Document ID: EPA-HQ-OPP-2008-0854-0004
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2009-09-23T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C.  20460

OFFICE OF

PREVENTION, PESTICIDES, AND

TOXIC SUBSTANCES

MEMORANDUM

DATE:		17-MAR-2009

SUBJECT:		Meptyldinocap (DE-126/Dinocap II): PP# 7E7294.  Tolerances on
Fresh and Processed Imported Grapes.  Human-Health Risk Assessment.  

PC Code:  036000	DP Barcode:  348877

Decision No.:  386467	Registration No.:  XXX

Petition No.:  7E7294	Regulatory Action:  Section 3 Registration

Risk Assessment Type:  Single Chemical/Aggregate	Case No.:  NA

TXR No.:  NA	CAS No.:  131-72-6

MRID No.:  NA 	40 CFR:  §180.XXX

FROM:		Mary Clock-Rust, Biologist

		         Robert Mitkus, Ph.D., Toxicologist

		         Thomas Bloem, Chemist

		         Risk Assessment Branch 1 (RAB1)

Health Effects Division (HED, 7509P)

THROUGH:	Ray Kent, Risk Assessment Review Committee (RARC) Reviewer

			P.V. Shah, RARC Reviewer

			Dana M. Vogel Branch Chief

					George F. Kramer, Ph.D., Senior Chemist

					RAB1/HED (7509P)

TO:			Tamue Gibson, (RM 21)

Mary Waller

Insecticide – Fungicide Branch

Registration Division (7505P)

The Registration Division (RD) of OPP has requested that HED evaluate
toxicology and residue chemistry data and conduct dietary assessments to
estimate the risk to human health from the proposed use of the fungicide
meptyldinocap on grapes imported into the United States.  An assessment
of human risk resulting from the proposed use of meptyldinocap is
provided in this document.  The hazard assessment was provided by Robert
Mitkus of RAB1, the residue chemistry data review and dietary exposure
analysis by Thomas Bloem, and the risk assessment by Mary Clock-Rust. 
There are currently no registered food/feed uses or tolerances for
meptyldinocap in the U.S.  No drinking water, residential or
occupational risk assessment was provided as only dietary exposure from
food is expected.  

Table of Contents

  TOC \o "1-3" \h \z \u    HYPERLINK \l "_Toc225068407"  1.0  Executive
Summary	  PAGEREF _Toc225068407 \h  3  

  HYPERLINK \l "_Toc225068408"  2.0  Ingredient Profile	  PAGEREF
_Toc225068408 \h  5  

  HYPERLINK \l "_Toc225068409"  2.1  Summary of Registered/Proposed Uses
  PAGEREF _Toc225068409 \h  5  

  HYPERLINK \l "_Toc225068410"  2.2  Structure and Nomenclature	 
PAGEREF _Toc225068410 \h  6  

  HYPERLINK \l "_Toc225068411"  3.0  Hazard Characterization/Assessment	
 PAGEREF _Toc225068411 \h  7  

  HYPERLINK \l "_Toc225068412"  3.1	  Hazard and Dose-Response
Characterization	  PAGEREF _Toc225068412 \h  7  

  HYPERLINK \l "_Toc225068413"  3.1.1  Studies Considered in the
Toxicity and Dose-Response Evaluation	  PAGEREF _Toc225068413 \h  8  

  HYPERLINK \l "_Toc225068414"  3.1.2	  Mammalian Toxicology	  PAGEREF
_Toc225068414 \h  8  

  HYPERLINK \l "_Toc225068415"  3.2	  FQPA Considerations	  PAGEREF
_Toc225068415 \h  10  

  HYPERLINK \l "_Toc225068416"  3.3  Hazard Identification and Toxicity
Endpoint Selection	  PAGEREF _Toc225068416 \h  11  

  HYPERLINK \l "_Toc225068417"  3.3.1 	 aRfD/aPAD – All Populations	 
PAGEREF _Toc225068417 \h  12  

  HYPERLINK \l "_Toc225068418"  3.3.2	  cRfD/cPAD	  PAGEREF
_Toc225068418 \h  12  

  HYPERLINK \l "_Toc225068419"  3.4  Recommendation for Aggregate
Exposure Risk Assessments	  PAGEREF _Toc225068419 \h  13  

  HYPERLINK \l "_Toc225068420"  3.5  Classification of Carcinogenic
Potential	  PAGEREF _Toc225068420 \h  13  

  HYPERLINK \l "_Toc225068421"  3.6  Endocrine Disruption	  PAGEREF
_Toc225068421 \h  13  

  HYPERLINK \l "_Toc225068422"  4.0  Public Health and Pesticide
Epidemiology Data	  PAGEREF _Toc225068422 \h  13  

  HYPERLINK \l "_Toc225068423"  5.0  Dietary Exposure/Risk
Characterization	  PAGEREF _Toc225068423 \h  14  

  HYPERLINK \l "_Toc225068424"  5.1  Pesticide Metabolism and
Environmental Degradation	  PAGEREF _Toc225068424 \h  14  

  HYPERLINK \l "_Toc225068425"  5.1.1  Metabolism in Primary Crops	 
PAGEREF _Toc225068425 \h  14  

  HYPERLINK \l "_Toc225068426"  5.1.2  Metabolism in Rotational Crops	 
PAGEREF _Toc225068426 \h  14  

  HYPERLINK \l "_Toc225068427"  5.1.3  Metabolism in Livestock	  PAGEREF
_Toc225068427 \h  14  

  HYPERLINK \l "_Toc225068428"  5.1.4  Analytical Methodology	  PAGEREF
_Toc225068428 \h  14  

  HYPERLINK \l "_Toc225068429"  5.1.5  Comparative Metabolic Profile	 
PAGEREF _Toc225068429 \h  15  

  HYPERLINK \l "_Toc225068430"  5.1.6  Toxicity Profile of Major
Metabolites and Degradates	  PAGEREF _Toc225068430 \h  15  

  HYPERLINK \l "_Toc225068431"  5.1.7  Pesticide Metabolites and
Degradates of Concern	  PAGEREF _Toc225068431 \h  16  

  HYPERLINK \l "_Toc225068432"  5.1.8  Drinking Water Residue Profile	 
PAGEREF _Toc225068432 \h  16  

  HYPERLINK \l "_Toc225068433"  5.1.9  Food Residue Profile	  PAGEREF
_Toc225068433 \h  16  

  HYPERLINK \l "_Toc225068434"  5.1.10  International Residue Limits	 
PAGEREF _Toc225068434 \h  18  

  HYPERLINK \l "_Toc225068435"  5.2  Dietary Exposure and Risk	  PAGEREF
_Toc225068435 \h  18  

  HYPERLINK \l "_Toc225068436"  5.2.1  Acute Dietary Exposure/Risk	 
PAGEREF _Toc225068436 \h  18  

  HYPERLINK \l "_Toc225068437"  5.2.2  Chronic Dietary Exposure/Risk	 
PAGEREF _Toc225068437 \h  18  

  HYPERLINK \l "_Toc225068438"  5.2.3  Cancer Dietary Risk	  PAGEREF
_Toc225068438 \h  19  

  HYPERLINK \l "_Toc225068439"  5.3  Anticipated Residue and Percent
Crop Treated Information	  PAGEREF _Toc225068439 \h  19  

  HYPERLINK \l "_Toc225068440"  6.0  Aggregate Risk Assessments and Risk
Characterization	  PAGEREF _Toc225068440 \h  19  

  HYPERLINK \l "_Toc225068441"  7.0  Cumulative Risk
Characterization/Assessment	  PAGEREF _Toc225068441 \h  19  

  HYPERLINK \l "_Toc225068442"  8.0  Data Needs and Label Requirements	 
PAGEREF _Toc225068442 \h  20  

  HYPERLINK \l "_Toc225068443"  8.1  Residue Chemistry	  PAGEREF
_Toc225068443 \h  20  

  HYPERLINK \l "_Toc225068444"  8.2  Toxicology	  PAGEREF _Toc225068444
\h  20  

 1.0  Executive Summary

HED is conducting a risk assessment for meptyldinocap, in support of the
establishment of permanent tolerances on grapes imported into the U.S. 
Dow AgroSciences requests a grape tolerance of 0.3 ppm for residues of
meptyldinocap per se.  

The current assessment is in support of a meptyldinocap tolerance on
grapes without a U.S. registration (first food/feed use for
meptyldinocap).  Meptyldinocap is one of the six isomers found in the
older fungicide dinocap (dinocap is 22% meptyldinocap and 77% remaining
five isomers).  Based on a comparison of the toxicological databases,
HED has determined that meptyldinocap and dinocap are toxicologically
different with meptyldinocap being less toxic.  Neither meptyldinocap
nor dinocap are proposed or registered for application in the U.S.
(i.e., tolerances only, no U.S. registration).  However, dinocap
tolerances in/on apple and grape at 0.1 ppm are currently established
(40 CFR 180.341; tolerances without a U.S. registration); since a
portion of these residues can be assigned to meptyldinocap, the current
assessment did consider the dinocap tolerances.  

Because there are no proposed or existing residential uses for
meptyldinocap, and the proposed use is limited to crops exported to the
U.S., no occupational, residential or drinking water exposure assessment
is necessary.

Hazard Assessment

Meptyldinocap technical caused no deaths following acute oral [LD50
>2000 mg/kg body weight (bw)] or dermal (LD50 >5000 mg/kg bw) exposures.
 No abnormal clinical observations were recorded following dermal
exposure other than erythema/edema at the dose site at 5000 mg/kg bw
beginning on day 1 and persisting through days 4-9.  Meptyldinocap is
minimally irritating to the eye (Category III) and slightly irritating
to the skin (Category IV) and exhibited a skin sensitization potential
under the conditions of the local lymph node assay.  Short-term (90-day)
exposure of rats to meptyldinocap led to decreased body weight,
body-weight gain, and food consumption in both sexes at the highest dose
tested (113 mg/kg bw/day).  Dogs treated with low doses of meptyldinocap
(approximately 4 mg/kg bw/day) for the same length of time showed
evidence of hepatic toxicity, specifically as significantly increased
ALT (alanine aminotransferase) and AST (aspartate aminotransferase)
levels that were sustained throughout the treatment period.  However,
unlike the parent mixture dinocap, there was no evidence of ocular
toxicity in dogs with meptyldinocap during the 90-day treatment period,
or when treatment of these dogs was extended to one year.  No adverse
effects were observed in mice treated with meptyldinocap for 28 days. 
Meptyldinocap was tested in a number of developmental toxicity studies
in several species.  Unlike dinocap, which was teratogenic in mice and
rabbits, meptyldinocap caused no developmental toxicity in any species
tested.  Meptyldinocap was negative in two in vitro mutagenicity
studies, as well as in one in vivo and one in vitro clastogenicity
assay.

Long-term toxicity studies in rodents, including carcinogenicity
studies, and studies designed to assess male and female fertility were
not performed with meptyldinocap.  However, the hazard database for
meptyldinocap, in conjunction with the dinocap hazard database, is
adequate for the purposes of this action on imported grapes.  

Residues of Concern

The HED Residues of Concern Knowledge-Base Subcommittee (ROCKS)
determined that the residues of concern in grapes for tolerance
enforcement and risk assessment are meptyldinocap and 2,4-DNOP (D362393,
B. Daiss, 3-18-2009).  Based on available information, the 2,4-DNOP
metabolite may be more toxic than the parent compound based on a
comparison of acute toxicity data for both compounds.  However, the
toxicity/hazard of this metabolite is not a significant concern for risk
assessment at this time because exposure to 2,4-DNOP for the existing
and proposed uses is very low relative to the parent compound (i.e.,
2,4-DNOP accounts for <10% of TRR).  If future uses of meptyldinocap
indicate the potential for greater exposure to 2,4-DNOP, additional
toxicity data on this metabolite may be required.  

Dietary/Aggregate Risk Estimates 

A chronic dietary risk assessment was conducted using the Dietary
Exposure Evaluation Model - Food Consumption Intake Database
(DEEM-FCID™, ver. 2.03) which incorporates the food consumption data
from the USDA’s Continuing Surveys of Food Intakes by Individuals
(CSFII; 1994-1996 and 1998).  Based on toxicological considerations,
acute and cancer dietary risk assessments are unnecessary.  The chronic
analysis assumed tolerance-level residues and 100% crop treated.  Since
22% of technical dincocap is meptyldinocap and since the proportion of
dinocap residues occupied by meptyldinocap is unknown, the analysis
assumed that 100% of the dinocap residues on imported apples and grapes
were meptyldinocap.  Based on the submitted dinocap processing studies,
the default grape juice and wine processing factors were reduced to 1
(D355561, T. Bloem, 12-Feb-2009); the raisin, apple juice, and dried
apple default processing factors were retained as processing data for
these commodities were not submitted.  Note that the reduction in the
grape juice and wine processing factors were based on a dinocap
processing study (a meptyldinocap processing study was not submitted);
however, HED anticipates that dinocap and meptyldinocap will partition
in a similar manner during processing.  The resulting exposure estimates
are less than HED's level of concern (≤35% chronic population-adjusted
dose (cPAD); children 1-2 years old were the most highly exposed
population subgroup).  

There are no proposed or existing residential uses for meptyldinocap. 
The proposed use is limited to grapes exported to the U.S. only.  The
exposure/risk assessment is limited to dietary food only.  Therefore,
the dietary risk represents aggregate risk.

Environmental Justice Considerations

Potential areas of environmental justice concerns, to the extent
possible, were considered in this human health risk assessment, in
accordance with U.S. Executive Order 12898, "Federal Actions to Address
Environmental Justice in Minority Populations and Low-Income
Populations,"
http://www.hss.energy.gov/nuclearsafety/env/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 CSFII 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.

  

Recommendations 

Dow AgroSciences requests a grape tolerance of 0.3 ppm for residues of
meptyldinocap per se.  Provided the petitioner submits analytical
standards (meptyldinocap and 2,4-DNOP) to the EPA National Pesticide
Standards Repository, submits a revised Section F, and agrees to submit
a grape to raisin processing study in the future, HED concludes that the
toxicological and residue chemistry databases supports the establishment
of the following tolerance for the combined residues of meptyldinocap
(2-(1-methylheptyl)-4,6-dinitrophenyl (2E)-2-butenoate) and 2,4-DNOP
(2,4-dinitro-6-(1-methyheptyl)phenol) expressed as meptyldinocap in/on
grape - 0.20 ppm.

2.0  Ingredient Profile

Meptyldinocap (2-(1-methylheptyl)-4,6-dinitrophenyl (2E)-2-butenoate) is
a dinitrophenol fungicide which interferes with fungal respiration by
acting as an uncoupler of oxidative phosphorylation (Fungicide
Resistance Action Committee (FRAC) Group 29).  Meptyldinocap is one of
the six isomers found in the older fungicide dinocap; dinocap contains
three isomers of 2,4-dinitro-6-octylphenyl crotonate (2,4-DNOPC) and
three isomers of 2,6-dinitro-4-octylphenyl crotonate (2,6-DNOPC).  In
dinocap, the octyl groups for both the 2,4-DNOPC and 2,6-DNOPC isomers
are a mixture of 1-methylheptyl, 1-ethylhexyl, and 1-propylpentyl
isomers.  Meptyldinocap consists of >90% of the
2,4-dinitro-6-(1-methylheptyl) isomer with the remaining five isomers
each being present at <1% while dinocap consists of 22% of the
2,4-dinitro-6-(1-methylheptyl) isomer with the remaining five isomers
being present at 77% of the total (see Table B.9 in Appendix B for
structures).

2.1  Summary of Registered/Proposed Uses

cated that all uses are standardized on the following parameters: 
preharvest interval (PHI) of ≥21 days, a maximum application rate of
0.21 kg ai/ha (excluding post-harvest application to vines in Chile),
and a maximum of 4 applications per season.  A summary of the
registered/pending meptyldinocap grape application scenarios is provided
in Table 2.1 below.  The submitted information concerning the
proposed/registered application scenarios are adequate for establishment
of the proposed import tolerance on grapes.  

Table 2.1.  Summary of Directions for Use of Meptyldinocap on Grapes.

