Australian Pesticides and Veterinary Medicines Authority AUSTRALIAN PESTICIDES AND VETERINARY MEDICINES AUTHORITY AUSTRALIA CHEMICAL REVIEW PROGRAM REVIEW OF THE MAMMALIAN TOXICOLOGY AND METABOLISM/TOXICOKINETICS OF PARAQUAT Supplement I: TOXICOLOGY Prepared by the Agricultural and Veterinary Chemical Assessment Team Office of Chemical Safety of the Department of Health Canberra October 2016 Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 1 - Australian Pesticides and Veterinary Medicines Authority PREFACE This report was undertaken by the Office of Chemical Safety (OCS) at the request of the Australian Pesticides and Veterinary Medicines Authority (APVMA), under the Chemical Review Program. Paraquat is under review because of concerns over the potential risks to human health and the environment. A draft toxicology review was submitted to APVMA in 2003 and was subsequently revised and updated in 2009, to address industry responses and to include additional published data. A report focussing on neurotoxicity was requested of the OCS and submitted to the APVMA in 2010 with additional neurotoxicity studies assessed and incorporated in 2014. In 2015, the complete review of the toxicology of paraquat was finalised and the report structured into three parts: • Summary Report - Comprises an overview of all relevant data available on paraquat relating to human health. • Supplement I: Toxicology - Comprises a detailed technical report on paraquat toxicology (excluding neurotoxicity) [this report]. • Supplement II: Neurotoxicity - Comprises a detailed technical report on paraquat neurotoxicity. These three reports should be considered together. This report ‘Supplement I: Toxicology’, is a complete evaluation of the data provided to the OCS, together with new information available in the public domain since the commencement of the review, with the exception of neurotoxicity. Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 2 - Australian Pesticides and Veterinary Medicines Authority TABLE OF CONTENTS 1 ABBREVIATIONS ................................................................................................................ 5 2 EXECUTIVE SUMMARY ................................................................................................... 8 2.1 TOXICOLOGY HAZARD PROFILE ............................................................................................... 9 3 SUMMARY TOXICOLOGY REPORT............................................................................ 11 3.1 INTRODUCTION ....................................................................................................................... 11 3.2 METABOLISM AND TOXICOKINETICS ...................................................................................... 11 3.2.1 Oral Administration ............................................................................................................... 11 3.2.2 Inhalational Administration ................................................................................................... 12 3.2.3 Intrabronchial Administration ............................................................................................... 13 3.2.4 Ocular Administration ........................................................................................................... 13 3.2.5 Percutaneous Absorption....................................................................................................... 13 3.2.6 Human studies ....................................................................................................................... 14 3.2.7 In vitro Metabolism ............................................................................................................... 15 3.3 ACUTE STUDIES ...................................................................................................................... 15 3.4 SHORT-TERM STUDIES ............................................................................................................ 16 3.4.1 Oral administration ............................................................................................................... 16 3.4.2 Inhalational Administration ................................................................................................... 18 3.4.3 Dermal Administration .......................................................................................................... 18 3.4.4 Subcutaneous Administration ................................................................................................ 18 3.5 SUBCHRONIC STUDIES ............................................................................................................ 18 3.6 CHRONIC STUDIES .................................................................................................................. 19 3.7 REPRODUCTIVE STUDIES ........................................................................................................ 21 3.8 DEVELOPMENTAL STUDIES .................................................................................................... 23 3.9 GENOTOXICITY STUDIES ........................................................................................................ 25 3.10 NEUROTOXICITY STUDIES ...................................................................................................... 