DRAFT CRITERIA DOCUMENT*
                  FOR ORTHO-DICHLOROBENZENE,
                    META-DICHLOROBENZENE,
                     PARA-DICHLOROBENZENE
                        FEBRUARY 1984
                    HEALTH EFFECTS BRANCH
               CRITERIA AND STANDARDS DIVISION
                   OFFICE OF DRINKING WATER
             U.S. ENVIRONMENTAL PROTECTION AGENCY
                   WASHINGTON, D.C.  20460
*This draft criteria document contains information on three
 dichlorinated benzenes.  At this time, an RMCL for 1,4-
 dichlorobenzene (p-DCB) is being proposed.  Ortho- and meta-
 (1,2- and 1,3-) dichlorobenzene will be examined for possible
 inclusion in Phase II of the revised regulations.

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                       TABLE OF CONTENTS

                                                      Page



   I.     Summary	 1-1-10

  II.     General Information and Properties	 II-1-3

 III.     Sources of Human Exposure	 III-l-
           (To be developed by STB)

  IV.     Toxicokinetics 	 IV-1-17

   V.     Health Effects in Non-humans	  V-l-88

            Plants	  V-l-2

            Microorganisms	  V-2-3

            Phytoplankton	  V-3-6

            Insects	  V-7

            Birds	  V-7

            Non-human mammals	  V-7-88


  VI.     Health Effects in Humans	VI-1-7

 VII.     Mechanism(s) of Toxicity	VII-1-6

VIII.     Quantification of Toxicological Effects..VIII-1-23

  IX.     References	IX-1-12

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I.  SUMMARY






     There are three isomers of dichlorobenzene:  ortho (1,2-),



meta- (1,3-) and para- (1,4-).  The major uses of ortho-dichloro-



benzene (o-DCB) are as a process solvent in the manufacture of



toluene diisocyanate and as an intermediate in the synthesis of



dyestuffs, herbicides and degreasers (Ware and West, 1977).  The



bulk of para-dichlorobenzene (p-DCB) usage is in direct application



as an air deodorant or insecticide which accounts for 90% of its



total consumption  (Lowenheim and Moran, 1975; Ware and West,



1977).  The use of o- and p-DCB as deodorizers in industrial



wastewaters and toilet bowl waters would suggest that increasing



amounts of these substances will be found in waters throughout the



country in the future.  No documented uses for meta-dichlorobenzene



(m-DCB) were found in the literature.



     Ortho- and para- DCB are produced in considerable quantity;



production volumes of o-DCB equalled 22,000 kkg and of p-DCB



equalled 34,000 kkg in 1981 (USITC, 1981).  Environmental releases



of the dichlorobenzenes have been estimated at 30,000 kkg (or 57%



of production).  Approximately 7,000 kkg of o-DCB are released after



solvent use and 22,000 kkg p-DCB are released from moth balls and



space deodorants (SAI, 1980).  Meta-DCB gets into the environment



as a breakdown product of certain pesticides and as a byproduct



of the manufacture of other chlorinated benzenes.



     All three isomers of dichlorobenzene have been detected in



drinking water supplies from both ground and surface waters, in



quantities ranging from less than 0.5 ug/1 to greater than  9 ug/1



(EPA, 1975; EPA, 1978a).

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                                1-2





     No studies have been  reported which  determine  the percentage



of a dose of dichlorobenzene  is  absorbed  following  oral  or inhalation



exposure.  However, for  the purpose  of  regulation development,



based  upon the  absorption  characteristics of  benzene  and the smaller



chlorinated ethanes and  ethylenes, it will be assumed  that 100%



of an  oral dose of any of  the isomers of  dichlorobenzene is



absorbed and that 30% of an inhalation  dose is  absorbed  when



exposure persists for longer  than one to  three  hours.



     After oral administration  to rabbits, the  DCBs are  oxidized



principally to  phenols.  Ortho-  and  m-DCB also  form catechols  (Azouz,



e_t al_., 1955; Williams,  1959).   The  metabolites are excreted as



free phenols or catechols  to  a  slight degree,  but in greater



percentage as conjugates of glucuronide or sulfate.  Ortho-  and



meta-DCB form mercapturic  acids  as well,  but  p-DCB  does  not



(Williams, 1959).  The dichlorophenols  appear to be the  principal



metabolic products of the  DCS isomers in  man  (Hallowell,  1959;



Pagnatto and Walkley, 1965).



     The ortho- and para-  isomers have  been shown to be  quite



lipophilic, and can be expected  to bioaccumulate in tissues  with



high fat content during  prolonged, continuous  exposures.   Para-DCB



has been detected in human adipose tissue and  all three  isomers



have been detected in blood (Dowty,  et  al., 1975; Morita,  et al.,



1975;  Morita and Ohi, 1975).



     Reports have appeared in the literature  describing  poisoning



-incidents resulting from exposure to the  dichlorobenzenes.



Girard, e_t al_.  (1969) reported  four  cases of  leukemia  in patients



purportedly exposed to varying  quantities and mixtures of dichloro-

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                               1-3
benzene, although each solution contained the ortho  isomer.



     Hallowell (1959), Gadrat, et aU  (1962), Girard, et al_.



(1969) and Campbell and Davidson  (1970) all described cases  in



which individuals suffered from moderate to severe anemia following



exposure to the DCBs.  Several instances of skin lesions developing



after contact also have been reported  (Downing, 1939; Frank  and



Cohen, 1961; Nalbandian and Pearce, 1965).



    In cases where moderate exposure to the DCBs was documented,



patients complained of vomiting,  headaches, irritation of the eyes



and upper respiratory tract with  profuse rhinitis and periorbital



swelling (Dupont, 1938; Cotter, 1953;  Campbell and Davidson,



1970).  Anorexia, nausea, vomiting, weight loss, yellow atrophy



of the liver and blood dyscrasias were reported for  higher exposure



concentrations (Petit and Champeix, 1954; Cotter, 1953; Wallgren,



1953; Weller and Crellin, 1953; Hallowell, 1959).  Liver damage



sometimes was accompanied by prophyria (Hallowell, 1959).



     The dichlorobenzenes produce sedation, analgesia and



anesthesia after acute oral or parenteral administration.  Rela-



tively high doses are needed to produce acute effects, but chronic



effects may occur at relatively low levels.  Acute poisoning is



characterized by signs of disturbance  of the central nervous sys-



tem:  hyperexcitability, restlessness, muscle spasms or tremors.



The most frequent cause of death  is respiratory depression.  Acute



exposure at high levels also may  result in kidney and/or liver




damage.  Liver damage may be manifested as necrosis/degeneration



or porphyria, depending upon the  isomer to which the individual




has been exposed.

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                               1-4
     The LDso for o-DCB in rats after oral administration  ranged



from 500 mg/kg (NIOSH, 1978) to 1,500 mg/kg  (Hollingsworth,  et



al . , 1958).  In the guinea pig, the oral LDso was 2,000 mg/kg



(Hollingsworth, £t al_., 1958).  The oral LDso for p-DCB ranged  from



500 mg/kg to 2,500 mg/kg  (Hollingsworth, et  al_., 1956) in  the rat



and was 3,220 mg/1 in  the mouse (Varshavskaya,  1968).  The lowest



published lethal oral  dose in guinea pigs was 2,800 mg/kg  (Hol-



lingsworth, et al_., 1956).  Irie, et al_. (1973) reported an  LDso



of 5,145 mg/kg for a subcutaneous dose of p-DCB in the mouse.



    Dogs exposed to 2  cc/m^ (0.04%) o-DCB by inhalation showed  no



adverse effects, whereas  0.08% produced somnolence (Riedel,



1941).  Histological studies following the administration  of



acute and subacute doses of o-DCB showed damage to the liver and



kidney.  Exposing mice to the same concentrations caused CNS stimu-



lation for about 20 minutes followed by CNS  depression, muscular



twitching, slow and irregular respiration, -cyanosis near the end



of an hour and death within 24 hours.  Rats  appeared to be



slightly more resistant than mice to the toxic  effects of  o-DCB.



     Inhalation of o-DCB by rats at 80 ppm for 11-50 hours was



irritating to the eyes and nose, produced slight changes in



the tubular epithelium of the kidney and resulted in confluent



necrosis of the liver  (Cameron, et_ al_., 1937).



     Rabbits, rats, and guinea pigs exposed  for 20-30 minutes



daily to 100 mg DCBAiter of air for 5-9 days showed marked  irrita-



tion of the eyes and nose, muscle twitching, tremors, CNS  depression,



nystagmus and rapid but labored breathing, but  recovered within




30-180 minutes after being removed from the  p-DCB-rich atmosphere

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                                1-5
(Zupko and Edwards, 1949).  Body weight decreased in 11/14 rabbits



and in 6/9 guinea pigs.  In rats, CNS depression was observed



to be greater than in rabbits.  There was complete narcosis with



attendant tremors and muscular twitching with each exposure.  The



observation that many of the test animals of all three species



developed granulocytopenia is an important one.  This condition is



considered to be a precursor to leukemia.  However, in these experi-



ments, when the animals were removed from exposure to p-DCB, the



decrease in granulocytes was reversed and the level returned to nor-



mal within three to four weeks.  The question arises as to whether



this condition was due to the DCB or to contamination by benzene or



other substances.



     Fourteen-day repeated dose gavage studies in mice and rats



were conducted with both o- and p-DCB in the prechronic testing



phase of the NTP bioassay on these two substances (Battelle-Columbus,



1978 a,b,d,e,f,g,h) .  In addition to early deaths and lack of body



weight gain at the higher doses, animals exhibited histopathology



indicative of hepatic centrolobular necrosis and degeneration,, occa-



sionally with cyto- and karyomegaly, as well as lymphoid depletion



of the spleen and thymus.



     Gavage doses of o-DCB given to rats and mice over a



thirteen-week schedule of 5 days/week resulted in liver path-




ology indicative of necrosis and porphyria  (Battelle^Columbus,



1978c, i).  Serum SGPT levels were increased in mice exhibiting



liver histopathology at the highest dose level.  Some mice also



exhibited myocardial and skeletal muscle mineralization and lymp-




hoid depletion of th£ thymus and spleen and necrosis of the spleen.

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                               1-6
Rats also showed kidney pathology as characterized by tubular




degeneration.



     Hollingsworth, et al_. (1958) gave rats a series o.f 138



doses of o-DCB over a period of 192 days (18.8, 188 or 376 mg/kg/



day, five days a week) by intubation.  No adverse effects were



detected at the lowest dose.  With the intermediate dose, a



slight increase in the weights of the liver and kidneys was noted.



At the highest dose, there was a moderate increase in the weight



of the liver, a slight decrease in the weight of the spleen and



cloudy swelling of the liver.



     Hollingsworth, et al. (1958) also measured the effects of



multiple inhalation exposures of o-DCB on rats, guinea pigs,



mice, rabbits and monkeys.  A range of concentrations was used,



seven hours a day, five days a week, for six to seven months.  No



adverse effects were observed in rats, guinea pigs or mice exposed



to 49 ppm (0.29 mg/1), or in rats, guinea pigs, rabbits and monkeys



exposed to 93 ppm  (0.56 mg/1).



     Oral doses of 10, 100 or 500 mg/kg p-DCB, five days a week,



for  20 doses, produced marked cloudy swelling and necrosis in



the  central area of the liver nodules only with the highest dose.



No effects were observed  at the other doses (Hollingsworth,



et al., 1956).



     •thirteen week exposures by gavage to p-DCB resulted in liver



pathology similar  to that observed with o-DCB, but at somewhat



higher doses  (necrosis, degeneration and porphyria)  (Battelle



Columbus, 1979a,b, 1980a,b).  The spleen and thymus  also exhibited



histopathology similar to that observed after o-DCB.  In mice and

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                                1-7
rats, hematopoietic hypoplasia of the bone marrow occurred  in  sur-



vivors at the highest dose (1500 mg/kg/day).  Rats at the two



highest dose levels also exhibited epithelial necrosis of the



nasal turbinates and small intestine and villar bridging of the



mucosa of the latter tissue.  Again, the rats exhibited renal



pathology, with multifocal degeneration or necrosis of the corti-



cal tubular epithelium.



     Oral doses of 188 or 376 ing p-DCB/kg, five days a week,



for 192 days (138 doses) in rats induced an increase in the weights



of the liver and kidneys (Hollingsworth, et al., 1956). At 376



mg/kg, increased splenic weight, slight cirrhosis and focal necrosis



of the liver were observed.  No adverse effects were seen with a



18.8 mg/kg dose.



     Inhalation studies also were carried out with p-DCB (Hollings-



worth, et al., 1956),  The concentrations used were 96, 158, 173,



314 and 798 ppm (0.58, 0.95, 1.04, 2.05 and 4.8 mgAt respectively.-



Exposure occurred for 7 hours/day, 5 days/week for 6-7 months.



Adverse effects observed included liver and kidney histopathology



with increased organ weights, pulmonary edema and congestion,



splenic weight changes and reversible non-specific eye changes.



