REUBER REPORT
April 9, 1976
REVIEW OF TOXICITY TEST RESULTS
SUBMITTED IN SUPPORT OF PESTICIDE
TOLERANCE PETITIONS
by
Melvln D. Reuber, M.D.
for
U.S. Environmental Protection Agency
Office of Pesticide Programs
This repo.rt has been reviewed by the
Off-tec of Pesticide Programs and
approved-jfor publication. Approval
does not signify that the contents
necessarily reflect the views and
policies of the Environmental
Protection Agency.
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• EPA STATEMENT. REGARDING DR. -REUEER'S REPORT
Oyer the past several weeks, Dr. Melvin D. Reuber, an independent
pathologist and EPA consultant, has been examining a small selection
of the thousands of pesticide toxicity test reports in EPA's files.
Such reports on testing with laboratory animals are submitted in support
of pesticide registration applications and petitions for establishment
of tolerances, i. e., maximum permissible limits, for pesticide
residues in or on raw agricultural commodities. An interim report
on Dr. Reuber's review is attached. Appended to it is information
regarding his professional qualifications and experience.
Dr. Reuber examined reports on chronic feeding studies in rats.
In such studies, rats are fed diets containing a pesticide for extended
periods (two years in most of the studies covered by Dr. Reuber). Such
studies, together with several others required for registration and
tolerance-setting, are designed to provide some indication of the health
risks that may be associated with human exposure to pesticides.
Test reports on 23 pesticide active ingredients were examined.
These were selected from among the 275 active ingredients for which
there are pesticide residue tolerances applicable to raw agricultural
commodities. Tne ones selected are among those for which tolerances
have been established for particularly large numbers of food
commodities. As an unintended consequence, all the tests selected were
performed between 1950 and 1970. This is due to the fact that, in
general, the longer a pesticide has been registered, the more
tolerances are likely to have been established. EPA will be examining
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more recent reports in an auditing program it expects to start in the
near future; until this is done, no definitive statement can be made
on the extent to which more recent reports suffer from the same
deficiencies, that Dr. Reuber found.
lest reports such as those Dr. Reuber examined are reviewed by
EPA scientists when the reports are first submitted; often, they are
examined again when they are cited in support of subsequent registration
applications or tolerance petitions. EPA reviewers may request
explanations or clarifications, ask for additional data, suggest additional
tests, or recommend that registrations be issued or tolerances set on the
basis of the submitted reports. Dr. Reuber was not asked to examine the
reviewers' comments. Neither did he have an opportunity to ask questions
of the testing laboratories. His examination was limited to the reports
originally submitted.
Examination of the reviewers' comments on some of the same reports
has indicated that they frequently included criticisms similar to those
made by Dr. Reuber. The following examples of reviewers' criticisms
were culled from EPA files:
— Low survival rate of experimental animals.
— Lack of data on statistical significance of observed differences
between experimental and control animals.
-- Inadequate histopatholical examinations; failure to report
findings on all tissues studies; failure to report on
examination of .tumors in control animals; describing tumors as
benign without histopathological examination.
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Such deficiences commonly are the basis for EPA requests that
petitioners provide additional information and/or clarification.
Dr. Reuber was not asked to perform, and he did not perform, a
detailed or definitive evaluation of the safety of each of the 23
pesticides. Neither did he perform a definitive evaluation of each test
report. His charge was simply to make a general qualitative assessment
of the adequacy of the test reports from the perspective of a knowledgeable
and experienced scientist.
Dr. Reuber's report reflects his own extensive experience as a
pathologist specializing in cancer research. Thus, in providing his
assessment of the test reports he reviewed, he has made detailed comments
on their adequacy for purposes of making judgements on the tumor-inducing
potential of the pesticides. His report suggests that the National Cancer
Institute's recently published "Guidelines for Carcinogen Bioassay in
Pats" may be used in some respects in planning and performing chronic
feeding studies. EPA certainly agrees that testing of the tumor-inducing
potential of pesticides is essential; indeed, such testing is required
for pesticide registration and tolerance-setting. It is important to
recognize, however, that many other factors must be taken into account
in planning, performing, and evaluating toxicity testing. For example,
EPA scientists believe that data on animals' blood chemistry and behavioral
patterns are useful in making a comprehensive assessment of chronic
toxicity; in such respects, EPA scientists are not in agreement with
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Dr. Reuber's comments. In essence, while EPA scientists share many of the
concerns expressed by Dr. Reuber, they have recommended that the reports
he reviewed also be examined by scientists trained and experienced in other
biomedical disciplines. EPA will undertake to have this more comprehensive
review performed.
