&EPA
United States
Environmental Protection
Agency
Office of Pesticides Programs
Washington, DC 20460
EPA-540/9-85-020
June 1985
Hazard Evaluation Division
Standard Evaluation Procedure
Toxicity Potential (Guidance for
Analysis and Evaluation of Subchronic
and Chronic Exposure Studies)
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EPA-540/9-85-020
June 1985
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
TOXICITY POTENTIAL:
GUIDANCE FOR ANALYSIS AND EVALUATION OF SUBCHRONIC AND CHRONIC
EXPOSURE STUDIES
Prepared by
Orville E. Paynter, Ph.D., D.A.B.T.
Jane E. Harris, Ph.D.
Gary J. Burin, M.P.H.
Robert B. Jaeger, 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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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.
^^
.^axmn W. Melone, Director
Hazard Evaluation Division
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TABLE OF CONTENTS
PREAMBLE
I. ANALYSIS AND EVALUATION OF ADVERSE EFFECTS IN
EXPERIMENTAL ANIMAL SUBCHRONIC AND CHRONIC
EXPOSURE STUDIES
A. Definitions and Concepts 4
B. Documentation and Data Acceptance 13
C. Major Considerations for Analysis and
Evaluation 18
1. Mortality/Survival 19
2. Clinical Observations 22
3. Body Weight and Food Consumption 23
4. Hematologic, Clinical Chemistry and
Urinary Measurements 25
5. Organ Weights and Body Weight Ratios ... 29
6. Postmortem Observation 30
D. Consideration of Auxiliary Evidence 32
E. Completion of Analysis 32
II. EVALUATION OF WEIGHT-OF-EVIDENCE
REFERENCES 36
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Preamble
Of all the chemicals to which humans might be exposed,
pesticides are unique by reason of their deliberate introduction
into the environment to kill or otherwise control life forms
considered detrimental to human welfare. Experimental animals
have served as useful models for detection of potential human
responses to these poisonous substances. The Environmental
Protection Agency has published regulations relating to acceptable
practices for conducting and reporting animal studies-*-, as
well as guidelines^ that suggest acceptable and useful experi-
mental designs (protocols) for evaluation of adverse health
effects (hazards) relating to pesticidal agents.
The subchronic oral study has been designed to permit
determination of toxic effects associated with repeated exposure
for a period of 90 days^. This type of study can provide
information relating to toxic effects and potential health
hazards likely to arise from repeated exposures over a limited
time period. Data from this type of study are also useful in
predicting potentially important toxicity end points, identifying
potential target organs and systems, and in establishing the
dose regimen in chronic exposure studies.
The objective of chronic exposure studies 4 is the
determination of toxic effects and potential health hazards
following prolonged, repeated exposure. This type of study is
generally used for substances, and sometimes their metabolic or
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breakdown products, when repeated exposure to humans is likely
to take place over a significant portion of their life span as
is potentially the case with pesticide residues in the diet.
The purpose of this document is to present a very general
guidance framework for analysis and evaluation of data from
subchronic and chronic dietary exposures of rodents to pesticidal
agents. it does not; pretend to take the place of or mimic the
many excellent textg on the subjects of toxicology, clinical
chemistry and pathology, nor does it attempt to consider all
specific effects and the multiplicity of effect patterns likely
to be encountered in subchronic or chronic exposure studies.
However, what is discussed is equally applicable to studies
using other continuous routes of exposure, other species, and
other types of chemical agents.
This document can and should ba used in concert with the
Core Classification system in determining study acceptability.
The proper use of the Core Classifieation system requires an
understanding of the underlying basis for the various Core
"requirements" and assumes a knowledge of which study parameters
should be construed as requirements and which are merely suggest-
ions. Guidance is provided in this document on such topics as
the Maximum Tolerated Dose, the No Observed Effect Level, and
the utility of analyzing blood and urine.
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A definition of chemical oncogenicity and discussion of
implications pertaining thereto are presented by Paynter^.
This definition and discussion should be considered as part of
the guidance offered by this document.
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! Analysis and Evaluation of Adverse Effects in Experimental
Animal Subchronic and Chronic Exposure Studies
A. Definitions and Concepts
Both subchronic and chronic exposure studies, regardless of
routes of administration, share many common toxicity end points
used for hazard identification and risk assessment.
Prior to discussion of these end points, some comment on
terms and concepts presented in this document is appropriate.
Toxicity means the intrinsic capacity of a chemical substance
or a mixture of substances to induce injury. Hazard means the
observed toxic manifestation(s) induced by a known quantity or
quantities of a substance under known exposure conditions.
Risk means the probability that the identified hazard(s) will
or will not be encountered under anticipated exposure conditions.
The identification of hazard and assessment of the risk potential
of a given substance are informed judgments. Such judgments
are usually based on data relating to toxicity, proposed uses,
and anticipated exposure conditions. Use and expected exposure
conditions define the type, probable duration and quantity of
exposure, as well as the size and composition of the exposed
population. A particular pesticide product may have one
or several potential risks depending on use(s) and attendant
exposure conditions.
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The relationship of toxicity, hazard, and risk was perhaps
first articulated by Paracelsus (1493-1541) as, "All substances
are poisons; there is none which is not a poison. The right
dose differentiates a poison and a remedy."-* In 1975 the
National Academy of Sciences restated this principle thus, "A
chemical--any chemicalis a poison only as a consequence of the
quantity with which the host must deal."6 This concept is a
fundamental principle of toxicology and hazard assessment. The
risk of a pesticide to man and the environment is related to
exposure conditions and cannot be rationally equated per se with
the intrinsic toxicity of any substance. To illustrate this
point imagine two containment systems: (a) a perfect system
which absolutely prevents any exposure of man and the environment
to a substance having a dermal or oral toxic dose of 0.001 ug/kg
of body weight and (b) an extremely imperfect system which allows
high human and environmental exposure to the same substance. In
system (a) the exposure is zero and the risk to man and the
environment is also zero although the toxicity of the substance
remains unchanged. In system (b) the exposure is potentially
large and the risks of intoxication and other adverse effects to
man or the environment are potentially very great.
The term dose refers to a stated quantity or concentration of
a substance to which an organism is exposed and dose-response-
relationship means the correlative association existing between
the dose administered and the response (effect) or spectrum of
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responses that is obtained. The concept expressed by these latter
terras is indispensable to the identification, evaluation, and
interpretation of most pharmacological and toxicological responses
to chemicals. it is therefore important to understand the basic
assumptions which underlie and support the concept.
The primary assumption is that a dose-response-relationship
is firmly based on knowledge or a defensible presumption that the
response (effect) observed is a result of exposure to a known
substance. Correlative assumptions are: (a) the observed response
is a function of the concentration at a site, (b) the concentration
at a site is a function of the dose, and (c) response and dose
are causally related.7
The essential purpose of chronic exposure studies is the
detection of valid biological evidence for a toxic and/or an
oncogenic potential of the substance being investigated.
