-&EPA
United States
Environmental Protection
Ayenoy
Office cf Festic-des Programs
Washington, DC 20460
July 1966
Hazard Evaluation Division
Standard Evaluation Procedure
Avian Reproduction Test
Support Document 54
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HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
AVIAN REPRODUCTION TEST
Prepared by
Dennis J. McLane, B.S
Standard Evaluation Procedures Project Manager
Stephen L. Johnson
Hazard ^Evaluation ;B,i vision
Office of Pesticide Programs
United States Environmental Protection Agency
Office of Pesticide Programs
Washington, D.C. 20460
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STANDARD EVALUATION PROCEDURE
PREAMBLE
This Standard Evaluation Procedure (SEP) is one of a set of
guidance documents which explain the procedures used to evaluate
environmental and human health effects data submitted to the
Office of Pesticide Programs. The SEPs are designed to ensure
comprehensive and consistent treatment of major scientific topics
in these reviews and to provide interpretive policy guidance
where appropriate. The Standard Evaluation Procedures will be
used in conjunction with the appropriate Pesticide Assessment
Guidelines and other Agency Guidelines. While the documents were
developed to explain specifically the principles of scientific
evaluation within the Office of Pesticide Programs, they may also
be used by other offices in the Agency in the evaluation of
studies and scientific data. The Standard Evaluation Procedures
will also serve as valuable internal reference documents and will
inform the public and regulated community of important consider-
ations in the evaluation of test data for determining chemical
hazards. I believe the SEPs will improve both the quality of
science within EPA and, in conjunction with the Pesticide Assess-
ment Guidelines, will lead to more effective use of both public
and private resources.
Fohn W. Melone, Director
Hazard Evaluation Division
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TABLE OF CONTEMTS {Continued)
Page
III. REPORTING REQUIREMENTS (Continued)
E. Body Weight and Food Consumption ............ 8
F, Raw Mortality Data 8
G. Gross Necropsy - . 8
H. Other Observations 8
I. Statistical Analysis ........................ 9
J. Acceptable Protocols 9
IV. REVIEWER EVALUATION
A* Review of Materials and Methods . * 10
1. Test Substance 10
2. Diet 10
3. Body Weight and Consumption 10
4, Raw Data 10
B. Verification of Statistical Analysis 10
C. ri.scuss.ion of Results ....................... .. . 11
1. Reproductive Effect, Mortality, and
Behavioral Observations .....,,.. 11
2. Implications of Dose/Effect Response ... 11
3. Gross Necropsy ......................... 11
4. General Comments ...,,...........,»... 11
D. Reviewer's Conclusions 11
.1.. Category .........*.... ........................ .11
2. Rationale' ..-.,...,*...........».......-.< "12
3.. Repairabi 1 ity '. . -... - ^ ............, - ................. .13
.E.. References .... ...........-.....* 13
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AVIAN REPRODUCTION TEST
I. INTRODUCTION
A. When Required
Avian reproductive effects are required to support the regis-
tration of an end-use product which meets one or more of the follow-
ing criteria:
0 Its labeling contains,-d4rections for using the product
under conditions where birds may be subject to repeated or
continuous exposure to the pesticide or any of its major
metabolites or degradation products, especially preceding
or during the breeding season;
0 The pesticide or any of its major metabolites or degrada-
tion products are stable in the environment to the extent
that potentially toxic amounts may persist in avian feed;
0 The pesticide or any of its major metabolites or degrada-
tion products is stored or accumulated in plant or animal
tissues, as indicated by the partition coefficient of
lipophilic pesticides, metabolic release and retention
studies, or as indicated by structural similarity .to known
bioaccumulative chemicals; and/or
0 Any other information, such as that derived from mammalian
reproduction studies, that indicates reproduction in terres-
trial vertebrates may be adversely aff.e.cted by the antici-
pated use of the pesticide product.