Dr. Reuber's findings do not necessarily mean that any of the 23
pesticides is dangerous to human health. Further investigation will be
necessary before EPA can make a conclusive determination on this issue.
In accordance with the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA), all pesticide products previously registered must be
reregistered by October 21, 1977. Each pesticide product will be reviewed,
and a determination made as to whether or not to register it for particular
uses, and whether each use should be classified as general or restricted.
This process is being conducted in accordance with EPA regulations issued
July 3, 1975 (40FR282U2). Of the 23 pesticides involved in Dr. Reuber's
review, 17 have already been identified as requiring additional.chronic
toxicity testing before they can be considered for full reregistration. .
See 41FR7218, dated February 17, 1976. Only one of the 23 was identified
as a candidate for full reregistration; review of the other five has not
been completed.
In addition, EPA is planning to start an auditing program, which
will involve examinations of the laboratory records related to many of
the test reports that Dr. Reuber reviewed. FPA will also audit laboratory
records related to many other toxicity test reports already submitted,
as well as many new reports, including reports submitted for purposes of
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reregistration. Depending on audit results, EPA action will be taken in
accordance with the existing regulations regarding pesticide registration,
reregistration, and classification.
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REVIEW OF TOXICITY TEST RESULTS SUBMITTED
IN SUPPORT OF PESTICIDE TOLERANCE PETITIONS
BY
MELVIN D. REUBER, M.D.
Prior to registering pesticides or establishing tolerances for
pesticide residues, the Environmental Protection Agency (EPA) requires
that data be submitted regarding potential hazards to human health.
Prospective registrants test pesticides in their own laboratories or
in commercial or university laboratories.
I was asked by Office of Pesticide Programs to do a preliminary
review of a series of reports submitted to EPA by various chemical
companies which involved chronic animal exposure to certain chemical
pesticides. On the basis of that review it has been recommended, and
I concur, that a group of scientists carry out a more extensive, in-
depth study as a follow up to my review. This report is based on a
preliminary review of the data and includes my-observations and con-
clusions from a biological medical viewpoint.
The reports of chronic rat studies for 23 pesticides were given to
I/
me for review. This report of my evaluation of these studies follows
the following format: (1) plan of the experiment; (2) pathology
protocol; (3) analysis of pathology and (4) statistical analysis; and
finally, (5) remarks and conclusions that could be made from the
studies.
]_/ Even though these reports were designated as chronic studies,
they also were generally regarded by the laboratories as oncology studies,
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Because of the severe time restrictions placed on my effort, it
was not possible to request further information from the chemical
companies about their research laboratories, nor to write an in-depth
review of each chemical, nor to analyze raw data in those instances when
they were included. I was not made aware of any criticisms or
conclusions of the reports which may have been made by EPA staff, nor
was I aware of any requests for additional studies which EPA staff or
officials may have made.
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1. Plan of the Experiment
Charles River and Carworth Farm albino rats were most often
used in the chronic rat studies; although sometimes the general term
"albino" was given rather than the specific strain. Choice of strain is
very important, and in fact one report stated: "The rats used . . .
were of the type that were prone to develop tumors." Results are,often
more difficult to interpret when the controls develop a high rate of
spontaneous tumors and/or other lesions.
All experiments used both males and females. The number of
rats per sex and per dose level varied from 10 to 35, with one study
using 50. Most often the number was 25, 30, or 35. Fifty rats of each
y
sex per group are usually recommended for chronic feeding studies.
Most studies did not mention how animals were chosen for the control
and experimental groups, i.e..whether the selections were random. One
study apparently replaced rats that died during the study with extra
rats.
