 

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 Section 3
New Use for Proposed Dog Spot-On Use 

PC Code:  577501	DP Barcode:  D410552

MRID No.:  Not Applicable	Registration No.:  1007-OT

Petition No.:  Not Applicable	Regulatory Action:  Section 3 Registration

Assessment Type:  Single Chemical, Non-Aggregate	Reregistration Case
No.:  Not Applicable

TXR 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)

THROUGH:	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)

Table of Contents

  TOC \o "1-3" \h \z \u   HYPERLINK \l "_Toc353457984" 1.0	Executive
Summary	  PAGEREF _Toc353457984 \h  4  

 HYPERLINK \l "_Toc353457985" 2.0	HED Recommendations	  PAGEREF
_Toc353457985 \h  6  

 HYPERLINK \l "_Toc353457986" 2.1	Data Deficiencies	  PAGEREF
_Toc353457986 \h  6  

 HYPERLINK \l "_Toc353457987" 2.2	Tolerance Considerations	  PAGEREF
_Toc353457987 \h  7  

 HYPERLINK \l "_Toc353457988" 2.3	Label Recommendations from
Occupational and Residential Assessment	  PAGEREF _Toc353457988 \h  7  

 HYPERLINK \l "_Toc353457989" 3.0	Introduction	  PAGEREF _Toc353457989
\h  7  

 HYPERLINK \l "_Toc353457990" 3.1	Chemical Identity	  PAGEREF
_Toc353457990 \h  7  

 HYPERLINK \l "_Toc353457991" 3.2	Physical/Chemical Characteristics	 
PAGEREF _Toc353457991 \h  7  

 HYPERLINK \l "_Toc353457992" 3.3	Pesticide Use Pattern	  PAGEREF
_Toc353457992 \h  8  

 HYPERLINK \l "_Toc353457993" 3.4	Anticipated Exposure Pathways	 
PAGEREF _Toc353457993 \h  8  

 HYPERLINK \l "_Toc353457994" 3.5	Consideration of Environmental Justice
  PAGEREF _Toc353457994 \h  8  

 HYPERLINK \l "_Toc353457995" 4.0	Hazard Characterization and
Dose-Response Assessment	  PAGEREF _Toc353457995 \h  9  

 HYPERLINK \l "_Toc353457996" 4.1	Summary of Toxicological Effects	 
PAGEREF _Toc353457996 \h  9  

 HYPERLINK \l "_Toc353457997" 4.2	Considerations of Toxicity to Children
  PAGEREF _Toc353457997 \h  10  

 HYPERLINK \l "_Toc353457998" 4.2.1	Completeness of the Toxicology
Database	  PAGEREF _Toc353457998 \h  10  

 HYPERLINK \l "_Toc353457999" 4.2.2	Evidence of Neurotoxicity	  PAGEREF
_Toc353457999 \h  11  

 HYPERLINK \l "_Toc353458000" 4.2.3	Evidence of
Sensitivity/Susceptibility in the Developing or Young Animal	  PAGEREF
_Toc353458000 \h  11  

 HYPERLINK \l "_Toc353458001" 4.2.4	Residual Uncertainty in the Exposure
Database	  PAGEREF _Toc353458001 \h  12  

 HYPERLINK \l "_Toc353458002" 4.3	Toxicity Endpoint and Point of
Departure Selections	  PAGEREF _Toc353458002 \h  12  

 HYPERLINK \l "_Toc353458003" 4.3.1	Dose-Response Assessment	  PAGEREF
_Toc353458003 \h  12  

 HYPERLINK \l "_Toc353458004" 4.3.2	Recommendation for Combining Routes
of Exposures for Risk Assessment	  PAGEREF _Toc353458004 \h  13  

 HYPERLINK \l "_Toc353458005" 4.3.3	Cancer Classification and Risk
Assessment Recommendation	  PAGEREF _Toc353458005 \h  13  

 HYPERLINK \l "_Toc353458006" 4.3.4	Summary of Points of Departure and
Toxicity Endpoints Used in Human Risk Assessment	  PAGEREF _Toc353458006
\h  13  

 HYPERLINK \l "_Toc353458007" 5.0	Residential (Non-Occupational)
Exposure/Risk Characterization	  PAGEREF _Toc353458007 \h  15  

 HYPERLINK \l "_Toc353458008" 5.1	Residential Handler Exposure	  PAGEREF
_Toc353458008 \h  15  

 HYPERLINK \l "_Toc353458009" 5.2	Residential Post-Application Exposure	
 PAGEREF _Toc353458009 \h  16  

 HYPERLINK \l "_Toc353458010" 5.3	Combined Residential Risk Estimates
(Multiple Exposure Scenarios)	  PAGEREF _Toc353458010 \h  18  

 HYPERLINK \l "_Toc353458011" 5.4 	Residential Risk Estimates for Use in
Aggregate Assessment	  PAGEREF _Toc353458011 \h  18  

 HYPERLINK \l "_Toc353458012" 5.5	Residential Bystander Post-Application
Inhalation Exposure	  PAGEREF _Toc353458012 \h  18  

 HYPERLINK \l "_Toc353458013" 6.0	Aggregate Exposure/Risk
Characterization	  PAGEREF _Toc353458013 \h  19  

 HYPERLINK \l "_Toc353458014" 7.0	Cumulative Exposure/Risk
Characterization	  PAGEREF _Toc353458014 \h  19  

 HYPERLINK \l "_Toc353458015" 8.0	Occupational Exposure/Risk
Characterization	  PAGEREF _Toc353458015 \h  19  

 HYPERLINK \l "_Toc353458016" 8.1	Occupational Handler Exposure/Risk
Estimates	  PAGEREF _Toc353458016 \h  19  

