Toxic Effects of Pesticides

1. Biochemical and Molecular Toxicology UNC ENVR/TOXC 442 November 8th, 2011 Toxic Effects of Pesticides Matthew T. Martin, Ph.D. National Center for Computational Toxicology Office of Research & Development U.S. Environmental Protection Agency RTP, NC 27711 Email: martin.matt@epa.gov http://www.epa.gov/ncct/

2. Pesticides • Prevent, destroy, repel or mitigate any pest ranging from insects, animals and weeds, to microorganisms such as fungi, molds, bacteria and viruses • Fungicides, Rodenticides, Herbicides, Fungicides, Antimicrobials • Inert & Other Ingredients make up final pesticide formulation • OPP (US) & PMRA (Canada) Regulate Pesticides • FIFRA, FFDCA, FQPA – Covers Most of US Pesticide Legislation • Why have a lecture on pesticide toxicology? OPP= USEPA Office of Pesticide Programs PMRA=Pest Management Regulation Agency FIFRA =Federal Insecticide, Fungicide, Rodenticide Act FFDCA=Federal Food, Drug, and Cosmetic Act FQPA =Food Quality Protection Act

3. Pesticides Pesticide Mass • Toxicity data rich chemicals • Potential for high human exposure • Designed to be bioactive • What makes a pesticide different from a commodity chemical? • Designed to be bioactive • Is any pesticide just a pesticide? (“biocide”) • Statutory authority to require toxicity tests • Indirect & direct application to food/crops • What makes a pesticide different from a pharmaceutical? • No direct human exposure • Design intentions (destroy vs treat)… Are they really different? • Molecular target potencies & efficacy differences

4. Pesticides (by the numbers) • About 8800 total pesticidal ingredients • About 4200 active ingredients • 1800 conventional pesticides • 300 antimicrobial pesticides • 250 biopesticides • 400 food-use (direct or indirect contact with the food supply) • Remaining are unsupported • About 4600 inert or other ingredients • ~200 Chemical class represented

5. Pesticides in the News August 23, 2009 Debating How Much Weed Killer Is Safe in Your Water Glass By CHARLES DUHIGG For decades, farmers, lawn care workers and professional green thumbs have relied on the popular weed killer atrazine to protect their crops, golf courses and manicured lawns. But atrazine often washes into water supplies and has become among the most common contaminants in American reservoirs and other sources of drinking water. Now, new research suggests that atrazine may be dangerous at lower concentrations than previously thought. Recent studies suggest that, even at concentrations meeting current federal standards, the chemical may be associated with birth defects, low birth weights and menstrual problems. Laboratory experiments suggest that when animals are exposed to brief doses of atrazine before birth, they may become more vulnerable to cancer later. An investigation by The New York Times has found that in some towns, atrazine concentrations in drinking water have spiked, sometimes for longer than a month. But the reports produced by local water systems for residents often fail to reflect those higher concentrations.

6. Pesticides in the News October 7, 2009 Regulators Plan to Study Risks of Atrazine By CHARLES DUHIGG The Environmental Protection Agency plans to conduct a new study about the potential health risks of atrazine, a widely used weedkiller that recent research suggests may be more dangerous to humans than previously thought. Atrazine — a herbicide often used on corn fields, golf courses and even lawns — has become one of the most common contaminants in American drinking water. For years, the E.P.A. has decided against acting on calls to ban the chemical from environmental activists and some scientists who argued that runoff was polluting ecosystems and harming animals. More recently, new studies have suggested that atrazine in drinking water is associated with birth defects, low birth weights and reproductive problems among humans, even at concentrations that meet current federal standards. The E.P.A. is expected to announce on Wednesday that it will conduct a new evaluation of the pesticide to assess any possible links between atrazine and cancer, as well as other health problems, such as premature births. The E.P.A. may determine that new restrictions are necessary.

