United States Prevention, Pesticides EPA712-C-95-177
Environmental Protection and Toxic Substances August 1996
Agency (7101)
&EPA Residue Chemistry
Test Guidelines
OPPTS 860.1380
Storage Stability Data
-------
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."
-------
OPPTS 860.1380 Storage stability data.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.), and the Federal Food, Drug, and
Cosmetic Act (FFDCA) (21 U.S.C. 301 et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP test guideline 171-4, Results of
Tests on the Amount of Residue Remaining, Including A Description of
the Analytical Methods Used (Pesticide Assessment Guidelines, Subdivi-
sion 0: Residue Chemistry, EPA Report 540/9-82-023, October 1982).
This guideline should be used in conjunction with OPPTS 860.1000, Back-
ground.
(b) Purpose. These studies are required to validate the stability or
rate of decomposition of the total toxic residue (TTR) in or on the raw
agricultural commodity (RAC) (or processed commodity) between the time
of harvest or sample collection and the final analysis of the residue.
(c) General. (1) In most instances samples collected in magnitude
of the residue and nature of the residue (metabolism) studies are stored
for a period of time prior to their analysis. During this storage period resi-
dues of the pesticide and/or its metabolites may be lost by processes such
as volatilization or reaction with enzymes. Therefore, in order to be certain
that the nature and level of residues that were present on samples at the
time of their collection are the same at the time of analysis, controlled
studies are needed to assess the effect sample storage has on the TTR.
In other words, registrants need to show that pesticide residues are stable
during storage of analytical samples or show the degree to which residues
are lost during that period.
(2) The term "storage stability" in this document does not address
manufacturing use product or end use product storage stability data re-
quired under the product chemistry subpart of 40 CFR part 158 or the
storage of food commodities under typical commercial conditions, e.g. dur-
ing the storage and transport of produce prior to its reaching the consumer.
Studies addressing the latter are examples of "reduction of the residue"
or "anticipated residue" studies that are occasionally required to obtain
a more realistic estimate of residues in food at the time of consumption.
The purpose of the present document is to address storage of analytical
samples, in most cases under frozen conditions. For this reason a better
name for the study might be that proposed by the Food and Agriculture
Organization of the United Nations (FAO)—Stability of Pesticide Residues
in Stored Analytical Samples (see paragraph (h)(4) of this guideline).
(3) Storage stability data will be required in conjunction with most
magnitude-of-the-residue studies, e.g. crop field trials, processing studies,
or livestock feeding studies. The Agency will make the following excep-
-------
tion: Unless a pesticide/residue of concern is otherwise known to be vola-
tile or labile, storage stability data will not be needed for samples stored
frozen for less than 30 days. The judgment as to what constitutes volatile
or labile will be based on information such as basic physical properties
and the results of metabolism studies.
(4) Storage stability requirements for nature of the residue or metabo-
lism studies are discussed in paragraph (e) of this guideline.
(5) Other considerations—(i) Need for concurrent studies. (A) It
is preferable that storage stability data be obtained as part of a magnitude-
of-the-residue study, not independent from it. Placing samples with known
residue levels into storage along with the treated commodity samples rep-
resents quality assurance similar to verifying the identity of test material.
If the treated samples were subjected to erratic storage conditions due to
loss of electrical power, the samples with known residue levels could be
used as a direct measure of any effects that temperature fluctuations might
have on residues. Thus, use of concurrent storage stability samples rep-
resents simple good laboratory practice.
(B) The Agency prefers that storage stability studies be conducted
concurrently with the corresponding magnitude-of-the-residue study when
possible. While this may not be possible for data needed to support com-
pleted field trials used for reregistration purposes, it should be possible
in conjunction with new magnitude-of-the-residue studies being initiated
in support of registration or reregistration. However, concurrent storage
stability studies will not be required in many cases (see paragraph (h)(l)
of this guideline). Provided that the pesticide residues are found to be sta-
ble in the matrices of interest, a storage stability study run in a separate
freezer at a different time period will be acceptable if the storage condi-
tions (especially temperature) are similar to those in the corresponding
magnitude-of-the-residue study. For pesticides whose residues are known
or suspected to be unstable or volatile, concurrent studies may be needed.
