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).
                                A-l

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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.
                             A-2

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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.
                             A-3

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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.
                                A-4

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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.
                              A-5

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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.

                               B-l

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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.
                             B-2

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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.
                              B-3

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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.
                             B-4

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                           Table  1.  Freshwater  fish  acute values  for  hexachlorocyclopentadiene
                                                                              Adjusted
CO
 I
ui
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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03
I
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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ro
i
                           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
03
 I
00
           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.
                             B-9

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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).
                             B-10

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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
                            C-l

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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-
                              C-2

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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
                             C-3

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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.
                              C-4

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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.

                             C-5

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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  ,
                             C-6

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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).
                              C-7

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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.
                             C-8

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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.
                              C-9

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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.
                             C-10

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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

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    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.
                                C-23

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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).
                             C-30

-------
    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.
                              C-31

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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

-------
      (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

-------
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

-------
                                     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

-------
    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

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    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

-------
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

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    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

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    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

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                            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

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    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

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                                               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

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                                              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

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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

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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.
                             C-72

-------
                    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

-------
    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

-------
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

-------
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

-------
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

-------
Carter, M.R.  1977a.  Legal affidavit filed in State of



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                            C-82

-------
Industrial Bio-Test Laboratories, Inc.  1977.  Mutagenicity
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                            C-83

-------
Konunineni, C.  1978.  Internal memo dated February 14, 1978,
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                             C-84

-------
Marks, D.R.  1977.  Letter to Donald I. Mount,  Director,
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                            C-85

-------
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                             C-86

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Treon, J.F., et al.  1955.  The toxicity of  hexachlorocylo-
pentadiene.  Arch. Ind.  Health  11: 459.
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Ungnade, H.E., and E.T. McBee.   1958.   The chemistry of
perchlorocylopentadienes  and  cyclopentadienes.   Chem. Rev.
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U.S. EPA.  1977.  Early warning  report on hexachlorocylopenta-
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Off. Toxic Subst.,U.S. Environ.  Prot.  Agency,  Washington, D.C.


U.S. EPA.  1978.  Derivation  of  freshwater criterion for
hexachlorocyclopentadiene.  Unpublished memo,   Water Quality
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Whitacre, D.M.  1978.  Letter to R. A.  Swing,   Battelle
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                                               •'0 fu .rii Dci^om Surest
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