Health Advisory on Hexachloroethane
Authors:
Loretta Gordon, M.S.
William R. Hartley, Sc.D.
Welford C. Roberts, Ph.D.
Project Officer:
Krishan Khanna, Ph.D.
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC 20460
January 1991

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HEXACHLOROETHANE
Health Advisory
Office of Drinking tfater
U.S. Environmental Protection Agency
Washington, DC 20460

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PREFACE
This report was prepared in accordance with che Memorandum of Understanding
between the Department of the Army, Deputy for Environment Safety and Occupa-
tional Health (QASA(I&L)), and the U.S. Environmental Protection Agencv (EPA),
Office of Drinking Vater (ODW), Criteria and Standards Division, for che
purpose of developing drinking water Health Advisories (HAs) for selected
environmental contaminants, as requested by che Army.
Health Advisories provide specific advice on the levels of contaminants in
drinking water at which adverse health effects would not be anticipated and
which include a margin of safety so as to protect the most sensitive members
of the population at risk. A Health Advisory provides health effects guide-
lines, analytical methods and recommends treatment techniques on a case-by-
case basis. These advisories are normally prepared for One-day, 10-day,
Longer-term and Lifetime exposure periods where available toxicological data
permit. These advisories do not condone the presence of contaminants in
drinking water; nor are they legally enforceable standards. They are not
issued as official regulations and they may or may not lead to the issuance of
national standards or Maximum Contaminant Levels (MCLs).
This report is che product of the foregoing process. Available toxicological
data, as provided by the Army, on the munitions chemical hexachloroethane
(HCE) have been reviewed and relevant findings are presented in this report in
a manner so as to allow for an evaluation of the daca without continued
reference to the primary documents. This report has been submitted to
critical internal and external review by the EPA.
A companion document, "Data Deficiencies/Problem Areas and Recommendations for
Additional Data Base Development for Hexachloroethane" is included in this
report.
I would like to thank the authors, Ms. Lorecta Gordon, Dr. William Hartley and
Dr. Welford Roberts who provided the extensive technical skills required for
che preparation of this report. 1 am grateful to the members of the EPA Tox-
Review Panel who took time to review this report and to provide their
invaluable input, and I vould like to thank Dr. Edward Ohanian, Chief, Health
Effects Branch, and Margaret Stasikowski, Director, Criteria and Standards
Division, for providing me with the opportunity and encouragement to be a part
of this project.
The preparation of this Advisory was funded in part by Interagency Agreement
(IAG) 85-PP5869 between the U.S. EPA and the U.S. Army Medical Research and
Development Command (USAMRDC). This IAG was conducted with the technical
support of the U.S. Army Biomedical Research and Development Laboratory
(USABRDL).
Krishan Khanna, Project Officer
Office of Drinking Water

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TABLE OF CONTENTS
?ACI
LIST OF TABLES		ii-
LIST OF APPENDICES		i-
EXECUTIVE SUMMARY 		ES-I
I.	INTRODUCTION		I-L
II.	GENERAL INFORMATION AND PROPERTIES		II-L
III.	OCCURRENCE			Ill -1
IV.	ENVIRONMENTAL FATE		17-1
V.	PHARMACOKINETICS		V-L
A.	Absorption		v-L
B.	Discribucion		V-l
C.	Excretion		V-4
D.	Metabolism		V-6
VI.	HEALTH EFFECTS		VI-1
A.	Health Effects in Humans		VI-1
B.	Health Effects in Animals		VI - L
1.	Short-term Exposure		VI-1
a.	Skin and Eye Irritation, Dermal Sensitization . .	VI-4
b.	Six-week Studies		VI-5
c.	Inhalation Toxicity		VI-6
2.	Longer-term Exposure		VI-3
a.	Thirteen-week Studies		VI-8
b.	Sixteen-week Studies 		VI-9
c.	Lifetime Exposure		VI  10
3.	Reproductive Effects		VI-13
4.	Developmental Effects		VI-13
5.	Carcinogenicity		VI-14
6.	Genocoxicity		VI-15
VII.	HEALTH ADVISORY DEVELOPMENT		VII-1
A.	Quantification of Toxicological Effects		VII-3
1.	One-day Health Advisory		VII -4
2.	Ten-day Health Advisory 		VII-4
3.	Longer-term Health Advisory		VII-5
4.	Lifetime Health Advisory		VII-6
B.	Quantification of Carcinogenic Potential		VII-8
VIII.	OTHER CRITERIA, GUIDANCE AND STANDARDS 		VIII-1
continued-
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Table of Concencs - continued
PACE
IX.	ANALYTICAL METHODS	 IX-1
X.	TREATMENT TECHNOLOGIES 		X-L
XI.	CONCLUSIONS AND RECOMMENDATIONS	 XI-1
XII.	REFERENCES	 XII-L
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LIST GF 7A3LZS
11 -1 General Chemical and Physical Properties of
Hexachloroechane 	 II-2
V-I Percentage of ^C-HCE Recovered in 48 Hours
in Rats and Mice Following Multiple Doses	 V-2
V-2 Concentration of HCE in Tissues
Following Single (Sheep) or Multiple (Rats) Oral Doses 	 V-3
V-3	Tissue-to-Blood Concentration Ratio
after Oral Administration of HCE to Rats	V-5
VI-1	Incidence of Hepatocellular Carcinoma
in B6C3F1 Mice Orally Dosed with HCE for up to 78 Weeks 	 VI-16
VI-2 Incidence of Renal or Renal Tubule Lesions
in Male Rats Orally Dosed with HCE for 2 Years	VI-17
LIST OF APPENDICES
Data Deficiencies/Problem Areas and Recommendations for
Additional Database Development for Hexachloroethane 	 Al-1
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EXECUTIVE SUMMARY
Hexachloroethane (HCE), a chLorinated alkane, is a colorless to white
crystalline material with a camphor-like odor. HCE can be dehalogenated by
mecals, alkalis, etc., co form spontaneously explosive chloroacetylenes. AC
300 to iOO'C, its vapor, in Che presence of metal filings, decomposes co form
phosgene and hydrogen chloride. Burning temperatures for the smoke mixtures
range from 700 Co 1,100C and the products of pyrolvsis are carbon and
chlorine via carbon cetrachloride, cecrachloroechylene and hexachlorobenzene.
Hexachloroethane is manufactured by the chlorination of tetrachloroechylene in
the presence of ferric chloride. Although the majority of HCE is imported,
HCE is produced as a co-product in industrial chlorination processes.
Hexachloroethane has been used by the military in the production of pyro-
technic devices and screening smoke. The primary nonmilitary use of HCE has
been as a precursor in the production of fluorocarbons which are used in dry-
cleaning, aerosols and refrigerants. Other uses include pressure lubricants,
rubber and insecticidal formulations, moth repellents, fire extinguishing
fluids, fermentation retardant, chemical precursor, rodenticide, veterinary
medicine and submarine paints.
The major transport mechanism of HCE from water and soil to the atmosphere is
volatilization. Biotransformation is the major transformation process. Once
in the atmosphere, HCE is thermodynamically stable. Adsorption to sediment
and bioaccumulation of HCE may occur. Bioconcentration in aquatic species has
been demonstrated.
Pharmacokinetics
Hexachloroethane can be absorbed upon inhalation, ingestion or dermal contact.
It is preferentially accumulated in fat and the concentration in the kidney is
higher in male rats than female rats. Several metabolites have been demon-
strated both in vivo and io vitro and a reductive dechlorination has been
proposed as the method of metabolism. Tetrachloroethene has been identified
as the principle metabolite under anaerobic conditions with liver microsomal
fractions. The main route of excretion appears to be expired air.
Health Effects in Humans
Data on the effects of HCE in humans is limited. HCE has been reported to
adversely affect the central nervous system and the eye. The neurologic
effects are mild, generally reported as an inability to close the eyelid,
while direct effects on the eye include irritation, tearing, inflammation and
photophobia.
Health Effects in Animals
Oral LD50S in rats range between 5,160 and 7,690 mg/kg in males and 4,460 and
7,080 mg/kg in females. The oral LDjq in male guinea pigs was reported at
4,970 mg/kg. Toxic signs include tremor, ataxia, signs of gasping, and the
appearance of a red exudate around the eyes.
ES-1

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An animal study of sheep indicated HCE produced liver toxicity at doses of
0.5, 0.75 and 1.0 g/kg. Kidney lesions, liver degeneration and necrosis and
decreased body weight gain were exhibited in rabbits dosed at 320 and
1,000 mg/kg/day for 12 days. A significant decrease in body weight gain and
development of gross liver and kidney lesions occurred in male rats when
orally dosed with HCE at 200 and 500 mg/kg/day for 16 days. A No-Observable-
Adverse-Effect-Level (NOAEL) of 50 mg/kg/day was determined based on this rat
s tudy.
A skin irritation study indicated that HCE is a mild skin irritant in rabbits
at a dose of 0.5 g of the dry technical grade of HCE. An eye irritation study
indicated that moderate corneal opacity, iritis, swelling and discharge
occurred upon exposure of the cornea to direct contact with 0,1 g of the dry
technical grade of HCE.
Exposure by inhalation at doses up to 200 ppm over a period of 6 weeks caused
lesions of the nasal passage, trachea and lungs of rats and an increase in
nasal mucous production in quail.
In a 16-week study, male rats administered HCE at doses up to 100 mg/kg/day
developed kidney lesions. A NOAEL of 1.3 mg/kg/day was indicated based on
this study.
No information is available on the effects of HCE on reproduction in animals.
A study of developmental effects on rats administered HCE indicated that it
was not teratogenic, although maternal and fetotoxic effects were observed,
indicating a NOAEL of 100 mg/kg/day.
In a 78-week bioassay study for carcinogenicity with rats and mice, the mice
developed hepatocellular carcinomas after exposure to HCE by oral Intubation
at doses of 590 and 1,179 mg/kg/day. The rats developed histopathological
lesions that were limited to the kidney. However, there were no significant
neoplastic lesions reported. No evidence of mutagenicity was detected in
genotoxicity assays.
Quantification of Toxicologlcal Effects
The 10-day Health Advisory (HA) is 5 mg/L for a 10 kg child based on the NOAEL
of 50 mg/kg/day determined from the 16-day rat study. It is recommended that
the 10-day HA be used as an estimate for the One-day HA because of lack of
suitable data to calculate a One-day HA. The Longer-term HA is calculated to
be 100 ug/L for the 10 kg child and 450 ug/L for the adult, based on a NOAEL
of 1.3 mg/kg/day determined by the 16-week study of rats. The NOAEL of
1.3 mg/kg/day was also used to calculate a Reference Dose (RfO) of
1 ug/kg/day, a Drinking Water Equivalent Level (DWEL) of 40 ug/L and a
Lifetime HA of 0.0007 mg/L or 1 ug/L.
Hexachloroethane is classified as a Group C carcinogen by EPA's guidelines,
indicating it is a possible human carcinogen.
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I.	INTRODUCTION
The Healch Advisory (HA) Program, sponsored by che Office of Drinking water
(ODW), provides information on che healch effeccs, analytical methodology and
creacmenc technology thac would be useful in dealing with the contamination c:
drinking water. Health Advisories describe nonregulatory concentrations of
drinking water contaminants at which adverse health effects would not be
anticipated to occur over specific exposure durations. Healch Advisories
contain a margin of safety to protect sensitive members of the population.
Health Advisories serve as informal technical guidance to assist Federal, State
and local officials responsible for protecting public health when emergency
spills or contamination situations occur. They are not to be construed as
legally enforceable Federal standards. The HAs are subject to change as new
information becomes available.
Health Advisories are developed for one-day, ten-day, longer-term (approxi-
mately 7 years, or 10 percent of an indivi4ual's lifetime) and lifetime
exposures based on data describing noncarcinogenic end points of toxicity. For
chose substances that are known or probable human carcinogens, according to the
Agency classification scheme (Group A or B), Lifetime HAs are noc recommended.
The chemical concentration values for Group A or B carcinogens are correlated
with carcinogenic risk estimates by employing a cancer potency (unit risk)
value together with assumptions for lifetime exposure and the consumption of
drinking water. The cancer unit risk is usually derived from the linear
multistage model with 95 percent upper confidence limits. This provides a low-
dose estimate of cancer risk to humans that is considered unlikely to pose a
carcinogenic risk in excess of the stated values. Excess cancer risk estimates
may also be calculated using the one-hit, Weibull, logit or probit models.
There is no current understanding of the biological mechanisms involved in
cancer to suggest that any one of these models is able to predict risk more
accurately than another. Because each model is based upon differing assump-
tions, the estimates that are derived can differ by several orders of magni-
tude .
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II
GENERAL IN'FORMAT I ON' AND PROPERTIES
Hexachloroethane (HCE), a chlorinated alkane, is a colorless zo white rhombic
crystalline substance with a camphor-like odor detectable at 0.15 ppm. At
higher temperatures, >46C and >7lC, its crystalline form is triclinic and
cubic, respectively (Santodonato, 1985). Since the 1930's. HCE has been used
primarily by the military, in combination with zinc oxide and aluminum, in
pyrotechnic devices and as a screening smoke. It has had more limited usage in
extreme pressure lubricants, rubber and inseccicidal formulations, moth
repellents and fire extinguishing fluids, and has also been used as a fermen-
tation retardant, a chemical precursor and rodenticide. The antihelminthic
properties of HCE have resulted in its use in veterinary medicine. It has also
been included as a component of submarine paints, paper, wood and related
products (Sax, 1986).
\
While nonflammable, HCE can be dehalogenated by metals, alkalis, etc. to form
spontaneously explosive chloroacetylenes (Sax, 1986). At 300 to 500"C, its
vapor, in the presence of metal filings, decomposes to form phosgene and
hydrogen chloride (1:8 at 450*C in the presence of iron) (Sjoberg, 1952 as
cited in Dacre et al., 1979). Burning temperatures for the smoke mixtures
range from 700 to 1,100C and the products of pyrolysis are reported to be
carbon and chlorine via carbon tetrachloride, tetrachloroethylene and hexa-
chlorobenzene (Jarvis, 1970 as cited in Dacre et al.. 1979). The general
chemical and physical properties of HCE are presented in Table II-l.
First produced commercially in the United States in 1921, HCE is manufactured
by the chlorination of tetrachloroethylene (perchloroethylene)in the presence
of ferric chloride at temperatures ranging between 100 to 140*C. When the
process is 50 to 60% complete, the reaction is stopped, neutralized with alkali
and the HCE is crystallized and separated via centrifugation (Kirk-Othmer, 1964
as cited in NIOSH, 1981). Under pressure and at lover temperatures, <60*C, HCE
may also be produced by the photochemical chlorination of the tetrachlorinated
ethylenes (Archer, 1979 as cited in Davidson et al., 1988). Smaller quantities
of the highly purified compound have been produced by the action of chlorine
on barium chloride (Jondorf et al., 1957). Current production in the U.S. is
generally limited to its formation as a co-product in the manufacture of other
chlorinated ethanes, followed by its recycling as feedstock or its thermal
oxidation (Archer, 1979 as cited in Santodonato, 1985). The U.S. Department of
Commerce reported an average annual import of approximately 1.6 million pounds
of HCE from several European sources between 1973 and 1979 (NIOSH 1981).
Smoke compositions containing HCE, at levels currently ranging from 43.5 to
46.5%, include smoke pots and canisters, grenades, cartridges and projectiles.
The HCE, used as a chlorine carrier, is premixed with zinc oxide with aluminum
added and blended prior to loading (Davidson et al., 1988). Laboratory
analysis of the by-product gases from HCE-containing smoke pots indicate that
perchloroethylene (tetrachloroethylene) is the major component (3 to 17% of
reagent weight) while carbon tetrachloride (1-3%), hexachloroethane (0.3-5%),
II-l

