HEXACHLOROCYCLOPENTADIENE
Ambient Water Quality Criteria
Criteria and Standards Division
Office of Water Planning and Standards
U.S. Environmental Protection Agency
Washington, D.C.
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CRITERIA DOCUMENT
HEXACHLOROCYCLOPENTADIENE
CRITERIA
Aquatic Life
For hexachlorocyclopentadiene, the criterion to protect
freshwater aquatic life, as derived using the Guidelines
is 0.39 pg/1 as a 24-hour average and the concentration
should not exceed 7.0 jig/1 at any time.
For saltwater aquatic life, no criterion can be derived
using the Guidelines, and there are insufficient data to
estimate a criterion using other procedures.
Human Health
For the prevention of adverse effects due to the organ-
oleptic properties of hexachlorocyclopentadiene in water,
the criterion is 1.0 ^ug/1.
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CRITERION DOCUMENT
HEXACHLOROCYCLOPENTADIENE
Hexachlorocyclopentadiene (Hex; C-56; 1,2,3,4,5,5-hexa-
chlorocyclopentadiene) is a pale to greenish yellow liquid
with the molecular formula, C,-Clg. Other physical properties
include a molecular weight of 272.77; a solubility in water
of 0.805 mg/1; a vapor pressure of 1 mm Hg at 78-79 degrees
C and a density of 1.7119 (20°/4° C)(Lu, et al., 1975; Ungnade
and McBee 1958).
Hex was used as a chemical intermediate in the manufacture
of numerous widely used chlorinated pesticides (Kirk-Othmer,
1964). Recent governmental bans on the use of chlorinated
pesticides have restricted the use of Hex as a pesticide
intermediate to the endosulfan(Thiodan) and decachlorobi-
2,4-cyclopentadiene-l-yl (Pentac) industries. Currently,
the major use of Hex is as an intermediate in the synthesis
of commerically important flame retardants (Sanders, 1978:
Kirk-Othmer, 1964). Hex, though commercially important
as a chemical intermediate (production levels approximate
50 million pounds per year), has no end uses of its own
(Bell, et al. 1978).
Environmental monitoring data for Hex are lacking except
for measured levels in the vicinity of industrial sites.
Hex has been identified and/or quantified in wastewater,
receiving streams, rivers, fish, soil, sediment, and air
surrounding pesticide plants (Spehar, et al. 1977: Swanson,
1976: Carter, 1977a). A recent incident involving the improper
disposal of hex-containing industrial wastes in a Louisville,
Kentucky sewer system, resulted in no apparent widespread
environmental release of the chemical (Carter, 1977b).
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On the basis of the Louisville incident and laboratory
and industrial observations, Hex has proven to be a potent
irritant. Industrial workers have experienced irritation
of the eye, irritation of the upper airway passages and
headaches upon exposure to Hex vapors and burns upon contact
of skin with the liquid. (Ingle, 1953; Carter, 1977b).
Long-term exposure to hazardous concentrations results in
systemic poisoning of laboratory animals (Treon, et al.
1955).
In the laboratory, Hex has exhibited toxicity to fish
and mammals (Spehar, et al. in press; Treon, et al. 1955).
Hex has been reported to be nonmutagenic in laboratory tests
(NCI, 1977; IBT, 1977; Litton Bionetics, 1978a,b) and there
are no data available to evaluate the carcinogenicity of
the compound.
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REFERENCES
Bell, M.A., et al. 1978. Review of the environmental effects
of pollutants XI. Hexachlorocyclopentadiene. Unpublished
report by Battelle Columbus Lab. for U.S. EPA Health Res.
Lab., Cincinnati, Ohio.
Carter, M.R. 1977a. Legal affidavit filed in State of
Georgia, Fulton Co. dated June 14, 1977. Testimony concerning
estimates of total daily discharge of hex from Velsicol
Chemical Corp. Memphis plant and calculations of estimated
hex concentration in Mississippi River resulting from said
discharge.
Carter, M.R. 1977b. The Louisville Incident. Internal
report (unpublished), Serveillance and Analysis Division,
Region IV, U.S. EPA, Athens, Ga.
Industrial Bio-Test Laboratories, Inc. (IBT) 1977. Mutageni-
city of PCL-HEX incorporated in the test medium tested against
five strains of Salmonella typhimurium and as a volatilate
against tester strain TA-100. Unpublished report submitted
to Velsicol Chemical Corp.
Ingle, L. 1953. The toxicity of chlordane vapor. Science
118: 213.
Kirk-Othmer Encyclopedia of chemical technology. 2nd ed.
1964. Interscierice Publishers, New York.
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Litton Bionetics, Inc. 1978a. Mutagenicity evaluation
of hexachlorocyclopentadiene in the mouse lymphoma forward
mutation assay. Unpublished report submitted to Velsicol
Chemical Corp.
Litton Bionetics, Inc. 1978b. Mutagenicity evaluation
of hexachlorocyclopentadiene in the mouse dominant lethal
assay: Final rep. Unpublished. Submitted to Velsicol Chemical
Corp.
Lu, PY., et al. 1975. Evaluation of environmental distribu-
tion and fate of hexachlorocyclopentadiene, chlordane, hepta-
chlor, and heptachlor epoxide in a laboratory model ecosystem.
Jour. Agric. Food Chem. 23: 967.
National Cancer Institute. 1977. Summary of data for chemical
selection. Unpublished internal working paper, Chemical
Selection Working Group. U.S. Dep. Health Educ. Welfare,
Pub. Health Serv. Washington,D.C.
Sanders, H.J. 1978. Flame retardants. Chem. Eng. News:
April 24, 22.
Spehar, R.L., et al. (In press). Toxicity and bioaccumulation
of hexachlorocyclopentadiene, hexachloroonorbornadiene and
heptachloronobornene in larval and early juvenile fathead
minnows, Pimephales promelas. Bull. Environ. Contain. Toxicol.
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Swanson, D. 1976. Discharges from Hooker Chemical Company.
Internal Staff Report (unpublished), Toxic Mater. Section,
Water Quality Div., Environ. Prot. Bur. Mich. Dep. Nat.
Resour.
Treon, J.F., et al. 1955. The toxicity of hexachlorocyclo-
pentadiene. Arch. Ind. Health 11: 459.
Ungnade, H.E., and E.T. McBee. 1958. The chemistry of
perchlorocylopentadienes and cyclopentadienes. Chem. Rev.
58: 240.
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AQUATIC LIFE TOXICOLOGY*
FRESHWATER ORGANISMS
Introduction
Acute and embryo-larval data are available for the
fathead minnow and hexachlorocyclopentadiene. However,
no data of any type are known for invertebrate species.
Acute Toxicity
Henderson (1956) exposed the fathead minnow under three
different conditions using two dilution waters. One had
a hardness of 40 mg/1 and pH of 7.4 and the other had a
hardness of 400 mg/1 and pH of 8.2. Two tests with hard
water were conducted to evaluate the method used to add
the chemical to the dilution water. The latter comparison
was important since hexachlorocyclopentadiene is quite volatile
and has an extremely low solubility in water. The chemical
was added in a 0.01 percent acetone solution and a 0.001
percent suspension of an emulsion prepared in a blender.
The effect of hardness, if any, was slight with unadjusted
96-hour LC50 values of 104 pg/1 in soft water and 78 pg/1
in hard water (Table 1). The test results comparing methods
of addition were 78 and 59 jig/1 (Table 1) indicating little
difference. Spehar, et al. (In press) determined the 96-
hour LC50 value for larval fathead minnows. This result,
using flow-through procedures and measured concentrations,
*The reader is referred to the Guidelines for Deriving Water
Quality Criteria for the Protection of Aquatic Life C43
FR 21506 (May 18, 1978) and 43 FR 29028 (July 5, 1978)3
and the Methodology Document in order to better understand
the following discussion and recommendation. The following
tables contain the appropriate data that were found in the
literature, and at the bottom of each table are the calcu-
lations for deriving various measures of toxicity as described
in the Guidelines.
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is 7.0 jug/1 (Table 1). This difference between the data
of Henderson (1956) and Spehar, et al. (In press) may be
due to differences in test methods or in relative sensitivity
of different life stages of the fathead minnow. The -Final
Fish Acute Value for hexachlorocyclopentadiene is 7.0 ^ig/1
based on the geometric mean of adjusted LC50 values divided
by the Guidelines sensitivity factor of 3.9. The lowest
value from a flow-through test with measured concentrations
is also 7.0 yug/1 (Table 1). Since there are no data for
invertebrate species, the Final Fish Acute Value of 7.0
yug/1 is also the Final Acute Value.
Chronic Toxicity
The chronic value for the fathead minnow embryo-larval
test by Spehar, et al. (In press) is 2.6 jug/1 (Table 2).
This concentration is not that much lower than the 96-hour
LC50 value (7.0 ^jg/1) for larval fathead minnows (Table
1). The Final Fish Acute Value is equal to 2.6 ;ug/l divided
by the sensitivity factor of 6.7 or 0.39 jug/1. This also
becomes the Final Chronic Value.
Plant Effects
No data are available on the effects of hexachlorocyclo-
pentadiene on.freshwater algae or plants.
Residues
The bioconcentration factor for whole-body fathead
minnows is 11 (Table 3) after a 30-day exposure (Spehar,
et al. In press). No Residue Limited Toxicant Concentration
can be determined since there is no permissible residue
concentration available.
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Miscellaneous
Applegate, et al. (1957) exposed sea lamprey, rainbow
trout and bluegill to concentrations of hexachlorocyclopenta-
diene of 1,000 and 5,000 ;ag/l (Table 4). Death or distress
was observed in one-half to one hour. The 30-day LC50 value
for the fathead minnow (Spehar, et al. In press) is 6.7
jig/1 which result is only slightly lower than the 96-hour
LC50 value of 7.0 ug/1 determined by the same investigators.
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CRITERION FORMULATION
Freshwater - Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two signif-
icant figures.
Final Fish Acute Value = 7.0 pg/1
Final Invertebrate Acute Value = no available
Final Acute Value = 7.0 /ag/1
Final Fish Chronic Value = 0.39 /ig/1
Final Invertebrate Chronic Value = not available
Final Plant Value = not available
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 0.39 ;ig/l
0.44 x Final Acute Value = 3.1 /ig/1
The maximum concentration of hexachlorocyclopentadiene
is the Final Acute Value of 7.0 /ig/1 and the 24-hour average
concentration is the Final Chronic Value of 0.39 jig/1.
No important adverse effects on freshwater aquatic organisms
have been reported to be caused by concentrations lower
than the 24-hour average concentration.
CRITERION: For hexachlorocylopentadiene the criterion
to protect f-reshwater aquatic life as derived using the
Guidelines is 0.39 jug/1 as a 24-hour average and the concen-
tration should not exceed 7.0 /ag/1 at any time.
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Table 1. Freshwater fish acute values for hexachlorocyclopentadiene
Adjusted
CO
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Bloaeaay Teet Time
Organ ism Method* cone.** (hrap|
Fathead minnow (larva), FT M 96
Pimephales promelas
Fathead minnow, S U 96
Pimephales promelas
Fathead minnow, S U 96
Pimephales promelas
Fathead minnow, S U 96
Pimephales promelas
LC50
(uq/il
7
104
78
59
LC50
(ug/lj
7.0
56.9
42.6
32.3
deference
Spehar, et al. In press
Henderson, 1956
Henderson, 1956
Henderson, 1956
* S = static, FT = flow-through
** U = unmeasured, M «• measured
Geometric mean of adjusted values = 27,2 iig/1 *«•'» = 7.0 ng/1
Lowest value from a flow-through test with measured concentrations = 7.0 i>g/l
27.2
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cn
Table 2. Freshwater fish chronic values for hexachlorocyclopentadiene (Spehar, et al. In press)
Chronic
Limits Value
Organism Test* (ug/i)
Fathead minnow (larva), E-L 3.7-7.3 2.6
Pimephales promelaa
* E-L = embryo-larval
Geometric mean of c
Lowest chronic value = 2.6 iig/1
9 £
Geometric mean of chronic values = 2.6 Mg/1 -r- =0.39 wg/1
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Tatle 3. Freshwater residues for hexachlorocyclopentadiene (Spehar, et al. In press)
.Time.
Organism Eioconceutration Factor (days;
Fathead minnow (juvenile), 11.0 30
Ptmephales promelas
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Table 4. Other freshwater data for hexachlorocyclopentadiene
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Organism
Sea lamprey (larva),
Petromyzon niarinus
Sea lamprey (larva),
Petromyzon marinus
Rainbow trout
(fingerling),
Salmo gairdneri
Rainbow trout
(fIngerling).
Salmo gairdneri
Test
Duration Ettect
24 hrs Death In 1 hr
24 hrs Distress In % hr
24 hrs Death in % hr
24 hrs Death in 1 hr
Result
(uq/il
Fathead minnow (larva). 30 days LC50
Pimephales promelas
Bluegill (flngerling), 24 hrs Death In fc hr
Lepomis macrochlrus
Bluegill (fingerling). 24 hrs Distress in % hr
Lepomis macrochlrus
5,000 Applegate, et al. 1957
1,000 Applegate. et al. 1957
5,000 Applegate, et al. 1957
1,000 Applegate, et al. 1957
6.7 Spehar, et al. In .press
5,000 Applegate, et al. 1957
1,000 Applegate, et al. 1957
Lowest value =6.7 pg/1
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SALTWATER ORGANISMS
Introduction
No data on the effects of hexachlorocyclopentadiene
on saltwater organisms are available.
Criterion Formulation
CRITERION: For saltwater aquatic life, no criterion
for hexachlorocyclopentadiene can be derived using the Guide-
lines, and there are insufficient data to estimate a criterion
using other procedures.
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REFERENCES
Applegate, V.C., et al. 1957. Toxicity of 4,346 chemicals
to larval lampreys and fishes. U.S. Fish and wild. Serv.
Spec. Rep. Fish. No. 207. U.S. Dep. Inter. Washington,
D.C.
Henderson, D. 1956. Bioassay investigations for International
Joint Commission. Hooker Electrochemical Co., Niagara Falls,
N.y. U.S. Dep. Health Educ. Welfare, Robert A. Taft Sanitary
Eng. Center, Cincinnati, Ohio. 12 p.
Spehar, R.L., et al. Toxicity and bioaccumulation of hexa-
chlorcyclopentadiene, hexachloronorbornadiene and heptachloro-
norbornene, in larval and early juvenile fathead minnows,
Pimephales promelas. Bull. Environ. Contain. Toxicol.
(In press).
