EPA 73S-B-?Z-0oi
PESTICIDE REREGISTRATION
REJECTION RATE ANALYSIS
RESIDUE CHEMISTRY
Follow-up
Guidance for:
Conducting Plant and Livestock
Metabolism Studies
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
JUL 16 1992
OFFICE OF
PESTCDESANOTOXC
SUBSTANCES
MEMORANDUM
SUBJECT:
FROM:
Additional Guidance for Conducting Plant and Livestock
Metabolism Studies. _
Ed Zager, Chief {VW~W(/'
Chemistry Branch II: Reregistration Supj
Health Effects Division (H7509C)
and
Debra Edwards, Ph.D., Acting Chief
Chemistry Branch I: Tolerance Support
Health Effects Division (H7509C)
!rt<
TO:
D. Barolo, Director
Special Review and Reregistration Division (H7508W)
THRU:
P. Fennjeno
Health Mf
sp, Ph.D. , Director
Division (H7509C) '
Earlier this year a Rejection Rate Analysis for Residue Chemistry
studies carried out by CBRS and CBTS and SRRD along with input from
NACA representatives, indicated the need for the Agency to provide
additional guidance on conducting plant and livestock metabolism
studies. The attached paper addresses the points determined to need
clarification. This paper was written by Dr. R. B. Perfetti, with
thoughtful and welcome comments from a number of HED senior staff.
This paper is not intended to replace existing documents but to
expand upon them. It will be utilized by the Chemistry Branches in
future reviews of metabolism studies.
We recommend that this paper be provided to the appropriate
parties.
cc: P. Caulkins
Printed en Recycled Paper
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Introduction
In the Rejection Rate Analysis conducted in the past year for
List A chemicals, certain common reasons for rejection of residue
chemistry studies were identified. The Agency then sought
industry comment on the adequacy of the guidelines. With regard
to metabolism studies industry provided numerous comments such as
the need for EPA to provide more direction with respect to bound
residues and to clearly distinguish between the terms "identifi-
cation" and "characterization". As a result it has become
evident that clearer guidance is needed on how to conduct metabo-
lism studies and that is the purpose of this memorandum.
It should be emphasized that this memo is not intended to replace
existing documents, but to expand upon them. In particular, it
clarifies the guidance in the Residue Chemistry Guidelines (1982)
and in the 7/25/89 Richard Schmitt memo entitled "Guidance on
When and How to Conduct Livestock Metabolism Studies" (published
in the 1989 FIFRA '88 Phase 3 Technical Guidance). Although
portions of the present memo emphasize plant metabolism, the
principles discussed also apply to livestock metabolism studies
and should be considered thus.
Application of Radiolabeled Pesticide
The first consideration in designing a metabolism study is
radiolabeling. The radiolabel should be positioned in the
molecule so that potentially significant toxicological moieties
can be tracked. This should involve ring labeling (preferred) or
even double labels, i.e. molecules containing two rings are
labeled in both or each ring is labeled in separate experiments.
Carbon-14 is the preferred isotope when possible. The use of
tritium as a label is strongly discouraged.
Other initial considerations include the method of application
and the application rate of radiolabeled pesticide to be used.
Since the primary purpose of a metabolism study is to identify
the chemical components of the residue, the application rate must
be high enough to result in sufficiently high radioactivity
levels to allow for characterization/identification of the
residue. A rate of at least IX (the registered application rate)
should generally be used for plant metabolism or dermal livestock
metabolism studies. In the case of oral livestock metabolism
studies, the dose should, at a minimum, approximate the maximum
anticipated dietary burden, but in no instance should the level
be less than 10 ppm in the diet (i.e. 10 jag per kg of feed) as
stated in the 7/25/89, R. Schmitt memo. However, for certain
pesticides/uses it is necessary to apply radioactive material at
exaggerated rates. The decision as to what rate to utilize is
contingent upon several factors. For example, in the case of
herbicides, phytotoxicity which may stress or even kill the
plant(s) may limit the exaggerated rate which can be used. For
all pesticides, the minimum application rate required to allow
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adequate characterization/identification of residues (up to a
maximum of 10X as discussed further below) must be utilized in
plant metabolism studies unless reasons such as phytotoxicity
prevent this. Safety concerns when using large amounts of
radioactivity must also be considered. In addition, the follow-
ing should be considered when selecting the dosing material, a
method of application and an application rate or dosage for plant
or livestock metabolism studies:
• The plant should be treated with parent only.