     It is difficult to reconcile the results of the previously-



described studies with those of Varshavskaya  (1968).  Rats



received daily oral doses of 0.001, 0.01 or 0.1 mg/kg o-DCB in



sunflower oil for nine months.  No adverse effects were noted  at



the lowest dose, but varying degrees of inhibition of mitosis  in



the bone marrow, as well as neutropenia, abnormal conditioned

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                                1-8
reflexes and adrenal hypertrophy occurred at  the  two higher  dose



1 evel s.




     Studies employing long-term or chronic exposures to o-  and



p-DCB were designed to evaluate the substances' chronic toxicity



and carcinogenic potential.  Preliminary assessment of the data



from the NTP bioassay performed with o-DCB by gavage suggests



that, under the conditions of the study, this substance is not a



carcinogen in  Fischer 344  rats or B6C3F1 mice (NTP, 1982).   No



non-neoplastic lesions were noted in either the mice or the  rats,



suggesting that the maximum tolerated dose was not achieved.



     The results of the NTP gavage bioassay with  p-DCB are not



available to ODW at this time.  A long-term inhalation study



revealed no increase in tumor incidence or type following exposure



to p-DCB in Alderly Park Wistar rats (Riley,  et al_», 1980a) .  At



the high dose  (500 ppm), changes indicative of non-neoplastic effects



were observed:  an increase in liver, kidney, heart and lung weights



 (both sexes) and an increase in urinary protein and coproporphyrin



output  (in males).



          No teratogenicity studies were found in the peer-reviewed  "



literature for any of the  three isomers of dichlorobenzene.  However,



studies are underway to evaluate the teratogenic  potential of o-DCB



in rats and rabbits (Dow,  1981).  In addition, results of a  study



by Hodge, _et al_. (1977) suggest that maternal exposure to atmospheric



levels of p-DCB up to 500  ppm on Days 6-15 of pregnancy does not



result in any embryotoxic, fetotoxic or teratogenic effects  in the



offspring.

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                              1-9
     Para-dichlorobenzene induces abnormal mitotic division in
                                       \


higher plants.  Effects seen include shortening and thickening



of chromosomes, precocious separation of chromatids, tetraploid



cells, binucleate cells and chromosome bridges (c-mitosis) (Sharma



and Battacharya, 1956; Sharma and Sarkar, 1957; Srivastava, 1966;



Gupta, 1972).  Ortho-DCB was shown to produce abnormal mitotic



division in the onion, Allium cepa (Ostergran and Levan, 1943).



     Ortho-dichlorobenzene and para-dichlorobenzene were not



mutagenic when tested in a culture of histidine-requiring mutants



of Salmonell a typhimurium or in the E_. col i WP2 system (Anderson,



ert al_. , 1972; Anderson, 1976; Simmon, et al_. , 1979).  However, all



three isomers increased the frequency of back mutation of the



methionine-requiring locus in the fungus, Aspergillus nidulans



(Prasad and Pramer, 1968; Prasad, 1970).  In addition, the meta



isomer was shown to increase mitotic recombination in the Sac-



charomyces cerevisiae C3 yeast system (Simmon, et al., 1979).



The results with the para isomer were ambiguous.  These investi-



gators also showed that both o- and m-DCB interacted with and



damaged bacterial DNA in the E_. col i W3110 polA+/p3478 polA"



differential toxicity assay system.



     No evidence of mutagenicity in animals has been published to



date.  Guerin, et_ al. (1971) showed that DCB did not produce a



significantly different number of mitoses in rat lung cell cultures



Cytogenetic studies with rat bone marrow cells and a dominant




lethal study in CD-I mice following exposure to p-DCB were all



negative (Anderson and Richardson, 1976; Anderson and Hodge,  1976).

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                               1-10
     In the few reports available on  the carcinogenic  potential



of the DCBs, the results are negative, although one  report  of



four cases of leukemia in humans attributed  to o-DCB or  a mixture



of all three isomers has been published  (Girard,  et^  al_., 1969).



Hollingsworth, el: al_.  (1956, 1958)  exposed several species  of



animals to various oral and inhalation exposures  of  ortho-  and



para-dichlorobenzene for six to seven months.  No pathological



changes indicative of cancerous changes were observed.   In  a some-



what inconclusive study, Parsons (1942) suggested that p-DCB pro-



duced a transplantable sarcoma in an  irradiated mouse.   Prelimi-



nary assessment of the data from the  NTP carcinogenicity bioassay



performed with o-DCB suggests that, under the conditions of the



study, this substance  is not a carcinogen in Fischer 344 rats or



B6C3F1 mice (NTP, 1982).  The bioassay with  p-DCB has  not been



reported as yet.  A long term inhalation study revealed  no  increase



in tumor incidence or type following  exposure to  p-DCB in Alderley



Park Wistar rats  (Riley, e_t al_., 1980a).
                             -12-

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                                II-l
II.  GENERAL INFORMATION AND PROPERTIES



     Chemical and Physical Properties



     Three isomers of dichlorobenzene (DCB) exist:  ortho (1,2-),



meta (1,3-) and para (1,4-).  Very little information is avail-



able on the meta isomer.  Therefore, unless otherwise noted, the



properties of this isomer will be assumed to be identical to those



of the ortho isomer.



     At room temperature  (20-25°C), ortho- and meta-DCB are colorless



neutral liquids; p-DCB is a colorless crystalline solid which



readily sublimes (Irish,  1963).  All isomers have a molecular



weight of 147.01.  Each is nearly insoluble in water, but readily



soluble in many organic solvents, including ethanol, benzene and



diethyl ether, as well as lipid.  Freed, e_t al_. (1979) determined



the solubility of p-DCB in water at 25°C to be 79 ppm (79 mg/1).



All isomers are heavier than water  (specific gravity = 1.306,  1.288,



1.458 at 20°C for o-, m-  and p-DCB, respectively).  Thus, each would



tend to sink in standing  water.  The ortho isomer has a vapor



pressure of 1.56 mm Hg at 25°C.  All three isomers are combustible.



     In air, 1 ppm = 6.01 mg/m^ and 1 mg/1 = 166.3 ppm, at 25°C



and 760 mm Hg (Irish, 1963).



     Sato and Nakajima (1979) determined the water/air, blood/air,



olive oil/air and olive oil/water partition coefficients for o- and



m-DCB.  These are listed  in Table II-l.  With the logs of their



oil/water partition coefficients approaching 4, both  isomers appear



to be quite lipophilic.   This is confirmed by the findings of  Freed,



et al. (1979) who established that  p-DCB has a log P  (n-octanol/



water) of 3.38.  It would be expected that these substances would

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                                II-2
tend to bioaccumulate in fatty tissues during prolonged, continuous

exposures.


                 Table II-l. Partition Coefficients

                  (after Sato and Nakajima, 1979)

        Partition Coefficient      Ortho-DCB      Meta-DCB
        Water/Air  (W)
        Blood/Air  (B)
        Olive oil/Air  (0)
        Olive oil/Water  (0/W)
        Olive oil/Blood  (0/B)
        W-0
        Log P (0/W)
     9
   423
 39920
  4436
    94
359280
     3.65
     5.5
   201.4
 27080
  4924
   134
148940
     3.69
     The odor threshold for o-DCB in air is 2-4 ppm (AIHA, 1964).

At 10-15 ppm, the smell becomes very noticeable, and at 25-30 ppm,

it is considered unpleasant.  Eye irritation becomes a problem at

the same concentration range, while at exposures of 60-100 ppm, eye

and mucous membrane irritation may be very painful.  The odor thres-

hold for p-DCB in air is 14-30 ppm in unacclimated persons (AIHA, 1964)

Eye irritation begins at 50-80 ppm and becomes painful at 100 ppm.

Kolle (1972) determined the odor threshold in water to range from

0.01-0.03 mg/1 for the three DCB isomers.

     Production and Use

     o-Dichlorobenzene and p-dichlorobenzene are produced in

considerable quantity.  In 1981, the USITC reported that production

volume for o-DCB was 22,000 kkg and for p-DCB, 34,000 kkg.  At

least 70 million pounds of p-DCB are used each year for moth control

and as a space odorant (Brown, et al., 1975).  Apparently only two

U.S. companies produce m-DCB (West and Ware, 1977), and in 1974,

31,000 pounds were imported (USITC, 1974).  Loss of o-DCB and p-DCB

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                               II-3
during their manufacture amounts to at least a million pounds a



year for each isomer  (Brown, e_t al . , 1975).  Meta-DCB is lost as a



by-product of the manufacture of monochlorobenzene.  It also gets



into the environment  through its being a breakdown product of cer-



tain pesticides such  as lindane.



     The major uses of o-DCB are as a process solvent in the



manufacture of toluene diisocyanate, and as an intermediate in the



synthesis of dyestuffs, herbicides and degreasers (West and Ware,



1977).  The bulk of p-DCB usage is in direct application as air



deodorants and insecticides which  account for 90% of its total



consumption (Lowenheim and Moran,  1975; West and Ware, 1977).  Use



of o- and p-DCB as deodorizers in  industrial wastewaters or in



toilet bowl waters would suggest that increasing amounts of these



chemicals will be found in waters  throughout-the country in the



future.  No documented uses of m-DCB were found in the literature.

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                              III. HUMAN  EXPOSURE

     Humans may  be exposed  to  dichlorobenzene in  drinking  water, food,  and
air.   Detailed  information concerning the  occurrence  of and exposure to  di-
chlorobenzene in  the environment  is presented  in  another document  entitled
"Occurrence of Dichlorobenzenes  in  Drinking Water,  Food,  and  Air"  (Letkiewicz
et al. 1983).  This  section  summarizes the  pertinent  information  presented in
that document in order to  assess the relative  source  contribution  from drink-
ing water, food, and air.

Exposure Estimation
     This  analysis  is  limited to  drinking  water,  food, and  air,  since  these
media  are  considered to be  general  sources common  to all individuals.   Some
individuals  may  be  exposed  to  dichlorobenzene  from  sources  other  than  the
three  considered  here,  notably  in  occupational  settings  and  from the use of
consumer  products  containing dichlorobenzene.   Even in  limiting the  analysis
to  these  three  sources, it  must be recognized that individual exposure  will
vary  widely  based  on  many  personal  choices  and several  factors over  which
there  is  little  control.   Where  one lives,  works, and  travels,  what  one  eats,
and physiologic characteristics  related to age, sex, and health  status can all
profoundly affect  daily  exposure and intake.   Individuals living  in  the  same
neighborhood  or  even in  the same  household  can experience  vastly  different
exposure patterns.
     Unfortunately,  data  and  methods to estimate exposure  of  identifiable
population  subgroups  from  all   sources simultaneously  have  not  yet   been
developed.   To  the  extent  possible, estimates are  provided  of the  number of
individuals  exposed  to  each  medium  at various  dichlorobenzene  concentra-
tions.  The 70-kg male is used for estimating intake.

a.  Water
     Cumulative  estimates  of the  U.S. populations  exposed to  various o- and
p-dichlorobenzene  levels in  drinking water  from  public  drinking water systems
are  presented  in  Tables  IV-I  and  IV-II, respectively.   The values  in  these
tables were obtained using  Federal  Reporting  Data Systems data  on populations
served by primary water supply systems (FRDS 1983) and the estimated number of
these water systems that contain a given  level of o- or p-dichlorobenzene.
                                       1

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      Table IV-I.  Total Estimated Cumulative Population (in Thousands)
                Exposed  to  o-Dichlorobenzene in  Drinking  Water
                    Exceeding the Indicated Concentration
System type
Groundwater
Surface water
Total
(% of total )
Number of
people served
in U.S.
(thousands)
73,473
140,946
214,419
(100%)
Cumulative population (thousands)
exposed to concentrations (ug/1 ) of:
2.0.5
156
1,431
1,587
(0.7%)
>5
0
0
0
(0.0%)
      Table IV-II.   Total  Estimated Cumulative Population  (in  Thousands)
                Exposed to p-Dichlorobenzene in Drinking  Water
                     Exceeding the  Indicated Concentration
System type
Groundwater
Surface water
Total
(% of total )
Number of
people served
in U.S.
(thousands)
73,473
140,946
214,419
(100%)
Cumulative population (thousands)
exposed to concentrations (ug/1) of:
XL 5
775
859
1,634
(0.8%)
>5
0
0
0
(0.0%)
     An estimated 1,587,000 individuals (0.7% .of the population of 214,419,000
using  public  water supplies)  are exposed to  levels  of  o-dichlorobenzene  in

drinking water  at  or  above  0.5  ug/1.   Mo  individuals  are estimated  to  be

exposed  to  levels  above  5  ug/1.    Of the  approximately  1.6 million  people

exposed to levels ranging from 0.5-5 ug/1, 1.4 million (90%) obtain water from

surface water supplies.