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TABLE II-1 General Chemical and Physical Properties of
Hexachloroechane
CAS Number
Synonyms
Molecular Weight
Chemical Formula
Structure
Physical State
Boiling Point
Melting Point
Density
Vapor Pressure
Solubility
Octanol/vater partition coefficient
Log Kow
Odor Threshold
Conversion Factor (air)
67-72-1
carbon hexachloride, perchloroechar.e
ethanehexachloride, 1,1,1,2,2,2 -hexa
chloroethane, hexachlorethylene,
avlothane, distokal, distopan, dis:=p:-.
egitol, falkitol, fasciolin, hexorar:,.
phenohep
236.74
C2C16
CI CI
I I
CIC	CCI
I I
CI CI
white solid, rhombic crystal
186.8*C (triple point)
186.8187.4#C (sealed tube) (sublines,
2.091 (20*C)
0.4 mm Hg (20*C)
50 mg/L in water (22*C), very soluble Lr.
alcohol, ether; soluble in benzene,
chloroform and oils
3.34 (measured)
0.15 ppm
1 ppn - 9.68 mg/rn^ (25*C; 760 mm Hg)
References: IARC, 1979; Amoore and Hautala, 1983 as cited in Santodonato, 19S5.
1986; Verschueren, 1983; Weast, 1986; Windholz, 1983
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phosgene (0.1-1%) and hexaehlorobenzene (0.4-0.9%) are the other major gases
produced. A burn efficiency of 70% is assumed (Katz et al., 1980). Zinc
chloride is the major mecallic by-product of chese tests wich a reagent veisht
mass of approximately 48% (Novak et al., 1987). Average annual use of HCE in
the manufacture of smoke devices was reported to be approximately 193,000 lbs
in the period between 1966 and 1977. If smoke device production was increased
to full capacity (e.g., to support a full military mobilization), the use o
HCE could increase to a rate of approximately 750,000 lbs/month (Kitchens et
al., 1978)
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III. OCCURRENCE
Hexachloroethane is not known co occur in nature (IARC, 1979), however, its
introduction into the environment can arise from its military and industrial
uses and during its manufacturing processes. It has been heavily used by the
military since World War II in the production of various smoke and pyrotechnic
devices. Its primary nonmilitary use had been as a precursor in the
production of fluorocarbons which are used in drycleaning, aerosols,
refrigerants and similar products (Kitchens et al.t 1978).
While the majority of the HCE currently used in U. S. industries is imported,
with the last report of U.S. manufacture being in 1967 (IARC, 1979), it is
produced as a co-product in industrial chlorination processes and is utilized
in the manufacture of C2-chlorinated hydrocarbons (Archer, 1979 as cited in
Santodonato, 1985). In 1987, DeMarini et al. indicated that the U.S. EPA
(1984) had measured HCE at a level of approximately 560 ug/g of hazardous
waste sample from a petrochemical manufacturing plant.
In 1978, Kitchens et al. reported that the production rate for HCE containing
devices at Pine Bluff Arsenal, the major facility for the production of
pyrotechnic devices in the U.S., required the use of approximately 15,000 lbs
of HCE/month. This use rate represents approximately 2% of the production
rate possible under full mobilization capacity. Losses of the HCE during the
production process, which includes mixing, transfer, pressing and sealing of
the canisters, has been estimated at 1 to 2% of the total chemical used or
approximately 150-300 lbs/month. Washdown of the assembly areas also
contributes to this loss but is relatively minor. Up to 1979, the Pine Bluff
facility produced approximately 117,000 gallons of wastewater/8 hr day from 23
buildings in the pyrotechnic area of the facility. The estimated average
concentration of HCE In the wastewater during this time was 168 mg/L with most
of the wastes eventually discharged into the Arkansas River in which the
substantial flow rate provided a large volume of dilution water. The
installation of a new pollution abatement facility, placed online in 1979,
resulted in no HCE being detected in wastewater samples from five areas of the
facility (Fortner et al., 1983 as cited in Davidson et al., 1988). Burning of
off-specification formulations and collected solid wastes was also a source of
entry of HCE into the environment. Installation of a new incineration
facility is anticipated to reduce this source of pollution (Kitchens et al.,
1978).
Its use by the military in chemical obscurant and screening smoke compositions
is another potential major source of HCE in the atmosphere with its subsequent
entry into the water supply via atmospheric fallout, runoff and soil or
sediment leaching. Although the production of HCE-containing smoke devices is
limited to the Arkansas facility, it is widely used by numerous training bases
both within and outside the U.S. While HCE represents approximately 44.5% of
the chemical composition of smoke pots and similar devices, analysis of its
combustion products during smoke generation indicates that the major product
of such combustion is zinc chloride (ZnCl2>. Hexachloroethane represents
approximately 0.3 to St of the gaseous products of this combustion.
Shinn et al. (1985) estimated maximum environmental concentrations of smoke
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from che various HCE devices following detonation and determined a range o:
10-110 mg/m for diffusing devices such as che smoke pots, and a range of
220-660 mg/m^ for grenade and howitzer diffusing devices. Concentrations of
HCE vapor, at a distance of approximately 15 cm from the mouth of a typical
HCE smoke pot device, was measured at approximately 40 ppm ( 385 mg/m . Katz
et al., 1980).
The area most significantly impacted during the detonation of such devices is
generally local, usually limited to isolated base areas 5,000-15,000 m (3.1 zo
9.3 miles) from populated areas, with vapor carrying to those areas
approximately 10-15 km (6.2-9.3 miles) downwind (Novak et al., 1987). The
minimum area impacted for HCE diffusing devices was estimated by Shinn et al.
(1985) to have a range of 50-1,200 nr (slightly more than one quarter of an
acre, maximum) for howitzers and grenades and a range of 9,500-65,000 m^
(approximately 16 acres, maximum) for smoke pots.
In the U.S., HCE has been detected in the effluent from a chemical plant, a
chlorinated sewage treatment plant (Shackelford and Keith, 1976) and a kraft
paper mill (Keith, 1976). It also has been found in river water and in
surface water from only 1 of 204 sites near heavy industrial areas (Ewing et
al., 1977). HCE was detected in drinking water from 4 of 13 cities sampled
(Keith et al., 1976) and in eight samples of finished drinking water (Shackel-
ford and Keith, 1976 as cited in IARC, 1979). It has also been detected in
soil from the Love Canal chemical dump site and in the troposphere (Davidson
et al., 1988).
In an ongoing EPA study as well as other studies, HCE has been measured in
manufacturing effluent and wastewaters from industrial and publicly-owned
treatment facilities at levels ranging from 0.9-1,405.6 ug/L (Ewing et al.,
1977; Keith, 1976 as cited in Davidson et al., 1988). In 1982, Oliver and
Nicol (as cited in Oliver and Niimi, 1983) reported that environmental
concentrations of HCE in Lake Ontario were measured in the range of 0.02 ng/L
while Oliver and Kaiser (1986 as cited in Davidson et al., 1988) reported
levels of 0.02 to 1,700 ng/L in St. Clair River water and from 1.4 to 530 ng/L
in suspended sediment. Levels between 1-3 ug/L have been measured in two
surface water samples from 204 sites near 14 heavily industrial river basins
in che U.S. (Ewing et al., 1977 as cited in Davidson et al., 1988).
Contamination of ground water with HCE has been reported to have occurred from
leachate arising from a pesticide manufacturing waste dump in Hardeman County.
Tennessee. The U.S. EPA (1979) measured HCE in 19 of 31 samples taken from
private wells in the contaminated area with levels ranging from trace to
4.6 ug/L and a median level of 0.26 ug/L (Clark et al., 1982). Keith et al.
(1976 as cited in IARC, 1979) reported levels of 0.03 to 4.3 ug/L in drinking
water in 4 of 13 cities while Suffet and Radziul (1976 as cited in Davidson ec
al., 1988) reported the presence of HCE in the Torresdale drinking water
supply for the city of Philadelphia which comes from the tidal waters of che
Delaware River.
In 1984, Silkworth et al. (as cited in Davidson et al., 1988) reported a level
of 1.0 ug/g in the soil taken from the dump site at the Love Canal and
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0.3-49 ug/m^ in air samples from a cage containing the HCE-contamir.ated soil
Finally, HCE has been measured in che troposphere of che northern hemisphere
at 0.17 parts per trillion (ppc) by volume and in the southern hemisphere at
0.25 ppt by volume. The primary source of this contamination has been
reported to be from the production of chlorinated C2 hydrocarbons (Singh et
al., 1980; Class and Ballschmitter, 1986 as cited in Davidson et al., 1988).
111*3

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IV- ENVIRONMENTAL FATE
Relatively liccle data are available on che environmental fate of HCE in
water. Experimental data indicate that volatilization is an important process
in the transport of HCE from water and soil to the atmosphere and that
biotrans- formation will also occur. Once transported to the atmosphere, it is
predicted that HCE will be stable. Absorption to sediment and bioaccumula-ior.
have also been demonstrated.
Callahan et al. (1979) reported on two experiments conducted by Dilling et ai.
(1975) and Dilling (1977) in which an experimental half-life for volatil-
ization of HCE from water was reported to be 45 minutes, based on a concen-
tration of 1 mg/L in an open, 250 mL container. Evaporation rate was directlv
proportional to the rate of stirring. In a continuation of this study, an
evaporative half-life of 40.7 minutes for 0.72 mg/L in stirred water was
reported. This second experimental result corresponded well with the
theoretical equation of MacKay and Leinonen (1975) as cited in Callahan et al.
(1979) which predicted a half-life of 38 minutes. In 1985, Spanggord et al.
calculated a volatilization rate constant for HCE of 0.01 hr"^ for a body of
water 180 cm deep with an estimated half-life of 70 hours. The Henry's Law
constant of 6,100 L-torr mole*1 indicates a rapid volatilization rate in
shallow, turbulent water.
In a series of experiments designed to determine if biotransformation of HCE
occurs in water, under either aerobic and anaerobic conditions, Spanggord et
al. (1985) determined that, in an open container of aerated pond water,
sterile and non-sterile HCE rapidly disappeared, indicating that volatil-
ization may be a competitive process. When sediment was present in the pond
water, an additional peak developed after 6 days of incubation, indicating
chat biotransformation was probably occurring. Under anaerobic conditions,
the pond water, either with sediment or with the addition of glucose plus
yeast extract, lost 90% of its HCE content in 18 days while the HCE content of
sterile pond water remained unchanged.
when organisms from the pond water sample were transferred to incubation
media, the formation of metabolites was observed, along with a decrease in che
HCE concentration under both aerobic and anaerobic conditions. The authors
concluded that biotransformation is a viable process for HCE and that the
process is more rapid under anaerobic than under aerobic conditions, where
volatilization is the predominating factor. Spanggord et al. (1985) also
confirmed that HCE is biotransformed in soil and suggested that biotrans-
formation is che major transformation process, while volatilization is the
major transport process of HCE from soil and water.
In 1986, Criddle et al. studied the fate of HCE in an unconfined sand aquifer
and determined a half-life of approximately 40 days, with aerobic conditions
prevailing. Additional laboratory studies indicted that the HCE was reduc-
tively biotransformed to tetrachloroethylene in aerobic cultures containing
either municipal wastewater microflora or aquifer microbial materials. In the
wastewater study, the transformation was reported to be constant with time.
In the aquifer study, the combined concentrations of HCE and tetrachloro-
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ethylene were less than the amounts added at the start of the study. The
combined concentrations decreased with time which indicated the possibility o:
a slow increase in sorption with time. The conversion of HCE to PCE was not
uniform in all aquifer samples, suggesting uneven distribution of the active
agents, and autoclaving of the sample did not prevent transformation although
it appeared to enhance sorption of the HCE.
Bioconcentration of HCE in aquatic species has been confirmed. In 1982,
Barrows et al. determined a bioconcentration factor (BCF) of 139 for HCE in
bluegill sunfish after an exposure for 28 days in water with a mean HCE
concentration of 6.17 ug/L. The BCF is defined as the quotient of the mean
measured residues of a compound in whole body fish tissue during the
equilibrium period divided by the mean measured concentration of the compound
in exposure water. Bioconcentration factors greater than unity indicate that
accumulation of the chemical has ctcurred in the tissue. Uhen the exposed
fish were transferred to an aquar- containing fresh water, a half-life of
<1 day was determined, indicatine it biological persistence is low.
Bioconcentration of HCE and othe Lorinated hydrocarbons from water at
environmental concentrations (r ange) was demonstrated by Oliver and Niimi
(1983) in rainbow trout. The . was designed to determine if the BCF was
dependent on the concentration che chemical in water, particularly when
exposure was at ambient or only slightly higher than environmental levels.
Additionally, the BCF at the higher test level averaged 2.4 times that at the
lower level. In this study, control water contained approximately 0.03 ng/L
of HCE, low level test water, 0.32 ng/L and high level exposure water,
7.1 ng/L. The BCF was 510 and 1,200 for the low and high exposure tests,
respectively. Furthermore, the experimental BCF developed in this study was
used to predict the concentration of HCE in fish in Lake Ontario water where
the concentration of HCE had been measured at 0.02 ng/L (Oliver and Nicol,
1982 as cited in Oliver and Niimi, 1983). The predicted value of 0.01 ng/g
for HCE correlated well with the mean concentration of 0.03 ng/g of HCE in
Lake Ontario fish.
Spanggord et al. (1985) also determined a relatively low soil sorption
partition coefficient and suggested that HCE should migrate fairly rapidly in
soil. No chemical transformation processes that dominate in soil, water or
air were identified in this study. Curtis et al. (1986) as cited in Davidson
et al. (1988) reported an uptake for HCE in laboratory studies that was
characterized by an initial rapid sorption step (<2 hrs) that accounted for
approximately 50% of the total sorption, followed by a gradually decreasing
rate. An equilibration time of 3 days was determined and the sorption and
desorption isotherms were reported to be linear and reversible at concen-
trations ranging between 1-50 ug/L. The authors indicated that partitioning
of HCE into organic matter was the observed mechanism for the sorption
process.
The photooxidation of HCE by ultraviolet light in the presence of water was
studied by Knoevenagel and Himmelreich (1976) as cited in Davidson et al.
(1988). A half-life of 93.7 hours for 237 mg of HCE in 900 mL of water at a
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temperature of 90 co 95'C under aerobic conditions vas determined. The
photodegradation was measured by the amount of carbon dioxide formed. Under
anaerobic conditions, the cleavage occurs at the C-Cl bond.y
Vhen released to the atmosphere by volatilization, HCE is thermodynamically
stable, no degradation by hydroxyl radicals is expected, and the tropospheric
lifetime is expected to be quite long (Davidson et al., 1988). A total
transport within the atmosphere and between the atmosphere and oceans of
approximately 1 kton/yr was predicted by Class and Sallschmitter (1986 as
cited in Davidson et al., 1988).
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pharmacokinetics
Hexachloroethane, which can be absorbed following inhalation, ingestion or
dermal contact, is preferentially accumulated in fat. In male racs. kidnev
levels are significantly increased over that of females. Several metabolites
have been demonstrated both jji vivo and vitro. and a reductive dechlor-
ination has been proposed as the method of metabolism. Tetrachloroethene has
been identified as the principle metabolite under anaerobic conditions with
liver microsomal fractions. The main route of excretion appears Co be expired
air with urinary excretion playing a relatively minor role.
A.	Absorption
Absorption of HCE by ingestion was demonstrated by Fowler (1969) in Scottish
Blackface male sheep administered, by drenching bottle, a dose of 0.5 g/kg of
HCE in olive oil emulsified with water. Absorption was slow with HCE first
detected in the blood of an anesthetized male after 27 minutes post-dosing.
Hexachloroethane was detected in the bile 15 minutes after dosing. Total
recovery (percent of dose) was not determined in this study. In a study by
Jondorf et al. (1957) in rabbits fed HCE at a level of 0.5 g/kg in the diet,
only 19 to 29% of the administered radioactivity was accounted for in 3 days
with 5% recovered in the urine and the remainder in expired air.
The maximum tolerated dose (MTD), as determined in the NCI (1978) bioassay of
HCE, was administered to male Osbome-Mendel rats and male B6C3F1 mice. They
were dosed by intubation 5 days/week for 4 weeks, followed by a single dose of
radiolabeled HCE. Between 93-95% of the administered radioactivity was
recovered over 48 hours (Mitoma et al., 1985). Doses in this study were
500 mg/kg/day (2.11 mmol/kg) for rats and 1,000 mgAg/day (4.22 mmol/kg) for
mice. Table V-l compares the recovery in both species. In contrast to the
Fowler (1969) study in sheep, between 65-72% of the radioactivity administered
to rats and mice was recovered in expired air while only 6-16% was recovered
in the combined excreta.
B.	Distribution
Studies on the distribution of HCE have been reported in two male Scottish
Blackface sheep following an oral dose of 0.5 g/kg (Fowler, 1969) and in male
and female COF Fisher 344 rats (4/dose) after 16 weeks on test diets
containing HCE at levels of 1, 15 or 62 mg/kg/day (Gorzinski et al., 1985).
In both studies, the majority of the HCE was recovered from the fat.
Table V-2 compares the tissue levels, in ug HCE/g tissue, in rats receiving
the high dose of 62 mg/kg/day for 16 weeks and in sheep at 8.5 hours after a
single dose of 500 mg/kg.
In the rat study, the concentration of HCE in the kidneys of the male rat was
higher than in females and was proportional to dose (Table V-3). Tissue
levels in the kidneys of females receiving the low dose amounted to approx-
imately 27% of the level detected in the males while the levels in
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Table V-l Percentage of ^C-HCE Recovered in 48 Hours
in Rats and Mice Following Multiple Doses3
Rat	Mouse
(500 mg/kg/day)	(1,000 mg/kg/day)c
Total Recovery
93.3
95.5
Expired Air
64.6
71.5
C02
2.4
1.8
Excreta
6.3
16.2
Carcass
20.0
5.9
Reference: .Mitoma et al. (1985)
a Standard deviacions omitted,
k 2.11 mmol/kg
c 4.22 mmol/kg
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Table V-2 Concentracion of HCE in Tissues
Following Single (Sheep) or Multiple (Rats) Oral Dosesa

