United States       Prevention, Pesticides     EPA712-C-96-182
          Environmental Protection    and Toxic Substances     August 1996
          Agency         (7101)
&EPA    Residue Chemistry
          Test Guidelines
          OPPTS 860.1480
          Meat/Milk/Poultry/Eggs

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                           INTRODUCTION
     This guideline is one  of a  series  of test  guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental  Protection Agency for use  in the testing of
pesticides and toxic substances, and the  development of test data that must
be submitted to the Agency  for review under Federal regulations.

     The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through  a process of harmonization that
blended the testing  guidance  and requirements that  existed in the Office
of Pollution Prevention and  Toxics  (OPPT) and appeared in Title  40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR),  the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and the guidelines pub-
lished by the Organization  for Economic Cooperation and Development
(OECD).

     The purpose of harmonizing these  guidelines  into a single set of
OPPTS guidelines is to minimize  variations among the testing procedures
that must be performed to meet the data  requirements of the U. S. Environ-
mental Protection Agency  under  the Toxic  Substances  Control Act  (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin   Board.   By  modem  dial   202-512-1387,  telnet   and   ftp:
fedbbs.access.gpo.gov    (IP     162.140.64.19),    internet:     http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks  or paper copies.
This guideline is also available electronically in ASCII and PDF (portable
document format) from the EPA  Public Access Gopher  (gopher.epa.gov)
under the heading "Environmental Test  Methods and Guidelines."

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OPPTS 860.1480   Meat/milk/poultry/eggs.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing  requirements   of both  the  Federal  Insecticide,  Fungicide,   and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et  seq.} and the Federal Food,
Drug, and Cosmetic Act (21 U.S.C. 301, et seq.}.

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is  OPP 171-4 Results  of Tests  on the
Amount of Residue Remaining, Including A Description of the Analytical
Methods Used (Pesticide Assessment Guidelines, Subdivision  O: Residue
Chemistry, EPA Report 540/09-82-023, October 1982) and Addendum No.
8 on Data Reporting, Residues in Meat, Milk,  Poultry, and Eggs: Livestock
Feeding Studies (Pesticide Assessment Guidelines, Subdivision O: Residue
Chemistry, EPA Report 540/09-89-010, 1989). This guideline should be
used in conjunction with OPPTS 860.1000, Background.

     (b) Purpose. Whenever pesticide residues  are detected in feed items,
data on the transfer of residues to  meat, milk, poultry, and eggs  are re-
quired.  These studies are  also required if a pesticide is to be applied di-
rectly to animals.  Data from these studies are used to determine which
components of the total toxic residue (TTR)  are present and at what con-
centrations secondary residues  could appear in meat,  milk, poultry,  and
eggs in order to set appropriate tolerances.

     (c) Data requirements. Data  must be  submitted to show the level
of residues that will result in ruminant meat (muscle), meat byproducts
(liver,  kidney) and fat, poultry (muscle, fat,  liver),  eggs, or milk. These
data are needed whenever a pesticide is to be applied directly to livestock
or residues occur  on a livestock feed. Based  upon the residue level in
the feed item and the residue resulting in meat, milk, poultry, and  eggs
in these  feeding  studies,  the  use  must  be classified  as specified in
40 CFR 180.6(a).  Category 1 of § 180.6(a)  applies to cases  where it is
shown that residues will occur in animal products, Category 2 applies if
there is a reasonable expectation of residues  in animal products, and Cat-
egory 3  applies if there is no  reasonable  expectation of residues. Since
tolerances for residues in animal products are required when the use is
judged to be Category 1 or 2,  the  animal  feeding  studies must not only
show whether residues transfer, but  may  also need to serve  as a basis
for setting appropriate tolerance levels for the  animal products.

