Fortification of dried distillers grains plus solubles with grape seed meal in the diet modulates methane mitigation and rumen microbiota in Rusitec.

The role of dried distillers grains plus solubles (DDGS) and associative effects of different levels of grape seed meal (GSM) fortified in DDGS, used as both protein and energy sources in the diet, on ruminal fermentation and microbiota were investigated using rumen-simulation technique. All diets consisted of hay and concentrate mixture with a ratio of 48:52 [dry matter (DM) basis], but were different in the concentrate composition. The control diet contained soybean meal (13.5% of diet DM) and barley grain (37%), whereas DDGS treatments, unfortified DDGS (19.5% of diet DM), or DDGS fortified with GSM, either at 1, 5, 10, or 20% were used entirely in place of soybean meal and part of barley grain at a 19.5 to 25% inclusion level. All diets had similar DM, organic matter, and crude protein contents, but consisted of increasing neutral detergent fiber and decreasing nonfiber carbohydrates levels with DDGS-GSM inclusion. Compared with the soy-based control diet, the unfortified DDGS treatment elevated ammonia concentration (19.1%) of rumen fluid associated with greater crude protein degradation (~19.5%). Methane formation decreased with increasing GSM fortification levels (≥ 5%) in DDGS by which the methane concentration significantly decreased by 18.9 to 23.4 and 12.8 to 17.6% compared with control and unfortified DDGS, respectively. Compared with control, unfortified DDGS decreased butyrate proportion, and GSM fortification in the diet further decreased this variable. The proportions of genus Prevotella and Clostridium cluster XIVa were enhanced by the presence of DDGS without any associative effect of GSM fortification. The abundance of methanogenic archaea was similar, but their composition differed among treatments; whereas Methanosphaera spp. remained unchanged, proportion of Methanobrevibacter spp. decreased in DDGS-based diets, being the lowest with 20% GSM inclusion. The abundance of Ruminococcus flavefaciens, anaerobic fungi, and protozoa were decreased by the GSM inclusion. As revealed by principal component analysis, these variables were the microorganisms associated with the methane formation. Grape seed meal fortification level in the diet decreased DM and organic matter degradation, but this effect was more related to a depression of nonfiber carbohydrates degradation. It can be concluded that DDGS fortified with GSM can favorably modulate ruminal fermentation.

The effects of inhalation anaesthetics on common clinical pathology parameters in laboratory rats.

UNLABELLED: Effects of common anaesthetics such as ether, methoxyflurane, isoflurane, carbon dioxide (at 100%, 80% or 60% admixed with O(2)) on toxicity and clinical pathology parameters in rats were investigated. Ether, methoxyflurane and 100% CO(2) induced toxicity in some animals. Erythrocyte, haemoglobin and haematocrit were reduced in females by 100% CO(2), methoxyflurane and isoflurane. Glucose was increased by 60% CO(2), 80% CO(2), ether, isoflurane and methoxyflurane in males. Chloride was reduced by isoflurane and all CO(2) concentrations in females. Serum proteins were reduced by isoflurane and methoxyflurane. Sodium, inorganic phosphate, calcium and magnesium were reduced by methoxyflurane and isoflurane, but increased by all CO(2) concentrations. Potassium was reduced by ether, methoxyflurane or isoflurane. Triiodothyronine and thyroxine were reduced by all anaesthetics. Prolactin was reduced by methoxyflurane, but raised by ether and isoflurane. Erythrocyte cholinesterase (E-ChE) activity is markedly reduced (20-40%) after anaesthesia with all CO(2) concentrations in both sexes. E-ChE was unaffected by ether, methoxyflurane, or isoflurane. Serum and brain cholinesterase activities were not affected. E-ChE inhibition correlated with decreased blood pH, suggesting that this was caused by acidosis. This is of practical relevance in the risk assessment of cholinesterase inhibitors. CONCLUSIONS: Clinical pathology data were affected by all anaesthetics. CO(2)/O(2) (80%/20%) and isoflurane are the most suitable anaesthetics. If E-ChE activity is to be determined, isoflurane is the anaesthetic of choice.

Determination of pulmonary irritant threshold concentrations of hexamethylene-1,6-diisocyanate (HDI) prepolymers by bronchoalveolar lavage in acute rat inhalation studies according to TRGS 430.