 HYPERLINK \l "_Toc353458017" 8.2 Occupational Post-Application Risk	 
PAGEREF _Toc353458017 \h  22  

 HYPERLINK \l "_Toc353458018" Appendix A.  Toxicology Profile and
Executive Summaries	  PAGEREF _Toc353458018 \h  23  

 HYPERLINK \l "_Toc353458019" A.1	Toxicology Data Requirements	  PAGEREF
_Toc353458019 \h  23  

 HYPERLINK \l "_Toc353458020" A.2	Toxicity Profiles	  PAGEREF
_Toc353458020 \h  24  

 HYPERLINK \l "_Toc353458021" A.3  Hazard Identification and Endpoint
Selection	  PAGEREF _Toc353458021 \h  27  

 HYPERLINK \l "_Toc353458022" A.4	Executive Summaries	  PAGEREF
_Toc353458022 \h  29  

 HYPERLINK \l "_Toc353458023" Appendix B.  Physical/Chemical Properties	
 PAGEREF _Toc353458023 \h  47  

 

1.0	Executive Summary

Demiditraz, 2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazole is an
acaricide which acts on the octopamine nervous system in invertebrates
and acts on neurotransmitter systems, 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 currently no active registrations for demiditraz.  A
human health risk assessment was previously conducted for the proposed
dog spot-on product which identified risks of concern for the use.  This
document updates the prior assessment with incorporation of additional
exposure and toxicity 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 (residential) 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 by 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 days),
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).   

alpha-2 (α2) adrenergic receptor in mammals. There 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 requirements for a non-food use
have been satisfied.  

A database uncertainty factor (UFDB) 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.  Accordingly, the
additional UFDB 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/genetic toxicity were determined to
be negative and, therefore, there is no concern for mutagenicity/genetic
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 selected 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 activity,
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, UFLOAEL, 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., dermal, 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
application (Day 0) residues were used to assess all durations of
residential post-application exposure.  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 Days 0 to 30 (i.e., the proposed product re-treatment interval).

Residential post-application adult dermal, and combined children 1 to <
2 years old exposures are not of concern for all durations of exposure
(i.e., adult dermal MOEs are > 300; and children 1 to < 2 years old ARIs
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. 

 (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 be negligible.  

Residential Aggregate Exposure and Risk Estimates:  An aggregate
exposure assessment, which combines exposures from different sources and
routes, is typically conducted for non-food/ non-feed chemicals when
there is potential for human exposure through water and residential
pathways.  Demiditraz currently has no registered food/feed uses and no
drinking water residues are expected to result from the proposed pet
use; therefore, aggregate exposure includes only 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.  

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); therefore, 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. 
Label directions 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 should 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 Formula	C13H16N2

Common Name	Demiditraz

Company Experimental Names	 PF-3814927

CAS Name	 2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-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 physicochemical 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 concern for
bioaccumulation in lipophilic matrices.  Technical grade demiditraz is a
solid at room temperature and, therefore, the volatility of the chemical
was not determined as it is not anticipated to have significant
volatility.  

  

3.3	Pesticide Use Pattern

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 available 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 summarized 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 dog
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 applying 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 justice concerns, to the extent
possible, were considered in this human health risk assessment, in
accordance with U.S. Executive Order 12898, "Federal Actions to Address
Environmental Justice in Minority Populations and Low-Income
Populations," ( HYPERLINK
"http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf"
http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf .  As a part of
every pesticide risk assessment, OPP considers a large variety of
consumer subgroups according to well-established procedures.  In line
with OPP policy, HED estimates risks to population subgroups from
pesticide exposures that are based on patterns of that subgroup’s food
and water consumption, and activities in and around the home that
involve pesticide use in a residential setting.  Extensive data on food
consumption patterns are compiled by the 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 analyzed and
categorized by subgroups based on age, season of the year, ethnic group,
and region of the country.  Additionally, OPP is able to assess dietary
exposure to smaller, specialized subgroups and exposure assessments are
performed when conditions or circumstances warrant.  Whenever
appropriate, non-dietary exposures 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 currently in development as
OPP has committed resources and expertise to the development of
specialized software and models that consider exposure to bystanders and
farm workers as well as lifestyle and traditional dietary patterns among
specific subgroups.

4.0	Hazard Characterization and Dose-Response Assessment

4.1	Summary of Toxicological Effects

The nervous system is the primary 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); subchronic dermal in rats;
subchronic oral in dogs; 2-generation reproduction in rats; and rat and
rabbit developmental.  Demiditraz effects on the nervous system 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 sways when walking;
hunched posture; hypoactivity; sitting with the head held low; a
flattened body; piloerection; tremors/convulsions; prostration; wet and
yellow urogenital area; shallow 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
increased 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 observed in the startle response 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, maternal toxicity was observed in
the rat and rabbit, as evidenced by mortality, clinical signs of
toxicity, and decreased body weight.  Developmental toxicity was
observed in the rat and rabbit, as evidenced by decreased fetal body
weight and associated delayed ossification of the skeleton in rats and
slight increases in the incidence of 27th presacral vertebrae and 13th
full ribs in the rabbit.  In the rat reproductive toxicity study,
mortality, clinical signs, 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.  Reproductive
toxicity was not observed.  In the developmental neurotoxicity study in
rats, maternal toxicity was observed, as evidenced by clinical signs of
toxicity and 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 Chinese 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/genetic toxicity for demiditraz. 
Demiditraz was assessed in a guideline immunotoxicity study, and no
specific 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 F1
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 excreted
within 48 hours post dose in the rat. The half-life in rats following
oral exposure was determined to be approximately 30 minutes.  In the
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 currently available to
determine the potential for demiditraz to affect the nervous system by
this route of exposure.  It should 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 tolerances 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 assessed the potential for increased susceptibility for
infants and children, and determined that no 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	Completeness 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
acceptable:  rat and rabbit developmental toxicity studies; rat
2-generation reproduction; rat acute and subchronic neurotoxicity; rat
developmental neurotoxicity; and rat immunotoxicity study.  

