United States
Environmental Protection
Agency
Office of Health
and Ecological Effects
Washington DC 20460
EPA-600/1-78-061
September 1978
Research and Development
Assessment of Health
Effects of Benzene
Germane to
Low-Level Exposure
i;
>
e.P 600/1
78-061
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EPA-600/1 -78-061
September 1978
Assessment of Health Effects
of Benzene Germane
to Low-Level Exposure
U.S ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Ecological Effects
Washington, D.C. 20460
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DISCLAIMER
This report has been reviewed by the Office of Health and
Ecological Effects, Office of Research and Development, U.S.
Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
11
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PREFACE
The Environmental Protection Agency has prepared three
documents concerning the health effects of benzene on the general
population:
1. A health effects assessment
2. An environmental exposure assessment
3. A population risk assessment, based on the data presented
in the first two documents.
This report is the health effects assessment; it will be
used by the Environmental Protection Agency's Office of Air and
Waste Management and by the Administrator to determine the
scientific basis for possible actions regarding benzene under
the Clean Air Act.
Earlier drafts of this report have been reviewed by many
individuals and organizations. On January 18, 1978, the
Environmental Health Advisory Committee of the Environmental
Protection Agency's Science Advisory Board held a special "readers'
meeting" concerning the report. Members of the Environmental
Health Advisory Committee who served as special readers are:
Dr. James H. Sterner (chairman), College of Medicine,
University of California (Irvine);
Dr. Samuel S. Epstein, School of Public Health, University
of Illinois (Chicago);
Dr. Bernard D. Challenor, College of Physicians and Surgeons,
Columbia University;
Dr. Jenifer L. Kelsey, Yale University School of Medicine.
111
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Drafts of the three benzene assessment reports were reviewed
by the Environmental Health Advisory Committee in public session
on February 3, 1978. The members of EHAC, in addition to those
listed above, are:
Dr. Norton Nelson (chairman), New York University Medical
Center;
Dr. William J. Darby, The Nutrition Foundation, Inc.;
Ms. Dorothy B. Hood, Haskell Laboratory for Toxicology and
Industrial Medicine;
Dr. Bailus Walker, Jr., Department of Environmental Services,
Government of the District of Columbia;
Dr. James L. Wittenberger, School of Public Health, Harvard
University;
Dr. Gerald N. Wogan, Department of Nutrition and Food
Science, Massachusetts Institute of Technology.
Drafts of this report were also reviewed at an interagency
meeting on December 13, 1977. Representatives of other Executive
Branch departments attending the meeting included:
G.W. Siebart, representing the Department of Defense,
Office of the Secretary of Defense;
Joan Cloonan, representing the Department of Justice;
Gene Lehr, representing the Department of Transporation;
Steven Bayard, representing the Consumer Product Safety
Commission, Office of Engineering Sciences and Health
Sciences;
Dorothy Canter, representing the Consumer Product Safety
Commission, Office of Engineering Sciences and Health
Sciences; and
B. Osheroff, representing the Department of Health, Education,
and Welfare.
IV
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Review copies of this document have also been provided to
other government agencies and to industrial and public interest
groups as the result of a notice that appeared in the Federal
Register November 9, 1977, on page 58440.
All comments and criticisms received at these meetings and
in response to the Federal Register notice have been reviewed and
incorporated into the document as deemed appropriate.
v
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CONTENTS
Page
PREFACE iii
ACKNOWLEDGMENT vii
SUMMARY AND CONCLUSIONS 1
1. INTRODUCTION 4
2. BENZENE METABOLISM; CYTOGENETIC AND EMBRYONIC EFFECTS 6
Benzene Metabolism 6
Cytogenetic and Embryonic Effects 10
3. CHRONIC BENZENE TOXICITY IN ANIMALS 29
Exposures by Inhalation 29
Exposure by Other Routes 34
Evaluation and Comments 37
4. BENZENE TOXICITY IN MAN 47
Introduction 47
Pancytopenia 48
Leukemia 65
Other Benzene-associated Disorders 89
Summary 93
VI
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ACKNOWLEDGMENT
This document was prepared by EPA's Office of Research and
Development with extensive help from a team of consultants led by
Bernard D. Goldstein, M.D. Major contributions were by Carroll
A. Snyder, Ph.D., Robert Snyder, Ph.D., and Sandra R. Wolman,
M.D. The views represented in this document are those of the EPA
and not necessarily those of the consultants.
VII
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SUMMARY AND CONCLUSIONS
This report presents the research findings on benzene
toxicity that are relevant to assessing human health risks at en-
vironmental exposure levels. Following are the principal con-
clusions to be drawn from this report.
1. Benzene exposure by inhalation and other exposure
routes is strongly implicated in three pathological conditions
that may be of public health concern at environmental exposure
levels:
0 leukemia, especially acute myelogenous leukemia
0 pancytopenia (including aplastic anemia)
0 chromosomal aberrations.
2. Given the epidemiological data obtained in occupational
exposure studies, one can argue convincingly that benzene is a
human leukemogen. The exposure data in these studies do not
allow a scientific derivation of a dose-response curve. Most
studies in which exposure levels were determined involved doses
in the range of 100 to 500 ppm, though in some the benzene
concentrations were lower.
3. Since most studies concern middle-aged to elderly males
who were occupationally exposed to benzene, no conclusions can
be drawn about differences in susceptibility of other populations.
4. Currently there is no convincing evidence that benzene
causes neoplasias, including leukemia, in animals. Failure to
induce leukemia in animals could be due to an as-yet-unknown
cocarcinogen required to evoke the leukemogenic response initiated
by benzene.
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5. Hematotoxicity, particularly pancytopenia, has been
observed in both humans and animals following exposure to benzene.
The toxicity does not follow exposure to other compounds such as
toluene and xylene commonly associated with benzene environmen-
tally.
6. Humans who develop hematologic abnormalities due to
benzene exposure have a greatly increased probability of devel-
oping leukemia and aplastic anemia, a finding consistent with the
thesis that benzene is leukemogenic.
7. Long-term occupational exposures of workers to benzene
at levels as low as 20 ppm, but generally at levels greater than
100 ppm, have resulted in various signs of hematotoxicity.
8. Two effects, as yet unconfirmed, of potential signifi-
cance have been reported at occupational exposure levels of 3 to
15 ppm. The effects are 1) an increase in red blood cell levels
of deltaaminolevulinic acid, a precursor in the heme biosynthetic
pathway, and 2) a decrease in the mean serum complement of the
blood.
9. Available data from studies in which measurements ranged
from 25 to 150 ppm strongly suggest that chromosome breakage and
rearrangement can result from chronic exposure to benzene; in at
least one study, significant effects were noted at 2 to 3 ppm
(time-weighted average). These aberrations have been observed
to persist in lymphoid and hematopoietic cells after removal from
benzene exposure. Since a favored mechanism for leukemia
development is somatic mutation, the persistence of chromosomal
aberrations, coupled with clinical observations of chromosomal
abnormalities in human leukemic cells, support the thesis that
benzene is a leukemogen. A dose-response relationship has not
been demonstrated for benzene-induced chromosome aberrations.
This lack may result from variations in individual susceptibility,
10. Benzene toxicity probably occurs via a toxic metabolite.
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11. In animals, benzene accumulates in lipoid tissue such as
fat and bone marrow, and benzene metabolites concentrate in the
liver and bone marrow. The concentration of metabolites in the
bone marrow exceeds that in the blood.
12. The accumulation of benzene metabolites in bone marrow
along with the coincidental covalent bonding of benzene to solid
residues of bone marrow is consistent with a phenomenon of
toxico- and carcinogenesis shared by many other chemicals.
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SECTION 1
INTRODUCTION
There is substantial evidence that concentrations of benzene
encountered in the work place (in the United States and else-
where) have caused diseases of the blood and bone marrow in
general (e.g., blood dyscrasia, pancytopenia) and leukemia in
particular (especially acute myelogenous leukemia). Because
current policy of the Environmental Protection Agency (EPA)
states that there is no zero risk level for carcinogens, benzene
has been listed by EPA under Section 112 of the Clean Air Act as
a hazardous air pollutant.
This report is an assessment of the health effects of
benzene germane to low-level exposure; it is largely a review
and evaluation of the scientific literature relevant to deter-
mining the human health effects of environmental exposures to
benzene. Most of what is known concerning the effects of benzene
on human health has been learned by studies of persons exposed
to benzene in the workplace. Virtually no information is
available that describes the health effects of nonoccupational
exposures of the general populace to benzene. Our evaluation
of potential environmental health effects, then, must be based
upon what we know of the mechanisms of benzene toxicity and its
genetic implications and of the effects of benzene on animals
and on human beings. This report is structured accordingly.
Section 2 introduces some of the major biomedical concepts
that are pertinent to assessment of the health effects of benzene,
Following a brief discussion of benzene metabolism in animals
and in humans, the cytogenetic effects of benzene are considered,
particularly its effects on chromosomes.
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The major portion of the report deals with assessments of
benzene toxicity in animals (Section 3) and in man (Section 4).
These latter analyses focus on two forms of benzene-induced
disorders: 1) pancytopenia, defined as the diminution of all
formed elements in the blood, and 2) leukemia, defined as a
proliferation and accumulation of mature and immature white blood
cells (leukocytes) in blood and/or in bone marrow, leading to the
impairment of normal function.
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SECTION 2
BENZENE METABOLISM; CYTOGENETIC, AND EMBRYONIC EFFECTS
BENZENE METABOLISM
Metabolism in Animals
Most of the benzene that enters the body is excreted via the
lungs in exhaled air. ' Study of the distribution of benzene
and its metabolites in animal organs shows that free benzene
accumulates in lipoid tissue such as fat and bone marrow. High
concentrations of benzene metabolites can be observed in liver
tissue and in bone marrow. It is particularly significant that
the concentration of metabolites in bone marrow exceeds that in
blood. Repetitive administration of benzene leads to accumula-
tion of both benzene and its metabolites in these organs and to
covalent binding of benzene metabolites to liver and to solid
..... 60
residues in bone marrow.
The metabolic pathway of benzene in liver is shown in
Figure 1. The initial step appears to be a reaction mediated
21
by the mixed-function oxidase. This enzyme is inducible, so
that pretreatment with benzene, phenobarbital, or 3-methylcho-
lanthrene can increase the rate of benzene metabolism. ' '
The direct product of the interaction of benzene with mixed-
function oxidase is probably an arene oxide that is highly
reactive. It can spontaneously rearrange to form phenol, undergo
enzymatic hydration followed by dehydrogenation to form catechol
or a glutathione derivative (phenylmercapturic acid), or bind
covalently with cellular macromolecules. Formation of hydroquinone
or of trihydroxylated compounds probably is the result of several
reactions with hydroxylating enzymes.
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Benzene Metabolism in Man
Most metabolic studies of benzene in man have been concerned
either with uptake and excretion of unchanged benzene via the
breath or with measurements of benzene metabolites in urine.
44
Nomayama and Nomiyama exposed volunteers to a series of solvent
vapors and found that among six subjects exposed to benzene at 52
to 62 ppm for 4 hours, retention of benzene in the respiratory
system decreased and then became constant after 3 hours at 30.2
percent of the inspired dose. There was no distinction attribut-
able to sex of the subjects. Excretion, as measured in exhaled
air after removing the subject from the benzene-laden atmosphere,
was about 16.8 percent. Net uptake, i.e., the sum of uptake and
43
excretion, was 46.9 percent. These authors went on to show
that when the logarithm of the benzene concentration in expired
air was plotted against time, the excretion pattern described a
hyperbole that could be expressed mathematically and that yielded
three rate constants to describe the phenomenon. The subjects
continued to excrete benzene in the exhaled air for as long as 15
27
hours. Hunter, in studies of people exposed to 100 ppm benzene,
detected benzene in the expired air 24 hours later. He suggested
that measurements of benzene in expired air could be used to
estimate benzene content of the inspired air by extrapolation.
Phenol content of the urine is often measured after benzene
exposure. Maximum concentrations are thought to occur within 2
27
hours after exposure. The major conjugated form appears to be
ethereal sulfate until phenol levels of the urine reach 400
58
mg/liter, at which point glucuronide begins to appear.
64
Teisinger et al, who exposed humans to benzene at 100 ppm
for 5 hours, reported that 46 percent of the dose was retained.
Of that amount, 61 percent was recovered as phenol, 6.3 percent
as catechol, and 2.4 percent as hydroquinone. In these studies
the major monohydroxylated metabolite and the two major dihydrox-
ylated metabolites observed by Parke and Williams in rabbits
were also observed in man.
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Relationship of Benzene Metabolism to Benzene Toxicity
46
Since Parks and Williams suggested in 1954 that a metabo-
lite of benzene is responsible for benzene toxicity, evidence to
42
support that hypothesis has mounted. Nomiyama demonstrated
that inhibition of benzene metabolism protected rats against
benzene-induced leukopenia. Andrews reported that when benzene
metabolism was inhibited with toluene the subjects were protected
against benzene-induced reduction of red cell production.
Animals have been protected against benzene toxicity when pre-
13 28
treated with phenobarbital, ' probably because phenobarbital
stimulates benzene metabolism in liver, which leads to detoxifi-
cation and thereby reduces the amount of benzene available for
formation of the toxic agent in bone marrow. The specific
metabolite that produces benzene toxicity has not yet been
identified, but likely candidates are benzene oxide, catechol,
and hydroquinone, or the corresponding semiquinones.
The demonstration that reduction of red cell count during
benzene treatment is accompanied by accumulation of benzene
metabolites in marrow and coincidental covalent binding of
benzene to solid residues of marrow suggests a phenomenon in
toxico- and carcinogenesis shared by a variety of other chemicals,
such as acetaminophen, bromobenzene, hydrazine derivatives,
4.1,- 41 A ^u 29
parathion, and many others.
Although further studies are required to prove the hypo-
thesis, it seems likely that benzene, like many other chemicals,
exerts its toxicity by formation of a toxic metabolite.
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CYTOGENETIC AND EMBRYONIC EFFECTS
Concepts
Mutagens and Carcinogens—
Benzene is believed to affect chromosomes, and chromosomal
aberrations have been sought as indications of a biologic
response to benzene for logical reasons. Somatic mutation has
long been accepted as a critical event in the initiation or
maintenance of malignant change, although the concept is not
unchallenged. Focus on sites of genetic damage is based partly
on observations of the prolonged delay from the time of exposure
to a carcinogen until the advent of malignancy, such delay being
consistent with perpetuation of the original damage in the
genetic system. Further, many lines of evidence indicate that
most, if not all, carcinogens are mutagens.
Rapid, convenient, accurate, and inexpensive systems for
mutagen testing are available for evaluation of point mutations
4
in prokaryotic cells; nevertheless, the assessment of damage to
mammalian chromosomes is probably more directly relevant to
estimations of human health hazards from mutagens. If a cell
shows sufficient chromosome alteration that further cell division
is interrupted, then from a reproductive point of view that cell
is dead and the damage is toxic. If, on the other hand, the
chromosome alteration does not interfere with cell division and
can be replicated, then it constitutes a mutation, a structural
change in the genome that presumably alters cell function.
Chromosomal breaks, which may be repaired, are not mutational
events (in the sense of being heritable). However, each occur-
rence increases the probability of formation of a structural
aberration and therefore of a mutation.
Investigations aimed at evaluating effects of benzene have
appropriately concentrated on changes in cell nuclei, metabolism
of deoxyribonucleic acid (DNA), cell division, and chromosome
10
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alterations. All these constitute direct measures of changes in
quantity, structure, organization, or function of the cellular
DNA. Moreover, some of these changes are heritable and imply
permanent changes in the genome of the affected cell.
Clastoqens and Mitotic Poisons—
The use of chromosome studies to monitor possible environmen-
tal mutagens should not be limited to evaluations of chromosome-
breaking or "clastogenic" effects on cells arrested in metaphase.
If the cells are analyzed without conventional pretreatment with
mitosis-arresting agents or hypotonic solutions, abnormalities in
the anaphase can be identified. These include multipolar
mitoses, imperfect or unequal separation of chromosomes, and
bridges interfering with reconstitution of the daughter nuclei.
Some abnormalities may be detectable only in cells recovering
from the effects of a chemical. During exposure the affected
cells may be totally blocked from entering mitosis. Thus, in
evaluating the potential action of a chemical as a chromosomal
mutagen the investigator must look for both clastogenic and
antimitotic effects. The latter may be especially important in
chemicals that do not induce point mutations.
The effectiveness of benzene as a mitotic poison has been
amply demonstrated. Decrease in DNA synthesis has occurred in
12 34
cultured human cells ' and in bone marrow of rats and rabbits
after treatment in vivo.9'33'31'39'62'63 The total numbers of
nucleated cells, and, in some cases, the mitotic indices have
declined. Inhibition of cell proliferation has been shown most
often by decrease in uptake of radioactive-labelled thymidine, a
DNA precursor. Although these may not be the most sensitive of
indices, they are clearly and directly relevant to cell survival
and reproductive fitness. Furthermore, both numerical and
structural chromosome aberrations have been described that could
be interpreted as either toxic or mutational damage. These
11
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include loss or gain of parts of chromosomes, whole chromosomes,
or chromosome sets, in addition to exchanges that result in
morphologically aberrant chromosomes.
Anaphase studies on human cells have not yet been reported
but are under way in several laboratories. Morishima and his
colleagues have described appropriate conditions for testing
human material.
Cytogenetic Aberrations in Leukemia—
The assumption that chromosomal mutation is etiologically
important in the development of leukemia has been strengthened by
observations of abnormalities in human leukemic cells. The close
association of the Ph translocation with chronic myelogenous
leukemia is well-known, ' and specific chromosomal abnormali-
ties have been reported with other forms of leukemia. ' '
These abnormalities appear to be specific to each disease entity,
confined to the leukemic cells, and clonal (indicating a probable
single-cell origin). Therefore, it is clearly important to
•• nvestigate the actions of potential leukemogens with particular
emphasis on their ability to cause site-specific chromosomal
lesions. It is, however, even more likely that the initial
damage caused by most carcinogens is nonspecific, causing a
genetically more variable population of cells. This, in turn,
increases the probability that an abnormal proliferative state
will arise (or be selected).
