FINAL
ECAO-CIN-424
United Stares September, 1968
Revised July, 1991
*vEPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
HEXACH10R0BENZENE
Prepared for
OFFICE OF WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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DISCLAIMER
This document has been reviewed In accordance with the U.S. Environ-
mental Protection Agency's peer and- administrative review policies and
approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use. -
11
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Final Drinking Water Criteria Document for
HEXACHLOROBENZENE
5. ruWLiNG .NLM52.75
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Environmental Criteria and Assessment Office (ECAO)
Office of Health and Environmental Assessment (OHEA)
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ECO/OHEA
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! ing the MCLG. To achieve this objective, data on pharmacokinetics, human exposure,
' acute and chronic toxicity to animals and humans, epidemiology and mechanisms of
toxicity are evaluated. Specific emphasis is placed on literature data providing
dose-response information. Thus, while the literature search and evaluation per-
• formed in support of this document has been comprehensive, only the reports con-
sidered most pertinent in the derivation of the MCLG are cited in the document.
The comprehensive literature data base in support of this document includes
information published up .to 1987; however, more recent data may have been added
during, the review process. Final revisions and editorial changes were made in
1 1991.
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FOREWORD
Section 1412. (b)(3)(A) of the Safe Drinking Water Act, as amended In
1986, .requires the Administrator of the Environmental Protection Agency to
publish maximum contaminant level goals (HCLGs) and promulgate National
Primary Drinking Water Regulations for each contaminant, which. In the
judgment of the Administrator, may have an adverse effect-on public health
and which is known or anticipated to occur in public water systems. The
MCLG-Is nonenforceable and is set at a level at which no known or antici-
pated adverse health effects in humans occur, and which allows for an
adequate margin of safety. Factors considered in setting the MCLG Incltide-
health effects data and sources of exposure other than drinking water.
This document provides the health effects basis to be .considered in
establishing the MCLG. To achieve this objective, data on pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemi-
ology and mechanisms of toxicity are evaluated. Specific emphasis Is placed
on literature data providing dose-response information. Thus, while the
literature search and evaluation performed In support of this documenL has
been comprehensive, only the reports considered most pertinent in the deri-
vation of the MCLG are cited In the document. The comprehensive literature
data base in support of this document includes Information published up .to
1987 ; however, more recent data may have been added during the review
process. Final revisions and editorial changes were made in 1991.
When adequate health effects data exist. Health Advisory values for less
than lifetime exposures (1-day, 10-day and longer-term, -10% of an
individual's lifetime) are included in this document. These values are not
used in setting the MCLG, but serve as informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
Tudor Davis, Director
Office of Science
and Technology
James Elder, Director
Office of Ground Water
and Drinking Water
111
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DOCUMENT DEVELOPMENT
John Clcmanec, Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Special Note:
This document was developed from the comprehensive Informa-
tion found In the Health Assessment Document for Chlorinated
Benzenes (EPA 600/8-84-015F).
Scientific Reviewers and Contributors
Larry D. Anderson
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC
Henry A. Peters
Department of Neurology
University of Wisconsin
Madison, Wisconsin
W. Bruce Pelrano
Oavld J. Relsman
Michael L. Dourson
Franklin L. Mink
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Tom Sutter (revised Chapter VII)
Becky Clark
University of Cincinnati, College of
Medicine
Cincinnati. Ohio
Douglas L. Arnold
Health and Welfare Canada
Ottawa, Ontario
Canada
Larry R. Valcovlc
Reproductive Effects Assessment Group
U.S. Environmental Protection Agency
Washington, DC
William E. Pepelko
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Washington, DC
Document Preparation
Kevin Garrahan
Exposure Assessment Group
U.S. Environmental Protection Agency
Washington, DC
Technical Support
Office, Cincinnati
Services Staff, Environmental Criteria and Assessment
1 v
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TABLE OF CONTENTS
Page
I. SUMMARY . 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES. .............. II-1
CHEMICAL ANALYSIS ....... II-5
III. TOXICOKINETICS. - . . III-l
ABSORPTION. III-l
DISTRIBUTION III-3
METABOLISM. ........ . 111-16
EXCRETION . ..... Ill-21
SUMMARY .......... 111-20
IV. HUMAN EXPOSURE. IV-1
(To be provided by the Office of Drinking Water)
V. HEALTH EFFECTS IN ANIMALS V-l
ACUTE TOXICITY V-l
SUBCHRONIC TOXICITY V-2
CHRONIC TOXICITY V-l5
MUTAGENICITY V-19
CARCINOGENICITY . . V-2G
Hamster Studies V-20
Mouse Studies V-27
Rat Studies. V-33
Discussion of Rat Studies. V-48
Other Studies. ........ V-5Q
Carcinogenicity Summary. ........... V-52
REPRODUCTIVE AND TERATOGENIC EFFECTS V-55
SUMMARY V-61
VI. HEALTH EFFECTS IN HUMANS VI-1
EPIDEMIOLOGIC STUDIES . . VI-1
ACCIDENTAL INGESTION IN TURKEY. . VI-4
SUMMARY . VI-9
v
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TABLE OF CONTENTS (cont.)
Page
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
INTRODUCTION VIII-1
QUANTIFICATION OF NONCARCINOGENIC EFFECTS 1. . . . VIII-9
Derivation of 1-Day Health Advisory VIII-9
Derivation of 10-Day Health Advisory . VIII-9
Derivation of Longer-Term Health Advisory VI11-15
Assessment of Lifetime Exposure and Derivation
of DWEL VIII-18
CARCINOGENIC EFFECTS VII1-20
Procedures for the Determination of Unit Risk VIII-23
Summary of Quantitative Estimation . . VIII-40
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDAROS. VIII-40
Occupational . VIII-40
Food VI11 -41
Water . VI11-41
SPECIAL GROUPS AT RISK VII1-42
SUMMARY VI11-43
IX. REFERENCES IX-1
v1
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LIST OF TABLES
No, Title Page
II-l Synonyms, Trade Names and Identification Numbers
of Hexachlorobenzene. 11-3
11-2 Physical Properties of Hexachlorobenzene. ......... 11-4
III-l Storage and Excretion of 14C-HCB Administered Orally
In Arachls Oil in Rats. .. . 111 -4
III-2 Tissue Concentration (ppm) of 14C-Hexachlorobenzene and
Its Metabolites 1n Sprague-Dawley Rats 111 -6
III-3 Tissue Levels of HCB (ppm) 1n Adult Female Rhesus Monkeys . III-7
III-4 HCB Concentrations In Tissues of Male Beagles Receiving
Single Intravenous Doses of 1 mg/kg bw 1n Olive Oil ... . 111-10
III-5 Mean (+SE) Hexachlorobenzene Radioactivity {dpm/g) of-
Selected European Ferret Tissues. . 111 -14
111-6 Mean (+SE) HCB Radioactivity {dpmxl03) of European
Ferret Kits 111-15
III-7 Concentrations of HCB and Us Metabolites (mg/kg) 1n the
Liver and Kidneys of Male and Female Rats ......... 111-19
111-8 Hexachlorobenzene and Its Major Metabolites In the
Excreta of Different Animal Species ....... 111-22
V-1 Summary of Toxicity Studies on Hexachlorobenzene. ..... V-3
V-2 Tumor Incidence In Hamsters Given HCB In the Diet V-22
V-3 Effect of HCB on Hamsters: Liver Tumors and Other Liver
LesIons . V-26
V-4 Liver Tumor Incidence In Mice Fed HCB . ^ . . . V-28
V-5 Tumor Data on Mice Fed HCB. V-29
V-6 Body Weights of Female Agus Rats Fed Hexachlorobenzene
for 90 Weeks V-34
V-7 Growth Rates for Female Agus Rats on a Diet Containing
100 ppm HCB V-35
V-8 Dosage Levels 1n the Chronic Feeding Study of Hexa-
chlorobenzene 1n Sprague-Dawley Rats. ..... V-39
v 11
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LIST OF TABLES (cont.)
No. T1 tie Page
V-9 Liver and Kidney Tumors In Sprague-Dawley Rats Given
Hexachlorobenzene In the Diet for up to 2 years . V-40
V-10 Adrenal Tumors In Sprague-Dawley Rats Given Hexachloro-
benzene In the Diet for up to 2 Years V-42
V-ll Exposure Levels In the Chronic Feeding, 2-Generatlon
Study of Hexachlorobenzene 1n Sprague-Dawley Rats V-44
V-12 Tumors 1n Organs that Showed Statistical Differences
from Control 1n F-| Sprague-Dawley Rats Treated with
Hexachlorobenzene . . . V-46
V-13 Parathyroid and Adrenal Pheochromocytomas 1n Sprague-
Dawley Rats Maintained on Synthetic Diets of Varying
Vltmaln A Content and With or Without Hexachlorobenzene . . V-47
V-14 Qualitative Comparison of Tumor Development In Rats
Following Hexachlorobenzene Administration in
Different Studies V-51
V-15 Significantly Increased Incidence of Tumors In Animals
Given Hexachlorobenzene In Diet . V-54
VI-1 Results of Blood and Urine Analysis in Hen Employed In
a Chlorinated Solvents Plant, 1974-1977 . VI-3
VI-2 HCB Plasma Levels 1n Exposed Individuals and Controls . . . VI-5
VI-3 Clinical Signs and Symptoms 1n Humans 25 Years After
Exposure to Low Levels In HCB 1n Turkey, 1955-1959 VI-8
VI-4 Porphyrin Levels In Patients and Controls ......... VI-10
VI-5 Laboratory Test Results of Turkish Patients . VI-11
VII-1 Porphyrin Content and Uroporphyrinogen Decarboxylase
Activity In the Liver Cytosol of Female Rats Pretreated
with 100 mg/kg HCB Every Other Day for 6 Weeks VII-9
VII-2 Analysis of the Excreta from Rats Administered Hexa-
chlorobenzene After an Initial Treatment with D1 ethyl-
stllboestrol.... VI1-12
v111
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LIST OF TABLES (cont.)
No. Title Page
VIII-1 Summary of Toxicity Studies on Hexachlorobenzene. ..... V111-10
VIII-2 Tumor Incidences 1n Male and Female Hamsters GWen_
Hexachlorobenzene In Diet 1 . VI11-32
VIII-3 Incidence of Liver Cell Tumors 1n Male and Female Swiss
Mice Given Hexachlorobenzene Diet . . . ' VIII-33
VI11-4 Liver and Kidney Tumor Incidence Rates In Male and Female
Sprague-Dawley Rats Given Hexachlorobenzene In Diet .... VI11-34
VIII-5 Incidence Rate of Adrenal Pheochromocytoma In Female
Sprague-Dawley Rats (F^ generation) In a 2-Generatlon
Feeding Study VI11-35
VI11-6 The Carcinogenic Potency of Hexachlorobenzene, Calculated
on the Basis of 14 Data Sets, Using the Linearized
Multistage Model. ................ VIII-37
VI11-7 Summary of the Data for Hexachlorobenzene Used to
Derived HAs and DWEL VI11-44
1 x
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LIST OF ABBREVIATIONS
CCV-
4
CRAVE
DUEL
GC/MS
GLC/MS
HA
1 .m.
l.p.
1. v.
L050
LOAEL
MTD
NOAEL
NOEL
. PCT
RBC
RfD
TLC
UV
Carbon tetrachloride
Carcinogen Risk Assessment Verification Endeavor
Drinking water equivalent level
Gas chromatography/mass spectrometry
Gas liquid chromatography/mass spectrometry
Health advisory
Intramuscular
Intraperitoneal
Intravenous
Dose lethal to 50% of recipients
Lowest-observed-adverse-effeet level
Maximum tolerated dose
No-observed-adverse-effect level
No-observed-effeet level
Porphyria cutanea tarda
Red blood eel 1
Reference dose
Thin-layer chromatography.
Ultraviolet
x
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I. SUMMARY
Hexachlorobenzerie Is imported but not produced commercially In the
United States, and occurs as a by-product 1n the synthesis of nine other
chlorinated hydrocarbons; 2-5 million pounds may be generated each year.
Hexachlorobenzene Is a colorless crystallIne (monocllnlc prism) solid
with a reported water solubility of 0.005 mg/i at 25°C. In general, hexa-
chlorobenzene has low water solubility, high octanol/water partition coeffi-
cient, low vapor pressure at 25°C, low flammabl11ty and Is photochemically
stable. Chemical analysis of hexachlorobenzene 1n water generally Involves
solvent extraction followed by a cleanup method being used to produce
organic extracts suitable for GC/MS analysis.
The pharmacokinetics of hexachlorobenzene In a number of mammalian
species have been studied in detail following oral administration and, to a
lesser extent, following Intravenous or intraperitoneal Injection. No
Information was found regarding hexachlorobenzene metabolism following
inhalation or topical application. Absorption of hexachlorobenzene from the
intestinal tract appears to depend on the vehicle used during test material
administration. Thus, when hexachlorobenzene is 'administered 1n olive oil,
-80% of the dose is absorbed; when it is administered in an aqueous
solution, in 1% methyl cellulose or in a crystalline form, relatively little
{<20%) is absorbed. Intestinal absorption of hexachlorobenzene occurs
primarily through lymphatic channels, with only a minor portion being
absorbed into the portal circulation.
02550
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09/14/88
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Following absorption, hexachlorobenzene Is distributed to tissues that
have a high lipid content. The adipose tissue accumulates the greatest con-
centrations of hexachlorobenzene in all species studied, although bone mar-
row and skin, which contain large amounts of lipids, also accumulate hexa-
chlorobenzene. The adrenal cortex accumulates hexachlorobenzene at concen-
trations approaching those of fat. Other body constituents (e.g., liver,
kidneys, lungs, heart, spleen and blood) generally contain much lower
amounts of hexachlorobenzene. Intravenous Injection of hexachlorobenzene
results In a tissue distribution similar to that seen following oral admin-
istration. Hexachlorobenzene 1s transported via the placenta and 1s
distributed 1n fetal tissue as Indicated by studies with rabbits, rats,
mice, mink and ferrets.
Hexachlorobenzene Is metabolized slowly Into other chlorinated benzenes,
chlorinated phenols and other minor metabolites and forms glucuronlde and
glutathione conjugates. Tissues were found to contain mainly unchanged
hexachlorobenzene together with small amounts of metabolites. Similarly,
only small amounts of hexachlorobenzene metabolites were detected In feces,
whereas most of the metabolites were excreted In the urine together with
small amounts of unchanged hexachlorobenzene. There are Indications that
females produce and excrete more hexachlorobenzene metabolites than do males.
The excretion of hexachlorobenzene from treated animals Is slow and
occurs mainly through the feces with relatively little being excreted In the
urine. It 1s characterized by an Initial rapid phase followed by one or
more slow phases. This slow phase of excretion can be enhanced by the
administration of mineral oil, paraffin or n-hexadecane. Both biliary and
02550
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09/14/88
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Intestinal excretion contribute to fecal excretion. A three-compartment
manrnlllary model has been reported for the behavior of hexachlorobenzene In
beagles and rhesus monkeys following a single l.v. Injection. Radioactivity
was not detected In exhaled air following l.p. Injection of 14C-hexa-
chlorobenzene. Hexachlorobenzene has been detected 1n the' milk of nursing
mammals.
The acute oral toxicity of hexachlorobenzene has been found to be low
with LDj.0 values ranging from 1700-10,000 mg/kg. Subchronlc oral toxicity
studies with a number of mammalian species Indicated a significant Increase
In liver and kidney weights as well as other organ weights .In hexachloro-
benzene-treated animals. The livers from hexachlorobenzene-exposed animals
have shown histologic changes .such as irregularly shaped and moderately
enlarged liver mitochondria and Increases in the size of the centrllobular
hepatocytes. Chronic toxicity studies revealed similar effects to those
seen in the subchronlc studies, plus hexachlorobenzene associated life-
shortening and various hepatic and renal pathologies. These subchronic and
chronic effects were usually dose-related. Other effects Included multiple
alopecia and scabbing, together with neurologic effects in rats, mice and
dogs. A dose-related histopathologic change In the ovaries of monkeys has
also been reported.
Increased porphyrin levels in the liver and In urine have been reported
for all species studied except the dog. Hexachlorobenzene was found to
cause the accumulation of 0-H-stero1ds, which Induce porphyrin biosynthesis,
and to Inhibit uroporphyrinogen decarboxylases. Indications are that
females are more susceptible to hexachlorobenzene-lnduced porphyria than are
02550
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males, which may be related to the females estrogen levels and greater
hexachlorobenzene metabolism. Hexachlorobenzene was reported to produce a
mixed-type Induction of cytochromes resembling that produced by a
combination of phenobarbltal (P-450) and 3,4-benzpyrene (P-448). In
addition, the activities of several hepatic microsomal enzymes were found to
be Induced by hexachlorobenzene.
Hexachlorobenzene did not Induce dominant lethal mutations In two stud-
ies but was reported to be mutagenic In a yeast, cerevlslae. assay at a
concentration of 100 ppm. Hexachlorobenzene possessed no detectable levels
of mutagenic activity In the Salmonella hlstldlne reversion assay. Chronic
feeding studies provide sufficient evidence for the carcinogenicity of hexa-
chlorobenzene In animals (U.S. EPA welght-of-evldence classification of B2)
since there was an Increased Incidence of malignant tumors of the liver In
two species (haemangloendothelloma In hamsters and hepatocellular carcinoma
In rats) as well as reports of hepatoma In mice, rats and hamsters.
Hexachlorobenzene given to pregnant mice was found to produce cleft palates
and renal agenesis In exposed pups. Certain chemicals were found to alter
the toxicity of hexachlorobenzene In mammals, whereas hexachlorobenzene
pretreatment was reported to Increase CC1 ^ toxicity and alter the tmmune
responses of treated animals.
A few epidemiologic studies with occupatlorvally-exposed workers have
been reported, together with studies conducted In Turkey and In the United
States (I.e., Louisiana) on the general population following accidental
exposure to hexachlorobenzene. These studies qualitatively support the tox-
icity of hexachlorobenzene, but give little dose-response Information. Bio-
logic monitoring of plasma levels clearly shows more hexachlorobenzene In
02550 1-4 09/14/88
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the plasma of exposed compared with nonexposed Individuals, although no bio-
logically significant adverse health effects .were seen during the observa-
tion periods. The exposure of humans to hexachlorobenzene In Turkey during
1955-1959 caused an epidemic of hexachlorobenzene-lnduced -porphyria cutanea
tarda (PCT), also known as porphyria turcica, which Is manifested by dis-
turbed porphyrin metabolism, cutaneous lesions and hyperplgmentatlon. The.
authors estimated that from 0.05-0.2 g/day were Ingested. In exposed
children under 1 year of age, pink sore was observed as well as 95% mortal-
ity In these Infants.
Follow-up studies conducted with Turkish patients 20-25 years after the
onset of porphyria showed that a few subjects still had active porphyria,
whereas >50% exhibited hyperplgmentatlon scarring, as well as other
dermatologlc, neurologic and skeletal features of hexachlorobenzene
toxicity. Hexachlorobenzene residues were also found In the blood, fat and
breast milk of some patients.
A correlation was found between hexachlorobenzene' levels In blood and
the number of years worked In a chlorinated solvents plant. The concentra-
tion of urinary uroporphyrins ' and coproporphyr.ins ranged from 21-37 and
67-101 ug/i, respectively, for the period between "1974- and 1977. An
epidemiologic survey conducted with 86 residents In the vicinity of this
chlorinated solvents plant showed elevated hexachlorobenzene residues in
plasma. Higher levels of hexachlorobenzene residues were found In males
than 1n females, but these were not associated with race or food consumption.
02550
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09/14/88
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The quantitative risk assessment for hexachlorobenzene Is based on
animal data since human data are Insufficient. Data are Insufficient for
the derivation of 1-day and 10-day HAs for a 10 kg child; therefore, the
longer-term HA for a 10 kg child of 0.05 mg/a, 1s recommended as the 1-day
and 10-day HAs for a 10 kg child. The longer-term HA for a 70 kg adult Is
0.2 mg/i. The lifetime DWEL derived from chronic toxicity data for a 70
kg adult Is 0.03 mg/i. The RfO Is 0.0008 ug/kg by/day and 1s based on
the results of a 130-week study In-rats and was verified In Hay 1988.
The 95% upper bound lifetime cancer risk associated with 1 pg/i of
hexachlorobenzene In drinking water Is estimated to be 4.6xl0~s. Levels
of 2.0xl0*5, 2.0x10"* and 2.0x10~3 mg/1 of hexachlorobenzene In
drinking water correspond to the 95% upper bound lifetime cancer risks of
10"6, 10"s and 10"*, respectively. These values were verified by the
CRAVE workgroup In March 1989. Hexachlorobenzene has been classified as a
B2, probable human carcinogen.
02550 1-6 07/11/91
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II. PHYSICAL AND CHEMICAL PROPERTIES
Hexachlorobenzene Is not manufactured as a commercial product In the
United States, but an estimated 2-5 million pounds were produced each year
during the synthesis of several chlorinated chemicals {Mumma and Lawless,
1975). Hexachlorobenzene 1s also an Ingredient In a fungicide of which
-200,000 pounds are Imported each year [IARC, 1979). Hexachlorobenzene Is
resistant to blodegradatlon, accumulates In the biologic environment and has
been detected In ambient air, drinking and surface water, sediments,
cropland and food (U.S. EPA, 1985).
Hexachlorobenzene 1s a colorless crystalline (monoclinlc prisms) solid.
Its water solubility was reported as 0.005 mg/i at 25°C (Yalkowsky and
Valvanl, 1980). Hexachlorobenzene 1s sparingly soluble in cold alcohol and
soluble In benzene, chloroform and ether {NLM, 1979). Impure cormierclal
preparations may contain pentachlorobenzene (10-81,000 ppm), octachlorodl-
benzo-p-dloxln (0.05-212 ppm) and octachlorodlbenzofuran (0.35-58.3 ppm)
(Vllleneuve et al., 1974). The chemical structure of hexachlorobenzene is
shown In Figure II-l. Synonyms, trade names and. IdentlfIcatlon numbers for
hexachlorobenzene are listed In Table II-l.
Some physical properties of hexachlorobenzene are shown In Table II-2.
In general., hexachlorobenzene has low water solubility, high octanol/water
coefficient, low vapor pressure at 25°C and low flammabl11ty. Hexachloro-
benzene has been demonstrated to be photochemically stable (Korte et at. r
1978; Hustert et al.( 1981).
02560 11-1 04/05/91
-------
CI
CI
FIGURE II-l
Chemical Structure of Hexachlorobenzene
02560
r i-2
07/13/84
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TABLE 11-1
Synonyms, Trade Names and
Identification Numbers of Hexachlorobenzene
Identification Numbers Synonyms and Trade Names
CAS No. 118-74-1 Esaclorobenzene (Italian)
TSL No. 0A2975000 Amatln
EPA Haz Waste No. U127 Antlcarle
Bunt-Cure
Bunt-No-More
Co-op Hexa
Granox NH
HCB
HEXA C.B.
Hexachlorobenzol (German)
Hexachlorobenzene
Julln's Carbon Chloride
No Bunt
No Bunt 40
No Bunt 80
No Bunt Liquid
Pentachlorophenyl Chloride
Perchlorobenzene
Phenyl Perchloryl
Sanodde
Smut-Go
Sn1ec1otox
~Source: National Library of Medicine (NLM), Toxicology
Data Bank (TDB)
02560
11-3
07/1.3/84
-------
)
TABLE 11-2
Physical Properties of Hexachlorobenzenea
Molecular Weight 284.79
Melting Point " 230°C
Boiling Point 322.9°C at 760 mm
Density at 23°C 1.57 g/ma
b
Henry's Law Constant x 10~3 0.12 atm. m3 mol 1
Log P°b 5.8
Water Solub111tyC 0.005 mg/i at 25°C
Flash Point 468°F
Vapor Pressure (ram Hg) 1 at 144.4"C^
1.68 x 10~* at 25°Fe
1.089 x 10*5 at 20°CF
Specific Vapor Density (air = 1) 9.84^
aData are from the National Library of Medicine (NLM), Toxicology Data
Bank (TDB), except as noted.
bMackay et al. p 1979
cYalkowsky and Valvanl, 1980
dWeast, 1981
eLeoni and D'Arca, 1976
fFarmer et al., 1980
9Verschueren, 1977
P° = Partition coefficient 'at 25°C
02560
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04/05/91
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Chemical Analysis
Chemical analysis of hexachlorobenzene In water generally Involves
solvent extraction followed by a cleanup method being used to produce
organic extracts suitable for GC/MS analysis. The U.S. EPA (1982) (Method
612) has recommeded the use of Florlsll column chromatography as a cleanup
step before the quantification of the samples by GC with electron capture
detector. This recommended method Is applicable for the determination of
hexachlorobenzene In drinking water and wastewater. The recovery of hexa-
chlorobenzene by this method was found'to be 95%.
A method for the determination of hexachlorobenzene In soil and chemical
waste disposal site samples has been developed by DeLeon et al. (1980). The
procedure Involves methane extraction followed by temperature-programmed GC
analysis using electron capture detection. Recoveries of samples spiked at
the 10, 100 and 300 *ig levels were 96.5% (+3.6), 93.1% (+8.1) and 78.0%
(+2.6), respectively. The lower detection limit for this method Is around
10 yg/g.
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III. TOXICOKINETICS
Absorption
Absorption of- hexachlorobenzene from the gut has been studied 1n detail;
however, no Information has been found In the available literature on hexa-
chlorobenzene absorption through the lungs or skin. Absorption of hexachlo-
robenzene from the Intestinal tract appears to depend on the solvent vehicle
used during test material administration. Thus, when hexachlorobenzene Is"
administered In olive oil, -80% of the dose Is adsorbed; when 1t Is admin-
istered 1n an aqueous solution, 1n 1% methyl cellulose, or In a solid
crystalline form, relatively little (<20%) Is absorbed. Intestinal absorp-
tion of hexachlorobenzene occurs primarily through lymphatic channels
(Iatropoulos et al.t 1975), with only a minor portion being absorbed Into
the portal circulation.
Ingebrlgtsen et al. (1981) Investigated the absorption of [14C]hexa-
chlorobenzene (TO mg In peanut oil) administered to male, blle-duct-cannu-
lated Wlstar rats by gastric catheter. Four days after dosing, 24.8% of the
administered 14C had been recovered 1n the feces, Indicating that at least
75% of the administered hexachlorobenzene was absorbed.
Albro and Thomas (1974) studied the absorption, of.hexachlorobenzene In a
squalane/cotton seed oil vehicle by male rats following administration of a
single dose by stomach Intubation. The results Indicated that at doses of
12 and 30 mg/kg, -82 and 72%, respectively, were absorbed within 96 hours.
Koss and Koransky (1975) compared the absorption rates of [14C]hexa-
chlorobenzene 1n female Wlstar rats following oral administration of olive
02570
111 -1
04/12/88
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oil solutions or suspensions In 6% gum arable In water (4, 20, 50.5, 60 and
180 mg/kg). Approximately 00% of the dose was absorbed from the olive oil
solutions; however, only 6% was absorbed from the aqueous suspension.
Similarly, Zablk and Schemmel (1980) found that, when -hexachlorobenzene
(32 mg/kg/day) was administered In the diet, h1gh-fat (45.3% w/w) diets
resulted 1n greater accumulation of hexachlorobenzene In the tissues and
less hexachlorobenzene excreted In the feces than did high-carbohydrate
diets (67.7% w/w). The female rats received 32 mg hexachlorobenzene/kg body
weight/day for 6, 12 or 18 days. Although this study did not Include a
control group receiving a balanced diet, the data suggest, that high-fat
diets Increase the absorption of hexachlorobenzene.
Sundlof et al. (1982) administered seven consecutive dally oral doses of
10 or 100 mg crystalline hexachlorobenzene/kg body weight to male laboratory
beagles. The results from the 100 mg/kg group Indicated that hexachloro-
benzene can continue to be absorbed from the Intestines for up to 1 week
following the cessation of dosing.
Bleavins et al. (1982) fed female European ferrets (Mustela putorlus
furo) a single dose of 57.6 yg hexachlorobenzene (14C-1abeled) 1n,7.5 g
of standard mink diet (22% fat) and calculated that 98.5% of the hexachloro-
benzene dose was absorbed by the ferrets. They made this calculation based
on predicted hexachlorobenzene excretion as extrapolated from this study,
and owing to a food passage time In the female ferret of Just over 3 hours.
02570
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Distribution
Following Intestinal absorption, hexachlorobenzene, which Is lipophilic,
distributes to tissues that are rich In lipid content. The adipose tissue
accumulates the greatest concentrations of hexachlorobenzene In all vspedes
studied, although bone marrow and skin, which contain Targe amounts of
lipids, also accumulate hexachlorobenzene. The adrenal cortex accumulates
hexachlorobenzene at concentrations approaching those of fat. Other tissues
(e.g., liver, kidneys, lungs, heart, spleen and blood) generally contain
lower amounts of hexachlorobenzene. Intravenous injection of hexachloroben-
zene results In a tissue distribution similar to that following oral admini-
stration. Hexachlorobenzene Is transported via the placenta and is distri-
buted 1n fetal tissue.
Mehendale et al. (1975) studied the disposition of ldC-hexachloroben-
zene by adult male rats following a single oral dose of 5 mg/kg. 14C-
Hexachlorobenzene was mixed with arachls oil and administered by stomach
Intubation. The animals were sacrificed.7 days later and the tissues and
organs radloassayed. Forty-three percent of the total radioactivity
administered was present in fat tissue 7 days after 14C-hexachlorobenzene
administration. In addition, muscle and skin tissues each contained -9% of
the radioactivity, whereas the other 12 tissues analyzed contained -5%
combined (Table 111-1).
When 1AC-hexachlorobenzene was suspended In 1% methyl cellulose and a
single oral dose containing 150 u9 of hexachlorobenzene was administered
to Sprague-Oawley rats, the absorption of lJlC-hexachlorobenzene by the
walls of the stomach and duodenum 1 hour later was relatively low: -1.0 and
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TABLE III-l
Storage and Excretion of 14C-HC8 Administered Orally
in Arachls 011 In Ratsa
Percent of Total
Organ or Tissue Radioactivity
Administered
Fatb
42.81
+ 6.14
Husclec
9.41
+ 1.17
Sklnd
8.64
7 1.21
Liver
3.01
+ 0.23
Small Intestine
2.43
7 0.47
Bonee
1.04
7 0.09
Kidneys
0.76
+ 0.11
Large Intestine
0.43
+ 0.08
Stomach
0.36
+ 0.04
Blood
0.24
7 0.04
Lungs
0.24
7 0.04
Testes
0.21
+ 0.04
Heart
0.18
+ 0.03
Brain
0.17
+ 0.03
Spleen
0.04
7 0.002
Total 1n tissues
70.09
+ 5.48
Excretion
+ 2.31f
Feces
16.02
Urine
0.85
7 Q.13f
Gut contents
2.48
i 0.45
Total recovery
89.44
+ 10.57
aSource: Mehendale et al., 1975
bBased on body weight as fat
cBased on 50% body weight as muscle
dBased on 16% body weight as skin
eBased on 10% body weight as bone
^Cumulative total for 7 days
Adult male rats were given 5 mg/kg of hexachlorobenzene.
HC8 = Hexachlorobenzene
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irr-4
07/13/84
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0.6 ppm were found 1n the stomach and duodenum, respectively (Iatropoulos et
al.( 1975). Increased radioactivity was found In the Jejunum and Ileum as
well as the lymph nodes and adipose tissues 3 hours after administration
(Table III-2). Although the radioactivity also Increased.In the liver and
kidneys, this Increase was relatively low compared with that found in the
lymph nodes and adipose tissue. Moreover, the radioactivity in the liver
and kidneys decreased within a 2-day period, whereas the radioactivity In
the lymph nodes and fat remained relatively constant. These results Indi-
cate that the portal venous* transport of hexachlorobenzene to the liver
appears to be a minor pathway, whereas the major part of the ingested hexa-
chlorobenzene Is absorbed by the lymphatic system In the duodenum and
Jejuno-lleum and deposited In the fat, bypassing the systemic circulation
and the excretory organs.
Knauf and Hobson (1979) Investigated the tissue distribution of hexa-
chlorobenzene In six female rhesus monkeys following the gastric administra-
tion of dally doses of hexachlorobenzene [0 (one monkey), 8 (one monkey), 32
(one monkey), 64 (one monkey), or 128 (two monkeys) mg/kg/day] In T% methyl
cellulose for a period of 60 days. The highest concentrations of hexachlo-
robenzene were located in tissues with high lipid content. Tissue levels
correlated more with body fat content than with dose, with the monkey that
had the least adipose tissue producing the highest nonfat tissue and serum
values (Table 111-3).
The highest levels of hexachlorobenzene residues were found in fat
tissue (215-930 ppm) and bone marrow (175-1700 ppm), and selectively higher
levels were found In the adrenal cortex (30-325 ppm) than In the adrenal
02570
111-5
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TABLE III-2
Tissue Concentration (ppm) of 14C-Hexachlorobenzenea and Its Metabolites In Sprague-Dawley Rats'*
Time (hours)
1
3
5
12
48
Tissue
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Stomach
0.6
1.6
0.8
1.0
1.1
0.5
0.1
0.1
0.1
0.1
Duodenum
0.6
0.6
1.4
1.0
0.2
0.3
0.1
0.1
0.1
0.1
Jejuno-Ileum
0.1
0.2
0.6
0.B
1.0
0.3
0.3
0.3
0.2
0.1
Cecum
0.1
0.1
0.1
0.2
0.1
0.1
0.1
.0.2
0.1
0.1
Colon
0.1
0.1
0.1
0.2
0.4
0.1
0.1
0.1
0.2
0.1
Liver
0.1
0.4
0.5
0.5
0.2
0.3
0.2
0.2
0.1
0.2
Mesenteric
lymph node
0.1
0.6
0.4
1.3
2.0
1.0
1.5
1.0
1.9
2.1
Adipose
tissue
0.1
0.2
1.7
1.2
2.3
1.5
1.3
1.1
2.6
2.7
Kidneys
0.1
0.2
0.4
0.3
0.5
0.2
0.2
0.1
0.2
0.1
Lungs
0.1
0.3
0.3
0.4
0.2
0.2
0.1
0.1
0.1
0.2
/
d 150 pg hexachlorobenzene was administered by stomach tube suspended In IX methyl cellulose.
bSource: Iatropoulos et al., 1975
-------
Tissue Levels of HCB
. TABLE
(ppm) 1n
111-3
Adult Female
Rhesus
Monkeysa'b
Monkey No.