27 3.11 HUMAN STUDIES .................................................................................................................... 27 3.11.1 Occupational Exposure ......................................................................................................... 27 3.11.2 Poisoning Incidences ............................................................................................................. 29 3.12 MECHANISITIC STUDIES ......................................................................................................... 29 3.12.1 Antidote Studies ..................................................................................................................... 31 4 MAIN TOXICOLOGY REPORT ...................................................................................... 33 4.1 INTRODUCTION ....................................................................................................................... 33 4.2 TOXICOKINETICS AND METABOLISM ...................................................................................... 33 4.2.1 Metabolic Pathway ................................................................................................................ 33 4.2.2 Mice ....................................................................................................................................... 34 4.2.3 Rats ........................................................................................................................................ 36 4.2.4 Rabbits ................................................................................................................................... 44 4.2.5 Monkeys ................................................................................................................................. 47 4.2.6 Percutaneous absorption ....................................................................................................... 48 4.2.7 Human Poisonings ................................................................................................................. 55 4.2.8 In vitro Studies ....................................................................................................................... 60 4.3 ACUTE STUDIES ...................................................................................................................... 60 4.3.1 Acute Oral, Dermal and Inhalation Studies .......................................................................... 60 4.3.2 Eye and Skin Irritation and Skin Sensitisation Studies .......................................................... 65 4.4 SHORT-TERM REPEAT DOSE STUDIES ..................................................................................... 71 4.4.1 Oral Administration ............................................................................................................... 71 4.4.2 Inhalational Administration ................................................................................................... 99 4.4.3 Dermal Administration ........................................................................................................ 108 4.4.4 Subcutaneous Administration .............................................................................................. 115 4.5 SUBCHRONIC STUDIES .......................................................................................................... 118 4.5.1 Oral Administration ............................................................................................................. 118 4.6 CHRONIC STUDIES ................................................................................................................ 128 4.6.1 Mice ..................................................................................................................................... 128 Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 3 - Australian Pesticides and Veterinary Medicines Authority 4.6.2 Rats ...................................................................................................................................... 138 4.6.3 Dogs ..................................................................................................................................... 153 4.7 REPRODUCTION STUDIES ...................................................................................................... 156 4.7.1 Rats (oral) ............................................................................................................................ 156 4.7.2 Mice (ip) .............................................................................................................................. 166 4.7.3 Rats (dermal) ....................................................................................................................... 168 4.8 DEVELOPMENTAL STUDIES .................................................................................................. 169 4.8.1 Mice ..................................................................................................................................... 169 4.8.2 Rats ...................................................................................................................................... 182 4.8.3 Rabbits ................................................................................................................................. 