4.2.2	Evidence of Neurotoxicity

There is evidence of neurotoxicity throughout the database, but
neuropathology was not observed.  Altered gait and posture, impaired
mobility, decreased rearing, in-coordination, lower body temperature,
and decreases in motor activity were observed in both sexes in the acute
neurotoxicity study in rats.  Several clinical signs of neurotoxicity,
which included subdued appearance, rocks/lurches/sways when walking,
hunched posture, hypoactivity, wet and yellow urogenital area, shallow
and/or decreased respiration, lacrimation, and dilated pupils, and
increased motor activity were observed in both sexes in the subchronic
neurotoxicity study in rats.  Decreased motor activity and altered
grooming behavior were observed in both sexes in   TC \l3 "3.3.2
Evidence of Neurotoxicity the 90-day dermal toxicity study in rats.  In
the developmental toxicity studies, clinical 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/sways while walking,
hypoactivity, tremors, clonic convulsions) suggestive of neurotoxicity
were observed.  In the developmental neurotoxicity study in rats,
decreased/increased motor activity and increased startle response
amplitude and reduced latency were observed at the high dose where a
delay in overall development was observed, mainly in males, as evidenced
by 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 mortality and clinical signs of
toxicity and decreased body weights.  Developmental toxicity was
observed in the rat and rabbit, as evidenced by decreased fetal body
weight and associated delayed ossification of the skeleton in rats and
slight increases in the incidence of 27th presacral vertebrae and 13th
full ribs in the rabbit.  The developmental effects were observed 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/sways while walking, hypoactivity, 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, hypoactivity, a flattened body, slightly
drooping eyelids, decreased respiration, clear material around
mouth/salivation, lacrimation, and/or dilated pupils) was observed at a
dose where no effects were observed in the offspring.  

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
assessment. 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 use pesticide.

The incidental oral endpoint (all durations) for risk assessment was
from the oral subchronic neurotoxicity study in the rat with a NOAEL of
5 mg/kg/day.  At the study LOAEL of 25 mg/kg/day, clinical signs of
neurotoxicity (subdued appearance, rocks, lurches, or sways when
walking, hunched posture, hypoactivity, shallow/decreased respiration,
lacrimation, dilated pupil), decreased motor activity, and decreased
body weights were observed in both sexes.  Inhalation hazard is assumed
to be equivalent 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. 
This 90-day duration 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 were 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 (UFLOAEL→NOAEL).  The 3X factor is considered appropriate for
dermal exposure assessment (all durations).  Application of the 3X
factor results in an extrapolated dermal NOAEL of 33 mg/kg/day.  Based
on a dermal absorption factor of 31%, the orally equivalent dose is
about 10 mg/kg/day.  This is consistent with the 2-generation
reproduction 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 UFDB of 10X retained in the previous human health risk assessment
was removed since the data requirements for a rat developmental
neurotoxicity study and a dog subchronic oral toxicity study have been
satisfied, and the dog was not found to be more sensitive than the rat
to the neurotoxic 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 been submitted
and are not required for the current non-food/non-feed use.  All studies
conducted for mutagenicity/genetic toxicity were determined to be
negative and, therefore, there is no concern for mutagenicity/genetic
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 Risk 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 Durations)	LOAEL  = 100

mg/kg/day	UFA= 10X

UFH= 10X

UFLOAEL= 3X	MOE = 300	Subchronic dermal toxicity study 

LOAEL = 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 Durations)	NOAEL= 5 mg/kg/day	UFA= 10X

UFH= 10X	MOE = 100	Subchronic neurotoxicity study with a LOAEL = 25
mg/kg based on clinical signs of neurotoxicity.

Inhalation 

(All Durations)

	NOAEL= 5 mg/kg/day	UFA= 10X

UFH= 10X	MOE = 100	Subchronic neurotoxicity 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. 
UFA = extrapolation from animal to human (interspecies).  UFH =
potential variation in sensitivity among members of the human population
(intraspecies).  UFLOAEL = additional factor for extrapolation from
LOAEL to NOAEL.  MOE = margin of exposure.  LOC = level of 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 Assessment	Study and Toxicological Effects



Dermal 

(All Durations)	LOAEL ≤ 100

mg/kg/day	UFA= 10X

UFH= 10X

UFLOAEL= 3X	MOE = 300	Subchronic dermal toxicity study 

LOAEL = 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	UFA= 10X

UFH= 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 point or an estimated point that is
derived from observed dose-response data and used to mark the beginning
of extrapolation to determine risk associated with lower environmentally
relevant human exposures.  NOAEL = no observed adverse effect level. 
LOAEL = lowest observed adverse effect level.  UF = uncertainty factor. 
UFA = extrapolation from animal to human (interspecies).  UFH =
potential variation in sensitivity among members of the human population
(intraspecies).   UFLOAEL = 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 proposed demiditraz dog
spot-on use.  Residential handler exposures are anticipated from
application of the proposed product to dogs, and residential
post-application exposures are expected to occur from contact with dogs
previously 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 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 Handler 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 differently by HED as homeowners are assumed to
complete all elements of an application without use of any 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 applying the proposed demiditraz products to
dogs.  Intermediate- and long-term exposures are not likely 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
product.

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 document which supports this
memorandum.

