Association of canine hypothyroidism with a common major histocompatibility complex DLA class II allele.

Dogs exhibit a range of immune-mediated conditions including a lymphocytic thyroiditis which has many similarities to Hashimoto's thyroiditis in man. We have recently reported an association in Doberman Pinschers between canine hypothyroidism and a rare DLA class II haplotype that contains the DLA-DQA1*00101 allele. We now report a further series of 173 hypothyroid dogs in a range of breeds where a significant association with DLA-DQA1*00101 is shown.

Cerebral and conjunctival haemorrhages associated with von Willebrand factor deficiency and canine angiostrongylosis.

A case of angiostrongylosis is described in a 14-month-old golden retriever bitch. Conjunctival haemorrhage and neurological signs, referable to a space-occupying cerebral lesion, were associated with defective primary haemostasis caused by low levels of von Willebrand factor. Full clinical recovery followed treatment with desmopressin, fresh whole blood transfusion, fenbendazole and supportive care. The magnetic resonance image of the suspected organising haematoma is described. Similarities to the human condition, acquired von Willebrand syndrome, and a possible role for aberrant larval migration in haematoma formation are suggested.

A comparative study of the sensitivity of the 3-induction and 9-induction Buehler test procedures for assessing skin sensitisation potential.

Assessment of skin sensitization potential is a mandatory requirement for the registration or notification of most types of chemicals and products. Until recently, two methods using the guinea pig as test model were the most widely accepted; the guinea pig maximisation test and the Buehler test. In the case of agrochemical formulations, which constitute the final end use product in contact with operators, industry and also some regulatory authorities consider the Buehler method more appropriate as the methodology is more relevant to likely exposure in the field. However, certain European regulatory authorities have become concerned about the sensitivity of the Buehler test for this purpose and have requested that a modified method is used in which additional applications of test materials are used during the induction phase of the protocol (a total of 9 rather than the normal 3). This study was designed to assess whether this modification was justified. Six reference substances (formaldehyde, alpha-hexylcinnamaldehyde, fragrance mix, thimerosal, mercaptobenzothiazole and phthalic anhydride); all mild to moderate skin sensitizing chemicals, were assessed in a study, which compared the use of 3 and 9 induction applications. The results of this study demonstrated that, although most of these sensitisers were detected by both protocols, the modified method (9 induction applications) was no more sensitive than the standard method (3 induction applications). As the modified protocol is also potentially more stressful to the animals, it is concluded that the use of additional induction applications in the Buehler test cannot be justified from either a scientific or an animal welfare perspective.

Tumorigenic potential of carbaryl in the heterozygous p53 knockout mouse model.

The heterozygous p53 knockout mouse model was used to assess whether vascular tumors noted in a 2-year carcinogenicity study in CD-1 mice with carbaryl were induced through a genotoxic mechanism. This knockout mouse model was selected for carbaryl because of the high sensitivity of this model to genotoxic events and its low spontaneous incidence of tumors until 9-12 months of age. Carbaryl was administered continuously via the diet to groups of 20 male heterozygous p53 knockout mice at concentrations of 0, 10, 30, 100, 300, 1000 and 4000 ppm for 180 days. Histopathological examinations revealed no evidence of carbaryl-induced neoplasms of any type. In particular, no neoplastic or preneoplastic changes were noted in the vascular tissue of any of the organs examined. Only neoplasms, recognized as those that occur spontaneously in untreated mice of this strain, were sporadically observed in a few animals from the intermediate dose groups with no evidence of a dose- or treatment-related effect. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) of carbaryl for neoplastic changes in male mice was 4000 ppm (around 716 mg/kg body weight/day). We conclude: (1) carbaryl does not appear to be a genotoxic carcinogen at least in male mice; (2) if the vascular tumors observed in the CD-1 mice are treatment-related, they could have been induced by a non-genotoxic mechanism; (3) the response in transgenic animals may provide useful complementary results to better assess carbaryl's potential genotoxic hazard to humans.

Critique of the paper: could pesticide toxicology studies be more relevant to occupational risk assessment? (by Ross et al., 2001).

The toxicology studies required for the registration of a pesticide are not necessarily well adapted to user safety assessment. This problem can be overcome in some cases by improving the guideline studies. In other cases the guidelines are based on animal models which are poor models for man; the most obvious example is rat percutaneous penetration. In other cases it may simply be impractical to have a model which can be extrapolated to all exposure situations. This is the case for intermittent exposure which is infinitely variable. The general conclusion is that the entire data package should be re-examined to make it more relevant to risk assessment.

The development of methods for assessing the in vivo oestrogen-like effects of xenobiotics in CD-1 mice.