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Mammalian Toxicology and Human Health Effects
EXPOSURE
Hexachlorocyclopentadiene, hereafter referred to as
hex, is a highly reactive, highly chlorinated compound which
is the key intermediate in the manufacture of many commer-
cially important organochlorine pesticides and flame retar-
dants for organic polymers (Kirk-Othmer, 1964). Although
it has also been suggested for use as an intermediate in
the manufacture of dyes, Pharmaceuticals, resins, and germi-
cides, these latter uses account for only a very small per-
centage of hex production. Historically, hex has been pro-
duced in the United States by two companies, Hooker Chemi-
cal and Plastics Corporation (Montague, Michigan) and Velsi-
col Chemical Corporation (Memphis, Tennessee). In 1977,
Hooker discontinued hex manufacture at the Montague plant,
making Velsicol's Memphis plant the only current U.S. pro-
ducer. Hex is produced at several facilities outside the
U.S. Hex was formerly used in the manufacture of aldrin,
endrin and dieldrin at the Shell Chemical Company plant
in Denver, Colorado (Zavon, 1978).
Hex has been used as a chemical intermediate in the
production of numerous chlorinated pesticides, several of
which have enjoyed very large usage. The list includes
chlordane, aldrin, dieldrin, heptachlor, isodrin, endrin,
R R
mirex, Kepone, endosulfan (Thiodan ), and Pentac . With
the exception of endosulfan and Pentac, both of which are
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in current use, the usage of hex-based pesticides has been
banned, suspended, or severely restricted by governmental
action. Although current production estimates are uncertain
and highly variable, one estimate has placed annual produc-
tion as high as 50 million pounds (25,000 tons) per year
(Bell, et al. 1978). Recent bans or restrictions on many
of the chlorinated pesticides have led to a decline in the
use of hex as a chemical intermediate in the manufacture
of these products; simultaneously, the use of hex in the
manufacture of flame retardants has increased. Currently,
a major use of hex is in the manufacture of flame retardant
compounds such as chlorendic acid and chlorendic anhydride
which are produced by reacting equimolar quantities of hex
and maleic anhydride. These and other hex-derived chlori-
nated organic compounds confer flame retardant properties
to plastics, including polypropylene, polyethylene, nylon,
rigid polyurethane foams, unsaturated polyesters, and other
polymers including epoxy resins (Sanders, 1978).
Although hex is a commercially important chemical inter-
mediate with high annual production, it has essentially
no end uses of its own. Consequently, hex concentrations
in the environment should be negligible and limited data
suggest that this indeed is the case. Small amounts of
hex are occasionally present as impurities in pesticides
made from it and some has undoubtedly entered the environ-
ment in this way. The most likely route of entry into the
environment arises from the manufacture of hex or hex-con-
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taining products. Discharge of these industrial wastes
appears to constitute the only documented sources of measur-
able hex in environmental samples.
Due to its infrequency in the environment and its low
profile as a chemical intermediate, there have been few
studies of the behavior of hex in the environment or in
biological systems. By the same token, until recently,
hex was not recognized as a major environmental problem
nor a potential threat to humans (except for those occupa-
tionally exposed). A recent incident in which scores of
workers at a sewage treatment plant in Louisville, Kentucky,
experienced a variety of toxic symptoms following the im-
proper disposal of hex manufacturing wastes has created
a great demand for information concerning the effects of
hex exposure on humans.
Several literature reviews on the health and environ-
mental effects of hex are available. These include reviews
of Equitable Environmental Health, Inc. (1976); U.S. Envi-
ronmental Protection Agency (1977); National Academy of
Sciences (1977); and Bell, et al. (1978). Although each
of these reports is different in emphasis, they each note
the unfortunate absence of epidemiologic studies of hex-
exposed workers and the lack of suitable chronic exposure
studies of animals (especially with respect to carcinoge-
nicity). Until these types of information are available,
proposed environmental criteria will necessarily be based
on extrapolation of animal data to humans, a practice which
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is invariably speculative and prone to error. Perhaps more
importantly, in the absence of suitable chronic exposure
studies, recommendations must be based on avoidance of rela-
tively overt manifestations of toxicity (e.g., irritation)
rather than less easily detectable effects (e.g., abnorma-
lities in physiologic tests, increased incidence of neo-
plasms, etc.) which may manifest themselves only after years
of exposure. Since effects of the latter type tend to be
elicited at doses lower than those causing acute toxicity,
criteria based on acute responses may fail to provide ade-
quate protection. Consequently, the criterion levels sug-
gested in this document are presented with the understanding
that they are based on decidedly inadequate chronic effects
data and should be reassessed upon completion of appropriate
chronic studies.
Hex, Cc-Clg, is not present in the environment under
natural conditions. It may be prepared in the laboratory
by several alternative methods which have been described
in a review article by Ungnade and McBee (1958). Commercial
technical hex is a pale yellow, nonflammable liquid having
a very pungent odor. It is soluble in a number of organic
solvents but is relatively insoluble, about 2.0 mg/1 in
water. Hex boils at 239 C (462 F) and it is sufficiently
volatile at ambient temperatures to have a tendency to dis-
perse into the atmosphere. This tendency to volatilize
undoubtedly accounts in part for the failure to detect hex
in many environmental samples.
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Six active chlorines and two double bonds make hex a
highly reactive compound which readily undergoes substi-
tution and addition reactions. Its versatility is based
upon its reactivity as a diene with a variety of decompo-
sition. Data from the iso-octane solutions revealed no
degradation after 24 hours, but a multi-peak spectrum indi-
cating the presence of degradation products was obtained
after 7 to 21 days' exposure. This spectrum suggested to
the investigators that the compound may be susceptible to
atmospheric oxidation and/or photodecomposition (National
Cancer Institute, 1977).
In using hex as an intermediate in the manufacture of
various chlorinated pesticides (chlordane,dieldrin, hep-
tachlor, etc.), it appears that although yields in all reac-
tions are good, they are not quantitative. Thus, there
is reason to suspect that in some cases free hex may have
been present in the marketed pesticide products. An early
study by Ingle (1953) provided good evidence that the reported
vapor toxicity of chlordane to mice was not attributable
to chlordane, but to some unreacted intermediate, chief
of which was hexachlorocyclopentadiene. It is suspected
that small quantities of unreacted hex may be present in
other related pesticides as well.
Because of the widespread use of hex as an intermediate,
and the belief that hex may comprise as much as 1 percent
of commercial chlordane (a pesticide with extremely great
usage), laboratory studies have of olefins and polynuclear
aromatic hydrocarbons in the Diels-Alder reaction.
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Rieck's report (1977a) provides evidence of the volati-
14
lization of hex from soil. Vapors of C-hex were evolved
from treated soil to the extent of 11, 13, 15, 16, 17, and
19 percent (cumulative) of the applied amounts 1, 2, 3,
5, 7, and 14 days respectively after treatment. One could,
therefore, deduce that there is volatility from treated
soil and that the rate decreases with time.
Another distinguishing feature of hex is that it ap-
pears to be strongly adsorbed to soil or soil components.
Studies of hex-treated soil (Rieck, 1977a, 1977b) have demon-
strated poor extractability from soil, which provides indi-
rect evidence of strong adsorption. In one study (1977b),
14
soil which had been extracted was then combusted to C02*
14
Any residual but unextracted .C was then measured directly.
14
Unextracted C was found in these samples and thus was
accounted for as a "bound" residue. Had it not been accounted
for, it would have probably been assumed to have volatilized.
Hex, unlike some of the pesticides derived from it,
degrades rapidly by photolysis, giving water soluble degra-
dation products. Tests on its stability towards hydrolysis
at ambient temperature indicated a half-life of about 11
days at pH 3-6, which was reduced to 6 days at pH 9.
In December, 1975, hex was qualitatively identified
as a contaminant in the discharge of a pesticide production
plant in Memphis. Later, (May, 1977) the compound was iden-
tified in the air at the Hooker plant in Montague, Michigan
(56 ppb), in its aqueous discharge (0.170 mg/1), and in ,
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fish tissue from the receiving stream (14-18 ppb) (Spehar,
et al. 1977). Hex has also been reported to have been pre-
sent in soil and bay sediments in the vicinity of a Virginia
pesticide plant long after production was discontinued (Swan-
son, 1976).
Data on environmental concentrations of hex are minimal
except for industrial discharges. Velsicol Chemical Cor-
poration' s Memphis plant has been issued a National Pollu-
tant Discharge System (NPDES) permit. Monitoring activities
in connection with the discharge permit indicate that hexa-
chlorocyclopentadiene, hexachloronorbornene, and hexachloro-
bornadiene are being discharged into the City of Memphis
wastewater collection system (Bennett, 1977; Marks, 1977).
Sampling for the month of January, 1977 (31 consecutive
days), revealed hex concentrations in wastewater ranging
from 0.156 to 8.240 mg/1. U.S. Environmental Protection
Agency's Water Surveillance Branch sampled Velsicol1s dis-
charge February 2-3, 1977. Hex was detected at 18 mg/1.
Based on the average monthly discharge by the Velsicol Chemi-
cal Corporation during February, 1977 (3.16 million gallons
per day), 474 pounds of hex was believed to have been dis-
charged through Velsicol1s discharge outfall into the City
of Memphis Wastewater Collection System and then into the
Mississippi River during the period February 2-3, 1977.
Calculated on the basis of the flow rate above, this dis-
charge caused a concentration of hex in the Mississippi
River of 0.0006 mg/1 (Carter, 1977a).
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In a recent, well-publicized incident, an estimated
6 tons equivalent of hexachlorocyclopentadiene (hex) and
octachlorocyclopentadiene (octa) dispersed in No. 4 fuel
oil were dumped into the Louisville, Kentucky, municipal
sewer system's Western Outfall sewer. The contaminated
sludge entered the Morris Forman Wastewater Treatment Plant
on March 26, 1977, causing illness among sewage treatment
plant workers. Toxic effects associated with this episode
forced closure of the plant with subsequent diversion of
105 million gallons per day of raw sewage into the Ohio
River. There was no evidence of environmental release (out-
side the immediate environs of the sewage treatment plant
and contaminated sewer lines). It was, however, necessary
to decontaminate the sewer.system and the treatment plant.
This incident is reported in further detail in the section
on "Effects - Human Studies".
Several general conclusions can be made regarding the
various modes of hex exposure.
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Ingestion from Water
Very little is known regarding potential hex exposures
through ingestion of contaminated food or water. Hex has
been detected in specific bodies of water near points of
industrial discharges. Except for such source-directed
sampling, it appears that there is little information on
hex concentrations in surface waters. Hex is usually not
detectable in water samples. Due to its low solubility
and tendency to volatilize, one would not expect it to remain
in flowing water. Moreover, there are no data on hex levels
in drinking waterror the extent to which hex in raw (untreated)
water would be passed through the water treatment process
to human consumers.
Hex has been identified in a few samples of fish taken
from waters near the Hooker plant in Michigan (Spehar, et
al. 1977). Frequently, however, hex residues have not been
detected in eoTble fish deliberately exposed to hex in labora-
tory experiments. According to the same investigator, the
inability to recover hex in fish samples probably results
from losses by vaporization during sample extraction. No
reports concerning hex contamination of other foods could
be located.
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A bioconcentration factor (BCF) relates the concentration
of a chemical in water to the concentration in aquatic organ-
isms, but BCF's are not available for the edible.protions
of all four major groups of aquatic organisms consumed in
the United States. Since data indicate that the BCF for
lipid-soluble compounds is proportional to percent lipids,
BCF's can be adjusted to edible portions using data on percent
lipids and the amounts of various species consumed by Americans,
A recent survey on fish and shellfish consumption in the
United States (Cordle, et al. 1978) found that the per capita
consumption is 18.7 g/day. From the data on the 19 major
species indentified in the survey and data on the fat content
of the edible portion of. these species (Sidwell, et al.
1974), the relative consumption of the four major groups
and the weighted average percent lipids for each group can
be calculated:
Consumption Weighted Average
Group (Percent) Percent Lipids
Freshwater fishes 12 4.8
Saltwater fishes 61 2.3
Saltwater molluscs 9 1.2
Saltwater decapods 18 1.2
Using the percentages for consumption and lipids for each
of these groups, the weighted average percent lipids is
2.3 for consumed fish and shellfish.
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A measured steady-state bioconcentration factor of 11
was obtained for hexachlorocyclopentadiene using fathead
minnows containing about 8 percent lipids (Spehar, et al.
In press). An adjustment factor of 2.3/8.0 = 0.2875 can
be used to adjust the measured BCF from the 8.0 percent
lipids of the fathead minnow to the 2.3 percent lipids that
is the weighted average for consumed fish and shellfish.
Thus, the weighted average bioconcentration factor for hexa-
chlorocyclopentadiene and the edible portion of all aquatic
organisms consumed by Americans is calculated to be 11 x
0.2875 = 3.2.
Inhalation
The heaviest and most chronic exposure to hex undoubtedly
occurs among persons engaged directly in the manufacture
of hex and among production workers fabricating hex-contain-
ing products. Although several cohorts of hex-exposed workers
C-ll
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can be specifically identified (employees of Hooker Chemi-
cals and Plastics, Michigan and Niagara Falls plants; Velsi-
col Chemical Corporation, Memphis plant; Shell Chemical
Company, Denver and Pernis, Netherlands, plants; an Israeli
company, Makhteshim; and the Hooker plant at Genk, Bel-
gium) , there have been no reports of epidemiologic studies
of these workers. Inhalation of hex is the primary mode
of occupational exposure. Accidental spills and illegal
discharges of hex represent the primary mode of acute human
exposure (e.g., the Louisville incident). Inhalation ap-
pears to be the most important mode of exposure in these
cases as well.
Dermal
According to Hooker Chemical and Plastic Corporation's
Material Safety Data Sheet, hex is readily absorbed through
the skin. Prolonged or repeated contract can lead to burns
and manifestations of systemic toxicity not unlike those
caused by inhalation. The hazards of skin contact are well
recognized and industrial workers are provided with imper-
vious clothing to prevent dermal contact (Hooker, 1972).
Thus, dermal exposure should not be anticipated among workers
familiar with hex. Persons outside the chemical industry
can be exposed to hazardous contacts as a result of acci-
dental spills or improper disposal of hex.
C-12
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PHARMACOKINETICS
Only two studies which address the pharmacokinetics
of hex could be located (Mehendale, 1977; Kommineni, 1978).
The latter study focuses upon absorption and elimination
of hex while the Mehendale (1977) study is more concerned
with the disposition of hex within the body and modes of
elimination.
The Kommineni (1978) study consisted of two parts.
The first consisted of a study of rats exposed to various
doses of hex by gavage while the second portion examined
guinea pigs exposed to varying doses of hex via dermal appli-
cation. Inferences regarding patterns of absorption, meta-
bolism, and excretion are based on gross pathology findings
and histopathologic findings at necropsy.
In the first series, a total of 10 female rats were
exposed to 0, 50, 100, 150, 200, and 300 mg/kg of hex by
gavage. All animals were sacrificed 24 hours post treat-
ment. The rats were necropsied and lungs, liver, spleen,
kidneys, adrenals, heart, stomach, and intestines were saved
for histopathology evaluation.
