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Fundamental Toxicological Sciences (Fundam. Toxicol. Sci.) 195 Vol.3, No.5, 195-204, 2016 Original Article Acute toxicity of phorate oxon by oral gavage in the Sprague-Dawley rat Thomas H. Snider1, Kevin G. McGarry1, Michael C. Babin1, David A. Jett2, Gennady E. Platoff Jr.3 and David T. Yeung2 1Battelle, 505 King Avenue, JM-7, Columbus, Ohio 43201-2693, USA 2National Institutes of Health/National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, USA 3National Institutes of Health/National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA (Received June 29, 2016; Accepted July 11, 2016) ABSTRACT — The oral toxicity of phorate oxon (PHO), with emphasis on gender- and age-related effects, was characterized in the Sprague-Dawley rat. The oral LD50 (95% fiducial limits) for PHO in corn oil was 0.88 (0.79, 1.04) mg/kg in males and 0.55 (0.46, 0.63) mg/kg in females with a probit slope of 15. Females had higher baseline blood cholinesterase titers, but males were significantly more toler- ant. Younger rats generally had lower absolute cholinesterase blood titers. However as PHO challenges increased, baseline-normalized cholinesterase inhibition was independent of age and gender. Butyrylcho- linesterase (BChE) and especially acetylcholinesterase (AChE) in brains of younger females were affected more than that in either males or older females. In summary, while female rats, especially older females, had higher titers relative to males, female rats were more susceptible in terms of absolute cholinesterase inhibition and 24-hr lethality data, but the differences were not observed when titers were normalized to baseline levels. Key words: Phorate oxon, Pesticide, Toxicity, Rat, Oral INTRODUCTION butyrylcholinesterase (BChE), carboxylesterase (CaE), etc. leading to an over-accumulation of the neurotransmit- Phorate (Thimet® 20G, AMVAC, Los Angeles, CA, ter acetylcholine (ACh) at neural synapses. Cholinolergic USA) is an extremely toxic broad use insecticide and crisis can occur if the resultant increase in brain tissues ascaricide, commonly applied as a granular agricultur- and peripheral nerves acetylcholine levels are not rap- al product for long-term release and systemic uptake by idly controlled (De Bleecker et al., 1994; Rusyniak and plants to control specific pests, such as sucking insects on Nañagas, 2004; Newmark, 2004). Clinical manifestation peanut and nematodes in soybean plants. Phorate is rap- of toxicity includes miosis, increased and uncontrolled idly metabolized by plants, insects, and mammals into secretions, lacrimation, urination, defecation, fascicula- several organophosphorus (OP) anti-cholinesterase (anti- tions, seizures, convulsions, respiratory distress, and even ChE) intermediates that are even more toxic than the par- death (Vale and Lotti, 2015). ent compound, specifically phorate-sulfoxide, phorate-sul- Adverse environmental consequences associated fone, phorate oxon (oxygen analog metabolite; also known as phoratoxon (PHO); O,O-diethyl S-(eththiomethyl) phosphorothioate; CAS RN 2600-69-3; Fig. 1), phorate oxon-sulfoxide, and phorate oxon-sulfone (pubchem.ncbi. nlm.nih.gov). These metabolites prevent ChE-mediated hydrolysis and inactivation of the neurotransmitter acetylcholine (ACh). Consequently, the mechanism of phorate toxicosis is similar to other OP compounds, i.e., nondis- criminatory inhibition of acetylcholinesterase (AChE), Fig. 1. Phorate oxon (Phoratoxon). Correspondence: Michael C. Babin (E-mail: [email protected]) Vol. 3 No. 5 196 T.H. Snider et al. with the agricultural use of phorate have been reported subcutaneous and topical LD85 challenge levels were then (Baburaj, 2013; Puschner et al., 2013; Lisker et al., 2011; used to assess the therapeutic efficacy of several oxime Holme et al., 2016). Additionally, phorate also presents a AChE reactivators administered in conjunction with the challenge to public health. For example, deliberate and/ U.S. FDA-recommended pre-hospital level of atropine or accidental human exposure to phorate and its metab- (Snider et al., 2015). Although some of the tested oximes olites is most probable through inhalation of ambient air demonstrated efficacy against PHO in the subcutaneously in areas recently treated with the pesticide (Han, 2011; exposed study (therapies given ~1 min after PHO admin- Mission, 2006), ingestion of contaminated food istration), none was therapeutically effective when expo- (Khatiwada et al., 2012; Zhou and Zhao, 2015) or liq- sure occurred dermally (treated at onset of clinical evi- uids (Zhang et al., 2012; Salas et al., 2003), and/or der- dence of toxicosis). mal contact through occupational exposure (Kashyap et Unfortunately, very little else is known about PHO al., 1984; Young et al., 1979). Owing to its high level of toxicity by other, more human-relevant routes of expo- toxicity and