**UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460**  **OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION** 

MEMORANDUM DATE: 4 /11 /2013 SUBJECT: Demiditraz : Human Health Risk Assessment to Support Sec tion 3 New Us e for Proposed Dog Spot - On Use PC Code: 577501 DP Barcode: D 410552 MRID No.: Not Applicable Registration No. : 1007 - OT Petition No.: Not Applicable Regulatory Action: Section 3 Registration Assessment Ty

pe: Single Chemical , Non - Aggregate Reregistration Case No.: Not Applicable T XR No.: Not Applicable CAS No.: 944263 - 65 - 4 FROM: Wade Britton, MPH, Risk Assessor and ORE

Linda Taylor , Ph . D . , Toxicologist Risk Assessment Branch V/VII Health Effects Division (HED; 7509P) TH ROUGH: William H. Donovan, Ph.D., Chemist

And Michael Metzger, Branch Chief Risk Assessment Branch V/ VII Health Effects Division (7509P) TO: Bewanda Alexander , Risk Manager

Richard Gebken, PM 10 Insecticide Branch Registration Division ( RD; 7505P)Demiditraz Human Health Risk Assessment D410552

Page **2** of **47 Table of Contents**  1.0 Executive Summary ................................ ................................ ................................ ............. 4 2.0 HED Recommendations ................................ ................................ ................................ ....... 6 2.1 Data Deficiencies ................................ ................................ ................................ ............. 6 2.2 Tolerance Considerations ................................ ................................ ................................ . 7 2.3 Label Recommendations from Occupational and Residential Assessment ..................... 7 3.0 Introduction ................................ ................................ ................................ .......................... 7 3.1 Chemical Identity ................................ ................................ ................................ ............. 7 3.2 Phy sical/Chemical Charac teristics ................................ ................................ ................... 7 3.3 Pesticide Use Pattern ................................ ................................ ................................ ........ 8 3.4 Anticipated Exposure Pathway s ................................ ................................ ....................... 8 3.5 Consideration of Environmental Justice ................................ ................................ .......... 8 4.0 Hazard Characterization and Dose - Response Assessment ................................ .................. 9 4.1 Summary

of Toxicologica l Effects ................................ ................................ .................. 9 4.2 Considerations of Toxicity to Children ................................ ................................ .......... 10 4.2.1 Completeness of the Toxicology Database ................................ ................................ 10 4.2.2 Evidence of Neurotoxicity ................................ ................................ .......................... 11 4.2.3 Evidence of Sensitivity /Susceptibility

in the Developing or Young Animal ............. 11 4.2.4 Residual Uncertaint y in the Exposure Database ................................ ......................... 12 4.3 Toxicity

Endpoint and Point of Departure Selections ................................ .................... 12 4.3 .1 Dose - Response Assessment ................................ ................................ ....................... 12 4.3.2 Recommendation for Combining Routes of Exposures for Risk Assessment ........... 13 4.3.3 Cancer Classificati on and Risk Assessment Recommendation ................................ .. 13 4.3.4 Summary

of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment ................................ ................................ ................................ ................................ 13 5.0 Residential (Non - Occupational) Exposure/Risk Characterization ................................ .... 15 5.1 Residential Handler Exposure ................................ ................................ ........................ 15 5.2 Residential Post - Applicat ion Exposure ................................ ................................ .......... 16 5.3 Combined Residential Risk Estimates (Multiple Exposure Scenarios) ......................... 18 5.4 Residential Risk Estimates for Use in Ag gregate Assessment ................................ ...... 18 5.5 Residential By stander Post - Application Inhalation Exposure ................................ ....... 18 6.0 Aggregate Exposure/Risk Characterization ................................ ................................ ....... 19 7.0 Cumulative Exposure/Risk Characterization ................................ ................................ ..... 19Demiditraz Human Health Risk Assessment D410552

Page **3** of **47** 8.0 Occupational Exposure/Risk Characterization ................................ ................................ .. 19 8.1 Occupational Handler Exposure/Risk Estimates ................................ ............................ 19 8.2 Occupational Post - Application Risk ................................ ................................ ................... 22 Appendix A. Toxicology Profile and Executive Summaries ................................ ....................... 23 A.1 Toxicology

Data Requirements ................................ ................................ ...................... 23 A.2 Toxicity

Profiles ................................ ................................ ................................ ............. 24 A.3 Hazard Identification and Endpoint Selection ................................ ................................ .. 27 A.4 Executive Summaries ................................ ................................ ................................ ..... 29 Appendix B. Phy si cal/Chemical Properties ................................ ................................ ................. 47Demiditraz Human Health Risk Assessment D410552

Page **4** of **47 1.0**  **Executive Summary** Demiditraz, 2 - [(1S) - 1 - (2,3 - Dimethylphen yl )eth yl ] - 1 H - imidazole is an acaricide which acts on the octopamine nervous system in invertebrates a nd acts on neurotransmitter sy stems, such as the alpha 2 adrenergic receptor in mammals. Demiditraz is proposed by the submitter, Pfizer Animal Health, as a spot - on liquid product for dogs to control ticks and mites. There are currentl y no active registr ations for demiditraz. A human health risk assessment was previousl y conducted for the proposed dog spot - on product 1 which identified risks of concern for the use. This document updates the prior assessment with incorporation of additional exposure and t oxicity data submitted since the prior assessment , and uses the 2012 Health Effects Division (HED) Residential Standard Operating Procedures (SOPs) to assess residential exposure and risk. Proposed Use Profile: The proposed dog spot - on product, 1007 - OT , contains two active ingredients; demiditraz (14%) and fipronil (4.8%). This human health risk assessment only addresses human health risk from potential exposures to demiditraz. The proposed product is labeled for application to four dog weight ranges: small, 11 to 20 pounds (lbs); medium, 21 to 33 lbs; intermediate, 34 to 50 lbs; and large, 51 to 66 lbs. Exposure Profile: Non - occupational (r esidential) use of the dog spot - on product is anticipated to result in dermal exposures for adult handlers. In addition, residential post - application dermal exposures are expected for adults and children, and incidental oral exposures for children. Occupational dermal exposures are expected from use of the proposed spot - on product b y veterinarians, veterinary assistants, and groomers. Inhalation exposures are not anticipated from either occupational or residential use of the spot - on product. Current HED policy

requires assessment for occupational handler and residential post application exposures of short - ( 1 to 30 day s), intermediate - (1 to 6 months), and long - term (greater than 6 months) exposures from pet spot - on products due to the preventative nature of pet products and the potential for extended usage in more temperate parts of the country . Residential handler exposure is assumed to be short - term due to the intermittent nature of homeowner spot on applications (i.e., once monthly treatment). Toxicity /Hazard: The nervous sy stem is the p rimary target organ for demiditraz . Pfizer Animal Health mainta ins that demiditraz affects neurotransmitter sy stems such as the alpha - 2 (α2) adrenergic receptor in mammals. The re are no chronic studies for demiditraz; however, the toxicological database is adequate for selecting endpoints for a human health risk assessment and is considered complete because all the data requirement s for a non - food use have been satisfied. A database uncertainty

factor (UF DB ) of 10X was retained in the previous human health risk assessment of the proposed demiditraz dog spot - on product based on the lack of developmental neurotoxicity

(DNT) and non - rodent (dog) studies. The data requirement has been satisfied with submission of rat developmental neurotoxicity and dog subchronic oral toxicity studies, and the dog was not found to be more sensitive than the rat to the neurotoxic effects of demiditraz . 1 W. Britton. Demiditraz: Human Health Risk Assessment for Proposed Dog Spot - On Us e . D378783. 11/30/2010.Demiditraz Human Health Risk Assessment D410552

Page **5** of **47** Accordingl y, the additional UF DB has been removed due to satisfaction of the required study requirements. Demiditraz has not been classified for carcinogenic potential. Rat and mouse carcinogenicity studies with demiditraz have not been submitted , but are not required for the proposed non food/non - feed use. All studies conducted for mutagenicity /g enetic toxicity

were determined to be negative and, therefore, there is no concern for mutagenicity /g enetic toxicity

for demiditraz. There are also no reproductive toxicity concerns. No specific immunotoxicity was identified in a guideline immunotoxicity study . The database for demiditraz is complete, which includes a developmental neurotoxicity assessment; and there is no evidence of increased susceptibility and no residual uncertainties with regard to pre - and/or postnatal toxicity following in utero exposure to rats or rabbits and pre - and/or postnatal exposures to rats. An incidental oral endpoint for risk assessment of all exposure durations was se lected from the oral subchronic neurotoxicity study in the rat with a no observed adverse effect level (NOAEL) of 5 mg/kg/day . At the study lowest observed adverse effect level (LOAEL) of 25 mg/kg/day , clinical signs of neurotoxicity , decreased motor acti vity , and decreased body weights were observed in both sexes. The total uncertainty factor applied for assessment of incidental oral exposures and risks is 100X. Dermal endpoints for all durations of risk assessment were selected from the route - specific subchronic dermal toxicity study in the rat with a LOAEL of 100 mg/kg/day. A NOAEL was not identified. Alterations in motor activity and grooming behavior were observed at all dose levels. An additional uncertainty factor, UF LOAE L , of 3X has been retained due to the lack of a NOAEL (i.e., LOAEL to NOAEL extrapolation). The total uncertainty factor applied for assessment of dermal exposures and risks is 300X. Residential Exposure and Risk Estimates: There is a potential for residential exposures from the use of the proposed demiditraz spot - on for dogs. Short - term residential handler dermal exposure and risk was assessed and is not of concern (i.e., MOES are > 300). Inhalation exposures for residential handler and from post - application exposures to the proposed spot - on product are assumed to be negligible. Residential post - application dermal and incidental oral exposures (all durations) were combined for all children 1 to < 2 years old exposure scenarios assessed. Children’s combined exposures were presented using the aggregate risk index (ARI) approach. This approach was required to assess combined post - application exposure and risks for children 1 to < 2 years old because the LOCs are not the same for dermal and oral routes of exposure (i.e., derm al, 300; and incidental oral, 100). For adults, only post - application dermal exposure is anticipated from contact with a demiditraz treated dog. Pfizer Animal Health submitted a pet residue transfer study in support of the proposed demiditraz spot - on use . These data were used in conjunction HED’s 2012 Residential SOPs to refine the assessment of residential post - application exposures to demiditraz. Day of applicationDemiditraz Human Health Risk Assessment D410552

Page **6** of **47** (Day 0) residues were used to assess all durations of residential post - application expo sure. For the purpose of characterizing longer - term exposures and risks (i. e., intermediate and long - term), multi - day exposure risk estimates were also quantified by use of the average of percent residue using transfer values predicted from Day s 0 to 30 ( i.e., the proposed product re - treatment interval). Residential post - application adult dermal, and combined child ren 1 to < 2 years old exposures are not of concern for all durations of exposure (i.e., adult dermal MOE s are > 300; and children 1 to < 2 ye ar s old ARI s are > 1) with use of Day 0 residue data. Exposures estimated for longer term exposures using 30 day average residue data are approximately 7X below (MOEs 7X greater) those estimated for all durations using Day 0 data. Occupational exposures to veterinarians, veterinary assistants, and groomers may occur from the application of the proposed spot - on product to dogs. D ermal exposure assessment s (all durations) w ere conducted to assess occupational handlers appl yi ng the demiditraz spot - on to dog s. Occupational handlers of the proposed product could potentially treat up to 170 dogs per day without resulting in risks of concern for all durations assessed (i.e., MOEs are ≥ 300). Occupational handler inhalation exposure is expected to be negligible and was not quantitatively assessed. Further, a quantitative assessment of occupational post - application exposure from the proposed demiditraz spot - on product was not conducted. Occupational post - application activities are expected to be significantly less than residential post - application exposures. That is, dogs are expected to be treated and returned to their owners such that post - application contact will b e negligible. Residential Aggregate Exposure and Risk Estimates : An aggregate exposure assessment, which combines exposures from different sources and routes, is ty picall y conducted for non - food/ non feed chemicals when there is potential for human exp osure through water and residential pathway s. Demiditraz currentl y has no registered food/feed uses and no drinking water residues are expected to result from the proposed pet use; therefore, aggregate exposure include s onl y the exposure from the dog spot - on use. Environmental Justice: Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment. Only non - dietary

exposures were considered. **2.0 HED Recommendations**  HED has evaluated the exposure and toxicology databases , and assessed occupational and residential exposure and risk from the proposed demiditraz dog spot - on use and no issues have been identified which would preclude granting the Section 3 registration of the proposed use. **2 .1**  **Data Deficiencies**  There are no exposure or toxicology data deficiencies to prohibit product registration.Demiditraz Human Health Risk Assessment D410552

Page **7** of **47 2.2**  **Tolerance Considerations** The dog spot - on product is the only use proposed for demiditraz and is classified ( a non food/non - feed use ); ther efore, there are no tolerances to evaluate. **2.3 Label Recommendations from Occupational and Residential Assessment**  As currently proposed, 1007 - OT labeling does not include directions regarding the manner in which the spot - on should be applied to dogs. L abel d irections should include the following: how the application tube is prepared for treatment (i.e., snap or twist open) ; whether preparation of the application site is required (e.g., parting dog’s hair coat); and where on the dog ’s body the product s hould be applied (i.e., single or multiple application points). Such language is necessary for appropriate use of the proposed product. **3.0 Introduction**  **3.1 Chemical Identity**  **Table 3.1. Demiditraz Nomenclature** Chemical Structure Empirical Form

ula C 13 H 16N 2 Common N ame Demiditraz Company E xperimental N ames PF - 3814927 CAS N ame 2 - [(1S) - 1 - (2,3 - Dimethylphenyl)ethyl] - 1 H - imidazole CAS Registry Number 944263 - 65 - 4 End Use Product/EP 1007 - OT, 14.4% SC Chemical Class Acaricide Known Impurities of Concern None **3.2 Physical/Chemical Characteristics**  The phy sicochemical properties of the technical grade of demiditraz are summarized in Appendix B, Table B . 1. The log of the octanol/water partition coefficient is 2.8, which indicates no special concer

n for bioaccumulation in lipophilic matrices. Technical grade demiditraz is a solid at room temperature and, therefore, the volatility of the chemical was not determine d as it is not anticipated to have significant volatility .Demiditraz Human Health Risk Assessment D410552

Page **8** of **47 3.3**  **Pesticide Use Pat tern**  The proposed spot - on product (EPA Reg. No. 1007 - OT) has been formulated by Pfizer Animal Health for the control of fleas and ticks on dogs and puppies ages 8 weeks and older. The liquid product contains 14.4% demiditraz and 4.8% fipronil and is avai lable in four dog weight ranges: small, 11 to 20 lbs (1.0 ml); medium, 21 to 33 lbs (1.5 ml); intermediate, 34 to 50 lbs (3.0 ml); and large, 51 to 66 lbs (4.0 ml). The spot - on product is not proposed for use on cats. Details of the proposed use are summ arized in Table 3.3. **Table 3.3. Summary of Proposed Demiditraz Spot -**  **On Use EPA Reg.**  **No. Use Site**  **Application Rate Use Restrictions** 