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     An estimated  1,634,000 individuals (0.8% of the  population  using public
supplies) are exposed  to  levels of p-dichlorobenzene  in  drinking  water at or
above  0.5  ug/1 ,  while  no individuals  are  estimated  to be  exposed  to levels
above  5  ug/1.   Fifty-three  percent  of the  approximately  1.6  million  indivi-
duals  exposed  to  levels  ranging  from 0.5-5  ug/1  obtain water from  surface
water supplies,  while 47% obtain water  from groundwater supplies.
     No individuals are estimated to be exposed to levels of m-dichlorobenzene
above 0.5 ug/1.
     No  data  were  obtained on  regional  variations  in  the concentration  of
dichlorobenzene in drinking water.  The highest concentrations  are  expected to
occur near sites of production  and use  of dichlorobenzene.
     Daily intake  levels  of  o-  and  p-dichlorobenzene  from drinking water were
estimated  using   various  exposure  levels  and the  assumptions  presented  in
Tables IV-III and  IV-IV,  respectively.   The data  suggest that  the  majority of
the  persons  using public drinking  water  supplies would  be  exposed  to intake
levels for the dichlorobenzene  isomers  below 0.014 ug/kg/day.

      Table  IV-III.  Estimated  Drinking Water Intake of o-Dichlorobenzene
                         Persons using supplies
                       exposed to indicated levels
Exposure level                          % of Total
    (ug/1)	Population	population	Intake (ug/kg/day)
    _>0.5                 1,587,000          0.7%                  _>°-014
    >5.0                        0          0.0%                  >0.14
Assumptions:  70-kg man, 2 liters of water/day.
      Table  IV-IV.  Estimated Drinking Water Intake of p-Dichlorobenzene

                          Persons  using supplies
                       exposed to indicated levels
Exposure level                          % °f Total
     (ug/1)             Population	population	Intake (ug/kg/day)
>0.5
>5.0
1,634,000
0
0.8%
0.0%
ML 014
>0.14
Assumptions:  70-kg man, 2 liters of water/day.

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     An  indication  of  the overall  exposure of  the  total population  to di-
chlorobenzene can be obtained through the calculation of population-concentra-
tion  values.   These values  are a  summation  of the individual  levels  of the
dichlorobenzene isomers to which each member of the population is exposed.  An
explanation of the derivation of these values is presented in Appendix C.
     Population-concentration  estimates  for   o-dichlorobenzene  in  drinking
water  were  7.9 x  105  ug/1  x  persons (best case),  4.4  x 106 ug/1  x persons
(mean  best  case),  1.1  x 108 ug/1  x persons (mean worst case),  and  1.1  x 108
ug/1  x persons  (worst case).   Assuming  a consumption  rate  of 2  liters  of
water/day,  population-exposure  values  of  1.6  x  106  ug/day  x  persons  (best
case), 8.8 x 106 ug/day x  persons  (mean best case), 2.2 x  108 ug/day x persons
(mean worst case), and 2.2 x 108 ug/day x persons (worst case) were derived.
     Population-concentration estimates  obtained  for  m-dichlorobenzene  were  0
(best  case)  and  1.1  x 108  (worst case).   A median  case value  could  not  be
calculated  due to the  absence  of positive values  in groundwater.  Using these
figures, population-exposure values of  0  ug/day x persons  (best  case) and 2.2
x 108  ug/day x persons  (worst case)  were  derived.
     Population-concentration  estimates  for   p-dichlorobenzene  in  drinking
water  were  8.2 x  105  ug/1  x  persons (best case),  4.6  x 106 ug/1  x persons
(mean  best  case),  1.1  x 108 ug/1  x persons (mean worst case),  and  1.1  x 108
ug/1  x persons  (worst  case).   The population-exposure  estimates derived from
these  values were  1.6  x 106 ug/day x persons  (best  case), 9.2  x 106 ug/day  x
persons  (mean  best  case),  2.2  x  108 ug/day x  persons  (mean  worst  case), and
2.2 x  108 ug/day x persons  (worst  case).

b.  Diet
     Data  on  levels of  dichlorobenzenes in  foods  in the United States were
limited to  concentrations  of the  chemicals  in  trout  from the Great Lakes and
in  mother's  milk.   These  data  are insufficient for determining the intake of
dichlorobenzene in the U.S. diet.

c.  Air
     Exposure to dichlorobenzene in the atmosphere  varies  from one  location to
another.   The  highest  level  of o-dichlorobenzene  reported  in  the  atmosphere

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was  19,000  ng/m3  (19 ug/m3)  (Bozzelli  and Kebbekus  1979  cited  in Brodzinsky
and Singh 1982).  High levels, averaging greater than 1,000 ng/m3 (1.0 ug/m3),
have  been  detected  in   other  areas.   Normal  levels,  however,  are  somewhat
lower.   Brodzinsky  and  Singh  (1982)  calculated median  air levels  of  o-di-
chlorobenzene for  rural/remote areas, urban/suburban areas,  and  source  domi-
nated areas of  0.0 ng/m3 (0.0  ug/m3),  6.6 ng/m3 (0.0066 ug/m3),  and 350  ng/m3
(0.35 ug/m3), respectively.
     The highest  level   of m-dichlorobenzene reported  in  the atmosphere  was
16,000  ng/m3  (16  ug/m3)  (Wallace 1981 cited  in Brodzinsky and Singh  1982).
Average  levels  greater  than   1,500  ng/m3 (1.5  ug/m3)  have been  reported  in
other areas.   The following median  concentrations  were calculated for  m-di-
chlorobenzene:    rural/remote  areas,  0.0 ng/m3  (0.0  ug/m3);  urban/suburban
areas,  36  ng/m3  (0.036  ug/m3);  and  source  dominated areas, 560  ng/m3  (0.56
ug/m3).
     The maximum  level   reported  for p-dichlorobenzene  in  the atmosphere  was
60,000  ng/m3  (60  ug/m3)  (Bozzelli  and  Kebbekus 1979 cited  in Brodzinsky  and
Singh  1982).    Mean  levels of  p-dichlorobenzene  above  1,000 ng/m3 (1 ug/m3)
were  found in  two  locations.    Median  air  levels  of  p-dichlorobenzene  for
rural/remote  areas,  urban/suburban  areas,  and source  dominated areas  were  0.0
ng/m3 (0.0 ug/m3), 280  ng/m3  (0.28  ug/m3), and  0.0  ng/m3  (0.0 ug/m3), respec-
tively.
     The monitoring  data  available  are  not sufficient  to  determine  regional
variations  in exposure   levels for  the  dichlorobenzenes.    However, urban  and
industrial  areas generally appear to contain higher  levels, as expected.
     The daily  respiratory intake of  each of the isomers  of  dichlorobenzene
was  estimated using  the  assumptions  presented  in  Tables   IV-V through  IV-VII
and  the median and  maximum levels  for  the dichlorobenzenes reported above.
The  estimates in  Tables  IV-V  and IV-VI  indicate that th-e  daily intake  of  o-
and  m-dichlorobenzene  from air  for  adults  in source  dominated  areas   is
approximately  0.1  and  0.2 ug/kg/day,  respectively.    A  similar  value  (0.09
ug/kg/day)  was  calculated  for  p-dichlorobenzene in  urban/suburban areas (Table
IV-VII).   In  contrast,  the  intakes  calculated  using the maximum  o-,  m-,  and
p-dichlorobenzene  levels  reported  are 6.3,  5.3,  and  20  ug/kg/day,  respec-
tively;  few  if any  persons are believed  to  be  exposed  at  those  levels.   The
values  presented   do not  account  for  variances  in individual  exposure  or
uncertainties in the assumptions used to estimate exposure.
                                       5

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        Table IV-V.   Estimated Respiratory Intake of o-Dichlorobenzene
        Exposure (ug/m3)                              Intake (ug/kg/day)
     Rural/remote (0.0)                                     0.0
     Urban/suburban (0.0066)                                0.0022
     Source dominated (0.35)                                0.12
     Maximum (19)                                           6.2
Assumptions:  70-kg man, 23 m3 of air inhaled/day (ICRP 1975).
        Table  IV-VI.   Estimated  Respiratory  Intake of m-Dichlorobenzene
        Exposure (ug/m3)                              Intake (ug/kg/day)
     Rural/remote (0.0)                                     0.0
     Urban/suburban (0.036)                                 0.012
     Source dominated  (0.56)                                0.18
     Maximum (16)                                           5.3
Assumptions:  70-kg man, 23 m3 of air inhaled/day (ICRP 1975).
       Table IV-VII.  Estimated Respiratory Intake of p-Dichlorobenzene
        Exposure (ug/m3)                              Intake (ug/kg/day)
     Rural/remote (0.0)                                     Q Q
     Source dominated (0.0)a
     Urban/suburban  (0.28.)                                  0.092
     Maximum (60)                                          20
aValue  reported for source  dominated areas was  lower  than  that reported for
 urban/suburban  areas.
Assumptions:  70-kg  man,  23 m3 of air  inhaled/day (ICRP 1975).

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     In addition to the available monitoring data, Systems Applications (1982)
has provided  estimates  of atmospheric  levels  of o- and  p-dichlorobenzene  by
applying  air  dispersion  models  to  dichlorobenzene  emission  sources.    The
computed  average  concentrations  of  the  dichlorobenzenes and  the number  of
individuals estimated to  be  exposed to these  concentrations  are  presented  in
Tables IV-VIII and  IV-IX.  Specific point sources in  these tables are indivi-
dually identified  sources with  known  locations  and modes and  rates  of  emis-
sions.   These are  generally  manufacturing plants.  General  point sources  are
sources  that  are  too numerous,  small,  or  of  uncertain  location to be treated
individually.   However, these sources produce isolated patterns of significant
concentration.  Area sources are sources that are numerous and emit only  small
concentrations of the chemical  (e.g.,  home chimneys,  automobiles).   The  esti-
mates  presented  for o-dichlorobenzene  in Table  IV-VIII  suggest  that only  a
small  number  of individuals  (less than  700,000)  are exposed  to o-dichloro-
benzene  concentrations  greater  than  250 ng/m3  (0.25  ug/m3).   Estimates  for
p-dichlorobenzene   (Table  IV-IX)   suggest  that  62,000,000  individuals   are
exposed  to p-dichlorobenzene levels at  or above 250 ng/m3 (0.25 ug/m3).
     Tables   IV-VIII  and   IV-IX  also  present total  population-concentration
estimates  for o-  and  p-dichlorobenzene  (6.45 x  106  and  5.14 x  107  ug/m3  x
persons,  respectively).    Assuming an  inhalation rate of  23 m3  of  air/day,
population-exposures  of  1.48 x  108 and 1.18 x  109 ug/day x  persons, respec-
tively,  were  calculated.

SUMMARY
     Tables IV-X,  IV-XI,  and IV-XII present a general  view of the total amount
of o-, m-, and p-dichlorobenzene,  respectively, received by an adult male from
air  and  drinking  water.    Insufficient data were  obtained  on levels of  di-
chlorobenzene in foods to  assess the  relative intake from that source.
     The data presented have been  selected from an infinite number of possible
combinations  of concentrations  for the  two sources.   The  actual  exposures
encountered  would  represent  some  finite  subset of  this  infinite  series  of
combinations.   Whether  exposure occurs at any  specific  combination of levels
is not known; nor  is  it possible to determine the number of persons that would
be  exposed to  dichlorobenzene  at  any  of the combined exposure  levels.   The
data  presented  represent  possible  exposures  based  on  the occurrence data and
the estimated intakes.

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                       Table  IV-VIII.   Exposure and  Dosage  Summary  for Airborne  o-Dichlorobenzene
Population
Concentration Specific
level point
(ug/m ) source
50 2
25 25
>10 234
5 917
2.5 5,406
1 36,787
0.5 93,389
0.25 172,270
0.1 426,427
0.05 626,291
0.025 839,531
0.01 1,525,505
0 6,113,449
General
point
source
0
0
0
0
0
0
0
0
0
0
0
800
--
exposed (persons)
Area source
0
0
0
0
0
0
0
505,140
9,149,730
33,072,205
81,759,648
142,928,535
158,679,135
U.S. total
2
25
234
917
5,406
36,787
93,389
677,410
9,576,157
33,698,495
82,599,179
144,454,840
--
Specific
point
source
91
892
3,610
8,410
23,600
69,500
110,000
137,000
178,000
192,000
200,000
210,000
224,000
Dosage (ug/m x persons)
General
point
source
0
0
0
0
0
0
0
0
0
0
0
9
2,460
Area source
0
0
0
0
0
0
0
232,451
1,772,052
3,479,775
5,056,481
6,121,131
6,225,594
U.S. total
91
892
3,610
8,410
23,600
69,500
110,000
369,451
1,950,052
3,671,775
5,256,481
6,331,140
6,452,054
Note:  The use of "--" as an entry indicates that  the  incremental
       not significant (relative to the last entry in  that  column
       that the exposure of the same population may be counted  in

Source:  Systems Applications 1982
increase in the population exposed or the dosage is
or to an entry in another column at the same row)  or
another column.