• -Livestock metabolism studies should reflect feeding of
one compound, usually the parent. If the plant metabo-
• lites are also found to be animal metabolites, then
additional livestock metabolism experiments which
involve dosing with plant metabolites will not general-
ly be required. However, if a plant metabolite com-
prises a major portion of the TRR on a feed item or is
not found to be an animal metabolite, additional live-
stock metabolism studies involving dosing with the
plant metabolite may be required.
• The specific activity of the labeled material should be
as high as possible. In cases where there has been
little or no characterization/identification of the
residue, in crops or animal tissues because of low
levels of activity, the Agency will make a determina-
tion as to the adequacy of efforts the Registrant has
made to maximize specific activity such that applica-
tion rates would yield characterizable/identifiable
levels of radioactivity in edible plant parts.
• In cases where low levels of radioactivity are observed
even at exagggerated rates, utilization of adjuvants or
typical inerts may enhance absorption of the active
ingredient into the plant or animal (dermal).
• Selection of specific crops and use patterns should
reflect the situation where the highest amount of
radioactivity would be expected in the edible portions
of the plant at harvest. If a pesticide has two dis-
tinct use patterns that could lead to different meta-
bolic situations (e.g., preplant soil application and a
foliar treatment), then two metabolism studies may be
required.
• If exaggerated application rates of a phytotoxic herbi-
cide are necessary to achieve sufficient radioactivity
for characterization/identification of residues, and
the required rate causes phytotoxicity in the plant,
metabolism information on the "sick" plant is prefera-
ble to having no information due to lack of sufficient
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radioactive residue.
Sampling of Plant Parts
Samples of all raw agricultural commodities (racs) as defined in
Table II of Subdivision O of the Pesticide Assessment Guidelines
should be obtained for characterization/identification of resi-
dues. In some cases, collection of samples of immature plant
parts not in Table II may be considered as an aid to facilitate
the characterization/identification of residues when low residue
levels are expected in the mature plants. Although collection of
immature plant parts not in Table II (Note that materials such as
corn forage are immature plant parts but are considered to be
raes.) is not required, it may facilitate characteriza-
tion/identification of residues in cases where the "trigger"
values (discussed below) are exceeded, but residues present
unusual difficulties in characterization/identification due to
low residue levels or the nature of the metabolites. These data
may provide adequate information to allow conclusions to be drawn
about the identity of residue in mature parts of the plant.
Registrants may also wish to use mature but inedible crop parts
(e.g., apple leaves, potato foliage) to help identify residues on
the mature rac. However, if this information is to be used in
support of the study, evidence of similar chromatographic pro-
files for mature edible and inedible plant portions is preferred.
Analytical Phase
In the analytical phase of a plant/livestock metabolism study,
the plant/animal parts to be analyzed are sampled, chopped or
homogenized, total radioactivity is determined and the samples
are extracted with a series of solvents and/or solvent systems
(including aqueous) with various polarities and other character-
istics depending on the nature of the expected residues. These
initially obtained residues are defined as •xtractable residues.
The required characterization/identification of extractable
residues is summarized in Figure 1 (This is a diagram of "trig-
ger" values described in the 7/25/89, R. Schmitt memo with some
modifications.).
Before discussing Figure l in greater detail, the terms charac-
terization and identification of residues will be defined as
follows%
• Identification refers to the exact structural determi-
nation of components of the total radioactive residue.
Typically, this is accomplished by comparing chromato-
graphic behavior to that of known standards and/or
actual spectroscopic analyses (MS, NMR, etc.).
