United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-022

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.2400:

Wild Mammal

Toxicity Testing
i

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.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
Page 1 of 24

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 (LD50) or dietary median lethal 

concentration (LC50), 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 LD50 or LC50 studies are specifically designed 

to allow calculation of the LD50/LC50, 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 LD50, or an acute dietary LC50, 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
Page 2 of 24

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 (LD50) 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 LD50 for Testing Vertebrate Control Agents (see 

paragraph (i)(2) of this guideline).

(ii) Dietary LC50 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 LC50 study in 

OCSPP 850.2200. See also McCann et al., 1981 (see paragraph (i)(4) of this 

guideline). 

(iii) Rat acute (LD50 and LC50) 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 LD50, 

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 LC50 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)(1) 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. 
Page 3 of 24

(4) Definitive test―

(i) Acute oral LD50 and dietary LC50 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 (LD50

or LC50), respectively, and standard error estimates and 95 percent (95%) 

confidence limits. An additional objective is to determine the slope of the doseresponse 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 LC50 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 LC50 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 
Page 4 of 24

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 LD50 study. Single oral dose followed by 14 days of observation 

after dosing.

(ii) Dietary LC50 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 toxicologically 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 

toxicologically 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 
Page 5 of 24

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 
Page 6 of 24

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., LC50 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. 
Page 7 of 24

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 

arabic (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) 
Page 8 of 24

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% carboxymethylcellulose, 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 LD50 and dietary LC50 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., LD10 to LD90; LC10 to LC90) is adequately characterized. If a rangefinding 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., LC50), 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. 
Page 9 of 24

(C) Limit Test – Acute Oral LD50. 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 lb 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.

  

 

















 



AW TW

C

Limit dose mga i kg bw diet 0.953 ( . ./ )

max Equation 1

where:

for a pesticide use with a single application per year:

 240  C max ApRate diet  Equation 2

for a pesticide use with more than one application per year:

  

    

















 

































n

i

n erval

halflife

diet C ApRate e

1

1 int 0.6931

max 240 Equation 3

ApRate = maximum single application rate (in lb a.i./acre);

Halflife = the foliar halflife (default is 35 days);

Interval = the minimum application interval (in days);

i = 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.
Page 10 of 24

(D) Limit Test – Acute Dietary LC50. 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./ kg  diet)  ApRate240 Equation 1

for a pesticide use with more than one application per year:

  

  

























































 

 















n

i

n erval

halflife Dietary Limit Concentration ApRate e

1

1 int 0.6931

240 Equation 2

ApRate = maximum single application rate (in lb a.i./acre);

Halflife = the foliar halflife (default is 35 days); 

Interval = the minimum application interval (in days); 

i = 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.
Page 11 of 24

(6) Number of test organisms and replicates―

(i) Acute oral LD50 and dietary LC50 tests. For a regression-based endpoint test 

(i.e., LD10, 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 
Page 12 of 24

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 
Page 13 of 24

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 
Page 14 of 24

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 
Page 15 of 24

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 LD50 value. At test termination, if no animals die at the 

limit dose, the acute oral LD50 is considered to be greater than the limit 

dose (i.e., LD50 > 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 LD50 is at or below the limit 
Page 16 of 24

dose is <0.001. Conversely the probability of seeing one or more dead 

mammals if the true LD50 is at or below the limit dose is >0.999.

(B) Acute dietary LC50 value. At test termination, if no animals die at 

the dietary limit concentration, the acute dietary LC50 is considered to be 

greater than the limit dose (i.e., LC50 > limit dose, typically 5,000 ppm). 

Binomial theory holds that when 10 organisms are tested, the probability 

of seeing no mortality if the true LC50 is below the limit dose is <0.001. 

Conversely the probability of seeing one or more dead mammals if the 

true LC50 is below the limit dose is >0.999.

(ii) Proportion of mortality ( pˆ ). The Binomial Theorem also can provide both 

an estimate of the true proportion of mortality ( pˆ ) 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 ( pˆ = 0), the 

upper 99% confidence bound on the estimate of pˆ 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 pˆ 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/kgbw, 5,000 ppm or the maximum EEC for either the acute oral or dietary 

study, whichever is greater), a definitive LD50 or LC50 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 LD50 or LC50 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 
Page 17 of 24

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 LD50 and dietary LC50 tests. Statistical procedures are employed 

to calculate the LD50 or LC50 (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 LC50, 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., chisquare 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 LD50 and dietary LC50 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 
Page 18 of 24

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.
Page 19 of 24

(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.
Page 20 of 24

(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 lbs 

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.
Page 21 of 24

(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).
Page 22 of 24

(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 LD50 or 

LC50, 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.
Page 23 of 24

(xvii) For acute oral and dietary tests, description of statistical method(s) used for 

point estimates, including software package, for determining the LD50 or LC50

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, 2

nd 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, D.J. 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 
Page 24 of 24

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