.2, 10.0 and 11.3 days in muscle, plasma and mucus respectively. The distribution study demonstrated a high quantity of radioactivity in mucous membranes (gastrointestinal tract, gills) throughout the observation period (56 days). Activity was high in the epiphysis, hypophysis and olfactory rosette throughout the study. The highest activity was observed in the bile, indicating this to be an important route for excretion. The distribution study confirmed the results from the elimination study with respect to concentrations in blood, skin mucous and muscle.

Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment.

Various methods of exposure assessment, such as questionnaires, sometimes combined with pictures of cooked meat, have been employed in investigations on the relationship between heterocyclic amines (HA) and health effects. However, as the content of heterocyclic amines vary greatly with cooking conditions, it is difficult to obtain an accurate estimate of the exposure. To improve the exposure assessment, the use of biomarkers has been investigated. The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is well characterised. In humans, the major part of the dose is excreted in urine within 24-48 h following a meal. A few percent is excreted as parent compounds, whereas the major part is metabolites. Urinary level of parent HA reflects only recent exposure. However, the pattern of excreted metabolites might indicate the capacity to activate or detoxify HAs. The excretion of glucuronide conjugates of N-hydroxy-PhIP and N-hydroxy-MeIQx could be a marker for the N-hydroxylation capacity and the dose of the proximate metabolites. Recently, we proposed 5-OH-PhIP as a marker for the ultimate reactive metabolite of PhIP, since it is formed from this compound as a by-product along with the formation of PhIP-DNA adducts. In a search for biomarkers reflecting exposure over some time, blood protein adducts with a longer lifespan have been investigated, and PhIP adducts of serum albumin and haemoglobin from meat-consuming humans were recently reported. Many compounds, like drugs, nicotine and narcotics, bind to melanin in hair and give information on exposure for longer time periods. In mice, PhIP is irreversibly incorporated in a dose-dependent manner into hair, and in humans exposed to an ordinary diet, it was found to vary from <50 to 5000 pg PhIP/g hair. The incorporation is also dependent on the content of eumelanin. The use of PhIP in hair as a biomarker of exposure is promising, but needs validation, using other methods of exposure assessment.

Disposition and depuration of lindane (gamma-HCH) and polychlorinated biphenyl-110 (2,3,3'4'6-pentachlorobiphenyl) in cod (Gadus morhua) and bullrout (Myoxocephalus scorpius) after single oral exposures.

The disposition and depuration of lindane (gamma-hexachlorocyclohexane [HCH]) and polychlorinated biphenyl (PCB)-110 (2,3,3',4',6-pentachlorobiphenyl), orally administered to cod (Gadus morhua) and bullrout (Myoxocephalus scorpius), were investigated using whole-body autoradiography, liquid scintillation counting, and gas chromatography with electron-capture detection. Both gamma-HCH and PCB-110 were distributed mainly to lipid-rich organs after absorption from the gastrointestinal tract of cod and bullrout. Compared to bullrout liver, the cod liver contained higher concentrations of both compounds, reflecting the distribution of fat in the two species. In both species, the depuration time for gamma-HCH was shorter than for PCB-110. Both substances were excreted via bile and urine, largely as water-soluble metabolites. The water-soluble bile metabolites, together with PCB-110 metabolites associated to endogenous macromolecules, strongly indicate that this compound is metabolized in both species.

Influence of o'p-DDD on the physiological response to stress in Arctic charr (Salvelinus alpinus).