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• Characterisation refers to the elucidation of the
general nature/characteristics of the radioactive
residue short of metabolite identification. Terms used
to characterize residues include organosoluble, water
or aqueous soluble, neutral, acidic or basic, polar,
non-polar, non-extractable, etc. Characterization may
also involve descriptions of chemical moieties known to
be present in the molecule based on conversion to a
common structure or due to reactivity with particular
reagents. The degree of characterization refers to how
close the assignment comes to structural identifica-
tion. When identification of radioactive residues is
not accomplished, the degree of characterization re-
quired for a portion of the total radioactivity will
depend on several factors including the amount of
residue present, the amount of the total radioactive
residue already identified, the importance of the crop
part as a food or feed, toxicological concern over a
class of compounds, the suspected significance of the
residue as determined by characterization already
performed and the capability of analytical methods to
detect characterized (i.e., by conversion to a common
moiety) but unidentified residues. (This radio-valida-
tion of the method would be important both for future
development of enforcement methodology and in a case
where a significant amount of radioactivity is observed
in a matrix but it consists of a large number of indi-
vidual moieties at levels below "trigger" values but
which can be converted to one or two .distinct compounds
by procedures such as oxidation or hydrolysis.).
Therefore, the terms characterization and identifica-
tion clearly have different meanings and should not be
used interchangeably.
Identification of metabolites must be established using two
different analytical techniques except when (a) unambiguous
identification is made using a spectroscopic method such as
GC/MS, or (b) the metabolite is determined to be of minimal
importance due to its low absolute level (<0.05 ppm) or percent-
age of the total radioactive residue (<10% of TRR). In the case
of (b), identification by one technique such as co-elution with
standards will be acceptable. These trigger values are meant as
rough guidance and may not apply to situations where a metabolite
is suspected to be of particular toxicological concern, or where
<10% of the TRR represents a high absolute residue level. In
general, the Agency will not consider chromatographic techniques
utilizing the same stationary phase with two different solvent
systems to be adequate two-method verification of metabolite
identity.
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Figure 1; Strategy for Identifieation/Characterization of Extra eta t? 3
Residues from Plant Metabolism Studies.
u
C-Rmw Agricultural Commodity
combustion
Total uc Residue
<10 ppb
No metabolism work
<10 ppb
No
metabolism
work
>10 ppb
Extraction (with organic-and/or aqueous
solvent systems).
u
Total
C-Extractables
Non-extractable: See Figure 2
10-50 ppb
partition
>50 ppb
Chromatographic
Analysis
' of
Organosolubles
Characterize
and
Identify
Activity in
Both Organic
and Aqueous
Phases
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gtrateerv For Determining When Identification Of Metabolites Is
fle.eded.
The strategy illustrated in Figure 1 for «xtractable polar and
non-polar residues was developed by Ciba-Geigy and applied
primarily to animal metabolism studies in the Schmitt memo of
7/25/89. The radioactivity trigger values shown in Figure l
reflect the characterization/identification required for each
rac. If total activity in a crop/animal part is eO.Ol ppro (10
PPk) or less, no differentiation of the radioactivity would be
required. For activity greater than BO.01 ppro, the sample should
be extracted with solvents and/or solvent systems (including
aqueous) of various polarities. The levels of extractable and
non-extractable activity should then be quantitated to determine
the degree of characterization that is needed. If the extract-
able activity represents «0.01 ppm or less, it need not be
examined further. For extractable activity of «0.01-0.05 ppm,
the partitioning behavior between aqueous and organic solvents
should be determined followed by chromatographic (TLC, HPLC)
analysis of the organosoluble activity. The chromatographic •
behavior of this activity can be compared to that of the parent
pesticide and likely metabolites (characterization and/or identi-
fication) . When the extractable activity exceeds «0.05 ppro,
complete characterization and identification should be attempted
for both organic and aqueous activity. It is important that the
components of the aqueous soluble portions of the radioactivity
be identified since they may contain toxic compounds. Given the
present state of HPLC columns and detectors, this type of re-
search is much simpler than previously. For the aqueous soluble
portion of the activity however, the "trigger" values for charac-
terization and identification would be levels down to 0.05 ppm or
10% of the TRR whichever is greater. The exception for this
would, of course, be toxicology concerns over potential residues
which might occur at lower levels. Identities of metabolites
should be confirmed with a second technique, spectroscopic if
possible, as discussed above.
