Human & Experimental Toxicology

Trends in survival and body weight were evaluated in 2140 control Sprague-Dawley-derived [Crl: COBS-CD(SD)BR and Crl: COBS-VAF CD(SD)BR] rats used for 24-month rat carcinogenicity studies between 1979 and 1991, Body weight and survival were remarkably stable in the CD-COBS rats used during 1979-1987: at 24 months, the mean survival in males was 68± 5%, and 60± 5% in females, With the CD-COBS-VAF rat, a variant of the CD-COBS strain used between 1988 and 1991, the survival at 24 months dropped to 41± 3% in males, and 44± 7% in females compared to the CD-COBS. The CD-COBS-VAF rat had a significantly reduced life span (P<0.001 at 24 months), a significant increase in mean body weight (males at 6 months : 672± 24 g vs. 536± 6 g; females : 359± 7 g vs 308± 3 g; P<0.001 ) and food consumption (males at 6 months : 31.3± 3.3 vs. 25.4± 2.1 g d^-1 ; females: 22,0± 2.7 g v. 20.3± 2.0 g d^-1 ; P<0.001). CD-COBS-VAF rats which failed to survive up to study termination had individual body weights at 3, 6 and 12 months which were significantly higher (P<0.001 ) than those which survived until 24 months. Our historical data base of control rats (CD-COBS and CD-COBS-VAF) in carcinogenicity studies revealed a significant (males:P<0.001; females:P<0.01) and inverse linear relation between mean 3-month body weight and 24-month survival. When compared to CD-COBS animals, CD-COBS-VAF rats showed an increase in the incidence of pituitary tumours in males, mammary fibroadenomas in females, an increase in the incidence and severity of glomerulonephrosis, and a greater incidence of animals which died without any obvious pathology. It is concluded that, in our Sprague-Dawley substrains, both the individual and the group mean body weights in early adult life appear predictive for the individual and group life expectancy. The decrease in longevity in the CD-COBS-VAF rat is principally due to disease and degeneration processes associated with fast growth and high body weight.

1 The preclinical animal safety testing strategy used in pharmaceutical development is generally successful, with the large majority of new medicines not causing serious toxicity in man. The use of the dog has significantly contributed to this achievement by aiding the selection of safe compounds for human trials.

2 The dog should be selected as the non-rodent species for pharmacodynamic, pharmacokinetic, metabolic and other scientific reasons, not solely for practical considerations.

3 The dog is an extremely useful laboratory animal, particularly for procedures requiring extensive handling, detailed observation or biological sampling.

4 Refinements in the use of the dog have been achieved, but further progress is desirable. Retrospective analysis of pharmaceutical toxicity data could encourage the regulatory flexibility necessary for further progress.

Two new cases of Veratrum poisoning are described. Clinical symptoms occurred quickly, within 30 min. Vomiting, a fall in blood pressure and bradycardia were observed. The outcome was favourable in both cases, producing a cure without sequellae. Examination of the literature showed that such cases are nearly always accidental, resulting from the difficulty in distinguishing Veratrum album and Gentiana.

1 Renal cortical slices isolated from the kidneys of male Wistar rats were used as an experimental model for studying the nephrotoxicity induced by 1,2-dichloropropane.

2 The solvent causes a depletion of renal reduced glutathione content and slight, but significant, lipid peroxidation. The block of the oxidative pathway with carbon monoxide prevents glutathione content depletion, and shows that this conjugation is the major step in 1,2-dichloropropane metabolism.

3 Loss of organic anion accumulation and release into the incubation medium of tubular enzymes, mainly from the soluble fraction, are the toxic effects of the solvent. The brush border is only slightly affected.

4 The mechanism of nephrotoxicity appears to occur via mercapturic acid metabolism. Acivicin and aminooxyacetic acid, inhibitors of gammaglutamyltransferase and β-lyase activity, respectively, partially but significantly prevent the loss of organic anion accumulation induced by 1,2-dichloropropane. Furthermore, α-ketobutyrate, an activator of β-lyase, enhances the effects of 1,2-dichloropropane on the target, but is itself toxic for organic anion accumulation.

1 A retrospective study, 1980 to 1989 inclusive, of hospital admission cases due to insect and scorpion stings or bites was carried out in the six main central hospitals of Zimbabwe's four main cities.

2 A total of 92 cases were recorded and analysed. This constituted 1.5% of all hospital admissions for poisoning which were analysed during that time.

3 Of the 92 cases the highest number of victims were seen in the 2-to-5-year age group (29.3%), followed by the 6-to-10-year age group (23.9%). In other age groups the incidence of poisoning was distributed approximately evenly with the 41 to 50 and the over 60-year age groups having the least number of victims, (2.2%).

4 The main groups of insects associated with poisoning were bees, 44.6%; wasps, 8.7%; and spiders 8.7%. A further 32.6% of the insect stings were not specified. Scorpion stings accounted for 5.4% of incidences.

5 No fatalities occurred.

6 Treatment consisted mainly of the administration of promethazine, as an antihistamine in 35% of the cases, steroids (16.5% of the cases), perhaps for their anti-allergic effects, and paracetamol, a mild analgesic, (14.7% of the cases). Interestingly, 22.6% of the cases were given an antibiotic (14.1 % of which were penicillins). Adrenaline was administered in only 1.7% of the cases.