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                        Table  IV-IX.   Exposure and  Dosage  Summary for Airborne p-Dichlorobenzene
Population
Concentration
level
(ug/m3)
50
25
10
5
2.5
1
0.5
0.25
0.1
0.05
0.025
0.01
0.005
0
Specific
point
source
2
8
42
127
389
1,691
3,879
10,792
36,631
126,422
384,501
888,210
--
2,341,084
General
point
source
0
0
0
0
0
0
0
0
0
0
0
3,400
19,600
--
exposed (persons)
Area source
0
0
0
0
505,140
9,149,730
26,976,292
61,583,693
--
--
--
—
--
158,679,135
U.S. total
2
8
42
127
505,529
9,151,421
26,980,171
61,594,485
--
--
--
--
--
--
Specific
point
source
118
328
815
1,420
2,350
4,330
5,930
8,400
12,200
18,400
27,400
35,200
--
41,400
o
Dosage (ug/m x persons}
General
point
source
0
0
0
0
0
0
0
0
0
0
0
50
160
3,360
Area source
0
0
0
0
1,917,818
14,620,149
26,029,918
37,167,988
49,590,816
--
--
--
--
51,363,678
U.S. total
118
328
815
1,420
1,920,168
14,624,479
26,035,848
37,176,388
49,603,016
--
--
--
--
51,408,438
Note:  The use of "--" as an entry indicates that the incremental
       not significant (relative to the last entry in that  column
       that the exposure of the same population may be counted  in

Source:  Systems Applications 1982
increase in the population exposed or the dosage is
or to an entry in another column at the same row) or
another column.

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              Table IV-X.  Estimated  Intake of o-Dichlorobenzene
               from the Environment by Adult Males in ug/kg/day
                            (% from Drinking Water)
Concentration in   	Concentration in air	
 drinking water    Rural/remote   Urban/suburban  Source dominated   Maximum
.   (ug/1)	(0.0 ug/m3)    (0.0066 ug/m3)    (0.35 ug/m3)    (19 ug/m3)

    0               0.0 (--)        0.0022  (0%)      0.12 (0%)     6.2  (0%)

    0.5a            0.014  (100%)    0.016 (88%)      0.13 (11%)    6.2  (0.2%)

    5.0b            0.14  (100%)     0.14  (100%)      0.26 (54%)    6.3  (2.2%)


Intake from each source (see  Sections  5.1-5.3):

Water:    0.5 ug/1:          0.014 ug/kg/day
          5.0 ug/1:          0.14  ug/kg/day

Air:      0.0    ug/m3:     0.0 ug/kg/day
          0.0066  ug/m3:     0.0022 ug/kg/day
          0.35   ug/m3:     0.12  ug/kg/day
          19      ug/m3:     6.2 ug/kg/day

Food:     Not included

 al,587,000  individuals using  public drinking water systems are estimated  to be
  exposed to  levels >_ 0.5  ug/1 (0.7% of population  using public water  supplies).
 bNo   individuals   using  public drinking  water  systems are   estimated  to  be
  exposed to  levels >  5.0  ug/1.
                                       10

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             Table  IV-XI.   Estimated  Intake  of m-Dichlorobenzene
               from the Environment by Adult Males in ug/kg/day
                            (%  from Drinking  Water)
Concentration in   	Concentration in air	
 drinking water    Rural/remote  Urban/suburban  Source dominated   Maximum
   (ug/1)	(0.0 ug/m3)   (0.036 ug/m3)     (0.56 ug/m3)    (16 ug/m3)

    0               0.0 (—)       0.012 (0%)       0.18 (0%)     5.3 (0%)

    0.5a            O.OH (100%)   0.026 (54%)      0.19 (7.4%)   5.3 (0.3%)


Intake from each source (see Sections  5.1-5.3):

Water:   0.5 ug/1:          0.014 ug/kg/day

Air:      0.0   ug/m3:      0.0 ug/kg/day
          0.036 ug/m3:      0.012 ug/kg/day
          0.56  ug/m3:      0.18 ug/kg/day
         16     ug/m3:      5.3 ug/kg/day

Food:    Not included

aNo  individuals  using  public drinking  water  systems  are  estimated  to  be
  exposed to  levels _>_ 0.5  ug/1.
                                       11

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             Table  IV-XII.   Estimated  Intake  of  p-Dichlorobenzene
               from the  Environment  by  Adult  Males in ug/kg/day
                            (% from Drinking Water)

Concentration in Rural
drinking water
(ug/1)
0
0.5a
5.0b
Intake from each source
Water: 0.5 ug/1 :
5.0 ug/1 :
Concentrati
/remote Source dominated
(0.0 ug/m3)
0.0 (-)
0.014 (100%)
0.14 (100%)
(see Sections 5.1-5.3) :
0.014 ug/kg/day
0.14 ug/kg/day
on in air
Urban/suburban Maximum
(0.28 ug/m3) (19 ug/m3)
0.92 (0%) 20 (0%)
0.11 (13%) 20 (0.07%)
0.23 (61%) 20 (0.7%)

Air:      0.0  ug/m3:        0.0 ug/kg/day
          0.28 ug/nr:        0.092 ug/kg/day
         60    ug/m3:       20 ug/kg/day

Food:     Not included

al,634,000 individuals using public drinking water systems are estimated to be
 exposed to levels >_ 0.5 ug/1  (0.8% of population using public water supplies).

bNo  individuals  using  public  drinking  water  systems  are  estimated  to  be
 exposed to levels > 5.0 ug/1.
                                       12

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     Brodzinsky and  Singh  (1982) calculated median  urban/suburban  air levels
of o-,  m-, and  p-dichlorobenzene of  0.0066,  0.036, and  0.28  ug/m3,  respec-
tively,  based  on air  monitoring data.   Assuming those air  levels,  drinking
water would be the predominant  source  of exposure in the adult male at drink-
ing water  levels  above 0.08,  0.42,  and 3.2 ug/1, respectively.   An  accurate
assessment of  the  number  of individuals for which drinking  water  is  the pre-
dominant source of exposure  cannot  be  determined from the  data since specific
locations  containing  high  concentrations of the dichlorobenzenes  in  drinking
water  and  low  concentrations of  the dichlorobenzenes  in ambient  air  and food
are unknown.
     Population-exposure  estimates  for  o-  and  p-dichlorobenzene  in  drinking
water  and  air  were  reported previously.  Estimates  for o-dichlorobenzene  in
drinking water ranged  from  0.016-2.2  x 108 ug/day x  persons; the estimate for
ambient  air  was  1.48 x 108  ug/day  x  persons.   These population-exposures are
comparable.    Estimates  for p-dichlorobenzene  in drinking water  also ranged
                   o
from 0.016-2.2 x 10° ug/day  x persons; the estimate for ambient air was 1.18 x
10^  ug/day x  persons.   These  estimates  suggest that  ambient  air may  be  a
slightly  greater  source of  exposure to  p-dichlorobenzene  than  drinking water
on a general population basis.   Comparison of these estimates, however, may be
deceiving  since  the  same population-exposure level can  occur  if:   1)  a whole
population  is  exposed  to  moderate levels of a  chemical or  2) some segments of
the same  population  are exposed to  high levels and others  to low levels.  The
population-exposure  values   presented give no  indication of  the relative pre-
dominance  of drinking  water and air as specific sources of o- and p-dichloro-
benzene  on  a site-by-site or subpopulation basis.
     The  relative  source  contribution data are  based  on estimated intake and
do not  account for a possible differential absorption  rate for dichlorobenzene
by  route of exposure.   The  relative  dose  received may vary  from the  relative
intake.    In addition,  the   relative effects of  the  chemical on  the  body may
vary by  different  routes of  exposure.

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                               IV-1
IV.  TOXICOKINETICS



     Absorption/Retention



     The absorption and excretion of the chlorinated benzenes



take place by simple diffusion.  The compounds can be absorbed from



the lungs, the gastrointestinal tract and through the skin.  The



dichlorobenzenes are poorly soluble in water/ but possess varying



degrees of high lipid solubility {Neely, _et al_., 1974; Lu and Met-



calf, 1975; Brown, e_t al_. , 1975).  Thus, these substances cross



most of the barrier membranes,  including brain and placenta.



Little information is available which demonstrates the percentage



of a dose of DCB absorbed  and  retained following exposure in any



environmental medium.   Yano  (1979) administered 2.5 mg p-DCB to



mice orally  (125 mg/kg  for a 20 g mouse).  Measuring carcass



content of the compound  (except for the gastrointestinal tract,



hair, skin and tail), he  determined the rate of absorption over



a  24-hour period  (Figure  IV-1). The rate reached a maximum at 6



hours after  dosing, falling  to near zero after 8 hours.  The



author concluded  that the absorption rate was 11 ± 2.9%.  The



percentage of the  dose  absorbed was not identified in this study.



     Based upon what  is  known  about the absorption character-



istics of benzene  and the smaller chlorinated aliphatics  (ethanes



and  ethylenes),  it will  be assumed that 100% of any oral dose of



a  dichlorobenzene  is  absorbed, while 30% of  any DCB isomer  inhaled



over a period of  one  to several hours  is absorbed  and  retained.

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                             IV--2
Figure IV-1.
Absorption  rate of DCB, TCB  and  HCB  in  mice
(•.-/hole body, except for the  digestive tract,
hair, skin  and tail)  after oral  administration
(DCB or TCB = 2 . Smg/niouse ; MCB=1 . 25 mg/mouse)

Source:  Yano (1979)

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                                IV-3
Distribution



     Hawkins, et al . (1980) described the tissue distribution



of p-DCB in adult female CFY rats following  inhalation, oral or sub-



cutaneous exposure.  The animals received 10 consecutive daily expo-



sures to p-dichloro  (14C) benzene either via inhalation at 1,000 ppm



(1^/6,000 mg/m^) for  3 hours/day or to a range of doses orally or



subcutaneously.  The compound was administered in sunflower oil at



levels of 50,  125, 250, 375 or 500 mg/kg.  The authors concluded



that oral or subcutaneous doses of 250 mg/kg would yield tissue con-



centrations in  fat similar to those observed after the 1,000 ppm in-



halation dose.



     Table IV-1 shows tissue concentrations of radioactivity in



animals killed  24 hours after two or ten consecutive daily doses.



During inhalation exposure, concentrations reached their maxima



after six days  of exposure, remaining the same or falling slightly



thereafter.  At the  maximum point, the highest levels were found



in the fat, liver and kidney.  Lung and muscle concentrations



reflected the levels in plasma.  The highest tissue concentrations



following the  250 mg/kg oral doses were reached after four doses.



Again, the highest concentrations were found in liver, kidney and



fat, with muscle and lung being similar to plasma.  After ten doses,



all concentrations had fallen somewhat, with all but fat being simi-



lar to the plasma levels.

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                                                    Table IV-1
    Tissue concentrations of 14C in female rats after dail
    subcutaneous (s.c.) doses (250 mgAg)  of p-dichloro (]
    and results expressed as ppm represent the mean of results from two animals.
                                                      v atmospheric (inhal.)  exposure  (1,000 ppm)  and oral or
subcutaneous (s.c.) doses (250 mgAg) of p-dichloro (C)  benzene.   Animals were  killed  at  24h  after dosing
           Liver              Kidneys
                                             Lungs
Muscle
Fat
ELasma
. of
ses
2
4
6
8
|LO
inhal .
14
22
28
16
18
oral
11
18
14
15
9
s.c.
21
22
24
21
20
inhal .
24
40
43
28
27
oral
27
29
23
18
16
s.c.
30
32
47
41
32
inhal.
9
12
11
10
10
oral
7
13
10
11
9
s.c.
18
12
14
21
17
inhal .
5
6
7
7
3
oral
5
6

-------
                               IV-5
      Tissue concentrations after subcutaneous dosing at 250
mg/kg were variable, often being higher after two doses than after
four or six doses, with a slight rise after six or eight doses be-
fore dropping slightly after  10.  The pattern of distribution was
the same as after oral or inhalation exposure.
     Figure IV-2 shows concentrations of radioactivity over a 24-
hour period following cessation of exposure by inhalation.  Except
for the lungs, concentrations were highest at 1 hour, falling there-
after.  At 120 hours, the concentration in fat had fallen to about
5 ppm from 2,400 ppm at 1 hour.  Concentrations in all other tissues
were below the level of detection (0.2 ppm).  Disappearance of
radioactivity from the same six tissues was monitored in selected
animals for up to 192 hours after cessation of exposure for 10
days by all three routes  (Table IV-2).  After oral dosing, maximum
plasma concentrations occurred 2-4 hours after cessation of
exposure.  Peak tissue concentrations also occurred at this
time, being highest in fat.   Concentrations declined rapidly at
all sites to undetectable levels by 120 hours.  After subcutaneous
dosing, peak plasma levels were reached within 1-2 hours, with
peak tissue concentrations occurring at 2 hours, again highest
in fat.  Concentrations fell  more slowly than after the other
two routes of exposure, with  detectable levels remaining after
192 hours.

Metabolism
     Figures IV-3-IV-5 depict the metabolic pathways proposed for
each of the three DCS isomers.  The figures reflect a composite of
work done in several laboratories.