B. '"Purpose
The data from this study are used to:
0 Establish the effects of the active ingredient on bird
reproduction;
0 Determine the stage of the reproductive cycle affected for
the tested species;
0 Compare toxicity information with measured or estimated pes-
ticide residues in the terrestrial environment in order to
assess potential impact on avian wildlife;
0 Determine susceptibility differences between waterfowl and
upland game;
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0 Provide support for precautionary label statements to ml
mize adverse effects to avian wildlife; and ni-
0 Indicate the need for further testing and/or field studies
C . Test Substance
1 , Technical Grade
This study must be conducted with the technical grade active
ingredient (a.i.). If more than one a,i. constitutes a technical
"duet, then the technical grade of each a.i, must be tested separately
II. MATERIALS AND METHODS: TESTING STANDARDS/RECOMMENDATIONS
A. Acceptable Protocols
Because avian reproduction testing is an established technique
for assessing toxicity of a chemical to avian wildlife,, much of the
methodology for performing these studies, as well as the procedures
for statistical analysis of results, have been carefully outlined
and documented in the published literature. Kotably, the infor-
mation to be discussed in this Standard Evaluation Procedure (SEP}
is presented in greater detail in the following reference.
Ecological Effects Branch 1982. Pesticide Assessment
Guidelines Subdivision E, Hazard Evaluation? Wildlife
and Aquatic Organisms, EPA-540/9-82-024 . pp. 48-57.
B. Test^ Org_anisms_
1, Acceptable Species/Age /Physical Condition
Testing must be done on both a wild waterfowl species, prefer-.
ably mallard duck (Anas platyrhynchos) and an upland game species,
preferably bobwhite quail (Colinus vj_rg_inianus) . Birds approaching
their first breeding season should be "used.
Test birds should be pen-reared and phenotypically indistingui
shable from wild birds. It is recommended that the birds to be
used are selected only from colonies that have had breeding his-
tories maintained for them. This history should include lighting
practices during rearing, disease record, drugs and any other medi-
cation administered, and exact age. If shipped, all birds should
be examined following shipment for possible physical injury that
have been encountered in transit. If deemed necessary, several_-
birds may be randomly selected for pretreatment necropsy at a diag-
nostic laboratory to assess the state of health upon arrival.
Sickness, injuries, excessive mortality of hatchlings or any
abnormal observations of birds are all signs that the affected
may not be adequate for testing.
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These species were chosen because of their commercial
availability, ecological significance, broad geographical distri-
bution, commercial and recreational importance, ease of handling
in the laboratory, and their past use in toxicity testing and known
susceptibility to chemical exposure. The test species should be
verified by its scientific name.
2. Source/Acclimation
Within a given test all organisms must be from the same source
(this includes laboratory or commercial stocks). It is desirable
to have a two- to six-week health observation period prior to selec-
tion of birds for treatment.
C. Test Conditions
1. Number Per Level
For either bobwhite quail or mallard ducks a minimum of three
test groups should be used. One group should serve as a control
and two groups as treated birds. For bobwhite quail one male and
two females per pen by random distribution, replicated by a minimum
of 12 pens, should be used per group.
For mallards two males and five females per pen by random
distribution, replicated by five or more pens, should be used per
group. For either species if individual pairs (one male and one
female) are to be used per pen, considerably more pens, greater
than 12 per test group, should be used to provide similar sensiti-
vity to the group testing design. Control and treated birds should
be kept under the same experimental conditions.
When other test data reveal bioaccumulative potential., the
number of test animals in the test group should be increased suffi-
ciently to partly offset animal deaths or data-gathering problems
associated with morbidity or with tissue residue determinations.
2. Pen Facilities
Pen conditions should conform to good husbandry practices.
This implies clean pens, adequate room, clean food and water,
heated areas for young birds, and protection from excessive distur-
bance. Pens should also be arranged to prevent cross-contamination.
Pen construction materials should not be toxic, capable of excessively
adsorbing test substance, dissolve in water, or loosened by peck-
ing . For example, stainless steel, galvanized steel and materials
coated with perfluorocarbon plastics can be used. The temperature
and relative humidity should be controlled throughout the reproductive
test and should be recorded. Recommended levels are 21°C and 55 per-
cent relative humidity. Ventilation is necessary. It is desirable
to offer mallards water in which to bathe. '
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3. Photoperiod
Since light is extremely important, both during rearing and
during the egg laying period, all birds should be maintained for
the first eight weeks under a regime of seven hours of light per
day for maximum egg production.