App. Timing, Type,

and Equip.	Formulation	App. Rate

(kg ai/ha)	Max. No. App. per Season	Max. Seasonal App. Rate

(kg ai/ha)	PHI

(days)	Use Directions and Limitations

Maximum Application Rate in Several Europe, Africa, and Asian Countries

Broadcast foliar - BBCH 13 (3 leaves unfolded) to 81 (beginning of
ripening); ground equipment	350 g/L EC	0.21	4	0.84	21	Retreatment
interval (RTI) = 10 days, RTI of 5 days when used as an eradicant late
in the season (only two applications may be made as a late season
eradicant )

Chile

Broadcast foliar - swollen-bud to preflower and postharvest; ground
equipment	350 g/L EC	preharvest - 

0.21 postharvest -0.41	3	not indicated	60	RTI = 7 days.

2.2  Structure and Nomenclature

The meptyldinocap nomenclature is summarized in Table 2.2.1; the
meptyldinocap physicochemical properties are summarized in Table 2.2.2. 

Common name	Meptyldinocap (also referred to as
2,4-dinitro-6-(1-methylheptyl) crotonate)

Company experimental name	DE-126; RH-23,163

IUPAC name	(RS)-2-(1-methylheptyl)-4,6-dinitrophenyl crotonate 1

CAS name	2-(1-methylheptyl)-4,6-dinitrophenyl (2E)-2-butenoate

CAS registry number	131-72-6

Molecular weight	364.40

End-use product (EP)	350 g/L EC (GF-1478 Fungicide; 33.2% EC)2

1  Previously referred to as 2,4-dinitro-6-(1-methylheptyl)phenyl
crotonate.

2  The TGAI contains >90% of the 2,4-dinitro-6-(1-methylheptyl) isomer;
the related isomers from dinocap are each present at <1%.



Table 2.2.2.  Physicochemical Properties of Technical Grade
Meptyldinocap.

Melting point	-22.5°C

pH	not applicable

Relative density	1.11

Water solubility (mg/L) at 20°C	0.151

Solvent solubility (g/L) at 25°C	Acetone	>252

Ethyl acetate	>256

1,2-Dichloroethane	>252

Xylene	>256

n-Heptane	>251

acidic: 	240 nm (ε=16727), 310 nm (ε=2155)

basic:	260 nm (ε=6376), 372 nm (ε=12980), 405 nm (ε=10969)

neutral:	240 nm (ε=16727), 310 nm (ε=2155)

Source: MRID 47304901

3.0  Hazard Characterization/Assessment

3.1	  Hazard and Dose-Response Characterization

Meptyldinocap (DE-126; Dinocap II) is the 2,4-dinitro-6-(1-methylheptyl)
phenyl crotonate isomer of the previously registered fungicide dinocap,
which was composed of six structurally related isomers in varying
proportions.  Meptyldinocap constituted approximately 1/5 of the mass of
the dinocap mixture.

Meptyldinocap technical caused no deaths following acute oral (LD50
>2000 mg/kg bw) or dermal (LD50 >5000 mg/kg bw) exposures.  No abnormal
clinical observations were recorded following dermal exposure other than
erythema/edema at the dose site at 5000 mg/kg bw beginning on day 1 and
persisting through days 4-9.  Meptyldinocap is minimally irritating to
the eye (Category III) and slightly irritating to the skin (Category IV)
and exhibited a skin sensitization potential under the conditions of the
local lymph node assay.  Short-term (90-day) exposure of rats to
meptyldinocap led to decreased body weight, body-weight gain, and food
consumption in both sexes at the highest dose tested (113 mg/kg bw/day).
 Dogs treated with low doses of meptyldinocap (approximately 4 mg/kg
bw/day) for the same length of time showed evidence of hepatic toxicity,
specifically as significantly increased ALT and AST levels that were
sustained throughout the treatment period.  However, unlike the parent
mixture dinocap, there was no evidence of ocular toxicity in dogs with
meptyldinocap during the 90-day treatment period, or when treatment of
these dogs was extended to one year.  No adverse effects were observed
in mice treated with meptyldinocap for 28 days.  Meptyldinocap was
tested in a number of developmental toxicity studies in several species.
 Unlike dinocap, which was teratogenic in mice and rabbits,
meptyldinocap caused no developmental toxicity in any species tested. 
Meptyldinocap was negative in two in vitro mutagenicity studies, as well
as in one in vivo and one in vitro clastogenicity assay.

3.1.1  Studies Considered in the Toxicity and Dose-Response Evaluation 

Data from the following studies were used to evaluate the hazard
potential of meptyldinocap.  

Acute:  Oral and dermal LD50 studies

Subchronic:  Three oral toxicity studies (mouse, rat, dog)

Long-term:  One oral toxicity study (dog)

Developmental:  Three developmental toxicity studies (mouse, rat,
rabbit) with meptyldinocap; two developmental toxicity studies with
various isomers of dinocap (mouse)

Genotoxicity:  Four mutagenicity/clastogenicity assays

ADME:  Three disposition studies (rat or mouse) 

Long-term toxicity studies in rodents, including carcinogenicity
studies, reproductive toxicity studies, and studies designed to assess
male and female fertility were not performed with meptyldinocap.

3.1.2	  Mammalian Toxicology

Absorption, Distribution, Metabolism, and Excretion

cid α-and β-oxidation.  Excretion of meptyldincap was primarily
through the feces (62-73%-confirm administered dose) and urine (15-20%
administered dose) following repeated dosing with 6-11% administered
dose accounted for in the cage wash.

Acute Toxicity

Meptyldinocap technical caused no deaths following acute oral (LD50
>2000 mg/kg bw) or dermal (LD50 >5000 mg/kg bw) exposures.  Animals
dosed orally at 1750 and 2000 mg/kg bw exhibited abnormal clinical
observations such as reduced fecal volume and/or anogenital or ventral
staining beginning on day one and continuing through days 2-4, after
which they were active and healthy.  The toxicological significance of
these observations is unknown.  No abnormal clinical observations were
recorded following dermal exposure other than erythema/edema on the dose
site at 5000 mg/kg bw beginning on day 1 and persisting through days
4-9.  Meptyldinocap is minimally irritating to the eye (Category III)
and slightly irritating to the skin (Category IV) and exhibited a skin
sensitization potential under the conditions of the local lymph node
assay.   

Short-term Toxicity

A summary of the toxicity database for meptyldinocap is found in
Appendix A.2.  No adverse effects were observed in mice treated with
meptyldinocap for 28 days.  Short-term (90-day) exposure of rats to
meptyldinocap led to decreased body weight, body-weight gain, and food
consumption in both sexes at the highest dose tested (113 mg/kg bw/day).
 Dogs treated with low doses of meptyldinocap (approximately 4 mg/kg
bw/day) for the same length of time showed evidence of hepatic toxicity,
specifically as significantly increased ALT and AST levels that were
sustained throughout the treatment period.  However, unlike the parent
mixture dinocap, there was no evidence of ocular toxicity in dogs with
meptyldinocap during the 90-day treatment period, or when treatment of
these dogs was extended to one year.  Ocular toxicity was chosen as the
primary endpoint measured in this one-year extension study with
meptyldinocap in the dog, because dinocap, a mixture of isomers,
including meptyldinocap, had caused adverse ophthalmoscopic changes,
including retinopathies, at similar doses in a previous 2-year toxicity
study in dogs, as well as in the current 90-day dog study when included
as a positive control.  No adverse effects were observed on several
other measured parameters in the dog after one year, including
mortality, clinical signs, or gross or microscopic pathological
examinations of the tibial nerve and heart.  However, because control
animals were not kept on study beyond 90 days, it was not possible to
assess whether there were treatment-related effects on body weight, body
weight gain, or food consumption over one year.

Developmental toxicity including postnatal development

Meptyldinocap was tested in a number of developmental toxicity studies
in several species.  Unlike dinocap, which was teratogenic in mice and
rabbits, meptyldinocap caused no developmental toxicity in any species
tested.  In the developmental toxicity study with meptyldincocap in
mice, no external alterations were observed in fetuses and there was no
effect on otoconial development up to the highest dose tested (500 mg/kg
bw/day).  [Development of a specific structure in the inner ear involved
with balance (otoconia) was shown previously to be adversely affected by
dinocap.  This teratogenic effect led to tilting of the head
(torticollis) and behavioral abnormalities in mouse offspring that were
exposed to dinocap as fetuses.] As expected, mice treated with dinocap
as a positive control in the study developed cleft palate and almost
complete otoconial agenesis.  No toxicity was observed with
meptyldinocap in maternal mice up to 500 mg/kg bw/day.  In two
additional special studies in mice with the various isomers of dinocap,
it was determined that the characteristic developmental toxicity of
dinocap was due to its 2,6-dinitro-4-(1-propylpentyl)-phenyl crotonate
isomer. 

In the developmental toxicity study in rabbits, no developmental
toxicity, including effects on otoconial or ocular development, was
observed with meptyldinocap at doses up to 48 mg/kg bw/day.  This was
the same dose at which dinocap was observed to cause malformations of
the neural tube, spine, and skull when tested previously under a similar
protocol.  Dinocap also induced cataracts in rabbits previously at a
similar dose.  In the present study in rabbits, meptyldinocap induced
decreased body-weight gains and food consumption but no cataracts in
maternal animals at the highest dose tested.  In a guideline
developmental toxicity study in rats, no developmental toxicity was
observed with meptyldinocap at doses up to 150 mg/kg bw/day.  Maternal
toxicity (decreased body weight/gains and food consumption) was observed
at this same dose, and dams treated with 500 mg/kg bw/day were
sacrificed early due to excessive toxicity. 

Genotoxicity

Meptyldinocap was negative in two in vitro mutagenicity studies, as well
as in one in vivo and one in vitro clastogenicity assay.

Immunotoxicity

The current toxicological database supports the conclusion that
meptyldinocap is less toxic than dinocap.  An immunotoxicity study has
not been conducted with meptyldinocap.  However, an in vivo
immunotoxicity study with additional in vitro measurements (Smialowicz,
et al., 199) has been conducted with dinocap in mice and published in
the open literature.  Immune function, cellularity, and organ weights
were measured over several doses in the study.  Because a well conducted
immunotoxicity study with dinocap was performed previously, and since
meptyldinocap is considered less toxic than dinocap, HED’s RARC
determined that the requirement for an immunotoxicity study with
meptyldinocap has been satisfied by the literature study with dinocap
and that selected endpoints are protective of immunotoxicity; although
an acute endpoint has not been selected for meptyldinocap,
immunotoxicity is not relevant for an acute endpoint.

3.2	  FQPA Considerations

The toxicology database used to assess increased sensitivity of the
young to meptyldinocap is adequate for the purposes of this import
tolerance.  Acceptable toxicological studies include guideline
developmental toxicity studies in rats and rabbits, as well as two
non-guideline developmental toxicity studies in mice, one of which also
tested for postnatal developmental effects.  Results of these studies
have been summarized in Section 3.1.2 and executive summaries are
available in Appendix 1 of this risk assessment.  There was no evidence
of increased susceptibility of offspring following prenatal exposure of
either rats or rabbits.  In both the rat and rabbit developmental
toxicity studies, toxicity to offspring was not observed, whereas
maternal toxicity was observed at the highest dose tested in both
studies.  Although a limited number of developmental endpoints was
measured in the non-guideline developmental toxicity studies in the
mouse with meptyldinocap (MRIDs 47289132 and 47304907), meptyldinocap
failed to cause either offspring or maternal toxicity in either study. 
Therefore, no evidence of increased susceptibility of offspring was
found in any of four relevant toxicity studies with meptyldinocap.  As
stated earlier, although a guideline reproductive toxicity study was not
performed with meptyldinocap, postnatal toxicity to offspring was
assessed, and not observed, in a special developmental toxicity study
with meptyldinocap in mice.  These results contrast with those for
dinocap, which was used as a positive control in the study and caused
developmental toxicity as well as adverse postnatal effects.

Despite the lack of developmental toxicity with meptyldinocap, the
compound caused hepatic toxicity in a 90-day oral toxicity study in the
dog.  Because this study was the most relevant and available study with
meptyldinocap, it was used to derive the cRfD.  Longer-term studies with
meptyldinocap would have been more relevant for this exposure scenario. 
Since longer guideline studies were lacking (including reproductive
toxicity studies), the risk assessment team recommends that the FQPA SF
for meptyldinocap toxicity be retained in the form of a UFS.  

However, the risk assessment team recommends that the magnitude of the
SF be reduced to 3X (from 10X) for the following reasons:

There was no effect of treatment on mortality, clinical signs,
ophthalmological examinations, or select gross or microscopic pathology
in dogs treated for one year with meptyldinocap;

While levels of serum hepatic enzymes in dogs were increased
significantly over controls throughout the 90-day exposure period, they
did not become more severe over time;

There is no evidence of offspring susceptibility with meptyldinocap in
any of four developmental toxicity studies across three species tested;

There was no evidence of neurotoxicity or neuropathology in any of the
submitted studies for meptyldinocap.  These results contrast with those
for dinocap in which minor neuropathology was noted in dogs treated with
dinocap as a positive control for 90 days.

Use of a 3X SF with the NOAEL of 1.51 mg/kg bw/day from the 90-day
toxicity study in the dog yields an effective NOAEL of 0.5 mg/kg bw/day
for meptyldinocap.  This value is virtually identical to the NOAEL used
for the cRfD for dinocap (0.4 mg/kg bw/day).  Use of a larger SF for
meptyldinocap would yield a lower effective NOAEL than that for dinocap,
which makes little sense, given that meptyldinocap is considered less
toxic than dinocap;

Meptyldinocap is considered less toxic than dinocap based on the lack of
developmental and ocular toxicities with meptyldinocap at approximately
5X the doses contained in dinocap.

3.3  Hazard Identification and Toxicity Endpoint Selection

The current regulatory request is for a tolerance for residues of
meptyldinocap on imported grapes.  The toxicological data requirements
for an import tolerance are the same as those for a domestic tolerance
in the U.S., with the exception that dermal and inhalation toxicology
studies are not required
(http://www.epa.gov/oppfead1/international/naftatwg/guidance/nafta-guida
nce.pdf).  Since there is no residential exposure when an import
tolerance is requested, potential human exposure is by the dietary route
only.  Therefore, acute and chronic oral exposures are the only relevant
exposure scenarios.  A summary of the toxicological endpoints and doses
chosen for the two relevant dietary exposure scenarios for human health
risk assessment is found in Table 3.3.

Table 3.3.  Summary of Toxicological Doses and Endpoints for
Meptyldinocap for Use in Dietary Human Health Risk Assessment.

Exposure Scenario	Point of Departure	Uncertainty/FQPA Safety Factors
RfD, PAD, LOC for Risk Assessment	Study and Relevant Toxicological
Effects

Acute Dietary (All populations)	N/A	N/A	N/A	An endpoint of concern
(effect) attributable to a single dose was not identified in the
database. Quantification of acute risk to all populations is not
necessary.

Chronic Dietary (All populations)	NOAEL = 1.51

mg/kg bw/day	UFA = 10X

UFH = 10X

UFFQPA = 3X

(includes UFS) 	cRfD = cPAD = 0.005 mg/kg bw/day	90-day oral (dog;
dietary) 

LOAEL = 3.58 mg/kg bw/day based on sustained increased ALT and AST
levels in males

Cancer	Carcinogenicity studies with meptyldinocap not performed. 
Classification of dinocap: “Group E, Evidence of non-carcinogenicity
in humans” (1994, TXR# 0011076)

Abbreviations: UF = uncertainty factor, UFA = extrapolation from animal
to human (interspecies), UFH = potential variation in sensitivity among
members of the human population (intraspecies), UFFQPA = FQPA Safety
Factor, UFS = extrapolation from subchronic to chronic exposure, NOAEL =
no-observed adverse-effect level, LOAEL = lowest observed adverse effect
level, RfD = reference dose (a = acute, c = chronic), PAD =
population-adjusted dose, MOE = margin of exposure, LOC = level of
concern.

3.3.1 	 aRfD/aPAD – All Populations

A toxicological endpoint of concern (effect) attributable to a single
dose was not identified in the database.  Quantification of acute risk
to all populations is not necessary.  