Pulmonary irritant threshold concentrations of two hexamethylene-1,6-diisocyanate (HDI)-based prepolymers (I: polymeric emulsfier modified and II: oligomeric allophanate modified) were determined in acute inhalation studies according to TRGS 430 (Dangerous Substances Technical Rule, isocyanates, Germany), based on benchmark extrapolation of bronchoalveolar lavage fluid (BALF) total protein. It was also investigated if the method is robust enough to be transferred to an independent laboratory. Five male Wistar rats per group were exposed nose-only to the test substances as liquid aerosols to concentrations of 0, 0.5, 3, 15 mg/m(3) for both test substances with an additional test group at 50 mg/m(3) for test substance I. The duration of the exposure was 6h, followed by serial sacrifices 1 day, 3 days and 7 days post exposure. BALF was analyzed for biochemical and cytological markers indicative for injury of the bronchoalveolar region. The exposure of rats to test substance I and II caused dose depended lung irritation with BALF total protein concentration being the most sensitive indicator of pulmonary effects. The extrapolated no observed adverse effect level of test substance I was 1.1 mg/m(3) and that of test substance II 2.3 mg/m(3). The acute pulmonary irritant threshold concentrations were found to be similar to those reported by [Pauluhn, J., 2004. Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage. J. Appl. Toxicol. 24, 231-247] for HDI-homopolymers and other HDI-based polyisocyanates, and were at least 30 times higher than the MAK (occupational exposure limit) value for the HDI monomer (0.035 mg/m(3)). Thus the EBW (exposure assessment value) for these two HDI-based prepolymers can be established at 10x MAK, i.e. at 0.35 mg/m(3).

Investigations on the subchronic toxicity of 2-methoxypropanol-1(acetate) in rats.

Wistar rats were exposed to 2-methoxypropylacetate-1 (2-MPAc-1) vapours in concentrations of 0, 110, 560 and 2800 ppm for (equiv. to 0; 0.6; 3.0 and 14.9 mg/L) for 4 weeks in chambers (6 hours/day; 5 days/week; five male and five female animals per group). The top concentration was equivalent to a 95% vapour saturation at 20 degrees C and the animals reacted to this with a moderate respiratory irritation during the 6 hours exposure times; at 560 ppm these effects were only slight. The top dose was also associated with a significantly reduced body weight development and some hematologic and biochemical alterations of little specificity. The most prominent effect was thymic atrophy. No effects were noted on the testes or on the cellularity in blood or bone marrow. 560 ppm were without systemic effects. Furthermore, 2-methoxypropanol-1 (2-MP-1), 2-MPAc-1 and 2-ethoxyethanol (EE) were administered in parallel by gavage to groups of five male Wistar rats daily for 10 days at near equimolar dose levels (1800, 2600 and 1800 mg/kg per day, respectively). At the end of the administration period the testes were investigated. There was a pronounced testicular atrophy in animals exposed to EE, whereas no adverse effects were observed with 2-MP-1 and 2-MPAc-1. The results of these studies indicate that 2-MP-1 and 2-MPAc-1 which previously had been shown to exert pronounced prenatal toxicity in rabbits and weak prenatal effects in rats are devoid of other forms of systemic toxicity in rats that are typically observed with ethoxyethanol and methoxyethanol.

Subchronic and chronic studies of the effects of oral administration of acrylic acid to rats.

In a 3-month study, groups of 10 male and 10 female Wistar rats were dosed by gavage, 5 times per week, with acrylic acid at doses of 150 or 375 mg/kg body weight. Five male and five female rats in the low-dose group died and six males and nine females given 375 mg/kg died. Pathological examination revealed a dose-dependent pronounced irritation in the forestomach and glandular stomach with ulcerations and purulent rhinitides and tubular necroses. Therefore, in comparison with drinking water administration using approximately equivalent doses (2000 or 5000 ppm; see below), administration by gavage was determined not to be suitable for long-term studies using as high as possible doses. In a 12-month study, Wistar rats (20 rats/group/sex) were given drinking water containing 120, 800, 2000 or 5000 ppm acrylic acid (providing doses of about 9, 61, 140 and 331 mg/kg body weight/day, respectively). Satellite groups (10 rats/group/sex) were treated concurrently for 3 months. Acrylic acid at 5000 ppm, and temporarily also at 2000 ppm, led to reduced drinking water consumption in male rats and, to a slight extent, also in female rats. In the males, feed consumption was reduced at 5000 ppm and body weight gain was retarded at 5000 ppm and marginally also at 2000 ppm. These findings indicate palatability problems and their consequences. There were no indications of systemic toxicity and/or any carcinogenic potential. In a carcinogenicity study, Wistar rats (50/group/sex) were given acrylic acid in the drinking water at concentrations of 0, 120, 400 or 1200 ppm (8, 27 or 78 mg/kg body weight/day, respectively) over 26 (males) or 28 (females) months. The concentrations were chosen on the basis of the interim results from the 12-month drinking water study, which had started earlier, and taking into account the longer study duration and geriatric effects to be expected. This carcinogenicity study did not reveal any toxic changes or indications of a carcinogenic potential of acrylic acid with the exception of slightly reduced (statistically not significant) consumption of drinking water.