The photoperiod should then be increased to 16-17 hours of
light per day and either maintained at this level or increased by
15 minutes per week for the following 12 weeks. (The 12-week
period may vary depending upon the time required for the onset of
egg production.) An illumination intensity of six footcandles at
the bird level during the lighting phase of the reproductive study
is adequate. Avoid the use of shorter wavelength "cool white"
fluorescent lights which do not emit the daylight spectrum.
4. Body Weights
Body weights should be recorded at test initiation and at
biweekly intervals up to week eight or up to the onset of egg laying
and at termination. During egg laying, body weight recording is
discouraged because of the adverse effects that handling may have
on egg production.
5. Food Consump-tion
Food consumption should be recorded at least at biweekly inter-
vals throughout the study.
Estimates of average consumption for each concentration level
must be reported (grams per day). Provisions for minimizing food
spillage and pr^evesting air icontamination by volatile chemicals
should foe reported.
6. Dose Preparation
Concentrations for the test substance should be based on
measured or calculated residues expected in the diet from the
proposed use pattern(s). The concentrations should include an
actual or expected field residue exposure level and a multiple
level such as five. The highest nonlethal level may be estimated
from data developed from the avian dietary LC5Q.
The test material should be added to table grade corn oil or
another appropriate vehicle and premixed with an aliquot of basal
diet, utilizing a mortar and pestle or mechanical blender. It is
recommended that the aliquot of basal diet used for the premix be
screened to remove large particles of diet before blending in the
corn oil and test material. The final diet should be a uniformly
mixed composition consisting of 98 to 99 parts by weight of basal
diet and one or two parts by weight of corn oil. The basal diet
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should be a commercial game bird breeder ration (or its equivalent)
that is treated with an equivalent amount of. vehicle. The premix
should be stored under conditions which maintain stability. Test
diets should be analyzed for pesticide concentrations at inter-
vals during the tests. If other long-term animal tests have demon-
strated a propensity for the test chemical to persist or bioaccumu-
late, the degree of bioaccumlation in birds should be determined by
measurement of tissue residues in the birds from an extra pen group
put through the reproduction test. Two or three tissues should be
selected for residue analysis at the end of the exposure period,
based on tissues known from other studies to hold highest residues.
Control diets must contain the maximum amount of vehicle avail-
able to treatment birds.
7. Feeding and Husbandry
All birds should receive the appropriate diet ad libitum for
the duration of the study. Water is to be provided~ad"~ITFitum.
The test chemical should be administered for at least ten weeks
prior to the onset of egg laying.
8. Egg Collection, Storage, and Incubation
All eggs should be collected daily, marked according to pen
from which collected, and stored at 16°C and 65 percent relative
humidity. Eggs should be set at weekly intervals for incubation in
a commercial incubator. All eggs should be candled on day 0 for
eggshell cracks; on approximately day 11 for bobwhites and day 14
for mallards to measure fertility and early death of embryos; and
on day 18 for bobwhite and day 21 for mallards to measure embryo
survival. For hatching, transfer of the eggs to a separate commer-
cial incubator or hatcher should be made on day 21 for bobwhites
and day 23 for mallards.
Recommended temperatures and relative humidity during hatching
phase are 39°C and 70 percent, respectively.
»
9. Observations
a. Bobwhite Chicks
On day 24 of incubation, the hatched bobwhite chicks should be
removed, hatchability recorded, chicks housed according to the
appropriate parental grouping, and maintained on control diet for
14 days. The time period should be extended if mortality occurs
appreciably late. The diet should be a commercial bobwhite starter
diet or its equivalent.
b. Mallard Ducklings
On
be removed
day 27 of incubation, the hatched mallard ducklings should
red, hatchability recorded, ducklings housed according to
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the appropriate parental grouping, and maintained on control diet
for 14 days. The time period should be extended if mortality
occurs appreciably late. The diet should be commercial mallard
starter diet or its equivalent.