Racs (62
mg/kg/day)
Sheep (500 mg/kg)b

Male
Female
Male
Liver
0.71
0.63
0.2
Kidney
95.12
2.01
0.1
Fat
176.1
162.1
1.7
Brain
..(c)

0.2
Muscle


0.04
Blood
0.74
0.61
0.2
References:
Gorzinski
et al. (1985)
Fowler (1969)
a Concentration in ug HCE/g tissue for 4 rats/sex and 1 sheep
k anesthetized for bile studies
c not analyzed
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females at che two higher doses were only about 2-3% of the levels in males
The increase of HCE concent of the fat in both species was proportional to
dose. A disproportionate increase in HCE content of the liver and blood was
evident in both species. Table V-3 compares the tissue-to-blood concentration
ratios for both sexes. When compared in this manner, a dose-related increase
of the HCE in the kidneys of male rats is clearly evident while that in the
female kidney appears to decrease slightly with increasing dose. Concen-
tration ratios for liver in both sexes are remarkably similar. In contrast,
che concentration ratio for HCE in the fat of the female rats, while similar
to males at the low and high dose, is >4 times higher than males at the mid-
dose leVel. This phenomena may be due, at least in part, to the low concen-
tration of HCE in the blood of females at this dose level.
Gorzinski et al. (1985) treated an additional group of male CDF Fischer-344
rats with the high dose of HCE (62 mg/kg/day) in the diet for 8 weeks followed
by 3, 6, 13, 22 or 31 days on control feed to obtain data on tissue clearance
rates. Samples of fat, liver, kidney and whole blood were analyzed for HCE
content. The concentrations of the HCE were found to decrease in the tissues
in an apparent first-order manner, with tissue half-lives between two and
three days. With an average half-life of 2.5 days, the level of HCE in the
rat was calculated to have reached a 99% steady state concentration in 17
days. The authors indicated that the data did not seem to suggest accumula-
tion in the tissue with increasing duration of treatment.
C. Excretion
Jondorf et al., (1957) reported that rabbits excreted 5% of the administered
radioactivity in the urine in three days following the feeding of 0.5 g/kg of
^C-HCE in the diet. Between 14-24% of the radioactivity was recovered in "he
expired air during the same time period with the remainder unaccounted for.
In contrast, rtitoraa et al. (1985) indicated that Osborne-Mendel rats and
B6C3F]_ mice excreted 64.6 and 71.5%, respectively, of the administered
radioactivity in expired air following oral dosing with ^C-HCE 5 days/week
for 4 weeks. Recovery in the combined excreta was 6.3 and 16.2% for rats and
mice, respectively, 48 hours after the last dose. Doses for this study, the
MTDs, were 500 mg/kg/day for rats and 1,000 mg/kg/day for mice.
Fowler (1969) measured HCE recovery from urine and feces in cvo sheep that
were given a single oral dose, 0.5 g/kg, of the chemical. More than 80% of
the total HCE measured in the feces, an average of approximately 1,000 ug, was
recovered within the first 24 hours. Recovery from the urine was approxi-
mately 6% of that found in the feces and was also largely excreted in che
first 24 hours. Recovery from expired air was not reported.
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Table V-3 Tissue - to-Blood Concentration Ratioa
after Oral Administration of HCE to Rats
Dose	Liver	Kidney	Fat
mg/kg/dayk	Male Female Hale Female Male Female
1.0	3.7	3.9 17.2	5.5 39.9 38.6
(0.079/0.067)
15	2.9	2.9	40.8	4.2	63.6 279.4
(0.596/0.162)
62	1.0	1.0 128.2	3.3 237.3 264.4
(0.742/0.613)
Adapted from Gorzinski et al. (1985)
4 ug/g tissue per ug/mL blood.
k The numbers in parentheses are the concentrations of HCE (in ug/mL) in the
blood of male/female rats, respectively.
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D. Metabolism
Fowler (1969) reported the occurrence of two metabolites of HCE in the hexar.e
extracts of both the urine and feces of sheep following its oral administra-
tion at a dose of 0.5 g/kg. Tetrachloroethylene (perchloroethylene) was the
major metabolite, present in amounts approximately equal to unmetabolized HCE
in the feces (1,050 ug) but urinary recovery was roughly 50% that of HCE in
the feces. The second metabolite, pentachloroethane, was present at levels
from trace to approximately 450 ug in the feces and at levels similar to TCE
(25 ug) in the urine. Analysis was by gas-liquid chromatography.
Trichloroethanol and trichloroacetic acid were reported as the major metaboli-
tes in rats and mice following the oral administration of ^C-HCE at the MTD
of 500 and 1,000 mg/kg/day, respectively, for 5 days/week over 4 weeks (Mitoma
et al., 1985). Urinary metabolites, extracted with methanol, were analyzed by
high performance liquid chromatography (HPLC) coupled to a radioactive flow
detector. The authors reported that between 24 and 29% of the HCE was
metabolized, as measured by the combined recovery in the excreta, carcasses
and as C02-
Jondorf et al. (1957) reported the recovery of six metabolites in the urine of
rabbits following the oral administration of 0.5 g/kg of ^4C-HCE in the diet.
The metabolites, ranging from 0.1 to 1.3% in the urine were identified by
chromatographic and isotope-dilution techniques as trichloroethanol, dichloro-
ethanol, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid and
oxalic acid. In expired air, recovered metabolites were identified as carbon
dioxide, tetrachloroethylene, and 1,1,2,2-tetrachloroethane along with
unmetabolized HCE.
In studies conducted under anaerobic conditions using the liver microsomes
from phenobarbitone-pretreated rats, HCE was reductively dehalogenated by
NADPH. The process was found to be dependent upon the presence of cytochrome
P450 (Nastainczyk et al., 1982). The authors further postulated that the
reaction proceeds via a two-electron reduction mechanism producing first a
radical and then a carbanion. The metabolites were identified as tetrachloro-
ethene (99.5%) and pentachloroethane (0.5%). Additional metabolites seen in
ill vivo systems may arise from further reduction processes.
Town and Leibman (1984) further described the conditions for the reductive
dehalogenation process. The reaction occurs, for the most part, in the
microsomes; it is inhibited in the presence of oxygen, and the optimum pH is
7.0-7.5. Inhibition of cytochrome P450 resulted in an inhibition in the
formation of the tetrachloroethylene. No tetrachloroethylene could be
detected when rabbit liver microsomes were used. In a study by Salmon et al.
(1985), dechlorination of halocarbons using a vesicular reconstituted system
of cytochrome P450 enzymes from rabbit liver was similar to that catalyzed by
rat liver microsomes, indicating that the system requires both reductase and a
phenobarbital inducible form of cytochrome P450.
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VI. HF.Al.TH EFFECTS
Few data are available on the effects of HCE in humans.	Laboratory studies i-
animals have identified the kidney as the principle site	of toxicity. No da:a
are available on the reproductive effects of HCE, but it	does not appear to be
teratogenic nor genotoxic. Evidence is presented on the	carcinogenic effects
of HCE in mice.
A.	Health Effects in Humans
In 1978, the National Institute for Occupational Safety and Health (NIOSH), in
a National Occupational Hazards Survey conducted from 1972-1974, estimated
that 1,500 workers were potentially exposed to HCE in the workplace (Parker ec
al., 1979). As is the situation with most chloroethanes, HCE has been
reported to adversely affect both the central nervous system (CNS) and the
eye. Its neurologic effects are mild, generally reported as an inability to
close the eyelid. Its direct effects on the eye include irritation, tearing,
inflammation and photophobia upon exposure to fumes from heated HCE (Grant,
1986).
No data on the acute or chronic toxic effects of HCE exposure in humans were
found in the English language literature. Plotnikov reportedly ingested 30 g
of HCE in three days while Sokolov ingested 48 g in 4 days, with reduced skin
sensitivity the only reported effect (Plotnikov and Sokolov, 1947, cited in
Dacre et al., 1979). Saric and Knezevic (1957 as cited in Dacre et al., 1979)
reported irritation as the most frequent complaint among industrially exposed
workers. A change in the serum albumin:globulin (AG) ratio was the only
clinical finding.
A report of death and significant injury due to inhalation of fumes from an
HCE smoke grenade (Fischer, 1969 as cited in Dacre et al., 1979) was
attributed to the presence of zinc chloride (ZnC^), a major ingredient of the
smoke product.
B.	Health Effects in Animals
l. Short-term Exposvre
Early studies in sheep indicated that HCE produced some degree of liver
toxicity, but similar findings were not seen in rats following a single oral
dose. When male rabbits were similarly treated for 12 days, both liver and
kidney toxicity were seen at the two high dose levels. Exposure of male and
female rats for 16 days to HCE in the diet resulted in kidney and liver
toxicity in the males. Exposure by Inhalation caused lesions of the nasal
passage, trachea and lungs of rats and an Increase in nasal mucous production
in quail, but no similar effects in dogs or guinea pigs.
Initial reports on the oral toxicity of HCE to animals were associated with
the use of HCE in the treatment of mature liver flukes (Fasciola hepatica) in
sheep and cattle. Olsen (1947) as cited in Fowler (1969) reported that
VI-1

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adverse reactions among Che created sheep included intoxication, incoordina-
tion, muscle tremors and death. Using castrated and uncastrated male Scott isr.
Blackface or Cheviot cross sheep (15-23 kg), Fowler (1969) studied the effects
of HCE. The HCE was dissolved in olive oil (15% w/v) and emulsified with
water in the presence of gum acacia (12.5% w/v) and gum tragacanth (0.7% w/v)
The solution was orally administered by drenching bottle at doses of 0.5, 0.75
or 1.0 g/kg (10, 1 and 1 animals/dose level, respectively). Hepatic toxicity
was evaluated by measuring plasma levels of glutamate dehydrogenase (GD),
sorbitol dehydrogenase (SD), ornithine carbamoyl transferase (OCT) and
aspartate aminotransferase (GOT). A bromsulphthalein (BSP) dye clearance test
also was conducted to assess liver function.
The doses were veil tolerated vith slight tremor of the facial muscles
reported in three of the animals within the first 4 hours after dosing but
with no effect on their ability to eat. Administration of HCE resulted in a
simultaneous increase in all of the plasma enzymes, vith maximum levels
evident at 48 hours after dosing and a return to normal levels by 4 to 5 days
post-dosing. Increases averaged from three to six times normal levels for CD
and SD and two to ten times for OCT. The GOT levels increased slightly. The
BSP clearance test indicated the HCE had no affect on the uptake from plasma
to liver cells, but a marked reduction in the transfer from liver to bile was
evident at 72 hours after dosing with HCE. This study indicated that HCE,
orally administered to sheep, resulted in a degree of hepatic dysfunction.
The elevated plasma enzymes suggested to the authors that HCE caused cell
membrane permeability changes, probably at the intracellular level, while the
decreased BSP dye clearance and unchanged hepatic uptake rate Indicated a
decrease in the excretory capacity of the liver.
In a study conducted by Veeks and Thomasino (1976), six male Sprague-Dawley,
Vistar strain rats per level were administered a single oral dose of a 50%
solution of HCE in corn oil vith doses ranging from 2,510 to 10,000 mg/kg.
Animals were observed for the occurrence of toxic signs, mortality was
recorded, and the survivors were observed for 14 days after dosing. A gross
pathological examination vas performed on all dosed animals.
The acute oral LD50 level (95% confidence limits) in the male rats vas 5,160
(4,250-6,270) mg/kg. Toxic signs vere evident within 4-12 hours after dosing
and included the appearance, at all dosage levels, of a red exudate around the
eyes vhich persisted throughout the 14-day observation period. At doses of
5,010 mg/kg and higher, the animals developed tremors, ataxia and signs of
gasping. Mortality usually occurred vithin the first 12-24 hours to 4 days
after dosing vith one delayed death on Day 7. No gross lesions vere observed
in any of the surviving rats vhile one animal that died after dosing showed
evidence of degeneration of the cortex of the kidney. The acute oral LD50 for
female rats, calculated by the method of Finney (1971), vas reported to be
4,460 (3,900-5,110) mg/kg (Weeks et al., 1979). An approximate lethal dose
(ALD - first dose of the lovest series of three ascending doses, all of which
produced fatalities) of 4,900 mg/kg vas determined after oral administration
of HCE to male rats (Weeks and Thomasino, 1976) and of >1,000 mg/kg in male
rabbits (Weeks et al., 1979).
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Weeks et al. ( 1979) also reported che acute oral LD50 for HCE, dissolved ir.
methylcellulose (5% w/v), in male and female Sprague-Dawley racs co be
7,690 (6,3809,250) and 7,080 (6,240-8,040) mg/kg, respectively. An oral L2;-
of 4,970 (4,030-6,150) mg/kg was reported in male Hartley guinea pigs for HCE
dissolved in corn oil (50% w/v). Calculations were by the method of Finney
(1971).
The effects of HCE on the liver were studied in four male Charles River rats
(150-300 g) fasted for 16 hours prior to the administration by gavage of a
single dose of HCE in mineral oil at a level of 2,600 umoles/100 g animal
(equivalent to as 6 gAg) (Reynolds, 1972). No significant effects were seen
on the chemical and functional properties of the liver microsomes as compared
to the controls and measured at 1 to 2 hours after dosing.
The development of toxic signs and changes in. blood chemistry parameters were
studied in male New Zealand White rabbits (5/group) following the oral
administration of HCE (99.8% pure) suspended in 5% aqueous methylcellulose at
doses of 0, 100, 320 or 1,000 mg/kg/day for 12 days (Weeks et al., 1979).
Body weights were recorded daily and blood samples, drawn from the central ear
artery, were taken on Days 1, 4, 8, 12 and 4 days after termination of
treatment. Serum levels for glutamic oxaloacetic transaminase (SCOT),
glutamic pyruvic transaminase (SGPT), blood urea nitrogen (BUN), alkaline
phosphatase, bilirubin, total protein, potassium and sodium were determined.
The animals were sacrificed on Day 4 after the last dose administration, major
organs were weighed and samples of tissues were prepared for microscopic
examination.
At the high dose level, toxicity was evidenced by a significant reduction in
body weight, beginning at Day 7, with a significant increase in the relative
liver and kidney weight ratios at termination. At the 320 mg/kg/day dose
level, body weight reduction became significant by Day 10 of dosing. No
changes in body weight were seen in the low dose group (100 mg/kg/day) and no
other toxic signs were evident.
Administration of HCE ac che two higher doses resulted in a significant
decrease in the serum potassium and glucose levels. A trend toward increasing
serum alkaline phosphatase, SCOT and bilirubin levels was seen in the high
dosed group but no statistical significance was evident. No other blood
chemistry parameters were affected.
Upon necropsy, signs of liver degeneration and necrosis were seen in animals
receiving the two highest dose levels. Histopathological examination revealed
dose related changes in the liver to include fatty degeneration, coagulation
necrosis, hemorrhage and ballooning necrosis. Eosinophilic changes and
hemosiderin-laden macrophages and giant cells were also evident. No signs of
liver toxicity were seen at the lowest dose level.
Toxic tubular nephrosis of the convoluted tubules of the corticomedullary
region of the kidney were seen in the two high dosed groups but the response
was not considered to be dose related. Minimal tubular nephrocalcinosis was
also seen at these two levels. No toxic effects were observed in the kidneys
VI-3