Gross pathology of the rats exposed to 200 and 300 mg/kg
revealed brown discoloration around the nostrils and anus
of the rats. The urinary bladders of two of the four rats
contained brown fluid. Subserosal emphysema of the nonglan-
dular stomach was evident in one animal. On histopathologic
examination, the lungs showed atelectasis with moderate
thickening of the alveolar walls. The alveolar walls con-
C-13
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tained moderate numbers of macrophages and neutrophils.
Some bronchi contained denuded epithelium. No edema was
present in the lungs. Rats receiving lower dosages showed
similar, but milder, changes. The stomachs of rats receiving
dosages of 200 or 300 mg/kg showed coagulative necrosis
of the gastric squamous epithelium. The submucosa of the
nonglandular part of the stomach showed mild neutrophilic
infiltration. The supporting structures of the stomach
(submucosa, submuscularis, muscularis) showed moderate edema.
Epithelium of the glandular part of the stomach showed no
treatment-related changes. Animals receiving lower doses
showed similar changes in the stomach. Ulcers of the nonglan-
dular portion of the stomach were seen in several of the
animals. At all dosages, the other organs were unremarkable.
The author commented that these morphological changes
indicate that hex is absorbed through the squamous epithe-
lium of the nonglandular part of the stomach and that the
major route of elimination of hex is through the lungs.
In the second part of the study, four male guinea pigs
were painted on the skin (site unspecified) with hex at
dosages of 0, 300, 600, and 1200 mg/kg and sacrificed 24
hours after the exposure. All animals were necropsied and
the lungs, liver, pancreas, kidneys, adrenals, urinary blad-
der, heart, skin, stomach, and intestines were saved for
histopathologic evaluation.
C-14
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On gross pathology, subcutaneous edema was seen extending
from the inguinal area to the sternum. At the lowest dosage,
the lungs were highly expanded and showed rib impressions
on the parietal surface. Similar but more severe changes
were seen in the animal receiving 600 mg/kg. The animal
painted with 1200 mg/kg expired prior to sacrifice; the
trachea was filled with frothy fluid. Histopathologic exami-
nation of the lungs revealed atelectasis with thickened
alveolar walls containing moderate numbers of macrophages
and neutrophils. Intense congestion of all pulmonary blood
vessels and occasional alveolar edema was seen in the ani-
mal receiving the 1200 mg/kg dose. In the skin, moderate
to marked edema disrupted the collagen bundles. Focal pockets
of neutrophils were seen in the edematous dermis. Edema
extended throughout the thickness of the adipose tissue
layer. One animal showed partial thrombosis of medium size
veins situated deep in the dermis. The skin appendages
were normal.
Kommineni (1978) concluded, "Hex is absorbed through
the skin and probably is eliminated through the lungs.
Unlike the rat stomach, the squamous epithelium of the guinea
pig skin and its adnexa did not show necrotic changes.
This is probably due to two factors, surface area and transit
time."
In the Mehendale (1977) study, radiolabeled hexachloro-
14
cyclopentadiene ( C-hex) was administered by oral intuba-
tion to four male Sprague-Dawley rats in order to examine
C-15
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absorption, metabolism, and excretion of the compound fol-
14
lowing a single oral dose. After dosing with C-hex (5
ji moles, 1 juCi per animal), the rats were maintained in
metabolism cages for 7 days, during which daily urine and
fecal samples were collected. After 7 days, the animals
were sacrificed and the major organs were removed and radio-
assayed.
Urine and powdered fecal samples were radioassayed for
14
total C. An average of approximately 33 percent of the
total dose was excreted in the urine after 7 days. About
87 percent of that (approximately 28.7 percent of total
dose) was eliminated during the first 24 hours after the
administration of the compound. Fecal excretion accounted
for 10 percent of the total dose; nearly 60 percent of
the 7-day fecal excretion occurred during the first day.
Beyond the third day after treatment, only trace amounts
of the hex-derived C were eliminated in the feces. Tis-
sues retained only trace amounts of hex after 7 days. For
example, the kidney retained only about 0.5 percent of the
total dose and the liver less than 0.5 percent. Other organs
and tissues--fat, lung, muscle, blood, etc.--contained even
less of the radiolabel. Such findings suggest that at least
half of the administered hex was eliminated by routes other
than urine and feces. The author felt that the respiratory
tract is probably the major route of excretion.
C-16
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The nature of the radioactivity excreted in the urine
was examined searching for possible metabolites. It was
found that about 70 percent of the radioactivity in the
urine was extractable using a hexane:isopropanol (9:1) mix-
ture. The organic solvent was concentrated, applied to
thin-layer chromatography (TLC) plates, and developed in
three solvent systems. The radioactive spots were visual-
ized by auto-radiography on medical x-ray film. The results
suggested the presence of at least four metabolites; how-
ever, at the time of this writing they had not been identi-
fied and characterized.
Disposition and biliary excretion of C-hex.was stu-
14
died by injection of approximately 1 jjCi (5 >i mole) of C-
hex into the femoral vein of anesthetized rats. Timed sam-
ples of blood and bile were collected for 1 hour from the
femoral artery and common bile duct which had been cannu-
lated prior to dosing. Approximately 9 percent of the admini-
stered dose was excreted in the bile in 1 hour. Because
this quantity is equivalent to that excreted in the feces
over 7 days, enterohepatic circulation of this compound
is probable. The nature of the compound present in the
bile is not yet known.
At the end of the above experiments, the animals were
sacrificed and the liver and kidneys were removed. Tissue
homogenates from these organs were radioassayed and the
distribution of the radioactivity among the various subcel-
lular fractions was examined by assaying the various centri-
C-17
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fugation fractions. Kidney cytosol accounted for 93 percent
of the radioactivity in the total kidney homogenate. This
behavior is consistent with rapid urinary excretion. Simi-
larly, 68 percent of the radioactivity in the liver homo-
genate was associated with the liver cytosol fraction, once
again consistent with rapid excretion.
Pre-exposure of some of the rats to hex (50 mg/kg/day)
for 3 days prior to the experiment did not affect blood
decay curves and biliary excretion; however, an increased
concentration in the kidneys after a single challenge with
14
C-hex was observed.
Whitacre (1978) reported that Velsicol has contracted
14
an independent metabolism study in rats and mice using C-
hex. The metabolism of hex was determined both after single
acute dosing and repeated administration over a period of
about 30 days. The results of these studies have not yet
been officially reported although verbal appraisal of some
results has been provided to Velsicol.
It appears that results of this study do not agree closely
with the Mehendale study. The recent study shows hex to
be eliminated from mammals (mice and rats) mainly by the
fecal route and with no more than about 15 percent being
eliminated in urine. Further, these studies do not indicate
any significant amounts of pulmonary elimination of hex
or its metabolites. Whitacre (1978) believes that the poor
recoveries in feces in the Mehendale study may be the result
of volatility of hex or its metabolites before removal for
analysis. Losses during sample preparation undoubtedly
further complicate the analysis of fecal matter.
C-18
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EFFECTS
Acute, Sub-acute, and Chronic Toxicity
The classic studies of hex toxicity to mammals were
conducted in the mid 1950's by Treon, et al. (1955). This
series of investigations reported on both acute and subacute
toxicity of hex to various species of mammals under a vari-
ety of exposure regimens. Oral, dermal, and inhalation
modes of exposure were included in Treon's experiments.
Mammalian toxicity studies subsequent to the 1950's could
not be located in the open literature, probably due to the
rather low profile of hex relative to other pesticide chemi-
cals. More recent, proprietary studies of the oral and
dermal toxicity have now become available. In general,
these findings agree remarkably well with those of Treon.
It is most unfortunate that no truly long-term (i.e., =
6 months) studies of chronic effects have been conducted.
Until data on the potential effects of long-term, chronic
exposure (especially carcinogenicity) becomes available,
any recommendations regarding environmental criteria must
be regarded as tentative.
1) Acute Toxicity. a. Oral. Acute toxicity of hex
was determined by Treon, et al. (1955) by administering
dosages of 180, 280, 340, 420, 520, 620, 940, 1400, and
2100 mg/kg of hex in peanut oil directly into the stomachs
of several groups of rabbits and rats. The data on rabbits
indicate that the median lethal oral dose (LD50) administered
as described above, lies in the range between 420 and 620
mg/kg of body weight.
C-19
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Rats showed variation in minimum lethal dose depending
on sex. Male rats were somewhat more sensitive in that
the lethal dose was somehwat less than 280 mg/kg body weight,
whereas for females the dosage causing death was greater
than 280 mg/kg. The LD50 for male rats was determined to
be 505 mg/kg with 95 percent confidence limits of 387-623.
Also noteworthy is the fact that very few of the test ani-
mals survivied longer than a week after oral administration
of hex.
The International Research and Development Corporation
(IRDC, 1972) conducted similar studies of the acute oral
toxicity of hex. Twenty-five albino rats of each sex were
given hex dissolved in corn oil at dosages of 315, 500,
794, 1250, and 1984 mg/kg. Five rats of each sex were used
at each dosage level. An LD50 of 530 mg/kg was determined
for female rats and 630 for male rats. The combined oral
LD50 for both sexes was determined to be 584 mg/kg. Note
that this is the reverse of the sex differential reported
by Treon, et al. (1955). Naishstein and Lisovskaya (1965)
reported a LD50 of 600 mg/kg for white rats. This value
is comparable to the upper part of the range (420-620 mg/kg)
reported by Treon, et al. (1955). Thus, the true LD50
is probably about 600 mg/kg.
b. Dermal. In this series of experiments, 93.3 percent
hexachlorocyclopentadiene was applied to the intact skin
of rabbits using the technique of Draize et al., described
by Treon et al. (1955). It was determined that the lethal
dosage lies between 430 and 630 mg/kg body weight. Such
a finding is notable in that hex appears to be just as toxic
via dermal application as by ingestion.
C-20
(ft
-------
More recently, the irritant properties of hex were exa-
mined in a study conducted by the International Research
and Development Corporation (IRDC, 1972). These tests were
commissioned by Velsicol Chemical Corporation in accordance
with the regulations of the Federal Hazardous Substances
Act.
IRDC (1972) reported the results of an investigation
of acute dermal toxicity of hex to rabbits. Four male and
four female New Zealand white rabbits were used in this
test. The hair was removed from the back of each rabbit
with electric clippers. Two male and two female rabbits
were used at each of two dosage levels. The test compound
was applied in a single administration to the back of each
rabbit at a dosage of 200 or 2000 mg/kg body weight. The
area of application was wrapped with a gauze bandage and
occluded with Saran Wrap. Twenty-four hours later, the
bandages were removed and the backs were washed with water.
The rabbits were observed for mortality for a period of
14 days.
All of the animals which received 2000 mg/kg dosage
died withi-n 24 hours after application of the compound.
At the 200 mg/kg dosage, both male rabbits died but both
female rabbits survived although they both exhibited weight
loss over the 14-day period. The male rabbits that died
showed weight loss also. In addition, cachexia, marked
dermal irritation and hypoactivity was observed. Skin at
the site of application turned purple in color within a
C-21
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few hours after hex application. Based on these results,
hex was concluded to be "a highly toxic material by the
dermal route of exposure" in accordance with the criteria
established under the Federal Hazardous Substances Act.
c. Inhalation. Treon, et al. (1955) exposed various
animal species to vapors formed by bubbling a stream of
air through liquid hex contained in a bubbling tower. This
air was then mixed with clean air to achieve the desired
concentration. The stream of air, conditioned with respect
to temperature, dust content, and humidity, was then passed
into a plywood exposure chamber in which the test animals
were confined. A series of hex concentrations in the air
in the exposure chamber were used; these varied from 0.15
to 73.6 ppm. Test species were guinea pigs, rats, mice,
and rabbits.
The authors reported that hex vapors were very toxic
to all four species of animals. Exposure to the concentra-
tion of 13.0 ppm (an intermediate level in this experiment)
for 15 minutes produced fatalities in all species except
guinea pigs. Of the four species, rabbits appeared to be
the most susceptible. Mice, rats, and guinea pigs followed
in order of decreasing susceptibility. Table 1 depicts
that results of the inhalation experiments. The values
tabulated correspond to the concentration in ppm which:
(1) permitted all animals to survive; (2) killed 50 percent
of the animals; and (3) produced 100 percent lethal con-
di tions.
C-22
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TABLE 1
Dose Response Data: Inhalation of Hex Vapors
(Treon, et al. 1955)
Species of Animal
Guinea pigs . . .
Rats . . .
Mice ...
Rabbits . . .
Fatalities ,
Percent
0
50
100
0
50
100
0
40
100
0
67
100
Hex Concentration
of Test
1-Hour
Exposure
7.2
13.8
20. Oa
3.1
7.2
20. Oa
1.4
7.2
13.8
1.4
3.1
7.2
(in ppm) Lethal to Percent
Animals Indicated
3-1/2-Hour
Exposure
3.1
7.1
12.4
1.4
3.1
7.1
1.4d
3.1e
7.1
--
6.4
7.1
7-Hour
Exposure
1.5
3.2
6.7
1.5b
3.2C
6.7
--
1.56
3.2
--
--
7.5
^Duration of exposure was 1.25 hours.
25 percent of group died.
^75 percent of group died.
80 percent of group died.
e20 percent of group died.
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Animals of the following species died regularly when
exposed to hex vapors at the following concentrations and
durations: rabbits - 1.5 ppm for 7 hours; mice -1.4 ppm
for two 7-hour periods; rats - 1.0 ppm for five 7-hour
periods or 3.2 ppm for two 7-hour periods; and guinea pigs
- 3.2 ppm for two 7-hour periods.
IRDC (1972) also reported the results of acute inhala-
tion experiements in rats. The test animals were exposed
to atmospheric concentrations of approximately 176.2 and
17,624 ppm of the test compound for 4 hours. Ten rats were
tested at each dosage level. Due to the extremely high
dosages employed, little information could be derived from
the study. No justification of the choice of dosages was
given. All of the animals receiving the test compound at
either exposure level died within 48 hours. All rats at
the 17,624 ppm dosage level died during the 4-hour exposure
period. At the 176.2 ppm atmospheric concentration, one
rat died during the exposure period, eight more were dead
within 24 hours, and the remaining rat died on the second
day of observation.
Signs seen during the exposure period included eye squint,
dyspnea, cyanosis, salivation, lacrimation, and nasal dis-
charge. Gross necropsy showed gray coloration of the skin,
severe hemorrhage of the lungs, and hydrothorax among rats
exposed to 17,624 ppm. Rats exposed to 176.2 ppm revealed
congestion of the lungs in all cases.
