    

	 	UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 
                           WASHINGTON, D.C.  20460 
                                                                     OFFICE OF 
                                                           CHEMICAL SAFETY AND 
                                                          POLLUTION PREVENTION 
    
    
    	 
   MEMORANDUM 
    
   Date: September 26, 2018  
    
   SUBJECT: Amitraz. Draft Human Health Risk Assessment for Registration Review. 
    
	PC Code:  106201 	DP Barcode: D435892 
	Decision No.: 521840 	Registration No.: NA 
	Petition No.: NA 	Regulatory Action: Registration Review 
	Risk Assessment Type: Single Chemical Aggregate 	Case No.:  NA 
    TXR No.: NA 	CAS No.: 33089-61-1 MRID No.: NA 	40 CFR: 180.287 
    

            
              
                
                  
                
                
                  
                
                
                  
                
                
              
            
          	FROM: 	Danette Drew, Chemist/Risk Assessor 
	 	 	Kelly Lowe, Environmental Scientist 
	 	 	Sarah Dobreniecki, Ph.D., Toxicologist 
	 	 	Risk Assessment Branch V/VII 
	 	 	Health Effects Division (7509P) 
	 	 	Office of Pesticide Programs 
    
   THROUGH: Michael Metzger, Branch Chief 
	 	 	Risk Assessment Branch V/VII 
	 	 	Health Effects Division (7509P) 
	 	 	Office of Pesticide Programs 
    
   And:               RARC Review Team 1 
    
	TO: 	Veronica Dutch 
	 	Melanie Biscoe 
	 	 	Risk Management and Implementation Branch V 
	 	Pesticide Reevaluation Division (7508P) 
	 	 	Office of Pesticide Programs 
    
   Attached please find the HED draft human health risk assessment for amitraz to support Registration Review.  
					Table	of	Contents	
1.0  Executive Summary	4 
2.0  HED Recommendations	6 
2.1  Data Deficiencies	6 
2.1.1.  Hazard and Toxicology	6 
2.1.2 Residential and Occupational Exposure	6 
2.2  Tolerance Considerations	6 
2.2.1  Enforcement Analytical Method	6 
2.2.2  Recommended and Established Tolerances	7 
2.2.3  International Harmonization	8 
2.3  Label Recommendations	8 
3.0  Introduction	8 
3.1  Chemical Identity	8 
3.2  Physical/Chemical Characteristics	9 
3.3  Pesticide Use Pattern	9 
3.4  Anticipated Exposure Pathways	10 
3.5  Consideration of Environmental Justice	10 
4.0  Hazard Characterization and Dose-Response Assessment	11 
4.1 Toxicological Studies Available for Analysis	11 
4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)	12 
4.2.1   Dermal Absorption	13 
4.3  Toxicological Effects	13 
4.4  Safety Factor for Infants and Children (FQPA Safety Factor)	15 
4.4.2  Evidence of Neurotoxicity	16 
4.4.3  Evidence of Sensitivity/Susceptibility in the Developing or Young Animal	16 
4.4.4  Residual Uncertainty in the Exposure Database	16 
4.5  Toxicity Endpoint and Point of Departure Selections	16 
4.5.1  Recommendation for Combining Routes of Exposures for Risk Assessment	18 
4.5.2  Cancer Classification and Risk Assessment Recommendation	18 
4.6  Endocrine Disruptor Screening Program	20 
5.0  Dietary Exposure and Risk Assessment	21 
5.1 Residues of Concern Summary and Rationale	21 
5.2 Food Residue Profile	21 
5.3  Water Residue Profile	22 
5.4  Dietary Risk Assessment	22 
5.4.1  Description of Residue Data Used in Dietary Assessment	22 
5.4.2  Acute Dietary Risk Assessment	22 
5.4.3  Chronic Dietary Risk Assessment	22 
5.4.4  Cancer Dietary Risk Assessment	22 
5.4.5  Summary of Acute and Chronic Dietary Exposure and Risk	22 
6.0  Residential Exposure and Risk Estimates	23 
6.1  Residential Handler Exposure/Risk Estimates	23 
6.2  Residential Post-Application Exposure/Risk Estimates	25 
6.3  Residential Risk Estimates for Use in Aggregate Assessment	27 
7.0  Non-Occupational Spray Drift Exposure and Risk Estimates	27 
8.0  Non-Occupational Bystander Post-Application Inhalation Exposure and Risk 	
Estimates	27 
9.0  Aggregate Exposure/Risk Characterization	28 
10.0  Cumulative Exposure/Risk Characterization	28 
11.0  Occupational Exposure and Risk Estimates	28 
11.1 Occupational Handler Exposure/Risk Estimates	28 
11.2 Occupational Post-application Exposure/Risk Estimates	31 
12.0  Incident and Epidemiological Data Review	31 
13.0  References	32 
Appendix A.  Toxicology Profile and Executive Summaries	33 
Appendix B.  Physical/Chemical Properties	46 
Appendix C.  Review of Human Research	47 
Appendix D.  Summary of Amitraz US and International Tolerances and Maximum 	
Residue Limits (MRLs)	48 

    
    	 
    
    
	1.0 	Executive Summary 
    
   Amitraz [N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-Nmethylmethanimidamide] is an insecticide/acaricide currently registered for use in pet collars for control of ticks on dogs and as an impregnated strip for control of mites in beehives.  The Health Effects Division (HED) has conducted a draft human health risk assessment (DRA) to support the Registration Review of amitraz.  Assessments were performed for potential dietary, residential, and occupational exposures based on the dog collar and beehive uses.  
    
   Humans may potentially be exposed to amitraz in the diet from consuming honey from treated beehives.  Drinking water is not considered a source of dietary exposure as amitraz is not expected to enter the environment, including drinking water sources, through the current uses.  
    
   There is a potential for residential exposure to amitraz from dog collar uses, either from applying (handling) the collars or from post-application contact with the treated dog.  Short-term dermal and inhalation exposure may occur for adults applying the collars.  Short-, intermediate- and long-term post-application exposures to treated dogs may be via the dermal (adults and children) or incidental oral (children) exposure routes.  Post-application inhalation exposure is expected to be negligible for the collar uses.  Spray drift and volatilization exposures are also not anticipated from the current uses.  There is a potential for occupational handler (e.g., veterinarian) dermal and inhalation exposure resulting from applying dog collars. Significant occupational postapplication exposures from contact with treated dogs are not expected to occur and have not been quantitatively assessed. For the beehive strip uses, occupational and residential exposures are expected to be negligible based on the labeled use pattern and formulation.  
    
   For the pet collar use assessments, due to the uncertainty associated with whether pet collars release the active ingredient as a liquid and/or solid (dust), occupational/residential handler and residential post-application exposures are assessed assuming both liquid and dust formulations concurrently, with varying ratios of liquid to dust (99/1, 50/50, and 1/99 liquid to dust). The 1/99 liquid to dust assumption is considered the worst-case exposure scenario for collars as dust formulations result in higher exposures compared to liquid formulations.  
    
   The toxicological database is complete for amitraz except for the following deficiencies in the recently submitted extended one-generation reproduction toxicity study (EOGRTS) in rats: lack of reliable thyroid hormone data in early lifestage (postnatal day 5) and failure to identify a NOAEL for brain morphometric measurements.  
    
   Multiple studies in various mammalian species, including humans, support that the common effects of amitraz are those of neurotoxicity, with signs such as sedation, hypothermia, drowsiness, etc. Human subjects were shown to be more sensitive than any other species tested, followed by the rat and dog. 
    
    
   The point of departure (POD) for chronic oral, incidental oral, and dermal and inhalation exposures (all durations) is selected from the results of the EOGRTS in rats and is based on decreases in thyroxine (T4) levels in F1 adult female rats. This effect is protective of all other effects in the database, including effects observed in human studies. The Food Quality Protection Act (FQPA) safety factor (SF) of 10X is retained as a database uncertainty factor (UFDB) because of concern for the lack of thyroid hormone measurements in post-natal day 5 (PND 5) pups and brain morphometric measurements at the low and mid dose groups to assess potential qualitative or quantitative susceptibility.  The total uncertainty factor is 1000X (10X for intra-species variation, 10X for inter-species extrapolation, and 10X FQPA UFDB).  
    
   The acute oral POD is selected from the combined results of oral toxicity and metabolism studies conducted in humans and is based on clinical signs (dry mouth, drowsiness, disorientation, light headedness, slurred speech, slight pale appearance), and decreased temperature and heart rate in two human subjects in the metabolism study. The FQPA SF is reduced to 1X because, for acute exposures, there is a lack of concern for the inadequate thyroid hormone or brain morphometric data, given permutations in both parameters do not occur after a single acute exposure. The total uncertainty factor is 10X (1X to account for interspecies extrapolation due to the use of a human study, 10X for intra-species variation, and 1X FQPA SF).  
    
   Amitraz is classified as showing "Suggestive Evidence of Carcinogenicity" based on the presence of liver and lung tumors in a single species (mouse) and the lack of concern for mutagenicity.  Quantification of risk using a non-linear approach (i.e., reference dose (RfD)/ population adjusted dose (PAD)) is adequate to account for all chronic toxicity, including carcinogenicity, which could result from exposure to amitraz.  
    
   Unrefined acute and chronic dietary assessments were performed using tolerance-level residues in honey and assuming 100% of beehives are treated. Only food was included in the dietary assessment as amitraz is not expected to reach drinking water sources based on the current uses. For the U.S. population and all population subgroups, acute dietary exposure estimates (95[th] percentile of exposure) are <1% of the acute population adjusted dose (aPAD) and chronic dietary exposure estimates are <1% of the chronic population adjusted dose (cPAD) and are not of risk concern.   
    
   For the residential pet collar use, the combined (dermal + inhalation) residential handler margins of exposure (MOEs) were all less than the level of concern (LOC) of 1000 and were of concern; MOEs ranged from 2.6 to 100 (with the dermal route driving the risk estimates) for small and large collars, depending on the assumed ratio of liquid to dust in the pet collar product.  For residential post-application exposures, the adult dermal and the children (1 to <2 years old) combined (dermal + incidental oral) MOEs were all less than the LOC of 1000 and were of concern.  Even with the use of chemical-specific data reflecting the potential for transfer of amitraz from pet fur, adult dermal MOEs ranged from 3.0 to 76, and children (1 to <2 years old) combined (dermal + incidental oral) MOEs ranged from 1.0 to 28 (with the dermal route driving the risk estimates) depending on the assumed ratio of liquid to dust in the pet collar product.   
    
   Aggregate risk estimates combine residential exposures with background dietary exposures. Since the residential exposures result in risk estimates of concern, an aggregate assessment was not performed. Combining dietary exposures with residential exposures would result in even greater risk estimates of concern. 
    
   For the occupational handler pet collar uses, the combined (dermal + inhalation) MOEs were all less than the LOC of 1000 and were of concern.  The combined MOEs ranged from 0.68 to 96 (with the dermal route driving the risk estimates) for small and large collars, depending on the assumed ratio of liquid to dust in the pet collar product and the personal protective equipment (PPE) assumed to be worn (ranging from single layer and no gloves to double layer with gloves, and assuming no respirator worn).  Based on the currently registered labels, gloves are not required to be worn.  Note that while risk estimates are presented for a double layer of clothing, this option may not be practical for the use scenario being assessed (i.e., veterinarians and/or technicians applying pet collars to dogs).  Significant occupational post-application exposures from contact with animals treated with a pet collar are not expected to occur and have not been quantitatively assessed.   
    
   Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment (see Section 3.5). 
    
   See Appendix C for information regarding the use of human research data in this assessment. 
    
	2.0 	HED Recommendations 
    
	2.1 	Data Deficiencies 
    
   2.1.1.  Hazard and Toxicology 
    
   There is concern for the lack of thyroid hormone measurements in post-natal day 5 (PND 5) pups for all dose groups and brain morphometric measurements at the low and mid dose groups in the 
   EOGRTS in rats to assess potential qualitative or quantitative susceptibility. As a result, the FQPA SF of 10X is retained as a database uncertainty factor (UFDB). These data should be submitted in order to evaluate potential susceptibility and to determine if the factor may be reduced. 
   2.1.2 Residential and Occupational Exposure 
    
   There are no specific guideline exposure data deficiencies or requirements for amitraz; however, HED recommends that information be submitted related to the relative amounts of solids (dust) or liquids available for exposure as a result of the use of amitraz pet collars.  This information could be used to address the related uncertainty in the pet collar exposure assessment calculations, and may potentially refine the estimated risks.  An option for obtaining this information is conduct of a dust torsion study where the collar is manually twisted and pulled to generate a dust on the surface.  A study of this type would provide a worst-case estimate of the amount of dust that might be generated during use.   
    
	2.2 	Tolerance Considerations 
    
	2.2.1 	Enforcement Analytical Method 
    
   There are two adequate methods listed in the Food and Drug Administration's (FDA's) Pesticide Analytical Manual (PAM Vol. II) for purposes of data collection and enforcement of tolerances for residues of amitraz and its metabolites containing the 2,4-dimethylaniline (2,4-DMA) moiety.  Methods I (designed for animal tissues and milk) and II (designed for plant commodities) are both gas-liquid chromatography (GLC) methods with electron capture detection, and involve conversion of residues of amitraz and its metabolites containing the 2,4-dimethylaniline moiety to 2,4-DMA using acid and base hydrolysis.   
    
   The FDA PESTDATA database dated 11/01 (PAM Volume I, Appendix I) indicates that amitraz has low recovery (<50%) using Multiresidue Method Section 302 (Luke Method; Protocol D) and that metabolite BTS-27919 is completely recovered using Section 302.  The detection limits of the methods are 0.01 ppm for milk and 0.05 ppm for plant commodities and animal tissues.  
    
	2.2.2 	Recommended and Established Tolerances 
    
   The tolerance definition in 40 CFR 180.287 is as follows: 
    
   "Tolerances are established for residues of the insecticide amitraz (N'-[2,4-dimethylphenyl]-N[[(2,4-dimethylphenyl)imino]methyl]]-N-methylmethanimidamide), including its metabolites and degradates, in or on the commodities in the following table. Compliance with the tolerance levels specified is to be determined by measuring amitraz residues convertible to 2,4-dimethylaniline, expressed as the stoichiometric equivalent of amitraz, in or on the following raw agricultural commodities:" 
    
   HED recommends that tolerances for residues of amitraz in livestock commodities be revoked as there are no longer registered uses on livestock (dermal treatment) and there are no uses on livestock feedstuffs. 
    
Table 2.2.2 Tolerance Summary for Amitraz (40 CFR §180.287). 
                                  Commodity 
                         Established Tolerance (ppm) 
                                 Recommended 
                               Tolerance (ppm) 
                                   Comments 
                                        
Cattle, fat 
                                     0.1 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Cattle, meat 
                                     0.02 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Cattle, meat byproducts 
                                     0.2 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Hog, fat 
                                     0.1 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Hog, kidney 
                                     0.1 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Hog, liver 
                                     0.1 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Hog, meat 
                                     0.05 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Hog, meat byproducts 
                                     0.3 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
Honey 
                                     0.2 
                                     0.10 
                          Harmonization with Canada. 
Honeycomb 
                                      9 
                                     9.0 
      Corrected value to be consistent with HED rounding class practice. 
Milk 
                                     0.03 
                                    Revoke 
No U.S. registrations on livestock or 
Table 2.2.2 Tolerance Summary for Amitraz (40 CFR §180.287). 

                                  Commodity 
                         Established Tolerance (ppm) 
                                 Recommended 
                               Tolerance (ppm) 
                                   Comments 
                                        



                                 feedstuffs. 
Milk, fat 
                                     0.2 
                                    Revoke 
              No U.S. registrations on livestock or feedstuffs. 
    
	2.2.3 	International Harmonization 
    
   Canada, Codex, and the U.S. have essentially the same amitraz residue definition; metabolites containing the dimethylaniline moiety are regulated (see Appendix D).  There are no established Codex maximum residue levels (MRLs) for amitraz in honey or honeycomb.  Canada has established an MRL of 0.1 ppm in honey which is lower than the U.S. tolerance of 0.2 ppm. HED recommends that the U.S. tolerance for amitraz in honey be lowered to 0.10 ppm to harmonize with Canada's MRL. Canada and the U.S. are major partners in honey trade. 
    