Carcinogenicity and chronic toxicity of copovidone (Kollidon VA 64) in Wistar rats and Beagle dogs.

Kollidon VA 64 (copovidone, CAS-No. 25086-89-9) was administered in the diet to male and female Wistar rats (0, 700, 1400, and 2800 mg/kg body weight) for 24 months, and to male and female beagle dogs (0, 500, 1500, and 2500 mg/kg body weight/day) for 52 weeks. Clinical signs, body weight, food consumption, hematology, and gross and histopathological evaluations were conducted on both rats and dogs, and dogs also underwent hearing tests, ophthalmoscopic examinations, electrocardiograms, blood pressure measurement, and clinical chemistry and urinalysis evaluations. No adverse in-life observations related to treatment were observed in either species. The rats exhibited dark discoloration of the feces that was attributed to the intake and excretion of large amounts of test substance and was not considered to be an adverse effect. No treatment-related hematological changes, or gross or histopathological lesions were observed in either species that could be considered clinically relevant. Vacuolated histiocytosis in the mesenteric lymph nodes of four dogs that was not accompanied by inflammation or degenerative changes reflected histiocytic removal and degradation of the test article rather than a toxic effect. The results of these studies demonstrate the absence of any significant toxicological findings of high dietary levels copovidone in rats and dogs, resulting in no-observed-adverse-effect levels of 2800 mg/kg body weight/day in rats and 2500 mg/kg body weight/day in dogs, the highest doses tested.

Subchronic inhalation toxicity study of 2-ethylhexanol vapour in rats.

A 90-day subchronic inhalation toxicity study was performed on Wistar rats in accordance to OECD testing guidelines to evaluate the toxicological profile of 2-ethylhexanol, potential target organs, and a no-observable-adverse-effect-level (NOAEL). 10 males and 10 females per group were exposed to 2-ethylhexanol vapours at concentrations of 15, 40 and 120 ppm (the latter corresponding to the vapour saturation at 20 degrees C) 6 hours/day for 90 days. The respective controls inhaled clean air under the same conditions. No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, clinical biochemistry and haematological parameters including clotting time. Cyanide-insensitive palmitoyl-CoA oxidation, a marker for peroxisome proliferation, was found elevated in a subchronic study in Fischer 344 rats after gavage application of 500 mg/kg but not under the conditions of this 90-day subchronic inhalation study. There were no findings related to the treatment with 2-ethylhexanol either at necropsy or at histological examination. The highest concentration tested under these conditions (120 ppm) was found to be the NOAEL for male and female rats.

Long-term inhalation toxicity of N-vinylpyrrolidone-2 vapours. Studies in rats.

In previous subchronic studies inhaled N-vinylpyrrolidone-2 (NVP) was haemotoxic, hepatotoxic and irritant to the nose. In the first of two long-term studies, study A, Sprague-Dawley rats were exposed by inhalation to 0, 5, 10 or 20 ppm NVP (6 hr/day, 5 days/wk) for 24 months. Satellite groups were killed after 3, 12 or 24 months. In study B, female Sprague-Dawley rats were exposed to 0 or 45 ppm NVP for 3 months and killed at 3 or 12 and 24 months post-exposure. In study A, survival was unaffected, but reduced body weight gain, haemotoxicity, effects on clinical chemistry parameters indicative of hepatotoxicity, increased liver weight, hepatocellular carcinomas, necrosis, reparative hyperplasia, adenomas and adenocarcinomas of the nasal cavity, and squamous cell carcinomas of the larynx were seen. Increased tumour incidence was seen only in the liver and upper respiratory tract. In study B, the effect of NVP on body weight evident at 3 months disappeared before 1 yr, but effects on liver pathology persisted throughout the subsequent 21-month exposure-free period, and a few liver tumours were seen at 2 yr. As NVP gave negative results in a battery of in vitro and in vivo genotoxicity tests, it appears that the tumours that arose were manifestations of a non-genotoxic mechanism.

