&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-022
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.2400:
Wild Mammal
Toxicity Testing
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.2400: Wild mammal toxicity testing.
(a) Scope—
(1) Applicability. This guideline is intended to be used to help develop data to submit to
EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), 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. 346a).
(2) Background. The source material used in developing this harmonized OCSPP test
guideline is OPP 71-3 Wild Mammal Toxicity Test (Pesticide Assessment Guidelines
Subdivision E), and the Standard Evaluation Procedure: Wild Mammal Acute Toxicity
Test, EPA Report.
(b) Purpose. This guideline is intended for use in developing data, specifically either acute
toxicity data, such as the acute oral median lethal dose (LDso) or dietary median lethal
concentration (LCso), or longer term continuous or repeated exposure no-observed-effect level
(NOEL) or no-observed-effect concentration (NOEC) data from subchronic studies for wild
mammals of chemical substances and mixtures ("test chemicals" or "test substances") subject to
environmental effects test regulations. While the LDso or LCso studies are specifically designed
to allow calculation of the LDso/LCso, the study can be used to obtain information regarding
sublethal effects which are used in Agency evaluations. The Environmental Protection Agency
will use these and other data to assess the hazard and risks to wild mammals that these chemicals
may present through environmental exposure.
(c) Definitions. The definitions in OCSPP 850.2000 apply to this test guideline.
Frank sublethal effects for the purpose of this study refers to overt or frank toxicological
effects for mammals and include, but are not limited to, decreased body weight, loss of
coordination, or lethargy. Less significant sublethal effects such as muted color are not
considered frank toxicological effects.
(d) General considerations—
(1) Summary of the test.
(i) The wild mammal toxicity study, using a representative wild mammal, is
typically a laboratory study designed to determine either the acute toxicity of a
test substance such as an acute oral LDso, or an acute dietary LCso, or it is
designed to determine the subchronic toxicity of a test substance. The test
substance is usually administered orally (by capsule or gavage) or via mixing in
the diet. The test duration may be days to months depending on the specific study
objectives.
(ii) This guideline presents a general overview of many of the elements of an
acceptable wild mammal study; it does not address every possible aspect of all the
potential types of tests that might be conducted under this guideline.
(2) General test guidance. The general guidance in OCSPP 850.2000 applies to this
guideline except as specifically noted herein. Because the Agency intends that toxicity
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tests on wild mammals be conducted only to assess specific situations, no single test
methodology can provide adequate guidance for all cases. There are no widely accepted
protocols that include husbandry practices appropriate to a diversity of wild mammals.
Therefore, the test methodologies should be determined on a case-by-case basis.
Appropriate tests methods should be developed in consultation with the Agency.
However, the general guidance provided in this document may be used as a starting point
for protocol development.
(i) Acute oral (LDso) tests. Methods for oral toxicity tests may be adapted from
the avian acute LD50 study in OCSPP 850.2100 and ASTM E555, Practice for
Determining Acute Oral LDso for Testing Vertebrate Control Agents (see
paragraph (i)(2) of this guideline).
(ii) Dietary LCso and continuous or repeated exposure no-effect level tests.
Methods for dietary tests may be adapted from the subchronic oral dosing studies
for mammals in OCSPP 870.3100 and/or from the avian dietary LCso study in
OCSPP 850.2200. See also McCann et al., 1981 (see paragraph (i)(4) of this
guideline).
(iii) Rat acute (LDso and LCso) tests. The results of these tests (e.g., OCSPP
870.1100) have been used as acceptable substitutes (surrogates) for a terrestrial
wild mammals for assessing the toxicity of industrial chemicals (see paragraphs
(i)(7), (i)(8) and (i)(10) in this guideline). In addition to the rat acute oral LDso,
the results of other tests conducted for human health effects may be considered
during the assessment process. These results include data from single dose oral
LD50 tests with other species, acute inhalation LCso tests with rats, dermal
irritation tests with rabbits, and dermal sensitization tests with various species.
(iv) Toxicity studies for large and relatively scarce mammals. An example of
an acceptable protocol for toxicity studies with mammals that are large, relatively
scarce, or otherwise difficult to obtain is provided as a modification of a protocol
that appears in paragraph (i)(l) of this guideline. When the animals are large or
the species is relatively scarce, a study which determines only the approximate
maximum tolerated dosage for the test species may be acceptable.
(3) Range-finding test. If the approximate toxicity of the test substance is unknown, a
range-finding test can be conducted to determine the dosage levels of the test substance to
be used in a definitive test. The dose levels are selected based on a mathematical factor
associated with the expected terrestrial residues. This mathematical factor can be derived
by reviewing the slope of the dose-response line from other mammalian testing. If a test
substance is expected to be of low acute toxicity, it may be useful to first conduct a limit
test as described under paragraph (d)(5) of this guideline. If mortality occurs at the limit
dose or concentration level, then further range-finding at lower levels is suggested. The
results of the range-finding test then may be used to establish the definitive test dosage
levels. Results of range-finding tests should be reported along with the results of the
definitive test, if range-finding tests are conducted.
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(4) Definitive test—
(i) Acute oral LDso and dietary LCso studies. The goal of an acute oral or
dietary definitive study is to determine a dose-response relationship for
mammalian mortality after oral dosing or after dietary acute exposure,
respectively, to establish the respective median lethal dose or concentration (LDso
or LCso), respectively, and standard error estimates and 95 percent (95%)
confidence limits. An additional objective is to determine the slope of the dose-
response curve and its standard error and 95% confidence limits. The definitive
test consists of a minimum of five dose or dietary levels of the test substance, plus
appropriate controls. The dosage and dietary levels are confirmed by chemical
analysis under test conditions.
(ii) Subchronic continuous or repeated exposure NOEL/LOEL and
NOEC/LOEC studies. The objective of longer duration studies is to determine
the toxic effects, the dose-response relationship, and the NOEL or NOEC
associated with continuous or repeated exposure to a test substance. The
definitive test consists of a minimum of five dose or dietary levels of the test
substance, plus appropriate controls. The dosage or dietary levels are confirmed
by chemical analysis under test conditions.