Various toxicants have previously been held responsible for an impaired capacity of fish from polluted environments to elevate their cortisol levels in response to stress. In the present study we investigated the responses to stress in o'p-DDD [2-(chlorophenyl)-2-(4-chlorphenyl)-1,1-dichloroethane] exposed (given a single, oral dose of 75 mg o'p-DDD/kg fish) and unexposed Arctic charr. After o'p-DDD administration fish were left undisturbed and without being fed for 28 days, when they were subjected to an acute handling stress. At 1, 3, 7 and 23 h following stress, primary (ACTH and cortisol secretion) and secondary (plasma Cl levels and energy mobilisation) components of the stress response were monitored. As the nutritional state of wild fish may influence this potential biomarker response, the fish had been subjected to a restricted feed ration prior to o'p-DDD administration in order to obtain marked within-group variations in condition factor. No effects of o'p-DDD were observed on post-stress hormone secretion (i.e. peak post-stress plasma ACTH and cortisol levels), nor on plasma chloride levels. However, other results obtained provided evidence for a metabolic depression by o'p-DDD, witnessed by consistently lower plasma glucose levels before and after stress in these contaminated fish. This may be related to the finding that during the 30-day period between o'p-DDD administration and stress treatment, toxicant treated fish lost less weight in comparison to their sham-treated counterparts. Nutritional state did not appear to influence the performance of the charr in the present experiment, as correlations between the parameters measured and condition factor or lipid contents on an individual basis in all cases turned out non significant. Overall, the results contrast with those of previous in vivo and in vitro studies on fish, which concluded that comparable headkidney o'p-DDD levels impaired interrenal steroidogenesis. Although we conclude that the effects of o'p-DDD on Arctic charr metabolism were not associated with the stress response, we propose that they may well interfere with the animals' ability to cope with stress in the long term, or may compromise other physiological processes, such as smoltification. Finally, the high level of integration of components involved in the stress response complicates the identification of single stress-sensitive indices as biomarkers applicable in environmental monitoring programmes.

Plants as de-worming agents of livestock in the Nordic countries: historical perspective, popular beliefs and prospects for the future.

Preparations derived from plants were the original therapeutic interventions used by man to control diseases (including parasites), both within humans and livestock. Development of herbal products depended upon local botanical flora with the result that different remedies tended to develop in different parts of the world. Nevertheless, in some instances, the same or related plants were used over wide geographic regions, which also was the result of communication and/or the importation of plant material of high repute. Thus, the Nordic countries have an ancient, rich and diverse history of plant derived anthelmintic medications for human and animal use. Although some of the more commonly used herbal de-wormers were derived from imported plants, or their products, many are from endemic plants or those that thrive in the Scandinavian environment. With the advent of the modern chemotherapeutic era, and the discovery, development and marketing of a seemingly unlimited variety of highly efficacious, safe synthetic chemicals with very wide spectra of activities, herbal remedies virtually disappeared from the consciousness--at least in the Western world. This attitude is now rapidly changing. There is a widespread resurgence in natural product medication, driven by major threats posed by multi-resistant pest, or disease, organisms and the diminishing public perceptions that synthetic chemicals are the panacea to health and disease control. This review attempts to provide a comprehensive account of the depth of historical Nordic information available on herbal de-wormers, with emphasis on livestock and to provide some insights on potentially rewarding areas of "re-discovery" and scientific evaluation in this field.

Synthesis, purification and biodistribution of (205)Bi-DOTMP, visualizing bone deposition patterns with autoradiography.

A HPLC system has been developed for carrier free and rapid delivery in a physiological buffer of the alpha-particle emitting bone-seeking radiopharmaceutical (212)Bi-DOTMP. (205)Bi-DOTMP was synthesized and HPLC purified to mimic and visualize the deposition pattern in bony tissues of (212)Bi-DOTMP by autoradiography. Inhomogeneous bone deposits were found with the highest concentration in the bone matrix, the endosteum and in the growth zones of young mice. Analysis of urine samples showed that (205)Bi-DOTMP was cleared as an intact complex.

Placental transfer of the estrogenic mycotoxin zearalenone in rats.