Residential handler short-term dermal risk 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.

Table 5.1.  Residential Handler Short-Term Non-cancer Exposure and Risk
Estimates from Use 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 Rate1

(lb ai/pet)	Amount Handled Daily2	Dermal







	Dose (mg/kg/day)3	MOE4

Applying 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 proposed label (EPA Reg. No.1007-OT).

2	Based on HED’s 2012 Residential SOPs (Treated Pets) ( HYPERLINK
"http://www.epa.gov/pesticides/science/residential-exposure-sop.html"
http://www.epa.gov/pesticides/science/residential-exposure-sop.html ).

3	Dermal Dose = Dermal Unit Exposure (120 mg/lb ai) × Application Rate
(lb ai/ treatment) × Area 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	Residential 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 residential post-application exposures is based on the
following scenarios:  

1)  Post-application dermal (adults and children 1 to < 2 years old)
exposure from contacting dogs treated with demiditraz; and 

2)  Post-application incidental oral exposure (children 1 to < 2 years
old only) from contacting dogs treated with demiditraz.

The lifestages selected for each post-application scenario are based on
an analysis provided as an Appendix in the 2012 Residential SOPs.  These
lifestages are not the only 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 any 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 durations were assessed for the proposed
demiditraz spot-on product.  

A series of assumptions and exposure factors served as the basis for
completing the residential post-application risk assessment.  Each
assumption and factor is detailed in the 2012 Residential SOPs.  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 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 (FAR) in the
2012 SOPs.  HED used the average of Day 0 percent residue transfer
values predicted from regression analysis in order to assess exposures
and risks for all durations of post-application exposures from the
proposed demiditraz spot-on use.  Day 0 FAR 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 accurately 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 Days 0 to 30 (i.e., proposed product
re-treatment interval) was inputted.  The FAR 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
assessment 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 MOEs are > 300; and children 1 to < 2 years old year old
ARIs are > 1) with use of Day 0 FAR.  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 FAR.

Table 5.2.  Residential Post-Application Non-Cancer Exposure and Risk
Estimates from the Proposed Demiditraz Spot-On Product:  All Durations
(Day 0 FAR)

Lifestage	Post-application Exposure Scenario	Application Rate

(mg ai)1	Dose (mg/kg/day)2	MOEs3	ARI4

	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

	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) * Absorption
Factor (unitless)) / Body Weight (80 kg, adult; 11 kg, child 1 to < 2
years old years old)]

	Hand-to-Mouth Dose = [(Hand Residue Loading (mg/cm2) × Fraction of
Hand Mouthed (0.13) × Surface Area of 1 Hand (150 cm2) × Exposure Time
(1.5 hrs/day) × # of Replenishment Intervals/hr (4 int/hr) ×
(1-((1-Saliva Extraction Factor (0.5))^(Number of Hand-to-Mouth Events
per Hour (13.9 events/hr) ) ÷  ( # of Replenishment Intervals/hr))]  /
Body Weight (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 LOC; 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 generally not
be combined.  There is the potential for the same 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 individual assessment.

5.4 	Residential Risk Estimates for Use in Aggregate Assessment

An aggregate exposure assessment, which combines exposures from
different sources and routes, is typically conducted for food/feed
chemicals when there is potential for human exposure through drinking
water and residential pathways.  Demiditraz currently 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 the proposed use.  

5.5	Residential Bystander Post-Application Inhalation Exposure

Occupational and residential inhalation exposures from dog spot-on
treatments are considered negligible.

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
currently 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
common mechanism of toxicity finding as to demiditraz and any other
substances and demiditraz does not appear to produce a toxic metabolite
produced by other substances. For the purposes of this tolerance action,
therefore, EPA has not assumed that demiditraz 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 released 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 Exposure/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 significantly less than residential
post-application exposures.

 

8.1	Occupational Handler Exposure/Risk Estimates

Based on the anticipated use patterns and current labeling, types of
equipment and techniques that can potentially be used, occupational
handler exposure (i.e., veterinarians, veterinary assistants, and
groomers) could be expected from the demiditraz spot-on use.  The
quantitative exposure/risk assessment developed for occupational
handlers (adults) is based on dermal exposure from the application of
the spot-on product to dogs.  Inhalation exposures 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 residential 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 currently 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 residential 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 the 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 (Treated
Pets) SOP, as well as in the occupational and residential exposure and
risk assessment which supports this memorandum.

dogs per day (all durations) without resulting dermal risks of concern
(i.e., MOEs are ≥ 300).   It is highly unlikely that this large a
number of dogs could be treated daily 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 handler dermal
risk estimates is presented in Table 8.1.   Table 8.1.  Occupational
Handler Non-Cancer Exposure and Risk Estimates (All Durations) for
Demiditraz

Exposure Scenario	Target	Dermal Unit Exposure 

(mg/lb ai)1	Inhalation Unit Exposure 

(mg/lb ai)	Maximum

Application Rate 

(lb ai)2	Maximum Amount Handled Daily3	Dermal	Inhalation



Single Layer, No Gloves	No Respirator

	Dose (mg/kg/day)4	MOE5	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)

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, dogs are expected to be treated
and returned to their owners such that post-application 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 any 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
Assessment 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 Separately with
Three Different Formulated End-Use Products.” D409400, EPA MRID
48766704, 4/11/13.