10. Eggshell Thickness
One day every two weeks newly laid eggs should be collected
and measured for eggshell thickness. For consistency, the eggs
used for thickness determinations should be collected during weeks
one, three, five, seven, and nine of the egglaying period.^ An
accepted procedure is to crack open the eggs at the widestvpartion
(girth or waist), wash out all egg contents, air-dry the shells for
at least 48 hours, and then measure the thickness of the dried
shell plus the membranes at three or four points around the girth
using a micrometer calibrated to 0.01 mm units.
11. Withdrawal
If the test substance is toxic (reduced reproduction evident),
then a withdrawal study period should be added to the test phase.
The withdrawal period need not exceed three weeks. Continued
observations should be made on egg production, fertility, hatcha-
bility, and hatching survival.
12. Definitions
a. Eggs Laid
The total egg production during a breeding season (which is
approximately ten weeks).
b. Eggs Cracked .
Eggs determined to have cracked shells when inspected with a
candling lamp; fine cracks cannot be detected without utilizing a
candling lamp and if undetected will bias data by adversely affec-
ting embryo development.
c. Eggs Set
All eggs placed under incubation, i.e., total eggs laid minus
cracked eggs and those selected for eggshell thickness analysis.
d. Viable Embryos (Fertility)
Eggs in which fertilization has occurred and embryonic develop-
ment has begun. This is determined by candling the eggs six to 14
days after incubation has begun. It is difficult to distinguish
between the absence of fertilization and early embryonic death.
This distinction can be made by breaking out eggs that appear
infertile and examining further. This is especially important when
a test compound induces early embryo mortality.
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e . Live Three-Week Embryos
Embryo that is developing normally after thr«e weeks of incu-
bation. This is determined by candling the egg.
f. Hatchability
The percentage of embryos that mature, pip the shell, and
liberate themselves from their eggs as computed from the number of
fertile eggs. For quail this generally occurs on day 23 or 24 of
incubation, and for mallard on day 25, 26, or 27.
g . Fourteen-Day-Old Survivors
Birds that survive for 2 weeks following hatch.
h. Eggshell Thickness
The thickness of the shell and the membrane of the egg at the
girth after the egg has been opened, washed out, and dried for at
least 48 hours, at room temperature.
III. REPORTING REQUIREMENTS
A. Test Material
The purity of the test material must be stated and should
generally be "technical" grade unless otherwise required. The
percent a.i. must be stated.
B . Observable ^Effects Criteria
:Ttee -criteria for ^determining effects must be defined. In addi-
tion to any deaths and other acute effects, the following repro-
ductive parameters, as defined, must be reported:
0 Eggs laid/hen;
0 Eggs cracked (%};
0 Viable embryos of egg set (%);
0 Live three-week embryos (%);
0 Normal hatchlings of live three-week embryos (%);
0 14-day-old survivors per hen; and
0 14-day-old survivors of normal hatchlings {%).
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C. Statistically Significant Differences
Statistical methods must be presented which calculate the
differences between reproductive parameters.
D. Results of Chemical Analysis
If the concentration of the test material was measured, the
results should be reported. It is particularly important that
concentrations of pesticide in the diet be measured and results
reported when:
0 The test material was a dry powder and no vehicle was used;
0 The test material was volatile; or
° The test material was administered in the feed or water and
data from other subdivision(s) suggest that the actual
concentrations over the length of the test may decrease by
30 percent or more.
E. Body Weight and Food Consumption
Body weights and food consumption should be recorded at test
initiation. Both should be recorded biweekly thereafter except for
body weights. Weighing should be discontinued after week eight or
after the onset of egg laying. The average for each test group
should be provided.
F. Raw Mortality Data
Raw mortality data or percentage of death/effects must be re-
ported ...... The data ,must indicate the numbers ,of birds dying ;at each
dose or control level. The day of death/effects must be reported;
it is preferable to also report the time of death.
G. Gross Necropsy
Gross necropsies are preferred. When performed, all dead birds
should be examined, as well as a sufficient number of survivors in
order to provide a characterization of gross lesions. Inspections
of the GT tract, liver, kidneys, heart, reproductive organs, and
spleen should be made. Also, subcutaneous fat and muscles should be
examined for evidence of deterioration.