613C
618^
627e
817
1163
1826'
Dose (mg/kg/day)
128
128 ,
64
32
8
0
Body fat
930
215
540
250 '
580
1.1
Bone marrow
460
175
1700
255
350
1.6
Adrenal cortex
150
30
325
90
50
0.1
Adrenal medulla
12
9
285
35
4
<0.1
Liver
20
50
365
40
30
<0.1
Kidney
18
19
258
11
3
-------
medulla (4-285 ppm). Residues In serum, muscle, ovaries, brain, kidneys and
liver were relatively much lower (0.5-365 ppm).
Engst et al. (1976) reported the administration by gavage of 8 mg/kg of
hexachlorobenzene In 1 ml of sunflower oil to male Wlstar rats for a dura-
tion of 19 days. The animals were then sacrificed, and the liver, kidneys,
adrenals, heart, spleen and Intestinal fat were analyzed for hexachloroben-
zene residues. The following results were reported: fat tissue, 82 yg/g;
muscle, 17 yg/g; liver," 125 yg total; kidneys total 21 yg each; spleen
total 9 yg; heart total 1.5 yg; and adrenals total 0.5 yg each. High
levels of hexachlorobenzene residues In fat tissues also have been reported
for female rats receiving 50.0 mg/kg (177 ymoles/kg) of hexachlorobenzene
every second day for 10 weeks (Koss et al., 1980b).
Szymczynskl and Wal1szewsk1 (1981) analyzed human semen and testicular
and fat tissues, and identified several chlorinated pesticides that Included
hexachlorobenzene. The compound was not detected in testicular Lissue, but
was present In semen and fat tissues at concentrations of 0.001 and 0.128
yg/g, respectively. Similarly, hexachlorobenzene was one of several
chlorinated compounds found in semen collected in 1979 from 132 college
students (Dougherty et al.f 1981).
Sundlof et al. (1982) studied the distribution of 14C-hexachloroben-
zene or unlabeled hexachlorobenzene in male beagles following a single
Intravenous dose of 1 mg/kg In olive oil. Two dogs each were sacrificed
after 2, 4, 8, 16, 32 and 48 hours and after 12 weeks; hexachlorobenzene
concentrations were determined in 16 tissues and organs as well as In the
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111-8
04/23/91
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blood (Table III-4). Two hours after dosing, the highest concentration was
found In the lungs (36.14 ppm). This was considered to be a property of the
injection vehicle rather than a. property of hexachlorobenzene per ,se. That
1s, It was believed that the olive oil vehicle formed mlcroemboll 1n the
blood which became trapped 1n the capillaries of the lung. Residue levels
In the lungs then dropped (4.4 ppm).# and a concurrent Increase . In.
hexachlorobenzene was detected 1n fat tissues (10.32 ppm in subcutaneous,
perirenal and mesenteric fat) 4 hours postinfection. Residues 1n all
tissues, organs and blood declined during the 48 hours postinfection except
for fat tissue, which remained constant. Twelve weeks after dosing, tissue
concentrations were very low in all tissues, Including fat (>0.01-0.46 ppm),
Indicating significant excretion of the compound by that time.
Yang et al. (1978) studied the distribution of hexachlorobenzene In male
Sprague-Oawley rats and female rhesus monkeys following Intravenous Injec-
tion of 14C-hexachlorobenzene In 1,2-propanedlol:plasma (1:8). Rats
received 0.1 mg of 14C-hexachlorobenzene and then were replaced 1n meta-
bolic cages for 48 hours before sacrifice. About 0.2 and 1.0% of the admin-
istered dose was excreted In the urlrre and feces, respectively. No radio-
activity was exhaled from the animals. Over 20.-tissues from the rats were
analyzed and all were found to contain radioactivity. The highest levels
were In fat (-3 yg/g of tissue). The adrenal glands also contained a
relatively high level of radioactivity, whereas the other tissues contained
much lower levels, generally 1n the range of 1/12 to 1/300 of those in fat
tissue.
02570
III-9
01/31/85
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TABLE .111-4
HC8 Concentrations 1n Tissues of Male Beagles
Receiving Single Intravenous Ooses of 1 mg/kg bw In-Olive 011*
Tissue
HCB
Time
Concentration
Interval After
(ppm)
Doslnq
2 hours
48 hours
12 weeks
Lungs
36.14
0.08
<0.01
Adrenals
2.82
0.38
0.06
Subcutaneous fat
1.14
3.38
0.37 ¦
Perirenal fat
1.00
3.24
0.46
Mesenteric fat
0.56
2.40
0.41
Spleen
0.54
0.01
¦
<0.01
Liver
0.51
0.04
0.02
Thyroid
0.37
NR
0.02
Heart
0.28
0.04
0.01
Kidneys
0.18
0.02
0.01
Stomach
0.18
0.36
0.01
Pancreas
0.17
0.06
0.07
Brain
0.15
¦
0.02
0,02
Duodenum
0.12
0.02
0.02
Colon
0.12
"o.oi
<0.01
Small Intestine
0.07
0.02
0.01
Blood
0.07
0.03
0.01
•Source: Sundlof et al.# 1982
NR = Not reported
HCB = Hexachlorobenzene
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The tissue distribution of 14C-hexachlorobenzene In rhesus monkeys was
determined In Individual animals 100 days, 6 months and 1 year after Intra-
venous Injection'of ^C-hexachlorobenrene at 0.38, 0.32 and 0.22 mg/kg,
respectively. The results again Indicated that the highest levels were
present In fat (6069 ng/g on day 100 and 828 ng/g on day 365) and bone
marrow (1638 ng/g on day 100 and 373 ng/g on day 365) among the 30 tissues
analyzed In all three monkeys. The adrenal glands contained -1/6 to 1/8 of
the levels present 1n fat, whereas the other tissues contained radioactivity
levels ranging between 1/10 to <1/800 of those In fat.
The transplacental transfer of hexachlorobenzene from pregnant mice,
rats and rabbits has also been reported. Brandt et al. (1980) conducted a
qualitative study on the distribution of 14C-hexachlorobenzene and several
of Us sulfur-containing metabolites In pregnant mice. The mice were
injected 1.v. and sacrificed at Intervals ranging between 20 minutes and 32
days after injection. The animals were frozen, sectioned and submitted to
autoradiography. The autoradlograms showed a strong uptake of hexachloro-
benzene In the adipose tissues. Hexachlorobenzene was found to persist In
the adipose tissues for more than 1 month after the administration.
Radioactive hexachlorobenzene was also found to penetrate the placenta,
resulting In the blood and liver concentrations In the fetuses that appeared
to equal those of the dams.
VUleneuve and Hlerllhy (1975) studied the placental transfer of hexa-
chlorobenzene in Wlstar rats and reported that hexachlorobenzene crosses the
placenta and accumulates In the fetus 1n a dose-dependent manner. The
females were dosed orally dally (5, 10, 20, 40 and 80 mg/kg) from gestation
02570
III-ll
09/15/88
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day 6-16 and then sacrificed on day 22. Only liver, brain and whole fetal
residue levels were determined In this study. Fetal liver residues
{1.8-35.8 yg/g) were much lower than those of the dams (9.3-86.0 yg/g).
The fetal brain and whole fetal levels were 1.1-17.5 y.g/g and 1.5-18.9
yg/g, respectively.
Vllleneuve et al. (1974) also reported that the transplacental transport
of hexachlorobenzene In New Zealand rabbits was dose-dependent. Rabbits
were mated and then dosed orally with hexachlorobenzene from days 1-27 with
subtoxlc doses of 0, 0.1, 1.0 or 10 mg/kg. On day 28 the dams were killed
for fetal and maternal tissue analysis of hexachlorobenzene. In dams, the
hexachlorobenzene residue concentrations were highest 1n fat, followed by
the liver, heart, kidneys, brain, lung, spleen and plasma. Hexachloro-
benzene residues were higher In the fetal liver than 1n the maternal liver.
Courtney et al. (1976) reported on the distribution of hexachlorobenzene
(assayed 90.4% hexachlorobenzene and 9.6% pentachlorobenzene) administered
via oral Intubation on days 7-11 of gestation at a dose of 50 mg/kg/day in a
corn oil acetone mix to five pregnant .and two non-pregnant CD-I mice. They
found there were no remarkable differences in the hexachlorobenzene tissue
levels between the pregnant and non-pregnant animals sampled at day 12 of
pregnancy. The levels of pentachlorobenzene.In sampled tissues were low as
compared with the very high hexachlorobenzene levels detected in the thymus,
skin, fat and urinary bladder. No detectable levels of hexachlorobenzene or
pentachlorobenzene were found In the control mice.
0257,0
111-12
04/05/91
V
-------
Courtney et al. (1979) studied the tissue distribution of hexachloroben-
zene In the maternal and fetal tissues of CD rats and CD-I mice and reported
that placentas and fetuses of both species demonstrated a dose-dependent
relationship for hexachlorobenzene residues, with levels In the fetuses
being higher than those In their corresponding placentas. The dams were
treated via oral Intubation with single or multiple oral doses (10, 50 or
100 mg/kg 1n corn oil) at different periods during gestation. The hexachlo-
robenzene concentrations 1n mfce and rat fetuses at mid-gestation were very
similar. In mice, multiple low doses of hexachlorobenzene resulted In
higher concentrations of hexachlorobenzene 1n maternal and fetal tissues
than single doses of equivalent total doses. In another study, Courtney and
Andrews (1979) reported that In mice the fetus could be exposed to hexachlo-
robenzene from maternal body burdens, established before fetal Implantation,
and. was not limited to maternal exposure during the postlmplantatlon
gestation.
Bleavlns et al. (1982) studied the tissue distribution and transfer of a
single dose of hexachlorobenzene given to female European ferrets (Mustela
putorlus fu-ro). They gave a single S7.6, ng hexachlorobenzene (re-
labeled) dose In 7.5 g of standard mink diet (22.2% fat) to each of three
bred and five non-bred ferrets. The dosed ferrets and offspring were
observed for 5 weeks after the kits were born, at which time they were
killed and tissue 14C-hexachlorobenzene levels were determined (Table
.111-5). One ferret kit per litter was also collected at birth and at weeks
1, 2, 3 and 4 for whole body residue determinations (Table 111 -6). These
results Indicate that nursing mothers can significantly reduce their body
burdens of hexachlorobenzene, when compared with unbred female counterparts,
by transferring a large amount of the hexachlorobenzene to their offspring.
02570 111-13 04/05/91
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TABLE 111-5
Mean (+SE) Hexachlorobenzene Radioactivity (dpm/g)
of Selected European Ferret T1ssuesa»&
Tissues Group I Group II K1tsc
(n-3) (n=5) (n=3)
Blood
49
+
34.6d
166
+
26.8
--
Subcutaneous fat
4472
*
780.5®
19,525
1503.9
11,678
+
712.4f
Visceral fat
4429
867.6e
19.704
1666.0
—
Muscle
53
+
14.4^
384
*
64.0
561
f
204.8
Heart
34
9.2d
310
+
56.8
--
Kidney
105
31.le
611
80.4
209
*
37.2 -
Spleen
13
~
7 .5e
180
+
24.8
--
Liver
248
68.9e
1,445
¥
145.2
1,420
V
185.69
Lung
1
0.3e
241
18.4
—
Brain
61
+
30.0e
395
~
48.5
130
~
29.4
aSource: Bleavlns et al.f 1982
bat 62 days postdoslng from adult bred (group I) and unbred (group II)
female Ferrets exposed to a single 57.6 ^g dose of 14C-labeled hexa-
chlorobenzene and from offspring born to the bred females.
cK1t tissues, from 5-week-old offspring, were rontrasted only with mater-
nal (group I) tissues.
dS1gnlfIcantly different (p<0.05) from group II tissue of the same type.
eS1gn1f1cantly different (p<0.01) from group II tissue of the same type.
fS1gn1fIcantly different from maternal tissue {group I) at p<0.01.
9S1gn1fIcantly different from maternal tissue (group I) at p<0.05.
HCB = Hexachlorobenzene
02570
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TABLE III-6
Mean l+SE) HCB Radioactivity (dpm *10") of European Ferret Kits'*'*
Weeks Postpartum
Measure
Nunber
0
1
2
3
i
4
5
Per gran of kit
3
3.0
~ 0.19
2.7 ~ 0.57
4.3 ~
0.67
3.9 ~
0.73
3.5 » 0.50
2.7 i 0.14
Per whole kit
3
25.1
i 1.43
76.7 ~ M.35
311.4 ~
63.39
492.5 t
92.22
672.0 » 117.63
805.7 ~ 54.25
Increase over previous week
-
51.6
234
.7
181
.1
180.3
132.8
Milk (per at)
3
--
—
6.1 ~
0.66
2' t
0.45
1.8 i 0.17
0.8 ~ 0.20
'Source: Bleavlns et al., 1982
''Born to ftuli ferrets exposed to a single dose of "C-labeled hexachlorobenzena and the *IU produced by those dams
HCB - Heiachlorobeniene
-------
The mothers' milk contaminated with hexachlorobenzene seems to be a large
contributor to the kits' body burdens with a reported milk to placental
exposure ratio of 31:1. The distribution of hexachlorobenzene 1n ferrets
follows similar trends, as observed In the other mammals, where the highest
hexachlorobenzene levels were found 1n the lipid rich tissues.
The transfer of hexachlorobenzene to nursing Infant rhesus monkeys from
lactatlng mothers receiving via oral Intubation 64 mg/kg/day hexachloroben-
zene suspended In 1% methyl cellulose/ for 60 days was reported by Bailey et
al. (1980). M1lk concentrations were on the average 17-fold higher than
maternal serum levels, whereas Infant serum levels were about 2- to 5-fold
higher than serum levels of their mothers. Similarly, the Infants had
higher tissue residues than their mothers and hexachlorobenzene was concen-
trated In- the Infant fat, bone marrow, adrenals and lymph nodes.
Hexachlorobenzene residues also have been reported In human fat In the
United Kingdom (Abbott et al., 1981, Japan (Cur ley et al., 1973), and
Australia (Brady and Slyall, 1972) and In human milk collected In Sweden
(Westoo and Noren, 1978; Hofvander et al., 1981), Canada (Mes and Davles,
1979), Norway (Bakken and Se1p, 1976; Skaare, 1981), and Hawaii (Takahashl
et al., 1981).
HetabolIsm
The metabolism of hexachlorobenzene has been studied In male and female
rats following oral administration, rhesus monkeys and beagles following
1.v. Injection, and In rabbits following l.p. Injection (Renner, 1981).
Hexachlorobenzene 1s metabolized slowly Into other lower chlorinated
benzenes, chlorinated phenols and other minor metabolites and forms
02570 111-16 04/12/88
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glucuronlde and glutathione conjugates. Tissues were found to contain
mainly unchanged hexachlorobenzene together with small amounts of metabo-
lites. Similarly, only small amounts of hexachlorobenzene metabolites were
detected In feces, whereas most of the metabolites were excreted 1n the
urine together with small amounts of unchanged hexachlorobenzene.
Mehendale et al. (1975) studied the metabolism of hexachlorobenzene In
male Sprague-Dawley rats 7 days after oral Intubation administration of a
single 5 mg/kg dose In arachls oil. - The fat, liver, Intestines, kidneys,
lungs and brain were found to contain hexachlorobenzene primarily, along
with trace amounts of other chlorinated benzenes. Analysis of these chlori-
nated benzenes suggested the presence of pentachlorophenol, 2,4,5-trlchlor.o-
phenol, pentachlorobenzene and the tetrachlorobenzenes. Extraction and
analysis of fecal radioactivity, which accounted- for 16% of the dose, did
not reveal the presence of metabolites. Although urine contained only 0.85%
of the administered radioactivity, 1t provided the only evidence of hexa-
chlorobenzene metabolite excretion. Several unidentified metabolites were
evident following thin-layer chromatography (TLC) separation of urine, in
addition to 2,4,5-trIchlorophenol, pentachlorophenol and one spot was
reported to contain a mixture of chlorinated benzenes.
In vitro metabolism studies with homogenates of the liver, lungs, kid-
neys and small Intestines produced trace amounts of chlorobenzene metabo-
lites when Incubated with [ldC]-hexachlorobenzene In the presence or
absence of added cofactors. Liver microsomal preparations produced amounts
of one or more chlorophenols when fortified with NADPH; In the presence of
UDPGA, pentachlorophenol was reported to form the glucuronlde conjugate.
02570
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Fortification of kidney homogenates with glutathione resulted In the appear-
ance of unextractable radioactivity 1n the aqueous phases, Indicating that
glutathione conjugates of polar hexachlorobenzene metabolites mlgh-t also be
formed (Mehendale et al., 1975).
The metabolism of hexachlorobenzene In male and female Sprague-Dawley.
rats each receiving nine oral doses of 85.6 mg/kg hexachlorobenzene (99.7%
pure) In arachls oil over a period of 1 month was reported by Rlchter et
al. (1981). The animals were sacrificed 3, 24 and 52 days after the last
dose, and various tissues were analyzed for hexachlorobenzene and Its
metabolites by CDE/GIC and GLC/MS. In addition to hexachlorobenzene, the
following metabolites were also detected: pentachlorobenzene (PCS), penta-
chlorophenol (PCP), pentachlorothlophenol (PCTP) and 2,3,4,6- and 2,3,5,6-
tetrachlorophenol (TCP). The results reported for the liver and kidneys for
day 3 Indicated that the livers of the females contained significantly more
PCTP, a derivative of a glutathione conjugate, than those of the males
(Table III-7). However, It Is not known whether this Increase Is due to a
higher rate of PCTP production or to a lower rate of elimination.
Rlzzardlnl and Smith (1982) Investigated tfw sex differences In hexa-
chlorobenzene metabolism In young F344/N rats who had been Intubated every
other day for 103 days with 14 mg/kg hexachlorobenzene (analytical grade)
dissolved In arachls oil. Three hexachlorobenzene metabolites were analyzed
for: pentachlorobenzene, pentachlorothlophenol and 2,3,5,6-tetrachloroben-
zene-1,4-dlol, and all three were found to be produced In larger concentra-
tions 1n the-female rats during the first 10 weeks of hexachlorobenzene
treatment. The greater quantities of hexachlorobenzene metabolites being
formed In female rats was believed due to their body estrogen levels.
02570 111-18 ' 01/31/85
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TABLE III-7
Concentrations of HC8 and Us Metabolites (mg/kg)
1n the Liver and Kidneys of Male and Female Ratsa.b
T1.ssue/Sex
HCB
PCB
PCP
PCTP
TCP
Liver
Hales
192
0.05
3.16
0.23
0.62
Females
147°
0.03°
2.12C
0.36C
0.04C
Kidneys
Males
127
0.05
5.79
0.24
0.09
Females
m
0.01
3.69
0.10
0.08
aSource: Rlchter et al.f 1981
^Determined 1 3 days after the last of nine oral doses of 85.6 mg/kg HC8
given within 1 month Vn arachls oil
cStat1st1cally significant from males (p<0.05)
HC8 = Hexachlorobenzene; PCB = pentachlorobenzene; PCP = pentachlorophenol;
PCTP = pentachlorothlophenol; TCP = 2,3,5,6-tetrachlorophenol
02570
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Engst et al. (1976) detected several urinary metabolites In male Wlstar
rats receiving by gavage 8 mg/kg of hexachlorobenzene dally dissolved 1n
sunflower oil for 19 days. The results of this study were presented qua 11 —
tatlvely, and the authors reported that the major metabolic route for hexa-
chlorobenzene was to pentachlorophenol. In addition, the feces contained
mainly unchanged hexachlorobenzene together with traces of pentachloro-
benzene.
Koss et al. (1976) Investigated the metabolism of hexachlorobenzene 1n
female Wlstar rats given 2-3 1.p. doses of [14C]hexachlorobenzene (260 or
390 mg/kg total dose). At the end of 4 weeks, 7% of the administered radio-
activity was excreted In the urine, with >90% of this amount contained In
the major metabolites (pentachlorophenol, tetrachlorohydroqulnone, and
pentachlorothlophenol). An Isomer of tetrachlorothlophenol was detected as
a minor urinary metabolite. Twenty-seven percent of the administered radio-
activity was excreted In the feces, of which 70% was Identified as unchanged
hexachlorobenzene. Only pentachlorophenol and pentachlorothlophenol were
Identified as fecal metabolites of hexachlorobenzene. In the tissues of the
animals, only pentachlorophenol was" detected 1n measurable quantities,
accounting for 10% of the radioactivity In blood and <0.1% 1n body fat.
Total radioactivity contained 1n the metabolites detected In the animal
bodies and excreted at the end of the 4 weeks accounted for 16% of the
administered radloactlvlty.
In follow-up studies, Koss et al. (1978a) compared the formation of
hexachlorobenzene metabolites 1n rats, mice, guinea pigs, Japanese quail,
laying hens and rainbow trout. The only metabolites detected were penta-
02570 111-20 07/13/84
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chlorophenol, tetrachlorohydroqulnone and pentachlorothiophenol; however,
the species tested differed greatly 1n their ability to metabolize hexa-
chlorobenzene (Table III-8).
Gas-liquid chromatography of urine, bile and fecal extracts from male
beagle dogs receiving a single l.v. Injection of 14C-hexachlorobenzene at
1 mg/kg revealed that 96% of the fecal radioactivity occurred as the parent
compound. Hexachlorobenzene accounted for 4% of the biliary radioactivity,
but no parent compound was detected Inurlne (Sundlof et al., 1982).
Kohll et al. (1976) studied the metabolism of several chlorinated ben-
zenes, including hexachlorobenzene, 1n rabbits following l.p. injection.
The urine was collected for 10 days after injection and analyzed for metabo-
lites following extraction and gas-liquid chromatography, but no hexachloro-
benzene metabolites were found in the urine.
Excretion
The excretion of hexachlorobenzene from treated animals Is slow and
occurs mainly through the feces, with relatively little being excreted In
th^e urine. It is characterized by an Initial rapid phase followed by a very
slow phase. This slow phase of excretion can be enhanced by the administra-
tion of mineral oil, paraffin and n-hexadecane. Both biliary and intestinal
excretion contribute to fecal excretion. A three-compartment mammlllary
model has been reported for the behavior of hexachlorobenzene in beagles and
rhesus monkeys following l.v. injection of a single dose. Radioactivity was
not detected In exhaled air following l.p. Injection of 14C-hexachloro-
benzene.
02570
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04/12/80
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TABLE 111-8
Hexachlorobenzene and Its Major Metabolites
In the Excreta of Different Animal Species*
Total Amount
of Substances
Spec1esb
Total Oosec
(mMol/kg)
' HCB
PCP
TCH
PCTP
Rat
0.92
6.1d
2.0
0.4
1.8
Mouse
0.92
2.6
0.3
0.1
NO
Guinea pig
0.92
1.8
0.9
0.2
0.5
Japanese quail
2.76
7.5
trace
trace
3.2
Laying hen
0.92
0.6
0.1
0.07.
0.134
Rainbow trout
2.76
1.8
0.4
NO
ND
aSource: Koss et al., 1978a
b2-3 animals were used per each species Investigated
cHexachlorobenzene was dissolved In oil and administered Intraperitoneal^
^Figures are given In yMol/kg bw/day
NO = Not detected. The lower detection limit ofpthe metabolites was deter-
mined to be 0.03 nMol/mi urine or g feces.
HC8 = Hexachlorobenzene; PCP = pentachlorophenol; TCH a tetrachlorohydro-
qulnone; PCTP =, pentachlorothlophenol
02570
111-22
01/31/85
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Studies conducted by Mehendale et al. (1975) with rats receiving a
single oral dose Indicated that only 16.0 and 0.85% were excreted 1n the
feces and urine, respectively, 7 days after treatment (see Table III-l).
Ingebrlgtsen et al. (1981) reported that 4 days after Intragastric admin-
istration of 14C-hexachlorobenzene, a total of 24.8 and 2.IX of the admin-
istered radioactivity were recovered In the feces and urine, respectively.
In addition, an average of 3.6% of the dose was recovered In the bile of
b1le-duct-cannulated rats within 48 hours after dosing. Of the radioactiv-
ity excreted In the bile, only 2% was unchanged hexachlorobenzene, 1.8% was
pentachlorobenzene, 24% was pentachlorophenol and -72% was unidentified
metabolites.
Rozman et al. (1977) studied the excretion of hexachlorobenzene In
female rhesus monkeys receiving 110 yg 14C-hexachlorobenzene/day/monkey
via diet for 15 months. The excretion and storage patterns showed a very
slow approach to a saturation level, Indicating a high tendency for hexa-
chlorobenzene accumulation In rhesus monkeys. A total of S.8 and 3.6% of
the administered dose was excreted In the urine of male and female monkeys,
respectively, after 15 months, of which 50% was pentachlorophenol, 25%
pentachlorobenzene and the remaining 25% consisting of unidentified metabo-
lites with varying amounts of hexachlorobenzene. A total of 47.9 and 27.5%
of the dose was present In the feces of male and female monkeys, respec-
tively, of which 99% was hexachlorobenzene.
Koszoet al. (1978) administered hexachlorobenzene (0.2% 1n the diet) to
young male and female Wlstar rats for as long as 200 days and measured the
accumulation of hexachlorobenzene 1n the liver and fatty tissue and the
02570
111-23
07/13/84
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excretion of hexachlorobenzene and pentachlorophenol 1n the urine and feces.
The concentration of hexachlorobenzene 1n the liver and fat Increased stead-
ily throughout the treatment period. Pentachlorophenol appeared In both the
urine and feces In Increasing amounts throughout the treatment period, with
the excretion of other apolar and polar products being markedly enhanced
after 5-6 weeks. .
Rlzzardlnl and Smith (1982) Investigated the sex differences In hexa-
chlorobenzene metabolism and excretion of hexachlorobenzene metabolites 1n
young F344/N rats. These rats were Intubated with 14 mg/kg analytical grade
hexachlorobenzene dissolved 1n arachls oil every other day for 103 days and
were analyzed for the three main hexachlorobenzene metabolites, pentachloro-
phenol. pentachlorothlophenol and 2,3,5,6-tetrachlorobenzene-l,4-diol, 1n
urine and feces. Results Indicated that the combined urinary excretion of
metabolites was greater 1n the female rats, especially during the first 10
weeks, with pentachlorothlophenol being particularly high In the females.
No wide variations between the sexes were seen 1n the analyzed feces hexa-
chlorobenzene metabolites after 103 days of treatment. Combined urine and
feces excretion of metabolites at the'end of the study were found not to be
significantly different between males (2291+116 nmole/ 24 hours/kg) and
females (2425+182 nmole/24 hours/kg). It was stated, though, that the total
excretion of pentachlorothlophenol was always significantly higher In the
female rats.
Koss and Koransky (1975) studied the metabolism of hexachlorobenzene 1n
rats when the compound was orally administered 1n an aqueous suspension or
In olive oil. The animals received different amounts of 14C-hexachloro-
02570
H1-24
07/13/84
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benzene In a single dose, and the feces and urine were collected at varying
time Intervals and radloassayed. When administered In water, hexachloro-
benzene was not readily absorbed; 76-97% of the dose was excreted In the
feces, and <0.1-0.4% was excreted In the urine 1 day after administration.
When administered In oil, only 45-46% of the dose was excreted In the feces
and 2.1-3.8% was excreted 1n the urine after 14 days of treatment. Rats
receiving 4 mg/kg of 14C-hexachlorobenzene administered l.p. excreted a
total of 5 and 34% of the dose In .the urine and feces, respectively, within
14 days. About 4 and 80% of the ex-creted radioactivity In the urine and
feces, respectively, was unchanged hexachlorobenzene. Animals Injected l.p.
with 50.5 mg/kg [14C]hexachlorobenzene released no radioactivity in
exhaled air (Koss and Koransky, 1975).
Rozman et al. (1981) reported that administration of mineral oil or
n-hexadecane to female Sprague-Dawley rats or male or female rhesus monkeys
who were pretreated with 14C-hexachlorobenzene enhanced the fecal elimina-
tion of 14C-hexachlorobenzene. All animals were administered 14C-hexa-
chlorobenzene (100 mg/kg) 1n 1% methyl cellulose as a single oral dose
Intubation except for one monkey that received three consecutive dally doses'
and two monkeys that received 14C-hexachlorobenzene (0.11 mg/kg) in sugar
pellets daily for 750 consecutive days. Aliphatic hydrocarbons were adminV-
¦
stered to the treated animals 11-40 days after hexachlorobenzene treatment.
When mineral oil was added to the diet of the rhesus monkeys, fecal excre-
tion of hexachlorobenzene was enhanced 6- to 9-fold. Similarly, dietary
administration of hexadecane resulted 1n the same Increase In fecal excre-
tion of hexachlorobenzene in both the rhesus monkeys and rats. Residue
analyses Indicated an enhanced depletion of hexachlorobenzene from blood and
02570
111-25
04/12/88
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of stored hexachlorobenzene from adipose tissue. Enhanced fecal excretion
of hexachlorobenzene as a result of. dietary administration of aliphatic
hydrocarbons was primarily due to Increased hexachlorobenzene elimination In
the large Intestine.
Rlchter and Schafer (1981) studied the Intestinal excretion of hexa-
chlorobenzene In male Sprague-Dawley rats using the pendular perfusion
method. The animals were Injected'1.p. with hexachlorobenzene at 100 mg/kg
and, 1 and 4 weeks "after treatment, various parts of the Intestines were
perfused with paraffin or squalane for 5 hours. The largest amount of hexa-
chlorobenzene excreted was Into the Jejunum followed by the Ileum and
colon. The ratios of total hexachlorobenzene excreted during paraffin
treatment were: jejunum/1leum = 1.26 and jejunum/colon = 2.43. The authors
concluded that these results Indicate the Importance of Intestinal excretion
In the elimination of hexachlorobenzene, and that paraffin treatment can be
one of the measures by which a long-term stimulation of hexachlorobenzene
excretion can be achieved.
Beagle dogs receiving a single 1.*. dose of 1 mg/kg excreted 44 and <6%
of the dose In the feces and urine, respectively, during a 12-week period
» (Sundlof et al., 1982). Both biliary and Intestinal excretion contributed
to fecal excretion; however, the data Indicated that biliary excretion was
the major contributor to fecal excretion. A computer-assisted pharmaco-
kinetic analysis of blood, urine and fecal radloactlvlty levels during a
12-week period suggested a three-compartment model for the behavior of hexa-
chlorobenzene in beagles. The biologic half-life values were calculated for
the three dogs used and ranged from 6 weeks to 3 years.
02570
111-26
04/05/91
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Yang et al. (1978) reported that the elimination rate of hexachloroben-
zene from male Sprague-Dawley rats and rhesus monkeys Injected l.v. with
hexachlorobenzene was slow because hexachlorobenzene Is stored .In the Fat
tissue. The major route of excretion for the radlolabel 1ji treated monkeys
was. via the feces. About 17.1, 8.8 and 28.2% of the dose was excreted In
the feces after 100 days, 6 months and 1 year, respectively, after treatment,
of Individual monkeys, with -90% of the radioactivity determined to be
unchanged 14C-hexachlorobenzene. The cumulative urinary excretion of
hexachlorobenzene metabolites was determined to be 1.6% of the administered
dose after 1 year. An.open system, three-compartment mammlllary model was
found to fit the data for plasma, fecal and metabolized hexachlorobenzene 1n
the rhesus monkey.
Koss et al. (1983) administered 100 mg/kg hexachlorobenzene In olive oil
every other day, via stomach tube, to female Wlstar rats for a period of 6
weeks and then observed the rats for an additional 18 months. At cessation
of hexachlorobenzene treatment they tried to assess the biologic half-life
of hexachlorobenzene and determined a value of 8 days for the start of the
elimination phase, a value of 10 weeks when assessed 3 months later, and
finally a value of 1.5 years after 12 months. .The authors then concluded
that It Is not possible to establish a valid biologic half-life for- the,
total elimination phase of hexachlorobenzene in rats.
Bleavins et al. (1982) studied the excretion and transfer of hexachloro-
benzene given to female European ferrets (Mustela putorlus furo). Three
bred and five non-bred female ferrets were each given a single dose of 57.6
ug 14C-hexachlorobenzene 1n 7.5 g of standard mink diet (22.2% fat).
02570
111-27
04/05/91
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The Investigators Indicated that there were no significant differences In
the excretion of hexachlorobenzene metabolites, between bred and non-bred
groups, In urine for the entire 8-week study period or 1n feces during the
beginning of the study. The observed fecal excretion during the middle
weeks to the end of the study showed a leveling of the cumulative fecal
excretion In the bred females and a continued Increase In fecal excretion In
the non-bred female ferrets, although It was stated that this difference was
not statistically significant. Excretion of hexachlorobenzene or metabo-
lites 1n the milk was found to be an Important route of excretion for lac-
tatlng females, -20.3% of the Initial dose was eliminated by the fifth week
of lactation, and found to be a very Important route of exposure to nursing
offspring. The Importance of placental transfer and milk excretion Is fur-
ther presented by observing the time required for 50% of the Initial hexa-
chlorobenzene dose to be excreted. The bred females required 32 days to
excrete 50% while 41 days was required for the unbred females.
Summary
The pharmacokinetics of hexachlorobenzene In a- number of mammalian
species have been studied 1n detail fallowing oral administration and, to a
lesser extent, following l.v. or l.p. Injection.-- No Information was present
In the available literature on hexachlorobenzene metabolism following
Inhalation or topical application. Absorption of hexachlorobenzene from
the Intestinal tract appears to depend on the solvent vehicle used during
test material administration. Thus, when hexachlorobenzene Is admin-
istered 1n olive oil, -00% of the dose Is absorbed; when It 1s administered
In an aqueous solution, In 1% methyl cellulose or In a crystalline form,
relatively little (<20%) Is absorbed. Intestinal absorption of hexachloro-
02570
111-28
07/13/84
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benzene occurs primarily through lymphatic channels, with only a minor por-
tion being absorbed Into the portal circulation.
Following absorption, hexachlorobenzene distributes to -tissues that have
a high lipid content. The adipose tissue accumulates the greatest concen-
trations of hexachlorobenzene In all species studied, although bone marrow
and skin, which contain large amounts of lipids, also accumulate hexachloro-
benzene. The adrenal cortex accumulates hexachlorobenzene at concentrations
approaching those of fat. Other tissues (e.g., liver, kidneys, lungs,
heart, spleen and blood) generally contain much lower amounts of hexachloro-
benzene. Intravenous Injection of hexachlorobenzene results In a tissue
distribution similar to that seen following oral administration. Hexachlo-
robenzene 1s transported via the placenta and 1s distributed 1n fetal tissue
1n rabbits, rats, mice, minks and ferrets,
Hexachlorobenzene Is metabolized slowly Into other chlorinated benzenes,
chlorinated phenols and other minor metabolites and forms glucuronlde and
glutathione conjugates. Tissues were found to contain mainly unchanged
hexachlorobenzene together with small" amounts of metabolites. Similarly,
only small amounts of hexachlorobenzene metaboll-tes were detected 1n feces,
whereas most of the metabolites were excreted 1n the urine together with
small amounts of unchanged hexachlorobenzene. There are Indications that
females produce and excrete more hexachlorobenzene metabolites than do males.