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of che low-dosed group. Based on the data from this study, a dose of
100 mg/kg/day can be considered che No-Observed-Adverse-Effect-Level (N'OaEL'
for che effects of HCE on che kidneys of male rabbits.
In a 16-day tolerance study, Gorzinski et al. (1980a) fed male and female CD"
Fisher 3^4 rats (number per group not specified) a diet containing HCE at
levels of 0, 10, 50, 200 or 500 mg/kg/day. A decrease in body weight gain was
evident in both sexes at the high dose level. Male rats receiving both the
500 and 200 mg/kg/day diets showed an increase in kidney weight as well as
grossly observable lesions. Females were not similarly affected at either
dose level. Other effects included a trend toward increased liver weight in
both sexes and the occurrence of gross liver lesions in the high-dose males.
A NOAEL of 50 mg/kg/day is indicated under the parameters of this study.
Additional evidence for the renal toxicity of HCE was provided by a 16-day
study which was conducted in Fischer-344 rats as part of the National Toxi-
cology Program (NTP) investigation of this compound (NTP, 1989). Groups of
five rats of each sex were administered twelve doses of 0, 187, 375, 750,
1,500 or 3,000 mg/kg HCE in com oil by gavage over the 16-day period. The
animals were observed twice daily and weighed weekly. After sacrifice,
necropsy was conducted for all animals. Clinical signs of compound toxicity,
including dyspnea, lacrimation, ataxia and prostration, were seen in the three
highest dose groups while all of the animals in the two highest dose groups
died before the termination of the study (NTP, 1989). In the 750 mg/kg dose
group, 1/5 males and 2/5 females died prior to sacrifice; the surviving
animals had body weights which were 25% lower than the controls for the males
and 37% lower for the females. Hyaline droplets were identified in the
cytoplasm of the renal tubular epithelium in all the males; tubular cell
regeneration was apparent in males at doses of 187 and 375 mg/kg. Comparable
effects were not observed in the females.
a. Skin and Eve Irritation. Dermal Sensitization
There was no primary skin irritation to the intact or abraded skin of six New
Zealand Vhite rabbits at 24 or 72 hours after the application of 0.5 g of the
dry technical grade HCE for 24 hours. Uhen 0.5 g of the technical grade HCE
was applied as a paste in 0.5 mL of distilled water, no edema but a barely
perceptible erythema was seen on the intact skin at 24 hours. On the abraded
skin, moderate to slight erythemic reactions were seen while one rabbit showed
a barely perceptible edema, indicating that HCE produced a mild skin irrita-
tion (Weeks and Thomasino, 1976).
Evidence of a very slight degree of epithelial hyperplasia was seen in che
skin of rabbits exposed for 31 days to 20 applications of a saturated solution
of HCE in 95% ethanol (<10% solution). The irritation was grossly evidenced
by a slight hyperemia of the ear with scaliness and some faint follicular
enlargement (Adams, 1937). This concentration produced no visible irritation
when applied to the abdomen for 4 days. When slightly less than 1.5% of HCE
in propylene glycol was applied 21 times to the rabbit ear over a period of
34 days, no irritation was evident. However, this same solution held in
contact with the abdomen for the same period of time produced a faint hyper-
emia and exfoliation after three days which persisted unchanged throughout che
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34 days of exposure. When the HCE was applied to rabbit skin as solutions ir
either Diamond paraffin oil or Deo base oil, severe Lrritacion was produced
but was largely attributed to the vehicle itself. Based on this study, HCE
was not considered to be a particular skin hazard for man.
Moderate corneal opacity along with iritis and severe swelling and discharge
were evident in five of six male New Zealand White rabbits exposed to a single
application of 0.1 g of the dry technical grade HCE applied to the cornea and
allowed to remain in contact overnight (Weeks ec al., 1979), The irritation
was no longer evident after 72 hours.
No dermal sensitization was Indicated in male Hartley strain guinea pigs at
24 and 48 hours after a single challenge dose of HCE. The challenge dose was
administered tvo weeks after the completion of a series of ten intradermal
injections of 0.1 ml of a 0.1% solution of HCE in saline (Weeks and Thomasino,
1976). Hexachloroethane is not considered a sensitizing agent.
Weeks and Thomasino (1976) calculated a dermal LD50 -32 g/kg for male New
Zealand White rabbits (4/group) treated with a single dermal application of
granular HCE mixed into a paste with equal quantities of distilled water. The
doses, ranging from 1 gAs to 32 g/kg, were applied to the shaved skin for a
period of 24 hours. Signs of toxicity included weight loss but no skin
irritation was evident. Two deaths occurred at the 32 g/kg level at 4 days
after treatment. Gross examination of the surviving animals revealed a
congestion of the cortex of the kidney in tvo rabbits and a hyperemia of the
gastric mucosa in one treated at the highest dose level.
b. Six-week Studies
In an effort to determine the maximum tolerated dose for HCE, NCI (1978)
conducted tvo subchronic toxicity studies utilizing Osborne-Mendel rats
(5/sex/group) and K6C3F^ mice (5/sex/group). The HCE was administered by
gavage 5 days/week for 6 weeks followed by a 2-week observation period. The
rats were administered doses of HCE in corn oil at levels of 0, 178, 316, 562,
1.000 or 1,780 mg/kg/day while the mice received doses of 0, 316, 562, 1,000,
1,780 or 3,160 mg/kg/day. Food and water were available ad libitum. Body
weights were recorded weekly and the animals were observed for signs of
toxicity. Mortality was recorded. No other parameters were studied in this
experiment.
In the rat study, all animals receiving the high dose of 1,780 mg/kg/day died
before the end of the 8-week experimental period while all rats at the middle
dose level o 562 mg/kg/day survived. Body weights were comparable to
controls at doses of 316 mg/kg/day while 38% of the males and 18% of the
females developed a body weight depression at the 1,000 mg/kg/day level.
In the mouse study, survival remained at 100% for males up to 1,000 mg/kg/day
and for females up to 1,780 mg/kg/day. At the high dose level of
3,160 mg/kg/day, mortality was 80% for males and 60% for females. Mean body
weight gain was generally comparable to controls at doses up to
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1,000 mg/kg/day and was subscantially depressed at che high dose level. A
Lovesc-Observed-Adverse-Effecc-Level (LOAEL) or NOAEL for this study would no:
be appropriate due to an insufficient number of parameters to judge toxicity.
c. Inhalation Toxicity
No toxic signs were evident in a group of six male rats exposed at room
temperature (23'C) to a nominal concentration of 2.5 mg/L (260 ppm) of HCE for
a period of 8 hours. Body weight and organ/body weight ratios did not differ
significantly from controls and no gross or histopathological lesions were
evident 14 days after exposure (Weeks and Thomasino, 1976). Exposure at 50"C
to a nominal concentration of 57 mg/L (5,900 ppm) for 8 hours resulted in
severe toxicity that included death of two of the animals at 8 hours after
exposure. Other toxic signs included staggering gait and reduced body weight
gain as well as evidence of a subacute diffuse interstitial pneumonitis with
vascular congestion, minimal to moderate in severity, in two of the four
surviving rats. Exposure at this same temperature to 17 mg/L (1,756 ppm) for
6 hours produced evidence of toxicity as indicated by a staggered gait and a
reduced weight gain over the first 24 hours. No deaths occurred and no gross
nor histopathological lesions were reported under these conditions. The dose
of 17 og/L (1,756 ppm) can be considered the LOAEL for a single inhalation
exposure in rats.
Weeks et al. (1979) studied the effects of repeated exposure to vapor of HCE
(99.8% pure) in 25 male and 25 female rats, 4 male dogs, 10 male guinea pigs.
20 male or female quail and 22 pregnant rats per exposure group. An
additional 15 male rats were studied for behavioral effects following
inhalation exposure. Exposure periods were for 6 hours/day, 5 days/week for
6 weeks and doses were analyzed at 0, 15, 48 or 260 ppm of the HCE vapor
(equivalent to  0, 145, 465 or 2,515 mg/m^). All animals were weighed weekly
and observed daily for signs of toxicity. Toxicological evaluation was made
for histopathological changes (rats, dogs, guinea pigs and quail), behavioral
effects (rats), signs of sensitization (guinea pigs) and teratological effects
(rats). Half of the treated animals were sacrificed at the end of the 6-week
exposure period with the remaining animals sacrificed 12 weeks after
termination of the exposure study.
At the 260 ppm exposure level, Weeks et al. (1979) reported toxic signs in
dogs which included tremors, ataxia, hypersalivation, severe head bobbing and
facial muscle fasciculation. During the exposure period, the dogs kept their
eyelids closed and one dog convulsed, with death occurring after 5 hours of
exposure (25% mortality). The signs recurred intermittently from Day 1
throughout the 6-week exposure period with recovery usually occurring over-
night. Exposure at this level had no effect on the body weight of the dogs
nor on the blood parameters measured (not specified). No exposure related
lesions were observed upon histological examination.
The guinea pigs exposed at this 260 ppm dose level showed a significant
reduction in body weight gain after one week of treatment and two guinea pigs
died during each of the fourth and fifth weeks of treatment (40% mortality)
(Weeks et al., 1979). A significant increase in liver/body weight ratio was
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recorded. No sensitization response could be elicited and no histological
lesions were observed.
Toxic signs in rats exposed to the high dose of 260 ppm of HCE vapor included
tremors, ruffled pelt and the appearance of a red exudate around the eyes
(Weeks et al., 1979). These signs appeared during the fourth week of exposure
when two animals were found dead (4% mortality). Male rats showed a decrease
in body weight gain beginning after 2 weeks of exposure. A significant
increase in organ to body weight ratios was seen in the kidney, spleen and
testes of males and in the liver of females. No gross changes were evident a:
necropsy. Comparison of the grouped data from a series of oxygen consumption
studies indicated that exposure to 260 ppm of HCE vapor for 6 weeks resulted
in a significant decrease in oxygen consumption. While nonspecific, this tes:
was considered indicative of a lowered basal metabolic race, possibly
resulting from the prolonged inhalation of this upper respiratory irritant.
Histological examination revealed an increased incidence and severity of
mycoplasma-related lesions in the nasal turbinates, trachea and lungs of these
exposed rats. The lesions were subclinical and consisted of a mucopurulent
nasal exudate, lymphoid hyperplasia in the lamina propria of the trachea and
pneumonitis, only rarely associated with bronchiectasis.
Pregnant female rats exposed to HCE vapor at the 260 ppm level on Days 6
through 16 of gestation developed slight to moderate tremors from Days 12
through 16 of gestation and body weight gain was significantly lover than
controls starting on Day 8 of gestation (Weeks et al., 1979). At the 48 ppm
exposure level, weight gain was significantly decreased beginning on Day 14 of
gestation. No significant skeletal or soft tissue anomalies were detected in
fetuses. All other maternal and fetal parameters, Including a number of
corpora lutea, implantation sites, resorption sites and viable fetuses as well
as appearance, sex, weight and body length of viable fetuses, were within
normal limits. Upon histological examination of the dams, an increased
incidence of mucopurulent nasal exudate was seen in the 260 ppm (100% of the
animals) and 48 ppm (85%) dosage groups when compared to controls.
Exposure to HCE vapor (0, IS, 48 and 60 ppm) caused no toxic effects in quails
and no animals died after exposure (Weeks et al., 1979). Upon histological
examination an excess of mucus, without evidence of inflammatory cells, was
seen in the nasal turbinates in 20% of the high-dose group. This finding was
considered a transient effect, as evidenced by lack of tissue damage, and due
to direct exposure to the test compound.
Animals of all species sacrificed at 12 weeks after termination of the
exposure studies had body weights comparable to controls (Weeks et al., 1979).
No significant changes were seen upon gross necropsy and all toxic signs had
disappeared. Incidence of upper respiratory irritations were comparable to
controls.
Exposure to HCE vapor had no effect on the behavior of rats as measured by
avoidance latency and spontaneous motor activity when compared to controls
(Weeks et al., 1979). Pulmonary function studies conducted in dogs indicated
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chat inhalation of HCE had no effect on flow, tidal volume, transpulmonary
pressure, compliance or resistance. Mean values for pre- and post-test
compliance and resistance functions were comparable and were within normal
limits.
At the 15 and 48 ppm exposure levels, no toxic signs were evident in any of
the exposed groups except for the decreased weight gain in the pregnant dams.
Body and organ weights were comparable to controls and no treatment-related
changes were seen upon gross examination. A NOAEL of 48 ppm (5=465 mg/m^) for
exposure to HCE vapor for six weeks can be established for all species (Weeks
et al., 1979).
2. Longer-term Exposure
Studies were conducted in male and female rats fed HCE in the diet for 13 or
16 weeks, as well as in lifetime studies in rats and mice orally gavaged with
HCE. Kidney toxicity was the most consistent finding of these studies
although some non-neoplastic lesions were evident in the livers of the rats.
The data from the lifetime studies indicate that HCE produces hepatocellular
carcinomas in mice and renal cell carcinomas in rats.
a. Thirteen-week Studies
A 13-week study of HCE was conducted as a portion of the National Toxicology
Program investigation of this compound (NTP, 1989). Groups of 10 male and 10
female Fischer-344 rats were given daily doses of 0, 47, 94, 188, 375 or
750 rag/kg HCE in corn oil by gavage. The animals were observed twice daily
and weighed weekly. Following sacrifice, all animals were necropsied; key
organs were weighed and the tissues of the controls, the high-dose group
females, the penultimate-dose group males, and five of the high-dose group
males were subjected to histopathological examination. Urine samples were
collected from all animals at the end of the study.
Kidney changes were apparent in the males from all dose groups evaluated (NTP,
1989). The severity of the kidney damage increased with the dose. In five of
the male rats which died prior to the completion of the study, there was renal
papillary necrosis and degeneration accompanied by necrosis of the tubular
epithelium. Hyaline droplets in the epithelial cytoplasm, tubular regener-
ation, and tubular casts were seen in these animals, as well as the males in
the penultimate dose group. Urine specimens also contained renal casts. None
of these manifestations of renal toxicity were seen in the females.
Some hepatocellular necrosis was seen in the males and females of the 375 and
750 rag/kg dose groups and in females from the 188 mg/kg dose group (NTP,1989).
Both the males and females of the 750 mg/kg dose group had statistical
increases in the relative weights of liver, heart, kidney and brain. Body
weights were depressed in the males and females of the high-dose group.
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An increased sensitivity of che male to kidney damage from HCE was apparent ;r.
the resulcs of this study. The LOAEL for kidney effects in the male was 47
rag/kg; there was no corresponding NOAEL. In the females, the NOAEL was 94
mg/kg with a LOAEL of 138 rag/kg, based on the occurrence of hepatocellular
necrosis (MTP,1989>.
b. Sixteen-week Studies
Gorzinski et al. (1980b) conducted a 16-week feeding study in male and female
CDF Fischer 344 rats (10/sex/group) in which the HCE (99.4% pure) was incor-
porated into the diet ac target dose levels of 0, 3, 30 and 100 mg/kg/day
(actual dose levels were analyzed to be approximately 0, 1.3, 20 and
32 mg/kg/day). Due to an apparent sublimation of the HCE in the feed during
mixing, diets were changed daily and were made available Co the animals for
approximately 17 hrs/day (3 PM to 8 AM) with water available ad libitum.
Further analysis of the eating pattern of the rats along with measurement of
the time-related loss of HCE from the diets led to a conservative estimate of
dietary intakes of 0, 1. 15 or 62 mg/kg/day (Gorzinski et al., 1985). Body
weights were recorded weekly and food consumption was measured for one to two
nonconsecutive days/week. The animals were observed for signs of toxicity on
a twice-weekly basis. Hematological evaluations were conducted at approxi-
mately three months. Urinalyses were done after two months and three months
on the test diets. Clinical chemistry parameters were measured at termination
and included BUD, SGPT, creatinine and alkaline phosphatase. After
109-110 days on the test diets the animals were sacrificed by decapitation
following an overnight fast. Terminal body and organ weights were recorded,
blood was drawn for chemical evaluations, and the eyes were examined by
fluorescent light illumination and abnormalities were recorded. The animals
were examined for gross abnormalities and all major organs were prepared for
histopathological examination. The liver, kidney and adrenals of all test
animals were microscopically evaluated while the remaining tissues from only
che control and high-dose groups were examined.
So signs of toxicity were noted and no deaths occurred during the course of
this feeding study. Body weight gain and food intake levels were comparable
between control and test groups. Clinical chemistry parameters were generally
comparable with the only significant alteration being a very slight decrease
in the BUN in the male rats receiving the lowest dose level. Hematological
determinations and routine urinalyses were comparable to controls. Total
urine volume was Increased in both sexes at all dose levels but not in a dose-
related manner. Female rats showed no difference in excretion of urinary
creatinine, porphyrins or delta-aminolevulinic acid (delta-ALA) while total
creatinine excretion was elevated in all treated males at the 3-month
evaluation. Excretion of coproporphyrin was unaffected in the males but the
uroporphyrin excretion was increased in males receiving the two highest doses
as measured during both evaluation periods. Delta-AIA was increased in all
treated males but was not dose-related ac che 60-day evaluation.
Upon necropsy, the kidneys of the male rats receiving the high-dose diet were
pale and mottled and the absolute and relative kidney weights were signi-
ficantly increased. Only one female from the high-dose group displayed a
mottling of the kidney and this change was considered equivocal. The absolute
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and relative liver weights were significantly increased in the high-dose males
while only the relative liver weights were significantly increased in females
at this dose level.
Histopathological changes in the kidneys of the high-dose males included an
increased, slight to moderate, renal tubular atrophy and degeneration with or
without peritubular fibrosis, increased renal tubular cytoplasmic clumping ar.ci .
droplet formation, also slight to moderate in degree, and scattered or
isolated renal tubules with slight hypertrophy and/or dilation of the proximal
convoluted tubules. Similar lesions, less severe in degree, were evident in
the 30 mgAg/day test males. In female rats receiving the 100 mg/kg/day dose,
the kidney alterations were limited to an increased incidence of very slight
renal tubular atrophy and degeneration.
Liver lesions were seen in the high-dose males and consisted of an increased
incidence of slight swelling of the hepatocytes. A similar effect was
considered equivocal in the males receiving the 30 mg/kg/day diet. No other
significant changes were seen in the tissues examined.
Evaluation of the results of this study indicate that the kidney is the target
organ for HCE toxicity and that the male rats are more sensitive to this
effect than the females. Based on this study, a NOAEL of 1.3 mg/kg/day (the
analyzed low dose) is indicted for kidney toxicity in rats.
c. Lifetime Exposure
The National Cancer Institute (NCI, 1978) conducted a 78-week cyclic oral
intubation study in 50 male and SO female Osborae-Mendel rats followed by a
treatment-free observation period of 34 weeks for the purpose of developing a
database for the carcinogenic evaluation of HCE. The rats received HCE in
corn oil at a low and high dose of 2S0 and 500 mg/kg/day, 5 days per week for
a period of 22 consecutive weeks, followed by a 1-week, treatment-free
interval. Thereafter, until the end of the 78 weeks, the rats were intubated
for 4 consecutive weeks followed by 1 treatment-free week, in a cyclical
pattern, for a total of 66 weeks of HCE treatment. Based on this schedule,
time-weighted average doses for the 78-week period were reported to be 212 and
423 mg/kg/day. Two control groups consisting of 20 rats/sex/group were either
untreated or received corn oil by gavage. Vehicle control rats were started
six weeks prior to the remaining groups and were approximately two weeks older
(8 weeks vs 6 weeks) when receiving their first intubation. The rats were
housed in individual cages and food and water were available ad libitum.
Body weights and food consumption were recorded weekly over the first 10 weeks
and at monthly intervals through the end of the study. The animals were
observed for signs of toxicity, behavioral changes and tissue masses and
mortality was recorded. At the end of the 112-week study, surviving animals
were euthanized by exsanguination under anesthesia and all major organs, as
well as any gross lesions, were subjected to both a gross and microscopic
examination. All animals dying or sacrificed moribund during the course of
this study were similarly examined.
Toxic signs, generally comparable at both dosage levels during the first year
of the study, included a hunched appearance, reddened, squinted or lacrimating
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eyes and urine staining of the abdomen. High-dose females, beginning at
Week 4 through termination, had a higher frequency of the abdominal urine
staining. During the second year of the study, the appearance and behavior of
the animals in all groups generally were comparable. Mean body weights of che
treated males were depressed in a dose-related manner while only the high-dose
females showed signs of a slight body weight depression. Respiratory abnor-
malities and signs of aging were comparable between all test and control
groups. Toxic signs such as transient tremors, ataxia and vaginal discharge,
seen in several treated animals, were considered isolated and spontaneous.
Significant mortality was noted in the treated males with survival to 90 weeks
being 48 and 38% for the low and high dose, respectively, as compared to
70 and 55% for the untreated and corn oil-treated controls, respectively.
Similar findings of significant dose-related mortality were reported for che
females with survival to the end of the study (112 weeks) being 70% for both
control groups as compared to 54 and 48% for the low and high doses,
respectively.
Significant histopathological lesions in the created animals were limited to
the kidney and were described as a toxic tubular nephropathy characterized by
degeneration, necrosis and the presence of large hyperchromatic regenerative
epithelial cells. Chronic interstitial nephritis and fibrosis, focal
pyonephritis, tubular ectasia, cast formation and focal glomerulosclerosis
were also reported. Four low-dose male rats also developed renal tubular-cell
adenomas but the incidence was not statistically significant. The incidence
of toxic nephropathy in the male rats was 45% (22/49) and 66% (33/50) for the
low and high doses, respectively, and 18% (9/50) and 59% (29/49) for the low-
and high-dose females, respectively. No control rats developed this lesion.
Ten percent of the high-dose male rats (3/29) also showed an increase in the
incidence of interstitial cell tumors of the testes which were first observed
at 109 weeks. This incidence was significant by the Cochran-Armitage test but
not under the parameters of the Fisher exact test. Mo significant neoplastic
lesions were reported in the rat study. Significant pathology and mortality
at both dose levels in the male rats precluded the development of a NOAEL or
L0AEL for HCE in rats chronically exposed by oral intubation.
Chronic toxicity studies were also conducted by NCI (1978) in 50 male and
50 female B6C3F} mice intubated orally with HCE in com oil at Initial levels
of 500 and 1,000 mg/kg/day for 8 weeks with these doses increased to 600 and
1,200 mg/kg/day, respectively, for the remaining 70 experimental weeks. A
time-weighted average dose of 590 and 1,179 mg/kg/day for the low and high
doses, respectively, was reported. The animals were dosed 5 days per week for
a total of 78 weeks, followed by an additional 13-week observation period
without treatment. Two control groups (20/sex/group) were included, one group
receiving corn oil by gavage while the remaining control group remained
untreated. The untreated controls were started on the experiment approxi-
mately 6 weeks prior to the orally dosed animals. All other experimental
parameters and observation intervals were the same as in the rat study.
No specific signs of toxicity were observed in the treated mice over the first
34 weeks of treatment. Thereafter, a greater frequency of hunched or thin
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appearance was seen in che treated groups. The incidence of palpable nodules
was also slightly increased in che created groups. Variations in body weight
gain were not dose-relaced. No significant relationship was seen between dose
and mortality rate. Survival of male control groups was unexpectedly low with
rates of 25 and 5% for che vehicle and untreated controls, respectively, as
compared to 14% for the low-dose males and 58% for the high-dose males.
Survival to the end of the experiment among female mice was 85% for untreated
controls, 80% for low-dose females and corn oil controls and 68% for the high-
dose females.
Non-neoplastic lesions in the mouse experiment included a toxic nephropathy
which was comparable between both sexes and both treatment groups with an
occurrence rate between 92 and 100%. The lesion was characterized by degener-
ation of the convoluted tubule epithelium at the cortical-medullary junction.
Hyaline casts were occasionally present and, in some cases, the damaged cells
were replaced by enlarged dark-stainir: regenerative tubular epithelium.
Infiltration of the inflammatory cell. fibrosis and calcium deposition was
often seen.
A statistically significant incidence or hepatocellular carcinomas was seen in
both the male and female high and low dose groups when compared to the matched
vehicle control group from this study or a pooled vehicle control groups from
other concurrent studies. Details on carcinogenicity are presented in
Section VI.B.5. (Carcinogenicity). Due to the occurrence of hepatocellular
carcinoma, as well as non-neoplascic toxic nephropathy in both sexes at both
dose levels, neither a NOAEL nor a LOAEL can be determined for mice orally
exposed to HCE over their lifetime.
In an effort to collect data concerning the toxicity of HCE to the kidney and
to examine the carcinogenicity of this chemical, the NTP carried out a 2-year
bioassay using groups of 50 male and 50 female Fischer-344 rats (NTP, 1989).
The animals were dosed by gavage for 5 days per week over a 2-year period.
The doses administered to the males and females differed due to the difference
in cheir sensitivity to HCE as determined in shorter duration studies. The
male rats received doses of 0, 10 or 20 mg/kg while the females received doses
of 0, 80 or 160 mg/kg. The animals were observed twice daily; initial
weighings were conducted weekly but decreased to monthly measurements as the
duration of the study increased. Following sacrifice, all the animals were
necropsied. The tissues of the controls and high-dose animals were subjected
to histopathological examination. Selected tissues, including the kidney,
were examined from the low-dose group animals.
In this 2-year bioassay, HCE had minimal effects on the body weights of the
exposed animals (5-9% lower than vehicle controls) and their longevity (NTP,
1989). There was a dose-related increase in the severity of nephropathy and
hyperplasia seen in the male rats. The occurrence of nephropathy was also
increased in the female rats. Tubular cell degeneration, dilation, atrophy,
inflammation and regeneration were apparent in both sexes but were more severe
in males. Damage to the renal papillae and pelvic transitional epithelium
occurred in the males. There was a dose-related increase in the combined
incidence of adenomas and carcinomas in the males with an occurrence of 1 for
the controls, 2 for the low-dose group and 7 for the high-dose group.
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Adenomas or carcinomas were not present in exposed females. Pheochromocvtorras
of che adrenal gland were also present in some of -he males. The details on
"he carcinogenicity of HCE are discussed in Section VI.B.5 (Carcinogenicity).
Due to the occurrence of carcinomas in the males, this study is not
appropriate as the source of a NOAEL or LOAEL for long-term chronic exposure
to HCE.
NIP hypothesized that the increased sensitivity of males in the renal toxicicv
of HCE was a product of che accumulation of a^y-globulin in hyaline droplets
in the tubular epithelial cells. a^Q-Globulin is a protein which is
synthesized by che liver and secreted into che blood. Ic is then, apparently,
filtered chrough che glomeruli and partially reabsorbed through che proximal
tubules. In the presence of HCE, as well as several nonpolar hydrocarbons
such as decalin and gasoline, <22y globulin accumulates in hyaline droplets in
the kidney cells. Based on limited evidence, a^y-globulin is an excretory
protein in male but not female rats. This may explain the male susceptibility
to kidney damage from HCE.
3. Reproductive Effects
No information was found in the available literature regarding the
reproductive effects of HCE in animals.
t*. Developmental Effects
Weeks et al. (1979) conducted teratology studies in pregnant female Sprague-
Dawley rats (22/group) given oral doses of HCE (99.8% pure) by gavage from
Day 6 through Day 16 of gestation at levels of 0, 50, 100 or 500 mg/kg/day.
All animals were sacrificed by intracardiac injection of sodium pentobarbital
on Day 20 of gestation. Parameters examined included number of corpora lucea,
implancation sices and resorption sites. Viable fetuses were examined for
gross abnormalities and the sex, weight and body length were recorded. All
grossly abnormal fetuses along with eight normal fetuses per dam were further
examined for soft cissue abnormalities and skeletal malformations.
Pregnant females receiving the high dose of 500 mg/kg/day demonstrated a
significant reduction in body weight gain beginning at Day 8 of gestation.
Significantly lover gestation Indices were recorded among the fetuses from the
high-dose exposure group. The number of viable fetuses per dam were also
reduced while a higher fetal resorption rate was recorded. No significant
skeletal or soft tissue anomalies were seen in any of the HCE-exposed fetuses.
Maternal and fetal parameters were comparable to controls in the two lower
exposure groups. Histological examination of the dams revealed an increased
incidence (70%) of upper respiratory tract infection with subclinical
pneumonitis evident in 20%. Only 10% of control dams were similarly affected.
Based on this study, HCE is not a teratogenic agent at exposure levels up to
500 mg/kg/day. The high dose level was maternally toxic and resulted in a
slowing of fetal development. A NOAEL for maternal and fetotoxic effects of
100 mg/kg/day is indicated.
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5.
Carcinogenicity
In a study conducted by NCI (1978) in Osborne-Mendel rats and B6C3F^ mice
orally intubated with HCE in corn oil, a statistically significant incidence
of hepatocellular carcinoma was reported in the mice. The mice were dosed
5 days/week for a period of 78 consecutive weeks with an untreated observation
period of an additional 13 weeks to yield time-weighted average doses of 5.90
and 1,179 mg/kg/day in the low- and high-dose mice, respectively. Details on
the parameters for both experiments are contained in Section VI.B.2.b.
(Lifetime Exposure). Due to an unusually high mortality rate among the male
control groups, the incidence was statistically compared against both the
vehicle control group from this study as well as a pooled vehicle control
group from several concurrent studies. The incidence was significant by the
Cochran-Armitage test (p<0.001) for both sexes against both controls and by
the Fisher exact tests (p<0.008) for both sexes as compared to the pooled
controls. The hepatocellular carcinomas varied greatly in appearance with
some containing well-differentiated hepatic cells with a relatively uniform
arrangement of cords and others containing anaplastic cells with large
hyperchromatic nuclei often containing inclusion bodies with vacuolated pale
cytoplasm. The neoplastic liver cells varied in arrangement from short,
stubby cords to nests of hepatic cells. Occasionally a pseudo-acinar
formation was seen and mitotic figures were often present. Incidence figures
for the occurrence of hepatocellular carcinomas in the mouse study are
contained in Table VI-1. No treatment-related neoplasms were reported in the
rat study, although an increased incidence of interstitial cell tumors of the
testes was reported in 10% of the high-dose male rats. The incidence (3/29)
was significant by the Cochran-Armitage test but not under parameters of the
Fisher exact test.
Lifetime exposures to HCE resulted in renal carcinomas and adenomas in
Fischer-344 rats during a bioassay conducted by NTP (1989). Groups of
50 males and 50 females were dosed with HCE in com oil by gavage for 5 days
per week over a 2-year period. The doses administered to the males and
females differed due to a difference in their renal sensitivity to HCE. The
male rats received doses of 0, 10 or 20 mg/kg while the female doses were 0,
80 or 160 mg/kg. Following sacrifice all the animals were necropsied. The
tissues of the controls and high-dose animals were subjected to histopatho-
logical examination. Selected tissues, including the kidney, were examined
from the low-dose group animals. There was a dose-related Increase in the
combined incidence of adenomas and carcinomas in the males with an occurrence
of 1 for the controls, 2 for the low-dose group and 7 for the high-dose group.
Carcinomas were only identified in the high-dose group (3/50). Each carcinoma
was apparent on gross examination of the organ; one carcinoma had metas-
tasized. The number of tumors seen in the high-dose group was significantly
greater than that for both the concurrent controls (p>0.03, Fisher Exact Test)
and the historical controls. The historical incidence of renal tubular cell
adenomas is 1/300. The exposed females were not affected. Pheochromocytomas
of the adrenal gland were also present in some of the males. The renal
lesions were considered by NTP to be indicative of compound carcinogenicity
while the pheochromocytomas were judged to be supportive evidence for carcino-
genicity. On the basis of these data, NTP concluded that there was clear
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evidence of carcinogenicity for HCE in the male rat and no evidence of
carcinogenicity in female racs.
6. Cenocoxicisv
Simon and Kauhanen (1978) tested HCE dissolved in DMSO in the standard
Salmonella/microsome assay (strains TA1535, TA1537, TA1538, TA98 and TA100) a
concentrations ranging from 10 to 10,000 ug/plate, with and without addition
of a metabolic activation system (S-9 fraction). No mutagenic response was
obtained ac any concentration. A slight toxicity was seen at the
10,000 ug/plate concentration without metabolic activation. When tested with
Saccharomvces cerevislae D3 at a dose range of 0.1 to 5.0%, no dose-related
increase in the number of mitotic recombinants was seen.
No evidence of mutagenicity was seen in a series of jjj vitro microbial assays
conducted by Ueeks et al. (1979). Hexachloroethane was tested at doses
ranging from 0.1 to 500 ug/plate, with and without metabolic activation with
liver enzyme preparation, in Salmonella tvuhimurium (strain TA-1535, TA-1537,
TA-1538, TA-98 and TA-100) and in 2^. cerevisiae (strain D4).
Ndkaraura et al. (1987) tested HCE for its potential to induce DMA damage in a
newly developed test which is based on the ability of a chemical to induce
urm,! gene expression, as measured through cellular beta-galactosidase activity
This study utilized a new 5^ typhimurium tester strain, TAl535/pSK1002, in
which an umuC-lacZ fused gene had been introduced. The ability for HCE to
induce a genotoxic response was measured by the beta-galactosidase activity
produced by the fused gene. Vhen tested at concentrations up to 42 ug/mL,
with and without metabolic activation (S9), HCE vas considered nongenotoxic.
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Table VI-L Incidence of Hepatocellular Carcinoma
in B6C3Fj_ Mice Orally Dosed with HCE for up co 78 Weeks3
Dose	Male	Female
590 mg/kg/day
15/50b
2Q/50b
1,179 mg/kg/day
31/49b
15/49b
Pooled vehicle control
6/60
2/60
Matched vehicle control
3/20
2/20
Untreated control
1/18
0/18
Reference: NCI, 1978
a Number with lesion/number examined.
b Significant (p <0.001) by the Cochran-Armitage test, when compared vith
either the matched or pooled vehicle control.
c Combined controls from HCE and two additional concurrent studies.
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Table VI-2 Incidence of Renal or Renal Tubule Lesions
in Male Racs Orally Dosed wich HCE for Two Years