(5) Limit test for acute oral LD50 and acute dietary LCso tests. For test substances
expected to have relatively low toxicity, a limit test may be conducted with a single
treatment level at 5,000 parts per million (ppm) of diet or 2,000 mg/kg-bw (oral) or the
maximum expected environmental residue concentration, whichever is higher, plus a
control group. For pesticides, if the expected environmental residue concentration
exceeds 2,000 mg/kg-bw or 5,000 ppm the test should be conducted at a higher level
equivalent to the maximum expected environmental concentration (EEC) on food items
(see paragraph (e)(4)(iv)(C) and (e)(4)(iv)(D) of this guideline). Based on the results of
the limit test, the LCso or LD50 may be reported as greater than the limit concentration or
dose, respectively, provided that the following conditions are met: first the test substance
treatment and control groups each contain a minimum of 10 animals; second no mortality
or frank sublethal effects occurs in the limit treatment group; thirdly except for the
number of treatment levels the test procedures and duration are the same as in the
definitive test; fourth the limit treatment level is confirmed by chemical analysis under
test conditions; and fifth for pesticides, the limit dose is 2,000 mg/kg-bw or 5,000 ppm or
the maximum expected environmental residue concentration, whichever is higher. Signs
of intoxication, if any, should be reported. Conduct the full definitive test when any
mortality is observed at the limit treatment level. Additionally, if frank sublethal effect(s)
are observed in one or more mammals at the limit dose, despite an absence of mortality,
then a full definitive test may be necessary. For pesticides if frank sublethal effect(s) are
observed in one or more mammals and the limit dose tested was: 1) less than ten times
the maximum expected EEC, then a full definitive study is necessary; or 2) was at least
ten times the maximum EEC, but there is other evidence or data that indicate a risk to
mammalian species, e.g., pesticide use incident data, then a full definitive test is
necessary. If a full definitive test needs to be conducted at ten times the maximum
expected EEC, multiple the result of Equation 1 in paragraph (e)(4)(iv)(C) for acute oral
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studies or Equation 1 or 2 in paragraph (e)(4)(iv)(D) for acute dietary of this guideline by
10.
(e) Test standards—
(1) Test substance. The substance to be tested should be technical grade unless the test
is designed to test a specific formulation, mixture, or end-use product. For pesticides, if
more than one active ingredient constitutes a product, then the technical grade of each
active ingredient should be tested separately, in addition to the combination, if applicable.
The OCSPP 850.2000 guideline lists the type information that should be known about the
test substance before testing, and discusses methods for preparation for use in testing.
(2) Test duration. The duration of the test is case specific and depends on whether the
objective is to determine the acute oral toxicity (see paragraph (e)(2)(i)), the sub-acute
dietary toxicity (see paragraph (e)(2)(ii)), or the no-effect level for repeated exposure (see
paragraph (e)(2)(iv).
(i) Acute oral LDso study. Single oral dose followed by 14 days of observation
after dosing.
(ii) Dietary LCso Study. Exposure is to treated diet for 5 days followed by an
additional 3 days of observation post-exposure.
(iii) Extension of acute study duration. If lexicologically related symptoms are
evident at the end of the suggested observation period for acute testing in
paragraphs (e)(2)(i) or (e)(2)(ii) of this guideline, the observation period may be
extended in relevant increments. Extensions should continue until no apparent
lexicologically related symptoms are observed. For example, in an acute oral
dosing study (paragraph (e)(2)(i) of this guideline), if mortality occurs during the
last 3 days of the 14-day observation period, or if signs of intoxication are not
clearly in remission, or if the test substance is expected to have delayed effects,
then extend the observation period until additional mortality or signs of
intoxication are not observed for 72 hours. An example for the sub-acute dietary
study (paragraph (e)(2)(ii) of this guideline) would be if any test mammals die
during the second or third day of the post-exposure period, if toxic signs are
evident on the third day of the post-exposure period, or if the test substance is
expected to have delayed effects, the test period should be extended until there are
two successive post-exposure days without additional mortality and one day
without signs of intoxication.
(iv) Continuous or repeated exposure NOEL/LOEL and NOEC/LOEC tests.
Number of days of exposure is based on the persistence of the test substance in or
on food items. The test duration may be days to months depending on the specific
study objectives.
(3) Test organisms—
(i) Species. Testing should be performed on a mammalian species representative
of those found in the area(s) likely to be affected by the proposed use pattern(s) of
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the test substance. A rationale for the species selection should be presented in the
study report. In no case should Federally-listed endangered or threatened species
be used for testing.
(ii) Source.
(A) Test animals may be pen-reared or wild captured, but should be
phenotypically indistinguishable from a free-ranging wild mammal. In
general, a species from a captive breeding or commercial stock is
preferred. It is recommended that animals be obtained from breeding
stock that have been outbred periodically with genetically wild stock in
order to maintain a genetic composition that approximates the natural
heterogeneity of the species. All control and treatment animals used in a
test should be from the same source and breeding population, if possible.
Animals should be obtained only from sources whose stocks have known
breeding histories. Purchased animals should be certified as disease-free
or as bred from disease-free stocks.
(B) In some cases, a certification of the health status of the animals by a
veterinarian may be required. This requirement is usually associated with
large or scarce mammal testing. However, it could be required when the
use of the wild caught mammals are proposed for testing.
(iii) Age, gender, and size.
(A) When selecting the appropriate age of test animals, the mode of action
and the means for administration of the test substance should be
considered. All animals should be of the same age class. The rationale for
using the same age class stems from the need for consistency in testing
whereby reproducible results can be achieved. An "even" age class group
should have similar behavioral, metabolic, and physiological attributes,
which will allow comparisons between animals for recognition of
toxicological symptoms.
(B) Typically, test mammals should be of both sexes. However, there may
be situations, where, in order to address concerns associated with a
chemical-specific pattern of use or mode of action, mammals of only one
sex (e.g., breeding females) are more appropriate for testing. The need for
this testing should be determined on a case-by-case basis.
(C) Animals used in the test should not vary in weight by more than plus
or minus (±) 20% of the mean of the test population. If adult animals
which are relatively large or scarce are utilized in the test, it may not be
possible to apply the variance recommendation.
(iv) Acclimation. Acclimation to pen facilities and basal diet should be for a
minimum of 14 days. In some situations with certain test animals, this timeframe
may be insufficient. Personnel conducting the test should provide a written
account of how the test animals were determined to have acclimated to the test
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conditions. Standards that can be used for determining that acclimation has
occurred include but are not limited to:
(A) If a wild mammal lost weight during the transition for the wild to
penned conditions, it can still be an acceptable test organism if the weight
is reestablished during acclimation.
(B) Responses to external stimuli, which may be initially excessive, return
to "normal".
(v) Health status. All animals used in the test should be in apparent good health
and physical condition. The acclimation period allows for determination of the
suitability of the test animals. Generally, if 10% or more of the prospective test
group die during the acclimation period, their suitability as test organisms is
questionable. If the cause of death for these animals can be established and if the
causal effect is not expected to affect the rest of the population or will not affect
the expected mode of action of the toxicant, the test group could be utilized after
an additional holding/acclimation period. However, this may not be feasible
when the age test standard is for young animals (i.e.., LCso studies). For a
satisfactory test, when significant mortality occurs during the acclimation period,
a certification of the health status of the rest of the animals by a veterinarian
should be obtained.