The increasing awareness and concern about the potential health risks posed to the ecosystem and to man by endocrine disrupting chemicals with oestrogen-like activity in the environment has focused attention on the need for developing sensitive and specific methods for identifying these xenobiotics and to evaluate their degrees of toxic effects. We have conducted dose response studies in immature (21 days old) CD-1 female mice treated with four compounds, diethylstilboestrol (DES) (0.1 microg to 25 mg/kg body weight), alpha-zearalanol (0.5 mg to 25 mg/kg body weight), methoxychlor (0.5 mg to 500 mg/kg body weight) and bisphenol A (10 microg to 100 mg/kg body weight) administered subcutaneously daily for 3 days, and measured a number of uterine markers in treated and control (vehicle treated) mice. These were, in addition to the commonly measured changes in relative uterus weight and histopathological examination of uterine tissue, three other markers indicative of uterotrophic effects, namely, uterine luminal epithelium BrdU labelling index over the last 24 hr, peroxidase activity and lactoferrin expression. All of these markers showed clear dose-related increases in DES- and methoxychlor-treated animals. In the case of alpha-zearalanol treatment, relative uterine weight, peroxidase activity and lactoferrin expression showed dose-related increases at all the doses investigated. BrdU incorporation (an index of cell proliferation) also progressively increased at dose levels ranging from 0.1 mg to 5.0 mg/kg body weight, but apparently decreased at 25 mg/kg body weight. In contrast to these findings, bisphenol-A treatment showed no consistent changes in any of the four markers at the dose levels investigated. Additionally, studies were also conducted on a number of chemicals in CD-1 mice at one dose level. The chemicals investigated were: bisphenol A (1 g/kg body weight/day), naringenin (1 g/kg body weight/day) o,p'-DDT (500 mg/kg body weight/day), genistein (1 g/kg/day), coumestrol (0.5 mg/kg/day) and chlordecone (20 mg/kg/day) administered subcutaneously daily for 3 days. There was some variability in response of the markers perhaps indicating that the chemicals did not all act in the same way. The findings of our exploratory in vivo studies in CD-1 mice suggest that the measurement of a range of uterine markers, in addition to organ weight and histopathology, would provide useful information on the potential oestrogenicity of chemicals.

The p53 heterozygous knockout mouse as a model for chemical carcinogenesis in vascular tissue.

Heterozygous p53 knockout mice were investigated as a potential model for vascular tumor carcinogenesis. Groups of 20 male mice were exposed by gavage for 6 months to the vascular carcinogen urethane at 1, 10, or 100 mg/kg body weight/day. Wild-type and heterozygous p53 knockout control groups were exposed by gavage to the vehicle alone. Another group of 20 male mice received d-limonene by gavage (d-limonene is noncarcinogenic in mice). The high dose of urethane caused early mortality in the majority of mice associated with histopathologic evidence of toxicity and tumors, including a high incidence of benign and malignant vascular tumors, in all animals. At the intermediate dose, toxicity was less marked and 3 of 20 mice had tumors; mice that received the low dose did not have signs of toxicity or neoplasia. The two control groups had no tumors and the d-limonene group had one tumor of the prostate, which was considered spontaneous. We conclude that the p53 knockout mouse is a useful tool for investigating vascular tumorogenesis.

The significance of mouse liver tumor formation for carcinogenic risk assessment: results and conclusions from a survey of ten years of testing by the agrochemical industry.

A survey was performed on the results of 138 carcinogenicity studies conducted in various mouse strains by the agrochemical industry over the period 1983-1993. Data for liver tumor incidence, liver weight, and histopathology were collected along with data on genotoxicity. Studies were judged positive or negative for liver tumor formation on the basis of apparent dose response, malignancy, and difference from historical control values using a weight of evidence approach. Thirty-seven studies were judged to be positive for liver tumorigenicity in one or both sexes. There was no evidence showing an influence of the mouse strain and the duration of the study on the proportion of positive studies. Although 8 of the chemicals tested in the 138 studies were positive in the Ames test, only one of these was judged positive for carcinogenicity. Only 6 of the 37 positive chemicals had any other reported positive genotoxicity findings. A clear relationship between hepatomegaly at 1 year after exposure and a positive tumorigenic outcome at 18 months or 2 years after exposure was demonstrated. Whereas the average relative liver weight of top dose animals was 110% of control in negative studies, it was 150% in positive studies. Likewise, very few negative studies demonstrated significant pathological findings after 1 year, whereas the majority of positive studies had significant liver pathology. The implications of these findings for extrapolation to humans are discussed.

Methods for measuring dermal penetration of pesticides.