The excretion of hexachlorobenzene from treated animals Is slow and
occurs mainly through the feces with relatively little being excreted in the
urine. It Is characterized by an Initial rapid phase followed by one or
02570
111-29
01/31/85
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more slow phases. This slow phase of excretion can be enhanced by the
administration of mineral oil, paraffin or n-hexadecane. Both biliary and
Intestinal excretion contribute to fecal excretion. A three-compartment
mamminary model has been reported for the behavior of hexachlorobenzene 1n
beagles and rhesus monkeys following l.v. Injection of a single dose.
Radioactivity was not detected 1n exhaled air following 1.p. Injection- of-
14C-hexachlorobenzene. Hexachlorobenzene has been detected In the milk of
nursing mammals .•
02570
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IV. HUMAN EXPOSURE
This chapter will be submitted by the Science and Technology Branch,
Criteria and Standards Division, Office of Drinking Water.
02500
IV-1
01/28/85
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V. HEALTH EFFECTS IN ANIMALS
Acute Toxicity
Information on the acute toxicity of hexachlorobenzene was limited to
oral LDgg values determined with a few marranallan species. The following
LD(.Q values were reported In the available literature: rats, 3500-10,000
mg/kg; rabbits, 2600 mg/kg; cats, 1700 mg/kg; and mice, 4000 mg/kg (IARC,
1979; NAS, 1977; Saxt 1979).
Graef et al. (1979) reported that hexachlorobenzene blocked the activity
of rat hepatic 3-hydroxysterold dehydrogenase leading to the accumulation of
5B-H-stero1ds, which are known Inducers of porphyrin biosynthesis. Hexa-
chlorobenzene-lnduced porphyria has also been reported to occur as a result
of a deficiency 1n the uroporphyrinogen decarboxylation process that Is
catalyzed by porphyrinogen carboxylase. This enzyme Is the only one in the
heme pathway that exhibits a decrease In activity. The Inhibition of por-
phyrinogen carboxylase In liver homogenates from female Wlstar rats with
severe porphyria Induced by hexachlorobenzene was studied by Rios de Molina
et al. (1980). Hexachlorobenzene had no effect on enzyme activity at
10"3 M, whereas pentachlorophenol caused a 90% Inhibition at the same con-
centration. However, pentachlorophenol" did not inhibit the enzyme at a con-
centration of 10"s M. . It was concluded that a- concentration >10~5 M of
pentachlorophenol, possibly together with other hexachlorobenzene metabo-
lites, was needed to cause enzyme.Inhlbl11 on.
Hexachlorobenzene has also been reported to Induce the activity of
hepatic microsomal enzymes In male or female rats following subchronlc
administration (Carlson, 1978; Carlson and Tardlff, 1976; Chadwlck et al..
02590
V-l
09/15/88
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1977). Hexachlorobenzene produced a so-called "mixed-type" Induction of
cytochrome P-450 content 1n female rats resembling that produced by a com-
bination of phenobarbltal (cytochrome P-450) and 3,4-benzopyrene (cytochrome
P-448) (Goldstein et al., 1982; Debets et al.( 1980a).; In female rats,
hexachlorobenzene Increased the activities of ^-aminolevulinic add syn-
thetase and amlnopyrlne demethylase (Arlyoshl et al.# 1974), ethoxy-
resoruf1n-0-deethylase, amlnopyrlne demethylase, aryl hydrocarbon hydroxy-
lase, p-n1trophenol glucuronyl transferase, and NADPH-cytochrome c reductase
(Goldstein et al., 1982; Oebets et al., 1980a). Similarly, In male rats,
hexachlorobenzene Increased the activities of hepatic ethyl morphine N- and
p-n1troan1sol O-demethylases, aniline hydroxylase, and UOP glucuronyl trans-
ferase (Mehendale et al., 1975), acetanlllde hydroxylase, acetanlllde ester-
ase, procaine esterase, and arylesterase activities (Carlson et al., 1979;
Carlson, 1980).
Subchronlc Toxicity
Several oral subchronlc studies of hexachlorobenzene have been reported,
but no studies were located on the effects of hexachlorobenzene following
inhalation. In several animal species, hexachlorobenzene was found to cause
alopecia and scabbing, decreased body weight, --Increased liver and kidney
weights and Increased porphyrin levels In,the urine and In several organs.
Histopathologic changes were noted In the liver and kidneys of rats, gastric
lymphoid tissue of dogs, and ovaries of monkeys. When placed on untreated
diets, rats were able to recover from most of the toxic effects of hexa-
chlorobenzene treatment. Hexachlorobenzene was also reported to cause
certain neurologic effects (ataxia, paralysis, etc.) on rats, mice, hamsters
and female beagles, and to Induce an Increase In hepatic microsomal enzyme
activity. Toxicity data for hexachlorobenzene can be found 1n Table V-l.
02590
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I
o
ro
ui
to
o
TABLE V-l
Summary of Toxic 1ty Studies on Hexachlorobeniene
Species
Route
Oose
Duration
Effects
Reference
i
CJ
Rat
(females)
Rat
Rat
(females)
Rats
(females)
CO
Rat
(females)
oral
oral
(diet)
oral
(gavage)
oral
(gavage)
oral
(diet)
100 mg/kg every other
day
0.5 mg/kg/day
2.0 mg/kg/day
8.0 mg/kg/day
32.0 mg/kg/day
O.S mg/kg twice
weekly
2.0 mg/kg twice
weekly
6.0 mg/kg twice
weekly
32.0 mg/kg twice
week I y
100 mg/kg diet
up to 43 days
IS weeks exposed and
held to IB weeks
IS weeks exposed and
held to 48 weeks
IS weeks exposed and
held to 48 weeks
IS weeks exposed and
held to 48 weeks
SO mg/kg every other 15 weeks
day
29 weeks
29 weeks
29 weeks
29 weeks
98 days
Suggested covalent binding of hexachlorobemene
metabolites to cytosollc proteins
Transient Increases In liver porphyrin levels
In females after termination of exposure
Increases In liver porphyrin levels In females
after termination of exposure. Increased size
of centrI lobular hepatocytes
Increased liver weights. Increased liver,
kidney and spleen porphyrin levels In females
(porphyria), centrllobular liver lesions espe-
cially In females at 48 weeks
Increased mortality In females. Intension
tremors In males and females and ataxia In a
few females. Increased liver, kidney and
spleen weights. Increased liver; kidney and
spleen porphyrin levels In females (porphyria),
centrllobular liver lesions and splenomegaly
Increased liver, kidney, spleen and adrenal
weights, porphyria (Increased liver porphyrin
levels and Increased excretion of porphyrins
and precursors), tremors, hair loss and skin '
lesions
Increase In relative liver weight
Increase In relative liver weight, moderately
enlarged hepatocytes
Porphyria, markedly enlarged hepatocytes,
-Increase In relative liver weight
Porphyria, markedly enlarged hepatocytes.
Increase In liver weights
Porphyria (Increased liver lobe porphyrins),
decreased activity of uroporphyrinogen
decarboxylase
Koss et al..
1980a
Kulper-Goodman
et al., 1977
Koss et al.,
1978b
BBger et al.,
1979
Smith et al.,
1980
-------
o
IV)
Ln
10
o
lABlf VI (cont.)
o
N
O
cn
SpecIcs
Rat
Rat
Rat
Rat
Rat
(male)
Rdt
(female)
Rat
(fema1e)
Rdt
Rdt
(females)
Rat
(f emaIe i)
Route
ora I
(diet and
nursing)
oral
(diet dnd
nursIng)
ora)
(diet)
oral
(diet)
ora I
(diet)
oral
(diet)
oral
(gavage)
oral
(gavage)
oral
(gavage)
ora 1
(diet)
Oose
SO my/kg diet
ISO mg/kg diet
4. 20 or 100 mg/kg
diet
500. 1000 or 2000
mg/kg diet
2000 mg/kg diet
2000 mg/kg diet
3000 mg/kg diet
SO, 100 or 200 mg/kg
14 mg/kg every other
ddy
100 mg/kg every
other day
6 8 mg/kg/day
Ourat ton
ges tat Ion unt11
S weeks of age
gestation until
S weeks of age
gestation until
S weeks of age
3 weeks
Effects
Reference
10 weeks
100 days
11 weeks
120 days
103 days
6 weeks exposed and
held for additional
IB months
7S-90 weeks
Depressed resistance to L. monocytogenes and Vos et a)..
1. spiralis, enhanced thymus-dependent antibody 1979b
response
Increased serum IgM and IgG, depressed resis-
tance to L. monocytogenes and T. spiralis,
enhanced thymus-dependent antibody response.
Increased liver and adrenal weights
Increased IgH and IgG responses to tetanous Vos et al..
toxlod, delayed-type hypersensitivity reactions 1983a,b
to ovalbumin, noted accumulation of alveolar
macrophages; no change In T. spiralis resistance
Dose-related Increases In relative spleen, Vos et al.,
lymph nodes, liver, adrenals, thyroid, testes 1979a
and kidney weights, dose-related Increase In
serum IgH levels, no change In serum IgG
levels, dose-related pathologic changes In
liver, lymph nodes and spleen
Porphyria found microscopically at 5 weeks and Gralla et al..
grossly at 10 weeks using fluorescence 1977
Elevated hepatic enzymes by ) week and Increased Llssner
urinary porphyrin and ALA levels (porphyria) as et al., 197S
early as 40 days
Decreased uroporphyrinogen decarboxylase Elder et al.,
activity and porphyria after 4 weeks 1976
Dose- and time-dependent Increase In liver and Carlson, 1977
urine porphyrins (porphyria)
Porphyria In treated females, susceptibility of RWiardlnl and
females to porphyria may be related to estrogen Smith, 1982
levels
Porphyria (liver uroporphyrin levels peaked 7 Koss et al.,
months postexposure and levels had not returned 1983
to normal by 18 months), decreased liver proto-
porphyrin and coproporphyrIn levels. Inhibition
of uroporphyrinogen decarboxylase activity
until 18 months postexposure
Decline In.body weights, porphyria, enlarged' Smith and
livers and liver tumors Cabral, 1980
-------
TABLE V-l (conl. )
CD
r\j
L/l
U3
O
Specles
Rat
Ral
Hal
Ral
i
i/i
Route
lira I
(diet)
or a ]
(diet)
ora I
(dtel)
ora I
(diet)
oral
(diet and
nur sIng)
Dose
75 nig/kg diet
(4 5 my/kg/day)
150 mg/kg diet
(8-9.5 mg/kg/day)
75 or 150 mg/kg diet
800 mg/kg diet
0.32. 16. 8.0 or
40 mg/kg diet
0.32 or 1.6 mg/kg
diet
8.0 mg/kg diet
40 mg/kg diet
OuratIon
Effects
Reference
up to 2 years
up to 2 years
20 weeks
-130 days
gestation through
lifetime (130 weeks)
gestation through
1 If elIme (130 weeks)
gestation through
11felIme (130 weeks )
Porphyria, time-related appearance of severe Lambrecht el
hepatic and renal pathologies, after 1 year In- al.. 1983a,b
creases In hepatomas, hepatocarc1 nomas, bile duct
adenomas, renal adenomas and renal carcinomas
Decreased nerve conduction velocities 8 and Suftt et al.,
31% In 75 and 150 ppm groups, respectively; 1986
muscles showed signs of denervation,
fIbr11 lalIons and pseudomyotonla
Reduced nerve conduction velocities, no muscle Suflt et al.,
abnormalities as observed In 2-year study 1986
Hematologic changes at all dose levels In Arnold et al.
males. Increases In liver and heart weights In 1985
males at 8.0 and 40 ppm diets, no treatment-
related effects observed In bred females
Glycogen depletion In 1.6 mg/kg males; no
effects reported at 0.32 mg/kg
Increase In liver pathologies
Increased mortality as pups. Increase In liver
and kidney pathologies. Increase In adrenal
pheochromocytomas In females and parathyroid
tumors In males
Kal
oral
(diet)
o
o
i_n
10 or 20 mg/kg diet
40 mg/kg dtel
80 mg/kg diet
160 mg/kg diet
320 and 640 mg/kg
dtel
fg to fj generations
Fq to F4 generations
Fq to F4 generations
Fq to I 4 general Ions
Fq to F4 generations
No effects reported
Increases In liver weights and aniline
hydroxylase activity
Decreased body weights, F3 and F4 generations had
decreased lactation Index and postnatal viability
and Increased stillbirths
Increased mortality and decreased lactation
Index starting In F] generation
20 and 50% mortality in Fo 320 and 640 mg/kg
groups, respectively, greatly reduced fertility
Index and litter size and Increase In still-
births, viability Index zero in F|
Grant et al.,
1977
-------
TABLE V-1 (cont.)
ro
cn
vO
O
Spec les
Route
Dose
Duration
Effects
Reference
Rat
oral
(diet)
60. 80. 100, 120 or
140 ag/kg diet
fo lo fia and
general ions
Increased aortallty In all groups at 21 days,
21-day LDjn values for pups were 100 and 140
ag/kg for fja and Fn, generations, respectively
Kltchln
et al., 1982
Rat
oral
(diet)'
0 or 80 ag/kg diet
gestatIon and
nursing or cross
nursed with controls
Nursing exposure produced greater effects than
did gestational exposure, effects noted were:
smaller brains, hearts, kidneys and spleens.
Increased liver weights
Mendoia
el al.. 1978
Rat
oral
(diet)
80 ag/kg diet
2 weeks prior to
aatlng to 3S-36 days
after weaning
Increased porphyrin levels and decreased liver
esterase activity In daas. no changes In
gestation Indices or neonatal survival
Mendoia
el al., 1979
V-6
Rat
oral
(gavage)
10, 20. 40. 60. 80
or 120 ag/kg
days 6-21 of gesta-
tion
Maternal toxicity (weight loss, treaors and
convulsions) and reduced fetal weights at 120
and 80 ag/kg aaternal doses, dose-related In-
crease In Incidence of unilateral and bilateral
14th rib, sternal defects were also no'ted In
one experiment
Khera, 1974
House
oral
(diet)
2.5. 26 or 250
ag/kg diet
21 days
Dose-related Increase In liver and decrease In
prostate and sealnal vesicle weights, dose-
related alterations In testosterone aetabollsa,
altered hepatic eniyne levels
illssalde and
Clark, 1979
Mouse
(male)
oral
(diet)
10 ag/kg diet (8.4
(ag/aouse/24 weeks)
or SO ag/kg diet
(35.3 mg/nouse/
24 weeks)
24 weeks
Dose-related reduction In weight gain, no tumor
pathology observed
Shlral et al.,
1978
House
(male)
oral
(diet)
167 ag/kg diet
3-6 weeks
lapalraent In host resistance as aeasurad by
Increased sensitivity to S. typhosa and P.
berghel. and decrease In IgA levels
Loose et al.,
1978a,b
o
ic
\
House
oral
(diet)
6. 12. 24 and 36*
mg/kg/day
101-120 weeks
'(IS weeks exposed
held until 120
weeks)
Reduced growth rate at all dose levels, short-
ened lifespan associated with tremors and con-
vulsions In 24 and 36 ag/kg/day groups, dose-
dependent Increase In liver-cell tumors In the
12. 24 and 36 mg/kg/day dose groups
Cabral et al.,
1979
in
s
00
CO
House
oral
(gavage)
100 ag/kg/day to
pregnant mice
days 7-16 of
gestatIon
Increased aaternal livers and decreased fetal
body weights. Increased Incidence of abnormal '
fetuses per litter observed
Courtney
et al., 1976
Hamster
oral
(diet)
200 or 400 tuij/kg
diet
90 days
PrecIrrhol1c and cirrhotic hepatic lesions,
bile-duct hyperplasias and hepatomas
Laiubrecht
el al.. 19B2a
(¦
-------
TABLE V-l (conl.)
Species
Route
Dose
OuratIon
iffects
Reference
Hamster
oral
(illel)
4, 8 or lb nig/kg/day
1 Ifespan
Shortened lifespan In 16 ag/kg/day group. In-
crease In hepatomas at all dose levels. Increase
In liver hemangioendothelioma In Males and
females and an Increase In thyroid alveolar
-adenomas In Mies In 16 ng/kg/day group
Cabral et al.,
1977
CdlS
(breeding
females)
oral
(diet)
3 or 8.7 mg/day/cat
142 days
Height loss and Increased disease susceptibility
In bred females, dose-related decrease In litter
size and survival of offspring, hepatomegaly In
offspring
Hansen et al.,
1979
Hlnks
oral
(diet)
1 or 5 ag/kg diet
during gestation
until 17 weeks of
aye
Dose-related Increase In offspring mortality.
Induction of hepatic Hf0-enzymes In exposed
offspring
Rush et al.,
1983
Dog
(female)
oral
(capsule)
SO or ISO ag/kg/day
21 days
Liver and hepalocyte enlargement, dose-Induced
electroencephalogram dysrhythmias
Sundlof
el al., 1981
Dog
oral
(capsule)
1. 10, 100 or 1000
rag/day/dog
1 year
Increase In mortality, neutrophilia, and
anorexia In the 100 and 1000 mg dose groups,
dose-related nodular hyperplasia of gastric
lymphoid tissue In all treated animals
Gralla et al.,
1977
Monkey
(female)
oral
(gavage)
8. 32. 64 or 128
ag/kg/day
60 days
Dose-related pathology In liver, kidney, ovaries
and thymus
latropoulus
el al.. 1976
Honkey
oral
(nursIng)
7.51-106 ppm milk
60 days
2 of 3 Infants died as a result of exposures
Datley el al..
1980
-------
Iatropoulos et al. (1976) reported that five adult female rhesus monkeys
given dally gavage treatments, of hexachlorobenzene suspended In 1% carboxy-
methylcellulose at 8, 32, 64 or 128 mg/kg/day for 60 days, showed extensive
morphologic changes In the ovaries. These changes were dose-related.
Subchronlc studies conducted by Koss et al. (1960a) with groups of four
female rats treated orally (probably by gavage) with 100 mg/kg of hexachlo-
robenzene In olive oil every, other day, suggested that hexachlorobenzene
metabolites covalently bind to cytosollc proteins although no binding to
uroporphyrinogen decarboxylase was specifically demonstrated.
Ellssalde and Clark (1979) reported a significant Increase In the
vitro metabolism of 3H-testosterone by liver microsomes from male mice fed
diets containing 250 mg hexachlorobenzene/kg for 21 days. In addition,
decreases In the concentration of testosterone In the serum and In the
weights of seminal vesicles and ventral prostates were reported. Hexa-
chlorobenzene was also reported to cause certain neurologic lesions in male
and female rats, hamsters and mice fed diets containing various levels of
hexachlorobenzene for 13 weeks. These Included hyperemia, edema, arboriza-
tion and hemorrhages in the brain and meninges. The lesions extended to the
cerebrum, cerebellum, medulla, spinal cord and their meninges. The severity
of these lesions was higher In males and was dose dependent in both sexes
(Headley et al., 1981). Physiologic changes (electroencephalogram dys-
rhythmias) In the central nervous system were reported In 10 female beagles
receiving gelatin capsules containing doses of 50 or 150 mg/kg of hexa-
chlorobenzene for 21 days (Sundlof et al., 1981).
02590
V-8
04/11/88
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Kulper-Goodman et al. (1977) conducted a 15-week subchronlc feeding
study wherein groups of 70 male and 70 female COBS rats were fed diets, pro-
viding 0, 0.5, 2, 8 or 32 mg/kg bw/day of hexachlorobenzene originally
dissolved In corn ,011 (5%).and mixed with the feed. Female rats were more
susceptible to hexachlorobenzene than males, as Indicated by all the para-
meters studied, and a NOEL of 0.5 mg/kg/ day was suggested by the authors.
This NOEL may be better Interpreted as a NOAEL since a transient Increase In
liver porphyrin levels was observed In females 4 weeks after removal from
hexachlorobenzene. The Z mg/kg/day dose may be interpreted as a LOAEL since
this level caused Increases In liver porphyrin levels In females even 33
weeks after removal from hexachlorobenzene, and Increases In the relative
observed severity of centrllobular liver lesions as compared with control
rats. About 40% mortality occurred In females, but none In males at the
highest dose. Clinical signs Included Intention tremor, excessive Irrita-
bility, multiple alopecia, scabbing and ataxia, with hind leg paralysis at
the highest dose. There was a significant Increase in liver and kidney
weights at the higher doses. An Increase In liver weight was also found In
groups of 36 female Wlstar rats treated by gavage twice weekly with hexa-
chlorobenzene dissolved In olive oil at 32 mg/kg for 29 weeks (Boger et al.,
1979 )*. Similarly, Koss et al. ( 1978b) reported a.1.5- to 2-fold Increase In
the weights of the liver, spleen, kidneys and adrenal glands from female
Wlstar rats treated orally {esophageal tube) with 50 mg/kg of hexachloroben-
zene dissolved 1n corn oil every other day for 15 weeks. When hexachloro-
benzene-treated rats were placed on untreated diets, they no longer showed
signs of hexachlorobenzene toxicity, such as dermal lesions, and body and
organ weights returnedr to normal (Kulper-Goodman et al., 1977; Koss et al.,
1978b). Enlarged livers were reported 1n subchronlc studies with female
02590
04/05/91
-------
beagles (Sundlof et al., 1981] and male mice (Shlral et al.( 1978) admini-
stered hexachlorobenzene In diet.
A dose-dependent enlargement of hepatocytes was observed In groups of 36
female Wlstar rats receiving gavage treatments of olive oil containing hexa-
chlorobenzene (99.8%-pure) 0.5, 2.0, 8.0 and 32 mg/kg twice weekly for 29
weeks (Boger et al., 1979). This effect was associated with the prolifera-
tion of the smooth endoplasmic reticulum In the centrllobular area, and an
Increase In glycogen deposits; however, animals receiving 0.5 mg/kg did not
develop enlarged hepatocytes. In addition, atypical membrane complexes In
treated animals were noted and liver-cell mitochondria were moderately
enlarged and had Irregular shapes. Kulper-Goodman et al. (1976) also
reported significantly enlarged hepatocytes In male and female COBS rats
receiving hexachlorobenzene In diets, containing 5% corn oil, at the 8.0 and
32.0 mg/kg bw dose levels for 15 weeks. They observed that this hepatocyte
enlargement consisted to a large degree of proliferation of the smooth endo-
plasmic reticulum. In males this proliferation was often associated with
large whorls of compacted membranes surrounding lipid droplets. The nuclei
of enlarged hepatocytes were also enlarged while the mitochondria were very
small and sparse. They stated that this proliferation of smooth endoplasmic
reticulum was related to the increased drug metabolizing enzyme activity of
the liver and was considered an adaptive rather than toxic response to the
hexachlorobenzene, since the enzyme activity and liver morphology returned
to normal after exposures were discontinued. An Increase in the size of
centrllobular hepatocytes was also reported In male and female rats
receiving 2 mg/kg/day for 15 weeks, together with histopathologic changes In
the spleen (Kulper-Goodman et al.f 1977).
02590
V-10
04/05/91
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Nodular hyperplasia of gastric lymphoid tissue was reported In groups of
b male and 6 female beagles receiving dally gelatine capsules containing lt
10, 100 and 1000 mg hexachlorobenzene/dog/day for 12 months (Gralla et al.f
1977). Extensive dose-related histopathologic changes were, also observed 1n
ovaries from groups of two rhesus monkeys given dally methyl cellulose/
distilled water solutions containing doses of 8," 16, 32, 64 or 128 mg hexa-
chlorobenzene/kg of body weight by gavage for 60 days (Knauf and Hobson,
1979; Iatropoulas et al., 1976). "Sh1ra 1 et al. (1978) conducted a 24-week
study with male mice fed -diets containing 10 or 50 ppm of hexachlorobenzene,
followed by a recovery period of 14 weeks. Histologic examination revealed
no pathologic changes In the liver or any other organ.
Lambrecht et al. (1982a) fed male and female Syrian golden hamsters
hexachlorobenzene at doses of 0, 200 and 400 ppm In their diet for 90 days.
The hamsters were killed on day 91 and at 6-week Intervals through day 361.
No differences were seen In growth and food consumption between control and
exposed animals. The liver was reported as the most severely affected organ
exhibiting a variety of preclrrhotic and cirrhotic lesions, bile-duct hyper-
plasias and hepatomas. The Incidence of neoplasms found in this study will
be further discussed In the Carcinogenicity Section.
Hexachlorobenzene has been found to cause increased porphyrin levels In
the liver of male and female rats receiving the compound Incorporated Into
the diet at doses of 8 and 32 mg/kg/day for 15 weeks (Ku 1 per-Goodman et al.,
1977). Koss et al. (1978b) reported that female rats treated orally with 50
mg hexachlorobenzene/kg every other day for 15 weeks still showed increased
levels of porphyrin In the liver, 38 weeks after the last treatment. In
02590
V-11
04/05/91
-------
addition, porphyrin, ^-aminolevulinic acid, and porphobilinogen levels In
the urine gradually Increased during the 15-week treatment period, but sub-
sequently decreased to normal levels. Smith et al. (1980) reported that the
lobes of livers From female Agus rats fed diets containing 0.01% hexachloro-
benzene developed porphyria at different rates. During the Initial course
of treatment, porphyria In the caudate lobe developed at a significantly
slower rate than the median, left or right sections of the liver, but event-
ually, all lobes became equally po'rphyrlc. In contrast, porphyria was not
observed when viewed for hepatic fluorescence of porphyrins In male and
female beagle dogs treated dally with 0# 1, 10, 100 or 1000 mg/dog/day for 1
year (Gralla et al., 1977). Gralla et al. (1977) observed that female CD
rats fed 0.2% hexachlorobenzene were porphyrlc using this fluorescence
method.
Rlzzardlnl and Smith (1982) clearly confirmed that female rats are more
susceptible to hexachlorobenzene-lnduced porphyria than are male rats, and
that this difference In susceptibility Is probably associated with the
faster metabolism of hexachlorobenzene In females. They Intubated male and
female F344/N rats every other day for 103 days with 14 mg/kg (50 ymole/
kg) hexachlorobenzene dissolved in arachls oil and monitored the rats for
hexachlorobenzene metabolites and porphyrin levels. The results Indicated
that after 75 days of hexachlorobenzene treatment the excretion of urinary
porphyrins increased rapidly In the females and after 103 days the females
had urine and liver porphyrin levels 40- and 310-fold higher, respectively,
than did the males. During this treatment period the females were found to
excrete greater quantities of hexachlorobenzene metabolites, especially
pentachlorothlophenol, than the males. Estrogen levels seem to play an
02590
V-12
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-------
Important part In the Increased susceptibility of Females to hexachloroben-
zene-lnduced porphyria. When both male and female rats were pretreated
Intraperltoneally with four doses of 20 ymole/kg of dlethylstllboestrol
dlpropionate (an estrogenic drug), both sexes had stimulated excretion of
hexachlorobenzene metabolites.
Hexachlorobenzene pretreatment has been reported to cause altered Immune
responses. Vos et al. (1979b) studied the effect of hexachlorobenzene on
the Immune system after combined pre- and postnatal exposure. Wlstar rats
were fed diets containing 0, 50 or 150 mg/kg hexachlorobenzene during preg-
nancy and lactation. The pups were weaned after 3 weeks and continued on
the test diets until 5 weeks of age, when their immune system was function-
ally assessed. At the higher dietary level, hexachlorobenzene caused a
significant Increase In serum IgM and IgG concentrations.
Hexachlorobenzene treatment also caused a decreased resistance to Infec-
tion with Listeria monocytogenes (Vos et al., 1979b). The LD^g values
were reported to be 14x10s, 7.1x10s and 5.0x10s bacteria in pregnant
Wlstar rats receiving diets containing 0, 50 and 150 mg/kg# respectively.
Similarly, decreased resistance of Tr1ch1nella spiralis Infection, as indi-
cated by an Increase in the number of larvae found in muscle tissue, was
noted. Hexachlorobenzene also enhanced the thymus-dependent antibody
response to T^_ spiralis antigen and to tetanus toxoid. No effects were
observed on allograft rejection, mltogenlc response of thymus and spleen
cells, thymus-lndependent IgM response to Escherichia col 1 llpopolysaccha-
rlde, passive cutaneous anaphylaxis, or on the clearance of carbon particles
and L monocytogenes. The authors concluded that hexachlorobenzene sup-
pressed cellular icmiunlty and enhanced humoral Immunity In both test groups.
02590 V-13 04/05/91
-------
In a second combined pre- and postnatal hexachlorobenzene dietary study
Wlstar rats were similarly exposed to diets containing 0, 4, 20 or 100 mg/kg
hexachlorobenzene during gestation, nursing and until 5 weeks of age (Vos et
al., 1983atb). The primary and secondary IgM and IgG responses to tetanous
toxoid (humoral Immunity parameters) were observed to be significantly
Increased In all test groups compared with controls. Delayed-type
hypersensitivity reactions to ovalbumin (cell-mediated Immunity parameter)
were significantly enhanced In the 4 and 100 mg/kg groups and the 20 mg/kg
group was observed as markedly Increased (non-slgn1f1cantly) compared with
controls. No hexachlorobenzene Induced effects were observed on the
resistance to Trlchlnella spiralis» on the antibody response to ovalbumin,
and on the J_n vitro natural cytotoxic activity of spleen cells against YAC
lymphoma cells. Even at the 4 mg/kg diet level accumulation of macrophages
were noted In the lung alveoli of exposed rats. At the 4 mg/kg diet level
liver weights, morphology and microsomal enzymes were not altered, except
for an Increase In the activity of 7-ethoxyresorufIn-o-deethylase. These
results led the authors to conclude that the developing Immune system Is
particularly vulnerable to hexachlorobenzene exposure.
In contrast, hexachlorobenzene pretreatment of weanling rats did not
alter their cell-mediated Immunity, but did stimulate their humoral Irwnune
response and enhanced the Yn vitro responsiveness of spleen cells to dif-
ferent mitogens, which was mainly a result of an Increase In the number of
splenic lymphocytes. The rats received diets containing 1000 pg hexa-
chlorobenzene/g for 3 weeks after weaning, before assessing their Immune
system (Vos et al.# 1979a).
02590
V-14
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Loose et al. (1978a,b) found that hexachlorobenzene pretreatment also
resulted In Impaired host resistance. Male BALB/c mice received diets con-
taining 167 ug hexachlorobenzene/g for 3 or 6 weeks before assessing their
Immune functions. The concentration of IgA was significantly
decreased,whereas those of IgG and IgM did not exhibit consistent
significant altera- tlons as compared* with the controls.
Hexachlorobenzene-treated mice were more sensitive to gram-negative
endotoxin (Salmonella typhosaJ. showed a decreased resistance to a"malaria
challenge (Plasmodium berghel). and exhibited slgnflcantly depressed
antibody synthesis.
Chronic Toxicity
Cabral et al. (1977) studied the tumorlgenlclty of hexachlorobenzene In
6-week-old Syrian golden hamsters given 0, 50 (4 mg/kg/day), 100 (8 mg/kg/
day) and 200 (16 mg/kg/day) ppm hexachlorobenzene in their diets for their
remaining lifespan. Shortened lifespan was observed In the male and female
200 ppm dose groups after 70 weeks of exposure along with marked weight
reduction in the males. Neoplasms were Increased by the hexachlorobenzene
exposures and are reported In the . Carcinogenicity Section. No other
pathologies were reported In this study.
Cabral et al. (1979) studied the tumorlgenlclty of 6- to 7-week-old male
and female outbred Swiss mice given 0, 50 (6 mg/kg/day) 100 (12 mg/kg/day)
and 200 (24 mg/kg/day) ppm hexachlorobenzene for 101-120 weeks and 300 ppm
(36 mg/kg/day) hexachlorobenzene for 15 weeks and held until 120 weeks of
age. Results Indicated that shortened HFespan occurred In the 200 and 300
ppm dose groups starting after the 30th week of the test and that this
02590
V-l 5
04/05/91
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reduced survival was associated with tremors and convulsions. Reduction In
the rate of growth was observed In female mice In the 50, 200 and 300 ppm
dose groups and more pronounced growth rate reduction was observed in male
mice In the 100, 200 and 300 ppm dose groups. An Increase 1n neoplasms were
found as a result of hexachlorobenzene exposures and are discussed In the
Carcinogenicity Section. No other pathologies were reported 1n this study.
Smith and Cabral (1980) fed young female Agus or MRC Wlstar rats 100 ppm
(6-8 mg/kg/day) hexachlorobenzene In a diet containing 2% arachls oil for 90
weeks. Hexachlorobenzene exposure resulted 1n a steady decline In body
weights over the study period and In the exposed rats possessing less hair
than the controls. Tremors or other nervous symptoms were not seen during
this study. Onset of porphyria was observed In the hexachlorobenzene
treated rats after -3 months, as Indicated by urines fluorescing red under
UV light, and liver porphyria was confirmed at autopsy by a red fluorescence
of the liver. The livers were enlarged 2-fold In the hexachlorobenzene-
exposed females and were associated with multiple liver cell tumors, lnls
neoplastic Incidence will be discussed 1n the Carcinogenicity Section.
Male and female Sprague-Oawley rats were fed hexachlorobenzene diets for
2 years containing 0, 75 or 150 ppm hexachlorobenzene (Lambrecht et a1.f
1983a,b). Four rats per group were killed at weeks 0, 1, 2, 3, 4, 8, 16,
32, 48 and 64 of the study and liver and kidney evaluations were made.
Times of appearance of lesions were as follows: 4 weeks -- hepatic hyper-
emia, edema, parenchymal and hydropic degeneration, renal hyperemia, con-
gestion, swelling and parenchymal degenerations; 32 weeks -- renal tubular
nephritis with hyaline casts, severe parenchymal degeneration, epithelial
02590
V-l 6
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necrosis accompanied by proximal convoluted tubular regeneration, and pre-
neoplastic foci; and 36 weeks -- hepatic preneoplastic foci; and 64 weeks —
hepatic neoplasms and renal neoplasms. The Incidence of neoplasms will be
further discussed in the Carcinogenicity Section.
In a short communication by Suflt et al. (1986), hexacholorobenzene-
Induced nerve function detriments were reported 1n rats. Rats fed
hexachlorobenzene for 2 years .at 150 and 75 ppm diet were observed with
prolonged conduction time's from the sciatic nerve to the foot of 31 and 8%.
respectively. Needle electromyograph of the muscle showed signs of
denervation, fibrillation, and chronic repetitive discharges (pseudo-
myotonla). Hepatocarclnomas and other disorders were also seen but not
described. In a second study male Sprague-Oawley rats were fed 800 ppm
hexachlorobenzene In diet for 20 weeks prior to testing. They reported a
significantly (p=0.02) reduced nerve conduction velocity in the hexachloro-
benzene fed rats when compared to controls. Needle electromyograph of the
gastrocnemius showed no fibrillations or other abnormaltles. The authors
stated that hexachlorobenzene had a definite detrimental effect on nerve
function and suggested an axomal effect.