     (d) Conduct of studies—(1) Feeding  studies, (i)  In most cases only
the parent pesticide should be  fed to livestock. However, in  those cases
where the parent compound comprises only a minor proportion of the resi-
due of concern, it may be acceptable to feed a mixture  of parent and plant
metabolites. In cases where a unique plant metabolite exists (i.e. one  that
is not formed in livestock), a separate feeding study may be required dos-
ing with that metabolite.  Registrants considering dosing  with a mixture
or a unique plant metabolite should contact the Agency prior to initiation

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of such a study. The feeding study should include the level of intake ex-
pected (Ix) (see next paragraph), plus two exaggerated levels  of 3x and
lOx. The Ix level should represent the worst case estimate of the potential
livestock exposure based on the assumption of all components of the feed
having tolerance level residues. The exaggerated levels are especially im-
portant  in judging a  Category  3 use,  as well as to cover possible  future
tolerances for the pesticide on  additional feed items and  allow  estimation
of whether residue levels in tissues vary linearly with the level in the feed.
The dosage levels should be  expressed in terms of concentration  (in parts
per million) in the total  ration (dry weight basis for ruminant  studies),
so that  the Agency can relate  the dosage to that expected from  the pro-
posed use. It is also desirable to express the feeding level in terms of
milligrams per kilogram body weight.

    (ii) In selecting the dosage levels  based on total rations, the petitioner
should take into account the proportion in the diet of the feed item bearing
the residue and, in the case of ruminants, the percentage of dry matter
in the  feed.  Table  1  of OPPTS 860.1000 should  be used as a  guide in
determining the proportion of the diet of the various  food items. The cor-
rection  for percentage of dry matter is explained in  more detail  in para-
graph (e) of this guideline. For example, dried citrus  pulp (91 percent dry
matter content) may in  some circumstances comprise up  to 20  percent of
the total ration (dry weight basis) of dairy cattle.  If a tolerance of 5 ppm
of a given  pesticide were  proposed on dried citrus pulp, the total diet (dry
weight basis) should be fortified at the  1.10 ppm level, i.e.:

                       (5.0 ppm/0.91)x 0.20

to reflect the expected level of intake  (Ix).  If other feed items with toler-
ances could also be fed in combination with citrus pulp, the contribution
from these  feed items should  also be added in.  As  noted, two  dosages
at exaggerated levels are also  required, preferably threefold and tenfold
or higher where not precluded by toxicity of the pesticide.

    (iii) Separate feeding studies are  required for a ruminant and poultry
whenever residues occur on the feeds  of these classes of livestock,  or di-
rect animal treatment is proposed. The species of choice for these feeding
studies  are the cow and  chicken. In most cases the  results of the cattle
feeding study will be used to establish tolerances on goats, hogs, horses,
and sheep. Data will not be translated  from other meat animals to poultry.
However, within the  poultry group, data on chickens will usually be ac-
cepted in lieu of data on turkeys. Data on residues  in milk from  dairy
cows will usually apply as well to dairy goats. Data  on  tissue storage in
rats,  dogs,  or other small animals used in toxicology studies will not be
accepted in lieu of residue data  on livestock.

    (iv) In addition to  establishing  a baseline  or blank in a predosing
period,  control animals  should be carried through the experiment with

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treated animals. This is highly desirable, since values for control animals
have been observed to change during feeding studies. The number of ani-
mals carried at each  treatment level, and as controls, will  vary with the
circumstances but as a general rule a minimum of three animals should
comprise a  group in  a cattle feeding study.  For chicken feeding studies,
a minimum of 10 birds per group should be used. It is often advisable
to have additional animals on test that  can be used to  determine the rate
of decline of residues on the cessation of dosing, so that if residues above
tolerance are found,  data on the time necessary for residues to fall to the
tolerance level are available.

     (v) Animals should be dosed daily  for a minimum of 28 days or until
residues plateau  in milk or  eggs  if they have not done so in 28  days.

     (vi) If  a feed-through formulation  is specifically designed to change
absorption  characteristics within the digestive system, this formulation
should be employed in the feeding study.

     (2) Direct animal treatment, (i)  When a pesticide is proposed for
direct use on food animals, data are required to show the extent of residues
incurred by the use.  The experimental treatment should reflect as closely
as possible the conditions under which the pesticide will be  used commer-
cially.  Control animals should be carried along with treated animals. Fac-
tors such as whether  sheep passing through a dip tank were freshly  shorn
or unshorn  should be considered.  Generally, separate studies should be
carried out for each species of livestock to be  treated.

