Captan and Folpet

Chapter 90

Captan and Folpet Elliot B. Gordon Elliot Gordon Consulting, LLC

90.1  Introduction Captan and folpet are fungicides that have been in use for over 55 years. During this period there have been no reports of systemic toxicity, but there has been a low incidence of skin sensitization. This record of safe use is consistent with these compounds’ chemical and physical properties. Adverse findings in laboratory test systems consist of mutagenicity, carcinogenicity, sensitization, and eye irritation. Until 2004, the U.S. EPA classified both compounds as “probable human carcinogens.” In the 2004, U.S. EPA reclassified captan as “not likely” based on exposure levels from registered uses. The potential for eye irritation is the basis for farm worker re-entry restrictions. The U.S. EPA is currently re-evaluating folpet for human carcinogenicity classification; it is expected that a similar “not likely” classification be established. Both captan and folpet irritate the gastrointestinal tract of mice when administered at high dietary doses. Adenomas and adenocarcinomas develop primarily in the duodenum, following prolonged compensatory proliferation of duodenal crypt cells following damage to villi. These fungicides are mutagenic when tested in vitro (e.g., the Ames point mutation assay) but negative when tested in vivo (e.g., the micronucleus assay). This paradox reflects the rapid degradation of solubilized compounds in blood: captan degrades with a half-life of less than 1 s; folpet degrades with a half-life of less than 5 s. The margins of exposure (MOEs) based on the no-observed-effect levels (NOELs) for gastrointestinal irritation and subsequent tumor formation is at least 1,000,000 based on estimated human exposure. In practical terms, these high MOEs mean neither captan nor folpet pose a risk for tumors in humans. Draize rabbit studies show that these compounds are severe ocular irritants. Extensive experience, however, particularly with agricultural workers engaged in re-entry operations, has shown that this laboratory phenomenon is not Hayes’ Handbook of Pesticide Toxicology Copyright © 2010 Elsevier Inc. All rights reserved

predictive of human experience. Eye protection is indicated for operators who mix, load, and apply these fungicides. Captan and folpet remain efficacious fungicides that present low manageable risks to agricultural workers and consumers.

90.1.1  Overview Captan and folpet are broad-spectrum protectant fungicides. Their mode of action centers on their reaction with thiols. These compounds along with a third, captafol, are collectively called chloroalkylthio fungicides due to the presence of side chains that contain chlorine, carbon, and sulfur. Of the chloroalkylthio fungicides, captan and folpet predominate in agronomic practice today; captafol registrations in the United States were withdrawn in 1988. Related compounds associated with this fungicide class, but not registered in the United States, are dichlofluanid and tolylfluanid. These later two compounds have a fluorine atom substituted for one of the terminal chlorine atoms. Early investigations on captan and folpet focused on their mutagenicity. These assays, conducted in vitro, showed both to be mutagenic. Citing this mutagenicity, regulators ascribed a genotoxic basis to the development of mouse duodenal tumors. This, in turn, led to an initial cancer risk assessment based on a linear low-dose extra­polation. Developmental toxicity studies of folpet were initiated following the perceived association of folpet’s phthalimide moiety with the human teratogen S-thalidomide; these structures have since been shown to be toxicologically unrelated. Captan and folpet show developmental toxicity at maternally toxic doses; neither compound is a frank teratogen. A number of reviews have addressed the toxicology of the chloroalkylthio fungicides (Ecobichon, 1996; Edwards et al., 1991; Elder, 1989; IARC, 1983; Saunders and Harper, 1994; Trochimowicz et al., 2001; U.S. EPA, 1975). 1915

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The relatively stable degradates of captan (tetrahydroph­ thalimide, THPI) and folpet (phthalimide, PI) have low toxicity; thus, there is low risk of systemic toxicity to farm workers who mix, load, or apply these fungicides.

90.1.2  History and Use Captan was first registered in the United States on March 8, 1949, as a fruit tree spray (NPIRS, 1999a) and its properties were described in 1953 (Kittleson, 1953). This compound proved extremely efficacious, spurring chemists to turn out a series of analogues in an attempt to capitalize on the fungicidal properties of the trichloromethylthio moiety (Horsfall and Rich, 1957; Kittleson, 1953; Lukens, 1966). Folpet was synthesized after captan; captafol was the last to be developed. As preventative fungicides, they are efficacious when applied prior to the establishment of pathogenic fungi. Captan and folpet are often used in integrated pest management (IPM) programs in conjunction with other fungicides. Registrations cover both agricultural and industrial uses (NPIRS, 1999b; U.S. EPA, 1985b). Captan is also efficacious as a bacteriostat in cosmetics (Elder, 1989). The U.S. EPA issued registration standards for captan (U.S. EPA, 1986b), folpet (U.S. EPA, 1987), and captafol (U.S. EPA, 1984a). A special review for captafol (U.S. EPA, 1985a) concluded in 1988 with the voluntary withdrawal of all registrations. A special review for captan was completed in 1989 with the issuance of Position Document 4 (U.S. EPA, 1989). Reregistration Eligibility Decision documents (REDs) have now been promulgated for captan and folpet (U.S. EPA, 1999a,b).

90.1.3  Toxicological Overview The principle chemical reaction that governs the toxicity of captan and folpet is their rapid reaction with thiol groups (i.e., sulfhydryl, -SH groups). This reaction results in degradation of the parent compound. Thiophosgene is a key degradation product that also reacts with thiols as well as other functional groups. Thiophosgene is more reactive than captan or folpet as reflected by its half-life in blood of 0.6 s (Arndt and Dohn, 2004). The net result of these chemical interactions is that both captan and folpet elicit primary toxicological effects locally at the site of initial contact. In mice, dietary exposure results in local irritation of the gastrointestinal tract, predominantly in the duodenum. It is primarily at this site that tumors develop. Continued administration of high doses will lead to secondary effects such as decreased body weight gain or growth retardation of fetuses in developmental studies. Pursuant to the Food Quality Protection Act and subsequent guidance by the EPA, captan and folpet have been found to share a common mechanism of toxicity with regard to the development of duodenal tumors in mice (see

Hayes’ Handbook of Pesticide Toxicology

discussion in Section 90.4). This finding suggests that both compounds be considered for cumulative risk assessment; it also affords the toxicologist the opportunity to combine mechanistic data for each into one unified database. Captafol, in contrast, was found not to share a common mechanism of toxicity with captan or folpet with regard to elicitation of systemic tumors. The FQPA requires consideration of the special sensitivities to infants and children, and the potential for endocrine disruption (Gabriel, 2005; U.S. EPA, 2008). There is no indication that captan or folpet shows disproportionate toxicity to infants or children. The mechanism by which these compounds exert their toxicity would make such a distinction unexpected. The EPA has recognized this for captan but currently has assigned an additional threefold safety factor for folpet (see Section 90.3.4; U.S. EPA, 1999a,b). Captan or folpet shows little evidence of being endocrine disruptors; however, captan has been “strongly suspected” of acting as an antiestrogen (Okubo et al., 2004). A Tier I endocrine screening program is currently under development (Gabriel, 2005; U.S. EPA, 2008). Captan and folpet induce mutagenic and clastogenic effects in a variety of in vitro assays. Mutagenic effects in vivo, however, do not occur. This paradox is explained by the extremely rapid degradation of these compounds in the intact animal. Whereas the delivered dose is negligible, captan and folpet are classic examples of the adage, “the (delivered) dose makes the poison.” Despite the obvious potential for mutagenic events, the dose at sensitive targets in the intact animal, such as cellular DNA, is essentially zero. Although these fungicides have low acute toxicity, their interaction with biological tissues can cause irritation. Persons handling these materials should do so with appropriate respiratory and eye protection. The compounds are not considered reproductive toxins or selective developmental toxins. The appearance of duodenal tumors in mice fed diets admixed with captan or folpet was, until recently, a key toxicological finding central to the regulation of these compounds. Data show that a mode of action based on increased rates of cell proliferation, a threshold phenomenon, accounts for these tumors. This proliferative pressure promotes nascent tumor cells that are normally resident within the duodenal crypt compartment. EPA accepted this mode of action when it reclassified captan (U.S. EPA, 2004). EPA considered comments from the public regarding this reclassification and affirmed the science supporting their decision (Jennings, 2007; Kent, 2006; Koch, 2007). Cancer reclassification of pesticides is now incorporated into the Pesticide Registration Improvement Renewal Act (U.S. EPA, 2007); the work leading to captan’s reclassification helped make these reevaluations routine (Gordon, 2007). Farm workers who handle captan or folpet are not at risk for developing duodenal cancer, as there is no systemic exposure. Persons exposed to residues of captan

Chapter | 90  Captan and Folpet

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and/or folpet in their food are not at risk since the margins of safety for both compounds are approximately 1 million (see Section 90.5).

90.2  Physical properties and chemical reactions

but is subordinate to the side chain with regard to the fungicide’s toxicological properties. The phthalimide ring is aromatic and, as such, is a resonance structure; THPI has one double bond between carbons 3 and 4. This hexene imide, unlike phthalimide, is nonplanar (Fickentscher et al., 1977). Chemical

Structure

90.2.1  Overview

O

Cl N—S–C–Cl – –

The toxicology of the chloroalkylthio fungicides is dependent on their physical properties and chemical reactions. The structures of captan and folpet along with typical ring degradates are shown in Figures 90.1 and 90.2. The chemical identity and physical properties are noted in Table 90.1, and the rates of selected chemical reactions are shown in Table 90.2. The characteristic chemical moiety for captan and folpet is the trichloromethylthio side chain that is connected to an imide ring structure by way of a nitrogen-sulfur bond. Captan’s ring is tetrahydrophthalimide (THPI) and folpet’s is phthalimide. This ring imparts certain physical properties to the molecule,

Folpet

Cl

O O Phthalimide (PI)

NH O O NH2

Phthalamic acid

OH O O

Chemical

Structure O

Cl

– – Cl

O

O NH

4,5-cyclohexene-1,2-dicarboximide (THPI)

4,5-dihydroxy-1,2-dicarboximide (4,5-diOH THPI)

Parameter

Captan

Folpet

CAS number

133-06-2

133-07-3

Molecular weight

300.61

296.56

Formula A

C9H8Cl3NO2S

C9H4Cl3NO2S

Formula B

C6H8 (C O)2N— SCCl3

C6H4(C  O)2N— SCCl3

IUPAC name

1,2,3,6-TetrahydroN-(trichloromethyl thio) phthalimide

N-(trichloromethyl thio) phthalmide

CA name

3a,4,7,7aTetrahydro-2[(trichloromethyl) thio]-IH-isoindole1,3(2H)-dione

3-[(Trichloromethyl) thio]-IH-isoindol1,3(2H)-dione

O CNH2

Physical form

Crystals

Crystals

Melting point

178°C

177°C

COOH

Solubility, water

3.3 mg/l at 25°C

1 mg/l at 20°C

Solubility, acetone

3.0 g/100 ml

3.4 g/100 ml

log Kow

2.35

2.85

O NH

O O

O

OH

NH OH

O

OH O NH

7-hydroxy-4,5-cyclohexene-1,2-dicarboximide (ci/trans-3-OH THPI)

O

O 6-hydroxy-4,5-cyclohexene-1,2-dicarboximide (cis/trans-5-OH THPI)

OH NH O

1-amido-2-carboxy-4,5-cyclohexene (cis/trans-THPAM)

6-hydroxy-1-amido-2-carboxy-4,5-cyclohexene (3-OH THP-amic acid)

Figure 90.2  Folpet and its ring metabolites.

Table 90.1  Physical Properties of Captan and Folpet

O 4,5-epoxy-1,2-dicarboximide (THPI expoxide)

OH O CNH2 COOH

Figure 90.1  Captan and its ring metabolites.

OH O

N—S–C–Cl

Captan

OH

Phthalic acid

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Table 90.2  Rates of Chemical Reactions of Captan and Folpet Parameter

Captan

Folpet

Reference

pH 5

18.8 h

2.6 h

Captan: Pack (1987)

pH 7

4.9 h

1.1 h

Folpet: Ruzo and Ewing (1988)

Aqueous hydrolysis

pH 9

8.3 min

1.5 min

Reaction with blood thiols

18 s, 22°C

51 s, 22°C

Captan: Crossley (1967a) Folpet: Crossley (1967b)

Degradation half-life (acid conditions to minimize hydrolysis)

0.97 s, 37°C

4.9 s, 37°C

Gordon et al. (2001)

Decolorization of dithionitrobenzoic acid (DTNB)-thiol complex in blood

1 min

3 min

Liu and Fishbein (1967)

90.2.2  Physical Properties Captan and folpet have similar physical properties. They have low water solubility, low volatility, and melt at approximately the same temperature. Octanol–water coefficients are high for both, although folpet’s Kow is somewhat higher than that of captan.

90.2.3  Chemical Reactions Captan and folpet are unstable in aqueous solution, but the rate of hydrolysis is slow compared with their reaction with thiols. The key to their fungicidal efficacy is the balance between the reactivity of the trichloromethylthio moiety and the stability of the nitrogen–sulfur bond linking this moiety to the imide ring. Analogues with very stable bonds prove to be ineffective fungicides, whereas analogues with bonds that are overly labile degrade spontaneously (Horsfall and Rich, 1957; Lukens, 1966, 1967). The hydrolytic and thiol reactions serve to degrade the parent molecule and thus influence the toxicology outcome by effectively reducing or eliminating exposure.

90.2.3.1  Hydrolysis The rates of aqueous hydrolysis increase in alkaline conditions and are more rapid for folpet than captan at comparable

pH values (Table 90.2). At pH 5, for instance, captan is approximately eight times more stable than folpet; thus, in the acid conditions of the stomach, it would be expected that relatively more folpet degradation products would be present compared with captan. The higher hydrolytic rates for folpet are related to the higher standard free energy of the phthalimide ring structure compared with the THPI ring (Lukens, 1966).