A two-generation hexachlorobenzene (analytical grade) feeding study was
conducted using Sprague-Dawley rats fed diets containing 0 (64 males, 64
females), 0.32 (40 males, 40 females), 1.6 (40 males, 40 females), 8.0 (40
males, 40 females), or 40.0 (66 males, 66 females) ppm hexachlorobenzene
(Arnold et al., 1985). The parental rats (FQ) received their respective
test diets for 90 days before mating and until 21 days after parturition (at
weaning), at which time they were killed and evaluated for hexachloro-
02590
V-17
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benzene-Induced effects. The number of offspring {F^ generation) From
these matlngs wer-e reduced to 50 males and 50 females per dose group at 28
days of age and fed their respective parents' diets. Thus, the animals
were exposed to hexachlorobenzene and metabolites utero. from maternal
nursing and from their diets for the remainder of their lifetime (130 weeks).
The results from this two-generation study indicated no consistent
treatment-related effects upon growth or food consumption In either gener-
ation and no change Irr fertility, gestation or lactation Indices. A
decreased viability Index was noted 1n the 40.0 ppm group relative to con-
trols. No treatment-related effects were found in the Fg females. The
Fq males were found to have significantly increased liver, heart and brain
absolute weights In the 8.0 ppm group and significantly Increased liver and
heart absolute weights In the 40.0 ppm group. The Fq males were observed
to have various significant changes In hematologic parameters at all dose
levels. Neoplasms were seen In the F^ generation and are discussed In the
Carcinogenicity Section. In the F^ generation the following changes were
seen:
1) Centrllobular basophilic chromogenesls showed a significant
dose-related trend 1n both males and females. Additionally, at
doses of 8.0 and 40.0 ppm the Increases were significant in com-
parison with controls for both males and females.
2) Increases in perlblHary lymphocytosis were significant In the
0.32, 1.6 and 40.0 ppm male groups, while Increases In
perlblHary fibrosis were statistically significant In the 0.32
and 40.0 ppm male groups.
3) Increases 1n severe chronic nephrosis were observed which were
dose-related, but significant relative to controls only for the
40.0 ppm male dose group.
02590
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In a second study conducted by Arnold et al. (1985), 50 male Sprague-
Dawley rats per group were fed hexachlorobenzene (0 or 40 ppm) and various
levels of vitamin A diet (0:1, 1 or 10 times normal control levels). The
test groups were as follows: control diet; control diet plus 40 ppm hexa-
chlorobenzene; 1/10 vitamin A diet; 1/10 vitamin A diet plus 40 ppm hexa-
chlorobenzenet 10 times control vitamin A diet; and 10 times vitamin A diet
plus 40 ppm hexachlorobenzene. Five rats per group were killed and evalu-
ated both at 25 and 49 weeks and the remaining animals were killed and
evaluated after 119 weeks:
Results revealed that the animals on the 1/10 vitamin A diet had sig-
nificantly reduced body weights and survivability when compared with control
diet animals. The animals on 1/10 vitamin A plus 40 ppm hexachlorobenzene
diet had significantly decreased body weights and did not survive as long as
rats receiving the control diet plus 40 ppm hexachlorobenzene. Hematologic
evaluations revealed no consistent treatment-related effects. Neoplasms
were observed in the test animals and are discussed In the Carcinogenicity
Section. No significant differences were found In the Incidence of any
pathological lesions between the test groups.
Mutagenicity
In a dominant lethal mutation study (Simon et al., 1979), male rats
(strain not given) received by oral gavage 0, 70 or 221 mg hexachloroben-
zene/kg body weight dissolved In corn oil for 5 consecutive days. A dose-
dependent reduction in male reproductive performance was observed, but
hexachlorobenzene did not Induce dominant lethal mutations. Khera (1974)
also reported a lack of dominant lethal mutations In Wlstar rats following
02530
V-19
04/05/91
-------
oral administration of 0, 20, 40 or 60 mg hexachlorobenzene/kg In 0.25%
aqueous gum traqacanth for 10 consecutive days. In 14 sequential mating
trials, no significant differences In the Incidence of pregnancies, corpora
lutea, live Implants and declduomas between the treated and control groups
were observed. Mutagenic activity has been observed In a yeast, Saccharo-
mvces cerevlslae. assay (Guerzonl et aK, 1976).' The mutagenicity of hexa-
chlorobenzene was Investigated In three strains of cerevlslae using
reversion from hlstldlne and methionine auxotrophy, and hexachlorobenzene
was reported to be mutagenic at a minimum concentration of 100 ppm.
Lawlor et al. (1979) measured the activity of hexachlorobenzene In the
Ames assay, strains TA98, TA100, TA1535, TA1537 and TA1538, at five unspec-
ified dose levels both with and without metabolic acltlvatlon by Aroclor
1254 Induced rat liver microsomes. Hexachlorobenzene possessed no
detectable levels of mutagenic activity In any of the Salmonella strains
used either with or without microsomal activation. These results were
reported in- an abstract with few experimental details. In addition, this
result Is not unexpected because the Salmonella test system Is generally
Insensitive to chlorinated compounds (R-1nkus and Legator, 1980).
Carcinogenicity
Studies on the carcinogenic potential of hexachlorobenzene have been
carried out on hamsters, mice and rats.
Hamster Studies.
Cabral et al. (1977) — In one study on Syrian golden hamsters (Cabral
et al., 1977) hexachlorobenzene was administered In the diet at 50, 100 or
200 ppm. These concentrations correspond to dosages of 4, 8 and 16 mg/kg/
02590
V-20
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I
day based on body weight and food Intake averages. The hexachlorobenzene
was prepared by dissolution In corn oil which was then mixed with the feed.
The feed was analyzed periodically to Insure that the Intended level of
hexachlorobenzene was maintained (Mollnerr 1983), The .hexachlorobenzene
preparation used In this study was 99.5% pure. Impurities reported to be
present In some hexachlorobenzene preparations Include chlorinated dlbenzo-
furan and chlorinated dlbenzo-p-d1ox1n, both members of classes of compounds
that are carcinogens (Vllleneuve et al.t 1974). The dosages selected for
this study were chosen In order to be comparable with those believed to be
consumed by victims of accidental hexachlorobenzene Ingestion In Turkey.
In this study on hamsters 1t was difficult to determine from the pub-
lished report whether an MTD was reached or exceeded because the Information
on mortality and weight changes was not detailed enough for an unambiguous
evaluation. Although mortality was monitored, the Investigators only stated
that 71% of the treated animals were alive at 50 weeks and that at the
highest dose, 16 mg/kg bw/day, there was a reduced lifespan among treated
animals after 70 weeks. The study was run for the lifetime of the animals,
but the actual duration in weeks was- not given. Since the investigators
also reported "marked weight reduction" in the highest dose group one could
conclude that the MTD may have been reached. However, in the absence of
weight data definite conclusions cannot be made.
The tumor Incidence among the hamsters is given in Table V-2. The
Incidence of hepatomas In males and females was statistically significant
-------
mil v-2
Tuaor Incldonco In Haailort Sinn HCI In IN Dlit'
(ffoctlvo
TM
No,
Of Twori
Roro Than
Ono Tuaor
Thvrold
Mopaloaa
Haoaanaloondotho Moat*
UW Saloon
Olhor
Group
No.
No.
%
No.
por
Naatltr
No.
*
No.
*
No. «
No.
%
No.
*
No.
«
Control
39 f
40 H
S
3
12.0
1. S
9
3
0.13
0.00
0
0
0
0
0
0
0
0
0 0
0 0
0
0
0 .
0
1
0
2.S
0
4
3
10.2
7.S
SO ppa
(4 Kg/kg)
30 F
30 N
16
18
S3.3
60.0
21
2?
0.10 '
0.90
4
a
13.3
26.6
2
0
6.6
0
14 46.6
14 46.6
0
1
0
3.3
0
1
0
3.3
S
11
16.6
36.6
100 ppa
|0 ag/kg|
30 f
30 N
18
27
60.0
90.0
32
45
1.06
1.50
n
14
36.6
46.6
1
1
3.3
3.3
17 S6.6
26 06.6
2
6
6.6
20.0
3
3
10.0
10.0
9
9
30.0
30.0
200 ppa
(H ag/kg)
60 f
S? H
52
56
06.6
90.2
13
07
1.21
1.S2
IS
27
2S.0
47.3
3
•
S.O
14.0
SI OS.O
49 S5.9
7
20
11.6
3S.0
4
4
6.6
7.0
•
6
13.3
10.5
¦Source Cabral ot al., HI?
TIH - Tuaor-boarIng anlaalt
-------
h1gh-dose groups and In males In the middle-dose groups. There was a
significant, dose-related trend for both tumor types. Three instances of
metastases were found among the animals with liver haemangloendothelioma. No
hepatoma metastases were found. One of the hepatomas In a female animal was
found at necropsy at 10 weeks; the Investigators did not Indicate which
dosage level this animal received.
Hamsters in the control group's showed no thyroid tumors but thyroid
alveolar adenomas were significantly Increased In the high dose males and
there was a significant dose-related trend. Thyroid tumors occurred In all
treated groups of females but were not statistically significant.
Chemical Induction of thyroid tumors has not been Identified with chem-
ically related compounds except for toxaphene, which 1s a mixture of chlori-
nated camphene derivatives.- Other chemicals associated with Induction of
thyroid tumors are thioureas, thlouradls, 3-am1no-4-ethoxyacetan11Ide,
amitrok, o-anisldlne, 2,4-d1am1nanisole sulfate, ethionamide, 4,4'-methylene
bis(n,n'-dimethyl) n,n'-dlmethylbenzenamlne, 1,5-naphthylened1amine, 4,4' -
oxydlanal1ne> pronetalol-HC1, 4,41-thlodlanalIne, Iodoform, dlbromomethane
and dlchloroethane (Krayblll, 1983; Welsburger, 1983). Hexachlorobenzene Is
In a different chemical class from these agents.
Induction of thyroid tumors In the animal studies is of particular
Interest because a very high Incidence of enlarged thyroids was found among
victims of accidental exposure to hexachlorobenzene In Turkey (Peters et
al., 1983). The Incidence among females, over 25 years after the Incident,
is 61.4% whereas the background Incidence in that geographic area for
02590
V-23
04/05/91
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females Is about 5% (Peters et a"!., 1983). The data and pathology reports
have not been made available yet, but 1t Is clear that the cohort exposed to
hexachlorobenzene has an unexpectedly high Incidence of enlarged thyroid.
It cannot be stated at present what percentage if any. of the enlarged
thyroids was the result of tumorlgenesls.
This hamster study provides strong positive evidence of tumorIgenlclty
and evidence of carcinogenicity of hexachlorobenzene, as Indicated by the
significant Increase In hepatomas, significant Increase of thyroid adenomas
In males and the occurrence of metastasizing liver haemangloendothellomas In
treated but not In control animals. Although not reported 1n detail In this
one page publication, the authors noted an Increase In adrenal neoplasms as
well. The data presented show that the tumor Incidence Is positively dose-
dependent In most Instances and that this is true not only of the number of
animals with tumors of all sites but also for the number of tumors per ani-
mal. The authors also Indicated that latency period was reduced,' but actual
supporting data was not presented. Although strong evidence for carcinogen-
icity was provided In the hamster study, a cautionary note should be added
regarding the results of this study and possibly other hexachlorobenzene
studies as well. The hexachlorobenzene used was reported to be 99.5% pure.
However, chlorinated dlbenzofuran and chlorinated d1benzo-p-dloxin, both
very potent carcinogens, have been reported 1n the past to be present In
some samples of hexachlorobenzene. Very small amounts of such contaminants
could Influence results.
Lambrecht et al. (1982a) Hamster Study — Another study on hamsters,
carried out in a different laboratory, adds further suggestive evidence for
02590
V-24-
04/05/91
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the tumorlgenlclty of hexachlorobenzene In hamsters (Lambrecht et al.,
1982a). This study, reported only In abstract form, was also carried out 1n
the Syrian golden hamster. In this study the animals were exposed for only
90 days to the hexachlorobenzene. On day 91, half of the Initial exposed 50
animals were sacrificed. The remaining animals were sacrificed periodically
until the end of the 1-year study. The exposure*levels used were 200 or 400
ppm hexachlorobenzene 1n the diet. Assuming that the hamsters from the
Cabral et al. (1977) study were comparable 1n weight and dietary consump-
tion, these ppm figures would be approximately equal to and twice those of
the high dose used In the lifetime studies of Cabral et al. ( 1977 ).
Lambrecht et al. (1982a) reported the Incidence of hepatoma at the 200 ppm
level to be 7.7% In males and 6.7% In females; at the 400 ppm level the
Incidence was 5% In females and 14.3% in males. These figures are based on
the numbers of animals at risk at the time of the earliest observed tumor.
The time to first tumor was relatively late In the study, 276 days for males
and 255 days for females of the lower dose and 153 days for males and 299
days for females at the higher dose. Since the test animals were
systematically sacrificed from 3 months onward, the time to tumor figures
should be reasonably close to actual, time to tumor. Table V-3 shows the
results reported by Lambrecht et al. (1982a).
The tumorlgenlclty and carcinogenicity of hexachlorobenzene has been
demonstrated by one lifetime study In hamsters. Additional suggestive evi-
dence for tumorlgenlclty Is found In a 90-day study 1n another laboratory.
In both cases hepatomas resulted. The longer period of exposure also
produced thyroid adenomas and metastatic liver haemangloendothel1omas.
02590
V-25
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TABLE V-3
Effect of HCB on Hamsters: Liver Tumors and Other Liver Lesions3
Sex
HC8
PC+C&
BDHC
Oay First
Hepatomas
Day First
(ppm)
IncIdence
Incidence
Observed
Incidence
Observed
M
0
3/50
0
0
200
48/49
0
1/13
276
400
50/50
1/25
101
1/20
153
F
0
10/43
0
0
200
48/49
1/6
340
1/15
255
400
45/45
2/20
174
1/7
299
aSource: Lambrecht et al., 1982a
bRrec1rrhot1c + cirrhotic
cB111ary duct hyperplasia
HCB = Hexachlorobenzene
02590
V—26
04/05/91
-------
House Studies.
Cabral et al. f 1979) — Cabral et al. (1979) reported that outbred
Swiss mice were fed hexachlorobenzene (99.5% purity) In their diets for up
to 120 weeks. The hexachlorobenzene content of the diet was monitored
periodically during the study and the diet was found to be free of ana-
toxins.. The exposure levels used were 50, 100 and 200 ppm corresponding to
dosages of 6, 12 or 24 mg/kg/day based on body weight and food Intake
averages. One other test group was" given 300 ppm (36 mg/kg/day) for only 15
weeks and retained on art hexachlorobenzene-free diet for the remainder of
the study.
Growth rates were monitored but not given In detail In the published
report. The investigators stated that among female mice there was a reduced
growth rate for all doses except In the 12 mg/kg/day dose group and among
males for all doses except In the 6 mg/kg/day dosage group.
Survival times were reported In detail. Survival was essentially
unaffected in the two lower dosage level groups at 50 weeks, while at that
time it was down by 60% of the orlglna.l number In the females and 52% of the
original number In the males In the highest dosage group. By 70 weeks on
test the survival was decreased In the two lower dose groups as well, and In
the highest dose group 1t was down to 14% in females and 10% In males. At
90 weeks there were only four surviving males out of the 50 and no surviving
females In the highest dosage group as compared with 96 and 100% survival In
the female and male controls.
The yield of tumors in this study Is given In Tables V-4 and V-5. In
Table V-4t the effective number of animals Is the number of animals alive at
02590
V-27
04/05/91
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TABU V-4
Liver Tumor Incidence 1n Mice Fed HCBa
Group
Effective1*
No.
Animals
Mice
with LCT
Node !
Size (mm)
Multiplicity
Aqe at Death
i
(weeks)
No,
X
<8
>8
Single
Multiple
Range
Average
HCB 100
F 12
3
25
2
1
1
2
87-104
98
H 12
3
25
1
2
2
1
83-98
89
HCB 200
F 26
14
54
5
9
3
11
47-85
67
H 29
7
24
4
3
2
5
46-101
73
HCB 300
F 10
1
10
1
1
_ __
101
101
(15 weeks
M 3
1
33
--
1
---
1
97
97
exposure)
^Source: Cabral et al., 1979
^Survivors at time first LCT was observed In each group
LCT = Liver cell tumors
HCB = Hexachlorobenzene
-------
TABLE V-5
Tumor Data on Nice Fed HCBa
o
ro
u>
vO
o
Animals with Tumors
<•
Lymphomas
Luna
Initial
Effective'*
TBA£
Liver
-cell
Gonads
Other
Group
No.
No.
Average Age
Average Age
Animals
Animals
No.
X
No.
X
at Death
No.
X
at Death
No.
X
No.
X
No.
X
-
(weeks)
(weeks)
'
Control
f
50
49
39
BO
21
43
89.6
14
29
89.0
0
0
3
6
gd
18
H
50
47
22
47
12
26
BO.8
13
28
83.8
0
0
0
0
4e
9
HCB
SO
f
30
30
21
70
16
S3
69.8
4
13
84.5
0
0
2
7
2f
7
N
30
30
IS
SO
13
43
73.7
4
13
87.0
0
0
0
0
0
0
HCB
100
F
30 <
30
13
43
5
17
94.4
6
20
83.5
3
10
1
3
39
10
N
30
29
10
34
7
24
70.4
0
--
3
10
0
0
lh
3
HCB
200
F
SO
41
19
46
5
12
58.2
2
5
66.5
14 .
34
1
2
1»
2
N
50
44
12
27
4
9
53.2
4
9
82.5
7
16
1
2
0
0
HCB
300
F
30
26
20
77
8
31
97.7
4
IS
91.2
1
4
3
12
el
31
(15
weeks)
N
30
16
5
31
3
19
68.6
2
13
83.5
1
6
0
0
0
0
'Source: Cabral et al., 1979
''Number of survivors at moment of appearance of first tumor at any site In each group
cIn relation to the effective number
dSk1n fibrosarcoma, uterine haemangloendothelloma, one skin haemangloendothelloma, two adrenal adenoma, two mammary adenoma
'Urinary bladder transition cell carcinoma, one liver haemangloendothelloma, one skin haemangloendothellooa, one skin fibrosarcoma
fpne uterine haemangloendothelloma, one skin fibrosarcoma
OTwo skin fibrosarcoma, one skin haemangloendothelloma
h0ne skin squamous-cel1 carcinoma 1
g 'One Intestinal leiomyosarcoma
lone skin fibrosarcoma, two liver haemangloendothelloma, one r.ecum carcinoma, one stomach papilloma, one skin haemangloendothelloma, one
uterine adenoma, one mammary adenoma
CO
oo
HCB » Hexachlorobemene
-------
the earliest time a liver cell tumor was observed In each group while In
Table V-5 the effective number of" animals Is that number of animals alive at
the earliest appearing tumor for any site in the body within that group.
There was a statistically significant elevation In the Incidence of liver
cell tumors at the high dose In females and a marginal Increase In high-dose
males, with a positive dose-related trend 1n both cases. There was also a
dose-dependent decrease In latent period and a dose-dependent Increase In
the size and multiplicity of liver" cell tumors (see Table V-4). The liver
cell tumors were subsequently defined as hepatomas (Cabral, 1983).
In this study there was a high Incidence of both lymphoma and lung
tumors In control mice. A dose-related decrease 1n the Incidence of lympho-
mas appears In the treated groups. The Investigators attributed this to the
decreased survival time of hexachlorobenzene-treated animals. This seems
reasonable but does not explain the reduction In lung tumors in the 50 ppm
(6 mg/kg/day) group when they are compared to controls, since there was not
an appreciable reduction of lifespan In this low dose group.
This study by Cabral et al. (1979) demonstrates the tumorlgenlc'1 ty of
hexachlorobenzene In Swiss mice by the significant Increase In liver cell
tumors in both sexes and by the demonstration of dose-dependency In the
response with respect to tumor Incidence, tumor size, multiplicity and
latent period duration. Tumorlgeniclty was detected as low as 12 mg/kg
bw/day (100 ppm) for lifetime exposure but not at 6 mg/kg bw/day (50 ppm).
Lambrecht et al. (1962b) -- Swiss mice exposed to hexachlorobenzene
for only 90 days at levels of 100 and 200 ppm In the diet showed degenera-
02590
V-30
04/05/91
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tlve changes of liver and kidneys when examined at various Intervals after
they were removed from the hexachlorobenzene-contalnlng diet (Lambrecht et
al., 1982b). Although liver tumors were not reported, treated animals
showed lymphosarcomas in both dosage groups 1n both sexes at-levels signifi-
cantly above those of controls. Exposure to hexachlorobenzene In this
Instance produced leukemogenlc changes. The animals were not permitted to
live beyond selected Intermediate sacrifice dates, so It was not possible to
determine whether survivors would have developed liver or other tumors. The
method of preparation of the hexachlorobenzene-contalnlng diet may have been
different In the Cabral et al. (1979) and Lambrecht et al. (1982b) studies,
but detailed Information was not presented in the Lambrecht "et al. (19B2b)
abstract.
.Mice may be somewhat less sensitive than hamsters to hexachlorobenzene
as evidenced by the difference In Incidence of hepatoma formation at various
doses. These animal species may differ In the distribution of the hexa-
chlorobenzene Into various tissue compartments (Lambrecht et al., 1981), and
differ In rates of metabolism and absorption. Administration of the same
levels of hexachlorobenzene in the feed can be expected to give different
effective dosages.
Shlral et al. (1978) — Sh1ra 1 et al: (1978) administered hexachloro-
benzene to male ICR mice (35 animals/group) at levels of 10 or 50 ppm In the
diet for periods of 24 weeks. PolychlorInated terphenyl was given alone to
another group at 250 ppm, and In combination with 50 ppm hexachlorobenzene
to a third group. Animals were examined histologically at 40 weeks.
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Final body weights were slightly lower In the hexachlorobenzene-treated
groups while liver weights were higher. Examination of the livers showed
that the hexachlorobenzene-treated groups had hypertrophy of the centrl-
lobular area at both doses. No liver tumors were Found .1n either group.
The total Intake of hexachlorobenzene was calculated to be 8.4 and 35.3
mg/mouse over 24 weeks In the 10 ppm and 50 ppm groups, respectively.
PolychlorInated terphenyl alone, at 250 ppm (total dose 207.4 mg/mouse)
gave 3/28 (10.7%) nodular hyperplasia. When this same level of polychlorl-
nated terphenyl was given along with hexachlorobenzene at 50 ppm (total dose
36.9 mg/ mouse) there were 23/26 (88.556) nodular hyperplasia and 8/26
(30.8%) hepatocellular carcinoma. This response Indicates that hexachloro-
benzene can enhance the carcinogenic potency of polychlorlnated terphenyl.
The duration of administration, 24 weeks, In this mouse study and the
doses used were below those used In the Cabral et al. (1979) study on Swiss
mice and also below the levels used In the 13-week study by Lambrecht et al.
(1982b) on Swiss mice. Therefore, It Is not surprising that hepatomas were
not found when hexachlorobenzene was given alone. The occurrence of liver
lesions, however, does Indicate the liver Is a target organ.
These three studies In mice demonstrate the tumorIgenlcity of hexa-
chlorobenzene with respect to the Induction of hepatomas, the leukemogenlc
effect of subchronlc exposure and the ability of hexachlorobenzene to
enhance the carcinogenic effect of another compound.
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Rat Studies.
Smith and Cabral (1980) — The carcinogenic potential of hexachloro-
benzene was tested In several different laboratories In rats. In one study
(Smith and Cabral, 1980) small numbers of female Agus rats,. and even smaller
numbers of female Wlstar rats, were used. There were 12 control and 14
treated Agus rats and 4 control and 6 treated Wlstar rats. The hexachloro-
benzene was analytical grade (99.5% purity) dissolved In arachls o 11 and
mixed with the Feed to give 100" ppm In the diet. This dietary level
supplied an average daily'dose of 6-8 mg/kg/day to the rats.
In this study the Agus rats showed signs of porphyria after 3 months
exposure to hexachlorobenzene, but other toxic manifestations were not
found. The investigators stated that "there was a steady decline in body
weight to eventually 80% of control animals" (Table V-6). Examination of
the weight data presented In the publication Indicates that this Interpre-
tation is based upon comparison of "final" average weight in control
(286+19 g) and treated (225+16 g) animals (see* Table V-6), representing a
21% difference In weight. This method of comparison can be misleading since
the final weights represent accumulaied differences in growth rates and
varying composition of the groups because of animal deaths. An effect pro-
duced, even transiently, at an early age, may persist in the figures, even
though all subsequent growth may be normal. Growth rates, rather than
absolute difference in weights provide a more suitable picture of the animal
response. Growth rates for the time Intervals reported were calculated
based on the data given in the publication and are shown in Table V-7. Ihe
equation used was:
R _ weight at end of Interval - weight at start of Interval
weight at start of Interval
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TABLE V-6
Body Weights of Female Agus Rats Fed Hexachlorobenzene for 90 Weeks3
Body' Weight (g)
Weeks of Diet % Difference
Control HCB
0
46
+•
6
(8)
45
24 |
[9)
2
10
191
+
5
180
17
6
30
236
13
212
~
13b
10
50
257
+
17
221
+
19c
14
90
286
19
(8)
225
~
16 1
!7)c
21
aSource: Smith and Cabral, 1980
^Significantly different from controls as assessed by Student's t-test
p<0.01
cp<0.001
Female Agus rats were fed HCB (100 ppm) In MRC 41B diet for 90 weeks and
then killed. Weights are means (no. of animals in parentheses) * S.D.
HCB =• Hexachlorobenzene
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TABLE V-7
Growth Rates for Female Agus Rats on a Diet Containing 100 ppm HCB*
Interval (on diet)
Averaae Growth
Control
Rate Vweek
Treated
0-10 weeks
31.5
30.0
10-30 weeks
1.2
0.89
30-50 weeks
0.45
0.22
50-90 weeks
0.28
0.05
*Source: Calculated from Smith and Cabral, 1980
HCB = Hexachlorobenzene
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According to this calculation Doth groups of animals grew during each time
interval.
The survival of the treated Agus rats was good; one. test animal was
sacrificed at 52 weeks and a second one died of pneumonia at 70 weeks. Both
of these animals had liver cell tumors found by histologic examination.
Another five treated animals were sacrificed at 75 weeks and the remaining
seven treated animals lived until the end of the experiment at 90 weeks.
Among controls, one was Killed at 63 weeks and three more at 75 weeks. The
remaining eight were killed at 90 weeks.
No control animals had liver pathology. In contrast, 14/14 (100%) of
the treated Agus rats had liver tumors; the earliest of these was detected
at 52 weeks. The livers of the treated animals were grossly enlarged and
some of the tumors were 1.5-2 cm In diameter. Although one liver cell tumor
was described as pedunculated, histopathology detail was not given, except
to note the absence of metastases In all cases. Four of the six (67%)
Wis tar rats also had liver cell tumors and none of the four .controls showed
such pathology at 75 weeks.
In this rat study hexachlorobenzene was a potent Inducer of liver
tumors, causing a 100% Incidence with the earliest tumor observed at 52
weeks. It is Important to determine whether the magnitude of the effect 1s
all attributable to the hexachlorobenzene or whether contaminants, unusual
characteristics of the test animals, or procedural factors were operative In
this study. In this context the following points are noted.
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First, historical control data on tumor Incidence for Agus rats were not
available, but, according to Cabral (1983), the Agus rat Is a strain partic-
ularly sensitive to porphyria and hepatic.tumors. In regard to the question
of contaminants, peanut oil is generally believed to be free of anatoxins
[they are destroyed in processing (NAS, 1977)] and the feed was analyzed for
both anatoxins and dlbenzofurans and found to be free of both (Cabral,
1983). Absorption is another factor to consider. The absorption of the
hexachlorobenzene In these animal*'might be enhanced by dissolution In the
arachis oil.
Lambrecht et al. (1983a.b. T984) -- Another study on rats was carried
out by Lambrecht et al. (1983a,b, 1984). In this study 94 Sprague-Oawley
rats of each sex for each dosage and control groups were used. Four animals
of each group were sacrificed at each of 10 Intervals: 0, 1# 2, 3, 4, 8,
16, 32, 48 and 64 weeks. The remaining 54 animals of each group were
allowed to continue until they died, or to the end of the 2 years. Ihe
number of animals at risk was considered to be those that survived at least
12 months, since this was the earliest time to tumor. This number would be,
at minimum, 54 plus some animals from the last sacrifice time.
The hexachlorobenzene was highly purified and the prepared diet moni-
tored for hexachlorobenzene levels periodically. The preparation was also
analyzed for anatoxins and found to be negative. The test diet was
prepared by mixing the hexachlorobenzene with dextrose and Wayne laboratory
feed (1.5 g hexachlorobenzene + 98.5 g dextrose 9.9 kg lab chow to give
150 ppm hexachlorobenzene). Half the amount of hexachlorobenzene was used
In the mix for the 75 ppm hexachlorobenzene level. This oil-free vehicle Is
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different from the vehicle used by both Smith and Cabral (1980) and Arnold
et al. (1985). The hexachlorobenzene was well absorbed as demonstrated by
progressive accumulation In fat which was measured In this study.
Based on an average food consumption of 22.6 g/rat/day for males and
16.5 g/rat/day for females, and on an average adult weight for females of
265 g and for males of 400 gf the low dose was calculated to be 4-5 mg/kg/
day and the high dose, 8-9.S mg/kg/day. In order to compare the results
obtained In this study with those obtained in Sprague-Dawley rats by Arnold
et al. (1985), more detailed calculation of doses at different time periods
on test are given In Table V-8.
The administration of hexachlorobenzene In the diet at these doses In
the Lambrecht et al. (1983a) chronic feeding study 1n rats resulted In liver
pathology Just before the appearance of hepatoma or hepatocellular carci-
noma. Pathology observed at the early sacrifice time Included parenchymal
degeneration, preneoplastic fod and adenoma. At 48 and 64 weeks of the
test females had gross liver tumors which measured between 1 and 2 mm*.
Porphyria was also detected.
Rats that lived 12 months or longer showed a significant increase In
hepatoma Incidence In both sexes. A statistically significant Increase In
the Incidence of hepatocellular carcinoma was found at both doses In the
females, and In males a slight non-slgnlfleant Increase was found. None of
the liver cell tumors metastasized. Table V-9 summarizes the findings.
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TABLE V-8
Dosage Levels In the Chronic Feeding Study of Hexachlorobenzene
In Sprague-Dawley Ratsa
(mg/kg/day)
Hales Females
Time on D1etb
(weeks) 75 ppm 150 ppm 75 ppm . 150 ppm
0
19.5
37.0
16.1
32.2
26
3.2
7.1
3.7
8.7
52c
3.3
6.4
3.0 ,
8.0
79
3.4
6.7
3.5
8.4
99
6.2
10.0
4.3
10.6
aSource: Calculations and data provided by Lambrecht, 1984
bThe an Imals were 3 weeks old when placed on test
cAt 52 weeks on test the males consumed an average of 24.7 g of the diet/
day and weighed an average of 553.7 g. The females consumed an average of
16.0 g diet/day and weighed an average of 311.7 g.
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. TABLE V-9
LWer and Kidney Tumors In Sprague-Oawley Rats Given Hexachlorobenzene
In the Diet for up to 2 years^.b
Exposure
Level
Heoatoma
Hepatocellular
Carcinoma
Renal Cell
Adenoma
Renal Cell
Carcinoma
M
F
M
F
n
F
H
F
0
0/54
0/52
0/54
0/52
7/54
1/52
0/54
1/52
percentage
0
0
0
0
13
2
0
2
75 ppm
10/52
26/56
3/52
36/56
41/52
7/56
0/52
2/56
percentage
19
46
6
64
79
13
0
4
150 ppm
11/56
35/55
4/56
48/55
42/56
15/54
0/56
2/54
percentage
20
64
7
87
75
28
0
4
aSource: Lambrecht et al., 1983a,b; Lambrecht, 1983
&The diet was prepared without solubilization of the hexachlorobenzene,
but by mixing 1t as a pulverized solid.
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Renal cell adenoma was found to be significantly elevated 1n both sexes
but with greater - frequency In males. In this study the control male group
had a high incidence of renal cell adenoma which was not explained; never-
theless, the Increase 1n the hexachlorobenzene-treated animals was statis-
tically significant. The Incidence of renal cell carcinoma In treated
animals was not significantly Increased over control animals In either males
or females.
In an updated report'from this laboratory (Peters et a!., 1983) hlsto-
pathology details were supplied. These data show that In addition to the
liver and kidney lesions there was an Increase 1n adrenal pheochromocy.toma
In female rats that was statistically significant at both 75 and 150 ppm.
Females also had elevated Incidences of adrenal cortical adenoma and
hemangioma In the treated groups. Among males the background Incidence of
adrenal pheochromocytomas Is high (76.5%), making It difficult to determine
whether the 90.6% Incidence found In the 150 ppm group has any biologic
significance. Other adrenal neoplastic and nonneoplastic lesions were
detailed: hyperemia and/or congestion, cortical hyperplasia, preneoplastic
foci, cysts, lipoma and adenocarcinoma; none of ,these were elevated in the
treated animals. The adrenal tumor incidences are given In Table V-l0L
One point to consider In the Interpretation of the results, particularly
1n terms of their application to risk assessment, is the form in which the
hexachlorobenzene was administered In the diet. The absorption from a
particulate form Introduces an additional possible exposure route, namely,
from the food preparation by inhalation. This consideration does not
invalidate the study, but raises the question of the actual exposure levels
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TABLE V-10
Adrenal Tumors In Sprague-Dawley Rats Given Hexachlorobenzene
In the Diet for up to 2 Yearsa'b
HALES
Days on diet
400-599
600+
Exposure ppm
hexachlorobenzene
0
75
150
0
75
150
Number of tissues
examined
17
23
28
34
25
23
Cortical adenoma
(X)
3
2
6
6
3
4
Pheochromocytoma
{%)
3
(17.6)
6
(26.1)
9
(32.1)
26
(76.5)
17
(68)
21
(91.3)
Hemangioma (%)
0
0
0
0
0
0
FEMALES
Days on diet
400-599
600+
Exposure ppm
hexachlorobenzene
0
75
150
0
75
150
Number of tissues
examined
12
5
13
35
47
32
Cortical adenoma
(%)
0
3
2
2
(5.7)
11
(23.4)
6
(18.8)
Pheochromocytoma
{%)
0
0
2
5
(14.3)
31
(66)
29
(90.6)
Hemangioma (%)
0
0
2
3
(8.5)
8
(17)
5
(15.6)
aSource: Peters et al., 1983
bThe diet was prepared without solubilization of the hexachlorobenzene,
but by mixing It as a pulverized solid.