(vi) Care and handling. Animals should be shielded from excessive noise,
activity, or other disturbance during holding, acclimation, and testing. They
should be handled only as much as is necessary to conform to test procedures.
(vii) Diet and feeding.
(A) A standard commercial diet appropriate and familiar to the test species
should be used. Conventional laboratory diets may be used, if appropriate.
Animals should be fed ad libitum in most studies. For acute oral studies, it
may be appropriate to withhold feed from all test groups for a few hours
prior to administration of the test substance but feed should be available
ad libitum during the study.
(B) Feed should not be used past its normal shelf life. Antibiotics or other
medication should not be used in the diet during the acclimation period or
the test. It may not be possible to obtain feed that is completely free of
pesticides, heavy metals, and other contaminants; however, diets should be
analyzed periodically, as described under paragraph (e)(9)(i) of this
guideline, and selected to be as free from contaminants as possible. Extra
precautions should be taken when fish meal or oil is a major ingredient,
since fish are often contaminated with high levels of chlorinated
hydrocarbons.
(viii) Water. Clean water should be available ad libitum. Only clean,
unmedicated water should be offered during the acclimation and testing periods.
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Water bottles or automatic watering devices are recommended. If water pans or
bowls are used, water should be changed at least once a day.
(4) Administration of test substance. The test substance is administered either by
incorporation into the diet (sub-acute dietary test or the no-effect repeated exposure test,
see paragraphs (e)(2)(ii) and (e)(2)(iv), respectively) or orally by capsule or gavage
(acute oral or the no-effect repeated exposure tests, see paragraphs (e)(2)(i) and (e)(2)(iv),
respectively), or by other acceptable methods.
(i) Incorporation in basal diet.
(A) A standard commercial diet appropriate and familiar to the test species
should be used in preparation of the test diets. The test substance is mixed
into the diet, if possible, without a vehicle in a manner that will result in
even distribution of the test substance throughout the diet. Diets can be
mixed by commercial mechanical food mixers. Recommend that the test
substance be added to a small quantity of the basal diet and thoroughly
mixed. This small batch of treated basal diet is subsequently mixed with
larger quantities of the basal diet to achieve a pre-determined nominal test
level. Treatment diets should then be analyzed to determine if the nominal
test levels are achieved and are homogeneous throughout the diet.
(B) If possible, test substance should be added to the diet without the use
of a vehicle. If a vehicle is needed, the preferred vehicle is distilled water,
but water should not be used as a vehicle for test substances known to
hydrolyze readily. When a test substance is not water soluble, it may be
dissolved in a reagent grade evaporative vechicle (e.g. acetone or
methylene chloride) and then mixed with the test diet. The vehicle should
be completely evaporated prior to feeding. Other acceptable vehicles may
be used; these include table grade corn oil, propylene glycol, and gum
arable (acacia). The maximum amount of vehicle in the diet should not
exceed 2% of the diet by weight. Use of a vehicle necessitates the
addition of an equivalent amount of the vehicle into the vehicle control
diet. If the vehicle is some substance other than water, then a clean basal
diet (e.g., without vehicle or test substance) is maintained for the negative
control group. This clean basal diet, when fed to the negative control, aids
in determining the effects of the vehicle on the results of the test. The
negative control and vehicle control diet consumption are compared to
each other and to the consumption of treated diets.
(ii) Oral dosing.
(A) Dosing by gelatin capsule is preferred, but when dosing with capsules
is not feasible, doses may be administered by gavage. Doses are based on
the individual body weight (bw) of each animal. The test substance
should be accurately weighed for each test organism at each dosage level.
Body weights are typically determined at the time of dosing, but may be
taken, especially for capsules, within 24 hours prior to dosing. If
necessary, an evaporative vehicle (such as acetone or methylene chloride)
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may be used in preparing the test substance for use in capsules. For a
satisfactory test, this evaporative vehicle should be completely evaporated
at room temperature prior to placing the test substance in the capsules.
(B) When intubation or gavage techniques are used for oral dosing, a
vehicle is often necessary. If dosing is by gavage and a carrier is used to
administer the test substance, the preferred carrier is distilled or deionized
water unless the test substance is known to hydrolyze readily. Other
acceptable carriers include corn oil, propylene glycol, 1% carboxymethyl-
cellulose, and gum acacia. Materials with known toxic or emetic
properties should not be used. In all preparations of the test substance, the
minimum amount of carrier necessary should be used. Under no
circumstances should the maximum vehicle amount per dose exceed 1.0%
of the body weight. In addition, the vehicle should be used on a constant
volume-to-body weight basis.
(iv) Treatment levels—
(A) Acute oral LDso and dietary LCso tests. At a minimum five dosage
or five dietary treatment levels are tested in the definitive test, plus the
controls. These dosage or dietary test substance levels should be spaced
geometrically in such a manner so that the entire dose-response curve
(e.g., LDio to LDgo; LCio to LCgo) is adequately characterized. If a range-
finding test is conducted, treatment levels should be spaced so that at least
three cause mortality between, but not including, 0% and 100%. For a
scientifically sound estimate of a point on the curve (e.g., LCso), responses
should immediately bracket the point estimate of concern. For some test
substances, it may be necessary to use more than five dosage or dietary
levels to achieve these results. If a range finding test is not conducted, the
number of dose levels should be increased to assure that the endpoint of
the definitive test is bracketed by known levels.
(B) Continuous or repeated exposure NOEL/LOEL and
NOEC/LOEC tests. A minimum of five doses or five dietary test
substance concentrations are tested in the definitive test, plus the controls.
With NOEL/LOEL (NOEC/LOEC) studies, the dose levels should bracket
the desired endpoints. If a range finding test is conducted, the endpoint
value that is depicted is used as the median dose level. Two geometrically
spaced values above and below the endpoint value are determined. With
NOAEL/LOAEL and NOAEC/LOAEC tests, the additional test doses or
concentrations are split above and below the endpoint of the range finding
test. The geometric spacing of doses is very close to the range finder
endpoint. This is of particular importance when the endpoint is close to
the expected or measured residues. With the closer spacing of doses a
more accurate NOAEL/LOAEL and consequently MATC can be
determined.
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(C) Limit Test - Acute Oral LDso. For a limit test, there is single dose
level, plus the appropriate control (see paragraph (d)(5) of this guideline).