All studies except one for one year and another for 22 months were of
two years duration. However, since in many studies part of the rats
were killed after 26 and/or 52 weeks (with no indication given as to
the criteria used to select the animals killed early), the number of
y Sontag, J.N., Page, N.P. and Saffiotti, U.: Guidelines for
Carcinogen Bioassay in Small Rodents. NCI Technical Report Series
No.l.
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rats at the end of the experiment was often smaller than at the
I/
beginning. The number of animals surviving until the termination of the
experiment was also reduced in several of the studies because of
excessive deaths due to pneumonia or other infectious diseases. In
certain studies the cause of high mortality was not identified or stated.
Most studies included rather extensive hematology, urinalysis and
limited blood chemistries taken at varying time intervals. These
analyses provided little or no useful information,- however,they could
have been useful in cases of leukemia or bone marrow suppression.
Generally 3 different dose levels were administered. The rationale
for choosing the doses used was never given although this may be
documented elsewhere. Relevant background information concerning
the chemical being tested was rarely included. Laboratories rarely
confirmed the chemical used, the dose levels in the diets, or the
stability of the chemical in the diet. Although in some cases
laboratories may be testing compounds unknown to them, these factors
nonetheless affect the reliability.
_. _ v
Although a few studies indicated that animals were "individually
housed," most failed to include information concerning housing.
Information was rarely given concerning how the animals at the
various dose levels were identified. Housing of control rats
and promimity to test rats is of great importance, to avoid inadvertent
exchange of test and control rats and to reduce the exposure
3/ In. one study animals killed at 52 weeks had more histopathological
lesions than those at the termination of the study.
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of control rats to volatile test compounds. In some studies animals
apparently were not observed frequently enough to allow autopsy of
sick and moribund animals prior to development of advanced autolysls
(decomposition of tissues that interferes with microscopic diagnoses).
In contrast to the general lack of detail concerning important
matters such as housing conditions several studies presented in detail
relatively unimportant clinical and behavioral observations of the rats
during the course of the experiments. Since there were generally no
differences observed between the control and test animals the question
is raised as to the conditions, such as housing, under which the studies
were carried out. Animals receiving high dosages of toxic chemicals would
be expected to be more susceptible to minor diseases then normally healthy
animals. The observations were no doubt included in order to con-
tribute to an overall impression of thoroughness. Examples of these
observations include the following:
"During the first year of the study, moderate respiratory involve-
ment characterized by wheezing, nasal discharge, and rapid respiration
was noted among the rats in a}! groups, including the controls. This
is a common syndrome among laboratory rodents, and was unrelated
to the feeding of . . ." (emphasis added)
"During the second year of the study, a gradually increasing number
of animals in each group, including the control, had body sores, alopecia,
and/or inflamed, protruding, or squinted eyes. 'Spinning' or circling
to one side was noted in a few animals among the control and test groups.
These signs are not uncommon among aged rats and are considered to be
unrelated to the ingestion of.. . "(emphasis added)
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2. Pathology Protocol
Complete gross autopsies were usually done; however, in very
few studies were complete histologic examinations reported. Generally
the most complete histology was reported only for the highest dose
level test groups and the control groups, and then only at the
termination of the study and based primarily on gross observation.
Histology on lower dose level test rats and on rats killed after 26
and/or 52 weeks was extremely limited and sometimes completely absent.
This is not satisfactory, since it is known that in many instances
competing acute-and even chronic toxicity at the highest dose levels
may interfere with the health of the animals in such a way that fewer
lesions may develop in high dose animals than in animals fed at the
lower or intermediate dose levels. Furthermore, microscopic tumors
of organs, particularly of endocrine organs,will be missed completely
because they were not observed or recognized on gross examination.
