Document ID: EPA-HQ-OPP-2009-0032-0014
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2011-01-19T05:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C.  20460

	OFFICE OF CHEMICAL SAFETY

	AND POLLUTION PREVENTION

  SEQ CHAPTER \h \r 1 MEMORANDUM

Date:  		11/24/2010

SUBJECT:	Fluazinam.  Petitions for the Establishment of Tolerances and
Registration of New Uses on Apples and Carrots.  HED’s Conclusions
Regarding Registrant’s Response to Data Deficiencies.

PC Code:  129098	DP Barcodes:  D381302 and D381370

Decision Nos.:  437290	Registration No.: 71512-1

Petitions:  9E7570  Carrots

                  9F7571  Apples	Regulatory Action:  Section 3
Registration

Risk Assessment Type:  NA	Case No.:  NA

TXR No.:  NA	CAS No.:    SEQ CHAPTER \h \r 1 79622-59-6

MRID Nos.:  47949001, 48103501,

                       48103502, 48154701	40 CFR:  §180.574

		              									

FROM:  	Douglas Dotson, Ph.D., Chemist

		Risk Assessment Branch II

		Health Effects Division (7509P)	  SEQ CHAPTER \h \r 1 

		

THROUGH:	Dennis McNeilly, Chemist

		Richard Loranger, Ph.D., Senior Scientist

		Risk Assessment Branch II

		Health Effects Division (7509P)

TO:		Laura Nollen/Barbara Madden, RM Team 5, RIMUERB

		John Bazuin/Tony Kish, PM Team 22, Fungicide Branch

		Registration Division (7505P)		  SEQ CHAPTER \h \r 1   SEQ CHAPTER \h
\r 1 

		

Executive Summary

In early 2010, HED completed a human health risk assessment for proposed
uses of the fungicide fluazinam,
3-chloro-N-[3-chloro-2,6-dinitro-4-(trifluoromethyl)phenyl]-5-(trifluoro
methyl)-2-pyridinamine, on apples, carrots, head lettuce, leaf lettuce,
and the Bulb onion subgroup (13-07B) (Memo, D365940, D. Dotson, et al.,
3/31/2010).  HED recommended in favor of tolerances for head lettuce,
leaf lettuce, and the Bulb onion subgroup; however, because of major
data deficiencies, HED was not able to recommend in favor of tolerances
on apple and carrot commodities.  These data deficiencies are discussed
in detail in the residue chemistry summary document (Memo, D360840, D.
Dotson, 3/31/2010) that was written in support of the risk assessment. 
In addition, the deficiencies are discussed in a 75-day letter that the
Agency provided to the registrant, ISK Biosciences.  The deficiencies
related to an apple processing study, a cattle feeding study, and the
analytical method for the metabolites AMPA and DAPA in fat, liver, and
kidney.  The previous residue chemistry summary document addressed all
residue chemistry aspects associated with the tolerance petitions.  This
current memo addresses ISK’s responses to the cited data deficiencies
and HED’s conclusions concerning those responses.        

In PP# 9E7570, IR-4, on behalf of the Agricultural Experiment Stations
of California, Georgia, Texas, and Washington, requested the
registration of fluazinam for use on carrots.  IR-4 proposed a tolerance
of 0.8 ppm for carrot, roots.  In PP# 9F7571, ISK Biosciences
Corporation requested the registration of fluazinam for use on apples. 
ISK proposed the establishment of permanent tolerances for residues of
fluazinam and its metabolite AMGT in or on apples at 1.7 ppm and apple,
pomace, wet, at 5.0 ppm.  In addition, ISK proposed tolerances for
residues of fluazinam and its metabolites AMPA and DAPA in milk as well
as in the fat, meat, liver, kidney, and meat byproducts of cattle,
goats, horses, and sheep at 0.03 ppm.  This value is the combined limit
of quantitation (LOQ) of the proposed enforcement method.

Data Deficiencies

The data deficiencies referred to above are discussed below.

Analytical Enforcement Method:  The initial validation of Method 1676W
showed that it adequately recovered residues of fluazinam, AMPA, and
DAPA in milk, muscle, and fat; how-ever, recoveries of AMPA and DAPA in
liver and kidney were low.  The results of concurrent method validation
also showed similar low recoveries of the metabolites in liver and
kidney.  Mean recoveries of AMPA ranged from 48% to 93% in hydrolyzed
liver and from 36% to 73% in hydrolyzed kidney.  Mean recoveries of DAPA
ranged from 35% to 51% in hydrolyzed liver and from 9% to 33% in
hydrolyzed kidney.  Because of low recoveries, the petitioner reported
both the corrected and uncorrected values for these compounds in liver
and kidney in the dairy cattle feeding study.  Method 1676W was also
subjected to an independent laboratory validation (ILV).  In the first
trial, the method was successfully validated for fluazinam and AMPA in
milk, for AMPA in beef fat, and for AMPA in the liver non-hydrolysis
procedure.  In the second trial, analysis of fluazinam and DAPA in beef
fat was successfully completed after communication with the sponsor and
the method developers.  In the third trial, fluazinam, AMPA, and DAPA in
milk and non-hydrolyzed liver were successfully completed after minor
method modification.

