An evaluation of the carcinogenic potential of the herbicide alachlor to man.

Chronic bioassays have revealed that alachlor caused nasal, thyroid, and stomach tumours in rats but was not carcinogenic in mice. Significant increases in thyroid and stomach tumours were observed only at doses that exceeded the maximum tolerated dose (MTD). While nasal tumours were found at doses below the MTD, they were small and benign in nature. This publication describes the work undertaken by Monsanto to understand the carcinogenic mode of action of alachlor in the rat and to investigate the relevance to humans. The genetic toxicity of alachlor has been investigated in an extensive battery of in vitro and in vivo test systems. In addition, target-specific mutagenicity tests, such as the COMET assay and DNA binding in nasal tissue, were carried out to investigate any possible in-situ genotoxic action. The weight-of-evidence analysis of all available data clearly demonstrates that alachlor exerts its carcinogenicity in the rat by non-genotoxic mechanisms. In the rat, alachlor is initially metabolised primarily in the liver through the P-450 pathway and by glutathione conjugation. The glutathione conjugates and their metabolites undergo enterohepatic circulation with further metabolism in the gastrointestinal tract, liver, and then nasal tissue where they can be converted to a diethyliminoquinone metabolite (DEIQ). This electrophilic species binds to the cysteine moiety of proteins leading to cell damage and increased cell turnover. When comparisons of in vitro nasal metabolic capability were made, the rat's capacity to form DEIQ from precursor metabolites was 38 times greater than for the mouse, 30-fold higher than monkey, and 751 times greater than that of humans. This data is consistent with the results of studies showing in vivo formation of DEIQ-protein adducts in the nasal tissue of rats but not mice or monkeys. The lack of DEIQ nasal adducts in mice is consistent with the lack of nasal tumours in that species. When the differences between rat and humans in the capacity for initial glutathione conjugation by the liver and nasal tissue are also taken into account, the rat is found to be even more susceptible to DEIQ formation than man. Based on this, it is clear that the potential for DEIQ formation and nasal tumour development in humans is negligible. The mechanism of stomach tumour formation has been studied in the rat. The results demonstrated that the mechanism is threshold-sensitive and involves a combination of regenerative cell proliferation and a gastrin-induced tropic effect on enterochromaffin-like (ECL) cells and stem cells of the mucosal epithelium. The absence of a carcinogenic effect in mice and of any preneoplastic effect in monkeys treated with very high doses is indicative ofthe species-specific aspect of this mechanism of action. The results of studies on thyroid tumour production indicate that alachlor is acting indirectly through the pituitary-thyroid axis by increasing the excretion of T4 by enhanced glucuronidation and subsequent biliary excretion. The increased excretion reduces plasma T4 levels and a feedback mechanism leads to increased synthesis of TSH by the pituitary. Chronic stimulation of the follicular epithelium of the thyroid by TSH produces hyperplasia and ultimately tumour formation. This non-genotoxic, threshold-based mechanism is well established and widely considered to be not relevant to humans. In this work, the modes of action for the three types of tumours elicited in the rat by alachlor were investigated. All are based on non-genotoxic, threshold-sensitive processes. From all the data presented it can be concluded that the tumours detected in the rat are not relevant to man and that alachlor presents no significant cancer risk to humans. This conclusion is supported by the lack of mortality and tumours in an epidemiology study of alachlor manufacturing workers.

Neurotoxicity evaluation of rats after subchronic inhalation exposure to isobutanol.

The subchronic neurotoxic effects of isobutanol were studied by exposing Sprague-Dawley rats to isobutanol vapor concentrations of 0, 250, 1000, and 2500 ppm for 6 hrs/day, 5 days/wk, for 3 months. A comprehensive set of neurotoxicity tests (functional observational battery, motor activity, perfusion fixation neuropathology, and schedule-controlled operant behavior) including an assessment of complex behavior dependent on learning and memory was conducted. In addition, full histopathology and blood chemistry evaluations were conducted in order to assess any potential functional/behavioral effects in the context of other possible systemic toxicities. There were no morphological or behavioral effects indicative of a specific, persistent or progressive effect of isobutanol on the nervous system at exposure concentrations up to 2500 ppm. A slight decrease in response to external stimuli was observed during exposures at all concentrations. These effects are likely transient effects of acute exposure to isobutanol.