Tumors in several studies, particularly subcutaneous tumors and
pituitary tumors, were not examined histologically, because they
were considered to be "spontaneous" and of no importance. Not only
is.it necessary to section tumors histologically, but often multiple
sections of large masses are needed to make the correct diagnoses. The
subcutaneous tumors often were large and no doubt the cause of death
in some or all of those animals in which they occurred. In at least one
study these subcutaneous tumors were autolyzed and the diagnosis was
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difficult, if not impossible to make. All subcutaneous tumors are
not merely fibroadenomas or fibromas of the mammary gland which do
often occur in control as well as in test animals, but may be
adenocarcinomas or fibrosarcomas, leiomyosarcomas, rhabdomyosarcomas
or reticulum cell sarcomas. Pituitary tumors likewise may be
carcinomas and are not invariably adenomas. In one study histologic
diagnoses were not given for markedly enlarged spleens or for abnormal
pituitary glands. It" was pointed out in another study that:
"It is, of course, obvious that the actual incidence of mammary
gland tumors is greater than would be apparent from gross observation
only and that such tumors may not be detected in a routine microscopic
examination unless multiple or serial sections are examined. It is
also of particular interest that four of the six mammary gland tumors
detected in the rats fed the two highest levels of . . .in the
present studies appeared microscopically to be malignant."
Autolysis (decomposition) of tissues was a major problem since
such animals were either not autopsied, incompletely autopsied
I/
or no tissues were saved. Most studies reported:
"Severe autolytic changes in the tissues of several animals.
prevented microscopic examination of all grossly evident tissue masses."
"If marked autolysis was noted, only the lung, liver, and kidney
were examined."
"There are no significant alterations, aside from frequent congestion
and autolysis in the section of kidney, liver and spleen."
4/ The histologic sections of a mouse study done by FDA were
given to one company for pathological evaluation. An unspecified number
of animals in that study was excluded because of autolysis.
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"There is no evident drug effect, although autolysi's makes' a
critical parenchyma! examination impossible in' numerous cases."
"Autopsies were performed . . . from every . . . animal with the
exception of those in which, autolysis was advanced."
"Throughout this entire group the predominating findings seemed
to be as follows: a marked autolysis,. . ."
Microscopic and gross observations were usually not correlated.
The gross lesion was ignored or assumed to be inaccurate if a histologic
section did not confirm such a lesion. The same person probably does
not write the gross description and examine the histopathology. Gross
examination and description most likely were done by technicians and
the histopathology by pathologists, without additional correlation of
the two.
Failure to correlate the gross with the histopathology can result
in the misdiagnosis or lack of diagnosis of tumors. "An apparent hemorrhage
in the muscle of the hip" was not diagnosed histologically and could
have been a hemangioendothelial sarcoma. Bloody areas, cysts, or
apparent hemorrhages in other organs such as liver or lung, likewise
can be sarcomas. Another study described "among the females of each
group that of enlargement of hemorrhagic appearance of the pituitary"
without corresponding microscopic diagnoses. Records might indicate
further instances of descriptions of gross lesions without histologic
study.
Body weights, selected organ weights and sometimes food consumption
were often recorded and analyzed statistically. This data was
apparently included in order to support the conclusions that there were no
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statistically significant differences between control and test
animals. However, when there were significant differences, they
usually were not incorporated into the discussion of the results or
the conclusions. In a few instances in which "statistical outliers"
(organ weights or chemistry values that were out of line with the
remaining values) were excluded, this resulted in a finding of no
differences between the control and test rats.
The terminology for diagnosis varied considerably from one
laboratory to another. In addition, some diagnoses need to be clarified,
For example, large subcutaneous masses responsible for death were
diagnosed as "cystic fibrosis" or "adenosis" rather than as tumors.
In another instance a very large mass was diagnosed as "organizing
granulation tissue." The diagnoses for some spleen were "lymphocytic
tumor"; however, the greatly elevated white blood counts and the
markedly increased splenic weights strongly suggest leukemia. Such
diagnoses as metastasizing fibroadenoma "involving the left kidney
and surrounding structures" and cellular fibroma "involving urinary
bladder and ancillary tissues," were used even though invasion and
metastases are considered to be signs of malignancy. The wide variety
of terms used to diagnose lesions of the liver was illustrated by
"hyperdysplastic, hypodysplastic and hypertrophic" nodules.
Often lesions were described without efforts at explanation. Other
changes were explained away with poor or inappropriate reasoning.
Examples include (emphases added):
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"The swollen pale, "blown-up" cells probably represent
hydropic change and this is possibly due to prolonged congestion.
