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Archives of Toxicology (v.79, #11)

Use of the dog as non-rodent test species in the safety testing schedule associated with the registration of crop and plant protection products (pesticides): present status by Rainer J. Box; Horst Spielmann (pp. 615-626).

The results from a survey of the expert information that is publicly accessible on the use of the dog as test species during the regulatory evaluation of agricultural chemicals and pesticides are reported. Methods that are being used or considered in order to reduce the number of dogs used for this purpose are described. Regulatory evaluation aims at establishing threshold values for safe human exposure; it is based on no-observed-adverse-effect levels (NOELs) determined in animal studies. Current regulations require testing in two species, a rodent species (usually rat or mouse), and a non-rodent species (usually the dog). Subchronic (90-day) and chronic (12-month) repeated-dose feeding studies must be routinely conducted in dogs. This report first focuses on the results from a retrospective study analysing data on 216 pesticides kept on record by the Bundesinstitut für Risikobewertung, BfR (German Federal Institute for Risk Assessment), the competent regulatory authority in Germany. The study was sponsored and coordinated by SET, the German Foundation for the Promotion of Research on Replacement and Complementary Methods to Reduce Animal Testing (Stiftung zur Förderung der Erforschung von Ersatz-und Ergänzungsmethoden zur Einschränkung von Tierversuchen, Mainz) and conducted by the BfR. Since the data submitted for registration of a product is the property of the manufacturer, the study could only proceed with the collaboration of the German Association of Manufacturers of Agricultural Chemicals (Industrieverband Agrar, IVA). To ensure confidentiality, designated codes were used instead of the compounds’ proper names when the study was published. The results support two major conclusions. The use of the dog for the testing of pesticides is indeed necessary because the dog has proved to be the most sensitive species for about 15% of the compounds examined. However, chronic studies are only of limited value since they only provide essential information that cannot be obtained in sub-chronic studies in about 5% of cases. These conclusions are supported by several retrospective analyses using data on pharmaceutical drugs carried out in the context of the International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH). Over 90% of drugs elicited no toxic symptoms in 12-month studies in dogs in addition to those that had been recorded previously in studies conducted for 90 or 180 days in dogs and rats. Another approach comparing the results from pre-clinical animal studies with clinical studies noted that animal studies predicted about 70% of the effects observed in volunteers, and in about 94% of cases the effects occurred in animal studies lasting not more than one month. Furthermore, the report summarises the current methods under consideration that could refine or reduce the use of dogs in toxicity testing: industrial data sharing and harmonisation of guidelines, in vitro methods, human studies, computational prediction models, and integrated testing approaches. The integrated Agricultural Chemicals Safety Assessment (ACSA) testing scheme, which is currently being developed in an international project initiated by the International Life Sciences Institute (ILSI, USA), is of particular relevance, since an ambitious attempt is being made to design a new comprehensive test framework incorporating modern scientific approaches and covering most aspects of current regulatory testing requirements. The ACSA project has access to the pesticide database of the US EPA’s Office of Pesticide Programs (OPP). Preliminary results have confirmed the two major conclusions from the joint SET/BfR study conducted in Germany. Taking these results into account, it is recommended that the regulatory requirement for 12-month studies to be routinely carried out in dogs should be abandoned. While 90-day studies should be conducted in both rats and dogs, chronic studies should only take place in rats. If the dog is more sensitive than the rat in the 90-day study, an additional safety factor to the NOEL value obtained in the chronic rat study should be applied in order to set the threshold for safe human exposure, instead of conducting a 12-month study in dogs. This safety factor may be calculated from chronic NOEL data available in several pesticide databases. Chronic tests using dogs would then only be required if the test compound belongs to a new class of chemicals that has never been tested before. Thus, the report concludes that, according to current scientific knowledge, the routine 12-month studies in dogs are no longer required for agricultural chemicals and pesticides, and international regulations should be changed accordingly. Active international support of such measures is welcomed, from both an economical and an animal welfare perspective.

Keywords: Pesticides; Regulatory testing; Chronic toxicity studies; Study duration; NOEL; Dog

Ability of the Hershberger assay protocol to detect thyroid function modulators by Shuji Noda; Takako Muroi; Saori Takakura; Satoko Sakamoto; Mineo Takatsuki; Kanji Yamasaki; Susumu Tateyama; Ryoji Yamaguchi (pp. 627-635).