For such pesticides it is advisable to run a storage stability study in ad-
vance of the magnitude-of-the-residue studies to determine proper storage
conditions and maximum storage times before treated samples are placed
into storage.
(ii) Representative commodities to be analyzed. (A) Use of crop
grouping is acceptable. If residues are shown to be stable in a given com-
modity, the residues in other crops of the same group, as listed in 40
CFR 180.41, would be assumed to be stable for the same time period
under the same experimental conditions.
(B) Combining of the crop groups in 40 CFR 180.41 into larger
groups would generally be acceptable for the purposes of determining sta-
bility of residues in storage. For example, leaves of root and tuber vegeta-
bles could be combined with leafy vegetables (except Brassica). With re-
-------
gard to how many representative crops need to be analyzed (with residues
shown to be stable) before it can be assumed that residues are stable in
all crops, the Agency believes that at least five diverse crops need to be
tested. If a pesticide is to be applied to all types of crops, suggested crops
for a storage stability study are an oilseed (or soybean or nut), a nonoily
grain, a leafy vegetable, a root crop, and a fruit or fruiting vegetable. The
fruit/fruiting vegetable should be an acidic commodity, such as citrus or
tomatoes. Field corn grain is to be considered a nonoily grain as opposed
to an oilseed. The crop parts to be examined in these studies are those
used for food and feed, in other words, those on which residue data are
generated and tolerances established, e.g. wheat grain, wheat forage, and
wheat straw.
(C) The guidance on representative crops is directed toward a pes-
ticide that will be applied to all crop groups. Many pesticides are applied
to only a portion of these groups. Therefore, the five crops listed above
will not always be the most appropriate ones. Since the Agency can not
provide guidance for all the possible combinations of crops that might
be treated, registrants will need to use judgment as to which representative
commodities to use for storage stability studies. One example will be pre-
sented here. Suppose a pesticide is to be applied to only cucurbit vegeta-
bles and stone fruit. In this case storage stability data should be provided
on one crop from each of these groups. Registrants may contact the Agen-
cy if questions arise as to which commodities should be tested for a par-
ticular combination of treated crops.
(D) If residues are found to be unstable in any representative com-
modity, additional storage stability studies will normally be required on
additional commodities of that group if tolerances are being sought on
such crops. Under these circumstances the concept of combining crop
groups in 40 CFR 180.41 may no longer be applicable.
(E) There are three major types of crops for which the Agency re-
ceives magnitude-of-the-residue data for processed commodities: Oilseeds,
grains, and fruits/fruiting vegetables (mainly citrus, apples, and tomatoes).
Since some of the processed commodities (e.g. oils, juices) have matrices
quite different from the starting RAC, storage stability data are required
to support processing studies. If the residues of concern of a particular
pesticide have been shown to be stable in the processed commodities from
one each of the three types of crops cited above, additional storage stability
data will generally not be required on other processed commodities (pro-
vided that the storage conditions are similar and samples are not stored
longer than those of the representative processed commodities).
(F) As with crops, this guidance on processed commodities is directed
toward pesticides applied to all types of crops that have processed com-
modities in which residues may concentrate. For pesticides that are not
applied to all such crops, storage stability data may be needed on proc-
-------
essed commodities other than the three types mentioned above. For exam-
ple, if a pesticide is to be used on only root crops, storage stability data
should be generated on the processed fractions of potatoes or sugar beets.
(G) With respect to animal commodities, storage stability data are
normally required to support livestock feeding or dermal treatment studies.
The representative commodities to be examined should include muscle
(cattle or poultry), liver (cattle or poultry), milk, and eggs. If residues are
stable in these matrices, analyses of other tissues (fat, kidney) will not
be needed.