3.3.2	  cRfD/cPAD

There are no long-term guideline toxicity studies with meptyldinocap. 
Among the short-term oral toxicity studies with meptyldinocap, the
90-day oral toxicity study in the dog has the lowest defined NOAEL (1.51
mg/kg bw/day) and LOAEL (3.58 mg/kg bw/day).  The LOAEL was based on
significant and sustained increased serum ALT and AST levels in males. 
This study was considered the most relevant of the available studies
with meptyldinocap to assess chronic oral exposure of the general
population, because 1) the study was conducted with the chemical of
concern and 2) unlike dinocap for which ocular toxicity was observed at
2-fold lower doses when included as a positive control in the study, no
ocular toxicity was observed with meptyldinocap.  Because this
short-term study is being used for a long-term exposure scenario, the
FQPA SF, as a UFS, is being retained at 3X (see Section 3.2).  The
one-year oral toxicity study in the dog (extension of the 90-day study)
would have been a better choice for this exposure scenario.  However,
because a control group was not included and very few guideline
parameters were assessed in the one-year extension, it could not be used
for endpoint selection.  Other studies considered for the cRfD for
meptyldinocap included the traditional long-term studies with dinocap as
described in the dinocap toxicology chapter for the RED (2000).  A
comparison of these studies is made in Table 3.3.2 below.

Table 3.3.2.  Comparison of Dinocap and Meptyldinocap Toxicity.

Study	NOAEL	LOAEL	Toxicological Effects

Meptyldinocap:

90-day oral toxicity (dog)	1.51 mg/kg bw/day	3.58 mg/kg bw/day
Significant and sustained increased ALT and AST levels 

Dinocap:

Combined chronic toxicity/carcinogenicity study (rat)	1 mg/kg bw/day
(estimated)	10 mg/kg bw/day (estimated)	“non-neoplastic effects”
(multi-organ histopathology)

Dinocap:

Carcinogenicity study (mouse)	2.8 mg/kg bw/day	17.8 mg/kg bw/day
Decreased BW/BWG and FC

Dinocap:

2-year oral toxicity (dog)	0.375 mg/kg bw/day	1.5 mg/kg bw/day
Ophthalmoscopic changes, retinal atrophy

BW: body weight; BWG: body-weight gain; FC: food consumption. 

The chronic toxicity and/or carcinogenicity studies (rat and mouse) from
dinocap were not used as bridging studies for the chronic dietary
endpoint for meptyldinocap since:  1) the combined chronic
toxicity/carcinogenicity study in the rat with dinocap was classified as
Unacceptable and, therefore, not usable for risk assessment purposes; 2)
the dinocap carcinogenicity mouse study with a NOAEL of 2.8 mg/kg/day
has a higher NOAEL than the subchronic dog meptyldinocap study (NOAEL =
1.5 mg/kg/day).  In addition, the dinocap database shows that the rat
and mouse are less sensitive than the dog following chronic exposure. 
Having a point of departure (POD) for meptyldinocap that is comparable
to the most sensitive species (dog) in the worse-case chemical (dinocap)
gives confidence that this endpoint is protective.

3.4  Recommendation for Aggregate Exposure Risk Assessments

An aggregated exposure risk assessment is not necessary since there are
no residential uses for meptyldinocap at this time.  

3.5  Classification of Carcinogenic Potential

Meptyldinocap was negative in two in vitro mutagenicity studies, as well
as in one in vivo and one in vitro clastogenicity assay.  The
carcinogenic potential of meptyldinocap has not been tested.  The parent
mixture dinocap, however, was previously classified as “Group E,
Evidence of non-carcinogenicity in humans” (1994, TXR# 0011076).  The
risk assessment team in consultation with HED’s ToxSac concluded that
given the lack of developmental, ocular, and genetic toxicities with
meptyldinocap, dinocap toxicity represents a “worst case” scenario,
relative to meptyldinocap.  In addition, in the case of dinocap, the rat
and mouse were considered less sensitive than the dog.  Meptyldinocap
appears to follow the same pattern.  Therefore, since effects observed
in the most sensitive species (dog) are being used for meptyldinocap
risk assessment, and since these effects are not expected to become more
severe with longer exposure, chronic studies with meptyldinocap,
including carcinogenicity studies, are not requested.

3.6  Endocrine Disruption

EPA is required under the Federal Food, Drug and Cosmetic Act (FFDCA),
as amended by FQPA, to develop a screening program to determine whether
certain substances (including all pesticide active and other
ingredients) “may have an effect in humans that is similar to an
effect produced by a naturally occurring estrogen, or other such
endocrine effects as the Administrator may designate.”  Following
recommendations of its Endocrine Disruptor and Testing Advisory
Committee (EDSTAC), EPA determined that there was a scientific basis for
including, as part of the program, the androgen and thyroid hormone
systems, in addition to the estrogen hormone system.  EPA also adopted
EDSTAC’s recommendation that the Program include evaluations of
potential effects in wildlife.  For pesticide chemicals, EPA will use
FIFRA and, to the extent that effects in wildlife may help determine
whether a substance may have an effect in humans, FFDCA authority to
require the wildlife evaluations.  As the science develops and resources
allow, screening of additional hormone systems may be added to the
Endocrine Disruptor Screening Program (EDSP).

When additional appropriate screening and/or testing protocols being
considered under the Agency’s EDSP have been developed, meptyldinocap
may be subjected to further screening and/or testing to better
characterize effects related to endocrine disruption.

4.0  Public Health and Pesticide Epidemiology Data

Literature searches performed by R. Mitkus (January, 2009) did not yield
any epidemiology data for meptyldinocap.

 

5.0  Dietary Exposure/Risk Characterization

5.1  Pesticide Metabolism and Environmental Degradation

5.1.1  Metabolism in Primary Crops

The petitioner submitted apple (two studies), cucumber, and squash
metabolism studies.  All of the studies were conducted with
[14C-U-phenyl]-meptyldinocap except for one of the apple studies which
was conducted with [14C-U-phenyl] 2,6-dinitro-4-(1-methylheptyl)phenyl
crotonate.  The apple metabolism studies indicate that following a
single foliar application 0-21 days prior to harvest, the majority of
the residues were in/on the fruit surface or peel and were degraded via
photolysis and/or metabolism to a large number of low-concentration
metabolites.  Meptyldinocap was the only compound found at greater than
10% of the total radioactive residue (TRR; 12-75% TRR); 2,4-DNOP (2-6%
TRR) and several benzoxazole compounds (<1% TRR) were also identified
(see Table B.9 in Appendix B for structures).  The squash and cucumber
studies are considered scientifically unacceptable.  These studies
contain a number of deficiencies relating to the lack of adequate
information on the test substance, field trial procedures, and sample
storage conditions.  In addition, substantial proportions of the TRR in
both the cucumber and squash samples were not adequately
characterized/identified, and the identities of meptyldinocap and
2,4-DNOP were not confirmed using a second analytical method.  While the
squash and cucumber studies are inadequate, they do support the
identification of 2,4-DNOP as a minor metabolite of meptyldinocap.

5.1.2  Metabolism in Rotational Crops

Information concerning the nature of the residue in rotational crops was
not submitted.  Since there are no proposed/registered domestic uses,
these data are unnecessary.  

5.1.3  Metabolism in Livestock 

Information concerning the nature of the residue in livestock was not
submitted.  Since there are no significant feedstuffs associated with
the proposed use (see Organization of Economic Cooperation and
Development (OECD) guidance document -   HYPERLINK
"http://www.oecd.org/dataoecd/60/9/41808585.pdf" 
http://www.oecd.org/dataoecd/60/9/41808585.pdf ), these data are
unnecessary.  

5.1.4  Analytical Methodology

The petitioner has submitted adequate validation and independent
laboratory validation (ILV) data for a liquid chromatography/mass
spectrometry/mass spectrometry method (LC/MS/MS; Method No.
DOS/220-01R).  This method does not distinguish between meptyldinocap
and 2,4-DNOP.  Briefly, residues are extracted with acetone:MeOH:4 N HCl
(100:10:5, v:v:v) followed by base hydrolysis of meptyldinocap residues
to 2,4-DNOP.  Residues of 2,4-DNOP are quantified by summing the m/z
295.1→208.9 and 295.1→193.1 transitions (residues expressed in
parent equivalents).  Radiovalidation data were not submitted for this
method; based on the extraction solvent employed in this method and the
results from the meptyldinocap apple metabolism study (meptyldinocap and
2,4-DNOP only identified in the MeOH extract), radiovalidation is
unnecessary.  HED has determined that this method is adequate for
tolerance enforcement and forwarded the method to the Food and Drug
Administration (FDA) for publication in the Pesticide Analytical Manual
(PAM; D361115, T. Bloem, 2-12-2009).  

5.1.5  Comparative Metabolic Profile

ase, and aldehyde dehydrogenase to form the carboxylated metabolite,
2,4-dinitro-6-(1-methylheptanoate)phenol.  Further metabolism proceeds
via fatty acid α-and β-oxidation.  

The apple metabolism studies resulted in the identification of parent
(12-75% TRR), 2,4-DNOP (2-6% TRR), and several benzoxazole compounds
(metabolites B-E; all at <1% TRR; see Table B.9 in Appendix B for
structures).  The majority of the TRR appeared to be a complex mixture
of minor metabolites.  Based on these data, the parent appears to
undergo hydrolysis of the crotonate ester to form 2,4-DNOP.  The 2-nitro
group of 2,4-DNOP is reduced to an amine which can then react with a
variety of acids to form an amide; further reactions result in the
formation of the benzoxazole compounds.  

5.1.6  Toxicity Profile of Major Metabolites and Degradates

The ROCKS concluded that the 2,4-DNOP metabolite may be more toxic than
the parent compound (D362393, B. Daiss, 3-18-2009) based on the acute
toxicity of the metabolite compare with that of meptyldinocap.  Based on
acute and chronic dietary analyses, HED concludes that exposure to
2,4-DNOP is not of concern (see below); these analyses incorporated the
dinocap uses as it is likely that this compound will result in the
formation of 2,4-DNOP/2,6-DNOP.  If future uses of meptyldinocap/dinocap
indicate the potential for greater exposure to 2,4-DNOP/2,6-DNOP, then
additional toxicity data on these metabolites may be required.  Note
that the field trial and processing studies employed a method which did
not distinguish between meptyldinocap and 2,4-DNOP; therefore, residues
of 2,4-DNOP/2,6-DNOP were estimated using the results from the
meptyldincoap apple metabolism study.

Acute:  Based on the meptyldinocap apple metabolism study, the ratio of
combined meptyldinocap and 2,4-DNOP to 2,4-DNOP is 3.1:1.  A residue
estimate for 2,4-DNOP/2,6-DNOP was calculated by dividing the
meptyldinocap and dinocap tolerances (i.e., combined parent and
2,4-DNOP/2,6-DNOP residues) by 3.1.  The resulting residue estimates
were incorporated into an acute dietary analysis along with the grape
juice and wine processing factors (grape juice processing factor
translated to apple juice; 100% crop treated assumed).  The resulting
highest exposure estimate was for children 1-2 years at 0.0019
mg/kg/day; using the 2,4-DNOP LD50 of 211 mg/kg (D362393, B. Daiss,
3-18-2009), a MOE of 110,000 is calculated.  Therefore, HED does not
have a concern for the potential acute exposure to 2,4-DNOP/2,6-DNOP.

analysis resulted in exposures ≤35% cPAD (assumed tolerance level
residues (0.20 ppm) and 100% crop treated; see Table 5.2.3).  As
indicated above, the meptyldinocap apple metabolism study indicated a
ratio of combined meptyldinocap and 2,4-DNOP to 2,4-DNOP of 3.1:1. 
Dividing the average grape field trial residue (combined meptyldinocap
and 2,4-DNOP; 0.041 ppm) by 3.1 yields a residue of 0.013 ppm which is
15.4x lower than the residue estimate incorporated into the
meptyldinocap chronic analysis.  Since HED does not anticipate that the
chronic endpoint for 2,4-DNOP/2,6-DNOP to be 44x lower than that for
meptyldinocap, HED does not have a concern for the potential chronic
exposure to 2,4-DNOP/2,6-DNOP.  Note that the chronic analysis did not
incorporate the grape juice and wine reduction factors (factor of 1
assumed) and the average grape field trial residue was calculated
assuming LOQ residues for those <LOQ (65% <LOQ).  

5.1.7  Pesticide Metabolites and Degradates of Concern 

The HED ROCKS determined that, for purposes of this petition, the
available apple metabolism data are acceptable.  Based on these data,
the ROCKS concluded that the residues of concern in grapes for tolerance
enforcement and risk assessment are meptyldinocap and 2,4-DNOP (D362393,
B. Daiss, 3-18-2009).  

Table 5.1.7.  Summary of Proposed Residues of Concern for Risk
Assessment and Tolerances.

Matrix	Residues included in Risk Assessment	Residues included in
Tolerance Expression

Grape	meptyldinocap and 2,4-DNOP	meptyldinocap and 2,4-DNOP

Ruminant	N/A

Poultry

	Rotational Crops

	Water	No U.S. registrations; therefore, determination of the residue of
concern in water is not necessary.

5.1.8  Drinking Water Residue Profile

As there are no proposed/registered domestic uses, a drinking water
assessment is not necessary.  

5.1.9  Food Residue Profile

 

The petitioner submitted adequate meptyldinocap grape field trial
studies.  All of the field trials were conducted in Europe; additional
data in Chile, Africa, and/or Asia are not being requested for the
following reasons:  (1) based on the Chilean application scenario (3 x
0.21 kg ai/ha; PHI = 60 days) and that performed in the field trials (4
x 0.21 kg ai/ha; PHI = 21 days), HED concludes that the European field
trial data are sufficient (ChemSAC minutes, 17-Sep-2008) and (2) the
U.S. does not import a significant quantity of table grapes, wine, or
raisins from the relevant African and Asian countries.  

The European field trial data indicated that following four applications
at 0.19-0.24 kg ai/ha (1x single/seasonal rate; RTI = 5-32 days),
combined meptyldinocap and 2,4-DNOP residues ranged from <0.025 ppm to
0.12 ppm.  HED notes that the proposed minimum RTI for the final two
applications is 5 days (studies employed RTIs of 5-32 days), only single
samples were collected at 11 of the 16 trials rather than duplicates as
requested in the OPPTS 860.1500 guidelines, and the studies employed
only wine grape varieties.  

For the following reasons, HED concludes that additional data are
unnecessary:  (1) RTIs - Based on the residue decline data which
indicated a rapid decrease in residues as the PHI increased from 0 to 14
days and then a more slow decline thereafter and the proposed 21-day
PHI, HED concludes that the employed RTIs are acceptable; (2) single
sample - The number of field trials conducted in Europe (n=16) exceeds
the number required for establishment of a U.S. tolerance (n=12) and
they were conducted over two years (2005 and 2006); and (3) wine grape
varieties - The U.S. does not import a significant quantity of table
grapes from Europe and table grapes have larger berries than wine or
raisin grapes and would, therefore, have a smaller surface area per unit
volume ratio than wine or raisin grapes (most likely resulting in lower
residues).  

Since 65% of the residues were <LOQ (limit of quantitation), employment
of the North American Free Trade Act (NAFTA) maximum residue limit (MRL)
calculator is inappropriate.  Based on the submitted data, HED concludes
that a tolerance of 0.20 ppm in/on grapes for the combined residues of
meptyldinocap and 2,4-DNOP (expressed in parent equivalents) is
appropriate.  A revised Section F is requested.  

The dinocap grape processing studies indicated that residues decreased
in juice (<0.15x) and wine (<0.15x).  Due to structural similarities,
HED concludes that dinocap and meptyldinocap will partition in a similar
manner during processing.  Therefore, separate grape juice and wine
tolerances are unnecessary.  The petitioner has not submitted
raisin-processing data.  Approximately 10% of the U.S. raisin
consumption is derived from imports with Chile and the Republic of South
Africa occupying 52% (5% of U.S. consumption) and 13% (1% of U.S.
consumption), respectively, of the total imported (remaining imported
raisins derived from countries where meptyldinocap is not
registered/proposed for application to grapes).  The petitioner
indicated that preliminary residue data are available which indicate
only a slight concentration of residues in raisin (concentration factor
was not provided).  Based on this information and the limited amount of
raisins imported from the relevant countries (note that Chile has a
lower application rate and longer PHI than Europe), HED concludes that a
tolerance is unnecessary.  However, the raisin processing data should be
submitted to HED when finalized; pending the submission of these data,
the risk assessment should assume a default raisin processing factor.  