H. Other Observations
Signs of intoxication should be described as to what was
observed, when, and for how long. Technical terminology used to
name signs of intoxication should be adequately defined if these
terms are not in general use. The report should indicate whether
deaths are associated with specific signs of intoxication, and the
time of onset and remission of toxic signs. If affected birds
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recover, the time interval to recovery should be reported. Marked
observation of fecal material and urine should be reported.
I. Statistical Analysis
A statistical analysis of the mortality data is required.
Reproductive data consist of continuous variables (e.g., shell thick-
ness, and body weight data) and discrete variables (e.g., number of
eggs laid or 14-day-old survivors). For continuous variables,
experimental groups should be compared to controls by analysis of
variance. For the following discrete variables, survival percentages
should be computed: eggs cracked, viable embryos of eggs set, live
three-week embryos, normal hatchling of live three-week embryos,
14-day old survivors per hen, and 14-day old survivors of normal
hatchlings. These values should then be arcsine transformed prior
to analysis of variance. Alternately, a chi square analysis of
survival (contingency tables) may be used for discrete variables.
Analyses should include body weight, food consumption, eggs laid,
eggshell thickness, eggs cracked, viable eggs, fertility, live
three-week ducklings, 14-day old survivors (per number of eggs
hatched, per hen, and per number of eggs laid). Sample units are
generally the pens within each group.
In order that the concentration causing an adverse effect can
be identified, a test such as the Duncan^s Multiple Range Test
should be performed if the analysis of variance indicates statis-
tical differences.
J. Acceptable Protocols
EEB does not endorse any one protocol. It is sometimes neces-
sary an<3 desiratelse ±o alter the procedures presented in published
protocols to meet the needs of the chemical or test organisms used.
However, EEB does recommend some protocols as guidancefor developing
avian dietary toxicity tests. These protocols include:
Ecological Effects Branch, 1982. Pesticide Assessment
Guidelines Subdivision E, Hazard Evaluation: Wildlife
and Aquatic Organisms, EPA-540/9-82--024. pp. 48-57.
This referenced protocol provides flexible guidance to help resear-
chers design scientific protocols arid, to assist the reviewer in
validating studies. It is important to recognize that avian repro-
duction studies are validated based upon ^whether they fulfill guide-
line requirements and whether they provide scientifically sound
information on the reproductive toxicity of the test material to
avian wildlife that is useful in hazard assessments. This is more
important than whether they follow a referenced protocol step-by-
step. ;
IV. REVIEWER EVALUATION
The reviewer should identify each aspect of the reported proce-
dure that is inconsistent with recommended protocol. The signif-
icance of these deviations must be determined. The number of
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deviations and their severity will determine the validity of the
study and the interpretation of the results.
A. Review of Materials and Methods
1. Test Substance
When the technical grade is greater than 80% a.i., the reviewer
may elect not to adjust the concentration levels at which statis-
tically significant adverse effects occurred to reflect actual
amount of a.i.. Reviewers should check the accuracy of the reported
effect levels by recalculating the doses in ppm a.i. in treated
diets.
2. Diet
A review of the diet composition should be made specifically
to check for unfamilar ingredients or medications. Any such-
unfamiliar ingredient or suspected additive should be verified by
contacting the testing laboratory prior to classifying the study.
3. Body Weight and Food Consumption
Body weight and food consumption data can be used
effects, if any, on feeding and/or weight change.
to evaluate
Reduced food consumption and body weight may affect the ability
of a bird to reproduce. If reduced food consumption or body weight
occurs, the reproductive parameters should be viewed with this in
mind as a possible explanation for adverse effects.
Also, those items mentioned in the SEPs for avian LD5Q and LC50
should be incorporated into the design of this study.
4. Raw Data
The raw data must be checked to insure consistency with the
written report. In cases where the two do not agree, the discrepan-
cies must be explained in writing prior to classifying the study.
The control data must be checked and should be consistent with
other studies on like species.