Appendix A.  Toxicology Profile and Executive Summaries

A.1	Toxicology Data Requirements

The requirements (40 CFR 158.340) for food 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

870.1100    Acute Oral Toxicity	

870.1200    Acute Dermal Toxicity	

870.1300    Acute Inhalation Toxicity	

870.2400    Primary Eye Irritation	

870.2500    Primary Dermal Irritation	

870.2600    Dermal Sensitization		yes

yes

yes*

yes

yes

yes	yes

yes

no*

yes

yes

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)	

870.3700b  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		Yes

yes

yes	yes

yes

yes

870.6100a  Acute Delayed Neurotoxicity (hen)	

870.6100b  90-Day Neurotoxicity (hen)	

870.6200a  Acute Neurotoxicity Screening Battery (rat)	

870.6200b  90-Day Neurotoxicity Screening Battery (rat)	

870.6300    Develop. Neurotoxicity		no

no

yes

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 = conditionally required

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	LD50 = 5000 mg/kg	III

870.1200	Acute dermal - rat	47744107	LD50 > 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

870.2500	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.
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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	Subchronic 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 both sexes. At 100 mkd, all
dogs displayed hypoactivity. 

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 minutes 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
47744ㄱ‷㈨〰⤹䄍捣灥慴汢⽥畇摩汥湩൥牃㩬䑃匨⥄ㄍ
‰敷步⁳牰ⵥ慭楴杮」‬⸷ⰵ㔠ⰰㄠ〵洠摫万䅏䱅㴠㜠
㔮洠摫മ〱眠敥獫瀠敲洭瑡湩൧楶⁡慧慶敧

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 sexes), 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 ambulating; 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.

≈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	Mammalian cell cytogenetics 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	Bone marrow micronucleus assay	MRID 47755403 (2008)

Acceptable/Guideline

0-1000 mg/kg/day	There was no significant increase in the frequency of
micronucleated
polychrom瑡捩攠祲桴潲祣整⁳湩戠湯⁥慭牲睯愠瑦牥愠祮
琠敲瑡敭瑮琠浩⹥܇㜸⸰㌶〰䐇癥汥灯敭瑮污渠略潲潴
楸楣祴ഠ爨瑡‬灓慲畧ⵥ慄汷祥ഩ䴇䥒⁄㠴㘷㜶㌰⠠〲
㈱ഩ捁散瑰扡敬䜯極敤楬敮䌍汲䌺⡄䑓ഩ䑇㘠琠⁯䑌㈠
റⰰ㔠‬㔱‬〱‰歭ݤ慍整湲污丠䅏䱅㴠㔠洠摫മ牆浯
䜠⁄‶潴䰠⁄ㄲ

(≈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, slightly 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 motor
activity counts, decreased grip strength (PND 21 and PND 35), decreased
brain weight 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匨⥄」‬〱‬〳‬〱‰歭ݤ楶⁡慧慶敧⠠㠲搠祡⥳
不䅏䱅⠠祳瑳浥捩琠硯捩瑩⥹㴠㌠‰歭⹤

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-side 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.



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 values 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/sways when walking, hunched posture, hypoactivity, etc.) in both
sexes.  

UF(s):  A UF of 100 was applied to account for interspecies
extrapolation (10X) and intraspecies variation (10X).  

Comments on Study/Endpoint/UFs:  This study provides the lowest NOAEL in
the database considered to be valid for this exposure and all durations
and is protective for any 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 days.  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 systems 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 additional 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.  

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 LOAEL in
the database considered to be valid for this exposure and all durations
and is protective for any 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 systems 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, 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: 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/sways when walking, hunched posture, hypoactivity, 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) should be applied when calculating reference doses for
long-term exposure.

Comments on Study/Endpoint/UFs:  This study provides the lowest NOAEL in
the database considered to be valid for this exposure and all durations
and is protective for any 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 system 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 developmental neurotoxicity study because of the extensive
neurotoxicity evaluations carried out in the adult male and female for
up to 90 days.  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 systems 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 additional uncertainty 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	Combined 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 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 system
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) by 33% in the males during
Weeks 7-8.  Overall body weight gains were decreased (p<0.05) by 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 sways
when walking (6 males and 6 females); (iii) hunched posture (6 females);
(iv) hypoactivity (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 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 sporadically decreased (p<0.05) by 14-48% in the males
and by 17-56% in the females.  Overall (Weeks 0-13) body weight gains
were decreased (p<0.01) by 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 sways when walking (all
males and females); (iii) hunched posture (17 males and all females);
(iv) hypoactivity (11 males and 3 females); (v) wet yellow 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). 
Cumulative (session) total activity was increased by 29-32% in the males
at Weeks 1 and 3 and by 31-32% in the females throughout the study. 
Also at this dose, cumulative ambulatory activity was increased by
43-56% in the males at Weeks 1, 3, and 7 and by 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
daily dosing), brain weight and morphology, organ weights, gross
lesions, or microscopic lesion in either sex.

The 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.

The study is classified as acceptable/guideline and satisfies the
guideline requirement (OPPTS 870.6200b; OECD 424) for a subchronic
neurotoxicity study in rats.

	870.3150	90-Day Oral Toxicity – Dog

In a subchronic 90-day oral toxicity study (2009, MRID 48459302)
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, urinalysis,
electrocardiographic measurements, pathology and biokinetic data were
obtained.

There were no adverse effects on mortality, ophthalmology, hematology,
clinical chemistry urinalysis 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 males 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 91showed
a loss of weight of 0.2 kg.  Food consumption was statistically
significantly 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 hypoactivity.  The NOAEL in
males and females is 10 mg/kg bw/day.

Pharmacokinetic data indicated that demiditraz did not bioaccumulate and
there were no sex differences in the pharmacokinetics. 

This 90-day oral toxicity study in the dog is acceptable/guideline and
satisfies the requirement for a 90-day oral toxicity study (OPPTS
870.3150; OECD 409) in the dog.