   The European Union (EU) has also established an MRL for amitraz residues in honey and other apiculture products at 0.2 ppm.  New Zealand has amitraz MRLs of 0.2 ppm in honey and 1 ppm in other bee products. 
    
	2.3 	Label Recommendations 
    
   No specific label recommendations are being made; however, HED notes that there are residential and occupational handler, and residential post-application scenarios for the registered uses as dog collars that have risk estimates of concern.   
    
	3.0 	Introduction 
	3.1 	Chemical Identity 
    
Table 3.1 Amitraz Nomenclature 

Chemical structure 
         H3C
                  
                    
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                    
                  
                
CH3
N
                  
                    
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                          
                            
                              
                                
                                
                              
                              
                                
                                  
                                
                                N
                              
                            

                          
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                    
                  
                     CH3

                  
                    
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                      
                        
                          
                            
                              
                                
                                
                              
                              
                                
                                  
                                
                                C
                              
                            

                          
                        
                      
                      
                        
                          
                            
                              
                                
                                
                              
                              
                                
                                  
                                
                                H
                              
                            

                          
                        
                      
                      
                        
                          
                            
                              
                                
                                
                              
                              
                                
                                  
                                
                                3
                              
                            

                          
                        
                      
                      
                        
                        
                      
                      
                        
                        
                      
                    
                  
                NCH3
    
Common name 
Amitraz 



Molecular Formula 
C19H23N3 



Molecular Weight 
293.42 



IUPAC name 
N-methylbis(2,4-xylyliminomethyl)amine 


CAS name 
N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-Nmethylmethanimidamide 

CAS # 
33089-61-1 

    
Table 3.1 Amitraz Nomenclature 
PC Code 
106201 
    
	3.2 	Physical/Chemical Characteristics 
    
   Amitraz is soluble in xylene, acetone, and methanol, and insoluble in water.  It has a low to moderate vapor pressure, and exposure to the gaseous state should be negligible.  Amitraz is highly lipophilic/hydrophobic.  See Appendix B for more details. 
	3.3 	Pesticide Use Pattern 
    
   There are two currently registered products with amitraz as the active ingredient: Preventic(R) 
   Tick Collar for Dogs (EPA Reg. #2382-104) and Apivar(TM) (EPA Reg. #87243-1).  The 
   Preventic(R) registered dog collar contains 9% active ingredient and kills and detaches ticks for up to 3 months.  The label directs users to apply one 18" collar (18.5 grams) to dogs up to 60 lbs and one 25" collar (27.5 grams) for dogs over 60 lbs.  The product label does not require handlers to wear specific clothing or PPE.   
    
   The other registered product, Apivar(TM), is for use in beehives, and consists of strips for the inhive control of mites (varroatosis) on honey bees.  The strips contain 3.33% ai.  The label directs users to place two strips per brood chamber (i.e., one strip per 5 Frames of Bees (FoB)), and to leave the strips inside the hive for 42 days before removing. Strips must be removed after a maximum of 56 days, and cannot be reused. The strips may not be used with honey supers in place. The strips are to be hung in the hives in the spring and/or the fall if varroa mite infestations have reached treatment threshold, and are to be removed two weeks before the honey flow starts (two weeks before the supers are replaced).  The strips are not to be used more than two times a year (i.e., no more than once in the spring and once in the fall).  Handlers must wear coveralls over short-sleeved shirt and short pants, socks, footwear, and chemical-resistant gloves. 
    
Table 3.3.   Summary of Directions for Use of Amitraz. 

                                   Use Site 
                         Formulation  [EPA Reg. No.] 
                                 Applic. Rate 
                        Use Directions and Limitations 
                                                                      Beehives 

                                   Beehives 
                                  Apivar(TM) 
                                        
                                   87243-1 
                                        
                                3.33% amitraz 
                                    strip 
        Two strips per brood chamber or one strip per 5 Frames of Bees 
                                    (FoB) 
Leave strips in the hive for 42 days, then remove.  
Strips must be removed after maximum of 56 days. 
 
Do not use strips when honey supers are present. Wait 14 days after removing strips before replacing supers. 
 
Product label requires handler to wear coveralls over short-sleeved shirt and short pants, socks, footwear, and chemical-resistant gloves when handling the strips 
                                                                    Pet Collar 

Dogs up to 60 lbs 
(Small to 
Medium Dog) 
Preventic(R) Tick 
                               Collar for Dogs 
                                        
                                   2382-104 
                                        
                               9% ai pet collar 
18.5 g collar x 9% ai = 1665 mg ai/collar 
or 0.0037 lb ai/collar 
Use on dogs only.  Do not use on cats.  Place the collar around the dog's neck, adjust for fit and buckle in place.  Cut off and dispose of excess length.  Replace collar when effectiveness diminishes. 
 
Dogs over 60 lbs 
(Large Dog) 

27.5 g collar x 9% ai = 2475 mg ai/collar 
or 0.0055 lb ai/collar 

Table 3.3.   Summary of Directions for Use of Amitraz. 

                                   Use Site 
                         Formulation  [EPA Reg. No.] 
                                 Applic. Rate 
                        Use Directions and Limitations 



Product label does not direct the user to wear any specific clothing or PPE.   
    
	3.4 	Anticipated Exposure Pathways 
    
   Humans may potentially be exposed to amitraz in the diet from consuming honey from treated beehives.  Drinking water is not considered a source of dietary exposure as amitraz is not expected to enter the environment, including drinking water sources, through the current uses (pet collars and beehive strips).  
    
   There is a potential for residential exposure to amitraz from dog collar uses, either from applying (handling) the collars or from post-application contact with the treated dog.  Dermal and inhalation exposures may occur to adults applying the collars.  Post-application exposures to treated dogs may be dermal (adults and children) or incidental oral (children).  Post-application inhalation exposure is expected to be negligible for the collar uses.  Exposures from spray drift or volatilization are not anticipated for the current uses.   
    
   Per standard practice, HED considers the inclusion of PPE on a product label to be an indicator that a product is unlikely to be used by residential/consumer users.  In the case of the amitraz beehive product label, however, despite the product requirement for PPE (e.g., coveralls over short-sleeved shirt and short pants, socks, footwear, and chemical-resistant gloves when handling the strips), HED believes that because of the unique nature of the use pattern, the product may be used by both commercial and residential/hobby beekeepers.  Exposures and risks to residential/consumer users from the registered beehive use have been considered, but are assumed negligible for two reasons:  (1) the Apivar beehive strips come prepackaged and do not involve any mixing or loading of active ingredient; the only potential times for individual contact are during application, when the beehive keeper removes the strips directly from the packaging and places them in the hives, and during removal and disposal of the strips six weeks later, and (2) based on the cultural practices associated with hobby beekeeping, it is assumed that individuals involved in this practice would be wearing protective clothing regardless of label requirements which would further reduce any potential exposure. 
    
   There is potential for occupational handler dermal and inhalation exposures resulting from applying dog collars. Significant occupational post-application exposures from contact with treated dogs are not expected to occur and have not been quantitatively assessed.  
    
   Occupational exposures are expected to be negligible for the beehive strip use. Dermal exposure resulting from use of these prepackaged impregnated strips is not likely to occur since there is no mixing or loading and gloves are prescribed to be worn when handling the strips.  Since beehives are located outdoors, and amitraz has low volatility, there is negligible potential for inhalation exposure, at any time, to the applicator or during post-application activities.   
	3.5 	Consideration of Environmental Justice 
    
   Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to Address Environmental Justice in Minority Populations and Low-Income Populations," (http://www.archives.gov/federal-register/executive-orders/pdf/12898.pdf). As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups according to well-established procedures.  In line with OPP policy, HED estimates risks to population subgroups from pesticide exposures that are based on patterns of that subgroup's food and water consumption, and activities in and around the home that involve pesticide use in a residential setting.  Extensive data on food consumption patterns are compiled by the U.S. Department of Agriculture's (USDA's) National Health and Nutrition Examination Survey, What We Eat in America, (NHANES/WWEIA) and are used in pesticide risk assessments for all registered food uses of a pesticide.  These data are analyzed and categorized by subgroups based on age, season of the year, ethnic group, and region of the country.  Additionally, OPP is able to assess dietary exposure to smaller, specialized subgroups, and exposure assessments are performed when conditions or circumstances warrant.  Whenever appropriate, non-dietary exposures are evaluated, based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or playing on treated areas post-application.  Further considerations are currently in development, as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditional dietary patterns among specific subgroups. 
    
	4.0 	Hazard Characterization and Dose-Response Assessment 
    
 	Since the last human health risk assessment (D. Drew, 1/8/2013, D407721), an Extended One-Generation Reproduction Toxicity Study (EOGRTS) in the rat and a dermal absorption triple pack (in vitro dermal penetration studies in rats and humans and in vivo dermal penetration study in rats) have been submitted and integrated into this hazard assessment.  
	4.1 	Toxicological Studies Available for Analysis  
    
   Except for the lack of reliable thyroid hormone data in early lifestage (PND 5) and the failure to identify a NOAEL for brain morphometric measurements in the EOGRTS, the toxicological database for amitraz is complete. The EOGRTS has been submitted since the last (2013) risk assessment to assess the reproductive and neurotoxic potential of amitraz, along with other systemic toxicity including thyroid function, across multiple life stages. However, this study was unable to effectively measure PND 5 thyroid hormone levels. In addition, a NOAEL cannot be established for the adverse brain morphometric alterations found at the highest dose tested (HDT), since morphometric measurements were not conducted at the low- and mid-dose levels. Given these deficiencies, a 10X FQPA SF in the form of a database uncertainty factor will be retained for exposure scenarios that impact children and females of child bearing age until further data is available to assess these sensitive lifestages. These data (thyroid hormone measurements in PND 5 pups for all dose groups and brain morphometric measurements at the low and mid dose groups) should be submitted in order to evaluate potential susceptibility and to determine if the factor may be reduced. 
 
   Based on a weight of the evidence (WOE) approach and considering all the available amitraz hazard and exposure information, the Hazard and Science Policy Committee (HASPOC) recommended that the requirement for acute and subchronic oral neurotoxicity studies, a developmental toxicity study in the rabbit, and a subchronic oral toxicity study in dogs be waived (J. Van Alstine, TXR# 0056451, 2/5/2013). Previously, the subchronic inhalation study in rats was classified as unacceptable/guideline due to the lack of analytical concentration data. The HASPOC reviewed the need for an updated subchronic inhalation study and recommended that the available study be cited qualitatively, but not quantitively, in the risk assessment and recommended that an additional inhalation toxicity study not be required. Several deficiencies were noted within this study; however, the clinical signs of neurotoxicity observed were similar to other effects in the database and, therefore, the study was considered useful for qualitative risk assessment purposes (J. Van Alstine, TXR# 0056451, 2/5/2013). An updated literature search was performed for amitraz and produced no studies that would result in lower PODs than currently selected (see Appendix E). The database contains the following acceptable studies:  
    
 Developmental toxicity in rats  
 One-generation reproductive toxicity in rats 
 EOGRT in rats (excluding adequate PND 5 thyroid data and brain morphometric data mentioned above) 
 Subchronic inhalation in rats (for qualitative purposes only) 
 Chronic oral toxicity in rats and dogs 
 Carcinogenicity in rats and mice 
 Metabolism in rats, mice, baboons, and humans   
 In vitro and in vivo genotoxicity  
 Immunotoxicity study in rats  
 Dermal penetration in rats 
 Dermal absorption triple pack 
 Acute oral toxicity in humans [2] 
    
	4.2 	Absorption, Distribution, Metabolism, & Elimination (ADME)  
    
   In a human metabolism study (MRID 00160964 and MRID 46249601), two male volunteers were given a single dose of 0.25 mg/kg of amitraz by capsule and experienced dry mouth, drowsiness, disorientation, light headedness, slurred speech, decreased temperature, decreased heart rate, and slight pale appearance after treatment. Clinical signs were observed within 90 - 160 minutes after ingestion and persisted for up to 12 hours.  
    
   Urine accounted for 82% of the radiolabel excreted in humans with 44% and 63% being excreted in the first 12 and 24 hours, respectively. However, determination of radiolabel in the feces, expired air and tissues was not completed. Polar metabolites, likely representing conjugates (type not reported), were the predominant metabolites identified, contributing ~57% of the total, and 4formamido-3-methylbenzoic acid (BTS 39 098) and 4-acetamido-3-methylbenzoic acid (BTS 31 158) metabolites contributing 27% in humans.   
    
   Additional metabolism data are also available in rats, mice, and baboons. No major differences in the amount of radiolabel excreted between sexes or species were noted. The overall metabolic profile was similar across sexes and species.  
    
    
   4.2.1  Dermal Absorption  
    
   Triple pack data (rat in vivo, rat in vitro, and human in vitro) are available for amitraz at several concentrations (0, 20, 60, or 100 ug a.i./cm[2]). There is a second in vivo rat dermal absorption study available (MRID 42133501) conducted with a wettable powder formulation vehicle. After an exposure period of 120 hours, with a 10-hour skin wash, the dermal absorption was 7% (total absorbed in urine, feces, cage wash, carcass, and GI tract). However, the purity of the test material used in this study is unknown and without proper characterization of the test compound, it cannot be used to estimate a dermal absorption factor.  
    
   In the triple pack, the rat in vivo data (MRID 49724602) demonstrated continued absorption from the stratum corneum with time (6-72 hours). However, the continued absorption may be due to the chemical properties of the corn oil vehicle increasing the penetration of amitraz across the skin which was not used as a vehicle in the second rat in vivo study (MRID42133501). Regardless, the estimate of dermal absorption from the in vivo study (MRID 49724602) is the amount absorbed + potentially absorbed (skin application site, adjacent skin, tape strips [excluding tape strips 1 and 2], cage wash, urine, feces, and carcass). Dermal absorption ranged from 25-33% across all dose groups and time points. However, a dermal absorption value of 33% was found in the 100 ug/cm[2] group sacrificed at 24 hours in the in vivo study and is considered most appropriate and protective of dermal exposure to amitraz.  
    
   The in vitro dermal absorption of amitraz in corn oil ranged from 0.8-1% and 4-7% in human and rat skin, respectively. The in vitro dermal penetration study on rat and human skin completed at the same dose (100 ug a.i./cm[2]) using a corn oil vehicle (MRID 49724601) is most appropriate for comparison to the rat in vivo study.  However, the in vitro studies are not considered appropriate for use in risk assessment because skin exposure occurred for only 6 hours rather than 24 hours; therefore, the dermal triple pack methodology could not be applied. 
    
   The in vivo dermal absorption from the rat (33%) is currently the most appropriate for risk assessment (MRID 49724602). The agency recognizes that the rat in vivo study provides a highly conservative assumption of dermal absorption and that dermal absorption in humans may be lower. However, without proper characterization of the test compound from the second rat in vivo study (MRID 42133501), the in vivo study as part of the triple-pack data will be used for risk assessment (MRID 49724602).   
    
	4.3 	Toxicological Effects   
    
   Multiple studies in various mammalian species, including humans, support that the common effects of amitraz are those of neurotoxicity, with signs such as sedation, hypothermia, drowsiness, etc. In insects, the mechanism of action is also neurotoxicity via interaction with the octopamine receptor causing paralysis and death. Multiple mechanisms have been hypothesized for the effects observed in mammals including, -adrenergic agonist activity, inhibition of the histamine H1 receptor, inhibition of prostaglandin E2 (PGE2) synthesis, inhibition of monoamine oxidase, and sex steroid actions, among others. However, to date the MOA leading to toxicity in mammalian species has not been elucidated.  
    
   Based on available human and animal studies, human subjects were shown to be more sensitive than any other species tested, followed by the rat and dog. Multiple species display evidence of neurotoxicity following exposure to amitraz. Clinical signs of neurotoxicity were seen across species, sexes, and routes of administration. In the chronic toxicity study in dogs, signs of central nervous system (CNS) depression were observed during the first two days of dosing. In both the oral chronic study and the 21-day inhalation study in the rat, clinical signs indicative of neurotoxicity were observed including mild dyspnea, hyposensitivity to noise, hypersensitivity to touch, aggressive behavior, and body tremors. In the EOGRTS, clinical signs of neurotoxicity (salivation, urine stained fur, and vocalization in males), decreased motor activity and changes in brain morphometrics were noted.  
    