Based on the application in the Middle East, Europe, and Northern
Africa, a dinocap apple import tolerance of 0.1 ppm is currently
established (U.S. EPA Reregistration Eligibility Document (RED),
29-May-2003).  The OECD guidance document - (  HYPERLINK
"http://www.oecd.org/dataoecd/60/9/41808585.pdf" 
http://www.oecd.org/dataoecd/60/9/41808585.pdf ) indicates that in the
European Union, wet apple pomace is feed to beef (20% of the diet) and
dairy (10% of the diet) cattle.  HED concluded that this use will not
result in significant meptyldinocap residues in livestock commodities
for the following reasons (residue data are unnecessary):  (1) wet apple
pomace is a minor feed commodity; (2) meptyldinocap comprises ~22% of
technical dinocap; and (3) due to the perishable nature of wet apple
pomace, chronic exposure to commodities derived from ruminants feed wet
apple pomace is unlikely (acute endpoint for meptyldinocap was not
identified); and (4) there are no U.K. or EC tolerances for
meptyldinocap or dinocap in/on livestock.  

5.1.10  International Residue Limits

Based on the currently available data, HED recommends for a
establishment of the tolerance listed in Table 5.1.1 for the combined
residues of meptyldinocap and 2,4-DNOP in/on grapes.  There are
currently no established Codex, Canadian, or Mexican maximum residue
limits (MRLs) for meptyldinocap on grapes.  However, a grape MRL of 1
ppm for the combined residues of meptyldinocap and 2,4-DNOP is
established in the U.K. (  HYPERLINK
"https://secure.pesticides.gov.uk/MRLs/mrls.asp?page=1" \t "_blank" 
https://secure.pesticides.gov.uk/MRLs/mrls.asp?page=1 ).  The petitioner
anticipates that once the European Union reviews the meptyldinocap
residue data, the MRL will be lowered.  Since there are currently no
Canadian, Codex, or Mexican meptyldinocap grape MRLs, harmonization is
irrelevant. 

Table 5.1.10.  Tolerance Summary for Meptyldinocap.

Commodity	Proposed Tolerance (ppm)	Recommended Tolerance (ppm)	Comments;
Correct Commodity Definition

Grape	0.3	0.20	Revised Section F is requested (revise tolerance
expression and numerical value of the tolerance).

Grape, wine	0.3	None	Adequate data are available indicating that
residues do not conc. in grape juice or wine; therefore, separate
tolerances are not necessary

Grape, juice	0.3	None

	

5.2  Dietary Exposure and Risk

A dietary risk assessment was conducted using DEEM-FCID™, ver. 2.03,
which incorporates the food consumption data from the USDA’s CSFII
(1994-1996 and 1998).  

5.2.1  Acute Dietary Exposure/Risk

An endpoint of concern (effect) attributable to a single dose was not
identified in the database (all population subgroups).  Therefore, an
acute dietary risk assessment is not necessary.

5.2.2  Chronic Dietary Exposure/Risk

s level of concern (≤35% cPAD; children 1-2 years old were the most
highly exposed population subgroup; see Table 5.2.3.1).

  

5.2.3  Cancer Dietary Risk

A cancer dietary exposure analysis was not conducted for this action. 
Based on structural similarities and the demonstrated lower toxicity of
meptyldinocap as compared to dinocap, the cancer classification for
dinocap was extended to meptyldinocap (classification of dinocap:
“Group E, Evidence of non-carcinogenicity in humans;" 1994, TXR#
0011076).

™

Exposure (mg/kg/day)	% PAD

Acute Dietary Estimates

Acute Dietary (All populations)	An endpoint of concern (effect)
attributable to a single dose was not identified in the database.
Quantification of acute risk to all populations is not necessary.

Chronic Dietary Estimates

U.S. Population	0.005	0.000255	5.0

All infants (< 1 yr)

0.000939	19

Children 1-2 yrs

0.001734	35

Children 3-5 yrs

0.001035	21

Children 6-12 yrs

0.000353	7.0

Youth 13-19 yrs

0.000128	3.0

Adults 20-49 yrs

0.000119	2.0

Adults 50+ yrs

0.000138	3.0

Females 13-49 yrs

0.000133	3.0

Cancer Dietary Estimate

U.S. Population	Carcinogenicity studies with meptyldinocap not
performed.  Classification of dinocap: “Group E, Evidence of
non-carcinogenicity in humans” (1994, TXR# 0011076).

5.3  Anticipated Residue and Percent Crop Treated Information

The chronic analysis assumed tolerance level residues and 100% crop
treated.  

6.0  Aggregate Risk Assessments and Risk Characterization

There are no proposed or existing residential uses for meptyldinocap. 
The proposed use is limited to grapes exported to the U.S. only.  The
exposure/risk assessment is limited to dietary food only, and the
dietary risk reported in Section 5.2 represents aggregate risk.

7.0  Cumulative Risk Characterization/Assessment

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 meptyldinocap and any other
substances and meptyldinocap 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 meptyldinocap 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   HYPERLINK
http://www.epa.gov/pesticides/cumulative/.
http://www.epa.gov/pesticides/cumulative/. 

8.0  Data Needs and Label Requirements

8.1  Residue Chemistry

Revised Section F.

Grape to raisin processing data.

Submission of analytical standards (meptyldinocap and 2,4-DNOP) to the
EPA National Pesticide Standards Repository:

	USEPA (attn:  Theresa Cole)

	National Pesticide Standards Repository/Analytical Chemistry Branch/OPP

	701 Mapes Road

	Fort George G. Meade, MD  20755-5350

8.2  Toxicology 

Guideline acute and subchronic neurotoxicity studies, as part of the
updated toxicological data requirements (Part 158).Appendix A:  Hazard
Assessment

A.1  Toxicology Data Requirements

The requirements (40 CFR 158.340) for the use of meptyldinocap on food
at the time of data submission are listed in Table 1.  Use of the new
guideline numbers does not imply that the 1998 guideline protocols were
used.

Test 

	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		yes

yes

no

no

no	yes

yes

-

-

-

870.3700a  Developmental Toxicity (rodent)	

870.3700b  Developmental Toxicity (nonrodent)	

870.3800    Reproduction		yes

yes

no	yes

yes

no

870.4100a  Chronic Toxicity (rodent)	

870.4100b  Chronic Toxicity (nonrodent)	

870.4200a  Oncogenicity (rat)	

870.4200b  Oncogenicity (mouse)	

870.4300    Chronic/Oncogenicity		no

no

no

no

no	-

-

-

-

-

870.5100    Mutagenicity—Gene Mutation - bacterial	

870.5300    Mutagenicity—Gene Mutation - mammalian	

870.5xxx    Mutagenicity—Structural Chromosomal Aberrations	

870.5xxx    Mutagenicity—Other Genotoxic Effects		yes

yes

yes

yes	yes

yes

yes

yes

870.6100a  Acute Delayed Neurotox. (hen)	

870.6100b  90-Day Neurotoxicity (hen)	

870.6200a  Acute Neurotox. Screening Battery (rat)	

870.6200b  90-Day Neuro. Screening Battery (rat)	

870.6300    Develop. Neuro		no

no

yes

yes

no	-

-

no

no

-

870.7485    General Metabolism	

870.7600    Dermal Penetration		yes

no	yes

-

A.2  Toxicity Profiles

Table A.2.1.	Acute Toxicity Profile – Technical Meptyldinocap.

Guideline No.	Study Type	MRID(s)	Results	Toxicity Category

870.1100	Acute oral (rat)	47289116	LD50 > 2000 mg/kg bw (F)	III

870.1200	Acute dermal (rat)	47289118	LD50 > 5000 mg/kg bw (M&F)	IV

870.1300	Acute inhalation (rat)	N/A	N/A	N/A

870.2400	Primary eye irritation (rabbit)	47289120	Minimally irritating
III

870.2500	Primary dermal irritation (rabbit)	47289122	Slight irritant	IV

870.2600	Dermal sensitization (mouse)	47289124	Positive (LLNA)	N/A

Table A.2.2.  Subchronic and Chronic Toxicity and Genotoxicity Profile
–Meptyldinocap.

Guideline No. 	Study Type	MRID No. (year)/ Classification /Doses	Results

870.3050	28-Day oral toxicity (mouse)	47289127 (2005)
Acceptable/non-guideline

0, 100, 200, or 750 ppm (equivalent to 0/0, 16/19.3, 30.7/40.8, or
117/161.1 mg/kg bw/day [M/F])	NOAEL = 750 ppm (117/161.1 mg/kg bw/day
[M/F])

LOAEL not observed.

870.3100

	90-Day oral toxicity (rat)	47289128 (2005) Acceptable/guideline

0, 200, 650, or 2000 ppm (equivalent to 0/0, 11/13, 37/41, or 113/127
mg/kg bw/day [M/F])	NOAEL = 650 ppm (37/41 mg/kg bw/day [M/F])

LOAEL = 2000 ppm (113/127 mg/kg bw/day [M/F]), based on decreased body
weight, body-weight gain, and food consumption in both sexes.

870.3150	90-Day oral toxicity (dog)	47289129 (2005) Acceptable/guideline

0, 15, 60, or 120 ppm (equivalent to 0/0, 0.49/0.48, 1.51/2.14, or
3.58/3.89 mg/kg bw/day [M/F])	NOAEL = 60 ppm (1.51 mg/kg bw/day [M])

LOAEL = 120 ppm (3.58 mg/kg bw/day) based on significant, sustained
increased ALT and AST levels (M).

870.4100

	1-year oral toxicity (dog)

Extension of 90-day study without a control group	47289130 (2006)
Acceptable/non-guideline

120 ppm (equivalent to 3.31/3.22 mg/kg bw/day [M/F])

	No effect of treatment at 120 ppm on limited number of parameters,
including ophthalmological measurements.

870.3700

	Prenatal developmental (mouse)	47289132 (2005)

Acceptable/non-guideline

0, 100, 250, or 500 mg/kg bw/day)	Maternal NOAEL = 500 mg/kg bw/day

Maternal LOAEL not observed.

Developmental NOAEL = 500 mg/kg bw/day

Developmental LOAEL not observed.

No effect on external or otoconial development.

870.3700

	Prenatal developmental (rat)	47289134 (2005)

Acceptable/guideline

0, 50, 150, [or 500 (terminated)] mg/kg bw/day)	Maternal NOAEL = 50
mg/kg bw/day

Maternal LOAEL =  150 mg/kg bw/day based on decreased body weights,
body-weight gains, and food consumption.

Developmental NOAEL = 150 mg/kg bw/day

Developmental LOAEL not observed.

Developmental toxicity was not observed, incl. effects on otoconial
development.

870.3700

	Prenatal developmental (rabbit)	47289136 (2005)

Acceptable/guideline

0, 3, 12, or 48 mg/kg bw/day	Maternal NOAEL = 12 mg/kg bw/day

Maternal LOAEL =  48 mg/kg bw/day based on decreased body-weight gains
and food consumption.

Developmental NOAEL = 48 mg/kg bw/day

Developmental LOAEL not observed.

Developmental toxicity was not observed, incl. effects on otoconial
development.

Special study	Oral developmental toxicity with non-methylheptyl isomers
of dinocap (mice)	47289133 (2005)

Acceptable/non-guideline

5 or 10 mg/kg bw/day, depending on isomer	Developmental toxicity was
observed with the 2,6-dinitro-4-(1-propylpentyl)-phenyl crotonate (4-PP)
isomer of dinocap only.

Special study	Oral developmental and postnatal toxicity with
methylheptyl isomers of dinocap and dinocap (mice)	47304907 (1987)

Acceptable/non-guideline

25 mg/kg bw/day	No evidence of developmental toxicity, incl. cleft
palate or torticollis and no effects on fetal body weight, swimming
ability, or otolith development with either methylheptyl isomer or in
combination.

Also published in Rogers et al. 1987. 

Teratogenesis, Carcinogenesis, and 

Mutagenesis 7:341-346.

870.5100	Bacterial gene mutation	47289137 (2005)

Acceptable/guideline

0-4690 µg/plate (+/- S9)	Negative

870.5300	Mammalian cell gene mutation	47289138 (2005)

Acceptable/guideline

0-18.6 (g/mL (-S9)

0-92.9 (g/mL (+S9)	Negative

870.5375	In vitro mammalian chromosomal aberration	47289139 (2005)

Acceptable/guideline

0-929 µg/mL (+/-S9)	Negative

870.5395	In vivo mouse erythrocyte micronucleus assay	47289140 (2005)

Acceptable/guideline

0, 465, 929, or 1858 mg/kg bw	Negative

870.7485

	Metabolism and pharmacokinetics	47289141 (1996)

Acceptable/non-guideline

29.5 (mice) or 95.5 (rats) mg/kg bw (single dose)

	This study was performed to evaluate 

differences in rat and mouse urinary 

metabolism (only) of meptyldinocap as a 

surrogate for dinocap. Fatty acid β-oxidation 

was the primary metabolic pathway in both 

species; more metabolism occurred via fatty 

acid α-oxidation in mice (approximately 

32.9%) than in the rats (18.0%).  A 

total of 7 metabolites were identified in 

mouse urine that were not identified in 

rat urine; however, each compound 

represented only 1.23-2.78% of the 

total radioactivity isolated in the urine.   

Based on these results, 2,4-DNHPC undergoes 

esterase-mediated de-esterification followed by 

sequential metabolism by cytochrome P-450, 

alcohol dehydrogenase, and aldehyde 

dehydrogenase to form the carboxylated 

metabolite, 2,4-dinitro-6-(1-

methylheptanoate)phenol.  Further metabolism 

proceeds via the fatty acid pathways, which is 

the primary pathway for metabolism of 2,4-

DNHPC in both rat and mouse.  

870.7485

	Metabolism and pharmacokinetics	00153615, 47289142 (1976)

Acceptable/non-guideline

49 mg/kg bw/day (7 days; 1 rat/sex)	Absorption: 18-24% administered dose
throughout 7 days.

Distribution: largest concentrations of radioactive residues (excluding
the GI tract) included heart, carcass, liver, pelt, and thymus
(0.27-0.85% administered dose).

Metabolism: hydrolysis of the crotonate ester followed by oxidation of
the octyl side chain.  

Excretion: primarily through the feces (52-58% administered dose) and
urine (15-20% administered dose) throughout 7 days.

A.3  Executive Summaries of Toxicology DERs

28-Day Oral Toxicity - Mouse

In this 28-day oral toxicity study (MRID 47289127), 5 CD-1 mice/sex/dose
group were treated daily with Dinocap II (Meptyldinocap; 92.9% a.i.; Lot
No. SL1088R301) in the diet at nominal concentrations of 0, 100, 200, or
750 ppm (equivalent to 0/0, 16/19.3, 30.7/40.8, or 117/161.1 mg/kg/day
in males/females [adjusted for purity]) for 28 days.  

No adverse, treatment related effects were observed on mortality,
clinical signs, body weight, body-weight gain, food consumption,
hematology, clinical chemistry, urinary pH, ophthalmoscopic examination,
organ weights, gross pathology, or microscopic pathology.   Bright
yellow urine was noted in all male and female treated groups, but was
interpreted to be related to urinary elimination of the test compound
and/or its metabolites.  A marginal decrease in urinary pH was observed
in the 750 ppm males, and this was also considered related to
elimination of the parent compound and/or its metabolites.  Increased
absolute (NSS) and relative (P<0.05) liver weights were noted in the 750
ppm group, but this effect was considered adaptive in the absence of
corroborating evidence of hepatotoxicity.

The LOAEL was not observed.  The NOAEL is 750 ppm (equivalent to
117/161.1 

mg/kg/day [M/F], adjusted for purity).

This 28-day study in mice is acceptable/non-guideline and does not
satisfy the guideline requirement for a 28-day oral toxicity study
(OPPTS 870.3050; OECD 407) in rodents.  A LOAEL was not observed, and
the limit dose was not tested.  An additional, but minor, deficiency was
that blood clotting measurements were not made.

90-Day Oral Toxicity – Rat

In a subchronic oral toxicity study (MRID 47289128), 10 Sprague Dawley
rats/sex/dose group were exposed to Dinocap II (Meptyldinocap; 92.9%
a.i.; Lot No.: SL1088R301) for up to 3 months in the diet at
concentrations of 0, 200, 650, or 2000 ppm (equivalent to 0/0, 11/13,
37/41, or 113/127 mg/kg bw/day in males/females [adjusted for purity]).