7 The majority of the recorded cases, 49.4%, were hospitalized for at least a day and the average duration of hospitalization was 6.4 days.

Dimethylacetamide (DMAc) is a skin-penetrating solvent able to induce hepatic damage after chronic exposure. Previous research has indicated that metabolism may be saturated at its present TLV/TWA (10 ppm). Biological monitoring of monomethylacetamide (MMAc), the primary metabolite of DMAc, might therefore underestimate exposure to DMAC and related health hazards. We used the recirculating perfusion technique in isolated rat liver to evaluate DMAC metabolism. Medium concentrations starting at about 30, 50, 100 and 275 μM, respectively, were tested. Perfusate samples were taken regularly and analysed for DMAc; pharmacokinetic parameters (extraction ratio and clearance) were calculated for each perfusion. Inlet DMAc concentrations were calculated and concentration groups divided in 16, 36, 70, 160, 225 μM. The extraction ratio of the 16 μm group differed significantly from the other concentration groups tested. DMAc metabolism was saturated at a DMAc concentration of 36 μM. Extraction ratios were unaffected when cimetidine, an inhibitor of cytochrome P450 activity, was added to the perfusion medium or when cimetidine-pretreated animals were used. DMAc clearance was 2.20 ml min^-1 at a medium concentration of about 36 μM. Extrapolation of the observed (rat) liver clearance to man showed that airborne concentrations of 18 ppm would, under the presumptions used, lead to saturated metabolism of DMAc; however, saturation at even lower concentrations could not be excluded.

The subcutaneous administration of methyl isocyanate (MIC) to female rabbits, resulted in significant increases in haemoglobin concentration, erythrocyte volume fraction and leucocyte number in blood, as well as plasma total proteins, and urea. The present study was designed to investigate whether the hydrolytic products of MIC, methylamine (MA) and N,N'-dimethylurea (DMU) play any role in eliciting these changes. Both MA and DMU administered subcutaneously in an equimolar dose to that of 1.0 LD₅₀ MIC, 2.2 mmol kg^-1, had no influence on these parameters, although there was a marginal increase in the plasma urea level shortly after the administration of DMU. This study establishes that the observed haematological and biochemical changes induced by MIC intoxication in rabbits are mostly due to MIC.

Hyperlipidaemia commonly develops in both transplant recipients and experimental animals receiving cyclosporin A (CsA). However, the threshold of CsA induced-changes on lipoproteins and the role of parenteral vehicle (cremophor) has not been defined.

Male Wistar rats were classified into five groups of six animals each and received CsA in cremophor vehicle at doses of 5, 10 or 20 mg kg^-1 d^-1, s.c., vehicle alone or saline for 7 d. Blood was obtained 24 h after the last dose and plasma was analysed. Plasma very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein subfractions (HDL-2, HDL-3) were isolated by sequential ultracentrifugation and their content of cholesterol, triglyceride and phospholipid was determined. Whole blood and trough plasma CsA levels were measured by monoclonal radioimmunoassay.

Plasma lipids did not differ significantly among the five groups. At a dose of 20 mg kg^-1 d^-1 of CsA VLDL cholesterol rose significantly (P<0.05). Administration of either CsA or cremophor vehicle increased HDL-2 phospholipids (P<0.05) and decreased HDL-3 cholesterol. There was not a linear relationship between whole blood and plasma CsA levels and increasing CsA doses.

Short-term treatment with low doses of CsA have little influence on lipid profile in the rat. Changes on lipoprotein composition can be attributed mainly to cremophor vehicle, conceivably due to its ethanol content.

The histamine H₂-receptor antagonist drug, cimetidine (CM), was investigated to determine its effect on the metabolism of 'model' alkoxyphenoxazone substrates ethoxyresorufin (ER) and pentoxyresorufin (PR). The investigation was carried out under different conditions in rat liver microsomes from rodents pretreated with various classical cytochrome P450 inducers. CM exerted a dual effect on uninduced and PB-induced liver microsomal ethoxyresorufin-O-de-ethylase (EROD) activities; it was initially stimulatory but became inhibitory. However, when 3-MC-induced preparations were used, CM only exerted an inhibitory effect on EROD activity over the whole concentration range (0.01 μM-20 mM). Pre-incubating PB-induced and uninduced liver microsomes with NADPH before the addition of ER and CM decreased the stimulatory effect of CM and increased the inhibitory effect in the concentration range (5-20 mM). With 3-MC-induced preparations, the inhibition of EROD was only marginally potentiated. Overall conclusions for the diphasic effects of CM on biological activities were due to CM binding with differing affinities to different P450s. Subsequent decreases and increases in stimulation and inhibition, respectively, on pre-incubation with NADPH were thought to be due to an increased affinity of CM for reduced cytochrome P450.

