EPA 73S-B-?Z-0oi
        Guidance for:

Conducting Plant and Livestock
      Metabolism Studies


                        WASHINGTON, D.C. 20460
JUL 16 1992
                                             OFFICE OF
Additional Guidance for Conducting Plant and Livestock
Metabolism Studies.            _
Ed Zager, Chief             {VW~W(/'
Chemistry Branch II: Reregistration Supj
Health Effects Division  (H7509C)

          Debra Edwards,  Ph.D.,  Acting Chief
          Chemistry Branch  I:  Tolerance Support
          Health Effects  Division (H7509C)
D. Barolo, Director
Special Review and Reregistration Division (H7508W)
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
cc: P. Caulkins
                                                         Printed en Recycled Paper



 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

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

     •     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

          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

Before discussing Figure l in greater detail, the terms charac-
terization and identification of residues will be defined as

     •    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.).

      •    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

Figure 1; Strategy for Identifieation/Characterization of Extra eta t? 3
          Residues from Plant Metabolism Studies.
                        C-Rmw Agricultural Commodity

Total uc Residue
    <10 ppb
No metabolism work
     <10 ppb
      >10 ppb
         Extraction  (with  organic-and/or aqueous
                solvent systems).
Non-extractable: See Figure 2
         10-50 ppb
                    >50 ppb
           '  of
                 Activity  in
                 Both Organic
                 and  Aqueous

gtrateerv For Determining When Identification Of Metabolites Is

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,

 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


 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


actable /Bound Res
1 1



o *>
« O
O i-l

O Q)
Jj «^4


 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-

•  i

               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-

          (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.


  (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..."


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.