A limit dose of 2,000 mg/kg-bw is used unless environmental residues are
expected to result in a higher dosage. Equation 1 of this guideline can be
used to calculate the acute mammalian oral limit dose (mg a.i./kg-bw) for
spray applications of pesticides. The dietary residue estimates are based
on a nomogram that relates food item residues to pesticide application
rate; for an application rate of 1 Ib a.i./Acre the highest residue level
expected is with short grass (nomogram value of 240). The nomogram is
based on an EPA database called UTAB (Uptake, Translocation,
Accumulation, and Biotransformation), a compilation of actual measured
pesticide residue values on plants (see references in paragraphs (j)(3) and
(j)(5) of this guideline). If there are multiple uses this study is supporting
for registration, the limit dose for the study should be based on the one
resulting in the highest dose. If the resulting limit dose exceeds the
digestive capacity of the test organism, consult with the Agency prior to
conducting the study to determine the appropriate dose to use.
f(C VO 953 V
Limit dose (mga.i. /kg-bw)=\ V T;^*^ ' Equation 1
^ \AWjLW) )
where:
for a pesticide use with a single application per year:
Cm^diet = (ApRate X240 ) Equation 2
for a pesticide use with more than one application per year:
n ( ( f 0.693 1\ u. AY\
^— \\ / . \/ \ - («-l)(interva/)
* = Z (ApRate\7AQ\ e\™fl*} Equation 3
<=1 ^ V ))
ApRate = maximum single application rate (in Ib a.i./acre);
Halflife = the foliar halflife (default is 35 days);
Interval = the minimum application interval (in days);
/' = application event from 1 to n;
n = total number of applications;
AW = the body weight (in g) of the assessed mammal - for
pesticides, use 15 grams as this is most conservative value in
screening level assessments;
TW= the body weight (in g) of the test mammal; and
0.953 = percent body weight consumed daily for a 15 gram
mammal.
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(D) Limit Test - Acute Dietary LCso. For a limit test, there is single
dietary level, plus the appropriate control (see paragraph (d)(5) of this
guideline). A limit dose of 5,000 ppm is used unless environmental
residues are expected to result in a higher dietary concentration. To
calculate the dietary limit concentration (mg a.i./kg-diet) for spray
applications of pesticides Equation 1 of this guideline can be used where a
use is limited to a single application per year and Equation 2 of this
guideline can be used where there are multiple applications per year. If
there are multiple uses this study is supporting for registration, the limit
concentration for the study should be based on the one resulting in the
highest dietary exposure.
where:
for a pesticide use with a single application per year:
Dietary Limit Concentration(mga.i. I kg — diet} = (ApRateJ(24Q) Equation 1
for a pesticide use with more than one application per year:
' 0.693lX w AN
r (n-l)(mterva/)
Dietary Limit Concentration =
I
Equation 2
ApRate = maximum single application rate (in Ib a.i./acre);
Halflife = the foliar halflife (default is 35 days);
Interval = the minimum application interval (in days);
/ = application event from 1 to n; and
n = total number of applications
(5) Controls.
(i) Every test includes a negative control group. For a satisfactory test, negative
control animals should be from the same population as the test substance treated
groups. Vehicle controls are used in addition to negative controls when a vehicle
other than reagent water is used. There is rarely sufficient wild animal historical
data available with vehicles to justify waiving vehicle controls for vehicles other
than reagent water. Mammalian control and treated groups should be kept under
the same experimental conditions.
(ii) Controls serve as a monitor of husbandry practices and an indicator of
possible problems due to handling and test substance administration and aid in
separating treatment related effects from non-treatment related effects. Controls
are important in assessing background mortality and disease. Background
mortality is never presumed to be negligible.
(iii) For an acute test, a test is not acceptable if more than 10% of a control group
dies during the test period.
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(6) Number of test organisms and replicates—
(i) Acute oral LDso and dietary LCso tests. For a regression-based endpoint test
(i.e.., LDio, LD50), a minimum of 10 animals is generally recommended in each
treatment group, and in the controls. However, depending on the test endpoint,
and the mammal to be tested, the number of animals per level could increase or
decrease.
(ii) Continuous or repeated exposure NOEL/LOEL and NOEC/LOEC tests.
At a minimum four replicates is generally recommended in each treatment group,
and in controls. However, the number of replicates should be sufficient to
produce a statistically valid endpoint. The criteria for determining the number of
animals and replicates is based on the variability of each response measure of
concern and the necessary quantity of data points to achieve a statistically and
scientifically sound NOEL and LOEL values (or NOEC and LOEC values).
(iii) Pen/cage assignment. Animals used in the test should be assigned randomly
to treatment and control pens without respect to sex. Randomization may be done
either at the initiation of the acclimation period or at the time when the animals
are weighed prior to test initiation. The latter is recommended because it avoids
additional handling stress.
(7) Facilities, apparatus and supplies. Normal laboratory equipment and supplies, and
items especially listed in (e)(7)(i) through (e)(7)(iii).
(i) Facilities.
(A) When space permits, cages or pens should be kept indoors to control
lighting, temperature, and other environmental variables. If the test is
conducted outdoors, a sheltered area should be provided.
(B) Where feasible, it is recommended that pens/cages not be stacked
upon each other. If pens/cages are stacked, only one test substance is
allowed in any single stack. If a test substance volatilizes or otherwise
forms aerosols or vapors in the air, no more than one test substance should
be tested in a room in order to avoid cross-contamination. Otherwise
pens/cages should be randomly arranged within a room.
(ii) Environmental monitoring equipment. Under outdoor testing conditions, a
maximum recording thermometer and relative humidity apparatus will be
necessary.
(iii) Pens/Cages—
(A) Size. Generally, the animals' enclosures need to be large enough so
that they can exhibit normal behavior and movement. The housing and
maintenance conditions should be in accordance with acceptable animal
husbandry practices (e.g., USDA Animal Care Regulations). In some
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cases, visual screening needs to be placed between pens or cages to reduce
aggression.
(B) Construction materials. Pens/cages should be constructed of
galvanized metal, stainless steel, or perfluorocarbon plastics. Materials
that are toxic, may affect toxicity, or may adsorb test substances should
not be used.
(C) Placement to avoid cross-contamination. Pens or cages, such as
stacked, unmodified, commercial pens with external feeders, that allow
food to be spilled from one pen to a lower pen, should be avoided. Any
modifications that prevent cross contamination of concentration levels are
acceptable.
(D) Fresh drinking water. Additionally, the design of all pens or cages
should ensure that animals have a continuous source of fresh drinking
water.