Cytogenetic Studies of Animals
Studies of benzene effects have been conducted in many
species, including rats, mice, rabbits, and newts. These studies
have included whole-animal exposures and effects of benzene on
cells iri vitro; they have been based on either acute or chronic
and repeated exposures. The results of such studies are diffi-
cult to evaluate since they differ not only in the biologic
12
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end-point chosen, but also in species, routes of administration,
and dosage. Since few of the studies have involved inhalation
exposure, their relevance to problems of human disease may be
questioned.
Unpublished studies by Wolman et al evaluated chromosomal
findings in rats chronically exposed to 300 or 100 ppm benzene.
Within 10 weeks there was a striking and persistent increase in
chromosome breaks and aneuploidy (deviation from the normal
diploid chromosome number) in the bone marrow of treated (JOO
ppm) animals. The increase following 100 ppm exposure was not as
great and not of clear statistical significance.
Increased chromosome breakage in several species has been
reported. Rats exposed to benzene subcutaneously over a period
of 12 days showed highly significant increases in chromosome
aberrations of bone marrow cells over untreated and toluene-
treated controls. Classification of both gaps and breaks as
aberrations complicates interpretation of these findings (since
gap rates vary more with the interpreter and with preparation)
and inflates the aberration rate. For example, although signif-
icant increases in aberrations were also found in the toluene-
treated controls in this study, the benzene-treated group was the
only one in which breaks were more common than gaps as aberra-
tions. Exchange figures such as might result from abnormal
repair after breakage (i.e., ring forms, translocations, and di-
centrics) were rarely seen. Another, more acute exposure (2.0 ml
benzene/kg body weight for 12 to 72 hours) in rats produced
47
similar findings. Increased numbers of chromatid breaks were
found at almost every exposure interval, although the responses
of individual animals varied considerably. Chronic exposure to
injection of 0.2 mg/kg per day in rabbits (up to 18 weeks of
treatment) also resulted in a high frequency of aberrations;
since less than 15 percent of the aberrations reported were
33
breaks, the significance of this study is not established.
Again, exchange figures, dicentrics, and hyperploid cells were
13
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rare. In each of these animal-exposure studies only a single
dosage of benzene was used. Thus, although different exposure
times in different species can induce increases in chromosome
aberrations, there is no clear evidence for a dose-dependent
response to benzene exposure. Furthermore, none of these studies
presents data suggestive of mutational rather than toxic damage.
Very few experiments have addressed the question of direct
interference with benzene-induced abnormalities and possible
therapeutic routes.
Studies of dividing erythroblasts taken from the amphibian
52 53
newt (Molge vulgaris L.) ' are of particular interest because
of their demonstration of anaphase abnormalities. Young animals
injected with water-saturated solutions of benzene were bled 6 to
12 hours later and a drop of tail blood was used for coverslip
culture. At the time of sampling, 38 percent of mitoses were
arrested in late metaphase. Another 28 percent showed evident
anaphase abnormalities, of which 20 percent were migration .
arrests, 3 percent were anomalies of numerical distribution, and
3 percent were anaphase bridges. The remaining 2 percent showed
small subgroups of chromosome condensations outside the two poles
of the newly forming nuclei. Observations over several hours
showed that these mitotic abnormalities resulted in unequal
nuclear divisions, polynucleated cells, and atypical nuclei.
Prophase and early metaphase anomalies were never found at the
doses used in these studies (up to 54 mg of benzene per animal).
Cytogenetic Studies of Man
Experiments—
A few experimental observations have been made on the
responses of cultured human cells to addition of benzene to the
culture medium. Increased incidence of breaks and gaps was
observed in leukocytes (white blood cells) and cancerous cells
after brief exposure to 1.1 or 2.2 x 10 M benzene in vitro. '
14
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At the higher dose a decrease in DNA synthesis interfered with
clear correlation of dose to the incidence of breaks. These
23
findings were considered to be toxic damage. Another experiment
in which peripheral blood lymphocytes stimulated by phytohemag-
glutinin (PHA) were exposed to benzene during 72-hour culture
revealed both numerical and structural alterations in the treated
cells. Aneuploidy was seven times more frequent in the treated
populations than in controls, and chromosome breakage was seen in
11 percent of the treated cells as compared with 1 percent in the
controls.
Chromosome Studies and Hematologic Disease—
In contrast to the paucity of experimental data, there is an
abundance of reports on chromosome studies in exposed populations
and of case reports on leukemia patients. The case reports are
particularly difficult to evaluate and compare. Some individuals
were exposed to benzene vapor above permissible levels, but in
many cases the exposure levels were unknown. The total periods
of exposure ranged from brief and acute to as long as 22 years.
The times between exposure and the development of disease or
death also varied greatly. Most of all, the endpoints of disease
were not comparable. The various reports include diagnoses of
acute intoxication, death with massive bleeding and extramedul-
lary hematopoiesis, benzene leukemia, acute myeloid
luekemia, acute erythroblastosis, erythroleukemia, acquired
1149 1
aplastic anemia, ' acute lymphoblastic leukemia, myelofibrosis,
and chronic myelogenous leukemia. Chromosome abnormalities
have been present in industrial workers in association with
23 14
hematologic pathology or pancytopenia.
Prior blood transfusions, the use of PHA-stimulated lympho-
cytes in some cases and bone marrow in others, and the lack of
chromosome breakage rates in controls also hamper interpretation
of the results of chromosome studies. Nevertheless, certain
15
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trends appear amid this mass of data. Additional chromosomes
were identified in several cases, ' ' ' and in two cases the
additional chromosome was identified as a member of the C group.
Both were cases of acute leukemia, in which additional C-group
chromosomes (usually number 8) are frequently found; therefore
24
this does not, as one reviewer suggested, constitute evidence
of benzene etiology. Persistence of abnormal chromosomes long
after exposure and illness was also reported. Tetraploidy or
polyploidy was found in several instances. ' ' ' Increased
chromosome breakage was reported but not well-documented.
It is important to emphasize that the end stage of exposure
to benzene was as variable in alterations of the karotype, or
chromosome "package," as it was in clinical manifestations.
Indeed, the karyotypic changes may well have reflected the
disease state more than they reflected the (presumed) inducing
agents.
Occupational Exposures—
The clearest picture of the relationship between benzene
exposure and chromosome changes emerges not from experimental
studies but from studies of occupationally exposed workers.
Tough and Court-Brown observed unstable* chromosome damage in
cultured lymphocytes from workers exposed to benzene solvents.
They and their collaborators expanded the study to include
groups from three factories and sex-matched controls. The first
group of 20 men had been exposed to benzene at factory A for
periods of 1 to 20 years and were tested 2 or 3 years after
exposure ended. The second group of 12 had worked periods of 6
to 25 years in areas where benzene was present (factory B), the
exposure ending approximately 4 years prior to the study. The
third group of 20 had worked for periods of 2 to 26 years in a
* Unstable aberrations include open breaks, fragments, ring and
multicentric chromosomes, and exchange figures. Stable
aberrations include deletions, translocations, inversions,
trisomies, and other alterations of chromosome number.
16
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closed distillation plant (factory C). In each instance controls
were selected from nonexposed individuals in the same factories.
Available measurements of atmospheric benzene were 25 to 150 ppm
in factories A and B and approximately 12 ppm in factory C. The
results indicated significant increases in unstable aberrations
in exposed workers from factory A, in both exposed workers and
controls in factory B, and in neither group in factory C.
Furthermore, the exposed workers at factory A were older than
their controls, and the authors demonstrated significant in-
creases in aberrations in the general population with increasing
age.
Several other investigators have reported increases in
chromosome breakage or in stable and unstable aberrations in
healthy workers. ' ' In one report the atmospheric concen-
trations of benzene were less than 25 ppm. More compelling
17
results were obtained by Forni and co-workers, who compared
data on 34 workers in a rotogravure plant with those of matched
controls. The group of workers was subdivided; 10 individuals
had been exposed to benzene for periods of 1 to 22 years (measure-
ments of benzene in the plant during a brief single period ranged
from 125 to 532 ppm). These 10, with the remaining 24 workers,
were then exposed to toluene for periods up to 14 years at levels
ranging up to 824 ppm. The age- and sex-matched controls had no
history of exposure to either solvent. The findings in workers
exposed only to toluene were not significantly different from
those in the controls, but the group that had been exposed to
benzene showed increases in both stable and unstable chromosome
18
aberrations (p < 0.01). Another study of 25 subjects who had
recovered from clinical "benzene hemopathy" indicated that
increases in both types of aberrations persisted several years
after cessation of exposure, although there was some decrease in
the proportion of unstable aberrations. Average values of
17
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unstable abnormalities in the exposed group were 3 times greater
than in the controls, and of stable chromosome abnormalities, 30
o
times greater. Researchers in a Swedish study observed road-
tanker petrol delivery drivers, crew members of petrol-carrying
ship tankers, and employees at petrol filling stations to detect
chromosome breakage and compared their findings with those
observed in milk tanker delivery drivers and a group of indus-
trial gas workers exposed occupationally to benzene. The results,
though suggestive of a relationship between benzene (5 to 10 ppm)
and chromosome damage, are not conclusive and are not helpful in
demonstration of dose-related effects.
32
A very recent report by Dow Chemical contains data indi-
cating that occupational exposure of 52 workers to benzene for
periods from 1 month to 26 years (average exposure time, 56.6
months) induced in peripheral lymphocytes chromosome breaks,
dicentric chromosomes, translocations, and exchange figures at
rates 2 to 3 times those found in controls (preemployment
personnel). Although the figures were small, totalling 15
aberrations, the large number of cells examined (approximately
20,000) supports the significance of the differences (p = 0.005).
A difference in the average age of the worker cohort (39.3 years)
and of the controls (26.6) is not considered confounding since
the difference is small; further, the cytogenetic effect most
often associated with aging is chromosome loss rather than the
chromosomal abnormalities reported. The authors, significantly,
reported that the percentage of individuals with changes was
larger in the group with the increased incidence of changes
than in the unexposed control group. Thus the aberrations
apparently were not confined to a few individuals in the
exposed group. The authors estimate that the time-weighted 8-hour
average dosage was 2 to 3 ppm benzene; the average concentration
determined by 15-minute sampling was 25 ppm, and peak concentra-
tions were 50 ppm (as a consequence of certain specific operations
18
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such as sampling and repairs). Exposures could reach 100 ppm
if the employee failed to stand upwind while collecting samples.
This is a carefully conducted investigation, and the results
appear to be significant.
48
Picciano, who was involved in the Dow study, using the
same data, records a dose-response curve at benzene levels of
32
less than 1 ppm. His report, as well as that presented by Dow,
indicated a 10-fold increase (compared with controls) in the
percentage of benzene workers exhibiting chromosome breaks and
"marker" chromosomes (dicentric chromosomes, translocations,
and exchange figures).
Most of these industrial studies were systematic to some
degree, including controls and statistical evaluations of
results. These studies of workers from several European
countries all present similar results; that is, statistically
significant increases in both numerical and structural chromosome
alterations in populations exposed to benzene. PHA-stimulated
lymphocytes showed both stable and unstable chromosome changes in
the absence of detectable alterations of the bone marrow, and
aneuploidy or polyploidy was reported frequently. In studies
where little or no clinical symptomatology resulted from expo-
sure, there was considerable variation among individuals. For
19 17
example, in Girard's and Forni's studies a few individuals
within each benzene-exposed group were responsible for the
significantly higher chromosome breakage rates in the exposed
populations. Moreover, it is clear that these changes persisted
for many years after exposure, particularly in persons who showed
clear evidence of clinical illness from benzene. The persistence
of damage has been likened to that occurring after exposure to
ionizing radiation. The few reported instances of abnormal clone
18 51
formation ' are important in terms of possible leukemogenesis.
There is no correlation, however, between the degree or length of
exposure to benzene, the clinical symptoms, and the persistence
or extent of chromosomal aberrations.
19
-------
Embryonic and Teratogenic Effects
The literature contains few reports concerning the effects
20
of benzene on embryogenesis. In one study, groups of female
rats were exposed continuously for 10 to 15 days prior to
mpregnation to concentrations of benzene vapor that ranged from
0.3 to 209.7 ppm. In the animals exposed to 209.7 ppm no preg-
nancy occurred. In the animals exposed to 19.8 ppm, resorption
of embryos was observed in one of ten. The number of offspring
per female exhibited an inverse relationship to benzene exposure
levels. The weights of the newborn rats did not vary from those
of the controls, but the ratio of organ weight to body weight in
the 19.8-ppm exposure group was significantly higher than that in
the controls (liver, lung, spleen, and kidney). A study conducted
3
by Litton Bionetics, Inc., indicated a small increase in the
frequency of fetal resorptions in rats exposed to 10 to 40 ppm
benzene during days 6 to 15 of gestation. The incidence of
resorptions was too low to allow firm conclusions. Another study
with concentrations of benzene at 2200, 300, and 100 ppm showed
22
no evidence of increased fetal resorption. In sum, the reports
of effects of benzene on embryos are conflicting and inconclusive
and hence are not useful in evaluating the possibility that low,
ambient concentrations of benzene might have an effect on human
embryogenesis.
/- p
In another study, groups of pregnant mice were given a
single very high subcutaneous injection of benzene (3 ml/kilogram
body weight) on days 11 to 15 of gestation and the fetuses were
delivered by caesarian section on day 19. Malformations occurred
more frequently in fetuses of mice injected on day 13 than on
other days, and the anomalies included cleft palate, agnathia,
co 22
and micrognathia. Greene et al reported that at dosages of
2200 and 300 ppm benzene, skeletal deformities (missing sterne-
bra) were observed. Since reports of effects of benzene on
teratogenesis are few and the concentrations of benzene used are
very high, a role for benzene in teratogenesis cannot be pre-
dicated with confidence at this time.
20
-------
Summary
The available documentation strongly suggests that
chromosome breakage and rearrangement can result from exposure
to benzene and that damage may persist in hematopoietic and
lymphoid cells. The aberrations in human cells appear nonspecific;
that is, they are random within the genome and unrelated to the
aberrations associated with various forms of leukemia. A dose-
dependent relationship between exposure to benzene and amount of
chromosome damage has not been demonstrated. Evidence that
benzene causes disturbance in DNA synthesis suggests that its
mutagenic action could involve interference with mitosis.
Cytogenetic analysis of anaphase and postmitotic damage has not
been evaluated adequately.
Theoretical considerations and some clinical observations
suggest a relationship between chronic benzene exposure,
chromosome damage, and leukemia. Chromosomally aberrant clones
are typical of some but not all human leukemias, and aberrant
cells and clones have been observed in individuals exposed to
benzene who have later developed leukemia. Many authors have
suggested that the lack of an observed dose-response relation
in benzene-induced chromosome damage is due to variation in
individual susceptibility. Some studies have recorded biological
effects at (chronic) exposure levels below 25 ppm. The report
of a recent international workshop on the toxicology of benzene
has commented on this literature: "No dose-effect relationship
has so far been demonstrated for benzene-induced chromosome
aberrations. In workers chronically exposed to levels in the
range of 5 to 25 ppm of benzene, both positive and negative
reports involve small numbers of workers and confirmation of
negative data is required on larger groups." Increased
susceptibility to chemical clastogens has been found in human
cancer syndromes that are genetically determined. The variable
response to benzene may be attributed also to such possibilities
21
-------
as activation of virus, suppression of immune surveillance, or
cocarcinogenic activity of other chemicals.
More detailed evaluation of the cytogenetic effects of
benzene will require definitive data on dose/response relationships,
relating the frequency and severity of chromosome damage to the
amount and duration of benzene exposure. Both clastogenic and
antimitotic measures of chromosomal mutagenicity should be
evaluated. Benzene dosage should be correlated with clinical
effects as well as with the various measures of chromosome
damage. When an appropriate animal model becomes available, the
evolution and sequence of chromosome changes with initiation and
progression of leukemia may become clear.
22
-------
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28. Ikeda, M. Enzymatic studies on benzene intoxication.
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37. Matsushita, T. Experimental studies on the disturbance of
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In: Biological Reactive Intermediates, D.J. Jollow,
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Jollow, J.J. Kocsis^ R\ Snyder, H~! Vainio (Eds.) . Plenum
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human chronic granulocytic leukemia. Science, 132:1497,
1960.
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26
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49. Pollini, G., Colombi, R. Lymphocyte chromosome damage
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Le alterazioni cromosomiche dei linfochiti rilevate dopo
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28
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SECTION 3
CHRONIC BENZENE TOXICITY IN ANIMALS
The earliest report of experimental work on chronic benzene
32
toxicity was published by Santesson in 1897. He had been asked
to determine the toxic agent in an industrial solvent that had
caused purpura hemorrhagica in a group of female employees. He
succeeded in producing similar effects in rabbits after treating
them with benzene by poultice and by subcutaneous injection.
Selling, who had studied several cases of benzene-induced
purpura hemorrhagica, leukopenia, and anemia in young girls
exposed to benzene in a canning factory, extended his investiga-
tion of the disease to rabbits. He administered benzene daily by
subcutaneous injection and demonstrated a dramatic decrease in
leukocytes, a smaller decrease in red cells, and degenerative
changes in bone marrow. This study was a landmark because it was
the first in which the investigators measured the depleting
effect of benzene on numbers of circulating blood cells and
related the decreases to bone marrow damage.
In subsequent studies of benzene toxicity, the solvent
either has been administered as a vapor, which the animals
inhale in an exposure chamber, or has been injected as the pure
solvent or as a mixture with a carrier such as oil. The injec-
tions are often subcutaneous. The following discussion focuses
first on experiments with inhalation, which is the most common
route of industrial exposure, then compares results of exposures
by inhalation with those resulting from injection of benzene.
EXPOSURES BY INHALATION
32 35
Santesson and Selling were not successful in attempts to
45
produce benzene toxicity by the inhalation route. Weiskotten,
29
-------
who exposed rabbits to benzene at a concentration calculated to
be approximately 240 ppm, produced leukopenia, slight anemia,
and hemorrhages. Differential blood cell counts suggested that
small mononuclear white cells were depressed more than "polynuclear
amphophiles." The effects were observed within 2 weeks of ex-
posures for 10 hours per day.