C-24
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Based on these results, the investigators concluded
that hex is highly toxic material by the inhalation route
of administration. Table 2 summarizes the results of acute
toxicity studies of hex.
2) Subacute Toxicity. a) Oral. To date, there has
not been a satisfactory study of subacute or chronic oral
toxicity of hex. One portion of the Treon, et al. (1955)
study attempted to examine subacute/chronic oral toxicity
but reported that dosages of 180-2000 mg/kg were fatal within
such a short period of time that the investigators were
unable to establish an oral dosage which could be tolerated
without mortality over an extended period. Similarly, Naish-
stein and Lispvskaya (1965) reported that oral administration
as little as 20 mg/kg for 6 months was fatal .to 20 percent
of white rats.
b) Dermal. Treon, et al. (1955) examined effects
of sublethal concentrations of hex applied to the skin of
rabbits and monkeys. In rabbits, dosages as low as 250
mg/kg induced extreme irritation, purplish-black discolora-
tion of the skin and subcutaneous edema. Although the skin
lesions healed eventually, damage to the skin in the area
of application persisted for many days and the damage varied
in severity and extent with the amount (dosage) of the ma-
terial applied.
A slightly different procedure was employed in the cuta-
neous exposures of the monkeys. In this case, a series
of hex concentrations (0.001, 0.01, 0.1, 1.0, and 10.0 per-
C-25
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TABLE 2
Acute Toxicity of Hexachlorocyclopentadiene
By Various Modes of Exposure
(Treon, et al. 1955)
Oral administration'
Rabbits
Rats
Ratsd
Females
Males
Rats
Males
LD
50
420-620 mg/kg
505 mg/kg
530 mg/kg
630 mg/kg
Minimum Lethal Dose
280 mg/kg
Dermal application
Rabbits
LD
50
420-610 mg/kg
Inhalation0
- (Dosage Expressed as Vapor
Concentration, ppm)
Guinea pig
Rats
Mice
Rabbits
13.8 ppm
7. 2 ppm
7. 2 ppm
3.1 ppm
Hex dissolved in peanut oil, administered by gavage.
93.3 percent hex solution in Utrasene, applied to intact skin
for 24 hours.
Loop's based on 1-hour vapor exposure.
Based on data reported by International Research and Development
Corp. (1972). Hex dissolved in corn oil.
C-26
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cent) dissolved in Ultrasene were applied to five sites
of the abdominal skin. Dosage of each of the solutions
was 0.01 ml. No irritation or other changes were noted;
however, when 0.05 ml of the 10 percent solution was ap-
plied to the back of a monkey for three consecutive days,
the skin became severely irritated and necrotic. Subsequent
experiments used more concentrated solutions (20, 40, 60,
and 90 percent) which were applied (dosage of 0.05 ml) on
separate areas of the monkeys' backs. At all concentrations
there was discoloration of the skin, ranging from very light
to dark tan as the concentration increased. The discolora-
tion was followed by swelling which varied from slight to
severe, again depending on concentration. The highest concen-
tration caused cracking, oozing, and serious discharge from
the treated areas; intermediate concentrations produced
hardening and swelling of the skin.
Among guinea pigs, application of solutions containing
0.01, 0.10, and 1.0 percent hex caused no alterations of
the skin, but more concentrated solutions (40, 60, and 90
percent) resulted in discoloration, hardening, and necrosis
of the skin at the application site. Based on these tests,
it appears that the threshold concentration at which hex
in Ultrasene induces irritation of the intact skin lies
between 10 and 20 percent for monkeys and between 1.0 and
40 percent for guinea pigs.
C-27
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Hex was tested for eye irritancy by instilling 0.1 ml
of the "test compound" (which was presumably undiluted liquid
hex) into the eyes of New Zealand white rabbits (IRDC,
1972). The test material was placed into the conjunctival
sac of the right eye of each rabbit; the left eye served
as an untreated control. Damage to the eye was evaluated
by instillation of sodium fluorescein into the eye, fol-
lowed by examination of the corneal surface for evidence
of ocular damage under ultraviolet light. A graded scale
was used to quantify the extent and severity of damage.
The eyes of the rabbits were checked for corneal lesions
at periodic intervals (at 1, 24, 48, and 72 hours post-expo-
sure and at 7, 14, and 21 days post-exposure). Examina-
tions at 14 and 21 days were precluded by the deaths of
all of the rabbits on or before the ninth day of the obser-
vation period. IRDC investigators attributed the deaths
to the effects of the test compound, but unfortunately did
not conduct post-mortem examinations to rule out other pos-
sible causes of death.
Based on the severity of the ocular lesions produced
in the rabbits, hex was concluded to be "an extreme irri-
tant and probable corrosive substance" in the 5-minute test
and "an extreme irritant and corrosive substance" in the
24-hour wash test (IRDC, 1972). These classifications are
set in accordance with standards set under The Federal Haz-
ardous Substances Act, specifically Part 191, Hazardous
Substances Test for Eye Irritants, Food and Drug Admini-
stration.
C-28
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c) Inhalation. When mice, rats, rabbits, and guinea
pigs were exposed to 0.34 ppm in air for 7 hours a day for
5 days per week, none of the mice or rats survived more
than 20 such exposures (Treon, et al. 1955). Two-thirds
of the rabbits had died by the end of the twenty-fifth period;
however, the guinea pigs survived through 30 periods. At
0.15 ppm, some animals from all four species survived 150,
7-hour exposures over a period of 216 days. Eight percent
of the mice did not survive the prolonged intermittent expo-
sure. Details of these findings are discussed in the next
section under the heading "chronic toxicity".
3) Chronic Toxicity. a) Oral. In the Treon, et
al. (1955) study, rabbits and rats given various dosages
of hex ranging from 180-2100 mg/kg tended not to survive
long enough at these dosages to provide acceptable data
on chronic oral toxicity. Consequently, these investigators
were unable to establish an oral dosage which could be tol-
erated (e.g., without mortality) over an extended period
of time.
Studies in the Soviet Union reported by Naishstein
and Lisovskaya (1965) appear to provide the only source
of information on the effects of long-term, low-dose exposure
to hex. Daily administration of 1/30 of the median lethal
dose (20 mg/kg) for 6 months killed only 2 animals out of
10, even though the cumulative dose received was 1.5 times
the acute LD^QQ, and six times the LD50. Although some
changes were noted in the weight coefficients of internal
organs of the animals, the authors judged the cumulative
effects of hex to be weak. No observations of neoplasms
or other abnormalities were reported.
C-29
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b) Dermal. High concentrations, 430-6130 mg/kg, ap-
plied to the skin of rabbits were frequently fatal within
a few hours. Six rabbits who survived for 7-21 days after
application of hex were killed and autopsied. Degenerative
changes were seen in the brain, liver, kidneys, and adre-
nal glands of these animals in addition to chronic skin
inflammation, acanthosis; hyperkeratosis; and epilation.
Visceral lesions due to dermal hex application reported
by Treon, et al. (1955) are described in the section on
toxic symptoms and pathological effects.
Naishstein and Lisovskaya (1965) also investigated the
effects of multiple, low-dose dermal exposures to hex.
These experiements consisted of applying 0.5-0.6 ml of a
concentration of 20 ppm hex in aqueous solution to the shaved
skin of rabbits daily for a period of 10 days. No differ-
ences were detected between the skin of the experimental
animals and that of the controls.
Treon, et al. (1955) reported that dosages of less than
10 percent hex appeared to be tolerated without irritative
effects in monkeys and probably also in guinea pigs. Unfor-
tunately, neither investigation continued the low-dose regi-
men for a sufficient period to observe chronic effects.
c) Inhalation. Treon, et al. (1955) exposed guinea
pigs to hex vapors at a concentration of 0.34 ppm hex for
7 hours per day, 5 days a week. All of them survived until
they reached 30 periods of exposure in 6 weeks. Rats and
mice exposed to this concentration survived only five per-
iods of exposure; however, survival of the rabbits was
intermediate; two-thirds had died before the end of the
fifth week (25 exposure periods).
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A lower concentration, 0.15 ppm hex, was tolerated by
guinea pigs, rabbits, and rats throughout 150, 7-hour per-
iods of exposure extending over a period of approximately
7 months. Four of five mice died within this period. Al-
though guinea pigs, rabbits, and rats appeared to grow nor-
mally during this period, slight degenerative changes were
observed in the livers and kidneys of these animals. These
changes are discussed in the following section.
4) Toxic Symptoms and Pathologic Effects. a) Oral
Administration. Rats and rabbits exposed to hex in the
Treon, et al. (1955) acute toxicity study exhibited diarrhea,
lethargy, and retarded respiration. The odor of hex could
also be detected in the feces of these animals and on their
bodies, presumably from fecal contamination. Rabbits which
died following exposure to moderately high doses of hex
(280-2100 mg/kg hex in corn oil) showed diffuse degenera-
tive changes in the epithelium of the renal tubules. As
in the study of Kommineni (1978), the lungs of these animals
were congested and edematous. The same types of degenera-
tive changes were also noted in the rats. In addition,
some of the rats showed acute necrotic gastritis. Animals
which survived the oral tests and were later sacrificed
exhibited residual degenerative changes of the type described
above, suggesting that the pathological changes are persis-
tent. The severity of the lesions diminished as the length
of the post-exposure survival interval increased, however.
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Equitable Environmental Health (1976) reported the re-
sults of a 90-day subacute oral toxicity study in rats con-
ducted for Hooker Chemical and Plastics Corporation by Indus-
trial Bio-Test Laboratories (IBT). Equitable reported that
rats exposed to hex (mixed in food) at concentrations of
0, 30, 100, and 300 ppm showed no treatment-related effects
in any of the parameters measured: growth, food intake,
mortality, abnormal behavior, hematology, clinical blood
i
studies, and urinalysis. Gross pathologic examination also
failed to reveal any abnormalities which could be attributed
to ingestion of hex. Similarly, organ weights and ratios
and microscopic examination of tissues and organs failed
to show treatment-related abnormalities. The IBT study
is unusual in that hex was mixed into the animal's food
rather than administered by gavage (hex in corn oil solution).
Due to the volatility of hex, the actual dose present in
feed at the time of ingestion is unknown. Therefore the
dosages above must be regarded as maximum hex content; it
is likely that the dosages ingested were well below this.
Other factors, including more direct contact of the hex
solution with stomach tissues in the gavage experiments,
may also explain the apparent discrepancy in toxicity thresh-
old between the IBT study and the gavage experiments reported
earlier.
Naishstein and Lisovskaya (1965) reported results of
a chronic oral toxicity experiement on 90 white rats. Rats
were given daily peroral doses of 0.002, 0.0002, and 0.00002
C-32
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(0.04, 0.004, and 0.0004 mg/1) in aqueous solution
for a period of 6 months. The first dose was 30 times greater
than the threshold concentration with respect to aftertaste
and smell (0.0013 mg/1); the second dose corresponded to
the practical limit of detection by smell, and the third
dose was 10 percent of the second. No deviations were ob-
served in the behavior of the rats or in their weights through-
out the 6-month experimental period. Likewise, no signi-
ficant changes were seen in hemoglobin, red blood cells,
white blood cells, or peripheral reticulocyte counts in
the experimental groups as opposed to the controls. In
animals receiving the highest dose, 0.002 mg/kg, neutro-
penia and a tendency toward lymphocytosis were noted. The
peripheral blood of animals receiving the two lower dosages
did not show any alterations relative to controls. The
authors concluded that daily peroral administration of doses
of 0.0002 and 0.00002 mg/kg (0.004 and 0.0004 mg/1 in aqueous
solution) caused no changes in peripheral blood cells, ascor-
bic acid content, conditioned reflexes, or histologic struc-
ture of the organs. Based on these tests and the threshold
level for organoleptic noxious effects (smell and aftertaste
in water), Naishstein and Lisovskaya (1965) recommended
a maximum permissible concentration of 0.001 mg/1 hex in
water.
b) Dermal Application. Treon, et al. (1955) showed
that dermal application of very low dosages of hex (0.25
mg/kg) were extremely irritating and induced local discolora-
tion and edema. The skin became hard, encrusted, and fis-
sured several days after application. The extent of the
local damage varied directly with the size of the dose applied.
C-33
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At autopsy rabbits exhibited visceral lesions similar
in appearance to those seen after oral administration of
hex. Again, diffuse degenerative changes were seen in the
brain, heart, adrenals, liver cells, and kidney tubules.
Pulmonary hyperemia and edema were also noticed. Animals
killed 7-21 days post-application of the compound showed
evidence of the same type of degenerative changes.
Monkeys dosed with various concentrations of hex in
solution exhibited discoloration of the skin which increased
directly as the concentration of hex applied increased.
Swelling, oozing, and encrustation similar to that described
above for rabbits was seen. Healing eventually took place,
but scarring and hair loss in the area of application ap-
peared to be permanent.
Industrial Bio-Test Laboratories also reported results
of a 28-day subacute dermal toxicity study using Albino
rabbits (Equitable Environ. Hlth., 1976). Hex solution
was allowed to contact the shaved, unoccluded skin of rab-
bits for an unlimited period of time (test material not
washed off). The test animals were dosed 5 days a week
for a period of 4 weeks, or 20 applications in all. The
concentration of hex in Group I was 0.1 (percent weight/-
volume); in Group II, the concentration was 0.5. None
of the animals died and no pharmacotoxic symptoms were noted;
however, both hex solutions were extremely irritating to
the skin and slight losses in body weight occurred in some
of the rabbits receiving the higher concentration (Group
C-34
-------
II). No adverse effects were noted in hematological studies,
clinical blood chemistry studies, and urinalysis. No signi-
ficant gross or microscopic pathology was noted except the
local skin lesions. Gross skin changes were characterized
by fibrosis, escharosis (scarring), and slight-to-severe
desquamation. Microscopic examination revealed acanthosis
and hyperkeratosis involving the epidermis. This effect
was seen in a few of the animals in Group I and most of
the animals in Group II. Such findings were thought to
be attributable to the irritant action of hex.
c) Inhalation Exposures. Rats, rabbits, guinea pigs,
and mice exposed to vapors of hex showed signs of extreme
irritation of the eyes and mucous membranes (Treon, et al.
1955). At very high concentrations (46.5 ppm) animals re-
sponded by rubbing their noses with their forefeet, closing
their eyes and retracting their heads. This behavior was
accompanied by sneezing, tearing, and irregular breathing.
In less than 30-60 minutes the animals were gasping for
breath.
Lower concentration of hex vapor (12.4 and 13.8 ppm)
produced similar irritation of the mucous membranes, al-
though somewhat milder in degree. The same symptoms were
even seen at the low dosages (1.0 and 1.6 ppm), but the
symptoms developed over a period of hours rather than min-
utes. Exposure to very low concentrations (0.33 ppm and
0.15 ppm) resulted in some irritation of the eyelids and
increased respiratory rate. In the case of the latter dos-
C-35
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age (0.15 ppm), irritation was seen only in the mice, which
developed mild respiratory changes (Treon, et al. 1955).
