DRAFT CRITERIA DOCUMENT*
FOR ORTHO-DICHLOROBENZENE,
META-DICHLOROBENZENE,
PARA-DICHLOROBENZENE
FEBRUARY 1984
HEALTH EFFECTS BRANCH
CRITERIA AND STANDARDS DIVISION
OFFICE OF DRINKING WATER
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
*This draft criteria document contains information on three
dichlorinated benzenes. At this time, an RMCL for 1,4-
dichlorobenzene (p-DCB) is being proposed. Ortho- and meta-
(1,2- and 1,3-) dichlorobenzene will be examined for possible
inclusion in Phase II of the revised regulations.
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TABLE OF CONTENTS
Page
I. Summary 1-1-10
II. General Information and Properties II-1-3
III. Sources of Human Exposure III-l-
(To be developed by STB)
IV. Toxicokinetics IV-1-17
V. Health Effects in Non-humans V-l-88
Plants V-l-2
Microorganisms V-2-3
Phytoplankton V-3-6
Insects V-7
Birds V-7
Non-human mammals V-7-88
VI. Health Effects in Humans VI-1-7
VII. Mechanism(s) of Toxicity VII-1-6
VIII. Quantification of Toxicological Effects..VIII-1-23
IX. References IX-1-12
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I. SUMMARY
There are three isomers of dichlorobenzene: ortho (1,2-),
meta- (1,3-) and para- (1,4-). The major uses of ortho-dichloro-
benzene (o-DCB) are as a process solvent in the manufacture of
toluene diisocyanate and as an intermediate in the synthesis of
dyestuffs, herbicides and degreasers (Ware and West, 1977). The
bulk of para-dichlorobenzene (p-DCB) usage is in direct application
as an air deodorant or insecticide which accounts for 90% of its
total consumption (Lowenheim and Moran, 1975; Ware and West,
1977). The use of o- and p-DCB as deodorizers in industrial
wastewaters and toilet bowl waters would suggest that increasing
amounts of these substances will be found in waters throughout the
country in the future. No documented uses for meta-dichlorobenzene
(m-DCB) were found in the literature.
Ortho- and para- DCB are produced in considerable quantity;
production volumes of o-DCB equalled 22,000 kkg and of p-DCB
equalled 34,000 kkg in 1981 (USITC, 1981). Environmental releases
of the dichlorobenzenes have been estimated at 30,000 kkg (or 57%
of production). Approximately 7,000 kkg of o-DCB are released after
solvent use and 22,000 kkg p-DCB are released from moth balls and
space deodorants (SAI, 1980). Meta-DCB gets into the environment
as a breakdown product of certain pesticides and as a byproduct
of the manufacture of other chlorinated benzenes.
All three isomers of dichlorobenzene have been detected in
drinking water supplies from both ground and surface waters, in
quantities ranging from less than 0.5 ug/1 to greater than 9 ug/1
(EPA, 1975; EPA, 1978a).
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1-2
No studies have been reported which determine the percentage
of a dose of dichlorobenzene is absorbed following oral or inhalation
exposure. However, for the purpose of regulation development,
based upon the absorption characteristics of benzene and the smaller
chlorinated ethanes and ethylenes, it will be assumed that 100%
of an oral dose of any of the isomers of dichlorobenzene is
absorbed and that 30% of an inhalation dose is absorbed when
exposure persists for longer than one to three hours.
After oral administration to rabbits, the DCBs are oxidized
principally to phenols. Ortho- and m-DCB also form catechols (Azouz,
e_t al_., 1955; Williams, 1959). The metabolites are excreted as
free phenols or catechols to a slight degree, but in greater
percentage as conjugates of glucuronide or sulfate. Ortho- and
meta-DCB form mercapturic acids as well, but p-DCB does not
(Williams, 1959). The dichlorophenols appear to be the principal
metabolic products of the DCS isomers in man (Hallowell, 1959;
Pagnatto and Walkley, 1965).
The ortho- and para- isomers have been shown to be quite
lipophilic, and can be expected to bioaccumulate in tissues with
high fat content during prolonged, continuous exposures. Para-DCB
has been detected in human adipose tissue and all three isomers
have been detected in blood (Dowty, et al., 1975; Morita, et al.,
1975; Morita and Ohi, 1975).
Reports have appeared in the literature describing poisoning
-incidents resulting from exposure to the dichlorobenzenes.
Girard, e_t al_. (1969) reported four cases of leukemia in patients
purportedly exposed to varying quantities and mixtures of dichloro-
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1-3
benzene, although each solution contained the ortho isomer.
Hallowell (1959), Gadrat, et aU (1962), Girard, et al_.
(1969) and Campbell and Davidson (1970) all described cases in
which individuals suffered from moderate to severe anemia following
exposure to the DCBs. Several instances of skin lesions developing
after contact also have been reported (Downing, 1939; Frank and
Cohen, 1961; Nalbandian and Pearce, 1965).
In cases where moderate exposure to the DCBs was documented,
patients complained of vomiting, headaches, irritation of the eyes
and upper respiratory tract with profuse rhinitis and periorbital
swelling (Dupont, 1938; Cotter, 1953; Campbell and Davidson,
1970). Anorexia, nausea, vomiting, weight loss, yellow atrophy
of the liver and blood dyscrasias were reported for higher exposure
concentrations (Petit and Champeix, 1954; Cotter, 1953; Wallgren,
1953; Weller and Crellin, 1953; Hallowell, 1959). Liver damage
sometimes was accompanied by prophyria (Hallowell, 1959).
The dichlorobenzenes produce sedation, analgesia and
anesthesia after acute oral or parenteral administration. Rela-
tively high doses are needed to produce acute effects, but chronic
effects may occur at relatively low levels. Acute poisoning is
characterized by signs of disturbance of the central nervous sys-
tem: hyperexcitability, restlessness, muscle spasms or tremors.
The most frequent cause of death is respiratory depression. Acute
exposure at high levels also may result in kidney and/or liver
damage. Liver damage may be manifested as necrosis/degeneration
or porphyria, depending upon the isomer to which the individual
has been exposed.
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1-4
The LDso for o-DCB in rats after oral administration ranged
from 500 mg/kg (NIOSH, 1978) to 1,500 mg/kg (Hollingsworth, et
al . , 1958). In the guinea pig, the oral LDso was 2,000 mg/kg
(Hollingsworth, £t al_., 1958). The oral LDso for p-DCB ranged from
500 mg/kg to 2,500 mg/kg (Hollingsworth, et al_., 1956) in the rat
and was 3,220 mg/1 in the mouse (Varshavskaya, 1968). The lowest
published lethal oral dose in guinea pigs was 2,800 mg/kg (Hol-
lingsworth, et al_., 1956). Irie, et al_. (1973) reported an LDso
of 5,145 mg/kg for a subcutaneous dose of p-DCB in the mouse.
Dogs exposed to 2 cc/m^ (0.04%) o-DCB by inhalation showed no
adverse effects, whereas 0.08% produced somnolence (Riedel,
1941). Histological studies following the administration of
acute and subacute doses of o-DCB showed damage to the liver and
kidney. Exposing mice to the same concentrations caused CNS stimu-
lation for about 20 minutes followed by CNS depression, muscular
twitching, slow and irregular respiration, -cyanosis near the end
of an hour and death within 24 hours. Rats appeared to be
slightly more resistant than mice to the toxic effects of o-DCB.
Inhalation of o-DCB by rats at 80 ppm for 11-50 hours was
irritating to the eyes and nose, produced slight changes in
the tubular epithelium of the kidney and resulted in confluent
necrosis of the liver (Cameron, et_ al_., 1937).
Rabbits, rats, and guinea pigs exposed for 20-30 minutes
daily to 100 mg DCBAiter of air for 5-9 days showed marked irrita-
tion of the eyes and nose, muscle twitching, tremors, CNS depression,
nystagmus and rapid but labored breathing, but recovered within
30-180 minutes after being removed from the p-DCB-rich atmosphere
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1-5
(Zupko and Edwards, 1949). Body weight decreased in 11/14 rabbits
and in 6/9 guinea pigs. In rats, CNS depression was observed
to be greater than in rabbits. There was complete narcosis with
attendant tremors and muscular twitching with each exposure. The
observation that many of the test animals of all three species
developed granulocytopenia is an important one. This condition is
considered to be a precursor to leukemia. However, in these experi-
ments, when the animals were removed from exposure to p-DCB, the
decrease in granulocytes was reversed and the level returned to nor-
mal within three to four weeks. The question arises as to whether
this condition was due to the DCB or to contamination by benzene or
other substances.
Fourteen-day repeated dose gavage studies in mice and rats
were conducted with both o- and p-DCB in the prechronic testing
phase of the NTP bioassay on these two substances (Battelle-Columbus,
1978 a,b,d,e,f,g,h) . In addition to early deaths and lack of body
weight gain at the higher doses, animals exhibited histopathology
indicative of hepatic centrolobular necrosis and degeneration,, occa-
sionally with cyto- and karyomegaly, as well as lymphoid depletion
of the spleen and thymus.
Gavage doses of o-DCB given to rats and mice over a
thirteen-week schedule of 5 days/week resulted in liver path-
ology indicative of necrosis and porphyria (Battelle^Columbus,
1978c, i). Serum SGPT levels were increased in mice exhibiting
liver histopathology at the highest dose level. Some mice also
exhibited myocardial and skeletal muscle mineralization and lymp-
hoid depletion of th£ thymus and spleen and necrosis of the spleen.
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1-6
Rats also showed kidney pathology as characterized by tubular
degeneration.
Hollingsworth, et al_. (1958) gave rats a series o.f 138
doses of o-DCB over a period of 192 days (18.8, 188 or 376 mg/kg/
day, five days a week) by intubation. No adverse effects were
detected at the lowest dose. With the intermediate dose, a
slight increase in the weights of the liver and kidneys was noted.
At the highest dose, there was a moderate increase in the weight
of the liver, a slight decrease in the weight of the spleen and
cloudy swelling of the liver.
Hollingsworth, et al. (1958) also measured the effects of
multiple inhalation exposures of o-DCB on rats, guinea pigs,
mice, rabbits and monkeys. A range of concentrations was used,
seven hours a day, five days a week, for six to seven months. No
adverse effects were observed in rats, guinea pigs or mice exposed
to 49 ppm (0.29 mg/1), or in rats, guinea pigs, rabbits and monkeys
exposed to 93 ppm (0.56 mg/1).
Oral doses of 10, 100 or 500 mg/kg p-DCB, five days a week,
for 20 doses, produced marked cloudy swelling and necrosis in
the central area of the liver nodules only with the highest dose.
No effects were observed at the other doses (Hollingsworth,
et al., 1956).
•thirteen week exposures by gavage to p-DCB resulted in liver
pathology similar to that observed with o-DCB, but at somewhat
higher doses (necrosis, degeneration and porphyria) (Battelle
Columbus, 1979a,b, 1980a,b). The spleen and thymus also exhibited
histopathology similar to that observed after o-DCB. In mice and
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1-7
rats, hematopoietic hypoplasia of the bone marrow occurred in sur-
vivors at the highest dose (1500 mg/kg/day). Rats at the two
highest dose levels also exhibited epithelial necrosis of the
nasal turbinates and small intestine and villar bridging of the
mucosa of the latter tissue. Again, the rats exhibited renal
pathology, with multifocal degeneration or necrosis of the corti-
cal tubular epithelium.
Oral doses of 188 or 376 ing p-DCB/kg, five days a week,
for 192 days (138 doses) in rats induced an increase in the weights
of the liver and kidneys (Hollingsworth, et al., 1956). At 376
mg/kg, increased splenic weight, slight cirrhosis and focal necrosis
of the liver were observed. No adverse effects were seen with a
18.8 mg/kg dose.
Inhalation studies also were carried out with p-DCB (Hollings-
worth, et al., 1956), The concentrations used were 96, 158, 173,
314 and 798 ppm (0.58, 0.95, 1.04, 2.05 and 4.8 mgAt respectively.-
Exposure occurred for 7 hours/day, 5 days/week for 6-7 months.
Adverse effects observed included liver and kidney histopathology
with increased organ weights, pulmonary edema and congestion,
splenic weight changes and reversible non-specific eye changes.
It is difficult to reconcile the results of the previously-
described studies with those of Varshavskaya (1968). Rats
received daily oral doses of 0.001, 0.01 or 0.1 mg/kg o-DCB in
sunflower oil for nine months. No adverse effects were noted at
the lowest dose, but varying degrees of inhibition of mitosis in
the bone marrow, as well as neutropenia, abnormal conditioned
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1-8
reflexes and adrenal hypertrophy occurred at the two higher dose
1 evel s.
Studies employing long-term or chronic exposures to o- and
p-DCB were designed to evaluate the substances' chronic toxicity
and carcinogenic potential. Preliminary assessment of the data
from the NTP bioassay performed with o-DCB by gavage suggests
that, under the conditions of the study, this substance is not a
carcinogen in Fischer 344 rats or B6C3F1 mice (NTP, 1982). No
non-neoplastic lesions were noted in either the mice or the rats,
suggesting that the maximum tolerated dose was not achieved.
The results of the NTP gavage bioassay with p-DCB are not
available to ODW at this time. A long-term inhalation study
revealed no increase in tumor incidence or type following exposure
to p-DCB in Alderly Park Wistar rats (Riley, et al_», 1980a) . At
the high dose (500 ppm), changes indicative of non-neoplastic effects
were observed: an increase in liver, kidney, heart and lung weights
(both sexes) and an increase in urinary protein and coproporphyrin
output (in males).
No teratogenicity studies were found in the peer-reviewed "
literature for any of the three isomers of dichlorobenzene. However,
studies are underway to evaluate the teratogenic potential of o-DCB
in rats and rabbits (Dow, 1981). In addition, results of a study
by Hodge, _et al_. (1977) suggest that maternal exposure to atmospheric
levels of p-DCB up to 500 ppm on Days 6-15 of pregnancy does not
result in any embryotoxic, fetotoxic or teratogenic effects in the
offspring.
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1-9
Para-dichlorobenzene induces abnormal mitotic division in
\
higher plants. Effects seen include shortening and thickening
of chromosomes, precocious separation of chromatids, tetraploid
cells, binucleate cells and chromosome bridges (c-mitosis) (Sharma
and Battacharya, 1956; Sharma and Sarkar, 1957; Srivastava, 1966;
Gupta, 1972). Ortho-DCB was shown to produce abnormal mitotic
division in the onion, Allium cepa (Ostergran and Levan, 1943).
Ortho-dichlorobenzene and para-dichlorobenzene were not
mutagenic when tested in a culture of histidine-requiring mutants
of Salmonell a typhimurium or in the E_. col i WP2 system (Anderson,
ert al_. , 1972; Anderson, 1976; Simmon, et al_. , 1979). However, all
three isomers increased the frequency of back mutation of the
methionine-requiring locus in the fungus, Aspergillus nidulans
(Prasad and Pramer, 1968; Prasad, 1970). In addition, the meta
isomer was shown to increase mitotic recombination in the Sac-
charomyces cerevisiae C3 yeast system (Simmon, et al., 1979).
The results with the para isomer were ambiguous. These investi-
gators also showed that both o- and m-DCB interacted with and
damaged bacterial DNA in the E_. col i W3110 polA+/p3478 polA"
differential toxicity assay system.
No evidence of mutagenicity in animals has been published to
date. Guerin, et_ al. (1971) showed that DCB did not produce a
significantly different number of mitoses in rat lung cell cultures
Cytogenetic studies with rat bone marrow cells and a dominant
lethal study in CD-I mice following exposure to p-DCB were all
negative (Anderson and Richardson, 1976; Anderson and Hodge, 1976).
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1-10
In the few reports available on the carcinogenic potential
of the DCBs, the results are negative, although one report of
four cases of leukemia in humans attributed to o-DCB or a mixture
of all three isomers has been published (Girard, et^ al_., 1969).
Hollingsworth, el: al_. (1956, 1958) exposed several species of
animals to various oral and inhalation exposures of ortho- and
para-dichlorobenzene for six to seven months. No pathological
changes indicative of cancerous changes were observed. In a some-
what inconclusive study, Parsons (1942) suggested that p-DCB pro-
duced a transplantable sarcoma in an irradiated mouse. Prelimi-
nary assessment of the data from the NTP carcinogenicity bioassay
performed with o-DCB suggests that, under the conditions of the
study, this substance is not a carcinogen in Fischer 344 rats or
B6C3F1 mice (NTP, 1982). The bioassay with p-DCB has not been
reported as yet. A long term inhalation study revealed no increase
in tumor incidence or type following exposure to p-DCB in Alderley
Park Wistar rats (Riley, e_t al_., 1980a).
-12-
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II-l
II. GENERAL INFORMATION AND PROPERTIES
Chemical and Physical Properties
Three isomers of dichlorobenzene (DCB) exist: ortho (1,2-),
meta (1,3-) and para (1,4-). Very little information is avail-
able on the meta isomer. Therefore, unless otherwise noted, the
properties of this isomer will be assumed to be identical to those
of the ortho isomer.
At room temperature (20-25°C), ortho- and meta-DCB are colorless
neutral liquids; p-DCB is a colorless crystalline solid which
readily sublimes (Irish, 1963). All isomers have a molecular
weight of 147.01. Each is nearly insoluble in water, but readily
soluble in many organic solvents, including ethanol, benzene and
diethyl ether, as well as lipid. Freed, e_t al_. (1979) determined
the solubility of p-DCB in water at 25°C to be 79 ppm (79 mg/1).
All isomers are heavier than water (specific gravity = 1.306, 1.288,
1.458 at 20°C for o-, m- and p-DCB, respectively). Thus, each would
tend to sink in standing water. The ortho isomer has a vapor
pressure of 1.56 mm Hg at 25°C. All three isomers are combustible.
In air, 1 ppm = 6.01 mg/m^ and 1 mg/1 = 166.3 ppm, at 25°C
and 760 mm Hg (Irish, 1963).
Sato and Nakajima (1979) determined the water/air, blood/air,
olive oil/air and olive oil/water partition coefficients for o- and
m-DCB. These are listed in Table II-l. With the logs of their
oil/water partition coefficients approaching 4, both isomers appear
to be quite lipophilic. This is confirmed by the findings of Freed,
et al. (1979) who established that p-DCB has a log P (n-octanol/
water) of 3.38. It would be expected that these substances would
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II-2
tend to bioaccumulate in fatty tissues during prolonged, continuous
exposures.
Table II-l. Partition Coefficients
(after Sato and Nakajima, 1979)
Partition Coefficient Ortho-DCB Meta-DCB
Water/Air (W)
Blood/Air (B)
Olive oil/Air (0)
Olive oil/Water (0/W)
Olive oil/Blood (0/B)
W-0
Log P (0/W)
9
423
39920
4436
94
359280
3.65
5.5
201.4
27080
4924
134
148940
3.69
The odor threshold for o-DCB in air is 2-4 ppm (AIHA, 1964).
At 10-15 ppm, the smell becomes very noticeable, and at 25-30 ppm,
it is considered unpleasant. Eye irritation becomes a problem at
the same concentration range, while at exposures of 60-100 ppm, eye
and mucous membrane irritation may be very painful. The odor thres-
hold for p-DCB in air is 14-30 ppm in unacclimated persons (AIHA, 1964)
Eye irritation begins at 50-80 ppm and becomes painful at 100 ppm.
Kolle (1972) determined the odor threshold in water to range from
0.01-0.03 mg/1 for the three DCB isomers.
Production and Use
o-Dichlorobenzene and p-dichlorobenzene are produced in
considerable quantity. In 1981, the USITC reported that production
volume for o-DCB was 22,000 kkg and for p-DCB, 34,000 kkg. At
least 70 million pounds of p-DCB are used each year for moth control
and as a space odorant (Brown, et al., 1975). Apparently only two
U.S. companies produce m-DCB (West and Ware, 1977), and in 1974,
31,000 pounds were imported (USITC, 1974). Loss of o-DCB and p-DCB
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II-3
during their manufacture amounts to at least a million pounds a
year for each isomer (Brown, e_t al . , 1975). Meta-DCB is lost as a
by-product of the manufacture of monochlorobenzene. It also gets
into the environment through its being a breakdown product of cer-
tain pesticides such as lindane.
The major uses of o-DCB are as a process solvent in the
manufacture of toluene diisocyanate, and as an intermediate in the
synthesis of dyestuffs, herbicides and degreasers (West and Ware,
1977). The bulk of p-DCB usage is in direct application as air
deodorants and insecticides which account for 90% of its total
consumption (Lowenheim and Moran, 1975; West and Ware, 1977). Use
of o- and p-DCB as deodorizers in industrial wastewaters or in
toilet bowl waters would suggest that increasing amounts of these
chemicals will be found in waters throughout-the country in the
future. No documented uses of m-DCB were found in the literature.
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III. HUMAN EXPOSURE
Humans may be exposed to dichlorobenzene in drinking water, food, and
air. Detailed information concerning the occurrence of and exposure to di-
chlorobenzene in the environment is presented in another document entitled
"Occurrence of Dichlorobenzenes in Drinking Water, Food, and Air" (Letkiewicz
et al. 1983). This section summarizes the pertinent information presented in
that document in order to assess the relative source contribution from drink-
ing water, food, and air.
Exposure Estimation
This analysis is limited to drinking water, food, and air, since these
media are considered to be general sources common to all individuals. Some
individuals may be exposed to dichlorobenzene from sources other than the
three considered here, notably in occupational settings and from the use of
consumer products containing dichlorobenzene. Even in limiting the analysis
to these three sources, it must be recognized that individual exposure will
vary widely based on many personal choices and several factors over which
there is little control. Where one lives, works, and travels, what one eats,
and physiologic characteristics related to age, sex, and health status can all
profoundly affect daily exposure and intake. Individuals living in the same
neighborhood or even in the same household can experience vastly different
exposure patterns.
Unfortunately, data and methods to estimate exposure of identifiable
population subgroups from all sources simultaneously have not yet been
developed. To the extent possible, estimates are provided of the number of
individuals exposed to each medium at various dichlorobenzene concentra-
tions. The 70-kg male is used for estimating intake.
a. Water
Cumulative estimates of the U.S. populations exposed to various o- and
p-dichlorobenzene levels in drinking water from public drinking water systems
are presented in Tables IV-I and IV-II, respectively. The values in these
tables were obtained using Federal Reporting Data Systems data on populations
served by primary water supply systems (FRDS 1983) and the estimated number of
these water systems that contain a given level of o- or p-dichlorobenzene.
1
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Table IV-I. Total Estimated Cumulative Population (in Thousands)
Exposed to o-Dichlorobenzene in Drinking Water
Exceeding the Indicated Concentration
System type
Groundwater
Surface water
Total
(% of total )
Number of
people served
in U.S.
(thousands)
73,473
140,946
214,419
(100%)
Cumulative population (thousands)
exposed to concentrations (ug/1 ) of:
2.0.5
156
1,431
1,587
(0.7%)
>5
0
0
0
(0.0%)
Table IV-II. Total Estimated Cumulative Population (in Thousands)
Exposed to p-Dichlorobenzene in Drinking Water
Exceeding the Indicated Concentration
System type
Groundwater
Surface water
Total
(% of total )
Number of
people served
in U.S.
(thousands)
73,473
140,946
214,419
(100%)
Cumulative population (thousands)
exposed to concentrations (ug/1) of:
XL 5
775
859
1,634
(0.8%)
>5
0
0
0
(0.0%)
An estimated 1,587,000 individuals (0.7% .of the population of 214,419,000
using public water supplies) are exposed to levels of o-dichlorobenzene in
drinking water at or above 0.5 ug/1. Mo individuals are estimated to be
exposed to levels above 5 ug/1. Of the approximately 1.6 million people
exposed to levels ranging from 0.5-5 ug/1, 1.4 million (90%) obtain water from
surface water supplies.
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An estimated 1,634,000 individuals (0.8% of the population using public
supplies) are exposed to levels of p-dichlorobenzene in drinking water at or
above 0.5 ug/1 , while no individuals are estimated to be exposed to levels
above 5 ug/1. Fifty-three percent of the approximately 1.6 million indivi-
duals exposed to levels ranging from 0.5-5 ug/1 obtain water from surface
water supplies, while 47% obtain water from groundwater supplies.
No individuals are estimated to be exposed to levels of m-dichlorobenzene
above 0.5 ug/1.
No data were obtained on regional variations in the concentration of
dichlorobenzene in drinking water. The highest concentrations are expected to
occur near sites of production and use of dichlorobenzene.
Daily intake levels of o- and p-dichlorobenzene from drinking water were
estimated using various exposure levels and the assumptions presented in
Tables IV-III and IV-IV, respectively. The data suggest that the majority of
the persons using public drinking water supplies would be exposed to intake
levels for the dichlorobenzene isomers below 0.014 ug/kg/day.
Table IV-III. Estimated Drinking Water Intake of o-Dichlorobenzene
Persons using supplies
exposed to indicated levels
Exposure level % of Total
(ug/1) Population population Intake (ug/kg/day)
_>0.5 1,587,000 0.7% _>°-014
>5.0 0 0.0% >0.14
Assumptions: 70-kg man, 2 liters of water/day.
Table IV-IV. Estimated Drinking Water Intake of p-Dichlorobenzene
Persons using supplies
exposed to indicated levels
Exposure level % °f Total
(ug/1) Population population Intake (ug/kg/day)
>0.5
>5.0
1,634,000
0
0.8%
0.0%
ML 014
>0.14
Assumptions: 70-kg man, 2 liters of water/day.
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An indication of the overall exposure of the total population to di-
chlorobenzene can be obtained through the calculation of population-concentra-
tion values. These values are a summation of the individual levels of the
dichlorobenzene isomers to which each member of the population is exposed. An
explanation of the derivation of these values is presented in Appendix C.
Population-concentration estimates for o-dichlorobenzene in drinking
water were 7.9 x 105 ug/1 x persons (best case), 4.4 x 106 ug/1 x persons
(mean best case), 1.1 x 108 ug/1 x persons (mean worst case), and 1.1 x 108
ug/1 x persons (worst case). Assuming a consumption rate of 2 liters of
water/day, population-exposure values of 1.6 x 106 ug/day x persons (best
case), 8.8 x 106 ug/day x persons (mean best case), 2.2 x 108 ug/day x persons
(mean worst case), and 2.2 x 108 ug/day x persons (worst case) were derived.
Population-concentration estimates obtained for m-dichlorobenzene were 0
(best case) and 1.1 x 108 (worst case). A median case value could not be
calculated due to the absence of positive values in groundwater. Using these
figures, population-exposure values of 0 ug/day x persons (best case) and 2.2
x 108 ug/day x persons (worst case) were derived.
Population-concentration estimates for p-dichlorobenzene in drinking
water were 8.2 x 105 ug/1 x persons (best case), 4.6 x 106 ug/1 x persons
(mean best case), 1.1 x 108 ug/1 x persons (mean worst case), and 1.1 x 108
ug/1 x persons (worst case). The population-exposure estimates derived from
these values were 1.6 x 106 ug/day x persons (best case), 9.2 x 106 ug/day x
persons (mean best case), 2.2 x 108 ug/day x persons (mean worst case), and
2.2 x 108 ug/day x persons (worst case).
b. Diet
Data on levels of dichlorobenzenes in foods in the United States were
limited to concentrations of the chemicals in trout from the Great Lakes and
in mother's milk. These data are insufficient for determining the intake of
dichlorobenzene in the U.S. diet.
c. Air
Exposure to dichlorobenzene in the atmosphere varies from one location to
another. The highest level of o-dichlorobenzene reported in the atmosphere
-------
was 19,000 ng/m3 (19 ug/m3) (Bozzelli and Kebbekus 1979 cited in Brodzinsky
and Singh 1982). High levels, averaging greater than 1,000 ng/m3 (1.0 ug/m3),
have been detected in other areas. Normal levels, however, are somewhat
lower. Brodzinsky and Singh (1982) calculated median air levels of o-di-
chlorobenzene for rural/remote areas, urban/suburban areas, and source domi-
nated areas of 0.0 ng/m3 (0.0 ug/m3), 6.6 ng/m3 (0.0066 ug/m3), and 350 ng/m3
(0.35 ug/m3), respectively.
The highest level of m-dichlorobenzene reported in the atmosphere was
16,000 ng/m3 (16 ug/m3) (Wallace 1981 cited in Brodzinsky and Singh 1982).