90.2.3.2  Reaction with Thiols The fungicide-thiol reaction has been studied with glutathione (GSH), proteins, and other thiol-containing compounds. In general, the thiol group is oxidized (e.g., GSH → GSSG; cysteine → cystine). Common to both captan and folpet is the generation of thiophosgene during degradation. This chemical entity appears to be a contributing toxicophore in that it rapidly reacts with a variety of functional groups in addition to thiols (Lukens, 1969; Lukens and Sisler, 1958b; Sharma, 1986). A general scheme of degradation for captan and folpet is shown in Figure 90.3. The rate of hydrolysis is faster for folpet than for captan, whereas the reverse is true for thiol-mediated degradation. The reaction of captan and folpet with cysteine results in the formation of thiazolidine-2-thione-4-carboxylic acid (TTCA; Lukens and Sisler, 1958a). This compound is seen in mammalian metabolism studies (DeBaun et al., 1974) and has been suggested for use as a biological marker for human exposure assessment (Krieger and Dinoff, 2000; Krieger and Thongsinthusak, 1993; van Welie et al., 1991). TTCA can now be detected at a level of 40 pmol/ml urine (Amarnath et al., 2001). The fate of captan and folpet in human and rabbit blood has been investigated (Crossley, 1967a,b). Crossley added captan and folpet to human blood and measured the decline of the parent with time and, concurrently, the increase of the imide ring (THPI or phthalimide). At initial concentrations of 1 g/ml, captan degraded with a half-life of 18 s. The degradation of folpet was three times slower, with a half-life of 54 s. By measuring the generation of the imide rings, it was shown that the parent compounds actually degraded rather than complexed with blood constituents. These investigations were carried out at 22°C with unlabeled materials. Subsequent investigations of degradation rates employed radiolabeled captan and folpet and physiological temperatures (37°C). As predicted by the Q10 (Purves et al., 1992), increased temperature results in a higher rate of degradation. The new data demonstrate that at physiological temperatures, captan degrades rapidly in human blood, having a t1/2 of 0.97 s, whereas folpet degrades somewhat slower but still quite rapidly (i.e., t1/2  4.9 s; Gordon et al., 2001). These data, which demonstrate the rapid degradation of the two compounds, are of course a critical component in any exposure assessment and risk

Chapter | 90  Captan and Folpet

Reaction with thiols

1919

R—SCCl3 Captan/Folpet

Hydrolysis

2R’SH

R’SSR’

H2O

HCl

THPI (captan) PI (folpet) HCl

S C Cl Cl Thiosphosgene Reaction with thiols

Hydrolysis

which such effects are induced has not been elucidated. When 35S-captan was incubated with calf thymus DNA in buffer at pH 7.5 or 9.0, binding of appreciable amounts of radioactivity could not be demonstrated (Couch and Siegel, 1977). Captan reacts with guanine in vitro to produce 7-(trichloromethylsulfenyl) guanine (Elder, 1989), as reported by FAO/WHO (1990). In vivo DNA binding studies are discussed in Section 90.3.5.5.

90.2.3.5  Miscellaneous Chemical Reactions

evaluation modeling. With oral exposure, it is unlikely that captan, folpet, or thiophosgene (with a half-life of 0.6 s in blood) would survive long enough to reach systemic targets such as the liver, uterus, or testes. With dermal exposure and subsequent low absorption, captan will be eliminated in less than 7 s and folpet in less than 35 s. This determination reflects the kinetics noting compounds are essentially gone in seven half-lives (Medinsky and Klaassen, 1996).

Because captan and folpet are reactive, there are unlimited opportunities for chemical reactions in isolation. Absent data that indicate exposure in vivo or relevance to the intact animal, these observations remain ancillary, reflecting their chemical reactivity, but having little bearing on mammalian toxicity and human risk assessment. Captan and folpet react with p-nitrothiophenol via the thiol group (Liu and Fishbein, 1967). This differential rate of reaction was measured at 25°C and was 1.9  104 l/(mol min) for captan and 1.5  104 l/(mol min) for folpet. Other effects include the inhibition of Escherichia coli RNA polymerase (Elder, 1989), the inhibition of RNA synthesis by intact bovine nuclei (Elder, 1989), the inhibition of microsomal cytochrome P450 benzphetamine N-demethylase and aniline hydroxylase after intraperitoneal dosing (Dalvi, 1988, 1989), the inhibition of the Ca2 transport ATPase in human erythrocytes (Janik, 1986), and the inhibition of oxidative phosphorylation in rat liver mitochondria, correlated to mitochondrial swelling (Elder, 1989). Captan also disrupted the differentiation of cultured cells from the midbrains and limb buds of 34–36 somite rat embryos in vitro (Flint and Ortaon, 1984) and inhibited the attachment of tumor cells to polyethylene disks that were coated with concanavalin (Braun and Horowicz, 1983).

90.2.3.3  Reaction with Proteins

90.2.3.6  Thiophosgene

Investigations on the effects of captan and folpet with proteins generally have been carried out in vitro. Such studies identify potential interactions that may occur in the living animal; however, for captan and folpet, the rapid degradation of reactive species and the resultant limitation in exposure prevent many of these reactions from resulting in in vivo toxicological phenomena. Folpet reacts with thiol-containing proteins (e.g., glyceraldehyde 3-phosphate; Siegel, 1971a), non-thiol-containing proteins (e.g., -chymotrypsin; Siegel, 1971b), and nuclear histones (Couch and Siegel, 1972, 1977). These reactions are often pH-dependent.

Thiophosgene (CAS 463–71–8) is a very short-lived compound that has a broad spectrum of reactions with a variety of functional groups (Sharma, 1978, 1986). Although this compound hydrolyzes at a slower rate than its oxygen analogue, phosgene, the rate is sufficient to eliminate mutagenic activity in Salmonella typhimurium TA 100 when dimethyl sulfoxide (DMSO) is the solvent (Schuphan et al., 1981). Thiophosgene is a toxicophore of captan and folpet, although its role in their fungicidal properties has been questioned (Lien, 1969). It is volatile and reacts with water to form carbonyl sulfide (COS) and two molecules of hydrogen chloride. The carbonyl sulfide then reacts with another water molecule to form hydrogen sulfide and carbon dioxide (Figure 90.3). Thiophosgene has two reactive sites associated with the carbon atom. Whereas both chlorine atoms are electronegative, the carbon atom becomes positively charged, thus creating an electrophile. The reaction with cysteine is shown in Figure 90.4.

TTCA Cysteine + 2HCl

O

2RSH

2H2O 2HCl + CO2 + H2S

SO3=

RSCSR + 2HCl RSR +CS2

Reaction with sulfite DMS-Acid [O] DMS-O

Figure 90.3  General degradation scheme. For captan, R  THPI (Tetrahydrophthalimide); for folpet, R  PI (phthalimide); TTCA: thiazolidine-2-thione-4-carboxylic acid; DMS-Acid: dithio-bis-methanesulphonic acid; DMS-O: monosulfoxide of dithio-bis-methanesulphonic acid.

90.2.3.4  Reaction with DNA The reactions of captan and folpet with DNA are not well characterized. Captan and folpet induce point mutations and clastogenic changes in vitro, but the mechanism by

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– –

O

NH2 Cysteine

+

S Thiophosgene

– –

C

C–OH

S NH C S

+ 2HCl

–

OH

C– C

–

–

HS

– – –

Cl Cl

– – –

O

Thiazolidine-2-thione-4carboxylic Acid (TTCA)

Figure 90.4  Reaction of cysteine with thiophosgene.

When introduced into human blood, the half-life of thiophosgene is 0.6 s, a value that reflects its high reactivity (Arndt and Dohn, 2004),

90.2.4  Metabolism The fate of captan and folpet in mammalian systems is determined by an amalgam of nonenzymatic chemical reactions with thiols and subsequent enzyme-mediated metabolism that predominately involve the generation of ring metabolites. In the intestine, both hydrolysis and thiolmediated reactions occur. The rate of hydrolysis is particularly sensitive to pH, and the transition from the acid environment of the stomach to the neutral or basic conditions of the duodenum promotes the hydrolytic breakdown of these materials. These fungicides undergo a similar pattern of degradation (Figure 90.3). The side chain is either fully mineralized or forms by-products such as TTCA with cysteine. The respective imides, THPI and phthalimide, are initially formed either through hydrolysis or through reaction with thiols. These are subsequently metabolized to secondary products; the THPI hexene structure of captan is more extensively metabolized than the phthalimide structure of folpet (Figures 90.1 and 90.2).

90.2.4.1  Rat Metabolism Captan and folpet are rapidly eliminated when administered either orally or intraperitoneally. Multiple doses of captan or folpet do not alter subsequent excretory patterns, suggesting that liver enzymes are not induced by repeated exposure. This finding was expected because the parent molecules are not likely to reach the liver. There is no sex difference in the way these fungicides are metabolized. A 10-mg/kg dose of ring-labeled captan is rapidly excreted in the urine. After 24 h, approximately 75% of the administered dose is excreted in the urine and 6.5% is excreted in the feces. Nearly all radioactivity is excreted by 36 h (Trivedi, 1990a). Fourteen repeated single doses of 10 mg/kg followed by a dose of radiolabeled captan produced a similar excretory profile (Bratt, 1990). A dose of 6 mg/kg 35Scaptan given intraperitoneally to male rats was effectively eliminated within 72 h (Couch et al., 1977).

Captan at a 500-mg/kg dose resulted in a similar profile except that relatively more material was excreted via the feces. In 96 h, 68.8 and 23.1% was excreted via the urine and feces in males and 73.4 and 25.0% was excreted, respectively, in females (Trivedi, 1990b). Folpet demonstrates a similar pattern. Administration of 10 mg/kg results in approximately 96% of the radioactivity being excreted by 24 h (90% in urine; 6% in feces). With doses 50 times higher, only approximately 69% of the administered dose is cleared by 24 h (47% in urine; 22% in feces). The imide ring is relatively stable and is excreted along with additional ring metabolites. The side chain is unstable and reacts with thiols to form mineralized products such as CO2, HCl, and H2S. In addition, products of the reaction also include TTCA, dithiobis (methanesulfonic acid) and its disulfide monoxide derivative. The reactions of captan and folpet are identical with regard to the -[trichloromethylthio] side-chain reactions. The THPI generated from captan is more easily metabolized than the phthalimide from folpet. This is due to the carbonyl groups of captan that draw electrons away from the hexene double bond, creating a  charge at this site, thereby promoting substitutions. Administration of phthalimide results in metabolism to phthalamic acid (79%, in females) and phthalic acid (7%). Less than 1% of the original phthalimide is recovered in the urine (Chasseaud et al., 1974). Phthalamic acid accounts for 80% of the original dose when 14C-[carbonyl] folpet is given to rats (Chasseaud, 1980).

90.2.4.2  Effect on Glutathione Levels in the Duodenum Sulfhydryl groups are intimately involved with the degradation of captan and folpet. It is therefore of interest to see their effect on GSH. Swiss Webster mice fed captan at 4000, 8000, and 16,000 ppm for 35 days had GSH levels (“soluble thiols”) elevated by day 1 (Miaullis et al., 1980). The percent increase over controls ranged from 146 to 227%. Gavage treatment at a relatively high dose of captan (2000 mg/kg) induced an increase in GSH levels that was observable within 2 h of treatment, whereas a smaller dose (20 mg/kg) induced a measurable increase at 4 h (Katz et al., 1982; Sauerhoff et al., 1982). Folpet-induced increased GSH levels were demonstrated after both dietary administration and gavage (Chasseaud et al., 1991). Folpet, administered by gavage (7.6, 72, and 668 mg/kg), initially induced a decrease (30 and 60 min), which subsequently rebounded to a higher than normal level. The decrease was statistically significant for the 72-mg/kg dose: levels of 76, 54, 82, 155, and 130% (of control values) were observed at 0.5, 1, 2, 6, and 24 h, respectively. This rebound effect was also seen at 668 mg/kg: 72, 52, 94, 143, and 178% at the same time periods. Diethylmaleate produced a similar pattern of GSH loss and rebound. These data demonstrate that captan and folpet

Chapter | 90  Captan and Folpet

cause an initial lowering of GSH levels followed by an increase due to a homeostatic rebound. The generative process for GSH exceeds the loss, and a steady state of higher GSH levels is quickly reached.

90.2.4.3  Goat Metabolism Ring-labeled (14C)captan was administered to goats in gelatin capsules three times per day for 4 days. The total daily dose equaled approximately 50 ppm (Cheng, 1980). Most of the radioactivity was excreted in the urine, and the next highest excretion was via the feces. Five biochemical reactions were noted from this study: 1. Cleavage of the N—S linkage in the captan molecule to form THPI, either by hydrolysis or reaction with SH compounds 2. Ring hydroxylation of THPI to form 3-OH THPI 3. Isomerization of the 3-OH THPI to form 5-OH THPI 4. Epoxidation of THPI to form THPI-epoxide, which is subsequently hydrolyzed to form 4,5-diOH HHPI 5. Hydrolysis of THPI and/or its hydroxylated derivatives to form their corresponding THP-amic acid derivatives When radiolabeled folpet [14C-labeled on the trichloromethylthio side chain (TCM)] was administered via capsules to goats, radioactivity was recovered as expired 14 CO2 (31%; reflecting the breakdown of the TCM), in the feces (21%), and in the urine (10%; Corden, 1997). Recovered 14CO2 from the expired air (ca. 31%) reflected the breakdown of the trichloromethylthio moiety to CO2. Similarly, administration of ring-labeled folpet showed the same excretion pattern as discussed for captan [i.e., more excretion via the urine (58%) than the feces (35%), with the major urinary metabolite being phthalamic acid (49% of the dose)].

90.2.4.4  Hen Metabolism Laying hens metabolize captan in a similar way as mammals (Daun, 1988a,b). When tagged with 14C on the imide ring, the identified metabolites were THPI (15.8–68.9% of the tissue radioactivity), and 3-OH, and 5-OH-THPI, which represented 2.4–26% of the radioactivity. When tagged with 14C on the trichloromethylthio moiety TTCA, dithiobis-methanesulfonic acid and its monosulfoxide were seen. Parent captan was not present in the eggs or tissues.