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If an additional route of exposure was occurring In the same experiment
simultaneously with oral Ingestion. The effect of mixing the
hexachlorobenzene In the diet In an oil free form .may also affect absorption
and thereby the effective dose.
Arnold et al. (1985) — In this study hexactilorobenzehe (99% pure) was
administered to parental male and female Sprague-Dawley rats for 3 months.
These animals were mated at that time and the females continued to receive
hexachlorobenzene-contalnlng diets during pregnancy and throughout lacta-
tion. At weaning, 50 pups of each sex were separated and fed for the
remainder of their lifetime on hexachlorobenzene-contalnlng diets. Controls
were fed diets free of hexachlorobenzene. The range of doses used In this
study Is considerably lower than those used by either Smith and Cabral
(1980) or Lambrecht et al. (1983atb). Table V-ll shows the doses used In
the Arnold et al. (1985) study at particular points In time since the doses
were not adjusted throughout the study. These doses represent a greater
exposure to the test animals from the point of view of exposure duration,
since the F-j animals were exposed ]_n utero and during nursing In addition
to their exposure from feeding on an hexachlorobenzene-contalnlng diet.
Total doses cannot be calculated since the actual dose received during
nursing is not known.
Arnold et al. (1985) found no differences in treated animals when
compared to controls with respect to growth rates, food consumption or
hematology. The only observed difference was a decreased viability index
for pups In the 40.0 ppm dose group.
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TABLE V-ll
Exposure Levels "in the Chronic Feeding, 2-Generatlon Study of
Hexachlorobenzene In Sprague-Dawley Rats8,
(mg/kg/day)
Exposure Level
Time on D1et&
(weeks)
0.32 ppm
1.6 ppm
8.0 ppm
40.0 ppm
MALES
1
0.04
0.18
0.93
4.85
30c
0.01
0.06
0.29
1-5
70
0.01
0.05
0.25
1.3
FEMALES
1
0.04
0.17
0.84
4.64
30c
0.02
0.08
0.40
1.9
70
0.01
0.06
0.32
1.6
aSource: Calculations and data provided by Arnold, 1984
bThe animals were placed on feed at 6 weeks of age.
cThe mean body weight of male controls was 663 g and for the highest dose
group males 653 g. The mean weekly food consumption for male controls at
that time was 176 g and for the highest dose group 169 g. Females of the
same age weighed 351 g for controls and 353 g for the highest dose treated
group and the mean weekly food consumption was 113 and 118 g, respectively.
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histopathology showed that females had a significant elevation in
neoplastic 1Iver .nodules and 1n adrenal pheochromocytoma In the high dose
females compared to controls (Table V-12). There was also a significant
positive dose-related trend 1n the Incidence of these tumors In females.
Among F^ males. In the highest dose group parathyroid tumors were sig-
nificantly Increased; 25% (12/48) In the treated groups and 4.2% (2/48)
among controls. Females also showed a few parathyroid tumors In the two
highest dose groups but none Tn controls or In the two lowest dose groups.
The differences were not significantly different from controls. Table V-12
gives the tumor Incidences. Although kidney tumors were not reported to be
elevated, there was an Increased chronic nephrosis in the F^ treated
animals.
Arnold et al. (1985) — In another study by Arnold et al. (1985) which
was related to the 2-generatlon study, the effect of vitamin A, because of
Its supposed antltumorlgenlc properties, was tested In conjunction with
hexachlorobenzene. This was a 1-generatlon study and the level of
hexachlorobenzene was the same as the highest dose of the 2-generatlon
study, 40 ppm. There were six separate groups of 50 animals each and the
experiment ran for 119 weeks. At 29 weeks and at 49 weeks five animals from
each group were sacrificed and evaluated histologically. The six groups are
shown in Table V-13. The vitamin A did not apparently alter the effects of
hexachlorobenzene. The number of animals with parathyroid tumors and
adrenal pheochromocytomas was somewhat elevated In all the cases In which
hexachlorobenzene was admin!stered bcompared with the total cases with the
three levels of vitamin A and no hexachlorobenzene. The significance of
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£ TABLE V-I2
in
i f>
o lumors In Organs that Showed Statistical Differences from Control In F| Sprayue-Dawley Rats Treated with Hexachlorobenzene*
(Incidence (X)]
Parathyroid Adenoma Adrenal Pheochromocvtoma Hepatocellular Carcinoma Neoplastic Liver Modules
Dose at 30 weeks
(rag/kg bw/day) Hales females Males females Males females Males Females
Controls
2/48 (4.2)
0/49 (0)
10/48 (20.8)
2/49 (4.1)
0/48
(0)
0/49
(0)
2/48
*4.2)
0/49 (0)
0.01-0.02
4/48 (8.3)
0/49 (0)
12/48 (25.0)
4/49 (8.0)
2/48
(4.2)
0/«9
(0)
0/48
(0)
0/49 (0)
0.06-0.08
2/48 (4.2)
0/50 (0)
7/48 (14.6)
4/50 (8.0)
1/48
(2.1)
0/49
1/49
(0)b t
(2.0)b
0/48
(0)
2/50 (4.0)
0.29 0.40
1/49 (2.0)
1/49 (2.0)
13/49 (26.5)
4/49 (10.2)
3/49
(6.1)
0/50
(0)
2/49
3/49
(4 Db
(6.1)b
2/49 (4.1 )b
3/49 (6.1 )b
T1». 5-1.9
CT>
12/49 (24.5)
2/49 (4.1)
.17/49 (34.7)
17/49 (34.7)
0/49
(0)
0/49
1/49
(0)b
(2.0)b
1/49
(2 0)
10/49 (20.4)b
9/49 (18.4)b
Other statistical tests
I ARC trend test
p<0.0t
p<0.05
p<0.01
p<0.01
p<0.0l
Armltage time-related
trend test
p<0.01
p<0.05 :
p<0.05
p<0.0l
p£0.0l
fisher exact
treated vs. control
p<0.05
p<0.01
p<0.0l
aSource: Arnold et al., 1985; Arnold, 1984
''Different results of two different pathologists reading the same slides
v.
o
i_n
U5
-------
TABLE V-13
Parathyroid and Adrenal Pheochromocytomas 1n Sprague-Dawley Rats
Maintained on Synthetic Diets of Varying Vitamin A Content and
With or Without Hexachlorobenzene*
Group No. with No. with Adrenal
Parathyroid Tumors Pheochromocytoma
Controls on diet with normal
vitamin A content 3
Control diet* 40 ppm HCB 4
Diet with- 0.1 times normal vitamin A 0
Diet with 0.1 times normal 0
vitamin A * 40 ppm HCB
Diet with 10X vitamin A 1
Diet with 1 OX vitamin A + 40 ppm HC8 3
Total without HCB 4
Total with HCB 40 ppm 7
•Source: Arnold et al., 1985
HCB = Hexachlorobenzene
3
&
7
2
4
7
9
15
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these tumor Incidences cannot be determined by simple comparison because It
was also found In the study that vitamin A had an effect on the background
level of some common tumors and these data have not yet been completely
analyzed.
Discussion of Rat Studies. It seems appropriate to compare the find-
ings of Smith and Cabral (1980) In Agus and Wlstar rats, Lambrecht et al.
(1983a,b) and Arnold et al. (1985) In Sprague-Dawley rats. None of the
three studies agree precisely on all four of the tumor target organs: Smith
and Cabral reported liver tumors, Lambrecht reported lWer, adrenal and
kidney tumors and had some liver carcinomas not found by Smith and Cabral.
Arnold found adrenal and parathyroid tumors and neoplastic liver nodules but
no Increase In kidney tumors. We find that, although differences do occur,
the results are not contradictory for the following reasons:
1. The dosages used in the Arnold et al. (1985) study were below those used
by either Smith and Cabral (1980) or Lambrecht et al. (1983a,b). The
range of doses used by Smith and Cabral was given as 6-8 mg/kg/day and
those used by Lambrecht were 3-9 mg/kg bw/day. Those of Arnold were, at
most, between 1.5 and 2.0 mg/kg bw/day.
2. There were notable differences in the animals used: In the case of
Smith and Cabral the liver tumor susceptible strain of Agus rat was
used, although tumors were also found with Wlstar rats. We do not have
full data on historical tumor Incidences in these animals to allow for
more detailed evaluation.
02590
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3. The conditions of the Smith and Cabral study and those of Lambrecht were
both different from the 2-generation study of Arnold. Differences In
sensitivity due to prenatal exposure may occur because of rapid cell
division and/or differences In xenoblotlc metabolism compared with older
animals. The dose received ' transplacentally and from nursing Is also
uncertain.
4. The method of preparation of' the hexachlorobenzene In the diet was
different In that both Smith and Cabral and Arnold used arachls oil and
corn oil as hexachlorobenzene solvents while Lambrecht did not use an
oil vehicle. Absorption characteristics are known to depend upon the
vehicles used.
5. .The Sprague-Dawley animals used by Arnold may have more fat than those
used by Lambrecht as they were somewhat larger. Distribution into
different tissue compartments, especially Into fat where It is likely
the hexachlorobenzene is at least temporarily stored, 1s likely to alter
the effective concentration In target tissues. In this regard the hexa-
chlorobenzene is known to concentrate in adrenal tissue; the degree of
such concentration may well vary with strain or diet of the host animals.
In summary, orally administered hexachlorobenzene has Induced hepato-
cellular carcinoma in male Sprague-Oawley (S-D) rats as well as hepatomas in
female Agus and Wistar rats and In S-D rats of both sexes. At the lowest
dose used In any of the studies (40 ppm In the diet or 1.5 mg/kg/day), neo-
plastic nodules were induced in S-0 rats, whereas hepatocellular carcinomas
occurred in the same strain at a higher dose (4-5 mg/kg/day). Adrenal pheo-
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chromocytoma was significantly elevated In two separate studies In female
S-D rats. In the same strain one Investigator reported parathyroid tumors
and a different Investigator reported kidney tumors; neither of these
findings has been repeated by other authors. Table V-14 summarizes this
Information.
Other Studies. In addition to the studies described on hamsters, mice
and rats there are a few studies that cover specific kinds of tests other
than lifetime exposure and examination of all potential target tissues for
tumorlgenlc or carcinogenic response.
One such study was that of Thelss et al. (1977) 1n which the experiment'
was designed to detect only pulmonary tumors following 1.p. Injection of
organic chemicals found as contaminants of drinking water. In this assay
hexachlorobenzene was one of the chemicals tested. Strain A mice were given
three dosage levels of hexachlorobenzene with the top level as the MID. A
total of 24 Injections over a period of 8 weeks were given to 20 mice/group.
The total doses received were 190, 480 and 960 mg/kg. The lungs were the
only organ examined and hexachlorobenzene did not Increase tumor Incidence
>ln that organ. The study ran for 32 weeks. Although this assay has proved
useful in detecting some pulmonary carcinogens, It Is not designed to detect
other tumors.
In another study on beagle dogs (Gralla et al., 1977) In which
hexachlorobenzene was given 1n dally gelatin capsules to 30 animals of each
sex/dosage group the duration of the study was only 1 year. Although this
Is not a long enough period of time for a carcinogenicity study 1n dogs. It
02590
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o
ro
<_n
CO
o
TABU V-H
Qualitative Comparison of Tumor Development In Rats Following Hexachlorobenzene Administration In Different Studies
Strain/Sex
Dosage
(lowest dose that
produced tumor)
Liver
Kidney
Adrenal
Parathyroid
Reference
Agus/Fumale
100 ppm (6-8 mg/kg bw/day)
liver-cell tumor
IF)
NA
NA
NA
Smith and
Cabral. I960
Hlstar/Female
prepared by dissolving In
oil and mixing oil with food
liver-cell tumor
(F 1
NA
NA
NA
Smith and
Cabral, I960
Sprague-Dawley/
Hale and female
75 ppm (1-4 mg/kg bw/day) ,
prepared In feed sans oil
vehicle
hepatocellular
carcinoma (H&F)
hepatoma (HW)
renal cell
adenoma (H&F)
pheochromo-
cytoma (F)
cortical
adenoma (F)
NA
Lambrecht
et al., ,1983a,b
Sprague-Dawley/
Male and female
F) animals of
2-generatlon study
40 ppm (0.3-1.5 mg/kg bw/day)
prepared In oil and mixing
oil with food at weaning —
animals exposed In utero and
during nursing
neoplastic liver
nodules (f)
not found
pheochromo-
cytoua (F)
adenoma |N)
Arnold
et al.. 1985
NA » It Is not known whether or not these tissues were examined.
o
\
o
cn
\
cD
-------
Is of Interest to note that the doses of 100, 10, 1 and 0.1 mg/kg bw/day
produced a number of toxic man1festatlons In the liver Including bile duct
hyperplasia, hepatomegaly and liver necrosis. This study Is more appro-
priately considered under chronic toxicity.
Finally. Perelra et al. (1982) designed a study to determine whether
hexachlorobenzene Increased y-glutamyltranspept1dase-pos1tlve foci In
rats. These foci are believed to be preneoplastic In the liver. The assay
was designed to test 1n1tiat1on/promot1on In this case by employing diethyl-
N-nltrosamlne (DENA) as the Initiating agent and hexachlorobenzene as the
promoter. Unfortunately, there are some errors In reporting of the results
In the published paper and some Important controls were not Included-
(Perelra, 1983). We have not yet received a corrected manuscript.
Carcinogen 1c1tv Summary. In a lifetime study of hexachlorobenzene
administration to hamsters, hepatomas were Induced In both males and
females. The response at a dose of 4-5 mg/kg/day dissolved In corn oil and
mixed 1n the feed was 47% for both sexes and controls had no hepatomas. In
addition to hepatomas, hamsters responded to hexachlorobenzene treatment
with malignant liver haemangloendothel1omas and thyroid adenomas. The
Incidence of haemangloendothelloma was 20% In males (versus 0 In controls)
at 8 mg/kg/day and 12% In females (versus 0 in controls) at 16 mg/kg/day.
The thyroid adenoma occurred at 14% Incidence in males treated with 16 mg/kg
hexachlorobenzene (versus 0 1n controls).
Liver cell tumors, described as hepatomas, were also produced In both
sexes 1n Swiss mice. At 24 mg/kg/day the Incidence was 34% for females and
02590
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!
1634 for males and the response showed a dose-dependency not only In the
number of tumor-bearing animals but also In the latent period, multiplicity
and size of tumors. In ICR mice, hexachlorobenzene administered concur-
rently with polychlorInated terphenyl Induced hepatocellular carcinomas.
In rats target organs for hexachlorobenzene-lnduced tumors Included
liver, kidney, adrenal gland and parathyroid gland 1n various studies.
Liver tumors were found In three* studies that Included three different
strains of rat: Agus (a'Uver tumor sensitive strain), Wlstar and Sprague-
Dawley rats. These tumors were Induced with doses between 1.5 and 8 mg/kg/
day. The Incidence was as high as 100% In Agus rats but lower for the other
strains. Renal cell tumors were found In one study on Sprague-Oawley rats.
In two studies on Sprague-Oawley rats, significant Increases In adrenal
pheochromocytoma In females were found. In one of these studies the Inci-
dence of parathyroid tumors In males was significantly increased as well.
Table V-15 summarizes the tumor data for hamsters, mice and rats for
hexachlorobenzene experiments.
The data on hexachlorobenzene provide sufficient evidence of the carci-
nogenicity and tumorlgenlclty of hexachlorobenzene since there were in-
creased Incidences of malignant tumors of the liver In two species (haeman-
gloendothelloma In hamsters and hepatocellular carcinoma In rats) as well as
reports of hepatoma In mice, rats and hamsters.
The appearance of thyroid tumors 1n hamsters and adrenal pheochromocyto-
mas and parathyroid tumors In rats as a result of hexachlorobenzene exposure
02590
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TABLE
Significantly Increased Incidence of lumors
V-15
In Animals Given Hexachlorobenzene In Diet
An Ima I
(strain)
Organ
Tumor
% Treated/X Control
Males females
Lowest Oose
to Produce Tumor
(nig/kg bw/day)
Reference
Hamslers
Hamsters
Nice
Rats
(S.O.)
Rats
(SO.)
Rats
(S.O.)
Rats
(Wis tar)
Rats
(Agus)
Rats
(S.O.)
Rats
(S.O.)
Rats
(S.O.)
Rats
(SO.)
Hamsters
1 Iver
liver
I Iver
I Iver
1 Iver
1 Iver
I Iver
T Iver
adrenal
adrena1
kIdney
parathyroid
thyroid
hepatoma
haemangloendothelloma
hepatoma
neoplastic nodules
hepatoma
hepatocellular
carcI noma
hepatoma
hepatoma
pheochromocytoma
pheochromocytoma
renal cell adenoma
adenoma
adenoma
47/0
20/0
16/0
NS
19/0
NS
NS
NS
79/13
25/4
14/0
47/0
12/0
34/0
20/0
46/0
64/0
67/0
100/0
35/4
91/14
13/2
NS
8 In males
16 In females
24
1.5
4-5
4-5
6-8
6^8
1.5
4-5
4-5
1.5
16
Cabral et al.,
1977
Cabral et al..
1977
Cabral et al..
1979
Arnold et al..
19B5
Lambrecht
et al...1983a
Lambrecht
et al.. 1983a
Smith and
Cabral. 1980 .
Smith and
Cabral. 1980
Arnold. 1984;
Arnold et al.,
1,905
Peters el al..
1983
Lambrecht
et al.. 1983b
Arnold et al.,
1985
Cabral el al.,
1977
NS - Not stated
-------
Is particularly Interesting because oF the clinical association of adrenal
pheochromocy tomas. with parathyroid and thyroid tumors In humans (Fraumenl,
1974; Hill, 1974), and because follow-up of Individuals 1n Turkey, who were
accidentally exposed to hexachlorobenzene over 25 years ago, shows a marked
elevation 1n thyroid tumors. Only a few of these subjects have had their
thyroid tumors examined histologically and the pathology reports are not yet
available.
If the I ARC criteria- for the classification of carcinogens were used,
this animal evidence would be considered "sufficient." In the absence of
human evidence of carcinogenicity, hexachlorobenzene would be classed In
IARC category 2B, meaning that It has been demonstrated to be carcinogenic
In animals and Is probably carcinogenic In humans.
Reproductive and Teratogenic Effects
Hexachlorobenzene has been shown to cross the placenta Into fetal
tissues and to be present In the milk of nursing dams (see Chapter III).
The NOLL in a 4-generatlon reproduction study with rats was reported to be
20 ppm of hexachlorobenzene 1n the d1et. Pups from treated dams (receiving
diets containing 00 ppm hexachlorobenzene) recovered from elevated liver
weights when nursed by foster dams. Hepatomegaly and reduced survival was
reported In kittens from cats receiving 263 ppm of hexachlorobenzene in
their diets. Infant rhesus monkeys developed clinical signs of toxicity,
but histologic examination showed only mild effects. Fetal mice from dams
treated with, 100 mg/kg/day during days 7-16 of gestation exhibited terato-
genic abnormalities.
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Results from a 4-generatlon reproduction study with Sprague-Oawley rats
was reported by Grant et al. (1977). Weanling rats, In groups of 20 females
and 10 males, were fed diets containing 0, 10, 20, 40, 80t 160, 320 or 640
yg hexachlorobenzene/g and at 100 days of age the Fg generation was
mated to produce the F. generation. The F1a pups were weaned at 21
i a i a
days, and the Fg rats were rested for 14 days-and again mated to produce
the second litter, F^ animals. The animals were then used to
produce the next generation, and- this sequence was followed to the F^b
generation. The two highest doses (320 and 640 yg/g) were toxic to the
mothers and resulted In 20 and 50% mortality, respectively, before the first
whelping and 25% -In each high dose group before the second' whelping. In
addition, the fertility Index In these rats was greatly reduced 1n these two
dose groups and the average litter size was decreased In the F^, F^a
and Fgenerations. The pups exhibited no gross abnormalities, but there
was an Increased number of stillbirths and all pups born alive died within 5
days In the 320 and 640 yg/g diet groups.
At the 160 yg/g level, 55% of the pups survived to day 5 but survival
to day 21 was greatly reduced. The number of live births and survival was
normal for the first two generations at the 80 yg/g dietary level, but by
the third generation there were stillbirths and a low degree of postnatal
vlabllllty. In addition, birth and weanling body weights were consistently
less than those of the control group. At 40 -yg/g diet only the liver
weights of the 21-day-old pups were significantly Increased, while the
kidney, heart and brain weights were not affected. Tissue concentrations of
hexachlorobenzene were dose-related, with body fat having the highest
concentration. The NOEL was reported to be 20 ppm In the diet.
02590
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The effect of hexachlorobenzene on rat reproduction was also reported by
KUchin et al. -(1982). Female Sprague-Oawley rats (10 animals/treatment
group) were fed diets containing 0, 60, 80, 100, 120 and 140 yg'hexa-
chlorobenzene/g of diet. The females were mated with untreated males after
96 days and then bred a second time 12 days after weaning of the
Utter. Fertility and fecundity of treated females were not affected by
treatment; however, a dose-related 21-day Increase In mortality was observed
1n both litters and the L&5q values were determined to be 100 and 140
>ig/g (maternal dietary 'concentration) for the F. and F generation,
« i a id
respectively.
Mendoza et al. (1978) studied the effects of hexachlorobenzene on
preweanllng Wlstar rats after a reciprocal transfer between 5 treated and 5
control dams. A significant Increase in the liver weight over that of the
control was observed In pups nursed by dams fed diets containing 80 Pg
hexachlorobenzene/g for 2 weeks before mating until birth, but this effect
did not persist after the treated pups were transferred to a control foster
dam. Similarly, the pups nursed by treated dams had smaller brains, hearts,
kidneys and spleens than the controls, and these organs were larger In
treated pups nursed by control dams. The authors concluded that hexachloro-
benzene transmission via the milk had greater effects on the pups than
transmission via the placenta.
Mendoza et al. (1979) placed female Wlstar rats on diets containing 80
ug hexachlorobenzene/g beginning 2 weeks before mating until 35-36 days
after weaning. Results Indicated that there were no marked differences in
the external appearance, body weight, liver weight, gestation, or neonatal
02590
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survival between the hexachlorobenzene treated and control females. In
addition, there were no differences In the number of litters, average number
of pups/Utter, average number of pups at birth and gestation Index.
Hansen et al. (1979) studied the effects of hexachlorobenzene on repro-
duction 1n cats fed contaminated pork cakes for 142 days. These cakes con-
tained 90+51 yg hexachlorobenzene/g, equivalent to an Intake of 3 mg/day/
cat, and were obtained from gilts' fed diets containing 100 hexachloro-
benzene/g for 6-8 week* before slaughter. The positive and untreated
control groups received pork cakes from gilts fed diets that did not contain
hexachlorobenzene, with the positive control group receiving hexachloroben-
zene-splked cakes (263+120 ng/g equivalent to 0.7 mg/day/cat). These
females were mated with untreated males and the resulting kittens did not
receive hexachlorobenzene-contalnlng cakes. Effects on survival were noted
In kittens born to only those cats receiving hexachlorobenzene-splked cakes
and was apparently due to the kittens being too weak to survive the stress
of weaning. There was a tendency for reduced average litter sizes and
increased mortality of nursing positive control kittens, and statistically
significant hepatomegaly and reduction In positive control kitten survival
at weaning. Treated positive control females exhibited a net weight loss
and Increased susceptibility to disease but no changes In relative organ
weights, hematologic parameters, or fecal coproporphyrIn excretion.
Rush et al. (1983) fed adult male and female standard dark minks
(Hustela vision) diets containing 0, 1 or 5 ppm hexachlorobenzene and then
mated the males to the females In each of the respective study groups. The
progeny were fed .their parents respective diets after weaning from their
02590
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mothers. The effects of exposures to hexachlorobenzene j_n utero and from
nursVng milk resulted 1n Increased mortality In the hexachlorobenzene-
treated weanlings with mortality In the 0, 1 and 5 ppm groups being 8.2,
44.1 and 77.4%, respectively. The surviving kits from all three groups had
no observed alterations In whole body, kidney or liver weights and no
observed damage to the kidneys or livers at 17 weeks of age. Induction of
hepatic mixed-function oxidases was observed 1n the surviving hexachloro-
benzene-exposed kits without any observable frank hepatotoxlclty.
Bailey et al. (1980) studied the transfer of hexachlorobenzene to three
nursing Infant rhesus monkeys from three lactatlng mothers receiving by
gavage 64 mg/kg/day of hexachlorobenzene suspended In methyl cellulose for
60 days. The hexachlorobenzene concentrated In the mothers' milk ranged
from 7.51-186 ppm during the dosing schedule. One Infant, by day 22, had
developed symptoms of hypoactlvlty and lethargy that progressed to. ataxia
and death 1 week later. Necropsy revealed severely congested lungs. A
second Infant died on day 38 and necropsy revealed a subdural hematoma and
bilateral hemorrhagic pneumonia. This Indicated that the risk cf exposure
to nursing infants was greater than, the risk to their mothers. Blood
(0.42-49.44 ppm) and tissue levels In the Infants were higher than In their
mothers (0.41-16.16 ppm blood), and the Infants developed clinical symptoms
of toxicity while the mothers were asymptomatic.
Studies on the placental transfer of hexachlorobenzene in Wlstar rats
and New Zealand rabbits did not reveal any apparent adverse effects on fetal
development. The female rats were dosed dally with 5, 10, 20, 40 or 80
mg/kg from day 6-16 of gestation, whereas the rabbits were treated with 0,
02590
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0.1, 1.0 or 10 rug/kg from day 1-27 of gestation. The compound was dissolved
In corn oil and administered by means of a stomach tube (Vllleneuve et al.,
1974; Vllleneuve and Hlerllhy, 1975).
Khera (1974) conducted a teratogenicity study with groups of 7-16 female
Wlstar rats given single oral doses of 0, 10, 20, 40, 60, 80 or 120 mg
hexachlorobenzene/kg suspended In corn oil or 0.25% aqueous gum tragacanth
during gestation days 6-21. Maternal toxicity and reduction In fetal
weights resulted from the two higher doses. Maternal toxicity was charac-
terized by loss In body weight, hyperesthesia, tremors and convulsions. A
significant Increase In the Incidence of unilateral and bilateral 14th rib
was observed and was related to the duration of treatment (days 10-13, 6-16
or 6-21 of gestation) and the dose. Sternal defects were observed In only 1
of 4 experiments, which lead the authors to conclude that It was doubtful
that hexachlorobenzene caused the observed sternal defects. There were no
hexachlorobenzene-related effects on external morphology. Visceral abnor-
malities were not observed, and microscopic examinations did not reveal any
treatment-related change In the histology of the fetuses. Values for live
and dead fetuses, resorption sites, and fetal weight were within the control
1lmlts.
Courtney et al. (1976) studied the effects of Ingestion of 100 mg/kg/day
hexachlorobenzene on days 7-16 of gestation in 10 pregnant CD-I mice. This
study was undertaken to evaluate the possibility that hexachlorobenzene
could be responsible for fetal malformations seen In pregnant animals
exposed to hexachlorobenzene-contaminated pentachloronltrobenzene. The
results showed that the hexachlorobenzene-treated mice had significantly
02590
V-60
04/05/91
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Increased maternal liver-to-body weight ratios and decreased fetal body
weights. Also, a- significant Increase in the Incidence of abnormal fetuses
per litter were observed as compared with control mice. The abnormalities
that were observed In these affected fetuses were cleft palates, one
straight leg, small kidneys, one renal agenesis, and enlarged renal pelvis.
They concluded from this study that the teratogenic activity of contaminated
pentachloronltrobenzene was probably due to hexachlorobenzene.
Summary
The acute oral toxicity of hexachlorobenzene has been found to be low,
with LD^g values ranging from 1700-10,000 mg/kg. Subchronic oral toxicity
studies with a number of mammalian species Indicated a significant Increase
In liver and kidney weights In hexachlorobenzene-treated animals. Some
studies have shown Increases In other organs as well. The livers from
hexachlorobenzene-exposed animals have shown histologic changes such as
Irregular shaped and moderately enlarged liver mitochondria and Increases In
the size of the centr1 lobular hepatocytes. Chronic oral toxicity stuaies
revealed similar, effects to those seen 1n the subchronic studies plus hexa-
chlorobenzene-assoclated life-shortening and various hepatic and renal
pathologies. These subchronic and chronic effects were usually dose-
related. Other effects Included multiple alopecia and scabbing, together
with neurologic effects In rats, mice and dogs. A dose-related histopatho-
logic change In the ovaries of monkeys has also been reported.
Increased porphyrin levels In the liver and In urine have been reported
for all species studied except the dog, which does not exhibit Increased
porphyrin levels. Hexachlorobenzene was found to cause the accumulation of
02590
V - 61
04/05/91
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0-H-stero1ds, which Induce porphyrin biosynthesis, and to Inhibit uroporphy-
rinogen decarboxylases. The Inhibition of uroporphyrinogen decarboxylases
appears to be due to pentachlorophenol, a hexachlorobenzene metabolite.
Indications are that females are more susceptible to hexachlorobenzene-
induced porphyria than are males, which may be related to the female estro-
gen levels and greater hexachlorobenzene metabolism. Hexachlorobenzene was
reported to produce a mixed-type Induction of cytochromes resembling that
produced by a combination of phenobarbUal (P-450) and 3,4-benzpyrene
(P-448). In addition, the activities of several hepatic microsomal enzymes
were found to be Induced by hexachlorobenzene.
Hexachlorobenzene did not Induce dominant lethal mutations In two
studies but was reported to be mutagenic In a yeast, cerevlslae. assay at
a concentration of 100 ppm. Hexachlorobenzene possessed no detectable
levels of mutagenic activity In the Salmonella h 1 s11dlne reversion assay.
The chronic toxicity studies provide sufficient evidence of the carcino-
genicity of hexachlorobenzene 1n animals since there was an Increased Inci-
dence of malignant tumors of the liver 1n two species, haemangloendothelloma
In hamsters and hepatocellular carcVnoma In rats as well as confirmed
reports of hepatoma In both of these species. Hexachlorobenzene was found
to cause teratogenic effects 1n fetal mice whose mothers were Ingesting 100
mg/kg/day of hexachlorobenzene during days 7-16 of gestation.
02590
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VI. HEALTH EFFECTS IN HUMANS
The effects of hexachlorobenzene on humans as a result of accidental or
occupational exposure have been reviewed by Courtney (1979) and Currier et
al. (1980). A few reports of data collected on occupationally- exposed
workers have been reported with studies conducted In Turkey and In the
United States (I.e., Louisiana) on the general population following
accidental exposure to hexachlorobenzene. The exposure of humans to toxleo-
loglcally significant levels of hexachlorobenzene 1n Turkey from 1955-1959
by Ingestion of contaminated grain, as .reported by Cam (1959, 1960), Cam and
Nlgogosyan (1963) and Peters et al. (1966), caused an epidemic of
hexachlorobenzene-lnduced porphyria cutanea tarda (PCI), also known as
porphyria turcica.
Epidemiologic Studies
Burns et al. (1974) found 0-310 ppb hexachlorobenzene In blood samples
from 20 vegetable spraymen. There were no signs of PCTt and no correlations
were observed between hexachlorobenzene levels and urinary porphyrin excre-
tion, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic trans-
aminase or lactate dehydrogenase. Increased levels of urinary porphyrins
were detected in 1 of 54 men occupationally exposed to hexachlorobenzene
(Horley et al.f 1973).
A medical survey was conducted by Dow Chemical Company (Currier et al.,
1980) on 50 employees working at a chlorinated solvents plant in Louisiana,
to determine blood hexachlorobenzene levels and signs suggestive of PCT or
other adverse effects, as well as any changes In hematologic, clinical chem-
istry and urinalysis parameters. The results from this study are of limited
02600
VI-1
09/15/88
-------
value because the various parameters studied during the 4-year period were
analyzed by several laboratories using various methods and on different
Individuals. There was potential exposure to other substances also. During
various times of the study, the time-weighted-average airborne concentra-
tions of hexachlorobenzene ranged from <1-13 ppb, and wipe samples from
surfaces In the control, laboratory and clerical work areas ranged from
0.03-1.24 yg/100 cm*.
The- laboratory analyses and physical examinations performed on the 1977
study group and on a control group from a polyethylene plant did not reveal
any signs Indicative of PCT. Blood levels of hexachlorobenzene, urinary
porphyrin and coproporphyrln and the average years of exposure are listed In
Table VI-1. A statistically significant (p<0.05) correlation was found
between hexachlorobenzene levels In blood and the number of years worked In
the plant. For the other studied parameters no statistically significant
differences were noted between the 44 chlorinated solvents workers and the
44 control workers for 1977, except for higher protein levels and lower
hematocrit values In the former workers, which were not considered to be
biologically significant. In addition, significantly lower levels of
urinary coproporphyrIns and albumin were detected In white male workers with
hexachlorobenzene blood levels >200 ppb than 1n those with hexachlorobenzene
levels <200 ppb.
Burns and Miller (1975) studied plasma hexachlorobenzene residues of 86
residents living and/or working In an area exposed to the production, trans-
portation and disposal of "hex" wastes (hexachlorobenzene and other chlori-
nated hydrocarbons) In Louisiana. Plasma hexachlorobenzene levels were
02600
VI-2
09/15/88
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TABLE VI-1
S Results of Blood and Urine Analysis In Hen Employed 1n a Chlorinated Solvents Plant, 1974-1977a
a*
o
o
Study Group
Comparison Group
Parameter
1974
(n=50)
1975
(n=49)
1976
(n=49)
1977
(n=44)
1977
(n=44)
Blood HCB
(mq/D
310.7 ~ 287.7b
311.5 ~ 242.9b
159.9 + 142.7C
170.3 ~ 111.8C
0.1 + 0.6
Urinary
uroporphyrins
(pQ/l)
22.4 ~ 21.1
20.9 * HO
37.4 * 14.4
26.2 + 14.3
NR
Urinary
coproporphyrlns
(ng/i)
77.4 ~ 40.5
67.2 ~ 36.1
100.6 + 40.8
95.2 t 48.9
NR
Age
(years)
30.1 ~ 6.3
31.1 ~ 6.6
30.8 *_ 6.7
31.7 ~ 7.1
31.3 + 6.8
Plant-years
5.5 t 3.9
6.3 + 4.0
5.9 * 4.5
6.6 ~ 4.8
6.6 ~ 4.4
aSource: Currier et al., 1980; 1974-1975 results conducted by Bioscience Laboratories; 1976-1977 results
conducted by Pathology Laboratories (i Standard Deviation)
bIn plasma
cIn blood
N = Sample size
NR = Not reported
HCB = Hexachlorobenzene
-------
measured and correlated with demographic characteristics, occupational
hazards, food sample analyses and house dust analyses. Average plasma
levels of hexachlorobenzene ranged from 2.4-3.6 ppb In exposed subjects as
compared with 0.5 ppb 1n controls (p<0-001; Table VI-2).
