EPA-540/9-85-004
June 1985
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
WILD MAMMAL TOXICITY TEST
Prepared by
Russel Farringer, M.S.
Standard Evaluation Procedures Project Manager
Stephen L. Johnson
Hazard Evaluation Division
Office of Pesticide Programs
United States Environmental Protection Agency
Office of Pesticide Programs
Washington, D.C. 20460
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50272-10)
REPORT DOCUMENTATION
PAGE
1. REPORT NO.
3. R«cipi.rrt'm Accession No.
PB8fc 129 2-1 MS
4. Title and Subtitle
HAZARD EVALUATION DIVISION, STANDARD EVALUATION PROCEDURE
Wild Mammal Toxicity Test
5. Report Data
June 1985
7. AuttK>r
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STANDARD EVALUATION PROCEDURE
PREAMBLE
This Standard Evaluation Procedure (SEP) is one of a set of
guidance documents which explain the procedures used to evaluate
environmental and human health effects data submitted to the
Office of Pesticide Programs. The SEPs are designed to ensure
comprehensive and consistent treatment of major scientific topics
in these reviews and to provide interpretive policy guidance
where appropriate. The Standard Evaluation Procedures will be
used in conjunction with the appropriate Pesticide Assessment
Guidelines and other Agency Guidelines. While the documents were
developed to explain specifically the principles of scientific
evaluation within the Office of Pesticide Programs, they may also
be used by other offices in the Agency in the evaluation of
studies and scientific data. The Standard Evaluation Procedures
will also serve as valuable internal reference documents and will
inform the public and regulated community of important consider-
ations in the evaluation of test data for determining chemical
hazards. I believe the SEPs will improve both the quality of
science within EPA and, in conjunction with the Pesticide Assess-
ment Guidelines, will lead to more effective use of both public
and private resources.
John W. Melor.e, Director
Hazard Evaluation Division
11
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TABLE OF CONTENTS
Page
I. INTRODUCTION
A. When Required 1
B. Purpose 1
C. Test Substance 2
II. MATERIALS AND METHODS: TESTING STANDARDS/
RECOMMENDATIONS
A. Acceptable Protocols 2
B. Test Organisms 3
1. Species Selection 3
2. Acclimation 5
C. Testing Facility 6
1. Pen/Cage Description 6
2. Photoperiod 6
3. Ambient Temperature and Humidity 6
4. Laboratory Air Exchange System 7
D. Test Design 7
1. Number of Test Animals 7
2. Controls 7
3. Presentation of Test Substances 7
4. Test Levels 8
5. Test Duration 9
6. Body Weight Measurements 10
7. Toxicosis Evaluations 10
8. Basal Diets 10
9. Necropsies 10
III. REPORTING REQUIREMENTS
A. Test Protocol 11
1. Previously Accepted by EEB 11
2. Industry Designed 11
B. Test Substance 11
C. Suitability of Test Animals 11
1. Selection 11
2. Acclimation 11
D. Parameters Associated with Testing Facility .. 12
1. Physical 12
2. Biological 12
E. Test Exposure Parameters 12
1. Range Finding Test 12
2. Definitive Test 12
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TABLE OF CONTENTS (Continued)
Page
F. Criteria for End Point of Study 13
G. Necropsies 13
H. Body Weights and Food Consumption 13
I. References 14
III. REVIEWER'S EVALUATION
A. Review of Test Design 14
B. Verification of Statistical Results 14
C. Conclusions 14
1. Categorization of Results 14
2. Rationale 15
3. Repairability 15
D. Descriptive Classification 16
E. References 16
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WILD MAMMAL TOXICITY TEST
I. INTRODUCTION
A. When Required
Data on the toxicity of a pesticide to wild mammals are
conditionally required by 40 CFR §158.145 to support the registra
tion of an end-use product intended for outdoor application. The
toxicity data required by 40 CFR §158.135 for evaluating safety
to humans and domestic animals are normally adequate to indicate
risks to wild mammals. Under certain conditions, these data are
not sufficient to assess the potential risks to wild mammals.
Examples of circumstances when data on the toxicity of a pesticid
to wild mammals may be required by 40 CFR §158.145 include, but
are not limited to, the following:
O
O
When data (e.g., data required by 40 CFR §158.135)
indicate there is considerable variation in the sensi-
tivity of different mammalian species to the toxic
effects of a pesticide, and when there is evidence of
sufficient exposure (e.g., data required by 40 CFR
§158.130) to wild mammals through the proposed use
pattern;
When pesticides with bactericidal properties will be
applied to the forage of wild ruminants, and toxico-
logical data do not include information on possible
interference with rumen fermentation in domestic (or
wild) ruminants; and
When vertebrate pesticides which have known bioaccuma-
ation potential (e.g., most anticoagulants) and/or
persistence in the environment (e.g., data required by
40 CFR §158.130) are proposed for field use patterns
through which exposure to wild mammals is probable.