(E) Cleaning.
(1) Between tests, pens or cages should be disassembled (if
feasible) and thoroughly cleaned to prevent disease transmission
and cross-contamination. Steam cleaning of cages is
recommended. Cages may be hosed, brushed thoroughly and
hosed again, as an alternative method. The use of detergents or
bleach is acceptable, but other chemical disinfectants such as
quaternary ammonium compounds should not be used. When
disease vectors have to be controlled, hot or cold sterilization
techniques are recommended, as long as such techniques will not
leave chemical residues on the cages. For cold sterilization,
ethylene oxide is recommended.
(2) Depending upon the type of pens used, pens may be cleaned
during a test as needed to maintain good animal husbandry;
however, care should be taken to minimize disturbance of the
animals.
(8) Environmental test conditions. Environmental conditions during the test should be
are maintained as follows.
(i) Temperature and humidity. For a satisfactory test, the air temperature and
humidity within a laboratory should be typical of the selected mammalian
species' thermoneutrality range.
(ii) Lighting and photoperiod. If the test is conducted in a laboratory, the
preferred photoperiod is 12 hours of light per 24-hr period. The photoperiod
should coincide with the natural daylight hours for the region where the test is
being conducted. Lighting may be either incandescent or fluorescent. Pens and
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lights should be positioned so that all pens will receive approximately equal
illumination.
(iii) Ventilation. It is recommended that ventilation be sufficient to supply 10 to
15 air exchanges per hour.
(9) Observations. The following list is not exhaustive, but provides elements of the type
of observations made in an acceptable study.
(i) Analysis for test substance concentrations. Analytical confirmation of the
dosing media or the treated diet should be performed as described in OCSPP
850.2000 using analytical methods, verified before beginning the test. For dietary
studies, it is preferred that representative samples of test feed be taken at the
beginning, middle and end of the test. If not already available, data should be
generated to indicate whether or not the test substance degrades or volatilizes. If
it is observed that the stability or homogeneity of the test substance in the diet
cannot be maintained, care should be taken in the interpretation of the results and
note made that the results may not be reproducible.
(ii) Contaminants in feed. Contaminated feed may compromise study results,
therefore, feed should be analyzed periodically to identify background
contaminants such as heavy metals (e.g. arsenic, cadmium, lead, mercury, and
selenium) and persistent pesticides. A broader pesticide screen to include other
chemicals that are known to be acutely toxic to mammals may be useful.
(iii) Basal diet composition. A nutrient analysis of the basal diet should be
included in the test report. The analysis should include percent by weight of
protein, fat, fiber, ash, calcium, and phosphorus. In addition to these analyzed
components, a list of expected amounts of vitamins, minerals or other
supplements should also be reported. The composition of any vitamin or other
supplements should also be reported. Most commercial feed companies provide
both the analysis and the list of supplements on the label.
(iv) Environmental conditions—
(A) Air temperature and humidity. During the conduct of laboratory
tests, the air temperature and relative humidity are recorded on a daily
basis. At a minimum the maximum and minimum values for temperature
and relative humidity are recorded daily at representative locations. Under
outdoor testing conditions, a maximum recording thermometer and
relative humidity apparatus should be utilized at representative locations.
(B) Weather conditions. If testing is conducted outdoors the date and
amount of rainfall, snow, or any other climatic conditions that could affect
the results of the test are monitored and recorded.
(C) Light and photoperiod. If the test is being conducted in outdoor pens
or cages, the day length from the beginning of the acclimation period to
the termination of the test should be recorded. Depending on the total
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time frame that the test is expected to run, this measurement should be
conducted weekly or monthly.
(D) Ventilation and laboratory air exchange. If the laboratory is
equipped with an inside/outside air exchange system, the rate and volume
of exchange should be measured.
(v) Measures of effect—
(A) Monitoring of mammals. For oral dosing studies, mammals should
be monitored closely for the first 60 to 120 minutes after dosing. Any
regurgitation should be noted. Additional observations of test mammals
are made at a minimum three times on the day of dosing and at least daily
(where feasible, twice daily observations are recommended) throughout
the remainder of the test period. For dietary treatment studies observation
of test animals should be made, at a minimum, 3 times on the first day of
the exposure period. Observations also should be made at least daily
throughout the remainder of the test period; twice daily observations are
recommended, where feasible.
(B) Mortality, intoxication, and other abnormal behavior. Throughout
the test period, all signs of intoxication, other abnormal behavior, and
mortality are identified, counted, recorded by dosage level, by sex, and by
day. Signs of intoxication are those behaviors apparently due to the test
substance and may include a wide array of behaviors, such as labored
respiration, leg weakness, hemorrhage, convulsions, excessive salivation
and paralysis. Abnormal behaviors, such as excessive aggression,
hyperactivity etc., that may or may not be attributed to the test substance
should be recorded. Among survivors, remission of signs of intoxication
and cessation of abnormal behavior is identified and recorded by dose
level, by sex, and by day. When differential signs of intoxication are
observed within a dose level, an estimate of the number of animals
exhibiting such signs should be recorded.
(C) Body weight measurements. At a minimum, body weight
measurements are taken at the beginning and end of the study. However,
if the study is greater than two weeks in duration, weekly body weight
measurements are recommended. Body weights can indicate sublethal
effects of the toxicant. Control group body weights are used as a baseline
for comparisons to the vehicle controls and treatment group's body
weights.
(D) Food consumption. Food consumption is monitored daily for each
animal. This record should begin the day the animals are received at the
testing facility and/or the starting day of acclimation for the test.
(E) Gross pathology and necropsies. Whether a gross and/or
histological necropsy is necessary depends on the test substance and the
endpoint of the study. For acute testing, all animals that die during the test
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should be necropsied. For a satisfactory study, a representative group of
the survivors (usually 50%) should be necropsied for comparison to those
that died. The survivors necropsy will provide insight into possible
sublethal effects. A gross necropsy of organs and muscle tissue is usually
adequate. For tests where a NOEL/LOEL (NOEC/LOEC) and MATC are
to be determined, any animals that exhibited an effect should be
necropsied. Samples are taken from test organisms not visually affected,
and from control organisms. Both gross and histological necropsies (with
possible tissue residue analysis) are recommended.