Therefore, protocols should maximize the sensitivity of the test
without significantly altering the accuracy and interpretability
of the biological data obtained. The dose regimen has an extremely
important bearing on these two critical elements. The concept
of the maximum tolerated dose (MTD) has had a significant influence
on the selection of doses for long-term (chronic) exposure studies
and on the interpretation of observed dose responses. This
subject has been discussed in relationship to oncogenicity data
bases.8
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Conscientious attempts to accommodate the MTD concept in
chronic studies, regardless of species used, have frequently
caused dose level adjustments in one or more animal groups and
these have frequently introduced interpretational difficulties at
the termination of the study. Misinterpretation of the intent of
the MTD concept has occasionally caused the invalidation of an
otherwise valid study or has caused its classification to be
inappropriately reduced when applying the Core Classification
scheme criteria. Therefore, the characteristics of the highest
dose to be used in modern chronic exposure studies should be
reconsidered and more clearly defined.8,9 ideally, the dose
selection for chronic studies should maximum the detection of
potential dose response relationships and facilitate the extrapola-
tion of these to potential hazards for other species including
humans. Therefore the largest administered dose, the MTD, should
be one which produces signs of minimal toxicity that do not
compromise biological interpretability of the observed responses.
For example, the upper dose should not: a) alter survival in a
significant manner due to effects other than tumor production;
b) cause a body weight decrement from concurrent control values
of greater than 10-12%; c) exceed 5% of the total diet because
of potential nutritional imbalances caused at higher levels or;
d) produce severe toxic, pharmacologic or physiologic effects
that might shorten duration of the study or otherwise compromise
the study results.
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Although it can be argued that responses observed at doses
far in excess of levels experienced under real or potential
exposure conditions legitimately fall within the classical dose-
response concept, there is a developing suspicion, based on
growing scientific evidence, that such doses introduce biases of
considerable importance into the already difficult task of
evaluating animal dose responses and the assessment of their
relevance to human hazard identification and risk.8 High
doses which produce severe tissue damage (i.e., necrosis demyelin-
ation) and/or interfere in a significant manner with metabolic
pathways or storage and excretion patterns in animal groups
should be thought of as extremely toxic doses which can make
interpretation difficult.
Responses produced by chemicals in man and experimental
animals may differ according to the quantity of the substance
received and the duration and frequency of exposure. In mammals,
acute experimental exposure is usually thought of as a single
exposure or multiple exposures occurring within twenty four
hours or less. Such exposure, if the substance is rapidly absorbed,
usually produces a mixture of responses. However, with this
type of exposure, some toxic effects may be delayed (i.e., certain
types of neurotoxicity, sensitization). Responses to acute
exposures may be both qualitatively* and ..quantitatively different
from those produced by subchronic and chronic exposures and not
all observed responses within a study, irrespective of exposure
duration or frequency, will represent toxicity per se. They
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will usually encompass a range of effects from physiologic through
pharmacologic and toxicologic manifestations. Although it may
be difficult at times to make a clear distinction between these
responses, an attempt to do so must be made. When an evaluator
is uncertain of the type or the biological significance of a
response, he or she should not hesitate to obtain competent
advice for resolving the uncertainty. It is essential that all
relevant toxicity end points be identified for consideration
when evaluating data for the presence or absence of nontoxic
levels.
The following discussion presents the distinction, as made
in this document, between three major response types - physiological,
pharmacological, and toxic. Physiological responses vary within
limits which are in accord with the normal functioning of a living
organism. Examples of such response are the usual respiratory
and pulse rate increases associated with increased physical
activity; systemic changes associated with normal pregnancy, and
those associated with homeostatic mechanisms. The variations in
this type of response are usually referred to as "normal ranges"
in clinical chemistry and other observational data. Generally
these variable factors are not important toxicity end points in
subchronic and chronic exposure studies unless their fluctuations
are abnormally altered by a dose regimen. If such alterations
occur at a specific dose or are part of a dose response relation-
ship, they should be correlated with other toxicity end points
which may be present.
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Pharmacological responses are altered physiologic functions,
are reversible, and are of relatively limited duration following
removal of the stimulus. While some of these responses may be
undesirable under certain circumstances, they are distinguished
from toxic (adverse) responses by generally not causing injury.
An example of this type of response is the increased activity
of the hepatic cytochrome P-450 containing mono-oxygenase systems
(enzyme induction) caused by exposure to many pesticides,
industrial chemicals, and drugs.
Toxic responses may be reversible or irreversible but are
distinguished from other types of responses by being injurious
and therefore adverse and harmful to living organisms. The
reversibility or irreversibility of a toxic response in animals
and humans will depend on the ability of the injured organ or
tissue to regenerate. For example, liver has a relatively great
ability to regenerate and many types of injury to this organ are
reversible. By contrast, differentiated cells of the central
nervous system are not replaced and many types of injury to the
CNS are irreversible.
An important concept, which has had several alterations in
nomenclature over the last decade, is here designated as the "No
Observed Effect Level" (NOEL). It is the dose level (quantity)
of a substance administered to a group of experimental animals
which demonstrates the absence of adverse effects observed
or measured at higher dose levels. This NOEL should produce
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no biologically significant differences between the group of
chemically exposed animals and an unexposed control group
of animals maintained under identical conditions.
Some implications of this definition need further discussion
and elaboration. Its acceptability and usefulness depend entirely
on the scientific rationale supporting the existence and demonstra-
bility of a threshold for almost all responses produced by biologi-
cally active agents. As used here, the term "threshold" designates
that level of a stimulus which comes just within the limits of
perception, and below which level a recognizable response is
not elicited. The earlier quotes of Paracelsus and the National
Academy of Science are based on this fundamental concept. Its
importance to the establishment of dose response relationships is
discussed by Paynter-8
The National Research Council10 has recently clarified
the concept of risk assessment and distinguished two essential
elements as follows:
Regulatory actions are based on two distinct elements?
risk assessment, the subject of this study, and risk
management. Risk assessment is the use of the
factual base to define the health effects of exposure
of individuals or populations to hazardous materials
and situations. Risk management is the process of
weighing policy alternatives and selecting the most
appropriate regulatory action, integrating the
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results of risk assessment with engineering data and
with social, economic, and political concerns to
reach a decision. Risk assessments contain some
or all of the following four steps:
0 Hazard identification: The determination of whether
a particular chemical is or is not causally linked to
particular health effects.
0 Dose-response assessment; The determination of the
relation between the magnitude of exposure and the
probability of occurrence of the health effects in
question.
0 Exposure assessment; The determination of the
extent of human exposure before or after application
of regulatory controls.