The term "complete characterization and identification" for
extractable residues above 0.05 ppm does not necessarily mean
that individual components at this level need to be identified.
Low level (in terms of both pom and % of total residue! individu-
al residues do not typically need to be identified if the major
components of the residue have been identified. For example, if
the total activity in a crop part is 3 ppm and 75% of that has
been firmly identified, it is unlikely that identification of a
series of individual residues in the 0.05-0.1 ppm range would be
required. On the other hand, extensive efforts toward identifi-
cation of 0.05-0.1 ppm residues would be expected when the total
activity is only 0.3.ppm.
The radioactivity levels shown in Figure 1 apply regardless of
the application rate used in plant metabolism studies. However,
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this is not meant to discourage use of exaggerated application
rates necessary to provide sufficient radioactivity for adequate
delineation of the plant metabolism. If application rates are
used which are insufficient to provide adequate radioactivity for
characterization/identification of residues, additional studies
may be required at increased application rates up to the point of
unacceptable plant phytotoxicity. The maximum exaggerated rate
which will be required for a plant metabolism study is 10X (The
use of highly exaggerated doses in livestock metabolism studies
is discussed in the 7/25/89 R. Schmitt memo for situations where
low residues are present on feed items.). It is important to
note that plant metabolism studies with little or no identifica-
tion of residues will not normally be acceptable to support new
uses which reflect different kinds of treatments, especially
modes of applications that result in higher residues.
One recent technique which, depending on the circumstances, may
be appropriate to utilize as an alternate extraction procedure
prior to the techniques suggested in the next section is super-
critical fluid extraction. .
Release of Nonextraetable/Bound Residues
The remainder of this discussion will pertain to non-extract-
able/bound radioactive residues and will provide guidance on what
steps need to be taken to provide enough information to allow the
Agency to draw conclusions as to the terminal residue of concern
in plants/animals.
There are three situations in which radioactive residues are
observed to be "non-extractable" in plants/animals.
(1)~ Incorporation into bioroolecules (i.e., amino acids, sugars,
etc.) which occurs when the test compound is degraded into
small (usually l or 2) carbon units which enter the carbon
pool, and which the plant uses to build new compounds.
(2) Chemical reaction with appropriate moieties in biomolecules
to form bound residues which can be released via other
chemical reactions (e.g., enzymatic or acid/base hydroly-
sis) .
(3) Physical encapsulation or integration of radioactive resi-
dues into plant/animal matrices (such as cellulose and
lignin for plants). Release of residues in this situation
may require solubilization of the tissue, usually by drastic
treatment with base, although use of surfactants may allow
the radioactive residue to be released under less severe
conditions.
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The following general "road map" for dealing with non-extract-
able/bound residues is intended to provide clarification of
Agency policy as well as more specific guidance regarding charac-
terization/identification of these residues.
The extracted solid plant/animal material from Figure 1 should be
assayed and, if radioactivity is present down to the "trigger"
values of 0.05 ppm or 10% of the TRR whichever is greater,
release of the activity should be attempted (See Figure 2). It
is emphasized that, if toxicology expresses concerns over poten-
tial residues at lower levels, the "trigger" values will not
necessarily apply. Treatments may be performed on either sub-
samples or sequentially. The types of treatments include dilute
acid and base at ambient temperatures (Note that these procedures
should be employed initially for both metabolism and method
development considerations.), surfactants, enzymes and 6N acid
and/or ION base with reflux. It should be kept in mind that the
milder procedures provide more accurate assignments of metabolite
structures released, i.e., acid/base reflux would probably
release moieties as their final hydrolysis products which could
have only a minor relationship to the conjugated form of the
radioactivity. An ambient temperature acid treatment followed
by ambient temperature base treatment will provide a mild hydro-
lysis of conjugated moieties, and again possibly release any
biomolecules containing incorporated radioactivity. The use of
surfactants may release physically encapsulated or membrane bound
residues. Because membrane and/or cell wall disruption may
improve substrate accessibility to the enzyme, a sonication step
should be employed followed by a carefully chosen enzymatic
battery (Note: In each case the activity of each enzyme utilized
should be confirmed using standard substrates and controls.