Subchronic inhalation and oral toxicity of N-vinylpyrrolidone-2. Studies in rodents.

N-Vinylpyrrolidone-2 (NVP) is a monomeric compound used as an industrial intermediate. Nine of 11 studies previously reported involved exposure of rats (two different strains), mice or hamsters to NVP by the inhalation route at concentrations of up to 120 ppm (6 hr/day, 5 days/wk) over a period of 1 wk to 12 months. The remaining two studies involved exposure of rats to NVP through the drinking water or by gavage at dose levels of up to 100 mg/kg body weight/day. Reduced body weight gain was seen in rats exposed by inhalation to 5 ppm or more for 3 months and in mice and hamsters exposed to 45 ppm for only 1 day. Effects were seen on haematological (reduced haemoglobin, erythrocyte count, haematocrit) and clinical chemistry parameters (specially raised gamma-glutamyltransferase activity and decreases in plasma protein), liver weight increase and liver lesions (centrilobular single-cell necrosis and foci of hepatocellular alteration) in rats and mice but not hamsters. Rats exposed to 40 mg/kg body weight/day NVP or more for 3 months by gavage developed similar liver changes. Atrophy of olfactory epithelium and hyperplasia of nasal respiratory epithelium was seen in rats exposed by inhalation to 5 ppm NVP for 7 wk but not in response to 1 ppm for 13 wk (no observed-adverse-effect level, NOAEL). These studies indicated that the upper respiratory tract and the liver are the main targets for NVP toxicity.

Thirteen-week oral toxicity study of synthetic lycopene products in rats.

Synthetic crystalline lycopene provides an alternative to extracts of naturally occurring lycopene for use in dietary supplements and functional foods. BASF Lycopene 10 CWD and Lyco Vit 10% formulated products each contain approximately 10% synthetic lycopene. These products were evaluated for toxicological and behavioral effects during a 13-week oral dosing study with male and female Wistar rats. Doses of 0, 500, 1500 and 3000 mg/kg body weight/day Lycopene CWD and 3000 mg/kg body weight /day Lyco Vit, as well as 3000 mg/kg body weight /day of the matrices used to formulate and stabilize each product, were administered by gavage to 10 rats/sex/day. A satellite group of five rats/sex received 0 or 3000 mg/kg body weight /day of each formulated product for an interim evaluation at 4 weeks of feeding. No statistically significant, dose-related effects on body weight, body weight gain, food consumption, hematology, urinalysis, clinical chemistry or ophthalmoscopic parameters were seen in any of the lycopene product or lycopene formulation matrix groups in comparison to the vehicle control group after 4 or 13 weeks of dosing. No deaths attributed to the test articles occurred during the study and the only clinical finding and at necropsy was the presence of red pigment in the feces and gastrointestinal tract that was associated with the red-pigmented test materials. No significant or dose-related abnormalities were found at necropsy or in microscopic evaluations of tissues collected at termination. Rats evaluated in home cages or in open field tests for behavioral and sensorimotor effects during the final week of the study showed no signs of treatment-related effects. The no-observed-adverse-effect level (NOAEL) for this study was concluded to be 3000 mg/kg body weight/day for both Lycopene CWD and Lyco Vit. The results of this study thus demonstrate the absence of any significant toxicological findings with Lycopene CWD and Lyco Vit products even at very high dose levels.

The effects of styrene on lung cells in female mice and rats.