Average levels greater than 1,500 ng/m3 (1.5 ug/m3) have been reported in
other areas. The following median concentrations were calculated for m-di-
chlorobenzene: rural/remote areas, 0.0 ng/m3 (0.0 ug/m3); urban/suburban
areas, 36 ng/m3 (0.036 ug/m3); and source dominated areas, 560 ng/m3 (0.56
ug/m3).
The maximum level reported for p-dichlorobenzene in the atmosphere was
60,000 ng/m3 (60 ug/m3) (Bozzelli and Kebbekus 1979 cited in Brodzinsky and
Singh 1982). Mean levels of p-dichlorobenzene above 1,000 ng/m3 (1 ug/m3)
were found in two locations. Median air levels of p-dichlorobenzene for
rural/remote areas, urban/suburban areas, and source dominated areas were 0.0
ng/m3 (0.0 ug/m3), 280 ng/m3 (0.28 ug/m3), and 0.0 ng/m3 (0.0 ug/m3), respec-
tively.
The monitoring data available are not sufficient to determine regional
variations in exposure levels for the dichlorobenzenes. However, urban and
industrial areas generally appear to contain higher levels, as expected.
The daily respiratory intake of each of the isomers of dichlorobenzene
was estimated using the assumptions presented in Tables IV-V through IV-VII
and the median and maximum levels for the dichlorobenzenes reported above.
The estimates in Tables IV-V and IV-VI indicate that th-e daily intake of o-
and m-dichlorobenzene from air for adults in source dominated areas is
approximately 0.1 and 0.2 ug/kg/day, respectively. A similar value (0.09
ug/kg/day) was calculated for p-dichlorobenzene in urban/suburban areas (Table
IV-VII). In contrast, the intakes calculated using the maximum o-, m-, and
p-dichlorobenzene levels reported are 6.3, 5.3, and 20 ug/kg/day, respec-
tively; few if any persons are believed to be exposed at those levels. The
values presented do not account for variances in individual exposure or
uncertainties in the assumptions used to estimate exposure.
5
-------
Table IV-V. Estimated Respiratory Intake of o-Dichlorobenzene
Exposure (ug/m3) Intake (ug/kg/day)
Rural/remote (0.0) 0.0
Urban/suburban (0.0066) 0.0022
Source dominated (0.35) 0.12
Maximum (19) 6.2
Assumptions: 70-kg man, 23 m3 of air inhaled/day (ICRP 1975).
Table IV-VI. Estimated Respiratory Intake of m-Dichlorobenzene
Exposure (ug/m3) Intake (ug/kg/day)
Rural/remote (0.0) 0.0
Urban/suburban (0.036) 0.012
Source dominated (0.56) 0.18
Maximum (16) 5.3
Assumptions: 70-kg man, 23 m3 of air inhaled/day (ICRP 1975).
Table IV-VII. Estimated Respiratory Intake of p-Dichlorobenzene
Exposure (ug/m3) Intake (ug/kg/day)
Rural/remote (0.0) Q Q
Source dominated (0.0)a
Urban/suburban (0.28.) 0.092
Maximum (60) 20
aValue reported for source dominated areas was lower than that reported for
urban/suburban areas.
Assumptions: 70-kg man, 23 m3 of air inhaled/day (ICRP 1975).
-------
In addition to the available monitoring data, Systems Applications (1982)
has provided estimates of atmospheric levels of o- and p-dichlorobenzene by
applying air dispersion models to dichlorobenzene emission sources. The
computed average concentrations of the dichlorobenzenes and the number of
individuals estimated to be exposed to these concentrations are presented in
Tables IV-VIII and IV-IX. Specific point sources in these tables are indivi-
dually identified sources with known locations and modes and rates of emis-
sions. These are generally manufacturing plants. General point sources are
sources that are too numerous, small, or of uncertain location to be treated
individually. However, these sources produce isolated patterns of significant
concentration. Area sources are sources that are numerous and emit only small
concentrations of the chemical (e.g., home chimneys, automobiles). The esti-
mates presented for o-dichlorobenzene in Table IV-VIII suggest that only a
small number of individuals (less than 700,000) are exposed to o-dichloro-
benzene concentrations greater than 250 ng/m3 (0.25 ug/m3). Estimates for
p-dichlorobenzene (Table IV-IX) suggest that 62,000,000 individuals are
exposed to p-dichlorobenzene levels at or above 250 ng/m3 (0.25 ug/m3).
Tables IV-VIII and IV-IX also present total population-concentration
estimates for o- and p-dichlorobenzene (6.45 x 106 and 5.14 x 107 ug/m3 x
persons, respectively). Assuming an inhalation rate of 23 m3 of air/day,
population-exposures of 1.48 x 108 and 1.18 x 109 ug/day x persons, respec-
tively, were calculated.
SUMMARY
Tables IV-X, IV-XI, and IV-XII present a general view of the total amount
of o-, m-, and p-dichlorobenzene, respectively, received by an adult male from
air and drinking water. Insufficient data were obtained on levels of di-
chlorobenzene in foods to assess the relative intake from that source.
The data presented have been selected from an infinite number of possible
combinations of concentrations for the two sources. The actual exposures
encountered would represent some finite subset of this infinite series of
combinations. Whether exposure occurs at any specific combination of levels
is not known; nor is it possible to determine the number of persons that would
be exposed to dichlorobenzene at any of the combined exposure levels. The
data presented represent possible exposures based on the occurrence data and
the estimated intakes.
-------
Table IV-VIII. Exposure and Dosage Summary for Airborne o-Dichlorobenzene
Population
Concentration Specific
level point
(ug/m ) source
50 2
25 25
>10 234
5 917
2.5 5,406
1 36,787
0.5 93,389
0.25 172,270
0.1 426,427
0.05 626,291
0.025 839,531
0.01 1,525,505
0 6,113,449
General
point
source
0
0
0
0
0
0
0
0
0
0
0
800
--
exposed (persons)
Area source
0
0
0
0
0
0
0
505,140
9,149,730
33,072,205
81,759,648
142,928,535
158,679,135
U.S. total
2
25
234
917
5,406
36,787
93,389
677,410
9,576,157
33,698,495
82,599,179
144,454,840
--
Specific
point
source
91
892
3,610
8,410
23,600
69,500
110,000
137,000
178,000
192,000
200,000
210,000
224,000
Dosage (ug/m x persons)
General
point
source
0
0
0
0
0
0
0
0
0
0
0
9
2,460
Area source
0
0
0
0
0
0
0
232,451
1,772,052
3,479,775
5,056,481
6,121,131
6,225,594
U.S. total
91
892
3,610
8,410
23,600
69,500
110,000
369,451
1,950,052
3,671,775
5,256,481
6,331,140
6,452,054
Note: The use of "--" as an entry indicates that the incremental
not significant (relative to the last entry in that column
that the exposure of the same population may be counted in
Source: Systems Applications 1982
increase in the population exposed or the dosage is
or to an entry in another column at the same row) or
another column.
-------
Table IV-IX. Exposure and Dosage Summary for Airborne p-Dichlorobenzene
Population
Concentration
level
(ug/m3)
50
25
10
5
2.5
1
0.5
0.25
0.1
0.05
0.025
0.01
0.005
0
Specific
point
source
2
8
42
127
389
1,691
3,879
10,792
36,631
126,422
384,501
888,210
--
2,341,084
General
point
source
0
0
0
0
0
0
0
0
0
0
0
3,400
19,600
--
exposed (persons)
Area source
0
0
0
0
505,140
9,149,730
26,976,292
61,583,693
--
--
--
—
--
158,679,135
U.S. total
2
8
42
127
505,529
9,151,421
26,980,171
61,594,485
--
--
--
--
--
--
Specific
point
source
118
328
815
1,420
2,350
4,330
5,930
8,400
12,200
18,400
27,400
35,200
--
41,400
o
Dosage (ug/m x persons}
General
point
source
0
0
0
0
0
0
0
0
0
0
0
50
160
3,360
Area source
0
0
0
0
1,917,818
14,620,149
26,029,918
37,167,988
49,590,816
--
--
--
--
51,363,678
U.S. total
118
328
815
1,420
1,920,168
14,624,479
26,035,848
37,176,388
49,603,016
--
--
--
--
51,408,438
Note: The use of "--" as an entry indicates that the incremental
not significant (relative to the last entry in that column
that the exposure of the same population may be counted in
Source: Systems Applications 1982
increase in the population exposed or the dosage is
or to an entry in another column at the same row) or
another column.
-------
Table IV-X. Estimated Intake of o-Dichlorobenzene
from the Environment by Adult Males in ug/kg/day
(% from Drinking Water)
Concentration in Concentration in air
drinking water Rural/remote Urban/suburban Source dominated Maximum
. (ug/1) (0.0 ug/m3) (0.0066 ug/m3) (0.35 ug/m3) (19 ug/m3)
0 0.0 (--) 0.0022 (0%) 0.12 (0%) 6.2 (0%)
0.5a 0.014 (100%) 0.016 (88%) 0.13 (11%) 6.2 (0.2%)
5.0b 0.14 (100%) 0.14 (100%) 0.26 (54%) 6.3 (2.2%)
Intake from each source (see Sections 5.1-5.3):
Water: 0.5 ug/1: 0.014 ug/kg/day
5.0 ug/1: 0.14 ug/kg/day
Air: 0.0 ug/m3: 0.0 ug/kg/day
0.0066 ug/m3: 0.0022 ug/kg/day
0.35 ug/m3: 0.12 ug/kg/day
19 ug/m3: 6.2 ug/kg/day
Food: Not included
al,587,000 individuals using public drinking water systems are estimated to be
exposed to levels >_ 0.5 ug/1 (0.7% of population using public water supplies).
bNo individuals using public drinking water systems are estimated to be
exposed to levels > 5.0 ug/1.
10
-------
Table IV-XI. Estimated Intake of m-Dichlorobenzene
from the Environment by Adult Males in ug/kg/day
(% from Drinking Water)
Concentration in Concentration in air
drinking water Rural/remote Urban/suburban Source dominated Maximum
(ug/1) (0.0 ug/m3) (0.036 ug/m3) (0.56 ug/m3) (16 ug/m3)
0 0.0 (—) 0.012 (0%) 0.18 (0%) 5.3 (0%)
0.5a O.OH (100%) 0.026 (54%) 0.19 (7.4%) 5.3 (0.3%)
Intake from each source (see Sections 5.1-5.3):
Water: 0.5 ug/1: 0.014 ug/kg/day
Air: 0.0 ug/m3: 0.0 ug/kg/day
0.036 ug/m3: 0.012 ug/kg/day
0.56 ug/m3: 0.18 ug/kg/day
16 ug/m3: 5.3 ug/kg/day
Food: Not included
aNo individuals using public drinking water systems are estimated to be
exposed to levels _>_ 0.5 ug/1.
11
-------
Table IV-XII. Estimated Intake of p-Dichlorobenzene
from the Environment by Adult Males in ug/kg/day
(% from Drinking Water)
Concentration in Rural
drinking water
(ug/1)
0
0.5a
5.0b
Intake from each source
Water: 0.5 ug/1 :
5.0 ug/1 :
Concentrati
/remote Source dominated
(0.0 ug/m3)
0.0 (-)
0.014 (100%)
0.14 (100%)
(see Sections 5.1-5.3) :
0.014 ug/kg/day
0.14 ug/kg/day
on in air
Urban/suburban Maximum
(0.28 ug/m3) (19 ug/m3)
0.92 (0%) 20 (0%)
0.11 (13%) 20 (0.07%)
0.23 (61%) 20 (0.7%)
Air: 0.0 ug/m3: 0.0 ug/kg/day
0.28 ug/nr: 0.092 ug/kg/day
60 ug/m3: 20 ug/kg/day
Food: Not included
al,634,000 individuals using public drinking water systems are estimated to be
exposed to levels >_ 0.5 ug/1 (0.8% of population using public water supplies).
bNo individuals using public drinking water systems are estimated to be
exposed to levels > 5.0 ug/1.
12
-------
Brodzinsky and Singh (1982) calculated median urban/suburban air levels
of o-, m-, and p-dichlorobenzene of 0.0066, 0.036, and 0.28 ug/m3, respec-
tively, based on air monitoring data. Assuming those air levels, drinking
water would be the predominant source of exposure in the adult male at drink-
ing water levels above 0.08, 0.42, and 3.2 ug/1, respectively. An accurate
assessment of the number of individuals for which drinking water is the pre-
dominant source of exposure cannot be determined from the data since specific
locations containing high concentrations of the dichlorobenzenes in drinking
water and low concentrations of the dichlorobenzenes in ambient air and food
are unknown.
Population-exposure estimates for o- and p-dichlorobenzene in drinking
water and air were reported previously. Estimates for o-dichlorobenzene in
drinking water ranged from 0.016-2.2 x 108 ug/day x persons; the estimate for
ambient air was 1.48 x 108 ug/day x persons. These population-exposures are
comparable. Estimates for p-dichlorobenzene in drinking water also ranged
o
from 0.016-2.2 x 10° ug/day x persons; the estimate for ambient air was 1.18 x
10^ ug/day x persons. These estimates suggest that ambient air may be a
slightly greater source of exposure to p-dichlorobenzene than drinking water
on a general population basis. Comparison of these estimates, however, may be
deceiving since the same population-exposure level can occur if: 1) a whole
population is exposed to moderate levels of a chemical or 2) some segments of
the same population are exposed to high levels and others to low levels. The
population-exposure values presented give no indication of the relative pre-
dominance of drinking water and air as specific sources of o- and p-dichloro-
benzene on a site-by-site or subpopulation basis.
The relative source contribution data are based on estimated intake and
do not account for a possible differential absorption rate for dichlorobenzene
by route of exposure. The relative dose received may vary from the relative
intake. In addition, the relative effects of the chemical on the body may
vary by different routes of exposure.
-------
IV-1
IV. TOXICOKINETICS
Absorption/Retention
The absorption and excretion of the chlorinated benzenes
take place by simple diffusion. The compounds can be absorbed from
the lungs, the gastrointestinal tract and through the skin. The
dichlorobenzenes are poorly soluble in water/ but possess varying
degrees of high lipid solubility {Neely, _et al_., 1974; Lu and Met-
calf, 1975; Brown, e_t al_. , 1975). Thus, these substances cross
most of the barrier membranes, including brain and placenta.
Little information is available which demonstrates the percentage
of a dose of DCB absorbed and retained following exposure in any
environmental medium. Yano (1979) administered 2.5 mg p-DCB to
mice orally (125 mg/kg for a 20 g mouse). Measuring carcass
content of the compound (except for the gastrointestinal tract,
hair, skin and tail), he determined the rate of absorption over
a 24-hour period (Figure IV-1). The rate reached a maximum at 6
hours after dosing, falling to near zero after 8 hours. The
author concluded that the absorption rate was 11 ± 2.9%. The
percentage of the dose absorbed was not identified in this study.
Based upon what is known about the absorption character-
istics of benzene and the smaller chlorinated aliphatics (ethanes
and ethylenes), it will be assumed that 100% of any oral dose of
a dichlorobenzene is absorbed, while 30% of any DCB isomer inhaled
over a period of one to several hours is absorbed and retained.
-------
IV--2
Figure IV-1.
Absorption rate of DCB, TCB and HCB in mice
(•.-/hole body, except for the digestive tract,
hair, skin and tail) after oral administration
(DCB or TCB = 2 . Smg/niouse ; MCB=1 . 25 mg/mouse)
Source: Yano (1979)
-------
IV-3
Distribution
Hawkins, et al . (1980) described the tissue distribution
of p-DCB in adult female CFY rats following inhalation, oral or sub-
cutaneous exposure. The animals received 10 consecutive daily expo-
sures to p-dichloro (14C) benzene either via inhalation at 1,000 ppm
(1^/6,000 mg/m^) for 3 hours/day or to a range of doses orally or
subcutaneously. The compound was administered in sunflower oil at
levels of 50, 125, 250, 375 or 500 mg/kg. The authors concluded
that oral or subcutaneous doses of 250 mg/kg would yield tissue con-
centrations in fat similar to those observed after the 1,000 ppm in-
halation dose.
Table IV-1 shows tissue concentrations of radioactivity in
animals killed 24 hours after two or ten consecutive daily doses.
During inhalation exposure, concentrations reached their maxima
after six days of exposure, remaining the same or falling slightly
thereafter. At the maximum point, the highest levels were found
in the fat, liver and kidney. Lung and muscle concentrations
reflected the levels in plasma. The highest tissue concentrations
following the 250 mg/kg oral doses were reached after four doses.
Again, the highest concentrations were found in liver, kidney and
fat, with muscle and lung being similar to plasma. After ten doses,
all concentrations had fallen somewhat, with all but fat being simi-
lar to the plasma levels.
-------
Table IV-1
Tissue concentrations of 14C in female rats after dail
subcutaneous (s.c.) doses (250 mgAg) of p-dichloro (]
and results expressed as ppm represent the mean of results from two animals.
v atmospheric (inhal.) exposure (1,000 ppm) and oral or
subcutaneous (s.c.) doses (250 mgAg) of p-dichloro (C) benzene. Animals were killed at 24h after dosing
Liver Kidneys
Lungs
Muscle
Fat
ELasma
. of
ses
2
4
6
8
|LO
inhal .
14
22
28
16
18
oral
11
18
14
15
9
s.c.
21
22
24
21
20
inhal .
24
40
43
28
27
oral
27
29
23
18
16
s.c.
30
32
47
41
32
inhal.
9
12
11
10
10
oral
7
13
10
11
9
s.c.
18
12
14
21
17
inhal .
5
6
7
7
3
oral
5
6
-------
IV-5
Tissue concentrations after subcutaneous dosing at 250
mg/kg were variable, often being higher after two doses than after
four or six doses, with a slight rise after six or eight doses be-
fore dropping slightly after 10. The pattern of distribution was
the same as after oral or inhalation exposure.
Figure IV-2 shows concentrations of radioactivity over a 24-
hour period following cessation of exposure by inhalation. Except
for the lungs, concentrations were highest at 1 hour, falling there-
after. At 120 hours, the concentration in fat had fallen to about
5 ppm from 2,400 ppm at 1 hour. Concentrations in all other tissues
were below the level of detection (0.2 ppm). Disappearance of
radioactivity from the same six tissues was monitored in selected
animals for up to 192 hours after cessation of exposure for 10
days by all three routes (Table IV-2). After oral dosing, maximum
plasma concentrations occurred 2-4 hours after cessation of
exposure. Peak tissue concentrations also occurred at this
time, being highest in fat. Concentrations declined rapidly at
all sites to undetectable levels by 120 hours. After subcutaneous
dosing, peak plasma levels were reached within 1-2 hours, with
peak tissue concentrations occurring at 2 hours, again highest
in fat. Concentrations fell more slowly than after the other
two routes of exposure, with detectable levels remaining after
192 hours.
Metabolism
Figures IV-3-IV-5 depict the metabolic pathways proposed for
each of the three DCS isomers. The figures reflect a composite of
work done in several laboratories.
-------
IV-6
I
I
1
I
i
Figure IV-2.
Concentrations of radioactivity in the plasma
and tissues of rats after repeated daily in-
halation exposures of p-dichloro (^4C) benzene
atmospheres for ten days.
Source: Hawkins, et al. (1980)
-------
1V-7
Table IV-2
Tissue concentrations of 14C in female rats at different times after consecutive daily atmospheric (inhal.)
exposures (1,000 ppm) and oral or subcutaneous (s.c.) doses (250 mgAg) of p-dichloro (14C) benzene for ten
days. Results are expressed as ppm and represent single animals.
Liver
Kidneys
Lungs
Muscle
Fat
Plasma
ne of
orifice inhal .
(h)
0.5
I
2
4
B
4
8
6
0
2
83
97
59
51
66
14
16
0.2
0.2
0.2
oral
117
82
75
90
101
31
7
2
0.2
4
s.c.
23
35
37
16
30
28
22
15
7
1
inhal,
172
304
89
86
138
21
15
2
0.2
0.2
. oral
74
56
81
149
123
31
3
2
0.2
0.2
s.c.
-
45
54
66
58
34
31
38
21
7
2
inhal .
178
84
58
42
39
9
8
1
0.2
0.2
oral
58
43
347
106
75
13
3
2
4
0.2
s.c.
24
22
9
22
18
18
15
9
3
0.2
inhal .
43
96
18
16
35
3
5
0.2
0.2
0.2
oral
12
22
NS
NS
23
11
0.2
0.2
0.2
0.2
s.c.
11
NS
9
25
18
16
NS
25
4
0.2
inhal .
1300
2434
1133
1307
1477
425
233
12
5
6
oral s.
401
421
630
1423
1385
559
56
8
0.2
0.2
c. inhal. oral s.c.
347
335
809
622
481
476
424
199
64
14
38
34
26
40
48
10
9
0.2
0.2
0.2
38
38
46
48
43
18
2
0.2
0.2
0.2
26
42
38
29
26
24
22
9
5
1
NS=No Sample
Source: Hawkins, et al. (1980)
-------
Cl
*Main Oxidation Product
Cl
3.4
dichlorophcr.yl
mercapturic acid
ethereal suifata: •''
r*69%
glucuronides
oo
hxcrc'jon Products (Rabbit):
5% mercapturic acids
40% phenols
A% catechols
GJ% conjugates
0% air
Figure IV - 3 . Proposed Metabolic Pathway-for o-Dichlorobenzene
-------
rncrcapturic acid
->-
Gtherosl
sulfates
glucuronides
Figure IV -4 . Proposed Metabolic Pathway for m-Dichiorobenzene
Excretion Products (Rabbit):
"1% morcapturic acids
27% SOq + gluC.
O
25% phenols
3% catechols
0% air
-------
Mo mercapturic
acids
Cl
(Hydro) Quino (L)
Cl
Figure IV - 5 . Proposed Metabolic Pathway for p-Dichlorobenzcne
No catechois
ctvic.'cai
sulfate
glucuroniuc
o
Exertion Products (Rnbbit):
0% n-.crcapturic acids
3ii% phenols
G7o quinols
637o SOg + tjluc, conjugates
0% air
-------
IV-11
In rabbits after oral administration (0.5 g/kg), all
dichlorobenzenes were slowly metabolized by oxidation, mainly to
dichlorophenols. The phenols and their conjugation products were
excreted in five to six days. The major phenolic metabolite of
0-DC& \*&ST 3,4-dichlorophenol (30% of the total dose), of m-DCB,
2,4-dichlorophenol (24% of the total dose) and of p-DCB, 2,5-
dichlorophenol (35% of the total dose). About 3-11% of the ortho
and meta isomers were excreted as dichlorocatechols. Para-DCB did
not seem to form this metabolite. The ortho and meta isomers
formed mercapturic acids (5-10% of the total), but p-DCB did not
(Williams, 1959). The orientation of the mercapturic acid was
the same as for the major phenolic product of each isomer. The
phenolic metabolites were excreted as conjugates of glucuronic
and sulfuric acid (Azouz, et al., 1953; Azouz, et al., 1955;
Parke and Williams, 1955).
Kitamura, _e_t al_. (1977) studied the metabolism of meta-
and para-dichlorobenzene in the mouse. After an intraperitoneal
dose (level not stated) of either compound, urinary metabolites
were characterized by GC-MS. The metabolites were the same as
noted in Figures IV-4 and IV-5, with the exception that an unquantified
amount of unchanged compound also was detected.
The dichlorophenols appear to be the principal metabolic
product of the DCB isomers in man. In a study of industrial expo-
sure to p-DCB, Pagnatto and Walkley (1965) used the urinary meta-
bolite, p-dichiorophenol, as a measure of exposure to p-DCB. In a
case of accidental ingestion of an unknown quantity of p-DCB
crystals by a three-year old boy, analysis of urine specimens
-------
IV-12
yielded four abnormal phenols as well as 2,5-dichloroquinol. These
were shown to be conjugated with glucuronic and sulfuric acids
(Hallowell, 1958).
Non-mammalian animal species have been shown to metabolize
the chlorinated benzenes, although the proportion of the products
may differ from mammals. Safe, e_t al_. (1976) investigated the
metabolism of chlorinated benzenes in the frog, Rana pipiens. A
solution of each substance studied (80 mg in 4-5 ml vegetable oil)
was administered in four equal aliquots by intraperitoneal injection
into each of four frogs. Para-DCB yielded trace amounts of 2,5-di-
chlorophenol.
Microorganisms also have been observed to metabolize the
halogenated benzenes. This factor may be of some importance if the
organisms inhabit the drinking water sources or waste waters which
may eventually contaminate these sources.
The oxidative degradation of chlorinated benzenes by
Pseudomonas was investigated by Ballschmiter and Scholz (1980).
They showed that each dichlorobenzene was oxidized to one or more
dichlorophenol, then to one or more dichlorocatechol. Action of
the organisms on o-DCB produced primarily 3,5- and 4,5- dichloro-
catechol; 3,5-dichlorocatechol was the principal product formed
from p-DCB.
Garrison and Hill (1972) studied the effects of biological
action on the lower chlorinated benzenes. Ortho- and p-DCB
volatilized completely from aerated mixed cultures of aerobic
organisms in less than one day. Midwest Research Institute (MRI,
1974) reported that p-DCB was degraded by biological organisms
-------
IV-13
to 2,5-dichlorophenol, dichloroquinol and conjugates. Gubser
(1969) reported that o-DCB was degraded by sewage sludge organisms,
but the degradation products were not given.
In a study with benzene-acclimated activated sludge, m-DCB
was oxidized to a greater extent than either of the other two isomers
after 192 hours (Malaney and McKinney, 1966). -The following organ-
isms were found in the sludge: Protozoa: Paramecium, Norticella
and Episylis; bacteria: Flavobacterium 1 actis, Achromobacter sul-
fureum, A. superficial is, Alcaligens marshallis and Rhizobium lupina.
A large number of rotifers also were found during the early
stages of the study.
Chlorinated Benzenes as Breakdown Products of Pesticides
Chlorinated benzenes have been identified as degradation
products of lindane metabolism by various plant and animal species.
Considering the once widespread use of lindane as a pesticide, some
of these chlorobenzenes must have entered the environment as lin-
dane degradation products.
Gamma-pentachloro-1-cyclohexane (r-PCCH) is a known breakdown
product of lindane. When corn and pea seedlings were exposed to a
concentration of 25 mg -PCCH/500 ml water, both varieties converted
the -PCCH to m-DCB, 1,2,4,5-tetrachlorobenzene and 2,4,5-tri-
chlorophenol. In addition, 1,2,4-trichlorobenzene, 1,2,3,4-tetra-
chlorobenzene and 2,3,5-trichlorophenol were formed by the corn
seedlings; 1,2,3-trichlorobenzene and 2,4,6-trichlorophenol were
formed by the pea seedlings (Mostafa and Moza, 1973; Moza, et al.,
1974). Gamma-PCCH, m- and p-DCB were identified in the roots
of mature wheat plants that had been grown from seeds treated
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IV-14
treated with 14C-lindane (Balba and Saha, 1974).
Mathur and Saha (1977) incubated a mineral soil and an
organic soil with 14C-lindane for eight weeks. While most of the
lindane was recovered unchanged from the soils (70-89%), those
degradation products that were identified included m- and p-DCB.
Six chlorobenzenes were "tentatively" identified as products
of lindane metabolism in the rabbit (Karapally, ^t al., 1971).
Ortho-DCB was among those detected. Chicken liver homogenates were
shown to degrade lindane to all three isomers of DCS as well as some
higher chlorinated benzenes (Foster and Saha, 1978). The pheasant
showed a similar metabolism pattern.
Degradability and Bioaccumulation Potential
In general, the halogenated benzenes may be broken down in
the environment. The extent to which they do break down depends
upon a number of factors, including the type of halogen and the num-
ber and position of halogens in the ring. The lower halogenated
compounds tend to be less resistant to biodegradation. The resis-
tence of the chlorinated hydrocarbons to chemical and physical
degradation and the marked ability of these compounds to accum-
ulate in fatty tissues are the most important factors in controlling
the fate and distribution of these compounds.
Both o- and p-DCB are resistant to auto-oxidation by the
peroxy radical (RO2) in water and by ozone in air (Brown, et al.,
1975). The dichlorobenzenes are reactive to hydroxy radicals (OH)
in air with a half-life of about three days.
-------
IV-15
Ortho- and para-DCB, in the presence of sunlight and a
dilute aqueous alkali, react with chlorine to form 1,2,4-trichloro-
benzene (photochlorination) (Kirk and Othmer, 1963). At moderate
temperature, o-DCB is resistant to alkaline hydrolysis.