Mechanism of thiazopyr-induced effects on thyroid hormone homeostasis in male Sprague-Dawley rats.

Chronic administration of thiazopyr in the diet at dose levels of 1000 and 3000 ppm, but not 100 ppm, has demonstrated an increase in thyroid follicular cell tumors in male Sprague-Dawley rats. In the studies reported here we have evaluated the mechanism of thiazopyr-induced thyroid tumors by studying the effect of thiazopyr on a number of endpoints that indicate hypothalamic-pituitary-thyroid homeostasis. At a dose level of 3000 ppm, thiazopyr caused a marked depression in circulating levels of T4 as soon as 7 days after commencement of treatment. Concurrent with this decrease in T4 was an increase in TSH levels, an increase in thyroid and liver weights, a three- to sixfold increase in hepatic T4-uridine diphosphate glucuronosyl transferase (UDPGT) activity, and increases in thyroid follicular cell hypertrophy and hyperplasia. Dose-related changes associated with thiazopyr treatment were significant increases in liver weight, thyroid weight, and hepatic T4-UDPGT activity at high doses. Increased levels of serum TSH, T3, and rT3, decreased levels of T4, and an increased incidence of thyroid follicular cell hypertrophy and hyperplasia were observed 56 days after the initiation of 3000 ppm thiazopyr. All the changes, except thyroid weight, were partially or completely reversible upon removal of thiazopyr from the diet. Increased thyroid T4 elimination, primarily via increased hepatic conjugation by T4-UDPGT, resulting in decreased serum T4, appeared to be responsible for the increased TSH levels. The sustained increase in TSH by thiazopyr appears responsible for the stimulation of the thyroid follicular cells resulting in follicular cell hypertrophy, hyperplasia, and ultimately neoplasia. In summary, evidence is presented for a hormonally mediated, threshold-dependent process for the development of thyroid follicular cell tumors from high-dose thiazopyr administration in male rats. This mechanism is not considered to be relevant to humans, since the thyroid of humans is much less sensitive to this pathogenic phenomenon than rodents.

Mode of action of thyroid tumor formation in the male Long-Evans rat administered high doses of alachlor.

Chronic administration of alachlor in the diet at a level of 126 mg/kg/day has previously been shown to cause an increase in benign thyroid follicular cell tumors in male Long-Evans rats. Studies were conducted to elucidate the mechanism of the alachlor-induced thyroid tumors in the male rat by evaluating changes in parameters that are collectively associated with a hormonally mediated mode of action for thyroid neoplasia. Male Long-Evans rats were administered 126 mg alachlor/kg body wt/day via the diet for up to 120 days. One group of animals was removed from alachlor-treated diet after 60 days and received untreated diet for an additional 60 days. Liver and thyroid weights and serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH), as well as hepatic uridine diphosphate glucuronosyl transferase (UDPGT) activity, were determined at 7, 14, 28, 60, and 120 days of treatment. Liver and thyroid weights, hepatic UDPGT activity, and circulating levels of TSH were significantly increased in animals administered alachlor. These increases were seen as early as 7 days after alachlor administration. Circulating levels of T4 in alachlor-treated animals were significantly decreased compared with controls at 7 days, but had returned to control levels by 60 days. T3 levels were elevated at all time points except at 28 days. The changes in TSH and T3 levels, hepatic UDPGT activity, and liver weights were all reversible on elimination of alachlor from the diet. Thyroid weights did not completely return to control levels after removal of alachlor from the diet, although some recovery was evident. The results of this study clearly suggest that alachlor-induced thyroid neoplasia, observed in previous chronic bioassays with alachlor, was associated with increases in circulating TSH levels. Increased metabolism of T4 via hepatic enzymatic conjugation (i.e., T4-UDPGT) appeared to be responsible for the increased TSH levels. These effects were shown to be reversible on cessation of exposure to alachlor. In summary, evidence is presented for a hormonally mediated process for the development of thyroid follicular cell tumors.

A study of the mechanism of butachlor-associated gastric neoplasms in Sprague-Dawley rats.