In order to study the possible placental transfer of the Fusarium mycotoxin zearalenone (ZON), Sprague Dawley rats were treated with a single dose (0.74 mg/kg b.w.) of ZON i.v. on day 12 or day 18 of pregnancy, or intragastrically (i.g.) on day 18 of pregnancy. Samples of placenta, foetus, and maternal liver and spleen were collected for chemical analyses 0.3 h after treatment on day 12, and 0.3, 4, and 24 h after treatment on day 18. Three rats were used for each pregnancy day, administration route, and exposure time. The concentrations of ZON and its metabolites alpha- and beta-zearalenol (-ZOL) were determined quantitatively by high-performance liquid chromatography (HPLC) after incubation with beta-glucuronidase and purification on immunoaffinity columns. Tissue distribution was studied by means of whole body autoradiography at 4 and 24 h after treatment with tritiated ZON (750 microCi/kg b.w; 7.4 mg/kg b.w.) on day 18 of pregnancy. ZON and alpha-ZOL were transferred into the foetus on both gestational days. However, a delay in distribution into the foetus, relative to the maternal tissue, was observed. Beta-ZOL was below the detection limit in the foetus. No specific site of foetal accumulation of ZON or its metabolites was apparent. In the maternal tissues, the highest levels of ZON and of alpha- and beta-ZOL were found in the liver.

Disposition of 14C-flumequine in eel Anguilla anguilla, turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus after oral and intravenous administration.

The absorption, distribution and elimination of 14C-labelled flumequine were studied using whole body autoradiography and liquid scintillation counting. Flumequine was administered to eel Anguilla anguilla, turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus intravenously and orally as a single dose of 5 mg kg(-1), corresponding to 0.1 mCi kg(-1). The turbot and halibut studies were performed in salt water (salinity of 32%) at temperatures of 16 +/- 1 degrees C (turbot) and 9.5 +/- 0.5 degrees C (halibut). The eel study was conducted in fresh water at 23 +/- 1 degrees C. In the intravenously administered groups flumequine was rapidly distributed to all major tissues and organs. After oral administration flumequine also appeared to have rapid and extensive absorption and distribution in all 3 species. After the distribution phase, the level of flumequine was higher in most organs and tissues than in the blood, except in muscle and brain. The most noticeable difference between the species was the slow elimination of flumequine from eel compared to turbot and halibut. In orally administered eels, substantial amounts of flumequine remained in all major organs/tissues for 7 d. At 28 d significant levels of flumequine were present in liver, kidney and skin (with traces in muscle), and at the last sampling point (56 d) in eye, bone, bile and posterior intestine. In orally administered turbot significant levels of flumequine were observed over 96 h in bile, urine, bone, skin, intestine and eye, and traces were detected over 28 d in bone and eye in addition to a significant level in bile. In orally administered halibut, significant levels of flumequine were observed in bile, skin, intestine and eye over 96 h. Traces were present in skin and eye over 7 d. The maximal flumequine concentrations in blood were calculated to be 2.5 mg equivalents l(-1) (eel at 12 h), 0.8 mg l(-1) (turbot at 6 h) and 0.6 mg l(-1) (halibut at 6 h) after oral administration.

Accumulation of the lipophilic environmental contaminant lindane in metacercariae of Bucephaloides gracilescens (Trematoda, Bucephalidae) in the central nervous system of bullrout Myoxocephalus scorpius.

Bucephaloides gracilescens is a common parasite in the intestine of the angler fish Lophiuspiscatorius, and the metacercariae have been recorded from a number of gadoid intermediate hosts. In a toxicokinetic study of lindane (gamma-hexachlorocyclohexane) in bullrout Myoxocephalus scorpius, metacercariae of B. gracilescens were found in the central nervous system (CNS). Furthermore, the metacercariae accumulated concentrations of lindane that were higher than in the surrounding CNS tissue. This is the first report of metacercariae of B. gracilescens in bullrout. In addition the present results contribute knowledge of the disposition of an important environmental contaminant in the metacercarial stage of a trematode that has been pointed out as a promising sentinel species of pollution in the marine environment.

In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar).