A concern with the dairy cattle feeding study is that the storage
stability data for animal commodities showed instability of DAPA and
AMPA in liver and kidney.  This apparent instability could have been a
result of problems with the analytical method.

Method 1676W was forwarded to ACB/BEAD for method validation.  ACB/BEAD
reported that the lab that performed the ILV made major modifications to
the original method.  As a result, HED requested that the registrant
submit a revised method that incorporated the revisions made by the ILV
lab.  ISK Biosciences revised the method and re-submitted it.  ACB/BEAD
performed a brief review of the revised method.  ISK incorporated the
modifications made by the ILV lab.  However, the revision eliminated the
cleanup steps that were in the original method, and the only recovery
data for the revised method were data provided by the ILV lab.  ACB/BEAD
was concerned that the elimination of the cleanup steps could lead to
dirty samples which would, in turn, lead to analytical problems, such as
interference.  ACB/BEAD performed no further validation of the method. 
As the revised method contained major revisions to the original method,
and ACB/BEAD believed it was very likely that the revised method still
was not adequate for sample analysis or tolerance enforcement, HED
recommended that ISK submit an ILV for the revised method.

Multiresidue Method Testing Data:  Multiresidue method testing data for
the regulated animal metabolites, AMPA and DAPA have not been submitted.

Dairy Cattle Feeding Study:  Although the in-life phase of the dairy
cattle feeding study was considered to be adequate, there were problems
with the analytical method.  The poor recoveries obtained in the feeding
study are discussed above.  In addition, AMPA and DAPA were apparently
unstable in liver and kidney in the storage stability studies.  HED
recommended that the registrant submit the results of an acceptable
dairy cattle feeding study.  Residues of the metabolites AMPA and DAPA
in animal commodities needed to be successfully quantified if they were
present at levels above the limit of quantitation.   

Apple Processing Study:  HED also identified a deficiency with the apple
processing study.  In apples, the metabolite AMGT is a residue of
concern for risk assessment.  In the processing study there were no
quantifiable residues of AMGT in raw apples.  In several of the field
trials that were performed, however, raw apples contained AMGT residues
at, or above, the limit of quantitation.  Of the 42 field trial values,
32 were below the LOQ of 0.01 ppm, 6 were 0.01 ppm, 3 were 0.02 ppm, and
1 was 0.03 ppm.  The Series 860 Residue Chemistry Guidelines state that
in the processing study, if residues in the RAC are below the LOQ, but
quantifiable residues occurred in the RAC in field trials performed at
the maximum requested label rate, that the processing study should be
conducted at exaggerated rates in order to ensure that quantifiable
residues will be present in the RAC.  Quantifiable residues are needed
in the RAC in order to determine a reliable concentration factor.  As a
result, the processing study needed to be repeated at a sufficiently
high rate that residues of both parent and AMGT were quantifiable in the
RAC.

Summary of Data Deficiencies

HED concluded that an analytical method was not available for
enforcement of fluazinam tolerances in animal commodities.  HED
recommended that the registrant submit an ILV of the revised method. 
The method needed to be adequate for analysis of parent fluazinam as
well as the metabolites AMPA and DAPA in animal commodities for which
tolerances would be established.  HED recommended that ISK Biosciences
submit multiresidue method testing data for the regulated animal
metabolites, AMPA and DAPA.  HED also recommended that the registrant
submit the results of a new dairy cattle feeding study.  Residues of the
metabolites AMPA and DAPA in animal commodities needed to be
successfully quantified if they were present at levels above the limit
of quantitation.  Finally, HED recommended that the registrant submit
the results of a new apple processing study.  The study needed to be
performed at a sufficiently high rate that residues of both fluazinam
and AMGT were quantifiable in the RAC.

Regulatory Recommendations and Residue Chemistry Deficiencies

With the exception of the multiresidue method testing data for the
regulated animal metabolites, AMPA and DAPA, ISK Biosciences has
resolved the data deficiencies associated with the apple and carrot
tolerance petitions.  There are no outstanding data deficiencies that
would preclude establishment of tolerances and conditional registration
for apple, carrot, and animal commodities.  Although analyte-specific
methods are available for AMPA and DAPA in animal commodities,
multiresidue method testing data continue to be required for these
regulated animal metabolites.  For any future uses that involve major
feed items and significantly higher dietary burdens, more vigorous
extraction steps might be required in the method for kidney and liver
along with radiovalidation data to demonstrate improved extractability. 

The recommended tolerances are listed in Table 4.  The registrant needs
to submit a revised Section F in which the recommended tolerances and
correct commodity definitions listed in Table 4 are proposed.

α,α,α-2-nitro-trifluoro-p-toluidino)-3-chloro-5-(trifluoromethyl)
pyridine), DAPA
(3-chloro-2-(2,6-diamino-3-chloro-α,α,α.-trifluoro-p-toluidino)-5-(tr
ifluoromethyl)-pyridine), and their sulfamate conjugates.”

Detailed Considerations

Structures of Fluazinam and Metabolites

The nomenclature and structures for fluazinam and its metabolites AMGT,
AMPA, and DAPA are presented in Table 1.  