No adverse, treatment-related effects were observed on mortality,
clinical signs, FOB parameters, motor activity, ophthalmoscopic
evaluation, hematology, clinical chemistry, urinalysis, organ weights,
gross pathology, or microscopic pathology.  At 2000 ppm, decreased body
weights were observed in both sexes throughout the study (decr 3-9%),
and were statistically significant (p<=0.05) in females at Days 50, 57,
and 64 (decr 7-8%).  Decreased (not compared statistically) overall
(Days 1-92) body-weight gains were also noted in the 2000 ppm group
(decr 15-18%).  Decreased food consumption was generally observed in
males (decr 2-7%) and females (decr 1-11%), and was statistically
significant (p<=0.05) in females at the intervals of Days 1-8, 15-22,
43-50, and 50-57 (decr 8-11%).  This effect corresponded to the effect
observed on body weight.  The following findings were also observed at
2000 ppm, but were not considered toxicologically significant due to the
slight or very slight magnitude of difference from the control groups
and the lack of corroborating evidence of toxicity:  (i) slight
increases in total protein, albumin, and cholesterol in females; (ii)
increased relative (to body) liver and weights in both sexes and
increased relative thyroid and kidney weights in females; and (iii) very
slight hepatocellular hypertrophy in both sexes.  All treated rats had
bright yellow urine (presumably due to excretion of the test compound in
the urine); 9-10 rats/sex at 2000 ppm and two 650 ppm males had
discolored fur (from contact with their urine).

 

The LOAEL is 2000 ppm (equivalent to 113/127 mg/kg/day in
males/females), based on decreased body weight, body-weight gain, and
food consumption in both sexes.  The NOAEL is 650 ppm (equivalent to
37/41 mg/kg/day in males/females).

This study is classified as acceptable/guideline and satisfies the
guideline requirements (OPPTS 870.3100; OECD 408) for a subchronic oral
toxicity study in the rat.

90-Day Oral Toxicity - Dog

In a subchronic oral toxicity study in dogs (MRID 47289129), Dinocap II
(DE-126; 92.9% a.i.; Lot # SL1088R301) was administered in the diet to
four beagle dogs/sex/dose group at doses of 0, 15, 60, or 120 ppm
(equivalent to 0/0, 0.49/0.48, 1.51/2.14, or 3.58/3.89 mg/kg bw/day in
males/females [adjusted for purity]) for at least 90 days.  A satellite
group of four dogs/sex was fed diets containing Dinocap II at a dose
level of 120 ppm (with no control group) and was to be maintained at
this level for up to one year primarily to evaluate the potential for
effects on the retinas of the eyes.  Additionally, four dogs/sex were
fed a diet containing Dinocap at a dose level of 60 ppm (equivalent to
1.78/1.92 mg/kg/day in males/females [adjusted for purity]) for at least
90 days to serve as a comparative control group.  Due to concerns over
the potential for reduced palatability of the test compounds, the 60 and
120 ppm Dinocap II and the Dinocap groups were exposed to lower doses of
the compound for one week up to the official start of the study. 
Details of these pre-study procedures are summarized in the Appendix to
the DER.

One 120 ppm male dog had elevated alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) on Day 42 (ALT ↑150%; AST ↑500%)
and Day 90 (ALT ↑143%; AST ↑206%).  These findings were considered
treatment-related and, based on the magnitude of the increases at two
time points and the limited number of animals tested, toxicologically
significant.  

The LOAEL in males is 120 ppm (equivalent to 3.58 mg/kg bw/day) based on
sustained increased ALT and AST levels.  The NOAEL in males is 60 ppm
(equivalent to 1.51 mg/kg bw/day).  The LOAEL was not observed in
females; the NOAEL in females was 120 ppm (equivalent to 3.89 mg/kg
bw/day).

The following findings were observed in dogs dosed with Dinocap at 60
ppm.  Significantly decreased body weights were noted in both sexes,
resulting in decreased mean overall body-weight gains (71% males, 67%
females).  The females had significantly decreased food consumption
generally during Days 1-36.  A treatment-related retinopathy
characterized by the following findings was observed by study
termination in both sexes:  (i) hyperreflectivity of the tapetal fundus
in 7/8 dogs; (ii) attenuation/constriction of retinal blood vessels in
5/8 dogs; (iii) pigment clumping in 2/8 dogs; and (iv)
hypopigmentation/depigmentation of the non-tapetal fundus in 3/8 dogs. 
One female presented with generalized decreased body fat and decreased
thymus size.  The decrease in body fat was consistent with the body
weight loss observed in this animal.  The decreased thymus size was
correlated with a slight atrophy of the cortex of the thymus observed
microscopically and may have been due to the body weight loss.  Very
slight to moderate bilateral atrophy of the retina was observed in 4/4
males and 3/4 females.  The atrophy was characterized by thinning of the
rod and cone layer, outer nuclear layer, and outer plexiform layer, and
was generally most prominent in the peripheral aspects of the retina. 
This finding was consistent with a previously performed chronic toxicity
study in dogs.  Very slight multifocal degeneration of individual nerve
fibers of the tibial nerve was noted in 3/4 males and 3/4 females. 
Slight atrophy of the cortex of the thymus was observed in one male and
one female that lost body weight over the course of treatment.  The
female was also noted with slight diffuse atrophy of the red pulp of the
spleen.

This study is classified acceptable/guideline and satisfies the
guideline requirements (OPPTS 870.3150; OECD 409) for a subchronic oral
toxicity study in dogs.  

One-year oral Toxicity – Dog

In a non-guideline one-year oral toxicity study (MRID 47289130), a group
of four Beagle dogs/sex was fed a diet containing Dinocap II (92.9%
a.i.; Lot # SL1088R301) at a dose level of 120 ppm (equivalent to
3.31/3.22 mg/kg/day in males/females [adjusted for purity]) for up to
one year.  These dogs were initially treated in a subchronic oral
toxicity study in dogs (MRID 47289129), in which Dinocap II was
administered in the diet to four beagle dogs/sex/dose group at doses of
0, 15, 60, or 120 ppm for at least 90 days, and the one-year dogs were
included as a satellite group.  The control group was sacrificed at the
end of the 90-day study and therefore was not included as part of this
one-year study.  Limited parameters examined in this non-guideline study
included clinical observations, ophthalmoscopic examinations, body
weights, food consumption, and gross and microscopic pathological
examinations of the eyes, tibial nerve, and heart.  The primary purpose
of this study was to evaluate Dinocap II for effects on the retinas of
the eyes following one year of treatment.

No effects of treatment were observed on mortality, clinical signs,
ophthalmological 

examinations, or gross or microscopic pathology.  Bright yellow urine
was noted in all 120 ppm males and females beginning on Day 57 and
continuing through Day 365.  This finding was interpreted to be related
to urinary excretion of the test material and/or its metabolites and was
also observed in short-term studies with meptyldinocap in rats.  Because
control animals were not kept on study beyond 90 days, it was not
possible to assess whether there were treatment-related effects on body
weight, body-weight gain, or food consumption over one year.  The eyes
were found to be unaffected by treatment following extensive
ophthalmological and histological examinations.  The heart and tibial
nerves were found to be normal following microscopic examinations.

This study is classified acceptable/non-guideline and does not satisfy
the guideline requirement for a chronic toxicity study in nonrodents
(OPPTS 870.4100).

Prenatal Developmental Toxicity Study – Mouse

The purpose of this non-guideline study was to determine if Dinocap II
was teratogenic in mice, with a specific focus on the development of
otoconia (calcium carbonate crystals of the inner ear) and cleft palate.
 In this developmental toxicity study (MRID 47289132), Dinocap II
(DE-126; stated purity of <98.5%; Lot No. 2003-03220-62) in 0.5% aqueous
methylcellulose was administered via daily oral gavage in a dose volume
of 10 mL/kg to 15 time-mated CD-1 mice/dose group at doses of 0, 100,
250, or 500 mg/kg/day from gestation days (GD) 6-17.  In prior studies,
Dinocap (the previous technical formulation) resulted in growth
retardation, cleft palate, precocious eyelid opening, and agenesis of
otoconia.  Therefore, a positive control group using Dinocap at 25
mg/kg/day was included.  On GD 18, all dams were euthanized; each
dam’s uterus was removed via cesarean section and its contents were
examined.  Fetuses were examined for external alterations, including
cleft palate.  Additionally, otoconia morphology was evaluated in all
fetuses.  Soft tissue and skeletal anomalies were not assessed in the
study.

All animals that were dosed with Dinocap II had bright yellow urine
starting as early as GD 7 and continuing until necropsy (GD 18).  This
finding was attributed to excretion of the test material and/or its
metabolites and was observed in several studies with meptyldinocap in
rodents and dogs.  Bright yellow urine was not observed in any animals
in the control or positive control groups.

At 500 mg/kg/day, one dam (#4587) prematurely delivered a litter on GD
18.  No fetal data were collected from this litter because several pups
had been cannibalized.  One dam (#4592) from the positive control group
died spontaneously on GD 18, just prior to necropsy; this dam was
pregnant with 16 dead fetuses.  A necropsy was performed on this dam,
although the gravid uterus weight was not recorded.  Because this death
occurred just prior to scheduled necropsy, all fetuses were weighed, and
external examinations and otoconia scoring were performed.  Gross
pathology of these two dams did not reveal any abnormalities.

Body weights and body-weight gains of the Dinocap II treated groups were
comparable to controls throughout the study.  In the Dinocap positive
control group, body weights were decreased (p<=0.05) by 18% on GD 18. 
Body-weight gains in this group were decreased (p<=0.05) by 42% for GD
12-15, by 64% for GD 15-18, by 39% for the overall (GD 6-18) treatment
period, and by 36% for the overall (GD 0-18) study.

The maternal LOAEL was not observed.  The maternal NOAEL is 500
mg/kg/day, the highest dose tested.

In the Dinocap II treated groups, there were no abortions or complete
litter resorptions and no effects of treatment on the numbers of
resorptions/dam, live fetuses/dam, or dead fetuses/dam.  Fetal weights
and post-implantation losses of the treated groups were comparable to
controls.  There were no fetal external variations.

In the Dinocap positive control group, the number of live fetuses/dam
was decreased (p<=0.05) by 28% compared to controls.  Additionally, the
number of dead fetuses/dam were increased in this group (2.9/dam)
compared to 0 controls.  Because resorptions in this group were
comparable to controls, the increase (p<=0.10) in post-implantation loss
(24.20% compared to 3.98% controls) could be attributed to the increased
number of dead fetuses.  This finding was primarily attributed to two
dams (#4590 and #4595) with 14/14 and 13/14 dead fetuses, respectively.

No treatment-related external malformations were observed in the groups
treated with Dinocap II.  Cleft palate was only found in 1 fetus at 100
mg/kg/day, and exencephaly was observed in one control fetus and one
fetus at 250 mg/kg/day.  No other malformations were observed in the
Dinocap II treated groups.  In the Dinocap positive control group, cleft
palate occurred in all 13 litters and affected 109/156 (70%) of the
fetuses.

Mean otoconia scores in the groups treated with Dinocap II (11.66-11.75)
were comparable to controls (11.79), indicating no effect of treatment
on the development of otoconia.  The mean otoconia score in the Dinocap
positive control group (0.82) was decreased (p<=0.05) by 93% compared to
the Dinocap II controls, indicating nearly complete agenesis of otoconia
in the positive controls.

The developmental LOAEL was not observed.  The developmental NOAEL is
500 mg/kg/day.

This study is classified as acceptable/non-guideline and does not
satisfy the guideline requirement for a developmental toxicity study
(OPPTS 870.3700; OECD 414) in rodents, since  soft tissue and skeletal
anomalies were not assessed in the study.

Prenatal Developmental Toxicity Study – Mouse

Technical grade Dinocap, a complex mixture of 6 isomers, is teratogenic
in CD-1 mice, causing cleft palate and otolith defects.  The purpose of
this non-guideline study was to compare the developmental toxicity
(specific endpoints) of two of these isomers
(2,4-dinitro-6-(1-methylheptyl)phenyl crotonate and
2,6-dinitro-4-(1-methylheptyl)phenyl crotonate) to the known
teratogenicity of the technical formulation.  In this developmental
toxicity study (MRID 47304907), groups of time-mated CD-1 mice were
administered 25 mg/kg/day of the 2,4-isomer, the 2,6-isomer, the two
isomers in combination (2:1 ratio; total 25 mg/kg/day), or Dinocap
technical formulation in corn oil daily via oral gavage from gestation
days (GD) 7-16.  A control group was also included and dosed with corn
oil.  On GD 18, some of the dams (n = 6-9) were euthanized for a
pre-natal study; each dam’s uterus was removed via cesarean section
and its contents examined.  Dead fetuses and resorptions were counted,
and live fetuses were weighed and preserved for examination for cleft
palate.  The remaining dams (n = 7-12) were allowed to give birth. 
Post-natal viability and growth, swimming behavior, torticollis, and
otolith formation were examined in the offspring.

In the teratology study, net maternal body-weight gain, the number of
live pups/litter, and the number of dead or resorbed pups/litter in all
treated groups were comparable to controls.  In the technical dinocap
group, fetal weights were decreased (p<=0.05) by 21% compared to
controls.  Additionally in this group, 7/8 litters examined contained
fetuses with cleft palate, affecting 48/84 fetuses (57%).  In contrast,
fetal weights in the 2,4 isomer group, the 2,6 isomer group, and in the
group exposed to both isomers were comparable to controls, and cleft
palate was not observed in any fetuses in these groups or in the
controls.

Technical grade Dinocap resulted in decreased fetal body weights and
cleft palate in the pre-natal study.  There were no effects of treatment
on any parameter in the 2,4-isomer group, the 2,6-isomer group, or the
2,4- and 2,6-isomer mixture (2:1) group.

In the Dinocap group, the number of live pups/litter was decreased by
37% (p<=0.05) on PND 3. Post-implantation loss was increased in this
group (5.0) compared to controls (0.5).  Mean pups weights were
decreased by 18-24% (p<=0.05) on PND 1, 3, 6, and 30.  In the 2,4 isomer
and 2,6 isomer groups, litter size and pup weights throughout the
post-natal period and post-implantation losses were comparable to
controls.

None of the pups in the 2,4-isomer or 2,6-isomer groups had torticollis,
were unable to swim, or were missing otoliths.  The otoliths scores in
these groups (4.00 each) were comparable to controls.  Technical grade
Dinocap caused torticollis in 20% of the pups, compromised swimming
behavior in 40% of the pups, and otolith agenesis in 60% of the pups. 
These findings were statistically significant (p<=0.05), and none of the
pups were affected in the 2,4-isomer, 2,6 isomer, or control groups.

Technical grade Dinocap resulted in decreased litter size, increased
post-implantation loss (as defined by the authors), and decreased pup
body weights when dams were treated from GD7-16.  There were no effects
of treatment on any parameter in the 2,4-isomer or 2,6-isomer groups.

A major deficiency of the study is that concentration, homogeneity, and
stability of the test formulations in corn oil were not reported.  

This developmental toxicity study in mice is classified as
acceptable/non-guideline and does not satisfy the guideline requirement
for a developmental toxicity study (OPPTS 870.3700; OECD 414) in
rodents.

Prenatal Developmental Toxicity Study – Mouse

The purpose of this non-guideline study was to identify the isomer(s) of
Dinocap responsible for the teratogenic effects (cleft palate and
agenesis of otoconia) in mice.  Of the six isomers that comprise
Dinocap, two have been previously demonstrated as not teratogenic,
either singly or in combination.  The remaining four isomers were
4-ethylhexyl (4-EH), 6-ethylhexyl (6-EH), 4-propylpentyl (4-PP), and
6-propylpentyl (6-PP).  In this developmental toxicity study (MRID
47289133), groups of 10 time-mated CD-1 mice were administered one of
these four Dinocap technical isomers in 0.5% aqueous methylcellulose via
daily oral gavage in a dose volume of 10 mL/kg at doses of 0 (vehicle
control), 5 mg/kg/day (for 4-EH and 4-PP), or 10 mg/kg/day (for 6-EH and
6-PP) from gestation days (GD) 6-17.  On GD 18, all dams were
euthanized; each dam’s uterus was removed via cesarean section and its
contents examined.  Fetuses were examined for external alterations,
including cleft palate.  Additionally, otoconia morphology was evaluated
in all fetuses.  Visceral and skeletal examinations were not conducted.