90.2.4.5  Dermal Absorption Dermal absorption of captan and folpet, as subsequently noted, is estimated at no greater than 0.5% per hour. Captan penetration of skin has been measured in vitro, comparing rat with human, and in vivo, using rats. The rat study measured absorption at 1, 2, 4, and 8 h at 19.4 g/cm2 (0.5 mg/kg) and 194 g/cm2 (5 mg/kg; Adir et al., 1982),

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and the data have been interpreted as indicating from 0.4% per hour absorption (Ghali, 1997) to 1.5% per hour (11.7% per 8 h; Thongsinthusak et al., 1999). The 11.7% per 8-h rate has been noted as overly conservative because the test sites were not occluded, allowing contamination of the urine and feces samples. Additionally, the absorption rate did not consider the difference between rat and human skin permeability (Fletcher et al., 1995). The in vitro rat/human comparisons showed that human skin was consistently less permeable than rat skin, but that the ratio of permeability was partly dependent on the concentration of captan applied and the solvent used. A study with 14C-folpet 50 WP in rats indicated a systemic absorption of 0.27% per hour (6.5% in 24 h; Wilson and Wright, 1990). This calculation was based on a least square analysis of 24-h urinary excretion at dose levels of 49, 460, and 4800 g/cm2 (13.2, 3.5, and 1.3%, respectively), matching the excretion to the approximate dermal exposure of 2400 g/cm2, based on the 50-WP formulation concentration. There was rapid uptake of folpet into the skin and 95 and 94% for dose levels 4800 and 460 g/cm2, respectively, were retained there at 24 h. The amount still in the skin after 24 h for the low dose was approximately 85% of the applied dose. A comparison with ring-labeled captan and sidechain-labeled folpet showed no difference in absorption between adult and young Fischer 344 rats, but a lesser amount of folpet was absorbed compared with captan (Shah et al., 1987). At 0.54 and 2.68 m/cm2, captan penetration in adult rats was 3.7 and 3.6%, whereas folpet penetration was 2.7 and 1.1%, respectively. These data were interpreted by the U.S. EPA to suggest 0.4% per hour (4.29% in 10 h) dermal absorption for folpet (Ghali, 1997; Levy et al., 1997). These data show high dermal adsorption, but low penetration rates for captan and folpet. There are differing interpretations of these data, but it is reasonable to conclude that the hourly dermal penetration rate is no greater than 0.5%. The normal sloughing of the stratum corneum serves to deplete the amount available for absorption.

90.2.4.6  Human Metabolism Humans appear to metabolize captan in a similar manner to other mammals (Krieger and Thongsinthusak, 1993). Both THPI and TTCA have been recovered after oral and dermal dosing. Comparable human studies with folpet have not been conducted, but are expected to yield similar results but with the urinary excretion of phthalamic acid (major) and phthalimide (minor).

90.2.5  Summary Captan and folpet are extensively altered in mammalian and avian systems through a combination of enzymatic and nonenzymatic chemical reactions. Two complementary

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processes, hydrolysis and thiol interactions, initially split the fungicides into their respective imide rings and trichloromethylthio complexes. Subsequent reactions, some of which may be enzymatic, produce a series of imide-based degradates and thiophosgene-mediated products. The reactions are rapid and nearly all material is eliminated from the animal within 24–48 h; there is no accumulation of either imide or side chain. Urinary metabolites from the rings differ between captan and folpet, but those associated with the trichloromethylthio side chain are common [e.g., TTCA and dithiobis(methanesulfonic acid)]. Captan or folpet do not survive in the systemic circulation, thus limiting their primary effects to areas of initial contact. Due to this rapid elimination, meat, milk, or eggs from livestock that might have consumed feed with residues of captan or folpet present would be devoid of the parent materials.

90.3  Toxicology 90.3.1  Acute Toxicology 90.3.1.1  Overview Both captan and folpet have low acute toxicity, except for the intraperitoneal route (Table 90.3). The reactivity to mucus membranes is high and the severe eye irritant finding is consistent with this property. When single 0.5-g doses are applied to the skin, the results show mild to low irritancy. Results of guinea pig sensitization studies give both positive and negative results; however, experience with handlers of these materials suggest that some persons (estimated at less than 10% of the population) are susceptible to sensitization.

90.3.1.2  Acute Oral Toxicity The low acute oral toxicity of captan and folpet reflects the rapid degradation of the fungicides once ingested and the absence of intact fungicide at sensitive biochemical targets. The LD50 values are above 5 g/kg for both technical and formulated products, placing them in Toxicity Category IV for U.S. EPA regulatory purposes. The captan 50W formulation LD50 is 8.4 g/kg, whereas the comparable folpet formulation LD50 is greater than 10 g/kg (Ben-Dyke et al., 1970). Results from other investigators consistently show LD50 values above 5 g/kg for both compounds (Boyd and Krijnen, 1968; Nelson, 1949). The imide ring degradates of captan and folpet, THPI and phthalimide, respectively, are stable compared with the parent compounds. Accordingly, some measure of their acute toxicity is in order. Both these compounds have low acute oral toxicity in mammals. The LD50 of THPI is 2 g/kg (Cavalli, 1970); for phthalimide it is greater than 8 g/kg (U.S. EPA, 1974). In contrast to mammals, aquatic organisms are particularly sensitive to captan and folpet, and offer a useful test system to compare the toxicity of parent and degradate. Comparative toxicity in trout show THPI to be approximately 3500-fold less toxic than captan (captan LC50  34 ppb versus THPI LC50  120,000 ppb; U.S. EPA, 1999a). A similar comparison for folpet shows a 3267-fold difference (folpet LC50  15 ppb versus phthalimide LC50  49,000 ppb; U.S. EPA, 1999b).

90.3.1.3  Acute Intraperitoneal Toxicity Intraperitoneal administered acute toxicity studies are not generally required for regulatory purposes because this route of entry affords little information for human risk assessment. The intraperitoneal route bypasses the intestine,

Table 90.3  Acute Toxicity and Captan and Folpeta Parameter

Captan

Reference

Folpet

Reference

Oral LD50, rat

5 g/kg

Gaines and Linder (1986)

5 g/kg

Gaines and Linder (1986)

8 g/kg 50W formulation

Ben-Dyke et al. (1970)

10 g/kg 50W formulation

Ben-Dyke et al. (1970)

Intraperitoneal LD50, rat

40 mg/kg, M 35 mg/kg, F

Copley (1985)

48–52.5 mg/kg

Dickhaus and Heisler (1983)

Dermal LD50, rabbit

2000 mg/kg

Thoa and Redden (1995)

5000 mg/kg

Korenaga (1982)

Irritation, eye

Severe

Thoa and Redden (1995)

Severe

0.5, EPA (1987)

Skin

Minimal

U.S. EPA (1975)

Minimal

U.S. EPA (1987)

Inhalation LC50, rat, 4 h exposure

0.72–0.87 mg/l

Thoa and Redden (1995)

1.89 mg/liter

Cracknell (1993)

Sensitization, guinea pig a

M, males; F, females.

Moderate

Thoa and Redden (1995)

Moderate

U.S. EPA (1987)

Chapter | 90  Captan and Folpet

although materials are still primarily absorbed by the portal system (Rozman and Klaassen, 1996). In the case of oral administration of captan or folpet, only trace amounts of parent material would enter the portal system and would be rapidly degraded. Intraperitoneal administration bypasses this degradation and thus affords an observation into the inherent toxicity of the materials. Male and female SPF Wistar rats treated with 92.7% captan administered by injection in 1% methylcellulose had LD50 values approximately 40 mg/kg for males and 35 mg/kg for females (Copley, 1985). This LD50, lower by over 100-fold compared with oral administration, indicates the inherent toxicity of captan. It also demonstrates the effective barrier provided by the intestine. Male and female Wistar rats injected with 87.5% folpet in 1% methylcellulose had a 24-h LD50 of 48.0–52.5 mg/kg. Deaths occurred between 24 h and 7 days, resulting in a 7-day LD50 of 36–40 mg/kg. There was a steep dose– response curve: at 30 mg/kg, one in 10 deaths were seen, but at 60 mg/kg, 10 in 10 deaths occurred by day 7 (Dickhaus and Heisler, 1983). The intraperitoneal LD50 values for captan and folpet are similar and reflect the rule that governs their toxicological profile: hazard in the absence of exposure limits adverse effects.

90.3.1.4  Acute Dermal Toxicity Captan and folpet pose little hazard of acute toxicity from dermal exposure. Limit doses of 2 g/kg are without effect in rabbits (Foster and Morgan, 1984; Gaines and Linder, 1986). A study with Phaltan 50W (50% folpet) showed that the LD50 was greater than 22.6 g/kg (Kay and Calandra, 1960).

90.3.1.5  Eye Irritation Captan and folpet irritate mucus membranes and there is the potential for damage when they contact the eyes. Bioassays, however, vary in their estimation of this hazard. Captan eye irritation studies show variable results. Minimal damage, as noted by no corneal or iris involvement, and low redness and swelling to the eyelids, has been reported (Harris, 1976). Conversely, severe damage, including corneal opacity, has also been reported (Rosenfeld, 1984). Washing the treated eyes after instillation of test material reduces irritation, as was observed in a study that employed a captan 50W formulation (Sauer and Seaman, 1980). An additional study employed a combination formulation that included captan (8%), folpet (44%), and captafol (8%), and showed conjunctival irritation but no corneal involvement (Cisson et al., 1983). Folpet Technical, in unwashed eyes, induced transient corneal opacity that progressed to vascularization of the cornea in two of six rabbits (Dreher, 1992a). A 100-mg instillation of Folpet Technical caused corneal opacity in some unwashed eyes and no opacity in eyes that were

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washed 30 s after instillation (Cisson et al., 1982). All eyes returned to normal by 7 days (washed) or 10 days (unwashed). Phaltan 500 Flowable formulation (50% folpet) instilled in rabbit eyes, followed by a 30-s wash after 30 s, resulted in no corneal opacity and minimal redness and swelling (Mercier, 1988). By 72 h, all swelling had subsided, but there was some residual redness and congestion.

90.3.1.6  Skin Irritation Both captan and folpet elicit very little irritation when applied as a single dose to either intact or abraded skin. Captan Technical applied to rabbit skin at 0.5 g showed no redness or edema at either 24 or 48 h for both intact and abraded test sites (Harris, 1976). Folpet Technical applied to rabbit skin at 0.5 g showed no redness or edema at observation periods up to 72 h (Rees, 1993). Doses of folpet as high as 22.6 g/kg produced only transient redness in rabbits (Kay and Calandra, 1960).

90.3.1.7  Acute Inhalation Toxicity Acute toxicity via the inhalation route of exposure varies somewhat with the specific formulation tested. For captan, the 4-h LC50 was 1.21 mg/l for males and 1.05 mg/l for females (Cummins, 1995). An earlier study reported these values as 0.90 mg/l for males and 0.67 mg/l for females (Blagden, 1991). For folpet, the 4-h LC50 for males and females was 1.89 mg/l (95% confidence limits 1.47–2.31 mg/l; Cracknell, 1993). The particle size mass median equivalent aerodynamic diameter was 4.6–5.2 m. Males were slightly more susceptible than females. The mortality for males was 0 in 5, 3 in 5, and 4 in 5 for dose levels 0.80, 1.60, and 1.99 mg/l, respectively. Females at these respective doses showed mortality of 0 in 5, 1 in 5, and 1 in 5.

90.3.1.8  Skin Sensitization Guinea pig bioassays show that both captan and folpet have the potential to induce delayed contact hypersensitivity reactions. Captan Technical was positive in the Magnusson and Kligman maximization guinea pig assays (Dreher, 1992b). Folpet Technical was tested using the Magnusson and Kligman protocol in which a 10% w/v preparation in propylene glycol was injected by the intradermal route along with a 1:1 preparation of Freund’s Complete Adjuvant. Subsequent topical inductions were made with 50% w/v folpet. Challenge with either 10 or 50% w/v folpet in propylene glycol resulted in positive reactions (LSR, 1993).

90.3.1.9  Human Experience Reports of adverse effects are limited to incidences of skin irritation or sensitization reactions (Guo et al., 1996; Peluso et al., 1991; U.S. EPA, 1989). In human patch tests, nine in 205 (4.4%) subjects showed a positive Draize reaction

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(Marzulli and Maibach, 1973), eight in 150 (5.3%) were sensitized by 1% topically applied captan (Jordan and King, 1977), and 24 in 200 were positive to chemicals in the fungicide category, predominantly the thiophthalimides (Lisi et al., 1986). In a patch test for photoallergens four of 12 patients who had positive reactions to a series of allergens reacted to folpet and captan (Mark et al., 1999). Additionally, a case of urticaria was associated with use of a captan-formulated product (Croy, 1973). However, a powder blush that contained 0.3% captan failed to sensitize any of 25 adult volunteers on which it was tested. This was in spite of the study design, which included repeated doses (five consecutive 300-mg induction exposures to the forearms) and occlusion of the application site for 48 h after each dose. The individuals were challenged 10 days later and all individuals were negative (Ivy Research Laboratories, 1981). Captan was noted as “the most common sensitizer” affecting five of 30 fruit and vegetable workers in Hamachi Prades, India (Verma et al., 2007). Regular exposure to captan, formulated in a shampoo at 7%, appears to be well tolerated (Guo, 2001). Although there is potential for eye irritation based on laboratory studies, there are no reports in the literature of adverse effects (NLM, 2001). Likewise, eye injuries in agricultural workers who carry out re-entry activities do not appear to be problematic (Krieger, personal communication). In an apparent suicide attempt, a 17-year-old ingested 7.5 g captan 50 WP. There were increases in creatine kinase and aspartate aminotransferase; resolution of all abnormalities occurred within 72 h (Chodorowski and Anand, 2003). In a second attempted suicide report by the same authors, a 22-year-old ingested 5 g captan and complained of nausea, weakness, numbness of the upper limbs and substernal pain (Chodorowski et al., 2004). In both cases, the mg/kg dose, using a standard 60 kg body weight, was well below the rodent LD50.

90.3.1.10  Summary Adverse skin reactions to captan and folpet due to delayed contact hypersensitivity are possible for mixers, loaders, and applicators, and may occur in low incidence. The potential for eye irritation exists, but extensive use experience suggests this problem is minimal. There is little acute risk from oral ingestion or dermal exposure to either product. The World Health Organization has classified folpet as unlikely to present an acute hazard in normal use (FAO/ WHO, 1996).

90.3.2  Subchronic Toxicity Mechanistic studies in mice have focused on changes to the duodenum and illuminate the mode of action for tumor formation. These studies confirm the irritant properties of captan and folpet, the effects of irritation to the duodenum,

and the reversibility of these effects upon cessation of treatment. Other observations in the mouse include reduced weight gain and depressed food intake at high doses; this is also seen as a general secondary effect in rat studies. Observations in rats also include hyperkeratosis and acanthosis of the esophagus and stomach, particularly for folpet. Dogs do not tolerate capsule-administered captan or folpet well; emesis is generally seen. Rabbits administered folpet dermally show marked skin irritation, and rats respond to repeated dermal application of folpet with severe skin irritation.