B. Purpose
When the Ecological Effects Branch (EEB) determines the
toxicity data requirements for humans and domestic animals are
insufficient to address the risks posed by, or the toxicity of,
the pesticide to wild mammals, this testing is required. By
the design of the test, the data generated under this requirement
will establish, at a minimum, one of the following:
° Acute toxicity levels (e.g., LC50 or LD50) of the
active ingredient through use of the technical product;
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° A no observable effect level (NOEL) or no effect level
(NEL); or
0 A maximum-acceptable-tolerated-concentration (MATC).
From the end points that are established by the data, EEB can:
° Compare toxicity information with expected or measured
amounts of pesticide in the terrestrial environment which
may be consumed by wild mammals, thereby assessing their
potential for adverse risks;
0 Defend precautionary label statement requests designed to
minimize adverse effects to wild mammals (including
threatened or endangered species); and
° Provide a better scientific rationale for additional wild
mammal tests.
C. Test Substance
The 40 CFR §158.145 indicates the test is to be conducted
with the technical grade material. If more than one active
ingredient is present, then testing with the technical grade of
each active ingredient can be required. The percentage of
active ingredient in the technical grade must be given. Lot
and batch numbers for the technical grade should be provided.
Under certain circumstances, the end-use product can be
required as the test substance (40 CFR §158.75(b)(7)). Examples
of circumstances include, but are not limited to;
° When the effects of the product on the rumen
fermentation process need to be determined;
° When primary toxicity to target and/or nontarget
organisms needs to be determined in order to address
bioaccumulation (e.g., a potential for secondary
poisoning); or
° When adverse behaviorial modifications are expected
from consumption of the product.
II. MATERIALS AND METHODS: TESTING STANDARDS/RECOMMENDATIONS
A. Acceptable Protocols
Based on the concerns or on the quest ions established
through the review of the data indicated in the Introduction
("When Required" paragraph), a protocol is designed to address
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the issues. EEB has not established a specific protocol for
all potential variables which would necessitate requiring wild
mammal toxicity testing. However, EEB does recommend the
following references as guidance for developing acceptable
protocols for wild mammal toxicity testing:
0 General
Office of Pesticide Programs. 1982. Pesticide Assessment
Guidelines Subdivision E, Hazard Evaluation: Wildlife
and Aquatic Organisms, EPA-540/9-82-024, pp. 43-48.
Office of Pesticide Programs. 1982. Pesticide Assessment
Guidelines Subdivision F, Hazard Evaluation: Human and
Domestic Animals, EPA-540/9-82-025, pp. 34-39, 66-76.
° Large and Relatively Scarce Mammals
Agr. Res. Service, USDA. Animal Disease and Parasite
Research Division. 1969. "The toxicity of some organic
herbicides to cattle, sheep, and chickens." A.R.S.
Production Research Report No. 106. U. S. Dept. Agri-
culture, Wash., D.C.
° Small Mammal Dietary LC50
McCann, J.A., Teeters, W., Urban, D.J., and Cook, N. 1981.
"A short Term Dietary Toxicity Test on Small Mammals",
Avian and Mammal Wildlife Toxicology: Second Conference,
ASTM STP 757, D.W. Lamb and E.E. Kenaga, Eds. American
Society for Testing and Materials, pp. 132-142.
0 Mammal Acute Oral LD50
"Standard Practice for Determining Acute Oral LD50 for
Testing Vertebrate Control Agents", ASTM Designation:
E 555-75 (Reapproved 1981).
When a registrant and/or a contract laboratory has designed a
protocol, EEB recommends submission of the protocol for approval
prior to initiating the study. When given this opportunity,
EEB can determine if the design is sufficient to address the
concerns that necessitated the request.
B. Test Organisms
1. Species Selection
Wild versus Captive: Selection of the test mammal is based
on those mammals exposed by the test substance's use pattern.
Generally, EEB prefers a species from a captive breeding or
commercial stock. These individuals are less likely to have
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been exposed to other toxicants prior to testing. The
history of their rearing and their age is usually documented.