40
Svirbely et al exposed rats, dogs, and mice to 1000 ppm
of benzene 7 hours per day, 5 days per week for 28 weeks.
Although measurements indicated intermittent leukopenia and
lymphocytopenia in the rats, the values returned to control
levels by the time the experiment ended. The dogs demonstrated
lymphocytopenia throughout the study. Hough et al exposed nine
dogs to a mean concentration of 800 ppm benzene for 4782 hours
over a 123-week period and observed a decrease in leukocyte
counts from 15,917 + 2863 (M+SD)* to 6272 + 3481, i.e., a reduc-
tion to 39 percent of control values. Differential cell counts
were not performed, and no anemia was observed.
24
Li et al investigated the effects of varying the protein
and fat contents of the diet of dogs on their susceptibility to
the depressant action of benzene on bone marrow. The dogs were
fed equicaloric diets described as 1) low fat, high protein; 2)
high fat, high protein; 3) low fat, low protein; and 4) high fat,
low protein. Controls included a group fed high fat, no protein
and a group that was not exposed to benzene and was fed high fat,
low protein. The exposed animals inhaled benzene at 600 ppm for
42 hours per week over varying periods of time depending on their
responses to benzene. Those least affected were continually
exposed for periods longer than 1 year, whereas others were
sacrificed when they became moribund after periods as short as 5
to 6 weeks. Monitoring of leukocyte and thrombocyte levels
indicated that benzene exposure reduced the numbers of both types
of cells but that protein deficiency produced greater reductions,
which were in turn exacerbated by high-fat diets.
* Mean value plus or minus the standard deviation.
30
-------
46
In a complex series of studies, Wolf et al used 7-hour
exposure periods. They exposed rats (9400 ppm, 1 to 10 times
over 1 to 19 days; 6600 ppm, 70 times over 93 days; 4400 ppm, 28
times over 38 days; 2200 ppm, 133 times over 212 days; 88 ppm,
136 times over 204 days), guinea pigs (two studies: 88 ppm, 193
times over 269 days; 99 ppm, 23 times over 32 days) and rabbits
(80 ppm, 175 times over 243 days). Although details of cell
counts were not reported, the authors claim to have observed
leukopenia in animals exposed to levels as low as 80 ppm and also
to have observed histopathological changes in bone marrow.
Deichmann et al exposed rats to benzene at various doses,
usually for 5 hours per day, 4 days per week. A decrease in
white cells was observed at benzene concentrations of 831, 65,
61, 47, and 44 ppm over periods ranging from 2 to 8 weeks. No
leukopenia was observed at concentrations of 31, 29, or 15 ppm.
Females were more sensitive than males, but differential counts
were not reported. Splenic hemosiderosis was a prominent, but
not dose-related, observation.
27
Nau et al exposed rats to benzene at three dose levels
(1000, 200, and 50 ppm) for various periods of time. At 1000
ppm, rats exposed for 23.5 hours per day seriously deteriorated
after 183 hours, showing distended stomach, empty gastrointestinal
tract, and engorgement of lungs, liver, kidneys, intestines, and
omental tissues. In addition, the leukocyte count dropped
markedly. Lymphocyte levels appeared to decrease, while polymor-
phonuclear leukocytes increased. Levels of DNA in the bone
marrow were depressed, and the proportion of red cell precursors
was increased. When exposure was reduced to 19 hours per day for
up to 1782 hours, similar effects were observed. When the rats
were removed from benzene exposure, the blood analyses showed a
return to normal, except that levels of DNA in the bone marrow
remained depressed. In exposures at 200 ppm for 8 hours per day,
5 days per week, leukopenia occurred after 750 hours, with equal
31
-------
reduction in lymphocytes and polymorphs. Myelocytic activity of
the bone marrow was reduced, and erythroid activity was increased.
Similar effects were observed after exposures at 50 ppm.
Jenkins et al detected no significant changes in leukocyte,
hemoglobin, or hematocrit values in rats, guinea pigs, or dogs
exposed to benzene concentrations of 255, 30, or 17.5 ppm during
repeated exposures for 8 hours per day for 30 days or during
continuous exposures for 90 or 127 days.
Ikeda exposed rats to benzene concentrations of 1000 ppm
for 60 days, 7 hours per day, 5 days per week. These exposures
indicate that age and sex may affect susceptibility to benzene
toxicity in rats, since the leukocyte levels decreased in the
following order: adult males, young males, adult females, young
1 ft 8
females. Both Ikeda and Ohtsuji and Drew et al reported that
pretreatment of rats with phenobarbital protected against
depression of leukocyte levels during exposure to atmospheric
benzene at levels of 1000 ppm and 1650 ppm, respectively. Boje
2
et al exposed rats to 400 ppm of benzene 7 hours per day for 13
weeks and observed marked leukopenia. Radioautography of samples
of bone marrow from benzene-exposed rats given tritiated thymi-
dine showed that benzene-treated animals displayed less incorpor-
ation of radioactivity than did control animals. These authors
hesitated to suggest a direct inhibition of DNA synthesis by
benzene because they recognized that cellular damage occurring at
any point in the cell cycle might be manifested as a reduction in
thymidine uptake.
41
Uyeki et al studied two parameters of benzene toxicity not
previously reported. The colony forming cell (CFC) assay is a
measure of granulocyte precursor activity of the bone marrow. In
these studies, the assay involved culturing marrow cells from
benzene-treated mice in an appropriate medium. The authors used
the number of colonies of granulocytic cells formed as a measure
of the number of granulocyte precursors in the marrow. The
32
-------
colony forming unit (CPU) assay, which measures stem cells, also
was used in these studies. The assay involved injecting marrow
cells from benzene-treated animals into X-irradiated mice and
evaluating the subsequent formation of colonies on the spleen of
the receptor mouse. One day after the mice were exposed to 4680
ppm of benzene for 8 hours, both CFC and CFU activity were
reduced to 40 to 45 percent of that measured in controls.
Repeated exposure by inhalation further reduced CFC activity.
The most recent studies on inhalation of benzene in rats and
mice, performed over a 2-year period by Laskin et al, are as
yet unpublished. Mice and rats were exposed to benzene at 100
and 300 ppm and were examined periodically for signs of change in
blood cell levels and for indication of aplastic anemia and/or
leukemia. Although evaluation of these data is not yet complete,
some observations may be made. Although depressions of white
cell levels were observed and animals that died during the study
often appeared to undergo depletion of bone marrow, other animals
that died displayed no signs of bone marrow toxicity. Of special
interest is the observation that decreased levels of white cells
generally were reflective of lymphocytopenia but not of granulo-
cytopenia. Further evaluation of the data is ongoing, and
additional useful information is expected. Preliminary assess-
ment indicates that in C-57 Black mice and Sprague-Dawley rats
that were exposed to 300 ppm of benzene for approximately 1 year,
lymphocyte counts were reduced to 25 and 60 percent of those in
controls, respectively, during the first 5 to 10 weeks of expo-
sure. The counts remained close to those levels for the remain-
der of the year. The mice, but not the rats, also displayed a
reduction in erythrocyte counts, which became apparent at the
same time as the lymphocytopenia and remained fairly constant at
about 68 percent of control levels throughout the remainder of
the period.
33
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EXPOSURE BY OTHER ROUTES
32 35
Both Santesson and Selling, administering benzene
subcutaneously as described above, produced benzene toxicity.
Selling administered the benzene dissolved in olive oil to
rabbits at a dose of 1 ml/kg per day. He monitored leukocytes
until they were reduced to a level of 200 to 800 cells per mm .
Because of differential sensitivity among the animals, periods of
4 to 9 days were required to reach these low levels. When
additional injections were given, most of the animals died.
Since these experiments were of short duration, red cell levels
did not vary significantly. In a similar series of studies
43
Weiskotten et al treated the animals until the white cell count
dropped to 1000 per mm . Studies of recovery after cessation of
35 44 3
treatment by Selling, Weiskotten et al, Brandino, and
34
Secchi showed that the white cell counts initially rise, then
go through a secondary depression, which Weiskotten described as
the deuterophase, and then return gradually to control values.
It is emphasized that Selling produced complete aplasia of the
bone marrow when giving benzene parenterally, an effect that
corresponds well with the observation of aplastic anemia in
humans exposed via inhalation to benzene in industrial environments,
9
Gerarde performed a similar set of experiments in which
rats were treated daily for 2 weeks with 1 ml/kg of benzene as a
50 percent solution in olive oil. He continued to observe the
animals for an additional 3-week period, during which treatments
were discontinued. Leukopenia was observed during the initial 2-
week treatment period and was accompanied by decreases in the
levels of nucleic acid and of nucleated cells in the marrow of
the femur. When administration of benzene was discontinued, each
22
of these three parameters returned to normal. Latta and Davies
treated rats with a solution of 50 percent benzene in olive oil
in doses of 2, 3, and 4 ml/kg daily for up to 60 days. Following
a temporary stimulation of white cell production, the circulating
34
-------
leukocyte levels dropped and the data suggested impairment of
leukocyte maturation. Lymphocyte production appeared to be more
dramatically impaired than was granulocyte production. At the
higher doses no myelocytes were observed after 24 days, and the
marrow was completely aplastic after 60 days.
39
Steinberg observed degeneration of bone marrow in benzene-
treated rabbits. This was followed by regeneration after the
benzene treatments were stopped. Although extrapolation of doses
from his data is difficult because the weights of the animals are
not given, it is calculated that the dose was approximately 2 ml
of benzene per day per rabbit. The animals were dosed subcutane-
ously for 6 to 70 days. Under these conditions leukocyte counts
were reduced but not in a dose-related fashion. Throughout the
treatment period white cell counts in the dosed animals averaged
22.6 +7.3 percent of control counts, the calculation being based
on all values from day 6 through day 70. As part of these
studies, Steinberg determined the ability of the marrow to
regenerate by comparing regeneration in rabbits from which
marrow had been surgically extracted with regeneration in rabbits
that had been intoxicated with benzene. Unlike control animals,
benzene-treated animals displayed regeneration only to the extent
of formation of primitive reticular cells.
28 29
Nomiyama, ' who performed daily subcutaneous injections
of rats with benzene (1 gm/kg), reported reduction of leukocyte
counts. In similar studies with several strains of mouse, he
found a strain-dependent variation in sensitivity to benzene.
19 20 26
Speck et al ' ' administered benzene to rabbits at doses
of 0.2 and 0.3 ml/kg per day and produced severe leukopenia
within 1 to 9 weeks. Although reductions in hemoglobin and red
cells were more pronounced, reductions in reticulocytes and
thrombocytes also were observed. Pancytopenia was a common
finding. Among 19 marrow smears, 21 percent were very hypoplas-
tic, 32 percent were hypoplastic, 26 percent appeared normal, and
21 percent were hypercellular. No correlation between cellularity
35
-------
and duration of exposure to benzene was apparent. In general,
myeloid precursors were diminished more than erythroid
cells. After treatment of the animals with tritiated thymidine,
radioautography of the marrow indicated that incorporation of
radioactivity into DNA was reduced. The authors interpreted
these findings to indicate that benzene toxicity leads to a
decrease in DNA synthesis. These authors also demonstrated
chromosomal aberrations in rabbits similarly treated with benzene.
Finally, they reported a decrease in incorporation of tritiated
cytidine into RNA, which they interpreted as an inhibition of RNA
synthesis.
Lee et al demonstrated that benzene depressed the incor-
59
poration of a radioactive iron isotope ( Fe) into circulating
59
erythrocytes. Radioactivity from Fe disappears from the blood
soon after parenteral administration and reappears when incorpo-
rated into hemoglobin. The rate of reappearance reflects the
maturation and proliferation of the components of bone marrow
cells. Single doses of benzene in corn oil at 400 mg/kg and 2200
mg/kg were given to mice. In measurements of 24-hour uptake, the
reappearance of radioactivity reflects only reticulocyte activity,
whereas measurements of 72-hour uptake reflect the entire cycle
from stem cell to mature red cells. Thus, the 24-hour uptakes
that were measured 24 or 48 hours after giving benzene were lower
than those of controls, whereas they were not lower when iron was
given 1, 12, or 72 hours after administration of benzene. Based
on current knowledge of red cell maturation in the mouse, these
data suggest that the cells most sensitive to benzene are early
precursors called pronormoblasts and normoblasts. In similar
experiments in which 72-hour uptakes were measured, the reduc-
tions in iron utilization were less dramatic, perhaps because the
longer uptake period allowed sufficient time for compensatory
mechanisms to come into play.
36
-------
38
In more recent studies not yet published, Lee et al
administered benzene to mice in multiple doses over several days.
They found that when mice were given a single subcutaneous dose
59
(440 mg/kg) of benzene per day, incorporation of Fe was reduced
50 to 60 percent in 20 days. When mice were given 880 mg/kg in
single daily doses, iron utilization decreased gradually to 80
percent of controls in 10 days. When doses of 440 mg/kg were
given twice per day (i.e., a total daily dose of 880 mg/kg given
as two doses) the reduction in iron uptake occurred more rapidly,
and by the tenth day almost no iron was taken up. Finally, when
two doses of benzene, at 880 mg/kg per dose, were given each day,
incorporation of iron appeared to terminate by the sixth day.
These data suggest that a dose-times-time relationship governs
the cumulative effects of benzene. Giving the dose twice per day
(440 mg/kg each) exacerbated the effect over that observed when
the same amount was given as a single dose, perhaps because the
single dose schedule allows for essentially complete excretion of
the benzene either as free benzene in exhaled air or as water-
soluble metabolites in the urine. When the dose is divided, the
animals apparently retain some benzene the morning after, to
which the next dose adds. Therefore, the animals accumulate more
and more benzene as the experiment progresses.
The oral route is the least investigated in studies of
46
benzene toxicity. Wolf et al administered 132 feedings of
benzene to rats at doses of 1, 10, 50, and 100 mg/kg over a 187-
day period. No effect was observed after the 1 mg/kg dose,
slight leukopenia was observed after the 10 mg/kg dose, and both
leukopenia and anemia were observed after the higher doses.
EVALUATION AND COMMENTS
The major problem in extrapolation of data obtained in
animal studies to benzene toxicity in humans relates to
37
-------
deficiencies in use of controls and in blood counting techniques
in the animal studies. Normal values of total leukocytes in
common laboratory animals such as the mouse, rat, and rabbit
3
range widely from approximately 4000 to 5000 cells per mm to
over 20,000, with occasional values in the rabbit as low as
33
3200. Thus, it may be argued that reductions in white cell
count within that range may not reflect true toxicity but may
reflect successful protective responses by the organism. In
contrast, decreases in leukocyte levels to below 2000 cells per
mm may reflect insults severe enough to impair the health of the
animal. In many reports that claim the production of leukopenia,
however, it is implied that leuko~enia is defined as a reduction
of white cell levels below some control value. When the control
values are set by measurements of the test animals prior to
exposure, an error may be introduced because of natural varia-
4
tions in white cell counts. For example, Cheng reported that
leukocyte counts in rabbits increase gradually from 2000 in the
first week of life to between 4500 and 6000 cells at 100 to 200
days of life. Considerable variability was observed among 240
adult rabbits in which white blood cell (WBC) counts averaged
7000 + 5000 cells. Although age may not be a factor in studies
with mice, the time of day of sampling, the site from which the
sample is taken, and the strain of mouse all affect the cell
count. The effect of age on the leukocyte count in rats remains
an open question. Reich and Dunning studied eight different
strains of rat, and Harris and Burke studied the Wistar rat, a
strain not examined by Reich and Dunning. Both groups reported
that age did not affect leukocyte counts. It was suggested that
the neutrophil-lymphocyte ratio increases with age in rats.
Recently, Laskin et al found that a relative decrease in
leukocytes occurs within the first 3 months of life of Sprague-
Dawley rats. These observations indicate that claims for
benzene-induced leukopenia should be supported by proper controls
for age, as well as for exposure.
38
-------
Although the extreme decreases in leukocytes observed by
35 44 20
Selling, Weiskotten and co-workers, and Kissling and Speck
clearly demonstrated leukopenia, the studies of Hough et al,
Wolf et al,46 Deichmann et al,5 Nau et al,27 Ikeda et al, fl
Drew et al, and others must be evaluated with respect to the age
effect and other factors.
A second question regarding changes in white cell counts
concerns the interpretation that reductions in numbers of white
cell must, of necessity, reflect bone marrow damage. Relatively
few investigators have performed differential counts of the white
blood cells. Laskin et al have shown that during extended
exposure to benzene, the total WBC counts in rats and mice
decreased but the numbers of granulocytes apparently did not.
Since granulocytopenia reflects bone marrow damage, these animal
models may not reflect the human disease in which granulocytopenia
is a prominent feature.
Even with proper controls and differential cell counts,
however, proper interpretation of the effects of benzene in
animal experiments will be difficult if evaluation of peripheral
blood factors remains the sole focus of attention. The problem
can be exemplified by comparing the results of Speck and asso-
1920 2
ciates ' with those of Boje et al. The Speck group treated
rabbits with benzene subcutaneously until white cell levels
decreased below 1000 per mm . Under these conditions mitosis in
bone marrow was impaired and uptake of tritiated thymidine into
2
DNA was reduced significantly. When Boje et al exposed rats to
benzene by inhalation at 400 ppm for up to 13 weeks, they
demonstrated a similar decrease in DNA synthesis in the bone
marrow, but in this case the white cell levels were decreased to
only 46 percent of controls, which represents a considerably
higher count than Speck's 1000 per mm . Thus, significant damage
to marrow may occur when leukocyte levels remain relatively high.
It must be argued then that reductions in white cell levels, even
39
-------
when the counts remain in the normal range, may be indicative of
marrow damage and it is not necessary to demonstrate leukocyte
levels below 2000 cells to suspect benzene-induced marrow damage.
Careful studies of the effects of benzene on circulating blood
cell elements should be supported with information concerning the
effects of benzene on bone marrow.