Higher levels of hexachlorobenzene residues, which were statistically
significant (p<0.05), were found 1n the male subjects (4.71 ppb) than In the
female subjects (2.79 ppb). These were not associated with race or exposure
to hexachlorobenzene through the consumption of homegrown vegetables and
animals. About 68% of the house dust samples contained an average hexa-
chlorobenzene concentration of 380 ppb as compared with 20 ppb In control
samples. When the hexachlorobenzene levels 1n dust were compared with the
mean plasma hexachlorobenzene levels for members of the same household, a
significant correlation was obtained (p<0.025). In addition, blood samples
from 11 workers employed for an average of 4.8 years (10 months to 15 years)
at the chemical plant contained an average of 78.6 (14-233) ppb
hexachlorobenzene.
Accidental Ingestion In Turkey
The hexachlorobenzene-lnduced PCT epidemic In Turkey, a result of expo-
sure during 1955-1959 1n individuals who used contaminated seed wheat for
food, has been reviewed by Courtney (1979). Cam and Nlgogosyan (1963) esti-
mated that 0.05-0.2 g of hexachlorobenzene was consumed per day. The method
of estimation was not described. PCT Is a disease of disturbed porphyrin
metabolism manifested by cutaneous lesions and Is commonly followed by
hypertrichosis (hairiness) and hyperplgmentatlon. The Induction of por-
phyria by hexachlorobenzene has been reviewed (DeMattels, 1967; Granlck,
1965; Tschudy and Bonkowsky, 1972; Courtney, 1979). Porphyrias are
02600 VI-4 04/12/88
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TABLE VI-2
HC8 Plasma Levels 1n Exposed Individuals and Controls3
Parameter
Exposedb
Co- :rolsb
Number of subjects
86
43
Age (years)
39.8 * 19.1
32.3 t 18.6
Black/white ratio
1.0
2.3
HCB plasma residues (ppb)
Range (ppb)
Percent positive
Percent >1 ppb
2.4 + 2.3C
0-23
99
99
0.5
0-1.8
95
5
aSource: Burns and Miller, 1975
^Values are mean + 1 SO
cLevel for random sample only, N=63 (3.6 + 4.3 for random and biased
samples, N=83)
HCB = Hexaclorobenzene
02600
VI - 5
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metabolic disorders of porphyrin metabolism that are characterized by
Increased excretion of porphyrins and their precursors. Normally,
A-amlnolevulinlc -acid synthetase Is the rate-limiting step 1n porphyrin
synthesis and heme acts as an end-product Inhibitor or an end-product
repressor of A-amlnolevul1n1c acid synthetase. In riexachlorobenzene-
Induced porphyria, 4-amlnolevul1n1c acid synthetase 1s Induced but heme
does not suppress or Inhibit the enzyme. The activity of uroporphyrinogen
decarboxylase Is decreased; consequently, porphyrin and Its precursors
(e.g., uroporphyrinogen, coproporphyrlnogen and occasionally series I
porphyrins) are excreted mainly In the urine but also In the feces.
Increased levels of porphyrins also can be measured In the liver, skin,
Intestinal tract and other tissues (Courtney, 1979). PCI appeared to occur
more frequently In children 4-16 years of age, whereas the number of adults
and children under 5 years of age reporting PCT was much lower {10-24% of
cases were individuals over 15 years of age and <5% were children below the
age of 4). A distinct disease described as "pink sore" was observed In
children under 1 year of age and reached an epidemic scale. The clinical
symptoms were weakness and convulsions and usually death In children whose
mothers had clinical symptoms of PCT or who had Ingested contaminated bread
during gestation and/or lactation. The presence of hexachlorobenzene In the
milk of nursing mothers suggested that pink sore was a manifestation of
hexachlorobenzene toxicity. The1 reviewer states that there was- a 95%
mortality 1n these Infants 1n addition to the very high Incidence of
stillbirths.
In a follow-up study, CMpps et al. (1981) examined 32 patients 20 years
after the onset. Porphyrins were determined In urine and stool specimens of
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29 patients and clinically significant porphyrin levels were observed 1n 5
patients. Clinical features such as hyperplgmentatlon, scarring, pinched
fades,, hypertrichosis, enlarged thyroid and distinctive arthritis were
present In about half of the patients.
A detailed follow-up study was also conducted with 161 Turkish patients
25 years after the Initial hexachlorobenzene Incident (Peters et al.t 1982).
The patient group studied Included* some of the patients previously examined
(Peters et al., 1966). Twenty-six patients were over 17 years of age at the
time of acute toxicity, whereas the average age of the remaining patients
was 7.1 years. An evaluation of the clinical signs and symptoms Is sum-
marized In Table V1-3.
.The chronic disease state was manifested by generalized hyperplgmenta-
tlon and hypertrichosis, scarring on the cheeks and hands, and tight sclero-
dermoid changes of the nose with perioral scarring. The most striking clin-
ical features 1n those patients who developed signs of hexachlorobenzene
toxicity, at an average age of 7 years consisted of palnles: arthritic
changes with osteoporosis of carpal_ metacarpal and phalangeal bones and
atrophy or failure to develop In the terminal phalanges. In addition,
neurologic symptoms Including weakness, paresthesias, myotonia, cogwheellng
and painless arthritic changes of the hands and feet, were observed In
50-70% of the patients examined. Since the signs and symptoms 20-25 years
later represented a continuum of signs and symptoms observed personally by
Peters and Gocmen (1959-1963), It was concluded that the symptoms repre-
sented the effects of both hexachlorobenzene toxicity and changes caused by
the Induced mixed porphyria. Control patients from the villages Inhabited
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TABLE VI-3
Clinical Signs and Symptoms In Humans 25 Years After Exposure to
Low Levels of HCB In Turkey, 1955-1959*
Clinical Signs/Symptoms
No. of Patients
with Symptoms**
Percent
PorDhvr1a--Neuroloa1cal
Weakness
117 (161)
73
Paresthesias
89 (161)
55
Sensory shading
75 (125)
60
Nervousness
39 (60)
65
Myotonia
35 (76)
46
"CogwheelIng"
34 (125)
27
Colic
84 (161)
52
Constipation
31 (161)
19
Recent red urine
17 (161)
11
Enlarged liver
10 (161)
6
Oermatoloqlc
Hyperplgmentatlon
125 (161)
78
Scarring
134 (161)
83
Hirsutism
81 (161)
50
Pinched fades
69 (161)
43
Fragile skin
62 (161)
39
Thyroid enlarqement
Total
64 (161)
40
Men
26 (98)
27
Women
38 (63)
60
Qrthooedlc and others
Arthritis
108 (161)'
67
Small hands
107 (161)
67
Short stature
74 (161)
46
aSource: Peters et al.. 1982
^Numbers In parentheses represent total number of patients examined for
this symptom
HCB = Hexachlorobenzene
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by these patients Included unaffected family members and clearly demon-
strated the uniqueness of this disorder that allowed for ready Identifica-
tion of affected patients. In addition the 60% Incidence of large thyroid
tumors In the females proved a sharp contrast to the 5% Incidence of thyroid
tumors in the geographical area. No conclusions were drawn as to the inci-
dence of cancer and mortality. Studies on these endpolnts are still in
progress and the length of time that has elapsed from the time of exposure-
may not yet be adequate for drawing conclusions.
A boy and three women of the exposed individuals treated In the early
19601s with l.v. and/or oral edetic acid (the metal chelating agent EDTA)
showed no active symptoms when examined, and skin pigmentation and scarring
were much less severe than In most of the other patients. Urine and/or
stool porphyrin studies showed that seven patients had clearly recognizable
increases In porphyrin levels (Table VI-4). Clinical chemistry and milk
residue data are summarized in Table VI-5. Percent i-amlnolevulinlc acid
values were found to be above the upper normal limit of 4 mg/i in 32/55
patients. The average residue levels in human milk samples from Turkish
mothers with porphyria was 0.51+0.75 ppm; 0.16+0.23 ppm was found in milk
samples from nonporphyrlc but hexachlorobenzene-exposed mothers.
Summary
A few epidemiologic studies with occupationally-exposed workers have
been reported, together with studies and surveys conducted in Turkey and in
the United States (i.e., Loulsana), on the general population following
accidental exposure to hexachlorobenzene. These studies qualitatively
support the toxicity of hexachlorobenzene but give little dose response
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TABLE VI-4
Porphyrin Levels In Patients and Controls*
Stool Iwg/q dry wetiihl)
Urine (ug/t)
CoproporphyrIn
Protoporphyrin
Uroporphyrin
CoproporphyrIn
Uroporphyrin
Controls
Turkey,
mean t SO
(N»33)
4.00 ~ 3.2
7.65 » 9.83
1.41 » 1.57
30.0 » 23.6
5.60 4.25
United States,
mi>an * SO
(N-40)
6.1 » 4.7
21.1 ~ 11.6
2.6 > 2.7
69.0 t 27.0
9.0 f 4.0
Heiachlorohemene-Faposed Pallents
Patients with active porphyria
(N.I5)
70.14
(1.0-837.6)
12.19
(0.7-61.8)
25.8
(0.7-189.2)
174.5
(32.6-779.3)
111.4
(16-1607)
Remainder
(N-146)
5.74
(0.5-4.1)
9.02
(0-103.4)
1.19
(0-12.6)
31.91
(0-198.4)
7.25
(0-29.5)
'Source: Peters et al.. 1982
-------
TABLE VI-5
Laboratory Test Results of Turkish Patients3
No. of
Test
Normal Range Patient Range
Abnormal
Resultsb
Urine
6-Amlnolevul1n1c acid, mq/1
<4
0.14-10.1
32 (55)
Porphobilinogen, mq/i
<1
0.11-1.04
0 (56)
Copper, ppm
0.01-0.06
0,01-0.046
0 (31)
Zinc, ppm
0.1-0.7
0.02-1.22
1 (31)
Serum
Copper, wg/dl
70-155
88-153
0 (30)
Zinc, yg/di
70-120
57-112
9 (29)
Creatine kinase, un1ts/l
women, <120
65-141
1 (8)
men, <150
51-318
4(11)
Iron, yg/dl
65-170
69-147
0 (29)
Thyroid function tests
5-11
2.2-10.1
women, 5
(10)
Thyroxine, yg/da
men, 2
(9)
Triiodothyronine uptake,
37-59
36-51.1
women, 1
(10)
percent
men, 1
(9)
Free thyroxine Index
1.85-6.5
0.9-4.6
women, 4
(10)
men, 0
(9)
Blood
Lead, erythrocyte, yg/dl
<35
2-17
0(11)
Uroporphyrinogen synthetasec
>20
12.4-34.8
5 (30).
Ml 1k hexachlorobenzene, ppmd
Patients with porphyria
NA
0.51 (0-3.12)
53 (56)
Patients without porphyria
NA
0.-16 (0-1.26)
16 (77)
aSource: Peters, et al.t 1982
^Numbers 1n parentheses represent total number of patient specimens
analyzed.
cValues expressed 1n nanomoles formed per milliliter of RBCs per hour
^Allowable limit In United States for cow's milk Is 0.02 ppm
NA = Not applicable
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Information. Biologic monitoring of plasma levels shows clearly more
hexachlorobenzene In plasma of exposed compared with nonexposed Individuals,
although no biologically significant adverse health effects were seen during
the observation periods.
The exposure of humans to hexachlorobenzene In Turkey from 1955-1959
caused an epidemic of hexachlorobenzene-lnduced PCT, also known as porphrya.
turcica, which is manifested, by disturbed porphyrin metabolism, cutaneous
lesions and hyperplgmentatlon. The authors estimated that 0.05-0.2 g/day
were Ingested. In children under 1 year of age, pink sore was observed as
well as 95% mortality 1n these Infants.
Follow-up studies conducted with patients 20-25 years after the onset of
porphyria showed that a few patients still had active porphyria, whereas
>50% exhibited hyperplgmentatlon scarring as well as other dermatologlc,
neurologic and skeletal features of hexachlorobenzene toxicity. Hexachloro-
benzene residues were also found 1n the blood, fat or breast milk of some
patients.
A correlation was found between hexachlorobenzene levels 1n blood and
the number of years worked In a chlorinated solvents plant. The concentra-
tion of urinary uroporphyrins and coproporphyrins In workers ranged from
21-37 and 67-101 vg/l, respectively, for the period between 1974 and
1977. An epidemiologic survey conducted with 86 residents In the vicinity
of this chlorinated solvents plant showed elevated hexachlorobenzene
residues In plasma. Higher levels of hexachlorobenzene residues were found
In males than in females, but these were not associated with race or food
consumption.
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VII. MECHANISMS OF TOXICITY
The exposure of humans to toxlcologically significant levels of hexa-
chlorobenzene occurred 1n Turkey from 1955-1959 by Ingestion of contaminated
grain, as reported by Cam (1959, I960), Cam and Nlgogosyan (1963), and
Peters et al. (1966), and caused an epidemic of hexachlorobenzene-lnduced
porphyria cutanea tarda (PCT), also known as porphyria turclcla. This
disease Vs characterized by a marked elevation 1n the levels of uropor-
phyrins In the urine. Also seen, but. to a lesser degree, are Increases In
coproporphyMns {Thiers, 1981). Photosensitivity Is associated with PCT
(Ellefson, 1982) and may be Involved In the manifestation of cutaneous
lesions and hyperplgmentatlon (Tonnukl et al., 1981; Crlpps et al., 1981). .
Although It Is clear that exposure to hexachlorobenzene adversely
affects the heme blosynthetlc pathway, the exact mechanism of action by
which hexachlorobenzene Induces hepatic porphyria remains unknown. In addi-
tion, only a small proportion of those people exposed to hexachlorobenzene
developed porphyria (Dogramad, 1964), but such adverse effects appear to be
long-term in both humans {Crlpps et al., 1984) and experimental animals
(Koss et al, 1983). As such, the toxicity of hexachlorobenzene poses a very
Interesting''problem whose solution will likely provide an increased under-
standing of human porphyria, heme biosynthesis and the influence of xeno-
blotlcs on each.
Mechanism of Porphyria
The Induction of porphyria by hexachlorobenzene has been extensively
reviewed (Granlck, 1965; OeMattels, 1967; Tschudy and Bonkowsky, 1972;
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and experimental models of hexachlorobenzene-lnduced
now exist In several animal species (reviewed by Elder,
Matteis, 1980).
It Is clear that exposure to hexachlorobenzene adversely affects the
heme blosynthetlc pathway, but the exact target(s) of Us toxic Insult Is
still unknown. Numerous studies have demonstrated that exposure to hexa-
chlorobenzene Impairs metabolism of uroporphyrinogen III by. decreasing the
enzymatic activity of uroporphyrinogen decarboxylase (Elder et al., 1976,
1978; Kushner et al., 1976; Strlk et al.. 1980; San Martin de Vlale et al.-,
1977; Koss et al., 1983). This loss of enzyme function Is associated with
decreased catalytic function and not with the concentration of uroporphyrin-
ogen decarboxylase (Elder and Sheppard, 1982; Elder and Urquhart, 1986). As
such, this decreased activity may be attributed to either the presence of an
Inhibitor or to modification of the enzyme Itself. Two hypotheses have been
presented to explain this decrease of uroporphyrinogen decarboxylase
(DeMattels, 1986). The first proposes that hexachlorobenzene is metabolized
to a reactive Intermediate capable of impairing enzyme function. As such,
inducers of drug metabolizing enzymes. Including hexachlorobenzene Itself,
can affect Its toxicity. The second suggests a more generalized effect of
hexachlorobenzene Involving oxidative damage to 'membranes and proteins via
the formation of peroxides and free radicals. This mechanism could directly
involve hexachlorobenzene, or alternatively, hexachlorobenzene could act to
Induce liver concentrations of other enzymes capable of generating such
reactive species (DeMattels, 1986).
Courtney, 1979),
hepatic porphyria
1978; Smlth and De
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Metabolism
Just as metabolism 1s Important to the conversion of hexachlorobenzene
to more polar compounds, hence to Its elimination and subsequent decreased
body burden (Sundlof et al., 1982), It also appears to be equally Important
for the Induction of the toxic effects attributed to this chemical (Koss et
al.t 1980a; Debets et al., 1980a; Walnstok de Calmanovlcl et al., 1984).
The metabolism of hexachlorobenzene has been reviewed (Renner, 1981;
Renner and Nguyen, 1984) and Is believed to have similar routes 1n rats and
humans (Stonard, 1974). Primary metabolites. Including pentachlorophenol
and tetrachlorohydroqulnone, are found 1n human urine, but pentachlorothlo-
phenol, a major rat metabolite, 1s not (Edgerton et al., 1981).
B1111 and coworkers have shown that certain of these metabolites can
Inhibit uroporphyrinogen decarboxylase j_n vitro (81111 et al.t 1986a,b) and
also are associated with Increased liver porphyrin concentrations vlvo
(B1111 et al., 1986b). Tetrachlorohydroqulnone and pentachlorophenol
decreased uroporphyrinogen decarboxylase activity 64 and 25%, respectively.
Hexachlorobenzene produced no effect (B1111 et al. 1986a). However, as
noted (BHH.et al,, 1986b), the effective Inhibitory concentration of these
»
metabolites was much greater than the concentration of these metabolites
found In the livers of rats treated with hexachlorobenzene {Koss et al.,
1978b). Others have shown that hexachlorobenzene Is porphyrlnogenlc when
added to primary chick embryo liver cells grown In culture. In this study
the parent compound was more effective than certain metabolites Including
pentachlorophenol, and was dependent upon the cytochrome P-450 content of
the cells (Debets et al., 1981).
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Using [14]C-labeled hexachlorobenzene It was shown that the binding of
laC to proteins 1s dependent upon cytochrome P-450 (Debets et al., 1981).
However, hexachlorobenzene 1s a poor substrate for cytochrome P-450 (Van
Ommen et al., 1986), but 1t does bind to cytochrome P-450 with high affinity
and does produce the type I spectrum characteristic of tytochrome P-450
substrates (Takazawa and Strobel, 1986). These studies suggest that metabo-
lism of hexachlorobenzene Is prerequisite to Its toxic effects, but the
discrepancy between the effective concentrations v1vq and the production
of reactive Intermediates In vitro Is not yet understood.
Oxidative Damage
An alternate mechanism has been proposed suggesting that exposure to
hexachlorobenzene results In oxidative damage and perturbation of lipid
membranes Including Increased permeability of liposomal membranes (Koszo et
al.t 1974) and direct membrane-fluldlzlng effects (Koszo et al.t 1982).
In the latter study, chronic dosing at 0*2% hexachlorobenzene In the
diet resulted In a significant decrease of the spin labeling order parameter
Indicative of Increased membrane fluidity. Since both 5-amlnolevulInic acid
and porphyrin must cross the mitochondrial membrane. It 1s conceivable that
»
alterations of membrane fluidity could alter the-'heme blosynthetlc pathway
(Koszo et al., 1982).
Observations supporting this theory have been reported (Nellson et al.,
1979, 1980), but It was also noted that both ethanol and hexachlorobenzene
exlblt membrane-fluldlzlng effects, while only the latter results In
significant Increases In porphyrin excretion (Nellson et al., 1980).
02610
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However, ethanol has been shown to potentiate certain effects of
hexachlorobenzene Including ones associated with damage to the plasma
membrane (Nlkolaev et al., 1986).
Other studies have shown that exposure to hexachlorobenzene causes no
Irreversible damage to mitochondrial membranes (Maslnl et al., 1984a,b,
1985) and suggest that the hexachlorobenzene metabolite, pentachlorophenol,
acts to uncouple mitochondrial oxidative phosphorylation. However, no clear
correlation exists between the concentration of pentachlorophenol and the
Increase of urinary and hepatic porphyrins (Mas 1n1 et al., 1985). Again It
Is unclear whether or not the metabolism of hexachlorobenzene. significantly
contributes to Us toxicity In vivo.
The role of glutathione has also been Investigated. Repeated exposure
of rats to hexachlorobenzene was shown to decrease the activity of
gTutathlone-S-transferase (Koss et al., 1980a). In addition,
dlethylmaleate, a compound known to deplete glutathione concentrations,
potentiates the action of porphyrinogens drugs (Puzynska et al., 1978).
With decreased glutathione or transferase activity, reactive, electrophll 1c
metabolites or oxygen-free radicals and peroxides have Increased opportunity
to react with sulfur-containing molecules Including proteins such as
uroporphyrinogen^ decarboxylase.
As recently reviewed (DeMattels, 1986), It Is possible that chemicals
like hexachlorobenzene affect porphyrin synthesis Indirectly by stimulating
the production of these oxygen-free radicals and peroxides, and that Iron
02610
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synerglstlcally enhances this effect by Its ability to catalyze these
reactions, Hexachlorobenzene may either stimulate the production of these
reactive species directly, or alternatively, may Induce enzymes that can
produce such reactive Intermediates (DeMattels and Stonard, 1977). With
respect to the latter hypothesis. It Is of interest that hexachlorobenzene
has been shown to Induce activities of NADPH-cytochrome P-450 reductase
(Goldstein et al., 1982; Debets et al., 1980a). This enzyme Is known to be
directly Involved in lipid peroxidation and superoxide formation (Morehouse
et al., 1984; Pederson arid Aust, 1972) by NAOPH and Iron-dependent (Ernster
and Nordenbrand, 1967) and hydroperoxlde-dependent (O'Brien and Rahlmtula,
1975) mechanisms. These can result In total destruction of microsomal P-450
and mitochondrial hemoprotelns (Hrycry and O'Brien, 1971), and also 1n the
Indirect Inactivation of other enzymes and the alteration of membrane
structure (Tappel, 1973).
It Is known that uroporphyrinogen decarboxylase contains many sulfhydryl
groups, at least one of which Is essential for catalytic activity (Sassj et
al., 1984). Recent studies have demonstrated that the effects of
hexachlorobenzene on uroporphyrinogen decarboxylase 1s to decrease Us
enzymatic activity, but the concentration of Immunoreactlve protein Is not
altered (Elder and Sheppard, 1982; Elder et al., 1976). It has been
suggested that reactive oxygen species could be responsible for the
alteration of uroporphyrinogen decarboxylase (Ferloll et al., 1984; Elder
and Urquhart, 1986). However, the specific mechanism(s) of this effect are
not yet understood, and this remains an Important and interesting example of
chemically-Induced pathology.
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Contributing Factors
. Several factors can contribute to the porphyrinogens action of
hexachlorobenzene. These Include the liver concentration of nonheme Iron,
as well as the sex (hormonal status) and the genetic background (strain) of
the animals used. Nonheme Iron has been shown to enhance the toxicity of
hexachlorobenzene, and as discussed above, this may be due to Us
contribution to oxygen-free radical formation (reviewed by Smith et al.,
1986).
Female rats are much more sensitive to the porphyrlnogenlc action of
hexachlorobenzene, and an Increase In sensitivity Is seen 1n males given
estrogens (R1zzard1n1 and Smith,. 1982). It Is especially significant that
In addition to the development of porphyria, female rats also show a higher
Incidence of liver tumors (Smith et al., 1986).
Finally, some strains of mice are more sensitive to the porphyrlnogenlc
effect of hexachlorobenzene. This difference may be associated with
differences of the Ah locus, a regulatory gene. Involved 1n the Induction of
several enzymes by polycycllc aromatic hydrocarbons. Nonetheless, It Is not
yet clear that a correlation exists between Ah responsiveness and. the toxic
effects oF hexachlorobenzene (Smith et al., 1986; llnko et al., 1986; Hahn
et al., 1986).
Time Course of Toxicity
Despite this understanding of hexachlorobenzene-lnduced hepatic
porphyria, 1t Is still difficult to explain the latency period between
exposure and onset of overt porphyria (reviewed by Koss et al., 1986).
02610
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Moreover, 1t 1s not known why this porphyria, once Induced, will persist
long after the exposure has stopped, but this Is true for both humans
(Crlpps et al., 1984) and experimental animals (Koss et al.f 1983).
As such, the study by Koss et al. (1983) provides an Important
understanding of the progression of hepatic porphyria. These researchers
administered 100 mg/kg hexachlorobenzene dissolved In olive oil every other
day for 6 weeks, through stomach tube, to female Wlstar rats and then
observed the rats for an additional 18 months. The rats were evaluated
during both the exposure period and the 18-month holding period for liver
hexachlorobenzene levels, levels of liver porphyrins, and the activity of
liver uroporphyrinogen decarboxylase. The results revealed a rapid Increase
In hexachlorobenzene liver levels, which reached a plateau after 10 days of
treatment and remained constant until exposure was terminated at 6 weeks.
The levels of liver hexachlorobenzene then decreased over time with no valid
biologic half-life determinable. The liver porphyrin levels, however,
started to rise slightly after 3 weeks of hexachlorobenzene exposure and
reached a maximum liver porphyrin concentration -7 months after the
exposures had ceased (Table VII-1). The liver porphyrin levels decreased to
a constant level -14 months after ceasing hexachlorobenzene exposures. At
18 months, after ceasing exposures, the treated rat's liver porphyrin levels
were still substantially higher than the levels 1n control rats. The
distribution pattern of the liver porphyrins was observed to be changed as
early as after the second hexachlorobenzene administration. The observed
changes were Increases In liver uroporphyrin levels and decreases In liver
protoporphyrin and coproporphyrln levels. The change In porphyrin patterns
was traced to the decreased activity of uroporphyrinogen decarboxylase
02610
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TABLE VII-1
Porphyrin Content and Uroporphyrinogen Decarboxylase Activity
In the Liver Cytosol of Female Rats Pretreated with 100 mg/kg HCB
Every Other Day for 6 Weeks*
Time After the
End of Treatment
Porphyrin "Content
(nmol/6 mil cytosol)fa
Enzyme Actlvlty
(pmol • mg"1 • m1n_1)c
1 day
14 + 3d
NDe
7 months
133 t 15
" ND
14 months
9 i 6
NO
18 months
8 > 5
0.3 i 0.2d
Controls
0.06 f 0.04
0.5 * 0.1
aSource: Koss et al., 1983
b6 mi cytosol correspond with 1 g liver tissue
cpmol coproporphyrlnogen I (determined as .coproporphyria) formed from uro-
porphyrinogen I In 1 mln by 1 mg cytosol protein
dMean (f SD) of three or four animals
eND = Not detectable. The lower detection limit was determined at 0.02
pmol • mg"1 • mln"1 coproporphyrin
HCB = Hexachlorobenzene
02610
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activity, which was found to be not detectable at the end of the 6-week
exposure period and the activity did not become detectable again until 18
months postexposure (see Table VI1-1J. These data led the Investigators
(Koss et al., 1983) to propose that there are four phases of hexachloro-
benzene-lnduced porphyria:
During the first phase an almost constant content of hexa-
chlorobenzene and a gradual decrease of uroporphyrinogen decarboxy-
lase activity Is achieved. In the second phase a noticeable accu-
mulation of porphyrins and a- practically complete Inhibition of
decarboxylase activity are conspicuous. In the third phase, which
occurs after hexach-lorobenzene .administration has been discon-
tinued, a further accumulation of porphyrins and a continuing Inhi-
bition of uroporphyrinogen decarboxylase activity can be seen, even
after extensive elimination of hexachlorobenzene. During the
fourth phase a decrease 1n porphyrin content and a return of
decarboxylase activity are clearly observable.
A possible reason for the continued Inhibition of uroporphyrinogen decar-
boxylase activity, even after substantial elimination of hexachlorobenzene
has occurred, was also discussed In this report. Koss et al. (1983) pre-
sented the scenario that once hexachlorebenzene had caused an Inhibition of
uroporphyrinogen decarboxylase activity and Increased liver porphyrin levels
that the accumulation of porphyrins could themselves maintain the Inhibition
of the enzyme activity.
Interactions
Certain chemicals have been shown to alter the toxicity and pharmaco-
kinetics of hexachlorobenzene In mammals. Pentachlorophenol and Iron
Increased the porphyrInogenlc effect of hexachlorobenzene, whereas
decachloroblphenyl had no effect. Hexachlorobenzene pretreatment resulted
In Increased CCl^ toxicity and altered Immune responses In hexachloro-
benzene-treated animals. In addition, hexachlorobenzene has been shown to
02610
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Induce hepatic xenoblotlc metabolism and thus has the potential to alter the
rate and extent of metabolism of other chemicals.
Debets et al. (1980b) studied the effect of oentachloroDhenol (PCP) on
hexachlorobenzene toxicity. Groups of female rats were fedidlets contalnlnq
1000 uq hexachlorobenzene/q, 500 uQ pentachlorophenol/q, or both chemi-
cals In the same amounts, and a fourth arouD served as the control. Penta-
chlorophenol accelerated the onset of hexachlorobenzene-induced porphyria,
as Indicated by an Increase In urinary excretion of uroporphyrin and a
decrease of porphyrins with two and three carboxyllc groups. This Increase
occurred.-3 weeks earlier In the hexachlorobenzene plus pentachlorophenol-
treated animals than In hexachlorobenzene-treated animals.
Rlzzardlnl and Smith (1982) Investigated d1ethylst1lboestrol (DES) pre-
treatment on hexachlorobenzene metabolite excretion In young male and female
F344/N rats. The rats were Injected 1.p. with four doses of DES dipropio-
nate 20 pmoles/kg dissolved In arachls oil over a 24-day period and then
given 14 mg/kg hexachlorobenzene by oral Intubation for 7 days. The results
Indicated that the DES pretreatment stimulated the excretion of hexachloro-
benzene metabolites, via urine and feces, in both males and females
(Table VII-2).
Blekkenhorst et al. (1980) reported that the simultaneous i.m. adminis-
tration of iron and hexachlorobenzene caused a marked potentiation of hexa-
y
chlorobenzene porphyrinogens effect in rats. This was shown by a decrease
In hepatic uroporphyrinogen decarboxylase activity and Increased urinary and
02610
VII-11
09/13/88
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TABLF VII-2
Analysts of the Excreta from Rats Administered Hexachlorobenzene
After an Initial Treatment wtth Dlelhylstllboestrola»b
Sex and Treatment Pentachlorophenol Tetrachlorobenzene-1,4-dlol Pentachlorothlophenol
(nmole/24 hours/kg bw)
Ur Ine
Hale *
oil
151
¥
19
3 1
23
»•
3
Hale t
DES
190
¥
22
17 * 2C
158
t
9C
Female
fr oil
174
«-
17
16 * 2d
142
«¦
12e
Female
t DES
453
105 f
35 i 9
176
«¦
7f
Feces
Hale t
oil
65
»¦
15
Trace
74
*
23
Hale t
DES
160
I-
23f
Trace
166
¥
33
Female
* oil
116
35,
Trace
65
¥
4
Female
» DES
279
I-
80
Trace
149
¥
13c
aSource: Rlzzardlnl and Smith, 1982
t*Hale and female rats (52-54 apd 71-73 days old, respectively) were given 20 pinole of DES dlproplo-
nate/kg dissolved In arachts oil (10 mg/mi) or oil alone by l.p. Injection on days 1, 4, 14 and 24.
From day 25 all rats were given 14 nig of hexachlorobenzene/kg by oral Intubation dally for 7 days. After
the last dose 24-hour samples of urine and feces were collected, hydrolyzed and analyzed. Results are
means S.E.H. (n=4/group).
c
Significance of differences from rats not given OES, p<0.001
''significance of differences from males, p<0.005
£
Significance of differences from males, p<0.001
fSIgn1fIcance of differences from rats not given DES, p<0.05
Total excretions of these metabolites were: male, 336^57; male t DES, 69H70 (p<0.01); female, 513^62;
female t DES, 1092H75 (p<0.025) nmole/24 hours/kg
-------
fecal porphyrin excretion. Conversely, simultaneous bleeding of hexachloro-
benzene-treated rats diminished the porphyrinogens effect of hexachloro-
benzene.
The role of Iron In Increasing the porphyrlnogenlclty of
hexachlorobenzene was noted In female Sprague-Dawley rats fed 0 or 400 ppm
of hexachlorobenzene In their diets for up to 118 days (Lambrecht et al.,
1986). Ethylenedlamlnetetracetlc acid (EOTA) was added to the study diets
at a concentration of 0, 0.5 or 1.0%, resulting In six study groups.
Dramatic Increases In the urinary excretion of uroporphyrin and
coproporphyMn were seen in the rats exposed to hexachlorob.enzene for 81
days or longer. EDTA In the diet significantly decreased the amounts of
porphyrins excreted In the urine. Hexachlorobenzene-lnduced liver porphyrin
fluorescence was also decreased In the EDTA groups. EDTA significantly
reduced the liver levels of Iron, zinc and copper, which were greatly
Increased In the hexachlorobenzene-exposed rats. EDTA had no effect on the
hexachlorobenzene-lnduced liver and kidney pathology observed. The authors
concluded that the liver metal levels may play an Important role In
hexachlorobenzene-lnduced porphyria but not the hexachlorobenzene-lnduced
pathology.
Goldstein et al. (1978) studied the comparative toxicity of pure hexa-
chlorobenzene (purity >99%) and technical hexachlorobenzene (purity 92%),
which was known to contain 200 ppm of decachloroblphenyl and 4 ppm of octa-
chlorodlbenzofuran, In female CD rats fed diets containing 0, 30, 100, 300
or 1000 jig hexachlorobenzene/g for up to 15 weeks. Neither grade con-
tained other chlorinated dlbenzofurans or dlbenzo-p-dloxlns. Both grades
02610
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09/19/88
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resulted in comparable effects (porphyria, cutaneous lesions, hyperexcit-
ablllty, changes In liver enzymes and morphologic liver changes) In treated
rats, although the technical grade appeared to be slightly more potent than
pure hexachlorobenzene In Its effects- on the pulmonary endothelium. The
Impurities did not appear to have a synergistic effect.
i
Kluwe et al. (1982) reported that pretreatment of male Sprague-Dawley
rats with hexachlorobenzene resulted 1n Increased CCl^ toxicity. The rats
received seven doses of hexachlorobenzene at 30 mg/kg In corn oil once every
72 hours followed by an l.p. Injection of CCl^ at 0.0, 0.03, 0.05, 0.25,
1.0 or 2.0 mi/kg In 4 ml/kg corn oil 24 hours after the last hexachloro-
benzene treatment. Hexachlorobenzene pretreatment Increased the CC1^-
Induced acute growth retardation, renal tubular functional impairment,
hepatocellular necrosis and further reduced the survival of the animals.