α,α,α-trifluoro-2-nitro-p-toluidino)-3-chloro-5-(trifluoromethyl)
pyridine

Compound	

Common name	DAPA

Chemical name
3-chloro-2-(2,6-diamino-3-chloro-α,α,α.-trifluoro-p-toluidino)-5-(tri
fluoromethyl)pyridine

In response to the data deficiencies discussed above, ISK Biosciences
submitted an analytical method and ILV for the analysis of fluazinam,
AMPA, and DAPA in fat, liver, and kidney.  In addition, ISK submitted
additional data for the AMGT metabolite in apples in order that the
apple processing study could be upgraded to acceptable.  Finally, ISK
submitted a rebuttal to the data deficiencies cited in the cattle
feeding study.  The ILV for the revised enforcement method, the
additional apple processing data, and the rebuttal to the request for a
new cattle feeding study are discussed in detail below. 

860.1340 Residue Analytical Methods

DER Reference:  47949001.DER.doc

The analytical methods for the analysis of fluazinam residues of concern
in plant and animal commodities are discussed in the previous residue
chemistry summary document prepared for the proposed uses (Memo,
D360840, D. Dotson, 3/31/2010).  HED concluded that an analytical method
was not available for enforcement of fluazinam tolerances in animal
commodities.  HED recommended that the registrant submit an ILV of the
revised method.  The method needed to be adequate for analysis of parent
fluazinam as well as the metabolites AMPA and DAPA in animal commodities
for which tolerances will be established.

ILV

In response to the deficiency cited in the analytical method for AMPA
and DAPA in liver and kidney, ISK submitted an analytical method
entitled “Enforcement Method for the Analysis of Fluazinam and its
Metabolites AMPA and DAPA in Milk and Meat (Document Number
IB-2009-JLW-005-01).”  ISK also submitted an ILV for this method.  The
method is written for LC/MS/MS analysis of fluazinam, AMPA, and DAPA and
their sulfamate conjugates in bovine liver, fat, and milk.  The reported
method LOQ was 0.01 ppm for each analyte in liver, fat, and milk.  The
method is described briefly below.

For beef kidney and liver, the sample was extracted with
acetonitrile:water (75:25, v/v) and 2 mL acetic acid.  The sample was
blended, and the mixture was vacuum filtered through glass fiber filter
paper with a layer of Celite on top.  The filter cake was rinsed twice
with acetonitrile:water (75:25, v/v).  The filter and filter cake were
extracted a second time with acetonitrile: water (75:25, v/v).  The
sample was blended and the mixture was vacuum filtered through a new
glass fiber filter into the same flask.  The bottle and filter cake were
rinsed with two 25 mL portions of acetonitrile: water (75:25, v/v).  The
contents of the Erlenmeyer flask were transferred to a 500 mL graduated
cylinder and were brought up to a final volume of 300 mL with
acetonitrile:water (75:25, v/v).  The sample was mixed well, and
approximately 1 mL was filtered through a 0.45 µm nylon syringe filter
into an autosampler vial.  This filtered extract was analyzed by
LC/MS/MS.

Fat Method (Trial 1):  For beef fat, the sample was mixed with Celite
and extracted with acetonitrile/acetic acid (75:2).  The sample was
blended, the mixture was vacuum filtered through glass fiber filter
paper, and the filter cake was rinsed with acetonitrile.  The filter and
filter cake were extracted, blended, filtered, and rinsed a second time.
 The contents of the graduated cylinder were brought up to a final
volume of 200 mL with acetonitrile and mixed well.  An aliquot of the
acetonitrile sample extract was transferred to a separatory funnel and
50 mL of acetonitrile saturated with cyclohexane were added.  The
separatory funnel was shaken, the phases were allowed to separate, the
acetonitrile layer was drained off, and the cyclohexane layer was
discarded.  The acetonitrile layer was added back to the separatory
funnel and the sample was extracted a second time following the same
procedure.  The acetonitrile layer was drained into a 500 mL round
bottom flask and the cyclohexane layer was discarded.  

The acetonitrile extract was rotary evaporated to dryness at
approximately 37°C.  The sample was re-dissolved in 5 mL of
acetonitrile and sonicated for approximately 5 minutes.  Five mL of
water were added to the sample.  The sample was mixed well and
approximately 1 mL was filtered through a 0.45 µm nylon syringe filter
into an autosampler vial.  This filtered extract was diluted with
acetonitrile:water (50:50 v/v), if necessary, and analyzed by LC/MS/MS.

Fat Method (Trial 2):  The samples were extracted as written above until
the rotary evaporation step.  The steps after the acetonitrile layer was
collected into a 500 mL round bottom flask are as follows:  The
acetonitrile extract was rotary evaporated down to approximately 10 mL
at approximately 37°C.  The sample was brought up to a 10 mL volume in
a graduated cylinder.  The round bottom flask was rinsed with 10 mL of
water and the rinse water was combined with the sample.  The sample was
then transferred to a 30 mL amber glass bottle.  The sample was mixed
well and approximately 1 mL was filtered through a 0.45 µm nylon
syringe filter into an 8 mL sample vial.  This filtered extract was
diluted with acetonitrile:water (50:50 v/v), if necessary, and analyzed
by LC/MS/MS.

In summary, the difference between Trials 1 and 2 is that in Trial 1,
the sample was rotary evaporated to dryness and in Trial 2, it was
evaporated to approximately 10 mL.  In Trial 1, the dried sample was
re-dissolved in solvent with the aid of sonication.