(d) Storage stability requirements for magnitude of residue stud-
ies—(1) General. Storage stability data normally are required for each
component of the TTR that is measured in the magnitude-of-the-residue
studies. In most cases this means all components included in the tolerance
expression. The Agency will allow representative components of the resi-
due to be employed when numerous compounds are included in the toler-
ance on a case-by-case basis. Registrants are advised to contact the Agency
when questions arise in this regard.
(2) Test compounds and analytical methods, (i) Samples could ei-
ther be from crops (or livestock) that have been treated with pesticides
in the field or from the spiking of control (untreated) samples with known
amounts of each analyte. In all cases, the storage stability samples should
be analyzed using the same analytical procedure that was employed in
the corresponding magnitude-of-the-residue studies. If not, data will be
needed to show that the method gives results equivalent to those obtained
by the method used in the magnitude-of-the-residue studies.
(ii) The samples used in the storage stability study could also be those
obtained from metabolism studies using radiolabeled material. If these are
to be used, the residues should be measured using the "cold" analytical
method that was employed in the magnitude-of-the-residue studies or an-
other method validated for quantitating the TTR. In other words, the stor-
age stability data should not be based on simply counting total radioactiv-
ity. (NOTE: The discussion in this paragraph does not refer to the storage
stability data needed to support a metabolism study. The latter involves
examining the chromatographic profile of all radiolabeled residues as de-
scribed in paragraph (e) of this guideline.)
(iii) In those instances where no detectable residues (or low levels
of residues close to the analytical method's limit of quantitation) are found
in field treated commodities, the Agency advises that spiked control sam-
ples be employed in the storage stability studies. Related to the latter point,
it is suggested that the minimum residue level to be used in storage stabil-
ity studies be lOx the method's limit of quantitation with the minimum
in any case to be 0.1 ppm. This will make it less likely that the stability
of the residues can not be ascertained due to highly variable recoveries.
-------
If typical residues observed in the magnitude-of-the-residue studies are
much higher than the minimum level suggested above, it is preferable (al-
though not required) for the storage stability study to employ comparable
residue levels.
(iv) Analytical methods yielding low and variable recoveries should
be avoided when conducting storage stability studies (as well as mag-
nitude-of-the-residue studies). Regardless of the method used, freshly for-
tified samples should be analyzed at each time point when storage stability
samples are removed from storage for analysis. This will allow for correc-
tion of observed residue values for the stored samples if recoveries are
significantly higher or lower than 100 percent for the freshly fortified sam-
ples.
(v) In those instances where the TTR consists of more than one com-
ponent, i.e. parent compound + metabolites, the storage stability samples
may be fortified with the mixture if the analytical method is capable of
measuring each component of the residue separately. In those cases where
the method converts all residues to a common moiety, spiking with mix-
tures or using field treated/weathered residues is discouraged. The type
of chemical and toxicity involved would determine the acceptability of
spiking with a mixture (or using field treated samples) when a common
moiety method is employed. For example, with pesticides where similar
chronic toxicity concerns exist over numerous components of the residue,
spiking with a mixture followed by use of a common moiety method is
probably acceptable. On the other hand, it would not be acceptable to use
a common moiety method for cholinesterase inhibitors where significant
differences in toxicity may occur as the parent compound oxidizes to as-
sorted metabolites. In other words, in the latter case the method would
need to detect each of the metabolites separately.
(3) Sample form, (i) It is preferred that the form of the commodity
(e.g. homogenate, coarse chop, whole commodity, extract) in a storage
stability study be the same as that in the corresponding magnitude-of-the-
residue study. In some cases the storage stability study may need to reflect
storage of more than one of the above forms. For example, if crop field
trial samples are stored as homogenates for several months, extracted, and
the extracts stored for several weeks prior to final analysis, the storage
stability samples should be handled in the same manner.