1007 - OT (14.4% ai) Small dogs from 11

to 20 lbs 0.00032 lb ai; 150 mg ai A single dose of 1007 - OT is

effective up to 6 weeks in controlling fleas and up to 4 weeks for ticks. Monthly application is recommended for effective control of fleas and ticks and to prevent infestation. Medium dogs from 21 to 33 lbs 0.00050 lb ai; 230 mg ai Intermediate dogs from 34 to 50 lbs 0.00099 lb ai; 450 mg ai Large dogs from 51 to 66 lbs 0.0013 lb ai; 600 mg ai **3.4 Anticipated Exposure Pathways**  The Registration Division (RD) has requested an assessment of human health risk to support the proposed new use of demiditraz as a d og spot - on product. Humans may be exposed to demiditraz from occupational and/or residential use of the proposed product. Exposures may occur from occupational and residential adult handlers appl yi ng the spot - on product to dogs, as well as from adult and child post - application contact with treated dogs. Because there are no proposed or existing food uses for demiditraz , no dietary or drinking water exposures are anticipated. **3.5 Consideration of Environmental Justice**  Potential areas of environmental j ustice 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 L ow - Income Populations," (http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf . As a part of every

pesticide risk assessment, OPP considers a large variet y 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 co nsumption patterns are compiled by the USDA under the Continuing Survey of Food Intake by Individuals (CSFII) and/or the CDC under the 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 anal yz ed andDemiditraz Human Health Risk Assessment D410552

Page **9** of **47** categorized b y subgroups based on age, season of the year, ethnic group, and region of the country . Additionally , OPP is able to assess die tary exposure to smaller, specialized subgroups and exposure assessments are performed when conditions or circumstances warrant. Whenever appropriate, non - dietary

exposures 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 are evaluated. Further considerations are currentl y in development as OPP has committed resources and expertise to the development of specialized software and mode ls that consider exposure to b ys tanders and farm workers as well as lifestyl e and traditional dietary patterns among specific subgroups. **4.0 Hazard Characterization and Dose**  **- Response Assessment**  **4.1 Summary of Toxicological Effects**  The nervous s ys tem is the p rimary target organ for demiditraz . Evidence of neurotoxicity after demiditraz exposure was observed in the following toxicity studies: acute neurotoxicity (ACN); subchronic neurotoxicity

(SCN); developmental neurotoxicity (DNT) ; subchroni c dermal in rats; subchronic oral in dogs ; 2 - generation reproduction in rats; and rat and rabbit developmental. Demiditraz effects on the nervous s ys tem were manifested as the following clinical signs which were observed within 30 minutes of dosing : altered gait and posture ; impaired mobility ; decreased rearing, in - coordination ; increased urination; lower body temperature; subdued appearance; rocks; lurches and sway s when walking; hunched posture; hy poactivity ; sitting with the head held low; a flattened body; piloer ection; tremors/convulsions; prostration; wet and ye llo w urogenital area; shall ow and/or decreased respiration; lacrimation; salivation; and dilated pupils . Decreased motor activity was noted in both sexes in the ACN and dermal toxicity studies, whereas i ncreased motor activity was observed in the SCN study . In the DNT, decreases and increases in motor activity were observed in the offspring at various ages; reduced grip strength, and increased amplitude and reduced latency were observe d in the startle re sponse test. Although the latter findings may be related to a general delay in offspring development rather than to neurotoxicity , they are consistent with the findings in other demiditraz toxicity studies. In the developmental toxicity studies, matern al toxicity was observed in the rat and rabbit, as evidenced b y mortality , clinical signs of toxic ity , and decreased body weight. Developmental toxicity was observed in the rat and rabbit, as evidenced b y decreased fetal body weight and associated delayed ossification of the skeleton in rats and slight increases in the incidence of 27 th presacral vertebrae and 13 th full ribs in the rabbit. In the rat reproductive toxicity study , mortality , clinical signs, and decreased body weight were observed in parenta l rats, and decreased survival, decreased brain weight, and decreased pup body weight were observed in the offspring. Reproductive toxicity was not observed . Inthe developmental neurotoxicity study in rats, maternal toxicity

was observed, as evidenced b y clinical signs of toxicity an d effects on maternal behavior. The effects observed in the offspring included a delay in preputial separation in males and changes in motor activity , startle response, and grip strength, which may be attributed to a general delay in development. The mutagenicity/ genetic toxicity database consists of a bacterial reverse mutation test (Ames test), a forward mutation test in the V79/HGPRT test, chromosome aberration test in ChineseDemiditraz Human Health Risk Assessment D410552

Page **10** of **47** Hamster Ovary

cells, and a mouse bone marrow micro nucleolus test. All studies were determined to be negative for mutagenicity and/or genetic toxicity , and there is no concern for mutagenicity

/g enetic toxicity

for demiditraz. Demiditraz was assessed in a guideline immunotoxicity

study , and no spec ific immunotoxicity was identified. In an absorption and distribution study in rats, evidence was obtained that showed a significant portion of the administered dose to the maternal rat partitioned into the milk and was available to the F 1 offspring through nursing. The amount of test material in the stomach of the offspring was similar to the concentration in maternal milk. Detectable levels of the test material were observed in neonatal plasma demonstrating exposure to the offspring following maternal exposure, which negated the need for direct exposure of the offspring in the developmental neurotoxicity

study . Based on limited metabolism data, demiditraz was rapidly absorbed and eliminated via the urine (71%) and feces (21%), with 92% being excrete d within 48 hours post dose in the rat. The halflife in rats following oral exposure was determined to be approximately 30 minutes. Inthe dog, the half - life was 4.7 hours (oral) and 347 hours (dermal), and bioavailability was 10.1% following oral exposure and 18.6% following dermal exposure. A dermal absorption study is available for demiditraz, which identified a dermal absorption factor of 30%. Since the dermal risk assessments for all durations of exposure are based on a route - specific study

(subchronic dermal toxicity study in rats), a dermal absorption factor is not needed. There is no subchronic inhalation toxicity study currentl y available to determine the potential for demiditraz to affect the nervous s ys tem by this route of exposure. It s hould be noted , however, that an inhalation study

is not relevant to the exposure pattern anticipated for the pet use , since significant inhalation exposure is not expected . **4.2 Considerations of Toxicity to Children**  Demiditraz does not require food to lerances and is considered to be a non - food/non - feed use chemical; therefore, it is not subject to the amendments to the Federal Food, Drug, and Cosmetic Act (FFDCA) promulgated under the Food Quality Protection Act (FQPA) of 1996. However, HED has asses sed the potential for increased susceptibility for infants and children, and determined that n o additional uncertainty factor is required for the protection of children based on considerations summarized in Sections 4.2.1 through 4.2.4, below. **4.2.1 Com**  **pleteness of the Toxicology Database** The toxicology

database for demiditraz is complete for the pet use being assessed. The following studies have been submitted and found to be a cceptable : rat and rabbit developmental toxicity studies ; rat 2 - generation reproduction ; rat a cute and subchronic neurotoxicity ; rat developmental neurotoxicity ; and rat immunotoxicity study .Demiditraz Human Health Risk Assessment D410552

Page **11** of **47 4.2.2**  **Evidence of Neurotoxicity** There is evidence of neurotoxicity throughout the database, but neuropathology was not observed. A lte red gait and posture, impaired mobility , decreased rearing, in - coordination, lower body temperature , and decreases in motor activity were observed in both sexes in th e acute neurotoxicity

study in rats. Several clinical signs of neurotoxicity , which inclu d ed subdued appearance, rocks/lurches/sway s when walking, hunched posture, hy poactivity , wet and ye llow urogenital area, shallow and/or decreased respiration, lacrimation, and dilated pupils, and increased motor activity

were observed in both sexes in th e subchronic neurotoxicity study in rats . Decreased motor activity and altered grooming behavior were observed in both sexes in the 90 - day dermal toxicity

study in rats. In the developmental toxicity studies, clini cal signs suggestive of neurotoxicity were observed in the maternal rats (rocking, lurching, or swaying while walking, piloerection, dilated pupils, and subdued appearance) and rabbits ( tremors, prostration, and/or clonic convulsions ) . In the 2 - generation reproduction study in rats, clinical signs ( subdued appearance, rocks/lurches/sway s while walking, h ypoactivity , tremors, clonic convulsions ) suggestive of neurotoxicity were observed. In the developmental neurotoxicity study in rats, decreased/increased motor activit y and increased startle response amplitude and reduced latency

were observed at the high dose where a delay in overall development was observed, mainl y in males, as evidenced b y decreased body weights throughout lactation in both sexes, and a delay

in preputial separation. **4.2.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal**  There is no evidence of increased susceptibility following in utero exposure to demiditraz in either the rat or rabbit developmental toxicity study , and there is no evidence of increased susceptibility

following in utero and/or pre - /post - natal exposure in the 2 - generation reproduction study in rats or in the developmental neurotoxicity study in rats . Demiditraz has been evaluated for potential developmental effects in the rat and rabbit (gavage administration). Maternal toxicity was observed in the rat and rabbit, as evidenced by mortalit y and clinical signs of toxicity and decreased body

weights. Developmental toxicity was observed in the r at and rabbit, as evidenced b y decreased fetal body weight and associated delayed ossification of the skeleton in rats and slight increases in the incidence of 27 th presacral vertebrae and 13 th full ribs in the rabbit. The developmental effects were obser ved at the same dose where maternal effects were observed in the rat and at a higher dose than where maternal effects were observed in the rabbit. In the rat reproductive toxicity study , mortality , clinical signs (subdued appearance, rocks/lurches/sway s while walking, hy poactivity , tremors, clonic convulsions), and decreased body weight were observed in parental rats, and decreased survival, decreased brain weight, and decreased pup body weight were observed in the offspring at the same dose levels. In the developmental neurotoxicity study in rats, maternal toxicity (sitting with head held low, hy poactivity , a flattened body , slightly drooping eyelids, decreased respiration, clear material around mouth/salivation, lacrimation, and/or dilated pupils) was obs erved at a dose where no effects were observed in the offspring.Demiditraz Human Health Risk Assessment D410552

Page **12** of **47 4.2.4**  **Residual Uncertainty in the Exposure Database** There is no residual uncertainty in the exposure database. Dietary (food and water) exposure assessments are not applicable to this a ssessment. Chemical - specific data are available for assessing exposure resulting from spot - on treatments of demiditraz; these data will not underestimate residential exposure. **4.3 Toxicity Endpoint and Point of Departure Selections**  **4.3.1 Dose**  **- Response**  **Assessment** A detailed description of the toxicity studies used for toxicity

endpoints and points of departure for various exposure scenarios is presented in Appendix A . No acute or chronic dietary assessments are required since demiditraz is a non - food u se pesticide. The i ncidental oral endpoint ( all durations ) for risk assessment w as from the oral subchronic neurotoxicity

study in the rat with a NOAEL of 5 mg/kg/day . At the study L OAEL of 25 mg/kg/day , clinical signs of neurotoxicity (subdued appearanc e, rocks, lurches, or sway s when walking, hunched posture, hy poactivity , shallow/decreased respiration, lacrimation, dilated pupil), decreased motor activity , and decreased body weights were observed in both sexes. Inhalation hazard is assumed to be equiv alent to oral hazard. The current risk assessment includes a chronic exposure assessment . The Point of Departure for assessing risks resulting from this duration of exposure was also the NOAEL from the subchronic neurotoxicity

study . Th is 90 - day

durat ion POD is acceptable for quantifying chronic risks for the long - term incidental oral and inhalation risk assessments because there is no evidence of cumulative toxicity ; i.e., rapid onset (within 30 minutes of exposure) and short duration (2 hours) of transient clinical signs . Dermal endpoints (all durations) for risk assessment were from the route - specific subchronic (90 - day) dermal toxicity

study in the rat with a LOAEL of 100 mg/kg/day. A NOAEL was not identified. Alterations in motor activity and grooming behavior we re observed at all dose levels. The same dermal study

and endpoints are appropriate for long - term dermal assessment because there is no evidence of cumulative toxicity . An additional uncertainty factor of 3X is applied for the lack of a NOAEL (UF LOAEL →NOAEL). The 3X factor is considered appropriate for dermal exposure assessment (all durations) . Application of the 3X factor results in an extrapolate d dermal NOAEL of 33 mg/kg/day . Based on a dermal absorption factor of 31%, the orall y equival ent dose is about 10 mg/kg/day . Th is is consistent with the 2 - gen eration repro duction study NOAEL of 7.5 mg/kg/day , which noted similar effects as observed in the dermal study at an oral dose of 50 mg/kg/day . The UF DB of 10X retained in the pre vious human health risk assessment was removed since the data requirements for a rat developmental neurotoxicity study and a dog subchronic oral toxicityDemiditraz Human Health Risk Assessment D410552

Page **13** of **47** study have been satisfied, and the dog was not found to be more sensitive than the rat to the neurotox ic effects of demiditraz. The demiditraz database is now considered complete. **4.3.2 Recommendation for Combining Routes of Exposures for Risk Assessment**  Since the dermal and oral endpoints are based on the same effects (neurotoxicity ) these routes of exposure may

be combined for purposes of this risk assessment. **4.3.3 Cancer Classification and Risk Assessment Recommendation**  Demiditraz has not been classified for carcinogenic potential. Rat and mouse carcinogenicity studies with demiditraz have not b een submitted and are not required for the current non food/non - feed use. All studies conducted for mutagenicity /g enetic toxicity

were determined to be negative and, therefore, there is no concern for mutagenicity /g enetic toxicity

for demiditraz. There are also no reproductive toxicity concerns. **4.3.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk**  **Assessment Table**  **4.3.4.1. Summary**  **of Toxicological**  **Doses and**  **Endpoints for**  **Demiditraz for**  **Use in**  **Non -**  **Occupational Human Health Ris**  **k Assessments Exposure/**  **Scenario Point of** 

**Departure Uncertainty** 

**Factors Level of** 

**Concern for Risk**  **Assessment Study and Toxicological Effects**  All dietary Exposures There are no current registrations for food uses. Therefore , no dietary endpoint has

been selected for demiditraz at this time.