90.3.2.1  Mice (a) Mechanistic Studies Male CD-1 mice (four or five per group) treated with 3000-ppm captan for 1, 3, 7, 14, or 28 days showed shortened duodenal villi due to damage by captan. This effect was observable in the crypts within 3 days of treatment initiation (Tinston, 1996). Immature cells were observed at the villi tips from day 7 onward, indicating a higher turnover of cells. There was some focal gastritis and parakeratosis noted in one mouse. When captan is fed to mice at 6000 ppm for 28–90 days, villus atrophy occurs, together with a crypt hypertrophy and crypt cell hyperplasia (Tinston, 1995). CD-1 mice administered captan for 56 days as 0, 400, 800, 3000, or 6000 ppm were evaluated for proliferative changes in the duodenum (Tinston, 1995). An assessment of the duodenum was made using histopathology and a bromodeoxyuridinelabeling index to measure crypt cell proliferation. Captan induced hyperplasia of the crypt cells, an increase in the crypt cell labeling index, and an increase in the number of cells in the crypt cell population. At 3000 ppm, the villus-to-crypt height ratio decreased from 5.4 in males and 5.9 in females to 1.4 and 2.6, respectively. These observed changes are consistent with an irritant action of captan on the duodenum. The no-observed-effect limit (NOEL) for duodenal hyperplasia is 400 ppm. Male CD-1 mice treated with 5000-ppm folpet for 28 days (Waterson, 1995) were observed to have proliferative changes in the duodenum proximal to the pyloric sphincter. An inflammatory response, similar to that noted with captan, was not seen. Treatment of CD-1 mice with folpet at 0, 150, 450, and 5000 ppm for 28 days resulted in duodenum proliferative effects (Milburn, 1997). Villi length was reduced and crypt compartments were expanded, reducing the villi-to-crypt ratio. The NOEL for hyperplasia in the duodenum for males was 450 ppm (69 mg/kg per day) and the NOEL for females was 150 ppm (29 mg/kg per day). (b) Subchronic Study B6C3F1 mice were fed folpet at 0, 1000, 5000, or 10,000 ppm for 4 weeks (Rubin, 1981). There was reduced food intake and body weight gain at 5000 ppm.

Chapter | 90  Captan and Folpet

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90.3.2.2  Rats

90.3.2.3  Rabbits

Oral Wistar rats treated for 4 weeks with captan at 0, 2000, 4000, 8000, or 12,000 ppm were observed to have a dose-related decrease in body weight gain and food intake (Til and Beems, 1979). At the top two doses, there were increases in basophilia of hepatocytes in the periportal area of the liver accompanied by increases in relative liver weight. A similar finding was observed in the females at the next lower dose (4000 ppm). The relative organ-to-body kidney weights were statistically increased at all doses. Folpet Technical admixed in the diet at 0, 2000, 4000, and 8000 ppm, and fed to Fischer rats for 13 weeks produced a treatment and dose-related decrease in body weight gain and hyperkeratosis/acanthosis of the esophagus and nonglandular stomach (Sela, 1982). The NOEL for decreased weight gain was 2000 ppm in males (136 mg/kg per day) and 4000 ppm in females (291 mg/kg per day). Irritation to the esophagus and forestomach occurred at all doses and in both sexes. A variety of hematological and clinical chemistry changes were noted, but the incidence and pattern did not indicate a clear target. Folpet Technical admixed in the diet at 0, 300, 1000, 3000, or 10,000 ppm and fed to Sprague-Dawley rats for 13 weeks, followed by a 2-week recovery showed similar signs of irritation in the forestomach, primarily at 10,000 ppm, but no irritation of the esophagus (Reno et al., 1981). Following a 2-week recovery period during which the rats received a control diet, the forestomach histology returned to normal. Folpet Technical fed to B6C3F1 mice for 28 days at levels of 0, 1000, 5000, and 10,000 ppm induced a reduced body weight gain in the top two doses (Crown, 1981).

A 21-day dermal study with captan in rabbits at 0, 12.5, 110, or 1000 mg/kg per day (6 h exposure per day) resulted in a dose-related desquamation of the skin by day 21, erythema and edema at the high dose, and acanthosis and hyperkeratosis of the treated skin at all doses (Johnson, 1987).

(a) Dermal A 28-day rat study with Folpet Technical applied mineral oil at 0, 1, 10, 20, and 30 mg/kg per day to the backs of Sprague-Dawley rats 6 h per day, 5 days per week. All dose levels elicited irritation that was more severe in males than females and resulted in decreased weight gain (Dougherty, 1988). The irritation was so severe in the 30-mg/kg per day male group that application was terminated after 10 days. All adverse effects noted were related to the skin irritation induced by repeated exposures to folpet. (b) Inhalation Captan has been tested in Wistar rats by nose-only inhalation at nominal dose levels of 0.1, 0.5, 5, and 15 g/l for 13 weeks (Hext, 1989). There were deaths in males at the high dose and dose-related effects on the larynx (e.g., squamous metaplasia, squamous hyperplasia, vacuolar degeneration of squamous epithelium). The no-observedeffect concentration (NOEC) for toxic effects (other than generalized irritation) was 0.6 g/l (measured).

90.3.2.4  Dogs Beagles, two per sex per treatment group, were administered captan by capsules at 0, 30, 100, 300, 600, or 1000 mg/kg per day for 4 weeks. The results included treatment-related emesis and a dose-related decrease in food intake and body weight gain (Blair, 1987). There was an increase in relative liver weight in males at 600 and 1000 mg/kg per day and relative kidney weight in females at 1000 mg/kg per day. Some fatty changes were seen in the kidney and liver of one male at 1000 mg/kg per day. Folpet administered to two beagle dogs per sex per group at 0, 20, 60, 180, and 540 mg/kg per day for 4 weeks induced emesis (Daly, 1983). Food intake and body weights were reduced in a dose-related manner. There were, however, no histopathologic changes noted. Folpet was administered to four beagles per sex at 0, 790, 1800, and 4000 mg/kg per day for 13 weeks with gelatin capsules (Barel et al., 1985). Daily doses of 4 g/kg were well above the maximum tolerated dose and resulted in severe deterioration of the males, all of which were killed for humane reasons. One of the females at the high dose was also terminated in moribund condition. Vomiting and diarrhea were noted clinical signs and both food intake and body weight gains were reduced in a dose-related fashion. Treatment resulted in irritation of the gastric mucosa, atrophied testes, thyroid degeneration, and muscular dystrophy.

90.3.2.5  Miscellaneous Studies Rats and mice administered 3000 ppm captan had reduced immune function as measured by sheep red blood cell antibody formation after treatment for 42 days (LaFargeFrayssinet and Decloitre, 1982). Captan was reported to suppress both B- and T-cell function in mice. Wistar rats had depressed lymphocyte count and lower relative thymus weight after 3 weeks of dietary administration of captan at 1000 ppm [50 mg/kg body weight (bw) per day], initiated as weanlings (Vos and Krajnc, 1983). Additionally, rats administered pre- and postnatal captan at 750 and 2000 ppm (37.5 and 100 mg/kg bw per day) showed a decrease in secondary IgG response to tetanus toxoid in the high-dose animals (Vos and Krajnc, 1983). Evaluation of these studies by Joint Meeting on Pesticide Residues (JMPR) concluded that captan may be an immunodepressant (FAO/WHO, 1990). These dose levels, however, are high and may not be relevant to anticipated human exposure scenarios. Three consecutive intraperitoneal administrations of captan at doses up to

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15 mg/kg to Swiss Albino CD1 mice impaired CTP-catalyzed metabolism (Paolini et al., 1999). This effect was attributed to captan metabolites; however, the route of administration is not relevant for human risk assessment. In another study in which captan was administered orally at 80 mg/kg (three doses) to rats there was no affect on any of the cytochrome P450 isoenzymes (Rahden-Staron et al., 2001).

90.3.3  Chronic Toxicity One mechanistic study was conducted in mice with captan. These data are also relevant for folpet because both share a common mechanism of toxicity. Duodenal tumors in mice were seen in both chronic and oncogenic studies (discussed below). Chronic administration of folpet produced evidence of irritation to the esophagus and stomach in rats.

90.3.3.1  Mice In a mechanistic study, male CD-1 mice were administered captan at dietary doses of 0 and 6000 ppm for 3, 6, 9, 12, 18, or 20 months (Pavkov and Thomasson, 1985). Mice were examined at the end of each dosing period and, in addition, various recovery periods were evaluated (Table 90.4). One group dosed with 6000 ppm for 6 months was held for an additional 6-month recovery period; another was held for a 12-month recovery period. One group dosed with 6000 ppm for 12 months was followed after a 6- and 8-month recovery. The most characteristic pathologic findings consisted of necrotizing and proliferative changes in the nonglandular portion of the stomach (after 3 months), dilation of the small intestine, and focal epithelial hyperplasia in the proximal part of the small intestine. Focal epithelial hyperplasia was also found in controls, but the incidence was lower compared with that of the treated animals, and the localization of these foci was more caudal

Table 90.4  Captan Mechanistic Study Designa

than was the case for captan-administered mice. Diffuse hyperplasia was found only in treated mice and was not considered prerequisite for the development of focal hyperplasia. Adenomas and adenocarcinomas also developed in the small intestines of treated mice with localization in the proximal 7 cm of the small intestine; the area of localization was the same as for the focal hyperplasia. Removal of captan from the diet resulted in a significant reduction in the incidence of focal epithelial hyperplasia as compared with the incidence in concurrent lifetime-treated mice and was no greater than that in concurrent controls. The incidence of neoplasia, however, in mice in the recovery group was not significantly different from that of concurrent lifetime-treated mice, but increased in mice treated for 6 months with a recovery period of 6 months and in mice treated for 12 months with a recovery period of 6–8 months, respectively, when compared with controls. The latter increase was not found in mice treated for 6 months with a recovery period of 12 months.

90.3.3.2  Rats Rats were administered folpet at dietary concentrations of 0, 250, 1500, and 5000 ppm (Crown et al., 1989). Body weight gain and food intake were decreased at 5000 ppm. The incidence and severity of diffuse hyperkeratosis in the esophagus and nonglandular epithelium of the stomach were increased in both sexes at 5000 ppm. The stomach was also affected at 1500 ppm. The NOEL was 250 ppm (12 and 15 mg/kg per day, males and females, respectively). A folpet combined chronic toxicity/oncogenicity study from which the EPA derived a NOEL for use in chronic dietary risk assessment employed dietary dose levels of 0, 200, 800, or 3200 ppm (Cox et al., 1985). The EPA selected the 200-ppm level (9 mg/kg per day) as the NOEL for conducting chronic dietary risk assessments, based on hyperkeratosis/acanthosis and ulceration/erosion of the nonglandular stomach at 800 ppm (equivalent to 35 mg/kg per day; U.S. EPA, 1999b).

90.3.3.3  Dogs

Time (months) Dose (ppm)

3

6

9

12

18

20

0

S

S

S

S

S

S

6000

T, S

6000

T

T, S

RS

RS

6000

T

T

T

T, S

RS

6000

T

T

T

T

T, S

6000

T

T

T

T

T

RS

T, S

T  treatment; S  sacrifice; RS  recovery sacrifice. a Reproduced with permission from Pavkov and Thomasson (1985).

Dogs were treated with captan at 0, 12.5, 60, and 300 mg/kg per day for 1 year (Blair, 1988). Only the high-dose animals differed from control in increased incidence of emesis and soft stool, increased relative liver weight, and decreased total serum protein and albumin. There were two 1-year dog studies conducted with folpet: the first at 0, 10, 60, and 140/120 mg/kg per day (Daly and Knezevich, 1986) and the second at 0, 325, 650, and 1300 mg/kg per day (Waner, 1988). In the first study, the 140 mg/kg per day was reduced to 120 mg/kg per day on day 50 due to unacceptable decreases in body weight gain and food intake. A NOEL was selected for the study at 10 mg/kg per day based on lowered body weight gain and food intake

Chapter | 90  Captan and Folpet

at 60 and 120 mg/kg per day. There were no clinical signs of toxicity noted, but clinical chemistry values showed a treatment-related decrease in total plasma protein parameters and cholesterol. Organ weights were not affected by treatment, nor was there evidence of macroscopic or microscopic changes as a result of treatment. In the second study, folpet induced incidences of diarrhea, vomiting, and salivation that were associated with reduced food intake and reduced body weight gain. Testes weights were reduced in males administered 1300 mg/kg per day when compared with controls on an absolute basis, but were similar to controls when measured on a relative body weight basis. The NOEL for this study was 325 mg/kg per day, based on decreased body weight gain. The WHO acceptable daily intake (ADI) is 0.1 mg/kg per day for both captan (FAO/WHO, 1990) and folpet (FAO/WHO, 1996).