Rats which survived the vapor exposure sessions lost weight
and many of these animals failed to regain their initial
weights as long as 6 to 8 weeks after cessation of the expo-
sures.
At autopsy Treon, et al. (1955) reported degenerative
changes similar to those described above (oral and dermal
administration experiments) in all species of animals tested.
Prolonged intermittent exposure to vapor concentrations
as low as 0.15 ppm hex induced slight degenerative changes
in the livers and kidneys in all species of animals employed.
Industrial Bio-Test Laboratories (1ST) also conducted
two vapor toxicity studies: an acute test and a 28-day
subacute test. Results of these studies were reported by
Equitable Environmental Health (1976). In the acute vapor
toxicity tests, Charles River rats were exposed for 4 hours
to varying concentrations of hex in air. An acute LD50
of 3.67 ppm was reported. Complete necropsies were performed
at death for those which died shortly after exposure and
at the termination of the study for those which survived.
Acute pneumonia was observed in the rats which died follow-
ing exposure to the test material. Treated rats which sur-
vived to the end of the study showed emaciation and chronic
proliferative inflammatory changes in the lungs.
C-36
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The second Industrial Bio-Test Laboratories study con-
sisted of a 28-day subacute vapor inhalation study using
albino rats (Equitable Environ. Hlth., 1976). Two groups
of 10 rats each were exposed to hex vapor for 6 hours per
day, 5 days per week for 4 weeks. A third group of 10 rats
served as untreated controls. One group was exposed to
vapor containing 0.529 ppm hex and the other experimental
group was exposed to 1.23 ppm. Hematologic and clinical
chemistry studies and urinalysis were performed on days
0 and 28. On the twenty-eighth day the animals were sacri-
ficed. No mention was made of mortality in either exposure
group so presumably all animals survived until the time
of sacrifice. Neither hematologic nor clinical studies
or urinalysis revealed any abnormalities directly attributed
to the hex vapor. Increases in absolute liver weight and
liver-to-brain weight ratios were seen among male rats and
increased liver-to-body weight ratios were seen for both
sexes of rats exposed to 1.23 ppm. Gross pathological exami-
nations failed to reveal abnormalities attributable to hex,
but microscopic examination of tissue revealed hepatocyto-
megaly and necrotizing hepatitis. These effects were thought
to be attributable to hex exposure.
Synergism and/or Antagonism
There does not appear to be any information available
on synergistic or antagonistic effects between hex and other
compounds.
C-37
-------
Teratogenicity
International Research and Development Corporation (Int.
Res. Dev. Corp., 1978) has recently completed a pilot tera-
tology study using pregnant Charles River (CD) rats. Nega-
tive findings with respect to teratogenic effects were re-
ported for oral hex dosages up to 100 mg/kg/day.
The test protocol employed in the pilot teratology study
involved administration of various dosages of hex to 30
female Charles River (CD) rats approximately 12 weeks of
age. Females were mated with male rats of the same strain.
After mating, the females were assigned to six groups, one
control and five treatment groups of five rats each. Hex
was dissolved in corn oil and administered by gavage from
day 6 through day 15 of gestation. Dosage levels of 3,
10, 30, 100, and 300 mg/kg/day were administered to the
test groups and the control group was given the vehicle
(corn oil) on a comparable regimen of 10 ml/kg/day.
During gestation, the females were observed for clini-
cal signs of toxicity, mortality, and body weight gains.
They were then sacrificed on gestation day 20 and the ute-
rine contents examined for viable and nonviable fetuses,
early and late resorptions, and total implantations. There
were no differences in the four treatment groups given 100
mg/kg/day or less when compared to the control group in
terms of number of viable or nonviable fetuses, resorptions,
implantations, or corpora lutea. Rats receiving doses of
3 or 10 mg/kg/day showed no treatment-related changes in
C-38
-------
appearance or behavior. Rats receiving 30 mg/kg/day or
higher showed staining of the anogenital area and reduced
body weight gains. The females in the 100 mg/kg/day group
had body weight losses during the first 3 days of treatment
and reduced weight gains for the remainder of the study.
Survival was 100 percent for all rats given 100 mg/kg/day
or less. All rats in the 300 mg/kg/day group were dead
by gestation day 10.
Various reproductive parameters examined in the pilot
teratology study are shown in Table 3.
Mutagenicity
Hex has. been tested for mutagenicity and reported non-
mutagenic in both short-term _in vitro mutagenic assays (Na-
tional Cancer Institute, 1977; Industrial Bio-Test Labora-
tories, 1977; Litton Bionetics, 1978a) and in a mouse domi-
nant lethal study (Litton Bionetics, 1978b).
The National Cancer Institute (1977) reported that preli-
minary results indicated that hex was non-mutagenic in Esche-
richia coli K12 (mutation site not specified) in the pre-
sence of a mammalian metabolic activation system containing
mouse liver microsomes.
Negative results were also reported by Industrial Bio-
Test Laboratories (1977) using a test protocol almost iden-
tical to the Ames Mutagenic Assay (Ames, et al. 1975).
The tests used four strains of Salmonella typhimurium with
and without metabolic activation. Hex was dissolved in
acetone and added to the microbial assay plates in dosages
C-39
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TABLE 3
Pilot Teratology Study in Rats: Cesarean Section Data
For Individual Females (Int. Res. Dev. Corp., 1978b)
Dosage Level
Dam Number
Control:
73662
77334
77336
77428
77428
Total
Mean
3 mg/ kg/day:
73642
77342
77343
77426
77428
JPotal
plean
10 mg/kg/day:
4K304
W309
77346
77427
77436
Total
Mean
30 mg/kg/day:
77310
77313
77350
77438
77450
Total
Mean
100 mg/kg/day:
73673
77302
77314
77415
K7439
"otal
Mean
2flO mg/kg/day;
Viable
Fetuses
13
14
12
11
15
65
13.0
16
17
16
12
15
76
15.2
17
13
11
12
15
68
13.6
14
13
6
11
12
56
11.2
16
14
15
11
12
68
13.6
Nonviable
Fetuses
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
Late
Resorptions
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
Early
Resorptions
1
1
1
1
0
4
0.8
0
0
0
1
0
1
0.2
0
0
3
0
0
3
0.6
0
1
0
0
0
1
0.2
0
0
2
0
0
2
0.4
Post
Implantation
Loss
1
1
1
1
0
4
0.8
0
0
0
1
0
1
0.2
0
0
3
0
0
3
0.6
0
1
0
0
0
1
0.2
0
0
2
0
0
2
0.4
Implan-
tations
14
15
13
12
15
69
13.8
16
17
16
13
15
77
15.4
17
13
14
12
15
71
14.2
14
14
6
11
12
57
11.4
16
14
17
11
12
70
14.0
Corpora
Lutes
14
15
13
22
16
80
16.0
16
17
16
18
15
82
16.4
18
13
14
13
15
73
14.6
14
16
7
14
14
65
13.0
16
14
17
11
12
70
14.0
r58 Died,
77324 Died,
77333 Died,
77417 Died,
77445 Died,
gestation day 9 - gravid
gestation day 10 - gravid
,gestation day 10 - gravid
gestation day 10 - gravid
gestation day 10 - gravid
C-40
-------
from 10-5000 pg/10 pi. Concentrations greater than 10 jug/10
pi produced a bacteriocidal effect in three of the four
strains tested; a possible lethal effect occurred at 2500
pg/10 pi or greater in the fourth strain. A repressive
effect was noted in three of the four strains at concentra-
tions below 10 pg/10 pi. Volitilate (volatile vapors) of
hex was also tested on one strain using the vapor from hex
concentrations of up to 2500 pg/10 pi and exposure times
of up to 2 hours. Results from two successive assays in
the absence of rat liver enzymes (hex concentrations 10,
25, 50, 75, and 100 pg/10 pi) were negative in all four
tester strains. Two assays using the same dosages in the
presence of rat liver microsomes were reported non-mutagenic;
similarly, negative results were obtained for the hex effu-
sate as well. The investigators expressed concern over
the repressive effect of hex on the test bacteria, stating
"It appears that hex is probably non-mutagnic and that some
toxic effect prevailed with respect to the tester strains
required for this assay. Analysis of variance and multiple
comparison of the data confirms this observation".
Litton Bionetics (1978a) conducted a mouse lymphoma
cell assay in order to evaluate the capability of hex in
inducing specific locus forward mutation. The indicator
cells used in the assay were Fischer mouse lymphoma cells
derived from cell line L5178Y. These cells are heterozygous
for a specific autosomal mutation at the TK locus and are
bromodeoxyuridine (BUdR) sensitive. Scoring for mutation
C-41
-------
is based on selecting cells which have undergone forward
mutation from a TK+/- to a TK-/- genotype by cloning them
in soft agar with BUdR. Cells were maintained in Fischer's
medium for leukemic mouse cells with 10 percent horse serum
and sodium pyruvate. The dosages used in the test were
predetermined by exposing the cells to a wide range of hex
concentrations and measuring the reduction of growth poten-
tial following a 4-hour exposure at each dose. The max-
imum dose selected was that which produced a 50 percent
reduction in growth. The actual hex dosages employed were:
.00040 >jl/ml;
.00048 >il/ml;
.00056 jJl/ml;
.00064 jul/ml; and
.00125 jjl/ml
in the activated series (mouse liver microsomes were added
to the growth medium). A nonactivated series using somewhat
lower dosages was included also.
Both negative and positive controls were used; the
negative control for both series was the solvent dimethyl-
sulfoxide (DMSO), whereas ethylmethane-sulfonate (EMS) and
dimethylnitrosamine (EMN) were used as positive controls
in the nonactivated and activated systems, respectively.
Hex was added to the cells in the growth medium for 4 hours.
The cells were then washed and allowed to express in the
growth medium for 3 days. After the expression period,
results were evaluated by counting the TK-/- mutants after
cloning the cells in a selection medium (soft agar with
BUdR).
C-42
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Hex dissolved in DMSO was evaluated over the concentra-
tion range of 0.0000025 jjl/ml to 0.00125 jjl/ml. Consider-
able toxicity occurred at concentrations greater than this
and the extent varied according to the presence of the mouse
liver activation system as shown in Table 4. No cells treated
with hex (at the concentrations shown) survived in the non-
activated system.
Hexachlorocyclopentadiene did not induce forward muta-
tion in L5178Y cells. The data presented in Table 4 show
the concentrations of the test compound employed, the number
of mutant clones obtained, surviving populations after the
expression period, and calculated mutation frequencies.
No dose-related trends in either absolute number of mutants
or mutant frequencies were observed, and at no level did
any of the test parameters increase significantly over the
spontaneous level. Consequently, hex was considered to
be nonmutagenic under the conditions of this assay.
The mutagenic properties of hex were also evaluated
in a dominant lethal study of mice (Litton Bionetics, 1978b).
The dominant lethal assay provides a means of determining
whether a compound is capable of inducing damage in the
germ cells of treated male mice. Dominant lethality is
manifested in various forms of fetal wastage, both pre-and
post-implantation. Positive dominant lethal assays indicate
that a compound is able to reach the developing germ cells.
Chromosome aberrations including breaks, rearrangements,
and deletions as well as ploidy changes and nondisjunction
C-43
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n
i
A.
B.
C.
NOTE:
TEST
TABLE 4
Summary of Mouse Lymphoma (L5^_gx) Results (Litton Bionetics, 1978a)
Name or code designation of the test compound: Hexachloropentadiene
Solvent: DMSO
Test date: 12/18/77
Concentrations are given in microliters (UL) or micrograms (UG) or nanoliters (NL) per milliliter.
Relative Relative
Suspension Total Total Cloning Percent
S-9 Daily Counts Growth (% Mutant Viable Efficiency Relative
Source Tissue (Cells/ml x 10ES) of control) Clones Clones (% of control) Growth*
NOHACTIVATION
Solvent control
Negative control
EMS .5UL/ML
ACTIVATION
Solvent control
Negative control
DMN .5UL/ML
Test compound
0.00002 UL/ML
0.00004 UL/ML
0.00008 UL/ML
0.00016 UL/ML
0.00032 UL/ML
mouse
mouse
mouse
mouse
mouse
mouse
mouse
mouse
liver
liver
liver
liver
liver
liver
liver
liver
1
16.8
13.2
9.0
15.2
14.2
7.2
16.8
13.0
12.4
13.6
18.2
2
10.2
12.0
9.2
9.6
13.0
7.6
9.0
12.4
9.8
13.8
9.0
3
13.8
15.0
11.8"
13.2
10.6
8.2
10.6
9.6
16.2
7.4
10.0
100.0
100.5
41.3
100.0
101.6
23.3
83.2
80.3
102.2
72.1
85.0
48.0
48.0
597.0
55.0
39.0
322.0
99.0
50.0
55.0
45.0
38.0
257.0
234.0
89.0
281.0
293.0
55.0
288.0
269.0
194.0
359.0
309.0
100.0
91.1
34.6
100.0
104.3
19.6
102.5
95.7
69.0
127.8
110.0
100.0
91.5
14.3
100.0
105.9
4.6
85.3
76.9
70.6
92.1
93.5
Mutant
Frequency**
(X 10E-6)
18.7
20.5
670.8
19.6
13.3
585.5
34.4
18.6
28.4
12.5
12.3
(Relative suspension growth X relative cloning efficiency: / 100
(Mutant clones / viable clones) X 10E-6
-------
are believed to produce positive results on this test.
Since substances capable of producing gross chromosomal
lesions are probably capable of producing more subtle bal-
anced lesions or specific locus mutations, the test also
provides suggestive evidence of nonlethal mutations transmis-
sible to future generations as well.
Litton Bionetics reported negative results, that is,
there was no evidence of significant dominant -lethal acti-
vity by hex in mice. The test protocol called for the assign-
ment of ten random bred male mice to one of five groups.
Three test groups received hex dosages of 1.0 mg/kg, 0.3
nig/kg, and 0.1 mg/kg, respectively. These dosages were
determined by deriving an LD50 level (1.0 mg/kg) and taking
one-third and one-tenth of that dose. A fourth group re-
ceived only the solvent and the fifth group served as a
positive control. Hex was mixed in the feed of the three
experimental groups and the solvent control group for five
consecutive days. The positive control group received a
known mutagen, triethylenemelamine (TEM) in a single intra-
peritoneal injection. Two days following treatment, each
male was caged with two unexposed virgin females. At the
end of seven days, these females were removed and replaced
by two unexposed virgin females. This mating cycle was
continued for seven weeks. Each pair of female mice was
killed two weeks after mating and necropsied. Their uterine
contents were examined for dead and living fetuses, resorp-
tion sites, and total implantations. All test parameters
C-45
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(fertility index, average implantations per pregnancy, aver-
age resorptions (dead implants) per pregnancy, proportion
of females with one or more dead implantations, proportion
of females with two or more dead implantations, and the
ratio of dead implantations to total implantations) were
within normal limits based on historical and concurrent
control levels for this test. Thus, there was no evidence
of dominant lethal activity in any of the hex treated groups.