(ii) If a storage stability study does not reflect the storage of extracts
prior to final analysis, the whole study need not be repeated. It would
be acceptable to spike extracts of untreated samples, hold them in storage
for the same time and under the same conditions as the corresponding
extracts in the magnitude of the residue samples, and then analyze them
to determine the stability of residues in the extract. To avoid this additional
study, registrants are advised to routinely include the storage of extracts
-------
in their storage stability studies unless their standard laboratory practice
is to analyze extracts on the same day as they are obtained.
(iii) In some cases magnitude of the residue samples are stored in
a whole state, while the storage stability samples are kept as homogenates.
(The latter is necessary to ensure the sample can be spiked uniformly.)
Provided the residues are found to be stable, the Agency will normally
accept such studies since the use of an homogenate in the storage stability
study is likely to represent a worse case versus the use of a whole com-
modity. The homogenization process can release enzymes, acids, and other
chemicals that react with the pesticide or its metabolites. If residues are
unstable in the homogenate, the Agency will decide on a case-by-case
basis whether to correct for loss of residues in the stored whole commod-
ities based on the results of the homogenate or take another course of
action (e.g. require field trials to be repeated with the samples stored in
a different form and/or analyzed closer to the time of collection.) The fac-
tors to be considered in making this decision include the degree of loss
observed in the homogenized samples and the current risk status of the
pesticide.
(iv) The FAO guidelines (see paragraph (h)(5) of this guideline) state
the following:
If prolonged storage is unavoidable, it is usually preferable to extract the
sample, remove most or all of the solvent and store the extracts at a low tempera-
ture, preferably at or below -20 °C. This removes the residue from contact with
enzymes which might degrade the pesticide and also prevents further possibility
of residues being "bound" in the tissue.
While the Agency does not believe this procedure should be the preferred
method of storing samples, it is an acceptable alternative to storing whole
samples or homogenates provided that the storage stability samples are
handled in the same manner.
(4) Sample container. As with most parameters in a storage stability
study, the sample container should be the same as that used for the mag-
nitude of the residue samples. However, the Agency has learned that the
standard practice by registrants is to store magnitude of the residue sam-
ples in plastic bags (for ease of handling and storing large samples that
may not be homogenized) and the storage stability samples in glass jars.
(The latter involve smaller, usually homogenized, samples that need to
be fortified with the TTR of concern in most cases.) The Agency has res-
ervations about this practice since the containers may differ in their
airtightness and pesticides might adsorb differently to the two materials.
However, as long as the pesticide is not volatile, studies will not be re-
jected solely due to the use of different containers.
(5) Storage conditions, (i) The Agency recognizes that magnitude
of the residue samples almost always require transport from the site of
-------
treatment to the laboratory prior to placement into storage until residue
analysis can be performed. Efforts should be made to keep samples cold
during transport, e.g. packed with dry ice, and to keep the transport period
as short as possible. The storage stability study should then simulate the
conditions (temperature, humidity, light) used in the laboratory for storage
of magnitude of the residue samples prior to their analysis. Storage tem-
peratures should be -20 °C or lower. For classes of pesticides with known
instability, petitioners should consider using even lower temperatures to
avoid or at least reduce loss of residues in storage. Samples should also
be kept in the dark to eliminate the possibility of photochemical reactions.
(While the focus of the present document is on the storage stability study,
the Agency wishes to emphasize that efforts should always be made to
assure the integrity of magnitude-of-the-residue samples from the time of
their collection until being placed into storage in the laboratory. Mag-
nitude-of-the-residue study reports should detail how samples are handled
and stored prior to receipt by the laboratory.)
(ii) In older magnitude-of-the-residue studies, the exact storage tem-
peratures may not be known, although samples were kept in a freezer.
If such studies are to be used in support of reregistration, the Agency
suggests storage stability studies be conducted at two temperatures (e.g.