In the environment, nonylphenol (NP) occurs predominantly as a degradation product of nonylphenol ethoxylate (NPE). They can be found in many types of products including detergents, plastics, emulsifiers, pesticides, and industrial and consumer cleaning products. As a consequence of their use in a variety of products, they are quite common in rivers and other aquatic environments that receive sewage discharges. Because of its enhanced resistance towards biodegradation, toxicity, estrogenic effects, and ability to bioaccumulate in aquatic organisms NP has been regarded as the most critical metabolite of APEs. We have studied the in vivo and in vitro metabolism and organ distribution of NP in juvenile salmon. Fish were exposed in vivo to waterborne [3H]-4-n-NP for a period up to 72 h or were administered a single oral dose of [3H]-4-n-NP. In vitro biotransformation of NP was studied by exposure of cultured salmon hepatocytes to [3H]-4-n-NP in the presence or absence of a CYP1A-inducer, beta-naphthoflavone (betaNF). Our results show that 4-n-NP was mainly metabolized in vivo, to its corresponding glucuronide conjugates and hydroxylates. The major route of excretion was the bile. The half-life of residues in carcass and muscle was between 24 and 48 h in both waterborne and dietary exposure. In whole body autoradiography, intragastric administered [3H]-4-n-NP was mainly present in the gastrointestinal tract and bile. NP-derived radioactivity in fish exposed via water was more evenly distributed in the organs compared to intragastric exposure and were observed in the intestinal contents, liver, kidney, gills, skin, abdominal fat and brain. In vitro pretreatment of hepatocytes with betaNF had no effect on rates or patterns of NP biotransformation. The in vitro metabolic rate of NP were 118 pmol NP metabolized/h/0.5x10(6) cells without betaNF, and 98 pmol NP metabolized/h/0.5x10(6) cells when betaNF was added to the culture medium.

Effects of enzyme induction on the distribution of the food carcinogen 2-amino-3,8-dimethyl-imidazo[4,5-ss]-quinoxaline (MeIQx) in Ah-receptor- responsive- and Ah-receptor-non-responsive mice.

The distribution of the food carcinogen 2-amino-3,8-dimethyl-imidazo[4,5-ss]quinoxaline (MeIQx) was studied in Ah-responsive-(C57BL/6J) and Ah-non-responsive mice (DBA/2N). The time dependent organ distribution of radioactivity after 14C-MeIQx (10 mg/kg) administration in C57BL/6J showed that at day 4 most of the radioactivity had been excreted and that the remaining radioactivity was found in liver, kidneys, lungs and spleen. C57BL/6J bound more radioactivity in the kidneys than the DBA/2N strain whereas approximately the same amount was left in the liver and lungs in both strains 4 days after MeIQx exposure. Liver microsomes of the two strains had approximately the same ability to activate MeIQx in the Ames Salmonella assay. beta-Naphthoflavone treatment of the animals greatly increased microsomal activating capacity, but only in the C57BL/6J strain. Isosafrole treatment of the animals only slightly increased the activating capacity, but particularly with microsomes from the DBA/2N strain, displacement of the putative inhibitory isosafrole metabolite greatly increased their activating capacity. In the whole animals pretreatment with beta-naphthoflavone, which induces P450IA only in the C57BL/6J strain, did not significantly change the amount of retained radioactivity in any of the strains. Isosafrole induces only P450IA2, the major N2-hydroxylating enzyme of heterocyclic amines, in both strains. Such pretreatment reduced the amount retained in the kidney of both strains whereas it reduced the retained amount of radioactivity in the liver with about 60% only in the Ah-non-responsive strain (DBA/2N). The effect of isosafrole did not persist when MeIQx was given three days after the last injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of iron on the induction of hepatic tumors and porphyria by octachlorostyrene in C57BL/10ScSn mice.

Octachlorostyrene (OCS) is an environmental contaminant, present in fish of Northern European waters and the Great Lakes of America. It has many distribution and toxic similarities to hexachlorobenzene (HCB). Administration of OCS at 0.01% of the diet to C57BL/10ScSn mice within iron overload for 18 months gave only a low incidence of hepatic nodular hyperplasia (2/10 survivors) and no hepatocellular adenomas or carcinomas. In contrast, with a similar regime, HCB causes severe liver cancer or nodules in all exposed mice. Whole body autoradiography of mice given [14C]OCS or [14C]HCB showed no gross variations in distribution or covalent binding of the radiolabelled compound to account for the difference between the chemicals in the development of tumours. In 12-week studies, the CYP1A subfamily was induced to a greater degree by HCB than OCS and iron-enhanced uroporphyria was significantly greater with HCB. The findings are consistent with the proposal that uroporphyria and liver cancer induced in mice by HCB are associated through related mechanisms, but occur to a significantly lesser extent with OCS.