90.3.4  Developmental and   Reproductive Toxicity 90.3.4.1  Developmental Studies (a) Rats Captan administered to Sprague-Dawley CD rats at 0, 18, 90, and 450 mg/kg per day resulted in decreased maternal weight gain and decreased food consumption at the high dose (Rubin, 1987). There were no effects on postimplantation loss or fetal survival. Fetal body weight was reduced and the incidence of “small” fetuses (3.0 g) was increased at the high dose. There were no increases in incidences of treatment-related malformations. The incidence of minor skeletal variations, including the presence of a fourteenth (lumbar) rib, incomplete fusion of vertebral hemicentra fusion, and reduced ossification of the pubes was increased at 450 mg/kg per day. The NOELs for maternal and developmental toxicity in this study were 18 and 90 mg/kg per day. In another study, folpet was administered by gavage to Sprague-Dawley CD rats at 0, 150, 550, or 2000 mg/kg per day from gestation days 6–15. Maternal toxicity in the form of decreased food intake and body weight gain was observed at the mid- and high dose. Fetuses showed slight developmental retardation at 150 mg/kg per day, suggesting the NOEL was slightly below this level (Rubin, 1983). Pups from rats treated with 400 mg/kg per day from gestation days 8–10 were normal (Kennedy et al., 1968) as were rats treated with 360 mg/kg per day from gestation days 6–19 (Hoberman et al., 1983). (b) Rabbits Captan did not induce any teratogenic effects when administered to New Zealand White (NZW) rabbits at 0, 10, 40, and 160 mg/kg per day from gestation days 7–19 (Rubin and Nyska, 1987). The highest dose was toxic to both dams and fetuses. An increased incidence of minor skeletal ­variations was seen at this dose. In another study, captan

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was administered to New Zealand White rabbits at 0, 10, 30, or 100 mg/kg per day (Tinston, 1991). The developmental NOEL was 10 mg/kg per day based on increased postimplantation loss, reduced mean fetal weight, and increased skeletal defects in fetuses (27 presacral vertebrae) at the maternally toxic dose of 30 mg/kg per day. THPI was tested at 5, 10, or 22.5 mg/kg/day (GD 6–28) and did not induce any adverse soft tissue or skeletal effects on fetuses (Blee, 2006b). Folpet was tested in both Dutch Belted and NZW rabbits for potential developmental toxicity. The original studies (Fabro et al., 1966; Kennedy et al., 1968; McLaughlin et al., 1969) were conducted at high doses ranging from 75 to 150 mg/kg per day during gestation days 7–12, 6–16, or 6–18. These studies consistently demonstrated the absence of adverse effects. One study reported five incidences of hydrocephaly at doses that were maternally toxic (Feussner et al., 1984; one at 20 mg/kg per day and four at 60 mg/kg per day). A second study in which doses of folpet were “pulsed” failed to replicated this finding (Feussner, 1985). The most recent study employed doses of 10, 40, and 160 mg/kg per day, and confirmed the absence of folpet-induced developmental effects (Rubin, 1985b). Although a weight-of-evidence (WOE) analysis concluded folpet is not a developmental toxin, the U.S. EPA has assigned this compound an FQPA uncertainty factor of 3 based on the initial Feussner study. Although the U.S. EPA pointed to one study where hydrocephaly occurred at maternally toxic doses of 20 (one instance) and 60 mg/kg per day (three fetuses in two litters; Feussner et al., 1984), a second “pulse dose” study (Feussner, 1985) failed to replicate this finding and other developmental studies in NZW rabbits showed no evidence of teratogenicity or hydrocephaly (Fabro et al., 1966; Kennedy et al., 1968; McLaughlin et al., 1969; Rubin, 1985b). The U.S. EPA cited the Feussner study as the basis for assigning an FQPA threefold uncertainty factor (U.S. EPA, 1999b), although a WOE analysis concluded that folpet is not a selective developmental toxin (Neal, 2000). Phthalimide was tested at 5, 15, or 30 mg/kg/day (GD 6-28) and did not produce any adverse soft tissue or skeletal effects in fetuses (Blee, 2006a). (c) Mice CD-1 mice administered folpet by gavage, subcutaneously 3 (100 mg/kg per day) or by inhalation (�������������� at 624 g/m ����������� ), showed no developmental abnormalities (Courtney et al., 1983). BL6 mice treated subcutaneously and orally with folpet at 100 mg/kg per day and AKR mice treated subcutaneously at 100 mg/kg per day were judged not to have adverse developmental findings (Bionetics Research Laboratories, 1968). (d) Other Species Captan is not teratogenic in beagles when administered in the diet at 60 mg/kg per day either throughout gestation or throughout gestation plus lactation (Kennedy et al., 1975b).

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Folpet was studied in Rhesus and stump-tailed macaques as part of research on thalidomide (Vondruska, 1969). There were no malformations with folpet at doses up to 75 mg/kg per day. Thalidomide at 10 mg/kg per day produced limb defects.

90.3.4.2  Reproductive Studies In a three-generation study, COBS CD rats were treated with captan at 25, 100, 250, and 500 mg/kg per day (Schardein et al., 1982). Nonreproductive parental toxicity was seen at 100 mg/kg per day and above in the absence of reproductive effects. Pup weights were lower by 7% compared with controls at 25 mg/kg per day. A subsequent one-generation study at 0, 6, 12.5, and 25 mg/kg per day showed no effect on pup weights at 25 mg/kg per day. The NOEL selected by the EPA for use in risk assessment was 12.5 mg/kg per day, based on the weight gain depression in the three-generation study (Ghali, 1997; Schardein and Aldridge, 1982). The reference dose employed by the U.S. EPA, 0.13 mg/kg per day, is based on this NOEL and the use of a 100-fold safety factor. Folpet administered to rats at 0, 250, 1500, and 5000 ppm showed diffuse hyperkeratosis of the nonglandular epithelium of the stomach (Rubin, 1986). The NOEL for this study was 250 ppm, which averaged 24 mg/kg per day. There were no reproductive effects noted. Other two- or three-generation studies in rats with folpet also showed no adverse reproductive effects (Hardy, 1985; Kennedy, 1967).

90.3.5  Mutagenicity 90.3.5.1  Overview The issue of mutagenicity has been controversial, but with mechanistic studies in place, disparate results in vitro and in vivo have been resolved. Throughout this chapter, the rapid degradation of captan and folpet in living systems has been central to understanding their toxicology. The pattern of mutagenicity is consistent with this degradation and provides examples of how such degradation diminishes adverse effects. In vitro studies show evidence that both captan and folpet have mutagenic potential. Captan appears more potent than folpet, and both their mutagenic activity is inversely proportional to the presence of thiols in reaction vessels. Once thresholds for complete degradation are reached, in vitro activity is abolished. The large reserves of thiols present in the intact animal and the near instantaneous reaction of captan and folpet with these thiols serve to ensure complete elimination of captan and folpet before they can reach sensitive DNA targets. The net result of this rapid degradation is the absence of mutagenicity in vivo. The mechanism by which captan and folpet effect their mutagenicity is not clear; however, data suggest that thiophosgene, in addition to the parent compounds,

Hayes’ Handbook of Pesticide Toxicology

is mutagenic (Arlett et al., 1975). For in vitro systems, both frame-shift and base-pair substitutions are seen. Cytogenetic effects are seen in vitro, but positive results are not as ubiquitous as point mutations. These clastogenic effects are reduced when enzyme-enhanced rat liver extract (S-9) is present and are generally absent in vivo. The weight of evidence shows that although captan and folpet possess inherent mutagenic potential, they are not mutagenic in vivo.

90.3.5.2  Mutations (a) In Vitro Assays Table 90.5 shows results from representative in vitro assays with Salmonella typhimurium, strain TA 100, a prokaryote organism. The greater potency of captan relative to folpet is noted. Other S. typhimurium strains showed similar results. A mutation index (the ratio between induced versus spontaneous revertants) of 7.3 for captan and 6.3 for folpet was seen for strain 104 (Barrueco and de la Pena, 1988). Positive findings were generally seen with strains 98, 1535, 1537, and 1538 (Carere et al., 1978; Shiau et al., 1981; Shirasu et al., 1976), but negative findings were seen with strain 1536 (Shiau et al., 1981). Where there were marginally positive results with captan, folpet was usually negative. Strain WP2 try–hcr of Escherichia coli showed a strong response to captan and a negative response to folpet (Nagy et al., 1975) or, where both were positive, the revertants per plate were greater for captan than for folpet (Shirasu et al., 1976). Both were positive with the WP2 try– hcr– strain (Nagy et al., 1975; Shirasu et al., 1976) as well as other tests with the WP2 strain (Bridges et al., 1972; Simmon et al., 1976). Tests with Bacillus subtilis strains TK 6321 and 5211 were positive for both compounds: captan showed a greater mutagenic response than folpet (Shiau et al., 1981). Captan also induced point mutations in Aspergillus nidulans (Martinez-Rossi and Azevedo, 1987). Assays with eukaryote organisms such as Chinese Hamster cells and mouse lymphoma cells are shown in Table 90.6. These data show that captan and folpet induce mutations when measured in vitro. THPI was tested with S. typhimurium strains TA 98, 100, 1535, and 1202 as well as E. coli WP2 uvrA, and was negative (Carver, 1985). Phthalimide is inactive in S. typhimurium as well (Rideg, 1982). (b) Effect of Exogenous Thiols on Mutagenicity Assays The rat liver S-9 fraction is added to in vitro systems to simulate the metabolic capability of intact organisms. In this way, compounds that are mutagenic only after they are metabolized by cell enzyme systems are detected. Metabolism, however, appears to play no role in the expression of mutagenicity of captan or folpet; on the contrary, the addition of S-9 serves to diminish mutagenic potency. The reduced mutagenic activity following the

Chapter | 90  Captan and Folpet

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Table 90.5  Prokaryote Reverse Mutation: Salmonella typhimurium, Strain TA 100 Compound

Resultsa

Reference

Captan

26.7 Revertants per 108 cells/nmol, −S-9

Shiau et al. (1981)

Table 90.6  In Vitro Eukaryote Mutation Assays Assay

Compound

Results

Reference

Chinese hamster V79/Hgprt

Captan

Positive only in the absence of serum from the culture media

Arlett et al. (1975)

 @ 50 g/plate (167 nmol), S-9

Mean number of resistant colonies: 0.3 and 0.6 at 5 and 10 g/ml captan; vapor emitted from sodium bicarbonate activated captan impregnated on filter paper above the test system also induced mutations

 @ 50 g/plate (167 nmol), S-9 Folpet

7.7 Revertants per 108 cells/ nmol, −S-9

Shiau et al. (1981)

 @ 50 g/plate (167 nmol), −S-9 – @ 50 g/plate (167 nmol), S-9 Captan

26 Revertants/nmol, −S-9

Folpet

8 Revertants/nmol, −S-9

Captan

93.7 Revertants/nmol, −S-9

Folpet

15.0 Revertants/nmol, −S-9

De Flora et al. (1984)

Moriya et al. (1983)

a  S-9: Rat liver homogenate included for “metabolism” of test material. −S-9: Incubation without rat liver homogenate.

addition of S-9 is an example of the general phenomenon of thiol-related degradation of captan and folpet. The presence of sufficient thiols abolishes mutagenic activity. The addition of S-9 or rat blood prior to the addition of captan or folpet reduces or abolishes activity (Table 90.7). When cysteine is added to either captan or folpet in varying ratios, the mutagenic activity declines as the ratio increases from 0.5 to 2.5. At a ratio of 5-m cysteine to 1-m captan or folpet, mutagenic activity is abolished (Moriya et al., 1978). Glutathione provided similar protective actions when added to assay vessels in ratios of 1 or higher compared with the fungicide (Rideg, 1982). Adverse tox­ icity as well as mutagenicity in Chinese hamster V79 cells is reduced when 10% fetal calf serum is used in the standard V79/Hgprt assay (Arlett et al., 1975). (c) In Vivo Assays Armed with knowledge of how these compounds degrade, it is not surprising that mutagenicity is absent in vivo (Table 90.8).

90.3.5.3  Cytogenetic Effects The effects on chromosomes mirror the pattern of activity for mutations: clastogenic findings are seen in vitro but are generally absent in vivo.

Chinese Captan and hamster folpet CHO/Hgprt

Both compounds were positive in the absence of S-9

O’Neill et al. (1981)

Mouse lymphoma L5178Y/TK

Positive in the absence of S-9

Oberly et al. (1984)

Captan

(a) In Vitro Assays Table 90.9 lists representative in vitro cytogenetic studies with captan or folpet. The addition of S-9 to assay vessels serves to detoxify captan and folpet as it does in the mutation assays. This action is expressed by a decrease in cytotoxicity with a resulting increase in tolerated dose levels. At some point, it is expected that the threshold for detoxification is exceeded and the remaining captan or folpet can act to affect the chromosomes. These data are mixed in that some positive and some negative findings are reported. (b) In Vivo Assays Table 90.10 lists representative in vivo cytogenetic studies with captan or folpet. Micronucleus assays were conducted in CD-1 mice with both compounds and yielded negative results. Captan was administered at 40, 200, and 1000 mg/kg (Jacoby, 1985b) and folpet was administered at 10, 50, and 250 mg/kg (Jacoby, 1985a). Chlorambucil, the positive control, resulted in a significant increase in micronuclei. Captan was also negative when tested in the wing spot test in Drosophila (Osaba et al., 2002). The work by Chidiac (Chidiac, 1985; Chidiac and Goldberg, 1987) provides valuable information with regard to the genotoxicity of these compounds. The basis for this

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Table 90.7  Comparison of Captan and Folpet Mutagenicity with the Addition of Exogenous Proteina Strain and dose

Component

Captan (rev/plate)

Folpet (rev/plate)

Commentb

E. coli

None

3200

1320

Captan is more active than folpet

WP2 hcr

S-9

30

50

S-9 decreases activity

0.15  M/plate

S-9 fraction

111

60

S-9 fraction decreases activity

(45 g/plate)

20-mM cysteine Rat blood

19 32

21 19

Cysteine abolishes activity (control rev/plate 30) Blood abolishes activity

S. typhimurium

None S-9

268 6

219 8

Captan is more active than folpet S9 abolishes activity (control rev/plate 17)

TA 1535

S-9 fraction

31

35

S-9 fraction decreases activity

0.15 M/plate

20 mM

4

6

Cysteine abolishes activity

(45 g/plate)

cysteine Rat blood

6

14

Blood abolishes activity

a

Reproduced from Moriya et al. (1978).

b

Captan (0.1 ml of 1.5 M/ml) or folpet (0.2 ml of 0.75 M/ml) was incubated for 10 min at 37°C with 0.5 ml of one of the following: S-9 (containing 0.3 ml S-9/ml), S-9 fraction (S-9 mix minus cofactors), 20-mM cysteine, or rat blood diluted twice with phosphate buffer or water as control. After incubation the tester strains (0.1 ml) and agar (2 ml) were added to the test tubes and plated out. Revertants/plate were read after incubation at 37°C for 2 days.

work drew upon evidence of mutagenicity and the tumorigenic effect captan has on the mouse duodenum. It was postulated that evidence of cytogenetic damage would be seen in the duodenum after exposure to captan. This mouse bioassay was validated with known carcinogens and noncarcinogens. Nuclear aberrations (NA) consisted of micronuclei and apoptotic bodies in the crypt cells of the duodenal epithelium. Xirradiation, 1,2-dimethylhydrazine, benzo(a)pyrene (B(a)P), and N-methyl-N-nitrosourea (MNU) induced tumors in the small intestine. Each led to a dose-related increase in the incidence of NA 24 h after administration to mice. Benzo(e)pyrene and methylurea, which are noncarcinogenic structural analogues of B(a)P and MNU, did not induce NA. Cells of the duodenum were harvested and examined for the presence of NA after a variety of captan dose regimens. Captan as well as THPI consistently failed to induce NA. Captan was administered to male CD-1 mice using a number of regimens, including a single bolus dose of 4000 mg/kg, dietary dose levels of 4000 and 16,000 ppm, and five repetitive doses totaling 5000 mg/kg (Table 90.11). In all cases, including pretreatment with l-buthionin-S,R-­sulfoximine (an inhibitor of glutathione synthesis), the investigators noted an absence of the expected signs of DNA damage. Folpet was tested in a study that replicated the Chidiac experimental design (Gudi and Krsmanovic, 2001). Mice were administered five consecutive daily oral doses of folpet at 2000 mg/kg per day. Nuclear aberrations in the duodenal crypt compartment were absent in folpet-treated mice, whereas mice administered a single dose (65 mg/kg) of dimethylhydrazine showed both apoptotic cells and micronuclei in the crypts.