Likewise, a larger number of "even" age individuals is usually
available from one source. These animals should be phenotypically
indistinguishable from a free ranging wild mammal.
One example in which wild captured mammals may be required,
due to a lack of a commercial source, is in the conducting
of a ruminant test. Usually the selected wild animal for the
ruminant test is an indigenous deer species associated with the
pesticide proposed use pattern.
If the study is to be conducted with rodents, a group of
animals could be trapped in the wild. These animals could be
bred, with the resulting offspring being utilized in testing.
Under no circumstances are threatened or endangered species
to be used in this test.
Under no circumstance is a animal with known previous pesticide
exposure to be used under this testing requirement.
Size: Based on Subdivision F testing requirements, the
animals used in wild mammal tests should not vary in weight more
than a + 20% of the mean of the test population. This variance
may be waived if adult animals which are relatively large or
scarce are utilized in the test.
Age: 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. Young mammals,
who recently completed weaning, are preferred for LC50 testing.
This age animal has a higher metabolic rate and usually will consume
more food on a body weight basis than an adult. Adult animals
are preferred for LD50 testing. This is primarily due to the oral
intubation of the test substance into the stomach of the animal.
For MTC or MATC testing, subadult or individuals not having
reached reproductive maturity are usually the preferred age
class. These animals have begun stablizing their weight gain,
and have matured to the extent they are behaviorally predictable.
Further, they are between the young and adult allowing greater
flexiblity and accuracy of extrapolation to other age groups.
NOEL or NEL testing is designed with either a LC50 or LD50 as an
additional end point, therefore the test animals will be of the
age class for the respective test.
Special concerns or testing procedures could mandate a
particular age class. An example of this would be:
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If the adults of a population were expected to be exposed
through dietary intake and the timing of application was such
that the young of year were subadult to adult, an LC50 study may
be requested with adult animals.
Sex: Sex of test animals must be specified. If females
are used, reproductive condition must be specified also.
Physical Condition; All animals should be in good health
and physical appearance. The holding or 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 holding/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 require-
ment is for young animals (e.g., LC50 studies).
In some cases, a certification of the health of the animals
by a veterinarian may be reguired as part of the study design.
This requirement is usually associated with large or scarce
mammal testing. However, it could be required when the use of
wild caught mammals are proposed for testing. Additionally,
when significant mortality occurs during the holding/acclimation
period, a certification as to the health of the rest of the
animals may be required.
2. Acclimation
Acclimation to pen facilities and basal diet should be for
a minimum of 14 days. In certain situations with certain test
animals, this timeframe may be insufficient. EEB requires
that the personnel conducting the test provide a written account
of how the test animals were determined to have acclimated to
the test conditions. The following depicts some, but not all
of the criteria that could be utilized for determining acclimation:
° If a wild mammal has lost weight during the
transition from the wild to penned conditions,
it can still be an acceptable test organism if the
weight is re-established during acclimation.
0 Responses to external stimuli, which may be initially
excessive, should return to "normal."
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C. Testing Facility
1. Pen/Cage Description
Generally, the animals' enclosure has to be large enough
that they can exhibit normal behavior and movement. The housing
and maintenance conditions should be in accordance with acceptable
animal husbandry practices (e.g., EPA Good Laboratory Practices and
USDA Animal Care Regulations). In some cases, visual screens
have to be placed between pens or cages to reduce aggression. If
the test is conducted outdoors, then the pens are required to
have a sheltered area. Additionally, all pens or cages must be
so designed that the animals have a continuous source of fresh
drinking water. Whether animals are housed separately or in
groups must be specified. If housed in groups, number per group
and sex-ratio per group must be specified.
2. Photoperiod
If the test is conducted in a laboratory, the preferred
photoperiod is 12 hours of light per 24-hour period. The
photoperiod should coincide with the natural daylight hours for
the region where the test is being conducted.
If the test is conducted in outdoor pens/cages, the day
length from the beginning of acclimation to the termination of
the test should be recorded. Depending on the total timeframe
that the test is expected to run, this measurement should be
conducted weekly or monthly.
3. Ambient Temperature and Humidity
The temperature and humidity within a laboratory must be
typical of the selected mammal's thermoneutrality range. During
the conduct of laboratory tests, the temperature and relative
humidity must be recorded on a daily basis. Variations during a
24-hour period must be measured.
Under outdoor testing conditions, a maximum recording
thermometer and relative humidity apparatus must be utilized.
The maximum and minimum values for temperature and relative
humidity have to be recorded daily.
Additionally, outdoor testing requires recording of rain-
fall, snow, or any other climatic condition which could affect
the results of the test.