Dermal (All D urations) LOAEL = 100

mg/kg/day UF A= 10X

UF H= 10X UF LO AEL= 3X MOE = 300 Subchronic dermal toxicity study L OAEL = 100 mg/kg/day, based on decreased motor activity in males and indications of ungroomed appearance. A NOAEL was not demonstrated since there was toxicity ( decreased motor activity) and ungroomed appearance at the lowest dose tested. Incidental Oral (All D urations) NOAEL= 5

mg/kg/day UFA= 10X

UFH= 10X MOE = 100 Subchronic n eur otoxicity study with a LOAEL = 25 mg/kg based on clinical signs of neurotoxicity.Demiditraz Human Health Risk Assessment D410552

Page **14** of **47 Table**  **4.3.4.1. Summary**  **of Toxicological**  **Doses and**  **Endpoints for**  **Demiditraz for**  **Use in**  **Non -**  **Occupational Human Health Ris**  **k Assessments Exposure/**  **Scenario Point of** 

**Departure Uncertainty** 

**Factors Level of** 

**Concern for Risk**  **Assessment Study and Toxicological Effects**  Inhalation (All D urations) NOAEL= 5

mg/kg/day UF A= 10X

UF H= 10X MOE = 100 Subchronic n eurotoxicity study with a LOAEL = 25 mg/kg, based on clinical signs of neurotoxicity. Cancer (oral, dermal, inhalation) There are no carcinogenicity studies with demiditraz. The carcinogenic potential of

demiditraz has not been evaluated. 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. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF LOAEL = additional factor for extrapolation from LOAEL to NOAEL . MOE = margin of exposure. LOC = level o f concern. N/A = not applicable. **Table 4.3.4.2.**  **Summary of**  **Toxicological Doses**  **and Endpoints**  **for Demiditraz**  **for Use**  **in Occupational**  **Human Health Risk Assessments Exposure/**  **Scenario Point of** 

**Departure Uncertainty** 

**Factors Level of** 

**Concern for Risk**  **Asses sment**  **Study and Toxicological Effects**

Dermal ( All Durations ) LOAEL ≤ 100

mg/kg/day UF A= 10X

UF H= 10X UF LO AEL= 3X MOE = 300 Subchronic dermal toxicity study L OAEL = 100 mg/kg/day, based on decreased motor activity in males and indications of ungroomed appearance. A NOAEL was not demonstrated since there was toxicity ( decreased motor activity) and ungroomed appearance at the lowest dose tested. Inhalation (All Durations) NOAEL = 5

mg/kg/day UF A= 10X

UF H= 10X MOE = 100 Subchronic Neurotoxicity study LOAEL = 25 mg/kg , based on clinical signs of neurotoxicity. Cancer (oral, dermal, inhalation) There are no carcinogenicity studies with demiditraz. The carcinogenic potential of

demiditraz has not been evaluated. Point of Departure (POD) = A data p oint 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. UF A = extrapolation from animal to human (interspecies). UF H = potential variation inDemiditraz Human Health Risk Assessment D410552

Page **15** of **47** sensitivity among members of the human population (intraspecies). UF LOAEL = extrapolation from LOAEL to NOAEL. MOE = margin of exposure. LOC = level of concern. **5.0 Residential (Non**  **- Occupational) Exposure/Risk Characterization**  Residential exposure is expected from the prop osed demiditraz dog spot - on use . Residential handler exposure s are anticipate d from application of the proposed product to dogs , and r esidential post - application exposures are expected to occur from contact with dogs previousl y treated with demiditraz. In assessing these exposures, the Health Effects Division’s (HED) 2012 Standard Operating Procedures (SOPs) for Residential Pesticide Exposure Assessment: Treated Pets 2 was used. Some of the data included in the 2012 Treated Pet SOP are proprietary and, thus, are subject to the data protection provisions of FIFRA . **5.1 Residential H**  **andler Exposure** HED uses the term “handlers” to describe those individuals who are involved in the pesticide application process. HED believes that there are distinct tasks related to applications and that exposures can vary

depending on the specifics of each task. Residential handlers are addressed somewhat differentl y b y HED as homeowners are assumed to complete all elements of an application without use of an y protective equipment. Residential handler dermal exposure is expected to occur from application of the proposed demiditraz spot - on product to dogs. Inhalation exposure of spot - on products is considered to be negligible. A short - term residential handler dermal exposure assessment was performed for homeowners appl yi ng the proposed demiditraz pro ducts to dogs. Intermediate - and long - term exposures are not likel y because of the intermittent nature (i.e., once monthly) of pet treatment by homeowners. No chemical - specific unit exposure data were provided in support of this submission . Therefore , HED used exposure values from the 2012 Residential SOPs ( Treated Pets ) as a surrogate to estimate handler exposures. Exposure data for spot - on applications (MRID 44433303) were used to estimate handler exposures for the proposed demiditraz spot - on produc t. The algorithms and inputs used to estimate exposure and dose for residential handlers can also be found in the occupational and residential exposure and risk assessment 3 document which supports this memorandum. Residential handler short - term dermal ri sk estimates are not of concern (i.e., MOEs > 300) for all dog weight ranges proposed. A summary of residential handler risk estimates is presented in Table 5.1.

2 http://www.epa.gov/pesticides/science/residential - exposure - sop.html 3 W. Britton. Demiditraz: Occupational and Residential Exposure and Risk Asse ssment for the Proposed Pet Spot On Use (1007 - OT). D400484,.D409676, D409677 . 4/11/13.Demiditraz Human Health Risk Assessment D410552

Page **16** of **47 Table 5.1**  **. Residential Handler Short**  **- Term Non**  **- cancer Exposure and Risk Estimates from Us**  **e of the Proposed Demiditraz Spot**  **- On**  **Exposure Scenario**  **Reg. No. Level of** 

**Concern Dermal** 

**Unit Exposure**  **(mg/lb ai) Inhalation** 

**Unit Exposure**  **(mg/lb ai) Maximum** 

**Application Rate**  **1 (lb ai/pet)**  **Amount**

**Handled Daily**  **2 Dermal** 

**Dose (mg/kg/day)**  **3 MOE**  **4**

Applyin g Demiditraz Spot - on 1007 - OT

(14.4% ai) Dermal:

300 120 Negligible 0.00032 (small) 2 Dogs 0.00097 100,000 0.00050 (medium) 0.0015 67,000 0.00099 (intermediate) 0.0030 34,000 0.0013 (large) 0.0040 25,000 1 Based on registered or propos ed label (EPA Reg. No. 1007 - OT ). 2 Based on HED’s 2012 Residential SOPs (Treated Pets) (http://www.epa.gov/pesticides/science/residential - exposure - sop.html ). 3 Dermal Dose = D ermal Unit Exposure ( 120 mg/lb ai) × Application Rate (lb ai/ treatment) × Ar ea Treated or Amount Handled ( 2 Dogs/day) × Dermal Absorption Factor ( 1.0 ) ÷ Body Weight ( 80 kg). 4 Dermal MOE = Dermal NOAEL ( 100 mg/kg/day) ÷ Dermal Dose (mg/kg/day). **5.2 Resid**  **ential Post -**  **Application Exposure** There is the potential for post - application exposure for individuals exposed as a result of contacting a dog previously treated with the proposed demiditraz spot - on product. The quantitative exposure/risk assessment for r esidential post - application exposures is based on the following scenarios: 1) P ost - application dermal (adults and children 1 to < 2 years old) exposure from contacting dogs treated with demiditraz; and 2) P ost - application incidental oral exposure (ch ildren 1 to < 2 ye ars old only) from contacting dogs treated with demiditraz. The lifestages selected for each post - application scenario are based on an analy sis provided as an Appendix in the 2012 Residential SOPs 4 . These lifestages are not the onl y that could be potentially

exposed for these post - application scenarios; however, the assessment of these lifestages is health protective for the exposures and risk estimates for an y other potentially exposed lifestages. Due to the preventative nature of pet products and the potential for extended usage in more temperate parts of the country , the potential exists for applications to extend beyond a short - term duration. Therefore, residential post - application exposures of short - , intermediate - , and long term d urations were assessed for the proposed demiditraz spot - on product.

4 Available: http://www.epa.gov/pesticides/science/residential - exposure - sop.htmlDemiditraz Human Health Risk Assessment D410552

Page **17** of **47** 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 Res idential SOPs. A chemical - specific pet fur residue transfer study (MRID 48766704) was submitted by Pfizer Animal Health in support of the proposed demiditraz spot - on use. HED has reviewed the exposure study 5 and this data was determined to acceptable for use for assessment of exposure and risk from the proposed use. The 2012 Residential SOPs ( Treated Pets ) recommends assessment of post - application exposures using day of application (i.e., Day

0) residue transfer -- defined as fraction application rate ( F AR) in the 2012 SOPs . HED used the average of Day 0 percent residue transfer values predicted from regression anal ys is in order to assess exposures and risks for all durations of post application exposures from the proposed demiditraz spot - on use. Day 0 F AR was determined to be 2.8%. A refined approach is also presented for the assessment of longer - term (i.e., intermediate - and long - term) post - application exposures. Per the 2012 Treated Pet SOP, the assessment of post application exposures to treated pets can be refined to more accuratel y reflect exposures over longer periods of time (e.g., several months). Instead of using the Day 0 percent residue transfer value, the average of percent residue transfer values predicted from Day s 0 to 30 (i.e., prop osed product re - treatment interval) w as inputted. The F AR for the 30 day interval is 0.38%. HED combines risk values resulting from separate routes of exposure when the hazard associated with the points of departure is similar across routes. A common toxicological endpoint, neurotoxicity , exists for dermal and incidental oral routes of exposure to demiditraz . Residential post - application inhalation exposure is expected to be negligible from the proposed spot - on product and, thus, a quantitative assess ment was not performed. Residential post - application adult dermal, and combined child 1 to < 2 years old years old exposures (all durations) are not of concern (i.e., adult dermal MOE s are > 300; and children 1 to < 2 years old ye ar old ARI s are > 1) wi th use of Day 0 F AR . Exposures estimated for the longer term exposures using 30 day average residue data are approximately 7X below (MOEs 7X greater) those estimated for all durations using Day 0 F AR . **Table 5.2. Residential Post -**  **Application Non -**  **Cancer E xposure and Risk Estimates from the**  **Proposed Demiditraz Spot -**  **On Product: All Durations (Day 0 F AR** **) Lifestage**  **Post -**  **application Exposure**

**Scenario Application Rate**  **(mg ai) 1**  **Dose**

**(mg/kg/day) 2**  **MOEs 3**  **ARI 4**  **Use Site Route of** 

**Exposure** Adult Dog Dermal 150 (small) 0.066 1,500 NA 230 (medium) 0.067 1,500 450 (intermediate) 0.096 1,000 600 (large) 0.099 1,000 5 W. Britton, Demiditraz: Data Evaluation Record for the Study “Determination of Transferable Residues of Demiditraz and Fipronil from the Hair of Dogs Following the Spot - on Treatment Separately with Three Different Form ulated End - Use Products .” D409400, EPA MRID 48766704 , 4/11/13 .Demiditraz Human Health Risk Assessment D410552

Page **18** of **47 Table 5.2. Residential Post**  **- Application Non**  **- Cancer E**  **xposure and Risk Estimates from the Proposed Demiditraz Spot**  **- On Product: All Durations (Day 0 F**  **AR )**  **Lifestage Post**  **- application Exposure** 

**Scenario Application Rate**  **(mg ai) 1**  **Dose**

**(mg/kg/day) 2**  **MOEs 3**  **ARI 4**  **Use Site Route of** 

**Exposure** Children 1 to < 2 Years Old Dermal 150 (small) 0.17 600 1.9 Incidental Oral 0.0017 3,000 Dermal 230 (medium) 0.17 590 1.8 Incidental Oral 0.0017 2,900 Dermal 450 (intermediate) 0.25 410 1.3 Incidental Oral 0.0025 2,000 Dermal 600 (large) 0.25 400 1.3 Incidental Oral 0.0025 2,000 1 Based on proposed label (Reg. No. 1007 - OT ) 2 Dermal Dose = [(Transfer Coefficient ( cm2 /hr) * (Application Rate (150, 230, 450, 600 mg ai) * Fraction Application Rate (0.028) ÷ Surface Area of Dog (cm2 )) * Exposure Time (hours/day) * A bsorption Factor (unitless)) / Body Weight (80 kg, adult; 11 kg, child 1 to < 2 years old ye ars old)] Hand - to - Mouth Dose = [(H and Residue L oading (mg/cm2 ) × Fraction of Hand Mouthed (0.13) × S urface Area of 1 H and (150 cm2 ) × Exposure Time (1.5 hrs/day) × # of Replenishment I ntervals/hr (4 int/hr) × (1 - ((1 - Saliva Extraction Factor (0.5))^(Number of H and - to - Mouth Events per Hour (13.9 events/hr) ) ÷ ( # of Replenishment Intervals/hr))] / Body W eight (11 kg child 1 to < 2 years old years old)] 3 MOE = Dermal, Incidental Oral POD (mg/kg/day) / Dose (mg/kg/day) 4. ARI = 1 ÷ [(1 / (Dermal MOE/Dermal L OC; 300)) + (1/(Incidental Oral MOE ÷ Incidental Oral LOC; 100))] **5.3 Combined Residential Risk Estimates (Multiple Exposure Scenarios)**  Residential handler and post - application scenarios should generall y not be combined. There is the potential for the sam e individual (i.e., adult) to apply a pesticide in and around the home and be exposed by

reentering a treated area in the same day ; however, combining both of these exposure scenarios would be inappropriate because of the conservative nature of each indivi dual assessment . **5.4 Residential Risk Estimates for Use in Aggregate Assessment**  An aggregate exposure assessment, which combines exposures from different sources and routes, is ty picall y conducted for food/feed chemicals when there is potential for huma n exposure through drinking water and residential pathway s. Demiditraz currentl y has no registered food/feed uses and no drinking water residues are expected to result from the proposed use; therefore, the aggregate exposure assessment consists only of th e proposed use. **5.5 Residential Bystander Post**  **- Application Inhalation Exposure**  Occupational and residential inhalation exposures from dog spot - on treatments are considered negligible.Demiditraz Human Health Risk Assessment D410552

Page **19** of **47 6.0**  **Aggregate Exposure/Risk Characterization** The demiditraz dog spot - on product is classified as a non - food/non - feed use with no proposed or existing food uses. Because demiditraz currentl y has no registered food uses and no drinking water residues are expected to result from the proposed pet use , the aggregate exposure assessment consists only

of exposures from the proposed use. **7.0 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 c ommon mechanism of toxicity finding as to demiditraz and any

other substances and demiditraz does not appear to produce a toxic metabolite produced b y other substances. For the purposes of this tolerance action, there fore, EPA has not assumed that demiditr az has a common mechanism of toxicity with other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy statements releas ed by

EPA’s Office of Pesticide Programs concerning common mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative. **8.0 Occupational Expos**  **ure/Risk Characterization** Occupational handler exposures are anticipated from the application of the proposed demiditraz spot - on product. Occupational post - application activities are expected to be significantl y less than residential post - application exp osures. **8 .1**  **Occupational Handler Exposure/Risk Estimates** Based on the anticipated use patterns and current labeling, typ es of equipment and techniques that can potentially

be used, occupational handler exposure (i.e., veterinarians, veterinary assistant s, and groomers) could be expected from the demiditraz spot - on use . The quantitative exposure/risk assessment developed for occup ational handlers (adults) is based on dermal exposure from the application of the spot - on product to dogs. Inhalation exposur es are expected to be negligible from application of the spot - on product. A series of assumptions and exposure factors served as the basis for completing occupational handler risk assessments. A number of these are the same used for assessment of reside ntial handler exposures. An assumption unique to the assessment of occupational handler exposures for the proposed demiditraz dog use is the number of animals treated daily. There is currentl y no recommendation for the number of animals treated by occupational handlers per application event. For the purpose of assessing occupational handler risk, the maximum number of dogs that could be treated with the proposed spot - on product without resulting in a risk of concern has been presented. Similar to resi dential use of the proposed spot - on product, occupational exposures are also expected to extend beyond a short - term duration due to temperate climates in some parts of theDemiditraz Human Health Risk Assessment D410552

Page **20** of **47** country and the preventative nature of spot - on pet products. For these reasons, it is expected that veterinarians, veterinary

assistants, and groomers could treat dogs with the proposed demiditraz spot - on product for all durations of exposure (short - , intermediate - , and long - term) for flea and tick prevention and/or treatment. The algorithms and inputs used to estimate exposure and dose for occupational handlers are the same as though used for residential handler exposure assessment , except for the number of pets treated explained above, and can be found in the 2012 Residential SOPs (Tre ated Pets) SOP, as well as in the occupational and residential exposure and risk assessment 6 which supports this memorandum. Only dermal exposures are expected from the proposed demiditraz spot - on use and, therefore, exposures were not combined from multiple routes. Occupational handlers of the proposed demiditraz spot - on product could treat up to 170 large dogs per day

(all durations) without resulting dermal risks of concern (i.e., MOEs are ≥ 300). It is highly unlikel y that this large a number of dogs could be treated dail y in an occupational setting and, therefore, no risk concerns are anticipated from occupational use of the proposed dog spot - on product . A summary of occupational han d ler dermal risk estimates is presented in Table 8.1.