In addition, since the changes are seen with Bouin's fixative
material more than with formalin it is felt that there is some element
of fixative artifact. In any case, the swollen cells certainly represent
chanoe which is reversible." (Hydropic change was seen in tissues
fixed in either fixative. There is no documentation that this change
is the result of congestion or is, in fact, reversible.)
"Occasional lenticular degenerative change was recorded but no.
significance can be attached to this observation because of the many
artifacts that appear during routine processing of this organ." (Some
effort should have been made to use more suitable methods of processing.)
"The atypical alteration in the epithelium of the urinary bladder
in animals . . . may reflect a toxic effect. . . This type of change
appears to be of a metaplastic rather than of a neoplastic nature."
(Is this change in the urinary bladder a prenoeplastic lesion that would
have become a tumor had the animals lived longer?)-
"Interpretation of this information is difficult, because of the
wide variation between animals in each group, and because the changes
involve hematopoeitic tissue, which cannot be accurately studiedusing
routine histologic techniques."(Routine histologic techniques may be
adequate; however, if not, wTTy not use suitable ones?)
"All of them, however, are of a benign nature and are not tumorous
in the^sensei.of the word but are tumor masses." (referrring to
tumors of the mammary gland)
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3. Analysis of Pathology
There was inadequate presentation, tabulation and analysis
of the data, and often there were insufficient data included to make
an accurate analysis possible.
The data were frequently presented in poorly designed tables and
in an incomplete manner; it was difficult or impossible in many instances
to ascertain total number of tumors versus total number of animals
with tumors in each group. Total numbers of animals at risk (numbers
necessary for proper analysis of data) were not always given. Tabulation
of lesions was often limited to tumors only, and not other lesions, and
often to "internal tumors" with the exclusion of subcutaneous or skin
tumors. Such data can be obtained only by requesting and reviewing
the raw data in greater detail.
Data presented in several tables were misleading or distorted.
Several studies included "palpable tissue masses, nodules or wart
like lesions in the skin" in the same category. The wart like lesions
weere not examined histologically. Tumors occurring in both control
and test animals were listed, whereas uncommon tumors that occurred
in only test animals were omitted from the tabular data.
Confirmation of the summary data by examination of the raw
data was not easy. Even without extensive examination, it was
apparent in a few cases that tumors had been included in the raw
data and yet were omitted from the tables. It was not possible to
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ascertain if clerical or arithmetic errors resulted in reports of
fewer tumors. It appears that little effort has been made by the
laboratories to verify information in the tables.
Benign versus malignant tumors, separation of tumors by dose
levels, correlation of lesions by sexes, single organ site verus
multiple organ involvement, and discussion of "unusual or rare"
tumors rarely or never seen in control animals of the same species and
strain, and total number of tumors are all important. The data were
usually not presented or analyzed in categories reflecting the above
important distinctions.
Benign tumors often occur in the controls, particularly in the
mammary gland or pituitary in females. Consideration should be given
to earlier tumor appearance, earlier animal death, increase in tumor
incidence, and multiple tumors at single sites when comparing such tumors
in test and control animals. Mammary gland tumors a'nd pituitary tumors
may also be malignant in test animals and benign in the controls.
In some organs benign tumors may be a stage in the development of malignant
tumors and this should be taken into consideration.
Discussions of tumor incidence usually did not distinguish between
sexes. This is significant because, for example, mammary gland tumors
and pituitary tumors generally occur more often in females than in males while
male rats are more susceptible to the development of tumors of the
liver. Thus, for a thorough and meaningful analysis it is important
to provide a complete break down by sex.
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The reports did not group tumors according to the organs
in which they were found. Further, the discovery of "unusual or rare"
tumors was not discussed. Tumors of the kidney, rarely seen in controls,
may be significant even in small numbers in test animals. Hemangioendothelial
sarcomas of the liver have not been reported in the literature in control
rats not treated by chemicals. If such rare tumors occur in the
controls, some evidence must be given to rule out the possibility that
experimental and control animals were mixed.