     (ii) When a pesticide may be applied in more than one type of formu-
lation or by more than one  mode of treatment, separate studies reflecting
the usage or combination of usages proposed are required. However, data
from dips or high pressure wetting sprays on cattle may be accepted in
lieu  of data from dust treatments  but  not vice versa. When the use  of
devices which permit unlimited access (e.g. backrubbers) is proposed, the
experiment  should be designed to  assure the maximum  exposure  of the
animal to  the pesticide. Data reflecting exaggerated treatments  are  desir-
able.

     (iii) If livestock are exposed to the pesticide  both in  feed and as  a
direct treatment, the magnitude of the residue study  should reflect the level
of residues to be  expected from the combined exposure scenarios. If sepa-
rate feeding and  direct treatment studies have been conducted, it is nor-
mally acceptable to add the residues from these studies to  determine the
appropriate  tolerances. However, this may result in higher than necessary
tolerances for animal commodities.

     (3) Agricultural premise use studies. When the use of pesticides
in agricultural buildings is such that restrictions cannot preclude the possi-
bility of residues in meat, milk, poultry or  eggs,  residue  studies should
be carried out reflecting the maximum conditions  of exposure. Separate

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studies  are  required for ruminants  (catties),  nonruminants (swine),  and
poultry  (chickens). The studies should reflect  all possible residue transfer
routes such as:

     (i)  Direct  absorption (dermal  or inhalation)  from sprays,  mists, or
fogs.

     (ii) Direct consumption (e.g. by the animal licking  surfaces treated
with sugar base baits, pick up of bait granules by poultry, or contamination
of feed, feed troughs, or water troughs).

     (iii) Direct contamination of milk from deposition on milking equip-
ment, treatment of milk rooms, etc.

     (4) Meat, milk, poultry, and egg sampling, (i) Milk and egg samples
should be taken twice  daily. Eggs from birds  within a dosage group may
be pooled if necessary so that adequate sample weight  is available  for
analysis and retained samples. Milk from animals within a dosage group
should not be pooled in order that data for individual animals are  available.
However, compositing the a.m. and p.m. milk from each  individual cow
in the ratio  of production is acceptable. Enough of the pooled daily milk
and egg samples should be  analyzed (preferably  at least twice weekly)
to allow for a  determination of trends in  storage of residues with time.
Three unique samples  of milk and  eggs should be analyzed at each time
point for each feeding level. Petitioners are advised to analyze the samples
from the highest feeding level first. If no quantifiable residues are observed
in any of these samples, samples from the lower feeding levels do not
need to be analyzed.

     (ii) If detectable residues occur in whole milk at any dosing level,
analyses of  a few samples of milk fat is advisable at some point to show
how residues partition  into that commodity. This information can be used
to determine if a specific tolerance value should be specified for milk fat
and to calculate dietary risk more accurately.

     (iii) Analysis of eggs should be conducted on the  egg  yolk and white
combined in one sample. The yolks  and eggs  may also be analyzed sepa-
rately provided the weights  of each are known so that the residue can
be calculated on a whole egg basis.

     (iv) Animals should be slaughtered within 24 hours of the last dosing
and tissue samples taken and frozen as  soon as possible.  Tissue residue
level results from animals slaughtered long after cessation of dosing cannot
be used in estimating tolerances, and thus if  only such samples are ana-
lyzed, the feeding study will have  to be  repeated.  The  commodities to
be analyzed in a feeding study include the following tissues that are used
as human food: Muscle,  fat, liver, and,  in the case of  cattle only, kidney.
For dermal  uses on  poultry or swine, skin should also  be analyzed.  As
noted above for milk  and eggs,  three  unique samples of edible tissues

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should be analyzed at each dose level to show the variability of residues
among different animals. In the case of cattle, this usually means one sam-
ple per animal as three cows are generally dosed at each level. For poultry,
tissue samples  from three to  four  birds may be composited  to generate
the three unique samples for each dosage group. If no quantifiable residues
of a pesticide are observed in  a tissue at the highest dose level, no further
analyses of that tissue at lower feeding levels are required.

    (v) Dermal treatment of livestock. Animals  should be sacrificed with-
in the preslaughter interval (PSI) prescribed on the product label. However,
PSIs longer than 3 days  are not considered to be practical by  the Agency
in most cases.  Since  it has been observed that residues may not peak in
tissues until a week or so after application, additional data reflecting longer
PSIs should be obtained to establish the maximum residue levels for toler-
ances.