-5 and -20 °C) to address the uncertainty regarding storage temperature
of the older samples. Samples stored at the higher temperature should be
analyzed first. If residues are stable at that temperature, the samples stored
at the lower temperature do not need to be analyzed.
(6) Frequency of sampling, (i) The Agency has no strict require-
ments on the number of sampling intervals that should be examined in
a storage stability study. There needs to be a sufficient number of time
points to establish that the residues are stable throughout the maximum
storage period used for magnitude of the residue samples or to show how
much of the residue is lost at various time points if it becomes necessary
to correct for such losses. In all cases the sampling points should include
zero time to establish the residue levels present at the time samples are
placed into storage. The minimum number of sampling times will vary
depending upon the stability of the residues and the maximum length of
the storage period for the magnitude of the residue samples. For example,
if the latter is only a few months, it may be sufficient to examine samples
stored that amount of time and some intermediate time (in addition to
the zero-time sample) if residues are stable. On the other hand, more time
points would be necessary if the samples are stored several years or if
residues are observed to decline significantly during the several months
of storage.
(ii) The following represent intervals suggested in FAO guidelines
(see paragraph (h)(4) of this guideline). These are not intended to be Agen-
cy requirements, but possibilities to be considered by registrants. If rel-
atively rapid degradation of residues is likely, sampling intervals such as
-------
0, 14, 28, 56, and 112 days could be chosen. For longer storage periods
involving stable residues, intervals of 0, 1, 3, 6, and 12 months are sug-
gested. In any case, the longest storage interval in the magnitude-of-the-
residue study needs to be included as discussed in the next section of
this document.
(iii) The storage intervals observed in a magnitude-of-the-residue
study typically will encompass a wide range. The corresponding storage
stability study does not have to include each and every sampling time
from the study. The Agency will usually interpolate results when correc-
tions for loss are necessary and the intervals from the two studies do not
match.
(iv) The Agency also has no strict requirements with regard to the
minimum number of samples per time point for each analyte. Although
one stored sample (in addition to the freshly spiked samples) may suffice
in many cases, the Agency strongly encourages registrants to have reserve
samples in case problems are encountered (e.g. poor recoveries observed
in freshly fortified samples or an apparently aberrant result (i.e. the avail-
ability of additional samples may provide justification for discarding such
a value)). Reserve storage stability samples are also useful if treated sam-
ples end up being stored longer than anticipated or additional analyses
of treated samples already in storage are requested by the Agency.
(7) Length of storage period, (i) The duration of a storage stability
study should normally be equal to or longer than the maximum storage
period for the corresponding samples in the magnitude-of-the-residue
study. However, for cases in which samples from storage stability studies
were stored for shorter intervals than samples from the corresponding mag-
nitude-of-the-residue studies, extrapolation of the storage stability data to
longer intervals will be considered on a case-by-case basis when minimal
losses have been observed at the shorter storage intervals. Such extrapo-
lation will be considered only in cases where the storage stability data
are available for at least 6 months and reflect at least 3 time points in
addition to the time-zero point.
(ii) Under some circumstances the Agency may also accept the analy-
ses of retained split samples from field trials as an alternative to the ex-
trapolation described above. In some cases the treated samples from field
trials or other magnitude-of-the-residue studies are split into several por-
tions, one portion analyzed quickly, i.e. within 30 days of harvest, and
the other portions placed in frozen storage. If analysis of the stored por-
tions after an extended period in the freezer shows the same residue level
as the portion analyzed within 30 days of harvest, the Agency will consider
using such analyses to support magnitude-of-the-residue studies.
(iii) It should be noted that the extrapolation process and use of split
samples discussed in the previous two paragraphs will normally not be
8
-------
applicable when residues of a pesticide have been found to be unstable
in any commodity. The available data on other crops need to show that
residues are stable for the Agency to consider these alternatives in support
of field trials on a particular crop.