Two fortification levels were used for the ILV:  the LOQ (0.01 ppm) and
10x the LOQ.  The LC/MS/MS method cites two transition ions for each
analyte in order to provide confirmation.  

Fortification Recoveries

In kidney, the overall mean recoveries for both the primary and
secondary ion transitions were:  fluazinam (86%), AMPA (92%), and DAPA
(92%).  All recoveries were within the acceptable range of 70% to 120%
with the exception of one fortification at the LOQ for parent fluazinam.

In liver, the overall mean recoveries for both the primary and secondary
ion transitions were:  fluazinam (84%), AMPA (91%), and DAPA (80%). 
Initially, all recoveries of parent fluazinam in beef liver fortified at
the LOQ were less than acceptable.  The poor recoveries were probably
due to suppression.  The standards and sample extracts were diluted 1 to
4 and reanalyzed for fluazinam.  Acceptable recoveries were obtained for
all but two samples.  All of the recoveries for AMPA and DAPA were
acceptable without dilution.

In the first method validation trial for fat, the overall mean
recoveries for both the primary and secondary ion transitions were: 
fluazinam (78%), AMPA (77%), and DAPA (36%).  Several recoveries for
beef fat for the analytes fluazinam and AMPA were below the acceptable
recovery range for the first method trial.  Recoveries for beef fat for
the analyte DAPA were all well below the desirable recovery range for
the first method trial.  In the second method validation trial for fat,
the overall mean recoveries for both the primary and secondary ion
transitions were:  fluazinam (89%), AMPA (91%), and DAPA (87%).  For the
second method trial for beef fat, all recoveries were within the
acceptable range of 70% to 120%.  

In all fortification trials, control sample residues were less than the
LOQ of 0.01 µg/g.  The correlation coefficients for the calibration
curves for fluazinam, AMPA, and DAPA were all greater than 0.99.  No
interferences were observed at the retention times of fluazinam, AMPA,
and DAPA.

The performing laboratory concluded that the method is suitable for use
on bovine kidney, liver, and fat.  That conclusion was based on the fact
that acceptable recoveries of fluazinam, AMPA, and DAPA were obtained in
beef kidney, liver, and fat down to a level of 0.01 ppm for each
compound.  In addition, correlation coefficients for the calibration
curves for all three analytes were all greater than 0.99, and no
interferences were observed at the retention times of fluazinam, AMPA,
and DAPA.

Radiovalidation

Radiovalidation data were not provided for the livestock analytical
method.  The procedure for liver and kidney includes an initial
extraction with 75:25 acetonitrile (ACN):water with 2 mL acetic acid
followed by a second extraction with 75:25 ACN:water.  For fat, the
first extraction uses 75:2 ACN:acetic acid followed by an extraction
with acetonitrile.  In the goat metabolism study, liver and kidney were
extracted with 1:1 ACN:water, and fat was extracted with a mixture of
1:1 ACN:water and hexane.  With the addition of acetic acid in the
proposed method’s initial extractions and the inclusion of a second
extraction of the tissues, HED considers these procedures to be
comparable to the initial extractions used in the goat metabolism study.
 Therefore, radiovalidation data will not be required for the proposed
uses that include only relatively minor feed items (apple pomace, cull
carrots) and 0.05 ppm tolerances for fat and meat byproducts.  HED notes
that additional extractions using enzymes (liver and kidney) and acid
hydrolysis (liver) released significant additional amounts of
radioactivity in the goat metabolism study.  Therefore, for any future
uses that involve major feed items and significantly higher dietary
burdens, more vigorous extraction steps might be required in the method
for kidney and liver, along with radiovalidation data to demonstrate
improved extractability.  

HED’s Conclusions

HED concludes that ISK Biosciences Corporation’s analytical method
entitled “Enforcement Method for the Analysis of Fluazinam and its
Metabolites AMPA and DAPA in Milk and Meat” is adequate as an
enforcement method for residues of fluazinam, AMPA, and DAPA and their
sulfamate conjugates in kidney, liver, and fat.  The method was
successfully validated based on the Series 860.1340 Residue Chemistry
Guidelines for fluazinam, AMPA, and DAPA in beef kidney, beef liver, and
beef fat.

860.1380 Storage Stability

The following storage stability information was included in the previous
residue chemistry summary document prepared for the proposed uses (Memo,
D360840, D. Dotson, 3/31/2010).  It is being reproduced here because it
is relevant to the data deficiencies being addressed in this memo.  

Storage Stability Data for Cattle Milk, Meat, and Meat Byproducts

Samples collected from the dairy cow feeding study (MRID 47756605) were
held under frozen storage conditions prior to residue analysis.  The
maximum storage durations of samples, from collection to analysis, were
146 days for milk, 185 days for cream, 179 days for skim milk, 157 days
for muscle, 203 days for fat, and 255 days for liver and kidney.  A
storage stability study was conducted to validate sample storage
conditions and durations.  The results showed mixed results.  In milk,
cream, and skim milk, residues of fluazinam, AMPA, and DAPA were found
to be stable for 183 days.  In muscle:  (i) fluazinam was stable for 1
day, but declined after 164 days to an average corrected recovery of
55%; (ii) AMPA was stable for 2 days, but declined after 161 days to an
average corrected recovery of 41%; and (iii) DAPA was stable for 2 days,
but declined after 161 days to an average corrected recovery of 10%.  In
fat:  (i) fluazinam and AMPA were stable for 205 days; and (ii) DAPA
showed relative instability with average corrected recoveries of 64%
after 205 days.  In liver and kidney:  (i) fluazinam was unstable,
average recoveries in liver were 26-34% after 210 days, and 41-44% in
kidney after 218 days; (ii) AMPA was stable in liver for 80/89 days, but
not in kidney where average corrected recoveries were 53-58% after 218
days; and (iii) DAPA is not stable with average corrected recoveries of
43-52% in liver after 209 days and 17-25% in kidney after 218 days.