    (vi) The components  of  the residue to be  analyzed in tissues, milk
and eggs should be those found to constitute the TTR in the animal prod-
ucts as determined in the livestock  metabolism study described in  OPPTS
860.1360. The analytical method  should be  described in detail  or ref-
erenced. Fortified samples  should be run concurrently with those from the
feeding study  to validate the  method. The required limit  of quantitation
for the  animal  products  will be related  to the toxicity of the compound
but should generally be  on the order of 0.01-0.05 ppm or less. Require-
ments for analytical methods are spelled  out in detail in OPPTS 860.1340.

    (5) Storage stability  data. Appropriate storage stability data are re-
quired on representative livestock commodities as outlined in  OPPTS
guideline 860.1380.

    (6) Waiver of livestock feeding studies. When the level of residues
in feed items is found to be low, registrants should refer to OPPTS guide-
line 860.1300 for a possible waiver  of conventional livestock feeding stud-
ies. In some cases, livestock metabolism studies will indicate that  feeding
studies and meat and milk tolerances are not necessary.

    (e) Guidance procedure  for calculating livestock dietary exposure.
(1) While feed percentages listed for ruminants (i.e. beef and dairy cattle)
in Table 1 of OPPTS 860.1000  and  in  the Harris Guide  (see paragraph
(g)(6) of this guideline) and the ANI  Report (see paragraph (g)(7) of this
guideline) are  listed on a dry matter basis, tolerances for these feed items
are established on  an as-fed basis. Hence, the  correct calculation of rumi-
nant dietary burden  includes  the conversion of the feed to a dry-matter
basis in the diet.

    (2) Percentages of the diet for poultry and swine  feeds in the Harris
Guide  are on  a dry matter basis. Poultry and swine listings  Table 1  of
OPPTS 860.1000  and the ANI Report are on  an  as-fed basis but since
almost all feeds for poultry and swine are in the dry category, the dietary

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burden  calculation for poultry and swine  using Table  1  of OPPTS
860.1000  does not require conversion of the  feed to a dry-matter basis.

     (3) The dietary burden calculation must also handle the situation that
arises when the feed items on which there are tolerances for a given chemi-
cal do not comprise a complete diet for the animal. For example, pesticide
A has tolerances on alfalfa forage (50 percent of beef cattle diet)  and  al-
falfa hay  (25  percent), but on no  other feed items. In this case, there is
no information on the feed items which would be used to round  out the
animal's diet.  If those additional feed items  are  wet, the residues on  an
as-fed basis will be diluted more than they  would be if the feed items
were dry.  Errors in the estimate of the dietary burden to the animal could
result.

     (4) These problems can be avoided if the burden is calculated in terms
of the weight  (as opposed to  concentration) of the pesticide consumed  by
the animal, and that  amount compared with  a standard amount of feed
consumed by  the animal.  The following equation, Equation A,  has been
derived  for such calculations. For ruminants, where feed percentages are
expressed on a dry matter basis, equation  A should be used to calculate
the total dietary burden.
        f dietary    \           (%diet [DM])            ( mg}
               [DM] \(ppm) = \	¥L x (tolerance).  —   (^)
        .burden     I             (%DM)              { kg }
        v         y        /     v     / /              V ° /
where

     (dietary burden [DM]) = estimation of total exposure of a pesticide
through feeds  on a dry-matter basis, expressed in ppm (mg pesticide per
kg feed)

     (% diet [DM])i = percentage in the animal diet of commodity / ex-
pressed on a dry-matter basis

     (%[DM])i = dry-matter percentage in feed commodity /

     (tolerance)i =  proposed or  existing  tolerance  expressed in mg/kg
(ppm)

     (5) The burden thus calculated is on a dry-matter basis, and ruminant
feeding and metabolism studies submitted to the Agency  must have their
feeding levels calculated on a dry-matter basis.  For  feeding  studies in
which the pesticide has been introduced via capsule, the petitioner should
report the feed items and intake of each  animal  so  that dietary burden
can be calculated on a dry-matter basis.