187 4.9 GENOTOXICITY STUDIES ...................................................................................................... 198 4.10 NEUROTOXICITY STUDIES .................................................................................................... 205 4.11 HUMAN STUDIES .................................................................................................................. 205 4.11.1 Occupational Exposure ....................................................................................................... 205 4.11.2 Poisoning Incidents ............................................................................................................. 216 4.12 MECHANISTIC STUDIES AND STUDIES ON TARGET ORGAN TOXICITY ..................................... 235 4.13 ANTIDOTE STUDIES .............................................................................................................. 259 5 REFERENCES .................................................................................................................. 270 5.1 EVALUATED STUDIES ........................................................................................................... 270 5.2 REFERENCES SEEN BUT NOT EVALUATED ............................................................................. 287 5.3 SECONDARY CITATIONS ........................................................................................................ 296 Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 4 - Australian Pesticides and Veterinary Medicines Authority 1 ABBREVIATIONS Time Weight d Day bw Body weight h Hour g Gram min Minute kg Kilogram mo Month mg Milligram wk Week µg Microgram s Second ng Nanogram yr Year wt Weight Length Dosing cm Centimetre id Intradermal m Metre im Intramuscular μm Micrometre inh Inhalation mm Millimetre ip Intraperitoneal Nm Nanometre iv Intravenous po Oral sc Subcutaneous mg/kg bw/d mg/kg body weight/day Volume Concentration L Litre M Molar mL Millilitre ppb Parts per billion µL Microlitre ppm Parts per million Clinical chemistry A/G Albumin/globulin ratio ALT Alanine aminotransferase (SGPT) ALP Alkaline phosphatase AST Aspartate aminotransferase (SGOT) BUN Blood urea nitrogen CPK Creatine phosphatase (phosphokinase) ESR Erythrocyte sedimentation rate GGT Gamma-glutamyl transferase Haematology APTT Activated partial thromboplastin time Hb Haemoglobin Hct Haematocrit LDH Lactate dehydrogenase MCH Mean corpuscular haemoglobin MCHC Mean corpuscular haemoglobin concentration MCV Mean corpuscular volume PCV Packed cell volume PT Prothrombin time RBC Red blood cell/erythrocyte Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 5 - Australian Pesticides and Veterinary Medicines Authority WBC White blood cell/leucocyte WBC-DC White blood cells – Differential count Chemistry ACTH Adrenocorticotrophic hormone DMSO Dimethyl sulfoxide DQ Diquat eCG Equine chorionic gonadotrophin EDTA Ethylenediaminetetraacetic acid GC Gas chromatography GLC Gas liquid chromatography hCG Human chorionic gonadotrophin HPLC High performance liquid chromatography LSC Liquid scintillation counting MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine MS Mass spectrometry NAD(PH) Nicotinamide adenine dinucleotide (phosphate) PQ Paraquat TGAC Technical grade active constituent TLC Thin layer chromatography Terminology AUC Area under the curve CI Confidence Interval F Female FEV Forced expiratory volume FVC Forced Vital Capacity GLP Good Laboratory Practice K Michaelis constant (substrate concentration at 1/2 V ) m max LC Median lethal concentration 50 LD Median lethal dose 50 LOEL Lowest Observed Effect Level M Male MLD Maximum lethal dose MTD Maximum tolerated dose NOEC No Observed Effect Concentration NOEL No Observed Effect Level QA Quality Assurance SCE Sister chromatid exchange SD Sprague-Dawley or Standard Deviation SEM Standard error of the mean SPF Specific pathogen free UDS Unscheduled DNA synthesis V Volume of distribution d V Maximum velocity max Organisations APVMA Australian Pesticides and Veterinary Medicines Authority EC European Commission Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 6 - Australian Pesticides and Veterinary Medicines Authority FAO Food and Agriculture Organization (United Nations) IARC International Agency for Research on Cancer MAFF Ministry of Agriculture, Forestry and Fisheries (Japan) OCS Office of Chemical Safety OECD Organization for Economic Co-operation and Development PIC Poison Information Centre US EPA United States Environmental Protection Agency WHO World Health Organization Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 7 - Australian Pesticides and Veterinary Medicines Authority 2 EXECUTIVE SUMMARY Paraquat is a non-selective contact herbicide belonging to the bipyridinium class of compounds which also includes the herbicide diquat. Both compounds share a similar phytotoxic mode of action which involves the inhibition of photosynthesis (specifically photosystem I) thereby generating superoxide free radicals, leading to lipid peroxidation and membrane damage. Plants die rapidly after treatment and subsequent exposure to light. The main findings of the toxicology assessment are that the mechanism of mammalian toxicity of paraquat, like its mode of action in plants, is via the generation of highly reactive free radicals and consequent peroxidation of membrane lipids, sulfhydryl