-------
                              IV-6
                   I
                   I
                   1
                   I
                   i
Figure IV-2.
Concentrations of radioactivity in the plasma
and tissues of rats after repeated daily in-
halation exposures of p-dichloro (^4C) benzene
atmospheres for ten days.

Source:  Hawkins, et al. (1980)

-------
                                                     1V-7
                                                          Table IV-2
      Tissue concentrations of 14C  in female rats at different times after consecutive daily atmospheric  (inhal.)
      exposures  (1,000 ppm) and oral or subcutaneous (s.c.) doses (250 mgAg)  of p-dichloro (14C) benzene for  ten
                           days.  Results are expressed as ppm and represent single animals.
            Liver
Kidneys
Lungs
Muscle
Fat
Plasma
ne of
orifice inhal .
(h)
0.5
I
2
4
B
4
8
6
0
2

83
97
59
51
66
14
16
0.2
0.2
0.2
oral

117
82
75
90
101
31
7
2
0.2
4
s.c.

23
35
37
16
30
28
22
15
7
1
inhal,

172
304
89
86
138
21
15
2
0.2
0.2
. oral

74
56
81
149
123
31
3
2
0.2
0.2
s.c.
-
45
54
66
58
34
31
38
21
7
2
inhal .

178
84
58
42
39
9
8
1
0.2
0.2
oral

58
43
347
106
75
13
3
2
4
0.2
s.c.

24
22
9
22
18
18
15
9
3
0.2
inhal .

43
96
18
16
35
3
5
0.2
0.2
0.2
oral

12
22
NS
NS
23
11
0.2
0.2
0.2
0.2
s.c.

11
NS
9
25
18
16
NS
25
4
0.2
inhal .

1300
2434
1133
1307
1477
425
233
12
5
6
oral s.

401
421
630
1423
1385
559
56
8
0.2
0.2
c. inhal. oral s.c.

347
335
809
622
481
476
424
199
64
14

38
34
26
40
48
10
9
0.2
0.2
0.2

38
38
46
48
43
18
2
0.2
0.2
0.2

26
42
38
29
26
24
22
9
5
1
                                                         NS=No Sample
Source:  Hawkins, et al. (1980)

-------
           Cl
*Main Oxidation Product
                                                                                            Cl
    3.4
    dichlorophcr.yl
    mercapturic acid
                                                                                                              ethereal suifata: •''
                                                                                                                             r*69%
                                                                                                              glucuronides
                                                                                                                                 oo
hxcrc'jon Products (Rabbit):
    5%  mercapturic acids
   40%  phenols
    A%  catechols
   GJ%  conjugates
    0%  air
 Figure IV - 3 .  Proposed Metabolic Pathway-for o-Dichlorobenzene

-------
                                                                            rncrcapturic acid
->-
                                                                                             Gtherosl
                                                                                             sulfates
                                                                                             glucuronides
Figure IV -4 .   Proposed Metabolic Pathway for m-Dichiorobenzene
                                                                       Excretion Products (Rabbit):
                                                                          "1% morcapturic acids
                                                                          27% SOq + gluC.
                                                                                  O
                                                                          25% phenols
                                                                           3% catechols
                                                                           0% air

-------
                                                          Mo mercapturic
                                                              acids
                                    Cl
                                      (Hydro) Quino (L)
                                                Cl
Figure IV - 5 .   Proposed Metabolic Pathway for p-Dichlorobenzcne
                                                                         No catechois
                                                                                                       ctvic.'cai
                                                                                                       sulfate


                                                                                                      glucuroniuc
                                                                                                                    o
Exertion Products (Rnbbit):
    0% n-.crcapturic acids
   3ii% phenols
    G7o quinols
   637o SOg + tjluc, conjugates
    0% air

-------
                              IV-11
     In rabbits after oral administration  (0.5 g/kg),  all



dichlorobenzenes were slowly metabolized by oxidation, mainly  to



dichlorophenols.  The phenols and their conjugation products were



excreted in five to six days.  The major phenolic metabolite of



0-DC& \*&ST 3,4-dichlorophenol (30% of  the total dose),  of m-DCB,



2,4-dichlorophenol (24% of the total  dose) and of p-DCB, 2,5-



dichlorophenol (35% of the total dose).  About 3-11% of the ortho



and meta isomers were excreted as dichlorocatechols.   Para-DCB did



not seem to form this metabolite.  The ortho  and meta  isomers



formed mercapturic acids  (5-10% of the total), but p-DCB did not



(Williams, 1959).  The orientation of the mercapturic  acid was



the same as for the major phenolic product of each isomer.  The



phenolic metabolites were excreted as conjugates of glucuronic



and sulfuric  acid  (Azouz, et al., 1953; Azouz, et al., 1955;



Parke and Williams, 1955).



     Kitamura, _e_t al_. (1977) studied  the metabolism of meta-



and para-dichlorobenzene  in the mouse.  After an intraperitoneal



dose (level not stated) of either compound, urinary metabolites



were characterized by GC-MS.  The metabolites were the same as



noted in Figures IV-4 and IV-5, with  the exception that an unquantified



amount of unchanged compound also was detected.



     The dichlorophenols  appear to be the principal metabolic



product of the DCB isomers in man.  In a study of industrial expo-



sure to p-DCB, Pagnatto and Walkley (1965) used the urinary meta-



bolite, p-dichiorophenol, as a measure of exposure to  p-DCB.   In a



case of accidental ingestion of an unknown quantity of p-DCB



crystals by a three-year  old boy, analysis of urine specimens

-------
                               IV-12
yielded four abnormal phenols as well as  2,5-dichloroquinol.  These
were shown to be conjugated with glucuronic and sulfuric acids
(Hallowell, 1958).
     Non-mammalian animal species have been shown to metabolize
the chlorinated benzenes, although the proportion of the products
may differ from mammals.  Safe, e_t al_. (1976) investigated the
metabolism of chlorinated benzenes in the frog, Rana pipiens.  A
solution of each substance studied (80 mg in 4-5 ml vegetable oil)
was administered in four equal aliquots by  intraperitoneal injection
into each of four frogs.  Para-DCB yielded trace amounts of 2,5-di-
chlorophenol.
     Microorganisms also have been observed to metabolize the
halogenated benzenes.  This factor may be of some importance if the
organisms inhabit the drinking water sources or waste waters which
may eventually contaminate these sources.
     The oxidative degradation of chlorinated benzenes by
Pseudomonas was investigated by Ballschmiter and Scholz (1980).
They showed that each dichlorobenzene was oxidized to one or more
dichlorophenol, then to one or more dichlorocatechol.  Action of
the organisms on o-DCB produced primarily 3,5- and 4,5- dichloro-
catechol; 3,5-dichlorocatechol was the principal product formed
from p-DCB.
     Garrison and Hill (1972) studied the effects of biological
action on the lower chlorinated benzenes.  Ortho- and p-DCB
volatilized completely from aerated mixed cultures of aerobic
organisms in less than one day.  Midwest Research Institute  (MRI,
1974) reported that p-DCB was degraded by biological organisms

-------
                               IV-13
to 2,5-dichlorophenol, dichloroquinol and conjugates.  Gubser



(1969)  reported that o-DCB was degraded by sewage sludge organisms,



but the degradation products were not given.




     In a study with benzene-acclimated activated sludge, m-DCB



was oxidized to a greater extent than either of the other two isomers



after 192 hours (Malaney and McKinney, 1966).  -The following organ-



isms were found in the sludge:  Protozoa: Paramecium, Norticella



and Episylis; bacteria: Flavobacterium 1 actis, Achromobacter sul-



fureum, A. superficial is, Alcaligens marshallis and Rhizobium lupina.



A large number of rotifers also were found during the early



stages of the study.



Chlorinated Benzenes as Breakdown Products of Pesticides



     Chlorinated benzenes have been identified as degradation



products of lindane metabolism by various plant and animal species.



Considering the once widespread use of lindane as a pesticide, some



of these chlorobenzenes must have entered the environment as lin-



dane degradation products.



     Gamma-pentachloro-1-cyclohexane (r-PCCH) is a known breakdown



product of lindane.  When corn and pea seedlings were exposed to a



concentration of 25 mg  -PCCH/500 ml water, both varieties converted



the  -PCCH to m-DCB, 1,2,4,5-tetrachlorobenzene and 2,4,5-tri-



chlorophenol.  In addition, 1,2,4-trichlorobenzene, 1,2,3,4-tetra-



chlorobenzene and 2,3,5-trichlorophenol were formed by the corn



seedlings; 1,2,3-trichlorobenzene and 2,4,6-trichlorophenol were



formed by the pea seedlings (Mostafa and Moza, 1973; Moza, et al.,



1974).  Gamma-PCCH, m- and p-DCB were identified in the roots



of mature wheat plants that had been grown from seeds treated

-------
                              IV-14
treated with 14C-lindane (Balba and Saha, 1974).




     Mathur and Saha (1977) incubated a mineral soil and an



organic soil with 14C-lindane for eight weeks.  While most of the



lindane was recovered unchanged from the soils (70-89%), those



degradation products that were identified included m- and p-DCB.



     Six chlorobenzenes were "tentatively" identified as products



of lindane metabolism in the rabbit (Karapally, ^t al., 1971).



Ortho-DCB was among those detected.  Chicken liver homogenates were



shown to degrade lindane to all three isomers of DCS as well as some



higher chlorinated benzenes (Foster and Saha, 1978).  The pheasant



showed a similar metabolism pattern.



Degradability and Bioaccumulation Potential



     In general, the halogenated benzenes may be broken down in



the environment.  The extent to which they do break down depends



upon a number of factors, including the type of halogen and the num-



ber and position of halogens in the ring.  The lower halogenated



compounds tend to be less resistant to biodegradation.  The resis-



tence of the chlorinated hydrocarbons to chemical and physical



degradation and the marked ability of these compounds to accum-



ulate in fatty tissues are the most important factors in controlling



the fate and distribution of these compounds.



     Both o- and p-DCB are resistant to auto-oxidation by the




peroxy radical (RO2) in water and by ozone in air (Brown, et al.,



1975).  The dichlorobenzenes are reactive to hydroxy radicals (OH)



in air with a half-life of about three days.

-------
                              IV-15
     Ortho- and para-DCB, in the presence of sunlight and a



dilute aqueous alkali, react with chlorine to form 1,2,4-trichloro-




benzene (photochlorination) (Kirk and Othmer, 1963).  At moderate



temperature, o-DCB is resistant to alkaline hydrolysis.



     Dichlorobenzenes are decomposed by radio frequency and



each DCS yields the other two isomeric DCBs (Rix and Still, 1966).



     Little information  is available relating to environmental



hydrolysis of chlorinated benzenes.  This is, of course, limited



by the insolubility of these compounds in water.  There is a



possibility that the mono- and polyhydric phenols could be



produced through hydrolysis.  If environmental hydrolysis occurs,



it must take place very  slowly to form phenols or conjugates



because of the insolubility of these compounds (Ware and West, 1977)



     Alexander and Lustigman (1966) found that the presence of



a chlorine atom on the benzene ring retarded the rate of biodegrada-



tion.



     Para-DCB has been shown to have accumulated in human blood



and adipose tissue.  Morita and Ohi (1975) reported an average



concentration of p-DCB in human adipose tissue of 2.3 ug/g (range



= 0.2-11.7, N=34).  Blood samples in six volunteers ranged from 4



ug/ml to 16 ug/ml and averaged 9.5 ug/ml.  In another study,



Morita, et al_. (1975) reported levels of p-DCB in human adipose



tissue of Tokyo residents.  The concentration in the fat ranged



from 0.02 ug/g to 9.90 ug/g, with a mean concentration of 1.7 ug/g.



Since Morita and Ohi had established that there was a relatively



high concentration of p-DCB in the atmosphere of the Tokyo



metropolitan testing area (2.1-4.2 ug/m3), they believed that

-------
                              IV-16
inhalation of p-DCB was probably the major route of entry of



the substance into the body.




     In a study conducted in the New Orleans area, all three



DCBs were detected in human blood samples (Dowty, e_t al_., 1975).



However, none of the chlorinated benzenes were detected in a 400



liter sample of air or in a sample of New Orleans drinking water,



although many other organic compounds were confirmed.  One might



speculate that the individuals showing p-DCB in their blood might



have been exposed to the substance during its use as a space



odorant or fumigant.



     Koenenmann and Leeuwen (1980) studied the kinetics of six



chlorobenzenes (including p-DCB) in guppies in an accumulation



and elimination experiment.  The other five compounds were more



highly substituted.  The fish were exposed for 19 days to a mix-



ture of the six substances, with the p-DCB concentration being



160 ng/ml in an aquarium through which the water flowed at a



rate of 27 1/hr.  At three-day intervals, three fish were removed



from the tank and chemical analyses for the six compounds per-



formed.  Concentrations of each in ug residue/g lipid weight



were determined.  Average fat content of the fish was 5.4 ±




2.0%.