6 W. Britton. Demiditraz: Occupational and Residential Exposure and Risk Assessment for the Proposed Pet Spot On Use (1007 - OT). D409676. D409677. 4/11/13.Demiditraz Human Health Risk Assessment D410552

Page **21** of **47 Table 8**  **.1. Occupational Handler Non -**  **Cancer Exposure and Risk Estimates (All Durations) for Demiditraz Exposure**  **Sc enario**  **Target Dermal Unit**  **Exposure (mg/lb ai)**  **1 Inhalation Unit** 

**Exposure (mg/lb ai)**  **Maximum**

**Application Rate**  **(lb ai) 2**  **Maximum**

**Amount Handled**  **Daily 3**  **Dermal Inhalation**  **Single Layer, No Gloves**  **No Respirator Dose**  **(mg/kg/day) 4**  **MOE 5**  **Dose**

**(mg/kg/day) MOE**  Applying Demiditraz Spot On Product Dogs 120 Negligible 0.00032 (small) 170

Dogs 0.082 1,200 Negligible 0.00050 (medium) 0.13 800 0.00099 (intermediate) 0.25 400 0.0013 (large) 0.34 300 1 Based on the “Occupational Pesticide Handler Unit Exposure Surrogate Reference Table” (March 2012); Level of mitigation: Baseline 2 Based on registered or proposed label (Reg. No. 1007 - OT) 3. Maximum number of dogs that could be treated per day and not result in risks of concern (i.e., short - , intermediate and long - term, MOEs = 300 ) 4 Dermal Dose = Dermal Unit Exposure (120 mg/lb ai) × Application Rate (lb ai/ treatmentl) × Amount Handled Daily (treatments/ day) × DAF (1.0) ÷ BW (80 kg) 5 Dermal MOE = Dermal NOAEL (100 mg/kg/day) ÷ Dermal Dose (mg/kg/day)Demiditraz Human Health Risk Assessment D410552

Page **22** of **47 8.2 Occupational Post**  **- Application Risk**  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 re entry exposure). For the proposed demiditraz pet collar use, occupational post - application activities are expected to be significantly less than residential post - application exposures. That is, d ogs are expected to be treated and returned to their owners such that post - a pplication contact will be negligible. As a result, no quantitative occupational post - application exposure and risk assessment has been performed. The residential post - application exposure and risk assessment (Section 5.2) is considered protective for an y potential occupational post - application exposures and risks. **References** W. Britton. Demiditraz: Human Health Risk Assessment for Proposed Dog Spot - On Use . D378783. 11/30/2010. W. Britton. Demiditraz: Occupational and Residential Exposure and Risk Ass essment for the Proposed Pet Spot - On Use (1007 - OT). D409676 . D409677. 4/11 /13. W. Britton. Demiditraz: Data Evaluation Record for the Study “Determination of Transferable Residues of Demiditraz and Fipronil from the Hair of Dogs Following the Spot - on Treatment Separatel y with Three Different Formulated End - Use Products .” D409400, EPA MRID 48766704 , 4/11/13 .Demiditraz Human Health Risk Assessment D410552

Page **23** of **47 Appendix A. Toxicology Profile and Executive Summaries**  **A.1 Toxicology Data Requirements**  The requirements (40 CFR 158.340) for f ood and non food uses for demiditraz are in Table A.1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used. **A.1. Summary of Toxicology Data Requirements -**  **Demiditraz Study**  **Technical Required**  **Satisfied** 87 0.1100 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 ................................ ...................... y es yes yes * yes yes yes yes

yes no* yes y es yes 870.3100 Oral Subchronic (rodent) ................................ ............... 870.3150 Oral Subchronic (nonrodent) ................................ ......... 870.3200 21 - Day

Dermal ................................ .............................. 870.3250 90 - Day

Dermal ................................ .............................. 870.3465 90 - Day

Inhalation ................................ .......................... CR CR no yes CR yes

yes no yes no 870.3700a Developmental Toxicity (rodent) ................................ ... 870.3 700b Developmental Toxicity (nonrodent) ............................. 870.3800 Reproduction ................................ ................................ . Yes yes yes yes

yes yes 870.4100a Chronic Toxicity (rodent) ................................ .............. 870.4100b Chronic Toxicity (nonrodent) ................................ ........ 870.4200a Oncogenicity (rat) ................................ .......................... 870.4200b Oncogenicity (mouse) ................................ .................... CR no CR CR no

no no no 870.5100 Mutagenicity

— Gene Mutation - bacterial ..................... 870.5300 Mutagenicity

— Gene Mutation - mammalian ................ 870.5xxx Mutagenicity

— Structural Chromosomal Aberrations ... Y es yes yes yes

yes yes 870.6100a Acute Delayed Neurotoxicity (hen) ............................... 870.6100b 90 - D ay

Neurotoxicity (hen) ................................ ........... 870.6200a Acute Neurotoxicity Screening Battery (rat) ................. 870.6200b 90 - Day

Neurotoxicity Screening Battery (rat) ............... 870.6300 Develop. Neurotoxicity ................................ ................. no no y es yes CR no

no yes yes yes 870.7485 General Metabolism ................................ ...................... 870.7600 Dermal Penetration ................................ ........................ 870.7800 Immunotoxicity ................................ ............................. CR CR yes no

no yes *Refer to review by R. Whiting dated May 21, 2010. There were technical problems in generating a test atmosphere that precluded completion of the acute inhalation toxicity study. CR = con ditionally requiredDemiditraz Human Health Risk Assessment D410552

Page **24** of **47 A**  **.2 Toxicity Profiles**  **Table A.2.1.**  **Acute Toxicity Profile –**  **Technical Demiditraz Guideline**  **No. Study Type**  **MRID(s) Results**  **Toxicity Category** 870.1100 Acute oral - rat 47744106 LD 50 = 5000 mg/kg III 870.1200 Acute dermal - rat 4 7744107 LD 50 > 5000 mg/kg IV 870.1300 Acute inhalation - rat None Waived Technical complications in conducting the study. Test material could not be generated as an aerosol. 870.2400 Acute eye irritation -

rabbit 47744109 Slight or non irritating IV 87 0.2500 Acute dermal irritation -

rabbit 47744110 Slight irritant IV 870.2600 Skin sensitization -

guinea pig 47744111 Not a sensitizer N/A **Table A.2.2. Demiditraz Study Details and Neurotoxicity Effects Observed**  **Guideline No.**  **Study Type MRID No. (yea**  **r)/ Classification Doses**  **Results**

870.6200a Acute neurotoxicity

(rat, Sprague - Dawley) MRID 47744113 (2008) Acceptable/Guideline 0, 15, 100, 600 mkd **NOAEL = 15 mkd.** 1 dose via gavage FOB/MA assessed ≈15 minutes post dose LOAEL = 100 mkd, based on altered gait and posture, impaired mobility, decreased rearing, in - coordination, increased urination, lower body temperature, decreased in motor activity in both sexes; at 600 mkd, clonic convulsions, hindlimb splay/dragging, ↓rotarod performance; low arousal.

870.6200b Subchronic

neurotoxicity (rat , Sprague - Dawley) MRID 47744114 (2008) Acceptable/Guideline 0, 5, 25, 125 mkd **NOAEL = 5 mkd.** 90 days via gavage FOB/MA assessed prior to dosing (wks 1, 3, 7, 12) LOAEL = 25 mkd, based on clinical signs (subdued appearance, rocks, lurches/sways when walking, hunched posture, hypoactivity, etc., both sexes), ↓BW, increased motor activity; 125 mkd increased incidence of same findings. 870.3150 Sub chronic oral

toxicity (dog , Beagle ) MRID 48459302 (2009) Acceptable/Guideline 0, 10, 30, 100 mkd **NOAEL = 10 mkd.** 90 days capsule assessed 30 minutes to 2 hrs post dose LOAEL = 30 mkd, based on hypoactivity in bothDemiditraz Human Health Risk Assessment D410552

Page **25** of **47 Table A.2.2.**  **Demiditraz Study Details and Neurotoxicity Effects Observed Guideline**  **No. Study Type**  **MRID No. (yea r)/**  **Classification Doses Results**  sexes. At 100 mkd, all dogs displayed hyp oactivity.

870.3250 Subchronic dermal toxicity (rat, Sprague - Dawley) MRID 47752701 (2008) Acceptable/Guideline 6 hr/day for 90 days 0, 100, 300, 1000 mkd **NOAEL not identified.** 6 hr/day for 90 days FOB/MA assessed prior to dosing during wk 10 and 20 minut es after removal of test material during wk 11 Dermal LOAEL = 100 mkd, based on decreased motor activity in both sexes at all dose levels. With 31% DAF, internal dose of 31 mkd (LOAEL)

870.3800 2 - generation

reproduction (rat , Sprague - Dawley) MRID 47744 117 (2009) Acceptable/Guideline Crl:CD(SD) 10 weeks pre - mating 0, 7.5, 50, 150 mkd **NOAEL = 7.5 mkd.** 10 weeks pre - mating via gavage assessed ≈30 minutes post dose LOAEL = 50 mkd based on numerous clinical signs (both sexes; both generations); subdued appearance; rocks, lurches, or sways as it walks; pupils dilated; and hypoactivity; at 150 mkd, increased incidences tremors (both sexe s), respiratory effects, and clonic convulsions (females), gasping (F1 male pups).

870.3700a Developmental toxicity (rat , Sprague - Dawley) MRID 47755402 (2008) Acceptable/Guideline GD 6 - 19 0, 5, 25, 100 mkd **NOAEL = 25 mkd.** GD 6 - 19 (14 days) via gavage assessed ≈30 minutes post dose LOAEL = 100 mkd, based on mortality (1 on GD 19) , clinical signs (behavioral findings of subdued appearance; rocking, lurching, or swaying while am bulating; rocking, piloerection, dilated pupils, sitting with head held low and appeared hypoactive but exhibiting normal behavior when stimulated), and wet clear material around mouth.

870.3700b Developmental

toxicity (rabbit) MRID 47755401 (2008) Acceptable/Guideline GD 6 - 29 0, 10, 50, 200 /150 mkd **Maternal NOAEL = 10 mkd.**  **Deve lopm**  **ental NOAEL = 50 mkd.**  GD 6 - 29 (24 days) via gavage assessed ≈30 minutes post dose LOAEL = 50 mkd, based on mortality (GD 16) , clinical signs ( tremors, prostration, and/or clonic convulsions); at 200 mkd, mortality GD 6, 9 @ 200 mkd , clinical signs ( tremors, prostration, and/or clonic convulsions). LOAEL = 200 mkd (developmental). 870.5100 Gene Mutation

Ames test MRID 47752704 (2008)

Acceptable/Guideline 0 - 5000μg/plate Negative. No evidence of induced mutant colonies over background 870.5300 Forward mutation

V79/HPRT MRID 47755404 (2008)

Acceptable/Guideline 0 - 9 μg/mL Negative. No evidence of induced mutant colonies

over background in the presence or absence of S9 - activation . 870.5375 M ammalian cell

cy togenetics assay MRID 47752705 (2008) Acceptable/Guideline 0 - 2000 μg/mL There was no evidence of chromosome aberrations induced over background in the presence or absence of S9 - activation. 870.5395 B one marrow

micronucleus assay MRID 47755403 (2008) Acceptable/Guideline There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in boneDemiditraz Human Health Risk Assessment D410552

Page **26** of **47 Table A.2.2.**  **Demiditraz Study Details and Neurotoxicity Effects Observed Guideline**  **No. Study Type**  **MRID No. (yea r)/**  **Classification Doses Results**  0 - 1000 mg/kg/day marrow after any treatment time.

870.6300 Developmental

neurotoxicity (rat , Sprague - Dawley) MRID 48766703 (2012) Acceptable/Guideline Crl:CD(SD) GD 6 to LD 21 0, 5, 15, 100 mkd Maternal **NOAEL = 5 mkd.** From GD 6 to LD 21 ( ≈40 days) via gavage Offspring **NOAEL = 15 mkd.** Until PND 72 via gavage ( ≈50 days) assessed ≈30 minutes and 2 hrs post dose Maternal LOAEL = 15 mkd , based on maternal clinical signs **(** sitting with the head held low, hypoactivity, a flattened body, slightl y drooping eyelids, decreased respiration, clear material around the mouth/salivation, lacrimation, and/or dilated pupils); at 100 mkd, same as above and slightly increased post - dosing incidences of findings indicative of diminished maternal care (dam away from the nest, but not eating, drinking, grooming, or tending to the litter, and 1 - 3 pups outside of the nest). Offspring LOAEL = 100 mkd, based on based on decreased postnatal survival, decreased pup body weights and body weight gains, increased maximum response to the auditory startle stimulus and reduction in response time on PND 60, decreased/increased total and ambulatory mo tor activity counts, decreased grip strength (PND 21 and PND 35), decreased brain w eight in PND 21 males, and delayed attainment of 870.7485 General metabolism MRID 47744136 (2008) MRID 47752712 (2008) MRID 47752713 (2009) MRID 47752714 (2009) MRID 47755406 (2009) 92% of administered oral dose recovered in 48 hours; 71% (urine); 21% (feces). Excretion nearly complete (84%) in 24 hours. i.v., oral, i.p., similar half - life of 0.28 to 0.5 hour. i.v., oral, dermal half

- lives of 1.06, 4.72, 347 hours; bioavailability reported to be 10.1% (oral); 18.6% (dermal).

870.7800 Immunotoxicity

(rat, Sprague - Dawley) MRID 47744122 (2009) Crl:CD (SD) 0, 10, 30, 100 mkd via gavage (28 days) NOAEL (systemic toxicity) = 30 mkd. assessed ≈30 minutes post dose Negative for immunotoxicity potential. 100 mkd (HDT). LOAEL (systemic toxicity) = 100 mg/kg/day , based on clinical signs of toxicity (rocking lurching, or swaying while walking; drooping eyelids; subdued appearance at cage - s ide observation; flattened body; hunched posture; clear material around mouth; and yellow material on various body surfaces). 870.7600 Dermal absorption (rat) MRID 47752709 (2008) 31% based on amount absorbed and remaining on skin after 168 hours.Demiditraz Human Health Risk Assessment D410552

Page **27** of **47 A**  **.3 Hazard Identification and Endpoint Selection A.3.1**  **Acute and Chronic Reference Doses (aRfD and cRfD) –**  **All Populations Study Selected**  **:** No studies were selected. These value s are calculated for dietary exposures, which currently

is not a relevant route of exposure for the proposed use. **A.3.2 Incidental Oral**  **– Short** **- and Intermediate**  **- Terms**  **Study Selected :**  Combined Subchronic Oral Toxicity and Neurotoxicity

Study **MRID No. :**  47744114 **Executive Summary :**  See Appendix A, Guideline [§ 870.3100 and 870 .6200b] **Dose and Endpoint for Establishing aRfD :**  The NOAEL of 5 mg/kg/day is based on clinical signs of neurotoxicity (subdued appearance, rocks, lurches/sway s when walking, hunc hed posture, hy

poactivity , etc.) in both sexes . **UF(s) :**  A UF of 100 was a pplied to account for interspecies extrapolation (10X) and intraspecies variation (10X). **Comments on Study/Endpoint/UFs :**  This study provides the lowest NOAELin the database considered to be valid for this exposure and all durations and is protective f or an y potential adverse effects in humans. The developmental neurotoxicity study in rats provided the same NOAEL of 5 mg/kg/day

in the maternal animals. The combined subchronic oral toxicity and neurotoxicity

study in rats was selected in preference to the developmental neurotoxicity study because of the extensive neurotoxicity evaluations carried out in the adult male and female for up to 90 day s. Most of the other oral toxicity studies gave similar values for the NOAEL: 15 mg/kg/day

in the acute neurotoxicity study in rats, 10 mg/kg/day

in the subchronic oral toxicity study in dogs, 7.5 mg/kg/day

in the 2 - generation reproduction study in rats, and 10 mg/kg/day for maternal toxicity

in the developmental toxicity study in rabbits. Demiditraz acts on neurotransmitter s ys tems in mammals. Clinical signs of neurotoxicity were the most sensitive endpoint and were observed in all the toxicity studies. The UFs included the standard accounting for interspecies extrapolation and intraspecies variation; no addi tional uncertainty factors were needed. There was no evidence provided which would indicate that children or more sensitive to the effects than adults. **A.3.3 Dermal**  **– All Durations**  **Study Selected :**  Subchronic Dermal Toxicity Study **MRID No. :**  47752701 **Executive Summary :**  See Appendix A, Guideline [§ 870.3250] **Dose and Endpoint for Establishing aRfD :**  A NOAEL was not established. The LOAEL ≤ 100 mg/kg/day is based on decreased motor activity and ungroomed appearance.Demiditraz Human Health Risk Assessment D410552