(f) Treatment of results—
(1) Body weight change and total food consumption. Observations on body weight
and feed consumption are used to provide a qualitative characterization of sublethal
effects. For body weight calculations are made on the change in body weight between
observation periods and the total change in body weight between test initiation and test
termination Total food consumption per animal between test initiation and test
termination is used to evaluate adversion or inhibitory effects of the test substance on
consumption of food. For longer duration tests, the weekly (or alternative frequency
interval) food consumption rate during the course of the test helps to identify impacts on
feeding patterns due to the test substance. If applicable determine effect on body weight
change for males and females, separately. For body weight gain and total food
consumption per animal calculate the percent inhibition (%I) as compared to the control
at each test substance concentration.
(2) Mortality. For mortality calculate the percent mortality, adjusted for control
mortality at test termination, at each test substance dosage or dietary concentration.
Cumulative number of dead for each dosage or dietary level and control group by
observation day should be summarized in tabular form.
(3) Appearance and behavior. Number of animals with appearance and behavioral
symptoms should be summarized in tabular form by time of observation, treatment and
sex (if applicable). Tabulate among survivors, remission of signs of intoxication and
cessation of abnormal behavior by dosage level and by observation day.
(4) Gross pathology and necropsies. Types of observed pathologies and number of
dead or examined surviving mammals with these lesions should be summarized in tabular
form by treatment and sex.
(5) Limit test—
(i) Acute oral or dietary tests—
(A) Acute oral LDso value. At test termination, if no animals die at the
limit dose, the acute oral LDso is considered to be greater than the limit
dose (i.e., LDso > limit dose, typically 2,000 mg/kg-bw). This is because
the Binomial Theorem predicts that when 10 organisms are tested, the
probability of seeing no mortality if the true LDso is at or below the limit
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dose is <0.001. Conversely the probability of seeing one or more dead
mammals if the true LDso is at or below the limit dose is >0.999.
(B) Acute dietary LCso value. At test termination, if no animals die at
the dietary limit concentration, the acute dietary LCso is considered to be
greater than the limit dose (i.e., LCso > limit dose, typically 5,000 ppm).
Binomial theory holds that when 10 organisms are tested, the probability
of seeing no mortality if the true LCso is below the limit dose is <0.001.
Conversely the probability of seeing one or more dead mammals if the
true LCso is below the limit dose is >0.999.
(ii) Proportion of mortality (p ). The Binomial Theorem also can provide both
an estimate of the true proportion of mortality (p) in the laboratory test
population as well as confidence bounds on that estimate (see Table A4 of the
reference in paragraph (i)(3) of this guideline). For small sample sizes the
interval may be large. For example, for no mortality in 10 mammals (p =0), the
upper 99% confidence bound on the estimate of p is (0.00,0.41) and 95%
confidence interval is (0.00, 0.31). Using the 95% confidence interval as an
example, the true (unknown) proportion of mortality will be covered by the
calculated confidence interval in 95% of repeated trials. For assessing risks, the
confidence in the proportion impacted is considered in determining acute effects
at environmental exposure levels. If the uncertainty in p is high at the limit
concentration, and the expected environmental exposure concentration is close to
the limit concentration, risks to threatened and endangered species may not be
able to be discounted.
(iii) Multiple-dose definitive testing.
(A) At test termination, if one or more mortalities occur among the 10
mammals at the limit concentration (which was conducted at 2,000 mg/kg-
bw, 5,000 ppm or the maximum EEC for either the acute oral or dietary
study, whichever is greater), a definitive LDso or LCso test should be
conducted. If frank sublethal effect(s) are observed in one or more
mammals at the limit dose, despite an absence of mortality, then a full
definitive test may be necessary. For pesticides if frank sublethal effect(s)
are observed in one or more mammals and the limit dose tested was: 1)
less than ten times the maximum expected EEC, then a full definitive
study is necessary; or 2) was at least ten times the maximum EEC, but
there is other evidence or data that indicate a risk to mammalian species,
e.g., pesticide use incident data, then a full definitive test is necessary.
(B) A multiple-dose definitive LDso or LCso test may be waived if, at test
termination: 1) the limit treatment group and control group each contain a
minimum of 10 mammals 2) no mammals died at the limit dose; 3) and
there are also no frank sublethal effects at the limit dose; 4) except for the
number of dosage levels the test procedures and duration are the same as
in the definitive test; 5) the dosage level is confirmed by chemical analysis
under test conditions; and 6) the limit dose was 2,000 mg/kg-bw, 5,000
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ppm or equivalent to the maximum expected environmental concentration
on food items for either the acute oral or acute dietary study, whichever is
higher.
(6) Multiple-dose definitive test—
(i) Acute oral LDso and dietary LCso tests. Statistical procedures are employed
to calculate the LDso or LCso (standard error and 95% confidence interval). If a
dose-response curve model (e.g., probit) was fit to the data to determine the LD50,
or LCso, the model parameters (e.g., slope) and their uncertainty estimates (e.g.,
standard error) should be recorded. A statistical test for goodness-of-fit (e.g., chi-
square test) should also be performed to determine how well the data fit the
computational model used.
(ii) Continuous or repeated exposure NOEL/LOEL or NOEC/LOEC and
MATC studies. A NOEC and LOEC are determined for each of the response
variables using appropriate statistical methods. All methods used for statistical
analysis should be described completely. Experimental units (replicates) are the
individual pens within each treatment level. The overall study NOEC and LOEC
are the lowest values (most sensitive) of all response variables considered.
(iii) Statistical methods. All methods used for statistical analysis should be
described completely.
(A) Acute oral LDso and dietary LCso tests. Data should be adjusted to
account for any control mortality. Statistical analysis may be conducted
by probit analysis by transforming the dose levels to logarithmic values
and the mortality response to probit units. Other standard statistical
methods are acceptable if they provide the same information listed above.
Observations on body weight and feed consumption may be used to
provide a qualitative characterization of sublethal effects.
(B) Continuous or repeated exposure NOEL/LOEL and
LOEC/NOEC and MATC tests. For studies for which a NOEL or
NOEC is to be determined, hypothesis testing (such as Williams test) is
recommended. Additional discussion about endpoints and statistical
procedures is found in OCSPP 850.2000.
(g) Test validity elements. A study would be considered to be unacceptable or invalid if one or
more of the conditions in Table 1 occurred. This list should not be construed as limiting the
reason(s) that a test could be found unacceptable or invalid. However, except for the conditions
listed in Table 1 and in OCSPP 850.2000, it is unlikely that a study will be rejected when there
are slight variations from guideline environmental conditions and study design unless the control
organisms are significantly affected, the precision of the test is reduced, the power of a test to
detect differences is reduced, and/or significant biases are introduced in defining the magnitude
of effect on measurement endpoints as compared to guideline conditions. Before departing
significantly from this guideline, the investigator should contact the Agency to discuss the reason
for the departure and the effect the change(s) will have on test acceptability. In the test report, all
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departures from the guideline should be identified, reasons for these changes given, and any
resulting effects on test endpoints noted and discussed.