Benzene and Leukemia in Animals
Leukemia is a general term for a group of diseases usually
characterized by large increases in numbers of white blood cells
in blood and/or bone marrow or the appearance of unusual leuko-
cyte precursors in the blood. Leukemia is associated with
neoplasms, i.e., new growths of tissue serving no physiologic
function. It is usually accompanied by a variety of defects in
the hematopoietic, or blood-cell forming, system. Leukemias are
known to occur spontaneously in some strains of mice, and there
is abundant evidence that chemicals can induce leukemia in both
mice and rats.6'12'14'25'30'36 Lignac25 reportedly produced
leukemia in mice by treating them with benzene subcutaneously for
17 to 21 weeks. Of the 44 mice that survived treatment, 8 were
described as having developed leukemia or lymphosarcoma. Failure
to include control mice and to provide details concerning the
strain of mouse and the diagnostic criteria leaves this report
open to question.
In a specific attempt to duplicate the results of Lignac,
Amiel treated mice of the AKR, DBA?, C^H, and C57B1 strains with
weekly injections of benzene (30 mg/kg) throughout their life-
times but observed neither aplastic anemia nor leukemia. Ward et
A O
al studied a group of C57B1 mice for a total of 104 weeks,
during which time the dosage schedule was varied but in general
was increased from 450 mg/kg to 1.8 gm/kg. Although a number of
mice died of the toxic effects of benzene, there was not a statis-
tically significant increase in incidence of neoplastic disease
in benzene-treated mice over that observed in controls.
40
-------
The evidence for production of leukemia in animals by
injection with benzene must be considered nonconclusive. More-
over, neither oral dosing, skin painting with benzene, '
nor inhalation ' had been demonstrated to produce leukemia or
any other type of neoplastic disease in rats, mice, guinea pigs,
or rabbits. Suggestive results, however, have been reported very
recently by Nelson*. Of 40 mice (strain CD-I) exposed over their
lifetimes to 300 ppm benzene, 2 died of leukemia (1 with chronic
myelogenous leukemia, 1 with acute stem cell leukemia of possible
myeloblastic type). Of 45 Sprague Dawley rats exposed similarly
to 100 ppm benzene, 1 died of chronic granulocytic leukemia.
There have been no published reports of spontaneous cases of
myelogenous leukemia, either acute or chronic, in CD-I mice or
Sprague-Dawley rats.
These findings, although the experiments were performed with
relatively small numbers of animals and at moderately high doses
of benzene, are consistent with epidemiological data in humans
indicating a relationship between benzene inhalation and leukemo-
genesis. The results, if confirmed, may indicate the possibility
of the much-desired animal model for leukemogenesis.
In contrast, with respect to possible benzene-associated
leukemia in humans, evidence from industries that have used
benzene heavily implies a direct relationship between benzene
exposure and development of leukemias. Furthermore, the
leukemias have been observed mainly in cohorts of workers among
whom many showed signs of benzene-induced bone marrow damage of
variable severity. Despite the inability of investigators to
demonstrate leukemia in groups of animals exhibiting bone marrow
damage, the studies of humans provide compelling evidence that
benzene is involved in leukemogenesis.
Several theories might be offered to explain the inability
of researchers to induce leukemia in animals by treatment with
benzene. Man may be the only species yet observed that is
*Nelson, N. Letter to R. Cortesi, EPA, March 13, 1978.
41
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susceptible to benzene-induced leukemia for a variety of reasons
such as 1) a novel metabolic pathway that produces a unique
reactive metabolite not formed in other animals, 2) relative
inefficiency in repair of DNA of benzene-induced damage, or 3)
relative ineffectiveness of immune surveillance following
benzene-induced insult. Also, the phenomenon may involve other
mechanisms of which we are not aware. It is possible that the
latency period in animals is long enough to preclude the appear-
ance of leukemia during the lifetime of the animal, or an as-yet-
unknown cocarcinogen may be required to evoke the leukemogenic
response initiated by benzene. If bone marrow damage is a
prerequisite for benzene-induced leukemia, the appropriate
animal experiments may not have been done. For example, it may
be necessary to induce bone marrow damage and then to allow for
recovery during the remainder of the life of the animal. Regular
monitoring might then show some animals with leukemia. The
researcher then should demonstrate that a similar control popula-
tion displays significantly fewer cases of leukemia. In any
attempts to disclose a cause and effect relationship between
benzene exposure and leukemia, it is essential that the studies
include sufficient numbers of control and treated animals. The
need for large numbers of animal subjects is underscored by the
exceedingly low incidence of benzene-induced leukemia in human
workers. Meaningful statistics on these cases have been collected
only when large groups of workers were observed.
42
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REFERENCES FOR SECTION 3
1. Amiel, J.L. Essai negatif d1induction de leucemies chez les
souris par le benzene. Rev. Franc. Etudes Clin. Biol.,
5:198-199, 1960.
2. Boje, H., Benkel, W., Heiniger, H.J. Untersuchungen zur
leukipoese im knochenmark der ratte nach chronischer benzol-
inhalation. Blut, 21:250-257, 1970.
3. Brandino, G. Osservazione istologische nel'intossicazione
acute e croniche da benzolo. Gazz. Med. Lomborda, 81:141,
1922.
4. Cheng, S.C. Amer. J. Hyg., 11:449, 1930.
5. Deichmann, W.B., MacDonald, W.E., Bernal, E. The hemipoie-
tic tissue toxicity of benzene vapors. Tox. Appl. Pharm.,
5:201-224, 1963.
6. Diwan, B.A., and Meier, H. Can. Lett., 1:249, 1976.
7. Drew, R.T., Fouts, J.R., Harper, C. The influence of
certain drugs on the metabolism and toxicity of benzene.
In: Symposium on Toxicology of Benzene and Alkyl-benzenes,
Braun, D. (Ed.). Industrial Health Foundation, Pittsburgh,
1974. pp. 17-31.
8. Drew, R.T., Fouts, J.R. The lack of effects of pretreatment
with phenobarbital and chlorpromazine on the acute toxicity
of benzene in rats. Tox. Appl. Pharm., 27:183-193, 1974.
9. Gerarde, H.W., Ahlstrom, D.B. Toxicologic studies on
hydrocarbons. XI. Influence of dose on the metabolism of
mono-n-alkyl derivatives of benzene. Tox. Appl. Pharm.,
9:185-190, 1966.
10. Goldstein, B.D., Laskin, S., Snyder, C.A. Unpublished
observations, 1977.
43
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11. Harris, C., Burke, W.T. Am. J. Path., 33:931, 1957.
12. Hartmen, H.A., Miller, E.G., Miller, J.A., Morris, F.K.
Can. Res., 19:210, 1959.
13. Hough, V.N., Guinn, F.D., Freeman, S. Studies on the
toxicity of commercial benzene and of a mixture of benzene,
toluene and xylene. J. Industr. Hyg., 26:296-306, 1944.
14. Huggins, C.B., Sugiyama, T. Proc. Natl. Acad. Sci. , 55:74,
1966.
15. Ikeda, M. Enzymatic studies on benzene intoxication.
J. Biochem., 55:231-243, 1964.
16. Ikeda, M., Ohtsuji, H., Imamura, T. In vivo suppression of
benzene and styrene oxidation by co-administered toluene in
rats and effects of phenobarbital. Xenobiotica, 2:101-106,
1972.
17. Jenkins, L.J., Jr., Jones, R.A., Siegel, J. Long-term
inhalation screening studies of benzene, toluene, o-xylene,
and cumene on experimental animals. Tox. Appl. Pharm.,
16:818-823, 1970.
18. Kirschbaum, A., Strong, L.C. Influence of carcinogens on
the age incidence of leukemia in the high leukemia F strain
of mice. Cancer Res., 2:841-845, 1942.
19. Kissling, M., Speck, B. Chromosome aberrations in experi-
mental benzene intoxication. Helv. Med. Acta., 36:59-66,
1972.
20. Kissling, M., Speck, B. Further studies on experimental
benzene induced aplastic anemia. Blut Z Gesamte Blut
Forsch, 25(2):97-103, 1972.
21. Laerum, O.D. Reticulum cell neoplasms in normal and benzene
treated hairless mice. Acta Path Microbiol. Scand.
81:57-63, 1973.
22. Latta, J.S., Davies, L.T. Effects on the blood and hemato-
poietic organs of the albino rat of repeated administration
of benzene. Arch. Path., 31:55-67, 1941.
23. Lee, E.W., Kocsis, J.J., Snyder, R. Acute effects of
benzene on 5^Fe incorporation into circulating erythrocytes.
Tox. Appl. Pharm., 27:431-436, 1974.
44
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24. Li, T.W., Freeman, S., Gunn, F.D. J. Physiol., 145:158-166,
1945.
25. Lignac, G.O.E. Die benzolleukamie bei menschen und weissen
mausen. Klin. Wschr., 12:109-110, 1932.
26. Moeschlin, S., Speck, B. Experimental studies on the
mechanism of action of benzene on the bone marrow (radio-
acutographic studies using ^n-thymidine). Acta Haemat.,
38:104-111, 1967.
27. Nau, C.A., Neal, J., Thornton, M. Arch. Env. Heal"h,
12:382, 1966.
28. Nomiyama, K. Studies on the poisoning by benzene and ils
homologues (6) oxidation rate of benzene and benzene poison-
ing. Med. J. Shinshu Univ., 7:41-48, 1962.
29. Nomiyama, K., Minai, M. Ind. Health, 7:54, 1969.
30. Ogui, T., Nakadate, M., Odashima, S. Can. Res., 36:3043,
1976.
31. Reich, C., Dunning, W.F. Cancer Res., 3:248, 1943.
32. Santesson, C.G. Ueber chronische vergiftungen mit stein-
kohlentheerbenzin; vier todesfalle. Arch. Hyg. Berlin,
31:336-376, 1897.
33. Schalm, O.W., et al. Veterinary Hematology, 3rd Edition,
Lea & Febiger, Philadelphia, 1975.
34. Secchi, P. Ricerche ematologiche nelle intossicazione acute
e chroniche da benzolo. Rif. Med., 30:995, 1914.
35. Selling, L. Benzol as a leucotoxin. Studies on the degener-
ation and regeneration of the blood and hematopoietic organs,
John Hopkins Hospitajl Reports, 17:83-142, 1916.
36. Shay, H. Gruenstein, M.G., Marx, H.E., Glazer, L. Can. Res.
11:29, 1951.
37. Snyder, R., Kocsis, J.J. Current concepts of chronic
benzene toxicity. CRC Critical Reviews in Toxicology, June
1975, pp. 265-288.
45
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38. Snyder, R., Lee, E.W., Kocsis, J.J., Unpublished, 1977.
39. Steinberg, B. Bone marrow regeneration in experimental
benzene intoxication. Blood, 4:550-556, 1949.
40. Svirbely, J.L., Dunn, R.C., von Oettingen, W.F. The chronic
toxicity of moderate concentrations of benzene and of
mixtures of benzene and its homologues for rats and dogs.
J. Industr. Hyg. Toxicol., 26:37-46, 1944.
41. Uyeki, E.M., Ashker, A.E., Shoeman, D.W., Bisle, T.U.
Toxicol. Appl. Pharm., 40L49, 1977.
42. Ward, J.M., Weisburger, J.H., Yamamoto, R.S., Benjamin, T.,
Brown, C.A., Weisburger, E.K. Long-term effect of benzene
in C57BL/6N mice. Arch. Environ. Health, 30:22-25, 1975.
43. Weiskotten, H.G., Schwartz, S.C., Steensland, J. Med.
Res., 33:127, 1915.
44. Weiskotten, H.G., Schwartz, S.C., Steensland, H.S. The
action of benzol. The deuterophase of the diphasic leuko-
penia and intogen-antibody reaction. J. Med. Res., 35:63-79,
1916.
45. Weiskotten, H.G., Schwartz, S.C., Steensland, H.S. J.
Med. Res., 41:425, 1920.
46. Wolf, M.A., Rowe, V.K., McCollister, D.D., Hollingsworth,
R.L., Oyen, F. Toxicological studies of certain alkylated
benzenes and benzene. AMA Arch. Ind. Health, 14:387-398,
1956.
46
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SECTION 4
BENZENE TOXICITY IN MAN
INTRODUCTION
The world medical literature contains hundreds of references
describing thousands of cases of human hematological toxicity asso-
ciated with benzene exposure. This literature has been reviewed
relatively recently by a number of authors.24'39'78'88'145'148'172
Although numerous hematological disorders have been reported in
association with benzene exposure, only two entities are clearly
related to benzene. These are 1) pancytopenia and its variants,
including anemia, leukopenia, thrombocytopenia and aplastic
anemia; and 2) acute myelogenous leukemia and its variants, such
as acute myelomonocytic leukemia and erythroleukemia. Unfortu-
nately, there is a relative paucity of information concerning the
doses of benzene to which affected individuals were exposed.
This section deals with the evidence presented in the
literature concerning the human hematological toxicity of benzene.
The emphasis is primarily on studies that have evaluated relative-
ly large numbers of occupationally exposed individuals, particu-
larly where measurement of dose has been attempted. Most of the
rest of the reports describe one or a few cases of hematotoxicity
associated with benzene exposure. Such reports do have cumula-
tive weight, particularly in relation to the causative role of
benzene in acute myelogenous leukemia. Individually, however,
they provide little information not given elsewhere and therefore
they are not discussed here.
47
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In the reported cases of benzene hematotoxicity, exposure
has usually occurred in a workplace where benzene was used as a
solvent or manufactured as a product. In some instances, benzene
was used because it is inexpensive and has excellent solvent
properties. In other instances, benzene was an inadvertent
contaminant of aromatic hydrocarbon solvents. Whatever the
reason for its use, in almost all cases in which benzene hema-
tctoxicity has been reported, the patient has been exposed also
to some other solvent or chemical product. That it is benzene,
rather than some other commonly associated agent such as xylene
or toluene, that is primarily responsible for hematotoxicity
appears well established. The major evidence supporting this
assertion includes the observation that benzene has been the
common denominator in many different occupational settings in
various parts of the world in which exposure to other chemicals
has varied widely. In addition, the pancytopenic effect of
benzene in man can be readily duplicated in experiments with a
variety of animal species. This is not true of the other common-
ly associated contaminants.
It is possible, however, that compounds inhaled along with
benzene alter the expression of benzene hematotoxicity. In
particular, it is conceivable that other aromatic hydrocarbons
may modify benzene metabolism in humans. This is an area in
which additional information on the effects of benzene in man
would be of great value.
PANCYTOPENIA
The term pancytopenia refers to a diminution of all formed
elements in the blood. In benzene exposure this diminution is
due primarily to an interference in the production of red cells,
white cells, and platelets in the bone marrow, although some
evidence suggests that survival of blood cells within the circu-
lation may be shortened also. Many of the cases associated with
48
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benzene exposure are described as "aplastic anemia." This term
classically denotes a condition in which the number of hemato-
poietic precursor cells within the bone marrow diminishes
markedly. Aplastic anemia is usually associated with a high
degree of pancytopenia and is observed particularly in severe or
fatal cases. However, a decrease in bone marrow cellularity is
not always noted in individuals with hematotoxicity associated
with benzene exposure nor in all animals treated with benzene.
This may be attributable to individual variation, to ineffective
response of poietic bone marrow to benzene, or to the sampling
error inherent in the presumption that a local aspirate of bone
marrow represents all hematopoietic tissue. For purposes of
discussion, cases of clear bone marrow aplasia are considered
under the heading of pancytopenia.
Numerous cases of individual cytopenias, e.g., anemia
without leukopenia or thrombocytopenia, also are reported in
benzene-exposed individuals. These have been observed mostly in
industries where severe benzene hematotoxicity in some workers
has led to evaluation of the entire work force. it should be
noted that there is wide variation in the normal red cell, white
cell, and platelet counts as well as appreciable reserve produc-
tion capability. A person in whom one of the formed elements
decreases to a level below the accepted normal value may also
experience an effect on the production of other cell types that
is not clinically apparent. Accordingly, the individual cyto-
penias are also discussed under the heading of pancytopenia.
Exposure to benzene clearly produces hematological toxicity
in animals and man. Evidence of a pancytopenic effect of benzene
was first noted in 1897 by Santesson, who reported four cases of
fatal aplastic anemia occurring in workers fabricating bicycle
tires. Since that time numerous case reports and surveys of
occupationally exposed groups of workers have documented this
association, and many reviews of these cases have
49
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appeared.24'39'78'85'88'92'145'148'165'172'173 The causal
relationship of benzene to pancytopenia in man is most clearly
supported by studies, described below, of groups of workers in
whom the appearance of pancytopenia was temporally related to the
inception of benzene use and in whom the outbreak of hematologi-
cal effects was ended by replacement of benzene with some other
solvents.
Current concepts of the hematopoietic system stress the role
of a pluripotential myeloproliferative stem cell in production of
erythrocytes, granulocytes, platelets, and perhaps fibroblasts
and other monocytic cells. This stem cell is believed to be able
to differentiate into the precursors of the various formed
elements in response to microenvironmental conditions. The
production of pancytopenia suggests that benzene is toxic to this
stem cell or to early hematopoietic precursors of red cells,
granulocytes, and platelets. Some incomplete evidence also
points to an even earlier pluripotential cell with capabilities
of differentiating into lymphocytic as well as myelocytic pre-
cursors. This may be pertinent to benzene toxicity in that
lymphocytopenia is frequently observed in pancytopenic indivi-
duals with a history of benzene exposure. In addition, chromo-
somal abnormalities in circulating lymphocytes have been reported
in association with benzene exposure (see Section 2), and
evidence suggests that such exposure increases the risk of
lymphocytic neoplasms.
The symptoms of pancytopenia in individuals exposed to
benzene are described by a number of authors who have investiga-
ted occupationally exposed groups. In milder cases, these tend
to be such nonspecific complaints as lassitude, tiredness, easy
fatigability, malaise, dizziness, headaches, palpitation, and
shortness of breath. Such symptoms tend to appear gradually, and
presumably reflect anemia. Occasionally, in more severe cases,
hemorrhagic manifestations due to thrombocythemia or decreased
50
-------
platelet function are also observed. In severe cases of pancyto-
penia, death is often due to hemorrhage or to overwhelming
infection, the latter reflecting the decrease in granulocytes.
In addition, patients with pancytopenia may subsequently develop
fatal acute leukemia, sometimes with an intervening period of
apparent recovery.