Page **28** of **47 UF(s)**  **:** A UF of 300 was applied to account for interspecies extrapolation (10X), intraspecies variation (10X), and the use of a LOAEL instead of a NOAEL (3X). **Comments on Study/Endpoint/UFs :**  This study provides the lowest LOAELin the database considered to be valid for this exposure and all durations and is protective for an y potential adverse effects in humans. Considering that the dermal absorption is 31%, the internal dose is expected to be 31 mg/kg/day . An uncertainty factor of 3X was applied for the use of a LOAEL instead of a NOAEL. Thus, the expected internal dose for the NOAEL is 10 mg/kg/day , which is similar to the results of the oral studies. As this was the case, it was preferable to use the dermal study for the estimation of a dermal endpoint rather than an oral study . Demiditraz acts on neurotransmitter s ys tems in mammals. Clinical signs of neurotoxicity were the most sensitive endpoint and were observed in all the toxicity studies. The UFs included the standard accounting for interspecies extrapolation and intrasp ecies variation, and an additional factor for using a LOAEL instead of a NOAEL(3X). There was no evidence provided which would indicate that children or more sensitive to the effects than adults. **A.3.5 Inhalation**  **– All Durations**  **Study Selected :**  Combine d Subchronic Oral Toxicity and Neurotoxicity

Study **MRID No. :**  47744114 **Executive Summary :**  See Appendix A, Guideline [§ 870.3100 and 870.6200b] **Dose and Endpoint for Establishing aRfD :**  The NOAEL of 5 mg/kg/day is based on clinical signs of neurotoxicity (subdued appearance, rocks, lurches/sway s when walking, hunc hed posture, hy

poactivity , etc.) in both sexes . **UF(s) :**  A UF of 100 was applied to account for interspecies extrapolation (10X) and intraspecies variation (10X). An additional factor (10X) s hould be applied when calculating reference doses for long - term exposure. **Comments on Study/Endpoint/UFs :**  This study provides the lowest NOAELin the database considered to be valid for this exposure and all durations and is protective for an y potential adverse effects in humans. No inhalation study was submitted, but inhalation exposures are not anticipated from either occupational or residential use of the spot - on product. Consequently , the oral study was selected, and complete absorption through the respiratory s ystem was assumed. The developmental neurotoxicity study in rats provided the same NOAEL of 5 mg/kg/day in the maternal animals. The combined subchronic oral toxicity and neurotoxicity

study in rats was selected in preference to the developm ental neurotoxicity study because of the extensive neurotoxicity

evaluations carried out in the adult male and female for up to 90 day s. Most of the other oral toxicity

studies gave similar values for the NOAEL: 15 mg/kg/day in the acute neurotoxicity

study in rats, 10 mg/kg/day

in the subchronic oral toxicity study in dogs, 7.5 mg/kg/day

in the 2 - generation reproduction study in rats, and 10 mg/kg/day for maternal toxicity in the developmental toxicity study in rabbits. Demiditraz acts on neurotransmitt er sy stems in mammals. Clinical signs of neurotoxicity were the most sensitive endpoint and were observed in all the toxicity

studies. The UFs included the standard accounting for interspecies extrapolationDemiditraz Human Health Risk Assessment D410552

Page **29** of **47** and intraspecies variation; no additional uncer tainty factors were needed, except when assessing long - term durations (10X). There was no evidence provided which would indicate that children or more sensitive to the effects than adults. **A.4 Executive Summaries**  **A.4.1 Sub**  **- chronic Toxicity**  **870.3100a C**  **om bined Subchronic Oral Toxicity and Neurotoxicity**  **– Rat**  In a subchronic neurotoxicity study (2008, MRID 47744114 ), PF - 3814927 (Demiditraz, 100% a.i.; Batch No. PFXU070002) in 0.5% methylcellulose and 0.1% Tween 80  was administered daily via gavage (5 mL/kg) to 22 Sprague - Dawley rats/sex/group at dose levels of 0, 5, 25, or 125 mg/kg/day

for 13 weeks. Ten rats/sex/dose were designated as the Phase I (general toxicity) group, and were evaluated for hematology , clinical chemistry , ophthalmology , organ weight, and gross and histopathology parameters. Neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) was performed on the remaining 12 rats/sex/group (Phase II) at pre - dosing and Weeks 1, 3, 7, and 12 (prior to admini stration of dosing). At study termination, all Phase II animals were anesthetized and perfused in situ for brain weights and morphometry . The tissues from 6 rats /sex/group of the perfused animals in the control and 125 mg/kg/day

groups were subjected to histopathological evaluation of central and peripheral nervous s ys tem tissues. At 25 mg/kg/day , body

weights were decreased (p<0.05) by 7 - 8% in the males during Weeks 8 - 13 and b y 7% each in the females at Weeks 7 and 10. Body weight gains were decrease d (p<0.05) b y 33% in the males during Weeks 7 - 8. Overall body weight gains were decreased (p<0.05) b y 11% in the males. At 30 minutes post - dosing, the following treatment - related clinical signs of toxicity

were observed (# affected/22 vs. 0/22 controls, unless otherwise stated): (i) subdued appearance at cage - side observation only (10 males and 9 females); (ii) rocks, lurches, or sway s when walking (6 males and 6 females); (iii) hunched posture (6 females); (iv) hy poactivity

(2 males); (v) wet yellow mate rial urogenital area (3 males and 6 females); (vi) lacrimation right/left eye (8 males and 19 females vs. 2 control females); (vii) dilated pupil right/left eye (5 males and 6 females); (viii) dried red material around mouth (4 males and 1 female vs. 1 control male); and (ix) wet red material around mouth (2 males). Cumulative ambulatory

activity was increased (p<0.05) by 41 - 45% in the males at Weeks 3 and 7. At 125 mg/kg/day , body

weights were decreased (p<0.01) by 9 - 14% in the males and by 7 - 10% in the females during Weeks 3 - 13. Additionally , body weight gains were sporadicall y decreased (p<0.05) b y 14 - 48% in the males and by 17 - 56% in the females. Overall (Weeks 0 - 13) body weight gains were decreased (p<0.01) b y 15 - 19% in both sexes. At 30 minutes po st - dosing, the following treatment - related clinical signs of toxicity were observed (# affected/22 vs. 0/22 controls, unless otherwise stated): (i) subdued appearance at cage - side observation only (all males and females); (ii) rocks, lurches, or sway s whe n walking (all males and females); (iii) hunched posture (17 males and all females); (iv) hypoactivity (11 males and 3 females); (v) wet ye llow material urogenital area (20 males and 21 females); (vi) shallow respiration (7 males and 1 female); (vii) decre ased respiration (10 males); (viii) lacrimation right/left eye (20 males and all females vs. 2 control females); (ix) dilated pupil right/left eye (20 males and 19 females); (x)Demiditraz Human Health Risk Assessment D410552

Page **30** of **47** dried red material around mouth (9 males and 11 females vs. 1 control male); and (xi) wet red material around mouth (8 of each sex). C umulative (session) total activity was increased by 29 - 32% in the males at Weeks 1 and 3 and b y 31 - 32% in the females throughout the study . Also at this dose, cumulative ambulatory activity was incr eased b y 43 - 56% in the males at Weeks 1, 3, and 7 and b y 49 - 65% in the females throughout the study . No compound - related effects were observed in mortality , food consumption, ophthalmoscopic examinations, hematology, clinical chemistry , FOB parameters (no te: FOB assessments were made prior to dail y dosing), brain weight and morphology, organ weights, gross lesions, or microscopic lesion in either sex. Th e LOAEL was 25 mg/kg/day based on multiple clinical signs of toxicity in both sexes, decreased body wei ght gains in males, and increased motor activity in males. The NOAEL is 5 mg/kg/day . The study is classified as acceptable/guideline and satisfies the guideline requirement (OPPTS 870.6200b; OECD 424) for a subchronic neurotoxicity study in rats. **870.3 150**  **90 -**  **Day Oral Toxicity –**  **Dog** In a subchronic 90 - day

oral toxicity

study (2009, MRID 484593 02) demidiraz (99.8% a.i., Lot # TCK08001K) was administered to 4 beagle dogs/sex orally in gelatin capsules at dose levels of 0 (control), 10, 30 or 100 mg/kg bw/ day . Clinical signs of toxicity , mortality , ophthalmologic , hematology , clinical chemistry , urinal ys is, electrocardiographic measurements, pathology and biokinetic data were obtained. There were no adverse effects on mortality , ophthalmology , hematology , clinical chemistry urinaly sis and electrocardiographic examinations . In the 30 mg/kg/day hypoactivity

was observed in 1 male and 1 female, and in all 8 dogs in the 100 mg/kg/day group. There was an increased incidence of vomitus containing food in 2 mal es and 2 females in the 30 mg/kg/day group and in 4 males and 3 females in the 100 mg/kg/day group . Body weight gain was statistically

significantly

decreased in males in the 100 mg/kg/ day group from predosing day 17 to Day 91 by 20% . Females in the 100 mg/kg day group from predosing day 17 to Day 91 showed a loss of weight of 0.2 kg . Food consumption was statistically significantl y decreased in males in the 100 mg/kg/day group from 36 - 80% . Three of 4 males in the 100 mg/kg/day group exhibited treatment related minimal cell infiltrates in the adrenal zona fasiculata. The LOAEL is 30 mg/kg bw/day in males and females, based on behavioral toxicity ; i.e., an increased incidence of h ypoactivity . The NOAEL in males and females is 10 mg/kg bw/day . Pharmac okinetic data indicated that demiditraz did not bioaccumulate and there were no sex differences in the pharmacokinetics. This 90 - day

ora l toxicity

study in the dog is acceptable/guideline and satisfies the requirement for a 90 - day

oral toxicity study

(OP PTS 870.3150; OECD 409) in the dog .Demiditraz Human Health Risk Assessment D410552

Page **31** of **47 870.3250**  **90 -**  **Day Dermal Toxicity –**  **Rat** In a repeated - dose dermal toxicity study (2008, MRID 47752701), PF - 3814927 (Demiditraz,  99.5 % a.i., L ot #s PFXA070001 and PFXU070002) in deionized water was applied to the shave d intact skin of 10 Sprague - Dawley rats /sex/dose at dose levels of 0, 100, 300, or 1000 mg/kg/day

(limit dose), 6 hours/day for 91/92 consecutive day s. Additional groups of 3 rats/sex/dose were exposed at the same doses for 87 consecutive day s. Blood was collected from the latter animals on Days 0 and 86 (approximately 6, 12 and 24 hours after application of the test material and the plasma was used for toxic okinetic exposure evaluations. Systemic exposure (as assessed b y C max and AUC 0 - 24 ) “ generally inc reased or remained unchanged with increasing dose. ” For example, at day 87 the mean plasma concentration (ng/ml) was 479±743, 1150±1540 and 1270±715 for the 100, 300 and 1000 mg/kg/day dose groups, respectively . T here is an apparent increase between the lowest dose and middle dose but the two highest doses are closer together. Clinical signs were consisted of increased incidences of wet and/or dried yellow material on various body

surfaces and dried red material around one or both eyes and nose in the 300 and 1000 mg/kg/day

males and females. During the handling observations of the FOB at Weeks 10 (prior to dosing) and 11 (after dose removal), unkempt appearance (slightl y soiled to very soiled fur) was noted in the 1000 mg/kg/day males and females. D uring Week 11 (after dose removal), total motor activity

was decreased (p<0.05) in all treated male groups during the 0 - 10 and 11 - 20 minute intervals and all treated female groups during the 11 - 20 minute interval. These decreases in the males resulted in decreases (not dose - dependent, p<0.05) in cumulative total activity of 26, 40, and 26% in the 100, 300, and 1000 mg/kg/day groups, respectively . The motor activity decrease is consistent with the decrease seen in the oral acute neurotoxicity study noted f ollowing dosing. No compound - related effects were observed in mortality, body weight, body weight gain, food consumption, ophthalmoscopic exams, hematology , clinical chemistry , absolute or relative organ weights, or gross or microscopic pathology in eithe r sex. Application site dermal effects were limited to very slight to slight ery

thema noted in the 300 and 1000 mg/kg/day

groups and are considered self limiting. The NOAEL (s ys temic) < 100 mg/kg/day . The LOAEL is 100 mg/kg/day based on decreased moto r activity

in both sexes at all dose levels tested. The LOAEL (local irritation) = 300 mg/kg/day based on ery

thema. The NOAEL is 100 mg/kg/day . This study

is classified as acceptable/guideline and satisfies the guideline requirement for a 90 - day der mal toxicity

study (OPPTS 870.3250; OECD 411) in rats .Demiditraz Human Health Risk Assessment D410552

Page **32** of **47 A.4.2**  **Prenatal Developmental Toxicity 870.3700a Prenatal Developmental Toxicity Study**  **– Rat**  In a developmental toxicity study ( 2008, MRID 47755402 ), Demiditraz technical ( PF - 3814927; 100% a.i.; Batch # PFXU070002) in 0.5% methylcellulose and 0.1% Tween TM 80 was administered dail y via gavage in a dose volume of 10 mL/kg to 25 time - mated presumed pregnant Sprague Dawley rat s/dose group at dose levels of 0, 5, 25, or 1 00 mg/kg/day from gestation day s (GD) 6 - 19 . B lood samples were collected from 4 randomly selected rat s per group 1 hour af

ter dose administration on GD 19 to determine test substance levels in plasma. On GD 29, all maternal rats were euthanized; each dam ’s uterus and ovaries were remove d via cesarean section and the contents examined. The fetuses were examined for external, visceral, and skeletal malformations and variations. Plasma concentrations . Demiditraz concentrations in plasma increased in a dose - dependent manner, with measureme nts of 2.41, 39.9, and 1020 ng/mL in the 5, 25, and 100 mg/kg/day groups. Maternal toxicity. At 100 mg/kg/day , one female (# 83309) was found dead approximately 0.5 hours following dose administration on GD 19. At the 0.5 hour post - dose observations, this female had: behavioral findings of subdued appearance upon cage - side observations; rocking, lurching, or swaying while ambulating on 3 to 5 occasions during GD 6 - 18; and a single occurrence of clear material around the mouth on GD 14. Although a caus e of death could not be determined at necropsy, this death was considered treatment - related because it occurred around the time of expected peak exposure (0.5 hours post - dose), and the indications of maternal toxicity (post - dosing clinical observations, de creased body weight gain, and decreased food consumption) were similar to the findings found in the rest of the animals in this group. All other dams survived until scheduled termination. At 100 mg/kg/day , the dams exhibited numerous clinical signs of to xicity at the 0.5 - hour post dosing examinations including rocking, lurching, or swaying while walking, piloerection, dilated pupils, subdued appearance upon cage - side observation only (sitting with head held low and appeared h ypoactive but exhibiting normal behavior when stimulated), and wet clear material around mouth. These findings were noted as early as the first day of dose administration (GD 6) and generally

continued throughout the treatment period. Additionally , salivation immediately prior to dos e administration was noted at this dose but was attributed to a response to the taste or irritating properties of the test material and was not considered adverse. At 100 mg/kg/day , body

weights were decreased by 5 - 6% (p<0.05) on GD 18, 19, and 20. Boy we ight gains were decreased (p<0.01) b y 58% during GD 6 - 10, by 16% for GD 14 - 20, and b y 20% for the overall (GD 6 - 20) treatment period. Gravid uterine weights were decreased b y 12% (p<0.01), and corrected body weight gain for the overall (GD 0 - 20) study wer e decreased b y 15% (p<0.05). Absolute and relative food consumption was decreased (p<0.01) by 14 - 15% for GD 6 - 10 and b y 6 - 11% for GD 6 - 20. Additionally at this dose, absolute food consumption was decreased b y 7% for GD 14 - 20.Demiditraz Human Health Risk Assessment D410552