Long term administration of butachlor to Sprague-Dawley rats in a previous bioassay, resulted in the induction of gastric neoplasms which occurred only in the highest dose group (3000 ppm in the diet), primarily in females and specifically in the fundic region. The tumors were a composite of highly undifferentiated enterochromaffin-like (ECL) cells and mucus producing cells with morphologic characteristics unlike those previously described in the rat stomach. Mucosal atrophy of marked intensity was a consistent feature of the gastric mucosa in animals from the highest dose group. An additional long term study was conducted in female Sprague-Dawley rats at dietary levels of 0, 100, 1000 and 3000 ppm to explore the mechanism(s) involved in the formation of these neoplasms. Cell proliferation was evaluated in both fundic and pyloric regions of the stomachs of rats at multiple time periods from 14 days to 26 months. Mucosal thickness was determined in the fundic region at the same time intervals as were used for cell proliferation studies. Gastric pH and gastric acid production were measured after approximately 21 months of exposure. Serum gastrin levels were analyzed at 14, 60, and 120 days and at 6, 18 and 20 months. Cholecystokinin (CCK)/gastrin receptor binding studies were conducted on samples of four tumors and pooled fundic mucosa from five animals in the control group. Cell proliferation was increased in both the neck and base regions of the fundic mucosa at nearly all time points measured from 14 days to 26 months. The magnitude of the changes in the base region were substantially greater than those in the neck region. Fundic mucosal thickness was decreased beginning at the 30-day time point and continued at all intervals, being less than one half that of controls at 20 and 26 months. Gastric pH in rats from the highest dose was elevated to nearly twice control levels at 21 months. Gastric acid secretion was dramatically decreased in animals from the 3000 ppm group and was moderately decreased in the 1000 ppm group at 21 months. Hypergastrinemia was observed at the 3000 ppm level only, beginning at 120 days with progression to extremely high levels by 18 months. CCK/gastrin receptor binding was demonstrated in all tumors studied, at levels comparable to or higher than that of the pooled control sample. All changes involved only the fundic region, the site of tumor formation. Tumors occurred only in animals from the 3000 ppm level, the only level at which hypergastrinemia occurred.(ABSTRACT TRUNCATED AT 400 WORDS)

Identity and pathogenesis of stomach tumors in Sprague-Dawley rats associated with the dietary administration of butachlor.

Macroscopic stomach tumors induced in Sprague-Dawley rats during two chronic bioassays with the acetanilide herbicide butachlor at a dietary concentration of 3000 ppm, were evaluated histologically and immunohistochemically in order to determine their identity and pathogenesis. The tumors, which occurred primarily in female rats, were a heterogeneous series, including a few consisting wholly or partly of classic solid or anaplastic epithelium, but with the majority containing diffusely distributed primitive neoplastic cells. The latter had either the general appearance of undifferentiated epithelium or presented a more "mesenchyme-like" pattern where the cells were epithelioid, blastema-like, neuroendocrine-like or sarcoma-like with fascicular disposition. Gastric glandular profiles were also present, usually located near the periphery of the tumors, but in some cases extending into the diffuse tumor tissue. Most of the tumors displayed variable immunohistochemical reactivity for cytokeratin, vimentin and neuron-specific enolase but were negative for muscle-specific actin or desmin except in the stromal tracts. Detailed examination of all available gastric tissue revealed the presence of additional microscopic neoplasms and precursor hyperplastic lesions. All of these were typical gastric neuroendocrine cell lesions (gastric carcinoids) originating in the fundic mucosa but occasionally invading submucosally, and consisting of epithelial cells in organized clusters, rosettes or primitive tubules. The enterochromaffin-like (ECL) nature of these microscopic neoplasms and precursor lesions was substantiated by strong immunohistochemical reactivity for cytokeratin, neuron-specific enolase and chromogranin A, and a negative reaction for vimentin. One microscopic tumor showed a transition from differentiated neuroendocrine type in the fundic mucosa to a dispersed "mesenchyme-like" pattern in the submucosal extension. An additional finding in the butachlor-treated male and female rats was atrophy of the fundic mucosa involving, in particular, reduction in the numbers of parietal cells. This effect was dose-related, being most severe in the high-dose (3000 ppm) females. On the basis of their morphological characteristics, coupled with the continuity evident in the microscopic lesions, it is concluded that the macroscopic stomach tumors associated with the dietary administration of butachlor are poorly differentiated gastric carcinoids, in some cases admixed with a non-neuroendocrine epithelial element. Fundic ECL and stem cells are known to be under the trophic influence of gastrin, which is apparently responsible for the induction of the tumors associated with butachlor administration. Gastric tumor development involving gastrin is recognized as a secondary, hormonal mechanism of carcinogenesis, demonstrating a dose-threshold phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicology of AMSA in beagle dogs and CDF1 mice.