0 Risk characterization; The description of the
nature and often the magnitude of human risk, including
attendant uncertainty.
In each step, a number of decision points (components)
occur where risk to human health can only be inferred
from the available evidence. Both scientific judgments
and policy choices may be involved in selecting from
among possible inferential bridges, and we have used the
term risk assessment policy to differentiate those judg-
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ments and choices from the broader social and economic
policy issues that are inherent in risk management deci-
sions. At least some of the controversy surrounding
regulatory actions has resulted from a blurring of the
distinction between risk assessment policy and risk
management policy. 10
The concept of separating risk assessment and risk management
functions, to the maximum extent feasible, allows evaluators to
concentrate on analysis, evaluation, and interpretation of
toxicological data according to sound scientific principles
and without regard for what potential regulatory desisions or
actions the results may portend.
B. Documentation and Data Acceptance
The quality, integrity, and completeness of reporting observa-
tional and experimental data are essential to the proper analysis
and evaluation of submitted studies. In essence, the "good
science" evaluations expected of EPA have their foundations in
the submitted evidential documentation. Therefore, qualitative
assessment of the acceptability of study reports has special
significance for hazard identification and other aspects of risk
assessment.
The following three important considerations address the
acceptability of subchronic and chronic exposure studies and
evidential documentation.
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1. The adequacy of the experimental design and other experi-
mental parameters such as: the appropriateness of the observational
and experimental methods; frequency and duration of exposure;
appropriateness of the species, strain, sex and age of the animals
used; choice of doses, and the conditions under which the substance
was tested.
There are no specific, internationally agreed upon scientific
rules or fixed checklists which make the judgment regarding the
acceptability of reports a standard routine procedure. However,
there are suggested guidelines concerning the mechanics of good
experimental design, reporting, and laboratory practice which are
aids not only to the evaluation of report and data acceptability
but also to the generation of scientifically valid data. These
may be found in the OECD and EPA guidelines and the EPA and FDA
Good Laboratory Practices Regulations.1 However, the evaluator
needs to be cautious when using the above guidelines as aids to
making an acceptability judgment for any study. The cardinal
question to be answered is how well does the study in toto
facilitate the identification of potential adverse effects, or
lack thereof, for the substance being evaluated, and not how
precisely it fits a prescribed recipe for performance. The
collective experience of HED evaluators can be very helpful in
resolving difficult questions of acceptability and should be
utilized whenever needed.
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The evaluator should carefully read through the report including
supporting data presentations, and make a tentative classifica-
tion according to the Toxicology Branch Core Concept Manual. If
there are obvious and significant deficiencies in the report
which would lead the reviewing toxicologist to consider the study
invalid, any further work would be a waste of resources. The
submitter of the report should be notified, through the Product
Manager, of the problem(s) as quickly and as accurately as possible
and any further review suspended until these deficiencies are
corrected.
Occasionally, the subsequent detailed analysis of the data will
indicate deficiencies which were not obvious during the initial
reading of the report. The deficiencies should be noted and the
analysis completed as far as possible. The submitter of the
document should be notified of the situation and provided with
any scientific questions and other identified data needs.
2. The competency and completeness with which the study was
conducted and reported.
Doubts on the part of the evaluator regarding the completeness
and/or competency with which a study was performed or reported
must be discussed with the evaluator's supervisor. If the doubts
are judged to be reasonable, the study should be nominated for a
laboratory and data base audit. Any further consideration of
the study should be suspended until the audit is completed,
reported, and evaluated.
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3. The effects of modifying factors which result in major
inequalities between control and treated animals.
This qualitative consideration has more to do with the evalua-
tion and interpretation of data than with acceptability of documen-
tation. It is placed here because determination of the various
factors influencing toxicological data, as may be indicated in
the submitted evidential documentation, needs to be made prior to
the detailed data analysis.
There are many factors influencing the responses of experi-
mental animals to chemical substances. Some of these are discussed
by Doull-'--'- and his presentation of this subject should be
reviewed. Some influences may be quite subtle as exemplified by
studies performed by Thompson et al.12 it had been noted
that acute pulmonary edema occurred in rats being used in immune
hypersensitivity studies and that the onset of this effect was
sudden and seasonal. The onset was coincidental with hair-coat
changes in laboratory rats as judged by shedding. Subsequent
studies demonstrated that sulfur deficiency, which occurs season-
ally in rats and which, according to the authors, primes the
animal for pulmonary edema onset, also changes glucose and glycogen
levels. The onset of acute pulmonary edema susceptibility was
apparently due to seasonal alterations (hair-coat changes) in
sulfur and carbohydrate metabolism as well as possible variations
in insulin and other hormone levels. Circadian rhythms and
seasonal physiological variations can subtly influence experimental
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results. Also the presence of idiosyncratic responses or disease
processes can complicate the evaluation and interpretation of
any toxicity study. The factors influencing animal responses
can be troublesome when their effects are confused with or mis-
interpreted as toxic. for further discussion of environmental
effects on experimental parameters see Herrington and Nelbach.13
The three qualitative considerations discussed above are
applicable to all experimental animal studies, no matter what
their intended purpose, and essentially establish the acceptability
not only of specific reports but also the acceptability of the
eventual evaluation, interpretation, judgments, and risk assess-
ments made by toxicologists.
Resolution of problems relating to qualitative or quantitative
considerations is not entirely the responsibility of the individual
evaluator. The submitter of the documentation may be requested
to assist. For difficult problems, the assistance of consultants
and/or the Science Advisory Panel may be utilized. Requests for
the latter type of assistance must be made through the appropriate
management level.
The acceptability of reports and other technical information
submitted to EPA is primarily a scientific judgment and only
secondarily a legal one. Therefore, EPA bears the burden of
defending and documenting the acceptance or rejection, in part or
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in whole, of the study report and data. The submitters of the
information deserve to know the rationale for any rejection of
data. The rationale should be succinctly stated in the evalua-
tion document.
C. Major Considerations for Analysis and Evaluation
Control animals must receive as much attention during the
analysis and evaluation process as do the treated ones. Any
untreated (control) animal or group may exhibit some signs of
abnormality or drift from the norm for that species or strain.
Table 1, taken from Weil and Carpenter,-^ presents examples of
abnormal values exhibited by control groups during long-term
studies which could complicate analyses of data. Because of the
real possibility that statistically significant differences
between chemically treated and untreated control groups are the
result of abnormal values among the controls, the authors concluded
that to be indicative of a true deleterious (adverse) effect, the
differences should be dose-related and should delineate a trend
away from the norm for that stock of animals.
Historical control data is useful when evaluating the accept-
ability of the "normal" values and observational data obtained
from control groups.8,15,16,17 Any departure from the norm
by the control group(s) must be discussed in the evaluation
document and taken into consideration, especially during any
statistical analysis.