Among the earliest systematic evaluations of the pancyto-
8 ?
penic effects of benzene are those of Greenburg et al,
79 80
Goldwater, and Goldwater and Tewksbury. These investigators
studied workers in rotogravure printing plants where benzene had
been in use for a period of 3 to 5 years. The benzene contents
of the ink solvents and thinners ranged from 10 to 80 percent.
Forty-eight analyses of benzene concentrations in the air of
pressrooms of three plants revealed levels ranging from 11 to
1060 ppm, with a median concentration of 132 ppm. The most
frequent clinical complaints were fatigue, dryness of the mucous
membranes, lethargy, dizziness, headache, and shortness of
breath. Hematological studies were performed in 332 exposed male
workers and 81 controls. Various degress of hematological
toxicity were observed in 65 of these workers, 23 of whom were
considered to be severely affected. As a result of these studies,
six individuals were referred for hospitalization. The remaining
workers continued on the job and, following replacement of
benzene with other solvents, hematological recovery was demon-
8 0
strated. In comparison with the control group, the most
frequently observed findings were anemia, macrocytosis, and
thrombocytopenia. An absolute lymphocytopenia was more common
than was neutropenia, which was rarely observed. Other relative-
ly infrequent findings were prolongation of the bleeding or
coagulation times, an increase in capillary fragility, and an
increase in serum bilirubin and reticulocytes. Osmotic fragility
was normal, as was the erythrocyte sedimentation rate. No
monocytosis, eosinophilia, or D^sophilia was observed.
51
-------
Several occupationally exposed groups were studied during
195
World War II. Wilson evaluated workers in an American rubber
factory in which peak benzene levels were said to be 500 ppm,
with an average of about 100 ppm. Of 1104 workers studied, 83
were observed to have mild hematological effects and 25 had more
severe pancytopenia. Nine of the latter group were hospitalized,
o c
and three died. Hamilton-Patterson and Browning reported
observations of 200 women in 13 aircraft factories in England as
compared to 200 controls. These workers had been involved in
rubber manufacture and had been exposed to solvents and adhesives
containing 5 to 20 percent aromatic hydrocarbons. In contrast to
82 79
the findings of Greenberg et al and Goldwater these authors
suggest that neutropenia is the earliest and most consistent
indicator of benzene toxicity. No significant difference in red
8 9
cell or lymphocyte counts was observed. Helmer in Sweden
evaluated 184 workers (169 women, 15 men) in a rubber raincoat
factory where benzene levels of 137 to 218 ppm were measured and
it was believed that levels had been higher in the past. Evi-
dence of hematological toxicity was observed in 60 individuals
(58 women, 2 men). Reevaluation 16 months after cessation of
benzene use revealed that 46 recovered, 12 still had significant
effects, and 2 had died. The most frequent symptoms were gradual
development of headache and tiredness. The author also notes the
frequency of cutaneous hemorrhages and stresses the finding of
93
thrombocytopenia. Hutchings et al studied 87 benzene-exposed
individuals in Australian air force workshops after the discovery
of a fatal case of aplastic anemia. The measured peak benzene
concentrations ranged from 10 to 1400 ppm, and were well above
100 ppm in most areas. For most of the time, however, the
concentrations ranged from 10 to 35 ppm. Solvents contained up
to 53 percent benzene. In addition, the worker exposure time was
studied in relation to atmospheric benzene concentrations.
52
-------
Little difference was observed in the hematological measurements
o± 8/ benzene-exposed workers as compared to those of 500 workers
exposed to other hydrocarbons and 300 unexposed controls, although
there was a tendency toward a lower hemoglobin and platelet
counts. The duration of benzene exposure in these workers is
unclear. Hutchings et al conclude that the worker who died of
aplastic anemia may have been unusually susceptible to the
effects of benzene.
140
Pagnotto et al report a study of benzene exposure in a
rubber coating plant where petroleum naphtha containing 1.5 to
9.3 percent benzene was in use. Atmospheric benzene levels were
generally less than 25 ppm but ranged up to 125 ppm. Correlation
between urinary phenol levels and measured concentrations of
benzene in air was excellent. In one plant, the hemoglobin
levels of 5 of 32 men studied were reduced. One of these men had
the second highest urinary phenol level measured in the plant
(480 mg per liter, equivalent to 58 ppm benzene exposure). In a
second plant/ only 1 of 9 individuals studied was anemic, but
this worker was severely affected requiring hospitalization. In
a third plant, none of six workers studied was anemic, nor was
leukopenia observed. Some follow-up data suggesting mild
persistent anemia are presented by the National Institutes of
Occupational Safety and Health (NIOSH).145
Several studies of groups occupationally exposed to benzene
174
have been reported in the past decade. Stewart et al (pub-
lished in abstract only) reported on ten persons with mild anemia
and macrocytosis who had recovered within 8 months following
104
apparent cessation of benzene exposure. Kliche et al reported
studies of 18 roof tilers exposed for an average of 17 years to
what is stated to be 15 ppm benzene. Six of these persons are
described as having mild early disease and seven as having
chronic benzene hematotoxicity. The author also presents indirect
evidence of qualitative abnormalities of platelets despite normal
platelet counts in these subjects.
53
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A number of Eastern European studies of benzene-exposed
workers have been reported in recent years. Doskin evaluated
365 workers employed for 3 years in an apparently new chemical
factory. Although detailed monitoring apparently was performed,
the measured concentrations are not given. The author states
that benzene levels exceeded the maximum permissible concentra-
tion 2 to 8 fold in 64 percent of the measurements in the first
year, 37 percent in the second year, and 3 percent in the third
year. The analytical method and total number of measurements are
not stated. We believe, although we have not been able to con-
firm, that the maximum permissible concentration of benzene in
the Soviet Union at that time apparently was 5 ppm. Pre-employ-
ment and serial hematological measurements were obtained during
the 3-year period and were compared with values from a control
group. Approximately 40 percent of the workers exhibited mild
hematological abnormalities during the first year, and this
percentage declined greatly in subsequent years. The most common
early sign of benzene hematotoxicity was mild thrombocytopenia
(96 to 155,000/mm ) followed by anemia, which was normochromic
with a tendency toward subsequent development of hyperchromia
(presumably because of an increase in mean corpuscular volume)
after 1 year of employment. An initial increase in WBC count was
followed in certain cases by leukopenia. In support of this
observation Doskin cites Soviet literature describing a phasic
91
response to benzene. Except for an early report by Hunter
suggesting increased erythropoiesis as an initial response to
benzene, Western investigators have not described such a phasic
response. Other somewhat different findings in Doskin1s study
include observations of lymphocytosis rather than lymphocytopenia
and relatively greater effects in younger subjects, in contrast
with Aksoy's finding of no effect of age on response to benzene.
Analysis of bone marrow of 30 workers showed hypercellularity,
particularly in subjects with leukocytosis, a decrease of mega-
karyocytes in subjects with thrombocytopenia, and an increase in
54
-------
lymphocytes in younger subjects. In addition, a decrease in the
phagocytic ability of leukocytes was noted. Although not
substantiated, these findings suggest that exposure of workers to
concentrations of 10 to 40 ppm benzene for less than 1 year
produces mild hematological effects. Information about the
benzene monitoring system would be of value in interpreting these
findings.
Kozlova and Volkova in the Soviet Union have also used
the phagocytic function of leukocytes as an indicator of benzene
hematotoxicity. They studied 252 workers exposed to benzene
during a 5-year period in which benzene concentrations initially
ranged from 47 to 310 ppm then, as control measures were improved,
decreased to average concentrations of 25 to 47 ppm by the end of
the period. The workers were classified in three groups depend-
ing upon exposure levels, the lowest being 24 to 39 ppm. All
groups showed a decrease in cell counts, and severity of the
changes was greatest with higher exposure levels. In the higher-
level exposure group, the extent of the hematological effects
correlated well with duration of exposure. The most prominent
findings were leukopenia, predominantly reflecting neutropenia,
and thrombocytopenia. Changes in red cells tended to occur
relatively late. In most cases a decrease in phagocytic activity
occurred earlier than other hematological effects.
Another study from Eastern Europe reported that 16 of 27
workers experienced an increase in levels of red cell delta-
aminolevulinic acid, a precursor in the heme biosynthetic path-
way. In 12 of the 16, earlier benzene exposures are stated to
have ranged from 6.4 to 15.6 ppm and more recent exposures were
to 1.6 ppm. The remaining four workers were exposed only to 1.6
ppm benzene. Blood counts were normal. The pertinence of these
findings must await confirmation of the authors' hypothesis
99 100
concerning the effect of benzene on porphyrin metabolism. '
Also of potential but unproven importance is the finding by
55
-------
Smolik et al of a decrease in the mean serum complement of 34
workers as compared to those of a control group. Benzene levels
ranged from 3.4 to 6.8 ppm, and duration of exposure, from 3
months to 18 years. Other investigators also have suggested that
benzene alters the immune system in man, including the frequent
observation of lymphocytopenia and reports of altered serum
immuno-globulin levels, the presence of antibodies against
circulating blood cells, and morphologically altered lymphocytes
, . 111,112,149,151 -, ., .-, ,
and monocytes. Based on the immune surveillance
hypothesis, it could be conjectured that a decrease in immune
function plays a role in benzene leukemogenesis. At present,
however, the evidence does not clearly delineate a primary effect
of benzene on immune function in man.
In a study performed in Korea, Chang evaluated hematological
toxicity in relation to measured benzene levels and urinary
41
phenol excretion in workers occupationally exposed to benzene.
The author presents data indicating hematological effects in
workers exposed to concentrations as low as 20 ppm; by extrapola-
tion he concludes that "benzene poisoning may occur when workers
are exposed to as low as 10.1 ppm benzene in the air." Unfortu-
nately, many details are missing in the published account of this
study. After eliminating workers with various disease states
from the study group, the author evaluated 119 individuals said
to be exposed to benzene in an unspecified industrial area.
Benzene levels were measured by a method based on ultraviolet
absorption in ethanol, but the number and duration of measure-
ments are not given. Urinary phenol was measured after 4 hours
of work by reaction with p-nitroaniline. The number of such
determinations for each worker is not specified. Of the 119
subjects, 28 showed hematological abnormalities: 21 with a
normochromic or hyperchromic anemia, 2 with leukopenia, and 5
with both anemia and leukopenia. In comparison with other
workers, those with hematotoxicity generally had been exposed for
a shorter duration at higher benzene levels. Extrapolation from
56
-------
a plot of benzene concentration (in ppm) against interval from
start of work until occurrence of hematotoxicity revealed an
0 2x
exponential function described as y = (82.5) (0.77 " ) + 10.1;
where y is ppm benzene and x is work duration in months (Figure
2). No hematological toxicity was observed in the 18 subjects
exposed to 10 to 20 ppm benzene. The graph suggests that 7 of
the 24 subjects exposed to 20 to 30 ppm benzene exhibited hema-
tological toxicity after 42 to 96 months of exposure. At the
higher benzene levels apparently 1 of 5 workers exposed to 100 to
120 ppm benzene and 4 of 13 workers exposed to 100 to 110 ppm
benzene developed hematological toxicity. Of interest is that
the average duration of work for all subjects was substantially
less at the higher benzene levels, perhaps implying an occupation-
ally related attrition. Studies of phenol excretion showed a
direct correlation with measured benzene levels (r = 0.469, p <
0.01). The levels of urinary phenol in this study were about 2
to 3 times lower than those reported by Elkins for equivalent
benzene concentrations. The author suggests that this disparity
may be due in part to his use of a 4-hour exposure period rather
than the 8-hour exposure used by Elkins. The urinary phenol
findings suggest that the author has not underestimated the
atmospheric benzene concentration at the time of the study. This
interesting study presents the most detailed evaluation of benzene
hematotoxicity versus dose available in the literature. Unfor-
tunately, because the information is incomplete, particularly as
to characterization of the work force and exposures, it is dif-
ficult to interpret the relevance of these findings to benzene
exposure in other populations.
Numerous cases of benzene hematotoxicity in Italy have been
reported, particularly by Vigliani, Saita, Forni, and their
n 58-62,155-162,189-194 m, . . , . .
colleagues. The accumulated case reports in
Milan and Pavia among shoe workers and other occupationally
191 193
exposed groups have been reviewed recently ' and are de-
scribed below. Measured benzene concentrations have ranged from
25 to 1500 ppm and often have been over 200 ppm.
57
-------
I 120
* 100
H-I
z 80
»-H
S 60
S 40
20
y = 82.5 x 0.77°'2x + 10.1
EXPONENTIAL LINE IS FOR
ABNORMAL BLOOD PICTURE.
20 40 60 80 100
DURATION OF WORK, MONTHS
Figure 2. Effect of benzene in air and duration
of work on abnormal blood picture.
58
-------
Among the many French investigators who have reported
benzene hematotoxicity, Girard and his colleagues present an
67-75
extensive series of investigations. Their studies include
the frequent observation of a decreased leukocyte alkaline
phosphatase in a group of 319 workers exposed to benzene levels
f\ 7 f\ fi
said to range from 10 to 25 ppm. ' They have also noted a
statistically significant occurrence of a history of occupational
benzene exposure in patients with aplastic anemia as compared to
72 73
a control group. '
Studies of Aksoy et al
Aksoy and his colleagues in Turkey have performed one of the
more extensive evaluations of individuals with benzene hemato-
toxicity. Most of the cases observed were in shoe workers
who between 1955 and 1960 began using an adhesive containing high
levels of benzene. Individual cases of aplastic anemia were
first noted in 1961. By 1977 Aksoy and his colleagues had studied
46 patients with this disorder, of whom 14 had died of aplastic
anemia, 5 had developed acute leukemia, 1 developed myeloid
metaplasia, 22 were in complete remission, 2 were still under
treatment, and 2 were lost to follow-up. This compilation
includes only patients personally observed by Aksoy et al.
Thirty-five were shoe workers and the remainder were also exposed
to adhesives containing 9 to 88 percent benzene. Exposure was
generally in small shops with poor ventilation. Where measured,
the benzene concentrations ranged from 150 to 650 ppm,
Aksoy and his colleagues also present a thorough study of
hematological findings in 217 apparently healthy male shoe
workers in comparison with 100 control subjects. Fifty-one of
the 217 exposed were considered to have a benzene-associated
hematological abnormality. Forty-one were afflicted with leuko-
penia and/or thrombocytopenia; the other ten are included on the
basis of various abnormalities including eosinophilia, basophilia,
giant platelets, lymphocytosis, and an acquired Pelger-Huet
anomaly. The latter is a morphological abnormality seen in
59
-------
leukemia and reported in association with benzene exposure. The
authors also report that 33 percent of the total work group was
anemic, but because the hematocrit readings returned to normal
following iron therapy in those treated, the anemia is not
definitively ascribed to benzene toxicity. Macrocytosis was not
observed, but the absence of this finding may be due to the
complicating iron deficiency anemia, which usually leads to
microcytosis. No difference in the relative incidence of benzene
hematotoxicity was noted in different age groups. Of note was a
tendency toward a higher incidence of hematotoxicity in workers
exposed for less than 1 year as compared with those exposed for
longer periods. The authors suggest that this could be due to
all hematological changes occurring or starting in the first year
of exposure, or to a loss of affected individuals from the work
force.
Aksoy's group has also detailed the hematological findings
in 32 patients with clinically significant pancytopenia. All
used adhesives containing benzene. Ambient levels of benzene
were said to range from "15 and 30 ppm outside working hours and
were recorded to reach 210 or, rarely, 640 ppm when benzene
containing adhesives were being used." Cellularity of the bone
marrow was decreased in 12 cases, normal in 12, and increased in
7. In the remaining case, giant erythroid precursors were
observed, perhaps indicating preleukemia. The severity of
disease and likelihood of fatal outcome was greatest in the group
with hypocellular bone marrows, although the duration of exposure
appeared unrelated to the bone marrow findings. Macrocytic
erythrocytes were observed.in 14 patients, most of those being
patients with hyperplastic bone marrows. Nucleated erythrocytes
were noted in the peripheral blood smear of six patients. Other
led cell abnormalities included a mild to moderate increase in
the osmotic fragility, with or without 24-hour preincubation, in
13 of 20 subjects tested; an increase in fetal hemoglobin in 20
of 24 patients; and a decrease in Hb A2 in 3 of 24 patients.
60
-------
This group has described additional studies suggesting an increase
4 9
in hemoglobin F in benzene henicttotox n.j. t^ . ' .^viaence of a
shortened red cell life span include'! a slight reticulocytosis,
mild hyperbilirubinemia, and increased urobilinogen in some
subjects, and a moderately shortened radioactive chromium sur-
vival in the one subject in whom this was measured. Except for
the latter finding, these observations could also be due to
ineffective erythropoiesis. White cell abnormalities included an
absolute lymphocytopenia in 24 subjects, a slight incieat.e in
monocytes in 4 subjects, and the development of a Pelgei-Hu<_-l
anomaly in 1 subject following recovery from pancytopema. A low
platelet count (thrombocytopenia) was observed in 28 individuals,
and the peripheral blood smears showed giant or morphologicalJy
abnormal platelets.
Miscellaneous Abnormalities in Benzene-induced Pancytopenia
There is evidence that in addition to producing a decrease
in number of circulating blood cells, benzene exposure causes
formation of abnormal red cells, platelets, and white cells. The
most common red cell alteration is macrocytosis, a condition that
also occurs in erythroleukemia and in vitamin deficiency states
associated with abnormal metabolism of nucleic acid. Megalo-
blastic and macroerythrocytic precursors have been noted in the
44 47 81
bone marrow of patients with benzene toxicity* ' ' Abnormal
red cell function is also suggested by the finding of
significant hemolysis in occasional cases of benzene
v, 4. 4. • -.. 19,55,57,92,121,139,151,158 , ^ . ,
hematotoxicity, ' ' by a report of abnormal
7 79
osmotic fragility in one but not another study, and by indirect
indications of altered heme synthesis, including changes in
levels of porphyrin in blood and urine and in levels of delta
aminolevulinic acid in red cells. ' ' A number of inves-
tigators report abnormal morphology and function of granulocytes.