Dichlorobenzenes are decomposed by radio frequency and
each DCS yields the other two isomeric DCBs (Rix and Still, 1966).
Little information is available relating to environmental
hydrolysis of chlorinated benzenes. This is, of course, limited
by the insolubility of these compounds in water. There is a
possibility that the mono- and polyhydric phenols could be
produced through hydrolysis. If environmental hydrolysis occurs,
it must take place very slowly to form phenols or conjugates
because of the insolubility of these compounds (Ware and West, 1977)
Alexander and Lustigman (1966) found that the presence of
a chlorine atom on the benzene ring retarded the rate of biodegrada-
tion.
Para-DCB has been shown to have accumulated in human blood
and adipose tissue. Morita and Ohi (1975) reported an average
concentration of p-DCB in human adipose tissue of 2.3 ug/g (range
= 0.2-11.7, N=34). Blood samples in six volunteers ranged from 4
ug/ml to 16 ug/ml and averaged 9.5 ug/ml. In another study,
Morita, et al_. (1975) reported levels of p-DCB in human adipose
tissue of Tokyo residents. The concentration in the fat ranged
from 0.02 ug/g to 9.90 ug/g, with a mean concentration of 1.7 ug/g.
Since Morita and Ohi had established that there was a relatively
high concentration of p-DCB in the atmosphere of the Tokyo
metropolitan testing area (2.1-4.2 ug/m3), they believed that
-------
IV-16
inhalation of p-DCB was probably the major route of entry of
the substance into the body.
In a study conducted in the New Orleans area, all three
DCBs were detected in human blood samples (Dowty, e_t al_., 1975).
However, none of the chlorinated benzenes were detected in a 400
liter sample of air or in a sample of New Orleans drinking water,
although many other organic compounds were confirmed. One might
speculate that the individuals showing p-DCB in their blood might
have been exposed to the substance during its use as a space
odorant or fumigant.
Koenenmann and Leeuwen (1980) studied the kinetics of six
chlorobenzenes (including p-DCB) in guppies in an accumulation
and elimination experiment. The other five compounds were more
highly substituted. The fish were exposed for 19 days to a mix-
ture of the six substances, with the p-DCB concentration being
160 ng/ml in an aquarium through which the water flowed at a
rate of 27 1/hr. At three-day intervals, three fish were removed
from the tank and chemical analyses for the six compounds per-
formed. Concentrations of each in ug residue/g lipid weight
were determined. Average fat content of the fish was 5.4 ±
2.0%.
The concentration of p-DCB in lipid reached its peak by
Day 2 (150 ug/g) and remained at that level for the remainder of the
exposure period. p-DCB residues were not measurable within three
days after termination of exposure. The investigators calculated
the log P oct to be 3.53 and established the bioaccumulation factor
at 1,800. Thus, while p-DCB was shown to be quite lipophilic with
-------
IV-17
preferential storage in 1ipid-containing tissues, it is rapidly
eliminated when exposure is terminated.
Neely, el: al_. (1974) estimated a steady-state bioconcen-
tration factor of 210 for p-dichlorobenzene using a short exposure
and duration study with the rainbow trout.
Bioconcentration by the bluegill has been studied using
14C-labeled dichlorobenzenes, with thin layer chromatography for
verification (U.S. EPA, 1978b). The bioconcentration factors were
89, 66 and 60 for 1,2-, 1,3-, and 1,4-dichlorobenzene, respectively.
Equilibrium occurred within 14 days and the half-life for each
dichlorobenzene was less than 1 day. These results confirm that the
dichlorobenzenes are unlikely to be a tissue residue problem in the
aquatic environment.
-------
V-l
V. HEALTH EFFECTS IN NON-HUMAN ORGANISMS
PI ants
Para-dichlorobenzene is added to field crop seeds to con-
trol seed storage insects. It was shown that while effective in
controlling pests, p-DCB significantly decreased the viability of
the seeds. This effect was more pronounced when seeds were stored
in a closed container rather than in an open bin (Day and Thompson,
1965). Oil seed crops, sorghum, corn and wheat were more seriously
affected by p-DCB than were grasses and legumes.
Seeds were stored in Mesa, Arizona, in open and closed Mason
jars (1 quart) containing 25 g p-DCB for up to eight years. No
attempt was made to control temperature or humidity. After four
years, none of the California Imperial flax seeds germinated from
either the open or closed test container. The open control regi-
stered 77% germination after four years, while the closed control
averaged 82%. Arivat barley seeds stored in the closed test jar
averaged 3% germination after eight years, while germination of
the barley seed in the open test jar was 54%. The control aver-
.aged 59% and 72% in the closed and open containers, respectively
After three years, none of the seed of the Pima S-l fuzzy cotton
from the closed container germinated (control= 58%). After eight
years in the open test container, 43% of the fuzzy cotton seeds
germinated (control= 68%).
EPA (1978b) determined 96-hour ECso values for the DCBs
on a fresh water algae, Sel enastrum capricornutum, and a marine
algae, Skeletonema costatum, in static bioassays. .In vivo
-------
V-2
chlorophyll content and cell number counts were the parameters
measured. In vivo chlorophyll content £€50 values in Selenastrum
were 91-6, 176 and 98.1 mg/1 for o-, m- and p-DCB respectively.
The ECso values for cell number count were 98 mg/1 for o-DCB, 149
mg/1 for m-DCB and" 96".Tmg/T for p-DCB. gfreretonenta was more
.sensitive to DCB toxicity. The EC50 values for .in vivo chlorophyll
count were 44.2 mg/1 for o-DCB, 52.8 mg/1 for m-DCB and 54.8 mg/1
for p-DCB. Cell number count EC$Q values were 44.1, 49.6 and
59.1 mg/1 for o-, m- and p-DCB, respectively.
Microorganisms
The chlorinated benzenes have been shown to exhibit toxic
effects upon a number of microorganisms. The antifungal vapor
phase activity in the soil of each chlorobenzene was studied by
Richardson (1968). A relationship between chemical structure
and action was noted. Three fungal species were exposed to a
range of eight vapor pressures (8-1,000 ppm). The percentage
retardation of radial growth was greatest for dichloro- (all
isomers) and trichlorobenzenes (both isomers).
Ortho-DCB is lethal to Mycobacterium smegmatis as a liquid
or a vapor (Crowle, 1958). Torres, et al. (1970) showed that at
a dilution of 1:800, o-DCB was active against Staphylococcus
aureus and Escherichi coli, in vitro. In the presence of organic
matter (10% defibrinated sheep blood), o-DCB was effective
against the spores of Bacillus anthracis and E_. col i. According
to Brown, et al. (1975), p-DCB is not toxic to Ustilago maydis.
-------
V-3
Boyles (1980) investigated the effect of ortho- and para-
DCB upon the selective permeability of the cell membranes of
Candida tropical is. A culture of organisms at a concentration of
1 g/lOOml was suspended in deionized water. One ml of the test
compound was shaken into this suspension and conductance measured.
Changes in conductance were measured across diptype bright platinum
electrodes. These changes reflect leakage of electrolyte salts
from the cells. The author concluded that solid hydrocarbons
diffuse into cells only very slowly since ortho-DCB (which is
liquid) was among the most active compounds tested (62% of internal
electrolytes lost in 6 hours), whereas p-DCB (a solid) had only a
very small effect (7.3% lost in six hours).
Boyles (1980) also studied the effect of a number of com-
pounds upon the growth rate of fast growing bacteria. Among the
compounds he tested was o-DCB. By employing an oxygen electrode
chamber, he measured the respiration rate of the colony, suspended
in a nutrient medium. Vibrio natriegens was diluted to a concentra-
tion of*» 10^ organisms/ml. Addition of increasing concentrations
of o-DCB to the solution caused a dose-dependent decrease in the
rate of growth. At^10 ppm DCB, there was a 15% decrease. At/-30
ppm, the rate decreased to 20% of control and at~45 ppm, the
rate was reduced further to 45% of control. At~60 ppm, growth
was arrested completely.
Phytoplankton
Ukeles (1962) conducted a laboratory study of the tolerance
of five species of marine phytoplankton to concentrations of various
toxicants including o-dichlorobenzene. o^Dichlorobenzene had no
-------
V-4
significant effect on growth of any of the tested species at 8 ppm.
At 13 ppm, none of the organisms grew, but all were viable. Lethal
concentrations were reached at around 80 ppm, and at 130 ppm all
species were dead (See Table V-l).
Ukeles pointed out that high concentrations of toxicants
used for predator control might be "safe" when used in shellfish
hatcheries, though hazardous under natural conditions. This is be-
cause plankton food is grown apart from the hatchery and periodi-
cally added to it. Hence, inhibition of phytoplankton growth would
not occur. In nature, phytoplankton blooms are important as a source
of food. Therefore, any alteration in growth of phytoplankton result-
ing from use of halogenated benzenes for predator control could have
consequences along the food chain.
Dawson, e_t al . (1977) determined the 96-hour LCso values
for o-DCB in marine and freshwater fish. Tests were conducted at
23°C in five-gallon containers with aeration at pH 7.6 - 7.9. The
estimated LC$Q value for the bluegill was 27 mg/1 and for tidewater
silverside, 7.3 mg/1.
EPA (1978b) determined 48-hour LC$Q values for the DCBs on
the water flea (Daphnia magna) in static bioassays. For o-DCB, m-
DCB and p-DCB, the values were 2.44, 28.1 and 11.0 mg/1, respectively
EPA (1978b) determined 96-hour LC$Q values on the marine mysid shrimp
in biostatic assays. For o-DCB, m-DCB and p-DCB, the values were
1.97, 2.85 and 1.99 mg/1, respectively. Ninety-six hour LC50 values
of each DCB on the bluegill were 5.59, 5.02 and 4.28 mg/1 for o-,
m-, and p-DCB, respectively. In the sheepshed minnow, the 96-hour
LC5Q values were 9.66, 7.77 and 7.40 mg/1 for o-, m- and p-DCB,
respectively. '
-------
V-5
Table V-l
Effects of o-DCB on Growth of Marine Phytoplankton
Concentration
(ppa) of
ODCB
1.3
7.6
13
130
Lindane
ppn
concentration
7.5
9
Protococcus
sp.
0.71
0.80
0.00*
0.00
0.75
1.0
Chlorella
S£.
0.82
0.95
0.00*
0.00
0.36
0.33
Dunarl iell a
euchlora
0.71
0.90
0.00*
0.00
0.73
0.60
Phaeodactylum
tricornutum
0.74
0.80
0.00*
0.00
0.00*
0.00*
Mjnochrysis
lutheri
1.00
0.65
0.00*
0.00
0.00
0.00
* no growth, but organisms viable
All numbers represent the ratio of optical density (o.d) of growth in the presence of
toxicants to o.d.
normal growth.
in the basal medium with no added toxicants. Hence 1 = approximately
Source: Ukeles, 1962.
-------
V-6
In a 30-day continuous flow chronic toxicity study, EPA
(1978b) determined the maximum acceptable toxicant concentration
(MATC) for o-DCB toward the fathead minnow to be greater than 4, but
less than 8 mg/1 with a geometric mean of 5.65 mg/1. The "no-effect"
%.-
level was then said to be 4.0 mg/1.
Several chlorinated and fluorinated benzenes are known to
be toxic to a variety of marine organisms including molluscs and
crustacean species. Because of this known toxicity, there have been
suggestions that mixtures of these substances be spread around oyster
beds to safeguard the crop from predators. Ortho-DCB, in unknown
concentration, was placed in the area of an oyster bed (Loosanoff,
et al., 1960). The oyster drills would not cross the barrier of
o-DCB and sand eight feet wide during a 13-month period. The
drills exhibited extreme swelling in the gastropods, immobility and
death, and curling of the tips of their rays. Crabs coming into
contact with the substance lost their equilibrium and went into
convulsions. In a study of the effects of o-DCB upon clam young
(Mercenaris mercenaris), Davis and Hidu (1969) found a TL^ value of
100 ppm in eggs exposed for 48 hours or larvae exposed for 14 days.
Oysters, themselves, are not immune from the toxic effects of
o-DCB. An exposure of 1 ppm was found to be the minimum concentra-
tion which inhibited the growth of young oysters (Crassosterca
Virginia) after 24 hours' exposure (Butler, e_t al^., 1960). No
bioaccumulation was noted; the DCB was excreted by the oyster when
the chemical was removed from the water.
-------
V-7
Insects
Both o- and p-DCB have been used as insecticides; of the
two, p-DCB has received more widespread application. However, only
a few toxicity studies are available for these substances.
The acute effects of insect exposure to chlorinated ben-
zenes are summarized in Table V-2.
Birds
The acute and subacute toxicity of p-DCB was studied by
Hollingsworth, e_t al_. (1956). Pekin ducks were fed a diet of 0.5%
p-DCB for 35 days. Growth was retarded in all animals, and three
of the animals died.
Non-human Mammals
The acute and chronic toxicities of the dichlorobenzenes
closely parallel those observed with chlorobenzene. A search of
the literature uncovered no data on the acute or chronic toxic
effects on m-DCB. However, a number of studies have been published
on the acute and long-term effects of the other two dichlorobenzene
isomers. In general, p-DCB found to be less toxic than the ortho-
isomer.
The dichlorobenzenes produce sedation, analgesia and
anesthesia after acute or parenteral administration. Relatively
high doses are needed to produce acute effects, but chronic effects
may occur at relatively low levels. Acute poisoning is character-
ized by signs of disturbance of the central nervous system (CNS).
There may be hyperexcitability, restlessness, muscle spasms or
tremors* followed by varying degrees of CNS depression. The most
-------
v—o
Table V-2
Acute Effects of Chlorobenzenes in Insects (West and Ware, 1977)
Chemical Organism
o-DCB Cimex lecturis
?o-DCB
p-DCB
p-DCB
p-DCB
Dendroctonus
pseudotsagae
Calandra
granaria
Aphis rumicis
Periplaneta
americana
p-DCB
p-DCB
Carabus memoralis
Calliphora
erthnocephala
Triatoma rubro-
fasciata
Cambarus virilus
Exposure
Fumigant
1 part o-DCB
5 parts diesel oil
Fumigant
Injection
Vapor
Allied.to nerve
sheath
Remarks
Nervous System
effects
100% mortality
Nervous System
effects
Narcosis
Nervous system
effects-increase
in activity followed
by a decrease and
spasmodic contractions
Respiratory and
nervous system
stimulated, then
depressed. Delayed
increase in CC«2
output at 4 hours,
then a decrease
Facilitation, then
depression of trans-
mission
Reference
Cameron, et al.,
1937
Gibson, 1957
Cameron, et al .,
1937
Tatterfield, et:
a±., 1925
Munson and Yaeger
1945
Punt, 1950
Punt, 1950
-------
Table V-2 (Continued)
Chemical
p-DCB
p-DCB
p-DCB
p-DCB
p-DCB
Organism
Larvae of: Tineola
bisselliella
Exposure
Vapor
Attagenus inegatoma
Attagenus piceus 2.3-3.2 nvg/1
Termites; Glypt-
otermeg dilatatus
Kalotermes
Lyctus africanus
Tyrophagus
dimidiatus
solid or
liquid
fumigant
(with TCBs)
25% solution
0.5% solution
v/v
Remarks
100% mortality
Reference
Batth, 1971
Low concentration Arnold, 1957
repelled larvae; high
concentration killed
100% after 4 days
Effective in
control
100% mortality
within 8 days
100% mortality
within 4 minutes
Dantharayana
and Fernando,
1970a, 1970b
Awad, 1971
Honma, 1967
-------
V-10
frequent cause of death is respiratory depression. Acute exposure
at very high levels also may result in liver or kidney damage. Cer-
tain of the halogenated benzenes, like some aliphatic hydrocarbons,
are thought to sensitize the myocardium to the effect of catechola-
mines, and thus set up conditions favoring ventricular arrythmias
(von Oettingen, 1955).
Acute Exposure
Several investigators have determined the acute lethal dose
levels after exposure to the dichlorobenzenes in several species.
These data are presented in Table V-3.
Varshavskaya (1968), in her comparative studies on the adverse
effects of the lower chlorinated benzenes, showed that, in the
acute exposures, o-DCB was slightly less toxic in mice and rats
than monochlorobenzene (MCB), and that p-DCB was even less toxic than
o-DCB or MCB. o-DCB was slightly more toxic than MCB in rabbits and
guinea pigs. The 1*059 values for acute oral doses of o-DCB were:
2,000 mg/1 for the mouse, 2,138 mg/1 for the rat, 1,875 mg/1 for the
rabbit and 3,375 mg/1 for the guinea pig. The LD50S for p-DCB were
found to be 3,220 mg/1 for the mouse, 2,512 mg/1 for the rat, 2,812
mg/1 for the rabbit and 7,593 mg/1 for the guinea pig.
Doses of 0.25-0.5 cc/kg (0.33-0.66 mg/kg) body weight of o-DCB
intravenously administered to rabbits were fatal within 24 hours;
doses of 1.0 cc/kg (1.31 mg/kg) were fatal within 20 seconds
(Cameron, et al., 1937).
Dogs exposed to 2 cc/m3 (0.04% or•*>400 g/m3) o-DCB via
inhalation showed no adverse effects, whereas 0.08% (-^800 g/m3)
produced somnolence (Riedel, 1941). Histological studies
-------
Table V-3
Acute Toxicity Data for o- and p-Dichlorobenzene
Animal
Route
o-Dichl
Rat
Rat
Mouse
Rabbit
Guinea
Guinea
Rat
Guinea
Guinea
p-Dichl
Rat
Rat
Rat
Mouse
Rabbit
Guinea
Guinea
Mouse
orobenzene
Pig
Pig
Pig
Pig
Oral
Oral
Oral
Oral
Oral
Oral
Inhal .
Inhal .
Inhal .
orobenzene
Pig
Pig
Oral
Oral
Oral
Oral
Oral
Oral
Oral
SC
LD50
500 mg/kg
2138 mg/1
2000 rag/1
1875 mg/1
3375 mg/1
2000 mg/kg
LCLp
821 ppm/7 hr
800 ppm/7 hr
800 ppm/24 hr
500 mg/kg
2500 mg/kg
2138 mg/1
3220 mg/1
2812 mg/1
7593 rng/l
2800 mg/kg (LDLO)
5145 mg/kg
Reference
NIOSH, 1978
Varshavskaya, 1968
Varshavskaya, 1968
Varshavskaya, 1968
Varshavskaya, 1968
Hollingsworth, et al.
1958
Hollingsworth, 1958
Hollingsworth, 1958
Cameron, et al., 1937
NIOSH, 1978
Holl ingsworth, et_ al^., 1956
Varshavskaya, 1968
Varshavskaya, 1968
Varshavskaya, 1968
Varshavskaya, 1968
Holl ingsworth, j5t al^., 1956
Irie, et al., 1973
-------
V-12
following the administration of acute and subacute doses of o-DCB
showed damage to the liver and kidneys. Exposing mice to similar
concentrations caused CNS stimulation for about 20 minutes followed
by CNS depression, muscular twitching, slow and irregular respiration,
cyanosis near the end of an hour, and death within 24 hours. Rats
appeared to be slightly more resistant than mice to the toxic
effects of o-DCB.
In the mouse, the LD5Q value for a subcutaneous dose of p-DCB
was found to be 5,145 mg/kg (Irie, et al., 1973).
Inhalation of o-DCB by rats at 800 ppm (4,800 mg/m3) concentra-
tion for 11-50 hours was irritating to the eyes and nose, produced
slight changes in the tubular epithelium of the kidney and resulted
in confluent necrosis of the liver (Cameron, e_t_ al_., 1937).
Rabbits, rats and guinea pigs exposed for 20-30 minutes daily
to 100+ g p-DCB/m3 of air for 5-9 days showed marked irritation of
the eyes and nose, muscle twitching, tremors, CNS depression,
nystagmus and rapid but labored breathing (Zupko and Edwards, 1949).
The animals recovered within 30-180 minutes after being removed
from the p-DCB-rich atmosphere. A definite granulocytopenia was
observed in 11 rabbits, a questionable change in three others and
no change in the remaining three. Body weight decreased in 14
animals; three rabbits showed an increase. In 13 rats, CNS de-
pression was observed to be greater than in rabbits. There was
complete narcosis with attendant tremors and muscle twitching
with each exposure. A definite granulocytopenia was observed in
eight rats, a questionable change in three, and no change in two
others. Nine rats showed a decrease in total white cell count
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V-13
while four showed an increase. In addition to exhibiting similar
symptoms to p-DCB inhalation as rats and rabbits, guinea pigs also
exhibited granulocytopenia in most cases. Five animals showed a
frank decrease in granulocytes, two showed a tendency toward lowering,
while two others showed no decrease. Six of the nine animals
suffered a weight loss while three did not.
The observation that many of the test animals developed granulo-
cytopenia is an important one. This condition is considered to be
a precursor to leukemia. However, in these experiments, when the
animals were removed from exposure to p-DCB, the decrease in granulo-
cytes was reversed and the level returned to normal within three to
four weeks.
Several animal studies document the effects of o- and p-DCB on
the eye and/or skin. The results are summarized in Table V-4.
Yang, et: _al_. (1979) showed that both o- and p-DCB alter
pancreatic function. They administered a single intraperitoneal
dose of each substance in sesame oil (5 mmol/kg: 735 mg/kg) to
fasted adult male Holtzman rats. Control animals received an equi-
valent volume of sesame oil (1 ml/kg). Experimental measurements
were made 24 hours later. After treatment with o-DCB, bile duct-
pancreatic flow (BDPF) was increased nearly ten-fold over that
observed in control animals (P<0.05). p-DCB did not alter the flow
significantly. Protein concentration of the bile effluent was re-
duced to about 25% after o-DCB (P<0.05), but was not changed after
p-DCB. However, p-DCB did increase significantly the chloride
content of the effluent, whereas o-DCB did not. Neither compound
affected the rate of bile flow or serum levels of SGPT. The authors
-------
Table V-4
Effects of Chlorinated Benzenes on the Eye and Skin
(modified from West and Ware, 1977)
Chemical
Animal
Exposure
Effects
Reference
o-Dichloro-
benzene
p-Dichloro-
benzene
p-Dichloro-
benzene
p-Dichloro-
benzene
Rabbits
Rabbits
Rabbits
Rabbits
2 drops in each eye
4.6 to 4.8 mg/1
(770-800 ppm) for
8 hours, inhalation
Inhalation. 5 gm
vaporized/2 days
5-47 days
Oral, 5 gms p-DCB
daily for 3 weeks
Pain slight. Conjunctival
irritation. Cleared in 7
days
Lateral nystagmus, transitory
edema of cornea. - edema of
optic nerve. Eye changes
reversible (17 days). No lens
changes or deposits in the
vitreous.
No opacity of the lens (liver
necrosis and death)
Opacity of lens slight and
mod
Hollingsworth,
et al . , 1958
Pike, 1944
Berliner, 1939
Berliner, 1939
-------
V-15
could not offer an explanation of the mechanism by which the observed
changes occurred, although they did conclude that the mechanism did
not involve secretin or cholinergic stimulation of the pancreas.
Thus, the significance of these findings remains unknown.
Effects on Porphyrin Metabolism
All of the chlorinated benzenes have been shown to produce
disturbances in prophyrin metabolism. The mechanism for this bio-
chemical lesion seems similar to that seen following administration
of other inducers of drug metabolism. It is also similar to that
seen in man following exposure to ethanol, synthetic estrogens and
progestins (Parke, 1972).
Rimington and Ziegler (1963) showed that in male albino rats
administration by gavage of 500 to 1,200 mg/kg/day for 5-15 days
of chlorinated benzenes (except pentachlorobenzene) leads to a hepatic
porphyria characterized by elevated levels of precursor porphyrins in
liver and feces. The investigators administered o-DCB in liquid par-
affin at maximum dose levels of 455 mg/kg/day for 15 days, and p-DCB,
also in liquid paraffin, at maximum dose levels of 770 mg/kg/day for
5 days. Doses were gradually increased until a level was reached
which yielded high porphyrin excretion, but few fatalities. The
first signs of intoxication were an increase in urinary copropor-
phyrin and porphobilinogen (PEG). An increase in aminolevulinic
acid excretion was a late effect. The most potent compound was
p-dichlorobenzene (see Table V-5). Mean peak values of urinary
coproporphyrin increased 10-15 fold after p-DCB to about 62 ug/day
when compared with control. o-DCB treatment elicited a 6-10 fold
increase in the same parameter (to about 43 ug/day). After p-DCB,
-------
V-16
Table V-5
Mean Peak Values of Urinary Porphyrin Precursors Following Treatment
With Maximum Doses Tested for each Chlorinated Benzene
(Rimington and Ziegler, 1963)
No.
of-
rats Compound
Controls*
3 Monochlorobenzene
3 1 , 2-Dichlorobenzene
3 1 , 4-Dichl orobenzene
3 1,2, 3-Trichl orobenzene
3 1,2,4-Trichlorobenzene
3 1 ,2,3, 4-Tetrachl oro-
benzene
6 1,2,4,5-Tetrachloro-
benzene
Sol-
vent
C
P
P
P
C
C
P
C
Max.
dose
(mgAg)
1140
455
770
785
730
660
905
Days
on
max.
dose
5
15
5
7
15
10
5
Copro-
porphyrin
(pg/day)
4.3-6.8
30.50
43.10
61.80
36.59
58.31
28.96
4.10
Uropor-
phyrin
(pg/day)
0.1-0.3
1.40
2.01
10.99
0.72
2.73
3.80
0.22
PEG
(pg/day)
2.5-6.5
26.70
26.65
1328.10
57.38
179.00
520.63
6.50
ALA
(pg/day)
38.7-51.6
56.40
11.32
437.41
36.26
145.70
315.78
18.08
ALA/PBG
'
2.98
2.11
0.36
1.01
1.06
0.61
6.69
P = liquid paraffin? C= 1% cellofas
PBG = porphobilinogen; ALA = /_ -aminolevulinic acid.
* Mean max. and min. of 5 rats during 41 days.
-------
V-17
a nearly 100-fold increase in urinary uroporphyrin levels occurred,
while o-DCB caused an increase of about 10-fold. Porphobilinogen
levels measured 1,328 ug/day after p-DCB, but only 26.65 ug/day
after o-DCB, increasing from a control level of 2.5-6.5 ug/day.
After p-DCB, a 10-fold increase (to 437 ug/day) in -ALA levels
were observed, while after o-DCB, levels actually decreased to
11.3 ug/day (normal range = 38.7-51.6 ug/day.)
Several parameters of liver function also were measured by
Rimiagton and Ziegler (Table V-6). In contrast to the change
observed in urinary coprophyrin excretion, liver coproporphyrin was
unchanged after p-DCB, and roughly doubled after o-DCB (to 10.05
ug/lOOg tissue vs. 4.5 ug/lOOg in the controls). Protoporphyrin
levels increased six-fold after p-DCB (60.5 ug/lOOg), but only
3.5-fold after o-DCB (to 34.8 ug/lOOg from a control level of 9.7
ug/lOOg). Catalase levels were unaffected by p-DCB, but more than
halved after o-DCB (0.85 meq/mg wet weight for control to 0.364
meq/mg wet weight after p-DCB exposure). These decreases in catalase
were observed only in animals in which marked histological changes
indicative of severe liver damage with large areas of necrosis were
observed. Thus, changes occurred in o-DCB-treated animals, but not
in those treated with p-DCB. In addition, p-DCB did not alter the
glutathione content of the liver. o-DCB was not tested. From
these data, the authors suggested that the mechanisms which produce
porphyria derangements may be different from those which lead to
liver necrosis.
Carlson (1977) studied chlorinated benzene induction of
liver porphyria in groups of five female rats receiving p-DCB,
-------
V-18
Table V-6
RDrphyrins, Etorphobllinogen and Catalase Activity
In Livers of Rats Treated With Chlorinated Benzenes
(Rimington and Ziegler, 1963)
No.
of,
rats Compound
2
2
2
2
2
5
3
2
Controls
Monochl orobenzene
1 , 2-Dichl orobenzene
1 , 4-Dichl orobenzene
1 , 2 f 3-Trichlorobenzene
1,2, 4-Trichl orobenzene
1,2,3, 4-Tetrachloro-
benzene
1,2,4, 5-Tetrachl oro-
benzene
Max.
dose
(mgAg)
1400
450
770
780
500
660
850
Days
on
max.
dose
5
15
5
7
10
10
5
Copro-
porphyrin
(ug/100 day)
4.5
Trace
10.05
5.07
2.65
45.56
35.04
6.30
Protopor-
phyrin
(ug/100 g)
9.7
13.00
34.80
60.35
3.55
55.60
56.57
9.90
Uropor- PBG
phyrin
(ug/100 g
uncorrected)
1.3
6.35
14.40
60.35
20.00
52.70 ++
41.32 -f
2.15
Catal ase
(meq/mg)
wet wt.