Some rare or unusual lesions or tumors observed in test animals
in the studies, but not noted as such by the authors, include hemangiosarcoma
of the kidney, reticulum sell sarcoma of the brain, nephroblastoma,
carcinomas of the pancreas, highly analplastic carcinoma of the kidney
with metastases to several other organs, adenocarcinoma of the stomach,
carcinoma of the prostate mesothelioma of the pleura, and hepatic
5/
vein thromboses sometimes with infarcts of the liver.
Tabulation and analysis of lesions other than tumors, particularly
necrosis of liver and kidney and chronic renal disease,were often
i/
omitted. Necrosis was difficult to observe in dead rats because
5/ Rare or unusual highly malignant tumors, including carcinoma
of the prostate were found in rats ingesting heptachlor epoxide .
Reuber, M.D.: Statement for testimony at Heptachlor/Chlordane hearings.
6/ Rats ingesting Mirex developed a statistically significant
incidence of renal disease Reuber M.D.: Statement for testimony at
Mirex hearings .
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"Part of this finding may have been due to an early
incidence of a disease syndrome which involved the lungs,
liver and heart. Certainly, the compound added its
effect to this already stressed condition.'" (increased
mortality)
"We must, however, qualify this statement by drawing
Attention to the fact that the general condition OTthe
animals was poor on account of the chronic infections
in the cages."(the study was reported as negative for
carcinogenicity)
"... life was maintained nearly entirely by the hypodysplastic
nodules (. . .) since the small fraction of parenchyma left
was severely altered by toxic reaction and compression by the
nodules. . ." (liver)
"Again, this is probably better referred to as a desmoplastic
mass of connective tissue producing atrophy of the mammary
gland."(mammary gland tumors)
"Externally palpable tissue masses, for the most part
associated with mammary glands, are a common finding in
aging laboratory rats."
"This is a common finding in the urine of aged albino rats."
(proteinuria]
"In addition to these rather subtle changes many of the
animals possessed abscesses that were observed grossly. . .
and were primarily located at the hiius of the liver."
(Abscesses of the liver are a rare finding)
"Histological findings do not indicate permanent tissue damage
produced by . . ., with the possible exception of changes in
the testes. Interpretation of these results is somewhat
complicated, inasmuch as the changes are equivocal and . . ."
"Organ weight findings suggest increase in size of liver
and kidney, but these changes do not appear to be significant
inasmuch as the weights of control organs were unusually low
and the weights of the organs obtained from the experimental
animals are essentially within normal limits."
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advanced autolysis had been allowed to occur. Most often chronic
renal disease was ignored. In one study it was described in great
detail and felt to be significant, but in other studies from the same
laboratory this lesion was given little attention. Lesions of the
testes, which were also observed in a few studies, were unexplained.
It is not always necessary to have a dose response with regard to the
development of tumors. Toxicity, particularly at higher dose
levels, interferes with the development of tumors by causing early
death, poor health, or development of other chronic diseases.
Furthermore, the target organ may be the liver at the higher doses, but
II
shift to the lungs or other organs at the lower dose levels.
Conclusions such as "the lack of a positive dose-related increase in all
tumors or tumors of any one tissue demonstrate the absence of a
carcinogenic effect attributable to continuous exposure to . . ." are
hot justified.
Discussions and summaries which attempt to belittle the significance
\
of such effects in test animals can be seen in the following statement5
(emphases added):
7/ Gross, A.: Statement for testimony at Dieldrin/Aldrin hearings
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"The micropathological findings in the organs or
tissues of the test animals including the relatively
small, widely scattered incidence of tumorous masses are appap-
ejitly of. coincidental nature and unrelated to the
ingestlon of . ! '. ."
"In spite of the large amount of abnormal findings, which
were more or less consistent throughout these animals,
there was no evidence in any of the tissues that were in
a better state of preservation which showed any change
that could be associated with the experimental material
either in the cells or in the tissues. It is therefore
believed that these animals died from the usual changes
associated with aging and not as the result of any experimental
material^. The control and experimental animals showed
similar changes."
"Some thyroid sections from both the control and test
groups showed. . .; these deviations were considered to
be of no significance."