Conclusions.  Adequate storage stability data are available to support
the storage conditions and durations of samples collected from the
magnitude of the residue studies on apples and carrots.  As residues of
fluazinam were found to be stable in tested crop matrices, storage
stability corrections do not need to be applied to the recommended
tolerances for fluazinam residues in/on apples and carrots.  Storage
stability data for the fluazinam metabolite AMGT in apple commodities
were also submitted and showed that residues in apple wet pomace were
reasonably stable for 12 months in apple wet pomace, but declined to an
average corrected recovery of 33% at the 37-month interval.  The
submitted storage data for bovine milk, meat, and meat byproducts showed
mixed results.  

860.1480 Meat, Milk, Poultry, and Eggs

DER Reference:  47756605.DER.doc

Because of the potential for residues to be present in animal
commodities, ISK submitted a dairy cattle feeding study with their
original submission.  Based on the poor recoveries of metabolites AMPA
and DAPA from liver and kidney and the mixed storage stability results,
HED determined that the residue data from the cattle feeding study were
not adequate to satisfy data requirements.  The feeding study data
(adjusted for residue decline) along with the calculated dietary burdens
for beef cattle (0.04 ppm) and dairy cattle (1.27 ppm) indicated that
tolerances might be needed for the combined residues of fluazinam and
its metabolites, AMPA and DAPA, and their sulfamate conjugates, in the
fat and meat byproducts of cattle and other ruminants to support the
proposed use on apples.  The feeding study also indicated that
tolerances probably would not be needed for milk and meat of ruminants,
as the expected combined residues in these matrices are below the
combined method LOQ of 0.03 ppm for the regulated compounds.  Based on
the transfer coefficients for livestock tissues and the relatively low
dietary burden for swine of 0.003 ppm for fluazinam, tolerances for hogs
are not needed.  A poultry feeding study is not required at this time
because of the low dietary burden for poultry (0.005 ppm).  HED
determined that, in order to support tolerances for apples and carrots,
the registrant needed to submit a new cattle feeding study in which a
method with acceptable recoveries of AMPA and DAPA from liver and kidney
was used.

In lieu of submitting a new cattle feeding study, ISK Biosciences
submitted a rebuttal to the data deficiency (MRID 47746605).  The
rebuttal is entitled “Rebuttal Response to Upgrade Magnitude of
Fluazinam Residues in Bovine Tissues and Milk from a 28-Day Feeding
Study.”  ISK explained that liver and kidney are metabolic matrices,
and as a result, residues are unstable during storage, and recoveries
are low when samples are analyzed.  ISK stated the following with
respect to this issue:  “In our experience it is often observed that
labile metabolites are unstable on long term storage and further
degraded during thawing and processing of liver tissue, during which
period enzyme activity is revived.  These processes are likely
responsible for the variable low recoveries obtained in the storage
stability and possibly with the concurrent fortification samples as some
degradation occurs during sample handling.  For example, our laboratory
(PTRL West) has analyzed another substituted aniline in many matrices
over the years and typical concurrent recoveries are 20-40%.”

In livestock, one of the nitro groups on fluazinam gets reduced to an
amine group to produce AMPA (see structures in Table 1).  The second
nitro group then gets reduced to form DAPA.  In its rebuttal, ISK stated
that aniline hydroxylases convert the amino groups to hydroxylamines,
which themselves are unstable and prone to further oxidation.  Variable
and generally poor recoveries of DAPA from liver and kidney in the
feeding study as well as the poor storage stability of this metabolite
are evidence of the well known reactivity of anilines to oxidative
environments, such as hydroxyl radical and peroxides in environmental
systems and P-450 and hydroxylase enzymes in liver and blood.

The dairy cattle feeding study included a depuration phase.  Residue
decline occurred for both AMPA and DAPA in liver and kidney.  AMPA
residues declined by at least 30% after one day of depuration and by at
least 70% after 7 days of depuration.  Decline occurred even more
quickly for DAPA.  Residues declined by at least 50% of their initial
value after one day of depuration and were non-detectable after 3 days
of depuration.  ISK Biosciences pointed out in the rebuttal that
detectable residues of AMPA and DAPA would only be found in dairy cattle
that had been fed a diet that contains treated wet apple pomace up to a
few days prior to slaughter.  For beef cattle, wet apple pomace is not a
significant feed item.  As a result, the point in time at which beef
cattle are fed treated wet apple pomace is not important to this
tolerance petition. 

The following table summarizes the storage stability, depuration, and
recoveries of AMPA and DAPA in liver and kidney.