       The concentration of p-DCB in lipid reached its peak by



Day 2 (150 ug/g) and remained at that level for the remainder of the



exposure period.  p-DCB residues were not measurable within three



days after termination of exposure.  The investigators calculated



the log P oct to be 3.53 and established the bioaccumulation factor



at 1,800.  Thus, while p-DCB was shown to be quite lipophilic with

-------
                              IV-17
preferential storage in 1ipid-containing tissues, it is rapidly



eliminated when exposure is terminated.



       Neely, el: al_. (1974) estimated a steady-state bioconcen-



tration factor of 210 for p-dichlorobenzene using a short exposure



and duration study with the rainbow trout.



     Bioconcentration by the bluegill has been studied using



14C-labeled dichlorobenzenes, with thin layer chromatography for



verification (U.S. EPA, 1978b).  The bioconcentration factors were



89, 66 and 60 for 1,2-, 1,3-, and 1,4-dichlorobenzene, respectively.



Equilibrium occurred within 14 days and the half-life for each



dichlorobenzene was less than 1 day.  These results confirm that the



dichlorobenzenes are unlikely to be a tissue residue problem in the



aquatic environment.

-------
                               V-l
V. HEALTH EFFECTS IN NON-HUMAN ORGANISMS

   PI ants
       Para-dichlorobenzene is added to field crop seeds to con-
trol seed storage insects.  It was shown that while effective in
controlling pests, p-DCB significantly decreased the viability of
the seeds.  This effect was more pronounced when seeds were stored
in a closed container rather than in an open bin (Day and Thompson,
1965).  Oil seed crops, sorghum, corn and wheat were more seriously
affected by p-DCB than were grasses and legumes.
     Seeds were stored in Mesa, Arizona, in open and closed Mason
jars (1 quart) containing 25 g p-DCB for up to eight years.  No
attempt was made to control temperature or humidity.  After four
years, none of the California Imperial flax seeds germinated from
either the open or closed test container.  The open control regi-
stered 77% germination after four years, while the closed control
averaged 82%.  Arivat barley seeds stored in the closed test jar
averaged 3% germination after eight years, while germination of
the barley seed in the open test jar was 54%.  The control aver-
.aged 59% and  72% in the closed and open containers, respectively
After three years, none of the seed of the Pima S-l fuzzy cotton
from the closed container germinated  (control= 58%).  After eight
years in the  open test container, 43% of the fuzzy cotton seeds
germinated (control=  68%).
       EPA (1978b) determined 96-hour ECso values for the DCBs
on  a fresh water algae, Sel enastrum capricornutum, and a marine
algae, Skeletonema costatum, in static bioassays.  .In vivo

-------
                                V-2
chlorophyll content  and  cell  number counts  were the parameters



measured.  In vivo chlorophyll  content  £€50 values  in Selenastrum



were 91-6, 176 and 98.1  mg/1  for  o-,  m- and p-DCB respectively.



The ECso values for  cell  number count were  98  mg/1  for o-DCB,  149



mg/1 for m-DCB and" 96".Tmg/T  for  p-DCB.   gfreretonenta was  more



.sensitive to DCB toxicity.  The EC50  values for .in  vivo chlorophyll



count were 44.2 mg/1  for o-DCB, 52.8  mg/1 for  m-DCB and 54.8 mg/1



for p-DCB.  Cell number  count EC$Q  values were 44.1,  49.6 and



59.1 mg/1 for o-, m-  and p-DCB, respectively.





Microorganisms



     The chlorinated  benzenes have  been shown  to exhibit  toxic



effects upon a number of microorganisms.  The  antifungal  vapor



phase activity in the soil of each  chlorobenzene was  studied by



Richardson (1968).   A relationship  between  chemical  structure



and action was noted.  Three  fungal  species were exposed  to a



range of eight vapor pressures  (8-1,000  ppm).   The  percentage



retardation of radial growth  was  greatest for  dichloro- (all



isomers) and trichlorobenzenes  (both  isomers).



     Ortho-DCB is lethal  to Mycobacterium smegmatis  as  a  liquid



or a vapor (Crowle,  1958).  Torres,  et  al.  (1970) showed  that  at



a dilution of 1:800,  o-DCB was  active against  Staphylococcus



aureus and Escherichi coli, in  vitro.   In the  presence  of  organic



matter (10% defibrinated sheep  blood),  o-DCB was  effective



against the spores of Bacillus  anthracis and E_.  col i.   According



to Brown, et al. (1975),  p-DCB  is not toxic to  Ustilago maydis.

-------
                               V-3
     Boyles (1980) investigated the effect of ortho- and para-



DCB upon the selective permeability of the cell membranes of



Candida tropical is.  A culture of organisms at a concentration of



1 g/lOOml was suspended in deionized water.  One ml of the test



compound was shaken into this suspension and conductance measured.



Changes in conductance were measured across diptype bright platinum



electrodes.  These changes reflect leakage of electrolyte salts



from the cells.  The author concluded that solid hydrocarbons



diffuse into cells only very slowly since ortho-DCB (which is



liquid) was among the most active compounds tested (62% of internal



electrolytes lost in 6 hours), whereas p-DCB (a solid) had only a



very small effect (7.3% lost in six hours).



     Boyles (1980) also studied the effect of a number of com-



pounds upon the growth rate of fast growing bacteria.  Among the



compounds he tested was o-DCB.  By employing an oxygen electrode



chamber, he measured the respiration rate of the colony, suspended



in a nutrient medium.  Vibrio natriegens was diluted to a concentra-



tion of*» 10^ organisms/ml.  Addition of increasing concentrations



of o-DCB to the solution caused a dose-dependent decrease in the



rate of growth.  At^10 ppm DCB, there was a 15% decrease.  At/-30



ppm, the rate decreased to 20% of control and at~45 ppm, the



rate was reduced further to 45% of control.  At~60 ppm, growth




was arrested completely.






Phytoplankton



     Ukeles (1962) conducted a laboratory study of the tolerance



of five species of marine phytoplankton to concentrations of various




toxicants including o-dichlorobenzene.  o^Dichlorobenzene had no

-------
                                V-4
significant effect on growth of any of the tested species at 8 ppm.



At 13 ppm, none of the organisms grew, but all were viable.  Lethal



concentrations were reached at around 80 ppm, and at 130 ppm all



species were dead (See Table V-l).



     Ukeles pointed out that high concentrations of toxicants



used for predator control might be "safe" when used in shellfish



hatcheries, though hazardous under natural conditions.  This is be-



cause plankton food is grown apart from the hatchery and periodi-



cally added to it.  Hence, inhibition of phytoplankton growth would



not occur.  In nature, phytoplankton blooms are important as a source



of food.  Therefore, any alteration in growth of phytoplankton result-



ing from use of halogenated benzenes for predator control could have



consequences along the food chain.



     Dawson, e_t al . (1977) determined the 96-hour LCso values



for o-DCB in marine and freshwater fish.  Tests were conducted at



23°C in five-gallon containers with aeration at pH 7.6 - 7.9.  The



estimated LC$Q value for the bluegill was 27 mg/1 and for tidewater




silverside, 7.3 mg/1.



     EPA  (1978b) determined 48-hour LC$Q values for the DCBs on



the water flea (Daphnia magna) in static bioassays.  For o-DCB, m-



DCB and p-DCB, the values were 2.44, 28.1 and 11.0 mg/1, respectively



EPA (1978b) determined 96-hour LC$Q values on the marine mysid shrimp



in biostatic assays.  For o-DCB, m-DCB and p-DCB, the values were



1.97, 2.85 and 1.99 mg/1, respectively.  Ninety-six hour LC50 values



of each DCB on the bluegill were 5.59, 5.02 and 4.28 mg/1 for o-,



m-, and p-DCB, respectively.  In the sheepshed minnow, the 96-hour




LC5Q values were 9.66, 7.77 and 7.40 mg/1 for o-, m- and p-DCB,




respectively.     '

-------
                                            V-5
                                         Table V-l

                     Effects of o-DCB on Growth of Marine Phytoplankton
Concentration
(ppa) of
ODCB
1.3
7.6
13
130
Lindane
ppn
concentration
7.5
9
Protococcus
sp.
0.71
0.80
0.00*
0.00

0.75
1.0
Chlorella
S£.
0.82
0.95
0.00*
0.00

0.36
0.33
Dunarl iell a
euchlora

0.71
0.90
0.00*
0.00

0.73
0.60
Phaeodactylum
tricornutum

0.74
0.80
0.00*
0.00

0.00*
0.00*
Mjnochrysis
lutheri

1.00
0.65
0.00*
0.00

0.00
0.00
 * no growth,  but organisms viable
All numbers  represent the ratio of optical density (o.d) of growth in the presence of
toxicants to o.d.
normal growth.
in the basal medium with no added toxicants.  Hence 1 = approximately
 Source:   Ukeles,  1962.

-------
                                V-6
     In a 30-day continuous flow chronic toxicity study, EPA

(1978b) determined the maximum acceptable toxicant concentration

(MATC) for o-DCB toward the fathead minnow to be greater than 4, but

less than 8 mg/1 with a geometric mean of 5.65 mg/1.  The "no-effect"
                                                                 %.-
level was then said to be 4.0 mg/1.

     Several chlorinated and fluorinated benzenes are known to

be toxic to a variety of marine organisms including molluscs and

crustacean species.  Because of this known toxicity, there have been

suggestions that mixtures of these substances be spread around oyster

beds to safeguard the crop from predators.  Ortho-DCB, in unknown

concentration, was placed in the area of an oyster bed (Loosanoff,

et al., 1960).  The oyster drills would not cross the barrier of

o-DCB and sand eight feet wide during a 13-month period.  The

drills exhibited extreme swelling in the gastropods, immobility and

death, and curling of the tips of their rays.  Crabs coming into

contact with the substance lost their equilibrium and went into

convulsions.  In a study of the effects of o-DCB upon clam young

(Mercenaris mercenaris), Davis and Hidu (1969) found a TL^ value of

100 ppm in eggs exposed for 48 hours or larvae exposed for 14 days.

Oysters, themselves, are not immune from the toxic effects of

o-DCB.  An exposure of 1 ppm was found to be the minimum concentra-

tion which inhibited the growth of young oysters (Crassosterca

Virginia) after 24 hours' exposure (Butler, e_t al^., 1960).  No

bioaccumulation was noted; the DCB was excreted by the oyster when

the chemical was removed from the water.

-------
                                V-7
Insects
     Both o- and p-DCB have been used  as  insecticides; of  the
two, p-DCB has received more widespread application.  However, only
a few toxicity studies are available for  these  substances.
     The acute effects of insect exposure to  chlorinated ben-
zenes are summarized in Table V-2.

Birds
     The acute and subacute toxicity of p-DCB was studied  by
Hollingsworth, e_t al_. (1956).  Pekin ducks were fed a diet of 0.5%
p-DCB for 35 days.  Growth was retarded in all  animals, and three
of the animals died.

Non-human Mammals
     The acute and chronic toxicities  of  the  dichlorobenzenes
closely parallel those observed with chlorobenzene.  A search of
the literature uncovered no data on the acute or chronic toxic
effects on m-DCB.  However, a number of studies have been published
on the acute and long-term effects of  the other two dichlorobenzene
isomers.  In general, p-DCB found to be less  toxic than the ortho-
isomer.
     The dichlorobenzenes produce sedation, analgesia and
anesthesia after acute or parenteral administration.  Relatively
high doses are needed to produce acute effects, but chronic effects
may occur at relatively low levels.  Acute poisoning is character-
ized by signs of disturbance of the central nervous system (CNS).
There may be hyperexcitability, restlessness, muscle spasms or
tremors* followed by varying degrees of CNS depression.  The most

-------
                                             v—o
                                           Table V-2

               Acute Effects of Chlorobenzenes in Insects (West and Ware,  1977)
Chemical   Organism

 o-DCB     Cimex lecturis
?o-DCB
p-DCB
p-DCB
p-DCB
Dendroctonus
pseudotsagae
Calandra
granaria
Aphis rumicis
Periplaneta
americana
 p-DCB
p-DCB
Carabus memoralis

Calliphora
erthnocephala

Triatoma rubro-
fasciata

Cambarus virilus
                     Exposure

                     Fumigant
                                1 part o-DCB
                                5 parts diesel oil

                                Fumigant
                                Injection
Vapor
Allied.to nerve
sheath
                     Remarks

                     Nervous System
                     effects

                     100% mortality
                                          Nervous System
                                          effects

                                          Narcosis
Nervous system
effects-increase
in activity followed
by a decrease and
spasmodic contractions

Respiratory and
nervous system
stimulated, then
depressed.  Delayed
increase in CC«2
output at 4 hours,
then a decrease

Facilitation, then
depression of trans-
mission
                       Reference

                       Cameron, et al.,
                       1937

                       Gibson, 1957
                                            Cameron, et al .,
                                            1937

                                            Tatterfield, et:
                                            a±., 1925

                                            Munson and Yaeger
                                            1945
Punt, 1950
Punt, 1950

-------
                                  Table V-2 (Continued)
Chemical
 p-DCB
 p-DCB
 p-DCB
 p-DCB


 p-DCB
Organism

Larvae of:  Tineola
                       bisselliella
Exposure

Vapor
            Attagenus inegatoma

Attagenus piceus            2.3-3.2 nvg/1
Termites;  Glypt-
otermeg dilatatus
Kalotermes
Lyctus  africanus
Tyrophagus
dimidiatus
solid or
liquid
fumigant
(with TCBs)

25% solution
0.5% solution
     v/v
Remarks
100% mortality
Reference
Batth, 1971
                 Low concentration      Arnold, 1957
                 repelled larvae; high
                 concentration killed
                 100% after 4 days
Effective in
control
100% mortality
within 8 days

100% mortality
within 4 minutes
Dantharayana
and Fernando,
1970a, 1970b
Awad, 1971
Honma, 1967

-------
                                V-10
frequent cause of death is respiratory depression.  Acute exposure



at very high levels also may result in liver or kidney damage.  Cer-



tain of the halogenated benzenes, like some aliphatic hydrocarbons,



are thought to sensitize the myocardium to the effect of catechola-



mines, and thus set up conditions favoring ventricular arrythmias



(von Oettingen, 1955).