     (6) The feed percentages for poultry and swine in Table 1  of OPPTS
860.1000  are  on an as-fed basis. Therefore,  no  correction will have to
be made for percent moisture; the dietary burden for poultry  and swine

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 will be simply calculated by Equation B as follows and the dietary burden
 in this case will be on an as-fed basis:
              (dietary}
                     J(ppm) = N  (%diet)i x {tolerance^ 	
                                                 \ ke
             I burden
             V     r         j                    \ 
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     (B) Without correcting for moisture, the same 2.1 ppm burden would
be calculated using Equation B. However, correcting for moisture, the bur-
den calculated by Equation A would be:

          (0.80)               (.20)
          	x (0.1 ppm} -\	x (10.0 ppm} = 8.1 ppm
          (0.88)              (0.25)

     (C) Thus, using only Equation B,  the dietary burden for beef cattle
would be seriously underestimated.

     (iii) Scenario 3 exists when not all feed items in the selected diet
have established tolerances.

     (A) Similar underestimation of an animal's dietary burden can occur
if the available  feed items do not comprise a complete diet.  Using the
example of Pesticide A for beef cattle,
alfalfa forage
alfalfa hay
50 percent of diet
25 percent of diet
35 percent DM
89 percent DM
2.0 ppm tolerance
8.0 ppm tolerance
     (B) The dietary burden, if calculated using Equation B without con-
version to a dry matter basis, is:

           [(0.50) x (2.0 ppm)] + [(0.25) x (8.0 ppm)] = 3.0 ppm

     (C) Using Equation A,  the  dietary burden is calculated  as follows:
            (0.50)              (0.25)
            	x (2.0 ppm) -\	x (8.0 ppm) = 5.1 ppm
            (0.35)              (0.89)

     (D) This latter number represents a worst-case scenario; thus, the bur-
den cannot be more than 5.1 ppm.

     (f) Data reporting format. The following describes a suggested order
and format for a report item by item. Other formats are  acceptable pro-
vided that the information described in this paragraph is included.

     (1) Cover page. Title page  and additional documentation requirements
(i.e., requirements for data submission and statement of data confidentiality
claims) if relevant to the study report, should precede the content of the
study formatted below. These  current requirements are described in PR
Notice 86-5 published by the Office of Pesticide Programs on July 29,
1986 (see reference in paragraph (g)(5).

     (2) Table of contents. The table of contents should provide page num-
bers on which are found the essential elements of the study.

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     (3) Introduction and summary, (i) This section should provide back-
ground and historical perspective for the study. It should include:
     (A) Registration history.
     (B) Proposed use of the pesticide.
     (C) Purpose of the study.
     (D) Summary of the results.
     (ii) The summary of the experiment should include:
     (A) A discussion of any unusual problems encountered and how these
were resolved.
     (B) A discussion of any deviation from the experiment's protocol and
the effect this may have had on the results.
     (C) A brief description of the study's  findings addressing such ques-
tions  as: Residues transfer, preferential accumulation in certain organs,
highest  residues,  and occurrence  of a plateau in residue concentration in
milk or  eggs.
     (iii) A comparison of the results to those of the  animal metabolism
studies would also be useful.  Such comparisons can also be presented in
a separate overview document.
     (4) Materials—(i) Test substance. (A) The pesticidal active ingredient
and/or its metabolites which are fed should be identified by:
     (7) Chemical name.
     (2) Common or trivial name (American National Standard Institute
(ANSI), British Standards Institution (BSI), International Standards Orga-
nization (ISO)).
     (3) Company developmental name/number.
     (4) Chemical Abstracts Service (CAS) number.
     (B) The source and purity of each compound  should be specified.
     (C) Chemical structure graphics of these compounds are also desired.
     (D) Rationale for feeding  compounds other than parent pesticide.
     (ii) Test facilities.  The animal housing should be described. Factors
to consider include: Sizes of enclosures, individual versus group housing,
food and water containers, temperature, lighting, and waste handling.
     (iii) Test animals.  (A) A description  of the test  animals should in-
clude: Species, breed, age, weight, and health status.

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     (B) The number of animals per feeding level must be specified.

     (C) The mode of identification should be noted (e.g. ear tags).

     (D)  Body weights and  egg/milk  production  should be reported for
both the acclimation and dosing periods.

     (E) Any  health problems, abnormal behavior, or unusual treatment
of animals should be reported and the  effect of these on  study results dis-
cussed.