In vivo screening methods for detection of thyroid function modulators are now under development in many research laboratories. We assessed the applicability of the Hershberger assay protocol to screen for thyroid function modulators. In experiment 1, castrated male BrlHan WIST@Jcl (GALAS) rats were administered a potent thyroid peroxidase inhibitor, 3-amino-1,2,4-triazole (AT), in doses of 0, 40, 200, and 1,000 mg/kg/day with gravimetric endpoint, and in experiment 2, castrated and intact male rats were administered in doses of 0, 40, and 200 mg/kg/day, with quantification of the extent of hypertrophy of the thyroid epithelium, to assess the effects of castration, by gavage to 8-week-old for 10 consecutive days. At necropsy of both experiments, the thyroid glands and hypophysis were collected and fixed with 10% neutral-buffered formalin. To avoid crushing during weighing because of their fragility, the thyroid glands and hypophysis were weighed approximately 24 h after fixation with 10% neutral-buffered formalin. All animals were sacrificed approximately 24 h after the final dose. In experiment 2, the thyroid glands of all animals were stained with hematoxylin and eosin for histological examination and morphometry of follicular epithelial height. In experiment 1, absolute and relative thyroid weights in all of the AT groups were statistically increased in a dose-dependent manner, regardless of the testosterone propionate (TP)-injection. In experiment 2, the results showed a significant increase in thyroid weight in the 200 mg/kg groups of both castrated and intact rats. Hypophyseal weight was unaltered by AT, but comparison of vehicle-treated groups showed that the hypophyseal weight of the castrated rats was greater than that of the intact rats. Enlarged thyroid glands were observed in the AT-treated rats at necropsy. Histological examination of the thyroid glands of all the AT-treated animals showed hypertrophy and hyperplasia of the follicular epithelial cells, and the height of follicular epithelium of the thyroid glands increased in a dose-dependent manner in both the castrated and intact rats. In experiment 1, assessment of the (anti-) androgenic action of AT in seminal vesicle weight revealed a significant increase in the 200 and 1,000 mg/kg + TP groups in a dose-dependent manner. These results suggest that the effect of AT can be detected by the Hershberger assay 10-day administration protocol and may be useful for screening for thyroid function modulators regardless of whether the animals have been castrated.

Keywords: Hershberger assay; Thyroid function modulators; 3-amino-1,2,4-triazole; Castrated rat; Intact rat

Effect of low-level lifetime exposure to cadmium on calciotropic hormones in aged female rats by Malgorzata M. Brzóska; Janina Moniuszko-Jakoniuk (pp. 636-646).

The effect of low-level lifetime exposure to cadmium (Cd) on calciotropic hormones and the possible association between the Cd-induced disorders in bone metabolism and these hormones were investigated on a female rat model of human environmental exposure in areas unpolluted by this metal. For this purpose, the concentrations of 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D (1,25(OH)₂D), calcitonin (CT) and parathormone (PTH) were measured in the serum of control and Cd-exposed (1 mg Cd/l in drinking water for 24 months) female rats. Calcium (Ca) and inorganic phosphorus (P_i) serum concentrations, renal tubular reabsorption of Ca (TRCa) and phosphate (TRP) and the glomerular filtration rate (GFR) were estimated as well. Moreover, 1,25(OH)₂D, metallothionein (MT) and Cd were determined in the kidney. The exposure to Cd led to a decrease in the serum concentrations of 25OHD and 1,25(OH)₂D (by 50 and 31%, respectively) and the concentration of 1,25(OH)₂D in the kidney mitochondrial fraction (by 55%). The serum concentrations of CT and PTH increased (5.2-fold and by 29%, respectively) and those of Ca and P_i were unchanged, whereas the TRCa, TRP and GFR decreased due to the exposure to Cd. The results give evidence that the low lifetime exposure to Cd disturbs the metabolism of calciotropic hormones and damages the reabsorptive and filtrative function of the kidney in aged female rats. Numerous correlations noted between calciotropic hormones and the indices of kidney function, and indices of bone turnover and bone mineral status (bone mineral content and density) of these females indicate a relationship between these hormones and the kidney functional status and bone metabolism. The results of the present study together with our previous findings on the bone status in the experimental model allow for the conclusion that the low lifetime exposure to Cd by affecting the metabolism and proper function of calciotropic hormones may contribute to the advancement of bone damage at the elderly.

Keywords: Cadmium; Calciotropic hormones; Bone metabolism; Kidney; Female rats

Detection of phthalate metabolites in human saliva by Manori J. Silva; John A. Reidy; Ella Samandar; Arnetra R. Herbert; Larry L. Needham; Antonia M. Calafat (pp. 647-652).