	870.3250	90-Day Dermal Toxicity – Rat 

In a repeated-dose dermal toxicity study (2008, MRID 47752701),
PF-3814927 (Demiditraz, (99.5% a.i., Lot #s PFXA070001 and PFXU070002)
in deionized water was applied to the shaved 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 days. 
Additional groups of 3 rats/sex/dose were exposed at the same doses for
87 consecutive days.  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 toxicokinetic exposure
evaluations.  Systemic exposure (as assessed by Cmax and AUC0-24)
“generally increased 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.  There 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 (slightly
soiled to very soiled fur) was noted in the 1000 mg/kg/day males and
females.  During 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 following 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
either sex.  Application site dermal effects were limited to very slight
to slight erythema noted in the 300 and 1000 mg/kg/day groups and are
considered self limiting.  

The NOAEL (systemic) < 100 mg/kg/day.  The LOAEL is 100 mg/kg/day based
on decreased motor activity in both sexes at all dose levels tested.   

The LOAEL (local irritation) = 300 mg/kg/day based on erythema.  The
NOAEL is 100 mg/kg/day. 

This study is classified as acceptable/guideline and satisfies the
guideline requirement for a 90-day dermal toxicity study (OPPTS
870.3250; OECD 411) in rats.

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% TweenTM 80 was administered daily via gavage in
a dose volume of 10 mL/kg to 25 time-mated presumed pregnant Sprague
Dawley rats/dose group at dose levels of 0, 5, 25, or 100 mg/kg/day from
gestation days (GD) 6-19.  Blood samples were collected from 4 randomly
selected rats per group 1 hour after 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 removed 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 measurements 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 cause 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,
decreased 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 toxicity
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
hypoactive 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 dose 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 weight gains were decreased (p<0.01) by 58% during GD
6-10, by 16% for GD 14-20, and by 20% for the overall (GD 6-20)
treatment period.  Gravid uterine weights were decreased by 12%
(p<0.01), and corrected body weight gain for the overall (GD 0-20) study
were decreased by 15% (p<0.05).  Absolute and relative food consumption
was decreased (p<0.01) by 14-15% for GD 6-10 and by 6-11% for GD 6-20. 
Additionally at this dose, absolute food consumption was decreased by 7%
for GD 14-20.  

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 by 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) compared to concurrent controls
(0%) and historical controls (0.0-1.1% fetuses).  Several other skeletal
variations characterized by 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 fetal body
weights at this dose.  These variations included:  (i) increases 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% fetuses; 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% fetuses) 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 LOAEL is 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 47755401),  Demiditraz
technical (PF-3814927; 100% a.i.; Batch # PFXU070002) in 0.5%
methylcellulose and 0.1% TweenTM 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 daily 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/day on GD 9, 10, or 11 for the
remainder of the study.  Blood samples were collected from 4 rabbits per
group 1 hour after dose administration on GD 28 to determine test
substance 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, visceral, 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 early in the study (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, body weights were decreased by 6% (p<0.05) on GD 14-16.
 At 200/150 mg/kg/day, body weights were decreased by 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 period (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 by 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 by 24% (p<0.05) for
GD 12-21.  Absolute and relative food consumption for the overall (GD
6-29) treatment period 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 necropsy.  The maternal LOAEL is 50 mg/kg/day based on
incidences of mortality/moribundity, clinical signs of toxicity, and
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 numbers of early resorptions, late resorptions, or live fetuses per
doe.  Sex ratio was unaffected by treatment.  There were no
treatment-related external, visceral, or skeletal malformations.

At the high dose, fetal body weights were decreased by 12% (not
significant [NS]) in the males and by 13% (p<0.05) in the females. 
Fetal and litter incidences of rabbits with 27 presacral 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% litters) 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 of 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 13th rib(s) were dose-dependently 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 13th rib(s) at
200/150 mg/kg/day were considered to be due to treatment.  However, it
should be noted that these findings (27 presacral vertebrae and 13th
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 13th 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 acceptable/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 for two
consecutive generations.  The P generation animals (30/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 generations, treatment-related generally 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 early 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 hypoactivity.  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 immediately 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.  

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 observed 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 (Days 0-126) was
decreased in these males.   Average food efficiency (PND 28-161) was
also decreased.  Decreased 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 either generation.  

The LOAEL for parental toxicity is 50 mg/kg/day based on:  clinical
findings indicating nervous system 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 NOAEL
is 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
sites 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.  Decreased 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 females) 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 sexes 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 separation were unaffected by treatment. Developmental
landmarks were not reported.  

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 NOAEL is
7.5 mg/kg/day.

There were no effects of treatment in either generation on:  estrous
cycle; sperm parameters; mating, conception, fertility, or gestation
indices; pre-coital interval; or gestation duration in either
generation.

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

MRID#47752704

Mecchi, M.S. (2008)	0-5000 µg/plate	Negative

870.5300

Mammalian cell gene mutation (CHO)

MRID #47755404

Stankowski, L.F. (2008)	0-900 µg/mL	Negative

870.5375

Mammalian cytogenetics assay (chromosomal aberration assay in human
peripheral blood 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	Negative



A.4.7	Neurotoxicity

		870.6200a Acute Neurotoxicity Screening Battery – Acute Rat 

In an acute neurotoxicity screening study (2008, MRID 47744113),
PF-3814927 (Demiditraz, 99.2% a.i., Batch # PFXU060001) in 0.5%
methylcellulose/0.1% polysorbate 80 was administered once via gavage (5
mL/kg) to 12 Sprague-Dawley rats/sex/group 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 days 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 system 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%) activity counts.  No statistically
significant differences in motor activity were noted on Days 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 females 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 moderately 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 slightly 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, consisted of no eye blink response, no pupil
response and no forelimb extension. 