     (iv) Feed. (A)  Diet of the animals during acclimation and the dosing
period should be described as to both:

     (7) The types  of feed (e.g., corn grain, layers mash, alfalfa pellets)
and liquids.

     (2) The quantities provided (i.e., specific amounts or ad libitum).

     (B) Feed  consumption (dry weight for ruminants) should be reported
on an individual or treatment  group basis throughout the study.

     (5) Methods—(i) Dosing. (A) The preparation of the  dose  should be
described  (mixing with feed  or concentrate ration, gelatin capsule, bolus,
etc.). The level of the test material in the total diet in parts per million
(mg/kg  feed)   (dry weight  basis  for  ruminants) is needed.  The  rec-
ommended doses are  Ix,  3x  and lOx the anticipated dietary intakes from
proposed usages of the pesticide. The calculation of these dietary burdens
based on Table 1 of OPPTS guideline 860.1000 and the procedure  in para-
graph (e) of this guideline should be explained. The petitioner should con-
sider possible  future uses of the  pesticide when determining the  dosages
to be fed.  Dosing schemes other than Ix, 3x, and  lOx are  acceptable pro-
vided a satisfactory rationale is given.

     (B) The date of dose preparation should be specified along with the
storage conditions prior to its  administration.

     (C) A brief description  of the method used to analyze spiked  feeds
and  the results of such  analyses should  be presented.  These analyses
should demonstrate  that the pesticide was stable in the feed or dosing ma-
terial throughout its entire storage period.

     (D) The frequency of dosing should be reported if the test material
is not incorporated into the total diet or feed.

     (E) The dates  of the initial  and  final  doses  (or the  total length of
the dosing period) should be indicated.

     (ii) Sample collection. (A) The collection  of milk  and eggs should
be described with any  differences from normal practice explained.  Any
compositing or pooling of samples should to be noted, although milk from

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animals within a dosage group should not be pooled. Compositing the a.m.
and p.m. milk from each individual cow in the ratio of production is ac-
ceptable.

     (B) The collection  dates for those samples which are analyzed for
the residue of concern should be reported.

     (C) The mode of sacrifice  and the time interval in hours between
the latter and the administration  of the last dose should be specified. An
explanation of intervals  longer than 24 hours should be presented along
with a discussion of their effect on residues.

     (D) The tissues taken after  sacrifice,  their type (e.g., thigh muscle,
omental fat, etc.), and their weights should be listed. Combining of sam-
ples from different animals should be noted (usually acceptable for poultry,
but not for ruminants).

     (iii) Sample handling and storage stability. (A) The storage and  han-
dling of tissues, eggs, and milk  between sample collection and analysis
should be described. Factors to consider are:

     (7) Sample preparation (e.g., chopping) prior to storage.

     (2) Containers.

     (3) How quickly the samples are put into storage.

     (4) Storage temperature.

     (5) Length  of storage (dates of collection,  shipping, analysis, etc.).

     (6) Mode of shipping, if applicable.

     (B) Evidence  should be presented showing that the storage  did not
affect the results of the study. Preferably, this is obtained by concurrently
spiking  control samples and storing  them under the same conditions as
samples from treated animals.  For guidance in this area refer to  OPPTS
860.1380. If such information is provided in another section of the overall
data report, the study may be referenced.

     (iv) Analysis of samples. (A) A detailed  description of the analytical
method  employed to measure residues should be provided along with a
statement as to which chemical species were measured (parent pesticide,
metabolites). When the method has been  submitted as a separate report
in the total data report (as is often the case), it may simply be referenced.
See OPPTS 860.1340 for assistance on how to describe methodology.

     (B) Recovery  data should be obtained concurrently with the residue
analyses to validate the method and establish its sensitivity (lowest reliable
quantitation limit).  The  experimental design of these validation studies
should be described, including:

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     (7) Identity of the test compounds and substrates (tissues, milk, and
eggs).

     (2) Magnitudes of fortification levels.

     (3) Number of replicates per test compound per level.

     (C) Dates of sample fortification,  extraction, and analysis of extracts
should be  listed. If extracts are not analyzed on the day  of preparation,
storage conditions should be described.

     (D) Raw data such as sample weights, final volumes of extracts, and
peak heights/areas should be  furnished for  control, fortified (including
those for storage stability data), and treated  samples to support reported
residue values and recoveries. Analytical  responses of standards  (xero-
graphic copies of calibration  curves)  are also needed.  See also OPPTS
860.1000 for more guidance on presentation of raw data.