The positive control group, however, did show the expected
dominant lethal activity.
Carcinogenici ty
Various types of evidence may be used in evaluating
the possible carcinogenic activity of a substance. In order
of preference, these include: (1) human data; (2) animal
data; (3) short-term (in vitro) tests; (4) metabolic pat-
tern; and (5) structure-activity relationships. This
section summarizes what is known about each of the above.
No epidemiologic studies or case reports examining the
relationships between exposure to hex and cancer incidences
could be found in the literature. As indicated previously,
Hooker Chemicals and Plastics Corporation reports that an
in-house study of the mortality patterns of hex-exposed
workers is now underway; however, the study is far from
being completed (Zavon, 1978, personal communication).
Other in-house studies of workers employed in the manufac-
ture of pesticides (including hex) are reportedly being
conducted by Velsicol Chemical Corporation. We were unable
to obtain any further information on the current status
or findings of these studies.
C-46
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The National Cancer Institute concluded that toxicologic
studies of hex in animals have not been adequate for evalua-
tion of carcinogenicity (National Cancer Inst., 1977).
Chronic toxicity studies as reported in section 4 Toxic
Symptoms and Pathologic Effects, were based on too few ani-
mals in some cases and/or the duration of the experiments
was too short for appropriate evaluation of chronic effects,
including carcinogencity.
Only one short term in vitro test of hex for carcino-
genic activity could be identified.
Litton Bionetics (1977) reported the results of a test
to determine whether hex could induce malignant transforma-
tion in BALB/3T3 cells in vitro. The cells and methodology
of the test were those of Dr. T. Kakunaga, described else-
where (Int. Jour. Cancer 12:463, 1973). The basic rationale
of the test and its validity as an indicator of carcinogenic
activity was described by the investigators as follows:
The endpoint of carcinogenic activity is deter-
mined by the presence of fibroblastic-like colo-
nies which are altered morphologically in com-
parison to the cells observed in normal cultures.
These (transformed) cells grow in criss-cross,
randomly oriented fashion with overlapping at
the periphery of the colony. The colony exhibits
dense piling up of cells. On staining, the foci
are deeply stained and the cells are basophilic
in character and variable in size. These changes
are not observed in normal cultures, which stain
uniformly.
Cell cultures with very little or no spontaneous trans-
formation are maintained for use in these tests. The data
generated at each dose level of the test material are ana-
lyzed using the t statistic. A significant set of data
C-47
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for any dose level may be sufficient to indicate a positive ,
response. Because this assay is still nonroutine, and defi-
nitive criteria for evaluation have yet to be developed,
scientific judgement and expert consultation are needed
for appropriate interpretation of results.
The BALB/3T3 cells used in the test were grown in Eagle's
minimal essential medium (EMEM) supplemented by 10 percent
fetal calf serum. Cultures were passaged weekly in 60 mm
culture dishes. Approximately 10,000 cells were seeded
into 50 ml sterile tissue culture flasks and incubated in
EMEM to permit attachment. After the cells were attached,
the control and test compounds were added to the plates.
Dosages of 0.00001 pi/ml; 0.00002 jul/ml; 0.000039 jul/ml;
0.000078 jul/ml; and 0.000156 pi/ml of hex were employed.
The maximal dosage, 0.000156 /il/ml, was determined by se-
lecting from preliminary cytotoxicity tests the maximum
dosage which permitted survival of at least 80 percent of
the cells. 3-Methylcholanthrene at 5 pg/ml was used as
a positive control and the test compound solvent was used
as a negative control. Ten replicates per dose level were
prepared and chemical exposure was maintained for 48 hours.
Plates were then washed free of the compound and replenished
with fresh growth medium. The plates were then incubated
for an additional 3-4 weeks with twice weekly medium changes.
Cell integrity was monitored by daily observations. Cells
were separated from the medium, washed with saline, and
stained. They were examined for stained foci; all poten-
tial foci were examined microscopically. Results were pre-
sented as the number of foci per set of replicate plates
at each dosage level.
C-48
-------
The test material was quite toxic to cells as indicated
in the preliminary range-finding tests. No significant
carcinogenic activity for hex was reported under the condi-
tions of this test. A low level of spontaneous transforma-
tion was observed on all of the plates. Only the 3-methyl-
cholanthrene treated plates showed a significantly higher
number of transformed foci than the negative control.
It should be noted that in this and other cell culture
tests, extremely low dosages of hex were used. Because
hex is relatively toxic to cells in culture and test proto-
cols normally require a high survival rate, the applicabi-
lity of test results to environmental conditions is unclear.
Taken together, however, the mutagenicity and carcinogeni-
city tests conducted by Litton (1977, 1978a) suggest that
outright toxicity, rather than chronic effects, is perhaps
the critical effect of hex, even at very low dosages. Ex-
tremely poor survival has also been problematic in several
subchronic tests of hex in mammalian species.
A very recent study involving chronic dietary exposure
of rats to hexachlorobutadiene also provides some insight
into the relationship between direct toxic effects and chro-
nic effects (i.e., carcinogenesis) in this related compound
(Kociba, et al. 1977).
Male and female Sprague-Dawley rats were maintained
on diets supplying 20, 2.0, 0.2, or 0 mg/kg/day of hexa-
chlorobutadiene (HCBD) for up to 2 years. Rats ingesting
0.2 mg/kg/day had no discernible ill effects that could
C-49
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be attributed to this dose level. Ingestion of the inter-
mediate dose level of 2.0 mg/kg/day caused some degree of
toxicity, affecting primarily the kidney in which increased
renal tubular epithelial hyperplasia was noted. Urinary
excretion of coproporphyrin was also increased at this dose
(level. Ingestion of the highest dose level of 20 mg/kg/day
(caused a greater degree of toxicity. Effects included de-
creased body weight gain and length of survival, increased
urinary excretion of coproporphyrin, increased weights of
kidneys, and renal tubular adenomas and adenocarcinomas,
some of which metastasized to the lung. In this study irre-
versible toxicological effects, such as the development
of neoplasms, occurred only at a dose level which caused
significant tissue injury and other manifestations of toxi-
city. No neoplasms resulted with dose levels which caused
no injury or only mild, reversible injury.
Little information is available on the metabolism of
hex. Although at least four metabolites were found in the
Mehendale (1977) study, at the time of this writing they
had not been identified. Thus, the metabolic pathway is
uncertain.
As far as structure/activity relationships are concerned,
the National Cancer Institute (1977) speculated that as
a cyclopentene vinyl halide, hex potentially may be meta-
bolized to an electrophile. In addition, hex is related
to the pesticides dieldrin, heptachlor, and chlordane which
have been found to induce liver tumors in mice following
oral administration (National Cancer Inst., 1977).
C-50
-------
Hex has recently been selected for testing in the Na-
tional Cancer Institute's test program (National Cancer
Inst., 1977). The reasons given for its selection include:
(1) its high potential for exposure (as an industrial inter-
mediate used in the manufacture of pesticides, flame retar-
dants and dyes, Pharmaceuticals, resins, and germicides);
(2) its suspect chemical structure; and (3) the relative
lack of information on the effects of chronic exposure to
this compound.
Extremely limited data are available concerning the
effects of hex exposure on humans. That which is known
about acute human toxicity is based largely upon isolated
spills or other accidental incidents involving pesticide
workers, laboratory technicians, or others having occupa-
tional contact with hex. A recent incident in which approxi-
mately 200 sewage treatment plant workers were exposed to
acutely toxic levels of hex from the illegal disposal of
large quantities of the compound has done much to elucidate
the potential health effects of acute human exposures.
Due to the accidental and episodic nature of these incidents
and the lag time in setting up environmental monitoring
equipment in response to the incidents, it has not been
possible to measure environmental concentrations of hex
at the exact time workers report symptoms (post-exposure
sampling results are sometimes available). Thus, while
there is information regarding the range and variety of
toxic responses, the exact dose which elicited a given re-
sponse remains unknown. It is obvious that reliable dose-
response estimates require accurate measurement of both
dose and response parameters.
C-51
-------
There is virtually no information regarding the health
effects of chronic exposure to hex. Despite the fact that
there are a number of potential cohorts for retrospective
epidemiologic studies (see section 2), studies of chronically
exposed workers have not been reported to date. Hooker
Chemicals and Plastics Corporation, a manufacturer of hex,
reports that they are presently conducting a mortality study
of hex-exposed workers, but the study is in its initial
stages and very likely would not be completed until 1980
(Zavon, 1978, personal communication).
Likewise, virtually nothing is known regarding the poten-
tial effects resulting from chronic exposure to environ-
mental sources of hex. Potential modes of environmental
exposure (e.g., through ingestion of contaminated air or
water) are uncertain at this time.
According to Hooker's material safety data sheet for
hexachlorocyclopentadiene (Hooker Industrial Chemicals Divi-
sion, 1972), the compound is very irritating to the eyes
and mucous membranes causing lacrimation, sneezing, and
salivation. Repeated contact with the skin can cause blis-
tering burns, and inhalation can cause pulmonary edema.
Hex is readily absorbed through the skin. Ingestion can
cause nausea, vomiting, diarrhea, lethargy, and retarded
respiration. Recommendations for safe use include: (1)
good general ventiliation plus local exhaust at points of
potential fume emission; (2) respiratory protection of
the organic vapor-acid gas canister type and full-face self-
C-52
-------
contained breathing apparatus for emergencies; (3) elbow
length neoprene gloves; (4) eye protection including chemi-
cal safety glasses, plus face shield where appropriate;
(5) protective clothing including full length clothing fas-
tened at neck and wrist, rubber safety shoes or boots, rubber
or other impervious clothing or aprons as needed for splash
protection.
According to Treon et al. (1955), a very faint odor
of hex was detectable in air by some individuals at concen-
trations as low as 0.15 ppm which was the lowest concentra-
tion employed in their experiments. At approximately twice
that concentration (0.33 ppm), a very pronounced, pungent
odor was present.
Treon, et al. (1955) observed that headaches developed
among laboratory workers following incidental exposure to
hex vapor from the respiratory chambers used for their vapor
inhalation experiments. The exact concentration of hex
escaping into the laboratory from the opening of the respi-
ratory chamber is unknown; however, the chamber was not
opened until the contaminated air had been exhausted and
the chamber flushed for some time with clean air. Thus,
the ambient concentration producing headhaches among the
laboratory workers was well below the dosages employed in
the animal experiments. Because no mention is made of any
other irritative symptoms (e.g., lacrimation, etc.), it
seems reasonable to speculate that the concentration of
hex present was somewhere in the range between 0.15 ppm-
1.0 ppm, above the detection threshold but below the level
producing acute symptoms of irritation.
C-53
-------
Irritant effects are elicited at a vapor concentration
greater than that shown to produce chronic toxicity in ani-
mals. Thus, Treon et al. (1955) concluded that the irri-
tant effects of hex vapors are not sufficiently pronounced
to serve as a warning that a hazardous level of hex vapor
is present and/or that hazardous exposure is taking place.
According to Naishstein and Lisovskaya (1965), hex may
be detected by taste and smell at very low concentrations
in water. They placed the threshold level for altering
the organoleptic qualities of water at 0.0014-0.0010 mg/1.
4) Epidemiologic Studies. To date, the only well
documented incident of the acute toxicity of hex to humans
occurred at the Morris Forman Wastewater Treatment Plant
(MFWTP) in Louisville, Kentucky. The problem apparently
began about the middle of March, 1977, when an unknown chemi-
cal, later identified as a mixture of hex and octachlorocyclo-
pentene (C-58), began entering the Morris Forman sewage
treatment facility. An exact date of initial appearance
at the plant, and hence, the initial date of worker exposure
is unknown. However, unusual odors became evident around
March 17, 1977.
The odor gradually intensified over the next 2 weeks.
From March 25-28, an odoriferous, sticky material entered
the plant and gummed the barscreens and grit collection
systems in the primary treatment area. Attempts to dislodge
the material with steam produced a blue gas which permeated
the grit removal and sludge handling areas. Workers exposed
C-54
-------
to this vapor complained of severe irritation of the eyes,
nose, throat, and lungs (Carter, 1977b). Approximately
20 workers sought medical treatment for tracheobronchial
irritation. These workers were treated in the local emer-
gency room; none was hospitalized (Singal, 1978).
A sample of the material from the Screen and Grit Build-
ing was sent to the U.S. EPA Laboratory in Athens, Georgia,
for analysis. The primary contaminants in the samples were
identified as hexachlorocyclopentadiene (hex) and octachloro-
cyclopentene (octa). (Octa is a waste by-product in the
manufacture of hex whose toxicity is presently unknown).
Table 5 shows the results of the analysis. Due to the ap-
parent potential toxicity of hex (and the unknown toxic
potential of octa), the sewage treatment plant was evacuated
and closed on March 29, 1977. Thereafter, until the partial
reopening in June, 1977, 105 million gallons per day of
domestic and industrial wastes were diverted directly to
the Ohio River.
Estimates of the extent of contamination indicate that
about 60 million gallons (25,000 tons) of hex-contaminated
material were present at the Morris Forman plant. Of this,
approximately 6 tons of hex and octa were thought to be
present in the contaminated waste. U.S. EPA's analysis
revealed hex concentrations up to 1000 ppm in the sewage
water at the time of the plant closure. The route of chemi-
cal contamination was traced to one large sewer line which
passed through several heavily populated areas. Wastewater
C-55
-------
TABLE 5
Analysis of a Sludge Sample Obtained in the
Screen and Grit Building on April 2, 1977,
Morris Forman Wastewater Treatment Plant,
Louisville, Kentucky
(Singal, 1978)
COMPOUND'
Concentration - % by weight
Octachlorocyclopentene
Hexachlorocyclopentadiene
Hexachlorobenzene
Pentachlorobenzene
Octachloronaphthalene
Heptachloronaphthalene
Hexachloronaphthalene
Mirex
9
4
0.
0.
0.
0.
0.007
(estimated)
(estimated)
(not quanti-
tated)
(estimated)
Analysis was conducted by the U.S. Food and Drug Administration,
Division of Chemical Technology, Chemical Industry Practices
Branch.
2
The sample was analyzed using gas chromatography interphased with
mass spectroscopy for positive identification of each compound.
C-56
-------
in this sewer showed hex and octa in concentrations ranging
up to 100 ppm. Samples from the sewer showed air concen-
trations ranging up to 0.4 mg/1 for hex and up to 0.03 mg/1
of octa. Thus, it was decided to investigate the health
of not only the workers at the sewage treatment plant but
also residents of the area surrounding the sewer line (Morse,
et al. 1978).