Observations include a decrease in phagocytic function, a
61
-------
loss in leukocyte alkaline phosphatase, ' an altered osmotic
143
fragility, a change in the fluorescence characteristics of
leukocyte nuclei, and a Pelger-Huet anomaly. ' ' '
Many studies suggest that benzene hematotoxicity results in
altered platelet function,29,44,56,97,104,138,160,161,164,171
perhaps leading to an increased susceptibility to bleeding.
Morphological abnormalities of circulating platelets and of
megakaryocytes have also been reported. ' ' No diT-»^J_
evidence of altered lymphocyte function is available, although
this has been suggested on the basis of apparent alterations in
. , , . .. ., n 111,112,149,150,168
immune function in benzene-exposed individuals.
At present, however, it is unclear whether the reported immuno-
logical findings are a primary effect of benzene, are secondary
to the formation of altered hematopoietic cells, or reflect some
unrelated situation. In addition, cytogenetic aberrations in
circulating lymphocytes have been observed (see Section 1).
Abnormal monocytes in the blood of workers exposed to benzene
also have been reported.
Many of these abnormalities in circulating red cells, white
cells, and platelets are reported to occur as relatively early
manifestations of benzene hematotoxicity. For this reason they
have been proposed for use in screening tests to detect early
benzene effects. An absolute lymphocytopenia is noted as a
3 79
relatively early indication of benzene hematotoxicity by some '
R ft Q n
but not other ' investigators. An increase in circulating
3 5 25 32 90 91
eosinophils and basophils is also sometimes observed. ' ' ' ' '
In addition, the serum levels of various enzymes ' are
reported to be altered in early benzene hematotoxicity. Further
systematic study of possible indicators of benzene hematotoxicity
would be of value in screening exposed individuals for the
detection of early damage to bone marrow.
62
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Although animal studies provide evidence of rapid inter-
ference in erythropoiesis following administration of benzene,
the relatively long normal red-cell survival period of 120 days
causes any diminution in red cell production to be manifest only
slowly as a decrease in circulating red cells. In contrast, the
normal period of platelet survival is 7 to 10 days, and survival
of granulocytes in the circulation is perhaps 24 hours. Although
a red cell count measures almost all mature red cells, about 30
percent of platelets are sequestered in the spleen, and the
measurable circulating granulocytes represent less than 20
percent of the total in the body. As a further complication,
there is a relatively large reserve of bone marrow, which is
capable of perhaps a sixfold greater output of mature cells in a
normal situation. Accordingly, counts of the circulating cells
do not give an adequate index of very early benzene hematotoxicity,
Long-term Evaluations
The prognosis in mild cases of pancytopenia is good if
benzene exposure is discontinued. In some individuals, however,
pancytopenia has progressed even after presumed cessation of
exposure, and apparent hematological recovery has sometimes been
followed by acute leukemia occurring as late as 15 and 27 years
49 98
after the initial findings. ' Relatively few longitudinal
assessments of occupational exposure groups have been performed.
90
One such study was reported by Hernberg et al, who reevaluated
a group of individuals exposed to benzene in a shoe factory,
originally studied by Savilahti. Benzene had been in use for
about 10 years, and levels close to 400 ppm were measured. The
original investigation disclosed abnormal blood counts, most
commonly thrombocytopenia, in 107 of 147 workers studied. Ten
of these individuals required hospitalization, one of whom died
with severe pancytopenia. Of note is that one of the more
severely affected individuals, whose peripheral blood counts
63
-------
returned to normal in 2 years, subsequently developed acute
leukemia. Hernberg et al restudied 125 of the original workers
in comparison with a nonexposed control group 9 years after
cessation of benzene exposure. There was definite improvement in
mean platelet count, which increased from 176,480 to 257,360.
However, the platelet count in the group exposed to benzene
remained significantly (p<0.01) lower than that of the control
level of 293,000 per mm . The mean erythrocyte count for male
workers was stable during this 9-year period and was also signif-
icantly lower than that of the control group (4.4 vs 4.7 million
per mm ; p<0.01). Improvement to control levels was observed in
the mean erythrocyte count of female workers and the mean leuko-
cyte count of the entire group exposed to benzene. These findings
suggest that recovery after benzene hematotoxicity may occur very
slowly or may be incomplete. Of interest is the observation that
the reported mean platelet and red cell counts in the exposed
population were within normal limits, and the observation of a
persistent benzene effect in this population required simultaneous
study of a normal control group. A possible inference from this
finding is that peripheral blood counts within the wide range of
normal do not preclude an effect of benzene on hematopoietic
90
tissue. A further observation by Hernberg et al is that the
severity of the original findings in 1955 did not correlate with
the extent of recovery in 1964. This analysis, however, was not
based on the blood counts of individual workers but rather on a
comparison of the initially severe with the initially mild cases.
Another long-term follow-up of an occupationally exposed
84
group has been reported by Guberan and Kocher. Their evalua-
tion of 216 of 282 workers 10 years after apparent cessation of
benzene exposure revealed two individuals with isolated thrombo-
cytopenia, one with anemia and thrombocytopenia, and one with
mild pancytopenia. They note that one worker had died of aplastic
anemia 9 years after cessation of benzene exposure. Few add"uion-
al details are provided. Further study of the long-term
64
-------
consequence of occupational exposure to benzene in well-defined
cohorts would be of great interest.
LEUKEMIA
Leukemia can be defined as a neoplastic proliferation and
accumulation of white blood cells in blood and/or bone marrow.
Hematologists generally agree concerning characterization of the
four main types of leukemia: acute and chronic myelogenous (also
known as granulocytic) leukemia, and acute and chronic lympho-
cytic leukemia. This classification represents diseases that
differ in terms of incidence, course, prognosis, and, presumably,
etiologic factors. In addition, there are other types of leukemia,
related to these four major types, about which there is some
disagreement concerning diagnostic criteria. Erythroleukemia,
acute promyelocytic leukemia, stem cell leukemia, and acute
myelomonocytic leukemia, all of which have been reported in
association with benzene exposure, are generally considered to be
variants of acute myelogenous leukemia. There is, however, some
question as to whether the relatively rare acute monocytic
leukemia (Schilling type) is an entity of its own or is related
to acute myeloblastic leukemia.
Chronic myelocytic leukemia has often been classified under
the heading of myeloproliferative syndrome, which includes
polycythemia vera, myelofibrosis and myeloid metaplasia, and
essential thrombocythemia. These disorders have in common a
neoplastic proliferation of the pluripotential stem cell respon-
sible for the formation of granulocytes, platelets, red blood
cells, and perhaps, fibroblasts and other monocytic cell types.
They also share a potential for the development of acute myelo-
blastic leukemia. Although the myeloproliferative disorders may
overlap clinically, chronic myelogenous leukemia does appear to
be distinct and readily separable on the basis of a characteristic
65
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chromosomal abnormality and other distinguishing features. The
cells identifiable in the blastic crises of chronic myelogenous
leukemia may be more closely related to lymphoblasts than to
myeloblasts, again consistent with the pluripotential nature of
the stem cell discussed above.
Investigators have associated exposure to benzene with
preleukemia. Preleukemia is very difficult to define, and there
is little agreement as to clinical criteria. In most cases, the
diagnosis is made in retrospect after the patient has developed a
clearcut acute leukemia, usually of the myeloblastic type. A
characteristic case history would be a benzene-exposed individual
with pancytopenia whose bone marrow demonstrates generalized
hypoplasia, but with a slight increase in somewhat atypical blast
cells. Over a period of weeks to years, repetitive bone marrow
measurements show a gradual increase in these blast forms until
frank acute myeloblastic leukemia is indicated by almost complete
blastic replacement of the bone marrow and by the presence of
these cells in the peripheral blood. Preleukemia is defined as
beginning at the point at which there is a reasonable expectation
that the patient would develop acute leukemia and ending at the
point at which acute leukemia is diagnosed. Despite much current
interest in the subject, no criteria are agreed upon to define
these two points. Accordingly, it is difficult to evaluate
literature concerning preleukemia in benzene-exposed
individuals beyond indicating that such a continuum is
f. . , 6,10,72,117,175
often reported. '
Relationship of Benzene to Leukemia
The evidence concerning the relationship of benzene and
acute leukemia has been reviewed by a number of authors and
panels in recent years. Most of these individuals and groups
accept causative role for benzene in human acute myelogenous
leukemia and it is so described in the routine hematological
66
-------
literature. The following quotations are from three standard
American textbooks on hematology:
"A variety of chemicals and drugs have been suggested
as possible leukemogenic agents in human leukemia, but only
benzol can be unequivocally implicated."
"Any chemical capable of producing myelotoxicity must
be regarded as a potential leukemogen, if the findings in
radiation-induced leukemia apply which indicate that cell
damage with depression of marrow function may produce
alterations leading to the transformation of damaged cells
into neoplastic ones. The only chemical which has been
clearly identified as one which increases the incidence of
myeloid leukemias in man is benzene in rather heavy occupa-
tional exposure. The development of stem cell, erythro-
blastic and myeloblastic leukemias and persistent chromosome
abnormalities in exposed individuals known to have had
neutropenia is especially significant."154
"Sporadic cases of acute leukemia have occurred after
exposure to chemical agents such as benzene, phenylbutazone,
and chloramphenicol. Aplastic anemia and acute myelogenous
leukemia have followed prolonged exposure to benzene. About
15% of the patients who develop hematologic abnormalities
due to benzene exposure also develop acute leukemia.
Leukemia is usually preceded by a period of bone marrow
aplasia."33
Other assessments of this relationship, however, leave some
room for doubt. Thus, a recent document from the National Cancer
109
Institute concluded:
"At best benzene must be considered as a suspect
leukemogen."
In evaluation of the causal relationship of benzene to acute
leukemia, three types of information are pertinent: the non-
leukemic effects of benzene, case reports of benzene-associated
leukemia, and results of epidemiological studies.
Nonleukemic Biomedical Effects of Benzene—
Benzene clearly damages hematopoietic tissue in man. That
this damage can be due to benzene alone is supported by observa-
tion of pancytopenia in diverse exposures in many different
67
-------
countries, as well as by animal studies in which administration
of benzene in the absence of any other solvent produces pancyto-
penia (see Section 3). A somewhat negative point is the absence
of conclusive evidence of leukemogenic action of benzene in
animals. Arsenic may be the only other compound for which there
is good epideroiological evidence of carcinogenesis in man in the
absence of an analogous animal model.
The fact that patients with idiopathic aplastic anemia or
aplastic anemia developing from other agents, most notably
chioramphenicol and phenylbutazone, may also progress to acute
leukemia ' provides inferential evidence that benzene-induced
damage to hematopoietic stem cells could lead to acute leukemia.
The mutagenic effects of benzene, particularly in relation to
chromosomal abnormalities in man, may also be considered inferen-
tial evidence supporting a causal role of benzene in leukemia
(see Section 2) .
Casp Reports of Leukemia Associated with Benzene--
Well over 100 cases of leukemia in benzene-exposed indivi-
duals have been described in the literature since the original
report in 1928. However, enumeration of case reports does not,
by itself, provide definitive evidence that benzene is a causa-
tive factor in acute leukemia. There is a finite probability
that each case represents a chance association with benzene
exposure in an individual whose leukemia is due to some other
cause. Reports of single cases and small series of cases gener-
ally lack information concerning the size of the population at
risk that is required for firm conclusions. On the other hand, a
number of factors would tend to lead to an underreporting of
benzene-associated leukemia. The general acceptance of this
relationship in the medical literature for some time would tend
to hinder the preparation or publication of manuscripts describ-
ing one further case. A long period often follows benzene
68
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exposure before the onset of acute leukemia; this delay could
lead to the relationship being overlooked by the patient or
. . . 6,49,83,98,116,150,162,166,193 „ .u .
physician. ' ' ' ' ' Furthermore, because
benzene is a ubiquitous component of our chemical age, exposure
may be unrecognized. Accordingly, simple quantitation of case
reports in the medical literature cannot provide definitive
information concerning the causal relationship of benzene to
acute leukemia.
These case reports, however, do provide presumptive evidence
of a leukemogenic effect of benzene. Of particular note is the
frequent description of persons suffering from benzene-associated
pancytopenia in whom evolution to acute leukemia was observed.
Idiopathic aplastic anemia is an uncommon disorder, reported far
less frequently than acute myelogenous leukemia. The relatively
frequent documentation of benzene-associated pancytopenia pro-
gressing to acute leukemia, which is in keeping with that
observed in other causes of aplastic anemia, further supports the
possibility that exposure to benzene increases the risk of
developing acute leukemia. Similarly, the case reports are also
pertinent for the frequency with which evolution ol the homa-
tological findings progress to or through an erythroleukemic
10,20,51,52,59,63,105,110,139,175 „, ,
stage. The relative occurrence
of the erythroleukemia variant in these case reports appears to
be more common than that observed in cases of acute myeloblastic
leukemia and its variants in the general population. Unless this
represents some unexpected bias in the reporting process, the
frequency with which erythroleukemia is observed in benzene-
exposed patients also supports a specific leukemogenic action of
benzene.
Epidemiological Studies—
Evidence supporting a role for benzene in leukemia has been
obtained in a number of epidemiological studies. Three general
approaches have been used. First, the discovery of a relatively
69
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large number of individuals with leukemia having a history of
occupational benzene exposure has led to assessment of the
likelihood of this occurrence. This has been done by estimating
the number of workers in the occupational group and computing the
incidence of leukemia in comparison with that expected in the
general population. A second approach has been to obtain
occupational histories of persons with leukemia and compare the
incidence of potential benzene exposure in these leukemics with
that of control populations. The third approach has been to
evaluate the mortality characteristics, including leukemia
deaths, of relatively large populations working in an industry
involving known benzene exposure. Any high incidence of leukemia
can be further analyzed in terms of occupational subgroup,
duration of employment, and other factors.
Each of these approaches entails advantages and disadvantages,
For example, the third approach, representing a standard epidemic-
logical technique, is by far the most thorough in its character-
ization of an entire work force; however, the validity of
information on leukemia incidence depends on the vagaries of
death certifications rather than on direct observation. In
contrast, enumeration of the population at risk in the first
approach represents a crude estimate, and little is known about
the population characteristics of the occupational group. This
approach does offer a high level of confidence in the diagnosis
of leukemia, in that the subjects have been observed by the in-
vestigators.
The relatively recent studies of Aksoy and his colleagues in
Turkey strongly support the causal relationship of benzene
exposure to acute leukemia. ' ' ' ' ' Their evidence
includes individual case reports of workers with aplastic anemia
which progresses through a preleukemic phase to frank acute
myeloblastic leukemia or erythroleukemia; an accumulation of
cases resulting in a statistically significant higher incidence
70
-------
of acute leukemia among shoeworkers; and an outbreak of leukemia
in this population that appears temporally related to the onset
of benzene use and that has subsided following replacement of
benzene as a solvent for adhesives. From 1967 to 1973 Aksoy et
al observed 26 patients with acute leukemia among shoe
workers. ' ' Analysis in selected work areas revealed that
"the concentration of benzene was found to reach a maximum of
210-650 parts per million when adhesives containing benzene were
in use." There were 14 cases of acute myeloblastic leukemia, 4
of preleukemia, 3 of acute erythroleukemia, 3 of acute lympho-
blastic leukemia, and 1 each of acute promyelocytic and acute
monocytic leukemia. Duration of benzene exposure ranged from 1
to 15 years. The authors state that there were 28,500 shoe
workers at risk in Istanbul and derived a leukemia incidence of
13 per 100,000 in this population. This incidence is statistically
significantly higher (p<0.02) than the risk of 6 per 100,000
assumed for the general population. Because this latter number
apparently is derived from the incidence of leukemia in developed
nations, rather than being specific to Istanbul, there is some
degree of uncertainty. This appears to be more than counterbal-
anced, however, by a number of factors. Foremost is that the
distribution of cases reported by Aksoy et al strongly differs
from that of leukemia in the general population. If the relative
incidence were computed solely for acute myeloblastic leukemia
and its variants, a magnification of the risk in benzene-exposed
shoe workers would be observed. Secondly, Aksoy et al apparently
have not age-adjusted their findings. In their series, the
average age at the time of diagnosis was 34.2 years. This is a
relatively low-risk age period for leukemia, with a reported
43
death rate about half of the overall incidence. Recalculation
of their data with an age factor would presumably increase the
statistical significance of the findings. In addition, Aksoy has
recently stated that his studies underestimate the relative risk
71
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of leukemia because the incidence of leukemia in the general
population of Turkey is 2.5 to 3.0 per 100,000 and because
shoeworkers with acute leukemia probably were admitted to other
Istanbul hospitals without his knowledge.
Recently, Aksoy presented his observations of acute leukemia
in shoe workers during the period 1967 to 1976. As shown in
Table 1, the peak incidence of leukemia in shoe workers occurred
between 1971 and 1973. This follows by a few years the appear-
ance of a notable incidence of aplastic anemia in this occupa-
tional group. The decline in cases since 1973 is temporally
related to a decrease in use of benzene as an adhesive solvent,
which began gradually in 1969. Aksoy also reports that pancyto-
penia was present in 27.5 percent of the cases before the onset
of acute leukemia, which occurred 6 months to 6 years later. ' The
hematological findings often indicated a period of recove'ry
before the onset of leukemia, a phenomenon also noted by other
investigators. Aksoy states that during this period, over 100
cases of aplastic anemia were observed that were either idiopathic
or associated with an agent other than benzene, and in none of
these cases did acute leukemia develop. He also states the
opinion that no blood dyscrasia is required before the onset of
leukemia and provides an example of a 23-year-old shoeworker who
was hematologically normal when studied 4 years before the onset
of acute erythroleukemia. As with other cases of leukemia
associated with benzene exposure in which the patient had no
detectable pancytopenia beforehand, the interval between hemato-
logical observations is too long to ensure that pancytopenia did
not in fact occur.