Page **33** of **47** The maternal LOAEL is 100 mg/kg/day based on mortality , clinical signs of toxicity , and on decreased body weights, body weight gains, and food consumption. The maternal NOAEL is 25 mg/kg/day . Developmental toxicity. There were no abortions, premature deliveries, complete litter resorptions, or dead fetuses. Furthermore, there were no treatment - related effects on the numbers of litters, early

resorptions, late resorptions, or live fetuses. Sex ratio and post implantation loss were unaffected b y treatment. Fetal variations were limited to the incidences of delayed ossification. At 100 mg/kg/day , fetal body weights were decreased by 11% (p<0.05) each in the males and females. The incidence of sternebra(e) #5 and/or #6 unossified was increased at 100 mg/kg/day (42.3% fetuses; 95. 7% litters) compared to concurrent controls (26.1% fetuses; 87.0% litters) and historical controls (0.2 - 23.1% fetuses). Similarly , the incidence of reduced ossification of the vertebral arches was increased at 100 mg/kg/day (1.9% fetuses; 17.4% litters) c ompared to concurrent controls (0%) and historical controls (0.0 - 1.1% fetuses). Several other skeletal variations characterized b y delayed ossification differed from concurrent controls at 100 mg/kg/day and, although the fetal incidences of these findings fell within the range of historical controls, they were considered to be treatment - related because they corresponded to the statistically significant, treatment - related decreases in fe tal body

weights at this dose. These variations included: (i) increas es in sternebra(e) #1, 2, 3, and/or 4 unossified at 100 mg/kg/day (1.1% fetuses; 17.4% litters) compared to concurrent (0.3% fetuses; 4.3% litters) and historical (0.0 - 1.3% fetuses) controls; (ii) increases in pubis unossified at 100 mg/kg/day (1.6% fetuse s; 4.3% litters) compared to concurrent (0%) and historical (0.0 - 2.3% fetuses) controls; (iii) increases in hyoid unossified at 100 mg/kg/day

(2.3% fetuses; 17.4% litters) compared to concurrent (1.4% fetuses; 17.4% litters) and historical (0.0 - 4.2% fetuse s) controls; and (iv) decreases in incidence of ossified cervical centrum #1 at 100 mg/kg/day (12.0% fetuses; 65.2% litters) compared to concurrent (19.7% fetuses; 78.3% litters) and historical (6.6 - 35.8% fetuses) controls. There were no treatment - related external, visceral, or skeletal malformations . The developmental LOAELis 100 mg/kg/day based on decreased fetal body weights and associated delayed ossification of the skeleton. The developmental NOAEL is 25 mg/kg/day . This study

is classified acceptable/guideline and satisfies the guideline requirement (OPPTS 870.3700a; OECD 414) for a developmental toxicity study in rats. **870.3700b Prenatal Developmental Toxicity Study –**  **Rabbit** In a developmental toxicity study (2008, MRID 4775 5401 ), Demiditr az technical ( PF - 3814927; 100% a.i.; Batch # PFXU070002) in 0.5% methylcellulose and 0.1% Tween TM 80 was administered via gavage in a dose volume of 5 mL/kg to 25 time - mated presumed pregnant New Zealand White rabbits/dose group dail y from gestation days ( GD) 6 - 28 . Initial doses were 0, 10, 50, and 200 mg/kg/day . Due to excess toxicity , the high dose level was lowered to 150 mg/kg/d ay

on GD 9, 10, or 11 for the remainder of the study . B lood samples were collected from 4 rabbits per group 1 hour after dos e administration on GD 28 to determine test substanceDemiditraz Human Health Risk Assessment D410552

Page **34** of **47** levels in plasma. On GD 29, all maternal rabbits were euthanized; each doe ’s uterus and ovaries were removed via cesarean section and the contents examined. The fetuses were examined for external, vis ceral, and skeletal malformations and variations. Plasma concentrations . Demiditraz concentrations increased in a dose - dependent manner, with measurements of 411, 1940, and 5390 ng/mL in the 10, 50, and 200/150 mg/kg/day animals. It was stated that this time point was the expected maximum plasma concentration (0.5 to 2.2 hours) based on the previously conducted range - finding study (Bowman, 2007, WIL - 344041). Maternal toxicity. The morbidity in the three animals at the high dose occurred earl y in the s tudy (GD 6 or 9) when the animals were receiving 200 mg/kg/day . The female that was euthanized in extremis at 50 mg/kg/day was observed with tremors and rales approximately 30 minutes following dose administration on GD 14, two days prior to euthanasia on GD 16. The deaths/euthanasia in the 50 and 200/150 mg/kg/day groups occurred along with clinical signs indicating neurotoxicity

(tremors, prostration, and/or clonic convulsions) approximately 30 minutes following dose administration. At 50 mg/kg/day , b ody

weights were decreased by 6% (p<0.05) on GD 14 - 16. At 200/150 mg/kg/day , body

weights were decreased b y 5 - 9% beginning on GD 8 and continuing throughout the remainder of the study ; with the exception on GD 26 and 27, these decreases were statistically significant (p<0.05). Statistically significant (p<0.05) body weight losses were noted in the 50 mg/kg/day

( - 11 g) and 200 mg/kg/day ( - 154 g) groups compared to a body weight gain in the control group of 35 g at the beginning of the dose administration p eriod (GD 6 - 9). For GD 9 - 12, body

weight gains were decreased by 88% (p<0.01), and a significant (p<0.01) body weight loss was observed at 200/150 mg/kg/day ( - 4 g) compared to a body weight gain in controls (58 g). For the overall treatment period (GD 6 - 29), a significant (p<0.01) body weight loss was noted at 200/150 mg/kg/day ( - 51 g) compared to a body weight gain in the controls (215 g). Absolute and relative (to body weight) food consumption was decreased (p<0.01) by 20 - 27% at 50 mg/kg/day

for GD 6 - 9 and 9 - 12 and b y 42 - 65% at 200/150 mg/kg/day for GD 6 - 9, 9 - 12, and 12 - 21. Additionally

at 50 mg/kg/day , absolute food consumption was decreased b y 24% (p<0.05) for GD 12 - 21. Absolute and relative food consumption for the overall (GD 6 - 29) treatment peri od were decreased (p<0.05) by 15 - 20% at 50 mg/kg/day and by 38 - 42% at 200/150 mg/kg/day . No treatment - related findings were found at necrops y. The maternal LOAEL is 50 mg/kg/day

based on incidences of mortality /moribundity , clinical signs of toxicity , an d decreased body weights, body weight gains, and food consumption. The maternal NOAEL is 10 mg/kg/day . Developmental toxicity. There were no complete litter resorptions or dead fetuses. Furthermore, there were no treatment - related effects on the number s of earl y resorptions, late resorptions, or live fetuses per doe. Sex ratio was unaffected b y treatment. There were no treatment - related external, visceral, or skeletal malformations . At the high dose, fetal body weights were decreased by 12% (not sign ificant [NS]) in the males and by 13% (p<0.05) in the females. Fetal and litter incidences of rabbits with 27 presacralDemiditraz Human Health Risk Assessment D410552

Page **35** of **47** vertebrae were dose - dependently increased at 50 mg/kg/day (15.0% fetuses, 54.5% litters) and 200/150 mg/kg/day

(29.6% fetuses, 64.7% li tters) compared to concurrent controls (5.0% fetuses, 26.1% litters). The fetal incidences of this variation at 50 mg/kg/day fell within the range of historical controls (4.8 - 15.6% fetuses); however, the incidence at 200/150 mg/kg/day exceeded the range o f historical controls. Therefore, the incidences of 27 presacral vertebrae at 200/150 mg/kg/day

were considered to be due to treatment. Similarly , the fetal incidences of rabbits with full 13 th rib(s) were dose - dependentl y increased at 50 mg/kg/day (42.9 % fetuses, 90.9% litters) and 200/150 mg/kg/day (57.2% fetuses, 88.2% litters) compared to concurrent controls (33.2% fetuses, 91.3% litters). The fetal incidences of this variation at 50 mg/kg/day fell within the range of historical controls (16.5 - 45.7% fetuses); however, the incidence at 200/150 mg/kg/day

exceeded the range of historical controls. Therefore, the incidences of full 13 th rib(s) at 200/150 mg/kg/day were considered to be due to treatment. However, it should be noted that these findings (2 7 presacral vertebrae and 13 th full ribs) comprise two of the three most commonly

observed skeletal variations. The developmental LOAEL is 200/150 mg/kg/day based on decreased fetal body weights and slight increases in the incidences of 27 presacral vertebrae and 13 th full rib(s). The developmental NOAEL is 50 mg/kg/day. Note: Apparent increases in the skeletal variations at the high dose were also evident in the 50 mg/kg/day dose but were within historical control range. This study

is classified accep table/guideline and satisfies the guideline requirement (OPPTS 870.3700b; OECD 414) for a developmental toxicity study in rabbits. **A.4.3 Reproductive Toxicity**  **870.3800 Reproduction and Fertility Effects –**  **Rat** In a two - generation reproduction toxicity study ( 2009, MRID 47744117 ), Demiditraz ( PF 03814927; 100 % a.i.; Batch #PFXU070002 ) in 0.5% methylcellose and 0.1% Tween ® 80 (10 mL/kg) was administered daily by gavage at dose levels of 0, 7.5, 50, or 150 mg/kg/day fo r two consecutive generations. The P generation animals (3 0 /sex/dose group) were dosed for 10 weeks prior to mating to produce the F1 litters. F1 offspring ( 30 /sex/dose group) were dosed after weaning for at least 10 weeks prior to mating to produce the F2 generation. In the P and F1 gener ations, treatment - related generall y transient clinical findings indicative of neurotoxicity

were noted with increased incidences in the 50 and 150 mg/kg/day group at approximately

0.5 hours following dose administration starting as earl y as study day 0 and lasting through study

day 115. The findings were numerous and included effects such as subdued appearance; rocks, lurches, or sways as it walks; pupils dilated; and hy poactivity . Additionally

at 150 mg/kg/day , increased incidences tremors in both sexes and respiratory effects (except in F1 females) were noted and clonic convulsions were noted in females. Gasping was noted especially in F1 male pups at 150 mg/kg/day . In the P generation, one 150 mg/kg/day female died, exhibiting clonic convulsions immed iately following dosing. In the F1 generation following weaning and starting gavage of the pups, the numbers of decedents were higher in the 150 mg/kg/day group (7/sex) than the controls (2/sex). The majority

of these deaths occurred during PND 23 - 26.Demiditraz Human Health Risk Assessment D410552

Page **36** of **47** In the P generation, decreases were observed in body weights and gains on LD 7, 14, and 21 at 50 mg/kg/day

and throughout the lactation period at 50 and 150 mg/kg/day . Decreased food consumption was noted at 150 mg/kg/day . Decreased food efficiency was o bserved at 50 and 150 mg/kg/day . In the F1 generation at 150 mg/kg/day , decreases were observed in body weights throughout the lactation period; however, the resulting decrease in body weight gain was not significant. Decreased food consumption was noted in the 150 mg/kg/day group. At 150 mg/kg/day , decreased body weights and gains were noted in the P and F1 generations males. Average food efficiency (Day s 0 - 126) was decreased in these males. Average food efficiency ( PND 28 - 161) was also decreased. D ecreased body weights were noted in the 150 mg/kg/day

females in the F1 generation up to PND 91 resulting in a decreased body weight gain for the pre - mating period. At 150 mg/kg/day , decreases in body weights throughout the gestation period in the P and F1 generations , resulting in a decreased overall (GD 0 - 20) body weight gain. Food efficiency was decreased during gestation in both generations. No adverse, treatment - related effects were noted on organ weights or gross or histological pathology in ei ther generation. The LOAEL for parental toxicity is 50 mg/kg/day

based on: clinical findings indicating nervous sy stem toxicity

in both sexes and generations; and decreased body weight, body

weight gain, and food efficiency

during the lactation period in the P generation. The NOAELis 7.5 mg/kg/day . At 150 mg/kg/day , decreases were noted in the number of pups born ( ↓13% and 16%) and born alive (both ↓18%) in both the F1 and F2 generations. There was also an increase in the number of implantation site s that did not result in the birth of a pup in both the F1 and F2 litters. The viability indices were decreased in the F1 ( ↓19%) and F2 (↓29%) generations. The lactation indices in the treated groups of both generations were similar to controls. Decreas ed postnatal survival (% per litter) was observed during PND 0 - 1 in the F1 and F2 generations and during PND 1 - 4 in the F2 generation. At 50 mg/kg/day

in the F2 generation, decreased body weights were noted in both sexes ( ↓8 - 11% in males and 8 - 12% in fem ales) throughout lactation, and decreased body weight gains (up to 12%) were observed in the males at PND 4 - 7, 7 - 14, and 14 - 21 and females at PND 4 - 7 and 7 - 14. At 150 mg/kg/day , decreased pup body weights (up to 31%) were observed during PND 1 - 21 in both s exes and generations. Except for the F1 males and females on PND 1 - 4, body weight gains during PND 1 - 21 were also decreased in both sexes and generations (up to 45%). Absolute brain weights were decreased in the F2 generation at 50 mg/kg/day ( ↓4 - 6%) in both sexes in the F2 generation. At 150 mg/kg/day absolute brain weight was decreased 6 - 13%. No treatment - related macroscopic findings were observed in the F1 and F2 generations. Pup sex ratio and time until vaginal opening or balano - preputial separatio n were unaffected b y treatment. Developmental landmarks were not reported.Demiditraz Human Health Risk Assessment D410552

Page **37** of **47** The LOAEL for offspring toxicity is 50 mg/kg/day

based on decreased pup body weights and body weight gains in both sexes in the F2 generation and slight decrease in brain weight in the F2 generation. The NOAELis 7.5 mg/kg/day . There were no effects of treatment in either generation on : estrou s cycle; sperm parameters; mating, conception, fertility , or gestation indices; pre - coital interval ; or gestation duration in either gene ration. The LOAEL for reproductive toxicity was not observed. The NOAEL is 150 mg/kg/day . This study

is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3800; OECD 416) for a two - generation reproduction study in the rat. **A.4.4 Chronic Toxicity**  **– No study available**  **A.4.5 Carcinogenicity**  **– No study available**  **A.4.6 Mutagenicity**  Demiditraz was negative in the mutagenicity studies. **Study Dose Levels**  **Result In vitro**  **studies 870.5100**  **Bacterial Reverse Mutation** M RID#47752704 Mecchi, M.S. (2008) 0 - 5000 μg/plate Negative **870.5300 Mam**  **malian cell gene mutation (CHO)** MRID #47755404 Stankowski, L.F. (2008) 0 - 900 μg/mL Negative **870.5375 Mam**  **malian cytogenetics assay (chromosomal aberration assay in**  **human peripheral bloo d lymphocytes)**  MRID #47752705 Murli, H. (2008) 0 - 2000 μg/mL Negative