A cooperative investigation involving Region IV U.S.
EPA (Surveillance and Analysis Division), Center for Di-
sease Control (CDC), National Institute for Occupational
Safety and Health (NIOSH), Jefferson County (Kentucky) Health
Department, and the Kentucky State Health Department was
initiated.
Information on both aspects of the investigation (i.e.,
community residents on one hand and exposed workers on the
other) is thus far unpublished but preliminary drafts of
reports were made available by Dale Morse, M.D., who headed
the initial epidemiologic studies conducted by the Center
for Disease Control (Morse, et al. 1978) and by Mitchell
Singal of the Hazard Evaluation and Technical Assistance
Branch of NIOSH who reported on the follow-up investigations
of workers during cleanup operations at the sewage treatment
facility (Singal, 1978, personal communication). Findings
from these drafts are reported below; however, they should
be regarded as preliminary.
C-57
-------
a) Plant Employee Health Effects Evaluation. i) Ini-
tial studies. The Center for Disease Control investigation
began by identifying all sewage treatment employees who
worked at the plant for 2 or more days during the period
from March 14-29, 1977. Health effects evaluations, includ-
ing mailed questionnaires, physical examination, and blood
and urine testing, were conducted appropriately to exposed
individuals who agreed to participate. The questionnaire
covered demographic information, a detailed work-area his-
tory, symptoms and history of chemical poisoning, personal
habits, and other sources of chemical exposure. Routine
tests were performed on blood and urine speciments. Addi-
tional samples were sent to NIOSH laboratories for potential
toxic chemical analysis.
Of 193 plant employees who had worked during the latter
half of March, questionnaire data was obtained from 145.
Seventy-five percent of the questionnaire respondents indi-
cated that they detected an unusual odor at the plant some-
time during March. A few individuals reported detecting
unusual odors as early as March 1, 1977; the percentage
reporting the odor by March 14 was noticeably increased.
From March 15 onward, the percentage of workers who reported
noticing the odor steadily increased until the plant was
closed on March 29.
A comparison between the time of odor detection and
the onset of eye irritation, the most common symptom, showed
that irritation developed on the same day in 45 percent
of individuals, within 1-5 days in 28 percent, and after
5 days in 21 percent. Only 6 percent of employees reported
onset of symptoms prior to noticing an unusual odor at the
plant.
C-58
-------
Eye irritation, headache, and throat irritation were
the most common symptoms, with 59 percent, 45 percent, and
27 percent of employees reporting these symptoms, respec-
tively. Data for these and other symptoms is reported in
Table 6. Of 41 workers physically examined, five had signs
of eye irritation (tearing and/or redness) and five had
signs of skin irritation.
Forty-two persons were interviewed and provided blood
and urine samples. This included 24 of 29 (83 percent)
of the workers who had been previously evaluated by local
physicians, 17 of 164 other plant employees (a 10 percent
random sample) as well as one non-employee accidentally
exposed to the contamined sludge.
Abnormalities were found in laboratory analysis of
some of the workers (e.g., LDH elevations in 27 percent
and proteinuria in 15 percent of those examined). These
results are suggestive of either a transitory abnormality
or a problem with the laboratory analysis. No LDH or urin-
alysis abnormalities were corroborated on repeat tests run
3 weeks later by another laboratory. Also, no abnormalities
were reported among individuals seen at the local hospital
or by the plant physician.
Analysis of data according to employee work areas revealed
that symptoms occurred in workers of all job categories
and in all work areas. Data for attack rates in employees
by main work area is reported in Table 7. Only small differ-
ences in case rates appeared by work area although the highest
C-59
-------
TABLE 6
n
i
o\
o
Symptoms of 145 Plant Employees, Louisville, Kentucky,
March, 1977 (Morse, et al. 1978)
Percent
Symptom With Symptom With Symptom
Eye irritation
Headache
Throat irritation
Nausea
Skin irritation
Cough
Chest pain
Difficult breathing
Nervousness
Abdominal cramps
Decreased appetite
Decreased memory
Increased saliva
86
65
39
31
29
28
28
23
21
17
13
6
6
59
45
27
21
20
19
19
16
14
12
9
4
4
-------
TABLE 7
Attack Rates in Employees, by
Louisville, Kentucky, March, 1977
Main Work Area,
(Morse, et al. 1978)
o
i
a\
Main Work Area
Primary treatment
Throughout plant
Vacuum filtration
Secondary aeration
chamber
Administration and
laboratory
Final effluent
pump station
Low pressure
oxidation
I ncineration
Number of
Employees
19
71
19
14
30
10
13
17
Number
Reporting
Symptoms
17
54
15
12
22
5
10
10
Percentage of
Employees Re-
porting Symptoms
89
76
79
86
73
50
77
50
Percentage of Cases
of Those Reporting
Symptoms
59
48
47
42
41
40
30
20
Totals
193
145
75
44
-------
attack rates occurred in workers in the primary treatment
area where the level of hex was presumably highest. Attack
rates were significantly higher by X test for individuals
who had been exposed to the screen and grit chamber (p =
.0001) and to the primary settling area (p = .02) than for
workers not exposed to these areas.
The initial investigation demonstrated that 64 of 145
(44 percent) of current employees questioned at the waste-
water treatment plant had experienced headache and mucous
membrane, skin, and respiratory tract irritation after expo-
sure to airborne hex. Highest attack rates occurred among
workers in the primary treatment area where exposure was
highest and ventilation poorest. In most cases symptoms
were transient, but in some workers, they persisted for
several days. This episode clearly demonstrates the volati-
lity of hex and its potential for having a toxic effect
on humans. Results of the follow-up investigation of the
sewage treatment plant workers and the community survey
are reported below.
ii) Follow-up Survey. After the initial health evalua-
tion survey was completed (April 3, 1977), NIOSH assumed
the responsibility for follow-up of the sewage treatment
workers exposed during the March, 1977, episode. NIOSH
was also responsible for medical monitoring of those involved
in the cleanup operations prior to reopening the Morris
Forman plant. NIOSH1s activities consisted of the following:
(1) administering follow-up questionnaires to all plant
C-62
-------
employees to determine how persistent symptoms had been
after the initial chemical exposure in March; (2) review
of the medical records of the 90 employees who had seen
the plant physician from late March through May 10, 1977;
(3) collection of repeat biologic samples on the 23 employees
who had shown some abnormality on the testing done by the
CDC physicians (March 31-April 2, 1977, tests); (4) biolo-
gical monitoring of EPA and NIOSH industrial hygienists
and environmental technicians exposed to the chemicals in
the sewer system during cleanup; and (5) medical monitoring
of Morris Forman plant employees who were actively involved
in the plant cleanup. Results of each of these aspects
of the investigation are reported below.
Usable responses were obtained from 182 individuals
on the follow-up questionnaire. The frequency of symptoms
among those who completed the questionnaire is shown in
Table 8. In decreasing order of frequency, these symptoms
included eye irritation, headache, fatigue, chest discomfort,
sore throat, cough, nausea, and skin rash. These symptoms
were surprisingly persistent. Except for eye irritation
and sore throat, 25-45 percent of those who exhibited symp-
toms during the last 2 weeks of March, 1977, still had them
6 weeks later. Although symptoms occurred in workers in
all areas of the plant, maintenance department personnel
consistently reported the highest number of symptoms.
C-63
-------
TABLE 8
Symptoms Reported on Follow-up Questionnaire,* Morris Forman Wastewater
Treatment Plant, Louisville, Kentucky (Singal, 1978)
Symptom
% with symptoms
Persistence of Symptoms** (% of those c symptoms)
Headache
Eye Irritation
Sore Throat
Cough
o
OA Chest Discomfort
Skin Irritation
Nausea
Fatigue
in last 2 weeks
of March**
55%
62%
30%
24%
34%
21%
22%
34%
Gone within
1 day
19%
36%
15%
14%
11%
18%
18%
8%
Gone within
1 Week
30%
23%
49%
27%
20%
18%
23%
16%
Gone within
2 Weeks
18%
16%
13%
16%
21%
10%
18%
24%
Still
at time
32%
15%
18%
36%
39%
46%
25%
45%
present
of survey
*Distributed and Collected last 2 weeks of May 1977
Excludes employees actively involved in cleanup, since their symptoms could relate to exposure
during cleanup instead of to exposure prior to the plant shutdown.
**% Persistences do not quite add to 100% due to some employee confusion about the need to fill
in questionnaire completely.
-------
A review of medical records of the 90 workers examined
by the plant physician (mid-March to May 10, .1977) revealed
symptom reports similar to those reported on the NIOSH and
CDC questionnaires. Fatigue, headache, and mucous membrane
irritation were the predominant complaints; respiratory
and skin problems were also reported. Seven of the 90 workers
reported transient memory loss ranging from a few minutes
to a few days. These are believed to represent a transient
state of confusion, rather than true amnesia (Singal, 1978,
personal communication). Although several workers reported
neurologic symptoms, the plant physician found no one with
any objective neurologic signs. Seven persons had rash
on exposed areas of face and arms. Respiratory tract symp-
toms, cough, and chest discomfort were commonly reported.
Twenty-eight persons, including those with respiratory symp-
toms, received chest x-rays. Essentially all of the x-rays
were normal. Sixteen persons received blood gas determina-
tions, none of which showed an elevated pCO^ or a pC^ below
70 mmHg. Pulmonary function tests were done on 22 indivi-
duals but no significant pattern of abnormalities was seen.
Cholinesterase levels on 27 workers were negative. Several
workers had elevated liver function tests; these were mainly
minor elevations of lactic dehydrogenase (LDH) and alkaline
phosphatase which are difficult to interpret. More specific
liver function tests such as serum glutamic oxalacetic trana-
minase (SCOT) and serum glutamic pyruvic transaminase (SGPT)
were elevated in three persons. Six elevations of bilrubin,
C-65
-------
two elevations of serum creatinine, and six mild proteinurias
were detected. Unfortunately/ the specimens were analyzed
by at least three different laboratories and comparison/inter-
pretation of these results is uncertain. Attempts to develop
a technique to isolate and identify concentrations of hex
in specimens of blood or urine at the time of the investiga-
tion were unsuccessful (Morse, et al. 1978).
Biological monitoring of NIOSH and U.S. EPA personnel
who were actively involved in the cleanup effort showed
no significant abnormalities.
Repeat laboratory tests were done on 20 of the 23 sewage
treatment plant workers who had abnormalities on the blood
and/or urine tests at the time of plant shutdown. Three
of these people continued to have persistent abnormalities
in liver function tests on one or more occasions but there
were no persistent urinary abnormalities.
Exposure levels of the cleanup crew were monitored by
taking samples of breathing zone concentrations (inside
masks) of hex and octa. These values are reported in Tables
9 and 10.
Biological monitoring of the cleanup crew was also car-
ried out by NIOSH. Due to continuous turnover of crew mem-
bers, it was not possible to obtain pre-exposure baseline
studies on more than 54 percent of the workers. Symptoms
reported by crew members were similar to those reported
on the NIOSH and CDC questionnaire surveys of the plant
employees in March. Headache and eye irritation were the
C-66
-------
TABLE 9
ms of Hexachlorocyclopentadiene (HCCPD) and
i the Grit Loading and Screen and Grit Buildings
ater Treatment Plant, Louisville, Kentucky, 1977
igal, 1978)
Sample Volume Airborne
ling
57 -
59 -
28 -
44 -
36 -
43 -
D5 -
08 -
43 -
D6 -
97 -
55 -
45 -
46 -
45 -
35 -
30 -
30 -
36 -
35 -
30 -
17 -
50 -
33 -
49 -
18 -
L7 -
46 -
58 -
Period
1322
1533
1919
2222
1907
2220
0905
1120
1519
0909
1120
1515
1803
1805
2253
0119
0440
0740
0120
0441
0741
0857
1112
1359
0851
1113
1405
1715
2234
liters
6
12
16
13
10
4
10
12
14
14
8
12
7
HCCPD
1.5
3
7
0.7
0.5
2.3
8
'
6
1
5
7
0.8
1.4
10
2
Concentration - ppb
OCCP
2.4
1.2
0.9
1.1
0.8
3.7
2
4
1.0
1.1
1.9
1.2
2.2
None Established
Loading Building and PBZ-SGB denotes personal breathing zone
sampled by volume.
suit in the breathing zone of the worker.
-------
TABLE 10
Personal Breathing Zone Concentrations of Hexachlorocyclopentadiene (HCCPD) and
Octachlorocyclopentene (OCCP) Measured Inside the Protective Suits Worn by Persons
Involved with the High Pressure Water Washdown of the Screen and Grit Building,
Morris Forman Wastewater Treatment Plant, Louisville, Kentucky, 1977 (Singal, 1978)
o
1
o>
00
Sample Date
4-22
4-22
4-22
4-23
4-23
4-23
4-25
4-25
4-25
Environmental
Sample No.
CR-058
CR-059
CR-060
CR-078
CR-079
CR-080
CR-081
CR-082
CR-083
Criteria
Sampling Period
0945 -
1225 -
0946 -
1225 -
0947 -
1226 -
0850 -
1248 -
0851 -
1253 -
1045 -
1252 -
1245 -
1438 -
1308 -
1438 -
1246 -
1117
1534
1122
1540
1120
1540
1145
1600
1145
1601
1145
1604
1401
1534
1405
1537
1402
Sample Volume
liters
12
15
13
18
9
8
32
23
29
Airborne Concentrations - ppb
HCCPD
0.8
0.6
0.7
0.5
1.0
1.0
0.3
0.4
0.3
10
OCCP
4
0.9
1.1
0.8
1.8
1.4
0.4
0.6
0.5
None Established
"Parts of contaminant per billion parts of contaminated air sampled by volume.
-------
predominant symptoms; sore throat, fatigue, nausea, dizzi-
ness, chest discomfort, cough, and skin irritation were
also reported. Physical examinations on cleanup crew members
were unremarkable except for conjunctival irritation in
workers wearing half-face respirators.
Of 97 crew members tested, 18 (19 percent) showed some
elevation on one or more of the five occasions testing was
done. These elevations were generally small (see Table
11), but once they appeared, they tended to persist over
several weeks (Table 12). A small number of abnormalities
appeared on renal function tests but generally these were
small and non-reproducible on serial testing. Likewise,
abnormalities in complete blood counts were also minor and
non-reproducible.
It should be noted that the laboratory results on cleanup
workers are difficult to interpret due to lack- of adequate
controls. Essentially all of the plant employees, including
many of the cleanup workers, had been exposed in March prior
to the plant shutdown. As indicated, there were no environ-
mental samples taken at the time of the acute exposure episode.