The quantitation of percutaneous absorption of pesticides is required as part of the registration, re-registration or hazard assessment process. There is a paucity of regulatory guidelines in this area. This paper presents three protocols that can be used to quantitate percutaneous absorption, primarily as a result of continuous skin exposure over a period equivalent to a working day (8 hr). A rat in vivo protocol, an in vitro protocol and a human in vivo protocol are described. None of these protocols is considered to be ideal and/or to represent a preferred method. The final choice of protocol must take into account the toxicity and physicochemical properties of the test molecule as well as cost and resource/technical ability. Nevertheless, the protocols described allow percutaneous absorption to be quantitated, and it is believed that, if adopted, they will prove useful in the regulatory and research areas for the acquisition of data under standard defined conditions.

Oral and dermal pharmacokinetics of triclopyr in human volunteers.

Blood levels and urinary excretion of triclopyr, the active ingredient in Garlon herbicides, were followed in six volunteers given single oral doses of 0.1 and 0.5 mg/kg body weight. Five of these volunteers later received dermal applications of Garlon 4 herbicide formulation equivalent to 3.7 mg triclopyr/kg body weight applied to the forearm. Following oral administration blood levels peaked at 2-3 h and declined to undetectable levels within 48 h; more than 80% of the dose was found as unchanged triclopyr in the urine. A two-compartment pharmacokinetic model was used to describe the time-course of triclopyr clearance; half-lives for the rapid initial and slower terminal phases were 1.3 h and 5.1 h respectively, and were independent of dose. Due to the slow half-life for dermal absorption (t1/2 = 16.8 h) the rapid initial elimination phase was obscured and the pharmacokinetics could be simplified by a one-compartment model. An average of 1.37% of the applied dose was recovered in the urine; when corrected for recovery after oral administration this was equivalent to an absorption of 1.65%. Triclopyr is slowly absorbed through skin and is rapidly eliminated. It has very low potential to accumulate in man or to be absorbed through the skin in acutely toxic amounts.

1,1,1-Trichloroethane: medium-term toxicity to carp, daphnids, and higher plants.

The toxicity of 1,1,1-trichloroethane to carp (Cyprinus carpio) and daphnids (Daphnia magna) and the effects of gaseous-phase exposure on the growth of higher plants (Sorghum bicolor and Brassica napus) were investigated. The test systems were designed to minimize loss of the volatile material during the exposure period. During an exposure period of 14 days, there were no mortalities or other symptoms of toxicity in carp exposed to a mean measured 1,1,1-trichloroethane concentration of 7.7 mg/liter. The survival and reproduction of daphnids over a test period of 17 days were not affected at a measured concentration of 1.3 mg/liter. Growth of emergent seedlings was not inhibited at measured gaseous-phase concentrations of 18 mg/liter for S. bicolor and 6.9 mg/liter for B. napus. The simple modifications made to standard techniques proved sufficient to maintain the exposure concentrations of this volatile chemical. Mean measured concentrations were generally greater than 60% of the nominal values.

Assessment of hazards to workers applying pesticides.

Exposure to pesticides as a result of their use in agriculture will vary according to the type of formulation, the method of application and the protective measures used. Quantitation of external exposure does not on its own predict the amount absorbed nor does it allow the toxic hazard to be assessed; information on skin penetration is also required. With the use of a suitable generic database for exposure, the assessment of many compounds would only require the measurement of skin penetration. With the knowledge of human dermal pharmacokinetics a field study can be performed which measures the absorbed dose directly and avoids the need for exposure measurement.

The experimental administration of lead to rats in neuropathological and behavioural studies.

The experimental investigation of the effects of lead have been studied for nearly 20 years. In that time, a variety of neurobiological processes have been reported to be altered following ingestion of lead. Most routes of administration and a bewildering number of doses have been employed to administer lead to experimental animals. However, inadequate characterization of dose regimes has impaired understanding of any effects, and their correlation to conditions of human exposure. As an index of lead burden, blood lead remains the most reliable means of assessing recent lead absorption. It is clear that a large number of model systems and dosing regimes do not address the problems of human clinical or subclinical lead intoxication. It is also clear from brain lead measurements in experimental animals that lead is selectively sequestered into specific areas of the control nervous system.

Minireview: the health implications of water treatment with ozone.

Ozone is a highly efficient disinfectant which may have significant advantages in water treatment compared to chlorine. It has, however, been shown that mutagenic and possibly carcinogenic byproducts may be produced under certain conditions of ozonation. Light chlorination following ozonization may meet the highest standards of disinfection. In addition the destruction of much of the organic matter by prior ozone treatment may well result in less harmful chlorinated and brominated products in the finished water. In many cases ozone treatment alone may suffice. It would be desirable to test with long term in vivo experiments which of the alternatives produces the best combination of microbiologically clean and pleasant water with minimum mutagenic and carcinogenic effect.