   In a human double-blind randomized crossover study (MRID 43283101), 6 male volunteers were given single oral doses of amitraz (purity = 98.2%) by capsule, at 0, 0.0625 or 0.125 mg/kg in lactose, over multiple phases. In Phase 1 of the study, 4 subjects received placebo and 2 received a single 0.0625 mg/kg dose of amitraz. During Phase 2 conducted 14 days after Phase 1, two subjects received placebo, two subjects received a single low dose, and two subjects received a single high dose.  In Phase 3 conducted 14 days after Phase 2, three subjects received placebo and four subjects received a single high dose. The research was designed so all subjects received placebo, low-dose, and high-dose during the course of the study. Vital signs (pulse, respiration rate, blood pressure, and body temperature) and ECGs were taken. Pupil responsiveness and psychomotor performance were also evaluated. Urine and blood were collected for urinalysis, clinical chemistry, and hematology examination. Vital signs and ECG parameters were within normal limits. No clinically significant changes were seen in pupil response or psychomotor performance. One subject had a slightly elevated total bilirubin at the post-study screen, but it returned to normal limits within three weeks' completion of the study. No treatment-related effects were found on hematology or urinalysis parameters. The NOAEL was 0.125 mg/kg, the HDT; a LOAEL was not established. However, as discussed in Section 4.2, the human metabolism study noted multiple signs of neurotoxicity after treatment with amitraz at 0.25 mg/kg/day.  
    
   No developmental toxicity was seen at the HDT (30 mg/kg/day) in an acceptable pre-natal developmental study in rats, while maternal effects occurred at 15 mg/kg/day and included decreased body weight. In the EOGRTS, parental effects were noted at the HDT (7.5 mg/kg/day) and included decreased body weight and clinical signs in males in addition to decreased motor activity in both sexes. At the same dose level, offspring toxicity manifested as increased PND 14 pup death and decreased pup body weight in both sexes and reproductive toxicity included an increased number of females in persistent diestrus with an increase in the number of primordial follicles. Developmental effects, including changes in brain morphometrics (decreased hypothalamus area, corpus collosum thickness, and distance across hippocampal pyramidal layers in males only) also occurred at 7.5 mg/kg/day; however, a NOAEL could not be established for this lifestage since brain morphometrics were not reported for the low or mid dose groups. F1 adult toxicity occurred at a lower dose level than all other lifestages, 1.5 mg/kg/day, and included a 25% decrease in thyroxine (T4) levels.  
    
   In an immunotoxicity study in rats no systemic toxicity or immunotoxicity was observed up to the HDT (8 mg/kg/day) which satisfies the immunotoxic portion of the EOGRTS. 
    
   Acute toxicity studies in various laboratory animals indicate that amitraz is moderately toxic (Toxicity Category II  -  category reflects the highest dose tested in the study and not dermal toxicity at 200 mg/kg) via the dermal route, slightly toxic (Toxicity Category III) via the oral and not acutely toxic (Toxicity Category IV) via inhalation routes of exposure. Further, it is not a skin or eye irritant, nor is it a skin sensitizer. 
    
   A summary of existing toxicity studies can be found in Appendix A of this risk assessment document. 
    
	4.4 	Safety Factor for Infants and Children (FQPA Safety Factor)  
    
   The amitraz risk assessment team recommends that the 10X FQPA UFDB be retained for chronic dietary, incidental oral, dermal and inhalation exposure scenarios to account for inadequate PND 
   5 thyroid hormone data and the failure to identify a NOAEL for brain morphometric measurements within the EOGRTS. A decrease in T4 levels (↓25%) was observed in F1 adult female rats. Given the low number of PND 5 female control animals that reported values above the lower limit of quantification (LLOQ) (n=3) the interpretation of T4 levels at all dose levels was not possible. The lack of thyroid-stimulating hormone (TSH) measurements for PND 5 pups, and all other exposed lifestages, makes the interpretation of thyroid hormone levels more difficult. In addition, a NOAEL cannot be established for the adverse brain morphometric alterations found at the HDT, since morphometric measurements were not conducted at the low- and mid-dose levels. The FQPA SF for acute dietary exposure scenarios may be reduced to 1X; the brain morphometric alterations and thyroid hormone changes in the EOGRTS were not attributed to a single exposure to amitraz; therefore, the database to assess acute exposure is considered protective and complete for assessing acute dietary exposure.  
    
	4.4.1 	Completeness of the Toxicology Database 
    
   The amitraz database contains acceptable developmental, one-generation reproductive, and 
   EOGRT studies in the rat for consideration of sensitivity to infants and children (discussed is Section 4.3). The HASPOC recommended that the requirement for acute and subchronic oral neurotoxicity studies, a developmental toxicity study in the rabbit, and a subchronic oral toxicity study in dogs be waived at this time. The HASPOC also reviewed the need for an updated subchronic inhalation study and recommended that the available study be cited qualitatively, but not quantitively, in the risk assessment and an additional inhalation toxicity study is not recommended (J. Van Alstine, TXR# 0056451, 2/5/2013). However, as discussed in Section 4.4, there is still concern for the lack of thyroid hormone measurements in PND 5 pups and brain morphometric measurements at the low and mid dose groups for the determination of qualitative or quantitative susceptibility; therefore, the 10X UFDB should be retained for chronic dietary, incidental oral, dermal and inhalation exposure scenarios. The database to assess acute exposure to amitraz is considered complete.  
    
    
	4.4.2 	Evidence of Neurotoxicity 
    
   Various mammalian species, including humans, rats, and dogs, in multiple studies have demonstrated signs of neurotoxicity as discussed in Section 4.3, ranging from drowsiness to decreases in activity. However, the concern for neurotoxicity is low because the selected endpoints are protective of all observed effects and the acute dietary endpoint, based on neurotoxic signs in humans (see Section 4.2), is protective of all other observed neurotoxic signs in the database. 
    
	4.4.3 	Evidence of Sensitivity/Susceptibility in the Developing or Young Animal 
    
   In the acceptable pre-natal developmental and one-generation reproductive toxicity studies in rats, there was no evidence of sensitivity or susceptibility. However, as discussed in Section 4.4, there is still concern for lifestage sensitivity in PND 5 pups due to the lack of adequate thyroid hormone data, the most sensitive endpoint in the EOGRTS study, at this lifestage. In addition, a NOAEL cannot be established for the brain morphometric changes observed at the HDT in the EOGRTS, because brain morphometry assessments were not conducted at the low- or mid-dose. 
   Therefore, the amitraz risk assessment team recommends the retention of the 10X FQPA SF as a UFDB for the lack of acceptable thyroid hormone measurements in PND 5 pups and brain morphometric data at the low and mid-dose for the relevant exposure scenarios. As previously discussed, neither the thyroid hormone changes nor the brain morphometry findings are considered to be the result of an acute/single exposure. Consequently, the 10X FQPA factor to account for uncertainty in the NOAEL/LOAEL for these parameters is reduced to 1X for the acute dietary exposure assessment. 
    
	4.4.4 	Residual Uncertainty in the Exposure Database 
    
   HED has used high-end assumptions in the dietary exposure assessment, including the use of 100% beehives treated assumptions and tolerance-level residue values. In addition, the residential exposure assessment was conducted using inputs and assumptions from the 2012 Residential Standard Operating Procedures (SOPs) such that residential exposure will not be underestimated.  
	4.5 	Toxicity Endpoint and Point of Departure Selections 
    
   Since the last (2013) risk assessment, points of departure have been updated to reflect the submitted EOGRTS and dermal triple pack data. The previous assessment selected the combined results of the human oral toxicity and metabolism studies as the POD for incidental oral, dermal and inhalation exposure scenarios. In light of the adverse thyroid hormone effects observed in the EOGRTS, which were not measured in the human oral or metabolism studies, the PODs were updated in the current assessment to be protective of the observed thyroid effects. Although certain NOAEL/LOAELs within the toxicity profile tables contain results that are no longer considered adverse based upon current practices (e.g. decreased body weight gain in the absence of decreased absolute body weight), NOAEL/LOAELs were not updated since the last risk assessment because it would not impact endpoint selection. 
    
   Toxicity endpoints and points of departure (PODs) for dietary, residential, and occupational exposure scenarios are summarized below and in Table 4.5.3.1 - Table 4.5.3.2.  
    
   Acute dietary (all populations including females 13+): The POD and endpoint of concern were selected from the combined results of the oral toxicity and metabolism studies conducted in human subjects (discussed in Sections 4.2 and 4.3). The combined NOAEL and LOAEL from these two studies are 0.125 mg/kg (human oral study) and 0.25 mg/kg (human metabolism study), respectively, based on clinical signs (dry mouth, drowsiness, disorientation, light headedness, slurred speech, slight pale appearance), and decreased temperature and heart rate, in two human subjects in the metabolism study and the lack of adverse effects in the human oral study. Clinical signs appeared as early as ninety minutes after dosing and are therefore considered appropriate for the acute duration of exposure. The aPAD of 0.0125 mg/kg/day is derived from the NOAEL of 0.125 mg/kg/day and a total uncertainty factor of 10X (1X to account for interspecies extrapolation due to the use of a human study, 10X for intra-species variation, and 1X for FQPA SF). The FQPA SF was reduced to 1X because there is a lack of concern for inadequate thyroid hormone or brain morphometric data, given permutations in both parameters do not occur after a single acute exposure.  
    
   Chronic dietary (all populations including females 13+): The EOGRTS is appropriate for chronic dietary exposures to all population groups including females 13+ (NOAEL = 0.5 mg/kg/day; LOAEL = 1.5 mg/kg/day) based on decreases in T4 levels in F1 adult female rats. This effect is protective of all other effects in the database, including effects observed in human studies, and is appropriate for the duration of exposure. The cPAD of 0.0005 mg/kg/day is derived from the NOAEL of 0.5 mg/kg/day and a total uncertainty factor of 1000X (10X to account for interspecies extrapolation, 10X for intra-species variation, and 10X for FQPA UFDB).  
    
   Incidental oral: The EOGRTS is appropriate for incidental oral exposures (NOAEL = 0.5 mg/kg/day; LOAEL = 1.5 mg/kg/day) based on decreases in T4 levels in F1 adult female rats. This effect is protective of all other effects in the database, including effects observed in human studies, and is appropriate for the duration of exposure. The LOC for MOE = 1000 (10X for intra-species variation, 10X for inter-species extrapolation, and 10X FQPA UFDB). 
    
   Dermal exposure (all durations): The EOGRTS was considered appropriate for all dermal exposure scenarios (NOAEL = 0.5 mg/kg/day; LOAEL = 1.5 mg/kg/day based on decreases in T4 levels in F1 adult rats) since the endpoint of concern, decreases in T4 levels, was not measured in the human route-specific dermal toxicity study. This endpoint is protective of all other effects in the database, including all effects observed in the human studies, and is appropriate for the duration of exposure. The LOC for MOE = 1000 (10X for intra-species variation, 10X for interspecies extrapolation, 10X FQPASF (residential uses)/UFDB (occupational uses)). Dermal absorption factor = 33%.   
    
   Inhalation exposure (all durations): The EOGRTS was also considered appropriate for all inhalation exposure scenarios (NOAEL = 0.5 mg/kg/day; LOAEL = 1.5 mg/kg/day based on decreases in T4 levels in F1 adult rats) since the endpoint of concern, decreases in T4 levels, was not measured in the route-specific study and it was determined that the rat subchronic inhalation study should not be used for quantitative purposes. This endpoint is protective of all other effects in the database, including all effects observed in the human studies, and is appropriate for the duration of exposure. Since an oral study was used, the agency assumed equivalent oral and inhalation toxicity for route-to-route extrapolation. The LOC for MOE = 1000 (10X for intraspecies variation, 10X for inter-species extrapolation, 10X FQPA SF (residential uses)/UFDB (occupational uses)).     
    
	4.5.1 	Recommendation for Combining Routes of Exposures for Risk Assessment 
    
   When there are potential occupational and residential exposures to a pesticide, the risk assessment must address exposures from three major routes (oral, dermal, and inhalation) and determine whether the individual exposures can be combined if they have the same toxicological effects/target organs. For both the occupational and residential amitraz risk assessment, it is possible to combine all applicable routes of exposure (oral, dermal, and inhalation) due to common toxicological endpoints (changes in thyroid hormone levels).  
    
	4.5.2 	Cancer Classification and Risk Assessment Recommendation 
    
   Amitraz is classified as "Suggestive Evidence of Carcinogenic Potential" based on the occurrence of tumors (liver and lung) in a single species (mouse). The cancer classification of amitraz is based on the following considerations: (1) no carcinogenic response was seen in an acceptable rat cancer study; (2) the tumors found in the mouse are commonly seen in this species and were only found at a dose that appears to have been excessive given the other adverse effects seen in the animals; (3) amitraz is not mutagenic; and (4) although there is limited positive mutagenicity data and equivocal evidence of cancer for a minor amitraz metabolite, 2,4dimethylaniline, that equivocal cancer evidence was present only at high doses in the metabolite study and was not consistent with the tumors seen in the amitraz study. Quantification of risk using a non-linear approach (i.e., RfD/PAD) for amitraz will adequately account for all chronic toxicity, including carcinogenicity, which could result from exposure to amitraz and its metabolites (J. Kidwell, TXR# 0054272, 7/18/2006). 
    
   4.5.3 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment 
    
Table 4.5.3.1      Summary of Residential Toxicological Dose and Endpoints for Amitraz Risk Assessment 
Exposure Scenario 
Dose Used in Risk Assessment, 
UF 
Study and Toxicological Effects 
Acute Dietary 
(All Populations including females 13+) 
NOAEL = 0.125 mg/kg 
 
UFH = 10X  
UFA =1X 
FQPA SF (UFDB) = 1X 
 
Acute RfD = 0.0125 mg/kg 
Acute PAD = 0.0125 mg/kg 
Combined results from human oral toxicity and human metabolism studies (MRID 43283101, MRID 00160964 and MRID 46249601; TXR# 0052737) 
 
LOAEL = 0.25 mg/kg based on clinical signs (dry mouth, drowsiness, disorientation, light headedness, slurred speech, slight pale appearance) and decreased temperature and heart rate 
Table 4.5.3.1      Summary of Residential Toxicological Dose and Endpoints for Amitraz Risk Assessment 
Exposure Scenario 
Dose Used in Risk Assessment, 
UF 
Study and Toxicological Effects 
Chronic Dietary 
(All Populations including females 13+) 
NOAEL= 0.5 mg/kg/day 
 
UFH = 10X 
UFA =10X 
FQPA SF (UFDB) = 10X 
 
Chronic RfD = 0.0005 mg/kg/day 
Chronic PAD = 0.0005 mg/kg/day 
EOGRTS Rat (MRID 49994401; TXR# 0057767) 
 
EOGRTS F1 adult toxicity  
 
LOAEL = 1.5 mg/kg based on decreased T4 levels (↓25%; females only) 
Incidental Oral  
(All durations) 
NOAEL= 0.5 mg/kg/day 
 
LOC = MOE of 1000X  
(UFH = 10X, UFA =10X, FQPA SF 
(UFDB) = 10X) 
  
EOGRTS Rat (MRID 49994401; TXR# 0057767) 
 
EOGRTS F1 adult toxicity  
 
LOAEL = 1.5 mg/kg based on decreased T4 levels (↓25%; females only) 
Dermal (All durations)  
NOAEL= 0.5 mg/kg/day 
 
LOC = MOE of 1000X  
(UFH = 10X, UFA =10X, FQPA SF 
(UFDB) = 10X) 
  
 
DAF = 33% 
EOGRTS Rat (MRID 49994401; TXR# 0057767) 
 
EOGRTS F1 adult toxicity  
 
LOAEL = 1.5 mg/kg based on decreased T4 levels (↓25%; females only) 
Inhalation (All durations)  
NOAEL= 0.5 mg/kg/day 
 
LOC = MOE of 1000X  
(UFH = 10X, UFA =10X, FQPA SF 
(UFDB) = 10X) 
  
 
EOGRTS Rat (MRID 49994401; TXR# 0057767) 
 
EOGRTS F1 adult toxicity  
 
LOAEL = 1.5 mg/kg based on decreased T4 levels (↓25%; females only) 
Cancer (oral, dermal, and inhalation) 
 "Suggestive Evidence of Carcinogenicity"   
Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UFA = extrapolation from animal to human (interspecies). UFH = potential variation in sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. UFDB =database uncertainty factor.   PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LOC = level of concern. 
    