90.3.5.4  Dominant Lethal Assays Dominant lethal assays have generally been negative (Table 90.7). However, positive findings for both compounds have been reported (Collins, 1972a,b). In spite of these positive findings, it appears that the compounds do not induce dominant lethal effects. This conclusion is based on: (1) the absence of positive micronucleus assays; (2) the absence of adverse effects in two-generation rat reproductive studies; (3) the lack of negative dominant lethal effects in other studies [captan: Kennedy et al. (1975a), Rideg (1982), Shirasu et al. (1978), Tezuka et al. (1978); folpet: Bradfield (1980), Calandra (1971), Kennedy et al. (1975a), Rideg (1982)]; and (4) the consistency of the findings with the rapid degradation of these compounds. A less than 1-s (captan) or less than 5-s (folpet) half-life in blood argues against the possibility of parent molecules reaching the testes. Thiophosgene is considered to be more reactive than captan or folpet, but also would not reach the testes.

90.3.5.5  DNA Interaction Captan was negative for inducing unscheduled DNA synthesis (UDS) in human diploid fibroblasts in vitro (Mitchell, 1975). It was also negative for UDS in primary liver cells (Probst et al., 1981; Rocchi et al., 1980). The nature of the captan and folpet molecules imparts difficulties in conducting in vivo DNA binding studies. Two approaches, using radiolabeled test material, have sought to determine if captan covalently binds with duodenal DNA of the mouse. In both cases, the trichloromethylthio side chain was labeled because it is the chemically

Chapter | 90  Captan and Folpet

1931

Table 90.8  In Vivo Mutagenicity Assays

Table 90.9  In Vitro Cytogenetic Assays

Assay

Compound Results

Reference

Assay

Compound

Results

Reference

Somatic cell mutation

Captan

Negative, oral

Nguyen (1981)

Chinese hamster V79

Captan

Tezuka et al. (1980)

(Mouse spot test)

Captan Folpet

2.2% frequency after intraperitoneal dose of 15 mg/ kg Negative, oral

Imanishi et al. (1987) Moore (1985)

Positive for sister chromatid exchange and chromosomal aberrations

Captan, folpet

Positive in the absence of S-9

Ishidate et al. (1981)

Negative

Mollet and Wurgler (1974)

Chinese hamster lung fibroblasts Chinese hamster ovary (CHO)

Folpet

Positive, but higher concentrations required with S-9

Loveday (1989)

Human blood

Captan

Negative

Pilinskaya (1983)

Lymphocytes

Folpet

Negative (5 g, 2-h exposure)

Bootman et al. (1987)

Human lymphoid cell line

Captan

Positive in the absence of S-9

Sirianni and Huang (1978)

Human diploid fibroblast cell line

Captan

Negative

Sasaki et al. (1980); Tezuka et al. (1978)

Human embryonic lung and rat kangaroo cell lines

Captan

Positive

Legator (1969)

Somatic mutation and recombination (Drosophila SMART test)

Captan

Mouse heritable translocation assay

Captan

Drosophila sex-linked

Captan

Recessive lethal assay

Folpet Captan Captan Folpet Folpet

Negative

Negative

Negative Negative Weakly mutagenic Weakly mutagenic Negative

Mouse dominant

Captan

Negative

Lethal assay

Folpet Captan

Negative Negative

Folpet Captan Folpet Folpet

Negative Negative Negative Negative

Captan

Negative

Simmon et al. (1977)

Kramers and Knaap (1973) Mollet (1973) Valencia (1981) Vogel and Chandler (1974) Jorgenson et al. (1976) Kennedy et al. (1975a) Rideg (1982) Epstein et al. (1972) Simmon et al. (1977)

Table 90.10  In Vivo Cytogenetic Assays Assay

Compound

Results

Reference

Captan

Negative

Jacoby (1985b)

Folpet

Negative

Jacoby (1985a)

Captan

Negative

Tezuka et al. (1978) Fry and Fiscor (1978) Chidiac and Goldberg (1987) Esber (1983)

Rat dominant

Captan

Positive

Collins (1972a)

Micronucleus

Lethal assay

Folpet Folpet

Positive Negative

Collins (1972b) Bradfield (1980)

Chromosomal aberration

active portion of the molecule and is expected to participate in DNA binding if it occurs. The first study used 14Ccaptan (Selsky and Matheson, 1981); the second study used 35S-captan (Provan et al., 1995). When the carbon atom of the trichloromethylthio moiety is labeled, it enters the C-1 carbon pool via CO2 that is

Negative Negative (see Table 22)

Heritable translocation

Folpet

Negative

Captan

Negative

Jorgenson et al. (1976)

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1932

formed as the molecule degrades. As such, a low level of ubiquitous labeling appears throughout the mouse. When the sulfur atom of the trichloromethylthio moiety is labeled, sulfur exchange occurs, resulting in a low level of incorporation of 35S into proteins. Histones, in turn, are associated with DNA and result in a low level of associated radioactivity (Provan et al., 1995). Investigators have concluded that covalent binding of captan to DNA has not been demonstrated (Pritchard and Lappin, 1991; Provan et al., 1992, 1993; Selsky and Matheson, 1981). An experimental design

Table 90.11  Nuclear Aberration Study with Captana Treatment

Dose levels

Results

Single bolus dose

0 and 4000 mg/kg

Negative

Single bolus dose after pretreatment with BSOb

0 and 4000 mg/kg

Negative

Dietary administration, 7 days

0, 8000, and 16,000 ppm

Negative

Five daily doses

Single 0 20 200 1000

Negative

Cumulative 0 100 1000 5000c

a

Reproduced with permission from Chidiac and Goldberg (1987). BSO: L-buthionin-S,R-sulfoximine, an inhibitor of glutathione synthesis. c Doses: Day 1, 2000 mg/kg; day 2, dosing suspended due to toxicity; days 3–5, 1000 mg/kg. b

that “proves the negative,” however, has not been achieved and the EPA holds that in vivo DNA binding has not been ruled out (Hsu and McCarroll, 1998). The polyps, adenomas, and adenocarcinomas that develop in the mouse duodenum as a result of continuous oral administration of captan or folpet arise from the crypt cell compartment. Figure 90.5 depicts a simplified anatomy of the duodenum. Should mutagenicity play a role in the development of these tumors, it must be consistent with this anatomy and the nature of the chemical reactions associated with these compounds. There are two factors that suggest mutagenicity cannot be involved in the etiology of these tumors: first, nearly all absorption takes place through the villi; second, the degradation rates of captan and folpet prevent them from reaching the crypt compartment through diffusion. Material that is absorbed through the villi enters blood or lacteal vessels and is transported away from the crypts. Crypt cells receive blood supply from arterial vessels rather than the portal system. The remaining molecules that start to diffuse down to the crypt compartment must first pass through mucus and then diffuse through approximately 16 epithelial cells before reaching the stem cells located in position T4 from the base of the crypt (Potten and Loeffler, 1990). Mutational events in cells distal to the stem cells (some of which may still be dividing) are of no import because these cells migrate up the villi and are shed within 2–4 days. The duodenal mucosal cells are rich in glutathione, having a concentration of approximately 8 mmol in CD-1 mice (Chasseaud et al., 1991); thus, the degradation of parent molecules is promoted. Whereas the half-lives of captan and folpet are very short, the exponential loss of captan virtually eliminates all molecules in short order.

Migration of cells

Mucus

> 99% Absorption through villi

Villus

<1% Absorption through crypts

Crypt Basal cell location (T4)

To portal circulation Arterial supply Figure 90.5  Schematic of duodenal villi and crypts.

Chapter | 90  Captan and Folpet

A study of DNA damage in agricultural workers using the Comet assay showed the mean tail intensity and tail moment was greater for 134 agricultural workers compared with control populations. The mean levels of THPI were elevated in these workers (0.14 g/ml) compared with controls (0.078 g/ml); however, specific pesticide exposure levels were not obtained and the thrust of this work was to demonstrate the feasibility of rapid studies of DNA damage using this technique (McCauley et al., 2008). In summary, captan and folpet are chemically active molecules that can induce mutations and cytogenetic effects if they are positioned to interact with sensitive targets. The very nature of this reactivity coupled with mammalian anatomy, however, precludes such interaction in vivo. This conclusion is supported by the weight of evidence (including chemical fate), although instances of positive results in vivo have been reported. Captan and folpet are judged not to act as mutagens or genotoxins in the intact animal.

90.3.6  Carcinogenicity 90.3.6.1  Overview Both captan and folpet induce duodenal tumors in mice when fed at high doses. Rodent bioassay data are robust: there is a treatment and dose relationship of tumor incidence in mice, but such a relationship is absent in rats. Rat studies, however, have shown increased incidences of some tumors, but these are judged to be not treatment-related (Gordon et al., 1994). While this finding was not initially embraced by the U.S. EPA (Quest et al., 1993; U.S. EPA, 1999a,b), the current classification of captan agrees with this finding (U.S. EPA, 2004) and concludes: The new cancer classification considers captan to be a potential carcinogen at prolonged high doses that cause cytotoxicity and regenerative cell hyperplasia. These high doses of captan are many orders of magnitude above those likely to be consumed in the diet, or encountered by individuals in occupational or residential settings. Therefore, captan is not likely to be a human carcinogen nor pose cancer risks of concern when used in accordance with approved product labels.

Similar analysis has been completed for folpet (Cohen, 2008) and these data are being submitted to the U.S. EPA under PRIA for cancer reclassification. Reliance on tumor incidence reported in U.S. EPA Data Evaluation Records (DERs) in the absence of weight of evidence MOA analysis can result in questionable data. For example, the U.S. EPA’s ToxRefDB notes folpet induces multisite tumors in rats and captan induces multisite tumors in mice (Martin et al., 2009).

90.3.6.2  Mouse Bioassays An early captan study combined both gavage and dietary administration (Innes et al., 1969). These investigators

1933

dosed neonatal F1 hybrid mice by gavage with 215 mg/kg per day for 3 weeks and followed by dietary administration of 560 ppm for 18 months. This study was negative. However, the dietary concentration, in hindsight, appears to be below the threshold necessary for tumor induction. The National Cancer Institute administered captan to B6C3F1 mice at 8000 and 16,000 ppm for 80 weeks (NCI, 1977). Duodenal tumors were evident at the high dose (three in 46 males; three in 48 females). There was one in 43 males at the 8000 ppm that also had a tumor. Two other studies confirmed the treatment relationship of captan and duodenal tumors (Daly and Knezevich, 1983; Wong et al., 1981). The tumor incidence is shown in Table 90.12. The NOEL for duodenal tumors in mice (based on proliferative changes in the duodenum) is 400 ppm. Captan has also been evaluated by intraperitoneal and dermal administration. A study that treated two different “strain A” mice intraperitoneally with captan (along with 64 other chemicals previously tested by the National Cancer Institute) indicated a slight increase in lung tumors in males in one strain (Maronpot et al., 1986), but the significance of these data was questioned due to lack of interlaboratory consistency and lack of correlation to the standard rodent bioassays (FAO/WHO, 1990). A dermal study using the two-stage carcinogenesis model concluded that captan was neither a complete skin carcinogen nor a promoter, although at high doses (450 mg/kg three times per week for 3 weeks, followed by croton oil factor A1 three times per week for 51 weeks) there was some evidence it may act as a weak initiator (Antony and Mehrota, 1994). Tissue damage rather than mutagenic effect might account for this finding, however, because the control, DMSO, did not replicate the irritation effects of captan. The carcinogenic effect of folpet on the mouse duodenum is similar to that of captan (Table 90.13). The first two bioassays had doses of 1000, 5000, and 10,000 ppm (Rubin, 1985a), and 1000, 5000, and 12,000 ppm (Wong et al., 1982). In both cases, there was a low incidence of tumors at 1000 ppm. This finding triggered a third study with lower doses (East, 1994). The NOEL for tumors in the third study was established at 450 ppm. Although the primary site of gastrointestinal tumors is the duodenum in mice, a low incidence of tumors is seen in the stomach with folpet. There were some tumors noted with captan, but the incidence was low, not dose-related, and not obviously treatment-related. The differential aqueous stability in acid conditions of the stomach between captan and folpet may account for this finding. Captan elicits effects in the stomach, but these are restricted to polyp formation. The blockage from the stomach to the duodenum seen in some mice that resulted from the presence of polyps and tumors located just after the pyloric sphincter was suggested as a contributing cause of stomach tumors (Nyska et al., 1990). This blockage was thought to result in an increased concentration of folpet and folpet degradates

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Table 90.12  Captan Duodenal Tumor Incidence in Micea Dose (ppm and mg/kg/day) 0

100

400

800

6000

8000

10,000

16,000

0

15

61

123

925

NC

NC

NC

Adenoma

2/91

3/83

0/93

1/87

4/84

Carcinoma

0/91

0/83

0/93

0/87

2/84

Adenocarcinoma

0/9

Reference

Males Daly and Knezevich (1983)a

3/43

5/46

NCI (1977) Wong et al. (1981)b

Duodenal neoplasms 2/74

20/73

21/72

39/75

0

100

400

800

6000

8000

10,000 16,000

0

18

70

142

1043

NC

NC

Adenoma

3/85

1/82

1/83

7/81

3/91

Carcinoma

0/85

0/83

0/83

0/81

1/91

Adenocarcinoma

0/9

NC

Females Daly and Knezevich (1983)c

0/49

3/48

NCI (1977) Wong et al. (1981)b

Duodenal neoplasms 2/72

24/78

19/76

26/76

a

NC: not calculated. Incidence reported reflects pathology reevaluation of slides (Robinson, 1993). c The total tumor incidence combines both benign and malignant tumors. b