(iv) During reregistration, questions may arise with respect to the
need for conducting new crop field trials versus conducting storage stabil-
ity studies to support old field trials. The decision as to which studies
should be conducted will normally be based on which can be completed
in a shorter time frame. For example, field trials may be available for
a given crop, but the samples were stored 4 years and no storage stability
data are available. In this case, in order to expedite reregistration, the
Agency would want new crop field trials to be carried out since they could
be completed in a much shorter time than a 4-year storage stability study.
(8) Use of storage stability results, (i) If a storage stability study
shows limited decline of residues during the storage period observed for
the corresponding magnitude-of-the-residue study, correction factors will
generally be used to determine the residue levels that were present at the
time of sample collection in the study. However, if extensive dissipation
of residues has occurred during storage, the study may need to be repeated
with samples analyzed closer to their time of collection. As a rule of
thumb, correction factors will be applied to losses in storage up to 30
percent. Beyond that point, the Agency will consider corrections on a case-
by-case basis taking into account factors such as the absolute (parts per
million) and relative (percent of TTR) residue levels of the component
that is unstable in storage.
(ii) The degree of loss will normally be adjusted or corrected for ana-
lytical method recoveries before applying the 30 percent rule of thumb.
In other words, the apparent residue level of an analyte after storage should
be divided by the analytical method recoveries obtained for freshly for-
tified samples analyzed at the same time. For example, a storage stability
sample was originally prepared by spiking at 1.0 ppm (level confirmed
by zero-day analysis after correcting for method recovery of 75 percent
on a freshly fortified sample). After a given period of storage, a portion
of the sample is analyzed and found to contain only 0.63 ppm (an apparent
loss of 37 percent). If the method recoveries for freshly fortified samples
analyzed at the same time are 70 percent, the corrected residue level in
the stored sample is 0.63 ppm/0.70 = 0.90 ppm. Thus, the corrected degree
of loss in storage is 10 percent (or corrected recovery of 90 percent for
the stored sample).
(iii) Regardless of the degree of loss in storage, registrants should
not report just the corrected results in magnitude-of-the-residue studies.
Such adjustments should be left for the Agency to perform. This comment
applies to corrections for both storage losses and analytical method recov-
eries. However, it would be acceptable for registrants to propose correction
-------
factors and report corrected results provided that the uncorrected residues,
correction factors, and corrected results are all clearly presented in the
report.
(e) Storage stability requirements for metabolism studies, (i) The
Agency needs to make a determination as to whether sample integrity was
maintained during collection, preparation, and storage of samples in plant
and livestock metabolism studies. In light of the difficulty of spiking sam-
ples before the identity of the residue is known and the length of time
needed for metabolism studies, the present Agency position is that storage
stability 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 appropriate storage of matrices
and extracts during the analytical portion of the study.
(ii) 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 sub-
strates 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 particular HPLC
peak or TLC spot), additional analyses or another metabolism study with
a shorter collection to analysis interval may be required.
(f) Data reporting. (1) As stated in the Agency report "Effects of
Storage (Storage Stability) on Validity of Pesticide Residue Data" (see
paragraph (h)(2) of this guideline), reports on storage stability studies
should include a detailed description of
* * * the commodities that were stored (whether raw or processed); the
test compounds; the experimental design and storage conditions (e.g. freezer tem-
perature, length of storage, type of containers, etc.); residue methods and instru-
mentation; storage stability results and reporting of the data; statistical analysis;
and quality control measures/precautions taken to ensure the validity of these
operations, including the dates for each step above.
In light of some of the earlier discussion in this document, it is especially
important for registrants to describe how samples are prepared (e.g. coarse-
ly chopped, homogenized) and the containers in which they are placed.
Differences between these and the sample preparation/containers used in
the corresponding magnitude-of-the-residue studies should be pointed out
and data or a rationale provided as to why they should not invalidate the
studies. If known, the Master Record Identification (MRID) numbers of
the corresponding magnitude-of-the-residue studies should be provided.
