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 Testingi **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 ac ronym (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 p rovides 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 o r 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 th at 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 situat ions. 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**

**O CSPP**  **850.24 00**  **: 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 T est (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 (LD 50) or dietary median lethal concentration (LC 50), or longer term continuous or repeated exposure no - observed

- effect level (NOEL) or no - observed

- effect concentration (NOE C) data from subchronic studies for wild mammals of chemical substances and mixtures (“test chemicals” or “test substance s”) subject to environmental effects test regulations. While the LD 50 or LC 50 studies are specifically designed to allow calculation o f the LD 50/LC 50 , 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 ty pically a laboratory

study designed to determine either the acute toxicity o f a test substance such as an acute oral LD 50 , or an acute dietary LC 50 , or it is designed to determine the subchronic toxicity o f a test substance

. The test substance is usually

administered orally (b y 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 o f the elements of an acceptable wild mammal study; it does not address every possible aspect of all the potential ty

pes of tests that might be conducted under this guideline. (2) **General test**  **guidance**

. The general guidance in OCS PP 850.2000 applies to this guideline except as specifically noted herein. B ecause the Agency intends that toxicityPage **2** of **24**

tests on wild mammals be conducted only to assess specific situations, no single test methodology

c an provide adequate guidance for all cas es. There are no widely accepted protocols that include husbandry practices appropriate to a diversity o f 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 Agenc y. However, the general guidance provided in this document may be used as a starting point for protocol development. (i) **Acute oral**  **(LD 50** **) tests.**  Methods for oral toxicity tests may

b e adapted from the avian acute LD 50 study in OCSPP 850.2100 and ASTM E555, Practice for Determining Acute Oral LD 50 for Testing Vertebrate Control Agents (see paragraph (i)(2) of this guideline). (ii) **Dietary LC**  **50 and**  **continuous or**  **repeated exposure**  **no -**  **effect level**  **tests.** Methods for dietary

tests may

b e adapted from the subchronic oral dosing studies for mammals in OCSPP 870.3100 and/or from the avian dietary LC 50 study

in OCSPP 850.2200. S ee also McCann et al ., 1981 (see paragraph (i)(4) of this guideline). (iii) **Rat acute**  **(LD 50**  **and LC**  **50 )**  **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 o f industrial chemicals (see paragraphs (i)(7), (i)(8) and (i)(10) in this guideline). In addition to the rat acute oral LD 50 , the results of other tests conducted for human health effects may b e considered during the assessment process. These results include data from single dose oral LD 50 tests with other species, acute inhalation LC 50 tests with rats, dermal irritation tests with rabbits, and dermal sensitization tests with various species. (iv) **Toxicity studies**  **for large**  **and relatively**  **scarce ma**  **mmals.** An example of an acceptable protocol for toxicity studies with mammals that are large, rel atively 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 o f 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 b e 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 b e 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) **D efinitive test** 

― (i) **Acute oral**  **LD 50**  **and dietary**  **LC 50**  **studies.** The goal of an acute oral or dietary definitive study

i s 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 (LD 50 or LC 50), 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 l evels are confirmed by chemical analys is under test conditions. (ii) **Subchron ic**  **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 l evels of the test substance , plus appropriate controls. The dosage or dietary l evels are confirmed by chemical analysis under test conditions. (5) **Lim it**  **test for**  **a cute**  **oral LD**  **50 and**  **acute dietary**  **LC 50**  **tests.** For test substance s expected to have relatively l ow toxicity, a limit test may b e 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 LC 50 or LD 50 may bereported 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 analys is 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

. S igns of intoxication, if any, should be reported. C onduct 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 b e necessary
. For pesticides if frank sublethal effect( s ) are observed in one or more mammals and the limit dose te sted was: 1) less than ten times the maximum expected EEC, then a full definitive study i s 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 oralPage **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. F or 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 ty pe 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 repeate d exposure (see paragraph (e)(2)(iv). (i) **Acute oral**  **LD 50**  **study**

. Single oral dose followed by 1 4 days of observation after dosing. (ii) **Dietary LC**  **50 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 sy mptoms 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 b e extended in relevant increments. Extensions should continue until no apparent toxicologically

related sy

mptoms are observed. F or example, in an acute oral dosing study

(paragraph (e)(2)(i) of this guideline), if mortality o ccurs 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 o f 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**  **NO EL/LOEL**  **and NOEC/LOEC**  **test s**  **.** 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) ofPage **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

b e pen- reared or wild captured, but should be phenoty pically indistinguishable from a free