Styrene has been shown to cause an increase in the incidence of lung tumors in CD-1 mice following chronic exposure at 40 and 160 ppm, whereas no treatment-related increase in tumors in any organ was seen in rats chronically exposed to up to 1000 ppm styrene. So far most of the mechanistic studies have been performed with male animals. The aim of the present study was to further elucidate the target cell population in mouse lungs exposed to styrene, and to investigate possible differential in vivo effects (e.g., glutathione depletion, increased lipid peroxidation, and oxidative DNA damage). Groups of female CD-1 mice were exposed to styrene at concentrations of 0, 172 or 688 mg/m3 (0, 40 or 160 ppm) for 6 h per day on 1 day, 5 consecutive days or for 20 days during a 4 week period. Groups of female Crl:CD rats were exposed to styrene at concentrations of 0, 688 or 2150 mg/m3 (0, 160 or 500 ppm) for a single 6 h period or for 6 h per day on 5 consecutive days. No signs of lung toxicity were observed in rats. The cytology of cells in lung lavage fluid provided no signs of an inflammatory response in either rats or mice. In mice, both exposure levels caused decreased CC16 protein concentrations in lung lavage fluid after 1 and 5 exposures and in mouse blood serum throughout the study, suggesting that styrene may cause destruction of Clara cells in mice. Degenerative lesions in mouse Clara cells (vacuolar cell degeneration, cell necrosis) were revealed by electronmicroscopy. After 5 and 20 exposures of mice at 160 ppm, cellular crowding, expressed as an irregular epithelial lining and indicative of a very early hyperplasia was noted. Although a depletion of glutathione was noted in mouse lung homogenates after 20 exposures, there was no evidence of oxidative stress as indicated by unchanged concentrations of 8-OH-deoxyguanosine. Malondialdehyde, an indicator of lipid peroxidation, was slightly increased in mice after 1 exposure at 160 ppm only.

Subchronic toxicity studies of 3-methyl-1-butanol and 2-methyl-1-propanol in rats.

1. 90-day subchronic toxicity studies with 3-methyl-1-butanol (MEB) and 2-methyl-1-propanol (MEP) were performed on rats to evaluate the toxicological profile of the compounds under conditions of drinking water studies, to identify the potential target organs, and to determine no-observable-adverse-effect-levels (NOAELs) respective of the substances. The test substances were administered to groups of 10 male and 10 female Wistar rats in drinking water at concentrations of 0, 1000 p.p.m. (about 80 mg/kg/d), 4000 p.p.m. (about 340 mg/kg/d) and 16,000 p.p.m. (about 1250 and 1450 mg/kg/d of MEB and MEP respectively). 2. 16,000 p.p.m. was found to be the maximal concentration for both alcohols applicable to rats in drinking water. Higher concentrations had an influence on palatability and could thus not be tested in drinking water studies. 3. At 16,000 p.p.m. MEB a marginal increase in the red blood cell count as well as a slight decrease in the mean corpuscular volume and the mean corpuscular hemoglobin content was observed in males only. These changes are considered to be treatment-related, although the toxicological significance of these findings is unclear. No other substance-related effects were found on body weight (b.w.), mortality, various parameters of clinical chemistry, organ weights, gross pathology and histopathology. 4000 p.p.m. MEB did not cause any substance-induced changes. Therefore, the NOAEL of MEB was defined as 4000 p.p.m. for male and 16,000 p.p.m. for female rats under conditions of oral application via drinking water. 4. MEP concentrations up to and including 16,000 p.p.m. did not induce any signs of toxicity and were therefore defined as the NOAEL respective of this substance for rats under conditions of drinking water application.

Aniline: early indicators of toxicity in male rats and their relevance to spleen carcinogenicity.

Early indicators of aniline hydrochloride (AH) toxicity were investigated in male Fisher 344 rats for 1 or 4 weeks at dietary dose levels of 10, 30 or 100 mg/kg body weight (bw)/day (actual intake at least 6, 17 and 57 mg/kg). The doses were based on earlier studies that had shown spleen toxicity and carcinogenicity in male rats at 100 mg/kg/day but not at 10 mg/kg/day. In the present study a dose-related formation of haemoglobin adducts and Heinz bodies was found from 10 and 30 mg/kg bw/ day, respectively, onwards. Signs of anaemia (decreased red blood cell counts and increased reticulocytes) were recorded from 30 mg/kg onwards. At 100 mg/kg, an overt haemolytic anaemia was associated with increases in serum transferrin concentration and total iron binding capacity in the blood reflecting major perturbations in iron metabolism. At this dose there was an increase in peripheral neutrophil leucocytosis in the blood, indicating an inflammatory process in the spleen. Histopathologic evaluation showed a focal perisplenitis and haemosiderin deposition in sinusoidal Kupffer cells of the liver at 100 mg/kg. These results corroborate the contention that carcinogenic doses of aniline cause early effects on haematological parameters, inflammatory reaction in the spleen and perturbations in iron metabolism as a result of haemolytic anaemia. Accordingly, the carcinogenicity of aniline may be linked to definable threshold-related processes.