Although exposure levels of the cleanup workers were well
below the current occupational standard for hex (0.01 ppm) ,
one cannot rule out the possibility that abnormalities among
the cleanup crew are reflective of earlier, unspecified
exposures. Interpreting the significance of variations
in liver function tests of the magnitude seen in this group
of workers is difficult. First, many of the abnormalities
C-69
-------
TABLE 11
Abnormalities in Lab Tests on Cleanup Workers,
Morris Forman Wastewater Treatment Plant,
Louisville, Kentucky (Singal, 1978)
Lab Test
Ranges
of values
Number of Persons Normal
Results in Range
Range
1 SCOT -
(serum glutamate-
oxalacetic acid
transaminase)
2 Serum alkaline
phosphatase
3 Serum total
Bilirubin
4 Serum LDSH
(lactate
dehydrogenase)
5 Serum creatinine
40-49
50-59
60-69
70-79
80-89
90-99
100-109
110-119
120-129
1.0-1.9
230-239
1.3-1.9
5
1
4
0
1
1
3
1
1
7-40 mp/ml
30-100 mp/ml
0.15-1.0 mg%
100-225 mjj/ml
0.5-1.3 mg/dl
C-70
-------
TABLE 12
Liver Function Abnormalities in Cleanup Workers, Morris Forman Wastewater
Treatment Plant, Louisville, Kentucky (Singal, 1978)
Patient No.
4/8
4/12
4/20
Date of Visit
5/5
Did
5/19 Hours spent Lab Abnormality
in cleanup result in removal
from cleanup?
1
2
3
4
? 5
H 6
7
8
9
10
11
12
13
14
15
16
17
18
SCOT 461,,
Bili 0.9
SCOT 48
SCOT 35
Bili ., 1.6
Alk phos 117
SCOT 47
SCOT 66
LDH4 239
Bili 1.4
SCOT
SCOT
Alk phos
Alk phos
LDH
SCOT
31
43
105
88
232
59
SCOT 51 SCOT
SCOT
SCOT
SCOT 44
Alk phos 96
Alk phos
LDH
SCOT
SCOT
63
31
52
103
159
42
54
SCOT
Alk phos
SCOT
Alk phos
SCOT
SCOT
Alk phos
Alk phos
SCOT
SCOT
45
100
42
113
60
39
120
101
87
46
SCOT
SCOT
SCOT
Alk phos
SCOT
SCOT
SCOT
43
63.
39
129
93
47
48
40
56
115
150
11
100
5
80
110
40
80
60
80
15
32
108
40
140
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
No
No
Yes
Yes
No
Yes
No
Yes
1 SCOT = Serum glutamate-oxaloacetate transterase in mJJ/ml - Normal range = 7-40 mJU/ml
2 Bili = Total serum bilirubin in mg% - Normal range = 0.15-1.0 mg%
3 Alk phos - Serum Alkaline phosphatase in mJJ/ml - Normal range = 30-100 mJU/ml
4 LDH = Serum Lactate dehydrogenase in mjU/ml - Normal range = 100-224 mJJ/ml
-------
seen are relatively nonspecific, that is such changes may
be caused by a variety of conditions and thus are not neces-
sarily attributable to exposure. Second, there is little
consensus concerning what constitutes the normal range in
some of these tests. Despite these problems in analysis,
Dr. Singal expressed the opinion that these data suggest
that exposure to the mixture of chemicals contaminating
the sewage treatment plant may be associated with some mild
liver injury (Singal, 1978).
iii) Community Survey. CDC workers administered a ques-
tionnaire to a systematically selected sample of residents
in a 48-block area surrounding the contaminated sewer line
(Morse, et al. 1978). One home per block was surveyed by
administering a questionnaire to the head of each household.
In all, 212 occupants of the 48-block area were surveyed.
Questions were asked concerning basic demographic data,
history of unusual odors, and any symptoms noted by household
members within the past 2 weeks.
Results of the community survey were essentially negative.
Eight of the 212 persons (3.8 percent) reported noticing
an unusual odor at some time during the preceding 2 weeks.
While some of the respondents reported symptoms compatible
with hex exposure (headache, 4.7 percent; burning or watering
eyes, 4.7 percent), no symptom occurred at greater than
background rates. Symptoms not associated with hex were
reported just as frequently as those possibly related to
exposure. Furthermore, there was no association between
symptom rates and distance from the sewer line. Subsequent
air sampling failed to show a significant ambient concentra-
tion of hex in the sewer line area.
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CRITERION FORMULATION
Existing Guidelines and Standards
The Occupational Safety and Health Administration (OSHA)
has not set a standard for occupational exposure to hex.
On the other hand, the American Conference of Governmental
Industrial Hygienists (ACGIH) has adopted both a threshold
limit value (TLV) and a Short Term Exposure Limit (STEL)
for hexachlorocyclopentadiene. The current occupational
TLV for hex is set at 0.01 ppm (0.11 mg/m ), which, according
to ACGIH "represents a time-weighted average concentration
for a normal 8-hour workday or 40-hour workweek to which
nearly all workers may be repeatedly exposed, day after
day, without adverse effect" (Am. Conf. Govt. Ind. Hyg.,
1977). The Short Term Exposure Limit (STEL) for hex is
set at 0.03 ppm (0.33 mg/m ). This level represents the
maximal concentration to which workers can be exposed for
a period up to 15 minutes without suffering from irrita-
tion; chronic or irreversible tissue damage; or narcosis
of sufficient degree to increase accident proneness, impair
self-rescue, or materially reduce work efficiency. The
STEL should be considered a maximum allowable concentration
or absolute ceiling not to be exceeded at any time in the
15 minutes. Up to four excursions up to the STEL are permit-
ted per day provided that at least 60 minutes between excur-
sions up to the STEL (Am. Conf. Govt. Ind. Hyg., 1977).
C-73
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In selecting the TLV and STEL values for hex, the ACGIH
emphasizes that these particular levels were selected on
the basis of preventing irritant effects rather than chronic
toxicity. The U.S.S.R. has recommended a tenfold lower
limit (0.001 ppm) for occupational exposures.
No nonoccupational exposure limits have been established
or recommended except for one Soviet study which proposed
a maximum concentration of 0.001 mg/1 in water to prevent
"organoleptic effects" (i.e., adverse effects on the taste
and odor of water). There is a serious lack of data to
support nonoccupational exposure limits or environmental
criteria for hex. Specifically lacking are: (1) epidemio-
logic studies of individuals having known and quantifiable
hex exposures; (2) long-term animal studies (e.g., 2-year
chronic feeding studies) suitable for evaluating chronic
effects, especially carcinogenicity; (3) data on current
levels of human exposure from various media; and (4) suit-
able methods for interpreting the significance of in vitro
assays and their applicability to actual environmental condi-
tions. Without these essential data it is not possible
to use the model proposed by U.S. EPA's Carcinogen Assessment
Group (CAG) to derive recommended exposure criteria for
humans. In fact, the CAG states that "there is insufficient
evidence to categorize this compound as a carcinogen or
non-carcinogen." Consequently, other toxic endpoints must
form the basis for recommended exposure criteria until a
more adequate information base on hex is developed.
C-74
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Special Groups at Risk
As indicated earlier, it is presently unknown whether
ingestion or inhalation of hex (through ingestion of hex-
contaminated food, water, or air) constitute significant
sources of exposure among the general population. Although
it is not likely this is the case, present data on the envi-
ronmental occurrence of hex are so sketchy that this possi-
bility cannot be ruled out.
Occupational exposures appear to constitute the only
documented source of human exposure to hex. Oral contact
does not appear to be a likely mode of occupational exposure.
However, dermal and inhalation exposures are recognized
hazards for the following groups: (1) workers engaged di-
rectly in hex manufacture; (2) those engaged in the formula-
tion and use of other, related pesticides where hex may
be present as an impurity; (3) flame retardant workers;
(4) those having "quasi-occupational" exposures such as
sewage treatment workers, industrial hygienists, etc.
Basis and Derivation of Criterion
Notwithstanding the obvious data deficiencies, some
tentative recommendations can be made in consideration of
the levels of hex which produce chronic toxicity in labora-
tory experiments.
As indicated earlier, there are no epidemiologic studies
nor suitable chronic toxicity studies in mammals from which
threshold levels for chronic effects could be derived.
Very little is known regarding potential hex exposures through
C-75
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ingestion of contaminated food or water. In the environment
hex has been detected only in specific bodies of water near
points of industrial discharges. There are no data on hex
levels in drinking or untreated water.
Based on the available and cited literature, there is
insufficient evidence to categorize this compound as a carci-
nogen or non-carcinogen. There has not been a satisfactory
study of the effects of chronic oral exposure to hex. A
single study of chronic oral toxicity reported by Naishstein
and Lisovskaya (1965). The test consisted of only one spe-
cies (rats) and the duration of exposure was only six months.
No neoplasms were reported, however the duration of the
study would not have been sufficient for a proper evaluation
of carcinogenicity.
Hex has been tested for mutagenicity and reported non-
mutagenic in both short-term in vitro mutagenic assays (NCI,
1977, IBT, 1977 and Litton Bionetics, 1978a) and in a mouse
dominant lethal study (Litton Bionetics, 1978b). No epidemi-
ologic studies or case reports examining the relationship
between exposure to hex and cancer incidences could be found
in the literature. Therefore, there is virtually no informa-
tion regarding the carcinogenic potential of chronic exposure
to hex. In selecting hex for future chronic toxicity testing,
National Cancer Institute (1977) recognized these data voids.
f
Although one study (Treon, et al. 1955) reported on
the effects of chronic low-dose inhalation of hex, its appli-
cability in deriving water quality guidelines is unclear.
C-76
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Furthermore, with the exception of very limited data on
hex in water near points of discharge, there appears to
be no information on hex levels in water bodies. What is
needed is a method for converting the results of respiratory
exposure experiments into equivalent dosages from water.
Stokinger and Woodward (1958) describe a model by which
the threshold limit values (TLV's) for industrial substances
in air may be used in establishing drinking water standards.
The model assumes that, for any given inhaled dose, an equiva-
lent ingested dose from ingested water can be derived using
reasonable estimates of daily air and water intakes and
corresponding respiratory and gastrointestinal absorption
rates. In the absence of suitable chronic ingestion studies
of hex, the Stokinger and Woodward (1958) model will be
used to estimate suitable limits for hex in water based
on the established threshold limit value expressed as milli-
grams per cubic meter of air.
The threshold limit of 0.11 mg/m (0.01 ppm) hex repre-
sents what is believed to be a maximal concentration to
which a worker may be exposed for 8 hours per day, 5 days
per week over his working lifetime without hazard to health
or well-being (Amer. Conf. Gov1t. Ind. Hyg. , 1977). To
the TLV, Stokinger and Woodward apply terms expressing respi-
ratory volume during an 8-hour period (assumed to be 10
m ) and a respiratory absorption coefficient appropriate
to the substance under consideration. As in the case of
hex where absorption rates are unknown, 100 percent absorp-
C-77
-------
tion is assumed. In addition, the 5-day-per-week occupa-
tional exposure is often converted to a 7-day-per-week equiva-
lent in keeping with the more continuous pattern of exposure
to drinking water.
According to the model, the amount of hex that may be
taken into the bloodstream and presumed to be noninjurious
and which, hence, may be taken in water each day is:
0.11 mg/m3 X 10 m3 X 1.0 X 5/7 week = 0.7857 mg/day
(TLV) Respiratory Respiratory Proportion Maximum
Intake Absorption of week Noninjurious
Term Coefficient Exposed Intake
To calculate the equivalent amount of hex in ambient water,
the model assumes a maximal daily intake of 2 liters of
water per day, the consumption of 18.7 grams of fish/shell-
fish per day, a bioconcentration factor of 3.2 for fish
and 100 percent absorption.
(X) x (2 + 3.2(0.0187)) x 1.0 = 0.7857
Upper Oral Gastrointestinal Maximum
Intake Intake Absorption Noninjurious
Limit — Term Coefficient Intake
Solving for X, the value derived is 0.38 mg/1 or 380 jug/1.
According to Stokinger and Woodward (1958), "This derived
value represents an approximate limiting concentration for
a healthy adult population; it is only a first approximation
in the development of a tentative drinking water criterion....
several adjustments in this value may be necessary...Other
factors, such as taste, odor and color may outweigh health
considerations because acceptable limits for these may be
below the estimated health limit."
C-78
-------
It should also be noted that the basis for the above
recommended limit, the TLV for hex, is set on the basis
of avoidance of irritation, rather than chronic effects
(Am. Conf. Govt. Ind. Hyg., 1977). Should chronic effects
data become available, both TLV's and recommendations based
on them will warrant reconsideration.
A single study of chronic oral toxicity in white rats
reported no adverse effects (specifically changes in peri-
pheral blood cells, ascorbic acid content of the adrenals,
conditioned reflexes of the animals, or histological struc-
ture of the organs) following daily oral administration
of doses up to 4 jag/1 of hex in aqueous solution (Naishstein
and Lisovskaya, 1965). Animals receiving the highest dosage,
40 jag/1, showed neutropenia and lympho-cytosis which the
investigators thought possibly attributable to mobilization
of the protective forces of the organism in response to
this dose. Such findings imply adverse effects at levels
as low as 10 percent of the tentative drinking water standard
based on the Stokinger and Woodward (1958) model.
Naishstein and Lisovskaya (1965) found that hex in concen-
trations of 1.4 to 1.6 pg/1 in water is capable of altering
the smell and taste of water. Based on these organoleptic
effects, these investigators proposed a maximum permissible
concentration of 1 /ag/1. Stokinger and Woodward (1975)
themselves noted that oftentimes "other factors, including
taste, odor and color may outweigh health considerations
because acceptable limits for these may be well below the
estimated health limit."
C-79
-------
Because chronic effects in a mammalian species (rats)
have been documented at water concentrations of hex as low
as 40 jag/1, it is obvious that an acceptable water quality
criterion should be well below this level. Thus, a reason-
able safety factor of 10 to 100 applied to 40 ;ig/l would
place an appropriate criterion recommendation in the range
of 4.0 - 0.4 jag/1 in water. The level recommended by Naish-
stein and Lisovskaya (1965) based on smell and aftertaste
falls well within this range.
No adverse effects on humans or mammals have been reported
to be caused by hex concentrations lower than approximately
1.0 jug/1. Therefore, based on avoidance of alteration in
smell and aftertaste in water, a criterion of 1.0 jig/1 of
hex in water is tentatively suggested. This level should
be adequate for protection of public health. It is to be
stressed that this criterion is based on inadequate chronic
effects data and should be re-evaluated upon completion
of chronic oral toxicity studies.
C-80
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C-81
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C-82
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C-83
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14
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/D'XJ.cn- ;1
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•'0 fu .rii Dci^om Surest
C-88
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