"One early death was due to ' diseased kidneys' in one
female rat ... cannot be attributed to the material
under study."
"No significant histopathologic changes were observed
in any of the test rats that could be attributed- to the
feeding of . . .All changes observed were considered
to be the effects of spontaneous disease in rats."
"It is possible but not likely that these were produced
by . . ."(thyroid lesions).
"As seen in the table above, the increased or decreased
organ weight data for the test groups did not follow a
consistent pattern and are not meaningful."
"Spontaneous microscopic alterations were seen in nearly
all of the organs examined from the animals on this study,
but neither their incidence or severity was greater in the
test animals than in the controls."
"Microscopic examination of tissues from the test rats
sacrificed at termination of the study did not reveal
any histopathological alterations Jthat could be attributed
to the ingestion of the test material.Degenerative or
neoplastic lesions encountered in control and test rats
were compatible with those commonly occurring in rats of this
strain and age." '. '
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4. Statistical Analysis
Statistical analysis of the pathological findings is, in
all but one study, absent. This omission, in view.of the poor
presentation, tabulation and analysis of the raw data, is hardly
surprising. In one study in which there were multiple tumors,
statistics were used to show that the study was negative. However, the
original data were reviewed by another independent investigator, in
addition to myself (an extremely time consumino review) and we both
concluded that there was evidence to suggest an increase in tumors
• 8/
caused by the chemical.
The statistical analysis of the pathological findings should include
not only the total incidence of tumors, but also the incidence of tumors
in the other categories discussed under Analysis of Pathology (see
page 12) as well.
One important factor in statistical analysis is the number of
animals in any one test or control group considered to be at risk.
In many studies, animals that die early in the study from infectious •
diseases have not been on the test compounds long enough to develop
tumors or other lesions. These animals should not be included in the
final results.
The only way to analyze numbers of animals in any one study is to
include only those animals surviving for a period of time equal to,
or longer than,the time of appearance of the first tumor as being
8/ The independent corroboration of that study was done by
Sidney M. Wolfe of the Health Research Group.
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at risk for that tumor. This factor has been ignored in some studies
which thereby 9ive misleading low percentages of tumors because the
experimental animals tend to die earlier than the controls.
No pattern was apparent in the studies as to which animals were
included in the results. Some included all animals; others included
those that lived longer than one year. In one study which included
only two year survirors, some of the test groups had only three surviving
rats, but some animals in all groups, including controls, had tumors.
The conclusions that there were no differences between the control and
test animals were not warranted. The histopathological data on the
rats that did not survive two years either was not available or was not
made available.
It is necessary to distinguish and separate hyperplastic nodules
from carcinomas of the liver. One the one hand the incidence of
carcinomas of the liver will be decreased if carcinomas are included
with nodules. On the other, the number of carcinomas will be increased if
hyperplastic nodules are counted as carcinomas. The overall effect
can be misleading in several ways. In the first instance, the incidence
of malignant tumors would be lowered in experimental animals. In the
second instance, the incidence of tumors of the liver in controls
animals would be increased. The overall effect would make a chemical
9/
appear less carcinogenic for the liver. ~
9/ This is particularly true in studies using mice that may develop
nodules of the liver spontaneously,-whereas the test mice develop carcinomas
Reuber, M.D.: Statement for testimony for hearings on Dieldrin/Aldrin
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Some studies included animals that had advanced autolysis
(decomposition of tissues) and therefore no histologic evaluation.
One study stated:
"Strictly speaking, the analysis should have been
based on only those animals receiving a full post-mortem.
However, since the groups received equitable treatment in
this respect, using all deaths within a period. . ."
and "The results showed a somewhat higher incidence of
tumors in animals that underwent complete necropsy."
There is no valid reason for the exclusion of statistical outliers
(very large values that were out of line with the remaining values)
from the data. In two studies:
"In several instances single organ weights or
organ-to-body weight ratios did not conform to the
other values in the groups in which they occurred,thus
causing the means of the groups to be widely
distorted; therefore, these values could be justifiably
deleted from the data."
". . . and in these summaries some of the means shown
in the detailed tables have been recalculated after
excluding one or two very large values."