NTA and Fe(III)NTA: differential patterns of renal toxicity in subchronic studies.

Differential patterns in terms of nephropathology and 8-hydroxyguanine formation in the course of oral 28-day studies were observed with nitrilotriacetic acid (NTA) and FeNTA. FeNTA, but not NTA, caused enhanced 8-hydroxyguanine formation in kidney DNA after oral and intraperitoneal administration. Enhanced lipid peroxidation in the kidney homogenate was observed with FeNTA as well as with NTA. For NTA, the low dose (9 mg/kg per day) was without adverse effect. The kidney toxicity of oral FeNTA (50, 200, and 1000 mg/kg per day) was only mild, 50 mg/kg per day; however, it still led to an increased 8-hydroxyguanine content. The relevance of Iron(III) (Fe(III)) or Fe(III)NTA formation as a relevant mediator of NTA-related toxicity was excluded on the basis of these data. Also, a thermodynamic consideration presented here, supports the view that zinc (Zn), and not Fe, is likely to mediate the tubular cell cytotoxicity of NTA.

The comparative toxicology of 4-chloro-2-methylphenoxyacetic acid and its plant metabolite 4-chloro-2-carboxyphenoxyacetic acid in rats.

4-Chloro-2-carboxyphenoxyacetic acid (CCPA) residues have occasionally been observed in crops treated with 4-chloro-2-methylphenoxyacetic acid (MCPA). The oral toxicity of MCPA and CCPA was compared in a 4-week rat study at a dietary concentration of 2000 ppm. CCPA was also given at 12,000 ppm (equivalent to 1g/kg bodyweight/day). MCPA at 2000 ppm caused reduced food consumption and body weight gain and increased water consumption in females only. Changes in clinical chemistry confirmed the liver as a target organ. Increased serum creatinine and urobilinogen, degenerated transitional epithelial cells in the urine showed that the kidney was also affected. Response to CCPA was confined to the 12,000 ppm dose. The target organs were liver and kidney as for MCPA. Microscopic examination revealed an increased severity of basophilic tubules and calcification at the outer/inner medulla transition in the kidneys. The results demonstrate that CCPA is less toxic than MCPA, that CCPA has no different toxicological end points when compared to MCPA, and that any risks associated with consumption of CCPA will not be underestimated if the CCPA residue is treated as if it were parent MCPA. Based on the MCPA-CCPA comparison, criteria for read across and minimal information requirements are proposed.

Association of putrescine, spermidine, spermine, and GABA with structural elements of brain cells.

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of "high-affinity uptake" showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg(2+) did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.

Detection of endocrine-modulating effects of the antithyroid acting drug 6-propyl-2-thiouracil in rats, based on the "Enhanced OECD Test Guideline 407".

The antithyroid acting drug propylthiouracil (PTU) was administered to male and female Wistar rats at 0, 0.1, 1, or 10mg/kg body weight for 4 weeks according to the draft protocol of the "Enhanced OECD Test Guideline 407" (enhanced TG 407) in order to investigate its suitability to detect endocrine-mediated effects. The study was conducted with two identical subsets of five animals per sex and dose each to provide data on sensitivity. The modified protocol includes the investigation of additional organ weights, pathology, and histopathology, of thyroid hormones, of spermatozoa, and of estrus cycle. At time of sacrifice, all females were in the diestrus stage as prescribed. Adverse effects were observed in the thyroid gland (hypertrophy/ hyperplasia) and the pituitary gland (hyperplasia of basophilic cells, hypoplasia of acidophilic cells) together with dose-related decreased serum triiodothyronine (T3) and thyroxine (T4) levels and increased thyroid stimulating hormone (TSH) levels. Other effects of PTU included decrease of organ weights, anaemia, impaired blood coagulation, and reduced activity of enzymes. Hence, some of the additional examined endpoints of the enhanced TG 407, e.g., examination of pituitary gland and thyroid hormones, were suitable to detect endocrine-modulating effects of propylthiouracil. Treatment of five animals provides sufficient sensitivity to detect the described adverse effects of propylthiouracil. The enhanced TG is currently under investigation in several laboratories, evaluation of all the results will allow determining its practicability as well as the most suitable additional endpoints.