Vehicle
Control
10 mg/kg
20 mg/kg
Hyperplasia
2/50
4/50
11/50
Adenoma
1/50
2/50
4/50
Carcinoma
0/50
0/50
3/50
Adenoma or carcinoma
1/50
2/50
7/50
Reference: NTP, 1989
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VII
HEALTH ADVISORY DEVELOPMENT
Available data on Che toxic effects of HCE in several species, for periods
ranging from single dose oral LD5QS to continuous exposure studies in the dier
of rats (16 weeks), have been evaluated. Carcinogenic studies in rats and
mice, developmental toxicity studies in rats and genotoxicity assays are also
included. Studies on the reproductive effects of HCE exposure are not
available. Exposures to HCE by inhalation are reported for several species.
Human exposure data is limited, with "irritation" the only effect reported
following industrial exposure.
Toxic effects of HCE (12 days to 16 weeks) are generally related to its
effects on the kidney, although liver toxicity has also been indicated in
several studies (Weeks et al., 1979; Gorzinski et al., 1980a,b; NIP, 1989).
In rats and sheep, acute exposure has resulted in CNS effects which include
incoordination, tremor, ataxia and signs of gasping (Fowler, 1969; Weeks and
Thomasino, 1976). Exposure by inhalation resulted in an increased incidence
of mucopurulent exudate in rats and quail, and CNS-related effects in rats and
dogs (Weeks et al., 1979). Lifetime administration by oral intubation to rats
and mice resulted in a significant increase in neoplastic lesions in the
livers of mice (NCI, 1978). Skin and eye irritation studies indicated that
HCE is a mild irritant in rabbits (Weeks and Thomasino, 1976; Weeks et al.,
1979), but no dermal sensitization was demonstrated in guinea pigs (Weeks and
Thomasino, 1976). Dermal toxicity is low.
Indications of the toxic effects of HCE in the kidneys were first seen by
Weeks and Thomasino (1976) in the cortex of a male rat that died while part of
an LD5Q study. A non dose-related toxic tubular nephrosis was reported in New
Zealand white rabbits following 12 days of oral intubation with HCE at levels
of 320 and 1,000 mg/kg/day (Weeks et al., 1979). Increased kidney weight and
evidence of gross lesions were seen in male rats following 16 days of feeding
with HCE at 200 and 500 mg/kg/day. Females were not similarly affected
(Gorzinski et al., 1980a). In 1980(b), Gorzinski et al. reported the
development of pale and mottled kidneys, with a significant increase in
absolute and relative weight, in male rats fed HCE for 16 weeks at a level of
100 mg/kg/day. The histological changes were described as slight to moderate
renal tubular atrophy and degeneration, slight to moderate tubular cytoplasmic
clamping and droplet formation, and scattered to isolated hypertrophy and/or
dilatation of the proximal convoluted tubule. Similar lesions, less severe in
degree, were also reported in the rats fed at the 30 mg/kg/day test level.
These levels were analyzed to contain actual amounts of 82 and 20 mg/kg/day,
respectively. High dosed females exhibited only a very slight renal tubular
atrophy and degeneration.
No kidney lesions were seen following inhalation exposure of dogs, rats,
guinea pigs and quail at levels up to 260 ppm (equivalent to approximately
2,515 mg/nr). Exposure duration was for 6 hours/day, 5 days/week for 6 weeks.
In a lifetime bioassay in rats and mice, NCI (1978) reported the development
of histopathological lesions in the kidneys of rats orally dosed at a time-
weighted average of 212 and 423 mg/kg/day. Exposure by oral intubation took
VII-1