0.85
0.502
0.364
0.880
0.857
0.747
0.772
0.838
-------
1,2,4-trichlorobenzene or hexachlorobenzene. Each substance was
suspended in corn oil and administered orally at Of 50, 100 or 200
ing/kg/day for 30, 60, 90 or 120 days. After the last dose, the
animals were placed in metabolism cages for collection of 24-hour
urine samples. At the end of the 24-hour period, the animals were
sacrificed and liver and urinary porphyrins measured. The results
after exposure to p-DCB can be seen in Table V-7. The data show that
this substance has little potential for causing porphyria, thus con-
firming the observations of Rimington and Ziegler (1963). After 30
and 60 days, liver weight increased in a dose-dependent manner.
Even after 120 days, only slight increases in liver porphyrins
occurred. Urinary excretion of -ALA, porphobilinogen (PEG) or
porphyrins was not increased over control levels.
Subacute to Longer-term Exposures
A number of reports have appeared in the literature that
describe subacute to longer-term exposures of experimental
animals to the ortho- and para- isomers of dichlorobenzene
(see Table V-8 for a summary of these data).
Ortho-Dichlorobenzene
Hollingsworth, et_ al_. (1958) gave rats a series of 138
doses of o-DCB over a period of 192 days (18.8, 188 or 376 mg/kg/
day, five days a week) by intubation. No adverse effects were
detected at the lowest dose. With the intermediate dose, a slight
increase in the weight of the liver and kidneys was noted. At the
highest dose, there was a moderate increase in the weight of the
liver, a slight decrease in the weight of the spleen and cloudy
swelling of the liver.
-------
V-20
Table V-8
Effect of 1,4-Dichlorobenzene p.o. on Porphyrin Production
and Excretion in Female Rats.
(Carlson, 1977)
Dose Liver wt(g) Liver porphyrins Urine porphyrins
(mg/kg) (ng/g) (ug/24h)
30 days of administration
0 6.8 + 0.3a 246 +_ 21a 1.5 + 0.2a
50 6.6 + 0.4a 269 + 20a 1.9 + 0.3a
100. 7.0 + 0.23 251 i 22a 1.4 + 0.2a
200 8.0 + 0.3b 276 + 18a 1.4 + 0.2a
60 days of administration
0 6.7 + 0.3a 381 + 20a 1.9 + 0.4a
50 7.2 + 0.4a 448 + 17a'b 2.0 +_ 0.4a
100 7.6 + 0.3a'b 435 ± 19a'b 1.6 ^ 0.2a
200 8.4 + 0.3b 472 _+ 30b 1.7 + 0.3a
90 days of administration
0 6.8 + 0.6a 541 + 33a 0.9 +_ 0.2a
50 7.0 +_ 0.6a 527 + 30b 1.0 + 0.2a
100 6.6 ^ 0.4a 555 +_ 20a 1.3 + 0.3a
200 7.2 i 0.3a 548 ^ 36a 0.8 +_ 0.2a
120 days of administration
0 6.9 + 0.2a 354 + 10a 1.4 ^ 0.2a
50 8.1 ^ 0.3b 391 +_ 18b 1.8 +_ 0.4b
100 7.3 + 0.6a'b 411 _+ 9b'c 1.6 ± 0.4a
200 7.5 + O.I-3'13 440 -f 8^ 1.0 + 0.2a
a~c Values with same superscript are not significantly different
(P>0.05).
-------
Table V-8
Summary of Subacute to Longer-term Toxicity Data
deal Species Dose
Duration Route Effects Observed Remarks
CNS Blood Liver Kidney Lung Bone
Marrow
Reference
B
Rat
18.8,188
or 376
mgAg/day
Rat
0.001,0.01
or 0.1 mg/
kg day
Rat(20) 49 ppm
Guinea
pig (8)
Mouse (108)
Rat(20)
Guinea
pig (8) 93 ppn
Rabbit (9)
Monkey (28)
5 days/week,
138 doses
192 days
Oral
9 mos.
Oral
7 hrs/day;
5 days/
week;
6-7 months
7 hrs/day;
5 days/week;
6-7 months
Inhala-
tion
Inhala
tion
No adverse effects
at 18.8 mgAg*
Slight increase in
kidney and liver
weight at 188 mgAg.
Moderate increase in
liver weight and
cloudy swelling,
decrease in spleen
weight at 376 mgAg.
No adverse effects
at 0.001 mgAg
Dose-dependant
changes in
conditioned reflexes,
depression of
hematopoietic system
at 0.01 and 0.1 mgAg.
No adverse effects
observed
Holl i ngsworth ,
et aU (1958)
Varshavskaya
(1968)
Hoi1i ngsworth,
et al. (1958)
No adverse effects
observed
)= Nuraber of animals tested
-------
Tabel V-8 (Continued)
Sunnmary of Subacute to Longer-term Toxicity Data
arnical Species Dose
Duration Foute Effects Observed
CNS Blood Liver Kidney Lung Bone
Marrow
Remarks
Reference
OCB Guinea pig 125 or 250 10-11 days Intra-
rog/day as muscular
? 50% soln (I.M)
or 125 rogAg
in olive oil.
Guinea pig 125 mgAg 20 days I.M. 4-
Guinea pig 125 (m)g/ 20 days I.M.
(kg)
Guinea pig 125 (m) g/ 21 days I.M.
(kg)
Rat
10, 100 or 5 days/week; Oral
500 mgAg 20 doses
as 10%
soln.
Intense steatosis
of liver; weight
loss; decreased
hepatic glycogen
Weight loss;
serum transaminase
increased
11.4% weight loss;
serum transaminase
increased
Frada and Cali,
(1958)
Totaro, (1961)
Totaro and
Licari (1964)
Increase in reaction Coppola, et_ al.
and clotting (1963)
formation time
No adverse effects
observed at 10 or
100 mgAg;
cloudy swelling and
centralobular
necrosis of liver;
cloudy swelling of
renal tubular
epithelium
Hoi1ingsworth,
et al. (1956)
-------
Table V-8 (Continued)
Summary of Subacute to Longer-term Toxicity Data
Chemical Species Dose
Duration Route Effects Observed Remarks
CNS Blood Liver Kidney Lung Bone
Marrow
Reference
Pekin duck 0.5% in 35 days
Diet
Oral
Rabbit
Rat
500 rog/kg 5 days/week; Oral
day 263 doses
1000 mgAg 5 days/week; Oral
day 92 doses
over 219 days
18.8,188 5 days/week; Oral
or 376 138 doses
mgAg/day in 192 days
Growth retardation; Hollingsworth,
no cataract^; 3 et^ ai_. (1956)
deaths after 4 weeks
on diet
Swelling and "
focal necrogis
in liver
Weight lossj "
tremors and weak-
ness; necroqis and
cirrhosis in liver
No adverse Affects "
observed at
18.8
Increased l|ver and
kidney weights at
188 and 376 mgAg.
Decreased spleen
weight; liv^r
necrosis and cirrhosis
at 376 mgAg.
-------
V-24
Table V-8 (Continued)
Summary of Subacute to Longer-term Toxicity Data
Chemical Species Dose Duration Route Effects Observed Remarks Reference
CNS Blood Liver Kidney Lung Bone
Marrow
> Rat 100 mgA 20 min/day; Inhala- 4- 4- + + +
(9M,9F) ( 16,000 5-9 days tion
ppm)
Guinea pig
(9M) +4-4-4-4-
Rabbit 30 min/day 4- 4- 4- 4-4-
(18M)
Rat 96 ppm, 7 hrs/day; Inhala-
(10M) 5 days/week; tion
Granulocytopenia in
8A8; tendency
toward same in 3A8
Granulocytopenia in
5/9; tendency toward
same 2/9; weight loss
in 6/9
Granulocytopenia in 11
Irritation of mucosa,
weight loss in 14A8;
Tremors rapid but
labored breathing,
death 12A8.
No adverse effects
observed
Zupko and
Edwards (1949)
M
A8
Holl ingsworth
et al.(1956)
16 days
(10)
(5)
158 ppm
173 ppm
Cloudy swelling or
granular degeneration
of liver
Slight interstitial
edema and congestion
in lung; slight
increase in liver and
kidney weights.
-------
Table V-8 (Continued)
Summary of Subacute to Longer-term Toxicity Data
mical Species Dose
» (19M) 798 ppm
(15F)
(20M) 341 ppm
Guinea pig 173 ppm
(5)
(16M) 798 ppm
( 7F)
(8) 96 ppm
(8) 158 ppm
Duration Route
CNS
8 hrs/day; +
5 days/week;
1-46 doses (M)
9-69 doses (F)
7 hrs/day;
5 days/week,
6 months
7 hrs/day; +
5 days/week;
16 days
8 hrs/day; +
5 days/week
1-23 doses (M)
11-20 doses (F)
7 hours/day;
5 days/week
157-219 days
n
Effects Observed Remarks Reference
Blood Liver Kidney Lung Bone
Marrow
+ + Tremors; weakness? cloudy "
(F) swelling of liver (M&F)
and kidney (F), Deaths:
2M, 2F
+ + Slight increase in liver "
and kidney weights
+ 4- 4- Slight decrease in Hollingsworth
(F) (M) spleen weight, slight etaU(1956)
edema and congestion
in lungs
4- Deaths: 2M "
No adverse effects "
observed
+ Increased liver "
(F) weight (F);
Body weight loss
-------
Table V-8 (Continued)
Sunmary of Subacute to Longer-term Toxicity Data
Chemical Species
(8M)
(8F)
T
Rabbit
(1M)
(IF)
(8M)
(8F)
(1M)
(IF)
Mouse
(10)
Monkey
(18)
Dose Duration toute
CNS
314 ppm 7 hours/day; +
5 days/week;
6 months
173 ppm 7 hours /day;
5 days/week;
16 days
798 ppm 8 hours/day; +
5 days/week;
1-62 exposures
158 ppm 7 hours/day;
5 days/week;
157-219 days
96 ppm 7 hours/day;
5 days/week;
157-219 days
158 ppm
158 ppm
Effects Observed Remarks
Blood Liver Kidney Lung Bone
Marrow
+ Cloudy swelling,
fatty degeneration,
focal necrosig,
slight cirrhosis of
liver
+ Slight edema and
congestion
of lungs
+ -f Reversible non-
specific eye
changes
No adverse effects
observed
No adverse effects
observed
No adverse effects
observed
No adverse effects
observed
Reference
ii
»
Hollingsworth
et aK (1956)
n
H
n
ii
-------
V-27
Hollingsworth, et al. (1958) also measured the effects of
multiple inhalation exposures of o-DCB on rats, guinea pigs,
mice, rabbits and monkeys. A range of concentrations was used,
seven hours a day, five days a week, for six to seven months. No
adverse effects were observed lit rats-, guii-fesr pig» or ia*ce> exposed:
to 49 ppm, or in rats, guinea pigs, rabbits and monkeys exposed to
93 ppm (0.56 mg/1).
Oral administration of o-DCB to white rats for nine months
was conducted at doses of 0.001, 0.01 or 0.1 mg/kg/day (Varshavskaya,
1968). Effects were observed at the two higher doses similar to those
described for monochlorobenzene. The author reported an inhibition
of mitosis in the bone marrow, as well as neutropenia and abnormal
conditioned reflexes. These changes in the blood profile may be
important in that they could be precursors to pancytopenia or
leukemia. In this report, however, Varshavskaya concluded that
no carcinogenic activity was observable at the doses studied. She
measured tissue DPN, TPN, glucose-6-phosphate and alkaline phospha-
tase since it has been claimed that an increase in the activity of
these enzymes is indicative of carcinogenicity (Burstone, 1965).
At the 0.1 and 0.01 mg/kg doses, o-DCB caused an increase in acid
phosphatase but a decrease in alkaline phosphatase.
The 0.1 mg/kg dose of o-DCB caused a marked increase in the
amount of 17-ketosteroids found in the urine. This increase is
most likely due to hyperplasia of the adrenal cortex, since an
increase in the weight of the adrenals and decrease in the ascorbic
acid concentration of the adrenals also were observed. The 0.001
mg/kg dose had no observable effects on any of the parameters studied.
-------
V-28
Para-Dichlorobenzene
Intramuscular injection of 125 or 250 mg p-DCB/kg into guinea
pigs over a 10 or 20 day period resulted in effects typical of
p-DCB toxicity (Frada and Cali, 1958; Totaro, 1961; Totaro and
Licari, 1964; Coppola, et al^. , 1963). The observed changes included
weight loss, liver changes and blood clotting time increases.
Oral doses of 10, 100 or 500 mg p-DCB/kg, five days a week, for
20 doses produced marked cloudy swelling and necrosis in the central
area of the liver lobules only with the highest dose (Hollings-
worth, et al_.,1956). No effects were observed at the other doses.
Pekin ducks, fed 0.5% p-DCB in their diets for 35 days exhibited
depression of body weight gain. Three animals died after the fourth
week.
Oral doses of 188 or 376 mg p-DCB/kg, five days a week,
for 192 days (138 doses) in rats induced an increase in the weights
of the liver and kidneys (Holl ingsworth, et_ al_. , 1956). At 376
mg/kg, increased splenic weight, slight cirrhosis and focal necrosis
of the liver also were observed. There was also cloudy swelling of
the renal tubular epithelium. No adverse effects were seen with the
18.8 mg/kg dose. Rabbits receiving 500 mg/kg/day oral doses, 5
days a week, for a total of 263 doses showed swelling and focal
necrosis of the liver. Other rabbits receiving a total of 92 oral
doses at 1000 mg/kg/day over a 219-day period exhibited necrosis
and cirrhosis of the liver. CNS effects were evident as well, as
both tremors and weakness were noted. Loss of body weight also
occurred.
The toxicity of p-DCB also has been studied via the
inhalation route of exposure. Zupko and Edwards (1949) exposed
-------
rats, guinea pigs and rabbits to a high level of compound (100 mg/1
or 16,000 ppm) 20-30 minutes/day for 5-9 days. In all species,
adverse effects were observed on- the CNS, liver, kidney and lung.
Among the rats (9 males and 9 females), eight animals exhibited
granulocytopenia, with three others showing a tendency toward the
same. In the guinea pigs (9 males), granulocytopenia was observed
in five, with a tendency towards this condition in two others. In
addition, six of the nine showed a weight loss. Eleven of the 18
male rabbits exposed developed granulocytopenia. Weight loss and
mucosal irritation occurred in 14 of the 18. CNS effects manifested
as tremors and rapid and labored breathing also occurred. Twelve of
the 18 animals died.
Holl ingsworth, et al_. (1956) conducted a series of inhalation
studies with p-DCB, at various levels and durations of exposure,
in rats, guinea pigs, monkeys, rabbits and mice. The concentrations
used were 96, 158, 173, 314 and 798 ppm (0.58, 0-95, 1.04, 2.05 and
4.8 mg/1, respectively). Rats exposed to 96 ppm seven hours a day,
five days a week, for 16 days showed no abnormalities. A 157-219
day exposure at this level in guinea pigs and mice yielded no
adverse reactons. With the same protocol, inhalation levels of 158
and 173 ppm caused cloudy swelling or granular degeneration of the
liver, slight increase in the weight of the liver and kidneys and
some interstital edema and congestion in the lungs of rats and
guinea pigs. Monkeys and mice showed no adverse effects at these
levels. Rats exposed to 341 ppm for six months showed evidence of
a slight increase in the weight of the liver and kidneys. Guinea
pigs exposed to 173 ppm for 16 days showed a slight decrease in
-------
V-JU
splenic weight and some lung edema and congestion. At 314 ppm, for
six months, the liver became slightly cirrhotic with focal necrosis,
cloudy swelling and fatty degeneration. Rabbits, exposed to an
atmosphere of 158 ppm for 157-219 days, were unaffected. At 173
ppm for 16 days, slight edema and lung congestion were observed,
and at 798 ppm, for 1-62 exposures, reversible non-specific eye
changes were apparent.
Effects Upon Drug-Metabolizing Enzymes
Most mammals possess a group of enzymes that specializes
in the biotransformation of foreign compounds. These enzymes are
located in the endoplasmic reticulum of the liver cells. The
metabolic transformation of foreign compounds usually leads to the
conversion of lipophilic materials into more polar compounds, which
are eliminated more readily from the hepatocyte and excreted from
the body. Thus, compounds which are of little nutritive value
are prevented from accumulating in cells and tissues. In this way,
undesired effects may be avoided (see reviews in Williams, 1959;
Parke, 1968).
The endoplasraic reticulum (ER) of the cell is the location
of many enzymes such as glucose-6-phosphatase, glucuronyl trans-
ferase, the hydroxylases and protein synthetases (Parke, 1972).
The enzymes concerned with the metabolism of drugs and other xeno-
biotics are referred to frequently as mixed function oxidases or
monooxygenases. All of the monooxygenase drug metabolizing enzymes
require reduced nicotinamide adenine dinucleotide phosphate (NADPH2),
molecular oxygen and a cytochrome, usually P-450. The ER is
associated not only with the oxidation of drugs, but also the
-------
V-31
biosynthesis of cholesterol, the catabolism of bile acids, the
oxidation of fatty acids and the oxidation of prostaglandins. In
addition to mixed function oxygenase activities, the hepatic endo-
plasmic reticulum contains a number of reductases, some of which
utilize cytochrome P-450 and NADPH2 (Williams, 1959; Parke, 1968). *.
The biotransformation of drugs and xenobiotics appears to
occur in two distinct phases. The first phase includes reactions
classified as oxidations, reductions and hydrolyses. In the second
phase, the reactions are referred to as syntheses or conjugations.
These enzyme reactions, expecially oxidations which require cyto-
chrome P-450, have been examined throughout the animal kingdom. Con-
trary to the suggestion of an evolutionary trend for the appearance
of cytochrome P-450 in mammals, this cytochrome is apparently ubiqui-
tous (Ahokas, et al_., 1976).
However, quantitative differences in metabolism exist,
for example, among livers from various species, among various
tissues from a single animal, and between the neonate and adult within
a species. It also has been well documented that enzymes of
biotransformation may be regulated (stimulated or depressed) by
xenobiotics or steroids (Parke, 1972). While there does appear to
be a ubiquitous distribution of cytochrome P-450, the concentration
in different animal species varies greatly, as can be seen in Table
V-9. Since these species have differing amounts of cytochrome^P-
450, they must have different abilities to manufacture toxic
intermediates. They also have varying abilities to metabolize
benzpyrene and halogenated benzenes.
-------
V-33
A number of investigators have suggested that a relationship
exists between the induction of -aminolevulinic acid synthetase
and the induction of drug or xenobiotic metabolizing systems by
compounds which are known to induce liver porphyria (Rimington and
lisglery l*6*>r Psfcsnd*, et ^_., 1971r Ariyoshi, et a^., 1975). The
effects of a series of chlorinated benzenes on hepatic -ALA
synthetase and cytochrome P-450 levels are summarized in Table V-10.
Poland et al. (1971) treated young female Sherman rats by
gavage with daily doses of m-DCB in peanut oil. Continuous daily
dosing with 800 mg/kg/day for nine days resulted in a biphasic
pattern of urinary coproporphyrin excretion (Figure V-6). ALA
synthetase was measured in animals dosed daily with 800 mg/kg/day
for 1, 3 or 5 days. Enzyme activity measured 24 hours after the
last dose showed a steep rise after Day 1, with lesser increases
seen after Days 3 and 5 (Table V-ll). Increases in liver size were
not sufficient to account for the changes observed. No histological
evidence of liver damage was noted in these animals.
The cyclic nature of the response suggests that an adaption
or tolerance to the compound is developing, perhaps because the
animal may be detoxifying the substance more rapidly. This possi-
bility was tested by observing the effects of the same dosing
regimen as described above on hexobarbital (150 mg/kg i.p) sleeping
time. Sleeping times we're significantly shortened after one dose of
m-DCB (from 180 minutes to less than 120 minutes), and after three
days, were only 20% of control times (180 minutes) vs. 30 minutes
(P<0.001). Acceleration of the rate of metabolism of bishydroxy-
coumarin (BHC) also occurred after treatment with m-DCB. After
-------
V-34
Table V-10
Effects of Chlorinated Benzenes on Aminol evul inic Acid
Synthetase and Cytochrome P-450 Content of Rat Liver
(Rimington and Ziegler, 1963; Poland etaU, 1971; Ariyoshi et aU, 1975).
(Modified from Ware and West, 1977)
Extent of
Conpound metabol ism
Control*
Monochlorobenzene
1 , 2-Dichlorobenzene
1 , 3-Dichl orobenzene
1 , 4-Dichlorobenzene
1,2, 3-Trichl orobenzene
1,2, 4-Trichl orobenzene
1,3, 5-Trichlorobenzene
1,2,3, 4-Tet rachl orobenzene
1,2,3, 5-Tetrachl orobenzene
1,2,4, 5-Tetrachl orobenz ene
Pentachlorobenzene
Hexachl orobenzene
High
High
High
High
High
High
Low
High
Low
Low
Very low
Very low
P-450
n moles/g
0.68
0.56
0.66
0.77
0.73
0.84
1.63
0.73
0.97
1.14
0.99
1.16
0.97
ALA
Synthetase
n moles/g/hr
22.6
49.2
36. 3+
29.8+
32.3
33.6
47.5+
34.8
33.3+
35. 4+
27.5
98. 7+
51.8
Prophyria1"
yes (1140)
yes ( 455)
yes ( 770)
yes ( 785)
yes ( 730)
—
yes ( 660)
—
-
-
yes ( 400)
* Rats were pretreated orally with 250 mgAg body
24 hours after the last dose.
+ Significantly increased over control.
for three days and were sacrificec
Numbers in parentheses indicate dose in mgAg body wt. used to produce porphyria
in 5 days. (Note: glutathione reduces porphyria produced by halogenated benzenes.)
-------
V-35
tor*
Fig. V-6. Effect of m-DCB on urinary coproporphyrin
excretion. Four.female rats (90-120 g) were
treated daily with 800 mg/kg m-DCB. The
24-hr urinary excretion of coproporhyrin
(mean +_ S.D.) is plotted versus time. The
first dose was given on day 0, and the uri-
nary coproporphyrin value for each day repre-
sents a urinary collection for the previous
24 hr. Urinary coproporphyrin excretion was
significantly lower on days 5 and 9 than on
day 3.
Source: Poland, e_t al_. (1971).
-------
V-36
Table V-ll
Effect of m-Dichlorobenzene on ALA
Synthetase Activity*
ALA synthetase (mmoles/g/hr)
Treatment Day 1 Day 3 Day 5
Control 13.3 ± 4.4 (7) 18.8 + 4.6 (6) 16.1 + 4.8 (7)
m-DCB 52.9 + 15.3 (7) 40.5 + 6.8 (7) 30.5 + 6.2 (8)
* Female rats were treated orally for 1,3 or 5 days with m-DCB (800 mgAg)
or peanut oil and sacrificed 24 hr after the last dose. All values
represent the means ± S.D. The number of animals in each group is given
in parentheses.
ALA synthetase activity is lower on day 5 in m-DCB-treated animals than
on day 1 (P<0.01) or day 3 (P<0.02).
Source: Poland, et al. (1971)
-------
V-37
five days of dosing, the serum BHC levels measured 82 +_ 24 ug/ml,
as compared with control levels of 133 +_ 10 ug/ml (P<0.025).
Poland et. al_. (1971) conducted further experiments designed
to test the hypothesis that m-DCB stimulates its own metabolism.
Liver and serum levels of m-DCB and 2,4-dichlorophenol (DCP), its
major metabolite, were determined after dosing daily for up to five
days, as described above. The serum m-DCB concentration was higher
on Day 3 (8.89 +_ 1.61 ug/ml) than on Day 1 (3.25 +_ 1.31 ug/ml), but
was significantly lower on Day 5 (5.91 +_ 2.02 ug/ml) than on Day 3
(P<0.02). The hepatic concentration rose steeply from Day 1 (13.01
+ 6.92 ug/g tissue) to Day 3 (44.17 +_ 6.31 ug/g). However, the dif-
ference between the concentration at Day 3 and 5 (32.1 ^ 16.18 ug/g)
was not quite statistically significant (0.10>P>0.05). In rats pre°
treated for four days with 40 mg/kg phenobarbital i,p., a known
inducer of drug metabolism, before receiving a single 800 mg/kg dose
of m-DCB, slightly lower concentrations of m-DCB in liver (8.49\+
2.37 ug/g) and serum (2.24 +_ 0.49 ug/g) were observed as compared
with those in rats receiving only a single dose of m-DCB. The dif-
ferences were not significantly different, however. The data from
this series of experiments add support to the hypothesis that m-DCB
does, in fact, stimulate its own metabolism.
Ariyoshi et al_- (1975) studied changes in certain liver
constituents, cytochrome contents, activities of some drug-metabo-
lizing enzymes and A-ALA synthetase in rats treated with each of
the three isomers of DCB. Animals received oral doses of 250 mg/kg/
day for up to three days. Activities of aminopyrine demethylase and
aniline hydroxylase were enhanced markedly by m-DCB, but cytochrome
-------
V-38
content was not altered significantly after any of the three
isomers. Delta-ALA synthetase activity was increased significantly
by treatment with o-, m- and p-DCB (63%, 32% and 42%, respectively).
However, significant parallel changes in the cytochrome P-450
content did not occur. All isomers increased microsomal protein
content of liver preparations. Microsomal inorganic P content was
also increased by 36% after treatment with m-DCB.
Carlson and Tardiff (1976) studied the effects of chlorobenzene,
1,4-dichlorobenzene, l-bromo-4-chlorobenzene, 1,2,4-trichlorobenzene
and hexachlorobenzene on adult male rats for 14 days at low doses
from 10 to 40 mg/kg body weight. All halogenated benzenes except
monochlorobenzene decreased hexobarbital sleeping time immediately
and/or 14 days following treatment. As can be seen in Table V-12,
cytochrome-c reductase, cytochrome P-450 content, 0-ethyl O-p-
nitrophenyl phenylphosphonothioate (EPN) detoxication, glucuronyl
transferase, benzpyrene hydroxylase and azoreductase were increased
to varying degrees. Administration of 1,4-di- and 1,2,4-trichloro-
benzene for 90 days resulted in an increase in EPN detoxication,
benzpyrene hydroxylation and azoreductase. The increases were still
apparent 30 days later. 1,2,4-Trichlorobenzene was the most potent
inducer of cytochrome reductase and cytochrome P-450. Either no
change or a decrease was reported for glucose-6-phosphatase and
isocitrate dehydrogenase activities.
These findings demonstrate the ability of halogenated aromatic
compounds at low doses to induce enzyme systems associated with the
metabolism of foreign compounds. This type of action may influence
the metabolism of endogenous steroids, other foreign compounds and
drugs.
-------
Table V-
Effect of Chlorinated Benzenes Administered Orally for 14 Days on Various
Parameters of Xenobiotic Metabolism
(Carlson and Tardiff, 1976)
(Modified from Ware and West, 1977)
Dose
Compound (mgAg/day)
Monochloro-
benzene
1,4-Dichloro-
benzene
l-Bromo-4-
chlorobenzene
1,2,4-Tri-
chlorobenzene
Hexachloro-
benzene
0
200
400
800
0
10
20
40
0
10
20
40
0
10
20
40
0
10
20
40
Cyrochrome c EPN1"
reductase Glucuronyl- detoxication Benzpyre
(nmol Cytochrome transf erase (pg hydroxy
cytocrorne c p-450 ( E/mg (nmol/min/mg p-nitrophenol/ (nmol/m
reduced/min protein x 10) protein) 50 mg/30 min) pro
mg protein)*
100 + 25
110 + 8
109 + 9
78+5
156 + 9
151 + 6
165 4- 8
176 + 10
149 -f 22
126 -f 6
123 + 4
141 + 5
103 4- 8
139 + 15
192 + 12
183 + 9
85+7
106 + 10
109 4- 7
100 + 7
236 + 20
209 + 10
197 4- 14
133 4- 15
178 + 20
174 + 15
167 + 11
193 + 13
205 + 35
180 4- 7
202 + 7
245 + 19
72 + 13
99 4- 18
212 + 45
268 4- 15
128 + 18
204 + 18
190 + 16
150 + 27
5.8 + 0.4
9.4 4- 0.6
12.2 4- 0.3
13.0 +_ 0.5
5.9 4- 0.6
7.8 4- 0.6
11.3 4- 1.0
9.6 +_ 0.6
5.8 4- 1.4.