Increase of ornithine decarboxylase activity elicited by reserpine in the peripheral and central monoaminergic systems of the rat.

Ornithine decarboxylase activity was increased about tenfold in adrenal glands and in brain regions preponderantly containing aminergic neurons, by a single dose of 16 mumol/kg of reserpine. Maximal enzyme activity in the adrenal glands was observed at about 8 hr after reserpine administration. The ornithine decarboxylase activity-time curves in the brain regions showed a concomitant polyphasic course, with the highest maximum at 12 hr postinjection. Ornithine decarboxylase induction is discussed as an early event in the cascade of molecular events preceding the induction of cell typic enzymes.

Tumor induction in mouse liver: di-isononyl phthalate acts via peroxisome proliferation.

Recently several chronic toxicity/carcinogenicity studies of di-isononyl phthalate (DINP) have been reported. These studies defined effect levels for liver tumors in male and female F344 rats at dietary levels exceeding 700 mg/kg/day; the no effect levels were 359 mg/kg/day in males and 442 mg/kg/day in females. Similar results were found in male B6C3F1 mice, but in female mice a significant increase in liver tumors was found at 336 mg/kg/day, making 112 mg/kg/day the NOAEL for liver tumors in that sex and species. DINP induces peroxisomal proliferation, and that, along with evidence that DINP is not mutagenic, is presumptive evidence for peroxisomal proliferation as the underlying mode of action for liver tumor development. To further explore the relationship between peroxisome proliferation and tumor induction in male and female mice, indicators of peroxisomal proliferation including liver weight, peroxisomal volume density, induction of peroxisomal enzyme activity, enhanced replicative DNA synthesis, and rates of apoptosis were measured at all of the dietary levels used in the chronic study in mice (500, 1500, 4000, and 8000 ppm, or approximately 100, 300, 800, and 1600 mg/kg/day). Liver weights, peroxisomal volume, and peroxisomal enzyme activity were significantly elevated in both male and female mice at the tumorigenic levels. Cell proliferation was also elevated in male and female mice, although the increases at levels below 4000 ppm in female mice were not significantly different from control values. Apoptosis was elevated at the 4000 and 8000 ppm levels, paralleling the increases in liver weight. These data along with previous results satisfy the criteria of the International Agency for Research on Cancer (IARC) and demonstrate that peroxisomal proliferation was indeed the mode of action for DINP-induced liver tumor induction in mice.

Prechronic toxicity studies on 2-ethylhexanol in F334 rats and B6C3F1 mice.

Data on the subchronic toxicity of 2-ethylhexanol (2EH) were required to establish the dose vehicle and dose levels for oncogenicity studies. In preliminary studies 2EH was given subacutely (11 days) to male and female Fischer 344 rats and B6C3F1 mice as an aqueous emulsion by oral gavage (0, 100, 330, 1000, and 1500 mg/kg/day). Clinical observations were made, body weights, food consumption, clinical chemistries, hematologies, and selected organ weights were measured, and gross and micropathologies were performed. Target organs were the central nervous system, liver, forestomach, spleen, thymus, and kidney in rats and the central nervous system, liver, and forestomach in mice. 2EH was then administered by oral gavage to male and female F344 rats 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 rat there was reduced body weight gain (6% male, 7% female), increased relative 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 and forestomach. There were no behavioral effects or effects on the spleen or thymus. A no-effect level for target organ effects in the rat was 125 mg of 2EH/kg/day. At 500 mg of 2EH/kg/day in the mouse the only effects were increased relative stomach weights in males (13%) and a low incidence of gross and microscopic findings in the forestomach (male and female) and liver (female). A no-effect level for target organ effects in the mouse was 125 mg of 2EH/kg/day. 2EH was a peroxisome proliferator in the rat 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/day for the absence of treatment-related effects in rats and mice, and 500 and 750 mg/kg/day, respectively, in rats and mice as high doses producing minimal toxicity without altering the life span.