(2) The values for individual samples (as opposed to just reporting
a mean) should be reported in all cases where multiple samples have been
analyzed at a given time point. A suggested tabular format for reporting
10
-------
the results that incorporates corrections for recoveries in freshly fortified
samples follows.
Commodity
Analyte
Residue
Level
Storage Pe-
riod
Fresh For-
tification
Recovery
Apparent
Recovery in
Stored Sam-
ple
Corrected
Recovery in
Stored Sam-
ple
(3) The values in the second column from the right represent the ap-
parent recovery in the stored samples. These can be divided by the recov-
eries obtained in the freshly fortified samples to determine the corrected
recovery, the measure of the stability of the residue in storage as discussed
in the previous section of this document.
(g) Data reporting format. The following describes a suggested
order and format for a report item by item. However, other formats are
also acceptable provided the information described in this paragraph is
included.
(1) Title/cover page. Title page and additional documentation require-
ments (i.e. requirements for data submission and procedures for claims
of confidentiality of data) if relevant to the study report should precede
the content of the study formatted below.
(2) Table of contents
(3) Summary/introduction. This section should include the following:
Purpose, introduction (include summary table of storage validation data),
sample preparation and fortification, storage and sampling procedures, ana-
lytical procedures, and methods of calculation.
(4) Materials—(i) Test substance. (A) If fortification is used, describe
the test substances (chemical/common/experimental/CAS names, including
the determination/check of the purity of the test compounds (parent plus
any metabolitess of special concern, all in reference standard form) and
preparation of standard solutions).
(B) If weathered residue samples are used, identify the nature and
amount of test substances in the sample at zero-time (defined as the begin-
ning of the storage stability testing).
(C) Any and all additional information the petitioner considers appro-
priate and relevant to provide a complete and thorough description and
identification of the test substances used in storage stability validation test-
ing.
11
-------
(ii) Test commodity. (A) Identification of the RACs (crop/type/variety/
botanical name) and the specific crop parts or processed commodity to
be used in storage stability testing.
(B) The development stagess, general condition (immature/mature,
green/ripe, fresh/dry, etc.) and sizes of the RAC samples used in storage
stability testing.
(C) Treatment/preparation of RAC or processed commodity samples
prior to storage stability testing (e.g. trimming, cleaning, or other means
of residue removal, compositing, subsampling, chopping, extraction, etc.,
reference to the FDA PAM, Vol. I, sections 141-142 for recommended
procedures (see paragraph (h)(6) of this guideline)).
(D) Sample identification number (source of samples, field trial iden-
tification number, control or weathered residue sample, coding and label-
ing information (should be the same as, or cross- referenced to, the sample
coding/labeling assigned at harvest).
(E) Any and all additional information the petitioner considers appro-
priate and relevant to provide a complete and thorough description of the
RACs.
(5) Methods—(i) Experimental design. Number of test commodities,
number of test substances, number and magnitude of test levels, number
of replicate samples per test compound per test level, number of sampling
intervals, representativeness of test commodities to the matrices of con-
cern, etc.
(ii) Test procedures. (A) Fortification (spiking) procedure, if used:
Detail the manner in which the test compounds was/were introduced to
the test substrates.
(B) Storage conditions: Temperature, humidity, lighting, container
types/size, crop form (extract/macerate/etc.), sample sizes/weights, dura-
tion, etc. should be provided.
(C) Sampling: Describe the sampling procedure at zero time and at
regular intervals thereafter. The duration of study should correspond to
the length of storage of the field trial samples collected for residue analy-
sis;
(D) Dates of sample preparation (maceration/extraction/etc.), spiking
or determining the type/amount of weathered residue (zero time), periodic
sampling intervals, end of storage, and residue analyses should be pro-
vided.
(E) Methods of residue analysis: (7) Title/designation/date and source
(PAM, Vol. II; scientific literature; company reports, etc.), or cross-ref-
12
-------
erence the analytical method section of submission if same methods used)
should be submitted.
