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 gastrininduced 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 of the 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.

New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48 mg/kg/day) and the 218 bp dsRNA (64 mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

Alachlor is the active ingredient in a family of preemergence herbicides. We assessed mortality rates from 1968 to 1993 and cancer incidence rates from 1969 to 1993 for manufacturing workers with potential alachlor exposure. For workers judged to have high alachlor exposure, mortality from all causes combined was lower than expected [23 observed, standardized mortality ratio (SMR) = 0.7, 95% CI, 0.4-1.0], cancer mortality was similar to expected (6 observed, SMR = 0.7, 95% CI, 0.3-1.6), and there were no cancer deaths among workers with 5 or more years high exposure and 15 or more years since first exposure (2.3 expected, SMR = 0, 95% CI, 0-1.6). Cancer incidence for workers with high exposure potential was similar to the state rate [18 observed, standardized incidence ratio (SIR) = 1.2, 95% CI, 0.7-2.0], especially for workers exposed for 5 or more years and with at least 15 years since first exposure (4 observed, SIR = 1.0, 95% CI, 0.3-2.7). The most common cancer for these latter workers was colorectal cancer (2 observed, SIR 3.9, 95% CI, 0.5-14.2 among workers). Despite the limitations of this study with respect to small size and exposure estimating, the findings are useful for evaluating potential alachlor-related health risks because past manufacturing exposures greatly exceeded those characteristic of agricultural operations. These findings suggest no appreciable effect of alachlor exposure on worker mortality or cancer incidence rates during the study period.

A broad array of in vitro and in vivo assays has consistently demonstrated that glyphosate and glyphosate-containing herbicide formulations (GCHF) are not genotoxic. Occasionally, however, related and contradictory data are reported, including findings of mouse liver and kidney DNA adducts and damage following intraperitoneal (ip) injection. Mode-of-action investigations were therefore undertaken to determine the significance of these contradictory data while concurrently comparing results from ip and oral exposures. Exposure by ip injection indeed produced marked hepatic and renal toxicity, but oral administration did not. The results suggest that ip injection of GCHF may induce secondary effects mediated by local toxicity rather than genotoxicity. Furthermore, these results continue to support the conclusion that glyphosate and GCHF are not genotoxic under exposure conditions that are relevant to animals and humans.

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