Table 1.—Test Validity Elements for Wild Mammal Toxicity Test
1. Animals were not randomly or impartially assigned to treatment and control pens or cages.
2. A vehicle control or negative control was not included in the test.
3. More than 10% of the organisms in any control treatment died or showed signs of disease or stress.
4. An incorrect vehicle was used as the dosing matrix.
5. A constant geometric progression between doses was not used in studies where only the LD50 or LC50
is obtained.
6. The dosage value per body weight factor exceeds 3.0 ml per 100 grams of body weight.
(h) Reporting—
(1) Background information. Background information to be supplied in the report
consists at a minimum of those background information items listed in paragraph (j)(2) of
the OCSPP 850.2000 guideline.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test, the reason for these changes, and any
resulting effects on test endpoints noted and discussed.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e. for pesticides, the identity and
concentration of active ingredient(s)), radiolabeling if any, location of label(s),
and radiopurity.
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance, stock solutions, and the
treatment dosages or dietary concentrations used in the range-finding and
definitive test, or limit test.
(iv) If a diluent is used to prepare stock or test substance provide: the name and
source of the diluent, the nominal concentration(s) of the test substance in the
stock solution, and the diluent concentration(s) used in the treatments and diluent
control. For oral dosing a description of the dosing volume of test substance plus
diluent for all animals with respect to individual body weights.
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(4) Test organisms.
(i) Name of species tested (including scientific name).
(ii) Information about the source: type, name, collection location, date, and
method if from the wild, breeding history if from a commercial source,
certification of disease status.
(iii) Sex and reproductive history and condition at test initiation.
(iv) Age of each animal at the beginning of the test and how determined.
(v) Acclimation procedures, conditions and duration, including housing and
environmental conditions (if different from test conditions) and feeding and
watering procedures.
(vi) Daily food consumption of each animal during the acclimation period.
(vii) Body weight of each animal at the beginning and end of the acclimation
period.
(viii) Health observations, daily behavior of each animal and mortality during the
acclimation period.
(ix) Body weight of each animal at the beginning of the test (typically determined
at the time of dosing for acute oral tests, but may be taken, especially for capsules,
within 24 hours prior to dosing) for each treatment and control group in each pen
or cage.
(5) Test methods and conditions. Description of the test system and conditions used in
the definitive or limit tests, and any preliminary range-finding tests. As a specific wild
mammal test may include additions or modifications to the test methods and conditions to
accommodate case-by-case issues to be addressed by wild mammal testing, the following
list is not exhaustive but provides examples of the type of test methods and condition
reporting elements for acute oral and dietary testing and some elements of longer
duration tests. All test methods and conditions reporting elements that will comprise the
report should be part of the submitted protocol reviewed by the Agency.
(i) Description of housing containers including: type, size, and materials of pen or
cage.
(ii) Description of feed and water dispensing apparatus.
(iii) Description of placement of pens to eliminate cross-contamination, due either
to food cross-contamination or volatility of test substance. Include a description
of any modifications of food dispensers that prevent cross-contamination.
(iv) Environmental conditions: test temperatures, approximate test room humidity,
photoperiod, and lighting source and intensity.
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(v) Detailed description of the basal diet including: source, type, nutrient analysis
containing percentages by weight of protein, fat, fiber, ash, calcium, and
phosphorus, a list of expected amounts of vitamins, minerals and other
supplements (if used). Composition of the vitamins, minerals, and supplements in
the diet.
(vi) Frequency and sample date(s) for documenting the contaminant status (heavy
metals, persistent or chlorinated pesticides) of the feed and tabulation of the
results of the analysis.
(vii) The number of pens or cages per test substance dosage or dietary level and
controls.
(viii) The total number of animals and the number of animals of each sex exposed
at test initiation per test substance dosage or dietary levels and controls.
(ix) Whether animals are housed separately or in groups. If housed in groups, the
number per group and sex-ratio per group.
(x) Method of marking each animal, if applicable.
(xi) Methods of assigning animals to test pens or cages, and test pens or cages to
dose or dietary treatment levels and the control groups.
(xii) The method of administering the test substance.
(xiii) In a definitive test, statement about the selection basis or source for the
highest dose or dietary test substance concentration such as the highest expected
environmental concentration based on the highest pesticide label rate (in Ibs
a.i./acre), accounting for multiple applications that occur in a season, or actual
highest measured test substance residue levels.
(xiv) Date of initial exposure of the animal to the test substance, dates of
exposure, duration of test, including length of exposure period (in days) and
length of any post-exposure period on "clean" feed (in days).
(xv) Methods and frequency of environmental monitoring performed during the
definitive or limit study for temperature, humidity, and air exchange.
(xvi) For tests conducted outdoors the methods and frequency of monitoring
performed during the definitive or limit study for weather conditions that may
affect the test.
(xvii) Description of any medication given during the test: type, frequency, and an
explanation of how it was administered and justification of why it was given.
(xviii) Methods and frequency of measuring test substance to confirm exposure
dosages or dietary levels, stability in the diet throughout the exposure period, and
homogeneity in a diet.
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(xix) For the definitive and limit test, all analytical procedures used. The
accuracy of the method, method detection limit, and limit of quantification should
be given.
(xx) Frequency and method of determining food consumption per pen and any
repellancy or food palatability issues.
(xxi) Methods and frequency of measuring number of dead animals, observing
signs of intoxication, including regurgitation, and other abnormal behavior,
including time of onset, duration, severity, and number affected at each dosage or
dietary level and control.
(6) Results. As a specific wild mammal test may include additions of effects measured
to accommodate case-by-case issues to be addressed by wild mammal testing, the
following list is not exhaustive but provides examples of the type of result reporting
elements for acute oral and dietary testing and some elements of longer duration tests.
All result reporting elements that will comprise the report should be part of the submitted
protocol reviewed by the Agency.
(i) For an oral dose test, tabulation of test substance dosage analytical results by
treatment (provide raw data).
(ii) For a dietary exposure test, tabulation of dietary test substance analytical
results by pen and treatment (provide raw data). Mean, standard error, minimum
and maximum test substance concentrations (mg a.i./kg-diet for pesticides) by
observation time and treatment level. Include a statement about the stability and
homogeneity of the test substance in the diet throughout the exposure period
based on results.
(iii) Environmental monitoring data results (temperature, humidity) in tabular
form (provide raw data for measurements not made on a continuous basis), and
descriptive statistics (mean, standard deviation, minimum, maximum). If study
was conducted outdoors, include a tabular summary of other weather conditions
monitored: precipitation, wind, etc.