     (E)  Xerographic  copies of representative chromatograms should be
supplied for control, fortified,  and treated  samples of each matrix (milk,
eggs, each edible tissue, etc.) along with a few sample calculations of resi-
due levels and percent recoveries using  the raw data.

     (6) Results and discussion,  (i) Recovery percentages (all values, not
just  averages  or ranges) for the pesticide and/or its metabolites should be
reported for tissues, milk, and eggs fortified with these compounds.

     (ii) Storage  stability data showing  the behavior of residues as a func-
tion of time in tissues, milk, and eggs  should  be submitted or referenced.
Storage duration and temperature of these samples  should  be specified in
the table.

     (iii) Levels  of the TTR should be reported for each tissue  for each
feeding level (including control (untreated)  samples).  The  tissues rec-
ommended for analysis include muscle,  fat, liver,  and kidney (latter not
required for poultry). The individual values  should be listed for all samples
(not merely  averages  or ranges). It should be clearly  indicated whether
or not residues have been corrected for recoveries.  If the parent  pesticide
and  its metabolites are measured separately,  the residues of each  should
be reported.

     (iv) Residues in milk and  eggs should be listed for each feeding level
including controls along with the dates  of sample collection. As with tissue
residues, the values for each sample should be reported (not just  ranges
or means).

     (v) Discussion should be presented as to whether the data indicate
that  residues  of the pesticide  transfer  to tissues, milk,  and eggs,  and if
so, when did residues plateau in milk and eggs? Do they preferentially

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accumulate in certain tissues? Are the results consistent with the 14C me-
tabolism studies?

     (7) Conclusions. A conclusion must be reached as to whether residues
of the pesticide transfer from feed items to meat, milk, poultry, and eggs.
If so, the extent of transfer should be discussed. The results can be summa-
rized in a table (the preferable format) showing either the ranges or maxi-
mum residues in type each of sample for each feeding level. Such a table
can then be used to determine appropriate tolerances each time  additional
feed items are registered.

     (9) Certification. A certification of authenticity must be provided by
the study director (including signature,  typed name, title, affiliation, ad-
dress, telephone number and date).

     (10) References. Any references cited in the report should be included
here.

     (11) Appendices. Reproductions of published reports that support the
submitted study may also be included here if, in the registrant's opinion,
it will increase the efficiency of its review by the Agency.

     (g) References. The following references should be  consulted for ad-
ditional background material on this test guideline.

     (1) Environmental Protection Agency. Pesticide Reregistration Rejec-
tion Rate Analysis—Residue Chemistry;  Follow-up Guidance for: Generat-
ing Storage Stability Data;  Submission of Raw Data; Maximum Theoreti-
cal Concentration Factors;  Flowchart Diagrams.  EPA Report 737-R-93-
001, February 1993.

     (2) Environmental Protection Agency. Pesticide Reregistration Rejec-
tion Rate Analysis—Residue Chemistry;  Follow-up Guidance for: Updated
Livestock Feeds Tables; Aspirated Grain Fractions (Grain Dust); A  Toler-
ance Perspective; Calculating Livestock Dietary  Exposure; Number and
Location of Domestic Crop Field Trials. EPA Report No. 737-K-94-001,
June 1994.

     (3) Environmental Protection Agency. Pesticide Reregistration Rejec-
tion Rate Analysis—Residue Chemistry;  EPA Report 738-R-92-001, June
1992.

     (4) Environmental Protection Agency. FIFRA Accelerated Reregistra-
tion—Phase 3 Technical Guidance. EPA  Report 540/09-90-078.

     (5) Environmental Protection Agency, Pesticide  Regulation Notice PR
86-5, Standard Format for  Data Submitted under the FIFRA  and Certain
Provisions  of the Federal Food,  Drug, and Cosmetic Act (FFDCA), May
3,1986.

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    (6) Harris, L. Guide for Estimating Toxic Residues in Animal Feeds
or Diets. (1975).

    (7) Update of Livestock Feed Consumption, Animal  Nutrition, Inc.,
1993  (available from National Technical  Information  Service,  order  #
PB94-107877).
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