These experiments should be documented.). These steps could
release chemically-bound residues including any biomolecules
containing incorporated radioactivity. The final release steps
would involve reflux acid and base hydrolysis which will likely
solubilize the plant part/ tissue. Radioactivity released at
this time would probably reflect amino acids, sugars and encapsu-
lated or conjugated compounds which may or may not have any
relationship to the original bound/encapsulated structures.
However, this step does provide evidence that residues of the
pesticide can be released, and may provide data on incorporated
radioactivity and limited information about the nature of the
metabolites (See discussion above.). In all cases, samples,
homogenates and extracts should be buffered and maintained at low
temperatures except during hydrolytic steps in order to reduce
degradation/artifact formation (See the discussion below regards-
ing storage stability in metabolism studies.).
Figure 2 provides a visual description of the steps discussed
above.
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Comments en Figures l and 2
1} At each step in Figure 2, the radioactivity of the released
residues should be quantitated; and if the "trigger" values
shown in Figure l for extractable residues are met, the
activity should again be partitioned against various sol-
vents/solvent systems and characterized and/or identified as
required. With respect to characterization, it should be
emphasized that the chromat©graphic behavior of the released
activity (including water solubles) should be compared to
that of the parent and likely metabolites which are close in
structure to the parent. This will indicate whether the
released activity is chemically different from the parent
molecule. If the remaining unextracted activity after a
given procedure is <0.05 ppm or <10% of the TRR further
attempted release of activity is not necessary.
2} The trigger values shown in Figure l are meant to negate the
need for characterization/identification of metabolites
present at very low and insignificant levels. However, in
many cases, a potentially important metabolite may partition
into multiple fractions because of solubility characteris-
tics, and/or because it is present in both free and conju-
gated forms. In order for the trigger values to apply,
particularly in cases where the TRR is distributed among
numerous fractions, it must be demonstrated (e.g. by HPLC
analysis of each fraction) that no single metabolite is
distributed among the various fractions in such amounts so
that the combined level (sum) of this component significant-
ly exceeds the trigger value.
3) Identification of specific radiolabeled amino acids, sugars,
phenolic compounds, nucleotides, etc, may alleviate the need
for further characterization of bound residues in many
instances, since this usually means that the pesticide has
been degraded into small carbon units which have entered the
carbon pool. This conclusion does not, however, apply to
tritium labeled compounds, or to pesticides in which the UC
label is incorporated at a labile site in the pesticide
molecule. This conclusion would also not apply in cases
where a single released metabolite, which comprises a sig-
nificant portion of the total radioactive residue (>lb% of
the TRR or >0.05 ppm), has not been identified.
4) When a fraction such as lignin, cellulose, or protein con-
tains radioactivity, the radioactivity does not necessarily
consist of radioactive amino acids or sugars. The radioac-
tivity may consist of biological macromolecules having
radioactive portions of the pesticide either chemically
conjugated onto them, or physically encapsulated within
them. This is an important distinction from having the
nacromolecules constructed from low molecular weight radiol-
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• i
11
abeled building blocks. The Registrant is responsible for
providing such determinations in a scientifically support-
able manner. The Agency will make an evaluation of the data
and, if not already provided, require definitive information
regarding which of the three conditions exist (i.e. incorpo-
ration, conjugation, or encapsulation).
Additional General Comments
The pathway described above should be viewed as a broad outline
of the type of information needed to determine that a
plant/animal metabolism study is acceptable. Different proce-
dures and methodologies may be appropriate in a given circum-
stance. The basic concepts regarding "trigger" values for
identification of radioactivity, methodologies required for
characterization/identification of radioactivity, and steps which
should be taken to assure adequate release of "non-extract-
able/bound" residues must be observed to assure that the submit-
ted study is adequate.