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Weil and McCollister18 analyzed toxicity end points, other
than oncogenicity, from short- and long-term tests and concluded
that only a relatively small number of end points are effective
in delineating the lowest dosage producing an effect in such
tests. Body weight, liver weight, kidney weight, and liver
pathology delineated this dosage level in 92% of test chemicals
in short-term (subchronic) studies and 100% in long-term (chronic)
studies. To reach 100% efficiency in short-term studies,
renal and testicular histopathology had to be included. There is
no implication that these criteria delineate all of the stress
markers or toxicity end points likely to appear at higher
dose levels. However, it is implied that toxicity effects in
these data areas are likely to appear earlier in a study and at a
lower dose than many other markers. Heywood-^ surveyed the
toxicological profiles of fifty compounds in rodent and non-rodent
species and confirmed the observations of Weil and McCollister.
For this reason these criteria of stress should receive careful
attention in the analysis and evaluation process.
1. Mortality/Survival
Death is a highly definitive, biological end point for analysis
regardless of the animal group or groups in which it is observed.
Reasonable efforts should be made to determine the cause of
individual deaths or to discover a defensible presumption of the
cause. The evaluation of pathological lesions or morphological
changes in unscheduled, belatedly observed deaths are very frequently
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complicated or hampered by postmortem autolysis. The separation
of deaths caused by factors unrelated to pesticidal agent exposure
(e.g., acute or chronic infections, age or disease dependent
degenerative processes, anatomical abnormalities, negligent
handling or accident) from toxicity induced deaths is important.
All data relating to the moribund or dead animals during their
study life, as well as the results of postmortem examinations,
should be scrutinized in an attempt to make this distinction.
Mortality analysis requires more than a statistical treatment
of incidence at termination of a study (e.g., Example A, Table
1). Survival/mortality data can be influenced by many factors
other than toxicity of the test substance. Changes in protocols
during the course of a study can complicate the analysis.
Alterations in dosage levels can produce a confusing mortality
pattern. This is also true of kills and especially unscheduled
kills during a study. The perturbation caused by both types
of changes during a study can be considerable and the resolution
of difficulties may not be a simple routine.^
Any unusual mortality pattern should be explained by the
data submitter on biological or toxicological grounds. If mortality
is high in toto for any short- or long-term study, or for a parti-
cular group within a study and a credible explanation is not
available, the study should be nominated for a laboratory and
data base audit.
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An analysis and evaluation of mortality patterns within each
group is important. Such patterns may indicate mortality is
clustered early or late in the course of the study; is intermittent
and scattered throughout the duration of the study; or has a
higher incidence in one sex than in the other. The analysis of
the cause of individual deaths will aid in determining the
toxicological significance of these various patterns. Early
deaths within treated groups (i.e., those occurring within the
first eight weeks of a subchronic study or within the first ten
months of a chronic study), can provide very valuable information
because they may represent the more susceptible animals among
the exposed population. However, Fitzhugh et al. , 20 found
that when the quantity of test substance in the diet is kept
constant, young rats ingest relatively more of the test substance
than do older rats. This growth dilution phenomenon is illustrated
for male rats in Figure 1 and for females in Figure 2. Compound
consumption, in mg/kg body weight per day, for each of the first
13 weeks and selected intervals thereafter is also presented for
males (Figure 1A) and females (Figure 2A). In these illustrations
it can be seen that for the first 13 weeks, a rapid weight gain
period for both sexes, the mg/kg of body weight per day consumption
of the compound is relatively high and tapers off to a relatively
stable value at approximately 40 weeks. Early deaths may therefore
be the result of the higher exposure, on mg/kg/day basis, of young
animals compared to older animals. Deaths which are clustered
at a specific time period may reflect a spontaneous epidemic
disease situation of limited duration. However, high mortality
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associated with infectious agents in treated groups, in the absence
of such evidence in the concurrent control group, may portend an
immuno-suppressive action on the part of the chemical being
tested.
2 . Clinical Observations
Generally, adverse clinical signs noted during the exposure
period should correlate with toxicity end points or disease
processes. These can frequently be used as supportive evidence
for dose-response-relationships and can play an important role in
determining the NOEL. However, not all adverse clinical signs
will correlate with pathological or morphological changes in
organs or tissues. Some will be caused by biochemical lesions or
shifts in mechanisms which require special methods for their
detection (i.e., incoordination, muscle twitching, tremor, or
diarrhea may indicate acetylcholinesterase inhibition without any
morphological changes being evident in nerve tissue).
Table 2 presents some of the clinical signs which may be
observed during the physical examination of individual animals.
Very few of these observations are made with the aid of instru-
ments. It is, therefore, essential that all deviations from the
"normal" observed in the control and treatment groups be adequately
and accurately described and recorded during the study and presented,
in like manner, in the study report.
Many of these qualitative signs can be counted, scored for
intensity, and tabulated as incidences. However, statistical
analysis is not of any real value in this area. The evaluator
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must, therefore, rely more on the number of individuals per
group exhibiting signs of a particular type, as well as the
intensity of the reponses, to gain an impression of a dose-
response-relationship.
Clinical observations such as those that relate to
palpable tumors or which might be associated with neoplastic
developments such as hematuria, abdominal distention, or impaired
respiration may be useful in defining the time a tumor was first
suspected as being present. Such signs might be an aide in
evaluation of decreased tumor latency in long-term rodent studies.
They may also aid in determining cause of death. A statement of
the correlations, or the lack thereof, between clinical signs and
specific toxicity end points should be made in the evaluation
document.
3. Body Weight and Food Consumption
Body weight changes (gains or losses) for individual animals
and groups of animals when compared to concurrent control changes
during the course of a study are a criterion of some impor-
tance .18,19f22 such changes are usually related to food intake
and analysis of one without an analysis of the other is of little
value. Weight decrement may not always be related to toxicity
per se.23 Occasionally the incorporation of the test substance
into the diet will cause the diet to be unacceptable (unpleasant
or not palatable) to many individuals in all treatment groups or
to the majority of individuals in the higher dietary level groups.
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This effect is usually evident during the first two or three
weeks of the study. Sometimes the majority of animals in
the affected groups(s) are able to accomodate and a gradual
increase in group weight gain will occur.24 jn subchronic
studies, the lag in group weight gain may persist, even though
the individual animal gains per gram of food consumed (food
efficiency) are favorable after the accommodation, and produce a
statistically significant difference between the affected
group and the concurrent controls which is not related to toxicity
of the test substance.25 This phenomenon is infrequently
encountered in chronic studies, since the problem can usually be
overcome by an appropriate method (e.g., intubation). Sometimes
the addition of the test substance will interact with one or
more essential nutritional elements in the diet thereby producing
weight gain decrements or alterations of toxic responses.26,27f28
This phenomenon may be encountered in subchronic studies and when
identified is usually overcome by acceptable means before a
chronic study is initiated. Infrequently seen is the control
effect illustrated by Example B in Table 1. This data represents
a situation in dogs where the control value is very low causing
the other value to appear unusually high, but it can be encountered
in rodents, where at one point in time the controls exhibit an
unusual weight difference when compared to the treated groups.