The toxic effects of AMSA in single-dose (x1) or five-daily-dose (x5) regimens were studied in male and female beagle dogs and CDF1 mice. AMSA was administered orally via gelatin capsules to dogs and as a Klucel suspension to mice. Suitable placebo controls were evaluated. Doses in dogs ranged from 62.5 to 1000 mg/m2 (lethal dose [LD]) for the x1 study and from 31.25 to 500 mg/m2 (LD) for the x5 study. Dogs given the LD (x1 and x5) had degenerative lesions in the gastrointestinal mucosa, depletion of bone marrow hematopoietic tissue, and lymphoid depletion. These lesions were not seen at doses lower than the LD in the x1 study but were present in the x5 study, with severity related to dose. Toxicity appeared to be reversible at the lower doses since animals killed after a 45-day observation period had none of the above lesions. Clinical signs of emesis, diarrhea, and weight loss correlated with the above lesions as did the depressed wbc counts. The toxicity in dogs was dose- and schedule-dependent. The 14-day LD10, LD50, and LD90 values (mg/m2) for mice in the x1 study were as follows: LD10, 440 in males and 475 in females; LD50, 810 in males and 728 in females; and LD90, 1489 in males and 1117 in females. Mortality was observed initially on Day 4. Single-dose toxicity studies were conducted in mice by oral administration of doses equal to one-half of the LD10, the LD10, and the LD50. The major drug-related lesions in mice included thymic degeneration and atrophy and bone marrow depletion at the higher dose levels, while the major drug-related lesions in dogs included enteric mucosal degeneration and generalized lymphoid and bone marrow depletion. The toxicity of AMSA was reversible in mice and dogs.

Determinants of the toxicity of L-alanosine to various organs of the mouse.

Toxicologic and biochemical properties of the antitumor antibiotic, alanosine [L-2-amino-3-(N-hydroxy,N-nitrosamino)propionic acid], were studied in mice. The LD50 of L-alanosine (given intraperitoneally) was approximately 2 g/kg; L-5178Y/AR tumor, small intestine, liver, and lung were the tissues more consistently or severely damaged by the drug. L-5178Y/AR tumor, small intestine, liver, and lung, which were more susceptible to damage by L-alanosine, showed high concentrations of the putative active antimetabolite of L-alanosine, "L-alanosyl-AICOR", and either high concentrations of SAICAR synthetase, which forms this conjugate or low specific activities of adenylosuccinate lyase, the enzyme believed to decompose it. In addition, a low specific activity of the enzyme, adenylosuccinate synthetase, appeared to predispose an organ to the toxicity of alanosine. These data are compatible with the hypothesis that "L-alanosyl-AICOR" is the molecule responsible both for the therapeutic and toxicologic effects of L-alanosine and suggest that it is the dynamic interplay of the synthesizing enzyme, the catabolizing enzyme, and the target enzyme which determines whether this anabolite accumulates to a concentration capable of inflicting cellular damage.

Preclinical toxicologic evaluation of dianhydrogalactitol in dogs and monkeys.

Thirty dogs in four different treatment schedules and 14 monkeys in a single multiple-treatment schedule were used to evaluate the toxicity of dianhydrogalactitol. Highest nontoxic, low toxic, high toxic, and lethal doses were established in single injection doses and five daily injections in dogs, and in five daily injections in monkeys. Dose ranges of 20-320 mg/m2 (single injection) and 5-80 mg/m2 (five daily injections) in dogs, and 3-96 mg/m2 (five daily injections) in monkeys were established. The monkeys were more sensitive than dogs to the low toxic dose and more tolerant to the high toxic dose in the repeated daily injections. The dose-response curves for the dogs and monkeys had similar slopes and inflection points. Because of the steep slope between the lethal dose and the highest nontoxic dose in both species, caution should be used in the initial clinical trials.