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4. Laboratory Air Exchange System
If the laboratory is equipped with an inside/outside air
exchange system, the rate and volume of exchange should be
measured. If the active ingredient is volatile, this information
is required.
D. Test Design
1. Number of Test Animals
EEB generally requires a minimum of ten test animals in
each treatment group and in controls and/or vehicle controls.
However, depending on the test end point and the mammal to be
tested, the number of required animals per level could increase
or decrease. The criteria for determining the number of animals
is based on the necessary quantity of data points to achieve a
statistically and scientifically sound end point.
2. Controls
EEB requires concurrent control groups to be used. Vehicle
controls are required in addition to negative controls, when a
vehicle other than distilled water is used. There is insufficient
wild mammal historical data available to justify waiving vehicle
controls for other vehicles.
3. Presentation of Test Substance
a. Vehicle
A vehicle is a carrier or a solvent which allows uniform
dispersion of the test substance in a basal diet or a means of
oral dosing when the test substance cannot be administered as
is. The solvent of choice is distilled water. However, not
all active ingredients are soluble in water. Table grade corn
oil is the preferred carrier for those compounds insoluble in
water. Other solvents and/or carriers would be accepted as
vehicles in the study design, if the proposal contained documenta-
tion as to their toxicity.
b. Basal Diet Incorporation
A standard commercial mammal diet appropriate and familiar
to the test species should be used in preparation of the test
diets. The test substance should be mixed into the diet, if
possible, without a vehicle. The test substance should 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. EEB recommends that all
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treatment level diets be chemically analyzed to determine if
nominal levels are achieved by this mixing of the toxicant in
the basal diet. If a vehicle is determined to be necessary for
uniform mixing of test substance and basal diet, then it should
conform to the above criteria. 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
controls, aids in determining the effects of the vehicle on the
results of the test. The negative and vehicle control diet
consumptions are compared to each other and to the consumption
of treated diets.
c. Oral Dosing
The test substance should be administered, if possible,
without a vehicle. The material should be accurately weighed
for each test organism at each dosage level. The use of gelatin
capsules, to hold the respective dose levels for each animal,
is acceptable. If necessary, an evaporative vehicle (such as
acetone or methylene chloride) may be used in preparing the
material for use in capsules. This evaporative vehicle must be
completely evaporated at room temperature prior to placing the
test substance in the capsules.
When intubation or gavage techniques are used for oral
dosing, a vehicle is often required. Section E.(3)a. of this
document indicates the acceptable vehicles. In all preparations
of the test substance, the minimum amount of vehicle necessary
must be used. Under no circumstances should the maximum vehicle
amount per dose exceed 1.0% of the body weight. Additionally,
the vehicle should be used on a constant volume/body weight
basis.
4. Test Levels
a. Range Finding Test
EEB recommends use of a range finding test. When the
definitive test is required, because the preliminary data
indicates varying sensitivities to the test substance, then the
range finding test is recommended. EEB recommends the use of
three dose levels with five animals per level. The dose levels
which are utilized should be based on a mathematical factor
associated with the expected terrestrial residues. This mathe-
matical factor can be derived by reviewing the slope of the
dose/response line from other mammal testing.
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Depending upon the required end point of the study, the
expected or measured residues could be the high (MATC), median
(LC50/LD50), or low (NOEL) dose level.
b. Definitive Test
The definitive test levels may not be necessary if the
range finding test end point indicates an acceptable margin of
safety between expected or measured residues and its end point
for MATC or NOEL. If the LC50 or LD50 values for the range
finding test exceed 5000 ppm and 2000 mg/kg, respectively, the
definitive test may not be required.
When a definitive test is necessary, EEB requires a minimum
of five dose levels. If a range finding test was not conducted,
then the number of dose levels should be increased to assure
that the end point of the definitive test is bracketed by known
levels. If a range finding test is conducted then the end
point value that is depicted is used as the median dose level.
Two geometrically spaced values above and below the end point
value are determined. The determination of the range of the
geometrically spaced dose levels should attempt to produce
mortality from 10 to 90 percent (LC50/LD50). With MATC's and
NOEL's the additional test levels are split above and below the
end point of the range finding test. The geometric spacing of
doses is very close to the range finder end point. This is of
particular importance when the end point is close to the expected
or measured residues. With the closer spacing of doses a more
accurate MATC or NOEL can be determined.