Variable results were reported In a study on the effect of hexachlorobenzene
pretreatment of male albino Sprague-Dawley rats on the \n vivo biotransfor-
mation, residue deposition, and elimination of 14C-aldrin, 1-naphthol,
DDT, hexachlorobenzene or mlrex (Clark et al.. 1901a). There was no evi-
dence of qualitative changes in the biotransformation of any test compound
that could be attributed to hexachlorobenzene pretreatment. Analysis of
residue deposition gave, mixed results: less r*C residues were found 1n
rats fed diets containing hexachlorobenzene and then treated with
14C-aldr1n, more laC residues were found after 1*C-DOT or 14C-mirex
treatment, and no difference was evident after 14C-hexachlorobenzene or
14C-1-naphthol treatment. Hexachlorobenzene also potentiates the effects
of stress on male Sprague-Dawley rats (Clark et al., 1961b). Rats fed 250
ppm hexachlorobenzene resulted in an increased severe loss of body weight
02610
VI1-14
04/12/91
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when placed Into crowded cages and compared with the weight loss of crowded
control rats. Crowded rats fed hexachlorobenzene had higher tissue residues
of hexachlorobenzene and higher mortality than the noncrowded hexachloro-
benzene-treated rats or the control rats.
Summary
Hexachlorobenzene (HCB) affects the heme biosynthesis pathway and this
Is the mechanism by which porphyria 1s Induced. Although many animal
studies have been performed, the exact target is unclear. From Yn v1 tro
experiments It is known that metabolites of HCB, such as pentachlorophenol,
Inhibit uroporphyrinogen dlcarboxylase but HCB Itself does not. An
alternate mechanism that has been proposed 1s that HCB and Us metabolites
cause oxidative damage to cellular lipid membranes. Despite extensive
Investigation of these two separate mechanisms of action the Issue has not
been resolved; In fact, other mechanisms have been proposed and are also
being explored.
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VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS
Introduction
The quantification of toxicologic...effects of a chemical consists of
separate assessments of noncardnogenlc and carcinogenic- health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarcinogenic health effects occur,,
while carcinogens are assumed to act without a threshold.
In the quantification of noncardnogenlc effects, a Reference Dose
(RfD), [formerly termed the Acceptable Daily Intake (AOI)] is calculated.
The RfD is an estimate (with uncertainty spanning perhaps an order magni-
tude) of a dally exposure to the human population (including sensitive
subgroups) that Is likely to be without an appreciable risk of deleterious
heal'th effects during a lifetime. The RfD Is derived from a no-observed-
adverse-effeet level (NOAEL), or lowest-observed-adverse-effeet level
(LOAEL), identified from a subchronlc or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfD is calculated as
follows:
(NOAEL or LOAEL)
RfD = * L = mg/kg bw/day
[Uncertainty Factor(s) x Modifying Factor]
Selection, of the uncertainty factor to be employed In the calculation of
the RfD is based upon professional Judgment, while considering the entire
data base of toxicologic effects for the chemical. In order to ensure that
uncertainty factors are selected and applied in a consistent manner,
02620
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the U.S. EPA (1991) employs a modification to the guidelines proposed by the
National Academy of Sciences (NAS, 1977, 1980) as follows:
Standard Uncertainty Factors (UFs)
Use a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor Is Intended to account for the variation
In sensitivity among the members of the human population. [10H]
Use an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
Inadequate. This factor 1s Intended to account for the uncer-
tainty in extrapolating animal data to the case of humans.
[IDA]
• Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there is no
useful long-term human data. This factor Is Intended to
account for the uncertainty in extrapolating from less than
chronic NOAELs to chronic NOAELs. [10S]
Use an additional 10-fold factor when deriving an RfO from a
LOAEL instead of a NOAEL. This factor Is intended to account
for the uncertainty in extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (MF)
Use professional Judgment to determine another uncertainty
factor (MF) that 1s greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of
the overall data base and the number of species tested. The
default value for the MF is 1.
The uncertainty factor used for a specific risk assessment is based
principally upon scientific judgment rather than scientific fact and
accounts for possible intra- and interspecies differences. Additional
considerations not Incorporated in the NAS/ODW guidelines for selection of
an uncertainty factor Include the use of a less than lifetime study for
deriving an RfD, the significance of the adverse health effects and the
counterbalancing of beneficial effects.
02620
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From the RfD, a Drinking Water Equivalent Level (DWEL) can be calcu-
lated. The DWEL represents a medium specific (i.e., drinking water)
lifetime exposure at which adverse, noncardnogenlc health effects are not
anticipated to occur. The DWEL assumes 100% exposure from drinking water.
The DWEL provides the noncardnogenlc health effects basls'for establishing
a drinking water standard. For Ingestion data, the DWEL 1s derived as
follows:
DWEL = (RfD) * (Body weight 1n kg) = ^
Drinking Water Volume in l/day
where:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 l/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for expo-
sures of shorter duration (1-day, 10-day and longer-term) are determined.
The HA values are used as Informal guidance to municipalities and other
organizations when emergency spills or contamination situations occur. The
HAs are calculated using an equation similar to the RfD and DWEL; however,
the NOAELs or LOAELs are Identified from acute or subchronlc studies. The
HAs are derived as follows:
HAs(NQAEL or LOAEL) x (bw) =-
(UF) x ( i/day)
Using the above equation, the following drinking water HAs are developed
for noncardnogenlc effects:
1. 1-day HA for a 10 kg child Ingesting 1 i water per day.
2. 10-day HA for a 10 kg child Inge-sting 1 i water per day.
3. Longer-term HA for a 10 kg child Ingesting 1 l water per day.
4. Longer-term HA for a 70 kg adult Ingesting 2 t water per day. ..
02620
VI11-3
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The 1-day HA calculated for a 10 kg child assumes a single acute
exposure to the chemical and Is generally derived from a study of <7 days
duration. The 10-day HA assumes a limited exposure period of 1-2 weeks and
is generally derived from a study of <30 days duration. The longer-term HA
Is derived for both the 10 kg child and a 70 kg adult and assumes an
exposure period of ~7 years (or 10% of an Individual's lifetime). The
longer-term HA Is generally derived from a study of subchronlc duration-
(exposure for 10% of animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chemical, based
on the overall weight-of-evidence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of
carcinogenicity 1n animals with limited (Group B1) or inade-
quate (Group B2) evidence In humans.
Group C: Possible Human Carcinogen. Limited evidence of
carcinogenicity 1n animals In the absence of human data.
Group D: Not Classified as to Human Carcinogenicity. Inade-
quate human and animal evidence of carcinogenicity or for which
no data are available.
Group E: Evidence of Noncarcinogenlclty for Humans. No
evidence of carcinogenicity in at least two adequate animal
tests In different species or in both -adequate epidemiologic
and animal studies.
If toxicologic evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the
Ingestion of the contaminant In drinking water. The data used In these
02620
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estimates usually come from lifetime exposure studies using animals. In
order to predict the risk for humans from animal data, animal doses must be
converted to equivalent human doses. This conversion includes correction
for noncontlnuous exposure, less than "11fetime studies and for differences
In size. The factor that compensates for the size difference Is the cube
root of the ratio of the animal and human body weights. It 1s assumed that
the average adult human body weight Is 70 kg and that the average water'
consumption of an adult human Is 2 l of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure from
ingestion of water. The cancer unit risk is usually derived from a linear-
ized multistage model with a 95% upper confidence limit providing a low dose
estimate; that Is, the true risk to humans, while not identifiable. Is not
likely to exceed the upper limit estimate and, In fact;, may be lower.
Excess cancer risk estimates may also be calculated using other models such
as the one-hit, Weibull, 1og1t and probit. There Is little basis in the
current understanding of the biologic mechanisms involved in cancer to
suggest that any one of these models is able to predict risk more accurately
than any other. Because each model Is based upon"differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an Inherent uncertainty that is due to the
systematic and random errors in scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there Is
02620
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uncertainty when the data are extrapolated to humans. When developing
cancer risk rate levels, several other areas of uncertainty exist, such as
the Incomplete knowledge concerning the health effects of contaminants In
drinking water, the Impact of the experimental animal's age, sex and
species, the nature of the target organ system(s) examines and the actual
rate of exposure of the Internal targets In experimental animals or humans.
Dose-response data usually are available only for high levels of exposure
and not for the lower levels of exposure closer to where a standard may be
set. When there Is exposure to more than one contaminant, additional
uncertainty results from a lack of Information about possible synergistic or -
antagonistic effects.
Noncarclnoqenlc Effects
The toxicity of long-term dietary exposure of humans to hexachloro-
benzene was demonstrated by the epidemic of porphyria cutanea tarda (PCT) In
Turkish citizens who accidentally consumed bread made from grain treated
with hexachlorobenzene (Cam and Nlgogosyan, 1963; Peters et al., 1966,
1982). The authors estimated that 0.05-0.2 g/day were Ingested. In
children less than 1 year of age, pink sore was observed as well as 95%
mortality. In addition to the PCT-assoclated symptoms of skin lesions,
hypertrichosis and hyperplgmentatlon, the exposure caused neurotoxicity and
liver damage. Follow-up studies reported PCT symptoms, reduced growth and
arthritic changes In the appendages of children who were directly or
Indirectly (I.e., through breast milk) exposed. Studies In rats have
demonstrated hexachlorobenzene' s ability to Increase the Incidence of
stillbirths, decrease fetal growth and decrease postnatal survival (Grant et
al., 1977; Khera, 1974). A study In rats reported that administration of
02620
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09/15/88
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hexachlorobenzene during gestation Increased significantly the number of
fetuses with extra ribs. A study In mice found that hexachlorobenzene given
on days 7-16 of gestation resulted In an Increased Incidence of fetal
abnormalities when compared with controls (Courtney et al., 1976).
The acute oral toxicity of hexachlorobenzene has been found to be lowt
with LD^0 values ranging from 1700-10,000 mg/kg (NAS, 1977 ; IARC# 1979;
Sax, 1979). Subchronlc oral toxicity studies with a number of mammalian
species Indicated statistically significant Increases In liver and kidney
weights In hexachlorobenzene-treated animals (Kulper-Goodman et al., 1977;
Boger et al., 1979; Koss et al.# 1978b; Sundlof et al., 1981; .Shlral et al.,
1978). Studies have shown Increases In other organs as well (EUssalde and
Clark, 1979; Koss et a!., 1978b). The livers from hexachlorobenzene-exposed
animals have shown histologic changes such as Irregularly shaped and mode-
rately enlarged liver mitochondria and Increases In the size of centrHobu-
1 ar hepatocytes (Kulper-Goodman et al., 1977; Boger et al., 1979 ; Lambrecht
et al., 1982a,b). Chronic oral toxicity studies revealed the same type of
effects seen In the subchronlc studies plus hexachlorobenzene-assoclated
life-shortening and various hepatic and renal pathologies (Cabral et al.,
1977, 1979; Smith and Cabral, 1980; Lambrecht et al., 1983a,b; Arnold et
al., 1985). These subchronlc and chronic effects were usually dose-related.
Multiple alopecia and scabbing, together,w1th neurologic effects, have also
been observed In rats, mice and dogs (Headley et al., 1981; Sundlof et al.,
1981). A dose-related histopathologic change 1n the ovaries of monkeys has
also been reported (Iatropoulos et al., 1976; Knauf and Hobson, 1979).
02620
VIII-7
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Increased porphyrin levels 1n the liver and 1n urine have been reported
for all species studied (Kulper-Goodman et al., 1977; Koss et al., 1978a,
1983; Smith et al.t 1980; Gralla et al., 1977; Rlzzardlnl and Smith, 1982)
except for the dog, which does not exhibit Increased porphyrin levels
(Gralla et al., 1977). Hexachlorobenzene was found to cause the accumula-
tion of 5B-H-stero1ds that Induce porphyrin biosynthesis (Graef et al.,
1979), and to Inhibit uroporphyrinogen decarboxylases (Graef et al., 1979;
Koss et al., 1903). The tnhlbttlon of uroporphyrinogen decarboxylases
appears to be due to pentachlorophenoT, a hexachlorobenzene metabolite (Rlos
de Molina et al., 1980). There 1s evidence that females are more suscepti-
ble to hexachlorobenzene-lnduced porphyria than are males," which may be
related to the female estrogen levels and greater hexachlorobenzene metabo-
lism (Rlzzardlnl and Smith, 1982). Hexachlorobenzene was reported to
produce a mixed-type Induction of cytochromes resembling that produced by a
combination of phenobarbltal (P-450) and 3,4-benzpyrene (P-448) (Goldstein
et al., 1982; Debets et al., 1980a). In addition, the activities of hepatic
microsomal enzymes were found to be Induced by hexachlorobenzene (Arlyoshl
et al., 1974; Mehendale et al., 1975; Carlson and Tardlff, 1976; Chadwlck et
al., 1977; Carlson, 1978, 1980; Carlson et al.. 1979).
»
Hexachlorobenzene has been shown to produce various types of tumors In
animals. Lifetime dietary administration of hexachlorobenzene to hamsters,
rats and mice Increased the Incidence of thyroid tumors in hamsters (Cabral
et al., 1977), liver tumors In hamsters (Cabral et al., 1977), mice (Cabral
et al., 1979) and rats (Smith and Cabral, 1980; Lambrecht, 1983; Arnold et
al., 1985), kidney tumors 1n rats (Lambrecht, 1983) and adrenal tumors 1n
rats (Arnold et al., 1985; Peters et al., 1983).
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Quantification of Noncardnogenlc Effects
Table VIII-1 presents a summary of the toxicity studies on hexachloro-
benzene that were considered for calculation of the 1-, 10-day and longer-
term Health Advisories (HAs) and the' lifetime Drinking Water Equivalent
Level {DWEL).
Derivation of 1-Day Health Advisory. Currently available evidence for-
the acute toxicity of hexachlorobenzene Is considered to be insufficient for
calculation of a 1-day HA for a 10 Jcg child. There are acute oral LD^Q
studies with hexachlorobenzene that" Indicate low acute toxicity, as LD^
values are >1700 mg/kgt for this material in rats, rabbits, cats and mice.
However, these studies do not provide an assessment of systemic toxicity
which can be applied to a.1-day HA calculation. Conversely, the Kuiper-
Goodman et al. (1977 ) study, which Is the basis for the longer-term HA
herein, gives a dose-response with effect and no-effect levels for systemic
toxicity in male and female rats given hexachlorobenzene In the diet. The
subchronic dosing pattern is a limitation In using the Kuiper-Goodman et al.
(1977) study specifically for a 1-day HA calculation. Nonetheless, adminis-
tration of hexachlorobenzene in the diet would more closely approximate
exposure patterns with drinking water" than bolus treatment via gavage or
capsules, which was the method of treatment in the acute as well as several
subchronic toxicity studies with hexachlorobenzene. Therefore, the longer-
term HA for a 10 kg child of 0.05 mg/l is also recommended for use as a
1-day HA.
Derivation of 10-Dav Health Advisory. Currently available oral exposure
toxicity data are considered insufficient for calculation of a 10-day HA for
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VI11-9
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TABLE VI11-1
Sumnary of Toxicity Studies on Hexachlorobenzene
Species
Route
Dose
DuratIon
Effects
Reference
Rat
(females)
Rat
Rat
(females)
Rats
(females)
oral
oral
(diet)
oral
(gavage)
oral
(gavage)
100 mg/kg every other
day
0.5 ag/kg/day
2.0 mg/kg/day
8.0 mg/kg/day
3?.0 ag/kg/day
Rat
(females)
oral
(diet)
O.S mg/kg twice
weekly
2.0 mg/kg twice
weekly
8.0 mg/kg twice
week 1 y
32.0 mg/kg twice
weekly
100 mg/kg diet
up to 43 days
IS weeks exposed and
held to 48 weeks
15 weeks exposed and
held to 48 weeks
15 weeks exposed and
held to 48 weeks
IS weeks exposed and
held to 48 weeks
50 mg/kg every other 15 weeks
day
29 weeks
29 weeks
29 weeks
29 weeks
98 days
Suggested covalent binding of hexachlorobenzene
metabolites to cylosollc proteins
Transient Increases In liver porphyrin levels
In females after termination of exposure
Increases In liver porphyrin levels In females
after termination of exposure, Increased size
of centrI lobular hepatocytes
Increased liver weights, Increased liver,
kidney and spleen porphyrin levels In females
(porphyria), centrllobular liver lesions espe-
cially In females at 48 weeks
Increased mortality In females, Intension
tremors In males and females an(l ataxia In a
few females. Increased liver, kidney and
spleen weights. Increased liver, kidney and
spleen porphyrin levels In females (porphyria),
centrllobular liver lesions and splenomegaly
Increased liver, kidney, spleen and adrenal
weights, porphyria (Increased liver porphyrin
levels and Increased excretion of porphyrins
and precursors), tremors, hair loss and skin
lesions
Increase In relative liver weight
Increase In relative liver weight, moderately
enlarged hepatocytes
Porphyria, markedly enlarged hepatocytes.
Increase In relative liver weight
Porphyria, markedly enlarged hepatocytes.
Increase In liver weights
Porphyria (Increased liver lobe porphyrins),
decreased activity of uroporphyrinogen
decarboxylase
Koss et al.,
1980a
Kulper-Goodman
et al,, 1977
Koss et al.,
1978b
BAger et al.,
1979
Smith et al.,
1980
-------
lABIt VIII 1 (cont.)
o
fv)
cr
f\J
o
Specles
Route
Oose
Our at Ion
Effects
Reference
Rat
ora 1
(diet and
nursIng)
Rat
Rat
\
Rat
Rat
(male)
Rat
(female)
Rat
(female)
Rat
Rat
(females)
Rat
(females)
ora 1
(diet and
nursing)
ora 1
(diet)
ora I
(diet)
oral
(diet)
oral
(diet)
ora 1
(gavage)
oral
(gavage)
ora 1
(gavage)
ora 1
(diet)
50 mg/kg diet
ISO mg/kg diet
4. 20 or 100 mg/kg
diet
S00. 1000 or 2000
mg/kg diet
2000 mg/kg diet
2000 mg/kg diet
3000 mg/kg diet
14 mg/kg every other
day
100 mg/kg every
other day
6-8 nig/kg/day
gestat Ion unt11
5 weeks of age
gestation until
5 weeks of age
gestatIon unt 11
5 weeks of age
3 weeks
10 weeks
100 days
11 weeks
50, 100 or 200 mg/kg 120 days
103 days
6 weeks exposed and
held for additional
16 months
75r90 weeks
Depressed resistance to L. monocytogenes and Vos et al.,
1- spiral Is. enhanced thymus-dependent antibody 1979b
response
Increased serum IgM and IgG, depressed resis-
tance to L. monocytogenes and T. spiralis,
enhanced thymus-dependent antibody response.
Increased liver and adrenal weights
Increased IgH and IgG responses to tetanous Vos et al.,
toxlod, delayed-type hypersensitivity reactions 1983a,b
to ovalbumin, noted accumulation of alveolar
macrophages;, no change In I. spiral Is resistance
Dose-related Increases In relative spleen. Vos et al.,
lymph nodes, liver, adrenals, thyroid, testes 1979a
and kidney weights, dose-related Increase In
serum IgH levels, no change In serum IgG
levels, dose-related pathological changes In
liver, lymph nodes and spleen
Porphyria found microscopically at S weeks and Gralla et al.,
grossly at 10 weeks using fluorescence 1977
Elevated hepatic enzymes by 1 week and Increased Llssner
urinary porphyrin and ALA levels (porphyria) as et al., 1975
early as 40 days
Decreased uroporphyrinogen decarboxylase Elder et al.,
activity and porphyria after 4 weeks 1976
Dose- and time-dependent Increase In liver and Carlson, 1977
urine porphyrins (porphyria)
Porphyria In treated females, susceptibility of Rizzardlnl and
females to porphyria may be related to estrogen Smith, 1982
levels
Porphyria (liver uroporphyrin levels peaked 7 Koss et al..
months postexposure and levels had not returned 1983
to normal by 18 months), decreased liver proto-
porphyrin and coproporphyrln levels. Inhibition
of uroporphyrinogen decarboxylase ac tlvlty
until 18 months.pos(exposure
Decline In body weights, porphyria, enlarged Smith and
livers and liver tumors Cabral, 1980
-------
o
r\j
cr>
l\J
o
Species Route
Dose
i
r\j
ro
Rat oral 75 mg/kg dtel
(diet) (4-5 mg/kg/day)
ISO rag/kg diet
(8-9.S nig/kg/day)
Rat oral 75 or ISO mg/kg diet
(diet)
Rat oral 800 mg/kg diet
(diet)
Rat oral 0.32, 1.6, B.O or
(diet) 40 mg/kg diet
oral 0.32 or 1.6 mg/kg
(dtet and dtet
nursing)
B.O mg/kg diet
40 mg/kg diet
Rat oral 10 or 20 mg/kg diet
(diet)
40 mg/kg diet
80 mg/kg diet
160 mg/kg diet
10 320 and 640 mg/kg
diet
TABLE Villi (cont.)
Dura I Ion
Effects
Reference
up to 2 years
up to 2 years
20 weeks
-130 days
gestation through
lifetime (130 weeks)
Porphyria, time-related appearance of severe Lambrecht et
hepatic and renal pathologies, after 1 year In- al.. 1983a,b
creases In hepatomas, hepatocarcInooias, bile duct
adenomas, renal adenomas and renal carcinomas
Decreased nerve conduction velocities B and
31% In 75 and 150 ppm groups, respectively;
muscles showed signs of denervation,
fibrillations and pseudomyotonta
Reduced nerve conduction.velocities, no muscle
abnormalities as observed In 2-year study
Hematologic changes at all dose levels In
males. Increases In liver and heart weights In
males at 8.0 and 40 ppm diets, no treatment-
related effects observed In bred females
Glycogen depletion In 1.6 mg/kg males; no
effects reported at 0.32°mg/kg
Suf11 'et al.
19B6
SufIt et a I.,
1986
Arnold et al.,
1985
gestation through
lifetime (130 weeks)
gestation through
1IfetIme (130 weeks)
Increase In liver pathologies
Increased mortality as pups. Increase In liver
and kidney pathologies. Increase In adrenal
pheochromocytomas In females and parathyroid
tumors In males
Fq to f( generations No effects reported
Fq to F4 generations
Fq to F4 generations
Fq to F4 generations
10 to F4 general Ions
Grant et al
19/7
Increases In liver weights and aniline
hydroxylase activity
Oecreased body weights. F3 and F4 generations had
decreased lactation Index and postnatal viability
and Increased stillbirths
Increased mortality and decreased lactation
Index starting in f| generation
20 and SOX mortality In Fy 320 and 640 mg/kg
groups. 'respec1Ively, greatly reduced fertility
Index and lltler sile and Increase In still
blrllis, viability Index zero In l|
-------
1ABLE VI11-1
(cont.)
o
fU
cr
Species
Route
Dose
DuratIon
Effects
Reference
fO
o
Rat
oral
(diet)
60. 80, 100, 1?0 or
140 mg/kg diet
Fo to f}a and fit
generations
Increased mortality In all groups at 21 days,
21-day ID50 values for pups were 100 and 140
mg/kg for F)a and F||, generations, respectively
Kltchln
el al.. 1982
Rat
oral
(diet)
0 or 80 mg/kg diet
gestation and
nursing or cross
nursed with controls
Nursing exposure produced greater effects than
did gestational exposure, effects noted were:
smaller brains, hearts, kidneys and spleens.
Increased liver weights
Hendoza
et al.. 1978
Rat
oral
(diet)
60 mg/kg diet
2 weeks pr lor to
mating to 35-36 days
after weaning
Increased porphyrin levels and decreased liver
esterase activity In dams, no changes In
gestation Indices or neonatal survival
Hendoia
et al.. 1979
Rat
oral
(gavage)
10. 20. 40. 60. 60
or 120 mg/kg
days 6-21 of gesta-
t Ion
Haternal toxicity (weight loss, tremors and
convulsions) and reduced fetal weights at 120
and 80 mg/kg maternal doses, dose-related In-
crease In Incidence of unilateral and bilateral
14th rib, sternal defects were also noted In
one experiment-
Khera, 1974
VI11-13
Mouse
oral
(diet)
2.5. 25 or 250
mg/kg diet
21 days
Oose-related Increase In liver and decrease In
prostate and seminal vesicle weights, dose-
related alterations In testosterone metabolism,
altered hepatic enzyme levels
illssalde and
Cl^rk. 1979
House
(male)
oral
(diet)
10 mg/kg diet (6.4
(mg/mouse/24 ueeks)
or 50 mg/kg diet
(35.3 mg/mouse/
24 weeks)
24 weeks
Dose-related reduction In weight gain, no tumor
pathology observed
Shlral et al.,
1978
House
(male)
oral
(diet)
167 mg/kg diet
3-6 weeks
Impairment In host resistance as measured by
Increased sensitivity to S. typhosa and P.
berqhel. and decrease In IgA levels
Loose et al.,
1978a.b
House
oral
(diet)
6. 12. 24 and 36'
mg/kg/day
101-120 weeks
*(15 weeks exposed
held until 120
weeks)
Reduced growth rate at all dose levels, short-
ened lifespan associated with tremors and con-
vulsions In 24 and 36 mg/kg/day groups, dose-
dependent Increase In liver-cell tumors In the
12. 24 and 36 mg/kg/day dose groups
Cabral et al..
1979
o
in
N
House
oral
(gavage)
100 mg/kg/day to
pregnant mice
days 7-16 of
gestation
Increased maternal livers and decreased fetal
body weights. Increased Incidence of abnormal
fetuses per litter observed
Courtney
el al.. 1976
tn
s
CO
oo
Hamster
oral
(diet)
200 or 400 mg/kg
diet
90 days
Preclrrhotlc and cirrhotic hepatic lesions,
bile-duct hyperplasias and hepatomas
Lambrecht
et al.. 1982a
-------
TABLE Wlll-l (conl.)
Species
Route
Dose
Durat ton
Effects
Reference
Hamster
oral
(diet)
4, B or 16 mg/kg/day
lifespan
Shortened lifespan In 16 mg/kg/day group. In-
crease In hepatomas at all dose levels. Increase
in liver haemangloendothelloma In males and
females and an Increase In thyroid alveolar
adenomas In males In 16 mg/kg/day group
Cabral et al.,
1977
Cats
(breeding
females)
oral
(diet)
3 or 8.7 mg/day/cat
14? days
Weight loss and Increased disease susceptibility
In bred females, dose-related decrease In litter
slie and survival of offspring, hepatomegaly In
offsprIng
Hansen et al..
1979
Hlnks
oral
(diet)
1 or 5 «g/kg diet
during gestation
until 17 weeks of
age
Dose-related Increase In offspring mortality.
Induction of hepatic NF0 enzymes In exposed
offsprIng
Rush et al.,
1983
Dog
(female)
oral
(capsule)
SO or ISO mg/kg/day
?1 days
Liver and hepatocyte enlargement. dose-Induced
electroencephalogram dysrhythmias
Sundlof
et al.. 1981
Dog
oral
(capsule)
1, 10. 100 or 1000
mg/day/dog
1 year
Increase In mortality, neutrophilia, and
anorexia In the 100 and 1000 mg dose groups,
dose-related nodular hyperplasia of gastric
lymphoid tissue In all treated animals
Gralla et al.,
1977
Monkey
(female)
oral
(gavage)
B. 3?. 64 or 1?B
mg/kg/day
60 days
Dose-related pathology In liver, kidney, ovaries
and thymus
latropoulus
et al.. 1976
Nonkey
oral
(nursIng)
7.S1-1B6 ppn milk
60 days
? of 3 Infants died as a result of exposures
Bailey el al..
1900
-------
a 10 kg child. Therefore, the longer-term HA for a 10 kg child of 0.05
mg/5, Is recomnended for use as a 10-day HA.
Derivation of Lonqer-Term Health- Advisory. The 15-week subchronlc
feeding study by Kulper-Goodman et al. (1977) is selected for the derivation
of the longer-term HA. The doses used In this study are on a mg hexachloro-
benzene/kg bw basis, which Is a preferred dose unit for HA calculations, and-
the data given In the report show dose responses with effect and no-effect
levels for the effects observed. In the"study by Arnold et al. (1985), FQ
males used as breeders for the generation had statistically significant
Increases In llver-to-body weight ratios and heart weights after exposure to
8 and 40 ppm hexachlorobenzene 1n the diet for 130 days. The study by
Arnold et al. (1985) using the FQ'males could also be proposed for calcu-
lation of a longer-term HA, but the organ weight and body weight data were
not reported to show the strength of the effects and the dose response, and
the selection of the exposure levels on a ppm dietary basis could result In
a less precise estimate of dose on a mg/kg bw basis.
Rush et al. (1983) observed a substantial reduction in the survival of
mink offspring from mothers exposed to dietary levels of 1 and 5 ppm hexa-
chlorobenzene from about 6 weeks before mating antll weaning of offspring.
Assuming a dally food consumption of 155 g and a body weight of 0.87 kg for
female minks (Bleavlns and Aulerlch, 1981), 1 and 5 ppm dose levels of hexa-
chlorobenzene would be equivalent to 0.155 and 0.775 mg/kg/day, respec-
tively. Thus, the mink Is quite sensitive to hexachlorobenzene effects on
reproduction. However, this study 1s not proposed for use in the longer-
term HA or DWEL calculations in that the mink is a species that has not been
02620
VIII-15
04-/12/91
-------
extensively Investigated as an animal model for toxicity testing. Further-
more, the gastrointestinal physiology of minks Is different from that of
humans.
In the Kulper-Goodman et al. (1977) study, groups of 70 male and 70
female Charles River (COBS) rats were fed diets providing 0, 0.5, 2.0, 8.0
or 32.0 mg/kg bw/day of hexachlorobenzene, dissolved 1n corn oil, for as-
long as 15 weeks. Female rats were found to be more susceptible to
hexachlorobenzene, as Indicated by all parameters studied, and an "apparent"
NOEL of 0.5 mg/kg/day was concluded by the authors. Increased liver
porphyrin levels In females and Increases in the size of centr1 lobular
hepatocytes along with the depletion of hepatocellular marker enzymes were
noted with higher doses. With the two highest doses, there were Increased
Hver-to-body weight ratios In males and females and Increased porphyrin
levels In the kidney and spleen. Exposure to the highest dose resulted in
decreased survival In females, splenomegaly in females. Increases In
spleen-to-body weight and kidney-to-body weight ratios in males and females,
intension tremors In males and females, ataxia in females, and decreased
body weight in males. Similar effects In liver were reported by Boger et
al. (1979) after treating groups of 36 female Wlstar rats twice weekly for
29 weeks with oral doses of 0.5, 2.0, 8.0 and 3*2.0 mg hexachlorobenzene/kg
bw in olive oil.
The longer-term HA for a 10 kg child and a 70 kg adult are calculated
using the NOEL of 0.5 mg/kg bw/day reported by Kulper-Goodman et al. ( 1977 )
as follows:
02620
VI11 -16
04/12/91
-------
For 10 kg ch11d:
(0.5 mq/kq bw/dav) (10 kcj) „
Longer-Term HA = J a—a LJ~i = 0.05 mg/i
y (100) (1 i/day)
where:
0.5 mg/kg bw/day = NOEL Identified In the* Kulper-Goodman et al.
(1977) study
10 kg = assumed weight of a child
100 = uncertainty - factor. In accordance with
'NAS/00W and Agency guidelines to account for
use of an animal study.
1 a/day = assumed water consumption by a child
For a 70 kg adult:
Longer-Term HA J°'S ^ (7° ^ = 0.175 mg/i
(100) (2 i/day)
(rounded to 0.2 mg/i)
where:
0.5 mg/kg bw/day = NOEL Identified in the Kulper-Goodman et al.
(1977) study
70 kg = assumed weight of an adult
100 - uncertainty "factor, in accordance with
NAS/0DW and Agency guidelines to account for
use of an animal study. -¦
2 i/day =. assumed water consumption of a 70 kg adult
Assessment of Lifetime Exposure and Derivation of DUEL. The deriva-
tion of the lifetime DWEL Is based on a 130-week study of Arnold et al.
(1985). This study Involved feeding male and female Sprague-Oawley rats,
the Fq generation, diets containing 0, 0.32. 1.6, 8.0 or 40 ppm of hexa-
chlorobenzene (analytical grade) for 90 days prior to mating and until 21
days after parturition (at weaning).
02620 " VI11-17 06/13/91
-------
The number of offspring (F-j generation) from these matlngs was reduced
to 50 males and 50 females per dose group at 28 days of age and fed their
respective parents' diets. Thus, the F animals were exposed to hexa-
chlorobenzene and metabolites J_n utero, from maternal nursing and from their
diets for the remainder of their lifetime (130 weeks). No hexachlorobenzene-
Induced adverse effects were reported in the 0^32 and 1.6 ppm hexachloro-
benzene groups. Indicating that these levels are NOAELs. Although
significant (p<0.05) Increases 1n" the Incidences of periportal glycogen
depletion (1.6 ppm), perlblllary lymphocytosis (0.32, 1.6 and 40 ppm), and
per 1 bl 11 ary fibrosis (0.32 and 40 ppm) were observed In the F^ male rat
groups, these effects are not being considered as hexachlorobenzene Induced
adverse effects because they were observed In a large number of F^ control
males as well. The 8.0 ppm hexachlorobenzene F^ groups were reporLed to
have an Increase (p<0.05) In hepatic centrllobular basophilic chromogenesls.
The 40 ppm hexachlorobenzene F^ groups were reported with Increases
(p<0-05) In pup mortality, hepatic centrllobular basophilic chromogenesls,
severe chronic nephrosis In males, adrenal pheochromocytomas -in females and
parathyroid tumors In males.
A lifetime DWEL for hexachlorobenzene Is calculated using a 100-fold
uncertainty factor, which represents two 10-fold factors to account for both
the Intra- and Interspecies variability to the toxicity of the chemical when
specific data are lacking. It Is difficult to estimate lifetime doses on a
mg hexachlorobenzene/kg bw basis in this study because of the Initial
exposure of the animals to hexachlorobenzene and Its metabolites j_n utero
and during lactation. However, In an attempt to estimate the lifetime
hexachlorobenzene doses on a mg/kg bw basis, the 1.6 mg/kg hexachlorobenzene
02620
VI11-18
06/13/91
-------
diet level. Interpreted from this study as the highest NOAEL level, was con-
verted to a dally Intake dose of 0.08 mg/kg bw/day by averaging the dosage
data provided by Arnold (1984). Using 0.08 mg/kg bw/day as a NOAEL from the
Arnold et al. (1985) study the RfD and DUEL for a 70 kg adult Is calculated
as follows:
Step 1 - RfD Derivation
where:
0.00 mg/kg bw/day = NOAEL Identified In the Arnold et al. (1985)
study
RfQ _ (0.08 mq/kq bw/day)
= 0.0008 mg/kg bw/day
100
= uncertainty factor. In accordance with
NAS/0DW and Agency guidelines to account for
use of an animal study.