Table 4.5.3.2      Summary of Occupational Toxicological Dose and Endpoints for Amitraz Risk Assessment 
Exposure Scenario 
Dose Used in Risk Assessment, 
UF 
Study and Toxicological Effects 
Dermal (All durations)  
NOAEL= 0.5 mg/kg/day 
 
LOC = MOE of 1000X  
(UFH = 10X, UFA =10X, UFDB = 10X) 
  
 
DAF = 33% 
EOGRTS Rat (MRID 49994401; TXR# 0057767) 
 
EOGRTS F1 adult toxicity  
 
LOAEL = 1.5 mg/kg based on decreased T4 levels (↓25%; females only) 
Inhalation (All durations)  
NOAEL= 0.5 mg/kg/day 
 
LOC = MOE of 1000X  
(UFH = 10X, UFA =10X, UFDB = 10X) 
  
 
EOGRTS Rat (MRID 49994401; TXR# 0057767) 
 
EOGRTS F1 adult toxicity  
 
LOAEL = 1.5 mg/kg based on decreased T4 levels (↓25%; females only) 
Cancer (dermal and inhalation) 
 "Suggestive Evidence of Carcinogenicity"   
Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UFA = extrapolation from animal to human (interspecies). UFH = potential variation in sensitivity among members of the human population (intraspecies). UFDB =database uncertainty factor.  MOE = margin of exposure. LOC = level of concern.  
	4.6 	Endocrine Disruptor Screening Program  
    
   As required by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Federal Food, Drug, and Cosmetic Act (FFDCA), EPA reviews numerous studies to assess potential adverse outcomes from exposure to chemicals. Collectively, these studies include acute, subchronic and chronic toxicity, including assessments of carcinogenicity, neurotoxicity, developmental, reproductive, and general or systemic toxicity. These studies include endpoints which may be susceptible to endocrine influence, including effects on endocrine target organ histopathology, organ weights, estrus cyclicity, sexual maturation, fertility, pregnancy rates, reproductive loss, and sex ratios in offspring. For ecological hazard assessments, EPA evaluates acute tests and chronic studies that assess growth, developmental and reproductive effects in different taxonomic groups.  As part of its reregistration decision for amitraz, EPA reviewed these data and selected the most sensitive endpoints for relevant risk assessment scenarios from the existing hazard database. However, as required by FFDCA section 408(p), amitraz is subject to the endocrine screening part of the Endocrine Disruptor Screening Program (EDSP). 
    
   EPA has developed the EDSP to determine whether certain substances (including pesticide active and other ingredients) may have an effect in humans or wildlife similar to an effect produced by a "naturally occurring estrogen, or other such endocrine effects as the Administrator may designate." The EDSP employs a two-tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify any adverse endocrine-related effects caused by the substance, and establish a dose-response relationship between the dose and the E, A, or T effect. 
    
   Under FFDCA section 408(p), the Agency must screen all pesticide chemicals. Between October 2009 and February 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. A second list of chemicals identified for EDSP screening was published on June 14, 2013 and includes some pesticides scheduled for registration review and chemicals found in water. Neither of these lists should be construed as a list of known or likely endocrine disruptors.  
    
   For further information on the status of the EDSP, the policies and procedures, the lists of chemicals, future lists, the test guidelines and the Tier 1 screening battery, please visit our website. 
    
	5.0 	Dietary Exposure and Risk Assessment  
	5.1 	Residues of Concern Summary and Rationale 
    
   Literature studies indicate that parent amitraz degrades quickly in honey with complete breakdown occurring within 10-15 days at ambient temperatures (Martel et al, 2002; Jimenez et al, 1997; Korta et al, 2001).  In beeswax, amitraz degraded in one day.  The degradation products in honey and beeswax were identified primarily as 2,4-dimethylphenyl formamide (referred to here as DMF, but also known as DMPF) and N-(2,4-dimethylphenyl)-N-methyl formamidine (DPMF), along with small amounts of 2,4-dimethylaniline (DMA).  DMF and DPMF were found in about equal proportions in honey in these studies.  These metabolites contain the dimethylaniline moiety and are covered by the current tolerance expression.  Parent amitraz and metabolites containing the dimethylaniline moiety are also the residues of concern for inclusion in the dietary risk assessment. 
    
   The qualitative nature of the amitraz residue in plants and animals has also been examined in plant metabolism studies with apples, beans, lemons, citrus, cotton, and pears, and animal metabolism studies with cattle and swine (dermal application) as well as cattle, goats and hens (oral dosing). Primary residues include parent amitraz and metabolites containing the dimethylaniline moiety. 
	5.2 	Food Residue Profile 
    
   The only current potential sources of amitraz residues in foods is honey and honeycomb from the strip use in beehives. Based on a study (MRID 48341001) in which samples were collected from honey supers of treated hives, total residues of amitraz in honey and beeswax were 0.059-0.092 ppm and <0.050-7 ppm, respectively, 10 days after treatment (DAT; days after strips were removed).  Residues at 15 DAT were <0.050-0.084 ppm and 1-4 ppm in honey and beeswax, respectively.  In the study, supers were installed on the same day that strips were removed. The U.S. label for beehive use requires a 14-day interval between removing the strips and installation of supers; therefore, this study may have resulted in higher residues than those expected to occur based on label directions. 
    
   Storage stability data are available indicating that total residues of amitraz are stable in honey stored at -20°C for up to 4 months and in honeycomb stored ambient for up to 1 month.  Total residues of amitraz are stable in cottonseed for 13.5 months, citrus for 18 months, and in animal muscle, fat, and milk for 12-15 months.  
	5.3 	Water Residue Profile 
    
   No residues of amitraz are expected in drinking water from the current uses. 
	5.4 	Dietary Risk Assessment 
    
	5.4.1 	Description of Residue Data Used in Dietary Assessment 
    
   Unrefined acute and chronic dietary (food only) exposure and risk assessments were conducted for amitraz using the Dietary Exposure Evaluation Model software with the Food Commodity Intake Database (DEEM-FCID) Version 3.16.  Recommended tolerance-level residues (0.10 ppm) and 100% of beehives treated were used. The only current potential sources of amitraz residues in foods are honey and honeycomb. The assessment includes only honey, as honeycomb is not reported as being consumed in the consumption database. Drinking water is not included in the dietary assessment as, based on current uses, amitraz is not expected to enter drinking water sources. 
    
	5.4.2 	Acute Dietary Risk Assessment 
    
   Acute dietary exposure estimates are <1% of the aPAD for the U.S. population and all population subgroups at the 95[th] percentile of exposure and are not of concern. 
    
	5.4.3 	Chronic Dietary Risk Assessment 
    
   Chronic dietary exposure estimates are <1% of the cPAD for the U.S. population and all population subgroups and are not of concern. 
    
	5.4.4 	Cancer Dietary Risk Assessment 
    
   Amitraz is classified as "Suggestive Evidence of Carcinogenic Potential."  Quantification of risk using a non-linear approach (i.e., RfD/PAD) for amitraz will adequately account for all chronic toxicity, including carcinogenicity, which could result from exposure to amitraz and its metabolites. A separate cancer dietary assessment is not necessary for amitraz. As chronic dietary exposure estimates are not of concern, cancer dietary risks are not of concern for amitraz. 
    
	5.4.5 	Summary of Acute and Chronic Dietary Exposure and Risk 
    
Table 5.4.5.  Summary of Dietary (Food Only) Exposure and Risk for Amitraz. 
                             Population Subgroup 
                        Acute Dietary (95th Percentile) 
                               Chronic Dietary 
                                    Cancer 

                                   Dietary 
                                   Exposure 
(mg/kg/day) 
 % aPAD 
                                   Dietary 
                                   Exposure 
(mg/kg/day) 
 % cPAD 
                                   Dietary 
                                   Exposure 
(mg/kg/day) 
                                     Risk 
General U.S. Population 
                                   0.00006 
                                    < 1 
                                   0.00001 
                                    < 1 
                                      NA 
                                      NA 
All Infants (<1 year old) 
                                   0.00002 
                                    < 1 
                                   0.00001 
                                    < 1 


Children 1-2 years old 
                                   0.00013 
                                    < 1 
                                   0.00003 
                                    < 1 


Children 3-5 years old 
                                   0.00011 
                                    < 1 
                                   0.00003 
                                    < 1 


Children 6-12 years old 
                                   0.00006 
                                    < 1 
                                   0.00002 
                                    < 1 


Youth 13-19 years old 
                                   0.00003 
                                    < 1 
                                   0.00001 
                                    < 1 


Adults 20-49 years old 
                                   0.00004 
                                    < 1 
                                   0.00001 
                                    < 1 


Adults 50-99 years old 
                                   0.00006 
                                    < 1 
                                   0.00001 
                                    < 1 


Females 13-49 years old 
                                   0.00004 
                                    < 1 
                                   0.00001 
                                    < 1 


    
	6.0 	Residential Exposure and Risk Estimates 
    
   There are existing residential uses of amitraz as a dog collar and as impregnated strips for use in beehives.  This assessment includes an updated pet collar methodology and assessment, with revised residential handler and post-application exposure assessments for adults and children coming into contact with dogs wearing pet collars.   
    
   Per standard practice, HED considers the inclusion of PPE on a product label to be an indicator that a product is unlikely to be used by residential/consumer users.  In the case of the amitraz beehive product label, however, despite the product requirement for PPE (e.g., coveralls over short-sleeved shirt and short pants, socks, footwear, and chemical-resistant gloves when handling the strips), HED believes that because of the unique nature of the use pattern, the product may be used by both commercial and residential/hobby beekeepers.  Exposures and risks to residential/consumer users from the registered beehive use have been considered, but are assumed negligible for two reasons:  (1) the Apivar beehive strips come prepackaged and do not involve any mixing or loading of active ingredient; the only potential times for individual contact are during application, when the beehive keeper removes the strips directly from the packaging and places them in the hives, and during removal and disposal of the strips six weeks later, and (2) based on the cultural practices associated with hobby beekeeping, it is assumed that individuals involved in this practice would be wearing protective clothing regardless of label requirements which would further reduce any potential exposure. 
    
	6.1 	Residential Handler Exposure/Risk Estimates 
    
   The quantitative exposure/risk assessment developed for residential handlers is based on applying a pet collar to a dog.  As described in the recent 2016 tetrachlorvinphos (TCVP) risk assessment (W. Britton, D436833, 12/21/2016), due to the uncertainty associated with whether pet collars release the active ingredient as a liquid and/or solid (dust), residential handler exposures for pet collar applications are assessed assuming the active ingredient could be present as liquid and solid (dust) formulations concurrently with varying ratios of liquid/dust (e.g., 99/1, 50/50, and 1/99 liquid/dust).   
    
   Residential Handler Exposure Data and Assumptions 
   A series of assumptions and exposure factors served as the basis for completing the residential handler risk assessments.  Each assumption and factor is detailed below. 
    
   Application Rate:  
   The maximum application rate for the collars (0.0037 lb ai/collar for small to medium dogs and 0.0055 lb ai/collar for large dogs as noted in Table 3.3) was used in the assessment. The application rate used represents the maximum amount of ai in the entire dog collar.  The amitraz pet collar label directs the user to cut off and dispose of any excess length once the product is fit and buckled into place.  As described in the 2012 Residential Treated Pet SOP, the removal of excess length is anticipated; however, because the exact length cannot be determined, the corresponding ai loss cannot be determined and exposure is assessed assuming the full collar length.   
    
   Unit Exposures and Area Treated or Amount Handled: 
   Unit exposure values and estimates for amount handled were taken from HED's 2012 Residential SOPs.  In the updated approach presented here where pet collar applications are assessed assuming the active ingredient in pet collars is released as both a liquid and solid (dust) concurrently with varying ratios of liquid/dust, the current UE for collars is used to assess the fraction that is released as a liquid formulation (using spot-on data as surrogate data), and the shaker can UE is used to assess the fraction that is released as a solid (dust) formulation. 
    
   Exposure Duration:   
   Residential handler exposure is expected to be short-term in duration.  Intermediate-term exposures are not likely because of the intermittent nature of applications by homeowners. 
    
   Combining Exposures/Risk Estimates: 
   Dermal and inhalation risk estimates were combined in this assessment, since the toxicological effects for these exposure routes were the same.  Dermal and inhalation risk estimates were combined using the following formula: 
    
    Total MOE = Point of Departure (mg/kg/day) / (Combined Dermal + Inhalation dose (mg/kg/day)) 
    
   Summary of Residential Handler Non-Cancer Exposure and Risk Estimates 
   For the pet collar uses, the combined (dermal + inhalation) residential handler MOEs were all less than the LOC of 1000, ranging from 2.6 to 100 (with the dermal route driving the risk estimates) for small and large collars, depending on the assumed ratio of liquid to dust in the pet collar product, and are of concern.   
    
Table 6.1.1.  Residential Handler Exposure and Risk Estimates for Use of Pet Collars Containing 9% Amitraz. 


                              Exposure Scenario 
                          Type of collar (g/collar) 
Application 
                            Rate (lb ai/collar)[1] 
                                    Amount 
                                   Handled 
                               (collar/day)[2]  
                          Dermal Dose (mg/kg/day)[3] 
                                  Inhalation 
                                     Dose 
(mg/kg/day)[4] 
                                    Dermal 
                                     MOE 
                                    (LOC = 
                                   1000)5  
                                  Inhalation 
                                     MOE 
                                    (LOC = 
                                   1000)[6] 
Combined 
                                     MOE 
                                    (LOC = 
                                   1000)[7] 
                                  99% Liquid/1% Solid (Dust) 


    
Table 6.1.1.  Residential Handler Exposure and Risk Estimates for Use of Pet Collars Containing 9% Amitraz. 


                              Exposure Scenario 
                          Type of collar (g/collar) 
Application 
                            Rate (lb ai/collar)[1] 
                                    Amount 
                                   Handled 
                               (collar/day)[2]  
                          Dermal Dose (mg/kg/day)[3] 
                                  Inhalation 
                                     Dose 
(mg/kg/day)[4] 
                                    Dermal 
                                     MOE 
                                    (LOC = 
                                   1000)5  
                                  Inhalation 
                                     MOE 
                                    (LOC = 
                                   1000)[6] 
Combined 
                                     MOE 
                                    (LOC = 
                                   1000)[7] 
                            Applying a pet collar  
                           Up to 60 lb Dog (18.5 g) 
                                    0.0037 
                                      2 
                                    0.0049 
                                   0.000017 
                                     100 
                                    30,000 
                                     100 

                          Above 60 lb  Dog (27.5 g) 
                                    0.0055 
                                      2 
                                    0.0073 
                                   0.000025 
                                      69 
                                    20,000 
                                      69 
                                  50% Liquid/50% Solid (Dust) 


Applying a pet collar  
                           Up to 60 lb Dog (18.5 g) 
                                    0.0037 
                                      2 
                                    0.0669 
                                   0.00083 
                                     7.5 
                                     610 
                                     7.5 

                           Above 60 lb Dog (27.5 g) 
                                    0.0055 
                                      2 
                                    0.0994 
                                   0.00123 
                                     5.0 
                                     410 
                                     5.0 
                                  1% Liquid/99% Solid (Dust) 


Applying a pet collar  
                           Up to 60 lb Dog (18.5 g) 
                                    0.0037 
                                      2 
                                    0.1288 
                                   0.00163 
                                     3.9 
                                     310 
                                     3.9 

                           Above 60 lb Dog (27.5 g) 
                                    0.0055 
                                      2 
                                    0.1915 
                                   0.00243 
                                     2.6 
                                     210 
                                     2.6 
 Based on registered label (Reg No. 2382-104) and percent ai (9%) in product. 
 Based on HED's 2012 Standard Operating Procedures for Residential Pesticide Exposure Assessment; Treated Pets 
 The dermal doses presented in the table are the combined (liquid and dust) dermal doses based on the assumed ratio.  For example, for the 99/1 liquid/dust ratio assumption, the dermal dose = (liquid formulation dermal dose * 0.99) + (dust formulation dermal dose * 0.01).  The liquid and dust dermal doses were calculated as follows:  Dermal Unit Exposure (mg/lb ai) x Application Rate (lb ai/collar) x Amount Handled (collars/day) x DAF (33%) / BW (80 kg), where the unit exposure for spot-ons was used (120 mg/lb ai) when assuming a liquid formulation, and the unit exposure for shaker cans was used (4,300 mg/lb ai) when assuming a solid (dust) formulation.   
 The inhalation doses presented in the table are the combined (liquid and dust) inhalation doses based on the assumed ratio.  For example, for the 99/1 liquid/dust ratio assumption, the inhalation dose = (liquid formulation inhalation dose * 0.99) + (dust formulation inhalation dose * 0.01).  The liquid and dust inhalation doses were calculated as follows:  Inhalation Unit Exposure (mg/lb ai) x Application Rate (lb ai/collar) x Amount Handled (collars/day) / BW (80 kg), where inhalation exposure is assumed negligible when assuming a liquid formulation, and the unit exposure for shaker cans was used (18 mg/lb ai) when assuming a solid (dust) formulation.   
 Dermal MOE = POD (0.5 mg/kg/day)/Dermal Dose (mg/kg/day). 
 Inhalation MOE = POD (0.5 mg/kg/day)/Inhalation Dose (mg/kg/day). 
 Combined MOE = POD (0.5 mg/kg/day)/(Dermal dose + Inhalation dose). 
    