7. Pesticides in the Scientific Literature Silent Spring Published in 1962

8. Pesticides in the Regulatory Process Major Amendment To FIFRA

9. Resource for High Quality Pesticide Chemical Use/Class Annotation http://www.alanwood.net/pesticides/

10. Pesticide Regulation • Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA, 1947) administered by USDA • Major amendments in 1972 and 1988 • Federal Food, Drug, and Cosmetic Act (FFDCA, 1954) established pesticide tolerances on food • Delaney Clause, forbade the use of carcinogens as food additives • Food Quality Protection Act (FQPA, 1996) reauthorized FFIFRA provisions • Tolerances reassessed as part of re-registrations • single, health-based standard • aggregate risk from all routes of non-occupational exposure • evaluating endocrine effects • extra tenfold uncertainty factor for children/in utero

11. Vulnerability of Children Greater exposure • On a caloric consumption:body-weight ratio Children are 2.5x adults. Diet less varied (fruit and milk) •  Hand to mouth activity • Skin surface area per body weight is double that of an adult •  Rate of respiration Greater physiological susceptibility • Period of rapid development of nerve cells • Loss of organ function can be permanently imprinted •  Absorption and  elimination of pesticides • Metabolizing enzymes not fully developed

12. Pesticide Testing- US EPA Harmonized Test Guidelines 810 - Product Performance Test Guidelines 830 - Product Properties Test Guidelines 835 - Fate, Transport and Transformation Test Guidelines 840 - Spray Drift Test Guidelines 850 - Ecological Effects Test Guidelines 860 - Residue Chemistry Test Guidelines 870 - Health Effects Test Guidelines 875 - Occupational and Residential Exposure Test Guidelines 880 - Biochemicals Test Guidelines 885 - Microbial Pesticide Test Guidelines 890 - Endocrine Distruptor Screening Program Test Guidelines http://www.epa.gov/ocspp/pubs/frs/home/guidelin.htm

13. http://www.epa.gov/opp00001/reregistration/status.htm

14. What is the acute and chronic Point-of-Departure for pesticide toxicity? Reference Dose (RfD) = NOAEL x UF

15. ToxRefDB website: http://actor.epa.gov/toxrefdb/

16. Chronic Rat & Mouse Endpoints

17. Reproductive Toxicity Profiling Systemic Toxicity & Delayed Sexual Maturation Decreased Reproductive Performance http://toxsci.oxfordjournals.org/cgi/reprint/kfp080

18. Pesticide Carcinogenicity • Roughly 50% of all conventional pesticides cause tumors in rodents • Generally pesticides are non-genotoxic carcinogens (screened out in development process) • Human relevance? • Site and tissue specificity (liver tumor in rodent vs lymphoma inc in humans) • Mechanistic relevance (peroxisome proliferators) • High dose vs real world exposure potential 248 Chemicals 122 w/ No Liver Pathology 126 w/ Liver Pathology

19. Chemicals Evaluated for Carcinogenic Potential by US EPA http://www.epa.gov/pesticides/carlist/

20. Is a pesticide an endocrine disruptor? • Estrogenic • (anti) Androgenic • Thyrotoxic • Other Panzica et al 2005

21. Endocrine Disruptor Screening Program • The Food Quality Protection Act (FQPA) of 1996 and subsequent amendments to the Federal Food, Drug, and Cosmetic Act (FFDCA) and Safe Drinking Water Act (SDWA) required EPA to “develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effect as the Administrator may designate.” • The first phase of EDSP assays are designated Tier 1 tests with a purpose of identifying chemicals that exhibit potential to interact with endocrine pathways or mechanisms (i.e. the estrogen, androgen, and/or thyroid hormone systems) and ultimately determine which chemicals should undergo more definitive in vivo testing (i.e., Tier 2). First test orders have been issued: http://www.epa.gov/endo/pubs/regaspects/testorders.htm

22. http://www.epa.gov/endo/pubs/regaspects/testorders.htm

24. Reproductive and Endocrine Organ Toxicity Endpoints from ToxRefDB

25. Interpretation of In Vitro Assay Results is Challenging ERa_TRANS ERE_CIS Concentration (µM)

26. Importance of Biotransformation ERa radioligand binding assay ERa cellular (HEK293) transactivation assay

27. Pesticidal MOA vs. Toxicological MOA *Methoxychlor was intended to be a replacement for DDT (“Silent Spring”) http://www.irac-online.org/wp-content/uploads/2009/09/MoA_Classification.pdf

29. Environmental Chemicals Molecular response Cellular response Tissue response Chemicals Molecular Response (Early) Cell fate Proliferation Death Apoptosis Necrosis Adverse Outcome Hyperplasia Tumor Cancer NR-sig Gene-reg. Transcription Pesticides Conazoles Pyrethroids Toxics DE-71 PCBs Phthalates PFOA/PFOS CAR PXR PPARa cis-reg. trans-reg. Xen. Met. Phase I Phase II Phase III Focus On a Mode of Action … NR activators stimulate intracellular processes that lead to hyperplasia Chronic stimulation increases the risk of neoplasms