Diet composition, food consumption, and body weight gains per
se can also have an important influence on many aspects of animal
responses including shifts in metabolic, hormonal, and homeostatic
-------
-25-
mechanisms29 as well as disease processes8,30f31f32
maturation^3 anc: should be considered when unusual effects
are observed in the absence of any indication of injury to organs
and other vital systems.
The resolution of difficulties in evaluation of body weight
changes and attendant effects may be aided by the graphing of
group body weight and food consumption and compound consumption
(on a mg/kg body weight basis). This allows a quick identification
of any unusual or sudden changes in gain or loss by any group.
In any case the evaluator should do some independent analysis of
body weight differences to determine whether an agreement or
disagreement with the submitters' conclusion or opinion can be
reached in an independent and defensible manner.
4. Hematologic, Clinical Chemistry, and Urinary
Measurements
The Pesticide Assessment Guidelines, Subdivision F, suggest
that certain measurements of hematologic, clinical chemistry,
and urinary parameters be routinely made in rodent and
non-rodent subchronic3 and chronic4 toxicity studies.
There is little doubt about the value to clinicians of such
data when treating or otherwise managing human and veterinary
patients and such data may also be of value to pesticide toxicol-
ogists when subchronic studies are being used to establish dose
regimens for longer term studies. Because of the automation of
both the routine clinical analysis and the statistical treatment
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-26-
of this type of data, evaluators will be forced to contend with
much "noise" in this area, and will frequently be presented with
scattered, statistically significant effects in the absence of any
evidence of clinically significant relationships to specific
toxicity end points. For example, Pearl et al. ,34 restrained
rats for six hours and followed SCOT and SGPT changes. These
transaminases were very much elevated and the SCOT did not return
to basal level within a period of six days, indicating an apparent
susceptability of these enzymes, particularly SCOT, to stress
factors.
Table 1, Examples C and D, presents examples of statistically
significant differences of lymphocyte counts and serum urea nitrogen
determinations which are not biologically significant because of
the control effect mentioned previously. These data also illustrated
the frequently observed random occurrence and non-dose-relationships
of this type of data. When using historical control data as an
aid to evaluation, it must be kept firmly in mind that "normal
values" in hematologic and clinical chemical measurements depend
heavily on the specific methods used to generate the data.
Therefore, only values produced by the identical methods from
the same laboratory are valid in such comparisons. Literature
values for normal ranges which do not specify the method by
which they were obtained must be used with caution.
Blood cytological and chemical data, with urinalysis, can be
valuable information in toxicity testing. Heywood,19 in
-------
-27-
surveying the correlation of sensitive criteria of target organ
toxicity across species, found that reduction of values relating
to red blood cells was a common effect recorded in all species
in his survey when the hemopoietic system was affected. Interim
elevations in serum enzyme levels of aspartate transminase (SCOT
or AST), alanine transaminase (SGPT or ALT) and alkaline phospha-
tase may be predictive of potential or actual hepatic lesions,
but should be confirmed by histopathological changes. Measurement
of specific isoenzymes of alkaline phosphatase may help distinguish
the site of a lesion, (i.e., bone, liver, placenta or intestine).
AST elevations may also suggest cardiac degeneration. Stress
and injury to the kidney may be reflected in increases in blood
urea nitrogen and creatinine levels which are generally correlated
with urinalysis data. Evaluation of lactic dehydrogenase may
indicate liver or cardiac injury and other myopathies. Another
indicator of cardiac or skeletal muscle lesions is an increase
of serum level of creatine phosphokinase. It is important to
understand that many of these types of serum enzyme tests and
urinalysis fail to detect minor injury or may reflect only
transient or reversible changes. Therefore, evaluation and
interpretation of the test results must be performed carefully
and correlated with more specific, sensitive, and reliable
histopathologic findings. Plaa35 discusses the conversion of
liver function data into quantal responses as well as the
quantitative problems involved in low-frequency adverse reactions
and the difficulty these present in the detection of liver injury
-------
-28-
in laboratory animals.
Sensitivity and specificity of the enzyme changes as diagnostic
of organ pathology are greatly influenced by the species selected
for testing.36 por example, in mammalian species, aspartate
transminase is not specific to any tissue and thereby elevated
plasma ACT activity may suggest damage to any one or many tissues.
In contrast, alanine transaminase is relatively specific to the
liver in the cat, dog, ferret, mouse, and rat, whereas in primates,
ALT is present in heart, skeletal muscle, and liver. Plasma alkaline
phosphatase measurement has been less useful in detecting liver
cell necrosis in the dog, sheep, cow, and rat but may be indicative
of other types of liver damage, particularly those of a cholestatic
nature in a number of species. It is evident that species differ-
ences are of great importance when specific clincial chemistries
are being selected for inclusion in toxicity studies.
When analysis and evaluation of clinical data indicate a dose
response relationship or a biologically important drift from
concurrent control values, the effects observed must be correlated
with other manifestation of toxicity. The evaluator should also
state that a correlation could not be made when that is the
situation.
Standard References (e.g., Reference 37) should be consulted
for evaluation of potential correlations between clinical chemistry,
hematologic, urinary data, and adverse effects.
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-29-
5. Organ Weights and Body Weight Ratios
Current EPA guideline protocols recommend that at least liver,
kidney and testes be weighed during necropsy of animals in sub-
chronic exposure studies3 and that, in addition to these,
brain weights be determined in chronic toxicity studies.1*
The most efficient criteria, according to Weil and McCollister,18
and Heywood,19 fOr evaluation of the lowest dosage producing an
effect in such studies are changes in liver, kidney, and body
weights.
Organ weight is usually reported both with and without a
consideration of body weight. The former is referred to as
absolute organ weight and the latter as relative organ weight.
Relative organ weight comparison is especially useful when body
weight is effected in a compound-related manner. Experimentally
controllable and uncontrollable factors (i.e., circadian rhythms,
food intake, nature of the diet, age of animals, organ workload,
stress, and method of killing) have an influence on organ and
body weights and the variability of such data. A review of this
subject, by Weil,38 should be read by all evaluators. The
most important influencing factor appears to be the method of
killing and the timing of necropsy. The killing method used not
only affects the appearance of the tissue, important in describing
gross necropsy observations, but also, in conjunction with the
timing of necropsies, may cause postmortem shifts in organ weights,
39^40 A uniform exsanguination technique has been described and
evaluated by Kaneva, et al.,41 which significantly (P<0.05)
reduced the absolute and relative liver and kidney weights with
-------
-30-
respect to these weights from animals that were not exsanguinated.