	6.2 	Residential Post-Application Exposure/Risk Estimates 
    
   Residential post-application exposures to amitraz can occur after collars are placed on a dog via dermal and incidental oral exposure routes.  As was noted in the residential handler section, because there is uncertainty whether pet collars release the active ingredient as a liquid and/or solid (dust), HED assumes that the active ingredient present in pet collars could be transferred in either form (liquid or dust) from the collar to the pet's fur and result in the potential for postapplication exposures from contact with the treated pet.  As a result, residential post-application exposures were evaluated assuming both liquid and solid (dust) formulations are present concurrently at varying ratios (e.g., 99/1, 50/50, and 1/99 liquid/dust).   
    
   The lifestages selected for each post-application scenario are based on an analysis provided as an Appendix in the 2012 Residential SOPs.  While not the only lifestage potentially exposed for these post-application scenarios, the lifestage that is included in the quantitative assessment is health protective for the exposures and risk estimates for any other potentially exposed lifestage. 
    
   Residential Post-application Exposure Data and Assumptions 
   A series of assumptions and exposure factors served as the basis for completing the residential post-application risk assessment.  Each assumption and factor is detailed in the 2012 Residential SOPs[9]. 
    
   Application Rate: The maximum application rate as noted in Table 3.3 was used in the postapplication assessment.  
    
   Exposure Duration:  Residential post-application exposure to pet collars is assumed to be short-, intermediate- and long-term due to the preventative nature of these products, the potential for extended usage in more temperate parts of the county, and the longer active lifetime of pet collar products.   
    
   Fraction Transferred: A chemical specific study has been submitted, reviewed, and determined to be acceptable for risk assessment that provides information related to transferable residues during the use of pet collars.  A total of 15 dogs were used in the study (all test dogs and no control dogs).  Amitraz residues found on layered cotton gloves were measured after 20 petting simulations to the treated dogs.  Each simulation consisted of three strokes (60 strokes total) conducted using a mannequin hand fitted with three cotton gloves over top of a nitrile glove for the pre-application samples and five cotton gloves over top of a nitrile gloves for the postapplication samples. Samples were collected from each dog prior to treatment, at 4 hours after treatment and at 1, 2, 4, 7, 14, and 28 days after treatment.  Average glove residue for each sampling interval showed that maximum residues occurred four hours after application of the collars at 1,954 ug/gloves (0.14% of applied dose and 0.395 ug/cm2 surface area of dog).  For the purpose of this assessment, a fraction transferred value of 0.14% was used to determine transferable residues. 
   Liquid/Dust Assumptions:  This approach uses the same methodologies described in the 2012 Residential SOPs for assessment of post-application exposure assessment for pet collar usage.  However, whereas the 2012 Residential SOPs recommend that pet collars be assessed as a liquid formulation, the present approach assesses pet collar exposures as both a liquid and solid (dust) form.  Transfer coefficients specific to each formulation were used in the assessment.  The individual dust and liquid formulation post-application risks were estimated, and then another step was included in the assessment where the liquid and dust risks were averaged assuming 99/1, 50/50, and 1/99 ratios.     
    
   Residential Post-application Non-Cancer Exposure and Risk Equations 
   The algorithms used to estimate residential post-application exposure and dose can be found in the 2012 Residential SOPs. 
    
   Combining Exposure and Risk Estimates 
    
   Since dermal and incidental oral exposure routes share a common toxicological endpoint, risk estimates have been combined for those routes.   
    
   Summary of Residential Post-application Non-Cancer Exposure and Risk Estimates 
   For the residential post-application scenarios, the adult dermal and the children (1 to <2 years old) combined dermal + incidental oral MOEs were all less than the LOC of 1000.   Adult dermal MOEs ranged from 3.0 to 76, and children (1 to <2 years old) combined (dermal + incidental oral) MOEs ranged from 1.0 to 28 (with dermal exposures driving the risk estimate) depending on the assumed ratio of liquid to dust in the pet collar product, and are of concern.   
    
Table 6.2.1. Residential Post-Application Exposure Estimates from Amitraz Pet Collars. 

 Lifestage 
                             Route(s) of Exposure 
 Pet Size 
 Maximum 
 Application 
                             Rate (mg ai/pet)[1] 
                           Dermal Dose (mg/kg/day) 
                           Dermal MOE (LOC = 1000) 
Incidental Oral 
                                     Dose 
 (mg/kg/day) 
Incidental Oral 
                                     MOE 
(LOC = 1000) 
                                   Combined 
                                   MOEs[1]  
 (LOC = 1000) 
                               99% Liquid/1% Solid (Dust) 

                                    Adult 
                                    Dermal 
 medium 
                                     1665 
                                    0.0069 
                                      72 
              NA 



                                    large 
                                     2475 
                                    0.0066 
                                      76 


                                    Child 
Dermal and 
                                     HTM 
 medium 
                                     1665 
                                    0.018 
                                      28 
                                    0.0015 
                                     340 
                                      26 


                                    large 
                                     2475 
                                    0.017 
                                      30 
                                    0.0014 
                                     360 
                                      28 
                              50% Liquid/50% Solid (Dust) 

                                    Adult 
                                    Dermal 
 medium 
                                     1665 
                                    0.077 
                                     6.5 
              NA 



                                    large 
                                     2475 
                                    0.073 
                                     6.9 


                                    Child 
Dermal and 
                                     HTM 
 medium 
                                     1665 
                                    0.197 
                                     2.5 
                                    0.053 
                                     9.4 
                                     2.0 


                                    large 
                                     2475 
                                    0.186 
                                     2.7 
                                    0.051 
                                     9.9 
                                     2.1 
                               1% Liquid/99% Solid (Dust) 

                                    Adult 
                                    Dermal 
 medium 
                                     1665 
                                    0.147 
                                     3.4 
              NA 



                                    large 
                                     2475 
                                    0.139 
                                     3.6 


                                    Child 
Dermal and 
                                     HTM 
 medium 
                                     1665 
                                    0.376 
                                     1.3 
                                    0.105 
                                     4.7 
                                     1.0 


                                    large 
                                     2475 
                                    0.356 
                                     1.4 
                                    0.100 
                                     5.0 
                                     1.1 
 Based on registered label (Reg No. 2382-104) and percent ai (9%) in product. 
 The dermal doses presented in the table are the combined (liquid and dust) dermal doses based on the assumed ratio.  For example, for the 99/1 liquid/dust ratio assumption, the dermal dose = (liquid formulation dermal dose * 0.99) + (dust formulation dermal dose * 0.01).  The liquid and dust dermal doses were calculated according to the 2012 HED Residential SOPs.   
 The incidental oral doses presented in the table are the combined (liquid and dust) incidental oral doses based on the assumed ratio.  
         For example, for the 99/1 liquid/dust ratio assumption, the incidental oral dose = (liquid formulation incidental oral dose * 0.99) + (dust formulation incidental oral dose * 0.01).  The liquid and dust incidental oral doses were calculated according to the 2012 HED Residential SOPs.     
 Dermal MOE = POD (0.5 mg/kg/day)/Dermal Dose (mg/kg/day). 
 Incidental oral MOE = POD (0.5 mg/kg/day)/Incidental oral Dose (mg/kg/day). 
 Combined MOE = POD (mg/kg/day)/(Dermal dose + Incidental oral dose) 
	6.3 	Residential Risk Estimates for Use in Aggregate Assessment 
    
   All residential risks associated with the registered pet collar use of amitraz are of concern (i.e., all MOEs are < the LOC of 1000).  Therefore, quantitatively aggregating residential exposures with food exposures would also result in risks of concern.   
    
	7.0 	Non-Occupational Spray Drift Exposure and Risk Estimates 
    
   Amitraz end use products are formulated as pet collars and beehive strips, and are unlikely to result in spray drift. 
    
	8.0 	Non-Occupational Bystander Post-Application Inhalation Exposure and Risk 
   Estimates 
    
   As noted above, amitraz is only formulated into pet collars and beehive strips.  There are no agricultural uses of amitraz.  The beehive strips are not anticipated to result in post-application inhalation exposure; therefore, a quantitative assessment for that use was not conducted.   
    
	9.0 	Aggregate Exposure/Risk Characterization 
    
   In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and risks from three major sources: food, drinking water, and residential exposures.  In an aggregate assessment, exposures from relevant sources are added together and compared to quantitative estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated.  When aggregating exposures and risks from various sources, HED considers both the route and duration of exposure.  For amitraz, since the residential exposures result in risk estimates of concern, an aggregate assessment was not performed. Combining dietary exposures with residential exposures would result in even greater risk estimates of concern. 
    
	10.0 	Cumulative Exposure/Risk Characterization 
    
   Unlike other pesticides for which EPA has followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to amitraz and any other substances and amitraz does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has not assumed that amitraz has a common mechanism of toxicity with other substances. In 2016, EPA's Office of Pesticide Programs released a guidance document entitled, Pesticide Cumulative Risk Assessment: Framework for Screening Analysis [https://www.epa.gov/pesticide-scienceand-assessing-pesticide-risks/pesticide-cumulative-risk-assessment-framework].  This document provides guidance on how to screen groups of pesticides for cumulative evaluation using a twostep approach beginning with the evaluation of available toxicological information and if necessary, followed by a risk-based screening approach.  This framework supplements the existing guidance documents for establishing common mechanism groups (CMGs) and conducting cumulative risk assessments (CRA).  During Registration Review, the agency will utilize this framework to determine if the available toxicological data for amitraz suggests a candidate CMG may be established with other pesticides.  If a CMG is established, a screeninglevel toxicology and exposure analysis may be conducted to provide an initial screen for multiple pesticide exposure.    
    
	11.0 	Occupational Exposure and Risk Estimates  
   11.1 Occupational Handler Exposure/Risk Estimates 
    
   Based on the anticipated use patterns and current labeling, types of equipment and techniques 
    
   that can potentially be used, occupational handler exposure is expected from the registered pet collar use.  The quantitative exposure/risk assessment developed for occupational handlers is based on applying pet collars to dogs.   
    
   The beehive strip use pattern does not indicate a significant potential for applicator dermal or inhalation exposure.  The Apivar beehive strips come prepackaged and do not involve any mixing or loading of active ingredient.  Occupational exposure resulting from use of these strips is not likely to occur, as the only potential times for worker contact are during application, when the beehive keeper removes the strips directly from the packaging and places them in the hives, and during removal and disposal of the strips six weeks later.  Also, the registered label requires the use of gloves when handling the strips which will further reduce the potential for exposure.   
    
   Occupational Handler Exposure Data and Assumptions 
    
   A series of assumptions and exposure factors served as the basis for completing the occupational handler risk assessments.  Each assumption and factor is detailed below on an individual basis. 
    
   Application Rate:  The maximum application rate for the collars (0.0037 lb ai/collar for small to medium dogs and 0.0055 lb ai/collar for large dogs as noted in Table 3.3) was used in the assessment.  As was noted above, the application rate used represents the maximum amount of ai in the entire dog collar.  The amitraz pet collar label directs the user to cut off and dispose of any excess length once the product is fit and buckled into place.  As described in the 2012 Residential Treated Pet SOP, the removal of excess length is anticipated; however, because the exact length cannot be determined, the corresponding ai loss cannot be determined and exposure is assessed assuming the full collar length. 
    
   Unit Exposures:  It is the policy of HED to use the best available data to assess handler exposure.  Sources of generic handler data, used as surrogate data in the absence of chemical-specific data, include PHED 1.1, the AHETF database, the Outdoor Residential Exposure Task Force (ORETF) database, or other registrant-submitted occupational exposure studies.  Some of these data are proprietary (e.g., AHETF data), and subject to the data protection provisions of FIFRA.  The standard values recommended for use in predicting handler exposure that are used in this assessment, known as "unit exposures", are outlined in the "Occupational Pesticide Handler Unit Exposure Surrogate Reference Table", which, along with additional information on HED policy on use of surrogate data, including descriptions of the various sources, can be found at the Agency website.  In the updated approach presented here where pet collar applications are assessed assuming pet collars could be liquid and solid (dust) formulations concurrently with varying ratios of liquid/dust, the current UE for collars is used to assess the fraction that is a liquid formulation and the shaker can UE is used to assess the fraction that is a solid (dust) formulation. 
    
   Area Treated or Amount Handled:  For occupational application of pet collars, HED assumes veterinarians could potentially treat one animal per hour for an 8-hour workday, or 8 collars handled per day.  
    
    
   Exposure Duration: Occupational exposure to amitraz is anticipated to be short- and intermediate-term in duration.  Since the short- and intermediate-term PODs are the same, the risk estimates presented herein are representative of both durations.   
    
   Mitigation/Personal Protective Equipment:  Estimates of dermal and inhalation exposure were calculated for "baseline," defined as a single layer of clothing consisting of a long sleeved shirt, long pants, shoes plus socks, no protective gloves, and no respirator.  The amitraz product label for pet collars does not list any recommended PPE. 
    
   Combining Exposures/Risk Estimates: 
   Since dermal and incidental oral exposure routes share a common toxicological endpoint, risk estimates have been combined for those routes.  Dermal and inhalation risk estimates were combined using the following formula: 
    
    Total MOE = Point of Departure (mg/kg/day) / (Combined dermal + inhalation dose (mg/kg/day)) 
    
   Summary of Occupational Handler Non-Cancer Exposure and Risk Estimates 
   For the pet collar uses, the combined (dermal + inhalation) occupational handler MOEs were all less than the LOC of 1000.  The combined MOEs ranged from 0.68 to 96 (with the dermal route driving the risk estimates) for small and large collars, depending on the assumed ratio of liquid to dust in the pet collar product and the personal protective equipment (PPE) assumed to be worn (ranging from single layer and no gloves to double layer with gloves, assuming no respirator worn).  Based on the currently registered labels, gloves are not required to be worn.  Note that while risk estimates are presented for a double layer of clothing, this option may not be practical for the use scenario being assessed (i.e., veterinarians and/or technicians applying pet collars to dogs). 
    