groups, proteins, and DNA, leading to membrane damage and cell death. The review confirmed the high acute toxicity of paraquat. The acute toxicity profile demonstrates that in laboratory animals, paraquat is of moderate to high acute oral toxicity and high acute inhalational toxicity, but low dermal toxicity. Paraquat is a severe eye irritant, moderate skin irritant, but not a skin sensitiser. Paraquat is moderately to highly acutely toxic to humans by the oral route. The review also confirmed that the repeat-dose toxicity of paraquat, when administered by the oral or inhalational routes, is predominantly characterised by pulmonary lesions due to the preferential uptake of paraquat by the lungs, with other effects seen in kidneys and liver also noted following acute exposures. From repeat dose studies there was no evidence to indicate that paraquat is carcinogenic in chronic animal studies. The weight-of-evidence indicates that paraquat is non-mutagenic and therefore is not considered to pose a genotoxic hazard to humans. Standard studies showed no reproductive or developmental toxicity in experimental animals. The neurotoxicity potential of paraquat is considered in detail in a separate report – Paraquat Toxicology Review Supplement II – Neurotoxicity. The weight of evidence is that paraquat is not neurotoxic by the oral route of exposure. Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 8 - Australian Pesticides and Veterinary Medicines Authority 2.1 Toxicology Hazard Profile Absorption, distribution, metabolism and excretion in mammals Poor; in the rat, approximately 10-18% of an orally Rate and extent of absorption administered dose is absorbed. Dermal absorption Low; 0.3% in humans Distributes to most organs of the body. The highest Distribution initial concentrations were found in the kidney and the lungs. No evidence of accumulation, other than potential Potential for accumulation accumulation in the lungs Rapid; in the rat 60-90 % in faeces with 10-20% in urine Rate and extent of excretion in 72 h, Not metabolised by rats. Generation of free radicals due Metabolism to redox-cycling, primarily in the lung, causing oxidative tissue damage. Toxicologically significant compounds Paraquat and 2,2’:6’,2”-terpyridine (an impurity) (animals, plants and environment) Acute toxicity (Manufacturing concentrate; approx 34% w/w paraquat cation) Note: Unless otherwise indicated, units are expressed as paraquat cation Rat oral LD (mg/kg bw) 100-249 50 Worst oral LD in other species (mg/kg 50 22 (guinea pigs) bw) Lethal human dose (mg/kg bw)† 50 -80 Rat dermal LD (mg/kg bw) >1448 (no deaths) 50 Worst dermal LD in other species 50 No data (mg/kg bw) Rat inhalation LC (mg/m3) 0.5 50 Worst inhalation LC in other species No data 50 Skin irritation Moderate irritant (rabbit) Eye irritation Severe irritant (rabbit) Skin sensitisation Non-sensitiser (Buehler method) (No evidence of sensitisation in humans) Acute toxicity of 2, 2’:6’, 2”-terpyridine (impurity) Rat oral LD (mg/kg bw) 2.17 - 2.61 50 Rat dermal LD (mg/kg bw) 4.31 - 5.04 50 Short-term toxicity Target/critical effect Lungs/oxidative tissue damage Lowest relevant oral NOEL 0.5 (13-week dietary study in dogs) (mg/kg bw/d) Lowest relevant dermal NOEL Not established – no reliable studies (mg/kg bw/d) Lowest relevant inhalation NOEC 0.01 (21-day rat whole body exposure) (mg/m3) Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 9 - Australian Pesticides and Veterinary Medicines Authority Clastogenic effects at cytotoxic concentrations due to Genotoxicity free radical damage. Negative in vivo. Some in vitro equivocal results. LOEL (mg/kg bw, single dose applied 6 (induction of micronuclei at this dose and higher in rat dermally) bone marrow micronucleus assay) Long-term toxicity and carcinogenicity Target/critical effect Lungs/oxidative tissue damage Lowest relevant NOEL 0.45 (pulmonary lesions in male dogs, 1-year study) (mg/kg bw/d) Carcinogenicity No evidence of carcinogenicity Reproductive toxicity No effect on reproductive performance. Toxic effects on Reproduction target/critical effect offspring only at maternotoxic doses (perivascular inflammatory cell infiltration in the lungs of pups). Lowest relevant reproductive NOEL 3.75 in pups (perivascular inflammatory cell infiltration (mg/kg bw/d, 3-Generation dietary study in the lungs) in rats) 1.25 for parental animals (focal alveolar histiocytosis) Developmental toxicity Minor effects (delayed skeletal development at Developmental target/critical effect maternotoxic doses) 1 - maternotoxicity (increased mortality, clinical signs Lowest NOEL and reduced body weight gain) (mg/kg bw/d, oral gavage in rats) 1 – foetotoxicity (reduced mean foetal and litter weights). <1 – maternotoxicity (effects on food consumption and Lowest NOEL body weight gain at all doses tested) (mg/kg bw/d, oral gavage in rabbits) 1 - foetal toxicity (increased incidence of post implantation losses and skeletal variations) Neurotoxicity Not neurotoxic by the oral route Delayed neurotoxicity No evidence of delayed neurotoxicity Immunotoxicity No data available † Dose level at which there are reports of human deaths (Pond, 1990) Note: Unless otherwise indicated ‘paraquat’ refers specifically to the paraquat cation Not to be used for commercial or registration purposes without the consent of the owner of the cited information - 10 -