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place 5 days/week in a cyclic manner virh '* weeks of dosing followed by
1 treatment:-free week for a total of 73 weeks. The lesions were described as
those found in toxic tubular nephropathy and were evident in both sexes at
both dose levels. They were characterized by signs of degeneration, necrosis,
and the presence of large hyperchromatic regenerative renal cells. Chronic
interstitial nephritis, fibrosis, focal pyonephritis, tubular ectasia, case
formation and focal glomerulosclerosis were also seen. Renal tubular cell
adenomas were reported in four of the low-dose male rats, but the incidence
was not statistically significant.
Similar findings of nephropathy were seen in male and female rats during a
2-year bioassay of HCE (NTP, 1989). The males received doses of 0, 10 and 20
mg/kg in corn oil by gavage 5 days per week for a lifetime; the corresponding
doses for females were 0, 80 and 160 mg/kg. In this study, the incidence of
renal adenomas plus carcinomas in the males was significantly greater than in
the controls; the non-neoplastic damage to the male kidneys was also more
pronounced than that seen in the females.
Toxic nephropathy was also reported in mice treated with time-weighted average
doses of 590 and 1,179 mg/kg/day over 78 weeks of continuous exposure. The
incidence, 92 and 100% for the low and high dose, respectively, was comparable
between sexes. Lesions were characterized by a degeneration of the epithelium
of the convoluted tubules. Calcium deposition, fibrosis and infiltration of
inflammatory cells was also seen.
Early indications of liver toxicity due to the oral administration of HCE were
first seen in studies with sheep (Fowler, 1969). The sheep were dosed with
HCE at 0.5 to 1.0 g/kg by drenching bottle. A significant increase in the
plasma enzymes indicative of liver toxicity was seen 48 hours after treatment.
Results of a BSP clearance test indicated that toxicity was due to a reduction
in the excretory capacity of the liver. No changes in the chemical or
function properties of liver microsomes were seen following a single oral dose
of 6 g/kg in rats (Reynolds, 1972). When New Zealand white rabbits were
administered HCE in methylcellulose at doses up to 1,000 mg/kg/day for
12 days, relative liver weights were significantly increased at the high dose
level (Weeks et al., 1979), with a non-significant increase in alkaline
phosphatase, SCOT and bilirubin. Coagulation necrosis, hemorrhage and fatty
degeneration were seen upon histopathological examination of livers from the
rabbits receiving doses of 320 and 1,000 mg/kg/day. Gross liver lesions were
observed in male rats fed HCE in the diet at 500 mg/kg/day for 16 days with a
trend toward increased liver weight apparent in both sexes (Gorzinski et al.,
1980a). Feeding of HCE to rats at levels up to 100 mg/kg/day for a period of
16 weeks resulted in an increased incidence of a slight swelling of the
hepatocytes at the highest dose level with equivalent changes at the next
highest dose of 30 mg/kg/day (actual levels analyzed at 82 and 20 mg/kg/day,
respectively; Gorzinski et al., 1980b).
No liver lesions were observed in dogs, rats, guinea pigs and quail exposed to
HCE by inhalation at levels up to 260 ppm (ss 2,515 mg/m ). Exposure was for
6 hours/day, 5 days/week for a period of 6 weeks. In a bioassay in rats and
mice (NCI, 1978), no liver lesions were seen in rats orally dosed with HCE at
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cime-weighted average doses up co 423 mg/kg/day for 78 weeks. Mice similar'.-/
created at time-weighted average doses of 590 and 1,179 mg/kg/day developed a
statistically significant incidence of hepatocellular carcinomas. The lesior.s
were seen ac both dose levels and were significant when paired to both the
matched vehicle controls and pooled vehicle controls from other studies.
An increased incidence of interstitial cell tuaors of the testes was reported
in 10% of the male rats orally dosed with HCE at a time-weighted average dose
of 423 mg/kg/day for 78 weeks in a cyclical pattern (NCI, 1978). The
incidence was significant by the Cochran-Annitage test but not the Fisher
exact test.
No reproductive data were located in the available literature but develop-
mental studies indicated that HCE is not teratogenic in rats. Exposure was
both by oral intubation and by inhalation on days 6 through 16 of gestation.
Doses were up to 500 mg/kg/day in the oral study and up to 260 ppm
(s2,515 mg/nr) in the inhalation study (Weeks et al., 1979). Indications of
maternal as well as fetotoxic effects were seen in the oral study, as
evidenced by a significant decrease in maternal body weight gain at Day 8, as
well as a significant decrease in gestation indices. These Included a
decrease in the number of viable fetuses per dam and higher fetal resorption
rate. Histological examination revealed an increased incidence of upper
respiratory infection with subclinical pneumonitis. Significant decreases in
maternal weight gain were seen in the inhalation study at both the 48 and 260
ppm levels. No other maternal or fetotoxic effects were affected by
inhalation of HCE although maternal rats displayed an increase in the
incidence of mucopurulent nasal exudate at the 260 ppm exposure level.
An increased incidence of mycoplasma-related lesions was also seen in male and
female rats exposed to HCE vapor at 260 ppm for 6 hours/day, 5 days/week for
6 weeks. The lesions were subclinical and consisted of mucopurulent exudate
affecting the nasal turbinates, trachea and lungs of the exposed rats. Excess
mucus was also evident in the nasal turbinates of quail similarly exposed
(Weeks et al., 1979).
No skin irritation was indicated in rabbits exposed to dry technical grade
HCE, but a mild irritation resulted from exposure to a HCE paste (Weeks and
Thomasino, 1976). No dermal sensitization was seen in guinea pigs. Dermal
toxicity was mild with an LD50 of >32 g/kg reported. Moderate corneal
opacity, as well as iritis and severe swelling and discharge, developed in
rabbits exposed to a single application of dry HCE to the cornea.
a. Quanciglsflti9n qf T
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where:
NOAEL or LOAEL - No- or Lowest-Observed-Adverse-Effecc Level
in mg/kg bw/day.
BW - assumed body weight of a child (10 kg) or
an adult (70 kg).
UF - uncertainty factor (10, 100 or 1,000), in
accordance with NAS/ODW guidelines.
	L/day - assumed daily water consumption of a child
(1 L/day) or an adult (2 L/day).
1.	One-dav Health Advisory
No data were located in the available literature that were considered suitable
for the calculation of a One-day HA. It is suggested that the Ten-day HA of
5 mg/L for the 10 kg child be used as a conservative estimate for the One-day
HA.
2.	Ten-dav Health Advisory
The study of Gorzinski et al. (1980a) has been selected to serve as the basis
for developing a Ten-day HA for the 10 kg child. Two studies are of appro-
priate duration to be considered for development of a Ten-day HA. Upon gross
examination, kidney lesions were seen in male rats fed HCE in the diet at 200
and 500 mg/kg/day for 16 days (Gorzinski et al., 1980a) and in New Zealand
white rabbits orally dosed with HCE at 320 and 1,000 mg/kg/day for 12 days
(Weeks et al., 1979). Liver degeneration and necrosis, as well as a signi-
ficant decrease in body weight gain, were also seen in the rabbits at both
treatment levels, while a significant decrease in weight gain and gross liver
lesions were seen in the male and female rats at the 500 mg/kg/day level. The
weight loss in rabbits receiving the 320 mg/kg/day dose developed on Day 10 of
this 12-day study. Initial results of these studies indicate apparent NOAELs
of 100 mg/kg/day in rabbits and of 50 mg/kg/day in rats. In view of the late
developing decrease in body weight gain in the rabbit study, the possibility
of this toxic sign also occurring over time in the rabbits at the
100 mg/kg/day level muse be considered. In addition, the lack of histo-
pathological data in the rat study warrants the use of caution in selection of
an appropriate NOAEL. It is, therefore, recommended that the more conser-
vative NOAEL of 50 mg/kg/day from the 16-day rat study be used to determine a
Ten-day HA which is calculated as follows:
For the 10 kg child:
Ten-day HA - (50 mg/ky/dav) (10 kg) - 5 mg/L (5,000 ug/L)
(100) (1 L/day)
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where:
50 mg/kg/day - NOAEL based on absence of effects on the liver,
kidney and growth rate of rats exposed to HCE in the
diet for 16 days.
10 kg - assumed weight of a child.
1 L/day - assumed water consumption by a 10 kg child.
100 - uncertainty factor; this uncertainty factor was
chosen in accordance with NAS/ODW guidelines in which
a NOAEL from an animal study is employed.
3. Longer-term Health Advisory
The only study of longer duration in which a NOAEL which applied to both sexes
could be determined was the 16-week study of Gorzinski et al. (1980b) in which
rats were fed HCE in the diet at target levels of 0, 3, 30 or 100 mg/kg/day.
An analysis of the diets indicated that actual levels of consumption were in
the range of 0, 1.3, 20 and 82 og/kg/day. Based on the occurrence of kidney
lesions in the male rats at the two highest dose levels, a NOAEL of
1.3 mg/kg/day was indicated. There was no NOAEL for male rats in the NTP
(1989) 13-week study in which kidney damage was apparent at doses of 47 mg/kg
and greater. The kidneys of female rats were only mildly affected by HCE
exposure at the highest dose level, and this change was considered equivocal.
Liver lesions were also seen in the high-dose males with equivocal changes in
the liver at the next lower level. Using the NOAEL of 1.3 mgAg/day. the
Longer-term HA is calculated as follows:
For the 10 kg child:
Longer-term HA - (1.3 mg/kg/dav) (10 kel - 0.13 mg/L
(100) (1 L/day)	(rounded to 100 ug/L)
where:
1.3 mg/kg/day - NOAEL based on absence of kidney and liver lesions in
racs exposed to HCE in the diet for 16 weeks.
10 kg - assumed weight of a child.
1 L/day - assumed water consumption by a 10 kg child.
100 - uncertainty factor: this uncertainty factor was
chosen in accordance with NAS/ODW guidelines in which
a NOAEL from an animal study is employed.
For the 70 kg adult:
Longer-term HA - <1.3 mg/ky/d&v^ (70 kg) - 0.455 mg/L
(100) (2 L/day)	(rounded to 450 ug/L)
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where
1.3 mg/kg/day - NOAEL based on absence of effects on the kidnev and
liver of rats exposed to HCE in che diec for 16
weeks.
70 kg - assumed weight of an adult.
2 L/day - assumed water consumption by a 70 kg adult.
100 - uncertainty factor; this uncertainty factor was
chosen in accordance with NAS/ODW guidelines in which
a NOAEL from an animal study is employed.
U. Lifetime Health Advisory
The Lifetime HA represents that portion of an individual's total exposure that
is attributed to ci .r.king water and is considered protective of noncarctno-
genic adverse heal-,:: effects over a lifetime exposure. The Lifetime HA is
derived in a three-step process. Step 1 determines the Reference Dose (RfD),
formerly called the Acceptable Daily Intake (ADI). The RfD is an estimate of
a daily exposure to the human population that is likely to be without
appreciable risk of deleterious effects over a lifetime, and is derived from
the NOAEL (or LOAEL) , identified from a chronic (or subchronic) study, divided
by an uncertainty factor(s). From the RfD, a Drinking Water Equivalent Level
(DWEL) can be determined (Step 2). A DWEL is a medium-specific (i.e.,
drinking water) lifetime exposure level, assuming 100% exposure from that
medium, at which adverse, noncarcinogenic health effects would not be expected
to occur. The DWEL is derived from the multiplication of the RfD by the
assumed body weight of an adult and divided by the assumed daily water
consumption of an adult. The Lifetime HA is determined in Step 3 by factoring
in other sources of exposure, the relative source contribution (RSC). The RSC
from drinking water is based on actual exposure data or, if data are not
available, a value of 20% is assumed. If the contaminant is classified as a
Group A or B carcinogen, according to the Agency's classification scheme of
carcinogenic potential (U.S. EPA, 1986), then caution should be exercised in
assessing the risks associated with lifetime exposure to this chemical.
Hexachloroethane is classified in Group C: Possible Human Carcinogen,
according to EPA's weight-of-evidence scheme for classification of carcino-
genic potential. Because of this, caution must be exercised in making a
decision on how to deal with possible lifetime exposure to this substance.
The risk manager must balance this assessment of carcinogenic potential
against the likelihood of occurrence of health effects related to noncarcino-
genic end points of toxicity. In order to assist the risk manager in this
process, drinking water concentrations associated with estimated excess
lifetime cancer risks over the range of one in ten thousand to one in a
million are provided in the following section. In addition, in this section,
a Drinking Water Equivalent Level (DWEL) is derived. The DWEL and estimated
cancer excess are determined for the 70 kg adult, ingesting two liters of
water per day. Also provided is an estimate of the excess cancer risk that
would result if exposure were to occur at the DWEL over a lifetime.
VII-6