Table 11.1.1.  Occupational Handler Exposure and Risk Estimates for Use of Pet Collars Containing 9% Amitraz. 
                              Exposure Scenario 
                                Type of collar 
                                 Application 
                            Rate (lb ai/collar)[1] 
                                    Amount 
                                   Handled 
                               (collar/day)[2]  
                     Combined (Dermal + Inhalation) MOEs 
                               (LOC = 1000)[3] 




                               Single layer/no 
                            Gloves + No Respirator 
                                Single layer/ 
                            Gloves + No Respirator 
                                Double layer/ 
                            Gloves + No Respirator 
                          99% Liquid/1% Solid (Dust) 
                            Applying a pet collar  
                         Up to 60 lb Collar (18.5 g) 
                                    0.0037 
                                      8 
                                      27 
                                      60 
                                      96 

                                 Above 60 lb 
                                    Collar 
                                   (27.5 g) 
                                    0.0055 
                                      8 
                                      18 
                                      40 
                                      64 
                         50% Liquid/50% Solid (Dust) 
Applying a pet collar  
                         Up to 60 lb Collar (18.5 g) 
                                    0.0037 
                                      8 
                                     2.0 
                                      35 
                                      48 

                                 Above 60 lb 
                                    Collar 
                                   (27.5 g) 
                                    0.0055 
                                      8 
                                     1.3 
                                      24 
                                      32 
                          1% Liquid/99% Solid (Dust) 
Applying a pet collar  
                         Up to 60 lb Collar (18.5 g) 
                                    0.0037 
                                      8 
                                     0.99 
                                      25 
                                      33 
Table 11.1.1.  Occupational Handler Exposure and Risk Estimates for Use of Pet Collars Containing 9% Amitraz. 
                              Exposure Scenario 
                                Type of collar 
                                 Application 
                            Rate (lb ai/collar)[1] 
                                    Amount 
                                   Handled 
                               (collar/day)[2]  
                     Combined (Dermal + Inhalation) MOEs 
                               (LOC = 1000)[3] 




                               Single layer/no 
                            Gloves + No Respirator 
                                Single layer/ 
                            Gloves + No Respirator 
                                Double layer/ 
                            Gloves + No Respirator 

                                 Above 60 lb 
                                    Collar 
                                   (27.5 g) 
                                    0.0055 
                                      8 
                                     0.68 
                                      17 
                                      22 
 Based on registered label (Reg No. 2382-104) and percent ai (9%) in product. 
 Based on an assumption that a veterinarian could treat one dog per hour for an 8hour workday. 
 Combined MOE = POD (0.5 mg/kg/day)/(Dermal dose + Inhalation dose). 
    
         The dermal dose is the combined (liquid and dust) dermal dose based on the assumed ratio.  For example, for the 99/1 liquid/dust ratio assumption, the dermal dose = (liquid formulation dermal dose * 0.99) + (dust formulation dermal dose * 0.01).  The liquid and dust dermal doses were calculated as follows:  Dermal Unit Exposure (mg/lb ai) x Application Rate (lb ai/collar) x Amount Handled (collars/day) x DAF (33%) / BW (80 kg), where the current unit exposure for collars was used when assuming a liquid formulation, and the unit exposure for a shaker can was used when assuming a solid (dust) formulation.  Unit exposures available: 
https://www.epa.gov/sites/production/files/2018-06/documents/opp-hed-pesticide-handler-surrogate-unit-exposure-table-june-
         2018.pdf 
    
         The inhalation dose is the combined (liquid and dust) inhalation dose based on the assumed ratio.  For example, for the 99/1 liquid/dust ratio assumption, the inhalation dose = (liquid formulation inhalation dose * 0.99) + (dust formulation inhalation dose * 0.01).  The liquid and dust inhalation doses were calculated as follows:  Inhalation Unit Exposure (mg/lb ai) x Application Rate (lb ai/collar) x Amount Handled (collars/day) / BW (80 kg), where inhalation exposure is assumed negligible when assuming a liquid formulation, and the unit exposure for a shaker can was used when assuming a solid (dust) formulation. Unit exposures available: https://www.epa.gov/sites/production/files/2018-06/documents/opp-hed-pesticide-handler-surrogate-unit-exposure-table-june2018.pdf  
   11.2 Occupational Post-application Exposure/Risk Estimates 
    
   HED uses the term post-application to describe exposures that occur when individuals are present in an environment that has been previously treated with a pesticide (also referred to as reentry exposure).  Such exposures may occur when workers enter previously treated areas to perform job functions, including activities related to crop production, such as scouting for pests or harvesting.  Post-application exposure levels vary over time and depend on such things as the type of activity, the nature of the crop or target that was treated, the type of pesticide application, and the chemical's degradation properties.  In addition, the timing of pesticide applications, relative to harvest activities, can greatly reduce the potential for post-application exposure. 
    
   Occupational post-application exposures are not anticipated for the registered uses (beehive strips and pet collars) of amitraz. 
    
	12.0 	Incident and Epidemiological Data Review 
    
   Amitraz incidents were previously reviewed in 2009 (M. Hawkins and S. Recore, D368729, 11/24/2009).  At that time, based on the low severity and frequency of cases reported to both IDS and NIOSH SENSOR-Pesticides, there was no risk of concern that warranted further analysis. 
    
   HED performed an updated Tier I review of human incidents for amitraz using the OPP Incident 
   Data System (IDS) and the Centers for Disease Control and Prevention/National Institute for 
   Occupational Safety and Health (CDC/NIOSH) Sentinel Event Notification System for Occupational Risk-Pesticides (SENSOR) databases (S. Recore and E. Evans, D448216, 8/2/2018).  
   For the Main IDS for the five years from January 1, 2013 to March 1, 2018, there were nine 
   cases reported that involved the active ingredient amitraz.  Of these nine case reports, four incidents involved the single active ingredient amitraz (only).  These incidents involved adults and were classified as moderate severity.  Three of the cases involved contact with a dog collar and the fourth involved ingestion of "dog flea medication."  Reported symptoms included headache, vomiting, malaise, ocular swelling, and respiratory distress.  The other five amitraz incidents reported involved multiple active ingredients. For Aggregate IDS for the same period,72 incidents reported involving amitraz.  Seventy incidents were classified as minor severity and two incidents had no or unknown severity.  
    
   A query of SENSOR-Pesticides from 1998-2014 identified 27 cases involving amitraz. Twenty-two cases were low in severity, four cases were moderate in severity and one case was high in severity.  The high severity case was a child who accidentally ingested a spoonful of flea dip mistaken by an adult for cough medicine (details in D448216).  Of the 27 amitraz cases, sixteen cases were occupational and eleven cases were non-occupational.  The occupational and non-occupational case exposure scenarios are delineated in D448216. The majority of all amitraz cases (n=19) involved the application of amitraz-containing products (including flea dips, collars, and spot-on products) onto pets. Eighty-one percent of amitraz cases reported in SENSOR were low in severity.  Ocular symptoms, primarily eye pain/irritation/inflammation, were most frequently reported among cases.  Dermal symptoms including skin rashes, skin redness and pain were also commonly reported.  Gastrointestinal symptoms including nausea and vomiting were reported in seven cases.   
    
   The Agricultural Health Study (AHS) is a federally-funded study that evaluates associations between pesticide exposures and cancer and other health outcomes and represents a collaborative effort between the US National Cancer Institute (NCI), National Institute of Environmental Health Sciences (NIEHS), CDC's NIOSH, and the US EPA.  Amitraz is included in the AHS, however; it is not currently included in any AHS publications and, therefore, this study does not provide information for this report. 
    
   Based on the continued low frequency of amitraz incidents reported to both IDS and SENSORPesticides, there does not appear to be a concern at this time.  The Agency will continue to monitor the incident data and if a concern is triggered, additional analysis will be conducted. 
    
	13.0 	References 
    
   D. Drew, et. al., 1/8/2013, Amitraz. Aggregate Human Health Risk Assessment for Section 3 New Use in Beehives. D407721. 
    
   D. Drew, 7/31/2018, Amitraz Acute and Chronic Dietary (Food Only) Exposure and Risk Assessment for the Registration Review Risk Assessment. D448220. 
    
   K. Lowe, 9/27/2018, Amitraz.  Occupational and Residential Exposure Assessment for Registration Review. D448221. 
    
   S. Recore, et.al., 8/2/2018, Amitraz: Tier I Update Review of Human Incidents and Epidemiology for Draft Risk Assessment. D448216. 
    
    
    
    
    
   Appendix A.  Toxicology Profile and Executive Summaries 
    
	A.1 	Toxicology Data Requirements 
    
  The requirements (40 CFR 158.500) for amitraz are in Table A1.1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used. 
Table A1.1     Toxicology Requirements for Amitraz   

 
Guideline Number and Toxicity Study 
 
870.1100 Acute Oral Toxicity ......................................................  870.1200 Acute Dermal Toxicity .................................................  870.1300 Acute Inhalation Toxicity.............................................  870.2400 Primary Eye Irritation ..................................................  870.2500 Primary Dermal Irritation .............................................  870.2600 Dermal Sensitization ....................................................  
 
                                   Required 
 
                                  Satisfied 

 
                             yes yes yes yes yes yes 
 
                             yes yes yes yes yes yes 
 
870.3100 Oral Sub-chronic (Rodent) ...........................................  870.3150 Oral Sub-chronic (Non-Rodent) ...................................  870.3200 21-Day Dermal .............................................................  
870.3250 90-Day Dermal .............................................................  870.3465 90/28-Day Inhalation ...................................................  
                                        
                                   yes yes yes 
                                      CR yes 
                                        
                                   yes [a] 
        yes b, d  yes c 
                                    -- yes d  
 
870.3700 Developmental Toxicity (Rodent) ................................  870.3700 Developmental Toxicity (Non-rodent) .........................  870.3800 Reproduction ................................................................  
 
                                   yes yes yes 
 
                                     yes 
                                 yes [d]   yes [a] 
 
870.4100 Chronic Toxicity (Rodent) ...........................................  870.4100 Chronic Toxicity (Non-rodent) ....................................  870.4200 Oncogenicity (Rat) .......................................................  870.4200 Oncogenicity (Mouse) ..................................................  
870.4300 Chronic/Oncogenicity ..................................................  
 
                               yes yes yes yes yes 
 
                               yes yes yes yes yes 
 
870.5100 Mutagenicity: Gene Mutation - bacterial .....................  870.5300 Mutagenicity: Gene Mutation - mammalian ................  
870.5375 Mutagenicity: Structural Chromosomal Aberrations ...  870.5550 Mutagenicity: UDS Assay ............................................  
 
                                 yes yes yes yes 
 
                                 yes yes yes yes 
 
870.6100 Acute Delayed Neurotoxicity (Hen) .............................  870.6100 90-Day Neurotoxicity (Hen) ........................................  870.6200 Acute Neurotoxicity Screening Battery (Rat) ..............  870.6200 90-Day Neurotoxicity. Screening Battery (Rat) ...........  870.6300 Developmental Neurotoxicity ......................................  
 
                                      CR 
                                    CR yes yes 
                                      CR 
 
                                      - 
   -     yes [a], d     yes [a], [d] yes a  
 
870.7485 General Metabolism .....................................................  
870.7600 Dermal Penetration .......................................................  
870.7800     Immunotoxicity....................................................................... 
 
                                     yes 
                                      CR yes 
 
                                   yes yes yes  
  a= Satisfied by the Extended One-Generation Reproduction Toxicity (EOGRT) study 
  b= Satisfied by the availability of a chronic toxicity study in this species c = Satisfied by the availability of a dermal study in human subjects d= HASPOC waiver recommendation (J. Van Alstine, TXR# 0056451, 2/5/2013) e = Available unacceptable studies in non-rodents does not indicate this species to be more sensitive than rats 
   	 
	A.2 	Toxicity Profiles 
    
   Table A.2.1 Acute Toxicity Data on amitraz technical 
 
Guideline No./ Study Type 
 
MRID No. 
 
                                   Results 
 
                                   Toxicity 
                                   Category 
 
870.1100 Acute oral toxicity 
 
                                   00041539 
 
LD50: 531 mg/kg (M) 515 mg/kg (F) 
 
                                     III 
 
870.1200 Acute dermal toxicity 
 
                                   00040862 
 
LD50: > 200 mg/kg 
 
                                      II 
 
870.1300 Acute inhalation toxicity 
 
                                   00029963 
 
LC50: 2.4 mg/L 
 
                                     IV  
 
870.2400 Acute eye irritation 
 
                                   00040861 
 
Non-irritating 
 
                                      IV 
 
870.2500 Acute dermal irritation 
 
                                   00040862 
 
Non-irritating 
 
                                      IV 
 
870.2600 Skin sensitization 
 
                                   00029965 
 
Not a sensitizer under conditions of study 
 
                                     N/A 
    
    
 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 
 
                             Sub-Chronic Toxicity 
870.3100 
MRID 
00051784 
 
90-Day Oral Toxicity - Rat  
 
0, 3, 12, 50 and 200 mg/kg/day 
 
Unacceptable/Guideline: No individual animal data for clinical signs or gross necropsy provided. No information on test material provided.  
NOAEL = 3 mg/kg/day 
LOAEL = 12 mg/kg/day based on irritability, excitability and reduced overall body weight gain. 
870.3150 
MRID 
00040345 
 
90-Day Oral Toxicity - Dog  
 
0, 0.25, 1.0, or 4.0 mg/kg/day 
 
Unacceptable/Guideline: only two animals per sex tested. 
 
Additional study waived by 
HASPOC (J. Van Alstine, TXR# 
0056451, 2/5/2013) 
NOAEL = 0.25 mg/kg/day 
LOAEL = 1.0 mg/kg/day based on CNS depression, decrease in pulse rate, increase in glucose in urine, hypothermia, neutrophilia of bone marrow, increased liver weights and increased extent of liver lesions. 
870.3200 
MRID 
00029972 
 
 
21-Day Dermal Toxicity  -  
Rabbits 
 
0, 50 or 200 mg/kg/day 
 
Unacceptable/Guideline: Too few animals per dose group, 
 
 
NOAEL = Cannot be determined 
LOAEL = 50 mg/kg/day based on clinical signs (sedation) and a decrease in food consumption in males.    

 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 


concurrent infections, lack of information on the test substance and limited histopathology. 

870.3465 
MRID 
00029964 
21-Day inhalation toxicity  -  Rats 
 
0, 0.01, 0.1 or 1.0 mg dust/L 
 
Acceptable/Non-guideline: This study may be cited qualitatively for risk assessment purposes but 
not quantitatively and therefore; NOAECs and LOAECs were not 
established (TXR# 0057752)  
 
Additional study waived by 
HASPOC (J. Van Alstine, TXR# 
0056451, 2/5/2013) 
 No effects observed at 0.01 mg/L/day 
 At 0.1 mg/L/day: mild dyspnea with occasional sneezing; intermittent blinking and licking of lips; hyposensitivity to noise; hypersensitivity to touch and aggressive behavior. Decreases in body weight gain were observed in both sexes (54 and 49% of control for males and females, respectively).   
 At 1.0 mg/L/day: labored movements, increased nasal secretion and polyuria with brown discoloration of the urine. In several rats, slight coma and body tremors. Decreases in body weight and body weight gain in both sexes. A significant reduction of packed cell volume (hematocrit), hemoglobin and erythrocyte count and an increase in the number of neutrophils in both sexes. A low mean corpuscular hemoglobin content and decrease in the number of lymphocytes of male rats. Significant reduction in total protein levels in both sexes. In males, a low albumin concentration and a decreased albumin/globulin ratio were observed. In females, a low urea level and a fall in the alpha and beta fractions of globulins were noted. 
 
 
                            Developmental Toxicity 
 
870.3700 
MRID 
00029959 
 
Developmental Toxicity - Rat 
(gavage) 
 
0, 1, 3, or 12 mg/kg/day 
 
Unacceptable/Guideline: Very limited data, lacking purity of test substance, dosage period was only from gestation days 8-20.  
 
Maternal NOAEL = 3 mg/kg/day 
Maternal LOAEL = 12 mg/kg/day based on decreases in body weight gain. 
 
Developmental NOAEL = 12 mg/kg/day 
Developmental LOAEL = > 12 mg/kg/day [HDT]. 
 
 
870.3700 
MRID  
44265902 
 
Developmental Toxicity- Rat 
 
0, 7.5, 15, or 30 mg/kg/day 
 
Acceptable/Guideline  
 
Maternal NOAEL = 7.5 mg/kg/day 
Maternal LOAEL = 15 mg/kg/day based on decreases in body weight and body weight gain. 
 
Developmental NOAEL = 30 mg/kg/day 
Developmental LOAEL = > 30 mg/kg/day [HDT]. 
 
