An Evaluation of the Common Mechanism Approach to the Food Quality Protection Act: Captan and Four Related Fungicides, a Practical Example

Abstract

Under the Food Quality Protection Act (FQPA) of 1996 (Act), the United States Environmental Protection Agency (EPA) is mandated to conduct cumulative risk assessment on pesticides that act through a common mechanism of toxicity. Incumbent on the Agency is the development of sound scientific principles upon which to evaluate compounds for the presence of a common mechanism. Using the currently available draft guidance criteria, this paper employs five fungicides of the same general class, typified by captan, to evaluate both the criteria and the available scientific data. Captan and folpet are two chloroalkylthio fungicides currently registered with EPA under Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) for agricultural use. As such, these compounds are subject to the provisions of FQPA. Three additional fungicidal compounds—dichlofluanid, tolylfluanid, and captafol—are not registered for use in the United States; however, these five compounds have chemical structure and biological toxicity similarities and differences that permit their utility as test cases to determine what the EPA would conclude, with regard to common mechanism, if these draft guidelines were applied to these compounds. The results of the analyses are consistent and support the conclusion that captan and folpet share a common mode of toxicity for mouse duodenal tumors as defined in the Act. This common mode of toxicity is not shared by dichlofluanid, tolylfluanid, or captafol. The basis for concluding a common mechanism exists between captan and folpet. They include 1. Structural Similarity—The compounds are structural analogs having the identical biologically active moiety (i.e., the-SCCl3 side chain). 2. Mechanisms of Pesticidal Action—The compounds have the same mechanism of action. The overwhelming body of evidence suggests they are active because of their reactions with thiols. Both compounds, in reacting with thiols, produce similar degradates. Differences in rates of reaction are attributable to physical-chemical properties of the two compounds. 3. General Mechanisms of Mammalian Toxicity—The compounds induce mammalian toxicity through the same mechanism that is responsible for their pesticidal action, reactions with thiols. Another, albeit less likely, mechanism (for both compounds) is cross-Unking of proteins with DNA, although the extremely short half-lives of these compounds (seconds) argues against this possibility. 4. Sites of Action—Both compounds express their primary toxicity as local rather than systemic effects. 5. Common Toxic Endpoint—These two compounds induce gastrointestinal tumors (in mice only). 6. Mode of Action—Both compounds express their common toxic endpoint through a nongenotoxic, compensatory proliferation mechanism. 7. Specificity of Action—For both compounds, the majority of tumors appear in the duodenum. Furthermore, these tumors are induced only in mice. Repeated carcinogenicity testing suggests that rats are refractive to the effects of captan and folpet. The significantly faster hydrolytic rate for folpet at the lower pH values (e.g., increased 8-fold at pH 5) encountered in the stomach is believed to account for the tumors of the stomach observed with folpet and not captan. 8. Other Toxic Endpoints—For other toxic endpoints where comparative data are available, captan and folpet show similar patterns of toxicity (e.g., mutagenicity, skin sensitization, and acute toxicity).

Carcinogenic mode of action of folpet in mice and evaluation of its relevance to humans

“…In CD-1 and B6C3F1 mice, the consistent finding is induction of glandular tumors (adenomas and adenocarcinomas) of the duodenum (Tables 1 and 2 1990; Quest et al�, 1993;Bernard and Gordon, 2000;EFSA, 2009aEFSA, , 2009bGordon, 2010)� The duodenum is the proximal portion of the small intestine� It transitions to the jejunum at the ligament of Treitz� The distal half of the small intestine is referred to as the ileum� In several of the short-and long-term studies involving folpet and related chemicals, some investigators have arbitrarily divided the small intestine into thirds� Regardless of whether the lesions occurred in what was stated as the duodenum or the jejunum, they occurred in the proximal portion of the small intestine, generally near the ampulla of Vater where bile and pancreatic secretions enter the intestine, with high concentrations of bicarbonate raising the pH of the intestinal luminal contents� There has also been an analysis of squamous cell tumors of the nonglandular, forestomach in mice (Figures 3-6)� It is suggested that one possibility is that the large duodenal tumors act Table 1. Tumor incidences of selective tissues in a study in CD-1 mice administered folpet in the diet (Wong, 1985 Table 2.…”

“…For the duodenal tumors induced in mice by folpet, the mode of action involves irritation-related cytotoxicity with consequent cellular regeneration ultimately leading to the development of tumors� The critical key event involves consumption of sufficiently high levels of folpet that yield a cytotoxic concentration of folpet and its degradation product, thiophosgene� Combined, these chemicals can cause cytotoxicity in the duodenum (Table 6)� In the stomach and duodenum there is cytotoxicity from the direct reaction of folpet with cellular components containing thiol groups as well as from the highly reactive thiophosgene, a hydrolysis product of folpet, which reacts with thiols as well as other cellular constituents (see Figures 1 and 2)� Folpet itself, like captan (Wilkinson et al�, 2004), is reactive with thiol groups, which generates thiophosgene (Figure 1)� However, thiophosgene is also reactive with thiol groups (Figure 2), with a half-life of less than 1 s (0�6 s in human blood)� Thiophosgene, thus, is generated either by the reaction of folpet with thiol groups or following hydrolysis of folpet� Direct interaction of folpet with thiol groups is the primary source of the thiophosgene� The hydrolysis of folpet to thiophosgene is highly pH dependent� The reaction occurs readily at neutral to alkaline pH but significantly less at low pH� Because of their marked reactivity, folpet and thiophosgene will react rapidly with thiol groups and not reach DNA or DNA-related targets (e�g�, histones) Liu and Fishbein, 1967;Lukens, 1966;Lukens et al�, 1965;Siegel, 1971aSiegel, , 1971bBernard and Gordon, 2000)� This is highlighted by a pair of key in vivo studies that show folpet and captan do not induce genotoxicity, as evidenced by the lack of increased nuclear aberrations in the tumor target site, the duodenum (Chidiac and Goldberg, 1987;Gudi and Krsmanovic, 2001)� Thus, DNA damage is highly unlikely and not detected in studies designed to assess this effect at the target tissue in vivo (Arce et al�, 2010)�…”