Table 2.  Comparison of Storage Stability, Recovery in Feeding Study,
and Depuration

AMPA

	Liver	Kidney

Storage Stability	80-89% Recovery after 210 days	53-58% Recovery after
218 days

Analytical Recovery in Feeding Study (Hydrolysis Method)	48-93%	36-75%

Depuration	30-50% Decline after 1 day

100% Decline after 3 days	42-100% Decline after 1 day

100% Decline after 3 days

DAPA

	Liver	Kidney

Storage Stability	43-52% Recovery after 209 days	17-25% Recovery after
218 days

Analytical Recovery in Feeding Study (Hydrolysis Method)	35-51%	9-33%

Depuration	60% Decline after 1 day

100% Decline after 3 days	50-100% Decline after 1 day

100% Decline after 3 days

HED’s Conclusions

The poor storage stability of AMPA and DAPA in liver and kidney, the
poor recovery from these matrices when the feeding study was performed,
and the results of the depuration study all support ISK’s assertion
that liver and kidney are metabolic matrices that cause the rapid
decline of AMPA and DAPA residues.  HED agrees with ISK Biosciences that
another animal feeding study does not need to be performed.  HED notes
that further justification for not requiring another study is the low
levels of AMPA and DAPA found in liver and kidney at the highest feeding
level.  Even at this high dose (28.84 ppm, 22.7x the maximum expected
dietary burden) and correcting for concurrent method recoveries, the
maximum combined residues of AMPA and DAPA in liver and kidney were less
than about 0.045 ppm.

HED previously determined that a tolerance of 0.05 ppm would be adequate
to cover residues in the fat and meat byproducts of cattle, goat, horse,
and sheep.  As a result, HED recommends in favor of the establishment of
tolerances of 0.05 ppm for the fat and meat byproducts of cattle, goat,
horse, and sheep.  The registrant needs to submit a revised Section F in
which these tolerances are proposed.

860.1520 Processed Food and Feed

DER Reference:  48103502.DER.doc

The results of an apple processing study are summarized in the previous
residue chemistry summary document prepared for the proposed use on
apples (Memo, D360840, D. Dotson, 3/31/2010).  HED originally concluded
that the apple processing study was not adequate to satisfy data
requirements.  In apples, the metabolite AMGT is a residue of concern
for risk assessment.  AMGT is not a residue of concern for tolerance
expression, however.  In the processing study there were no quantifiable
residues of AMGT in raw apples.  In several of the field trials that
were performed, however, raw apples contained AMGT residues at, or
above, the limit of quantitation.  Of the 42 field trial values, 32 were
below the LOQ of 0.01 ppm, 6 were 0.01 ppm, 3 were 0.02 ppm, and 1 was
0.03 ppm.  The Series 860 Residue Chemistry Guidelines state that in the
processing study, if residues in the RAC are below the LOQ, but
quantifiable residues occurred in the RAC in field trials performed at
the maximum requested label rate, that the processing study should be
conducted at exaggerated rates in order to ensure that quantifiable
residues will be present in the RAC.  Quantifiable residues are needed
in the RAC in order to determine a reliable concentration factor.

HED requested that the processing study be repeated at a sufficiently
high rate that residues of both parent and AMGT would be quantifiable in
the RAC.  Instead of performing a new processing study, however, the
registrant submitted additional data from the initial study.  When the
laboratory personnel originally analyzed the samples, they did not
quantitate samples in which the AMGT residue levels were below the LOQ. 
As a result, it was not possible to determine processing factors.  ISK
Biosciences submitted an amended report in which the residue levels that
were between the LOD and LOQ were measured.  As stated above, the LOQ
was 0.01 ppm.  The original electronic data were not saved by the
performing laboratory.  As a result, residues were quantitated by
manually measuring peak heights.  The residue values are given in Table
3, below.  The 860 Residue Chemistry Guidelines provide a value of >14x
as the maximum theoretical concentration factor for apples, based on
observed values for apple pomace.   

Table 3.  Residue Data from Apple Processing Study with Fluazinam.

Location

(City, State; Year)	Total Rate

(lb ai/A) 

[kg ai/ha]	PHI 

(days)	Commodity	Fluazinam residues

(ppm)

[Avg.]	Fluazinam Processing Factor	AMGT residues

(ppm)

[Avg.]	AMGT Processing Factor1

Williamson, NY; 1993	4.50

[5.04]	29	Fruit (RAC)	0.03, 0.02

[0.025]	--	0.0047, 0.0049

[0.0048]	--

	Wet pomace	0.07, 0.07

[0.07]	2.8x	0.0061, 0.0064

[0.00625]	1.3x

	Dry pomace	0.102, 0.082

[0.09]	3.6x	0.02, 0.01, <0.01

[<0.0133]	<2.8x

	Raw juice	<0.01, <0.01

[<0.01]	<0.4x	0.0025, 0.0025

[0.0025]	0.52x

	Cider	<0.01, <0.01, <0.01

[<0.01]	<0.4x	0.0038, 0.0025, 0.0038, [0.0034]	0.71x

1  NC = Not calculated; residues were below the LOQ (<0.01 ppm) in both
the RAC and the processed fraction.  Despite there being detectable
residues in dry apple pomace, as no detectable AMGT residues were
observed in the apple RAC, the actual processing factor for apple dry
pomace could also not be calculated.