1 Following daily administration to male albino rats for 2 weeks, β-naphthoflavone (25 and 60 mg kg^-1, i.p.) and phenobarbitone (100 mg kg^-1, p.o.) produced their characteristic patterns of induction of P450-related enzyme activities. β-naphthoflavone also induced p-nitrophenol glucuronidation by 160% over controls, while phenobarbitone produced only a 45% induction of this conjugation activity.

2 β-Naphthoflavone produced significant reductions in plasma thyroxine (T₄) concentrations on days 4 and 16 and also decreased tri-iodothyronine (T₃) on day 4. Thyroid-stimulating hormone (TSH) was significantly increased on day 16 by the higher dose of β-naphthoflavone. Thyroid weight was significantly increased at both dose levels on day 16 and liver weight was significantly increased on both days 4 and 16. No histopathological changes were seen in the liver or pituitary, but 30% of the animals showed minimal to mild follicular epithelial hypertrophy of the thyroid gland.

3 Phenobarbitone had no effect on T₄ concentrations, but significantly decreased T₃ on day 4. TSH increased by 60% on day 16, but was not statistically significant compared with controls. Thyroid weight was significantly increased on day 16 and liver weight was significantly increased on days 4 and 16. Mild to moderate thyroid follicular epithelial hypertrophy and moderate hepatocyte hypertrophy occurred in all animals. No histopathological changes were seen in the pituitary.

4 The early changes in T₄ and/or T₃ were probably due to increased hepatic clearance by induction of thyroxine glucuronidation with both compounds. Thyroid hypertrophy would be expected to follow as a result of activation of the hypothalamic-pituitary-thyroid axis. Data for the two compounds suggest some variation in the precise mechanism of action, which has not yet been fully elucidated.

The literature contains only one report of methylene chloride poisoning after ingestion.¹ Unfortunately carboxyhaemoglobin (COHb) levels were not obtained in this case. We now describe a fatal case where a similar amount of the solvent was ingested and after which toxicology screening was done.

Ninety-day-old Sprague-Dawley rats were intoxicated for 70 d with lead, given either as 0.3% lead acetate in drinking water or by inhalation as 5 mg m^-3 lead oxide. Direct or transmitted lead toxicity for the male reproductive system was assessed in the rats and their offspring from pituitary and genital organ weights after exposure, the numbers of Sertoli and germ cells, the number, motility and morphology of epididymal spermatozoa, the levels of plasma testosterone, LH and FSH and fertility tests. Whole blood lead levels were similar after lead ingestion and after inhalation (58.0 ± 1.7 μg dl^-1 vs. 51.1 ± 1.8 μg dl^-1). Lead acetate ingestion did not affect the reproductive system or fertility of rats. Inhalation of lead oxide did not affect fertility either, but seminal vesicle weight dropped significantly, which might suggest an alteration in the pattern of testosterone secretion. In the male progeny of sires that inhaled lead, the number of epididymal spermatozoa decreased but this did not interfere with fertility. Our results show that for the doses studied, lead inhalation and lead ingestion do not produce strikingly different effects on the male rat's reproductive system. Differences between the present findings and those of others might be due to difference of rat strain or of age at exposure.

1 This study assessed urinary creatine excretion as a marker for testicular atrophy.

2 Male rats received a single i.p. dose of 2-methoxyethanol at 0, 250 or 750 mg kg^-1 and were sacrificed 2 d later. Urinary creatine and creatinine excretion were measured on days 0, 1 and 2.

3 Decreased testicular weights and histopathological assessment revealed dose-related testicular damage.

4 On day 1, at both doses of 2-methoxyethanol, urinary creatine levels increased and creatinine levels decreased, resulting in a dose-related increase in the creatine/creatinine ratio. On day 2, the creatine/creatinine ratio was elevated relative to controls, but was less marked than on day 1.

5 The study confirmed that creatine excretion is a potential marker for acute testicular damage.

The ability of high doses of flumazenil to antagonize ethanol-induced sedation was assessed via ethanol-induced loss of the righting reflex (LRR) in rats. Ethanol (3.5-5 g kg^-1 i,p) produces a dose-dependent increase in the proportion of animals presenting a LRR response. Flumazenil (0.1 to 1 mg kg^-1 i.p) was ineffective in preventing the suppression of the righting reflex and even aggravated it for ethanol dosages of 3.5 and 4.0 g kg^-1. These data support the view that flumazenil has no effective 'anti-ethanol' properties. Conversely, the prolongation of mean sleep time provoked by the antidote could be related to the suppression of a putative endogenous inverse-agonist activity at the benzodiazepine receptor, which could be implicated in the mechanism of the acute tolerance to ethanol.

D-Glucaro(1,4)lactone, a potent β-glucuronidase inhibitor was attached via the carboxylic acid moiety to polyvinylbenzyl chloride (PVBC) using a bimolecular nucleophilic displacement reaction, First, the caesium salt of D-glucaro(1,4)lactone was prepared by titrating the carboxylic acid to neutrality with aqueous caesium bicarbonate. The polyvinylbenzyl D-glucaro(1,4)lactonate was obtained in maximum yields of between 50 and 60% when caesium D-glucaro(1,4)lactonate was incubated with PVBC in DMF at 50°C for 7 d in the presence of a catalyst, caesium iodide.