5. Test Duration
The duration of the test is dependent upon the end point
of the study. Generally, the following timeframes are used:
0 LD50 - 14 days of observation after dosing;
0 LC50 - 5 days on the treated diet followed by
3 days of observation; and
0 MATC and NOEL - number of days exposure is based
on the persistence of the test substance in or
on food items.
If toxicologically related symptoms are evident at the
end of the suggested observation period for acute testing, the
observation period should be extended in seven day increments.
These extentions should continue until no apparent toxicologically
related symptoms are observed.
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6. Body Weight Measurements
EEB requires the measurement of body weight as a means of
determining the suitability of the test animal to the test
conditions. Body weights can indicate sublethal effects of the
toxicant. Control group body weights are used as a baseline
for comparisons to vehicle controls and treatment group's body
we ights.
Generally, EEB requires the body weights be taken at the
beginning and end of the study. However, if the study is
greater than two weeks in duration additional body weight
measurements may be required.
7. Toxicosis Evaluation
EEB requires daily observation of all test animals in
order to record any toxicant related effects. The following
are a few examples of observable effects: hyperactivity, paralysis,
hematomas, lethargy, excessive salivation, and aggression.
Since EEB requires acclimation of the animals to the test
conditions, personnel monitoring acclimation should be able, by
comparison, to determine observable toxicological effects.
8. Basal Diets
° Analysis: EEB requires the basal diet be described in
terms of its constituents. Since commercial diets are
produced with a quality control of the constituents, the
producers of these diets have developed formulae with
the appropriate percentages of each constituent. At a
minimum, EEB requires a quantitative description of any
medications and vitamins in the diet. If there is an
indication that the diet may have biased the results of
the test, additional quantitative data on the diet may
be required.
° Food Consumption: EEB generally requires daily food
consumption be recorded 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. The recording of food consumption ends
on the last day of the test.
9. Necrops ies
Necropsies are generally required. Whether a gross and/or
histological necropsy is required depends on the test substance
and the end point of the study. The number of animals necropsied
depends on the number used at each dose level and the toxicosis
of the test substances. Necropsies are required on all dead or
affected animals and a portion of the unaffected animals (50 to
100%) .
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III. REPORTING REQUIREMENTS
A. Test Protocols
1. Previously Accepted By EEB
Where EEB has previously accepted the protocol, the report
should reference the protocol. The report must state any
deviations occurring while the study was conducted. In order
to substantiate the deviations a sound rationale must be provided.
2. Industry Designed
When EEB requires a wild mammal test, industry must design
a protocol to answer our concerns. Therefore, EEB requires
that the complete protocol and rationale for industry's test
design parameters be submitted with the results of the study.
B. Test Substance
The test substance must be accurately described. The per-
centage of active ingredient, at a minimum, has to be reported.
The batch and lot number can be useful information. If a
formulated product was used, then the confidential formula for
the product must accompany the report.
C. Suitability of Test Animals
1. Selection
If EEB did not specify the test organism of concern, beyond
the family level, then a rationale for the species selected
should be made. Confirmation that the selected organisms had not
been previously exposed to other toxicants is required. The
method of determining the age of the animals and their respective
ages is required. The health of the animals and means of determini
their health are required reporting.
2. Acclimation
The following observations are the minimum required reporting
i terns:
° daily food consumption;
° daily behavior of test animals; and
° body weights (beginning and end of acclimation).
Additional specific test requirements may be required based
on specific test designs.
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D. Parameters Associated with Testing Facility
1. Physical
The following are required reporting items:
° size and construction of pen or cage with description
of any special features;
° location of and availablity of drinking water;
° ventilation system in a laboratory; and
° identification of any special laboratory equipment
unique to the study.
2. Biological
The following are required reporting items:
° daily temperature measurements;
° daily relative humidity measurements;
° daily photoperiod; and
° daily laboratory air exchange rate.
E. Test Exposure Parameters
1. Range Finding Test
The following are required reporting items:
0 the number of animals used;
° the method used to determine dose levels;
° the raw data for determining the end point;
° treatment and observation period; and
° any statistical analysis utilized.
2. Definitive Test
The following are required reporting items:
° the number of animals used and a rationale for
using less than ten animals per treatment level,
if appropriate;
° the number of treatment levels;
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° if a vehicle was used;
° references to toxicity of vehicle;
° if controls and vehicle controls were used;
0 the raw mortality or effects data for each group;
° the duration of treatment and observation periods;
° the statistical analysis of the data;
° the methodology for oral dosing;
° the methodology for treatment diet preparation;
° toxicologically related behavioral changes; and
° analysis of basal diet.