(2) Discuss any deviations (in reagents, procedures, instrumentation,
operating parameters, etc.) from the Analytical Methods used for residue
analysis of field trial samples or processed commodities if same methods
is/are used.
(3) Detail the principles and stepwise procedures (extraction/clean-
up, derivatization, determination), including any modifications made,
chemical species determined, confirmatory techniques used, if any, etc.,
extraction efficiency (if pertinent).
(4) Instrumentation and operating parameters (make/model, type/spec-
ificity of detectors, columns (packing materials, size), carrier gases, flow
rates, temperatures, voltage, limit of detection and sensitivity, calibration
procedures, etc.) should be provided.
(5) Reagents or procedural steps requiring special precautions (to
avoid safety or health hazards) should be explained.
(6) Time required for analysis (to carry a sample/set completely
through the analytical procedure, including the determinative step) should
be submitted.
(7) Procedures for calculating residue levels and percent recoveries
(detail) should be reported.
Any other information the petitioner considers appropriate and rel-
evant to provide a thorough description of the analytical methodology and
the means of calculating the residue results should be provided.
(6) Results/discussion — (i) Residue results. Raw data, dilution
factorss, peak heights/areas, method correction factors applied, formula(e)/
standard curves used, ppm theoretical/found, recovery levels (range), per-
cent recovery vs. length of storage (dissipation data), appropriateness of
length of storage study, etc. should be provided.
(ii) Statistical treatments. Describe tests applied to the raw data.
(iii) Quality control. Report the control measures/precautions fol-
lowed to ensure the fidelity of storage stability validations.
(iv) Other. Any additional information the petitioner considers appro-
priate and relevant to provide a complete and thorough description of stor-
age stability validation results should be provided.
(7) Conclusion. Discuss conclusions that may be drawn regarding the
stability of the test compounds in the test matrices as a function of storage
time.
13
-------
(8) Certification. Certification of authenticity by the Study Director
(including signature, typed name, title, affiliation, address, telephone num-
ber, date) should be provided.
(9) Tables/figures, (i) Tabless of raw data from storage stability vali-
dation testing and a summary table of residue levels in stored samples
as a function of commodity and storage time should be submitted.
(ii) Graphs, figures, flowcharts, etc. (as relevant) may be included.
(10) References.
(11) Appendixes, (i) Representative chromatograms, spectra, etc.
should be provided.
(ii) Reprints of methods and other studies cited (unless physically lo-
cated elsewhere in the overall data submission, in which case cross- ref-
erencing will suffice) should be submitted.
(iii) Include any relevant material not fitting in any of the other sec-
tions of this report.
(h) 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, EPA Report 738-R-92-0001
(1992).
(2) Environmental Protection Agency, 1987, Pesticide Assessment
Guidelines, Subdivision O, Position Document, Effects of Storage (Storage
Stability) on Validity of Pesticide Residue Data, EPA Report 540/09-88-
002.
(3) Environmental Protection Agency. Pesticide Reregistration Rejec-
tion Rate Analysis - Residue Chemistry; Follow-up Guidance for: Gener-
ating Storage Stability Data; Submission of Raw Data; Maximum Theoreti-
cal Concentration Factors; Flowchart Diagrams. EPA Report 737-R-93-
001 (February 1993).
(4) United Nations Food and Agricultural Organization (FAO). Stabil-
ity of Pesticide Residues in Stored Analytical Samples. 1994 draft prepared
by Codex Committee on Pesticide Residues Working Group on Methods
of Analysis and Sampling.
(5) United Nations Food and Agricultural Organization (FAO).
Guidelines on Pesticide Residue Trials to Provide Data for the Registration
of Pesticides and the Establishment of Maximum Residue Limits—Part
1—Plants and Plant Products (1986).
14
-------
(6) Pesticide Analytical Manual. Food and Drug Administration. Vol-
ume I. (1994). Available from the National Technical Information Service,
Springfield, VA.
15
------- |