5.6 + 0.8
9.0 + 0.7
9.1 + 0.5
5.0 + 0.4
5.1 + 0.4
8.6 4- 1.6
8.3 +_ 0.7
6.3 + 1.0
4.5 + 0.5
6.8 + 0.3
6.6 4- 0.8
6.7 + 0.6
7.1 4- 0.5
8.4 + 0.9
7.4 + 0.7
6.7 + 0.6
7.2 4- 0.6
9.5 4- 1.1
9.0 +_ 0.5
9.5 + 0.8
11.4 + 0.9
12.4 + 1.1
14.4 + 0.6
5.8 + 0.3
10.3 + 0.9
12.0 + 0.8
15.4 + 0.4
5.3 + 0.9
15.0 + 1.1
18.4 + 0.8
18.9 + 0.7
1.39 + 0.43
0.89 + 0.05
0.92 + 0.04
0.78 + 0.16
2.36 + 0.49
1.75 + 0.13
3.53 + 0.68
2.15 +; 0.34
1.51 + 0.20
1.37 + 0.17
2.06 + 0.26
2.35 +_ 0.23
2.82 + 0.53
3.82 + 0.65
5.37 + 1.01
5.22 ± 1.14
2.79 + 0.54
4.76 + 0.58
4.08 + 0.37
4.24 + 0.82
Azoreduc-
tase
(ng/min/mg
protein)
63.9 + 2.8
67.5 + 2.5
71.4 + 4.3
79.2 +_ 6.6
59.1 + 2.5
60.9 + 2.6
69.7 + 4.2
79.4 + 2.7
72.4 + 11.1
102.0 + 2.7
91.1 + 4.6
130.7 + 5.3
122.0 + 24.9
287.7 +- 7.4
210.0 + 7.4
262.7 + 19.1
* Value is mean + S.E. for group of six rats except for benzpyrene hydroxylase group receiving 40 mgAg of I,2f4-
trichlorobenzeTie. In that group, there were five rats,
-------
V-39
Multiple Chemical Exposures; Actions of Combinations
Since many compounds, whether they are agricultural and
industrial chemicals or drugs, are handled similarly by the
cytochrome P-450 system, there would be many possibilities
for additive, synergistic as well as antagonistic actions.
Experiments to determine this should be carefully designed.
For example, in manr phenobarbital has been shown to stimulate drug
metabolism. This effect requires several days to reach a maximum
rate (Goodman and Oilman, 1975). If treatment continues, it appears
that the marked stimulation is lost. This has not been taken into
consideration in many animal studies, i.e., only the three to four
day effect has been studied.
From recent studies in man using identical or fraternal
twins, the role of the environment as an explanation for differences
in metabolism is being deemphasized in favor of genetic differences.
It has been postulated that people who have genetic susceptibility
to indueible drug-metabolizing enzymes may be more prone to adverse
effects, if there is a toxic intermediate or product formed ir\ vivo
(Goujon et al_. , 1972; Vesell et al_. , 1976). Goujon et aL_. (1972)
found that hexachlorobenzene differentially inhibits aryl hydrocar-
bon hydroxylase in genetically responsive and non-responsive mice.
Similarly, Vesell et al_- (1976) showed that the toxicity of chloro-
form to the kidney is different in genetically susceptible and non-
susceptible mice.
Goujon et aO^. (1972) and Vesell et al_. (1976) demonstrated
genetically controlled variations in the susceptibility of mice to
hexachlorobenzene and chloroform. Hence, exposure to a wide range of
-------
Multiple Chemical Exposures; Actions of Combinations
Since many compounds, whether they are agricultural and
industrial chemicals or drugs, are handled similarly by the
cytochrome P-450 system, there would be many possibilities
for additive, synergistic as well as antagonistic actions.
Experiments to determine this should be carefully designed.
For example, in man, phenobarbital has been shown to stimulate drug
metabolism. This effect requires several days to reach a maximum
rate (Goodman and Oilman, 1975). If treatment continues, it appears
that the marked stimulation is lost. This has not been taken into
consideration in many animal studies, i.e., only the three to four
day effect has been studied.
From recent studies in man using identical or fraternal
twins, the role of the environment as an explanation for differences
in metabolism is being deemphasized in favor of genetic differences.
It has been postulated that people who have genetic susceptibility
to inducible drug-metabolizing enzymes may be more prone to adverse
effects, if there is a toxic intermediate or product formed in vivo
(Goujon e_t al_. , 1972; Vesell et_ al_. , 1976). Goujon et al_. (1972)
found that hexachlorobenzene differentially inhibits aryl hydrocar-
bon hydroxylase in genetically responsive and non-responsive mice.
Similarly, Vesell et al_. (1976) showed that the toxicity of chloro-
form to the kidney is different in genetically susceptible and non-
susceptible mice.
Goujon €st aO_. (1972) and Vesell et al_. (1976) demonstrated
genetically controlled variations in the susceptibility of mice to
hexachlorobenzene and chloroform. Hence, exposure to a wide range of
-------
environmental contaminants including the halogenated benzenes could
affect the ability of these animals to detoxify xenobiotic substances.
Synergistic effects of the halogenated benzenes in combination or
with other environmental contaminants could be especially damaging
for the genetically susceptible individual.
There was one example of synergistic effects of halogenated
benzenes on a target organism found in the literature (Hinzer et
al., 1970). The antifungal activity of halogenated benzenes was
synergistic with organo-tin compounds. The mechanism of the toxicity
was not discussed. There were no studies found on synergistic effects
in mammals or other nonmammalian species.
Teratogenicity
No teratogenicity studies with the dichlorobenzenes alone
were found in the peer-reviewed literature. Studies to assess the
embryotoxic and teratogenic potential of o-DCB and p-DCB have been
initiated and/or completed under sponsorship of the chemical indus-
try.
Hodge, ^t al_. (1977) conducted a teratogenicity study of p-DCB
in rats. Groups of pregnant rats (32 animals per group) were
exposed to atmospheric concentrations of 0, 75, 200 or 500 ppm
p-DCB 6 hours/day on Days 6-15 of pregnancy. Data were collected
only from the first 20-24 animals in each group proven to be pregnant
at the time of sacrifice (Day 21). Nine animals littered sponta-
eously on Day 21 and thus were not included in the study results
(two at 75 ppmf two at 200 ppm and five at 500 ppm).
-------
V-42
Maternal weight gain was monitored over the 21 days of
>
pregnancy. Exposure to p-DCB did not alter the rate of weight gain
in any group when compared with controls.
On Day 21, the animals were sacrificed. The intact uteri
were examined for numbers of fetuses and resorptions. Corpora lutea
were counted. Upon dissection of the uteri and removal of the fe-
tuses, the resorptions which had occurred were classified as early or
late. Resorptions are designated as late when fetal tissues are
distinguishable. When abnormal fetuses were noted, maternal heart,
liver, lung, uterus, ovary, kidney, and adrenal were preserved for
histological examination. Liver and lung from at least ten animals/
group also were fixed for histology.
Each fetus was examined for viability, sex, weight and
presence of abnormalities. Half of each litter was eviscerated,
examined for abdominal abnormalities and stained with Alizarin Red
for subsequent skeletal examination for abnormalities and degree of
ossification.
Upon gross examination, uteri from three females exposed to 75
ppm contained excessive amounts of blood. In one case, this appeared
to be associated with a dead fetus. One female exposed to 200 ppm
had inflated lungs. Upon histological examination, no lesions were
observed which were attributable to exposure to p-DCB.
Exposure to p-DCB did not induce adverse effects on numbers
of implantations, viable fetuses, resorptions, corpora lutea or on
mean fetal weights, mean litter weights or on implantation efficiency
(number of implantations/number of corpora lutea). Sex ratios (male/
female) were within normal limits. There was no increase in the
-------
V-43
number of runts.
Three gross fetal abnormalities were noted, one in each exper-
imental group. From the 75 ppm group, there was one fetus with
gastroschisis and malrotation of the left hindlimb. In the 200 ppm
group, there was a single fetus with gastroschisis and*malratation
of the right hindlimb, and in the 200 ppm group, there was one fetus
with agnathia and cleft palate. One control fetus was found to be
anemic.
i
Upon examination for skeletal abnormalities, no evidence was
found that maternal exposure to p-DCB resulted in delayed ossifi-
cation of fetal bones or an increased incidence of minor abnormali-
ties. Occasionally, 14 ribs were noted, but, in almost all cases,
these were vestigial.
The results of this study suggest that maternal exposure to
atmospheric levels of p-DCB up to 500 ppm on Days 6-15 of pregnancy
does not result in any embryotoxic, fetotoxic or teratogenic effects
in the offspring.
Dow Chemical is completing studies in which pregnant rats
and rabbits are being exposed to o- and p-DCB via inhalation.
Final reports are not yet available to ODW. However, when they
are, they will be subjected to review and evaluation. Dow Chemical,
however, has submitted a review of the results of the dose range-
finding study to determine the maximum tolerated dose of o-DCB for
the full study, as well as the final protocol for that study (Dow,
1981). In the probe study, groups of 10 pregnant rats and seven
pregnant rabbits were exposed to nominal o-DCB concentrations of
0, 200, 400, or 500 ppm ( 0,^1200,^2400 or"* 3000 mg/m3). The
-------
V-44
animals were exposed six hours/day on Days 6-15 (rats) or Days
6-18 (rabbits) of gestation.
Severe maternal toxicity, as evidenced by significant decreases
in body weight, body weight gain and food consumption, increases in
relative liver and kidney weights and signs of systemic toxicity
at gross necropsy, was observed in pregnant rats exposed to 500 ppm
of o-DCB. Embryolethality, secondary to maternal toxicity, was ob-
served among rats in the 500 ppm exposure group. Increased relative
liver and kidney weights and decreased food consumption were observed
among pregnant rats exposed to 400 ppm of o-DCB. Exposure to 200
ppm of o-DCB did not produce any sign of toxicity among maternal
animals. No statistically significant effects on reproductive
parameters were observed among rats at any exposure level.
Among rabbits, slight maternal toxicity was observed among
pregnant animals exposed to 500 ppm of o-DCB. Non-significant de-
creases in maternal body weight gain, and absolute and relative liver
weights were observed among rabbits exposed to 500 ppm. Gross
observation at necropsy revealed hepatic changes indicative of mild
toxicity among pregnant rabbits exposed to 500 ppm. No adverse ef-
fects were observed among rabbits exposed to 200 or 400 ppm of o-DCB,
and no significant effects on reproductive parameters were observed
among rabbits at any exposure level.
Based on the results of the probe study, as summarized
above, exposure concentrations of 0, 100, 200 or 400 ppm o-DCB were
chosen as the test concentrations for the definitive teratology study
in rats and rabbits.
-------
V — >±O
Recently, Dow Chemical submitted to EPA the results of the
dose range-finding study for p-DCB exposure to pregnant rabbits
(Hayes, e_t al_., 1982). Pregnant females were exposed to 0, 300,
600 or 1,000 ppm (0, 1,800, 3,600 or 6,000 mg/m3). Each was ex-
posed for 6 hrs/day on Days 6-18 of gestation.
No maternal deaths occurred during the study and no signi-
ficant changes in gross appearance or demeanor were observed among p-
DCB-exposed rabbits. Evidence of slight maternal toxicity was ob-
served among pregnant rabbits exposed to 1,000 ppm of DCB. 'In this
group, a decrease in body weight gain and slight decreases in absolute
and relative liver weights were observed. In addition, histopatho-
logic examination of livers revealed decreased hepatocellular vacuoli-
zation suggestive of decreased glycogen deposition in the 1,000 ppm
group.
No significant effects on the incidence of implantations
undergoing resorption were observed in any of the exposed groups
when compared to controls indicating that p-DCB is not embryolethal
at exposure concentrations up to 1,000 ppm.
Based on the. results of this probe study, where evidence of
slight maternal toxicity was observed in the 1,000 ppm group, exposure
levels of 100, 300 and 800 ppm of p-DCB were selected for the defini-
tive teratology study in rabbits.
-------
V-46
Mutagenicity
Effects on Plants
Abnormal mitotic division of the onion, Allium cepa, after
treatment with several halogenated benzenes has been described by
Ostergran and Levan (1943). Ortho-DCB produced full c-mitosis abnor-
malities at 300 x 10~6 mol concentration with partial disturbances of
mitotic division at 100 x 10~6 inol.
Para-dichlorobenzene also induces abnormal mitotic divi-
sion in higher plants (Ostergran and Levan, 1943). Effects seen
include shortening and thickening of chromosomes, precocious separ-
ation of chromatids, tetraploid cells, binucleate cells and chromosome
/
bridges (c-mitosis). Available studies with p-DCB are summarized
in Table V-13.
Effects on Microorganisms
Anderson _et al_. (1972) evaluated 110 herbicides for their
ability to produce point mutations in a number of different microbial
systems. When tested in a culture of histidine-requiring mutants
of Salmonella typhimurium, both o-DCB and trichlorobenzene (isomer
not specified) gave a negative response, i.e., they were not
mutagenic. Pentachlorophenol, a metabolite of pentachlorobenzene,
also was negative in this test system. The metabolites of other
halogenated benzenes were not evaluated. No liver homogenates
containing the metabolic activating enzymes were added in order to
study the effect of conversion of the test compounds to active
intermediates.
Prasad and Pramer (1968) and Prasad (1970) investigated the
mutagenic effects of the three dichlorobenzene isomers. The
»>
chemicals were evaluated for frequency of back mutation of the
-------
V-47
Table V-13
Effects of p-DCB on Mitotic Division of Hants
(Modified after Ware and West, 1977)
)rganism
Treatment
Fragments
Persistence
Of
Fragments
Remarks
Reference
\Lliurft
Six species
of monocoty-
ledon angio-
sperms-root
tips
Nine species
of dicotyle-
don angio-
spe rate-root
0.05, 0.1, 0.25,
0.5, 1.5 g for
five days to
seeds in Petri
dish
same doses to
four-day old
seedlings for
four hours
4 1/2 hour soak
in sat. soln.
1-4 hours
depending on
plant
No signs of
mitosis at
three highest
doses
Polyploidy in
root cells at
0.5 and 1.5 g
Abnormal chromo-
some numbers;
lagging chromo-
somes and dumb
bell shaped
nuclei also
occasionally seen
Frequency of frag-
ments at metaphase
tended to decrease.
Frequency high-24,
48 hrs. and de-
creased at 72, 96
hrs. 2 plants still
highly fragmented at
96 hrs. 1 complete
recovery.
2 plants recovered
before 96 hrs. 1
plant died after 48
Carey and McDonough,
1943
Sharma and Rattacharya,
1956
Sharma and Bhattacharya,
1956
-------
Table V-13 (Continued)
Organism
Treatment
Fragments
Persistence
Of
Fragments
Remarks
Reference
Nothoscordum
fragrana Kunth
Root tips
Root tips
Pollen
(Flower Buds)
Three species
of yicieae-
root tips
3 1/2 hrs.
soak in sat.
soln.
6 hrs. sat.
6 hours
(3 1/2 hrs.
sat. soln.)
(6 hrs. sat,
sol.)
4-6 hours in
soln.
Fenugreek seeds 4-24 hours soak
in sat. soln.
not indicated
not indicated
not indicated
Erosion and frag-
mentation of chromo-
somes both at meta-
phase and anaphase
Erosion and stick-
iness increase in
fragments from 3
1/2 hour soak.
No fragmentation,
some diplochromo-
somes. Indication
of slight disturbance
in spindle mechanism
and failure in sepa-
ration.
(Meiotic division -
only stickiness of
chromosomes noted.)
(Lagging, non-dis-
junction and stick-
iness of chromosomes.
No fragments.)
c-Mitosis abnormali-
ties. Breaks associ-
ated with heterochro-
matic chromosome reg-
ions
No change in morph-
ology or cytology of
seed! inas
Sjparma and Sarkar,
1057
Sjjfiarma and Sarkar,
S^iarma and Sarkar,
SJiarma and Sarkar,
1957
Sharma and Sarkar,
1957
Srivastava, 1966
Qupta, 1972
-------
Table V-13 (Continued)
Organism
Treatment
Fragments
Persistence
Of
Fragments
Remarks
Reference
Fenugreek
seedlings
4 hours "exposure"
(corn)
Greater than 4
hours (not speci-
fied)
3 hour soak of
0.5 cm long seed-
lings in saturated
aqueous solution,
then growth allowed
for 7 more days
Lens escu- 4-48 hour soak of
lenta micro- root tips in saturated
sperma aqueous solution
4-48 hour exposure
of germinating
seeds to vapors of
25, 50, 100, 250,
500, 800-1,000 mg
of p-DCB crystals
4-48 hour soak of
germinating root
tips in saturated
aqueous solution
not Root tip chromo-
indicated somes contracted
and arrested at
metaphase
After "longer"
treatment, mitosis
appeared normal
Accelerated root
growth, cell divi-
sion; polypi oidy,
formation of chromo-
somal bridges and
laggards at anaphase.
Polarity of cell
changed to an angle
of 90° from normal.
Germiability and
growth inversely
proportional to
level and duration
of exposure. Mitotic
and chromosomal
anomalies observed:
chromosome contrac-
tion and condensa-
tion, fragmentation,
bridges, tetraploidy,
binucleate cells
Gupta, 1972
Gupta, 1972
Sharma and Agarwal,
1980
Sarbhoy, 1980.
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V-50
methionine-requiring (meth3) locus in the fungus Aspergillus
nidulans. The mutagenicity of the dichlorobenzenes increased
in the following order: o-DCB-5/106 spores, m-DCB-9/106 spores
and p-DCB-11/106 spores.
Anderson (1976) reported on a study with p-DCB designed
estimate the .mutagenic potential of this substance in the Salmonella
typhitnurium plate incorporation assay. Mutant strains TA 1535, TA
1538, TA 98 and TA 100 were exposed to varying concentrations of
p-DCB dissolved in DMSO in two separate studies. In the first
study, concentrations of 100, 500 and 2500 ug/plate were used. The
experiment was run three times. In the second study, concentrations
of 4 and 20 ug/plate were used in addition to the three used in the
first study. This experiment was run five times. Exposure to
p-DCB occurred both with and without metabolic activation with S-9
mix from Alodor-treated rats. In another series of experiments,
the tester strains were exposed to atmospheric concentrations of
p-DCB at levels of 94, 299 or 682 ppm, again with and without
metabolic activation. This protocol was employed four separate
times.
A greater than two-fold increase in the number of rever-
tants is the criterion by which a compound is considered to be muta-
genic in this assay system. After atmospheric exposure to p-DCB, no
significant increases were noted in any tester strains, except in one
of the four exposures to 682 ppm in TA 1535 with metabolic activation.
This increase was not observed in three follow-up experiments. In
the first series of experiments in which p-DCB was dissolved in DMSO
at three concentrations, the number of revertants in TA 1535 increased
-------
nearly 10-fold at 500 ug/plate, with metabolic activation. This in-
crease occurred in two of the three runs. This increase was not ob-
served in the subsequent series of five runs employing five doses
including the previously-described three. In spite of the few posi-
tive results, the data from all of the experiments together would sug-
gest that p-.D.CB is not mutagenic to tester strains used in this Sal-
monella typhimurium plate incorporation assay system, either when
dissolved in DMSO or in the gaseous phase.
More recently, Simmon et al. (1979) examined all three dichloro-
benzene isomers for mutagenic activity in both the standard Ames/
Salmonella assay and the ]2. col i WP2 system. In the Ames assay,
tester strains TA 1535, 1537, 1538, 98 and 100 were employed, with
and without metabolic activation. In the first of two experiments,
concentrations of 0.05-1.0 ul/plate (0.065-1.3 mg/plate) of o- or
m-DCB were added to each Salmonell a strain or IS. col i culture. No
reproducible dose-related increases in the number of revertants were
observed in either system. In a second experiment, each compound was
retested at lower levels, ranging from 0.0005 to 0.5 ul/plate. Again,
no significant changes were noted. Para-DCB was tested in the same
manner, but at higher dose levels: 50-1,000 ul/plate in the first
experiment and 0.5-500 ul/plate in the second. Again, no increases
in the number of revertants were observed.
Simmon, et al. (1979) also conducted tests for chromosomal
aberrations in yeast. All three isomers were tested for their poten-
tial to induce mitotic gene conversion and reciprocal recombination
in Saccharomyces cerevisiae C3, with and without metabolic activa-
tion. At doses ranging from 0.001-0.25%, o-DCB produced no effects.
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V-52
Toxicity was observed at 0.05%, with and without activation.
Meta-DCB was tested at doses ranging from 0.005 to 0.1% in two
experiments. In both, but only after activation, enhancement of the
recombination response was observed. Para-DCB, at dose levels rang-
ing from 0.005-5% in the first three experiments, appeared to increase
mitotic recombination, with a considerable variation in survival. In
two later experiments, toxicity occurred, but no increase in recombi-
nation was observed. The inconsistency of the results may be attri-
buted to the relative insolubility of the compound.
The differential toxicity of each of the three isomers was
evaluated in the DNA repair-proficient and repair-deficient strains
of E. coli (W3110 polA+/p3478 polA~) and Bacillus subtil is (H17 recV
M45 rec~) (Simmon, _et: al_., 1979). In three of four experiments, 20
ug/plate o-DCB was more toxic to the repair-deficient jE. coli (polA~)
than to the repair-proficient strain (polA+). There was no apparent
difference in toxicity to either strain of !3. subtil is. At the same
concentration (20 ul/plate), m-DCB was more toxic to the repair-
deficient strain of E. coli than to the polA+ strain in four of five
experiments. Para-DCB, at concentrations of 1 or 5 mg/plate, had no
effect on any of the four strains. This result was interpreted to
mean that either the compound was truly nontoxic under these test
conditions or that the substance was unable to diffuse away from the
impregnated filter paper disc in the culture dish.
In summary, the results of these studies indicate that the
dichlorobenzenes do possess mutagencity activity in certain of the
test systems. None were positive in the Ames/Salmonella assay system
or in the E. coli WP2 assay. However, m-DCB, both with and without
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V-53
metabolic activ-atrion, increased mitotic recombination in S_. cerevisiae.
The results with p-DCB were ambiguous. Both o- and m-DCB were shown
to interact with and damage bacterial DNA in the E. coli W3110 polA+/
p3478 polA" differential toxicity assay system.
Effects on Animals
There has been at least one in vitro study of the inhibitory
effect of dichlorobenzene (isomer not specified) on the number of mi-
toses in rat lung cell cultures (Guerin, _et al_., 1971). The dose of
5 ug did not produce any significantly different number of mitoses
than the control. In this test system, dichlorobenzene gave a nega-
tive result: it did not exert any inhibitory action on the cultures.
Cytogenetic studies have been conducted on rat bone marrow
cells following inhalation exposures to p-DCB (Anderson and Richard-
son, 1976). Three series of exposures were carried out: 1) one ex-
posure at 299 or 682 ppm for two hours, 2) multiple exposures at 75
or 500 ppm, five hours/day for five days and 3) multiple exposures to
75 or 500 ppm, five hours/day, five days/week for three months. Ben-
zene (at 10, 750 or 7,500 ppra) was used as the positive control in
the first experiment. Vinyl chloride (at 1,500 ppm) was used as the
positive control in the other two experiments. Negative controls
breathed fresh air alone. In Experiment 1, three rats were in each
treatment group, four in the negative control group. In the other
two experiments, there were two rats in each treatment group and two
rats in the negative control group. In Experiment 1, 50 cells from
each animal were examined; in Experiments 2 and 3, 100 cells from
each animal were examined.
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V-54
Animals were sacrificed 22 hours after termination of exposure,
following one hour after an intraperitoneal dose of colchicine.
Bone marrow cells from both femurs were stained with Giemsa
and surveyed for chromosome or chromatid gaps, chromatid breaks,
fragments or other complex abnormalities. In all three experiments,
exposure to p-DCB failed to induce any statistically significant
effects indicative of chromosomal damage when compared to the nega-
tive controls, whereas in all experiments, the positive controls
did produce damage at all levels employed. Also, there was no evi-
dence of a dose-response relationship to p-DCB exposure, whereas,
there was with benzene in Experiment 1. Thus, under the conditions
of these experiments, p-DCB does not appear to elicit chromosomal
damage.
Para-dichlorobenzene also was tested in a dominant lethal
study in the CD-I mouse (Anderson and Hodge, 1976). Fertile males
were exposed, by inhalation, to plain air or to one of three levels
of p-DCB, or, by other routes of exposure, to one of three sub-
stances known to produce dominant lethal effects in this assay
system. Groups of males were subdivided into the following experi-
mental groups. Numbers of animals in each group are shown in
parentheses.
-------
V-55
Group 1: Air (negative control)-(35)
Group 2: 75 ppm p-DCB, 6 hr/day for 5 days (16)
Group 3: 225 ppm p-DCB, 6 hr/day for 5 days (16)
Group 4: 450 ppm p-DCB, 6 hr/day for 5 days (16)
Group 5: 200 mg cyclophos^h.auiLide./k.g, once by i.p.
injection on Day 5 (13)
Group 6: 150 mg ethyl methanesulfone/kg,
orally once a day for 5 days (5)
Group 7: 2.5 mg nitrogen mustard/kg,
once by i.p. injection on Day 5
The mating protocol consisted of placing two virgin females
in a cage containing one fertile male. Five days later the females
were removed to a separate cage. Two days later, the male was
caged with two different virgin females. This process was repeated
until the males had been mated at weekly intervals eight times.
Females were sacrificed 13 days after the assumed date of fertili-
zation, or 15-16 days after caging with the male. Uteri of these
females were examined for live implants, early and late deaths.
Statistically, the data were analyzed by a simple Chi-square and a
week by week hierarchical analysis of variance.
Only one death occurred among males exposed to p-DCB. This
was a male in the 75 ppm group during Week 3. The investigators
suggested that the death was unrelated to p-DCB exposure. Five
males in the cyclophosamide positive control group died early,
three during Week 6 and one each in Weeks 7 and 8.
Frequency of mating was affected in only two of the groups.
At Week 7, in the 77 ppm-dosed males, the frequency of mating was
100%, but significantly fewer males mated with both females (P<0.05).
-------
V-56
The number and percentage of females becoming pregnant was
decreased significantly at Weeks 6 and 7 among those mated to
males exposed to 75 ppm (P<0.05). The mean total of implants per
pregnant female in each group also was determined. The ratio was
significantly reduced in the 75 and 450 ppm groups at Week ff" when
compared with the negative control. All positive controls showed
significant reductions at Week 1 (P<0.01), cyclophosaminde and
nitrogen mustard at Week 2 (P<0.01) and ethyl methanesulfone at
•
Week 8 (P<0.05) when compared with the negative controls.
Early fetal deaths were analysed in three ways. When
determining the number of females with at least one early death,
groups exposed to 225 ppm p-DCB showed an increase at Week 1
(P<0.05). All positive controls showed significant differences at
Weeks 1 and 2 (P<0.001 or 0.05). When comparing the mean number of
early fetal deaths/pregnancy, there were no significant differences
in any of the p-DCB-exposed groups. All positive control groups
exhibited differences in Week 1 and 2, with cyclophosamide also in
Week 3 (P<0.05 or less).
Comparison of mean percentages of early deaths per total
implants per pregnancy revealed significant differences in p-DCB
treated groups in Week 6 in the 225 ppm group (P<0.05). Values
for the 75 ppm group in Week 1 were higher than the negative controls,
but'not significantly by Dunnett's "t" test. No significant
differences were seen in late fetal deaths for any groups.
Utilizing the three methods of analysis, two indicated
significant differences between p-DCB-exposed groups and negative
controls. However, these changes were different at different times
-------
V-57
and they did not occur in a dose-related manner. Therefore, the
authors suggested that these changes were not biologically signifi-
cant. In addition, they concluded that p-DCB, at least at the
exposure levels tested, does not cause dominant lethal mutations in
germ cells, of CD-I mice.