In assessment of exposure to environmental contaminants, the use of unconventional matrices is becoming an increasingly important area of research. Saliva is one of the most promising alternative matrices because its collection is easy, noninvasive, and inexpensive. In this study, we measured the salivary concentrations of 14 phthalate metabolites in 39 anonymous adult volunteers using isotope-dilution, automated solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. Seven phthalate metabolites were detected at the concentrations ranging from below the limit of detection (<1 ng/mL) to 10.6 ng/mL for phthalic acid, 3.1 ng/mL for monomethyl phthalate (MMP), 91.4 ng/mL for monoethyl phthalate (MEP), 65.8 ng/mL for mono-n-butyl phthalate (MBP), 17.9 ng/mL for mono-iso-butyl phthalate, 353.6 ng/mL for monobenzyl phthalate, and 6.8 ng/mL for mono-2-ethylhexyl phthalate (MEHP). The frequency of detection was highest for MBP (85%) and lowest for MMP (8%). The median salivary MBP level in this group of adults was higher than the median serum MBP level in another non-occupationally exposed human adult population in the United States, whereas, the median salivary levels of MEP and MEHP were lower than the corresponding median serum levels. The frequency of detection and the salivary levels of each phthalate monoester in this study population were lower than the frequency of detection and urinary level of the same monoester in the general US population. Although urine is preferred for exposure assessment to non-persistent chemicals such as phthalates, the similar levels in serum and saliva suggest that saliva could be used as a surrogate matrix for measuring the bioavailable dose of phthalates in biomonitoring studies.

Acute effects of styrene inhalation on the neuroendocrinological system of rats and the different effects in male and female rats by Tomohiro Umemura; Norie Kurahashi; Tomoko Kondo; Yoko Katakura; Fumihiro Sata; Toshio Kawai; Reiko Kishi (pp. 653-659).

There have been several epidemiological and experimental studies about styrene from the neuroendocrinological viewpoint. Some reported that styrene exposure affected the neuroendocrinological system and enhanced prolactin (PRL) secretion, but others have denied those effects. It was assumed that styrene exposure caused depletion of dopamine (DA), which is a PRL inhibitor, and that, in consequence, the PRL level increased. However, not only DA but also many other factors control PRL secretion. Therefore, the mechanism of hypersecretion of PRL has not yet been clearly elucidated. In addition, effects of styrene on the female reproductive system have been reported, but the susceptibility needs to be further studied. Therefore, to investigate what causes hypersecretion of PRL and how different the susceptibility is in males and females, we studied acute effects of styrene exposure on the neuroendocrinological system in male and female rats. Immediately after exposure to 150 ppm styrene vapor for 10 days (8 h/day), male and female rats were killed, and blood and brain samples were collected. The styrene concentration in blood, hormones such as PRL, growth hormone (GH) and thyroid-stimulating hormone (TSH) in plasma and neurotransmitters in various brain regions were measured. The styrene concentration in the blood of female rats was higher than that in male rats, and the PRL level was significantly increased in female exposed rats compared with controls. No significant change was observed in male rats. We did not observe any significant changes in DA, 5-hydroxytryptamine (5-HT) or their metabolites. Because neurotransmitters were not affected in either male or female rats, the mechanism enhancing PRL secretion remains unclear. These results suggest that styrene exposure may cause hypersecretion of PRL and that the sensitivity to styrene exposure of the female may be higher than that of the male.

Keywords: Styrene; Prolactin; Dopamine; Inhalation; Sexual differences

Involvement of tumor necrosis factor-α in sulfur mustard-induced skin lesion; effect of topical iodine by Uri Wormser; Berta Brodsky; Elena Proscura; Julie F. Foley; Tinette Jones; Abraham Nyska (pp. 660-670).

Sulfur mustard (SM), also termed mustard gas, is a potent vesicant that elicits an inflammatory response upon exposure of the skin. Evaluation of mouse ear 3 h after SM exposure revealed acute inflammatory-cell aggregates in the vascular beds accompanied by strongly TNF-α-positive neutrophils. Eight hours after SM exposure, this phenomenon became intensified and associated with infiltration into the adjacent dermis. In ear skin topically treated with iodine, however, no inflammatory cells were observed 3 h after SM exposure; 8 h postexposure, blood vessels contained very few TNF-α-positive inflammatory cells. Since TNF-α induction was shown to be associated with reactive oxygen species production, we studied the effect of iodine on activated peritoneal mouse neutrophils. Iodine elicited a concentration-dependent reduction in the oxidative burst of activated neutrophils. Iodine also scavenged hydroxyl radicals generated by glucose oxidase in a concentration-dependent manner. The involvement of TNF-α in SM-induced skin toxicity was confirmed by reduction of 49 and 30% in ear edema following administration of 1 and 2 μg anti-TNF-α antibodies, respectively. These findings were corroborated by quantitative analysis of the histological findings showing 46% reduction in acute inflammation and no signs of subacute inflammation in the treated group, in contrast to the control group treated with SM only. Other epidermal (microblister formation, ulceration, and necrosis) and dermal (neutrophilia, hemorrhage, and necrosis) parameters also showed marked reductions in the antibodies-treated group in comparison to controls. The combination of iodine and antiTNF-α antibodies might constitute a new approach for treatment of SM-exposed individuals.