Tissue distribution and effects on hepatic xenobiotic metabolising enzymes of 2,3,3',4,4'-pentachlorobiphenyl (PCB-105) in COD (Gadus morhua) and rainbow trout (Oncorhynchus mykiss).

2,3,3',4,4'-Pentachlorobiphenyl, PCB-105 (IUPAC no. 105) was orally administered twice with a 4-day interval between dosings (total dose 10 mg kg(-1) body weight) to gonadally immature cod and rainbow trout of both sexes. The fish were killed 9 and 17 days after the first treatment, and the effects of PCB-105 on hepatic xenobiotic metabolising enzymes were determined by examining the cytochrome-P450-dependent ethoxyresorufin-O-deethylase (EROD) and aldrin epoxidase activities, and the EROD-catalysing P450 1A1 protein by indirect enzyme-linked immunosorbent assay (ELISA). Glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene was included. The hepatic levels of the compound were determined. In addition, the distribution patterns of radio-labelled PCB-105 were studied by whole-body autoradiography. In exposed rainbow trout EROD activity and P450 1A1 by enzyme linked immunosorbent assay (ELISA) were significant induced, while GST activity was significant reduced. Exposed cod did not show significantly different enzyme values from controls, but percentage fat in the liver was significantly reduced. The whole cod liver contained about 1000 times more PCB-105 than the corresponding trout liver, and on a fat-weight basis the PCB level was five to six times higher in cod liver than in the rainbow trout liver. The autoradiographical investigation revealed high concentrations of radiolabelled compound in the liver and the brain of cod, while in rainbow trout the radiolabel was mainly confined to extrahepatic fat depots. These results demonstrate that the mono-ortho chlorinated coplanar analogue, PCB-105, has a different distribution pattern in the two fish species and that the potential for induction of the hepatic xenobiotic metabolising enzyme system seems to be lower in the cod than in the rainbow trout.

Optimalized in vivo production of monoclonal antibodies in mouse ascitic fluid.

The note contains a brief summary of the method in use at the National Institute of Public Health for the production of monoclonal antibodies in mouse ascitic fluid. This method of obtaining large amounts of monoclonal antibodies is widely used. Refining the in vivo method and increasing the yield from each mouse could result in a reduction of the number of animals used.

Enrichment of metabolites in the cerebrospinal fluid of cod (Gadus morhua) following oral administration of hexachlorobenzene and 2,4',5-trichlorobiphenyl.

The disposition of 14C-labelled hexachlorobenzene (HCB) and 2,4',5-trichlorobiphenyl (triCB) was studied in cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss). For both compounds tape section autoradiography revealed substantial amounts of radiolabelled material in the central nervous system (CNS) of cod, whereas only traces of radioactivity were observed in the CNS of rainbow trout. Furthermore, an enrichment of radiolabelled compound in the cerebrospinal fluid (CSF) was observed in the cod, whereas no radioactivity could be detected in the CSF of rainbow trout. According to autoradiography, the CNS of cod dosed with HCB contained the parent compound, whereas the major part of radioactivity in CSF was due to HCB metabolites. Thin-layer chromatography of extracts from cod dosed with triCB showed the presence of parent compound in the CNS, whereas part of the radioactivity in the CSF was due to triCB metabolites. The activities of cytochrome P-450 and UDP-glucuronosyltransferase in the CNS of cod and rainbow trout were determined in microsomal and mitochondrial fractions. Both species expressed activities which were in the same order of magnitude as those reported for the corresponding fractions from rat brain. Incubation of triCB with cod brain mitochondria and microsomes resulted in the formation of two polar metabolites. It is suggested that cod may be more vulnerable than rainbow trout regarding neurotoxicological effects of HCB, triCB and related environmental pollutants.