- ranging wild mammal. In general, a spe cies 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

o f 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 f rom sources whose stocks have known breeding histories. Purchase d 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

b e required. This requirement is usually associated with large or scarce mammal testing. H owever, 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 whereb y 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 sy

mptoms. (B) Ty pically, 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 b e insufficient. P ersonnel conducting the test should provide a written account of how the test animals were determined to have acclimated to the testPage **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 penne d 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 o f 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. H owever, this may not be feasible when the age test standard is for young animals ( i.e ., LC 50 studies). F or a satisfactory

test, when significant mortality o ccurs during the acclimation period, a certification of the health status of the rest of the animals by aveterinarian 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 b e 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 m etals, and other contaminants; however, diets should be analyz ed 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 c apsule or gavage (acute oral or the no

- effect repeated exposure te sts, see paragraphs (e)(2)(i) and (e)(2)(iv), respectively), or b y 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. D iets can be mixed by c ommercial mechanical food mixers. Recommend that the test substance be added to a small quantity o f 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 analyz ed 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 substance s known to hydrolyz e readily. When a test substance is not water soluble, it may b e 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 glyc ol, and gum arabic (acacia)

. The maximum amount of vehicle in the diet should not exceed 2% of the diet by w eight. U se 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 di et 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 g avage
. 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 ty

pically determined at the time of dosing, but may b e taken, especially

f or capsules, within 24 hours prior to dosing. If necessary, an evaporative vehicle (such as acetone or methylene chloride)Page **8** of **24** may b e used in preparing the test substance for use in capsules. F or 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 c arrier is distilled or deionized water unless the test substance is known to hydroly ze readily. Other acceptable carriers include corn oil, prop yl ene glyc ol, 1% carboxymethyl

cellulose, and gum acacia. M aterials with known toxic or emetic properties should not be used. Inall 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**  **LD 50**  **and dietary**  **LC 50**  **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., LD 10 to LD 90 ; LC 10 to LC 90) is adequately characterized. If a range finding test is conducted, treatment levels should be spaced so that at least three cause mortality

b etween, but not including, 0% and 100%. F or a scientifically

sound estimate of a point on the curve ( e.g., LC 50), responses should immediately

bracket the point estimate of concern. F or some test substance s, it may

b e 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 b y 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 c lose 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/LOAE L and consequently MATC can be determined.Page **9** of **24** (C) **Limit Test** 

**– Acute**  **Oral LD**  **50 .**  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. E quation 1 of this guideline can be used to calculate the acute mammalian oral limit dose (mg a.i./kg

-bw) for spray a pplications of pesticides. The dietary residue estimates are based on a nomogram that relates food item re sidues 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 i s 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 Agenc y prior to conducting the study

to determine the appropriate dose to use. 

##      

      

   

### TW

AW C bw kg i mga dose Limit diet 

### 953

. 0 ) /

. . ( max **Equation 1**

where: for a pesticide use with a single application per ye ar:



##   

240 max 

### ApRate C diet 

### 

 **Equation 2** for a pesticide use with more than one application per year: 

##        

# 

         



 

###                

 n

i erval n halflife diet 

### e

ApRate C
1 int 1 6931

. 0

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 LC**  **50 .**  For a limit test, there is single dietary l evel, 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 c oncentration. T o calculate the dietary

l imit 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 ye ar and Equation 2 of this guideline can be used where there are multiple applications per ye ar. If there are multiple uses this study i s 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 ye ar: 

##    240 ) /

. . ( ApRate

diet kg i mga ion Concentrat Limit Dietary   **Equation 1** for a pesticide use with more than one application per year:



##       

###                            

 

# 

          n

i erval n halflife 

### e ApRate ion Concentrat Limit Dietary
1 int 1 6931

. 0 

### 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. F or 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 vehicle s to justify w aiving vehicle controls for vehicles other than reagent water. M ammalian 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. C ontrols are important in assessing background mortality and disease. B ackground 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 or ganisms and replicates** 

― (i) **Acute oral**  **LD 50**  **and dietary LC 50**  **tests.** For a regression

-based endpoint test ( i.e ., LD 10 , LD 50), a minimum of 10 animals is generally recommended in each treatment group, and in the controls. H owever, 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. H owever, the number of replicates should be sufficient to produce a statistically

v alid 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 o f 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 su**  **pplies**
**.** Normal laboratory e quipment and supplies, and items especially listed in (e)(7)(i) through (e)(7)(iii). (i) **Fa cilities.**  (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) **Environ mental**  **mon itoring**  **equipment.** Under outdoor testing conditions, a maximum recording thermometer and relative humidity a pparatus will be necessary
. (ii i) **Pens/Cages**