“…Captan shares a common mode of action with folpet (Bernard and Gordon, 2000;Gordon, 2010), It also produces gastrointestinal tumors in the duodenum in mice but does not induce treatment-related tumors in rats� In captan rat bioassays there was an increased incidence of kidney or uterine tumors, but these were deemed non-treatmentrelated by an independent third party (TERA, 2003) and subsequently confirmed by EPA (US EPA, 2004) and by EFSA (2009a)� Thus, both captan and folpet produce a carcinogenic effect in the gastrointestinal tract in mice, primarily in the Table 3. Tumor incidences of selective tissues in a study in Sprague-Dawley rats administered folpet in the diet (60 rats per group) (Cox et al, 1985 Table 4.…”

“…duodenum, but are not carcinogenic to rats (US EPA, 2004;Bernard and Gordon, 2000;Gordon 2010)� The incidences of mammary gland, thyroid, and lymphoid tumors in rats did not follow a distinct dose-response, and were not increased at statistically significant levels above the controls� Furthermore, statistical analysis for trend was not significant� The investigators in these studies stated that they did not believe these tumors were related to chemical treatment, based on statistical comparisons, and they also concluded that the tumors lacked biological relevance to humans� Lack of statistical significance in these studies was concluded on the basis of p >�05� Lack of statistical significance is even more strongly concluded if one utilizes the recommendation by the National Toxicology Program (NTP) (Haseman, 1983) that statistical comparison of common tumors in treated animals compared to controls in rodent bioassays should utilize a p value of <�025 or even p <�01 instead of the standard p <�05 recommended for uncommon tumors� This modified standard for statistical significance is now routinely used by the NTP for their bioassay assessments, and it is also used by pharmaceutical regulatory agencies worldwide, based on International Committee on Harmonisation (ICH) recommendations (Lin, 1998)� Thus, for folpet, it would be appropriate to use p <�05 for statistical significance for the duodenum and forestomach tumors, since these are relatively uncommon, but a lower p value should be utilized for the mammary gland, thyroid, and lymphoid tumors� This is also true for the testicular Leydig cell tumors� Regardless, the incidences of these three common tumors (mammary gland, thyroid, and lymphoma) in the rats in the 2-year bioassays with folpet were not statistically significant, even at p <�05� They certainly do not meet the criteria of a p value of <�025 and none of the tumors in any of the rat studies showed statistical significance for a trend analysis, even at p <�05� Furthermore, these tumors occur spontaneously quite frequently in control rats (Haseman et al�, 1998)� There does not appear to be biological significance for human risk assessment for any of these three tumor types in these strains of rats (Sprague-Dawley, F344)� Mammary gland fibroadenomas in female rats often occur spontaneously, especially in Sprague-Dawley rats where spontaneous mammary gland tumor incidences can exceed 75%� In addition, these fibroadenomas are benign tumors that are not predictive of malignant tumors in the rodent and they are not predictive of malignancy in humans (Russo and Russo, 1987;Russo et al� 1996)� Fibroadenomas are common lesions in the human breast, and are not considered precursor lesions for carcinomas (Rosen and Oberman, 1993;Russo et al�, 1990)�…”

“…Folpet has been evaluated in several 2-year rodent bioassays (Quest et al�, 1993;Bernard and Gordon, 2000), including three mouse studies (East 1994;Rubin and Nyska 1985;Wong, 1985) and three rat studies (Cox et al�, 1985: Crown et al�, 1985, 1989� In these dietary studies, folpet doses ranged in mice from 150 to 12,000 ppm (7�5-600 mg/kg/ day); and in rats, due to increased toxicity, the doses ranged from 200 ppm to a maximum of 5000 ppm (10-250 mg/kg/ day)� Despite its reactivity with thiol groups, folpet has been shown to be stable in the diet administered to the rats and mice (96-100% up to 3 weeks at 5000 ppm (Milburn, 1997); 96-115% of nominal levels for 12 weeks at 1000, 3000, or 10,000 ppm, but 59-67% at 300 ppm (Reno et al�, 1981)� All of the standard bioassays included high doses that met the criteria of maximum tolerated dose (MTD) and resulted in dose-related pathology� One study (East, 1994) was designed to establish a no observed adverse effect level (NOAEL) and included a high dose below the MTD but sufficient to cause tumors�…”