Extrahepatic disposition of 3H-aflatoxin B1 in the rainbow trout (Oncorhynchus mykiss).

Whole-body autoradiography in rainbow trout (Oncorhynchus mykiss) after oral and intravenous administration of 3H-labelled aflatoxin B1 showed labelling of several extrahepatic tissues, such as the uveal melanin and the vitreous humour of the eyes, the trunk and head kidney, the olfactory rosettes and the pyloric caecae. Liquid chromatography of extracts of the vitreous humour showed that unmetabolized 3H-AFB1 was the main labelled material present at this site. Liquid chromatography of extracts of the uveal melanin showed presence of aflatoxicol and aflatoxin B1 in proportions of about 3:1. The binding to the pigment is probably due to a hydrophobic type of interaction with the melanin. Microautoradiography showed that melanin-containing cells in the trunk and head kidney and in the olfactory rosettes also accumulated high amounts of radioactivity. In the trunk kidney there was, in addition, a labelling of the second segment of the proximal tubules and of the distal tubules and the collecting ducts. Studies in vitro with microsomal and 12,000 x g supernatant preparations of the trunk kidney showed formation of DNA- and protein-bound metabolites from the aflatoxin B1. It is probable that the bioactivation of the aflatoxin B1 is confined to the cytoplasm of the cells, may be related to excretion and/or absorption processes. Microautoradiography of the olfactory rosettes, showed labelling of the sensory epithelium, but not the indifferent epithelium. A low formation of protein-bound aflatoxin B1-metabolites was found in incubations with microsomal preparations of this tissue. The same observation was made in incubations with microsomal preparations of the head kidney. In the pyloric caeca bound metabolites were observed in vivo at a level comparable to that found in the trunk kidney. Our results suggest that retention and metabolism in some extrahepatic tissues might be of importance as concerns the toxicologic potential of aflatoxin B1 in the rainbow trout.

Tissue distribution of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in two strains of male rats.

Albino and hooded male rats were administered 14C-labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by gavage. The tissue distribution was investigated by means of whole-body autoradiography and liquid scintillation counting. MeIQx was rapidly absorbed from the alimentary tract and distributed to several tissues. The major predilection sites were the liver and kidneys. The amount of radioactivity decreased dramatically within a few days. However, unextractable radioactive material was still detectable in these organs 6 days after the administration.

Plasma concentration of paracetamol and its major metabolites after p.o. dosing with paracetamol or concurrent administration of paracetamol and its N-acetyl-DL-methionine ester in mice.

1. Single doses of paracetamol 400 (PAR 400) and 800 mg/kg (PAR 800), SUR 2647 combination (free paracetamol + paracetamol-N-acetyl-DL-methionate, paracetamol/methionine ratio 2:1) equivalent to PAR 400 (SURc 400) and PAR 800 (SURc 800) were given p.o. to male Bom:NMRI mice. 2. The objective was to compare the plasma concentrations of free paracetamol and the major metabolites paracetamol-sulphate and paracetamol-glucuronide for a 6 hr period after each test drug. 3. There was no significant difference between PAR 400 and SURc 400 with respect to plasma paracetamol, paracetamol-glucuronide and paracetamol-sulphate concentration with the exception of lower plasma paracetamol concentration (P less than 0.03) at 3 hr following PAR 400. 4. There was no significant difference between PAR 800 and SURc 800 with respect to plasma paracetamol, paracetamol-glucuronide and paracetamol-sulphate concentrations with the exception of lower plasma paracetamol-glucuronide concentration (P less than 0.03) at 4 hr after dosing following SURc 800. 5. Combining free paracetamol and its methionine ester does not seem to alter the pattern of plasma paracetamol, paracetamol-glucuronide and paracetamol-sulphate compared to equal doses of free paracetamol alone after p.o. administration of toxic doses to male Bom:NMRI mice.