― (A) **Size.** Generally, the animals’ enclosures need to be large enough so that they c an 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 somePage **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. M aterials that are toxic, may

affect toxicity, or may

adsorb test substance s 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. S team cleaning of cages is recommended. C ages may b e 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 l ong as such techniques will not leave chemical residues on the cages. F or cold sterilization, ethylene oxide is recommended. (2 ) Depending upon the ty pe of pens used, pens may b e 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) **T emperature**  **and humidity.**  For a satisfactory test, the air temperature and humidity within a laboratory

should be ty

pical of the selected mammalian species’ thermoneutrality range. (ii) **Light ing**  **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 b e either incandes cent or fluorescent. P ens andPage **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 1 0 to 15 air exchanges per hour. ( 9 ) **Observations.** The following list is not exhaustive, but provides elements of the ty pe of observations made in an acceptable study. (i) **Analysis for**  **test substance**  **concentrations.** Analy tical confirmation of the dosing media or the treated diet should be performed as described in OCSPP 850.2000 using analy

tical 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

o f 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) **C ontaminants**  **in feed** 
**.** Contaminated feed may c ompromise study

results, therefore, feed should be analyz ed periodically to identify

b ackground contaminants such as heavy metals ( e.g
. arsenic, cadmium, lead, mercury, and selenium) and persistent pesticides. Abroader pesticide screen to include other chemicals that are known to be acutely toxic to mammals may be useful. (ii i) **B asal**  **diet composition.**  A nutrient analysis of the basal diet should be included in the test report. The analysis should include percent by w eight 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. M ost commercial feed companies provide both the analys is and the list of supplements on the label. (iv) **Environm ental 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 rec orded daily atrepresentative 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

l ength from the beginning of the acclimation period to the termination of the test should be recorded. D epending on the totalPage **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 i s equipped with an inside/outside air exchange system, the rate and volume of exchange should be measured. (v) **Measures of effect**

― (A) **Monitoring of**  **ma mmals.**  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 o f the test period. F or dietary treatment studies observation of test animals should be made, at a minimum, 3 times on the first day o f 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

. S igns of intoxication are those behaviors apparently due to the test substance and may

include a wide array

o f behaviors, such as labored respiration, leg weakness, hemorrhage, convulsions, excessive salivation and paralysis. Abnormal behaviors, such as excessive aggression, hy peractivity 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 s ex, 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

. H owever, if the study

i s greater than two weeks in durati on, weekly b ody weight measurements are recommended. B ody w eights can indicate sublethal effects of the toxicant. C ontrol group body weights are used as a baseline for comparisons to the vehicle controls and treatment group’s body weights. (D) **Food con**  **sumption.** Food consumption is monitored daily f or 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 necrops y is necessary depends on the test substance and the endpoint of the study . For acute testing, all animals that die during the testPage **15** of **24** should be necropsied. F or a satisfactory study, a representative group of the survivors (usually 50%) should be necropsied for comparison to those that died. The survivors necrops y will provide insight into possible sublethal effects. Agross necropsy of organs and muscle tissue is usually adequate. F or tests where a NOEL/LOEL (NOEC/LOEC) and MATC are to be determined, any animals that exhibited an effect should be necropsied. S amples are taken from test organisms not visually affected, and from control organisms. B oth gross and histological necropsies (with possible tissue res idue 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. F or body

w eight 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 e ffects of the test substance on consumption of food. F or 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

. F or 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 c alculate the percent mortality, adjusted for control mortality at test termination, at each test substance dosage or dietary c oncentration. Cumulative number of dead for each dosage or dietary l evel and control group by observation day

should be summarized in tabular form. (3) **Appearance and**  **behavior.** Number of animals with appearance and behavioral sy mptoms should be summarized in tabular form by time of observation, treatment and sex (if applicable). T abul ate among survivors, remission of signs of intoxication and cessation of abnormal behavior by dosage level and by

observation day

. (4) **Gross pathology**  **and necropsies.**  Ty pes of observed pathologies and number of dead or examined surviving mammals with thes e lesions should be summarized in tabular form by treatment and sex. (5) **Limit test**

― (i) **Acute oral or dietary**  **tests**

― (A) **Acute oral**  **LD 50**  **value.** At test termination, if no animals die at the limit dose, the acute oral LD 50 is considered to be greater than the limit dose ( i.e ., LD 50 > limit dose, ty pically 2,000 mg/kg

-bw). This is because the Binomial Theorem predicts that when 10 organisms are tested, the probability

o f seeing no mortality

i f the true LD 50 is at or below the limitPage **16** of **24** dose is < 0.001. C onversely the probability

o f seeing one or more dead mammals if the true LD 50 is at or below the limit dose is > 0.999. (B ) **Acute dietary**  **LC 50**  **value.** At test termination, if no animals die at the dietary

l imit concentration, the acute dietary LC 50 is considered to be greater than the limit dose ( i.e ., LC 50 > limit dose, ty pically 5,000 ppm). Binomial theory

holds that when 10 organisms are tested, the probability of seeing no mortality

i f the true LC 50 is below the limit dose is < 0.001. Conversely the probability o f seeing one or more dead mammals if the true LC 50 is below the limit dose is > 0.999. (i i) **Proportion of**  **mortality (**  p ˆ **).** The Binomial Theorem also can provide both
an estimate of the true proportion of mortality ( p ˆ ) 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). F or small sample sizes the interval may

be large. For example, f or no mortality

in 10 mammals ( p

ˆ = 0), the upper 99% confidence bound on the estimate of p ˆ is (0. 0 0, 0.41 ) and 95% confidence interval is (0. 0 0, 0.31)