The standard deviations of the mean absolute and relative liver
weights were also significantly (P<0.05) reduced. Exsanguination,
in this study, did not appear to affect the absolute or relative
weights nor the standard deviations for heart, brain, and spleen.
Additionally, the use of fasted animal body weights can reduce
the variability of organ/body weight ratios. Adkins, et al.,42
discuss the standardization of the technique for determination
of testes weights to reduce variability.
The interpretation of organ weight changes must not be made
solely on the determination of a statistically significant
difference between the concurrent control value and a treatment
group value. A proper evaluation will also include consideration
of any correlation between organ weights, histopathologic and
metabolic/pharmacodynamic data. Such correlations if they exist
must be discussed in the evaluation documentation.
6. Postmortem Observation
The pathologist has a unique position in toxicological and
oncological evaluations. Such individuals perform a special
role in providing information on the differences in tissue and
organ morphology that will establish the presence or absence of
dose effect relationships for some lesions. This data is
critical to establishment of toxic and other effects produced
by a substance. Zbinden^3 discusses the role of the pathologist
in some detail. He also discusses the use of semi-quantitative
-------
-31-
methods as well as more accurate morphometric methods for rating
the severity of lesions, but cautions that even with their use, we
cannot be entirely satisfied with diagnostic labels for lesions
because of the lack of generally and internationally accepted
nomenclature in toxicological pathology- The problems created by
differing nomenclature are also discussed by Haseman, et al.17
To prevent this type of problem, an experienced pathologist will
describe each significant lesion type, at least once, in such
detail that any competent pathologist can perceive a good mental
picture of the lesion and form his own judgment as to its relevance
to the histopathology induced by the chemical being tested.
More detailed discussions of problems relating to complete
reliance in diagnostic terms and other aspects of evaluating
oncogenic potential are presented by Paynter.8 Age associated,
especially geriatric, influences can have an extremely important
effect on histopathologic as well as clinical chemistry, metabolic
and pharmacokinetic data bases;44 and therefore important
overt, and frequently subtle, influences on observed physiologic,
pharmacologic, and toxicologic response during the latter part
of any long-term study. As indicated earlier, spontaneous degener-
ative lesions, especially when misinterpreted as induced toxic
effects, can cause major difficulties in hazard evaluation and
risk assessment. It is essential in all cases where spontaneous
and/or age associated lesions are present, to differentiate
between such lesions and treatment induced lesions. References
such as Grice and Burek44, Benirschke and Jones4^ are very
helpful in this respect but are really not a substitute for
-------
-32-
advice from a competent and experienced pathologist. For
detailed descriptions of potential histopathological changes
induced by toxic substances, spontaneous or degenerative and
other diseases, and their incidences in experimental animals,
see Reference 45.
D. Consideration of Auxiliary Evidence
The usefulness of mammalian metabolism data and the
enhancement of our knowledge of response mechanisms by studies
of absorption, distribution and elimination patterns of a test
substance is briefly discussed by Paynter.^ The following
references cited in that document are of importance to the
evaluation and interpretation of subchronic and chronic exposure
study data: Wolf (1980), Anderson (1981), Smith and Hottendorf
(1980), Yacobi et a^. (1982), Park (1982), and Mitchel et al. (1982),
In addition, references in this document discuss dose-dependent
effects in the absorption process and biotransformation interac-
tions;^^ the potential difficulties presented by impurities
and the overloading of detoxification mechanisms;*^ and various
other important aspects of experimental considerations.^8
E. Completion of Analysis
At this point an evaluator should have formulated judgments
and supporting rationale concerning: a) the acceptability of
the data base; b) the existence of biologically important
toxic and/or oncogenic effects, c) the relevancy of any modifying
factors; and d) the likelihood that any of the observed effects
were induced by the administered substance.
-------
-33-
The evaluator should summarize, briefly and cogently, the
critical biological and auxiliary data together with any modifying
factors for all studies under review. Any rationale pertinent
to an evaluation of the toxic and oncogenic potential of the
substance should also be included in the summary. NOEL'S or the
absence thereof, should be clearly stated for each of the critical
biological and toxicological responses noted.
II. Evaluation of Weight-of-Evidence
The essential purpose of subchronic and chronic exposure
studies is the detection of valid biological evidence of the
toxic and/or oncogenic potential of the substance being investigated
In this document, the evaluation of the strength or weight of
evidence produced by toxicity studies is that process which
considers the cumulative observational and experimental data
pertinent to arriving at a level of concern about a substance's
potential adverse effects. It is composed of a series of judgments
concerning the adequacy, validity, and appropriateness of the
observational and experimental methods used to produce the data
base, and those judgments which bring into causal, complementary,
parallel, or reciprocal relationships, all the data considered.
Because our knowledge concerning toxic mechanisms is still
developing, because good epidemiological evidence is seldom
available, and because animal studies are not always conclusive;
all of the information available at a given time may provide
only "persuasive evidence" (i.e., not clearly robust; feeble),
suggestive of a defensible presumption one way or another about
-------
-34-
the potential health effects of a substance under given conditions
of exposure. It is therefore necessary to succinctly articulate
the rationale for judgments and conclusions contained in risk
assessments and the uncertainties pertaining thereto. This
becomes important when new data or new scientific knowledge
requires reevaluation of the data base or a change in a previous
risk assessment or regulatory action.
For the present there is no acceptable substitute for
informed judgment based on sound scientific principles in analyzing,
evaluating, interpreting, and weighing biological and toxicological
data derived from currently available animal toxicity study
protocols. The present universally accepted practice of estimating
a NOEL in subchronic and chronic animal studies is based on the
following procedure: (1) Identification of adverse effects induced
by a known quantity of a chemically and physically characterized
substance. Generally, a defensible presumption that the observed
adverse effects are induced by a known exposure to the substance
is based primarily on the detection of a trend away from the
normal for the species and strain of animals used (concurrent
control and/or historical control data) and a demonstration of a
dose-response relationship for an observed effect or spectrum of
effects; (2) Identification of an approximate threshold level
where the adverse effects observed at higher doses are just
perceptable (the lowest adverse effect level); and, (3) Identifi-
cation of a dose level which does not elicit the adverse effects
-------
-35-
observed at the threshold or higher levels (i.e., absence of
adverse effects). This includes the judgment that any other
effects observed at this level portend no biologically significant
consequences for the health and well being of the exposed
population.
It is also a universally accepted practice to apply uncertainity
factors to the NOEL derived from subchronic and chronic animal
studies when estimating a guide post, i.e., ADI as an aid in evalu-
ating the acceptability of actual or potential human exposure limits.