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Neither che cancer risk estimates nor The DwEL cake relative source
contribution into account. The risk manager should do this on a case-bv-case
basis, considering che circumstances of the specific contamination incident
chat has occurred.
The 16-week study of Gorzinski et al. (1980b) has also been selected to serve
as a basis for the Lifetime HA for HCE. The 78-week screening study by NCI
(1978), using mice and racs, and the NIP (1989) lifetime study in rats failed
to establish a NOAEL or LOAEL for HCE. A dose of 47 mg/kg/day (NTP, 1989) in
rats and 590 mg/kg/day in mice (NCI, 1978) produced significant signs of
toxicity in che liver and/or kidney of both species. In addition, a signi-
ficant increase in the incidence of hepato-cellular carcinomas was reported ac
590 mg/kg/day in the study in mice (NCI, 1978), and an increased incidence of,
renal neoplasms in male rats ac a dose of 20 mg/kg/day (NTP, 1989). Therefore
the NOAEL of 1.3 mg/kg/day (Gorzinski et al., 1980b) may be used to calculate
the Lifetime HA as follows:
Step 1: Determination of Reference Dose (RfD)
RfD - (1.3 mg/kg/dav) - 0.0013 mg/kg/day
(1000)	(rounded to 1 ug/kg/day)
where:
1.3 mg/kg/day - NOAEL based on the absence of effects on the kidney
and liver of rats exposed to HCE in the diet for 16
weeks.
1000 - uncertainty factor; this uncertainty factor was
chosen in accordance with NAS/0DW guidelines in which
a NOAEL from a less-than-lifetime animal study was
employed to derive an RfD.
Step 2: Determination of a Drinking Water Equivalent Level (DUEL)
DWEL - fP.QQI fig/kg/day) (70 - 0.035 mg/L
(2 L/day)	(rounded to 40 ug/L)
where:
0.001 mg/kg/day - RfD
70 kg - assumed body weight of an adult
2 L/day - assumed daily water consumption of an adult.
Step 3: Determination of the Lifetime Health Advisories
Lifetime HA - fQ.0351 (0.2) - 0.0007 mg/L
10	(rounded to 1 ug/L)
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where:
0.2 - Relative Source Contribution
10 - Extra uncertainty factor to account for equivocal
evidence of carcinogenicity, used in accordance with
ODW Policy for Group C chemicals.
B. Quantification of Carcinogenic Potential
Applying the criteria described in EPA's guidelines for assessment of
carcinogenic risk (U.S. EPA, 1986), HCE is classified in Group C: Possible
Human Carcinogen. This category applies to agents for which there is
inadequate evidence from human studies and sufficient evidence from animal
studies.
The risk manager must balance Che assessment of carcinogenic potential against
the likelihood of occurrence of health effects related to noncarcinogenic end
points of toxicity. In order to assist the risk manager in this process,
drinking water concentrations, associated with cancer risks over the range of
one excess tumor in populations of ten thousand (10*^) to one excess tumor in
populations of one million (10'6) for the 70 kg adult, drinking 2 liters of
water per day, are provided.
Technical grade hexachloroethane (98% pure) was administered by gavage to 50
each male and female Osborne-Mendel rats and B6C3F^ mice (NCI, 1978). Rats
were treated with either 250 or 500 mg HCE/kg bw/day, 5 days/week for
22 weeks. After this time animals were cyclically rested 1 week and gavaged
for 4 succeeding weeks up to Week 78; an observation period of 3334 weeks
followed. TWA final treatment doses were 212 and 432 mg/kg bw/day. There was
no evidence of hexachloroethane- induced neoplastic growth in rats.
The mice were administered 500 or 1,000 mg/kg/day, 5 days/week, continuously.
At Ueek 9, the doses were increased to 600 and 1,200 mg/kg/day and this dosage
was maintained until Ueek 78. The mice were observed for 12-13 weeks after
cessation of treatment. The TVA doses were 590 and 1,179 mg/kg bw/day. Mice
of both sexes showed a significant increase in the incidence of hepatocellular
carcinoma.
The multistage model was used for high-to-low dose extrapolation (Crump and
Watson, 1979; Howe and Crump, 1982), and the 95% upper confidence limit was
used to assess risk as an upperbound estimate. The multistage model conforms
to a biological model of tumor initiation and promotion (Crump et al., 1977).
The relationship of the concentration (ug/L) of a chemical in drinking water
to cancer risk is expressed as follows:
qi*
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where
qi* - (mg/kg/day)*1
R - risk (10*4, 10"5 . LO"6, ecc.)
C - concentration of chemical in ug/L
3 5,000 - conversion factor for mg to ug and assumption that a
70 kg adult drinks 2 L of water/day
The administered doses were TWA-adjusted for frequency of exposure (i.e., from
5 to 7 days). Human equivalent doses were adjusted for length of exposure
(546 days of a potential lifespan of 637) and weight of the animals (assumed
to be 0.032 kg). The vehicle control group incidence data were used in
modeling.
The human slope factor (q]_*) is 1.4 x 10" ^ (mg/kg/day) * ^ for the linearized
multistage model. The q^* is taken as an upper bound of potency of the
chemical co induce cancer at low doses below the experimental dose range.
Assuming that a 70 kg adult consumes 2 L of water a day over a 70-year
lifespan, the estimated cancer risk is as follows (the dose is rounded):
Level of
Human Risk
10*4	300
10*5	30
10-6	3
For comparison purposes, drinking water concentrations associated with an
excess cancer risk of 10"6were 1 ug/L, 200 ug/L, 5,000 ug/L, 50 ug/L, and
2 ug/L for the one hit, multihit, probit, logit, and Veibull models,
respectively. The parameters estimates for these models were calculated with
RISK-81 (Kovar and Krewski, 1981).
The estimated excess lifetime cancer risk associated with lifetime exposure to
drinking water containing hexachloroethane at 60 ug/L is approximately 2x10*^.
The estimated excess lifetime cancer risk associated with lifetime exposure to
drinking water containing hexachloroethane at 6 ug/L is approximately 2x10*.
This represents the 95% upper confidence limit on risk from extrapolation
using the linearized multistage model. The actual risk is unlikely to exceed
this value.
VII -9

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VIII. OTHER CRITERIA ffllTDANCE AND STANDARDS
The ACGIH (1986) 8-hour, cime-weighted, average threshold limit value (TVA-
TLV) for exposure to HCE is 10 ppm (100 mg/ar). In 1990, ACGIH proposed co
lower the TWA-TLV to 1 ppm (9.7 mg/m3) and to classify HCE as an A2 carcinogen
(suspect human carcinogen; based on limited evidence of carcinogenicity in
experimental animals) (ACGIH, 1990; Hexachloroethane, 1990). The Occupational
Safety and Health Administration permissible exposure limit (PEL) as indica-ed
in NIOSH (1985) remains at 1 ppm (sslO mg/nr) for an 8-hour TWA. NIOSH (1985)
recommends that HCE be created as a carcinogen and exposure be limited co the
lowest feasible limit.
The U.S. EPA (1980) published HCE ambient water quality criteria for human
health at 19 ug/L, 1.9 ug/L, and 0.19 ug/L associated with lifetime excess
cancer risks of 10 , 10 , and 10" , respectively. The criteria assumed that
contaminated aquatic organisms are consumed in addition to water.
In Russia, the maximum allowable concentration (MAC) in reservoir water is
0.01 ppm (Sax, 1986), while the recommended maximum contaminant level (RMCL)
is reported to be 10 g/L (Davidson et al., 1988).
VIII-1

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IX.
ANALYTICAL METHODS
Analysis for HCE in water generally involves an extraction step with an
organic solvent followed by analysis using various modifications of gas
chromatographic techniques.
In 1982, Otson and Williams described a modification of a purge and trap
technique for the analysis of organics in water. In this procedure, a 40 mL
aliquot water sample was injected into a modification of a Bellar and
Lichtenberg glass purge assembly which was immersed into a 40*C temperature-
controlled water bath for 20 minutes. Ultra high purity nitrogen was then
bubbled through the device at a rate of 40 mL/min for 30 minutes and on-column
trapo^ng was initiated. The Tenax GC column (60/80 mesh), held at 40*C during
the purging phase, was raised at a rate of 20*C/min to 100C and then at
8C/min to 190C where it was maintained for 6.75 minutes. Nitrogen carrier
gas, at a rate of 37 mL/min, was directed to either a flame ionization
detector (FID) or a Hall electrolytic conductivity detector (HECD).
Hexachloroethane was detectable to 1 ug/L of water on the HECD detector with
an electrolytic solvent of 2-propanol/water (50/50, v/v), hydrogen gas flow of
10 mL/min and a furnace temperature of 820*C. Retention time, relative to the
start of the temperature program, was 14.9 minutes and resulted in its
detection as a broad peak. A straight line was obtained when the concen-
tration was plotted against peak area for aqueous standards over the range of
0.23 to 16 ug/L. Precision, as determined from triplicate analyses of aqueous
standard samples at 16 ug/L, was reported as 12% relative standard deviation.
Concentrations above 16 ug/L were not evaluated. Purging efficiency using the
HECD, as percent recovery by comparison of peak area values from dynamic
headspace analysis of aqueous standards at 40*C and direct injection analysis
of equivalent methanolic standard solutions, was reported to be 82%.
A similar method for analysis of organics in water vas described by Pankow ec
al. (1982) and involved the adsorption of the organics from aqueous solutions
onto a polymeric sorbent followed by thermal desorption (ATD) with analysis by
capillary gas chromatography (GC). The problem of desorption from a large
volume cartridge [bed length 6.0 cm, inside diameter (i.d.) 0.69 cm] with a
flow rate of approximately 30 mL/min to a fused silica capillary column
(length 30 m, i.d. 0.25 ma) with a flow rate of approximately 1-2 mL/min was
handled by addition of an interface cartridge between the two systems. To
insure good chromatographic resolution without loss of sensitivity, the
authors used a cold-trap method to retain the analytes at the head of the GC
column until desorption was complete and GC analysis could proceed. A small,
fixed 35/60 mesh Tenax GC interface cartridge (bed length 5.0 cm, i.d. 0.40
cm) made of Pyrex glass was connected between the Pyrex glass sample cartridge
(packed with 35/60 mesh Tenax-GC) and the DB-5 (SE-54) fused silica capillary
column by means of a short piece of small inside diameter (0.5 mm) glass-lined
stainless steel tubing. During the desorption process, the column was
maintained at a subambient temperature of -30'C with liquid nitrogen to trap
the analytes at the column head. Analysis was carried out with a flame
IX-1