2  Average of triplicate analyses.

HED’s Conclusions

AMGT is not a residue of concern for tolerance expression.  As a result,
the results of the processing study are not relevant to tolerance
enforcement.  AMGT is a residue of concern for risk assessment, however.
 As a result, the results of the processing study have the potential to
affect the dietary risk estimates determined in the human health risk
assessment.

Although the peak heights for AMGT in raw apples and the processed
commodities were small, HED believes that they are large enough compared
to the noise level in the chromatograms that they can be quantitated to
at least 2-significant figure accuracy by manually measuring peak
heights.  Because of the small peak heights and the fact that the AMGT
residue levels are below the LOQ, there is a higher degree of
uncertainty in the processing factors for AMGT than there is for the
parent.  Regardless, the results for AMGT in apple pomace are comparable
to those of the parent.  That is, in juice and cider, both compounds
were reduced, whereas in wet pomace and dry pomace, residues
concentrated slightly.  In the Dietary Exposure Evaluation Model (DEEM,
Version 7.81), the maximum theoretical concentration factor for apple
juice is only 1.3x.  This default factor is based on the assumption that
when the juice is expressed from raw apples, all of the pesticide
partitions into the juice.  This is why it’s considered to be a
maximum concentration factor.  The 860 Residue Chemistry Guidelines
provide maximum theoretical processing factors for several commodities. 
A factor is not listed for apples; however, factors are listed for
grapes, tomatoes, and citrus.  The factor for grape juice is 1.2. 
Grapes have a similar consistency to apples.  The factors for tomato
juice and citrus juice are 1.4 and 2, respectively.  Considering that
these factors are all maximum theoretical factors, HED believes that the
empirical apple juice factor of 0.52 is reasonable.  In effect, although
there is a higher degree of uncertainty in the AMGT processing factors
than in the fluazinam processing factors, HED believes that the AMGT
processing factors are sufficiently precise for risk assessment
purposes.  The factors were determined to 2-significant figure accuracy,
and they are consistent with what would be expected based on separation
of apples into components.

The processed apple commodities that impact the dietary risk estimates
are wet apple pomace and apple juice.  Wet apple pomace is an animal
feed item.  A conservative tolerance level of 5.0 ppm was used to
calculate the maximum expected dietary burden.  AMGT is not a residue of
concern in animal commodities, though.  As for apple juice, HED believes
that the processing factor was determined precisely enough that AMGT
residues can be completely accounted for in apple juice.  Again, the
processing factor that was determined is consistent with what would be
expected.   

Regardless of the precision with which the AMGT processing factors were
determined, in most of the field trials that were performed, AMGT
residues were very low.  In 38 of the 42 field trial samples (or 90% of
them), AMGT residues were at or below the LOQ.  In 3 of the remaining 4
samples the AMGT residue level was 0.02 ppm, and only one sample had a
residue level of 0.03 ppm.  The processing factor for apple juice would
have to be considerably higher in order for the AMGT residues to have a
significant impact on the dietary risk estimates.  AMGT itself is not a
residue of concern in animal commodities.  The only way it would affect
the dietary burden is if it gets metabolized to AMPA and DAPA.  However,
AMGT residues in field trials were so low that any AMPA or DAPA that
would form would be negligible.

For the reasons discussed above, HED concludes that the residue data for
fluazinam and AMGT in the submitted apple processing study are
acceptable.  Residues of fluazinam concentrated in wet pomace
(processing factor of 2.8x) and dry pomace (processing factor of 3.6x),
but did not concentrate in juice and cider (processing factor of <0.4x
each).  Residues of AMGT concentrated in wet pomace (processing factor
of 1.3x) and dry pomace (processing factor of <2.8x), but did not
concentrate in juice and cider (processing factors of 0.52x and 0.71x,
respectively).  The submitted data satisfy the deficiency cited in the
previous residue chemistry summary document and risk assessment.

The highest average field trial value for fluazinam was 1.54 ppm. 
Multiplying this value by the wet pomace processing factor of 2.8x
yields a residue value of 4.3 ppm.  Based on this value, a tolerance of
5.0 ppm needs to be established for apple, wet pomace.  AMGT is not
included in the tolerance expression for apples.  As a result, AMGT
residues do not need to be accounted for in the tolerance.  A separate
tolerance is not needed for juice based on the fact that fluazinam
residues are lower in juice than they are in the RAC.  Although ISK
Biosciences submitted processing data for dry pomace and cider, the
Agency does not establish tolerances for these commodities.

Tolerance Harmonization

There are currently no established Codex, Canadian, or Mexican maximum
residue limits (MRLs) for fluazinam on apples or carrots.  These
tolerance petitions are being evaluated as a joint review with
Canada’s Pest Management Regulatory Agency (PMRA).  The USEPA and PMRA
will be harmonizing the tolerances for the apple and carrot commodities.
 The recommended tolerance for carrot, roots is 0.70 ppm.  The
NAFTA-harmonized tolerance generator recommends a tolerance of 1.5 ppm
for apples.  The USEPA and PMRA have agreed to establish the tolerance
at 2.0 ppm.  This increase is being made because in one of the field
trials, residue values were 1.31 and 1.49 ppm and in another field
trial, residues were 1.39 and 1.67 ppm.  These field trials were
performed in arid regions (Toppenish, Washington and Eckert, Colorado,
respectively).  The next highest field trial value after these four was
0.17 ppm.  The U.S. and Canada agreed that a tolerance of 2.0 ppm might
be needed to protect farmers who grow apples in arid regions.  The
recommended tolerance for apple, wet pomace is 5.0 ppm.