Acute Exposure




     Several investigators have determined the acute lethal dose



levels after exposure to the dichlorobenzenes in several species.



These data are presented in Table V-3.



     Varshavskaya (1968), in her comparative studies on the adverse



effects of the lower chlorinated benzenes, showed that, in the



acute exposures, o-DCB was slightly less toxic in mice and rats



than monochlorobenzene (MCB), and that p-DCB was even less toxic than



o-DCB or MCB.  o-DCB was slightly more toxic than MCB in rabbits and



guinea pigs.  The 1*059 values for acute oral doses of o-DCB were:



2,000 mg/1 for the mouse, 2,138 mg/1 for the rat, 1,875 mg/1 for the



rabbit and 3,375 mg/1 for the guinea pig.  The LD50S for p-DCB were



found to be 3,220 mg/1 for the mouse, 2,512 mg/1 for the rat,  2,812



mg/1 for the rabbit and 7,593 mg/1 for the guinea pig.



     Doses of 0.25-0.5 cc/kg (0.33-0.66 mg/kg) body weight of  o-DCB



intravenously administered to rabbits were fatal within 24 hours;



doses of 1.0 cc/kg (1.31 mg/kg) were fatal within 20 seconds




(Cameron, et al., 1937).



     Dogs exposed to 2 cc/m3 (0.04% or•*>400 g/m3) o-DCB via



inhalation showed no adverse effects, whereas 0.08% (-^800 g/m3)



produced somnolence (Riedel, 1941).  Histological studies

-------
                                    Table  V-3

                Acute Toxicity Data for o-  and  p-Dichlorobenzene
Animal
Route
o-Dichl
Rat
Rat
Mouse
Rabbit
Guinea
Guinea
Rat
Guinea
Guinea
p-Dichl
Rat
Rat
Rat
Mouse
Rabbit
Guinea
Guinea
Mouse
orobenzene




Pig
Pig

Pig
Pig
Oral
Oral
Oral
Oral
Oral
Oral
Inhal .
Inhal .
Inhal .
orobenzene





Pig
Pig

Oral
Oral
Oral
Oral
Oral
Oral
Oral
SC
LD50
                               500 mg/kg
                              2138 mg/1
                              2000 rag/1
                              1875 mg/1
                              3375 mg/1
                              2000 mg/kg
LCLp
                                            821  ppm/7  hr
                                            800  ppm/7  hr
                                            800  ppm/24 hr
                               500  mg/kg
                              2500  mg/kg
                              2138  mg/1
                              3220  mg/1
                              2812  mg/1
                              7593  rng/l
                              2800  mg/kg  (LDLO)
                              5145  mg/kg
Reference
                                           NIOSH,  1978
                                           Varshavskaya,  1968
                                           Varshavskaya,  1968
                                           Varshavskaya,  1968
                                           Varshavskaya,  1968
                                           Hollingsworth,  et al.
                                           1958

                                           Hollingsworth,  1958
                                           Hollingsworth,  1958
                                           Cameron,  et  al.,  1937
                                           NIOSH,  1978
                                           Holl ingsworth,  et_ al^.,  1956
                                           Varshavskaya,  1968
                                           Varshavskaya,  1968
                                           Varshavskaya,  1968
                                           Varshavskaya,  1968
                                           Holl ingsworth,  j5t al^.,  1956
                                           Irie, et  al.,  1973

-------
                                V-12
following the administration of acute and subacute  doses  of  o-DCB



showed damage to the liver and kidneys.  Exposing mice  to similar



concentrations caused CNS stimulation for about  20  minutes followed



by CNS depression, muscular twitching, slow and  irregular respiration,



cyanosis near the end of an hour, and death within  24 hours.   Rats



appeared to be slightly more resistant than mice to the toxic



effects of o-DCB.



     In the mouse, the LD5Q value for a subcutaneous dose of p-DCB



was found to be 5,145 mg/kg (Irie, et al., 1973).



     Inhalation of o-DCB by rats at 800 ppm (4,800  mg/m3) concentra-



tion for 11-50 hours was irritating to the eyes  and nose, produced



slight changes in the tubular epithelium of the kidney  and resulted



in confluent necrosis of the liver (Cameron, e_t_  al_., 1937).



     Rabbits, rats and guinea pigs exposed for 20-30 minutes daily



to 100+ g p-DCB/m3 of air for 5-9 days showed marked irritation of



the eyes and nose, muscle twitching, tremors, CNS depression,



nystagmus and rapid but labored breathing (Zupko and Edwards,  1949).



The animals recovered within 30-180 minutes after being removed



from the p-DCB-rich atmosphere.  A definite granulocytopenia was



observed in 11 rabbits, a questionable change in three  others  and



no change in the remaining three.  Body weight decreased  in  14



animals; three rabbits showed an increase.  In 13 rats, CNS de-



pression was observed to be greater than in rabbits.  There was



complete narcosis with attendant tremors and muscle twitching



with each exposure.  A definite granulocytopenia was observed  in



eight rats, a questionable change in three, and no  change in two



others.  Nine rats showed a decrease in total white cell  count

-------
                                V-13
while four showed an increase.  In addition to exhibiting similar



symptoms to p-DCB inhalation as rats and rabbits, guinea pigs also



exhibited granulocytopenia in most cases.  Five animals showed a



frank decrease in granulocytes, two showed a tendency toward lowering,



while two others showed no decrease.  Six of the nine animals



suffered a weight loss while three did not.



     The observation that many of the test animals developed granulo-



cytopenia is an important one.  This condition is considered to be



a precursor to leukemia.  However, in these experiments, when the



animals were removed from exposure to p-DCB, the decrease in granulo-



cytes was reversed and the level returned to normal within three to



four weeks.



     Several animal studies document the effects of o- and p-DCB on



the eye and/or skin.  The results are summarized in Table V-4.



     Yang, et: _al_. (1979) showed that both o- and p-DCB alter



pancreatic function.  They administered a single intraperitoneal



dose of each substance in sesame oil (5 mmol/kg:  735 mg/kg) to



fasted adult male Holtzman rats.  Control animals received an equi-



valent volume of sesame oil (1 ml/kg).  Experimental measurements



were made 24 hours later.  After treatment with o-DCB, bile duct-



pancreatic flow (BDPF) was increased nearly ten-fold over that



observed in control animals (P<0.05).  p-DCB did not alter the flow



significantly.  Protein concentration of the bile effluent was re-



duced to about 25% after o-DCB (P<0.05), but was not changed after



p-DCB.  However, p-DCB did increase significantly the chloride



content of the effluent, whereas o-DCB did not.  Neither compound



affected the rate of bile flow or serum levels of SGPT.  The authors

-------
                                             Table V-4

                        Effects of Chlorinated Benzenes on  the  Eye  and  Skin
                                (modified  from West and Ware, 1977)
Chemical
Animal
      Exposure
   Effects
                                  Reference
o-Dichloro-
  benzene
p-Dichloro-
  benzene
p-Dichloro-
  benzene
p-Dichloro-
  benzene
Rabbits
Rabbits
Rabbits
Rabbits
2 drops in each eye
4.6 to 4.8 mg/1
(770-800 ppm) for
8 hours, inhalation
Inhalation.  5 gm
vaporized/2 days
5-47 days

Oral,  5 gms p-DCB
daily for 3 weeks
Pain slight.  Conjunctival
irritation. Cleared in 7
days

Lateral nystagmus, transitory
edema of cornea. - edema of
optic nerve.  Eye changes
reversible (17 days).  No lens
changes or deposits in the
vitreous.

No opacity of the lens (liver
necrosis and death)
Opacity of lens slight and
mod
                                Hollingsworth,
                                et al . ,  1958
                                Pike,  1944
                                Berliner,  1939
                                Berliner,  1939

-------
                                V-15
could not offer an explanation of the mechanism by which the observed



changes occurred, although they did conclude that the mechanism did



not involve secretin or cholinergic stimulation of the pancreas.



Thus, the significance of these findings remains unknown.





Effects on Porphyrin Metabolism



     All of the chlorinated benzenes have been shown to produce



disturbances in prophyrin metabolism.  The mechanism for this bio-



chemical lesion seems similar to that seen following administration



of other inducers of drug metabolism.  It is also similar to that



seen in man following exposure to ethanol, synthetic estrogens and



progestins (Parke, 1972).



     Rimington and Ziegler (1963) showed that in male albino rats



administration by gavage of 500 to 1,200 mg/kg/day for 5-15 days



of chlorinated benzenes (except pentachlorobenzene) leads to a hepatic



porphyria characterized by elevated levels of precursor porphyrins in



liver and feces.  The investigators administered o-DCB in liquid par-



affin at maximum dose levels of 455 mg/kg/day for 15 days,  and p-DCB,



also in liquid paraffin, at maximum dose levels of 770 mg/kg/day for



5 days.  Doses were gradually increased until a level was reached



which yielded high porphyrin excretion, but few fatalities.  The



first signs of intoxication were an increase in urinary copropor-



phyrin and porphobilinogen (PEG).  An increase in aminolevulinic



acid excretion was a late effect.  The most potent compound was



p-dichlorobenzene (see Table V-5).  Mean peak values of urinary



coproporphyrin increased 10-15 fold after p-DCB to about 62 ug/day



when compared with control.  o-DCB treatment elicited a 6-10 fold



increase in the same parameter (to about 43 ug/day).  After p-DCB,

-------
                                                        V-16
                                                     Table V-5

                        Mean Peak Values of  Urinary Porphyrin Precursors Following Treatment
                               With Maximum  Doses Tested for each Chlorinated Benzene
                                            (Rimington and Ziegler,  1963)

No.
of-
rats Compound
Controls*
3 Monochlorobenzene
3 1 , 2-Dichlorobenzene
3 1 , 4-Dichl orobenzene
3 1,2, 3-Trichl orobenzene
3 1,2,4-Trichlorobenzene
3 1 ,2,3, 4-Tetrachl oro-
benzene
6 1,2,4,5-Tetrachloro-
benzene


Sol-
vent
C
P
P
P
C
C
P

C


Max.
dose
(mgAg)

1140
455
770
785
730
660

905

Days
on
max.
dose

5
15
5
7
15
10

5


Copro-
porphyrin
(pg/day)
4.3-6.8
30.50
43.10
61.80
36.59
58.31
28.96

4.10


Uropor-
phyrin
(pg/day)
0.1-0.3
1.40
2.01
10.99
0.72
2.73
3.80

0.22



PEG
(pg/day)
2.5-6.5
26.70
26.65
1328.10
57.38
179.00
520.63

6.50



ALA
(pg/day)
38.7-51.6
56.40
11.32
437.41
36.26
145.70
315.78

18.08




ALA/PBG
'
2.98
2.11
0.36
1.01
1.06
0.61

6.69

P = liquid paraffin? C= 1% cellofas
PBG = porphobilinogen; ALA = /_ -aminolevulinic acid.
*    Mean max. and min. of 5 rats during 41 days.