Even though the results of the pathology studies were not
analyzed statistically, there was excessive analysis of other data.
"The criteria chosen for statistical analysis were
survival at 26, 52, and 104 weeks; body weight gains
from 0-52 weeks; total food consumption from 0-13
weeks, organ weights and organ-to-body weight
ratios for those animals sacrificed at 26 and 104 weeks."
While I am not a statistician, my own experience with biological
and pathological aspects of medical research leads me to believe that
a qualified statistician should review the studies and confirm the
presence or absence of significant lesions.
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5. Remarks and Conclusions
Bias in the plan of an experiment, carrying out of an
experiment, or in the analysis of a study, in a lot of innocent or
minor appearing ways, can influence the final results and conclusions
of the study. Some of these biases which may enter into these studies
include the following:
failure to randomize animals for the various
groups;
choosing animals with lesions to be killed after
26 and/or 52 weeks;
- failure to section particular lesions histologically;
including autolyzed animals in the tissues examined
but failing to perform histopathology;
using terminal animals for bio-chemical and other
studies, but failing to include autopsy or histology
examination;
killing an animal with a skin tumor or mammary gland
tumor early, or excising, benign mammary gland tumors
so that they may not become malignant later in the
experiment;
failure to end a study at a Point where the test
animals have more tumors than the controls, or
failure to extend the duration of studies to a
point where the test animals may have a bhance to
develop more tumors than the controls;
choosing a strain of rat that has a high incidence
of spontaneous tumors;
choosing pathologists with insufficient or inappropriate
experience; IP/
Recommended decision of Chief Administrative Law Judge, Velsicol
Chemical Corporation FIFRA Docket No. 384, pp. 83-85.
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Most of the studies were reported as essentially negative
with little or no changes that could be attributed to the compound
tested. Only one study could be readily determined as satisfactory.
In many of the- studies no conclusions could be made from the study
as reported; however, it may or may not be possible to decide.if these
particular studies are negative or positive if further extremely
time-consuming analyses of the data are done. Indeed one study reported
as negative was probably positive for carcinogenicity. In several
other cases there was insufficient data to analyze, so that additional
data is needed before conclusions can be reached.
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CIRRICULUM VITAE
NAME: Reuber, Melvin Dwaine
TITLE: Consultant, Human and
Experimental Pathology
EDUCATION: University of Kansas - A.B. - 1952
University of Kansas,
School of Medicine - M.D. - 1958
Dip!ornate, American
Boards of Anatomical
Pathology - 1962
RESEARCH AND/OR PROFESSIONAL EXPERIENCE:
Exchange Scientist, NCI sponsored US - Japan Cooperative
Cancer Research Program, National Hygiene Institue,
Tokyo, Japan 1975
Consultant, Experimental Pathology - 1975
Associate Professor, University of Maryland School of
Medicine, Baltimore, Maryland - 1971-1974
Pathologist, National Cancer Institute, National Institutes
of Health, Bethesda, Maryland - 1963-1971
Assistant Pathologist, Beth Israel Hospital and Instructor
in Pathology, Harvard Medical School. Boston, Massachusetts 1962-1963
Research Fellow in Pathology, Beth Israel Hospital and
Harvard Medical School, Boston, Massachusetts 1961-1962
Experimental Pathology Trainee,University of Maryland
School of Medicine, Baltimore, Maryland 1960-1961
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REUBER, MELVIN D.
Resident in Pathology, University of Maryland School of
Medicine, Baltimore, Maryland 1959-1960
Intern in Pathology, Department of Pathology, University
of Maryland School of -Medicine, Baltimore, Maryland 1958-1959
Research Fellow. Department of Pathology and Oncology,
University of Kansas, School of Medicine, Kansas 1954-1958
SOCIETIES:
American Association of Pathologists and Bacteriologists
International Academy of Pathology
American Association for Cancer Research
American Society for Experimental Pathology
Japanese Cancer Association
Society of Toxicology
New York Academy of Science
Pan American Medical Association
Society of Pharmacological and Environmental Pathologists
CONSULTANT:
Environmental Protection Agency
National Cancer Institute
PUBLICATIONS:
List available on request
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