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),
considerable 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), moderately coarse (2 males and 1 female at 600 mg/kg), and
markedly 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 circling 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 were 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 physiological observations on Day 0, body temperatures ((C) were
decreased (p<0.01) at 100 and 600 mg/kg in both sexes (35.1 and 34.5
treated, respectively, in males vs. 37.2 controls, and 35.2 and 34.6
treated, respectively, in females vs. 37.8 controls).  

No compound-related effects were observed in mortality, body weight
(except of initial decrease in day 0-7 gain 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 acceptable/guideline 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 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 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 system
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) by 33% in the males during
Weeks 7-8.  Overall body weight gains were decreased (p<0.05) by 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 sways
when walking (6 males and 6 females); (iii) hunched posture (6 females);
(iv) hypoactivity (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 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 sporadically decreased (p<0.05) by 14-48% in the males
and by 17-56% in the females.  Overall (Weeks 0-13) body weight gains
were decreased (p<0.01) by 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 sways when walking (all
males and females); (iii) hunched posture (17 males and all females);
(iv) hypoactivity (11 males and 3 females); (v) wet yellow 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). 
Cumulative (session) total activity was increased by 29-32% in the males
at Weeks 1 and 3 and by 31-32% in the females throughout the study. 
Also at this dose, cumulative ambulatory activity was increased by
43-56% in the males at Weeks 1, 3, and 7 and by 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
daily dosing), brain weight and morphology, organ weights, gross
lesions, or microscopic lesion in either sex.

The 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.

The study is classified as acceptable/guideline and satisfies the
guideline requirement (OPPTS 870.6200b; OECD 424) for a subchronic
neurotoxicity study in rats.

	870.6300 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%
methylcellulose (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 (F0 females)
on GD 10 and GD 15 and on LD 10 and LD 20. All F0 females were allowed
to deliver and rear their offspring to LD 21. All surviving F0 females
were euthanized on LD 21 and subjected to a gross necropsy. Clinical
observations, body weights, and sexes were recorded for the F1 pups at
appropriate intervals. On postnatal day (PND) 4, litters were culled to
8 pups/litter, and a subset (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. From this
subset, 15 pups/sex/group were deeply 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 subset (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 deeply anesthetized and
perfused in situ on PND 21 for brain weight and brain measurement
evaluations; of these, 10 pups/sex/group were selected for
neuropathological and brain morphometric evaluations.  Indicators of
physical development (balanopreputial separation and vaginal patency)
were evaluated for all F1 selected pups in Subsets A and B. All F1 pups
not selected for neuropathological or behavioral evaluations were
euthanized and necropsied on PND 21. F1 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 F1 data.

One F0 female in the 100 mg/kg/day group was found dead on GD 21, and
cause of death could not be determined. All other F0 females survived to
the scheduled euthanasia.  One female each in the control and 100
mg/kg/day groups failed 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 F0 females and were observed at
approximately 15-30 minutes post-dosing throughout the treatment period.
 In both dose groups, these findings included sitting with the head held
low, hypoactivity, a flattened body, slightly drooping eyelids,
decreased respiration, clear material around the mouth/salivation,
lacrimation, and dilated pupils.  Additional 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 generally continued to the 2-hour
post-dosing observation period for the 100 mg/kg/day group F0 females,
but at decreased incidences. 

Treatment-related decrements in maternal care were observed mainly in
the 100 mg/kg/day group F0 females during the same interval post dose
when clinical signs were observed.  A decreased incidence of  F0 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 outside of the nest were all noted at the 100 mg/kg/day dose level.


During the detailed clinical observations (FOB) for F0 females, a
flattened posture or sitting with the head held low, low or very low
arousal, slight to moderately coarse tremors, lacrimation, 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.  Additionally, 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 F0 females at 15 mg/kg/day from GD
15 on. 

Mean body weights for F0 females in the 100 mg/kg/day group were
slightly (↓4.6%) lower than the control group by GD 20.  F0 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 reductions in food consumption during
GDs 6-12 (↓13-14).  Mean body weights for F0 females in the 100
mg/kg/day group were slightly (↓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 F0 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 generally similar to the control group
for the remainder of the lactation treatment period (LDs 4-21).  Mean
gestation length of F0 females was comparable among the groups, and
there were no treatment-related effects on the process of parturition or
macroscopic findings of the F0 females.

The maternal LOAEL is 15 mg/kg/day, based on clinical signs (sitting
with the head held low, hypoactivity, 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 clinical signs,
decrements in maternal care were observed during the same time interval
post dose when clinical signs were observed (decreased incidence of F0
females on the nest, a higher incidence 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 outside of the nest. 

The mean live litter size in the 100 mg/kg/day group was slightly lower
than the control group (14.0 vs. 15.1 for controls), 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 found 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, F1 pups of both sexes had decreased body weights [PND
1 (↓8%-9%); PND 4 (↓14%); PND 21 ↓16%)] and body weight gains (PND
1-4 (↓21%-29%); PND 4-7 (↓27%); PND 1-21 (↓16%-18%) generally
throughout the pre-weaning period, relative to their respective
controls.  Mean body weights of the F1 males at 100 mg/kg/day were
significantly lower than the control (PND 28-72 ↓10%-14%), and lower
mean body weight gains were noted for the 100 mg/kg/day F1 males
compared to the control throughout the post-weaning period (overall PND
28-72, ↓11%).  Mean body weights for the 100 mg/kg/day F1 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 F1 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 days) in the mean age of attainment of
balanopreputial separation was noted for the 100 mg/kg/day group F1
males (47.0 days vs. 44.3 days for controls) but mean body weights of
the F1 males on the day of attainment were not affected.  In contrast
the F1 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 F1 females
of the 100 mg/kg/day group.