Carcinogenicity
Few studies have been reported which address the carcinogenic
potential of the dichlorobenzenes. Some of these are inadequate
for judging this characteristic.
A study by Parsons (1942) gave somewhat inconclusive
results on the carcinogenic effects of p-DCB in mice. One group of
mice was irradiated with an unspecified source of radiation, then
given 0.2% intraperitoneal p-DCB in sesame oil in silica. One
animal showed ascites and a sarcoma-like growth. This animal tumor,
when grafted, gave 100% takes. In "control" mice, i.e., those that
were not irradiated, one animal developed a sarcoma.
Murphy and Sturm (1943) studied the effects of p-dichlo-
robenzene on induced resistance to a transplanted leukemia in the
rat. Forty rats were immunized by intraperitoneal injection of
either defibrinated rat blood., or chopped 15 day old rat embryo.
They were exposed to saturated p-dichlorobenzene vapors for two to
three hours daily for 14 days, and then injected with 0.2 cc of
leukemia cells.
Of the 40 animals in the immunized group exposed to p-dichloro-
benzene, 67.5% had tumors. An immunized group not exposed to p-DCB
recorded 20.5% tumors while the control (no immunization) was 84.2%
positive. It would appear that p-dichlorobenzene modified the
-------
V-58
induced resistance of the rats to the leukemia. However, there is
not sufficient evidence to state that there are possible immuno-
suppressant effects resulting from exposure to p-dichlorobenzene.
In the two studies by Holl ingsworth, _et al_. (1956, 1958)
described earlier, the investigators did a cursory survey for tumor
occurrence during the subchronic exposure to rats, rabbits and
guinea pigs. No tumors were reported in any species after exposure
to either substance (o- and p-DCB). Little confidence can be
placed in these results as the conditions of study were inadequate
for evaluating carcinogenic or mutagenic potential.
Guerin and Curzin (1961) found that dichlorobenzene (iso-
mer not specified) gave a slight response for carcinogenic activity
in mice, as measured by the sebaceous gland and hyperplasia tests.
In both cases, dichlorobenzene (1 g/100 cc solution in acetone)
was applied to the skin of Swiss mice three times (0.1 cc solution).
The sebaceous gland test was based upon the disappearance of the
glands after application of the test compound. The hyperplasia
test examined the thickening of the skin epithelium after applica-
tion. On an arbitrary scale of 0 to 4, (negative to strongly .
positive), dichlorobenzene scored 0.9 on the sebaceous gland test
and 0.7 on the hyperplasia test.
The National Toxicology Program (NTP) recently completed
carcinogenicity bioassays in two species of rodents (rat and mouse)
for ortho- and para-dichlorobenzene. The results of the chronic
gavage studies on the ortho isomer were presented to the NTP Board
of Scientific Counselors in a draft report last year (NTP, 1982).
However, the Board has not approved the report as yet. Nonetheless,
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V-59
thre: .results are summarized below. The chronic gavage studies on
p-dichlorobenzene are complete. The results of these latter studies
on p-DCB may be presented soon. The protocols for the chronic
studies are presented below. In addition, the results of the
studies conducted in support of the chronic experiments with both
isomers also are discussed.
Two 14-day repeated dose studies with o-DCB were conducted in
B6C3F1 mice as part of the prechronic test phase of the NTP bioassay
on this substance (Battelle Columbus, 1978a, 1978b). These studies
were designed to determine approximate doses for the three month
subchronic toxicity study. No acute toxicity study was conducted.
In the first study, gavage doses of 0, 250, 500, 1,000, 2,000 or
4,000 mg/kg in corn oil were administered daily to six groups of
five mice of each sex (Battelle Columbus, 1978a). By the end of
the study, 47 of the 60 mice had died, leaving 5 male and 8 female
survivors. All mice in the top two dose groups died by Day 3. One
female mouse in the 1,000 mg/kg group survived to the end of the
experiment. The same was true for the 500 mg/kg group. One male
and one female survived from the 250 mg/kg group. In addition, one
male mouse in the control group died on Day 4.
At gross observation, early death mice had pale livers,
beginning as early as Day 2 in the 4,000 mg/kg group and seen by
Day 7 in the 250 mg/kg females. Other gross lesions observed in
all dose groups included reddened lung areas and yellow-green or
red small intestines. Liver histopathology in the six male mice
receiving histopathological examination was characterized by mod-
erate to severe centrolobular necrosis at the 500 mg/kg level
-------
V-60
and milder (in one) or none (in two) at the 250 mg/kg level. At
500 mg/kg, one mouse exhibited lymphoid necrosis of the spleen,
and the other animal, marked lymphoid depletion of the thymus.
Of the seven females receiving histopathological examination, the
one at 1,000 mg/kg surviving to the end of the study showed no
pathology. Of the three examined from the 500 mg/kg group, one
of two early fatalities exhibited no pathology while the other had
moderate lymphoid depletion of the spleen. The one survivor had no
lesions. At 250 mg/kg, one early fatality exhibited mild centro-
lobular hepatic necrosis, the other, no histopathology. The one
survivor examined showed no pathology.
Since no no-effect dose level was determined from this
initial experiment, it was decided that a follow-up study would
i
be conducted, using a range of doses lower than that employed in
the first experiment. The protocol was identical to that of the
first study; however, the doses selected were 0, 30, 60, 125, 250
or 500 mg/kg in corn oil (Battelle Columbus, 1978b). In this
study, only two early deaths occurred: one male in the 500 mg/kg
group on Day 3 and one female in the 125 mg/kg group Day 8.
Body weight changes in the treated animals were not markedly dif-
ferent from the controls, although males in the 30 and 60 mg/kg
groups showed a 9% lag (a 6% increase vs. a 15.7% increase in the
controls). During the first three days of the study, all mice in
the highest dose group exhibited labored breathing, rough coats and
watery eyes. The signs then disappeared. Gross examination was
conducted on all animals. The male mouse dying on Day 3 had a pale,
mottled liver, enlarged stomach and reddened small intestine.
-------
V-61
His-tological examination of livers from four males and four fe-
males from the 500 mg/kg dose group revealed no changes in
two males, mild focal necrosis in one, and mild focal necrosis
as well as cytomegaly, karyomegaly and chronic moderate multi-
focal granulomatous hepatitis in the fourth male. Among the
four females, one exhibited moderate focal necrosis, another showed
mild focal necrosis, and the other two had mild centrolobular
degeneration with cyto- and karyomegaly. These changes were judged
to be treatment-related. On the basis of the results of this
second study, it was recommended that the subchronic study in
the mouse be performed at the same dose levels used in this
14-day study.
The subchronic toxicity gavage study with o-DCB in mice was
conducted to assist in dosage selection for the 104-week chronic
study (Battelle Columbus, 1978c). The doses used were as detailed
above (0, 30, 60, 125, 250 or 500 mg/kg/day). Treatment groups
consisted of five animals of each sex. Single gavage doses of the comp-
ound in corn oil were administered 5 days/week for 13 weeks. Weekly
individual body weights and cage food consumption rates were moni-
tored. Animals were sacrificed on Day 92 or 93, with full necrospy,
recording of organ weights and histological examination of various
tissues. Special studies also were performed near or at the end of
the exposure period. These included: urinalysis in the highest
dose group and controls, hematology and clinical chemistry. Organ/
body weight ratios were calculated and urine and liver porphy-
rin determinations made.
During the study, lethargy and rough coats were observed
-------
in both sexes at the four highest doses. However, by the final
week on test, only animals of both sexes at the 500 mg/kg dose
and males at 250 mg/kg showed these signs. Body weight gain was
affected in males at 500 and 250 mg/kg (-49% and -18%, respectively,
when compared with controls). Female mice receiving 500 mg/kg/day
exhibited a differential weight gain of -62% when compared with
controls. No other groups showed differential weight gains of
greater or less than 10% when compared with controls.
Hematological parameters evaluated included hemoglobin,
hematocrit, total and differential white counts, red cell and
platelet counts, mean corpuscular volume and reticulocyte counts.
The investigators did not perform any statistical analyses on
these data, but claimed that no clinically-significant treatment-
related changes occurred. The apparent differences in white cell
counts of treated males when compared with control was attributed
to relatively low counts among the controls. It was suggested
that these counts were below those typically observed in that
laboratory and others (3.4 x 103/mm3 in controls vs. 5.4-6.6 x 103/
mm3 in the treated groups). Since the individual data were not
available, the statistics cannot be done which would show whether
or not there were significant differences between the controls and
and the treated groups. In addition, information on the "normal"
counts is' not available for evaluation. But, since there is at
least anectodal evidence of a possible relationship between ex-
posure to ortho-dichlorobenzene and leukemias in humans, it would
be prudent to evaluate these results in greater depth.
-------
Blood samples were analyzed for alkaline phosphatase, SGPT and
gamma-glutamyl-transpeptidase (GGTP). No GGTP was detected in any
sample. Statistically significant dose-dependent changes in alkaline
phosphatase did not occur, although increased levels were noted in
males receiving 125 and 250 mg/kg/day. SGPT levels in the two
surviving males receiving 500 mg/kg/day were increased significantly
over control, due to the high value recorded in the animal exhibiting
the hepatocellular necrosis. The other animal showed no hepatic
histopathology.
The following parameters were monitored during the uri-
nal ysis: pH, glucose, protein, bilirubin, ketones, occult blood,
specific gravity and creatinine. The report stated that the
volume of urine collected from treated animals, especially the
males, was generally greater than that collected from controls.
The individual data were not available to corroborate this con-
clusion. No record of fluid intake was kept during the study.
Decreases in specific gravity and creatinine were noted, reflect-
ing dilution due to increased urine output. No other compound-
related effects were observed.
Uroporphyrin levels in treated males were generally
higher than in the controls. In females, coproporphyrin levels were
higher in the treated animals than in controls. Coproporphyrin levels
in treated males and uroporphyrin levels in females were not dif-
ferent from controls. Sex differences were seen in liver protopor-
phyrin levels, as a dose-dependent increase was observed in the
females but not in the males.
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V-64
Organs weighed were: heart, lung, kidney, testis, spleen,
thymus, brain, ovary and uterus. While no statistics were done,
it appeared that relative liver weights were increased in the
highest dose males and females. An increase of lesser magnitude
was seen in females receiving 250" mg/kg/day. No other differences
were noted.
All of the above-mentioned tissues and skeletal muscle from
control and highest dose animals were examined microscopically.
The liver, thymus, heart, spleen and thigh muscle were examined
from animals in the 250 mg/kg group and livers only from the 125
mg/kg group. Lesions were observed in all tissues from the highest
dose animals. These were considered to be treatment-related, since
they were absent or occurred less frequently in the controls.
Livers of the highest dose animals exhibited significant
centrolobular necrosis, hepatocellular necrosis and degeneration,
and deposition of yellow-green to golden pigment considered to be
hemosiderin. The heart showed multiple foci of mineralization in
the myocardial fibers. Skeletal muscle also exhibited mineraliza-
tion as well as necrosis and myositis. Some animals had lymphoid
depletion of the spleen and thymus. One female exhibited lymphocyte
necrosis in the spleen.
Among the animals receiving 250 mg/kg, only hepatocellu-
lar necrosis was noted in two males, with pigment deposition in
one male and hepatocellular degeneration in one male. No lesions
were noted in the group treated with 125 mg/kg.
The results of this study suggest that an oral no-effect
level can be identified in mice over 13-week exposure period of
125 mg/kg/day. Doses of 60 and 120 mg/kg/day were selected for use
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V-65
in the 104-week chronic study.
A draft NTP Technical Report on the bioassay of ortho-DCB
in mice and rats was presented to the NTP Board of Scientific
Counselors on September 22, 1982. This document does not become a
final report until it is reviewed and approved by the Technical
Reports Review Subcommittee of the NTP Board of Scientific Counsel-
ors. Therefore, the text that follows represents only a preliminary
assessment of the data from the bioassay.
The bioassay was conducted by administering 60 or 120 mg/kg
doses of o-DCB in corn oil, five days/week for 104 weeks. Groups
of 50 males and 50 females comprised each dosage group and a vehicle
control group, as well. The control group received equivalent vol-
umes of corn oil on the same schedule as the treated animals.
No difference in survival rates were observed between the
treated and control groups of either sex. There was no evidence
of compound-related nonneoplastic liver lesions, suggesting that a
higher dose may have been tolerated in the chronic study.
Statistically significant positive trends in the incidence
of malignant histiocytic lymphomas occurred in mice of both sexes
(P<0.05). However, the incidence of total lymphomas of all cell
types was not significantly increased above control. The draft
reports states that "since the histiocytic lymphoma is a controversial
diagnosis among different pathologists and since all types of
lymphomas have the same histiogenesis, an increase in this specific
type of lyraphoma in the absence of an increase in the total incidence
of all types of lymphoma is not considered to be biologically
significant."
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V-66
An increase in alveolar/bronchiolar carcinomas were ob-
served in male mice (control=4/5, 8% low dose=2/50, 4%, high dose=
10/50, 20%). This increase was shown to be significant when ana-
lyzed by the Cochran-Armitage test, but not the life-table or inci-
dental tumor test. Thus, this increase was discounted because the
combined incidence of males with alveolar/bronchiolar adenomas or
carcinomas was not significantly greater than controls by any of
the three tests (control=8/50, 16%, low dose=8/50, 16%, high dose=
13/50, 26%).
Male mice also exhibited a significant decrease in hepato-
cellular adenomas at the high dose (control=8/50, 16%, low dose=
5/49, 10%, high dose=2/46, 4%)-. This decrease was accompanied by
a negative dose-response trend using the Cochran-Armitage test.
When total incidence of adenoma or carcinoma in the high dose males
was evaluated, this decreased incidence was statistically significant
only by the life table test (control=19/50, 38%, low dose=14/49,
29%, high dose=ll/46, 24%).
The preliminary assessment suggests that, under the con-
ditions of this bioassay, ortho-dichlorobenzene was not carcino-
genic in the B6C3F1 mouse of either sex, but that the maximum tol-
erated dose was probably not achieved in the study.
A 14-day repeated dose study with o-DCB also was conducted
in Fischer 344 rats as part of the prechronic .test phase of the NTP
bioassay on this substance (Battelle Columbus, 1978d). Single oral
gavage doses of o-DCB in corn oil of 60, 125, 500 and 1,000 mg/kg
were selected. Five animals of each sex were placed in one of
five treatment or one control groups. All of 10 rats in the 1,000
group died early, the males by Day 4 and the females by Day 5.
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V-Of
No other- early deaths occurred. The percentage body weight gain
decreased with increasing dosage in both sexes. Males at 250 mg/kg
showed an -11% deficit, those in the 500 mg/kg group, a -16.6% dif-
ferential. Only females at 500 mg/kg showed a weight gain deficit
exceeding 10% (-11.8%).
No tissues from animals dying early were examined histo-
logically. Those in the highest dose group exhibited, upon gross
examination, pale and yellow livers, yellow and/or green or red
colored contents in the small intestine, similarly-colored fluids
in the urinary bladder, red fluid in the cecum and congestion of
the vasculature of the brain. The liver lesion was interpreted to
reflect hepatotoxicity. No toxic lesions were observed in other
dosage groups during gross examination. Tissues from two males
and two females receiving 500 mg/kg/day were examined microscopi-
cally, with no lesions indicating toxicity.
Based upon the results of this study, it was recommended that
the 13-week subchronic gavage study in Fischer 344 rats employ
doses of 25, 50, 100, 200 and 400 mg/kg/day, five days/week. The
doses actually administered in the subchronic study were 30, 60,
125, 250 and 500 mg o-DCB in corn oil (Battelle Columbus, 1978i).
Controls received corn oil. Special studies, as described for o-
DCB in mice and p-DCB in both species, also were conducted in this
study. No statistical analyses were performed on the data from
these special studies, except for group means and standard devia-
tions of those means.
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V-68
Of the animals used in this study, only four died early:
one male each in the 30 mg/kg (Week 11) and control (Week 10) groups
and two females in the highest dose group (Weeks 6 and 9). Only nine
males in the 250 mg/kg group completed the study; the tenth was
found to be a female during Week 10 and was removed from the group.
Food consumption did not vary more than 10% in treated
groups when compared with controls (down 1.5 mg/day in the highest
dose males). Lack of body weight gain increased with increasing
doses in both sexes. At 250 mg/kg and 500 mg/kg, this differential
exceeded 10%. No striking differences in hematological parameters
were noted, but without statistical analysis, it is difficult to
determine if the changes are truly statistically non-significant.
Those parameters which may have been altered significantly in the
highest dose males were hematocrit (down 5%) and red cell count
(down to 8.57 +^ 0.25 x 106 cells/mm3 from an average of 9.42 +_ 0.53
x 106 cells/mm3 in the control group). There appeared to be a
trend in platelet count, directly proportional to increasing dose
in the females, with levels of 300,000 in the controls to 365,000-
600,000 as the dose increased, with the 250 mg/kg group falling out
of sequence. This apparent change may have been due to what appeared
to be a lower-than-normal count in the controls.
Of parameters measured in the clinical chemistry analyses,
cholesterol levels were increased in males in 250 and 500 mg/kg and
in females at 125, 250 and 500 mg/kg. Triglycerides dropped in
high dose males. The combined alpha-globulin fraction appeared to
be increased in males receiving 250 and 500 mg/kg and in females
treated with 500 mg/kg. Of the parameters tested in the urinaly-
-------
sis, urine volume was altered significantly. Output increased an
average of 157% in treated males and 187% in treated females, when
compared with their respective controls. Decrease in urine creat-
inine accompanied the dilution occurring with the increased volume
output.
Porphyrin levels in the urine showed a striking increase in
the highest dose animals (3-6 fold), both in coproporphyrin and
uroporphyrin levels. Liver protoporphyrin levels were not altered
significantly at any dose level. Thus, there appeared to be ab-
normal excretion of the porphyrin, but not retention.
Organ weights and organ/body weight ratios were determined.
In rats of both sexes receiving 250 or 500 mg/kg, relative liver
weights were increased. The liver/body weight ratios in the other
treatment groups did not appear to differ from the controls. In
addition, ratios for the other organs (spleen, kidney, testis,
ovary, uterus, thymus, brain and heart) did not appear to have been
altered.
No consistent lesions were noted upon gross examination
at necropsy. Microscopic examinations were performed on tissues
of all animals in the control and 500 mg/kg groups and on the thy-
mus, liver and kidney of the animals from the 125 and 250 mg/kg
groups. A moderate degree of centrolobular hepatocellular necrosis
was seen in the livers of the highest dose rats-which died early.
Of the survivors in that group, most showed liver lesions, either
centrolobular degeneration or necrosis of individual hepatocytes.
This necrosis was characterized by randomly scattered cells that
were pyknotic or karyolytic, with shrunken, dark red cytoplasm.
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V-70
Some of the 500 mg/kg group males also exhibited renal tubular
degeneration and lymphoid depletion of the thymus. These latter
lesions were not seen in other treated groups or controls. However,
hepatocellular necrosis was observed in the 250 mg/kg group and in one
female treated with 125 mg/kg groups was considered to be hemosiderin-
since it was PAS- and Perls positive.
The liver lesions observed in the 250 and 500 mg/kg groups
were considered to be dose- and treatment-related, and probably
life-threatening, as were the renal and thymic lesions in the high
dose group. The reviewing pathologist recommended that a Maximum
Tolerated Dose (MTD) of 125 mg/kg (the apparent no-effect level)
be set for both sexes of rats in the 104-week chronic study.
Ultimately, doses of 60 and 120 mg/kg were chosen for this study.
As mentioned above, this assessment of the effects of o-
DCB in the bioassay is preliminary, pending acceptance by the NTP
Board of Scientific Counselors.
As with the mice, groups of 50 male and 50 female rats
received 60 or 120 mg/kg doses of o-DCB in corn oil, five days/
week for 104 weeks. Controls, also 50 of each sex, received an
equal volume of corn oil on the same schedule.
There was no significant difference in the survival rates
of female rats of either treatment group or low dose males when
compared with the controls. However, there was a significant de-
crease in the survival of the males at the high dose (P<0.001).
However, several of the males dying before the end of the study were
found to have amounts of corn oil or o-DCB in corn oil in their
lungs (3 in the control group, 8 at the low dose and 12 at the high
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V-71
dose). Therefore, gavage trauma may have contributed to their
deaths. The report suggests that the lower survival rate among
the dose males may not reflect that the maximum tolerated dose was
exceeded. In fact, as was seen in the mice, there was no increase
in the incidence of nonneoplastic lesions of the liver in rats
receiving either dose of o-DCB, again suggesting that a higher dose
might well have been tolerated.
The low dose males showed a significant increase in the
incidence of adrenal pheochromocytomas when compared with the con-
trol group by life table analysis (control=9/50, 18%, low dose=16/50,
32%, high dose=6/49, 12%). The report concludes that since this
incidence was significant only by one of the three tests, and this
tumor expressed no dose-response trend or high dose effects, and
there were no malignant pheochromocytomas, the increase seen in the
low dose group was not related to treatment with o-DCB.
Interstitial cell tumors of the testis in the males also
occurred with a significant positive trend when analyzed by the life
table test (control=47/50, 94%, low dose=49/50, 98%, high dose=
41/50, 82%), but with a significant negative trend when analyzed
by the Cochran-Armitage test. The reports states that "since this
tumor is not considered to be life-threatening, this increase
detected by the life table test was discounted."
Preliminary assessment of these data suggests that, under
the conditions of this bioassay, ortho-dichlorobenzene was not
carcinogenic in the Fischer 344 rat of either sex, but that the
maximum tolerated dose may not have been achieved.
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V-72
Para-dichlorobenzene
A 14-day repeated dose oral gavage study was conducted in
B6C3F1 mice to determine doses for the 13-week subchronic toxicity
study (Battelle-Columbus, 1978e). Doses of 250, 500, 1,000, 2,000
or 4,000 mg/kg in corn oil were administered to groups of five males
and five females. Controls received corn oil only on Day 1. All
mice receiving 4,000 mg/kg died by Day 3. At 2,000 mg/kg, four
males died by Day 7 and two females by Day 8. At 1,000 mg/kg, four
males and two females died by Day 8. At 500 mg/kg, four males died
by Day 8, but all females were dead by Day 5. At 250 mg/kg, three
males died by Day 1, three females by Day 6. Among controls, two
males and one female died on Day 3, presumably as a result of gavage
trauma.
Upon gross necropsy, a number of lesions were noted in
both sexes and at all dose levels. These included: livers of
abnormal color, either yellow or tan or reddish-chocolate, soft or
mushy small intestines from yellow-green through pink or red to
black in color, and pink to bright red lungs. Occasionally, kidneys
were pale, and there occurred enlarged, chalk-white mandibular sali-
vary glands. According to the investigators, no lesions were seen
microsopically in either sex or at any dose level which indicated
significant toxicity.
Since the study did not establish a no-effect level, a second
14-day repeated dose study was performed (Battelle Columbus, 1978g).
Dose levels of 60, 125, 250, 500 and 1,000 mg/kg in corn oil were
employed. The controls were untreated. Only one early death, a
male in the 125 mg/kg group, occurred, purportedly due to gavage
trauma. Only the 500 mg/kg females showed a decreased body weight
-------
gain (-10.9% vs. control). No gross pathology was noted at necropsy.
No histology was performed.
On the basis of these two 14-day studies, the principal
investigators recommended that the subchronic gavage studies in mice
be performed at doses of 125, 250, 500, 1,000 and 2,000 mg/kg/day,
5 days/week, for 13 weeks. However, the final doses actually employed '
in the first subchronic study were 600, 900, 1,000, 1,500 and 1,800
mg/kg/day (Battelle Columbus, 1979a). Ten males and ten females
were assigned to each group. Special studies included: urine and
liver porphyrin determination, calculation of organ/body weight
ratios, urinalysis, clinical chemistry and hematology.
Twenty-six animals (12 males and 14 females) died before
the end of the study. Seven males and females in the 1,800 mg/kg
died early, most within the first week. At 1,500 mg/kg, three males
and five females died early. One male in the 1,000 mg/kg group and
one control male died during the last week.
No effects on food consumption were noted in any group.
Differential body weight gains in treated males of all groups were
significantly different from controls. These differences ranged
from -50% at 1,500 mg/kg to -22.9% at 1,000 mg/kg. The differences
did not show a dose-related trend. Females showed a differential
weight gain of -39% at 600 mg/kg, with lesser changes as the dose
increased, but still > 10%. Since food consumption was not altered,
the weight gain reductions were apparently unrelated to dietary intake.,
The hematological parameters included: hemoglobin, hema-
tocrit, total and differential white cell counts, red cell counts,
mean corpuscular volume, platelet and reticulocyte counts. No
-------
statistical analyses were performed on these~ data. However, the
investigators stated that there appeared to be no significant dif-
ferences between treated groups and controls. However, a cursory
review of the group data suggest that there may have been a signifi-
cant decrease in platelet counts in the high dose males (445,000 4^
8,700 vs 691,900 ± 209,200 for controls), and a significant increase
in surviving females at the two highest doses (707,400 + 41,000 at
1,500 mg/kg, 707,500 at 1,800 mg/kg vs 492,000 + 86,631 for controls)
This latter observation may be the result of a lower than normal
platelet count among the control animals.
The following analyses were performed on blood samples
drawn at the time of sacrifice: serum glutamic pyruvic transami-
nase (SGPT), alkaline phosphatase, gamma-glutamyltranspeptidase
(GGTP), bilirubin, cholesterol, triglycerides, blood urea nitrogen
(BUN), glucose and total protein. While no statistical analyses
were performed on the data, it appeared that triglycerides were
increased in males at the two highest dose levels, and that choles-
terol was increased in all males except those in the 600 mg/kg
group. Total protein appeared to be increased in males in the
1,800 mg/kg group. Since there were no increases in hemoglobin
or hematocrit, this increase likely was due to an actual, rather
than a relative, increase in blood proteins.
Both SGPT and bilirubin may have been increased in females
in the two highest dose groups. No control values in females were
recorded for cholesterol, triglycerides, BUN, glucose or total pro-
teins. Thus, it is difficult to speculate whether or not the in-
crease in cholesterol values in these groups was significantly
-------
V-75"
greater "than control, as would be suggested by the dose-related
upward trend, the relationship observed in the males.
During urinalysis, the parameters measured were: pH, protein,
glucose, ketones, bilirubin, occult blood, specific gravity and
creatinine, as well as uroporphyrins and coproporhyrins in the
controls and two highest dose groups.
Ketonuria was noted in one of two pooled groups of males
at the 1,500 mg/kg dose level and in both pooled groups of females
at that dose. None was observed in urine of animals at other
doses or in the controls. In the males, this occurrence may have
been due to the severe body weight gain differential, according to
the investigators. However, this postulate does not hold up for
the females, since this severe weight gain differential was not
observed. The investigators suggested that this observation was
due to contamination of the urine samples. As was seen in the o-
DCB studies, increased urinary output occurred, especially in the
mal es.
Urinary coproporphyrin levels in both sexes appeared to
increase significantly at the 1,500 mg/kg/dose level but only in
females at the highest dose. No change was seen in coproporphyrin
levels in males, but appeared to be lower in the surviving female
at the highest dose. Dose-dependent increases in liver protopor-
phyrin in both males and females occurred. However, since no sta-
tistics were applied to the data, one cannot identify the lowest
dose level at which significant increases occurred.
-------
Of the organ/body weight ratios calculated, those for the
liver were increased at all dose levels for both males and females.
In addition, relative uterine weights of females decreased in a
dose-related manner, with the greatest decrease in the ratio observed
in mice receiving 1,800 mg/kg. No differences were apparent for
lung, heart, kidney, spleen, thymus, brain, testis or ovary.
The only lesions noted during necropsy were some pale and/or
yellow livers and pale kidneys in the highest dose group. No
hemosiderin was noted in any liver or kidney of mice from any
dosage group.
A number of lesions were noted upon histological examina-
tion. These included lesions of the spleen, thymus, bone marrow and
lymph nodes among animals from the two highest dose groups. These
were considered to be treatment-related. Hepatocellular changes
which were seen in treated animals of all groups were not seen in
the controls. In mice from the 1,500 mg/kg group which died before
the end of the study, but not those who survived to the end, there
was lymphoid depletion of the spleen, lymphoid necrosis in the
thymus and hematopoietic hypoplasia in the spleen and bone marrow.