Keywords: Sulfur mustard; Tumor necrosis factor-α; Neutrophils; Iodine; Mouse-ear model

Dose-dependent liver regeneration in chloroform, trichloroethylene and allyl alcohol ternary mixture hepatotoxicity in rats by S. S. Anand; M. M. Mumtaz; H. M. Mehendale (pp. 671-682).

The present study was designed to examine the hypothesis that liver tissue repair induced after exposure to chloroform (CF) + trichloroethylene (TCE) + allyl alcohol (AA) ternary mixture (TM) is dose-dependent similar to that elicited by exposure to these compounds individually. Male Sprague Dawley (S-D) rats (250–300 g) were administered with fivefold dose range of CF (74–370 mg/kg, ip), and TCE (250–1250 mg/kg, ip) in corn oil and sevenfold dose range of AA (5–35 mg/kg, ip) in distilled water. Liver injury was assessed by plasma alanine amino transferase (ALT) activity and liver tissue repair was measured by ³ H-thymidine incorporation into hepatonuclear DNA. Blood and liver levels of parent compounds and two major metabolites of TCE [trichloroacetic acid (TCA) and trichloroethanol (TCOH)] were quantified by gas chromatography. Blood and liver CF and AA levels after TM were similar to CF alone or AA alone, respectively. However, the TCE levels in blood and liver were substantially decreased after TM in a dose-dependent fashion compared to TCE alone. Decreased plasma and liver TCE levels were consistent with decreased production of metabolites and elevated urinary excretion of TCE. The antagonistic interaction resulted in lower liver injury than the summation of injury caused by the individual components at all three-dose levels. On the other hand, tissue repair showed a dose-response leading to regression of injury. Although the liver injury was lower and progression was contained by timely tissue repair, 50% mortality occurred only with the high dose combination, which is several fold higher than environmental levels. The mortality could be due to the central nervous system toxicity. These findings suggest that exposure to TM results in lower initial liver injury owing to higher elimination of TCE, and the compensatory liver tissue repair stimulated in a dose-dependent manner mitigates progression of injury after exposure to TM.

Keywords: Hepatotoxicity; Mixture toxicity; Risk assessment; Ternary mixture; Tissue repair

The effect of brevenal on brevetoxin-induced DNA damage in human lymphocytes by Andrew Sayer; Qing Hu; Andrea J. Bourdelais; Daniel G. Baden; James E. Gibson (pp. 683-688).

Brevenal is a nontoxic short-chain trans-syn polyether that competes with brevetoxin (PbTx) for the active site on voltage-sensitive sodium channels. The PbTxs are highly potent polyether toxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. Blooms of K. brevis have been associated with massive fish kills, marine mammal poisoning, and are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in beach-goers. Additionally, the consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). The purpose of the present study was to determine whether PbTx could induce DNA damage in a human cell type, the lymphocyte, and if so, whether the damage could be antagonized or ameliorated by brevenal, a brevetoxin antagonist. The DNA damage may occur through both endogenous and exogenous physiological and pathophysiological processes. Unrepaired or erroneously repaired DNA damage may result in gene mutation, chromosome aberration, and modulation of gene regulation, which have been associated with immunotoxicity and carcinogenesis. A single-cell gel electrophoresis assay, or comet assay, was used to determine and compare DNA damage following various treatments. The data were expressed as tail moments, which is the percentage of DNA in the tail multiplied by the length between the center of the head and center of the tail (in arbitrary units). The negative control tail moment was 29.2 (SE=±0.9), whereas the positive control (hydrogen peroxide) was 72.1 (1.5) and solvent (ethanol) was 24.2 (2.1). The PbTx-2 (from Sigma, St. Louis, MO, USA), 10⁻⁸ M was 41.3 (3.6), PbTx-9 (Sigma), 10⁻⁸ M was 57.0 (5.3), PbTx-2 (from University of North Carolina at Wilmington, UNCW), 10⁻⁸ M was 49.4 (9.9), and PbTx-3 (UNCW), 10⁻⁸ M was 64.0 (6.4). 1.0 μg/ml brevenal applied 1 h before the PbTxs protected the lymphocytes from DNA damage; PbTx-2 (Sigma), 31.3 (2.1); PbTx-9 (Sigma), 35.5 (2.9); PbTx-2 (UNCW), 33.9 (1.4); PbTx-3 (UNCW), 34.9 (1.25). The tail moment for 1.0 μg/ml brevenal alone was 30.8 (2.6). The results indicate that extensive genotoxic damage is induced by PbTx-2 and 9 (Sigma), and PbTx-2 and 3 (UNCW) in normal human lymphocytes, which is fully antagonized by brevenal. This suggests that the immune systems of individuals exposed to PbTx during harmful algal bloom (HAB) events may be at risk.

Keywords: Brevetoxin; Brevenal; Lymphocyte; Gel electrophoresis; Tail moment

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Archives of Toxicology (v.79, #11)