Dose response relationships during perinatal lead administration in the rat: a model for the study of lead effects on brain development.

Pregnant rats were dosed with 300, 600, 1000, 2000, 3000 or 4000 ppm inorganic lead through their drinking water during pregnancy and during lactation until 21 days post partum. An effect on live litter size was noted at the higher doses as was an increased tendency for sperm positive females to fail to produce a litter. Dose related responses were seen in the offspring for brain weight, body weight, body length and blood lead at birth and at age 21 days. In neonates from the dose groups above 1000 ppm Pb these 3 indices of growth indicated that there was an effect of lead on nutrition. The 300 ppm, 600 ppm and 1000 ppm dose groups showed no significant dose related effects for body length or brain weight and only slight effects on body weight. These low dose groups gave average blood lead values of 39.8 micrograms/100 ml blood, 54.6 micrograms/100 ml blood and 85.4 micrograms/100 ml blood respectively in the 21-day old neonates. These values correspond to blood lead values in children with so called "subclinical lead poisoning" and we suggest the use of this regime as a model to investigate effects of lead on development of the nervous system.

Effects of separate and combined chronic mercuric chloride and sodium selenate administration in rats: histological, ultrastructural, and x-ray microanalytical studies of liver and kidney.

The separate administration of mercuric chloride (HgCl2) and sodium selenate (Na2SeO4) to male rats in drinking water or a combined administration of both (50 ppm Hg, 15 ppm Se) caused different signs of toxicity over a 22 week period. The HgCl2 group showed histopathological and ultrastructural lesions as evidenced by periportal fatty degeneration and cell necrosis in the liver and tubular necrosis with proteinaceous casts in the kidney. The Na2SeO4 group showed the most severe depression of growth and food and water consumption, but no pathological changes were seen in the liver or kidney. Simultaneous administration of both toxicants produced a protective effect on weight loss and histopathology. These effects were associated with the formation of electron dense nuclear inclusions in kidney proximal tubule cells and similar electron dense formations in the reticuloendothelial cell cytoplasm and in the extracellular space of Disse in the liver. These formations were shown to contain both Se and Hg by energy dispersive X-ray microanalysis. The basis of the protective interaction of these two elements appears to result from an alteration of the chemical form or association of the mercury and selenium.

Early cellular effects of circulating cadmium-thionein on kidney proximal tubules.

Circulating cadmium-thionein (Cd-MT) is cleared from the mammalian circulatory system by filtration through the kidney glomerulus with subsequent reabsorption by kidney proximal tubules. Damage to the tubules results following uptake of Cd-MT, which is dependent upon time and the dose level of cadmium administered. Intravenous administration of 109Cd-MT at doses of 0.017 and 0.17 mg Cd/kg body weight with examination of total renal uptake of 109Cd at 0.5, 3, and 24 hr disclosed that the rate of clearance from the blood and uptake by the kidney was significantly more rapid at the 0.017 mg Cd/kg dose. Ultrastructural changes resulting from intravenous injection of either form A or B of Cd-MT were characterized by increased numbers of pinocytotic vesicles and small, dense lysosomal structures. There was no evidence of mitochondrial swelling or cell death at either 3 or 6 hr after injection. The subcellular distribution of cadmium in kidney tissue at various times after administration of Cd-MT was determined by using differential centrifugation techniques with 109Cd and in situ by using x-ray microanalysis. At 30 min after injection of Cd-MT, significant amounts of cadmium were present in lysosomal fractions indicating an interaction between the tubular lysosome system and Cd-MT prior to the onset of overt cellular toxicity. Results suggest that Cd-MT is reabsorbed and broken down by kidney tubule cells in a physiological manner with possible subsequent release of the toxic cadmium ion.

Effects of selenium, alone and in combination with silver or arsenic, in rats.

In two experiments, Sprague-Dawley rats were administered elemental selenium (Se; 10 ppm), silver (Ag; 1000 ppm) and arsenic (As; 50 ppm), either alone or in combination (Ag+Se or As+Se). Administration was via drinking water. Body weight, fluid intake, and food consumption were monitored weekly and measures of forelimb and handlimb strength were taken. Se depressed body weights in both experiments, as did As+Se. Food consumption relative to body weight tended to be increased by Se alone; none of the other treatments affected body weight, except for As+Se. Water consumption was depressed in all cases, which was attributed to a palatability effect. Limb strength was not affected by any treatment. The addition of Ag to the drinking water containing Se appeared to reduce the toxic effect of Se. However, As appeared to interact in a potentiating fashion with Se. There was 1 death in the Se alone group, none in the As group, but 7 out of 10 animals receiving As+Se had died by the end of the 18 week dosing period.