multiple avenues of exposure to this com- sure. Consequently, the purpose of the current study was pound, phorate (and its metabolites) represents a very real to 1) evaluate the oral/ingestion toxicity of PHO in the poisoning threat. As such, it is critical that gaps in under- Sprague-Dawley rat and 2) investigate and characterize standing its overall toxicity be addressed. any potential differences in susceptibility due to gender While animal toxicity data of the parent compound and age. phorate has been reported (pmep.cce.cornell.edu; pestici- deinfo.org), relatively little is known about its more toxic MATERIALS AND METHODS metabolites. Since OP oxons are typically more toxic than the parent pesticides (Sultatos et al., 1985; Natoff, 1967) PHO was synthesized in-house. Under argon, an and PHO is easily generated via a simple O-substitution 11.9-mL volume of triethylamine was slowly added to in the phosphorodithioate group (Fig. 2, inchem.org) of a suspension of 10.7 g of diethyl phosphite and 2.7 g of phorate, it was identified as the first metabolite to be char- solid sulfur in a two-necked round-bottom flask equipped acterized. Previous work involving topical challenges of with a reflux condenser and a rubber septum. After full bioactivated PHO on guinea pigs demonstrated a medi- conversion of the phosphite, as monitored by 31P NMR an lethal dose (LD50) of 98 mg/kg, and by the subcutane- spectroscopy, the suspension was diluted with diethyl ous route, 2.1 mg/kg (unpublished data). From the dose- ether to 100 mL and then washed with 100 mL aqueous lethality probit curves characterized in those works, the 1M HCl, dried over magnesium sulfate, concentrated Fig. 2. Metabolic pathways of phorate. Vol. 3 No. 5 197 Phorate oxon toxicity in rats under reduced pressure, and finally dried under high vac- uum. The resulting suspension was filtered over a small cotton plug to yield the diethyl S-hydrogen phosphorothi- olate. The yield was 10.48 g (80%) of product as a yellow oil. A 51-g volume of O,O-diethylthiolphosphoric acid was added to 35 g. of aqueous 33% formaldehyde solution and subsequently 20 g of ethyl sulfide with stirring at 30°C. The mixture was allowed to stand for 2 hr, after which the oily layer separated. The oily layer was extract- ed with chloroform, washed with 5% aqueous sodium carbonate and dried over sodium sulfate. The solvent was removed, and the residue distilled to give 10.7 g (15% yield). Purity was determined by 31P NMR, (99.6%), 1H NMR (97.6%), and GC/MS (99.4%), and 97.6% purity was used in all dose calculations. PHO was used as Fig. 3. Typical growth curves for Sprague-Dawley male and a 0.125 mg/mL solution in Mazola® corn oil (CAS RN female rats and selection of weight ranges. 8001-30-7; ACH Food Companies, Cordova, TN, USA). Male and female Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN, USA). Rats feeding needle (18G x 7.6-cm, 2-mm ball). The target were identified with ear tags and same-gender pair housed PHO dose administered varied across test days in order to before challenge in polycarbonate cages labeled with cage obtain relevant and valuable dose/lethality data for assess- cards. During the 7-day quarantine period, the rats were ing PHO toxicity. In Phase 1, the objective was to obtain weighed and randomized by body weight into test days. a median lethal dose (LD50) with 95% fiducial limits and For the first aim of the study (Phase 1), each of four test slope using probit analysis for each gender. Then in Phase days comprised of five treatment groups of either two rats 2, age and gender effects were investigated by repeating receiving PHO/corn oil or one vehicle control rat receiv- Phase 1 in rats varied both by age and gender. ing corn oil. Among the 72 rats used in this aim, body Clinical observations of survivors were recorded at weight means (minimum - maximum) on the day prior 0.25, 0.5, 1, 2, 4 and 24 hr post-challenge. After the final to challenge were 297 (270 - 315) g for males (approxi- observation, each surviving animal was euthanized with mately 12 weeks old) and 266 (212 - 306) g for females an IM injection of euthanasia solution. A 2-mL volume of (approximately 25 weeks old). Among the 24 male rats blood was collected via the intracardial route from eutha- used in the second aim of the study (Phase 2), body nized animals, placed into K3 EDTA tubes, and designat- weight means (minimum - maximum) on the day prior to ed as the “terminal” sample. Brains from animals surviv- challenge were 242 (233 - 233) g for males at 9.9 weeks ing to 24 hr were collected and bisected sagittally. One old and 360 (339 - 375) g for males at 15.9 weeks old. brain half was placed into a cassette and plunged into liq- These groups were roughly equidistant from the Phase 1 uid nitrogen and the other half was placed into 10% neu- males in body weight.

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