The hepatocellular changes observed in the various groups included
karyomegaly, cytomegaly, and occasionally, large prominent nuclei
with variations in their shape along with changes in the number of
centrolobular hepatocytes. The cytoplasm of these enlarged cells
was grainy, sometimes hazy and amphophilic. These changes were
dose-related.
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v-77
Since the hepatocellular changes were seen in animals from
all treated dose groups, no no-effect level could be established
from the study results. The changes observed at 600 mg/kg could be
characterized as minimal. Subsequently, a second subchronic study
was performed in the attempt to establish the maximum tolerated
dose for the chronic study and-a no-effect dosage level for sub-
chronic exposure (Battelle-Columbus, 1980a). In this second study,
target doses of 75, 150, 300, 600 or 900 mg/kg/day were chosen. As
shown later, because of an error in the preparation of the doses,
those actually administered were 84.4, 168.8, 337.5, 675 or 900
nig/kg/day, five days/week for 13 weeks. The protocol was other-
wise identical to the first subchronic study, except that no special
studies were performed. During the study, three males and seven
females died early. These deaths were not dose-related, but rather
attributable to gavage trauma. No significant differences were ap-
parent in dietary consumption or relative weight gains between
treated and control animals of either sex.
During gross necropsy, no consistent observations were
made which were considered significant. Microscopically, there
was a significant number of animals in the two highest dose groups
exhibiting centrolobular to midzonal hepatocytomegaly. Few changes
of this type were seen in animals treated at 337.5 mg/kg/day. The
investigators determined that 337.5 mg/kg/day was the maximum tol-
erated dose for this duration of exposure.
( 104-week results to be added later)
-------
Fourteen-day repeated dose and 13-week subchronic gavage
studies also were conducted in Fischer 344 rats as dose-range
finding efforts preliminary to the 104-week chronic bioassay for
p-DCB in this species. The first repeated dose study employed
single doses of 60, 125, 250, 500, or 1,000 mg/kg/day p-DCB in
corn oil (Battelle Columbus, 1978f). Doses were administered to
five males and five females per group. Controls were untreated.
Only one early death was recorded, a male at the 125 mg/kg
dose level on Day 8. Body weight gains were slightly suppressed in
the males, with the greatest difference being seen at the highest
dose (-9.2%). No gross or histological pathology was observed.
Because of the absence of significant signs of toxicity, it was
decided that the study be repeated at higher dose levels.
A re-run of the 14-day repeated dose study was conducted
according to the protocol of the first study. In this study, how-
ever, doses of 500, 1,000, 2,000, 4,000 or 8,000 mg/kg/day were
administered in corn oil to the rats (Battelle Columbus, 1978g).
Again, controls were untreated. No clinical pathology studies or
histological examinations were done.
By Day 3, all animals in the group receiving 2,000, 4,000 or
8,000 mg/kg had died. Gross pathology included pale livers,
discolored lungs and intestines and mandibular lymph nodes
and fluid discharge from the eyes and nose. One male rat receiving
1,000 mg/kg died on the first day, apparently due to gavage trauma.
Four females in that dose group died on Day 1 (1), Day 4 (1) and
Day 5 (2), with those dying on Day 5 showing slightly congested
livers. One female receiving 500 mg/kg died on Day 13 of gavage
trauma.
-------
At these higher doses, evidence of depressed body weight
gain appeared. Among males, those receiving 1,000 mg/kg/day had
a 22% reduced gain; those receiving 500 mg/kg/day showed a 24%
reduced gain. No significant differences were seen in the females
at any dose.
From the results of the two repeated dose studies it was
determined initially that a subchronic gavage study in the Fischer
344 rat be performed at dose levels of 60, 125, 250, 500 or 1,000
ntg/kg/ day. In fact, the first subchronic study utilized doses of
300, 600, 900, 1,200 or 1,500 mg/kg/day (Battelle Columbus, 1979b).
The protocol included use of ten animals/sex/dose or control group.
Animals received doses of p-DCB in corn oil, five days/week for
13 weeks. Vehicle controls received corn oil alone. In addition
to gross necropsy and histological examination, additional special
studies were performed as described for the subchronic studies on
o-DCB. These included: clinical chemistry and hematology on blood
samples drawn the day of sacrifice, urinalysis on animals at the
1,500 and 1,200 mg/kg groups and controls, porphyrin analysis in
urine and liver of the same groups and calculations of organ/body
weight ratios. Again, no statistical analysis of these data was
performed.
Eight early deaths occurred in the 1,500 mg/kg males, and five
in males at 1,200 mg/kg. One male receiving 900 mg/kg died during
Week I, and one control male during Week 10. Among females, 9 of
10 receiving 1,500 mg/kg died early, one of 10 at 1,200 mg/kg and
two of 10 at 900 mg/kg. The males tended to die earlier than the
females.
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V-80
Food consumption in the treated groups varied little
from control. The one remaining male at 1,500 mg/kg showed a
slight increase compared with controls. Male rats at 300 mg/kg
averaged slightly less than controls. A dose-dependent depression
of body weight gain was seen in both sexes. In females, a greater
than 10% difference was seen at the 900 mg/kg level and above.
All treated males exceeded the 10% differential when compared with
controls.
Of the hematological parameters analysed (hemoglobin,
hematocrit, mean corpuscular volume, red cell, reticulocyte and
platelet counts, total and differential white cell counts), the
hemoglobin and hematocrit levels appeared to be lower in the two
surviving males at 1,500 mg/kg. Hemoglobin levels were 17.6 jf
0.6 G/dl in controls vs. 15.3 +_ 0.2 G/dl in the treated rats.
These two males also exhibited mild anemia with average red cell
counts of 8.85 ^ 0.22 x 106 cells/mm3 compared with control levels
of 10.03 +_ 0.36 x 106 cells/mm3. Mean corpuscular volume also was
decreased slightly (51 ^ 2 in controls vs. 48+0 in the treated
rats). The investigators reported that no histological evidence of
bone marrow changes were apparent in these two rats, although one
other male and five females treated with this dose did exhibit bone
marrow hypoplasia. The single surviving high dose female showed
an increase in hemoglobin, lowered white cell count, increased
red cell count and decreased mean corpuscular volume when compared
with controls. However, one cannot establish the significance of
these observations, since they occurred in a single animal.
-------
Clinical chemistry analyses on blood drawn at sacrifice
included: gamma-glutamyl-transpeptidase, alkaline phosphatase,
bilirubin, cholesterol, triglycerides, total protein, blood urea
nitrogen, glucose and globulin fractions. Of these parameters,
there was evidence of a possible dose-related increase in alkaline
phosphatase in females, with a substantial increase in the lone
survivor at" the highest dose. Trends also appeared in both sexes
as an increase in cholesterol levels with increasing dose and an
increase in albumin levels at the highest doses. The total pro-
tein increases seen at the higher doses likely are due to the
increase in the albumin levels.
The investigators reported no significant changes in the
parameters measured in the urinalysis: pH, protein, glucose,
creatinine, ketones, bilirubin, occult blood and specific gravity.
As previously described for o-DCB, urine volume of rats treated at
the higher dose levels was significantly increased: 246% in males
at 1,200 mg/kg, 142% in females at this dose. The few survivors
from the 1,500 mg/kg groups had urine volumes 6-10 times greater
than control levels. Since drinking water intake was not moni-
tored, one cannot speculate on the consequences of this alteration
in output.
Porphyrin levels in urine and liver also were monitored
in the two higher dose group and control males_ Significant in-
creases in both coproporphyrin and uroporphyrin levels in the
urine were observed. A similar increase was seen in the urine of
the surviving highest dose female. No data for survivors at 1,200
mg/kg were presented. Liver protoporphyrin levels apparently were
-------
v-ai
not increased in either sex. On the contrary, the levels in the
survivors at the highest dose were depressed somewhat. Therefore,
there was no retention of porphyrins in treated animals, just as
was reported for o-DCB.
Several organs were weighed at the time of sacrifice. These
included: heart, liver, kidney, uterus, ovary, testis, brain,
thymus and spleen. The relative liver weights were increased clearly
in animals of both sexes at all doses except the lowest (300 mg/kg).
An increase at this dose level was seen in females, but it may not
have been statistically significant. Dose-related changes in brain/
and uterus/body weight ratios also were observed. The investigators
suggested that these differences might be due to the significant
lack of body weight gain in the affected groups at the higher dose
levels.
Histological examination was performed on tissues from
animals in the control and three highest dose groups. The kidneys
and lungs from males treated at 300 and 600 mg/kg also were examined.
Retinal atrophy was seen in almost all of the animals, the
degree of severity of which was inversely related to the time of
death. Thus, those dying early showed few signs; those dying later
had severe bilateral atrophy.
Pulmonary lesions related to the gavage procedure were
seen in animals of all tested groups. Presumably, aspiration of
the test material occurred.
Of significance was the degeneration and necrosis of the
hepatocytes, hypoplasia of bone marrow, lymphoid depletion of the
spleen and thymus, epithelial necrosis and villar bridging of the
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V-83
mucosa of the small intestine and epithelial necrosis of the nasal
turbinates that occurred in animals in the two highest dose groups.
None of the lesions were observed in animals receiving doses of 900
mg/kg or less. Thus, these changes were considered to be treatment-
and dose-related.
Most of the males surviving beyond the halfway point of
the study exhibited renal lesions characterized by multifocal
degeneration or necrosis of the cortical tubular epithelium. An
amorphous eosinophilic material was often present in the lumen of
thes'e tubules. Some thickening of the basement membrane of these
cells was visible. Occasionally, a dilatation of some tubules
could be observed at the corticomedullary junction. These tubules
also had degenerated epithelia and were filled with material simi-
lar to that described above.
The investigator describing the pathology stated that the
renal lesions are not unusual for male rats of this strain. How-
ever, he would have expected to have seen lesions of some magnitude
in the control animals, and to a lesser degree in treated ones.
Thus, he concluded that while the lesion was not clearly dose-related,
it might be at least partially treatment-related and for this reason,
no no-effect dose level could be established from the results of
this study. Thus, it was decided that the subchronic gavage study
in rats would be repeated, employing lower dose levels of 37.5, 75,
150, 300 or 600 mg/kg/day (Battelle Columbus, 1980b) . The protocol
employed was identical to that described above for the first sub-
chronic study, except that no special studies were done. A few
early deaths occurred during the second study. All were attributed
to gavage trauma.
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V-84
Na significant differences in food consumption were ob-
served. No dose group had a greater than -10% differential weight
gain when compared with controls. Histological examination was
performed on all control and 600 mg/kg dose animals, as well as
all early death animals, and kidney from males at the two lower
doses.
Microscopically, some lung pathology was noted which was
attributed to aspiration of the gavaged material. There also was
an increased incidence and severity of renal cortical degeneration
in males receiving the two higher doses, as had been observed in
the earlier study. Females showed no significant changes at any
dose level. Some rats also exhibited myocardial degeneration and
lymphoid hypoplasia in lung tissue. These changes were character-
ized as being normal for this strain and age of rat. The reviewing
pathologist suggested that the MTD for males be set at 150 mg/kg
and for females at 600 mg/kg for the chronic study. Therefore,
Battelle Columbus proposed doses for the chronic study of 75 and
150 mg/kg in males, 300 and 600 mg/kg in females. Tracor Jitco
suggested 150 and 300 in males, 300 and 600 mg/kg in females.
(104-week study to be added later.)
Other Carcinoqenicity Studies
Long term inhalation studies with p-DCB have been conducted
in mice and rats. Groups of Swiss strain mice (75 males and 75 fe-
males per group) were exposed to airborne concentrations of 0, 75
or 500 ppm p-DCB five hours/day, five days/week for 57 weeks for all
female groups and the 500 ppm males and for 61 weeks for the 0 and
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V-85
75-ppro males. Males were sacrificed at these times. Females were
held unexposed until sacrifice after 75 or 76 weeks.
The original objective of this study was to assess the
chronic toxicity and carcinogenic potential of p-DCB in the mouse.
However, because of fighting among the males during the early
stages of the study and a high background incidence of respiratory
disease in both sexes resulting in high mortality rates, the investi-
gators felt that this objective was not attained satisfactorily.
The following information was gathered from the study, however.
At termination, blood samples were drawn from at least ten
males in each group to determine the blood levels of urea and glucose
and serum alanine and aspartate transaminase activities. Because of
insufficient or clotted samples, no urea or glucose levels were ob-
tained from the 500 ppm group. No significant changes in blood
glucose or plasma alanine transaminase activity were observed in
the 75 ppm group. Blood urea concentrations appeared to be slightly
reduced in the 75 ppm group, but this likely was due to the rela-
tively high control levels observed. There was some evidence of
an increase in the plasma aspartate transaminase activities in both
treatment groups, but this increase was not statistically signifi-
cant.
Urinalysis was performed for pH, glucose, bilirubin, specific
gravity, protein and coproporphyrin, in males only. No significant
differences were observed in any of the parameters. Several hemato-
logical parameters were studied in the males: hemoglobin levels,
packed cell volume, total white cell count and differential,
platelet count and red cell morphology, red cell count, mean red
-------
cell count, mean hemoglobin concentration and raethemoglobin content.
Femoral bone marrow smears also were examined. Slight reductions
in hemoglobin concentration and packed cell volume were observed in
isolated males in all three groups. These animals also showed
slight increases in methemoglobin. Among the 500 ppm-dosed animals,
there was a slight, but not statistically significant, decrease in
the mean total white cell count. No bone marrow changes were
observed.
While tissues from animals of both sexes and all treatment
groups were examined grossly, only those from females sacrificed
when moribund or at the end of the study were examined histologi-
cally. The "epithelial repair" observed in the nasal sinuses and
the "resolving pneumonia" seen in the lungs of both test and con-
trol animals were said to be related to the high incidence of
respiratory disease in the colony thought to be caused by a Sendai
virus. Lesions in the liver (hepatitis) and the kidney (inflamma-
tion) were seen in similar quantities in both control and test
animals. No significant increases in numbers of neoplastic lesions
were observed in either treatment group when compared with the con-
trol group run concurrently or with historical controls from this
laboratory.
From the data available, it was concluded that the admini-
stration of p-DCB by inhalation at levels up to 500 ppm for longer
periods of exposure, followed by a period of recovery, did not pro-
duce any significant non-neoplastic lesions or increase the number
or types of neoplastic lesions in female mice.
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V-87
A similar long term inhalation study was conducted in
Alderly Park Wistar-derived albino rats (Riley, et al_., 1980a) .
Groups of animals (76-79 animals/sex/group) were exposed to airborne
concentrations of 0, 75 or 500 ppm p-DCB five hours/day, five days/
week for 76 weeks. Survivors at this time were left unexposed for
36 additional weeks. Interim sacrifices (5 animals/sex/group) were
conducted at 26, 52 and 76 weeks. Body weights were determined
weekly until Week 13 and then monthly thereafter. Food and water
consumption was monitored biweekly until Week 15, then every six
weeks thereafter. Urinalysis, clinical chemistry and histopatholo-
gical parameters as described above for the mouse study also were
analyzed in the rat study. Hepatic aminopyrine demethylase acti-
vity also was measured at the 52 week sacrifice. Organ weights
were measured at each sacrifice date. •
No consistently significant effects on mortality rates, food
and water consumption, body weight gain, clinical chemistry, hema-
tology or histopathology were observed in either sex, at either
exposure dose or at any of the sacrifice times when compared with
controls at the same times. Group mean body weights in the high
dose females were significantly depressed from Weeks 4-38 except
at Week 10, but were not significantly different at Week 50 or
later. Increase in liver and kidney weights (both sexes at Weeks
76 and 112), heart and lung (both sexes at Week 112) and urinary
protein and coproporphyrin output (in males) were noted in animals
exposed at 500 ppm. No increases in tumor incidence or types
were produced at either dose in either sex. The investigators
concluded that, under the conditions of this study, p-DCB was not
-------
carcinogenic to rats at doses up to 500 ppm. An increase in hepa-
tic hyperplasia was seen in treated females, but not in controls
or treated males. Hemosiderosis was increased in treated males at
both doses, but not in females. Focal chronic hepatitis with
infiltration was increased in all treated animals, but signifi-
cantly so only in the animals exposed at 500 ppm. A slight in-
crease in myocardial calcification was seen in high dose males.
Adrenal hyperplasia was increased only in the low dose males. The
investigators concluded that non-neoplastic changes of a minor
nature occurred in the high dose animals, but were absent or insig-
nificant in the low dose animals.
-------
VI-1
VI. HEALTH EFFECTS IN HUMANS
•
Most of the poisoning incidents reported in the literature
resulted from inhalation. Accidental inhalation can occur either
at home or at work. There are several cases, however, when a
chlorinated benzene was accidentally or deliberately ingested.
Table VI-1 summarizes the available literature for ortho-
and para-dichlorobenzene. Again, no reports appear in the litera-
ture concerning m-DCB.
o-Dichlorobenzene
One case of sensitization to o-dichlorobenzene was re-
ported for a man who regularly handled window sashes dipped in the
compound (Downing, 1939). When applied to the skin, there was a
burning sensation after 15 minutes and for the duration of expo-
sure. The site of application showed a reddish hue which increased
up to 24 hours later, when blisters formed. A brown pigmentation
formed later and persisted for three months. The man was forced to
seek alternative employment.
Dupont (1938) described the reactions of a group of sewage
workers performing cleaning operations in the sewer at a point di-
rectly below a pipe discharging sewage from a dry-cleaning establish-
ment. Inhalation of the fumes caused irritation of the eyes and
upper respiratory tract and vomiting. Apparently, no deaths occur-
red, but no clinical follow-up was described.
An 18-year old female employee of a dry-cleaning shop was
admitted to the hospital with severe acute anemia (Gadrat, et al.,
1962). She had been employed as an ironer in that shop for about
6 months prior to admission. During her employment, she was
-------
Vi—i!
Table VI-1
Report of Human Exposure to o- and p-Dichlorobenzene
(Modified from Ware, West, 1977)
Compound Subject
Exposure
Symptoms
Clinical Report
Follow-up Studies Reference
OrDCB Sewage o-DCB effluents
workers from dry-clean-
ing establish-
ment above sewer
Eye and upper res-
piratory tract ir-
ritation, vomiting
o-DCB intoxication Not indicated
Dupont, 1938
47 year
old male
18 year
old fanale
40 year
old male
53 year
old male
o-DCB in dipping
solution for
window sashes,
occupational
Dry-cleaning
and dyeing shop
1940-1950 occu-
pational expo-
sure to solvent:
80% o-DCB, 15%
p-DCB
1932-1961, glue
containing 2%
o-DCB, methyl
ethyl ketone
& cyclohexane,
(no benzene or
homologues)
Water blisters on
face, hands, arms
Pallor, tiredness,
headaches, vomit-
ing, violent gas-
tric pains
Weakness, fatigue
Weakness, fatigue
Eczematoid derma-
titis due to o-DCB
Severe hemolytic
anemia: 1.5 x
erythocytes/mm-*
Not indicated
Downing, 1939
Chronic lymphoid
leukemia
Chronic lymphoid
leukemia, periph-
eral and abdominal
adenopathy, spleno-
megaly
10 months later Gadrat et al.,
erthyrocytes 1962
"excellent" but
leucocyte equil-
ibrium showed
tendency to
neutropenia
Treatment ongoing Girard, et al.,
1969
Died 1968
Girard et al.,
1969
-------
Table VI-1 (Continued)
Compound Subject
Exposure
Symptoms
Clinical Report
Follow-up Studies
Reference
o-DCB
p-DCB
15 year
old
female
60 year
old male
62 year
old male
19 year
old
female
60 year
old male
Wife of
male
above
36 year
old
female
Cleaned clothes
with products
containing 37%
o-DCB, (no ben-
zene or toluene)
1930 to 1960
shipping mono,
o-di- and tri-
chlorobenzene
p-DCB in bath-
room
Preparation of
p-DCB for 18
months
Heavy p-DCB moth
ball vapor in
house for 3 to
4 months
As above
p-DCB moth killer
in house
Initially hos-
pitalized with
ret reel av icul a r
adenopathy
Weakness, tired-
ness
Asthenia, dizzi-
ness
Asthenia, dizzi-
ness, weight loss
Weight loss,
loose bowels,
tarry stools,
numbness, clumsi-
ness
Weight and
strength loss,
abdominal swell-
ing, jaundice
Periorbital
swelling, intense
headaches, profuse
rhinitis
Acute myeloblastic
leukemia
Anemia: 3 x 106
erythrocytes/mm-*
Light hyperchromic
anemia, after 1
month, increase in
anemia, hypogran-
ulocytosis
Slight anemia,
reactional hyper-
leucocytosis
Acute yellow
atrophy of the
liver (confirmed
by autopsy)
Acute yellow
atrophy of the
liver (confirmed
by autopsy), spleno-
megaly
Exposure to p-DCB
Died 10 months later
of 100% peripheral
1eukoblastosis
Not indicated
Girard et al.,
1969
General hematologi-
cal improvement but
increase in hypogra-
nulocytosis at 6
months
Not indicated
Girard et al.,
1969
Perrin, 1941
Developed ascites
and died
Petit and
Champeix,
1948
Cotter, 1953
Died 1 year after
initial exposure
Cotter, 1953
Symptoms subsided
within 24 hours
Cotter, 1953
-------
Table VI-1 (Continued)
Compound
p-DCB
>
Subject
34 year
old
fanale
52 year
old male
Exposure
Demonstrating
p-DCB containing
products
p-DCB exposure
in fur storage
plant
Symptoms
Tiredness, nausea
headache, vomiting
Weakness, nausea,
blood, vomiting,
jaundice.
Clinical Report
Subacute yellow
atrophy and cirrho-
sis of the liver
Subacute yellow
atrophy of the
liver
Follow-up Studies
Not indicated
4 years later
reported "in good
health-
Reference
Cotter, 1953
Cotter, 1953
20 year p-DCB manufacture Loss of weight
old male 1 to 7 months exhaustion, de-
(+ 26 exposure crease of appetite
workmates)
53 year
old
fanale
3 year
old
male
19 year
old
female
12 to 15 year Cough, progressive
exposure to p-DCB dyspnea, fatigue,
moth balls in mucoid sputum
house
Methemoglobinemia
and other blood
pathologies
Pulmonary granulo-
matosis? focal
necrosis of liver
Played with p-DCB Cough, listlessness. Acute hemolytic
crystals black urine anemia
4-5 p-DCB pel-
lets ingested
daily for 2-
1/2 years
Increased patchy
pigmentation of
skin
21 year
old
pregnant
female
Pica for p-DCB
toilet blocks
first trimester
General tiredness,
mild anorexia, diz-
ziness, edema of
ankles
Due to p-DCB inges-
tion. Unsteadiness
and tremors on ceas-
ing consumption
thought to be psycho-
logical, not physio-
logical
Hemolytic anemia
All workers trans-
ferred to other
working environ-
ment
Not indicated
Complete recovery
Pigment returned to
normal
Wallgren,
1953
Weller and
Crellin,
1953
Hallowell,
1959
Frank and
Cohen, 1961
Healthy child
delivered several
months later
Campbell and
Davidson,
1970
-------
VI-5
continuously exposed to fumes of cleaning solution containing 95%
o-DCB and 5% p-DCB. She exhibited pallor, weakness, headaches,
vomiting and severe pains. She was diagnosed as having a severe
hemolytic anemia (1,500,000 RBCs/mm3), accompanied by leukocytosis and
polynucleosis, and the presence of some immature elements belonging
to the granulocytic and erythrocytic series. Vigorous treatment
and a change of employment resulted in essentially a full recovery.
Girard, e_t al_. (1969) reported three cases of leukemia which
they attributed to chronic exposure to o-dichlorobenzene (o-DCB).
One man hospitalized for chronic lymphoid leukemia worked with a
solvent containing 80% o-DCB and 15% p-DCB for 10 years. A girl
hospitalized with acute myeloblastic leukemia died 10 months later
of peripheral leukoblastosis. She reportedly had a neurotic compulsion
to remove dirt and grease stains from her clothes, which she did
repeatedly with a product containing 37% o-DCB (no benzene or
toluene). Another man exposed to a glue containing 2% o-dichloro-
benzene, methyl ethyl ketone and cyclohexane for a period of 29
years died of chronic lymphoid leukemia. No further details of
these incidents were given.
Girard, et al . (1969) also reported the case of a 60-
year old male, who for 30 years had worked in a job during which
he had been in contact with mono-, o-di- and trichlorobenzene.
At the time he was seen by the authors, he exhibited symptoms
of weakness and tiredness. Clinical studies revealed that he
suffered from anemia, with an erythrocyte count of 3 million/mm3.
No follow-up of this individual was described.
-------
VI-6
In cases where moderate exposures to p-dichlorobenzene
were documented, patients complained of severe headaches, profuse
rhinitis and periorbital swelling for approximately 24 hours after
exposure (Cotter, 1953; Campbell and Davidson, 1970). Anorexia,
nausea, vomiting, weight loss and yellow atrophy of the liver were
reported for high exposure concentrations (Petit and Champeix,
1948; Cotter, 1953; Hallowell , 1959).
Wallgren (1953) reported loss of weight, exhaustion, decrease
of appetite and blood dyscrasias in 27 men who manufactured p-di-
chlorobenzene for 1 to 7 months. Cotter (1953) described the
case of a woman who demonstrated products containing p-DCB and
who complained of tiredness, nausea, headache and vomiting. Clinical
studies showed that she had subacute yellow atrophy and cirrhosis
of the liver.
Heavy use of p-dichlorobenzene as either a moth-repellent
or a deodorizer apparently resulted in weakness, nausea, vomiting
of blood and jaundice (Perrin, 1941; Cotter, 1953; Weller and Crellin,
1953). One man and his wife died within months of each other of
acute yellow atrophy of the liver (confirmed by autopsy). Their
house was apparently saturated with p-DCB moth ball vapor for a
period of at least three to four months (Cotter, 1953).
There are at least two reports of deliberate ingestion of
p-dichlorobenzene. One woman who developed a pica for p-DCB during
the first trimester of her pregnancy complained of general tired-
ness, mild anorexia, dizziness and edema of the ankles. She was
hospitalized with hemolytic anemia and delivered a healthy child
several months later (Campbell and Davidson, 1970). Another
-------
VI-7
wontanr who ingested 4 to 5 p-DCB pellets (size not indicated) daily
for 2 1/2 years complained about increased patchy pigmentation.
Unsteadiness and tremors occurred when she stopped taking the
pellets, but these symptoms were thought to be due to psychological
rather than physiological withdrawal (Frank and Cohen, 1961).
-------
VII-1
VII. MECHANISM(S) OF TOXICITY
Of the various toxic effects occurring after exposures
to the dichlorobenzenes, information on the mechanisms of toxi-
city is available only for the necrosis noted in the liver, which
perhaps also can be applied to similar changes in the kidney and
lung, and for the induction of porphyria via acceleration of
synthesis in the heme pathway.
Many workers have studied the possibility that cellular
damage caused by many drugs and xenobiotics is mediated via chemi-
cally reactive metabolites. Many of the metabolites formed are
chemically inactive, but certain of the metabolites such as the
arene oxides or epoxides may interact with physiological or bio-
chemical processes, causing either pharmacological or toxicologi-
cal effects.
Studies of halogenated benzenes, including the dichloro-
benzenes, demonstrate that hepatic necrosis produced on exposure
to these compounds results from their conversion to reactive toxic
intermediates. Reid and co-workers have shown that an increase in
toxicity can be correlated with an increase in covalent binding of
metabolites to proteins within liver cells. This relationship can
be seen both in. vivo and ijn vitro. In the presence of the result-
ing hepatic necrosis, an increase in mercapturic acid excretion can
be measured, as well as a decrease in available glutathione levels
(see Table VII-1). This suggests that with sufficient depletion of
glutathione (greater than 20-25%), the liver loses its ability to
"detoxify" the chemical by complexing with the substance to form a
less reactive substance, and, thus, proportionately more reactive
-------
Table VII-1
Covelent Binding, Hepatotoxicity and Mercapturic Acid Excretion oE
Halogenated Benzene Derivatives
(Reid et aU, 1971j Reid et aU, 1973; Reid and Krishna, 1973)
(After Ware and West, 1977)
Compound
Monobronobenzene
Monochl orobenzene
Monoiodobenzene
Monofl uorobenzene
o-Dichlorobenzene
p-Dichl orobenzene
(1 nMAg)
(1 nMAg)
(1 nMAg)
(1 nMAg)
(0.5 nMAg)
(0.5 nMAg)
Covalent binding
(nM/mg protein
+ S.E.)