870.3700 
MRID 
00029961 
 
Developmental Toxicity- Rabbit 
(gavage) 
 
0, 1, 5, or 25 mg/kg/day 
 
Maternal NOAEL = 5 mg/kg/day 
Maternal LOAEL = 25 mg/kg/day based on decrease in body weight gain and abortions 
 

 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 


 
Unacceptable/Guideline: Too few litters, unclear method of dosing, purity of test substance unknown.  
 
Additional study waived by 
HASPOC (J. Van Alstine, TXR# 
0056451, 2/5/2013) 
Developmental NOAEL = 5 mg/kg/day 
Developmental LOAEL = 25 mg/kg/day based on decreased litter size, decreased implantations, increased post-implantation loss, abortions and decreased mean fetal body weight. 
 
870.3700 
MRID 
44265901 
 
Developmental Toxicity - Rabbit  
 
0, 3, 6, or 12 mg/kg/day 
 
Unacceptable/Guideline: too few litters in two treatment groups and pre-existing maternal illness which may have confounded the results. 
Additional study waived by 
HASPOC (J. Van Alstine, TXR# 
0056451, 2/5/2013) 
 
Maternal NOAEL = not established 
Maternal LOAEL = 3.0 mg/kg/day based on clinical signs. 
 
Developmental NOAEL = 12 mg/kg/day 
Developmental LOAEL = > 12 mg/kg/day (HDT). 
 
                            Reproduction Toxicity 
 
870.3800 
MRID 
00029960 
 
One-generation Reproductive 
Toxicity - Rat  
 
0, 1, 3 or 12 mg/bw/day 
 
Acceptable/Non-guideline: only one-generation studied, does not provide purity of test compound. 
 
Parental/Systemic NOAEL = 3 mg/kg/day 
Parental/Systemic LOAEL = 12 mg/kg/day based on decreased body weight gain. 
 
Reproductive NOAEL = 12 mg/kg/day 
Reproductive LOAEL > 12 mg/kg/day (HDT). 
 
Offspring NOAEL = 3 mg/kg/day 
Offspring LOAEL = 12 mg/kg/day based on lower mean litter size at birth and on lactation day 4.  
870.3800 
MRID 
00029962 
Three-generation Reproductive 
Toxicity  -  Rat 
 
0, 1.29, 4.36, or 16.41 mg/kg/day males 
0, 1.58, 5.09 or 20.05 mg/kg/day females 
 
Unacceptable/Guideline: Limited data provided, mating was not 1 male to 1 female, no data on reproductive organs provided, litter data only provided for a few time points, and histopathology data not provided. 
Parental/Systemic NOAEL = 4.36/5.09 (M/F) mg/kg/day 
Parental/Systemic LOAEL = 16.41/20.05 (M/F) mg/kg/day based on decreased body weight gain during the F0 premating period. 
 
Reproductive NOAEL =16.41/20.05 (M/F) mg/kg/day Reproductive LOAEL > 16.41/20.05 (M/F) mg/kg/day (HDT). 
 
Offspring NOAEL = 1.29/1.58 (M/F) mg/kg/day Offspring LOAEL = 4.36/5.09 (M/F) mg/kg/day based on decreased survival and mean litter size during lactation.   

 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 
OECD 
443 
MRID 
49994401 
Extended One-Generation 
Reproduction Toxicity Study 
(EOGRTS)  -  Rat  
 
0, 0.5, 1.5, or 7.5 mg/kg/day 
 
Acceptable/Non-Guideline 
(OECD) 
Parental: 
LOAEL = 7.5 mg/kg/day, based on clinical signs of toxicity (salivation, urine stain fur, vocalization; males only), decreased body weights (males only), and decreased motor activity (both sexes) 
 
NOAEL=1.5 mg/kg 
 
Offspring: 
LOAEL = 7.5 mg/kg/day, based on increased PND 14 pup deaths and decreased body weights in the pups 
(males and females) 
 
NOAEL = 1.5 mg/kg/day 
 
F1 adult:  
LOAEL = 1.5 mg/kg/day, based on decreased T4 levels (↓25%; females only) 
 
NOAEL = 0.5 mg/kg 
 
Reproductive: 
The LOAEL = 7.5 mg/kg based on increased incidence of persistent diestrus and increase number of primordial follicles 
 
NOAEL = 1.5 mg/kg 
 
Developmental neurotoxicity: 
LOAEL = 7.5 mg/kg/day based on brain morphometrics (↓ hypothalamus area, ↓ corpus callosum thickness, ↓ in distance across hippocampal pyramidal layers; males only) 
 
NOAEL =not established since brain morphometrics were not reported for the mid or low dose groups. 
 
                               Chronic Toxicity 
870.4100 
MRID 
00044586 
Chronic Toxicity- Dog  
(2 years) 
 
0, 0.1, 0.25, or 1.0 mg/kg/day 
 
Acceptable/Guideline 
 
NOAEL = 0.25 mg/kg/day 
LOAEL = 1.0 mg/kg/day based on CNS depression during first two days of dosing. 
 
870.4300 
MRID 
00044585 
 
Combined Chronic 
Toxicity/Carcinogenicity - Rat (2 years) 
 
0, 0.77, 2.5, or 10.8 mg/kg/day males 
0, 0.97, 3.13 or 12.59 mg/kg/day females 
 
NOAEL = 2.5/0.97 (M/F) mg/kg/day 
LOAEL = 10.18/3.13 (M/F) mg/kg/day based on clinical signs (M and F) and decreased body weight gain (M). 
 
No evidence of carcinogenicity. 

 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 


 
 
Acceptable/Guideline  

 
870.4300 
MRID 
00139552 
 
Carcinogenicity - Mice 
(2 years) 
 
0, 2.31, 9.61, or 44.65 mg/kg/day males 
0, 2.63, 10.77 or 50.13 mg/kg/day females 
 
 
Acceptable/Guideline  
 
 
NOAEL = < 2.31/2.63 (M/F) mg/kg/day 
LOAEL = 2.31/2.63 (M/F) mg/kg/day based on a dose-related increase in the incidence of hyperplastic nodules, basophilic and telangiectatic foci in the liver of females, accompanied by an increased incidence of stomach hyperkeratosis and spleen hematopoiesis in males. 
Evidence of carcinogenicity: hepatocellular adenomas, carcinomas and combined; and lung adenomas, probably at dose levels above MTD. 
                                Neurotoxicity 
 
870.6200a 
NA 
 
Acute Neurotoxicity - Rat 
 
Waived by HASPOC (J. Van Alstine, TXR# 0056451, 2/5/2013) 
 
870.6200b 
NA 
 
Sub-chronic Neurotoxicity - Rat 
 
Waived by HASPOC (J. Van Alstine, TXR# 0056451, 
2/5/2013) 
 
870.6300 
NA 
Developmental  
Neurotoxicity - Rat 
 A component of the EOGRT study 
                                 Genotoxicity 
 
870.5100 
MRID 
00161009 
 
Reverse Gene Mutation in 
Salmonella typhimurium 
 
 
Negative up to 1 mg/plate, with and without metabolic activation.   
 
 
870.5300 
MRID 
00161008 
Forward Gene Mutation in mouse lymphoma cells 
 
 
 
Negative at 0.06-20 ug/ml with and without metabolic activation.  HDT is highest non-cytotoxic dose. 
 
870.5375 
MRID 
41795101 
In Vitro Cytogenetics (Human 
Lymphocytes) 
 
Negative up to cytotoxic and/or insoluble concentrations. 
870.5550 
MRID 
00161011 
UDS Assay 
(Human Embryonic Lung 
Fibroblast) 
 
Negative up to cytotoxic concentrations, with and without metabolic activation. 
NonGuideline In Vitro Study 
MRID 
00161010 
Cell Transformation 
 
Negative up to cytotoxic concentrations, with and without metabolic activation. 
                          Metabolism (Non-guideline) 
 
N/A 
MRID 
0016096 and 
MRID 
46249601 
 
Metabolism  -  Rat 
 
1, 10, or 100 mg/kg 
 
Acceptable/Non-guideline 
 
No sex differences in proportion of various metabolites recovered in 24-hour urine samples.  
Metabolic process saturated at the 100 mg/kg level.  
No unchanged parent material found in the urine. Major metabolites: N-(2,4-dimethylphenyl)-N-methyl formamidine, 4-formamido-3-methyl benzoic acid, 4-

 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 



acetamido-3-methyl benzoic acid and a polar fraction.  The polar fraction was labile to acid hydrolysis, yielding conjugates of 4-amino-3-methylbenzoic acid, N-(2,4-dimethylphenyl)-N-methyl formamidine, 4formamido-3-methyl benzoic acid and 4-acetamido-3methyl benzoic acid.   
 
Peak levels of amitraz reached in urine within 8 hours: 78% of the dose in the urine and 9% in the feces by 98 hours.  Peak levels of metabolite BTS 27271 (chemical structure not identified in study) reached in urine within 24 hours: 89% of the dose in the urine and 4% in the feces by 96 hours.  Highest residues of amitraz and BTS 27271 reported in the liver.  Blood residue levels were 17.9 ppb for amitraz and not detectable for BTS 27271.  Kidney residue levels were comparable with 18.0 and 24 ppb for amitraz and BTS 27271, respectively.  Degradation products of amitraz and its metabolite, BTS 27271 were similar 
N/A 
MRID 
00160964 
MRID 
46249601 
Metabolism  -  Mouse 
 
1, 10, or 100 mg/kg 
 
Acceptable/Non-guideline 
Urine accounted for 73% (male) and 66% (female) of the radiolabel excreted with 56-58% excreted in the first 24 hours. Determination of radiolabel in the feces, expired air and tissues was not done. Polar metabolites, likely representing conjugates (type not reported), were the predominant metabolites identified, contributing 59-64% of the total, and BTS 39 098/BTS 31 158 contributing 15-19%. When the dose was increased to 100 mg/kg, the percentage of polar metabolites decreased in mice and the metabolite BTS 27 271 increased.  This suggests metabolic saturation and shifting in the metabolic pathway.   
N/A 
MRID 
00160964 
MRID 
46249601 
Metabolism  -  Baboon  
 
10 mg/kg  
 
Acceptable/Non-guideline 
Urine accounted for 65% (male) and 83% (female) of the radiolabel excreted with 55-75% excreted in the first 24 hours. Determination of radiolabel in the feces, expired air and tissues was not done. Polar metabolites were the predominant metabolites identified, contributing 52-55% of the total, and BTS 39 098/BTS 31 158 contributing 19-26%. 
                                Dermal Studies 
 
870.7600 
MRID 
42133501 
 
Dermal Penetration  -  Rats 
 
1.0 mg/kg 
 
Unacceptable/Guideline  
 
The mean percent of dose absorbed: treatment site (2.98% at 24 hours, 1.41% at 120 hours); total absorbed (3.69% at 24 hours, 6.56% at 120 hours); total (6.67% at 24 hours, 7.79% at 120 hours).  
870.7600 
 
OECD 
428 
MRID 
49724601 
 
MRID 
49724602 
Dermal Triple Pack: In vitro dermal penetration in rats and humans (Acceptable/Nonguideline) & in vivo dermal 
penetration in rats 
(Acceptable/Guideline) 
 
The most conservative estimate of dermal absorption in the rat in vivo study was 38% for the 100 ug/cm[2] group evaluated 24 hours after dosing. This value represents the combined absorbed and potentially absorbable dose and was similar to other measurements in the different dose groups and sacrifice times. The maximal dermal absorption 
 
Table A.2.2    Toxicity Profiles for Amitraz 
 
                              Guideline  Number 
 
                                     MRID 
                                     No. 
 
                                  Study Type 
 
                                   Results 


0, 20, 60, or 100 ug a.i./cm[2] 
through rat skin in the in vitro study was only 7 +- 3%. Consequently, the in vitro study was not considered predictive of in vivo results; therefore, the dermal triple pack methodology could not be applied, and the Agency will use the in vivo dermal absorption without refinement for risk assessment.  
 
                             Immunotoxicity Study 
                                   870.7800 
MRID 
49611501 
Immunotoxicity  -  Rats 
 
0, 0.5, 2, or 8 mg/kg/day 
 
Acceptable/Non-guideline  
  
Systemic: 
NOAEL = 8 mg/kg/day (HDT) 
LOAEL = not established  
 
Immunotoxicity: 
NOAEL = 8 mg/kg/day (HDT) 
LOAEL = not established 
                        Non-Guideline Studies (Human) 
Non-
Guideline 
Human 
Study 
 
MRID 
43283101 
 
Acute Oral Toxicity 
 
0, 0.0625, or 0.125 mg/kg 
 
NOAEL = 0.125 mg/kg/day (HDT) 
LOAEL = not established  
 
Non-
Guideline 
Human 
Study 
 
MRID 
00160964 
46249601 
Metabolism Study 
 
0.25 mg/kg 
NOAEL = not established  
LOAEL = 0.25 mg/kg based on dry mouth, drowsiness, disorientation, decreased temperature, bradycardia, and slightly pale appearance. 
 
Humans appear to be most sensitive species. Above effects not seen in mouse, rat, or baboon, however, effects would be difficult to evaluate in these species.  
Non-
Guideline 
Human 
Study 
 
MRID  
44639401 
Acute Dermal Toxicity 
 
0, 8, 16 or 24 mg/kg 
 
NOAEL = 24 mg/kg (no LOAEL) 
 
    
   Executive Summaries for Endpoint Selection Studies  
    
              CITATION: Campbell, J.K. and D. Needham (l984). A comparison of the metabolism of [14]Camitraz in rat, mouse, baboon and human. FBC Limited, Chesterford Park Research Station, Saffron Walden, Essex CB10 1XL, January 17, 1984. MRID 00160964.  Unpublished. TXR# 0052737.  
    
              Campbell, J.K. and D. Needham (l984). Urinary excretion of [14]C-amitraz by two male humans following a single oral dose of 0.25 mg/kg body weight. FBC Limited, Chesterford Park Research Station, Saffron Walden, Essex CB10 1XL, February 4, 1984.  MRID 46249601. Unpublished. TXR# 0052737. 
    
   EXECUTIVE SUMMARY: In a metabolism study (MRIDs 00160964 and 46249601) radiolabeled Amitraz (1,5-di([14]C-2,4-dimethylphenyl)-3-methyl-1,3,5-triazapenta-1,4-diene) purity >95%, Batch #s REM-XLVI-85 and VA) was orally administered to rats, mice, baboons and humans.  Rats and mice were given 1, 10, or 100 mg/kg [[14]C]-Amitraz at 1, 10 or 100 mg/kg body weight in corn oil by gavage.  In addition, another group of mice were pretreated with 100 ppm and 400 ppm Amitraz in the diet for six weeks before being given a single gavage dose of 10 mg/kg of [[14]C]-Amitraz.  Baboons were given 10 mg/kg [[14]C]-Amitraz by gavage while human volunteers received a single 0.25 mg/kg dose of [[14]C]-Amitraz by capsule.   
    
   Humans appear to be the most sensitive species to the neurological effects of Amitraz as shown by dry mouth, drowsiness, disorientation, decreased temperature, bradycardia, and slightly pale appearance after treatment with 0.25 mg/kg.  These effects were not found in rats, mice, or baboons, but, subjective effects would be difficult to evaluate in animal species.  Following treatment, the majority of the radiolabel was excreted in the urine, with 54.9-73.7%, excreted within 24 hours of treatment.  In further studies on human urine samples, ~43.5% of the dose was excreted within 12 hours, with 62.9% excreted within 24 hours.  No major differences in the amount of radiolabel excreted between sexes or species were noted.  The overall metabolic profile was similar across sex and species.  Polar metabolites, likely representing conjugates (type not reported), were the predominant metabolites identified, contributing 52-64% of the total with BTS 39 098/BTS 31 158 contributing 15-27%.  
    
   The average proportion of metabolites identified in the urine following a single 1, 10, or 100 mg/kg oral dose of radiolabeled Amitraz was similar in rats and mice at doses of 1 mg/kg and 10 mg/kg.  When the dose was increased to 100 mg/kg, the percentage of polar metabolites decreased for both rats and mice and the metabolite BTS 27 271 increased.  This suggests metabolic saturation and shifting in the metabolic pathway.  In rats and mice pretreated with 100 ppm Amitraz in the diet for three weeks followed by three weeks of 400 ppm Amitraz in the diet, no effect on the relative concentrations of the metabolites were found.   
    