The following additional comments should be considered in carry-
ing out a plant/animal metabolism study.
(1) For a case where bound residues are present at levels down
to 0.05 ppm or more than 10% of the TRR whichever is great-
er, the Agency will require workup and attempted identifica-
tion.
(2) All unsuccessful attempts at releasing unextracted activity
and characterization and/or identification of the TRR should
be documented and submitted.•
(3) The Agency will not accept situations where the exaggeration
rate is used to calculate "trigger values". For example, if
a crop is treated with radiolabeled material at an exagger-
ated rate (e.g., 5X), the resulting radioactivity levels
should not be divided by the degree of exaggeration (e.g.,
5) to arrive at "trigger values".
However, the following example should be considered. Assume
the plant is treated at a 5X application rate resulting in
0.1 ppm radioactivity in an edible plant part. If the
registrant identifies one component as 0.05 ppm of the TRR,
and only characterizes 2 other components at 0.02 and 0.03
ppro, then consideration of the terminal residue to be regu-
lated would take into account the 5X treatment with respect
to the 0.02 ppm and 0.03 ppm materials.
(4) Consultation with the Agency prior to initiation and during
the metabolism study is appropriate and encouraged.
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(5) The discussion above is intended to provide guidance on how
a plant/animal metabolism study is to be conducted. Howev-
er, plant/animal metabolism studies are complex and defy a
review which follows strict adherence to established crite-
ria. The scientific techniques used to study xenobiotic
metabolism and conjugate formation, isolation of
plant/animal macromolecules and procedures for generating
monomers/oligomers are constantly advancing. It is, there-
fore, the responsibility of the Registrant to utilize state-
of-the-art techniques and provide citations of such tech-
niques when they are used. Flexibility in review is neces-
sary in determining whether a study is adequate for the
intended purpose of identifying the nature of the terminal
residue to be regulated. Plant/animal metabolism studies
will always be examined on a case-by-case basis, and will
frequently require scientific judgement to make sound con-
clusions and recommendations.
(6) The ideal result of a metabolism study is identification of
90% of the total radioactive residue in each raw agricultur-
al commodity. However, the Agency recognizes in many cases
this is not possible, especially when low total levels of
residue are present and/or when the pesticide is extensively
metabolized to numerous low level components. In the latter
case it is important for the registrant to demonstrate
clearly that numerous components are present and, as dis-
cussed above, attempt to characterize these residues by
conversion to a common moiety where feasible.
Storage Stability
The issue of storage stability in metabolism studies has been
discussed in earlier documents. For example, the Standard
Evaluation Procedure (SEP) on "Metabolism in Food Animals:
Qualitative Nature of the Residue" notes that "storage at freezer
temperatures for a month or less is acceptable". The SEP on
plant metabolism does not specify an acceptable storage period,
but reminds reviewers to make a determination as to whether
sample integrity was maintained during collection, preparation.
and storage.
In light of the difficulty of spiking samples before the identity
of the residue is known and the length of time needed for metabo-
lism studies, the present Agency position is that storage stabil-
ity data should not normally be required for samples analyzed
within 4-6 months of collection, provided evidence is given that
attempts were made to limit degradation of residues by appropri-
ate storage of matrices and extracts during the analytical
portion of the study, in other words, as stated in the SEP on
animal metabolism, "The reviewer should be convinced that storage
conditions have not invalidated the Registrant's results..."
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In those cases where a metabolism study can not be completed
within 4-6 months of sample collection, evidence should be
provided that the identity of residues did not change during the
period between collection and final analysis. This can be done
by analyses of representative substrates early in the study and
at its completion. Such analyses should show that the basic
profile of radiolabeled residues has not changed during that
time. If changes are observed (e.g., disappearance of a particu-
lar HPLC peak or TLC spot), additional analyses or another
metabolism study with a shorter collection to analysis interval
may be required.
Further clarification of storage stability requirements in
general will be provided in the near future in another paper.
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