(N=6)
0.534 + 0.050+
0.604 + 0.044+
0.323 + 0.054
0.060 + 0.004+
0.234 + 0.015S
0,021 + 0.002S
Hepatic
necrosis
Yes
Yes
Yes
No
Yes
No
Mercapturic Gtutathione
acid excretion concentration
(% of control)*
3 +
3 +
3 +
+
2 +
+
67
Not detennined
66f
82
48f
101
* GLutathione concentration determined 3 hours after administration of the hydrocarbon.
+ Killed at 24 hourg
t P<0.01 compared with control.
S Killed at 6 hours.
-------
VII-3
metabol'ite is available to interact with tissue proteins, resulting
in cellular and tissue damage. A threshold does seems to exist for
the halobenzene-induced necrosis. (Reid, et al_., 1971; Reid, et
al_., 1971; Reid and Krishna, 1973). The stimulation of metabolism
by pretreatment with phenobarbital potentiates hepatic damage in
rats, as can be seen in Table VII-2. Conversely, blocking metabolism
by SKF-525A (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride)
or piperonyl butoxide (a pesticide synergist) prevents their hepa-
toxicity (Reid et al_., 1973; Reid and Krishna, 1973).
Just as there are species difference in metabolism there
are also differences in the rate and degree of metabolism of a
halogenated benzene in different organs, i.e., lung may differ from
kidney which differs from liver, etc. Phenobarbital does not
increase covalent binding in the lung as shown for the liver if
administered before exposure to o-DCB (Reid, e_t al_., 1973; see
Table VII-3). Nevertheless, both the lung and kidney exhibit
pathology following exposure to the dichlorobenzenes (Hollingsworth,
_et al_., 1956; Battell e-Columbus, 1979b). The primary mechanism of
toxicity in these tissues is likely to be the same as that described
in the liver: necrosis due to binding of the reactive metabolite
to cellular and tissue proteins, thereby interfering with the
normal physiological and biochemical processes.
The halobenzenes, the dichlorobenzenes among them, also
have been si-town to i-nditee perptiyria (Rimington and Ziegler, 1963;
Carlson, 1977, Battelle-Columbus, 1978c, 19781, 1979a, 1979b).
This condition, a disturbance in porphyrin metabolism, is character-
ized by increased formation and excretion of porphyrin precursors,
-------
Table VI1-2
Effect of Phenobarbital and SKF-525A Administration on Cbvalent Binding of Halogenated
Benzene Derivatives to Rat Liver Protein Ir± Vivo 6 Hours After Administration
(Reid et aU, 1973; Reid and Krishna, 1973)
(After Ware and West, 1977)
Control
(nM/mg protein
+ S.E.)
(N=6)
Phenobarbital
(nM/mg protein
+_ S.E.)
(N=6)
Phenobarbital +
SKF-525A3
(rm/mg protein
_+ S.E.)
(N=6)
Monobromobenzene-l^C (1 mM/kg)
Monochlorobenzene-l^C (1 mM/kg)
Monoiodobenzene-14C (1 mMAg)
Monofluorobenzene-^C (1 mM/kg)
o-Dichlorobenzene-^c (Q.5
p-Dichlorobenzene-14C (0.5
0.267 +_ 0.034
0.364 jf 0.053
0.090 j- 0.015
0.085 HH 0.015
0.234 +_ 0.015
0.021 + 0.002
0.550 4; 0.031+
1.268 + 0.278^
0.545 ± 0.129+
0.054 + 0.008
0.308 + 0.038
0.012 + 0.001+
0.036 +_ 0.024f
0.438 + 0.144§
0.666 4 0.304
0.052 +_ 0.005
0.186 +_ 0.014§
0.006 + 0.001f
a Diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) (75 mg/kg i.p.) was given 1 hour
before the hepatotoxin.
+ P<0.01 compared with controls.
* P<0.01 compared with phenobarbital alone.
§ P<0.02 compared with phenobarbital alone.
-------
Table VI1-3
Binding of Aromatic Hydrocarbons in Rat Lung:
Effect of Phenobarbital*
(Reid et aK, 1973)
(Modified from Ware and West, 1977)
Compound
o-Dichlorobenzene-^C ,
0.5 mM/kg
p-Dichlorobenzene-l^C,
0.5 mMAg
Time of
Sacrifice
(hour)
6
24
6
24
Binding of Hydrocarbon in Lung
(mumole/mg protein)
Control
27.3 + 1.4
20.9 + 2.4
4.6 -f 0.2
3.4 + 0.4
Phenobarbital
18.9 +
15.3 +
3.4 4-
1.8 +
2.7 (p<0.05)
2.2
0.5
0.2
Values are the means -f SE of 6 animals.
-------
vii-b
cutaneous photosensitivity, frequent hemolytic anemia and spleno-
megaly. Acute abdominal and nervous system manifestations may also
occur. As was described in some detail earlier, exposure to the
dichlorobenzenes leads to the induction of the mitochondrial
enzyme, A -aminolevulinic acid synthetase, resulting in an increased
production from heme of aminolevulinic acid and then other constit-
uents of the heme synthetic pathway (Rimington and Ziegler, 1963;
Poland, ejt al_., 1971; Ariyoshi, £t al_., 1975). When these levels
exceed those needed for maintenance of the system, clinical
symptoms may be manifested.
-------
viii-i OCT 3 I S83
VIII. QUANTIFICATION £F TOXICOLOGICAL EFFECTS
The quantification of toxicological effects of a chemical
consists of an assessment of the non-carcinogenic and carcino-
genic effects. In the quantification of non-carcinogenic
effects, an Adjusted Acceptable Daily Intake (AADI) for the
chemical is determined. For ingestion data, this approach
is illustrated as follows:
Adjusted ADI = (NOAEL or MEL in mg/kg)(70 kg)
(Uncertainty factor)(2 liters/day)
The 70 kg adult consuming 2 liters of water per day is used
as the basis for the calculations. A "no-observed-adverse-effect-
level" or a "minimal-effect-level" is determined from animal
toxicity data or human effects data. This level is divided
by an uncertainty factor because, for these numbers which are
derived from animal studies, there is no universally acceptable
quantitative method to extrapolate from animals to humans,
and the possibility must be considered that humans are more
sensitive to the toxic effects of chemicals than are animals.
For human toxicity data, an uncertainty factor is used to
account for the heterogeneity of the human population in
which persons exhibit differing sensitivity to toxins. The
guidelines set' forth by the National Academy of Sciences
(Drinking Water and Health, Vol. 1, 1977) are used in estab-
lishing uncertainty factors. These guidelines are as follows:
an uncertainty factor of 10 is used if there exist valid
experimental results on ingestion by humans, an uncertainty
factor of 100 is used if there exist valid results on long-
-------
VIII-2
term feeding studies on experimental animals, and an uncertainty
factor of 1000 is used if only limited data are available.
In the quantification of carcinogenic effects, mathematical
models are used to calculate the estimated excess cancer
risks associated with the consumption of a chemical through
the drinking water. EPA's Carcinogen Assessment Group has
used the multistage model, which is linear at low doses and
does not exhibit a threshold, to extrapolate from high dose
animal studies to low doses of the chemical expected in the
environment. This model estimates the upper bound (95%
confidence limit) of the incremental excess cancer rate that
would be projected at a specific exposure level for a 70 kg
adult, consuming 2 liters of water per day, over a 70 year
lifespan. Excess cancer risk rates also can be estimated
using other models such as the one-hit model, the Weibull
model, the log it model and the probit model. Current
understanding of the biological mechanisms involved in cancer
do not allow for choosing among the models. The estimates
of incremental risks associated with exposure to low doses
of potential carcinogens can differ by several orders of
magnitude when these models are applied. The linear, non-
threshold multi-stage model often gives one of the highest
risk estimates per dose and thus would usually be the one
most consistent with a regulatory philosophy which would
avoid underestimating potential risk.
The scientific data base, which is used to support the
estimating of risk rate levels as well as other scientific
-------
VI11-3
endeavors, has an inherent uncertainty. in addition, in
many areas, there exists only limited knowledge concerning
the health effects of contaminants at levels found in drinking
water. Thus, the dose-response data gathered at high levels of
exposure are used for extrapolation to estimate responses at
levels of exposure nearer to the range in which a standard
might be set. In most cases, data exist only for animals; thus,
uncertainty exists when the data are extrapolated to humans.
When estimating risk rate levels, several other areas of
uncertainty exist such as the effect of age, sex, species
and target organ of the test animals used in the experiment,
as well as the exposure mode and dosing rates. Additional
uncertainty exists when there is exposure to more than one
contaminant due to the lack of information about possible
additive, synergistic or antagonistic interactions.
Non-Carcinogenic Effects
The principal toxic effects of the dichlorobenzenes in
humans and other animals from both acute and longer-term
exposures include central nervous system (CNS) depression, blood
dyscrasias (granulocytopenia, hemolytic anemia and leukemias),
lung, kidney and liver damage. In addition to liver
necrosis, the dichlorobenzenes also can produce porphyria.
The appearance and intensity of these and other adverse
effects are dependent upon dose and duration of exposure.
Death following high level acute exposure usually results
from the CNS effects (primarily, respiratory failure). Deaths
in humans have been reported following accidental exposure.
-------
VIII-4
Several investigators have determined the acute lethal
dose levels after exposure to ortho- and para-dichlorobenzene
in several species. These data are summarized in Table VIII-1
(same as Table V-3).
Varshavskaya (1968), in her comparative studies on the
adverse effects of the lower chlorinated benzenes, showed that,
when determining the LD50S, o-DCB was slightly less toxic in mice
and rats than was monochlorobenzene (MCB), and that p-DCB was
even less toxic than either MCB or o-DCB. o-DCB was slightly
more toxic than MCB and p-DCB in rabbits and guinea pigs. Thus,
in general, one may conclude that o-DCB is acutely more toxic
than is p-DCB.
A number of studies with o-dichlarobenzene and p-dichloro-
benzene are available in which dose-response data are described
and which allow the identification of no-observed-adverse-effect-
levels (NOAELs) following longer-term or lifetime periods of
exposure. Animals were exposed both by gavage and by inhalation
for periods of time constituting a subchronic or chronic
exposure. Since several gavage studies are available for
evaluation, only these, and not the inhalation studies, will
be used in the quantification of toxicological effects, as
this route of exposure is more appropriate for the development
of allowable exposure levels in drinking water. Comparable
-steadies with the meta- isomer of dichlorobenzene have not
been reported. Therefore, it will be assumed that ADIs
developed for o-dichlorobenzene also will be appropriate for
m-dichlorobenzene. This assumption is defensible for several
-------
VI I I — ±
Table VIII-1
Acute Toxicity Data for o- and p-Dichlorobenzene
Animal
Route
LCLp
o-Dichlorobenzene
Rat Oral
Rat Oral
Mouse Oral
Rabbit Oral
Guinea Pig Oral
Guinea Pig Oral
Rat
Guinea Pig
Guinea Pig
Inhal
Inhal
Inhal
500 mg/kg
2138 mg/1
2000 mg/1
1875 mg/1
3375 mg/1
2000 mg/kg
821 ppm/7 hr
800 ppm/7 hr
800 ppm/24 hr
Reference
NIOSH, 1978
Varshavskaya,
Varshavskaya,
Varshavskaya,
Varshavskaya,
Hollingsworth,
1958
Hollingsworth,
Hollingsworth,
Cameron, et al
1968
1968
1968
1968
et al
195,8
1958
., 1937
p-Dichlorobenzene
Rat
Rat
Rat
Mouse
Rabbit
Guinea
Guinea
Mouse
Pig
Pig
Oral
Oral
Oral
Oral
Oral
Oral
Oral
SC
500 mg/kg
2500 mg/kg
2138 mg/1
3220 mg/1
2812 mg/1
7593 mg/1
2800 mg/kg
5145 mg/kg
-------
VIII-6
reasons: 1) in general, in mutagenicity and other short-term
tests, the meta isomer behaved more like the ortho isomer
than like the para isomer, and 2) short- and longer-term
studies with o- and p-DCB suggest that the ortho isomer is
somewhat more toxic than the para isomer on a mg/kg basis.
Thus, to assume that the meta isomer is more similar to the
ortho isomer would be consistent with a regulatory philosophy
that seeks to avoid underestimating the potential risk to
human health.
o-Dichlorobenzene (and/or m-Dichlorobenzene)
Hollingsworth, e_t aJL. (1958) gave rats a series of 138
doses of o-DCB over a period of 192 days ( 18.8, 188 or 376
rag/kg/day, five days a week) by intubation. No adverse effects
were observed at the lowest dose. With the intermediate dose,
a slight increase in liver and kidney weight was noted. At the
highest dose, there was a slight decrease in the weight of the
spleen and a modest increase in the weight of the liver accompanied
by cloudy swelling.
If one were to assume that the results of this study were
appropriate for use in developing an acceptable daily intake
(ADI), it would be derived thusly:
18.8 mg/kg/day x 70 kg x 1.0 x 5 = 0.94 mg/day
100 x. 10 7
(for a 70 kg adult)
-------
VIII-7
Where: 18.8 mg/kg/day = NOAEL
70 kg = weight of protected individual
1.0 = ratio of administered dose absorbed
5/7 = conversion of 5 day/week dosing
regimen to 7 day/week
100 = uncertainty factor, appropriate
for use with NOAEL from animal
studies with no comparable human
data ( longer-term exposure duration)
10 = uncertainty factor, appropriate
for use with data from exposure
duration significantly less than
1 ifetime
Varshavskaya (1968) administered o-DCB orally to white
rats for nine months at doses of 0.001, 0.01 or 0.1 mg/kg/day.
Effects were observed at the two higher doses. The author
reported an inhibition of mitosis in the bone marrow, as well
as neutropenia and abnormal conditioned reflexes. These changes
in the blood profile can be important in that they could be
precursors to pancytopenia or leukemia. In this study, however,
no carcinogenic activity was observed. Also, at the two
higher doses, there was an increase in acid phosphatase and
a decrease in alkaline phosphatase. At the highest dose, a marked
increase in the amount of 17-ketosteroids in the urine occurred.
This was attributed to hyperplasia of the adrenal cortex, as an
increase in adrenal weight and a decrease in ascorbic acid content
of the adrenals also were observed. The O.OTTL mgAg/Say
dose had no observable effects on any of the parameters studied.
-------
VIII-8
If one were to assume that the results of the Varshavskaya
study were appropriate for use in developing an ADI for o-DCB,
it could be derived thusly:
0.001 mg/kg/day x 70 kg x 1.0 = 0.00007 mg/dav
100 x 10 *
(for a 70 kg adult)
Where: 0.001 mg/kg/day = NOAEL
70 kg = weight of protected individual
1.0 = ratio of administered dose absorbed
100 = uncertainty factor, appropriate
for use with NOAEL from animal
studies with no comparable human
data ( longer-term exposure duration)
10 = uncertainty factor, appropriate
for use with data from exposure
duration significantly less than
lifetime
Subchronic gavage studies with o-DCP in mice and rats
were conducted to assist in dosage selection for the NTP 104-
week carcinogenicity study (Battelle-Columbus, 1978c,i).
Single doses in corn oil were administered 5 days/week for
13 weeks. Treated mice received doses of 30, 60, 125, 250 or 500
nig/kg/day; treated rats received 30, 60, 125, 250 or 500 mg/kg/day.
Controls received corn oil. Other protocol details are
described in Chapter V.
In the mice, body weight gain was decreased significantly
in animals of both sexes at the 500 mg/kg dose level and in males
at the 250 mg/kg dose. Of the hematological parameters tested,
white cell counts of treated males were lower than those of
control males. It was suggested that this was due to lower
-------
than normal control values, as observed typically in that
laboratory. Since individual data were not available for
statistical analysis, it cannot be shown whether or not
the differences between the controls and the treated groups
were statistically significant. But, since there is at least
anecdotal evidence to suggest a possible relationship between
exposure to o-DCB and leukemia in humans, it would be prudent
to evaluate these results in greater depth.
Increased, but apparently not statistically-significant,
blood alkaline phosphatase levels were observed in males
receiving 125 and 250 mg o-DCB/kg/day. SGPT levels were
increased significantly in the two surviving males receiving
500 mg/kg/day, due to the high value for the one animal
exhibiting hepatocellular necrosis. Urine volume was greater
in the treated animals than in controls, but no record of
fluid intake was kept. The significance of this observation
is unknown, since no parameters of the urinalysis measured
were altered, except for the decreases in specific gravity and
creatinine levels.
Males receving 500 mg/kg exhibited higher uroporphyrin levels
than did male controls; females at that dose showed higher coproporphyin
levels. These parameters were not measured in the other dose
groups. In addition, a dose-dependent increase in liver proto-
porphyrin was observed in the females, but not in the males.
Even without statistical evaluation, one could observe that
liver weights in the highest dose group of both sexes were
increased significantly. An increase of lesser magnitude
-------
VIII-10
was" observed in the females at the 250 mg/kg/day dose level.
Histopathological examination of several tissues revealed
that no lesions were apparent in animals treated with 125 mg/kg/day
or lower doses of o-DCB.
Interpretation of the results of the study suggests that
an oral NOAEL of 125 mg/kg/day could be identified in mice, if
one discounts the observations concerning the white cell counts.
In the absence of the raw data, at this time, it will be
assumed that the investigators have interpreted this finding
correctly. Under these circumstances, if one were to assume
that the results of the mouse 90-day study were appropriate
for use in developing an ADI, it could be derived thusly:
125 mg/kg/day x 70 kg x 1.0 x 5 - 6<25 ma/day
100 x 10 x 7
(for a 70 kg adult)
Where: 125 mg/kg/day = NOAEL
70 kg = weight of protected individual
1.0 = ratio of administered dose absorbed
5/7 = conversion of 5 day/week dosing
regimen to 7 day/week
100 = uncertainty factor, appropriate for
use with NOAEL from animal studies
with no comparable human data
(longer-term exposure duration
10 = uncertainty factor, appropriate for
use with data from exposure duration
of significantly less than lifetime
In the rats, body weight gain was decreased significantly
at the two higher doses (250 and 500 mg/kg/day). Cholesterol
levels were increased in males at the two higher doses and in
females at the three higher doses. The combined alpha-globulin
-------
VHI-li " ' "
fraction appeared to be increased in females at the highest dose
and males at the two highest doses. As in the mice, urinary
output was increased substantially, with concomitant decreases
in specific gravity and creatinine-
Both uro- and coproporphyrin levels in the urine increased
significantly in aninmals receiving 500 mg/kg/day. No measurments
of these parameters were made in other dose groups. However,
liver protoporphyrin levels were not changed. Absolute and
relative liver weights were increased in the 250 and 500
mg/kg/day groups. Histopathological examination of tissues
revealed liver and kidney changes in the highest dose group,
and liver changes in the 250 mg/kg/day groups. As for the
mice, 125 mg/kg/day was identified as the NOAEL for the rats.
If one were to assume that the results of the study in which
rats were exposed to o-DCB subchronically were appropriate for
use in developing an ADI, it could be derived thusly:
125 mg/kg/day x 70 kg x 1.0 x 5 = 6.25 mg/day
100 x 10 x 7
(for a 70 kg adult)
Where: 125 mg/kg/day = NOAEL
70 kg = weight of protected individual
1.0 = ratio of administered dose absorbed
5/7 = conversion of 5 day/week dosing
regimen to 7 day/week
100 = uncertainty factor, appropriate for
use with NOAEL from animal studies
with no comparable human data
(longer-term exposure duration)
-------
VIII-16
Where: 337.5 mg/kg/day = NOAEL
70 kg = weight of protected individual
1.0 = ratio of administered dose absorbed
5/7 = conversion of 5 day/week dosing
regimen to 7 day/week
100 = uncertainty factor, appropriate for
use with NOAEL from animal studies
with no comparable human data
(longer-term exposure duration)
10 = uncertainty factor, appropriate
for use with data from exposure
duration significantly less
than lifetime
In the rats, no significant differences in food consumption
or body weight gain were observed between treated and control
animals of either sex at any dose. Microscopically, there
was an increased incidence and severity of-renal cortical
degeneration in males receiving the two highest doses, as had
been observed in the first subchronic rat study. Females showed
no significant changes at any dose level. The NOAEL for this
study was established at 150 mg/kg/day.
If the results of this rat subchronic study were considered
to be appropriate for use in developing an ADI, it could be
derived thusly:
150 mg/kg/day x 70 kg x 1.0 x 5 = 7.5 mg/day
100 x 10 7
(for a 70 kg adult)
-------
VIII-17
Where: 150 mg/kg/day = NOAEL
70 kg = weight of protected individual
1.0 = ratio of administered dose absorbed
5/7 = conversion of 5 day/week dosing
regimen to 7 day/week
100 = uncertainty factor, appropriate for
use with NOAEL from animal studies
with no comparable human data
(longer-term exposure duration)
10 = uncertainty factor, appropriate
for use with data from exposure
duration significantly less
than lifetime
Quantification of Non-carcinogenic Effects
Table VIIl-2 summarizes the ADIs derived from the
available gavage studies on o- and p-dichlorobenzene which
contain adequate dose-response data identifying NOAELs. As
can be seen, a wide range of numbers was derived. For o-DCR
(and m-DCB), the ADIs range from 0.00007 mg/day (Varshavskaya,
1968) to 60 mg/day (NTP, 1982; preliminary report). For
p-DCB, the ADIs range from 0.94 mg/day (Hollingsworth, et al.,
1956) to 16.9 mg/day (Battelle-Columbus, 1980a). However, a
rationale can be presented by which certain of these numbers
can be eliminated and others supported.
Ortho-dichlorobenzene (and, meta-dichlorobenzene)
While the Varshavskaya study suggests that effects can
be seen at very low doses when compared with the other studies,
little in the way of quantitative experimental detail was
presented in her publication. Therefore, it is difficult to
-------
VIII-19
assess fully the results presented, and one cannot conclude
that this paper should be the basis for the development of an
Adjusted Acceptable Daily Intake (AADI).
The fact that the ADIs generated from the chronic studies
in the NTP bioassay are larger than those derived from the
subchronic studies preceding them would suggest that the 10-
fold uncertainty factor used to estimate a chronic ADI from
subchronic data may be unnecessarily large for this compound.
However, the NTP Board of Scientific Counselors has not yet
approved the report prepared on the bioassay of o-DCB. Therefore,
it is prudent to reserve judgment on its validity until such
time as the report is approved.
The results of Hollingsworth, et al. (1958) suggest
an ADI of 0.94 mg/day while those of the subchronic studies
preceding the NTP bioassay suggest an ADI of 6.25 mg/day.
Each ADI was derived from an NOAEL ( 18.8 mg/kg vs. 125 mg/kg,
respectively). Since the highest NOAEL should be used to
derive an ADI, it is more appropriate to use the NOAEL established
in the NTP subchronic studies, than the NOAEL from the Hollingsworth
study. In addition, the NTP subchronic studies employed
additional doses {5 vs 3), thereby allowing for a more precise
identification of a NOAEL. It also should be noted that the
minimal effect dose identified in the Hollingsworth study
(188.8 mg/kg) is somewhat higher than the NOAEL established
in the NTP subchronic studies.
For o-DCB ( and, m-DCB), then, if one were to use the ADI
from the NTP subchronic studies to determine the AADI, it would
be derived thusly:
-------
AADI „ ADI = 6.25 mq/day = 3.125 mg/l/day
21 21
This AADI assumes that the protected individual (.a 70 kg
adult) drinks 2 liters of water/day and that the sole source
of exposure to o- or m-dichlorobenzene is via that drinking
water. [It is important to note that the odor threshold for
o-DCB and m-DCB in water has been identified as 0.01 and 0.02
ppm, respectively (Kolle, 1972). Therefore, any MCL for
these compounds may have to consider the asthetic, as well as
the toxic, consequences of exposure to these compounds in
drinking water].
p-Dichlorobenzene
The results of the Hollingsworth, et al. (1956) study
suggest an ADI of 0.94 mg/day while those from the subchronic
studies preceding the NTP bioassay suggest ADIs of 7.5 mg/day
(rats) and 16.9 mg/day (mice). It is apparent from these
three studies, as well as the acute toxicity studies described
earlier, that the rat is more sensitive to p-DCB toxicity
than is the mouse. Therefore, to be consistent with the
philosophy that one uses data from the most sensitive
animal species when estimating the potential risk to the
human, the data from the experiments in the rat should be
used in deriving an AADI.
The ADI derived from the Hollingsworth study was based
upon an NOAEL of 18.8 mg/kg; the ADI from the NTP subchronic
study in the rat was derived from an NOAEL of 150 mg/kg.
-------
Since the highest NOAEL should be used to derive an ADI, it
is more appropriate to use the NOAEL established in the NTP
subchronic study than the NOAEL from the Hollingsworth study.
The NTP subchronic study employed additional treatment doses
(5 vs. 3), thereby allowing for a more precise identification
of an NOAEL. In addition, it should be noted that the minimal
effect level identified in the Hollingsworth study (188 mg/kg)
was somewhat higher than the NOAEL established in the NTP
subchronic study.
As with o-DCB (and m-DCB), it may be that any ADIs derived
from the as yet unreported NTP chronic studies would be
larger than those derived from the subchronic studies preceding
them because the 10-fold uncertainty factor applied to accommodate for
the difference in duration of exposure may be unnecessarily large.
However, until the report has been approved by the NTP Board of
Scientific Counselors and published as final, it is appropriate
to develop an ADI and AADI based upon the subchronic data. The
AADI can be reevaluated at a later date.
For p-DCB, then, if one were to use the ADI from the NTP
subchronic study in rats to determine the AADI, it would be
derived thusly:
AADI _ ADI = 7.5 mq/day = 3.75 mg/l/day
~ 2 1 21
-------
VIII-18
Table VIII-2
Possible ADIs for the Dichlorobenzenes
Compound
Experiment
Possible ADI
o-DCB/m-DCB
Hollingsworth, et al
(1958)
subchronic rat
Varshavskaya (1968)
subchronic rat
Battelle-Columbus
(1978c)
subchronic mouse
Battelle-Columbus
(19781)
subchronic rat
NTP (1982)
chronic mouse
(preliminary report)
NTP (1982)
chronic rat
(preliminary report)
0.94 mg/day
0.07 ug/day
6.25 mg/day
6.25 mg/day
60 mg/day
60 mg/day
p-DCB
Hollingsworth, et al .
(1956)
subchronic rat
Battelle-Columbus
(1980a)
subchronic mouse
Batt el1e-Columbus
(1980b)
subchronic rat
0.94 mg/day
16.9 mg/day
7.5 mg/day
-------
VIII -22
i'M.;..<3 A ADI ^ssv.i-sos that <:ho p r: '".' '- •- ' ' I: e '1 i ; id L v i. ^I'er
the asthotic, as~ we 1.1 as the toxic, ociu-.3q.K- . cos of exL'0*ure
to p-dichlorobenxene in drinking water].
C_a i-^if. og o n ic_ E f_f -^c 1; s
Poth o-nCB ^-!id p-DCB have bo<-,-n tnstod by fja\',-ge
for their carcinogenic potential in F344 rats a iid Mfi(j3Fl i;'ii;o
in Lhe NT? Pioassay Prog can. A draft report of ti-.o ro-:-nl!:s
of the studios -vith o-DCB is a vr. i lab lo ( ^TP , 1^82). A •..•opo'.'t
ol: the results of the studies with p--DCB has not boen ;nade
a v.i i lab 1 e o s yet.
The preliminary assessment of the data from the studies
on o-DCB suggests that, under the tost conditions, o-DCB does
not possess carcinogenic potential- Kov.'cvoc, until the N;TP
Board of Scientific Counselors approves the draft no port ,
this asnosc^icnt nust remain preliminary.
Since no report, preliminary or oth-'i c.v/i. se , is available
on the results of the studies v/ith p-DCB, no asscsr.nont of
its carcinogenic potential can be made at this time.
Quantification of Carcinoge^nic^ !•: f_0 ^c t s_
Preliminary assessment of i:ha \'TP ^io.-.say on o-DCB
suggests that it was not carcinogenic u.-.dor the con-.! i i-.ions
-------
VTTI-23
o f. i-'"> '5 o x p e c i.
-------
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