In the 4-PP isomer group, four dams (#2185, 2187, 2189, and 2192) were
euthanized in moribund condition between GD 15-17.  The following
clinical signs of toxicity were observed in these dams (# animals
affected):  absent activity (4); decreased activity (4); eyelids fully
closed (1); cloudy eyes (1); eyelids partially closed (1); decreased
feces (5); cold to touch (2); muscle twitches (5); red (3) or brown (1)
vulvar discharge; shallow respiration (2); deep respiration (1);
ungroomed appearance (7); dehydration (1); and perineal soiling with
urine (2) or feces (2).  At necropsy, these animals also had general
decreased amounts of body fat, and two mice had multifocal ulcers in the
glandular mucosa of the stomach.  Dosing of the remaining members of
this group was halted at this time, with four animals at GD 15 and two
dams at GD 16.  All other animals survived until scheduled sacrifice,
and there were no clinical signs of toxicity observed in any other
treated groups.  Bright colored urine was noted in three animals in the
6-EH group; however, this finding was considered to be due to excretion
of the test material and not toxicologically significant.

In the 4-PP isomer group, body-weight gains were decreased by 61%
(p<0.05) for GD 12-15, by 43% for the overall (GD 6-18) treatment
interval, and by 40% for the overall (GD 0-18) study compared to
controls.  These decreases in body-weight gains resulted in a mean
absolute body weight decrease of 19% (p<0.05) on GD 18 compared to
controls.  Gravid uterine weights in this group were 25% lower (not
significant) than controls, and corrected terminal body weights were
decreased by (p<0.05)16%.  Body weights and body-weight gains in the
other isomers were comparable to controls throughout the study.

In the 4-PP isomer group, the number of live fetuses/dam was decreased
by 25% (NSS) compared to controls (9.0 treated vs 12.0 controls), and
the number of dead fetuses/dam was increased (2.9 treated vs 0 controls;
NSS).  Thus, post-implantation loss was higher (NSS) in this group
(38.77%) compared to controls (7.05%).  Additionally, fetal body weights
were decreased by 46% (NSS).  There were no abortions or complete litter
resorptions in any treated group.  Furthermore, in the 4-EH, 6-EH, and
6-PP treated groups, there were no adverse effects of treatment on fetal
weights, post-implantation loss, or on the numbers of litters, live
fetuses/dam, dead fetuses/dam, or resorptions/dam.

In the 4-PP isomer group, all 83 fetuses in all 6 litters had cleft
palate, whereas cleft palate did not occur in any other treated group or
in the controls.  Four of the fetuses from two litters in this group
also had ablepharia, also considered to be due to treatment with the
4-PP isomer.  No treatment-related external malformations were observed
in the 4-EH, 6-EH, and 6-PP treated groups.  There were no external
variations in any group.  Mean otoconia scores in the groups treated
with 4-EH, 6-EH, and 6-PP (11.59-11.76) were comparable to controls
(11.78).  However, in the 4-PP isomer group, the mean otoconia score
(1.78) was decreased (p<=0.05) by 85% compared to the controls.

This study is classified an acceptable/non-guideline and does not
satisfy the guideline requirement for a developmental toxicity study
(OPPTS 870.3700; OECD 414) in the rodent since skeletal and visceral
anomalies were not assessed.

Prenatal Developmental Toxicity Study – Rat

In a developmental toxicity study (MRID 47289134), Dinocap II (DE-126;
97.4%; Lot No. 2004-03140-24) in 0.5% aqueous Methocel® A4M was
administered via daily oral gavage in a dose volume of 4 mL/kg to 26
time-mated Sprague-Dawley rats/dose group at doses of 0, 50, 150, or 500
mg/kg/day from gestation days (GD) 6-20.  On GD 21, all dams were
euthanized; each dam’s uterus was removed via cesarean section and its
contents examined.  Fetuses were examined for external, visceral, and
skeletal malformations and variations.  It was stated that otoconia
(calcium carbonate crystals of the inner ear) are considered to be a
highly sensitive target of Dinocap and were therefore evaluated in
addition to the standard fetal parameters.

In the 500 mg/kg/day group, one dam died on GD 7 (#6521), and another
died on GD 9 (#6518). Prior to death, animal #6518 exhibited perinasal
and perioral soiling, labored respiration, decreased activity, splayed
hindlimbs, and crouched posture.  At necropsy, this dam had lung
congestion and general decreased amount of fat.  Dam #6521 showed no
clinical signs of toxicity prior to death and no visible lesions at
necropsy.  Both dams were pregnant with live fetuses.  Aside from these
two mortalities, only 14 other dams in this group had begun treatment
due to the cohort study design with staggered breeding dates.  Nine of
these 14 surviving animals had splayed hindlimbs, and three had crouched
posture that started between GD 7 and 9.  Body weights were decreased
(p≤0.05) by 14% on GD 9 compared to controls.  For GD 6-9, significant
(p≤0.05) body weight loss was observed at this dose (-17.4 g) compared
to a body-weight gain in controls (19.9 g), and food consumption was
decreased (p≤0.05) by 51%.  Due to the excessive toxicity observed at
500 mg/kg/day, this group was terminated on GD 7-11. At the time that
the 500 mg/kg/day group was terminated, dosing had only begun for 16 of
the 26 rats.  Fifteen out of 16 were pregnant with normal fetuses.  At
necropsy, all 16 of these rats had perineal urine soiling.  Aside from
this finding and the macroscopic findings associated with the mortality
at 500 mg/kg/day (Dam #6518), no other gross findings could be
attributed to treatment.

At 150 mg/kg/day, all animals survived until scheduled termination, and
no clinical signs of toxicity were observed.  Body weights were
decreased (p≤0.05) by 7% on GD 9 and remained decreased (p≤0.05) by
4-6% throughout the remainder of the study.  Body-weight gains for GD
6-9 were decreased (p≤0.05) by 72% at this dose.  Although not
statistically significant, body-weight gains were decreased by 7% for
the overall (GD 6-21) treatment period and by 6% for the overall (GD
0-21) study.  Food consumption was decreased (p≤0.05) by 27% for GD
6-9.  Additionally at this dose, food consumption was decreased by 10%
for GD 9-12 but was increased by 7% for GD 18-21.  Absolute and relative
(to body weight) liver weights were increased (p≤0.05) by 22-27% at
150 mg/kg/day.  However, without examination of histopathology or
clinical chemistry parameters, it is not possible to determine whether
the increases in liver weights reflected hepatotoxicity or were simply
an adaptive response to the test material.

 decreased (p≤0.05) by 8% on GD 6-9.  However, this decrease was
transient, and body weights and body-weight gains at this dose were
unaffected throughout the study.  Therefore, this finding was not
considered toxicologically significant.  The only other finding at this
dose was increased (9-10%; p≤0.05) absolute and relative (to body
weight) liver weights. However, the increases at this dose were
considered minor and were likely an adaptive response to the test
material.

Bright yellow urine was noted in all treated animals.  This finding was
considered to be due to excretion of the test material and/or its
metabolites and therefore not toxicologically significant.

The maternal LOAEL is 150 mg/kg/day based on decreased body weights,
body-weight gains, and food consumption.  The maternal NOAEL is 50
mg/kg/day.

At 500 mg/kg/day, excessive maternal toxicity resulted in early
termination and precluded assessment of developmental toxicity at this
dose.  There were no abortions, premature deliveries, complete litter
resorptions, or dead fetuses and no effects of treatment on the numbers
of litters, live fetuses, early resorptions, or late resorptions at any
dose.  Similarly, fetal weights, sex ratio, and post-implantation losses
of the treated groups were comparable to controls.  There were no
treatment-related external, visceral, or skeletal variations or
malformations.  The otoconia score of each treated group was comparable
to controls.

The developmental LOAEL was not observed.  The developmental NOAEL is
150 mg/kg/day.

This study is classified acceptable/guideline and satisfies the
guideline requirement for a developmental toxicity study (OPPTS
870.3700; OECD 414) in rats.

Prenatal Developmental Toxicity Study - Rabbit

In a developmental toxicity study (MRID 47289136), Dinocap II (DE-126;
97.4%; Lot No. 2004-03140-24) in 0.5% aqueous Methocel® A4M was
administered via daily oral gavage in a dose volume of 4 mL/kg to 26
time-mated New Zealand White rabbits at doses of 0, 3, 12, or 48
mg/kg/day from gestation days (GD) 7-27.  On GD 28, all surviving
maternal rabbits were euthanized; each doe’s uterus was removed via
cesarean section and its contents examined.  Additionally, weights of
the liver and kidneys were recorded.  Ten rabbits per dose group were
randomly selected for ophthalmoscopic examinations and histopathology of
the eyes.  Fetuses were examined for external, visceral, and skeletal
malformations and variations.  It was stated that otoconia (calcium
carbonate crystals of the inner ear) are considered to be a highly
sensitive target of Dinocap in mice and were therefore evaluated in
addition to the standard fetal parameters.

No treatment-related effects were observed on cesarean section
parameters, organ weight, or the eyes.  At 48 mg/kg/day, two animals
experienced long periods of decreased food consumption and inanition. 
One of these animals (#7956) aborted on GD 20, and the other (#7953) was
euthanized that same day.  During the clinical examinations, bright
yellow urine was noted at 3 mg/kg/day (2/26 does), 12 mg/kg/day (4/26
does), and 48 mg/kg/day (23/26 does).  During necropsy of the animals
that survived until scheduled termination, yellow discoloration of the
urinary bladder was observed at 3 mg/kg/day (1/26 does), 12 mg/kg/day
(10/26 does), and 48 mg/kg/day (10/26 does).  These non-adverse findings
were attributed to excretion of the test material and were observed in
other studies with rodents and dogs.  Near the beginning of treatment
(GD 7-10), a body weight loss occurred in the 48 mg/kg/day animals (-4.3
g) compared to a body-weight gain of 41.5 g in the controls. 
Body-weight gains for the overall (GD 7-28) treatment period were
decreased by 11% compared to controls.  Food consumption was decreased
by 17-26% during GD 12-13, GD 14-15, and GD 19-20.  

The maternal LOAEL is 48 mg/kg/day based on decreased body-weight gains
and food consumption.  The maternal NOAEL is 12 mg/kg/day.

There were no premature deliveries, complete litter resorptions, or dead
fetuses and no effects of treatment on the numbers of litters or mean
numbers of live fetuses or resorptions.  Sex ratio, fetal body weights,
and post-implantation losses of the treated groups were comparable to
controls.  There were no treatment-related external, visceral, or
skeletal malformations.  Mean otoconia scores in the treated groups
(11.71-11.92) were comparable to controls (11.93), indicating no effect
of treatment on the development of otoconia.

The developmental LOAEL was not observed.  The developmental NOAEL is 48
mg/kg/day.

This study is classified acceptable/guideline and satisfies the
guideline requirement for a developmental toxicity study (OPPTS
870.3700b; OECD 414) in rabbits.

Metabolism

I.  In a rat metabolism study (MRIDs 00153615 and 47289142),
14C-2,4-dinitro-6-(2-octyl) phenyl crotonate isomers (95% trans and 5%
cis; 100% radiochemical purity) in aqueous 0.5% methylcellulose was
administered by daily oral gavage for 7 consecutive days to 1 Wistar
rat/sex at an average daily dose of approximately 49 mg/kg bw.  Excreta
(urine, feces, and carbon dioxide) were collected until termination at 6
hours following the final dose.  Upon sacrifice, selected tissue/organ
samples, as well as the residual carcass, were collected.  All these
samples were radioassayed, and selected fecal and urine samples were
analyzed to determine a metabolic profile.  

Total recovery was 93-94% administered dose (AD).  Absorption was at
least 18-24% AD, based on residues isolated in the urine and tissues
(excluding unwashed digestive tract) of the animals.  Negligible amounts
of the radioactivity were isolated in the carbon dioxide traps (0.02% AD
in both sexes).  Excretion occurred primarily through the feces (52-58%
AD), and urine accounted for 15-20% AD.  Cage wash accounted for 6-11%
AD, and 3-4% AD was isolated in the tissues (excluding unwashed
digestive tract).  Organ tissue samples that had the largest amounts
(%AD) of radioactive residues (excluding the GI tract) included the
heart, carcass, liver, pelt, and thymus (0.27-0.85% AD).  Very low
tissue residues suggest that bioaccumulation may not be a concern after
7 consecutive daily treatments with approximately 49 mg/kg bw/day of the
test compound. 

In MRID 00153615, an apparent difference was noted in the metabolite
profile of the male urine compared to the female urine.  The major
metabolites in the Day 6 urine (as % of 14C-residue in urine) were
Metabolites E (35%) and F (13%) in males, and metabolite M (13%) in
females.  A large amount of the radioactivity from the urine samples was
not separated and remained at the origin in the TLC analyses (21% in
males and 46% in females).  The major metabolites in the Day 2 male
feces sample (as % of 14C-residue in feces) were Metabolite B (21%) and
M (20%), and only 7% remained unseparated at the origin.  Metabolite B
in the feces was tentatively identified as 2,4-dinitro-6-(2-octyl)
phenol, because this metabolite co-migrated with this compound
(authentic standard).  In MRID 47289142, up to 15 metabolites were
isolated in the feces, of which 3 compounds (Metabolites A, B, and P)
were identified.  The identified Metabolites A, B, and P accounted for
2, 5, and 4%, respectively, of the total radioactivity isolated in the
fecal sample.  Metabolite P isolated in the feces was found to be the
same compound as Metabolite E that was isolated in the urine.  Most of
the radioactivity found in the fecal sample remained unextracted (61%). 
Likewise, up to 15 metabolites were isolated in the urine, of which 2
(Metabolites E and F) were identified.  Most of the radioactivity
isolated in the urine sample was identified as Metabolite E (37%) and F
(27%).  The metabolism of Meptyldinocap in rats appears to proceed first
by hydrolysis of the crotonate ester followed by oxidation of the octyl
side chain.  

This metabolism study in the rat is classified acceptable/non-guideline
and does not satisfy the guideline requirement for a metabolism study
[OPPTS 870.7485, OPP 85-1] in rats.  An insufficient number of animals
was used in the study, and it was unknown whether standards similar to
GLP were followed in the study.  Data from this study are acceptable as
supplementary data and provide limited indications of test compound
absorption and excretion, tissue distribution, and differential
metabolite profile that would be more definitively addressed in a
guideline study. 

II.  Dinocap has been shown to be more toxic to mice than rats.  Because
2,4-DNHPC (meptyldinocap) was thought to be representative of dinocap
metabolism, this study was performed to evaluate differences in rat and
mouse urinary metabolism of meptyldinocap as a surrogate for dinocap. 
It was also stated that the metabolic profile for urine was more complex
than for feces, and it was thought that differences in the urinary, and
not fecal, metabolic profile between the mouse and rat might explain the
differences in dinocap toxicity.  In this non-guideline study (MRID
47289141) [14C]-DNHPC (Meptyldinocap; 95.4% radiochemical purity; Lot
nos. 742.0212 and 742.0211) in corn oil was administered in a single
gavage dose to male Sprague-Dawley rats and CD1 mice.  [14C]-DNHPC was
administered to 6 rats (95.5 mg/kg bw) and 15 mice (29.5 mg/kg bw). 
Urine was collected for 24 hour intervals up to 96 hours, and samples
collected on Day 1 were analyzed.  Urinary metabolites were quantified
and identified.

Total recovery in the urine (including urine funnel wash) was 30.9% in
rats and 58.3% in mice, and 93.1-95.2% of the radioactivity was isolated
in the Day 1 sample.  Approximately 89-99% of the urinary metabolites
that eluted as discrete peaks were identified.  An additional 6.51% of
the mouse urinary metabolites were apparently conjugates and were only
partially characterized.  Twelve rat metabolites and 13 mouse
metabolites were identified.  The major metabolites as a percentage of
total urinary metabolites were 2,4-dinitro-6-(1-methylpentanoate)phenol
in rats (58.2%) and mice (21.9%) and
2,4-dinitro-6-(1-methylbutanoate)phenol in rats (18.0%) and mice
(23.9%).  Additionally, 2,4-dinitro-6-(1-methylpropanoate)phenol and
2,4-dinitro-6-(1-methyl-β-hydroxypentanoate)phenol were isolated in the
mouse urine at 6.0 and 8.1%, respectively.  No other identified
metabolite occurred at ≥5% of the total urinary metabolites.  