For further discussion of this subject see Weil,49 Paynter and
Schmitt,50 and Dourson and Stara.51 The development of mathema-
tical models,52,53 may modify this process in the future.
-------
-36-
References
(1) Pesticide Programs; Good Laboratory Practice Standards.
Final Rule F.R. 46, No. 230,pp. 53946-53969, Tuesday 11/29/83.
(2) EPA (1982). Pesticide Assessment Guidelines Subdivision
F. Hazard Evaluation: Human and Domestic Animals. Office
of Pesticide and Toxic Substances. Washington, D.C.
(3) Ibid., p. 66.
(4) Ibid., p. 107.
(5) Casarett, L.J. and Doull, J. (1975). Toxicology, The Basic
Science of Poisons, frontis piece. Macmillin Pub. Co.,
New York.
(6) National Academy of Science (1975). Principles for evaluating
chemicals in the environment. Part 3, Human Health Effect,
Washington, D.C. p. 94.
(7) Klaassen, D. and Doull, J., (1980). Evaluation of Safety;
Toxicologic evaluation. Casarett and Doull, s Toxicology,
2nd Ed., p. 18, Macmillan Pub. Co., New York.
(8) Paynter, O.E., (1984). Oncogenic Potential Guidance for
Analysis and Evaluation of Long Term Rodent Studies.
Evaluation Procedure #1000.1 Office of Pesticide and Toxic
Substances, EPA, Washington, D.C.
(9) Haseman, J.K., (1985). Issues in carcinogenicity testing;
Dose selection. Funda. Appl. Toxicol. 5,pp. 66-78.
(10) National Research Council. (1983). Risk Assessment in the
Federal Governments Managing the Process, p.3 National Acad.
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(11) Doull, J., (1980). Factors influencing toxicology. Casarett
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(12) Thompson, G.E., Scheel, L.D. and Lowe, D., (1982). Seasonal
alteration in response to stress or physiological change.
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gland weights to growth and aging processes in rats ex-
posed to certain environmental conditions. Endorinology
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(14) Weil, C.S. and Carpenter, C.P-, (1969). Adnormal values
in control groups during repeated-dose toxicologic studies.
Tox. Appl. Pharma. 14, pp. 335-339.
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(15) Tarone, R.E. (1982). The use of historical control information
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between short-and-long term feeding studies in designing
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(19) Heywood, R.,(1981). Target organ toxicity. Toxicology
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(20) Fitzhugh, O.G. Nelson, A.A., and Bliss, C.I. (1944)
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energy and efficiency of feed utilization in rats
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(25) McLean, A.E.M. and McLean, E.K. (1969). Diet and toxicity.
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(26) Conner, M.W., and Newbern, P.M., (1984). Drug-nutrient
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(1974). Dietary enhancement of nitrosamine carciongenesis.
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of pesticide administration upon esterase activities
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(29) Kennedy, G.C., (1969). Interactions between feeding
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(32) Ross, M.H. and Bras, G., (1965). Tumor incidence patterns
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(33) Innami, S., Yang, M.G., Mickelsen, O. and Hafs, H.D., (1973).
The influence of high-fat diets on estrous cycles, sperm
production and fertility of rats. Proc. Soc. Exp. Biol.
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(34) Pearl, W., Balazs, T., and Buyske, D.A, (1966). Effects of
stress on serum transaminase activity in the rat. Life
Sci. 5, pp. 67-74.
(35) Plaa, G.L., (1976). Quantitataive aspects in the assessment
of liver injury. Environ. Health Perspec. 15, pp. 39-46.
(36) Clampitt, R.B., (1978). An investigation into the value
of some clinical biochemical tests in the detection of
minimal changes in liver morphology and function in the rat.
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(37) Todd-Sanford, Clinical Diagnosis by Laboratory Methods.
(1974) Eds. Davidson, I. and Henry, J.B., Saunders Co.,
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(38) Weil, C.S., (1970). Significance of organ-weight changes
in food safety evaluation. In Metabolic Aspects of Food
Safety. pp. 419-454, Academic Press.N.Y.
(39) Pfeiffer, C.J. and Muller, P.J., (1967). Physiologic
correlc.tes dependent upon mode of death. Toxicol. Appl.
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(40) Boyd, E.M. and Knight, L.M., (1963). Postmortem shifts
in the weight and water levels of body organs. Toxicol.
Appl. Pharma. 5, pp. 119-128.
(41) Kanerva, R.L., Alden, C.L., and Wyder, W.E., (1982). The
effect of uniform exsanguination on absolute and relative
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(42) Adkins, A.G., Alden, C.L., and Kanerva, R.L., (1982). Optimi-
zation and standardization of male gonad weight determinations
in rats. Toxicol. Pathol. 10, pp. 33-37.
(43) Zbinden, G. , (1976). The role of pathology in toxicity
testing. Progress in Toxicology 2, pp. 8-18, Springer
Verlag, New York.
(44) Age associated (geriatric) pathology: Its impact on long-
term toxicity studies. (1983). Grice, H.C. and Burek,
J.D., Ed. In Current Issues in Tox., Springer-Verlog,
New York.
(45) Benirschke, K., Garner, F.M., and Jones, T.C. (eds), (1978).
Pathology of Laboratory Animals. Vol I and II. Springer-
Verlag, N.Y.
(46) Levy, G., (1968). Dose dependent effects in pharma-
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(47) Munro, I.C., (1977). Considerations in chronic toxicity
testing: The chemical, the dose, the design. J. Environ.
Path. Toxical. 1, pp. 183-197.
(48) Dayton, P.G. and Sanders, J.E., (1983). Dose-dependent
pharmacokinetics: Emphasis on phase I metabolism. Drug
Metabol. Rev. 14, pp. 347-405.
(49) Weil, C.S., (1972). Statistics vs. safety factors and scien-
tific judgment in the evaluation of safety for man. Toxicol.
Appl. Pharmaco. 21, pp. 454-463.
(50) Paynter, O.E., and Schmitt, R., (1979). The acceptable daily
intake as a quantified expression of the accepatability of
pesticide residues. Advances in Pesticide Science, Part 3,
pp. 674-679, Pergamon Press Oxford.
(51) Dourson, M.L., and Stara, J.F., (1983). Regulatory history
and experimental support of uncertainty (safety) factors.
Reg. Toxicol. Pharmacol. 3, pp. 224-237.
(52) Grump, K.S., (1984). A new method for determining allowable
daily intakes. Fund. Appl. Tox. 4, pp. 851-871.
(53) Krewski, D.; Brown, C. and Murdock, D., (1984). Determining
"safe" levels of exposure: Safety factors or mathematical
models? Funda. Appl. Toxicol. 4, pp. S383 - S394.