**In vivo studies**  **870.5395 Mouse Bone Marrow Micronucleus**  **Assay** MRID#47755403 Xu, Y (2008) 0 - 1000 mg/kg bw NegativeDemiditraz Human Health Risk Assessment D410552

Page **38** of **47 A.4.7**  **Neurotoxicity**

**870.6200a Acute Neurotoxicity Screening Batte ry**  **– Acute Rat**  In an acute neurotoxicity

screening study

(2008, MRID 47744113 ), PF - 3814927 ( Demiditraz , 99.2 % a.i., Batch # PFXU060001) in 0.5% meth ylcellulose/0.1% polysorbate 80 was administered once via gavage (5 mL/kg) to 12 Sprague - Dawley rats/sex/gr oup at dose levels of 0, 15, 100, or 600 mg/kg. Neurobehavioral assessment (functional observational battery [FOB] and motor activity

testing) was performed on 12 rats/sex/group at pre - dosing and day s 0 (approximately

15 minutes post - dosing; time of peak effect), 7, and 14. At study termination, 6 rats /sex/group were perfused in situ for neuropathological examination. The tissues from the perfused animals in the control and 600 mg/kg groups were subjected to histopathological evaluation of brain and peripheral nervous s ys tem tissues. Motor Activity. Findings at 15 mg/kg were limited to decreased (p<0.05) mean total and ambulatory

activity counts in the females (decr 17 - 18%) during the 0 - 15 minute interval on Day 0. Mean total and ambulatory activity counts were decreased (p<0.001) by 90 - 96% in males and 88 - 96% in females at 100 mg/kg and 600 mg/kg. At the higher doses, these decreases resulted in decreases (p<0.001) in cumulative total ( decr 68 - 90%) and ambulatory ( decr 74 - 93%) activit y counts. No s tatistically

significant differences in motor activity were noted on Day s 7 or 14. Although these values for the low dose group were lower than the minimum historical control values provided, these decreases were not sufficient to affect cumulative total or ambulatory activity counts, as animals at this dose had higher (not statistically significant) values than the concurrent control group for the remainder of the 60 - minute test session. FOB evaluations on Day 0. At 100 and 600 mg/kg, numerous effects were noted but these findings were generally

resolved by

Day 7. During the home - cage observations, the following treatment - related effects (# affected/12 vs. 0/12 controls, unless otherwise noted) were reported: (i) flattened posture (9 males and 11 femal es at 100 mg/kg, and 10 males and 7 females at 600 mg/kg); (ii

) immobility (9 males and 7 females at 100 mg/kg, and 7 males and 4 females at 600 mg/kg); and (iii) eyelids slightly drooping (6 females at 100 mg/kg, and 6 males and 7 females at 600 mg/kg vs. 3 control males and 0 control females). Additionally at 600 mg/kg on Day

0, slight tremors were observed in one male and 3 females, and moderatel y coarse tremors were observed in 2 males. Clonic convulsions (whole body tremors) were also observed at 600 mg/kg in 2 males and 1 female. During handling observations, eyelids slightl y drooping were noted in 7 rats of each sex at 100 mg/kg and in 10 males and 6 females at 600 mg/kg (vs. 0 controls). Additionally at 600 mg/kg, 6 males displayed slight resistance to handling (vs. 1 control). During the sensory observations, increased numbers of animals displayed no reaction to the following stimuli: (i) approach; (ii) touch; (iii) tail pinch; and (iv) startle. Several animals in these groups also exhibited no olfactory response, were slightly

uncoordinated (3 - 6 males and 2 - 9 females) or landed on their sides (4 males and 1 female at 600 mg/kg only) during air - righting reflex assessment. Additional findings, occurring in 1 or 2 animals at 600 mg/kg, consiste d of no eye blink response, no pupil response and no forelimb extension.Demiditraz Human Health Risk Assessment D410552

Page **39** of **47** During the open - field observations, the following treatment - related effects were noted: (i) impaired mobility , slight (5 males and 6 females at 100 mg/kg, and 7 males and 11 females at 600 mg/kg), moderate (3 males at 600 mg/kg), and totally impaired (1 rat of each sex at 600 mg/kg); (ii) abnormal gait , body drags (5 males and 2 females at 600 mg/kg), hindlimbs splayed or dragging (6 males and 5 females at 600 mg/kg), hunched body (4 males and 3 females at 100 mg/kg, and 2 males and 1 female at 600 mg/kg), and ataxia (8 females at 100 mg/kg, and 7 males and 10 females at 600 mg/kg); (iii) gait score , slight impairment (6 rats each sex at 100 mg/kg, and 4 rats each sex at 600 mg/kg), c onsiderable impairment (1 rat each sex at 100 mg/kg, and 5 males and 6 females at 600 mg/kg), severe impairment (1 male), and marked impairment (1 female) at 600 mg/kg); (iv) tremors , slight (2 males and 3 females at 600 mg/kg), moderatel y coarse (2 males and 1 female at 600 mg/kg), and markedl y coarse (one 600 mg/kg female); and (v) arousal , low (6 of each sex at 100 mg/kg, and 9 of each sex at 600 mg/kg vs. 1 control female), and very

low (one 600 mg/kg male). Additionally , one 600 mg/kg male displayed c ircling and retropulsion. Rearing counts were decreased (p<0.01) in the males (0.6 to 1.8 treated vs. 5.8 controls) and females (1.5 to 2.9 treated vs. 7.6 controls). Rearing counts in these males remained decreased on Day 7; however, the differences wer e not statistically significant. Urination counts were increased (p<0.05) in the  100 mg/kg males (1.4 to 1.7 treated vs. 0.2 controls) and females (1.2 to 1.7 treated vs. 0.2 controls). During the neuromuscular observations on Day 0, treatment - related decreases (p<0.01) in hindlimb footsplay

(decr 31 - 48%) and rotarod performance (decr. 55 - 67%) were noted at 600 mg/kg in both sexes. During phy siological observations on Day 0, body temperatures (  C) were decreased (p<0.01) at 100 and 600 mg/kg in both s exes (35.1 and 34.5 treated, respectivel y, in males vs. 37.2 controls, and 35.2 and 34.6 treated, respectivel y, in females vs. 37.8 controls). No compound - related effects were observed in mortality , body weight (except of initial decrease in day 0 - 7 gai n at 600 mg/kg), brain weight and morphology , or gross and neuropathology in either sex. The LOAEL was 100 mg/kg based on FOB findings (predominantly altered gait and posture, impaired mobility , decreased rearing, incoordination, increased urination, and lower body temperature) and statistically significant decreases in motor activity in both sexes. The NOAEL is 15 mg/kg. The slight decrease in interval motor activity at 15 mg/kg is a possible threshold effect. The study is classified as a cceptable/gu ideline and satisfies the guideline requirement (OPPTS 870.6200a) for an acute neurotoxicity study in rats. **870.6200b Subchronic Neurotoxicity Screening Battery –**  **Rat** In a subchronic neurotoxicity study (2008, MRID 47744114 ), PF - 3814927 (Demiditraz, 100 % a.i.; Batch No. PFXU070002) in 0.5% methylcellulose and 0.1% Tween 80  was administered daily via gavage (5 mL/kg) to 22 Sprague - Dawley rats/sex/group at dose levels of 0, 5, 25, or 125 mg/kg/day

for 13 weeks. Ten rats/sex/dose were designated as the Ph ase I (general toxicity) group, and were evaluated for hematology , clinical chemistry , ophthalmology , organ weight, and gross and histopathology parameters. Neurobehavioral assessment (functional observationalDemiditraz Human Health Risk Assessment D410552

Page **40** of **47** battery [FOB] and motor activity testing) was performed on the remaining 12 rats/sex/group (Phase II) at pre - dosing and Weeks 1, 3, 7, and 12 (prior to administration of dosing). At study termination, all Phase II animals were anesthetized and perfused in situ for brain weights and morphometry . The tissues from 6 rats /sex/group of the perfused animals in the control and 125 mg/kg/day

groups were subjected to histopathological evaluation of central and peripheral nervous s ys tem tissues. At 25 mg/kg/day , body

weights were decreased (p<0.05) by 7 - 8% in the males during Weeks 8 - 13 and by 7% each in the females at Weeks 7 and 10. Body weight gains were decreased (p<0.05) b y 33% in the males during Weeks 7 - 8. Overall body weight gains were decreased (p<0.05) b y 11% in the males. At 30 minutes post - do sing, the following treatment - related clinical signs of toxicity

were observed (# affected/22 vs. 0/22 controls, unless otherwise stated): (i) subdued appearance at cage - side observation only (10 males and 9 females); (ii) rocks, lurches, or sway s when wal king (6 males and 6 females); (iii) hunched posture (6 females); (iv) hy poactivity

(2 males); (v) wet yellow material urogenital area (3 males and 6 females); (vi) lacrimation right/left eye (8 males and 19 females vs. 2 control females); (vii) dilated pup il right/left eye (5 males and 6 females); (viii) dried red material around mouth (4 males and 1 female vs. 1 control male); and (ix) wet red material around mouth (2 males). Cumulative ambulatory

activity was increased (p<0.05) by 41 - 45% in the males at Weeks 3 and 7. At 125 mg/kg/day , body

weights were decreased (p<0.01) by 9 - 14% in the males and by 7 - 10% in the females during Weeks 3 - 13. Additionally , body weight gains were sporadicall y decreased (p<0.05) b y 14 - 48% in the males and by 17 - 56% in the fe males. Overall (Weeks 0 - 13) body weight gains were decreased (p<0.01) b y 15 - 19% in both sexes. At 30 minutes post - dosing, the following treatment - related clinical signs of toxicity were observed (# affected/22 vs. 0/22 controls, unless otherwise stated): (i) subdued appearance at cage - side observation only (all males and females); (ii) rocks, lurches, or sway s when walking (all males and females); (iii) hunched posture (17 males and all females); (iv) hypoactivity (11 males and 3 females); (v) wet ye llow material urogenital area (20 males and 21 females); (vi) shallow respiration (7 males and 1 female); (vii) decreased respiration (10 males); (viii) lacrimation right/left eye (20 males and all females vs. 2 control females); (ix) dilated pupil right/left eye (20 males and 19 females); (x) dried red material around mouth (9 males and 11 females vs. 1 control male); and (xi) wet red material around mouth (8 of each sex). C umulative (session) total activity was increased by 29 - 32% in the males at Weeks 1 and 3 and b y 31 - 32% in the females throughout the study . Also at this dose, cumulative ambulatory activity was increased b y 43 - 56% in the males at Weeks 1, 3, and 7 and b y 49 - 65% in the females throughout the study . No compound - related effects were observed in mortality , food consumption, ophthalmoscopic examinations, hematology, clinical chemistry , FOB parameters (note: FOB assessments were made prior to dail y dosing), brain weight and morphology, organ weights, gross lesions, or microscopic lesion in eithe r sex. Th e LOAEL was 25 mg/kg/day based on multiple clinical signs of toxicity in both sexes, decreased body weight gains in males, and increased motor activity in males. The NOAEL is 5 mg/kg/day .Demiditraz Human Health Risk Assessment D410552

Page **41** of **47** The study is classified as acceptable/guideline and sati sfies the guideline requirement (OPPTS 870.6200b; OECD 424) for a subchronic neurotoxicity study in rats. **870.6300 Developmental Neurotoxicity Study** In a developmental neurotoxicity study (MRID 48766703), demiditraz (PF - 03814927) (100% purity ; Batch 1 Lot # TCK08005K and Batch 2 Lot # TCK08005K) was administered daily by oral gavage to 25 mated female Sprague - Dawley [Crl:CD(SD)] rats per dose at dose levels of 0 ( 0.5% meth yl cellulose (400 cps)/0.1% Tween® 80 in deionized water), 5, 15, or 100 mg/kg/day from gestation day

(GD) 6 through lactation day (LD) 20. Detailed clinical observations were conducted on all dams (F 0 females) on GD 10 and GD 15 and on LD 10 and LD 20. All F 0 females were allowed to deliver and rear their offspring to LD 21. All sur viving F 0 females were euthanized on LD 21 and subjected to a gross necrops y. Clinical observations, body weights, and sexes were recorded for the F 1 pups at appropriate intervals. On postnatal day

(PND) 4, litters were culled to 8 pups/litter, and a subse t (Subset A) of 20 pups/sex/group was assigned to the functional observational battery (FOB; PND 4, 11, 21, 35, 45, and 60), auditory

startle response (PND 20 and 60), locomotor activity (PND 13, 17, 21, and 61), and learning and memory (PND 62) groups. Fr om this subset, 15 pups/sex/group were deepl y anesthetized and perfused in situ on PND 72 for brain weight and brain measurement evaluations; of these, 10 pups/sex/group were selected for neuropathological and brain morphometric evaluations. A second subs et (Subset B) of 20 pups/sex/group was selected for learning and memory

(PND 22). A third subset (Subset C) of 15 pups/sex/group was deepl y anesthetized and perfused in situ on PND 21 for brain weight and brain measurement evaluations; of these, 10 pups/se x/group were selected for neuropathological and brain morphometric evaluations. In dicators of phy sical development (balanopreputial separation and vaginal patency) were evaluated for all F 1 selected pups in Subsets A and B. All F 1 pups not selected for neuropathological or behavioral evaluations were euthanized and necropsied on PND 21. F 1 pups selected for learning and memory assessment on PND 22 were necropsied following attainment of sexual developmental landmarks. The litter was used as the experimental unit for all F 1 data. One F 0 female in the 100 mg/kg/day group was found dead on GD 21, and cause of death could not be determined. All other F 0 females surviv ed to the scheduled euthanasia. One female each in the control and 100 mg/kg/day groups faile d to deliver and were determined to be non - gravid. Treatment - related clinical findings were observed in a dose - dependent manner in the 15 and 100 mg/kg/day

group F 0 females and were observed at approximately 15 - 30 minutes post - dosing throughout the treatme nt period. In both dose groups, these findings included sitting with the head held low, h ypoactivity , a flattened body, slightly drooping eyelids, decreased respiration, clear material around the mouth/salivation, lacrimation, and dilated pupils. Additio nal signs seen in the 100 mg/kg/day

dose group included rocking, lurching, or swaying while ambulating, piloerection, tremors/convulsions, and yellow material on various body surfaces. These findings generall y continued to the 2 - hour post - dosing observatio n period for the 100 mg/kg/day group F 0 females, but at decreased incidences.Demiditraz Human Health Risk Assessment D410552

Page **42** of **47** Treatment - related decrements in maternal care were observed mainl y in the 100 mg/kg/day group F 0 females during the same interval post dose when clinical signs were observed. A decreased incidence of F 0 females on the nest, a higher incidence of females that were away from the nest (not actively eating, drinking, grooming or tending to the litter), and higher incidences of scattered litters with 1 - 3 pups and more than 3 pups o utside of the nest were all noted at the 100 mg/kg/day dose level. During the detailed clinical observations (FOB) for F 0 females, a flattened posture or sitting with the head held low, low or very low arousal, slight to moderatel y coarse tremors, lacrim ation, salivation, piloerection, slightly drooping eyelids, decreased respiration, soft and flabby muscle tone, impaired mobility , ataxia, dragging bodies, and/or higher urination counts were observed for the 100 mg/kg/day

group at each time points. Addit ionally , a flattened posture or sitting with the head held low, ataxia, impaired mobility , and a higher urination count were observed in a smaller number of F 0 females at 15 mg/kg/day from GD 15 on. Mean body weights for F 0 females in the 100 mg/kg/day group were slightly