   These urine metabolism studies in the rat, mouse, baboon and human and the human metabolism study are classified Acceptable/Nonguideline and satisfy the intent of the studies.  
    
    
    
    
    
    
    
              CITATION: Cass, L (1992). T-344, Amitraz: Report of a double-blind tolerance study of amitraz in six adult healthy volunteers. Simbec Research Ltd., Merthyr Tydfil, Mid Glamorgan CF48 4DR, South Wales, United Kingdom. Laboratory Project RD 197/20170. June 8, 1992. MRID 43283101. Unpublished. TXR# 0052737. 
    
   EXECUTIVE SUMMARY: In a double-blind sequential dosing oral human tolerance study of 
   Amitraz, (Lot No. not reported, purity 98.2%), 7 adult male volunteers were given encapsulated 0, 0.0625, or 0.125 mg/kg in lactose over three 14-day periods. One volunteer was withdrawn from the study following development of a rash unrelated to treatment. The study subjects were 28.17 +- 3.54 years in age and weighed 67.15 +- 3.15 kg. The study was approved by the Simbec 
   Independent Ethics Committee and done according to the Helsinki and Hong Kong revisions (l989). Each dose was administered 30 minutes after breakfast with 150 ml water. Vital signs including: pulse, respiration rate, blood, pressure and temperature were taken at  - 1, -0.5, 1, 3, 6, 12, 24, and 36 hours after dosing. ECGs were performed at  - 1, 1, 3, 6, 12, 24, and 36 hours after dosing and pupil responsiveness and psychomotor performance were evaluated pre-dose and at 2.5 and 8 hours after dosing. Urine was collected at 0-36 hours and 36-60 hours after treatment and blood for clinical chemistry and hematology was collected prior to the start of the study and 36 hours after each phase of the study.  
    
   Two subjects reported tiredness, pre-dose (0.125 mg/kg) for 9 and 8h respectively, but this was determined to be unrelated to treatment. Vital signs and ECG parameters were within normal limits. No clinically significant changes were seen in pupil response. Psychomotor performance results showed no significant differences between pre- and post-treatment. One subject had a slightly elevated total bilirubin at the post-study screen but it returned to normal limits within three weeks completion of the study. No treatment-related effects were found on hematology or urinalysis parameters.  
    
   This oral toxicity study in humans is Acceptable/Nonguideline and satisfies the intent of the study.  

CITATION: 	Hoberman, A.M. (2016). Oral (Gavage) Extended One-Generation Reproduction 
              Toxicity Study of Amitraz in Rats. Charles River Laboratories, Inc., Horsham, PA.  Laboratory Project ID.:  20001081, August 12, 2016. MRID 49994401.  Unpublished. TXR# 0057767.  
 
EXECUTIVE SUMMARY:  In an extended one-generation reproduction toxicity study (MRID 49994401), Amitraz (98.8% a.i.; Batch # X080769/R5) in corn oil was administered daily via oral gavage in a dose volume of 5 mL/kg to 25 Sprague Dawley [Crl:CD(SD)] rats/sex/dose at dose levels of 0, 0.5, 1.5, or 7.5 mg/kg/day during premating, mating, littering, and post-partum.  The P generation males were dosed for 28 days prior to mating; P generation females were dosed for 15 days prior to mating. Following the birth of the F1 litters, the pups were randomly culled on postnatal day (PND) 4 to 12 pups/litter where possible (equal numbers of males and females if possible). The remaining F1 pups were administered the test substance beginning on PND 7 at the same dose level as their dam. On PND 21, the F1 pups were weaned and randomly assigned to one of four cohorts: Cohorts 1A and 1B to examine reproductive toxicity (Cohort 1B was not evaluated); Cohort 2A for evaluation of auditory startle (PND 24+-1), a functional observational battery (FOB; lactation day [LD] 17+-3 for females or PND 66+-3 for males), motor activity (FOB; LD 17+-3 for females or PND 66+-3 for males), and neurohistopathology (PND 90-92); and Cohort 2B for evaluation of neurohistopathology (PND 21). 
 
Parental animals:  In females, there were no effects of treatment observed on body weights, body weight gains, or food consumption during pre-mating, gestation, or lactation. In both sexes, no adverse effects were observed for mortality, hematology, clinical chemistry, urinalysis, T4 concentrations, organ weights, or macroscopic or microscopic examinations. 
 
Treatment-related effects were observed in the 7.5 mg/kg/day P males. Slight to moderate excess salivation, urine-stained abdominal fur, and vocalization to touch were observed (p<=0.01) at increased frequencies and in increased numbers of rats. Premating body weights of males were decreased (p<=0.01) by 6-8% during Days 15-28, resulting in a 46% decrease (p<=0.01) in premating (Days 1-28) body weight gains. Food consumption was decreased (p<=0.01) for every weekly interval during premating, resulting in an 11% decrease in premating food consumption. Following cohabitation, body weights were decreased (p<=0.01) by 9-14% during Days 36-85, resulting in a 43% decrease (p<=0.01) in overall (Days 1-85) body weight gains; overall food consumption was decreased (p<=0.01) by 12%. During the FOB observations, the number of males that appeared unkempt and had fecal and/or urine staining was increased (p<0.05). Males and females of the high dose group experienced decreased motor activity (ambulation and fine movements), particularly during the first several intervals, that were found to be statistically significant at individual intervals and over the entire treatment*time interval (where tested). Given the known neurotoxic effects of amitraz, the statistical significance, and the magnitude of the changes, the decrease in motor activity was considered adverse in both males and females.   
 
The LOAEL for parental toxicity is 7.5 mg/kg/day, based on clinical signs of toxicity (salivation, urine stain fur, vocalization; males only), decreased body weights (males only), and decreased motor activity (both sexes).  The NOAEL is 1.5 mg/kg/day. 
 
Offspring:  There were no effects of treatment on the number of pups delivered live or stillborn, birth or live birth indices, developmental landmarks, hematology, clinical chemistry, organ weights, or macroscopic examinations. At 7.5 mg/kg/day, an increase (p<=0.01) in pup deaths between PND 1-4 (3.9% treated vs. 0 control) resulted in a decreased (p<=0.01) viability index (95.1% treated vs. 99.7% control). Also at this dose, PND 21 body weights were decreased (p<=0.01) by 5% and 7% and PND 7-21 body weight gains were decreased (p<=0.01) by 8% and 10% in males and females, respectively. 
 
T4 levels in PND 5 males could not be evaluated at the high dose because only one of two available sample was above the LLOQ. Overall there appears to be no effect of treatment on T4 levels on PND 5 males at the low and mid doses. Thyroid hormone levels for PND 5 females are unable to be interpreted because of the low number of pups in the concurrent control group (n = 3) very near the LLOQ. There was no effect of treatment on T4 levels on PND 22 males or females. TSH levels could not be evaluated because most samples were below the assay's LLOQ.   
 
The LOAEL for offspring toxicity is 7.5 mg/kg/day, based on increased PND 1-4 pup deaths and decreased body weights in the pups (males and females).  The NOAEL is 1.5 mg/kg/day. 
 
F1 adults  -  Cohorts 1A and 1B:  There were no treatment-related effects on mortality, clinical signs, sexual maturation, hematology, clinical chemistry, urinalysis, organ weights, or macroscopic or microscopic findings. 
 
At 1.5 and 7.5 mg/kg/day there was a 24-25% decrease in female serum T4 levels. The changes were considered adverse due to the statistical significance and concerns for the number of animals near the LLOQ. At 7.5 mg/kg/day, body weights were decreased (p<=0.05) in the males by 4-13% during PND 42-98; body weights were decreased (p<=0.05) in the females by 4-6% during PND 28-77 and PND 91.  Adult body weight gains (PND 21-98) were decreased by 13% in the 
7.5 mg/kg/day males; adult body weight gains were unaffected by treatment in the 7.5 mg/kg/day females.  Food consumption was decreased (p<=0.01) by 5-14% for every weekly interval during PND 42-98 in the 7.5 mg/kg/day males. Males and females of the high dose group experienced decreased motor activity (ambulation and fine movements) that were found to be statistically significant at individual intervals and over the entire treatment*time interval. Given the known neurotoxic effects of amitraz, the statistical significance, and the magnitude of the changes, the decrease in motor activity was considered adverse in both males and females.   
 
The LOAEL for F1 adult toxicity is 1.5 mg/kg/day, based on decreased T4 levels (↓25%; females only). The NOAEL is 0.5 mg/kg/day. 
 
Reproductive toxicity:  No treatment-related effects were observed on sperm motility or density, or reproductive performance in either the P males and females or in the F1 males. 
 
Evaluation of estrous cyclicity found an increase incidence of females with persistent diestrus at the high dose (7/24 of the females; 29.1%). Evaluations of ovarian follicles in the F1 Cohort 1A females revealed the mean number of primordial follicles was increased (p<=0.05) in the 7.5 mg/kg/day females for both the left and right ovary and both ovaries combined.   
 
The LOAEL for reproductive toxicity was 7.5 mg/kg/day based on increased incidence of persistent diestrus and increase number of primordial follicles.  The NOAEL for reproductive toxicity is 1.5 mg/kg/day. 
 
Developmental neurotoxicity  -  Cohorts 2A and 2B:  
 
There were no effects of treatment on brain weights in the Cohort 2A or 2B rats. The Cohort 2A 
7.5 mg/kg/day males had an 11% decrease (p<=0.01) in terminal body weight that resulted in a 12% increase (p<=0.01) in relative brain weight; absolute brain weight was unaffected by treatment. 
 
In the 7.5 mg/kg/day Cohort 2A males (PND 90), the area of the rostral (preoptic) hypothalamus was decreased (↓8%; p<=0.05) compared to control. In the 7.5 mg/kg/day Cohort 2B (PND 21) males, decreases in corpus callosum thickness (↓18%; p<=0.05) and distance across the hippocampal pyramidal layers (↓5%; p<=0.001) were observed. Given the statistical significance of the above-mentioned parameters, the magnitude of change, and the lack of data for both the low and mid dose, these effects are considered adverse. 
     
In the 7.5 mg/kg/day Cohort 2A females (PND 90), a slight increase (↑3%; p<=0.05) in the thickness of the parietal cortex was noted, but this minor change was not considered adverse. There were no other findings in the microscopic measurements. 
 
The LOAEL for developmental neurotoxicity is 7.5 mg/kg/day based on brain morphometrics (↓ hypothalamus area, ↓ corpus callosum thickness, ↓ in distance across hippocampal pyramidal layers; males only). The NOAEL is not established since brain morphometrics were not reported for the mid or low dose groups. 
 
Initially, a Cohort 3 was integrated into the EOGRTS protocol to determine potential immunotoxic effects. This Cohort was not tested within the EOGRTS; however, an immunotoxicity study was submitted in which 10 female SD rats/dose were administered the test compound via gavage at 0, 0.5, 2, or 8 mg/kg/day for 27 days (MRID 49611501). The systemic and immunotoxic NOAEL was set at the highest dose tested, 8 mg/kg/day, with positive controls (cyclophosphamide monohydrate) acting accordingly. Given the lack of immunotoxicity in the stand alone study up to the highest dose tested in the EOGRTS, the immunotoxicity study (MRID 49611501), satisfies the immunotoxic portion of the EOGRTS. 
 
This study is classified as acceptable/non-guideline and does not satisfy the guideline requirements (OCSPP No guideline; OECD 443) for an extended one-generation reproduction toxicity study in the rat. Additional thyroid hormone data and brain morphometric data (discussed in deficiency section at the end of DER) is required to upgrade this study to guideline. 
 
 
  
 

 
Appendix B.  Physical/Chemical Properties 
 
  
 Table B.1.1  Physicochemical Properties of Amitraz. 
  
 Parameter 
  
                                    Value 
  
                                  Reference 
  
 Melting point/range 
  
                                  86-87 C 
  
 Amitraz RED, 3/1/1995 
  
 pH of 1% aqueous suspension 
  
 N/A (low solubility; decomposes in water) 
  
 Amitraz RED, 3/1/1995 
  
 Density or specific gravity 
  
                            1.128 g/mL at 20 C 
  
 Amitraz RED, 3/1/1995 
  
 Water solubility 
  
                           <1 ppm at 20-25 C 
  
 Amitraz RED, 3/1/1995 
  
 Solvent solubility 
  
 At 20-25 C xylene  	 acetone  	 methanol  
66.6 g/100 mL 
50.0 g/100 mL 
 2.38 g/100 mL 
  
 Amitraz RED, 3/1/1995 
  
 Vapor pressure 
  
                          3.4 x 10[-4] Pa at 25 C 
  
 Amitraz RED, 3/1/1995 
  
 Octanol/water partition coefficient (Kow) 
  
                       3.0 x 10[5] at 25 C (pH 5.8) 
  
 CBRS No. 3975, H. Fonouni, 7/21/1988 
 
 
 
 
 
 
Appendix C.  Review of Human Research 
 
This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical.  These data include an acute oral human study, a human metabolism study, and the Residential SOPs (Pet SOP), are (1) subject to ethics review pursuant to 40 CFR 26, (2) have received that review, and (3) are compliant with applicable ethics requirements.  For certain studies, the ethics review may have included review by the Human Studies Review Board (HSRB).  Descriptions of data sources, as well as guidance on their use, can be found at the Agency website[,].   
 
 
Appendix D.  Summary of Amitraz US and International Tolerances and Maximum 
Residue Limits (MRLs) 
 
Table D.1.  Summary of Amitraz US and International Tolerances and Maximum Residue Limits (MRLs) 
U.S. 
Canada 
Mexico 
Codex 
Residue Definition: 
40 CFR 180.287 
Amitraz (N'-[2,4-dimethylphenyl]N-[[(2,4dimethylphenyl)imino]methyl]]-Nmethylmethanimidamide) and its metabolites containing the 2,4dimethylaniline moiety calculated as the parent  
N,N-bis(2,4xylyliminomethyl)methylamine, including the metabolite N-(2,4dimethylphenyl)-N'-methyl formamidine 
 
Sum of amitraz and N-(2,4dimethylphenyl)-N'methylformamidine calculated as N-(2,4dimethylphenyl)-N'methylformamidine. 
Commodity Tolerance (ppm) /Maximum Residue Limit (mg/kg) 
Commodity 
U.S. 
Canada 
Mexico 
Codex 
Cattle, fat 
0.1 
 
 
 
Cattle, meat 
0.02[1] 
 
 
0.052 
Cattle, meat byproducts 
0.2[1] 
0.2 (kidney; liver) 
 
0.2 (edible offal)2 
Cotton, undelinted seed 
 
 
 
0.5 (cotton seed) 
0.05 (cotton seed oil) 
Hog, fat 
0.1[1] 
 
 
 
Hog, kidney 
0.1[1] 
0.2 
 
0.2 (edible offal)3 
Hog, liver 
0.1[1] 
0.2 
 
0.2 (edible offal)3 
Hog, meat 
0.05[1] 
 
 
0.052 
Hog, meat byproducts 
0.3[1] 
 
 
 
Honey 
0.2 
0.1 
 
 
Honeycomb 
9 
 
 
 
Milk 
0.03[1] 
 
 
0.01 (*)2 
Milk, fat 
0.2[1] 
 
 
 
Pear 
 
1 
 
0.5 (pome fruit) 
Cherry 
 
 
 
0.5 
Cucumber 
 
 
 
0.5 
Orange 
 
 
 
0.5 
Peach 
 
 
 
0.5 
Sheep, meat 
 
 
 
0.12 
Sheep, meat byproducts 
 
 
 
0.2 (edible offal)[2] 
Tomato 
 
 
 
0.5 
Apple 
 
0.5 
 
0.5 (pome fruit) 
Prepared 7/16/2018, D. Drew 
Appendix E. Search Parameters for Amitraz Toxicology Literature Review  
 
Date and Time of Amitraz Literature Search:   
4/18/2018; 08:50 am 
 
Search Details: 
((Amitraz)) AND (rat OR mouse OR dog OR rabbit OR monkey OR mammal) 
 
PubMed hits: 361 
Number of Swift Articles:  330 for Animal 
Number of Swift Articles:  165 for Human 
Number of Swift Articles:   1 for No Tag  
 