F. Criteria for End Point of Study
Since this study is developed around
questions which EEB has determined, based
other organisms and the use patterns, the
prior to testing.
G. Necrops ies
EEB requires necropsies for the acute, no effect level and
maximum-acceptable-tolerated-concentrat ion.
For acute testing, all animals that died during the test
must be necropsied. A representative group of the survivors
(usually 50%) must be necropsied for comparison to those that
died. The survivors' necropsy will provide insight to sublethal
effects. A gross necropsy of organs and muscle tissue is usually
adequate.
For tests where a NEL and a MATC are required, 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 necessary.
H. Body Weights and Food Consumption
The records for body weight and daily food consumption
should be presented by animal and level. The timeframe in
which body weights are taken is important. Body weights can be
used as an indicator of the physical condition of the animals.
The acclimation/treatment/observation period report on food
consumption will indicate the degree of exposure and if the
specific concerns or
on the toxicity to
end point is determined
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animals ate normally during the treatment phase.
I. References
Any references which were used in the development of the
study design must be cited.
IV. REVIEWER'S EVALUATION
A. Review of the Test Design
The reviewer determines if the test design was adequate to
address the EEB's concerns. Notations of deviations to a
previously approved protocol and a determination as to their
effect on the study are made. A review of any references cited
in the report is made in order to determine their applicability
to the test design.
B. Verification of Statistical Results
An integral part of the data evaluation process is the
verification of statistical analyses. The reviewer should
ensure that the end point has been properly derived by reanalyzing
the raw data in EEB's currently available statistical programs.
An acceptable acute toxicity test should provide additional
important information other than the LC50/LD50 values. Results
from a valid study should provide a NOEL and a slope of the
dose/mortality response line. These data can give further
insight into the toxicological characteristics of the test
substance such as whether the response is gradual over a wide
concentration range, or relatively rapid over a narrow range.
If the recalculated results differ substantially from the
submitted results, the reviewer should note and attempt to
explain the discrepancies.
C. Conclusions
1. Categorization of Results
The significance of inconsistencies in the test procedures
must be determined by the reviewer. Then the results of the
study can be categorized as to their usefulness in a risk
assessment and as to their fulfilling the Part 158 regulations.
Categories are described as:
0 Core: All essential information was reported and the
study was performed according to recommended guideline
protocols. Minor inconsistencies with standard methodolo-
gies may be apparent; however, the deviations do not
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detract from the study's soundness or intent. Studies
within this category fulfill the basic requirements of
Part 158 of the regulations and are acceptable for use
in a risk assessment.
° Supplemental: Studies in this category are scientifically
sound; however, they were performed under conditions
that deviated substantially from recommended guideline
protocols. Results do not meet regulatory requirements;
however, the information may be useful in a risk assessment.
Some of the conditions that may place a study in a
supplemental category include:
unacceptable test species;
inappropriate test material or diet; or
dosage levels were inadequate to substantiate
the end point.
° Invalid: These studies provide no useful information.
They may be scientifically unsound, or were performed
under conditions that deviated so significantly from
recommended guideline protocols that the results will
not be useful in a risk assessment. Failure to report
required information could place a test in this category.
Some of the conditions which place a study in an invalid
category include:
failure to report test substance;
use of non-indigenous species;
insufficient duration of acclimation/treatment/
observation period; and
significant control mortality.
2. Rationale
To support a supplemental or invalid category, the reviewer
must list and explain all test conditions that deviated from
the standard protocols.
3. Repairability
If any or all of the deviations can be re-examined and
found acceptable, then the study can be upgraded to a higher
category. The reviewer describes what is required by requesting
additional information about the study.
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D. Descriptive Classification
Tests which are acceptable for use in a risk assessment
can be categorically compared to other tests conducted with
other species or test substances by the following descriptive
classification:
ld50
Category
(mg/kg)
Description
< 10
very highly toxic
10-50
highly toxic
51-500
moderately toxic
501-2000
slightly toxic
> 2000
practically non-toxic
LC50
Category
(ppm)
Description
< 50
very highly toxic
51-500
highly toxic
501-1000
moderately toxic
1001-5000
slightly toxic
> 5000
practically non-toxic
These descriptive categories are for inter-chemical com-
parisons only and are not intended to reflect actual environmental
risks to mammalian wildlife.
E. References
EEB cites any references they used in the review and
validation of these studies. EEB indicates by citation if the
references used by the conducting laboratory were available for
review. A request for copies of pertinent citations may be
made before validation of the study.
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