( ↓4.6%) lower than the control group b y GD 20. F 0 females in the 100 mg/kg/day group had slightly

lower mean body weight gains throughout the gestation treatment period (initially ↓44%; overall GDs 6 - 20, ↓15%), and corresponding reducti ons in food consumption during GDs 6 - 12 ( ↓13 - 14). Mean body weights for F 0 females in the 100 mg/kg/day group were slightl y ( ↓4% to 6%) lower than the control group during LDs 4 - 17, but were similar to the control group on LD 21. A lower mean body weight gain was noted for F 0 females in the 100 mg/kg/day group during LDs 1 - 4 (67% less than controls), but mean body weight gains for this group were generall y similar to the control group for the remainder of the lactation treatment period (LDs 4 - 21). Mean g estation length of F 0 females was comparable among the groups, and there were no treatment - related effects on the process of parturition or macroscopic findings of the F 0 females. The maternal LOAEL is 15 mg/kg/day , based on clinical signs (sitting with the head held low, hy poactivity , a flattened body , slightly drooping eyelids, decreased respiration, clear material around the mouth/salivation, lacrimation, and/or dilated pupils) . The maternal NOAEL is 5 mg/kg/day . At 100 mg/kg/day , in addition to clini cal signs, decrements in maternal care were observed during the same time interval post dose when clinical signs were ob served (decreased incidence of F 0 females on the nest, a higher incidence that were away from the nest (not activel y eating, drinking, grooming or tending to the litter), and higher incidences of scattered litters with 1 - 3 pups and more than 3 pups outside of the nest. The mean live litter size in the 100 mg/kg/day group was slightl y lower than the control group (14.0 vs. 15.1 for contro ls), and mean postnatal survival for the 100 mg/kg/day group was lower than the control group during PND 0 - 1, 1 - 4 (pre - culling), and birth to PND 4 (pre - culling) (87.7% vs. 96.2% for the latter). At the 100 mg/kg/day dose level, a total of 24 pups were fo und dead and 20 others were missing (presumed cannibalized) compared with 11 found dead and 3 missing at the control level. At 100 mg/kg/day , F 1 pups of both sexes had decreased body weights [PND 1 ( ↓8% - 9%); PND 4 ( ↓14%); PND 21 ↓16%)] and body weight ga ins (PND 1 - 4 ( ↓21% - 29%); PND 4 - 7 ( ↓27%); PND 1 - 21 ( ↓16% - 18%) generally throughout the pre - weaning period, relative to their respectiveDemiditraz Human Health Risk Assessment D410552

Page **43** of **47** controls. Mean body weights of the F 1 males at 100 mg/kg/day were significantly

lower than the control (PND 28 - 72 ↓10% - 1 4%), and lower mean body weight gains were noted for the 100 mg/kg/day

F 1 males compared to the control throughout the post - weaning period (overall PND 28 - 72, ↓11% ). Mean body weights for the 100 mg/kg/day F 1 females were lower than the control during PND 28 - 72 ( ↓5% - 13%), and lower mean body weight gain was noted for the 100 mg/kg/day

group F 1 females during the first post - weaning week (PND 28 - 35, ↓8%), but mean body weight gains were similar to the control for the remainder of the post - weaning period and overall. A treatment - related delay (2.7 day s) in the mean age of attainment of balanopreputial separation was noted for the 100 mg/kg/day group F 1 males (47.0 day s vs. 44.3 day s for controls) but mean body weights of the F 1 males on the day of attainment were not affected. In contrast the F 1 females showed no treatment related effects on the mean age of attainment of vaginal patency , but a reduced mean body weight ( ↓11%) on the day of attainment of vaginal patency was noted for the F 1 females of the 100 mg/kg/day group. Mean forelimb grip strength for the 100 mg/kg/day group F 1 males and F1 females was significantl y lower than the control group on PND 21 and PND 35 but was no longer significantl y affected on PND 45 or PND 60. Although no statistically significant effects on locomotor activity or habituation were observed among the F 1 offspring, there were differences in response between the control and 100 mg/kg/day

groups and betwe en the sexes at 100 mg/kg/day . On PND 13, the concurrent control males displayed motor activity (MA) that was not consistent with the testing facility ’s historical control data, which resulted in a finding of decreased MA at all dose levels (males). However, when compared to the historical control (HC) mean, the PND 13 male s at 100 mg/kg/day showed an increase in MA (ambulatory ↑100%/total ↑45%) and the 5 and 15 mg/kg/day PND 13 males were comparable to the HC mean. PND 13 females at 100 mg/kg/day showed a reduction in MA (ambulatory ↓41%/total 20%) compared to the concurrent control. On PND 17, the 100 mg/kg/day

males displayed reduced MA and the 100 mg/kg/day females displayed MA that was comparable to the control. On PND 21, MA among the male groups was comparable, whereas the 100 mg/kg/day

females displayed an increase ( ambulatory ↑40%/total ↑2%) compared t o the control females. The male control and 5 and 15 mg/kg/day male dose groups showed the expected reduction in MA on PND 21 compared to PND 17 levels, but the 100 mg/kg/day males did not display

the expected reductio n in MA. On PND 61, males at 100 mg/kg/day displayed a reduced activity

(ambulatory ↓18%/total ↓14%), and females at 100 mg/kg/day were comparable to the control. Both sexes at 100 mg/kg/day displayed an increase in auditory startle response peak amplitude on PND 60 compared to the concurrent controls and the historical controls. The PND 60 males at 100 mg/kg/day

had a mean peak amplitude value that was outside the HC, whereas the PND 60 females at 100 mg/kg/day were within the HC range, although at the upper end. Additionall y, both sexes at 100 mg/kg/day displayed a reduction of 4 msec in latency time on PND 60 compared to the concurrent control and HC control.Demiditraz Human Health Risk Assessment D410552

Page **44** of **47** At 100 mg/kg/day , mean brain weight was significantly lower in PND 21 males compared to th e control, and relative brain weights were significantly increased in both se xes at 100 mg/kg/day on PND 21. Relative brain weight was significantly lower in the PND 60 males at 100 mg/kg/day . No treatment related effects were observed in the detailed c linical observations performed on PNDs 4, 11, 21, 35, 45, or 60 during the home cage, handling, open field, or sensory assessments. There were no treatment - related effects on the numbers of former implantation sites or unaccounted - for sites, the pup sex r atio at birth, pup survival following culling, clinical findings, swimming ability or learning and memory

assessments, macroscopic internal findings, gross or microscopic observations of the brain or peripheral nervous s ys tems, brain measurements at necrop sy , or morphometry taken from brain sections. The F 1 offspring LOAEL is 100 mg/kg/day , based on decreased postnatal survival, decreased pup body weights and body

weight gains, increased maximum response to the auditory startle stimulus and reduction in response time on PND 60, decreased/increased total and ambulatory motor activity

counts, decreased grip strength (PND 21 and PND 35), decreased brain weight in PND 21 males, and delayed attainment of balanopreputial separation. The corresponding F 1 offspring NOAEL is 15 mg/kg/day . This study

is classified Acceptable/Guideline and satisfies the guideline requirement for a developmental neurotoxicity study in rats [OCSPP 870.6300 ( § 83 - 6); OECD 426 (draft)]. **A.4.8 Metabolism**  **870.7485 Metabolism**  **- Rat**  No guideline study is available. **870.7600 Dermal Absorption**  **– Rat**  In a dermal penetration study ( 2008, MRID 47752709 ), [ 14 C] PF - 03814927 ( Demiditraz; 99.0 % radiochemical purity ; Code No. CFQ15145 Batch 1 ) formulated in dieth yl ene gl yc ol monobutyl eth er was applied to the shaved skin (10 cm 2 ) of Sprague Dawley rats. Doses of 0.0099, 0.0937, or 1.03 mg/rat were applied to the skin of each rat in volumes of 10 μl/cm 2 skin. Four males/dose/time point were tested using exposure durations and termination times of 0.5, 1, 2, 4, 10, or 24 hours post - administration. Additional groups of 4 male rats was exposed for 10 hours at the same dose levels, the application site was washed, and the rats were terminated at 168 after application. Analy tical recoveries were 76 - 108% of the applied dose (AD ) . The amount absorbed included residues found in the cage wash, blood, urine, feces, and carcass. After 24 hours exposure, there was 17.46%, 12.06% and 10.61% of the AD was determined to be absorbed for the low , mid an high dose exposure groups, respectivel y. The groups terminated at 168 hours post - application indicated additional absorption (25.8 - 28.1% AD) resulting from absorption from the skin depotsDemiditraz Human Health Risk Assessment D410552

Page **45** of **47** (skin at the application site and adjacent skin). The absorptio n rate constants were similar (0.055 - 0.089/hr) for each dose level. Thus, absorption was not saturated. Because absorption continued to occur from demiditraz remaining at the site of application after washing, the total absorbable dose (AD plus remainin g on the skin) should also be considered in a conservative estimate of absorption. The mid - dose provided the most conservative estimate of total absorbed (28.06%) and absorbable (3.09%) for a total of 31% and should be considered for a dermal penetration factor in risk assessment. Quantifiable residues were detected in the urine and feces b y 2 and 10 hours after application, respectivel y. Onl y minor amounts were found in the blood and cage wash. The carcass accounted for ~1 - 8% AD with the highest perce ntages in the low dose group. The majority of the residues eliminated in the feces and urine were excreted within 72 hours. Regardless of dose level, the elimination rate constants were similar in both the urine and feces (0.0227 - 0.0291/hr). The elimination half life was 28 - 31 hr in urine and 24 - 28 hr in feces. A conservative estimate of dermal absorption is 31% based on total absorbed and remaining on the skin after 168 hours. This study

is classified as acceptable/guideline and satisfies the guide line requirements (OPPTS 870.7600; OECD - none) for a dermal penetration study in rats. The study provides data that can be useful to derive a dermal absorption factor. Itmust be noted that the test material was applied in diethylene gl yc ol monobutyl eth er and the relationship of this vehicle to practical formulations in not known. **A.4.9 Immunotoxicity**  **870.7800 Immunotoxicity**  In an immunotoxicity

study (MRID 47744122 ), female Sprague Dawley Crl:CD(SD) rats (20 /dose group ) was administered daily via oral gavage with d emiditraz ( PF - 3814927; 99.5 % a.i.; Batch # PFXA070001 ) in 0.5% methylcellulose and 0.1% Tween TM 80 at dose levels of 0, 10, 30 , or 100 mg/kg/day with a dose volume of 10 mL/kg for 28 day s. Splenic antibody forming cell (AFC) assays were conducted on 10 rats/dose group (AFC Group) , and the remaining 10 rats/dose gr oup were used for N atural Killer (NK) cell activity assay , immune cell phenoty ping, and anti - CD3 stimulation assay (non - AFC group) . In addition, two positive control groups (10 rats/group) were included. For all assay s except for NK activity and NK cell enumeration, the positive control cyclophosphamide was administered b y i ntraperitoneal injection (50 mg/kg) on Day s 24 - 27 at a dose volume of 5 mL/kg. For NK cell activity and N K cell enumerat ion, the positive control, Anti - a sialo GM1 , was administered by intravenous injection (1 mL/animal of a 1:10 dilution ) approximately 24 hours prior to scheduled necrops y. For s ys temic toxicity , there were no treatment - related effects on body weights, body weight gains, food consumption, spleen weight, thy mus weight, or gross pathology .Demiditraz Human Health Risk Assessment D410552

Page **46** of **47** At 100 mg/kg/day , treatment - related clinical signs were noted in all treated animals 30 - minutes after dose administration. These findings consisted of rocki ng, lurching, or swaying while walking; drooping eyelids; and subdued appearance upon cage - side observation. Clear material around the mouth and yel low material on various body surfaces (anogenital area, urogenital area, ventral trunk, hindlimbs, and/or around the mouth) were also observed in the animals at 100 mg/kg/day . Additionally, flattened body was noted in 8 rats, and 2 rats had hunched posture at this dose. At 30 mg/kg/day , subdued appearance was observed in 5 rats in the AFC group for a total of 6 occurrences and in 4 rats in the non - AFC group for a total of 18 occurrences. Although considered treatment - related, in the absence of other clinical signs of toxicity , this finding was not considered adverse at this dose. The only findings noted at the time of dosing or during the daily examinations that were considered treatment - related were oral secretions and urogenital/anogenital staining. Positive control, cyclophosphamide, treated animals showed a few incidence of yellow material on various body surfaces (anogenital area, urogential area, ventral trunk, and hindlimbs ). S ignificant (p<0.01) mean body weight losses were observed for overall (day s 0 - 28) and day s 24 - 28 in the cyclophosphamide treated animals compared to the negative controls. For s ys temic toxicity , the L OAEL is 100 mg/kg/day based on clinical signs of toxicity (rocking lurching, or swaying while walking; drooping eyelids; subdued appearance at cage - side observation; flattened body ; hunched posture; clear material around mouth; and yellow material on various body

surfaces). The NOAEL for s ys temic toxicity is 30 mg/kg/day . For immunotoxicity , there were no significant differences among treated and control groups in thy mus and spleen weights and total spleen cell numbers. There were no significant treatment related effects on antibody forming cell response (humoral immune response) to the T - dependent antigen (SRBC) and on the NK cell activity (innate immunity) . Splenocy te surface marker differential anal ys es indicated that the absolute numbers of B cell s , total T cell s , T helper cell s, T cy totoxic cell s, natural killer cells, and monocy tes/macrophages in the treated groups were comparable to negative controls. There were no significant differences among treated and control groups in the cell - mediated typed proliferative response in spleen cell cultures with or without the anti - CD3 antibody stimulation. Positive control s , cyclophosphamide for antibody forming cell assay and Anti - asialo GM1 for NK cell activity

assay s, were adequately preformed to demonstrate the sensitivity of the assay s. Under conditions of this study , no immunotoxicity was observed. The NOAELis 100 mg/kg/day

(the highest dose tested). This immunotoxicity

study is classified acceptable/guideline and satisfies the guideline requirement for an immunoto xicity study (OPPTS 870.7800).Demiditraz Human Health Risk Assessment D410552

Page **47** of **47 Appendix B. Physical/Chemical Properties**  **Table B.1 Physicochemical Properties of Demiditraz** 

Parameter Value Reference Melting point/range 138.36 o C (range 137

– 142 o C )

MRID No . 477441 03 B. Kitchens. 8/31/10 pH (23 °C) Not applicable as technical grade demiditraz is not soluble in water . Density 0.15 g/cc Water solubility (mg/ml) < 0.1 mg/ml Solvent solubility (mg/ml) Acetone: 33 to 100 Acetonitrile: 33 to 100 Dichloromethane: 100 to 1,000 Dimethyl sulfoxide: 100 to 1,000 Ethanol: 100 to 1,000 Ethyl acetate: 10 to 33 Tol uene: 1 to 10 Vapor pressure Not applicable as technical grade demiditraz is solid at room temperature Dissociation constant (pK a ) Not applicable as technical grade demiditraz is a weak base and insoluble in water Oc tanol/water partition coefficient The mean log 10 P value reported is 2.78 UV/visible absorption spectrum methanol (nm) Shows maximum absorption at about 264 nm