(iv) For preliminary range-finding tests, if conducted, tabulate the number and
percentage of animals that died in each test pen or cage, treatment level and in the
control at each observation period. A description and count of any other
appearance or behavioral effects at each treatment level and in the control.
Tabulate the results of gross pathological examinations in dead and samples of
surviving animals.
(v) For a limit test, tabulate for the limit treatment level and the control the
number of dead animals in each pen or cage at each observation time during the
test (provide the raw data).
(vi) For the definitive test, tabulation by test pen and treatment of the number of
dead animals at each observation time during the test (provide the raw data).
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(vii) For the definitive test, tabulation of the percent dead by treatment group,
adjusted for control mortality.
(viii) Tabulation of body weights of each animal by pen and observation time
(provide raw data) and the body weight gain for each mammal by pen between
test initiation and termination. Descriptive statistics of body weight gain (mean,
standard deviation, standard error, 95% confidence limits, median, minimum, and
maximum) by treatment level and plot of mean, median, minimum and maximum.
For longer duration studies, tabulation and plot of the mean change in body
weight by observation interval and treatment level during the course of the test
(e.g., day 0 to 14, 14 to 21, 21 to 28, etc.).
(ix) For the limit and definitive test, daily food consumption for each animal and
wastage by treatment group (provide raw data) and the calculation of total food
consumption for each animal from test initiation to test termination. Indicate
whether food was regurgitated or spilled (wasted) and in which treatments.
Descriptive statistics of body weight gain (mean, standard deviation, standard
error, 95% confidence limits, median, minimum, and maximum) by treatment
level and sex, if applicable, and plot of mean, median, minimum and maximum.
For longer duration tests, tabulation and plot of the mean change in body weight
by observation interval and treatment level during the course of the test (e.g., day
0 to 14, 14 to 21, 21 to 28, etc.).
(x) For the limit and definitive test, description of signs of intoxication and other
abnormal behavior, including time of onset, duration, severity (including death),
and numbers affected in the different test substance treatments and controls each
day of the test (provide raw data).
(xi) For the limit and definitive test, tabulation by test pen of treatment gross
morphology and necropsy results of dead animals and samples of surviving
animals at test termination (provide raw data). The tabulation should include the
weight of the animal at the time death is observed.
(xii) Graphs of the dose-response data for percent mortality.
(xiii) For an acute oral or dietary limit test, provide conclusion about the LDso or
LCso, respectively, being above the limit level and the lack or presence of other
signs of toxic effects at the limit level.
(xiv) For the acute oral or dietary definitive test, where sufficient data exist to fit a
model (e.g. probit) the slope of the dose-response curve and its standard error and
95% confidence limits and any goodness of fit results.
(xv) If determined for the acute oral or dietary definitive test, the NOEL for
mortality.
(xvi) For longer exposure continuous or repeated exposure tests, the NOEL/LOEL
or NOEC/LOEC and MATC values for each measurement endpoint of concern.
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(xvii) For acute oral and dietary tests, description of statistical method(s) used for
point estimates, including software package, for determining the LDso or LCso
value, fitting the dose-response model, and the basis for the choice of method.
Provide results of any goodness-of-fit tests.
(xviii) Description of statistical method(s) used for NOEL and LOEL (or NOEC
and LOEC) determination, including software package, and the basis for the
choice of method.
(i) References. The following references are recommended for additional background material
on this test guideline.
(1) American Institute of Biological Sciences (AIBS), 1974. Analysis of Specialized
Pesticide Problems, Volume VI, Wildlife Toxicology Study, pages 4 to 9. EPA Contract
No. 68-01-2457, unpublished.
(2) American Society for Testing and Materials. ASTM E555-95, Standard Practice for
Determining Acute Oral LD50 for Testing Vertebrate Control Agents, Annual Book of
Standards, Vol 11.05, reapproved 1995, deleted in 2000 from Annual Book of ASTM
Standards, Section 11, Vol. 11.05.
(3) Conover, W. 1980. Practical Nonparametric Statistics, 2nd Edition. John Wiley &
Sons, Inc., New York, NY. 493 pp.
(3) Fletcher, J., J. Nellessen, and T. Pfleeger. 1994. Literature review and evaluation of
the EPA food-chain (Kenaga) Nomogram, an instrument for estimating pesticide residues
on plants. Environmental Toxicology and Chemistry 13(9): 1383-1391.
(4) McCann, J.A., W. Teeters, DJ. Urban, and N. Cook, 1981. A short term dietary
toxicity test on small mammals, in Avian and Wildlife Toxicology: Second Conference,
D.W. Lamb and E.E. Kenaga, eds., American Society for Testing and Materials, pp. 132-
142.
(5) Nellessen, J. and J. Fletcher. 1992. UTAB: A computer database on residues of
xenobiotic organic chemicals and heavy metals in plants. Journal of Chemical
Information and Computer Sciences 32:144-148.
(6) U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard
Evaluation Procedure: Wild Mammal Acute Toxicity Test. EPA-540/9-85-004. Office of
Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC, June 1985.
(7) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines
Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. Office of Pesticides
Programs, Washington, D.C. EPA-540/9-82-024, October 1982.
(8) U.S. Environmental Protection Agency, 1983a. Testing for Environmental Effects
Under the Toxic Substances Control Act. U.S. Environmental Protection Agency, Office
of Toxic Substances (now: Office of Pollution Prevention and Toxics), Health and
Environmental Review Division (now: Risk Assessment Division), Environmental
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Effects Branch (now: Existing Chemicals Assessment Branch [in part]), Washington,
D.C., 24 pp.
(9) U.S. Environmental Protection Agency, 1983b. Technical Support Document for the
Testing for Environmental Effects Testing Scheme. U.S. Environmental Protection
Agency, Office of Toxic Substances (now: Office of Pollution Prevention and Toxics),
Health and Environmental Review Division (now: Risk Assessment Division),
Environmental Effects Branch (now: Existing Chemicals Assessment Branch [in part]),
Washington, D.C., 31 pp.
(10) U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard
Evaluation Procedure, Wild Mammal Acute Toxicity Test. Office of Pesticides and
Toxic Substances, Washington, D.C. EPA 540/9-85-004.
(11) Smrchek, J.C. and M.G. Zeeman, 1998. Assessing risks to ecological systems from
chemicals. In Handbook of Environmental Risk Assessment and Management, P. Calow,
ed., Blackwell Science, Ltd., Oxford, UK, pp. 24-90, Chapter 3.
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