Mean forelimb grip strength for the 100 mg/kg/day group F1 males and F1
females was significantly lower than the control group on PND 21 and PND
35 but was no longer significantly affected on PND 45 or PND 60. 

 

Although no statistically significant effects on locomotor activity or
habituation were observed among the F1 offspring, there were differences
in response between the control and 100 mg/kg/day groups and between 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 males 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 to 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 reduction 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.  Additionally, 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. 

At 100 mg/kg/day, mean brain weight was significantly lower in PND 21
males compared to the control, and relative brain weights were
significantly increased in both sexes 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 clinical
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 ratio 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 systems, brain
measurements at necropsy, or morphometry taken from brain sections.  

The F1 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 F1 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	

	

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No guideline study is available.  

	870.7600	Dermal Absorption – Rat 

In a dermal penetration study (2008, MRID 47752709), [14C] PF-03814927
(Demiditraz; 99.0% radiochemical purity; Code No. CFQ15145 Batch 1)
formulated in diethylene glycol monobutyl ether was applied to the
shaved skin (10 cm2) 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/cm2 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.  

Analytical 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, respectively. The groups terminated at 168 hours
post-application indicated additional absorption (25.8-28.1% AD)
resulting from absorption from the skin depots (skin at the application
site and adjacent skin).  The absorption 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
remaining 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 by 2 and 10
hours after application, respectively.  Only minor amounts were found in
the blood and cage wash.  The carcass accounted for ~1-8% AD with the
highest percentages 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
guideline 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.  It must be noted that the test
material was applied in diethylene glycol monobutyl ether 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 demiditraz (PF-3814927; 99.5% a.i.; Batch # PFXA070001) in 0.5%
methylcellulose and 0.1% TweenTM 80 at dose levels of 0, 10, 30, or 100
mg/kg/day with a dose volume of 10 mL/kg for 28 days.  Splenic
antibody-forming cell (AFC) assays were conducted on 10 rats/dose group
(AFC Group), and the remaining 10 rats/dose group were used for Natural
Killer (NK) cell activity assay, immune cell phenotyping, and anti-CD3
stimulation assay (non-AFC group).  In addition, two positive control
groups (10 rats/group) were included.  For all assays except for NK
activity and NK cell enumeration, the positive control cyclophosphamide
was administered by intraperitoneal injection (50 mg/kg) on Days 24-27
at a dose volume of 5 mL/kg.  For NK cell activity and NK cell
enumeration, the positive control, Anti-asialo GM1, was administered by
intravenous injection (1 mL/animal of a 1:10 dilution) approximately 24
hours prior to scheduled necropsy.

For systemic toxicity, there were no treatment-related effects on body
weights, body weight gains, food consumption, spleen weight, thymus
weight, or gross pathology.

At 100 mg/kg/day, treatment-related clinical signs were noted in all
treated animals 30-minutes after dose administration.  These findings
consisted of rocking, lurching, or swaying while walking; drooping
eyelids; and subdued appearance upon cage-side observation.  Clear
material around the mouth and yellow 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). Significant (p<0.01)
mean body weight losses were observed for overall (days 0-28) and days
24-28 in the cyclophosphamide treated animals compared to the negative
controls.

For systemic toxicity, the LOAEL 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 systemic toxicity is 30
mg/kg/day.

For immunotoxicity, there were no significant differences among treated
and control groups in thymus 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).  Splenocyte surface marker differential analyses indicated
that the absolute numbers of B cells, total T cells, T helper cells, T
cytotoxic cells, natural killer cells, and monocytes/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 controls, cyclophosphamide for antibody forming cell assay and
Anti-asialo GM1 for NK cell activity assays, were adequately preformed
to demonstrate the sensitivity of the assays. 

Under conditions of this study, no immunotoxicity was observed.  The
NOAEL is 100 mg/kg/day (the highest dose tested).

This immunotoxicity study is classified acceptable/guideline and
satisfies the guideline requirement for an immunotoxicity study (OPPTS
870.7800).

Appendix B.  Physical/Chemical Properties

Table B.1  Physicochemical Properties of Demiditraz 



Parameter	

Value	

Reference

Melting point/range	138.36 oC (range 137 – 142 oC) 	MRID No.

47744103

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                         

Toluene:   1 to 10                               

	Vapor pressure 	Not applicable as technical grade demiditraz is solid
at room temperature

	Dissociation constant (pKa)	Not applicable as technical grade
demiditraz is a weak base and insoluble in water

	Octanol/water partition coefficient  	The mean log10P value reported is
2.78 

	UV/visible absorption spectrum methanol (nm)	Shows maximum absorption
at about 264 nm 

	

 W. Britton. Demiditraz: Human Health Risk Assessment for Proposed Dog
Spot-On Use. D378783. 11/30/2010.

 HYPERLINK
"%20http:/www.epa.gov/pesticides/science/residential-exposure-sop.html" 
http://www.epa.gov/pesticides/science/residential-exposure-sop.html 

 W. Britton.  Demiditraz: Occupational and Residential Exposure and Risk
Assessment for the Proposed Pet Spot-On Use (1007-OT). D400484,.D409676,
D409677.  4/11/13.

 Available:  HYPERLINK
"http://www.epa.gov/pesticides/science/residential-exposure-sop.html"
http://www.epa.gov/pesticides/science/residential-exposure-sop.html 

 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 Formulated End-Use Products.” D409400, EPA MRID
48766704, 4/11/13. 

 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

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Demiditraz Human Health Risk Assessment		D410552

Page   PAGE  47  of   NUMPAGES   47 