. U sing 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 ˆ 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 LD 50 or LC 50 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

b e 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 i s 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 LD 50 or LC 50 test may b e 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) ex cept for the number of dosage levels the test procedures and duration are the same as in the def initive test; 5) the dosage level is confirmed by chemical analys is under test conditions; and 6 ) the limit dose was 2,000 mg/kg

-bw, 5,000Page **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**  **LD 50**  **and dietary**  **LC 50**  **tests.** Statistical procedures are employed to calculate the LD 50 or LC 50 (standard error and 95% confidence interval). If a dose

-response curve model (e.g., probit) was fit to the data to determine the LD 50 , or LC 50 , the model parameters (e.g., slope) and their uncertainty e stimates (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 analys is 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**  **LD 50**  **and dietary**  **LC 50**  **tests.** Data shoul d be adjusted to account for any control mortality

. S tatistical analysis may b e conducted by probit analysis by

transforming the dose levels to logarithmic values and the mortality

response to probit units. Other standard statistical methods are acceptab le if they provide the same information listed above. Observations on body

weight and feed consumption may b e 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, hy pothesis 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 b e 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. H owever, 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. B efore 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, allPage **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 b etween doses was not used in studies where only the LD 50 or LC 50 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 o f 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 c oncentrations 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 organism**  **s.** (i) Name of species tested (including scientific name). (ii) Information about the source: ty pe, name, collection location, date, and method if from the wild, breeding history i f 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 b ehavior of each animal and mortality during the acclimation period. (ix) Body weight of each animal at the beginning of the test (ty pically 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 conditi** **ons.** 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 ty pe 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 b y the Agency. (i) Description of housing containers including: ty pe, 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 o f 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, ty pe, nutrient analys is 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 analys is. (vii) The number of pens or cages per test substance dosage or dietary l evel 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 grou

p 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) Ina

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 o f environmental monitoring performed during the definitive or limit study

for temperature, humidity, and air exchange. (xvi) For tests conducted outdoors the methods and frequency o f monitoring performed during the definitive or limit study f or weather conditions that may affect the test. (xvii) Description of any

medication given during the test: ty pe, frequency, and an explanation of how it was administered and justification of why it was given. (xviii) Methods and frequency o f measuring test substance to confirm exposure dosages or dietary

l evels, 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 analy tical 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 f ood palatability

issues. (xxi) Methods and frequency o f 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 ty pe 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 b y the Agency. (i) For an oral dose test, tabulation of test substance dosage analy tical results by treatment (provide raw data). (ii) For a dietary

exposure test, tabulation of dietary test substance analy tical results by p en and treatment (provide raw data). M ean, 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

o f 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 o f 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

. Adescription 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 l imit 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 ob servation 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 p en and observation time (provide raw data) and the body w eight gain for each mammal by p en between test initiation and termination. D escriptive statistics of body w eight 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 f ood 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 me an, 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 o f 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 l imit test, provide conclusion about the LD 50 or LC 50 , 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 NOELf or 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 LD 50 or LC 50 value, fitting the dose

-response model, and the basis for the choice o f method. Provide results of any goodness

- of

- fit tests. (xviii) Description of statistical method(s) used for NOEL and LOE L (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 S tudy, 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) C onover, W. 1 980. Practical Nonparametric Statistics, 2 nd Edition. John Wiley & Sons, Inc., New York, NY. 493 pp. (3) Fletcher, J., J. Nellessen, and T. Pfleeger. 1 994. 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 f or Testing and Materials, pp. 132

- 142. (5) Nellessen, J. and J. Fletcher. 1 992. UTAB: A computer database on residues of xenobiotic organic chemicals and heavy metals in plants. Journal of Chemical In formation 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 Agenc y, 1982. P esticide 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. T esting 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), EnvironmentalPage **24** of **24**

Effects Branch (now: Existing Chemicals Assessment Branch [in part]), Washington, D.C., 24 pp. ( 9 ) U.S. Environmental Protection Agency, 1983b. T echnical 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 As sessment 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 T est. O ffice of Pesticides and Toxic Substances, Washington, D.C. EPA 540/9

- 85

- 004. (1 1 ) 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.