31. HISTORY OF DDT1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane DDT was discovered to be an insecticide in 1939 by Paul Muller. He was a scientist working for Geigy, a Swiss firm that was focused on the chemical development of agricultural insecticides. Products with DDT entered the Swiss market in 1941. Seven years later, in 1948, Muller received the Nobel Prize for medicine and physiology in recognition for the lives DDT saved. • WWII – DDT was used by the allies to suppress a typhus epidemic in Naples • 1943-1944 DDT was applied directly to the head of humans to control lice • Success with DDT hastened the development of aldrin, dieldrin, endrin, chlordane, benzene hexachloride etc.

32. CURRENT STATUS: • No US registration, most uses cancelled in 1972, all uses by 1989 • No US production, import, or export • DDE (metabolite of DDT) is regulated as a hazardous air pollutant (Clear Air Act) • Priority toxic pollutant (Clean Water Act) DDT • DDT can take more than 15 years to break down • Found in animals far from where they were it is used • Bio-accumulates in fish and marine mammals. Found concentrations in these animals are many thousands of times higher than levels in water • DDT can be absorbed by some plants and by animals and humans who eat those plants • DDT is fat-soluble and is stored in adipose tissues of humans and animals • HUMAN EXPOSURE FROM: • Eating contaminated fish and shellfish • Eating imported food exposed to DDT • Infant exposed through breast milk • Eating products from crops grown in contaminated soil

33. Insecticide advantages of DDT • Low volatility • Chemical stability • Lipid solubility • Slow rate of biotransformation and degradation Disadvantages of DDT • Persistence in the environment • Bioconcentration • Biomagnification in food chain • Profound effects on wild life (“Silent Spring”) Health Effects of DDT • Paresthesia of tongue, lips, and face • Irritability, dizziness, vertigo, tremor, and convulsions • Hypersusceptibility to external stimuli (light, touch, and sound) • Hypertrophy of hepatocytes • Hepatic tumors • No epidemiological evidence linking DDT to carcinogenicity in humans • Low rate of absorption through the skin • Human health effects minor

34. Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001

35. Sites of DDT poisoning Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001

36. Neurotoxicity: Inhibition of choline esterase or action potential • Organochlorine Insecticides • Organophosphate Insecticides • Carbamates • Pyrethroid insecticides • Botanical Insecticides Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001

37. Most chemical insecticides act by poisoning the nervous system of the target organisms • CNS of insects are highly developed and similar to that of the mammal • Chemicals that act on the insect nervous system may have similar effects on higher forms of life Stenersen, J. Chemical pesticides: Mode of Action and Toxicology. CRC Press 2004

38. General Modes of Action Pesticides acting on the axon (impulse transmission): • Interference with transport of, Na+, K+, Ca2+, or Cl- ions Pesticides acting on synaptic transmission: • Inhibition of specific enzyme activities: GABA-ergic (inhibitory) synapses Cholinergic synapses • Contribution to the release or persistence of chemical transmitters at nerve endings Stenersen J, Chemical Pesticides Mode of Action and Toxicology, CRC Press 2004

39. Pyrethroid Insecticides • Newest class of insecticides • New analogs will be (hopefully): • More stable in light and air • Better persistence • Low mammalian toxicity Soderlund et al. (2002)

40. Importance of Structure-Activity-Toxicity Relationships Soderlund et al. (2002)

41. Pyrethroid Use • Household sprays • Flea preparations for pets • Plant sprays for home • Plant sprays for greenhouses Pyrethroid Poisoning • Similar to DDT • Not highly toxic in animals • Toxic ingredients • Chrysanthemic acid • Pyrethric acid

42. Figure 1. Nine neonicotinoid insecticides and four nicotinoids. The neonicotinoids are nitromethylenes (C==CHNO2), nitroguanidines (C==NNO2), and cyanoamidines (C==NCN). Compounds with 6-chloro-3-pyridinylmethyl, 2-chloro-5-thiazolylmethyl, and 3-tetrahydro-furanmethyl moieties are referred to as chloropyridinyls (or chloronicotinyls), chlorothiazolyls (or thianicotinyls), and tefuryl, respectively. The nicotinoids are naturally occurring [(−)-nicotine and (−)-epibatidine] and synthetics (ABT-594 and desnitroimidacloprid). Tomizawa & Casida (2004)

43. Tomizawa & Casida (2004)