Step 2 - DWEL Derivation
DWEL (Q.QQQ8 mq/kq bw/day) (70 kg)
(2 i/day)
= 0.028 mg/l
(rounded to 0.03 mg/a)
where:
0.0008 mg/kg bw/day = RfD
70 kg
= assumed weight of an adult
2 i/day
= assumed water consumption by an adult
02620
VI11-19
06/13/91
-------
Carcinogenic Effects
This quantitative section deals with estimation of the unit risk for
hexachlorobenzene as a potential carcinogen In water, and with the potency
of hexachlorobenzene relative to other carcinogens that have been evaluated
by the U.S. EPA Human Health Assessment Group (HHAG). The unit risk for a
water pollutant Is defined as the lifetime cancer risk to humans from dally,
exposure to a concentration of 1 pg/l In water by Ingestion. The U.S.
EPA HHAG has prepared the rationale and the calculation for the unit risk
estimate presented herein" for the U.S. EPA (1905) Health Assessment Document
for Chlorinated Benzenes. The HHAG has also classified hexachlorobenzene as
a B2, probable human carcinogen.
The unit risk estimate for hexachlorobenzene represents an extrapolation
below the dose range of experimental data. There 1s currently no solid
scientific basis for any mathematical extrapolation model that relates
exposure to. cancer risk at the extremely low concentrations, including the
unit concentration given above, that must be considered when evaluating
environmental hazards. For practltal reasons, the correspondingly low
levels of risk cannot be measured directly either by animal experiments or
by epidemiologic study. Therefore, low dose extrapolation must be based on
current understanding of the mechanisms of carcinogenesis. At the present
time the dominant view of the carcinogenic process Involves the concept that
most cancer-causing agents also cause Irreversible damage to DNA. This
position Is based In part on the fact that a very large proportion of agents
that cause cancer are also mutagenic. There 1s reason to expect that the
quantal response that Is characteristic of mutagenesis is associated with a
linear (at low doses) non-threshold dose-response relationship. Indeed,
there 1s substantial evidence from mutagenicity studies with both Ionizing
02620 VI11-20 07/11/91
-------
radiation and a wide variety of chemicals that this type of dose-response
model 1is the appropriate one to use. This Is particularly true at the lower
end of the dose-response curve; at high doses there can be an upward curva-
ture, probably reflecting the effects of multistage processes on the muta-
genic response. The linear non-threshold dose-response relationship Is also
consistent with the relatively few epidemiologic'studies of cancer responses,
to specific agents that contain enough Information to make the evaluation
possible (e.g., rad1at1on-1nduced leukemia, breast and thyroid cancer, sk 1 n
cancer Induced by arsenic In drinking water, liver cancer Induced by
anatoxins In the d1§t). Some supporting evidence also exists from animal
experiments (e.g., the Initiation stage of the two-stage carcinogenesis
model 1n rat liver and mouse skin).
Because Us scientific basis, although limited, Is the best of any of
the current mathematical extrapolation models, the nonthreshold model, which
is linear at low doses, has been adopted as the primary basis for risk
extrapolation to low levels of the dose-response relationship. The risk
estimates made with such a model should be regarded as conservative, repre-
senting the most plausible upper limit- for the risk (i.e., the true risk is
not likely to be higher than the estimate, but It.could be lower).
For several reasons, the unit risk estimate based on animal bioassays is
only an approximate indication of the absolute risk in populations exposed
to known carcinogen concentrations. First, there are Important species
differences in uptake, metabolism and organ distribution of carcinogens, as
well as species differences in target site susceptibility, Immunologic'
responses, hormone function, dietary factors and disease. Second, the con-
02620
VI11-21
06/13/91
-------
cept of equivalent doses for humans compared with animals on a mg/surface
area basis Is virtually without experimental verification in regard to
carcinogenic response. Third and finally, human populations are variable
with respect to genetic constitution and diet, living environment, activity
patterns and other cultural Factors.
The unit risk estimate can give a rough Indication of the relative
potency of a given agent as compared with other carcinogens. Such estimates
are, of course, more reliable when th'e comparisons are based on studies 1n
which the*test species, strain, sex and routes of exposure are similar.
The quantitative aspect of carcinogen risk assessment 1s addressed here-
because of Its possible value In the regulatory decision-making process,
e.g.., In setting regulatory priorities, evaluating the adequacy of technol-
ogy-based controls, etc. However, the imprecision of presently available
technology for estimating cancer risks to humans at low levels of exposure
should be recognized. At best, the linear extrapolation model used here
provides a rough but plausible estimate of the upper limit of risk -- that
is, with this model It is not likely that the true risk would be much more
than the estimated risk, but 1t could be considerably .lower. The risk esti-
mates presented in subsequent sections should not be regarded, therefore, as
accurate representations of the true cancer risks even when the exposures
Involved are accurately defined. The estimates presented may, however, be
factored into regulatory decisions to the extent that the concept of
upper-risk limits Is found to be useful.
02620
VI11-22
06/13/91
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Procedures for the Determination of Unit Risk'.
Low Dose Extrapolation Model — The mathematical formulation chosen to
describe the linear non-threshold dose-response relationship at low doses Is
the linearized multistage model (Crump and Watson, 1979). This model
employs enough arbitrary constants to be able to fit almost any monotonlc-
ally Increasing dose-response data, and It Incorporates a procedure for
estimating the largest possible linear slope (in the 95% confidence limit
sense) at low extrapolated doses that Is consistent with the data at all
dose levels of the experiment.
Let P(d) represent the lifetime risk (probability) of cancer at dose d.
The multistage model has the form:
P(d) = 1 - exp [-(qQ + q-jd +¦ q2d* + ...+ qkdk)]
where
Equivalently,
where
q1 > Gt and 1 = 0# 1, 2, k
Pt(d) = 1 - exp [-(q-|d * q2ds * ... qkdk)]
Pt(d) =
L 1 - P(0)
is the extra risk over background rate at dose d.
The point estimate of the coefficients q , 1 = 0, lf 2 k, and
consequently, the extra risk function, P^(d), at any given dose d, is
calculated by maximizing the likelihood function of the data.
02620
VI11-23
06/13/91
-------
The point estimate and the 95% upper confidence limit of the extra risk,
P^(d), are calculated by using the GL0BAL79 computer program developed by
¦Crump and Watson (1979). At low dosest upper 95% confidence limits on the
extra risk and lower 95% confidence limits on the dose producing a given
risk are determined from a 95% upper confidence limit, q^*, on parameter
. Whenever q^ > 0, at low doses the extra risk P^(d) has approxi-
mately the form P (d) = q^* x d. Therefore, q^* x d Is a 95% upper
confidence limit on the extra risk and R/q^* Is a 95% lower confidence
limit on the dose, producing an extra risk of R. Let Lg be the maximum
value of the log-likelihood function. The upper-limit q^* 1s calculated
by Increasing q^ to a value q^* such that when the log-Hkelihood is
remaxlmlzed subject to this fixed value q^* for the linear coefficient,,
the resulting maximum value of the log-likelihood satisfies the equation
2 (Lq - L1) - 2.70554
where 2.70554 is the cumulative 90% point of the chl-square distribution
with one degree of freedom, which corresponds to a 95% upper-limit (one-
sided). This approach of computing the upper confidence limit for the extra
risk Pt(d) is an Improvement on previous models. The upper confidence
limit for the extra risk calculated at low doses is always linear. This is
conceptually consistent with the linear non-threshold concept discussed
earlier. The slope, q Is taken as an upper-bound of the potency of the
chemical in Inducing cancer at low doses. [In the section calculating the
risk estimates, P {d) will be abbreviated as P.]
In fitting the dose-response model, the number of terms in the poly-
nomial Is chosen equal to (h-lj, where h Is the number of dose groups In the
experiment, including the control group.
02620
VI11-24
06/13/91
-------
Whenever the multistage model does not fit the data sufficiently, data
at the highest dose 1s deleted and the model Is refitted to the rest of the
data. This is continued until an acceptable fit to the data 1s obtained.
To determine whether or not a fit Is acceptable, the ch1-square statistic:
' ^ N1p1 d-Pl) "
1s calculated where Is the number of animals In the 1^ dose group,
X^ Is the number of animals 1n the 1th dose group with a tumor response,
1s the probability of a response In the 1th dose group estimated by
fitting the multistage model to the data, and h Is the number of remaining
groups. The fit Is determined to be unacceptable whenever X2 Is larger
than the cumulative 99% point of .the chl-square distribution with f degrees
of freedom, where f equals the number of dose groups minus the number of
non-zero multistage coefficients.
Selection of Data— For some chemicals, several studies in different
animal species, strains and sexes, each run at several doses and different
routes of exposure, are available. A choice must be made as to which of the
data sets from several studies to use In the model. It may also be appro-
priate to correct for metabolism differences between species and for absorp-
tion factors via different routes of administration. The procedures used In
evaluating these data are consistent with the approach of making a maximum-
Hkely risk estimate. They are as follows:
1. The tumor Incidence data are separated according to organ sites or tumor
types. The set of data (I.e., dose and tumor incidence) used in the
model 1s the set where the Incidence 1s statistically significantly
02620
VI11-25
06/13/91
-------
higher than the control for at least one test dose level and/or where
the tumor Incidence rate shows a statistically significant trend with
respect to dose level. The data set that gives the highest estimate of
the lifetime carcinogenic risk, Is selected in most cases.
However, efforts are made to exclude data sets that produce spuriously
high risk estimates because of a small number of animals. That Is, 1f
two sets of data show a similar dose-response relationship, and one has
a very small sample size, the set of data having the larger sample size
1s selected for calculating the carcinogenic potency.
2. If there are two or more data sets of comparable size that are Identical
with respect to species, strain, sex and tumor sites, the geometric mean
of q^*r estimated from each of these data sets, 1s used for risk
assessment. The geometric mean of numbers A^ 1s
defined as
, * a a , 1 /m
(A1 x A2 x ... x Affl)
3. If two or more significant tumor sites are observed in the same study,
and If the data are available, the number of animals with at least one
of the specific tumor sites under consideration is used as incidence
data In the model.
Calculation of Human Equivalent Dosages -- Following the suggestion of
Mantel and Schneiderman (1975), it Is assumed that mg/surface area/day Is an
equivalent dose between species. Since, to a close approximation, the
surface area Is proportional to the two-thirds power of the weight, the
2/3
exposure in mg/day of the weight 1s also considered to be equivalent
02620
VII1-26
06/13/91
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exposure. In an animal experiment, this equivalent dose Is computed In the
following manner.
Let
Le = duration of experiment
le = duration of exposure
m = average dose per day In mg during administration of the agent
(I.e., during le), and
W = average weight of the experimental animal
Then, the lifetime exposure 1s
la x m
d = —
Le x W2/3
Oral. Often exposures are not given 1n units of mg/day, and It
becomes necessary to convert the given exposures Into mg/day. Similarly, In
drinking water studies, exposure Is expressed as ppm In the water. For
example, in most feeding studies exposure 1s given 1n terms of ppm in the
diet. In these cases, the exposure In mg/day Is
m = ppm-x F x r
where ppm is parts per million of the carcinogenic agent in the diet- or
water, F is the weight of the Food or water consumed per day In kg, and r Is
the absorption fraction. In the absence of any data to the contrary, r is
assumed to be equal to one. For a uniform diet, the weight of the food
consumed Is proportional to the calories required, which in turn Is propor-
tional to the surface area, or two-thirds power of the weight. Water
demands are also assumed to be proportional to the surface area, so that
02620
VIII -27
06/13/91
-------
2/3
m a ppm x W x r
or
m
a ppfl)
rW2/3
As a result, ppm In the diet or water Is often -assumed to be an equivalent
exposure between species. However, this Is not justified In dose extrapola-
tion of laboratory animals to humans since the ratio of calories to food
weight Is very different- In the diet of man as compared with laboratory
animals, primarily due to differences In the moisture content of the foods
eaten. For the same reason, the amount of drinking water required by each
species also differs. It Is, therefore, necessary to use an empirically-
derived factor, f = F/Wf which 1s the fraction of an organism's body weight
that Is consumed per day as food, expressed as follows:
Fraction of Body Weight Consumed as
Species W ffood fwater
Man 70 0.02B 0.029
Rats 0.35 0.05 0.078
Mice 0.03 0.13 0.17
»
Thus, when the exposure is given as a certain dietary or water concentration
2/3
\n ppm, the exposure 1n mg/W 1s
_nL_ = mUL± = ppm x f x W __ ppm x f x ul/3
rw2/3 w2/3 w2/3
When exposure Is given In terms of mg/kg/day = m/Wr = s, the conversion Is
simply
02620
VIII-28
06/13/91
-------
—— = s x wl/3.
rW2/3
Calculation of the United Risk from Animal Studies -- The risk associ-
2/3
ated with d mg/kg /day Is obtained from GL0BAL79 and, for most cases of
Interest to risk assessment, can be adequately approximated by P(d) = 1 -
exp (-q *d). A "unit risk" 1n units X Is simply the risk corresponding to
an exposure of X = 1. This value Is estimated simply by finding the number
2/3
of mg/kg /day that corresponds to one unit of X, and substituting this
value Into the above relationship. Note that an equivalent method of calcu-
lating unit risk would be to use mg/kg for the animal exposures, and then to
increase the polynomial coefficient by an amount
(Wh/wa)J/3 J = 1 • 2 k-
and to use mg/kg equivalents for the unit risk values.
Adjustments for Less Than Lifespan Duration of Experiment. If the
duration of experiment L^ is less than the natural lifespan of the test
animal L, the slope q^*, or more generally the exponent g(d), Is increased
by multiplying a factor (L/L^)3. We assume that If the average dose d
Is continued, the age-specific rate of cancer will continue to Increase as a
constant function of the background rate. The age-specific rates for humans
increase at least by the third power of the age and often by a considerably
higher power, as demonstrated by Doll (1971). Thus, It Is expected that the
cumulative tumor rate would Increase by at least the third power of age.
Using this fact, it Is assumed that the slope q *, or more generally the
exponent g(d), would also increase by at least the third power of age. As a
02620
VI11-29
06/13/91
-------
result, 1f the slope q^* [or g(d)] Is calculated at age L , It Is
expected that If the experiment had been continued for the full lifespan L
at the given average exposure, the slope [or g(d)] would have been
Increased by at least (L/L0)3.
This adjustment Is conceptually consistent with the proportional hazard
model proposed by Cox (1972) and the time-to-tumor model considered by
Daffer et al. (1980), where the probability of cancer by age t and at dose d
1s given by
P(d.t) = 1 - exp [-f(t) x g(d)].
Unit Risk Estimates --
Data Available for Potency Calculation. Hexachlorobenzene has been
shown to Induce tumors in hamsters, mice and rats. The primary target organ
appears to be the liver In all three of these species. Liver haemangloendo-
thellomas in hamsters and hepatocellular carcinomas in rats were signifi-
cantly increased In the hexachlorobenzene-treated animals. The potency
estimate calculated on the basis of hepatocellular carcinomas in female rats
Is used to derive unit risk estimates for hexachlorobenzene in water. This
particular tumor site is selected for calculating unit risks because it is a
malignant tumor in the primary target organ and results in the highest
potency estimate.
Increased Incidences of thyroid, parathyroid, adrenal and kidney tumors
were also observed among these species. Fourteen data sets showing signifi-
cant tumor Incidences have been used herein to calculate the carcinogenic
02620
VI11-30
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-------
potency of hexachlorobenzene. These calculations provide a range of
estimates that, In part, reflect the uncertainties Inherent In the risk
assessment process. Tables VIII-2 through VIII-5 summarize the data used to
calculate the potency of hexachlorobenzene.
Choice of Low-Dose Extrapolation — In addition to the multistage
model currently used by the U.S. EPA HHAG for low-dose extrapolation, HHAG
also uses three other models, the probit, the Welbull and the one-hit
models, to estimate the risks from exposure to hexachlorobenzene using the
data for hepatocellular carcinoma In female rats. These models cover almost
the entire spectrum of risk estimates that could be generated from the
existing mathematical extrapolation models. These models are general ly
statistical In character, and are not derived from biologic arguments,
except for the multistage model which has been used to support the somatic
mutation hypothesis of carcinogenesis (Armltage and Doll, 1954; WhHtemore,
1978; Whittemore and Keller, 1970). The main differences among these models
is the rate at which the response function, P(d)p approaches zero or P(0) as
dose, d, decreases. For Instance, the prob1t model would usually predict a
smaller risk at low doses than the multistage model because of the
difference of the decreasing rate In the low-dose region. However, it
should be noted that one could always artificially give the multistage model
the same (or even greater) rate of decrease as the problt model by making
some dose transformation and/or by assuming that some of the parameters in
the multistage model are zero. This, of course, is not reasonable without
knowing, a priori, what the carcinogenic process for the agent 1s. Although
the multistage model appears to be the most reasonable or at least the most
general model to use, the point estimate generated from this model Is of
02620
VI11-31
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TABLE VI11-2
Tumor Incidences In Hale and Female Hamsters Given
Hexachlorobenzene In D1eta
Thyroid Hepatoma Liver Hemangioendothelioma
Doseb
(mg/kg/day)
Male
Hale
Female
Hale
Female
0
0/40
0/40
0/30
0/40
0/39
4
0/30
14/30
14/30
1/30
0/30
9
1/30
26/30
17/30
6/30
2/30
16
8/57
49/57
51/60
20/57
' 7/60
aSource: Cabral et al., 1977
bIf mg/surface area/day Is assumed to be equivalent between humans and
animals, the dose In mg/kg/day Is multiplied by a factor (0.1/70)^3,
where 70 and 0.1 kg are, respectively, the average body weights of humans
and hamsters.
02620
VIII-32
04/13/88
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TABLE VIII-3
Incidence of Liver Cell Tumors 1n Male and Female Swiss Mice
Given Hexachlorobenzene Diet3
Dose*3
Malec
Femalec
(mg/kg/day)
0
0/47
0/49
6
0/30
0/30
12
3/12
3/12
24
7/29
14/26
aSource: Cabral et al.t 1979
&If the equivalent dose between humans ,and mice Is assumed to be on the
basis of bodv surface area, the dose In mg/kg/day Is multiplied by a factor
(0.035/70)^3( where 0.035 kg and 70 kg are, respectively, the average
body weights of mice and humans.
cThe number of animals that survived at the first observed liver cell
tumor is used as the denominator.
02620
VII1-33
04/13/88
-------
TABLE VIII-4
Liver and Kidney Tumor Incidence Rates In Male and female
Sprague-Dawley Rats Given Hexachlorobenzene 1n Diet3
Sex
Doseb
Hepatocellular
Hepatoma
Renal Cell
(mg/kg/day)
Carcinoma
Adenoma
Male
0
0/54
0/54
7/54
4.24
3/52
10/52
41/52
8.48
4/56
11/56
42/56
Female
0
0/52
0/52
1/52
4.67
36/56
26/56
7/56
9.34
48/55
35/55
15/54
aSource: Lambrecht et al., 1983a,b. Additional data from this study on
adrenal pheochromocytoma has recently become available (Peters et al., 1983)
but was not available when quantitative estimates were made.
bThe dosages are calculated by the Investigator based on the average food
consumption of 22.6 g/rat/day and an average body weight of 400 g for'male
rats. For female rats, the average food consumption 1s 16.5 g/rat/day and
the average body weight Is 265 g. If the equivalent dose between humans and
mice Is assumed to be on the basis of body surface area, the dose presented
In the table Is multiplied by a factor {Wa770)1/3t where Wa Is the
body weight of ma 1e or female rats, and 70 kg Is the human body weight.
02620
VI11-34
04/13/88
-------
TABLE VIII-5
Incidence Rate of Adrenal Pheochromocytoma In Female Sprague-Dawley
Rats (F] generation) 1n a 2-Generatlon Feeding Study
Dosea -
(mg/kg/day)
Incidence Rateb
(used In calculations)
Revised Incidence Ratec
0
2/48
2/49
0.02
4/50
4/49
0.08 .
. 4/50
0.40
5/49
4/49
1.90
17/49
aIf the equivalent dose between humans and rats is assumed to be on the
basis of body surface, the dose In this table Is multiplied by a factor
(0.35/70)1 , where 70 kg and 0.35 kg are, respectively, assumed to be
the body weight of humans and rats.
^Source: Arnold et al.t 1985
cSource: Arnold, 1984. The amended 1984 data" were not available when
quantitative estimates were made.
02620
VI11-35
04/13/88
-------
limited value because It does not help to determine the shape of the
dose-response curve beyond experimental exposure levels. Furthermore, point
estimates at low doses extrapolated beyond experimental doses could be
extremely unstable and could differ drastically, depending on the amount of
the lowest experimental dose. Since upper-bound estimates from the
multistage model at low doses are relatively more stable than point
estimates. It is suggested that the upper-bound estimate for the risk [or
the lower-bound estimate for the dose) be used in evaluating the
carcinogenic potency of a suspect carcinogen. The upper-bound estimate can
be taken as a plausible estimate 1f the true dose-response curve Is actually
linear at low doses. The upper-bound estimate means that the risks are not
likely to be higher, but could be lower 1f the compound has a concave upward
dose-response curve or a threshold at low doses. Another reason one can, at
best, obtain an upper-bound estimate of the risk when animal data are used
Is that the estimated risk is a probability conditional to the assumption
that an animal carcinogen Is also a human carcinogen. Therefore, in
reality, the actual risk could range from a value near zero to an
upper-bound estimate.
Quantification of Carcinogenic Effects
Fourteen sets of tumor Incidences which show significant Increases (see
Tables VII1-2 through V111-5) are used herein to calculate the carcinogenic
potency of hexachlorobenzene. Since preparing these calculations additional
data from the Lambrecht et al. (1983a,b) study (adrenal pheochromocytoma)
and from the Arnold et al. (1985 ) study (neoplastic liver nodules) have
become available. Quantitative estimates have not been made using this
data. Using the multistage model for low-dose extrapolation, as shown 1n
02620
VIII-36
06/13/91
-------
Table VIII-6, the potency estimates calculated on the basis of these data
sets are approximately within an order of magnitude from each other, with
the exception of the thyroid tumor. These potencies provide a range of
estimates that reflects the uncertainties stemming from the differences In
species, tumor sites, solvent vehicles and composition of diet. The range
does not reflect uncertainty resulting from the use of different
extrapolation models.
To calculate the unit risks of hexachlorobenzene in water, HHA6 used an
estimate of carcinogenic potency based upon the data for hepatocellular
carcinoma 1n female rats, which Is the highest potency among the carcinoma
responses. This particular data set was preferred over the hepatoma only
response. A combining of hepatomas and carcinomas was not possible because
of a lack of Individual animal data.
Risk Associated with 1 uq/9. of Hexachlorobenzene in Drinking
Water — Under the assumption that daily water consumption for a 70 kg.
person is 2 i, the hexachlorobenzene Intake In terms of mg/kg/day is
d = 2 I x 1 yg/i x 10"3 mg/l/70 kg = 2.86 x 10~5 mg/kg/day.
{rounded to 2.9xlQ~5 mg/kg/day)
Therefore, the risk from drinking water containing 1 wg/i of hexachloro-
benzene is estimated to be
P = 1.7 x 2.86 x 1CT5 = 4.9 x 10~5.
02620
VI11-37
06/13/91
-------
o
ro
CT*
O TABLE VI11-6
The Carcinogenic Potency3 of Hexachlorobenzene, Calculated on the Basis of 14 Data Sets,b
Using the Linearized Multistage Model
Dose Is Assumed to be
Equivalent on the Basis of
Study Oate Base Reference
Body Uelght Surface Area
Hamster
Thyroid (male)
9.3
X
10"«
8.3
X
10"a
Cabral
Hepatoma:
et al., 1977
Male
1.9
X
10"*
1.7
f ema1e
1.5
X
10*
1.3
Hemangioendothelioma:
Male
3.2
X
10"»
2.8
X
10*
Female
1.1
X
10"'
1.0
X
10*
Mice
Liver cell:
Cabral
Male
1.7
X
10"a
2.1
X
10"*
et al.. 1979
Female
1.4
X
10"a
1.8
X
10*
O
CT
N
LO
lO
-------
o
\)
(7>
oj
O
TABLE VI11-6 (cont.)
Dose Is Assumed to be
I
CO
(D
Study
Date Base
Body Weight
Surface Area
Reference .
Rats
Renal cell:
.
Lambrecht et
Hale
2.5
X
10"»
1.4
al., 1983a,b
Female
4.2
X
10~a
2.6
x 10"*
Hepatocellular carcinoma:
Hale
1.8
X
10"a
1.0
x 10~*
female
2.7
X
10"*
1.7
Hepatoma:
Hale
4.7
X
10"a
2.6
x lO"1
Female
1.5
X
10~*
9.0
x 10~l
Rats
Adrenal
Arnold
2-generatlon
Pheochromocytoma
2.8
X
10"1
1.6
et al.. 1985
study
(female)
O
cr
CO
\
10
aqj* (mg/kg/day) 1 Is the 95% upper confidence limit of the linear component In the multistage model.
bS1nce preparing these calculations, additional data from Lambrecht et al. (1983a,b) study (adrenal
pheochromocytoma) and from Arnold el al. (1985) study (neoplastic liver nodules) have become available.
These data have not been evaluated.
-------
This calculation uses the carcinogenic potency q-j*- 1.7/(mg/kg/day),
based on the data on hepatocellular carcinomas In female rats, assuming that
dose per surface area Is equivalent between rats and humans. If the equiva-
lent dose 1s assumed to be on the basis of body weight, the unit risk, P,
would be reduced to 7.6x10"*.
Summary of Quantitative Estimation. Data on hepatocellular carcinomas
In female rats after oral Ingestion have been used to estimate the carcino-
genic potency of hexachlorobenzene and the risks associated with one unit of
the compound In drinking water. The upper bound cancer risks associated
with 1 ug/i of hexachlorobenzene In drinking water is estimated to be
4.9x10"3. Accordingly, upper bound cancer risks of 10"*, 10~5 and
10'4 would be associated with 0.02, 0.2 and 2 vq/l, respectively, of
hexachlorobenzene 1n drinking water. This estimate Is calculated on the
basis of the assumption that dose per surface area Is equivalent among
species. If the dose 1s assumed to be equivalent on the basis of body
weight, the corresponding risk would be reduced approximately by a factor of
6. The carcinogenic potencies of hexachlorobenzene are also estimated on
the basis of 13 other data sets, encompassing different tumor sites and
animal species. Except for the case of thyroid .tumors, these potency esti-
mates differ from each other within a single order of magnitude. The range
of the estimates reflects the uncertainties due to differences in species,
tumor sites, solvent vehicles, composition of diet, etc.
Existing Guidelines. Recommendations and Standards
Occupational. Workplace standards have not been established in the
United States. The USSR has established a TLV of 0.08 ppm (0.9 mg/m3)
02620
V.I 11-40
06/13/91
-------
(Verschueren, 1977). In 1978, NIOSH classified, hexachlorobenzene (HCB) as a
Group II pesticide and recommended criteria for standards for occupations In
pesticide manufacturing and formulating. These standards rely on engineer-
ing controls, work practices and medical surveillance programs, rather than
workplace air limits, to protect workers from the adverse effects of pesti-
cide exposure In manufacturing and formulating. NIOSH specifically chose
not to establish sclent IfIcally valid environmental (workplace air) 11m1ts
for pesticides (except those- already promulgated), because exposure via
other routes, especially -dermal, had proven to be of critical Importance for
many pesticides and betause NIOSH believed that "Immediate action" was
needed to protect workers in pesticide manufacturing and formulating plants
(NIOSH, 1978).
Food. USDA regulates the use of hexachlorobenzene as a seed treatment
for the control of wheat bunt (smut) under the Federal Seed Act (7 CFR 201).
The U.S. EPA (1991) verified a reference dose (RfO) In May 1988 for
hexachlorobenzene. The RfO Is BE -4 mg/kg/day. A carcinogenicity reference
dose has been verified through CRAVE and this value Is a slope factor of 1.6
per mg/kg/day.
Water. The U.S. EPA (1980), in an Ambient Water Quality Criteria
Document for Chlorinated Benzenes, determined that hexachlorobenzene is a
j
suspected human carcinogen and since there is no recognized safe concentra-
tion for a human carcinogen, the recommended concentration of hexachloro-
benzene in water for maximum protection of human health 1s zero. However, a
zero level was thought to be unfeasible In some cases; therefore, water
02620
VI11-41
07/11/91
-------
levels that may result in Incremental Increases In cancer risk of lifetime
exposure were estimated at 10"5 (one additional case of cancer for every
100,000 people exposed), 10~6 and 10'7. The corresponding recommended
criteria were 7.2, 0.72 and 0.072 ng/i, respect!vely,. assuming humans
consume 2 l of water and 6.5 g of fish and shellfish per day. The
criteria were calculated by using the hepatoma data on male hamsters in the.
Cabral et al. (1977) carcinogenicity study and the multistage model.
Using the tumor data "from the Cabral et al. (1979) carcinogenicity study
with Swiss rrtlce and the multistage model, the NAS (1983) averaged cancer
risks based on the male and female mouse data to obtain a 95% upper limit of
cancer risk of 1.85x10"* with lifetime daily consumption of 1 l of water
containing 1 yg of hexachlorobenzene. Earlier, the NAS (1980) used this
method and the tumor data in the Cabral et al. (1977) carcinogenicity study
with male and female hamsters to calculate a 95% upper limit of cancer risk
of 2.9xl0~5. The NAS (1980) also calculated a 7-day suggested no adverse
response level (SNARL) of 0.03 mg/i for hexachlorobenzene.
Special Groups at Risk
Infants and children (4-16 years of age) appear to be the most suscep-
tible to exposure to hexachlorobenzene. This susceptibility was observed
after the accidental Ingestion of hexachlorobenzene-contaminated grain
occurred in Turkey during 1955-1959 (Peters et al., 1982). A distinct
disease described as "Pink Sore", which reached an epidemic scale, was
observed 1n infants under 1 year of age. These infants' mothers had con-
sumed the hexachlorobenzene-treated grain during gestation and/or lactation.
This exposure has also been associated with the observed 95% 1-year-old
02620
VI11-42
06/13/91
-------
infant mortality and the high Incidence of stillbirths. During the same
Turkish accident-, children (4-16 years of age) were found to be more
susceptible to porphyria cutanea tarda .than the adults.
There are strong Indications that adult females may be more susceptible
to hexachlorobenzene-lnduced porphyria than adult males, which 1s the case.
In the rodent studies (Rlzzardlnl and Smith, 1982). According to these
researchers, this Increased susceptibility In adult females is related to
their estrogen levels and" faster metabolism of hexachlorobenzene.
Summary
Health advisories based on noncarclnogenlc toxicity data and the
carcinogenic risk assessment are given In Table VIII-7. Available data are
concluded to be Insufficient for calculation of 1-day and 10-day HAs, The
longer-term HA( of 0.2 mg/l for a 70 kg adult and 0.05 mg/i for a 10 kg
child, which 1s also being proposed as the 1-day and 10-day HA for 10 kg
child, Is based on the study by Kulper-Goodman et al. (1977) In which male
.and female Charles River rats were fed hexachlorobenzene In the diet for as
long as 15 weeks. An RfD of 0.0008 mg/kg bw/day and a DWEL of 0.03 mg/i
for a 70 kg adult Is based on noncarclnogenlc .effects In male and female
Sprague-Dawley rats exposed to hexachlorobenzene J_n utero. during lactation,
and In the diet for the remainder of their lifetimes. However, there Is
sufficient evidence for the carcinogenicity of hexachlorobenzene In
experimental animals. Based on a we1ght-of-ev1dence classification
hexachlorobenzene 1s rated as a B2, probable human corclnogen. By using the
95% upper limit with the multistage model and the hepatocellular carcinoma
data In female rats from the lambrecht et al. (1983a,b) hexachlorobenzene
02620
VIII -43
07/11/91
-------
carcinogenicity study, lifetime exposures to hexachlorobenzene In drinking
water at levels of 2x10"5 f 2xl0~4 and 2xl0~3 mg/2. are associated
with 10*6, 10"* and 10"4 cancer risks, respectively.
02620
VIII-44
07/11/9*1
-------
o
l\J
cr
ro
o
TABLE VIII-7
Summary of the Data for Hexachlorobenzene Used to Derive HAs and DUEL
Health
Advisory
Specles/Route
Oose Duration
(mg/kg bw/day)
Basts
Uncertainty
Factors
Value'
(rag/ft|
Reference
1-Day and
10-Day
Longer-tern HA rat/oral
DUEL
rat/oral
Cancer unit rat/oral
risk estimate
0.5
0.08
"linearized*
multistage
model
Insufficient data
Insufficient data
IS weeks NOAEL, higher doses cause 100
an Increase In liver, kidney
and spleen porphyrin levels
and centrtlobular liver
lesions
130 weeks NOAEL. higher doses cause 100
an Increase In liver and
kidney lesions
lifetime Hepatocellular carcinomas NA
0.05b
0.2C
0.03c
Kutper-Goodman
et al.. 1977
Arnold et al..
1965
2.0xl0~lC*d Lambrecht et
al., 1963a,b
'Hexachlorobenzene water solubility Is referenced as 0.005 mg/t 0 25*C
bFor a 10 kg chlId
cfor a 70 kg adult
duater concentration associated with an upper level excess lifetime cancer risk of 10~*. Values of 2xl0~4 and 2xl0~* mg/i are asso-
ciated with upper level excess lifetime cancer risks of 10~* and 10~*, respectively.
NA * Not applicable
-------
IX. REFERENCES
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chlorlne pesticide residues In human fat In the United Kingdom, 1976-7. Br.
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Albro, P.W. and R. Thomas. 1974. Intestinal absorption of hexachloroben-
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Arlyoshl, T., M. Arakakl, K. Ideguchl and Y. Ishlzuka. 1973". Relationship
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»
Arnold, D.L. 1984. Personal communication to Murlal M. Llppman, EHNACO,
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02630
IX-1
04/12/91
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Bailey, J., V. Knauf, W. Mueller and W. Hobson. 1980. Transfer of hexa-
chlorobenzene and polychlorlnated blphenyls to nursing Infant rhesus mon-
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B1111, S.C., G. Koss and L.C. San Martin de Vlale. 1986b. Ability of
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J.R.P. Cabral, Ed. IARC Scl. Publ. No. 77, Lyon, France.
Bleavlns, M.R. and R.J. Aulerlch. 1981. Feed consumption and food passage
time In mink (Mustela vlson) and European ferrets (Mustela putorlus furo).
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i
Bleavins, M.R., W.J. Breslln, R.J. Aulerlch and R.K. Ringer. 1982. Excre-
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Blekkenhorst, G.H., R.S. day and L. Eales. 1980. The effect of bleeding
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02630
IX-2
09/15/88
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BSger, A., G. Koss, W. Koransky, R. Naumann and H. Frenzel. 1979. Rat
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