Table 90.13  Folpet Duodenal Tumor (Adenoma/Carcinoma) Incidence in Mice Dose (ppm and mg/kg/day) 0 0 Males

47

1000 93

b

1350 151

5000a 502

1/87

2/61

8/67

0

150 16

0/42

450 51

1000 96

b

1350 154

5000 515

a

10,000

b

0/88

1/63

7/67

12,000 1284

19/52

Rubin (1985a) 38/73

1/50

Dose levels for the Rubin study were 5000 and 10,000 ppm for the first 21 weeks and then adjusted down to 3500 and 7000 ppm. Wong et al. (1982).

b

Wong et al. (1982) East (1994)

a

10/52

0/49

Reference Rubin (1985a)

0/44

2/52

0/49

25/52 38/71

1/51

0/96

12,000 1282

17/52

0/48

10,000a

b

4/52

0

a

16

450

0/52

0/89

Females

150

Wong et al. (1982) East (1994)

Chapter | 90  Captan and Folpet

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Table 90.14  Rat Bioassays with Captan and Folpet Findingsa

Reference

Osborne-Mendel 0, 2525, 6060 ppm (TWA)b mg/kg/day not calculated

Negative

NCI (1977)

Wistar (Cpb:WU) 0, 125, 500, 2000 ppm 0, 6.25, 24, 98 mg/kg/day

Negative (uterus)

Til et al. (1983)

Charles River CD1 0, 500, 2000, 5000 ppm 0, 25, 100, 250 mg/kg/ day

Negative (kidney)

Goldenthal et al. (1982)

0, 1000, 5000, 10,000 ppm mg/kg/day not calculated

Negative

Hazleton (1956)

Fischer (chronic toxicity study) 0, 250, 1500, 5000 ppm 0, 12.5, 75, 250 mg/kg/day

Negative

Crown et al. (1989)

CD 0, 200, 800, 3200 ppm 0, 10, 40, 160 mg/kg/day

Negative (thyroid, testes)

Cox et al. (1985)

Fischer 0, 500, 1000, 2000 ppm 0, 25, 50, 100 mg/kg/day

Negative (mammary glands, thyroid, lymphoma)

Crown et al. (1985)

Test material Experimental design Captan

Folpet

a The U.S. EPA notes the incidence of tumors (in tissues) “associated” with treatment in organs listed in support of B2 cancer classification for both captan and folpet (Quest et al., 1993). Weight of evidence analysis shows captan is not a rat carcinogen (Foster and Elliott, 2000) nor is folpet (Study Director conclusions). b TWA: time-weighted average.

in the stomach. Stomach tumors were evident, however, where no blockage was apparent and thus argue against this hypothesis (East, 1994).

1985, 1989). The U.S. EPA now concurs with these findings for captan (U.S. EPA, 2004) and is reevaluating the folpet data.

90.3.6.3  Rat Bioassays

90.3.6.4  Comparison of Rat and Mouse Response to Folpet

In contrast to mice, there is no consistent tumor response across studies with rats (Table 90.14). Evaluation of captan tumor incidence data for kidney and uterine tumors using appropriate statistics and proper tumor grouping shows no treatment effect (Foster and Elliott, 2000; Gordon et al., 1994). It is unlikely the kidney tumors are related to treatment with captan because there is no increase in malignant tumors (carcinomas), there is a small increase (in a single animal) in benign tumors (adenomas) only, there is no statistically significant increase or trend in kidney adenomas, and the finding of kidney adenomas is seen in one out of four rat bioassays with captan and one out of seven bioassays with both captan and folpet. It is unlikely the uterine sarcoma tumors are related to treatment with captan because there is no statistical significance when tumors and polyps are considered together, a consideration dictated by the etiology of uterine sarcomas (Leininger and Jokinen, 1990). The four tumors noted comprise three different cell types and this finding was not consistent with the other bioassay results. In evaluating this study, the JMPR found “no other effects” in addition to depression of food intake and body weight gain at 2000 ppm and a slight increase in relative liver weight in males (FAO/WHO, 1990). With folpet, the study director concluded that the incidence for mammary glands and thyroid tumors were not related to treatment (Crown et al.,

A stark difference between mice and rats exists when comparing their tumor response to captan and folpet. All strains of mice tested show a treatment and dose-related incidence of duodenal tumors. All strains of rats show neither duodenal tumors nor proliferative changes. This suggests that the physiology of the mouse and rat differ in specific toxicokinetic and/or toxicodynamic ways that account for this difference. A series of comparative studies in the CD-1 mouse and Sprague-Dawley rat were conducted with folpet at 50 and 5000 ppm in an attempt to uncover the reason or reasons for this difference (Chasseaud et al., 1991). Areas investigated included respective milligrams per ­ kilogram per day doses, transit times through the gut, glutathione changes with dosing, effects on enzymes, pH changes, GSH levels, and binding of 14C-folpet to intestinal components. There were a variety of quantitative differences between rats and mice, suggesting that differential amounts of available glutathione may influence the tumor response.

90.3.6.5  Relevance of Mutagenicity to Mouse Tumors and Human Risk Assessment The presence or absence of a mutagenic component in the etiology of mouse duodenal tumors determines the paradigm used to assess risk to humans. Currently, the U.S. EPA

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acknowledges that mutagenesis is not involved in tumor induction by captan; thus, a threshold-based margin of exposure risk assessment is appropriate. As folpet is still officially classified as a B2 carcinogen (pending ­ ongoing PRIA review), the linear multistage model (Pitot and Dragan, 1996) can be used to describe risk.

90.3.6.6  Mode of Action Leading to Duodenal Tumors in the Mouse There is a preponderance of data that point unambiguously to a proliferation-based nongenotoxic mode of action for captan and folpet. This mode of action is consistent with the chemical and physical properties of captan and folpet, and the effect these compounds produce with high dietary exposure in the mouse. A key component of this mode of action is that it is threshold-based; that is, at dietary doses below the threshold, tumors will not develop. The physical and chemical properties include the instability of captan and folpet in aqueous solution at physiological pH, the reaction of captan and folpet with thiols, the generation of thiophosgene from both hydrolysis and thiol interactions, the transient nature of thiophosgene due to its chemical reactivity, and the comparatively low toxicity of THPI and phthalimide. The mode of action for mouse duodenal tumors must be consistent with these properties and effects. This MOA must also be supported by generally accepted principles of carcinogenicity. Mouse duodenal tumors develop with oral administration above a threshold if maintained for at least 6 months (Pavkov and Thomasson, 1985). Histopathological analysis shows that tumors arise from the crypt compartment and show a continuum from hyperplasia to polyps, adenomas, and adenocarcinomas (Tinston, 1995, 1996). Histologic and proliferation studies have characterized the changes to the duodenum with exposure to captan and show two sequential events (Allen, 1994; Foster, 1994). First, epithelial cells that comprise the villi are damaged by exposure to captan and sloughed off into the intestinal lumen at an increased rate. The villi height is shortened. Second, basal cells in the crypt compartment that normally divide at a rate commensurate with the normal loss of villi cells from the tips of the villi increase their rate of proliferation to a hyperphysiologic state. Crypt depth is subsequently increased and the villi-to-crypt ratio (measured by their respective sizes) decreases. A small number of transformed cells exist in the duode­ num as evidenced by a low incidence of duodenal tumors in bioassay control (Tables 90.12 and 90.13) and historic­al control mice (Bomhard and Mohr, 1989; Chandra and Frith, 1992; Lang, 1995; Maita et al., 1988; Ward et al., 1979). It is postulated that these transformed cells are subject to proliferative pressure and, as a result of this continued pressure for at least 6 months, progress to tumors. The basis for this postulation is the body of data that

Hayes’ Handbook of Pesticide Toxicology

show abnormally high cell proliferation, which is not carcinogenic per se, but does play a role in tumor development (Butterworth et al., 1992; Ledda-Columbano et al., 1989; Pitot et al., 1991). The role of proliferation in thyroid tumors is well established (Chhabra et al., 1992) and the influence of proliferation on initiated liver cells is also known (Solt et al., 1977). Classically, the two-stage carcinogenesis model in the skin points to the importance of sustained proliferation in the promotion of initiated cells to tumors (Berenblum and Armuth, 1977). In addition to promoting the clonal expansion of nascent tumor cells in situ, abnormally high proliferation may increase fixation and expression of premutagenic DNA lesions, increase the number of spontaneously initiated cells during replication, perturb checkpoints in the cell cycle leading to mutagenic events, and increase the number of spontaneously initiated cells by blocking cell death/ elimination (Ledda-Columbano et al., 1989). Thus, there are two avenues for duodenal tumors to develop: promotion of nascent tumor cells and initiation of normal basal cells through disruptions in normal DNA replication. The progression to tumors under this mode of action is depicted schematically in Figure 90.6. A genetic component is neither required nor plausible. Thresholds have been established for the initial cellular response to captan or folpet administration: villi damage and crypt cell hyperplasia. The NOELs for captan and folpet are similar: 400 ppm (60 mg/kg per day) for captan and 450 (69 mg/kg per day; males) or 150 ppm (29 mg/kg per day; females) for folpet. Administration of captan or folpet below these thresholds will not lead to tumors, because the basis for tumor progression (hyperphysiologic cell division rate) is absent. This mode of action requires that the appropriate paradigm for assessing carcinogenic risk in humans is margin of exposure not linear low-dose * extrapolation ( q1 ).

90.3.6.7  Epidemiology In a limited retrospective cohort mortality study, 138 workers in a captan manufacturing plant who were employed for a minimum of 3 months during a 23-year period beginning in 1954 were followed for 30 years (Palshaw, 1980). These workers were exposed to captan at estimated air concentrations ranging from 0.83 mg/m3 for THPI operators to 1.54 mg/m3 for captan operators. Other workers had little or no exposure (originally ranked as 0, 1, 2, or 3 for none, low, moderate, or high, respectively). These data showed there were no increased deaths that resulted from captan exposure. An analysis of epidemiology data from the Agricultural Health Study concluded that observed cancer cases and follow-up times of 9.14 years, “though limited by low numbers,” did not provide evidence of increased cancer risk due to use of captan (Greenburg et al., 2008).

Chapter | 90  Captan and Folpet

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Captan/folpet

Shortened villi Villi cell loss

Enlarged crypts

Crypt cell proliferation Hyperplastic crypts Dose > 50 mg/kg/day

Normal duodenum

Removal of captan/folpet Rapid recovery

Normal duodenum Captan/folpet Continued irritation Adenoma

Adenocarcinoma

Proliferative pressure on spontaneously-transformed cells in situ. Figure 90.6  Mode of action for captan and folpet in the mouse duodenum.

90.3.6.8  Summary Captan and folpet at sufficiently high doses act locally on the duodenal mucosa and result in damage to villi. Epithelial cells of the villi are lost and the homeostatic feedback mechanism increases cell proliferation in an attempt to make up this loss. Transformed cells that reside in the crypt compartment are sensitive to this proliferative pressure and are promoted to frank tumors (Figure 90.6). This mode of action has no mutagenic component and has a clear threshold for the first event that leads to tumors: increased ­proliferation/ hyperplasia of the duodenal crypt compartment.

90.4  Common mechanism of toxicity 90.4.1  Captan and Folpet Captan and folpet show obvious similarities in structure and effects. The Food Quality Protection Act (U.S. Congress, 1996) formally recognized the existence of such similarities and mandated that the U.S. EPA consider common mechanisms of toxicity when conducting risk assessments. The U.S. EPA issued guidance on how to determine the presence of a common mechanism for two or more pesticides (U.S. EPA, 1999c). Their criteria include structure, adverse effects, and mode of action. Captan and folpet

share sufficient common characteristics to conclude that they have a common mechanism of toxicity (Bernard and Gordon, 2000). This finding is specific to the key toxicological endpoint, duodenal tumors in mice, but may apply as well to other nonspecific endpoints. The finding that a common mechanism of toxicity exists for captan and folpet is supported by the following determinations: 1. Structural similarity. The active side chains, —SCCl3, are identical. 2. Site of action. Toxicity is expressed at the site of contact for both chemicals (that is, they are local irritants as opposed to systemic toxicants). 3. Reactivity with thiols. Both react with thiols to produce similar degradates. Differences in rates of reaction are attributable to the physical/chemical properties of the two compounds and do not serve to diminish their commonality. 4. Mechanism of pesticidal action. Toxicity to fungi is mediated through reactions with both soluble and insoluble thiols in fungal conidia. These same reactions account for expression of the common toxic endpoint in mammals. 5. Common toxic endpoint. Gastrointestinal tumors in mice that generally are specific to the duodenum. 6. Mode of action. Both captan and folpet express their common toxic endpoint through a nongenotoxic compensatory proliferation mechanism.

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7. Specificity of action. For both materials, the majority of tumors appear in the duodenum, but with folpet some tumors are noted in the stomach. The hydrolytic rate of folpet is approximately eight times faster than that of captan at pH 5 and may promote the presence of active metabolites in the acid environment of the stomach. Tumors are restricted to the mouse; rats are refractory. 8. Other toxic endpoints. Both captan and folpet show a similar pattern of toxicity for mutagenicity and skin sensitization. Both compounds show nonspecific secondary endpoints such as developmental toxicity manifested as decreased fetal weights and ossification defects at maternally toxic doses. Finding a common mechanism of toxicity for captan and folpet will influence the way the U.S. EPA regulates these two fungicides under FQPA. It also will afford toxicologists an opportunity to integrate data from the individual compounds to generate a more robust database, which is particularly valuable for evaluation of noncarcinogenicity in rats and elucidation of the mode of action of carcinogenicity in mice.

90.4.2  Captafol Captafol (CAS 2939-80-2; Figure 90.7) differs from captan and folpet in a number of areas. The side chain differs in structure as well as chemical activity. The two-carbon tetrachloro moiety of captafol is able to produce an episulfonium ion that can act as a systemic alkylating agent (Figure 90.8). This ion, absent with captan and folpet, is

O

– –

Cl Cl

– –

N— S– C –C–H Cl Cl

O Captafol

–

–

– –

CH3

F

–

CH3–

–

–

CH3

Cl O O N— S—N— S– C –Cl –

–

Cl O O N—S—N— S– C –Cl –

CH3–

F

CH3 Tolylfluanid

Dichlofluanid

Figure 90.7  Captafol, dichlofluanid, and tolylfluanid.