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ionization detector at 300*C with helium as the carrier gas. The column vas
heated ballistically from -30 to 5CTC and then at a rate of 4C/min from 50
250C. Recovery efficiency for HCE by this method vas reported to be betveer.
62 and 75%. Detection limits for this procedure were not determined but were
predicted to be in the 1.8-3.8 ng/L range.
Eichelberger et al. (1983) compared the scope and limitations of two methods
of simultaneous identification and measurement processes for HCE and other
organics in a complex sample matrix. In Method 625, the analytes were twice
partitioned between pH adjusted water and methylene chloride in a separatory
funnel, first at pH 11 and Chen at pH 2. Extracts were analyzed separately
after drying and concentration to a low volume. An internal standard,
anthracene-d^g was a<*ded t0 each extract. Analysis was by gas chromatography
interfaced with mass spectrometry, with an all-glass transfer line and an all-
glass, jet-type enrichment device. The column was a 1.8 mx 2 mm i.d. glass
tube packed with 3% SP-2250 on 100/120 mesh Supelcoport. The injector
temperature was 260*C and the transfer line temperature was 240#C with helium
as the carrier gas at a flow rate of 30 mL/min. With this method, a 3.8 ug/L
concentration of HCE in water had a mean recovery of 76% and a standard
deviation of 23% of the mean recovery value.
Method 625.1 used essentially the same extraction procedure with the exception
that the first separation was at pH 7 and the second remained at pH 2. Three
internal standards, naphthalene-dg, anthracene-d^Q and chrysene-d^. were
added to the dried concentrated extract for GC analysis. In this method, the
capillary column was of fused silica (28 m x 0.25 mm i.d.) coated with SE 54.
The column temperature was maintained at 40*C for 4 minutes and programmed to
increase at 10*C/min to a temperature of 275*C. The injector and interface
oven temperatures were maintained at 260*C and the heliua carrier gas had a
linear velocity of 28 cm/sec. This method also interfaced with a mass
spectrometer. Mean recovery was 55% for a 10 ug/L sample of HCE in water,
with a standard deviation of 6.2% of the mean recovery value.
Fisher et al. (1985) used a modification of Standard Method 509A (APHA et al.,
1985) to analyze chlorinated organics in water. Pentane (HPLC grade)
extraction of a 50 mL water sample and concentration to 5 mL was followed by
analysis using GC and an electron-capture detector. Quantification was by
means of a Spectra-Physics integrator with standards prepared in the HPLC
grade pentane. The extract was injected onto a column (6 ft x 4 mm i.d.)
packed with 100/120 mesh Gas Crom Q support with an OV-210 stationary phase.
Injector temperature was 100*C; carrier gas was nitrogen at 48 cc/min. The
electron capture detector was sec at 350'C and the oven was programmed for an
initial one-minute hold at 80*C with an increase of 15*C/min to 160*C.
Following a seven-minute hold, the oven was cooled to the initial 80*C.
A linear correlation was evident between peak area and concentration when
levels were between 0.1 and 10 mg/L, with loss of linearity at higher levels.
Retention time for HCE was 3.17 minutes and limits of detection were reported
to be 0.01 mg/L for the concentrated pentane extracts of the 50 mL water
samples.
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X.	TREATMENT TECHNOLOGIES
No information was found in the available literature on technologies useful
for the removal of HCE from water. Studies of environmental fate indicate
that HCE is acted upon by bacteria in pond waters under both aerobic and
anaerobic conditions. Under anaerobic conditions, approximately 90% of the
HCE content was lost in 18 days (Spanggord et al., 1985). Additional studies
indicate that HCE is biotransformed to tetrachloroethylene in aerobic cultures
under laboratory conditions. Transformation was constant with time in the
presence oC municipal sludge wastewater (Criddle et al., 1986).
Volatilization is also a major transport process for HCE in water (Spanggord
et a., 1985). It is likely that these environmental processes can be utilized
to develop a method to remove quantities of HCE from water.
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XI
CONCLUSIONS AMD RECOMMENDATIONS
Based on the available animal data, the HA for One-day and Ten-day exposures
for a child is 5 mg/L. The Longer-term HA for the child is 130 ug/L and for
che adult is 450 ug/L. Evidence is presented that HCE may meet the EPA
criteria for classification as C, Possible Human Carcinogen, based on animaL
data. The DUEL for HCE is 40 ug/L for lifetime exposure. The lifetime HA is
1 ug/L assuming a 20% RSC and equivocal evidence of carcinogenicity (Group C)
As indicated in che companion report, "Data Deficiencies/Problem Areas and
Recommendations for Additional Data Base Development for Hexachloroethane"
(Appendix 1), standard reproductive toxicity studies were not available and
should be considered in future medical research plans.
XI-1

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XII.
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contaminants from water bodies to atmosphere. Environ. Sci. Technol. 9:1178-
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Metabolic disposition study of chlorinated hydrocarbons in rats and mice.
Drug Chem. Toxicol. 8(3):183-194.
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31(3):391-396.
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tributaries on the St. Clair River. Water Pollut. Res. J. Can. 21:344-350 as
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rainbow trout: Correlations with partition coefficients and environmental
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Olsen OW. 1947. Hexachloroethane-bentonite suspension for controlling the
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Pankow JF, Isabell LM, Kristensen TJ. 1982. Tenax-GC cartridge for
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APPENDIX 1
Data Deficiencies/Problem Areas and Recommendations for
Additional Database Development for Hexachloroethane
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INTRODUCTION
The Office of Drinking Water (ODW), Environmental Protection Agency (EPA), in
conjunction with the Department of the Army, has reviewed the available data
on hexachloroethane (HCE) for the purpose of developing a Health Advisory (HA,,
useful in dealing with contamination of drinking water, to include "state-of-
the-art" information on environmental fate, health effects and analytical
methodology.
OBJECTIVES
The objective of this appendix is to provide an evaluation of the data
deficiencies and/or problem areas encountered in the review process for HCE
and to make recommendations, as appropriate, for additional database
development. This document is presented as an independent analysis of the
current status of HCE technology, as related to its possible presence in
drinking water, and includes a summary of the background information used in
the development of the HA. For greater detail on the toxicology of HCE the
Health Advisory on Hexachloroethane should be consulted.
BACKGROUND
Hexachloroethane, a chlorinated alkane with a camphor-like odor, is used
primarily by the military in pyrotechnic devices and screening smokes. It has
more limited usage commercially in lubricants, insecticides, moth-repellents,
fire-extinguishing fluids and submarine paints and as a solvent, chemical
precursor and rodenticide. It is manufactured by the chlorination of
tetrachloroethylene in the presence of ferric chloride at temperatures between
100 and 140*C (NIOSH, 1981). Most HCE is imported, with current U.S.
production limited to its formation as a co-product in the manufacture of
other chlorinated ethanes (Archer, 1979 as cited in Santodonato, 1985).
Smoke compositions contain HCE at levels averaging approximately 45% (Davidson
et al., 1988). Once combusted, several by-product gases are formed with HCE
amounting to approximately 0.3 to 5% of these gases (Katz et al., 1980). Zinc
chloride is the major metallic by-product (Novak et al., 1987). Its presence
in the environment is largely due to its military and industrial uses, as well
as from the load and pack procedures for the smoke devices. It is not found
as such in nature. It has been measured in surface waters at 1-3 ug/L (Ewing
et al., 1977 as cited in Davidson et al., 1988), in drinking water at levels
of 0.03-4.3 ug/L (Keith et al., 1976 as cited in IARC, 1979), and in air at
0.3-49 ug/m^ (Silkworth et al., 1984 as cited in Davidson et al., 1988). Once
in water, volatilization and biotransformation have been shown to occur.
Volatilization half-lives between 40 minutes and 70 hours have been estimated
(Dilling, 1977 as cited in Callahan et al., 1979; Spanggord et al., 1985)
while a biotransformation half-life of approximately 40 days was determined in
an unconfined sand aquifer (Criddle et al., 1986). When released to the
atmosphere, HCE is relatively stable (Davidson et al., 1988).
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The pharmacokinetic properties of HCE have been studied in sheep, rabbits,
rats and mice. Absorption has been demonstrated after oral, dermal and
inhalation exposure, with distribution largely to fat. Concentration in the
kidneys of male rats has also been demonstrated (Gorzinski et al., 1985).
Excretion is mainly via the expired air with 65 to 71% of the administered
dose recovered following multiple oral doses in rats and mice (Jtitoma et al.
1985). Recovery in combined excreta ranged between 6 and 16%.
Tecrachloroethvlene is the major metabolite in sheep (Fowler, 1969) while
trichloroethanol and trichloroacetic acid were the major metabolites in mice
and rats (Mitoma et al., 1985). In an in vitro study with rat liver
microsomes, tetrachloroethene was the major metabolite (Nastainczyk et al.,
1982).
Data on the exposure of humans to HCE are very limited, with reports of mild
neurological effects as well as direct effects on the eye following acute
exposure (Grant, 1986). Irritation was the only effect reported after chronic
exposure in the workplace (Dacre, 1979).
Oral LD5QS in rats range between 5,160 and 7,690 mg/kg in males and 4,460 to
7,080 mg/kg in females, depending upon the vehicle used. The oral LDjq in
male guinea pigs was reported at 4,970 (Weeks et al., 1979; Weeks and
Thomasino, 1976). Toxic signs include tremor, ataxia, signs of gasping, and
the appearance of a red exudate around the eyes. Oral doses up to 1 g/kg in
sheep resulted in signs of liver toxicity as demonstrated by an increase in
plasma hepatic enzymes and a marked reduction in transfer from liver to bile
in the BSP clearance test (Fowler, 1969). No effects were demonstrated on the
chemical or functional properties of liver microsomes of rats that were orally
dosed with HCE at a level equivalent to approximately 6 g/kg (Reynolds, 1972).
Weeks et al. (1979) reported the development of a significant decrease in body
weight gain, increased relative liver and kidney weights, and liver and kidney
histopathology in rabbits orally administered HCE at doses up to 1,000
mg/kg/day for 12 days. A NOAEL of 10Q mg/kg/day was determined. In a 16-day
study in rats, dietary intake of HCE at doses up to 500 mg/kg/day also
produced a significant decrease in body weight gain, increased kidney weight
in males as well as gross liver and kidney lesions (Gorzinski et al., 1980a).
A NOAEL of 50 mg/kg/day was determined.
In a six-week screening study designed to determine the maximum tolerated dose
(NCI, 1978), toxicity was demonstrated at levels >562 mg/kg/day in rats, and
was evidenced by weight loss and mortality. In mice, toxicity was
demonstrated at doses >1,780 mg/kg/day. Doses in this study ranged between
178-1,780 mg/kg/day in rats and 316-3,160 mg/kg/day in mice.
Skin and eye irritation studies indicated that HCE is a mild skin irritant in
rabbits (Weeks and Thomasino, 1976), while moderate corneal opacity, iritis,
swelling and discharge occurred upon exposure of the eye to direct contact
with the dry technical HCE (Weeks et al., 1979). No skin sensitization was
evidenced in guinea pigs (Weeks and Thomasino, 1976). A dermal LO5Q of
>32 g/kg was also reported.
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Inhalation studies were conducted in dogs, racs, guinea pigs and quail over a
period of 6 weeks at doses up to 260 ppm (approximately 2,515 mg/m^).
Exposure was for 6 hours/day, 5 days/week (Weeks et al., 1979) The only toxic
sign was the appearance of a mucopurulent exudate in rats and quail, and mild
CNTS effects in dogs. When HCE was administered by inhalation on Days 6-16 of
gestation, maternal toxicity in rats was demonstrated by a significant
decrease in weight gain at both 48 and 260 ppm. No anomalies were detected ir.
the fetuses and no other changes occurred in maternal and fetal parameters.
Gorzinski et al. (1980b) reported toxic effects on the liver and kidneys,
demonstrated by histopathological changes, in rats administered HCE in the
diet at levels up to 100 mg/kg/day for 16 weeks. Kidney lesions were
characterized by a renal tubular atrophy and degeneration in the male rats at
doses of 30 and 100 mg/kg/day, while in females at the high dose, kidney
lesions were very slight. Liver lesions were characterized by a swelling of
the hepatocytes, and were observed in the high-dose males only. No effects
were seen on body weight, food intake and most clinical chemistry parameters
evaluated. Actual intake levels for this study were analyzed at 1.3, 20 and
82 mg/kg/day. A NOAEL of 1.3 mg/kg/day was indicated.
In a bioassay for carcinogenicity (NCI, 1978), rats and mice were exposed to
HCE by oral intubation at doses of 212 and 423 mg/kg/day in rats and 590 and
1,179 mg/kg/day in mice for 78 weeks of exposure. Kidney lesions were seen in
both species at both the low and high doses. An increase in the incidence of
interstitial cell tumors of the testes was reported in the high-dose rats. No
liver lesions were reported in the rat, while a significant increase in
hepatocellular carcinomas was reported in both sexes of mice at both dose
levels.
In a 2-year gavage study of HCE in Fischer-344 rats, an increased incidence of
hyperplasia, adenomas and carcinomas of the kidneys was seen in the males but
not the females (NTP, 1989). The incidence of nephropathy, however, was
increased in the females, although the severity of the lesions was mild. The
males in this study were given doses of 10 or 20 mg/kg while the females were
given doses of 80 and 160 mg/kg.
No information on the effects of HCE on reproduction in animals was found in
the available literature. Developmental effects were studied in rats orally
administered HCE on Days 616 of gestation at doses up to 500 mg/kg/day (Weeks
et al., 1979). Hexachloroethane was not considered teratogenic, although
signs of maternal and fetotoxic effects were indicated. Maternal rats
displayed a significant decrease in weight gain and significantly lower
gestations indices were reported. These included a decrease in the number of
viable fetuses and an increase in fetal resorption at the high dose. A NOAEL
for maternal and fetotoxic effects was 100 mg/kg/day.
Several analytical methods involving extraction with various GLC procedures
have been described (Otson and Williams, 1982; Pankow et al., 1982;
Eichelberger et al., 1983; Fisher et al., 1985). No treatment technologies
were found in the available literature.
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Based on the foregoing studies, HA values for One-day and Ten-day exposures
were recommended at 5 mg/L. The Longer-term HA for the 10 kg child is
100 ug/L and for che 70 kg adult, 450 ug/L. The Lifetime HA is 1 ug/L
assuming a 20% RSC and equivocal evidence of carcinogenicity (Group C) .
A DWEL of 40 ug/L was determined.
DISCUSSION
Available data on the pharmacokinetics, health effects and analysis of HCE
have been reviewed.
The pharmacokinetic properties have been studied in various species and
results indicate that HCE is absorbed following all exposure routes, is
distributed largely to fat with kidneys of male rats also containing
significant amounts. Excretion occurs mainly via the expired air with lesser
amounts in the combined excreta. Major metabolites have been variously
described. No data gaps are apparent; no additional studies are required.
Available studies on the health effects of HCE include oral LD50S in rats and
guinea pigs. A dermal LD50 has also been reported. Twelve- and 16-day
toxicity studies indicate that HCE exerts some toxic effects on the liver and
kidney over the short exposure duration, as well as causing significant
decreases in weight gain in rats and rabbits. Similar effects on the kidney
and liver were seen at lower doses in the diet of racs over a 16-week period,
although body weight was not affected in this study. Similar findings on che
kidney and liver were also evident after a lifetime exposure in rats and mice
Male rats~developed a significant increase in interstitial cell tumors of the
testes after a lifetime oral exposure to HCE. While this lifetime study did
not establish a N0AEL or LOAEL for noncarcinogenic effects of HCE, the
available data appears adequate for developing a Longer-term HA. No further
studies are recommended at this time.
No studies were found on the reproductive effects of oral exposure to HCE in
animals. Developmental toxicity studies indicated that HCE is not teratogenic
in racs but significant maternal and fetotoxic effects were demonstrated. In
view of the occurrence of interstitial cell tumors in the testes of rats, as
well as the apparent fecoeoxic effects, it is recommended that standard
reproduction studies be conducted to evaluate this parameter.
A 78-week bioassay study in mice and a 2-year bioassay in rats indicates that
HCE may be classified as a carcinogen, based on the development of a signi-
ficant incidence of hepatocellular carcinomas in mice and renal adenomas and
carcinomas in rats after oral exposure to HCE. Further evaluation of these
studies, as well as of the 78-veek bioassay in rats, is suggested before
recommendations for additional studies are made.
No evidence of mutagenicity was seen in several assays for genotoxicity. No
further assays are recommended at this time.
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Several methods for the analysis of HCE are available, limits of detection
indicate that they are adequate for the detection of HCE in water at levels
currently considered to be harmful to man. No further studies are required
No treatment technologies for the removal of HCE could be found in the
literature. It is recommended that studies be undertaken to develop such
methods in the event that HCE should enter the water supply at levels above
those currently found.
CONCLUSION/RECOMMENDATIONS
Based on the above discussion, the following conclusions/recommendations can
be made:
1.	The available studies on the toxicity of HCE are generally considered
adequate for development of a HA useful in dealing with potential
contamination of drinking water.
2.	In view of the toxic effects of HCE to the testes of male rats, and
considering its apparent maternal and fetotoxic effects, it is
recommended that a standard three-generation reproduction study be
undertaken to evaluate the effects of HCE on this parameter.
3.	It is recommended that methods be developed for the removal of HCE
from drinking water in the event that a high level contamination
occurs.
4.	Aside from the aforementioned data gaps, no other studies on HCE, as
related to its possible presence in drinking water, are recommended ac
this time.
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