A relationship between benzene exposure and leukemia is
stressed in a series of reports by Vigliani and his colleagues in
190 193
Northern Italy. ' In recent review articles, Vigliani and
Vigliani and Forni summarized their experience from 1942 to
72
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Table 1. ANNUAL NUMBER OF LEUKEMIA CASES aAMONG
SHOEWORKERS WITH CHRONIC EXPOSURE TO BENZENE
IN ISTANBUL BETWEEN 1967 AND 197516
Number of Number of
leukemic leukemia
Years shoeworkers Years shoeworkers
1967 1 1972 5
1968 1 1973 7
1969 3 1974 4
1970 4 1975 3
1971 6 1976 0
Four other leukemic workers with different jobs and two leukemic
individuals outside of Istanbul are not included in this series.
73
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189 190
1975. ' They observed 66 cases of significant benzene
hematotoxicity in Milan, of which 11 were cases of acute myelo-
blastic leukemia or its variants. All 11 leukemia patients died,
and 7 subjects died of aplastic anemia. In Pavia between 1959
and 1974 there were 135 patients with benzene hematotoxicity; 13
died of acute myeloblastic leukemia or its variants, and three of
aplastic anemia. All of the patients in Pavia were workers in
the shoe industry. Measured benzene concentrations in the
breathing zone of workers using glue usually were 200 to 500 ppm,
and ranged from 25 to 600 ppm. Many factory shoe workers also
worked at home, where presumably further benzene exposure
occurred. Shoe workers were also the largest single occupational
group with hematotoxicity in Milan, although many other working
groups were also affected. Of interest is an apparent outbreak
of benzene hematotoxicity, including eight cases of severe
pancytopenia and two cases of myelogenous leukemia, in the
rotogravure industry. This outbreak was temporally related to
the use of inks and solvents containing large amounts of benzene.
Ambient benzene levels were calculated to be between 200 and 400
ppm, with peaks up to 1500 ppm. In an earlier study, Vigliani
193
and Saita estimated the number of workers exposed to benzene
in Pavia and Milan and, based on the incidence of acute leukemia
in the general population of Milan, calculated a 20-fold higher
risk of acute leukemia in these workers.
An interaction between benzene and radiation in leukemo-
95
genesis is suggested by the study of Ishimaru et al. These
authors performed a retrospective study of survivors of the two
atomic bombings in Japan, evaluating the effect of occupation on
the incidence of leukemia. Controls were matched with patients
by age, sex, residence, and distance from the atom bomb explosion,
The occupational history of leukemic patients was obtained from
relatives. Of 492 cases of leukemia through 1967 in this popula-
tion, adequate histories and appropriate controls were obtained
74
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for 413, of whom 303 were adults. Comparison of the controls and
leukemia patients revealed that the risk of leukemia was 2.5
times higher in those with an occupational history potentially
related to benzene or X-rays. The risk was significantly higher
in those with 5 or more years of potential exposure but not in
those who had been employed in such occupations for less than 5
years. The relative risks were similar in Hiroshima and Nagasaki
and were higher for acute leukemia (2.9) than for chronic
leukemia (1.8). The major limitation of this study is the
possible inaccuracy of the occupational history of leukemic
subjects as compared with that of the living controls.
72 73
Girard et al ' have evaluated the frequency of a positive
history of benzene exposure in 401 hospitalized patients with
serious hematological disorders as compared with 124 patients
hospitalized for nonhematological problems. A statistically
2
significant increase (p < 0.05; x > 3.84) in history of benzene
exposure was noted for patients with aplastic anemia (10 of 48,
2 2
X = 12.2), acute leukemia (17 of 140; x = 5.6), and chronic
2
lymphocytic leukemia (9 of 61; x = 6.7) as compared with the
control group (5 of 124).
The rubber industry has long been characterized by exposure
to various solvents including benzene. Pancytopenia associated
with benzene exposure has been noted in workers in a number of
195
countries (see above). In the United States, Wilson reported
a large cohort of individuals exposed to benzene during the
expansion of rubber production early in World War II. Some
degree of cytopenia was noted in 83 of 1104 individuals studied.
Leukemia was not reported. Ascertaining the present vital status
and causes of death in this cohort, particularly in relation to
leukemia incidence, could be of great value.
Other studies of workers in the rubber industry include a
-i p c:
report of the U.S. Department of Health, Education, and Welfare.
This report cites a 54 percent increase in the death rate for
75
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cancers of the lymphatic and hematopoietic system in male rubber-
industry workers dying in 1950 as compared with workers in all
manufacturing industries. There were also a 30 percent increase
in death rate due to large bowel cancer, a 19 percent increase
due to cancers of the respiratory tract, and an 8 percent higher
overall cancer death rate.
119 120
Mancuso and his colleagues ' performed a series of
studies of occupational cancer rates, with particular emphasis on
the rubber industry, including evaluation of a cohort of 1977
workers. No apparent increase in hematopoietic cancers was
observed. There was, however, a higher incidence of tumors of
the gallbladder, bile duct, and salivary gland in occupational
subgroups not associated with high solvent exposure.
u • f *. T 17,18,126-129,182 ,. ..
A comprehensive series of studies of the
health status of rubber-industry workers has been performed by
the Occupational Health Studies Group of the University of North
Carolina. They have evaluated the 10-year mortality experience
of a large cohort of male workers (5106 deaths) at four tire
lanufacturing plants. The subjects were in the work force or
were retired in 1964. This series of studies was reviewed
18 2
recently by Tyroler. The mortality due to all cancers (1014)
was normal or slightly elevated, depending on the data base used
for comparison. Deaths due to cancer of the lymphatic and
hematopoietic system (total of 109) were 31 percent higher than
expected and were increased in cohorts of each of the four
companies. In the category of lymphosarcoma and Hodgkin's
disease, the standard mortality ratio (SMR) was 129 and an
increase in the expected number of deaths was observed in two of
the four company cohorts. Similarly, for deaths due to all forms
of leukemia the SMR was 130 and the increase was observed in
three of the four cohorts. When this latter category was further
subdivided, the overall SMR for lymphatic leukemia was found to
be 158 and the expected death rate was elevated in two of the
76
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four company cohorts. Of particular note is that the SMR for
deaths attributed to lymphatic leukemia was 291 in the age group
40 to 64.
Several approaches were followed in further study of the
increased incidence of lymphatic leukemia. Contrasting the work
history of 17 patients with lymphatic leukemia with those of
three matched controls for each case revealed that solvent
exposure increased the overall risk by a factor of 3.25. Further
classifying the groups according to high, low, and medium solvent
exposure, yielded a 5.5 factor for the high-exposure group. In
those patients first subjected to high exposure between 1940 and
1960, the factor for the relative risk of lymphatic leukemia was
9.0. The relationship of solvent exposure to lymphatic leukemia
was statistically significant at p < 0.025. The study also
showed an increase in the mean difference in years of work
history between lymphatic leukemia and the case controls. This
was inversely proportional to the extent of solvent exposure.
TyroLer has cited several epidemiologic reasons why these findings
TOO
must be interpreted with caution.' These include the depen-
dence on death certificates for diagnostic information, which
apparently is now being validated with clinical and pathological
findings. Tyroler notes good agreement between the death
certificate and medical information concerning deaths stated to
be due to leukemia. It wotild be pertinent to learn whether the
deaths certified as caused by lymphatic leukemia represent cases
of chronic lymphatic leukemia, a relatively common disorder in
this age group, or of acute lymphoblastic leukemia. The latter
is rare in adults and is sometimes difficult to distinguish from
the acute myeloblastic and stem cell leukemias that are clas-
sically associated with benzene exposure. Other conceivable
limitations to the studies by the University of North Carolina
group include the possibility that the association between work
history and nonfatal cases in this cohort might be systematically
77
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different from that observed in fatal cases, and that individuals
with leukemia and a work history of solvent exposure may also
have had uncontrolled exposures to radiation, drugs, or other
environmental agents that might cause the observed increase in
leukemia rates. A further limitation is the lack of historical
data concerning the benzene exposure or the concentrations of
other solvents. These studies do, however, strongly support the
possibility that long-term exposure to benzene in the U.S.
rubber industry leads to an increased risk of lymphatic leukemia.
Monson and Nakano also have recently studied mortality of
I o f- -I o "7
workers in the rubber industry. ' Evaluation of a cohort of
13,571 white males in one plant in Akron, Ohio, revealed an SMR
of 82 for all causes of death and an SMR of 94 for all malignant
neoplasms in comparison with U.S. mortality statistics. When
the causes of death were further subdivided by types of neoplasm,
the highest SMR (128) was observed for leukemia (43 expected, 55
observed). This high incidence was observed particularly in
workers in the tire (SMR 150) and processing (SMR 240) divisions,
but was also observed in other worker subgroups. The excess in
leukemia was notable in those who were less than 25 years of age
at the time of employment and in those who began work before
1935. However, reevaluating the data for the 7374 employees who
had worked at least 25 years gave no evidence of a decrease in
risk for those who began working after 1935. The SMR for non-
leukemic hematological neoplasms (lymphatic tumors and myeloma)
was 101 in the overall cohort and 160 in the workers employed in
the tire building area. Studies were also performed in three
additional cohorts from this plant; female workers, black male
workers, and salaried white males. Data on these groups
showed a tendency toward an increase in deaths due to lymphatic
tumors and myeloma (SMR's 111, 125, and 115, respectively).
Deaths due to leukemia were not greater than expected, although
generally higher than the SMR for all causes of death. Evalua-"-
tion of 574 deaths in white males working in other factory
78
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locations at the same rubber company revealed 14 deaths due to
all lymphatic and hematopoietic tumors as opposed to 11.8
expected.
Other epidemiological studies of individuals potentially
exposed to benzene have failed to reveal an increased incidence
of acute leukemia. Thorpe reported an extensive study of leu-
kemia mortality rates for the period 1962-71 in 36,000 employees
and annuitants of eight European affiliates of a major petroleum
178
company. The report is noteworthy for the extensive discus-
sion of methodological problems inherent in such retrospective
studies. These problems include the low incidence of leukemia in
the general population; the general inability to quantitatively
define benzene exposure in the selected population; the difficul-
ty of obtaining both accurate occupational histories and complete
follow-up of the workers; and the problems of verifying the
diagnoses of leukemia and of the subtype of leukemia.
In Thorpe's study four of the affiliates reported 18 cases
of leukemia, whereas the other four reported none. In only 6 of
the 18 cases was the subtype of leukemia known. Benzene exposure
was believed to have occurred in 8 of the 18 individuals although
the author points out the difficulty of clearly distinguishing
among the job categories relative to benzene exposure. The data
revealed no statistically significant increase in the leukemia
rate over that expected in the population. There was, however, a
tendency toward higher leukemia rate in the benzene-exposed as
compared to the unexposed work groups. This study has been
criticized by Brown and defended by Thorpe. The major
criticism concerns factors possibly leading to an underreporting
of leukemia incidence in this population.
Although benzene is associated with coke oven operations,
study of cancer mortality in coke plant workers has revealed no
146
statistically significant increase in leukemia incidence.
79
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This is also true for coke by-product workers, the occupational
subgroup expected to be subjected to benzene exposure. These
workers, however, constitute a relatively small group. Redmond
147
et al studied 345 individuals working 5 or more years in a
coke by-product area. They found only 10 fatal neoplasms, of
which 2 were of the lymph and hematopoietic tissue category (1.1
expected). Accordingly, no firm conclusions can be reached
concerning benzene leukemogenesis in coke oven workers.
A risk of benzene exposure might also be expected in other
occupations that are not well characterized epidemiologically.
Adelstein examined the standardized proportional mortality ratios
for various cancers in British males, 1959-1963, listing them
according to 27 occupational groups. The highest level of
leukemia deaths was in the group described as "professional,
technical workers, artists" (p < 0.01). This presumably would
include chemistry professors, laboratory technicians, and other
professional groups with relatively frequent exposure to benzene.
Artists may be particularly at risk because they use unknown
solvent mixtures in poorly ventilated and unregulated work
areas. As with other studies, however, an exposure to benzene
cannot be defined and it is possible that other factors, such as
radiation, may be responsible for the increased incidence of
leukemia in this occupational group.
In the United States, Li et al evaluated the cause of
death of 3637 members of the American Chemical Society who died
between 1948 and 1967. Among deaths occurring between ages 20
and 64, an increase in all malignant neoplasms was noted (444
observed, 354 expected, p < 0.001) as compared with a control
group derived from U.S. professional men. The increase in tumors
of the lymphatic and hematopoietic system (94 observed, 50 expected)
was the most highly statistically significant (p < 0.001) of all
tumor types. Further subdivision revealed that the major increase
was in nonleukemic hematopoietic and lymphatic tumors (61 observed,
34 expected, p < 0.001). The difference in number of leukemia
80
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cases (33 observed, 25 expected) was not statistically signifi-
cant. Among deaths occurring after age 64, in comparison with
U.S. white males, the highest level of statistical significance
again was observed for all tumors of the lymphatic and hemato-
poietic system (p < 0.001). In this analysis, the increased
incidence of both leukemic (16 observed, 9 expected) and nonleu-
kemic (17 observed, 9 expected) hematological neoplasms was
statistically significant.
A recent and relatively thorough epidemiological evaluation
of benzene-exposed workers was conducted by Infante et al of
94
NIOSH. These investigators evaluated the mortality of 748
white male workers exposed to benzene in manufacture of a rubber
product. The cohort consisted of those employed at any time
during the period 1940 through 1949, and the period of risk for
death was January 1, 1950, through June 30, 1975. The two
control groups were the general population of U.S. white males
and a group of 1447 white males employed in the same state and
time period for at least 5 years in manufacture of a fibrous
glass product. The vital status of this second control group was
determined as of June 1, 1972. In the benzene-exposed group,
data concerning the vital status as of June 30, 1975, were avail-
able for approximately 75 percent of the workers. The remaining
25 percent were assumed to be living. This assumption leads to
an underestimation of the determined risk of death due to
leukemia or other causes within this group and presumably
accounts for the observation of only 140 deaths as opposed to 187
expected. Among the 140 deaths, 9 were due to all lymphatic and
hematopoietic cancer as opposed to 3.45 based on expectations for
U.S. white males (p < 0.05) and 5.10 expected in the fibrous
glass industry control group (not statistically significant). Of
particular pertinence is that 7 of the 9 deaths in the benzene-
exposed group were of leukemia, as opposed to 1.38 and 1.48
expected on the basis of the respective control groups (p < 0.02),
81
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Four of the leukemias were reported as acute myelocytic, two as
monocytic, and one as chronic myelocytic. The latter case was
unusual in that the patient's age at death was 29 years,
relatively young for chronic myelocytic leukemia, and there was
only a 2-year period between initial exposure and death. In the
other six individuals, this period ranged from 10 to 21 years.
As pointed out above, the description of "monocytic" leukemia in
two cases most likely represents the myelomonocytic variant of
acute myelocytic leukemia, but this is uncertain in the absence
of review of the clinical material. Infante et al also cite an
eighth case who was not part of the cohort, having started
employment in 1950. This person died at age 28 of myelocytic
leukemia 3 years after the initial exposure.
The authors also use their data to determine the relative
risk for myeloid and monocytic leukemias, as opposed to
total leukemia, and conclude that the population studied had a
tenfold increased risk of death from these forms of leukemia.
This factor could be an overestimate due to lack of clinical
confirmation of the cause of death. Furthermore, the authors
state that the death data were recalculated on the basis of a
50.37 percent incidence of myelomonocytic types of leukemia in
this age group. Because chronic lymphatic leukemia is common in
this age group and is a relatively mild disease in which death
commonly occurs from some other cause, the percentage of deaths
caused by myelomonocytic types of leukemia may well be much
higher than the 50.37 percent value used in the analysis.
Counterbalancing this is the potential underestimation due to the
assumption that 25 percent of the cohort, for whom vital status
is unavailable, is living. The authors also discuss the presumed
dose, which is described below. In general, this epidemiological
study provides excellent confirmatory evidence of the causal
relationship of benzene exposure to acute myelocytic leukemia.
82
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Another recent epidemiological study is that of Ott et al*
who report the mortality experience of 594 individuals occupa-
tionally exposed to benzene in the chemical industry. The study
is notable for its stratification of the work force in terms of
benzene exposure levels and for relatively detailed descriptions
of the individual cases with hematological findings. The
cause-specific mortality rates for the 102 individuals identified
as deceased are said to agree well with those observed in a
cohort study of over 8000 employees in the same location. No
association with the benzene exposure dose was observed. Of note
are two deaths ascribed to anemia (one aplastic anemia, one
pernicious anemia), two deaths due to acute myelogenous leukemia,
and one death ascribed to bronchopneumonia with myeloblastic
leukemia listed as a significant other condition. The time-
weighted average benzene exposure of the three individuals with
leukemia was characterized as low (2 to 9 ppm) or very low
(< 2 ppm). The expected incidence of myelocytic leukemia in
the study population is said to be 0.8. Observation of three
cases is of borderline statistical significance. Considering
the relatively small number of deaths evaluated, it is not
surprising that the findings are inconclusive. Future reanalysis
of this well-characterized cohort would be of interest.
Relationship of Benzene Exposure Level to Leukemogenesis
The medical literature provides little information concern-
ing the dose of benzene inhaled by individuals who subsequently
developed acute leukemia. In those few reports where benzene
levels are cited, the duration of monitoring has been clearly
inadequate for estimation of individual exposure levels. This is
particularly true for assessment of the leukemogenic effects of
benzene as opposed to its effects in pancytopenia. Concerning
the latter, there are a number of studies in which a large
*0tt, M.G., J.C. Townsend, W.A. Fishbeck, R.A. Langner. Mortality
Among Individuals Occupationally Exposed to Benzene. Exhibit
154, OSHA Benzene Hearings, July 19 - August 10, 1977.
83
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percentage of the work force has developed benzene hematotoxicity.
In these instances the available monitoring information might
reasonably be used to estimate the average exposure. In con-
trast, because leukemia occurs in only a very small percentage of
the work force exposed to benzene, it is difficult to be certain
whether the occasional individual who does develop leukemia might
have undergone some high exposure, perhaps due to a specific job
or to faulty work habits, that is not typified by an area-wide
benzene monitoring system.
In the few studies of acute leukemia where benzene levels
are reported, the concentrations generally have been above 100
ppm. These include the studies of Aksoy et al and Vigliani et al
discussed earlier. In other reports, benzene levels associated
with acute leukemia have been 220 ppm and 63 to 517 ppm.