The U.S. is establishing tolerances of 0.05 ppm for the fat and meat
byproducts of cattle, goats, horses, and sheep.  Canada is not
establishing tolerances for livestock commodities.

Data Deficiencies

With the exception of the multiresidue method testing data for the
regulated animal metabolites, AMPA and DAPA, ISK Biosciences has
resolved the data deficiencies associated with the apple and carrot
tolerance petitions.  There are no outstanding data deficiencies that
would preclude establishment of tolerances and conditional registration
for apple, carrot, and animal commodities.  Multiresidue method testing
data continue to be required for the regulated animal metabolites, AMPA
and DAPA.

For any future uses that involve major feed items and significantly
higher dietary burdens, more vigorous extraction steps might be required
in the method for kidney and liver, along with radiovalidation data to
demonstrate improved extractability.  

The registrant needs to submit a revised Section F in which the
recommended tolerances and correct commodity definitions listed in Table
4 are proposed.

Recommended Tolerances

Although the NAFTA-harmonized tolerance generator recommends a tolerance
of 1.5 ppm for apples, the USEPA and PMRA have agreed to establish the
tolerance at 2.0 ppm.  This increase is being made because in one of the
field trials, residue values were 1.31 and 1.49 ppm and in another field
trial, residues were 1.39 and 1.67 ppm.  These field trials were
performed in arid regions (Toppenish, Washington and Eckert, Colorado,
respectively).  The next highest field trial value after these four was
0.17 ppm.  The U.S. and Canada agreed that a tolerance of 2.0 ppm might
be needed to protect farmers who grow apples in arid regions.  Although
the NAFTA-harmonized tolerance generator recommends a tolerance of 1.5
ppm, the OECD tolerance generator recommends a tolerance of 2.0 ppm for
this same data set.  The recommended tolerance for apple, wet pomace is
5.0 ppm.  The recommended tolerance for carrot, roots is 0.70 ppm.

HED recommends in favor of the establishment of tolerances of 0.05 ppm
for the fat and meat byproducts of cattle, goats, horses, and sheep. 
Although ISK Biosciences proposed tolerances of 0.03 ppm for the meat of
cattle, goat, horse, and sheep, these tolerances are not needed based on
the results of the cattle feeding study and the calculated dietary
burden for dairy cattle.  Although ISK Biosciences proposed tolerances
of 0.03 ppm for the liver and kidney of cattle, goat, horse, and sheep,
these tolerances are not needed either, because tolerances are being
established for the meat byproducts of cattle, goat, horse, and sheep. 
HED’s Chemistry Science Advisory Council (ChemSAC) determined that
individual tolerances are not needed for liver and kidney when a
tolerance is being established for meat byproducts (ChemSAC Minutes,
7/18/2007).

α,α,α-trifluoro-2-nitro-p-toluidino)-3-chloro-5-(trifluoromethyl)
pyridine), DAPA
(3-chloro-2-(2,6-diamino-3-chloro-α,α,α.-trifluoro-p-toluidino)-5-(tr
ifluoromethyl)-pyridine), and their sulfamate conjugates.”  

The recommended tolerances and correct commodity definitions are listed
in Table 4.

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be 1.5 ppm.  The USEPA and PMRA agreed to establish the tolerance at 2.0
ppm at such time as the data deficiencies were resolved.

Apple

Apple, pomace, wet	5.0	5.0	Adequate apple processing data are available
for both fluazinam and AMGT.

Apple, wet pomace

Cattle, fat	0.03	0.05	An adequate cattle feeding study is available. 
Based on the results of the study, the maximum expected residue in meat
byproducts and fat is 0.045 ppm.

Based on the results of the feeding study, tolerances are not needed for
milk and meat of cattle, goat, horse, and sheep.

Cattle, kidney	0.03	Not required

	Cattle, liver	0.03	Not required

	Cattle, meat	0.03	Not required

	Cattle, meat byproducts	0.03	0.05

	Goat, fat	0.03	0.05

	Goat, kidney	0.03	Not required

	Goat, liver	0.03	Not required

	Goat, meat	0.03	Not required

	Goat, meat byproducts	0.03	0.05

	Horse, fat	0.03	0.05

	Horse, kidney	0.03	Not required

	Horse, liver	0.03	Not required

	Horse, meat	0.03	Not required

	Horse, meat byproducts	0.03	0.05

	Milk	0.03	Not required

	Sheep, fat	0.03	0.05

	Sheep, kidney	0.03	Not required

	Sheep, liver	0.03	Not required

	Sheep, meat	0.03	Not required

	Sheep, meat byproducts	0.03	0.05

	

References

Fluazinam.  Petitions for the Establishment of Tolerances and
Registration of New Uses on Apples, Carrots, Lettuce, and Bulb Onion
Subgroup (3-07A), and a Request for a Reduced Tolerance on Bushberry
Subgroup (13-07B).  Summary of Analytical Chemistry and Residue Data,
D360840, D. Dotson, 3/31/2010

Fluazinam	Summary of Analytical Chemistry and Residue Data	DP#:  381302

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