B. Verification of Statistical Analysis
The reviewer should "validate" the statistical analysis of the
data. This may be done using EEB's "SAS" program "Bigbird" which
performs analysis of variance with and without arcsine transformed
variables. The significant groups are then identified by Duncan's
Multiple Range Test. Any differences from the control should be
noted and an explanation required from the registrant.
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Adjustments to the study's statistical parameters may be made
at this time to reflect the reviewer's estimate of the parameters.
These may be necessary to account for actual amount of a.i. con-
sumed, e.g., in cases where the test material a.i. content was lower
than 80% or insufficient diet was consumed, or to correct errors in
the authors' calculations or methodology.
C. Discussion of Results
1. Reproductive Effect, Mortality,
and Behavioral Observations
The reviewer should discuss the results in light of the observa-
tions of intoxication. Statistically and biologically significant
observations should be discussed if possible. Reproductive effects
and mortality which cannot be fully attributed to the effects of
the test material may be better understood if assessed in light of
behavioral observations. Also, signs of intoxication could aid in
understanding potential sublethal effects which could affect the
birds' ability to develop and survive in the wild.
2. Implications of Dose/Effect Response
Dose/effect response many times reveals important characteris-
tics about the toxicity of the test material, such as whether the
response is commensurate with the decline of the test level.
Pertinent data on dose/effect response should be included in
data evaluation records.
3. Gross Necropsy
The ^results of gross necropsies, when performed/ may be helpful
in evaluating the study. Lesions of reproductive systems may confirm
changes in the reproductive parameters.
4. General Comments
Reviewers should comment on the protocol used and the methods or
other aspects of the study which are irregular, unfamiliar, or unaccep-
table. Suggestions for improvements to the protocol can be made, as
well as giving rationale for rejecting certain methods or aspects of
the study. Of particular importance are the reviewer's comments on
any aspect of the study, such as condition ofthe birds, husbandry
practices, dose administration or rejection, toxic symptoms, or envi-
ronmental conditions, which could bear on the interpretation of the
results.
D.
1. Category
The reviewer should indicate under which of the three valida-
tion categories the study fits:
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0 Core: All essential information was reported and the study
was performed according to recommended protocols. Minor
inconsistencies with standard methodologies may be apparent;
however, the deviations do not detract from the study's
soundness or intent. Studies within this category fulfill
the basic requirements of current guidelines and are accep-
table for use in a risk assessment.
° supplemental: Studies in this category are scientifically
sound; however, they were performed under conditions that
deviated substantially from recommended protocols. Results
do not meet guideline requirements; however, the information
may be useful in a risk assessment. Some-©f the conditions
that may place a study in a supplemental category include:
- Unacceptable test species;
Inappropriate test material;
- Deviations from recommended diet preparation procedures;
or . '
- Dosage levels tested were below the expected concentra-
tion on avian food items when treated at recommended
label rates.
0 Invalid: These studies provide no useful information.
They may not be scientifically sound, or they were performed
under conditions that deviated so significantly from the
.recommended protocols, that the results will not be useful
in a risk assessment.
Examples of studies placed in this category include those
in which there were problems with volatility of the test
material or a dry chemical was mixed without the use of a
vehicle. Unless acceptable chemical analyses of actual
toxicant concentrations were performed in studies such as
these, the reviewer cannot be sure that test organisms were
actually exposed to nominally designated concentrations.
A study where the test material was not properly identified
can also be invalidated.
2. Rationale
Identify what makes the study supplemental or invalid. \It may
also be necessary to justify a higher category in spite of devia-
tions. That is, a study may have been called core or supplemental
even though there were substantial deviations from recommended
protocol. While all deviations should be noted, it may be that the
deviations did not actually alter the response of the test organism
to the test material. The reviewer is expected to exercise judgment
in this area.
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3. Repairability
Indicate whether the study may be upgraded or given a higher
validation category if certain conditions are met. Usually this
would involve the registrant submitting more data about the study.
E. References
The reviewer should reference any information used in the valida-
tion procedure. This should include protocol documents, statis-
tical methods, or information taken from "files of other Divisions
or Branches within HED. ^-"
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