There is some indication that the duration of benzene exposure is
longer for acute myelogenous leukemia than for pancytopenia. A
recent tabulation showed far more cases of pancytopenia ' ' '
than of acute myelogenous leukemia developing after less than 2
years of exposure. This interpretation is complicated by the ap-
parent lag period between cessation of benzene exposure and the
development of acute leukemia, an interval reported to be as long
49 98
as 15 and 27 years." Accordingly, the apparently longer
period of benzene exposure in cases oi cicute myelogenous leukemia
than in pancytopenia may not be a function of dose but rather of
the biological lag period between initiation of leukemogenesis
and its eventual clinical appearance. For instance, the two case
lan
166
78
reports of leukemia tabulated by Goldstein as having less than
2 years exposure to benzene wGi/e those ot Seliyei and Kelemen,
in which an 18-month exposure led to pancytopenia, development of
a Pelger-Huet anomaly, and, 7 years later, acute myelogenous
leukemia. Similarly, the case reported by Kinoshita et al
had an onset as aplastic anemia after 6 months of benzene
exposure, with subsequent development of acute leukemia. Also
84
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perhaps pertinent are the findings of Aksoy et al in Turkey. In
1972, tnese authors reported 32 cases of benzene-associated
pancytopenia and only 4 cases of acute myelogenous leukemia. '
In 1976 they reported 20 cases of acute myelogenous leukemia or
erythroleukemia, whereas the total of pancytopenia cases had
increased only to 46 by mid-1977. Of the 44 patients with
pancytopenia who were available for follow-up, 5 had subsequently
developed leukemia. Accordingly, it is not certain whether the
generally longer exposure period associated with leukemia as
opposed to pancytopenia is a reflection of benzene dose.
94
The study of Infante et al provides the most information
about dose. As described in detail above, in a determination of
vital status in 1975, these authors noted a statistically
significant increased incidence of leukemia (total of 7) in a
cohort of workers identified as having been employed any time
between 1940 and 1949 in a factory where benzene was used. The
exhaust ventilation was said to be excellent. Data concerning
benzene levels include a report in 1946 stating that "Tests were
made with benzol detectors and the results indicate that concen-
trations have been reduced to a safe level and in most instances
range from 0 to 10 or 15 parts per million" (emphasis added). It
is also stated that 112 surveys conducted between 1963 and 1974
"indicated that employees' benzene exposure was generally below
the recommended concentration in effect at the time of each
survey" (emphasis added). This frequency of survey is less than
once per month in the stated time period, and thus represents
grossly inadequate information for determining the benzene
exposure levels of the seven individuals who developed leukemia.
These individuals may in fact have been exposed to concentrations
of benzene well below the acceptable industrial hygiene limits,
which ranged from 100 ppm maximum allowable concentration in 1941
to 10 ppm as a time-weighted average in 1971. It is also
possible that these individuals were exposed to concentrations
85
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well above the permissible concentrations, which were not de-
tected in the relatively infrequent monitoring surveys. This
87
possibility is supported by recent testimony of Harris, who
cited a survey performed in 1973-74 of the pliofilm manufacturing
plant studied by Infante et al, with the following observation:
"The spreader and drying units are entered on an
intermittent basis to remove damaged film and rethread
rollers and to repair mechanical failures. It was reported
to the survey team that workers can spend up to 30 minutes
inside these units. Periodic inspections of shorter dura-
tion are also conducted inside the spreader. Samples inside
these units indicate a highly dangerous level of benzene,
ranging from 200 to 350 ppm. Chemical cartridge respirators
are required but are often not worn."
Short-term exposure levels of up to 30 ppm benzene were also
noted in other areas of this plant.
In summary, the available literature concerning the benzene
levels associated with the development of acute leukemia is
inadequate for generation of dose-response curves or for an
analysis of risk related to dose.
Possible Mechanisms of Leukemogenesis
The mechanisms by which benzene produces leukemia are
unknown. Of particular interest is whether leukemogenesis is
independent of the pancytopenic effect of benzene or is directly
or indirectly related to overt damage to stem cells. The former
possibility would put benzene into the category of other carcino-
gens for which the regulatory approach is to assume that no safe
level exists. Alternatively, if benzene leukemogenesis requires
preexisting damage to hematopoietic tissue, perhaps associated
with an error in repair, then it is possible that a true threshold
for leukemia could be determined. The prerequisite for bone
marrow toxicity might be sufficient damage to cause an overt
decrease in circulating blood cells. If this is true, then the
public could be protected against leukemia by a standard that
86
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precludes such overt toxicity. Furthermore, this would allow
extrapolation, with suitable care, from animal inhalation dose-
response studies of benzene hematotoxicity.
It is also possible, however, that leukemia results from
bone marrow damage that is too slight to produce clinically
recognizable cytopenias. This would not necessarily preclude use
of an animal model if the parameters of bone marrow toxicity are
sensitive enough. Both Vigliani and Aksoy ' describe
patients with apparent benzene-associated acute leukemia in whom
prior study revealed normal blood counts. The frequency of such
blood counts, however, was not sufficient to rule out an un-
detected pancytopenic phase. Furthermore, it is conceivable that
these were the individuals in the population whose leukemia was
unrelated to benzene exposure. The available information does
not clearly support any of the pathways of leukemogenesis dis-
cussed here. Obviously an animal model of benzene leukemogenesis
would be useful.
It has been suggested that benzene may act as a cocarcino-
gen, which would in part explain why only certain benzene-exposed
individuals develop leukemia. An effect of benzene as a
cocarcinogen or initiator might also explain cases in which
initial hematotoxicity led to cessation of exposure, followed
by a long delay before eventual development of acute leu-
, . 6,49,83,98,116,150,162,166,193 _ ., ... , . .
kemia. ' ' ' ' ' ' ' Evidence on this subject
is not firm.
A genetic predisposition has also been invoked to explain
the response to benzene. Erf and Rhoads note possible hemato-
toxicity in brothers. Aksoy and his colleagues present a
number of cases of benzene hematotoxicity developing in families.
These include two thalassemic brothers with pancytopenia, two
cousins with pancytopenia, a nephew and paternal uncle with acute
lymphoblastic and acute myeloblastic leukemia, a man with
releukemia whose father had died of a possible acute leukemia,
87
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and two maternal cousins, one of whom had preleukemia and one an
acute myelomonocytic leukemia. With one exception, a painter,
these persons all worked in the shoe industry. Although these
cases represent suggestive evidence of a genetic predisposition
to benzene hematotoxicity, the degree to which family groups
participate in the shoe industry has not been specified. If, as
in many similar crafts, it is likely that most of the male family
members will follow the same occupation, then the cases reported
by Aksoy et al may not be greater than what is expected by
chance. This would be true particularly if family groups were
large and if the members tended to work in the same shops with
the same benzene-containing adhesives. Accordingly, a genetic
predisposition to benzene toxicity is not yet proven. Genetic
factors also could be operative in determining the rate or
extent to which benzene is metabolized to its presumed hemato-
toxic intermediate. Also of interest is an increased incidence
of leukemia in a number of disorders that have in common an
increased predisposition to chromosomal damage.
The question of other individual host factors in suspecti-
bility to benzene has been raised by a number of authors.
82
Greenburg et al suggested that obesity predisposes to hemato-
toxicity, presumably reflecting the solubility of benzene in fat.
Although several investigators suggest that younger individuals
and females are more susceptible to benzene toxicity, ' '
this has not been confirmed in other studies. There is also
conflicting evidence as to whether persons with beta-thalassemia
minor, an inherited disorder of hemoglobin synthesis, may be at
increased risk for benzene hematotoxicity. ' ' ' If greater
susceptibility is confirmed, this might indicate that more
rapidly proliferating bone marrow, which is part of the thalas-
semia syndromes, increases the risk of hematopoietic cell damage
due to benzene. Environmental factors, such as high ambient
88
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108
temperatures, may also affect benzene toxicity. In addition,
the ingestion or inhalation of food, drugs, or chemicals might
modify the metabolism of benzene and hence its toxicity. At
present the information is too meager for clear identification of
individual host factors in development of pancytopenia or
leukemia resulting from benzene exposure. Thus, it is unclear
whether the marked variability in individual benzene hemato-
.toxicity observed in occupational settings primarily reflects
variations in benzene dose or operation of unknown host factors.
In summary, the principal mechanisms by which benzene could
act as a leukemogen are considered to be 1) overt pancytopenia,
2) inapparent damage to bone marrow, 3) cocarcinogenic action, 4)
response based on genetic predisposition, and 5) operation of
coincident host factors.
OTHER BENZENE-ASSOCIATED DISORDERS
In addition to pancytopenia and acute myelogenous leukemia,
which are the primary forms of toxicity associated with benzene
exposure, various other hematological disorders also have been
associated with benzene exposure, including some of the leukemia
variants discussed earlier. We now consider some of the evidence
relating benzene exposures to acute and chronic lymphatic
leukemia, other lymphoproliferative disorders including Hodgkin's
disease and multiple myeloma, chronic myelogenous leukemia, acute
monocytic leukemia, myelofibrosis and myeloid metaplasia,
thrombocythemia, paroxysmal nocturnal hemoglobinuria, and various
disorders involving organ systems other than hematopoietic
tissue.
Acute lymphoblastic leukemia is the usual form of childhood
leukemia but is relatively rare in adults, in whom acute myelo-
genous leukemia is more common. It is sometimes difficult to
distinguish acute lymphoblastic leukemia from other forms of
acute leukemia by standard morphological techniques. Aksoy et al
89
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noted four cases of acute lymphoblastic leukemia in a group of 34
leukemic individuals. The control population consisted of 50
leukemic individuals with no history of benzene exposure, of whom
26 had acute lymphoblastic leukemia. Goguel et al noted 2 cases
of acute lymphoblastic leukemia in 50 cases of leukemia in
77
benzene-exposed individuals. Individual case reports of acute
lymphoblastic leukemia associated with benzene exposure are also
. , 50,90
reported.
More substantial evidence is available concerning an
a ssociation of benzene exposure with chronic lymphatic leukemia.
129
As described in detail earlier, McMichael et al noted an
association of chronic lymphatic leukemia with solvent exposure
in a study of the cause of death of more than 6000 men employed
in the rubber industry. (They observed no increase in acute
leukemia.) In addition to a number of other individual case
reports34'37'54'69'102'142 and a collection of three cases by
Tareef et al in the Soviet Union, Girard and his colleagues in
France have noted a statistically significant increase in history
of benzene exposure in patients with chronic lymphatic leukemia
72-75
as compared to control subjects without this disease. In
contrast to these findings, however, Aksoy et al in Turkey and
189 193
Vigliani et al ' in Italy have not reported any cases of
chronic lymphatic leukemia in their relatively large series.
This apparent discrepancy in the observations of the. French
investigators and those in Italy and Turkey is puzzling. Chronic
lymphatic leukemia is a disease of the elderly. Because substan-
tial occupational exposure to benzene may have been discontinued
in France at an earlier time period, cases of chronic lymphatic
leukemia may eventually appear in the Italian and Turkish groups
having more recent benzene exposure. Another possible explana-
tion is that the risk of developing chronic lymphatic leukemia
due to benzene exposure is modified by the presence of other
solvents, which may have differed in France, Italy, and Turkey.
90
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The evidence of a relationship between benzene exposure and
chronic lymphatic leukemia is suggestive but not conclusive.
Lymphosarcoma, ' Hodgkin's disease, ' reticulum cell
sarcoma, and multiple myeloma ' also are reported in
association with benzene exposure. In none of these situations
have there been enough case reports to imply other than a chance
relation to benzene. Similarly, acute monocytic leukemia of the
Schilling type is rarely related to benzene exposure.
Various myeloproliferative disorders are reported in asso-
ciation with benzene. A recent review tabulated 27 cases of
7 8
chronic myelogenous leukemia. Thirteen were from a series by
77
Goguel et al evaluating leukemia observed in benzene-exposed
individuals from 1950 to 1965 in the Paris area. The authors
note no clinical differences from cases of chronic myelogenous
leukemia observed in the absence of benzene exposure. Many of
the other case reports are also from France. ' In addition,
Tareef et al in the Soviet Union reported five cases of chronic
myelogenous leukemia associated with benzene exposure. One
of these individuals eventually developed acute myeloblastic
leukemia, a not infrequent outcome of chronic myelogenous
leukemia.
Myelofibrosis and myeloid metaplasia is a relatively rare
myeloproliferative disorder in which fibrosis of the bone marrow
is accompanied by extramedullary hematopoiesis, particularly in
the spleen and liver. An increased incidence of this disorder in
atom bomb survivors is reported. Several case reports associate
this disorder with benzene exposure. ' ' ' Most recently,
12
Aksoy et al described a 35-year-old shoeworker with evidence of
myelofibrosis and myeloid metaplasia including a diagnostic
splenic aspiration. Nine years earlier, the patient had been
diagnosed as having a benzene-induced pancytopenia due to an 8-
year occupational exposure. Although these findings are suggestive,
there are too few case reports to document a causal relationship
between benzene and myelofibrosis and myeloid metaplasia.
91
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Aksoy et al recently reported another myeloproliferative
variant, thrombocythemia, as a transient phenomenon in a shoe-
maker whose benzene-associated pancytopenia evolved into a
disorder characterized by a high platelet count (540,000 to
1,600,000 per mm ) and then into acute leukemia. Polycythemia
vera, another myeloproliferative disorder, apparently has not
been described in association with benzene exposure.
Paroxysmal nocturnal hemoglobinuria is a hemolytic disorder
noted infrequently in benzene-exposed individuals. ' This is
an extremely rare condition that has been linked to both aplastic
anemia and acute myelogenous leukemia. The disease is paraneo-
plastic, being defined by a population of circulating red cells
that are abnormally sensitive to the hemolytic effect of comple-
ment. It often occurs in the setting of an aplastic anemia, may
evolve into acute myelogenous leukemia or particularly erythro-
leukemia, and is sometimes noted as a relatively transient
phenomenon during cases of acute myelogenous leukemia. Accord-
ingly, its observation in workers exposed to benzene is not un-
expected.
In addition to hematopoietic tissue, several other organ
systems are said to be affected by benzene. Effects on the
central nervous system following acute exposure to benzene have
been clearly documented in man. These have been reviewed by
Gerarde and by Browning, the latter noting 13 fatal cases in
Great Britain following inhalation of high levels of benzene in
enclosed areas. Reported effects following acute exposure
include headache, nausea, staggering gait, paralysis, convulsions,
and eventual unconsciousness and death. Recent reports of
fatalities have been presented by Tauber and by Bass.
Giddiness and euphoria also have been noted. These acute effects
are usually observed only at relatively high levels of benzene,
well above concentrations believed to be responsible for hemato-
logical effects following chronic exposure. Eastern European
92
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investigators have suggested recently, however, that effects on
the central nervous system, including changes recorded by
electroencephalograph and alterations in cerebral circulation,
may occur in occupational settings. ' There are also
unconfirmed suggestions in the literature that the human cardio-
vascular ' ' and gastrointestinal systems ' may be
affected by benzene.
SUMMARY
Benzene exposure has been clearly demonstrated to produce
hematotoxicity in man. The most commonly reported effect is a
decrease in one or more of the formed elements of the blood. In
more severe cases, this takes the form of pancytopenia, often
with aplastic bone marrow. Evaluation of occupationally exposed
groups reveals a wide spectrum of disease ranging from fatal
aplastic anemia to individual cytopenias and, in some studies,
qualitative abnormalities of blood cells in the presence of
normal peripheral blood counts. The evidence that benzene is
causally related to pancytopenia includes the observation that
benzene is the common denominator in outbreaks of pancytopenia
observed in many different occupational exposure settings
throughout the world, that detection of pancytopenia in a work
force is often temporally related to the use of benzene, and that
similar effects are observed in animals treated with benzene.
The causal relationship of benzene exposure to leukemia is
more controversial, particularly because an animal model of this
effect has not been clearly demonstrated. Recent studies,
however, have provided strong confirmatory evidence of a causal
relationship, which now appears to be beyond reasonable doubt for
acute myelogenous leukemia and its variants. The evidence
includes the many individual case reports of benzene-induced
93
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pancytopenia that proceed to acute myelogenous leukemia and
erythroleukemia, the accumulated case reports of leukemia
associated with benzene exposures, and, most importantly, the
epidemiological evidence indicating a greater risk for leukemia
among benzene-exposed individuals.
It would appear that the evidence indicating an increased
risk of leukemia on exposure to benzene for various periods of
time and at various concentrations is overwhelming. Unfortunate-
ly, the data are not adequate for deriving a scientifically valid
dose-response curve. Such a curve may be estimated on the basis
of various assumptions; these assumptions, however, usually
represent hypotheses that, although they may be valid, are not
yet proven. Hence the estimation of a dose-response curve is not
appropriate in this report, which deals with the currently
available scientific knowledge of health effects, but is under-
taken in the benzene risk assessment document.
94
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Ill
* U.S. GOVERNMENT PRINTING OFFICE: 1Q78—740-261/4164 Region No. 4
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1 REPORT NO
EPA 600/1-78-061
3. RECIPIENT'S ACCESSION NO.
4 TITLE AND SUBTITLE
ASSESSMENT OF HEALTH EFFECTS OF BENZENE GERMANE TO
LOW LEVEL EXPOSURE
5. REPORT DATE
September 1978
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Washington, B.C. 20460 (and various contractors)
10. PROGRAM ELEMENT NO.
1HA630
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/18
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report on the health effects of benzene assesses the teratagenicity,
mutagenicity, carcinogenicity, and hematopoietic effects from benzene
germane to low level exposure.
Epidemiological data strongly indicate that benzene is a human leukemogen
although no animal models have been developed.
This is one of three documents which will be used by EPA's Office of Air
and Waste Management, and by the Administrator, to determine what actions,
if any, should be taken against benzene under the Clean Air Act.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATl Field/Group
Dose-Response
Health Effects
Toxicology
Air Pollution
Exposure
Carcinogen
Environmental Pollution
Benzene
Leukemia
Blood Dyscrasias
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
120
20. SECURITY CLASS (Thispage}
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
112
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