-------
Table 1
Abnormal Values In Control Groups*
Example A
Mortality of Rats
Chemical in diet
Mortality
(g/kg)
0.50
0.10
0.02
0.00
Ratio
9/15b
8/14b
8/17c
15/15
Percentage
60
57
47
100
^Mortality or rats alive at 1.5 year of
doses during last half-year of inclusion
of UCCN lubricant 50-HB-5100 in the .diet
of rats for 2 years.
b0.05>P>0,OTJC0.01>F>0.001
Example C
Percentage of Lymphocytes in Dogsa
Chemical in diet
Number of doses 100 ppm
0 ppm
0
59
128
155
185
249
30.8
32.5
35.5
32.9
33. &
34.1
32.6
40.5
29.5
30.5
18.2
33.2
Percentage of group meanc
185
101
55
'Data from white cell differential blood
count during the inclusion of CRAG SEVIN
insecticide in the diet of dogs for 2
years (200 cells counted).
U.05>P>0.01.
Group mean does not include value at
significant period.
Example B
Body Weight Gain of Dogs
Chemical in diet
(ppm)
Mean body weight char.
(kg)
6400
1600
400
0
1.08b
0.70
0.70
0.03
Weight change during inclusion of TERGITl
anionic 08 in the diet of dogs for 1 yeai
b0.05>P>0.01.
Example 0
Serum Urea Nitrogen in Dogs3
Chemical in diet (g/kg?
Number of doses 0.009 0.000
0
67
138
195
209
243
255
261
23.9
24. 1
22. 8b
25.9
19.7
20.0
22. 4b
24.2
Percentage
26.1
21.2
17.0
21.5
17.6
18.3
16.7
20.0
of group meanc
138
255
99
98
82
80
change during inclusion of
anionic 08 in th« diet of dogs for 1 year
b0.05.P>0.01.
C group means do not Include values at
significant periods.
Example E
Tumor Incidence of Ratsa
Female rats with tumors
Chemical
Ratio
Percentage
0.50
0.10
0.02
0.00
4/18*
12/20
8/18
16/20
22
60
44
80
3Tumors in fenalo rats during second year of inclusion
of UCON lubricant 25-H-2005 in the diet of rats.
bO.OKP>0.001.
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Table 2
Physical examination in toxicity tests in rodents.
Organ system
Observation and
examination
Gannon signs of toxicity
CNS and
sonatonotor
Autonomic
nervous system
Respiratory
Cardiovasular
Gastrointestinal
Genitourinary
Skin and fur
Mucous
membranes
Eye
Other
Behaviour
Movements
Reactivity to various
stimuli
Cerebrial and spinal
reflexes
Muscle tone
Pupil.size
Secretion
Nostrils
Character and rate
of breathing
Palpation of cardiac
region
Events
Abdominal shape
Faeces consistency
and colour
Vulva, mammary
glands
Penis
Perineal region
Colour, turgor,
integrity
Conjunctiva, mouth
Eyelids
Eyeball
Transparency
Rectal or paw skin
temperature
Injection site
General condition
Change in attitude to observer,
unusual vocalization, restlessness,
sedation
Twitch/ tremor/ atoxia, catatonia,
paralysis/ convulsion, forced move-
ments
Irritability/ passivity/ anaesthesia,
hyperawsthesia
Sluggishness/ absence
Rigidity, flaccidity
Myosis, mydriasis
Salivation/ lacrimation
Discharge
Bradypnoea, dyspnoea/ Cheyne-
Stokes breathing/ Kussmaul
breathing
Thrill/ bradycardia, arrhythmia,
stronger or weaker beat
Diarrhoea/ constipation
Flatulence/ contraction
Unformed/ black or clay coloured
Swelling
Prolapse
Soiled
Reddening/ flaccid skinfold, erup-
tions/ piloerection
Discharge/ congestion, haemorrhage
cyanosis/ jaundice
Ptosis
Exophthalrous, nystagmus
Opacities
Subnormal/ increased
Swelling
Abnormal posture/ emaciation
Frcm (21) Table 3.1, p 53
-------
Figure 1.
Male Rat Body Weight in Grams and Compound Consumption
in mg/kg of Body Weight/day
The values for the selected weeks, in the compound consumption
graph, represent the percent of the first week compound consumption,
-------
900 r
700
600
500
400
300
>OQ
MALE
Body Weight
300 ppm
OOppm
10 ppm
13 26 40 52 66 78 92 104
WEEKS
CO
O
MALE
Compound Consumption
28-
28-
24-
22-
20-
18-
16-
14-
12-
10-
8-
6-
4-
2-
i
\
I
\
\
\
i
i
^ .^.
41^ ^'sS*-^.^ .>
i5f
\
4^---*- 37*
. . ...... i
WEEKS
-------
Figure 2.
Female Rat Body Weight, in Grams and Compound Consumption in
mg/kg of body weight/day
The values for the selected weeks, in the compound consumption
graph, represent the percent of the first week compound consumption.
-------
FEMALE
Body Weight
580
520
460
400-
340-
280-
220-
160
300 ppm
60 ppm
10 ppm
0 ,13 26 40 ,52 66 78 ,92 i104
'WEEKS
FEMALE
Compound Consumption
28^
26-
24^
122-
'20-
'18-
<§" ne-
'e*
^ 114-
112-
ijo-
6-
14-
i
\
\
\
\
\
V >,
169% *\^^
» ,^
'53% ^-^
47%
\'56%
w " "*" " *- - ~ -%-*- - ^ S% ~
1 60% ,40% 3^
^» - »
, .- . . ' , , ' . ,1 .- 1
1 113 '26 140 152 166 I78 92 1104
(WEEKS
-------
Figure 1A
Male Rat Compound Consumption in mg/kg/of body weight/day
The values for the selected weeks represent the percent of
the first week compound consumption.
Figure 2A
Female Rat Compound Consumption in mg/kg of body weight/day
The values for selected weeks represent the percent of the
first weeks compound consumption.
-------
MALE
FEMALE
28
26
24
22
20
18
1 16
112
110
B
e
4
'I
Compound Consumption n
\ mgAg bd. wt/day 2fl
\ M
N.^ ****" 22
\ ' £ H
\_'- . O
l«% ^'--x "«* 1e
% V. 2 M
V XN. '2
*n,' ^
»*
*~ ""^ - -«_ 4
; >; ; i 11 « T li'o in l« in in 40 so tea >7i «
^. Compound Consumption
'x mg/kg bd. wtVday
v^ «ppn I~_"I7J
63% * ^
4
""""""*""--.._ * 47» 36
' _ 70» 60% 40% 30
1 i i i i i i i i i i i i i ; ; ' f 1 ?
WEEKS
figure 1A
WEEKS
Rgure 2 A
------- |