Based on the identified urinary metabolites, a metabolic pathway for
meptyldinocap was inferred. 2,4-DNHPC undergoes esterase-mediated
de-esterification followed by sequential metabolism by cytochrome P-450,
alcohol dehydrogenase, and aldehyde dehydrogenase to form the
carboxylated metabolite, 2,4-dinitro-6-(1-methylheptanoate)phenol. 
Further metabolism proceeds via the fatty acid pathways, which is the
primary pathway for metabolism of 2,4-DNHPC in both rat and mouse.  

tes resulted from the following 4 pathways: (i) fatty acid β-oxidation
(67.3% of the radioactivity in the urine); (ii) fatty acid α-oxidation
(18.0%); (iii) fatty acid β-oxidation followed by nitroreduction and
N-acetylation (4.5%); and (iv) multiple monooxygenation of
2,4-dintro-6-(1-methylheptyl)phenol (4.4%).  Thus, approximately 71.8%
of the metabolic products underwent fatty acid β-oxidation.  

In the mouse, isolated metabolites resulted from the following 5
pathways: (i) fatty acid β-oxidation (45.0% of the radioactivity in the
urine); (ii) fatty acid α-oxidation (27.9%); (iii) fatty acid α- and
β-oxidation followed by conjugation (6.5%); (iv) multiple
monooxygenation of 2,4-dintro-6-(1-methylheptyl)phenol (1.1%); and (v)
one sulfate conjugate (1.2%).  Thus, approximately 46.5% of the
metabolic products underwent fatty acid β-oxidation.  

Fatty acid β-oxidation was the primary metabolic pathway in both
species; however, clearly more metabolism occurred via fatty acid
α-oxidation in mice (approximately 27.9%) than in the rats (18.0%).  A
total of 7 metabolites were identified in mouse urine that were not
identified in rat urine; however, each compound represented only
1.23-2.78% of the total radioactivity isolated in the urine.  Based on
these results, it is unclear whether the differential toxicity of
dinocap observed between mouse and rat species is explained by
differences in the urinary metabolites of meptyldinocap.  Because
dinocap is composed of five other isomers besides meptyldinocap, the
difference in species sensitivity may be due to one of these other
isomers.  The fact that rats and mice received different doses of
meptyldinocap in this study was an additional confounder.

This study in the rat is classified acceptable/non-guideline and does
not satisfy the guideline requirement for a metabolism study [OPPTS
870.7485, OECD 417] in rats.

Appendix B:  Metabolism Assessment

Livestock Metabolism and Confined Rotational Crop Studies

Livestock metabolism and confined rotational crop studies were not
submitted.  

Plant Metabolism Studies  

Apple (47289104.der.doc and 47289105.der.doc):  Apple trees (Golden
Delicious) were treated with a single foliar application of
[14C-U-phenyl]-meptyldinocap at 1.96 kg ai/hectare (1.75 lb ai/acre)
during the latter stages of fruit development.  Samples of leaves and
fruit were collected 0, 7, 14, and 21 days after treatment (DAT).  TRRs
in/on apple leaves were 195 ppm at 0 DAT and declined to 113 ppm by 7
DAT and 71 ppm by 21 DAT (see Table B.1).  For fruit, the majority of
radioactivity was associated with the peel fractions (93-95%) at each
sampling interval.  TRR levels in/on peel and pulp fractions were
respectively 16.74 and 0.25 ppm at 0 DAT and declined to 10.06 and 0.12
ppm by 21 DAT.  Based on the residues in/on peel and pulp, TRR levels
in/on whole fruit were equivalent to 2.90 ppm at 0 DAT and declined
steadily to 1.57 ppm by 21 DAT.  Radioactive residues in/on leaf and
pulp fractions were not further characterized, and only minimal
characterization was conducted on residues in whole fruit samples.  The
most extensive analyses were conducted on the peel samples.

For the whole fruit samples, methanol (MeOH) extraction released 97% of
the TRR at 0 DAT and this decreased to 62%, 43%, and 45% TRR for the 7,
14, and 21 DAT samples, respectively.  For the 7, 14, and 21 DAT
samples, a basic MeOH (0.1M NaOH in MeOH) extraction was performed which
resulted in an additional release of 21%, 42%, and 41% TRR,
respectively.  Residues in the post-extraction solids (PES) accounted
for 3% TRR in the 0 DAT sample and 9-14% TRR in the 7 to 21 DAT samples.
 Thin-layer chromatography (TLC) analyses of the hexane fraction derived
from the MeOH extracts detected meptyldinocap and 2,4-DNOP.  Parent
residues accounted for 75%, 21%, 12%, and 12% TRR in the 0, 7, 14, and
21 DAT samples, respectively.  Residues of 2,4-DNOP were similar at each
interval accounting for 2-6% TRR.  No further analyses were conducted on
the whole fruit samples.

The fractionation and distribution of residues in peel were similar to
whole fruit.  The initial MeOH extractions released 97%, 59%, 45%, and
40% TRR in the 0, 7, 14, and 21 DAT samples, respectively.  With the
decline in MeOH-soluble residues, there was an increase in residues in
the basic MeOH fractions from 33% TRR at 7 DAT to 47% TRR by 21 DAT. 
Residues in the PES fractions accounted for 3% TRR in the 0 DAT samples
and 8-13% of the TRR in the 7 to 21 DAT samples.  The MeOH and basic
MeOH fractions were TLC analyzed with residues of parent accounting for
73%, 23%, 11%, and 8% TRR in the 0, 7, 14, and 21 DAT samples,
respectively.  2,4-DNOP was also identified in peel accounting for 2-4%
of the TRR across all sampling intervals.  The only other compounds
identified in peel were five minor metabolites (metabolites A through E,
Table B.9.) that were each present at <0.5% of the TRR (identified in
the in the hexane fraction of the MeOH extracts).  From 7 to 21 DAT, the
majority of the residues in/on apple peel were comprised of a complex
mixture of more polar components (each ≤5% TRR).  Acid and base
hydrolyses did not result in the conversion of these residues into a
common moiety.

Although the level of identification of residues was limited and several
PES fractions containing >10% TRR were not analyzed further, the apple
metabolism data are classified as scientifically acceptable.  The
available data indicate that that aside from parent compound, the
majority of extractable residues consisted of a complex mixture of minor
metabolites.  Given the complexity of the metabolite profile for the
extractable residues, residues in the PES fractions would also be likely
consist of a complex mixture of minor metabolites.  Therefore, no
additional work is required on the PES fractions.  Tables B.2 and B.3
are summaries of these data.

Apple (47289103.der.doc):  Apple trees (Red Delicious) were treated with
a single foliar application of [14C-U-phenyl]-2,6-DNOPC at 2.0 kg
ai/hectare (1.78 lb ai/acre) during the latter stages of fruit
development.  To assess the translocation of residues, several fruit
bearing branches were covered with plastic bags during application
(translocation test).  After the spray had dried, several treated
clusters of fruit were also covered with paper bags.  Samples of treated
leaves and fruit were collected on the day of application (0 DAT) and at
7, 14, and 21 DAT.  The bagged fruit samples (dark treatment) were
harvested at 7 DAT, and leaf and fruit samples from the branches from
the translocation test were harvested at 21 DAT.  TRRs in/on apple
leaves were 129 ppm at 0 DAT and declined steadily to 89 ppm by 21 DAT
(see Table B.1).  TRR levels in/on fruit samples were variable over time
(2.59-4.28 ppm) showing no trend toward increasing or decreasing.  In
the translocation test, TRR levels were ~500-800x lower in the leaf
(0.105 ppm) and fruit (<0.007 ppm). 

The leaf samples were rinsed with acetonitrile (ACN) and dichloromethane
(DCM; 97%, 78%, 68%, 60%, and 38% for the 0, 7, 14, 21, and
21-translocation DAT samples, respectively).  Excluding the 0 DAT sample
which was not extracted further (3% PES), the rinsed leaf samples were
homogenized and extracted with ACN/water (9%, 12%, 17%, and 27% TRR for
the 7, 14, 21, and 21 translocation DAT samples, respectively). 
Excluding the 21-translocation DAT sample which was not extracted
further (24% TRR PES (0.026 ppm)), the leaf samples were also extracted
with 0.1N HCl (≤6% TRR) followed by refluxed with 1N H2SO4 containing
hexadecytrimethylammonium bromide (3-5% TRR; hemicellulose and protein
fractions).  Residues in the PES accounted for 7%, 11%, and 10% TRR for
the 7, 14, and 21 DAT samples, respectively.  The 14 DAT PES were
treated overnight under refrigeration with 72% H2SO4 with the resulting
suspension diluted with water and refluxed for 2 hours (<1% TRR;
cellulose fraction) followed by reflux overnight in 3% KOH in MeOH (8%
TRR; cutin fraction; 2% TRR PES).  

The leaf surface washes and ACN/water extract were HPLC
(high-performance liquid chromatograph) analyzed with the following
compounds identified:  (1) 2,6-DNOPC (radiolabeled test substance) -
95%, 47%, 38%, 30%, and 10% TRR in the 0, 7, 14, 21, and
21-translocation DAT samples, respectively and (2) 2,6-DNOP - <5% TRR. 
Unknowns accounted for <1%, 23%, 33%, 39%, and 22% TRR in the 0, 7, 14,
21, and 21-translocation DAT samples, respectively (individual unknowns
accounted for <10% TRR; see Table B.4).  

The fruit samples were rinsed with ACN and DCM (97%, 88%, 44%, 28%, and
30% for the 0, 7 dark, 7, 14, and 21 DAT samples, respectively).  The
samples were then separated into pulp (≤3% TRR; not analyzed further)
and peel (3-11% TRR for the 0 and 7-dark DAT samples; 55-70% TRR for the
7, 14, and 21 DAT samples).  The 7, 14, and 21 DAT peel samples were
homogenized and extracted with ACN/water (14-18% TRR), 0.1N HCl (≤9%
TRR), and refluxed with 1N H2SO4 containing hexadecytrimethylammonium
bromide (6-8% TRR; hemicellulose and protein fractions).  Residues in
the PES accounted for 36%, 40%, and 25% TRR for the 7, 14, and 21 DAT
samples, respectively.  The 21-DAT PES were treated overnight under
refrigeration with 72% H2SO4 with the resulting suspension diluted with
water and refluxed for 2 hours (2% TRR; cellulose fraction) followed by
reflux overnight in 3% KOH in MeOH (11% TRR; cutin fraction; 4% TRR
PES).  

The fruit surface washes and peel ACN/water extract were HPLC analyzed
with the following compounds identified:  (1) 2,6-DNOPC (radiolabeled
test substance) - 94%, 87%, 31%, 17%, and 16% TRR in the 0, 7-dark, 7,
14, and 21 DAT samples, respectively and (2) 2,6-DNOP - <5% TRR. 
Unknowns accounted for <1%, <1%, 19%, 23%, and 26% TRR in the 0, 7-dark,
7, 14, and 21 DAT samples, respectively (individual unknowns accounted
for <10% TRR; see Table B.5).  

Cucumber (47289106.der.doc):  Cucumbers were treated with a single
application of  [14C-U-phenyl]-meptyldinocap at 0.5 lb ai/acre
approximately 1-2 months prior to normal fruit maturation (information
concerning the method and type of application were not provided). 
Samples of whole fruits were harvested at 48 and 63 DAT.  

≤1.1% of the TRR (≤0.002 ppm).  In the 63-DAT sample, both
meptyldinocap (3.5% TRR; 0.0033 ppm) and 2,4-DNOP (4.5% TRR; 0.0042 ppm)
were detected in the organic fraction, along with several minor
unknowns, each accounting for ≤3.8% of the TRR (≤0.004 ppm; see
Table B.6).

The following deficiencies were noted in the study: (1) detailed
information on the test substance (e.g.; purity), how it was formulated,
and how it was applied were not provided; (2) details pertaining to the
field application were not provided; (3) no information was provided
concerning the sample storage conditions or duration; (4) the samples
were washed prior to analysis and the wash was not analyzed; (5)
radioactivity in the residual solids and aqueous fractions, which
accounted for 34-53% of the TRR (0.032-0.071 ppm), were not adequately
characterized/identified; and (6) the identity of 2,4-DNOP and
meptyldinocap were not confirmed using a second analytical method.  A
signed Quality Assurance Statement was not provided; a signed and dated
Good Laboratory Practice (GLP) statement was provided which stated that
the submitter was not the sponsor and does not know whether this study
was conducted according to GLP standards.  

Squash (47304905.der.doc):  Squash plants were treated with three
broadcast foliar applications of  [14C-U-phenyl]-meptyldinocap at 0.185
lb ai/acre (RTIs of 7 and 10 days) for a total of 0.56 lb ai/acre (0.63
kg ai/hectare).  Samples of leaves, stems/roots, and mature fruits were
collected at 0, 7 (immediately prior and after the second application),
17 (immediately prior and after the third application), 25, 32, 40, 53,
66, and 80 days after the first application (DAFA); samples of immature
fruit were collected at 53, 66 and 80 DAFA.  The mature fruit samples
collected 32, 40, 53, and 66 DAFA were separated into peel and flesh
fractions.  

At each sampling interval, TRRs were highest in leaves followed by
stem/roots, mature fruits, and immature fruits (see B.1).  For leaves,
TRRs were 36.9 ppm at 0 days after the last application (0 DALA = 17
DAFA) and declined to 11.9 ppm by 23 DALA and 1.7 ppm by 63 DALA.  For
stems/roots, TRRs levels also declined steadily from 3.7 ppm at 0 DALA
to 0.7 ppm by 49 DALA.  TRRs in mature fruits were more variable
overtime, but generally declined at longer post-treatment intervals. 
TRRs in/on mature fruits were highest at 8 DALA (0.41 ppm) and declined
to 0.18 ppm by 49 DALA.  TRRs levels were 2.4-3.9x higher in/on the
fruit peels than in the fruit flesh.  TRRs in/on immature fruits were
steady at 0.02-0.06 ppm from 36 to 63 DALA.

The 8 DALA leaf (19.4 ppm) and fruit (0.41 ppm) samples were used for
residue identification/ characterization purposes.  Benzene extraction
release 14% of the TRR from the 8 DALA leaf sample, while 86% of the TRR
remained in the PES.  1D-TLC analysis of the leaf extract detected
meptyldinocap (3.2% TRR, 0.62 ppm) and 2,4-DNOP (4.8% TRR, 0.93 ppm),
along with nine minor unknowns that were more polar than the parent
compound (0.3-1.0% TRR; see Table B.7).  Acetone extraction released 42%
of the TRR from the 8-DALA sample of mature fruit, with 58% of the TRR
remaining in the PES fraction.  Solvent partitioning separated the
extractable residues into organic (18.5% TRR; 0.08 ppm) and aqueous
(18.9% TRR; 0.08 ppm) fractions.  1D-TLC analysis of the organosoluble
residues detected meptyldinocap (5.9% TRR, 0.02 ppm) and 2,4-DNOP (1.3%
TRR, 0.01 ppm), along with six minor unknowns (0.4-5.5% TRR; see Table
B.8).

The following deficiencies were noted in the study:  (1) no information
was provided on the conditions or duration of sample storage; (2)
radioactivity in the residual solids and aqueous fractions, which
accounted for 19-86% of the TRR, were not adequately characterized; and
(3) the identity of 2,4-DNOP and meptyldinocap were not confirmed using
a second analytical method.  A signed Quality Assurance Statement was
not provided; a signed and dated GLP statement was provided which
indicated that the study was a non-GLP study. 



Table B.1.  TRRs.

Crop; Timing; Application Scenario	PHI (days)	Residues (ppm)1

Apple; later stages of fruit development; 1 x 1.75 lb ai/acre (treated
with meptyldinocap)2

leaves	whole apples	pulp	peel	whole fruit4

(pulp + peel)

	0	195 ± 5.0	1.37 ± 0.55	0.253 ± 0.059	16.74 ± 0.91 (93%)3	2.90 ±
0.15

	7	113 ± 12	2.38 ± 1.73	0.131 ± 0.029	12.83 ± 1.60 (95%)	2.28 ±
0.27

	14	76.3 ± 4.1	1.67 ± 1.09	0.155 ± 0.026	12.97 ± 2.64 (94%)	2.22 ±
0.43

	21	70.6 ± 9.9	1.57 ± 0.64	0.116 ± 0.088	10.06 ± 3.21 (94%)	1.57 ±
0.47

Apple; later stages of fruit development; 1 x 1.75 lb ai/acre (treated
with 2,6-DNOPC)

leaves	whole fruit

	0	129.12	2.59

	7	119.53	3.48

	7 (dark sample)	no sample	1.73

	14	92.77	4.28

	21	89.18	3.07

	21 (translocation test)	0.105	<0.007

Cucumber; 1 x 0.5 lbs ai/acre (treated with meptyldinocap)

fruit

	48	0.135

	63	0.094

Squash; 3 x 0.185 lb ai/acre (treated with meptyldinocap)

leaves	roots and stems	mature fruit	immature fruit

	0	4.2	not sampled	0.14	not sampled

	7	7.75, 44.06	1.55, 8.26	0.125, 0.366	not sampled

	17	10.75, 36.96	1.15, 3.76	0.175, 0.196	not sampled

	25 (8)8	19.4	3.2	0.41	not sampled

	32 (15)8	12.2	1.9	0.25 (0.58, 0.15)7	not sampled

	40 (23)8	11.9	1.8	0.21 (0.19, 0.08)7	not sampled

	53 (36)8	4.2	1.5	0.09 (0.70, 0.19)7	0.02

	66 (49)8	2.7	0.7	0.18 (0.15, 0.06)7	0.04

	80 (63)8	1.7	0.7	not sampled	0.06

1  Parent Equivalents.

2  Data are the average (±S.D.) of the radioassay of 12 subsamples from
whole fruit, 5 subsamples from leaves, and 3 subsamples each from peel
and pulp.  