N— S +–C–CCl2

–

Captafol

N— S– C –C–H – –

O

Cl Cl

Cl –

Cl Cl

– –

– – – –

Cl Cl

N—S–C–C–H

–

O

CHCl

Cl Cl

Episulfonium ion

Figure 90.8  Episulfonium ion formation by captafol.

able to enter the systemic circulation and may be carcinogenic (Williams, 1992). The spectrum of tumors in rodent bioassays is broad and affects both mice (Ito et al., 1984) and rats (Nyska et al., 1989; Quest et al., 1993), whereas the tumor spectrum of captan and folpet is narrow, focusing on the mouse duodenum (Gordon et al., 1994). Mutagenic results in some assays show a differing pattern of activity. For example, when tested in S. typhimurium, TA 102 and TA 104, captan was negative in strain TA 102 and positive in strain TA 104, whereas captafol was negative for TA 104 and positive for TA 102 (Barrueco and de la Pena, 1988). In S. typhimurium strains TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as well as E. coli strain WP2 hcr, captan and folpet were positive in all systems, whereas captafol was positive only in WP2 hcr and was “doubtful” in TA 100 (Moriya et al., 1983). Two results follow from the finding that captafol does not share a common mechanism of toxicity with captan and folpet. First, under FQPA, residues will not be combined for a cumulative risk assessment. Second, the “structural similarity” (Quest et al., 1993) of captafol should not be referenced when evaluating the carcinogenicity of captan or folpet. The first point is moot, because captafol is not registered in the United States; the second point avoids confounding comparisons.

90.4.3  Dichlofluanid and Tolylfluanid Dichlofluanid (CAS 1085-98-9) and tolylfluanid (CAS 73127-1) do not share a common mechanism of toxicity with captan or folpet with regard to mouse duodenal tumors, principally because they do not induce these tumors. Both compounds have a fluorine atom substituted for one of the three chlorine atoms on the trichloromethylthio moiety (Figure 90.7). They differ from one another by the addition of a methyl group on the benzene ring. Like captan and folpet, these compounds react with sulfhydryl groups (Schuphan et al., 1981). The monofluorodichloromethylthio moiety conveys more chemical reactivity to the parent as measured by the reaction rate with 4-­nitrothiophenol compared with the trichloromethylthio moiety. The reaction rate of dichlofluanid is over twice that of captan and folpet, but the trichloro dichlofluanid analogue is less reactive than either captan or folpet. Dichlofluanid and its bis(fluorodich loromethyl) disulfide degradate were reported to be negative for mutagenicity in S. typhimurium TA100, whereas the bis-(trichloromethyl) disulfide from captan and folpet was positive (Schuphan et al., 1981). The presence of the fluorine atom apparently lessens the mutagenicity of these compounds. Thiophosgene and its monofluorine analogue are postulated to be degradates of dichlofluanid. Either compound reacts with cysteine to form TTCA in a similar way as it is formed with captan or folpet. These compounds have been reviewed by the Joint Meeting of the FAO Panel of Experts on Pesticide Residues

Chapter | 90  Captan and Folpet

in Food and the Environment and the WHO Expert Group on Pesticide Residues (FAO/WHO, 1984, 1989). Dichlofluanid was negative for carcinogenicity when tested in mice at 5000 ppm. The levels that cause no toxicological effect in rats and dogs are 500 (30 mg/kg bw per day) and 1000 ppm (25 mg/kg bw per day), respectively. For tolylfluanid, the levels that cause no toxicological effect in rats and dogs are 300 ppm (15 mg/kg bw per day) and 12.5 mg/kg bw per day, respectively. The absence of duodenal tumors in mice suggests that the ability to induce these tumors is not a general property of the chloroalkylthio fungicides.

90.5  Human risk assessment 90.5.1  Cancer In contrast to the relatively high background duodenal tumor incidence seen in mice, the incidence in humans (Parkin et al., 1992) and rats (Goodman et al., 1979; Maekawa et al., 1983; Maita et al., 1987; McMartin et al., 1992) is low. This suggests that humans are closer to rats; that is, humans are refractory to tumors with captan or folpet because the number of transformed cells in situ is low. Nonetheless, prudence dictates that humans be considered similarly to the mouse for risk assessment purposes. The no effect levels for duodenal crypt cell proliferation, the prerequisite for tumor formation, are 400 ppm for captan and 150–450 ppm for folpet. The approximate equivalent doses are 30–60 mg/kg per day. For this assessment we used a NOEL of 50 mg/kg per day. Humans are exposed to captan and folpet predominantly by two routes: oral and dermal. Exposure via the oral route occurs through consumption of food that contains residues; exposure via the dermal route occurs through the use of products that contain these fungicides. Exposure from food is low and there are no contributions from water. Milk, which is both aqueous-based and metabolically-produced, was shown to have no captan or degradates present. A national milk survey for captan that was conducted over the course of 1 year and analyzed 224 samples from a statistically derived paradigm across four regions of the United States (North East, North Central, West, and South) found no detectable levels (LOQ  0.005 ppm) of captan, THPI, 3-OH-THPI, or 5-OH-THPI (Slesinski and Wilson, 1992). Exposure to oral residues only is considered relevant for human cancer risk assessment. Dermal exposure is not relevant for human cancer risk assessment, because dermal contact does not result in systemic exposure and captan has been found not to be a skin carcinogen (Antony and Mehrota, 1994). For both captan and folpet, the EPA has calculated the estimated exposure for cancer risk purposes as 0.00005 mg/kg per day (U.S. EPA, 1999a,b). The MOE for each of these fungicides, based on a NOEL for duodenal crypt cell proliferation of 50 mg/kg per day is 1,000,000. These MOEs suggest virtually no

1939

risk of cancer to persons who consume produce treated with either captan or folpet. It is unlikely that both compounds would be present on the same commodity at the same time because the uses of captan in the United States do not overlap those of folpet. Additionally, normal agronomic practice usually relies on one or the other, not both. Nonetheless, if the expected residues are combined for a cumulative risk assessment, the MOE is still satisfactory. This analysis shows that humans are not at risk for duodenal tumors from these fungicides, a position embraced by EPA for captan (U.S. EPA, 2004). This assessment is particularly relevant for re-entry workers such as strawberry harvesters who might be exposed dermally to captan residues. There are, however, instances of reported associations of captan with human cancer. In an epidemiology study that examined pesticide use and breast cancer among 30,454 farmers’ wives, there was a “possible” increase in risk associated with captan but the authors cautioned that further follow-up of this cohort was necessary to clarify the relationship (Engel et al., 2005). In a multicenter case– control study, it was reported that captan was associated with incidence of non-Hodgkin’s lymphoma (McDuffie et al., 2001). The odds ratio (OR) reported for individuals handling captan “greater than two days/year” was 2.80 (95% CI: 1.13–6.90). These associations are not discussed in light of biological feasibility. Since captan is not present systemically and since its degradate, THPI, has shown no evidence of carcinogenicity in rodent bioassays (through testing of its parent), other than local duodenal tumors, biologic feasibility for the association of captan and human cancer remains speculative.

90.5.2  Noncancer For noncancer risks, captan and folpet present an interesting challenge for risk assessors. The transient nature of these molecules coupled with their inherent low toxicity make it difficult to assign meaningful endpoints. Noncancer endpoint risk characterization requires the selection of relevant endpoints for nondietary and dietary exposure, and that NOELs be determined for both acute and chronic exposure. Nondietary exposure, in turn, comprises dermal exposure (including eye exposure) and inhalation. Three nondietary hazards associated with captan and folpet that are relevant to human safety are skin sensitization, eye irritation, and lung irritation. Only one of these, skin sensitization, appears to affect persons who come in contact with these materials. The incidence of sensitization reactions is below 10% in trials with captan and well below this incidence in actual use (Krieger, personal communication). Systemic toxicity from dermal exposure is lacking due to the labile nature of these molecules in the blood. Skin irritation from single-instance contact with captan or folpet is not expected. Repetitive dermal exposure, however, might induce progressive skin irritation, although it is not

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evident with the limited number of people who repeatedly use a shampoo containing 7% captan (Guo, 2001). Inhalation is a potential avenue for adverse effects, although the absence of adverse reports suggests that this is not an issue. The AIHGH has assigned a threshold limit value of 5 mg/m3 (5 g/l) for captan (ACGIH, 1998) and the same value has been suggested as appropriate for folpet (Seifried, 1996). In vitro studies with human bronchial epithelial cells (16HBE140-) note adverse effects due purportedly to lipid peroxidation and the generation of reactive oxygen species (ROS) at levels between 2.89 and 5.11 g/cm2 for two folpet commercial products (Canal-Raffin et al., 2007, 2008). These researchers note that folpet, used extensively in French vineyards, is relatively persistent when introduced to cell cultures. Average airborne residues in rural and urban settings associated with folpet usage in vineyards are reported to be 1.2 and 0.01 g/m3, respectively (Chretien, 2004b), which, when compared with a NOAEL of 0.0006 g/m3 (0.6 g/l) from a 90-day inhalation study with captan, provide high margins of exposure (folpet’s NOAEL is expected to be similar to that of captan). Higher average airborne residues are noted within vineyards during spray operations (40 g/m3, Chretien, 2004a), but appropriate respiratory protection is indicated for these operations. For acute dietary risk, the U.S. EPA has used the NOEL from developmental studies for both captan (U.S. EPA, 1995a) and folpet (Levy et al., 1997). For captan, this is 10 mg/kg per day, based on effects at 30 mg/kg per day (a maternally toxic dose) in a rabbit study. For folpet, this is 10 mg/kg per day, based on effects at 20 mg/kg per day in a rabbit study. This “default” selection is not ideal because the NOEL is based on multiple doses, it is based on effects on the fetus and not the individual, and it is specific for a subgroup (women of childbearing age) that comprises only part of the general population. A meaningful acute dietary risk assessment is dependent on an appropriately designed single-exposure oral toxicity study; such data are not currently at hand. Captan acute dietary exposure at the 99th percentile is estimated for the general U.S. population at 0.009512 mg/kg per day (Kidwell and Watters, 1999); the exposure for folpet is estimated at 0.00046 mg/kg per day (Petersen, 1997). The EPA estimates these acute dietary exposures at 0.036 mg/kg per day for captan at the 99.9th percentile (U.S. EPA, 1999a) and at 0.001532 mg/kg per day for folpet at the 99th percentile (U.S. EPA, 1999b). For chronic U.S. EPA estimates, the dietary exposure for the general population in the United States is at 0.000664 mg/kg per day for captan and at 0.000053 mg/kg per day for folpet (U.S. EPA, 1999a,b). For chronic dietary risk assessment, the captan NOEL of 12.5 mg/kg per day and the folpet NOEL of 9 mg/kg per day are used. Margins of exposure (NOEL ÷ exposure) for captan are 18,825 and for folpet are 169,811. The WHO ADI is 0.1 mg/kg per day for both captan (FAO/WHO, 1990) and folpet (FAO/WHO, 1996). This is approximately equal to the U.S. EPA’s cPAD for

captan, 0.13 mg/kg per day, and the EPA’s PAD (without the threefold FQPA safety factor) for folpet, 0.09 mg/kg per day. The JMPR reevaluated captan and folpet in 2007 and noted the ADI for both compounds at 0–0.1 mg/kg bw and the acute reference dose for captan at 0.3 mg/kg bw and for folpet at 0.2 mg/kg bw, both applicable only to women of childbearing age (FAO/WHO, 2007).

Conclusion Captan and folpet are structurally similar molecules that act through a common mechanism with regard to their ability to induce duodenal tumors in mice. The mode of action has been elucidated for these tumors and is dependent on irritation to and cell loss from the intestinal villi, followed by a compensatory increase in proliferation within the crypt compartment. This proliferative pressure, with time, promotes transformed cells that are normally resident in situ. The mode of action is not dependent on a mutagenic component nor are mutations within basal cells of the crypts a plausible occurrence. Captan and folpet are, however, mutagenic when tested in a variety of in vitro systems, and this observation has challenged investigators to solve the paradox that exists between in vitro and in vivo test results. The solution to this question is the finding that these compounds degrade extremely rapidly when thiols are present. In human blood, captan’s t1/2 is less than 1 s and folpet’s is less than 5 s. Thiophosgene, the reactive degradate that is formed from the trichloromethylthio side chain, reacts not only with thiols, but with other functional groups and its t1/2 is less than 0.6 s. The import of this rapid degradation is that systemic exposure to captan, folpet, or their common degradate, thiophosgene, is absent. This, along with the low estimated dermal absorption rate of 0.5% per hour, assures that adverse systemic risk in agricultural workers is absent. Local effects due to irritation, however, may occur. These include eye and skin irritation, skin sensitization, and irritation of the airways. Oral exposure at sufficient doses will irritate the mucus membranes of the gastrointestinal tract. Systemic effects noted in laboratory studies such as depressed weight gain or delayed development of fetuses and pups are secondary effects that result from the primary irritation of the gastrointestinal tract. Thus, captan and folpet, when used in the agricultural setting are characterized as follows: They have low acute toxicity. They are not carcinogenic, mutagenic, or teratogenic. l They are neither selective developmental toxins nor are they reproductive toxins. l l

Relevant hazards are the following: Irritation of mucus membranes Sensitization after repeated exposure

l l

Chapter | 90  Captan and Folpet

Irritation of the skin after repeated exposures (specifically for folpet) l Irritation of the airways l

These products have been in use for over 55 years and experience shows that eye irritation and sensitization reactions, particularly with re-entry operations, are not problematic. In addition, a limited survey of persons using a 7% captan-based shampoo indicates that repeated use does not cause skin irritation or skin sensitization reactions. Captan and folpet remain valuable fungicides as the risks are low and benefits high.

Acknowledgments Reports with MRID numbers are available from U.S. Environmental Protection Agency, Freedom of Information Office, Ariel Rios Building, 1200 Pennsylvania Avenue, N.W., Washington, DC 20460.

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Chapter | 90  Captan and Folpet

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Chapter | 90  Captan and Folpet

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