3  Percentage of whole fruit TRR accounted for by the peel fraction.

4  Whole fruit residue calculated form peel and pulp data.

5  Samples collected immediately prior to either the 2nd or 3rd
application.

6  Samples collected immediately after either the 2nd or 3rd
application.

7  Values in parenthesis are for peel and peeled fruit, repectively. 

8  Values in parenthesis are for DALA. 



Table B.2.  Summary of Characterization and Identification of TRR in
Whole Apple Fruit (meptyldinocap study).

Compound	0 DAT	7 DAT	14 DAT	21 DAT

	TRR = 1.37 ppm	TRR = 2.38 ppm	TRR =1.67 ppm	TRR = 1.57 ppm

	%TRR	ppm	%TRR	ppm	%TRR	ppm	%TRR	ppm

meptyldinocap	74.9	1.03	21.2	0.50	12.1	0.20	12.2	0.19

2,4-DNOP	4.2	0.06	3.4	0.08	2.3	0.04	5.6	0.09

TLC Origins1	8.6	0.12	14.1	0.34	6.3	0.11	7.4	0.12

unresolved radioactivity	4.1	0.05	7.2	0.17	3.0	0.05	ND	--

minor solvent fractions (<10% TRR)	5.4	0.08	24.8	0.59	8.2	0.14	18.0
0.28

major EtOAc fractions1

愀Ĥ

	

-

 

G

T

]

q

‘

Ï

Ö

ä

å

è

ê

ì

ø

%

&

(

/

1

F

H

‡

”

•

—

Ú

ï

਀ø

$

 h

 h

 h

 hF

	”

	”

	”

	”

	”

	”

	”

*	 h

\

?

鐇＋혈0ᴂ尀逍耤㼆

鐇＋혈0ᴂ尀逍耤㼆

鐇＋혈0ᴂ尀逍耤㼆

혈0ᴂ尀逍耤㼆

鐇＋혈0ᴂ尀逍耤㼆

鐇＋혈0ᴂ尀逍耤㼆

혈0ᴂ尀逍耤㼆

혈0ᴂ尀逍耤㼆

 hF

 hf{

䀀Ȧ摧筦

 hf{

hÞ

 hf{

h

”ÿ

 h0

”ÿ

 h¿

h¿

h¶

  h)

h)

h¢

h¶

h×

摧⤟

摧⤟

/

@

@

\

\

 hÉ&

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

愀Ĥ摧ᣴ>

摧ᣴ>

옍)

옍)

옍)

$̀Ĥ옍)

옍)

옍)

옍)

&̀Ĥ옍)

&̀Ĥ옍)

葞и葠﯈摧ᣴ>

摧ᣴ>

%

4

5

6

D

E

F

—

É

摧ᣴ>

摧ᣴ>

摧ᣴ>

6

>

	

ô

%

6

?

X

\

摧ᣴ>Ԁ>

?

A

C

E

F

P

S

\

]

y

‚

„

†

‡

Š

“

–

ž

Ÿ

 

¡

Á

Å

Í

Î

÷

ÿ

ô

f

y

‚

 

ô

ª

Á

Å

É

Í

ô

×

÷

ô

ô

ô

š

 h0

 h0

 h'

摧ᣴ>

Ø	@

Ø	@

 h@

 h@

€	Ò

¸

 

"

#

0

2

8

9

:

>

?

B

C

G

¸

¸

¸

¸

¸

¸

€	Ò

€	Ò

€	Ò

€	Ò

€	Ò



$

0

摧ᣴ>؀0

1

€	Ò

1

2

:

?

C

H

T

摧ᣴ>؀G

H

S

T

V

]

^

y

z

{

‡

‰

‘

¬

º

¼

Ã

Ä

Þ

ß

å

ç

î

ï

,

-

f

g

i

Ÿ

¤

¦

´

µ

ç

é

î

ð

þ

ÿ

H*

@T

U

€	Ò

U

V

_

d

h

{

‡

摧ᣴ>؀‡

ˆ

€	Ò

ˆ

‰

’

—

›

®

º

摧ᣴ>؀º

»

€	Ò

»

¼

Å

É

Í

à

å

摧ᣴ>؀å

æ

€	Ò

æ

ç

ð

ô

ø

€	Ò

€	Ò

H*

yzed in more detail during the analysis of peel samples (see Table 7).

2  Residues remaining after 0.1 M NaOH MeOH extractions.

3  Accountability = (Total extractable + Total unextractable)/(TRR from
combustion analysis; see Table 5) * 100.

NA = not applicable.

Table B.3.  Summary of Characterization and Identification of TRR in
Apple Peel (meptyldinocap study).  

Compound	0 DAT	7 DAT	14 DAT	21 DAT

	TRR = 16.74 ppm	TRR = 12.83 ppm	TRR =12.97 ppm	TRR = 10.06 ppm

	%TRR	ppm	%TRR	ppm	%TRR	ppm	%TRR	ppm

meptyldinocap	73.03	12.23	22.65	2.91	11.13	1.44	7.77	0.78

2,4-DNOP	2.15	0.36	3.66	0.47	2.43	0.32	1.96	0.20

minor Unknowns1

(each at <5% TRR)	8.42	1.41	39.14	5.02	45.86	5.95	49.37	4.97

TLC Origins1	12.68	2.12	21.93	2.81	20.05	2.60	17.53	1.76

total identified	75.18	12.59	26.31	3.38	13.56	1.76	9.73	0.98

total characterized	96.28	15.45	87.38	11.21	79.47	10.31	76.63	7.71

total extractable	96.73	16.19	93.82	12.04	89.99	11.67	89.46	9.00

unextracted (PES)2	3.27	0.55	7.50	0.96	12.10	1.57	12.80	1.29

accountability3	100	101	102	102

1  Attempts at identifying and characterizing minor organosoluble
unknowns and polar residue remaining at TLC origins included acid and
base hydrolyses and methylation and acetylation both before and after
hydrolysis of isolated fractions.  No components were identified and
hydrolyses did not convert residues into a common moiety.

2  Residues remaining after MeOH and 0.1 M NaOH MeOH extractions.

3  Accountability = (Total extractable + Total unextractable)/(TRR from
combustion analysis; see Table 5) * 100.

Table B.4.  Summary of Characterization and Identification of TRR in
Apple Whole Leaves (2,6-DNOPC study).

Compound/Fraction	0 DAT	7 DAT	14 DAT	21 DAT	21-DAT (Translocation)

	TRR = 129 ppm	TRR = 119 ppm	TRR = 92 ppm	TRR = 89 ppm	TRR = 0.105 ppm

	%TRR	ppm	%TRR	ppm	%TRR	ppm	%TRR	ppm	%TRR	ppm

2,6-DNOPC	95.3	123.0	47.0	56.2	37.7	35.0	29.6	26.3	10.3	0.011

unknown 1/2,6-DNOP1	1.5	1.9	10.7	12.7	7.1	6.5	5.9	5.3	3.7	0.037

minor unknowns2	0.2	0.3	23.0	27.4	33.4	30.8	38.8	34.6	21.7	0.2

minor solvent fractions3	NA	--	2.5	2.9	4.1	3.7	5.6	5.0	27.0	0.028

plant component fractions4

	hemicellulose/proteins	NA	--	2.7	3.3	4.9	4.6	5.0	4.4	NA	--

	cellulose	NA	--	NA	--	0.6	0.6	NA	--	NA	--

	lignin	NA	--	NA	--	1.5	0.4	NA	--	NA	--

	cutin	NA	--	NA	--	8.2	7.6	NA	--	NA	--

total identified	95.3	123.0	47.0	56.2	37.7	35.0	29.6	26.3	10.3	0.011

total characterized	97.0	125.2	85.9	102.6	97.5	89.1	85.0	75.6	62.7	0.293

total extracted	97.4	125.8	91.9	109.8	97.2	90.2	87.7	78.3	64.5	0.067

unextracted (PES)5	2.6	3.4	6.9	8.3	1.5	0.4	9.8	8.7	24.5	0.026

accountability6	100.2	99.2	98.5	97.7	88.6

1  Detailed analyses (HPLC, TLC and LC-MS) of Unknown 1 from the 21 DAT
leaf sample identified 2,6-DNOP as a being a minor component (<5% TRR)
of this fraction.

2  Minor unknowns each accounting for <10% TRR, includes multiple
component solvent front fraction.

3  Minor solvent and acid hydrolysis fractions that were not analyzed
further.

4  Residues remaining in solids following acid hydrolysis were
fractionated into natural plant components.

5  Residues remaining after extraction.

6  Accountability = (Total extracted + Total unextracted)/(TRR) * 100.

ND = not detected; NA = not applicable.

Table B.5.  Summary of Characterization and Identification of TRR in
Apple Whole Fruit (2,6-DNOPC study).

Compound/Fraction	0 DAT	7 DAT (Dark)	7 DAT	14 DAT	21 DAT

	TRR = 2.59 ppm	TRR = 1.73ppm	TRR = 3.48 ppm	TRR = 4.28ppm	TRR =  3.07
ppm

	%TRR	ppm	%TRR	ppm	%TRR	ppm	%TRR	ppm	%TRR	ppm

2,6-DNOPC	94.2	2.44	86.6	1.50	30.7	1.08	17.1	0.73	16.4	0.50

unknown 1 / 2,6-DNOP1	2.4	0.06	1.4	0.02	5.4	0.19	3.1	0.13	2.6	0.08

minor unknowns2	ND	--	ND	--	18.8	0.65	23.1	0.99	25.9	0.80

minor solvent fractions3	NA	--	NA	--	8.6	0.30	4.6	0.20	7.4	0.23

plant component fractions4

	hemicellulose/proteins	NA	--	NA	--	NA	--	0.4	0.02	0.5	0.02

	cellulose	NA	--	NA	--	NA	--	NA	--	1.5	0.05

	lignin	NA	--	NA	--	NA	--	NA	--	3.8	0.12

	cutin	NA	--	NA	--	NA	--	NA	--	11.5	0.35

total identified	94.2	2.44	86.6	1.50	30.7	1.08	17.1	0.73	16.4	0.50

total characterized	96.6	2.50	88.0	1.52	70.0	2.47	55.5	2.37	72.9	2.24

total extracted	96.8	2.50	88.2	1.52	73.9	2.59	59.2	2.53	72.2	2.22

unextracted (PES)5	3.2	0.08	11.9	0.21	37.3	1.30	42.4	1.81	6.9	0.21

accountability6	99.7	99.8	112	101	79.3

1  Detailed analyses (HPLC, TLC and LC-MS) identified a small portion of
this fraction as 2,6-DNOP.

2  Minor unknowns each accounting for <10% TRR, includes multiple
component solvent front fraction.

3  Minor solvent and acid hydrolysis fractions that were not analyzed
further.

4  Residues remaining in solids following acid hydrolysis were
fractionated into natural plant components.

5  Residues remaining after extraction.  The most extensive analysis of
the PES fraction was conducted on the 21-DAT sample.

6  Accountability = (Total extracted + Total unextracted)/(TRR) * 100.

ND = not detected; NA = not applicable.

Table B.6.  Summary of Characterization and Identification of TRR in
Cucumber.

Fraction/ Metabolite	Fruit, 48 DAT	Fruit, 63 DAT

	TRR = 0.135 ppm	TRR = 0.094

	%TRR	ppm1	% TRR	ppm1

acetone extracts (solvent partitioning)	47.5	0.064	59.9	0.056

organic fraction (pet-ether/DCM) - TLC analysis	7.6	0.010	23.5	0.022

2,4-DNOP	0.6	0.0008	4.5	0.0042

meptyldinocap	0.7	0.0009	3.5	0.0033

unknown C	0.9	0.0012	1.3	0.0012

unknown D	0.9	0.0012	3.3	0.0031

unknown E	1.1	0.0015	3.8	0.0036

other minor unknowns	1.6	0.0022	3.8	0.0036

TLC Origin	1.4	0.0019	5.0	0.0047

aqueous fraction (not analyzed further)	43.3	0.059	34.0	0.032

PES	52.5	0.071	40.1	0.038

1  Values were calculated by the reviewer using TRR data and information
from Tables II and III of the study report.

Table B.7.  Summary of Characterization and Identification of TRR in
Squash Leaves.  

Fraction/ Metabolite	Leaves, 25 DAFA (TRR = 19.4 ppm)1

	%TRR	ppm

benzene (1D-TLC analysis)	14.0	2.72

	2,4-DNOP	4.8	0.93

	meptyldinocap	3.2	0.62

	unknown 1	0.6	0.12

	unknown 2	0.3	0.06

	unknown 3	0.3	0.06

	unknown 4	0.3	0.06

	unknowns 5 and 6	1.0	0.19

	unknowns 7 and 8	0.4	0.08

	unknown 9	0.6	0.12

	TLC Origin/polar unknowns	1.7	0.33

PES	86.0	16.68

total Identified	8.0	1.55

total Characterized	13.2	2.56

1  The 25-DAFA sample was collected 8 days after the final application.

Table B.8.  Summary of Characterization and Identification of TRR in
Squash Fruit.  

Fraction/ Metabolite	Mature fruit, 25 DAFA (TRR = 0.41 ppm)1

	%TRR	ppm

acetone (solvent partitioning)	42.0	0.17

	petroleum ether/DCM (1D-TLC analysis)	18.5	0.08

		2,4-DNOP	1.3	0.01

		meptyldinocap	5.9	0.02

		unknown 3	0.4	0.002

		unknown 4	0.4	0.002

		unknown 5	0.8	0.003

		unknowns 6 and 7	5.5	0.02

		unknown 8/TLC origin	5.5	0.02

	aqueous Fraction	18.9	0.08

PES	57.6	0.24

total Identified	7.2	0.03

total Characterized	19.8	0.08

The 25-DAFA sample was collected 8 days after the final application.

Table B.9.  Chemical Structures.

Chemical name	Chemical structure	Matrices (%TRR)

meptyldinocap

2,4-dinitro-6-(2-octyl)phenyl crotonate

2,4-dinitro-6-(1-methyheptyl)phenyl crotonate 

	Apple fruit – 7 and 21 DAT (21.2% and 12.2%)

Squash leaves – 25 DAT (3.2% TRR)

Squash - 25 DAT

(5.9%)

Cucumbers – 48 DAT (0.6%)

2.4-DNOP

	Apple fruit – 7 and 21 DAT (3.4% and 5.6%)

Squash leaves - 25 DAT (4.8%)

Squash - 25 DAT (1.3%)

Cucumbers – 48 DAT (0.7%)

Metabolite A

	Apple peel – 14 DAT (0.3%)

Metabolite B

	Apple peel – 14 DAT (0.1%)

Metabolite C

	Apple peel – 14 DAT (0.1%)

Metabolite D

	Apple peel – 14 DAT (0.4%)

Metabolite E

1-methylheptyl isomer	1-ethylhexyl isomer	1-propylpentyl isomer	not
identified

   HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22S
mialowicz%20RJ%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_
ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Smialowicz RJ
,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22L
uebke%20RW%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Resu
ltsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Luebke RW ,  
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22R
iddle%20MM%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Resu
ltsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Riddle MM . 1992.
 Assessment of the immunotoxic potential of the fungicide dinocap in
mice.  Toxicology 75(3):235-47.

Meptyldinocap	      Human-Health Risk Assessment for Use on Imported
Grapes    	            D348877

Page   PAGE  1  of   NUMPAGES  47