-------
                                V-17
a nearly 100-fold increase in urinary uroporphyrin levels occurred,
while o-DCB caused an increase of about 10-fold.  Porphobilinogen
levels measured 1,328 ug/day after p-DCB, but only 26.65 ug/day
after o-DCB, increasing from a control level of 2.5-6.5 ug/day.
After p-DCB, a 10-fold increase (to 437 ug/day) in   -ALA levels
were observed, while after o-DCB, levels actually decreased to
11.3 ug/day (normal range = 38.7-51.6 ug/day.)
     Several parameters of liver function also were measured by
Rimiagton and Ziegler (Table V-6).  In contrast to the change
observed in urinary coprophyrin excretion, liver coproporphyrin was
unchanged after p-DCB, and roughly doubled after o-DCB (to 10.05
ug/lOOg tissue vs. 4.5 ug/lOOg in the controls).  Protoporphyrin
levels increased six-fold after p-DCB (60.5 ug/lOOg), but only
3.5-fold after o-DCB (to 34.8 ug/lOOg from a control level of 9.7
ug/lOOg).  Catalase levels were unaffected by p-DCB, but more than
halved after o-DCB (0.85 meq/mg wet weight for control to 0.364
meq/mg wet weight after p-DCB exposure).  These decreases in catalase
were observed only in animals in which marked histological changes
indicative of severe liver damage with large areas of necrosis were
observed.  Thus, changes occurred in o-DCB-treated animals, but not
in those treated with p-DCB.  In addition, p-DCB did not alter the
glutathione content of the liver.  o-DCB was not tested.  From
these data, the authors suggested that the mechanisms which produce
porphyria derangements may be different from those which lead to
liver necrosis.
          Carlson (1977) studied chlorinated benzene induction of
liver porphyria in groups of five female rats receiving p-DCB,

-------
                       V-18
                     Table V-6

 RDrphyrins, Etorphobllinogen and Catalase Activity
In Livers of Rats Treated With Chlorinated Benzenes
           (Rimington and Ziegler,  1963)
No.
of,
rats Compound
2
2
2
2
2
5
3
2
Controls
Monochl orobenzene
1 , 2-Dichl orobenzene
1 , 4-Dichl orobenzene
1 , 2 f 3-Trichlorobenzene
1,2, 4-Trichl orobenzene
1,2,3, 4-Tetrachloro-
benzene
1,2,4, 5-Tetrachl oro-
benzene
Max.
dose
(mgAg)
1400
450
770
780
500
660
850
Days
on
max.
dose
5
15
5
7
10
10
5
Copro-
porphyrin
(ug/100 day)
4.5
Trace
10.05
5.07
2.65
45.56
35.04
6.30
Protopor-
phyrin
(ug/100 g)
9.7
13.00
34.80
60.35
3.55
55.60
56.57
9.90
Uropor- PBG
phyrin
(ug/100 g
uncorrected)
1.3
6.35
14.40
60.35
20.00
52.70 ++
41.32 -f
2.15

Catal ase
(meq/mg)
wet wt.
0.85
0.502
0.364
0.880
0.857
0.747
0.772
0.838

-------
1,2,4-trichlorobenzene or hexachlorobenzene.  Each substance was



suspended in corn oil and administered orally at Of 50, 100 or 200



ing/kg/day for 30, 60, 90 or 120 days.  After the last dose, the



animals were placed in metabolism cages for collection of 24-hour



urine samples.  At the end of the 24-hour period, the animals were



sacrificed and liver and urinary porphyrins measured.  The results



after exposure to p-DCB can be seen in Table V-7.  The data show that



this substance has little potential for causing porphyria, thus con-



firming the observations of Rimington and Ziegler (1963).  After 30



and 60 days, liver weight increased in a dose-dependent manner.



Even after 120 days, only slight increases in liver porphyrins



occurred.  Urinary excretion of  -ALA, porphobilinogen (PEG) or



porphyrins was not increased over control levels.





Subacute to Longer-term Exposures



     A number of reports have appeared in the literature that



describe subacute to longer-term exposures of experimental



animals to the ortho- and para- isomers of dichlorobenzene



(see Table V-8 for a summary of these data).



     Ortho-Dichlorobenzene



     Hollingsworth, et_ al_. (1958) gave rats a series of 138



doses of o-DCB over a period of 192 days (18.8,  188 or 376 mg/kg/



day, five days a week) by intubation.  No adverse effects were



detected at the lowest dose.  With the intermediate dose, a slight



increase in the weight of the liver and kidneys was noted.  At the



highest dose, there was a moderate increase in the weight of the



liver, a slight decrease in the weight of the spleen and cloudy



swelling of the liver.

-------
                                 V-20
                              Table V-8

      Effect  of  1,4-Dichlorobenzene p.o. on Porphyrin Production
                    and Excretion in Female Rats.

                           (Carlson, 1977)
 Dose       Liver wt(g)     Liver porphyrins    Urine porphyrins
(mg/kg)                    (ng/g)              (ug/24h)


           30  days  of administration
   0        6.8 + 0.3a      246 +_ 21a             1.5 + 0.2a
  50        6.6 + 0.4a      269 + 20a             1.9 + 0.3a
 100.       7.0 + 0.23      251 i 22a             1.4 + 0.2a
 200        8.0 + 0.3b      276 + 18a             1.4 + 0.2a

           60  days  of administration
   0        6.7 + 0.3a      381 + 20a             1.9 + 0.4a
  50        7.2 + 0.4a      448 + 17a'b           2.0 +_ 0.4a
 100        7.6 + 0.3a'b    435 ± 19a'b           1.6 ^ 0.2a
 200        8.4 + 0.3b      472 _+ 30b             1.7 + 0.3a

           90  days  of administration
   0        6.8 + 0.6a      541 + 33a             0.9 +_ 0.2a
  50        7.0 +_ 0.6a      527 + 30b             1.0 + 0.2a
 100        6.6 ^ 0.4a      555 +_ 20a             1.3 + 0.3a
 200        7.2 i 0.3a      548 ^ 36a             0.8 +_ 0.2a

           120 days of administration
   0        6.9 + 0.2a      354 + 10a             1.4 ^ 0.2a
  50        8.1 ^ 0.3b      391 +_ 18b             1.8 +_ 0.4b
 100        7.3 + 0.6a'b    411 _+ 9b'c            1.6 ± 0.4a
 200        7.5 + O.I-3'13    440 -f 8^              1.0 + 0.2a
  a~c  Values with same superscript are not significantly different
      (P>0.05).

-------
                                                     Table V-8

                                  Summary of  Subacute to Longer-term Toxicity Data
deal   Species    Dose
              Duration    Route        Effects Observed            Remarks
                                CNS Blood Liver Kidney Lung Bone
                                                            Marrow
                                                                           Reference
B
        Rat
18.8,188
or 376
mgAg/day
         Rat
0.001,0.01
or 0.1 mg/
kg day
Rat(20)   49 ppm
Guinea
 pig (8)
Mouse (108)

Rat(20)
Guinea
 pig (8)  93 ppn
Rabbit (9)
Monkey (28)
5 days/week,
138 doses
192 days
                                 Oral
9 mos.
                                 Oral
                            7 hrs/day;
                            5 days/
                            week;
                            6-7 months
                            7 hrs/day;
                            5 days/week;
                            6-7 months
                          Inhala-
                          tion
                          Inhala
                           tion
No adverse effects
at 18.8 mgAg*
Slight increase in
kidney and liver
weight at 188 mgAg.
Moderate increase in
liver weight and
cloudy swelling,
decrease in spleen
weight at 376 mgAg.

No adverse effects
at 0.001 mgAg
Dose-dependant
changes in
conditioned reflexes,
depression of
hematopoietic system
at 0.01 and 0.1 mgAg.

No adverse effects
observed
Holl i ngsworth ,
et aU (1958)
Varshavskaya
(1968)
                                                                           Hoi1i ngsworth,
                                                                           et al.  (1958)
                                                    No adverse effects
                                                     observed
 )= Nuraber of animals tested

-------
                                                 Tabel  V-8 (Continued)

                                   Sunnmary of Subacute  to Longer-term Toxicity Data
arnical    Species    Dose
            Duration    Foute        Effects Observed
                              CNS Blood  Liver Kidney Lung Bone
                                                         Marrow
 Remarks
Reference
OCB    Guinea pig  125 or 250  10-11  days   Intra-
                   rog/day as                muscular
     ?             50% soln                 (I.M)
                   or 125 rogAg
                   in olive oil.

       Guinea pig  125 mgAg   20 days      I.M.    4-
       Guinea pig  125 (m)g/   20 days      I.M.
                   (kg)
       Guinea pig  125 (m) g/  21 days      I.M.
                   (kg)
       Rat
10, 100 or  5 days/week; Oral
500 mgAg    20 doses
as 10%
soln.
                                                               Intense steatosis
                                                               of liver; weight
                                                               loss; decreased
                                                               hepatic glycogen
                                                               Weight loss;
                                                               serum transaminase
                                                               increased

                                                               11.4% weight loss;
                                                               serum transaminase
                                                               increased
                      Frada and Cali,
                      (1958)
                      Totaro, (1961)
                      Totaro and
                      Licari (1964)
                                                               Increase in reaction  Coppola, et_ al.
                                                               and clotting          (1963)
                                                               formation time
No adverse effects
observed at 10 or
100 mgAg;
cloudy swelling and
centralobular
necrosis of liver;
cloudy swelling of
renal tubular
epithelium
Hoi1ingsworth,
et al. (1956)

-------
                                                     Table V-8  (Continued)

                                        Summary of Subacute to Longer-term Toxicity Data
Chemical   Species    Dose
           Duration      Route        Effects Observed            Remarks
                               CNS Blood Liver Kidney Lung Bone
                                                           Marrow
                     Reference
         Pekin  duck  0.5% in    35 days
                      Diet
                         Oral
         Rabbit
          Rat
500 rog/kg  5 days/week;  Oral
day        263 doses
                      1000 mgAg   5 days/week; Oral
                      day          92 doses
                                   over  219 days
18.8,188   5 days/week;  Oral
or 376     138 doses
mgAg/day  in 192 days
Growth retardation;  Hollingsworth,
no cataract^; 3      et^ ai_. (1956)
deaths after 4 weeks
on diet

Swelling and              "
focal necrogis
in liver

Weight lossj              "
tremors and weak-
ness; necroqis and
cirrhosis in liver

No adverse Affects        "
observed at
18.8
                                                                                       Increased l|ver and
                                                                                       kidney weights at
                                                                                       188 and 376 mgAg.

                                                                                       Decreased spleen
                                                                                       weight; liv^r
                                                                                       necrosis and cirrhosis
                                                                                       at 376 mgAg.

-------
                                                    V-24


                                            Table V-8 (Continued)

                              Summary of Subacute to Longer-term Toxicity Data
Chemical Species Dose Duration Route Effects Observed Remarks Reference
CNS Blood Liver Kidney Lung Bone
Marrow
> Rat 100 mgA 20 min/day; Inhala- 4- 4- + + +
(9M,9F) ( 16,000 5-9 days tion
ppm)
Guinea pig
(9M) +4-4-4-4-
Rabbit 30 min/day 4- 4- 4- 4-4-
(18M)
Rat 96 ppm, 7 hrs/day; Inhala-
(10M) 5 days/week; tion
Granulocytopenia in
8A8; tendency
toward same in 3A8
Granulocytopenia in
5/9; tendency toward
same 2/9; weight loss
in 6/9
Granulocytopenia in 11
Irritation of mucosa,
weight loss in 14A8;
Tremors rapid but
labored breathing,
death 12A8.
No adverse effects
observed
Zupko and
Edwards (1949)
M
A8
Holl ingsworth
et al.(1956)
                       16 days
(10)
(5)
158 ppm
173 ppm
Cloudy swelling or
granular degeneration
of liver

Slight interstitial
edema and congestion
in lung; slight
increase in liver and
kidney weights.

-------
             Table V-8  (Continued)



Summary of Subacute to Longer-term Toxicity Data
mical Species Dose
» (19M) 798 ppm
(15F)
(20M) 341 ppm
Guinea pig 173 ppm
(5)
(16M) 798 ppm
( 7F)
(8) 96 ppm
(8) 158 ppm
Duration Route
CNS
8 hrs/day; +
5 days/week;
1-46 doses (M)
9-69 doses (F)
7 hrs/day;
5 days/week,
6 months
7 hrs/day; +
5 days/week;
16 days
8 hrs/day; +
5 days/week
1-23 doses (M)
11-20 doses (F)
7 hours/day;
5 days/week
157-219 days
n
Effects Observed Remarks Reference
Blood Liver Kidney Lung Bone
Marrow
+ + Tremors; weakness? cloudy "
(F) swelling of liver (M&F)
and kidney (F), Deaths:
2M, 2F
+ + Slight increase in liver "
and kidney weights
+ 4- 4- Slight decrease in Hollingsworth
(F) (M) spleen weight, slight etaU(1956)
edema and congestion
in lungs
4- Deaths: 2M "
No adverse effects "
observed
+ Increased liver "
(F) weight (F);
                                            Body weight loss

-------
             Table V-8  (Continued)



Sunmary of Subacute to Longer-term Toxicity Data
Chemical Species
(8M)
(8F)
T
Rabbit
(1M)
(IF)
(8M)
(8F)
(1M)
(IF)
Mouse
(10)
Monkey
(18)
Dose Duration toute
CNS
314 ppm 7 hours/day; +
5 days/week;
6 months
173 ppm 7 hours /day;
5 days/week;
16 days
798 ppm 8 hours/day; +
5 days/week;
1-62 exposures
158 ppm 7 hours/day;
5 days/week;
157-219 days
96 ppm 7 hours/day;
5 days/week;
157-219 days
158 ppm
158 ppm
Effects Observed Remarks
Blood Liver Kidney Lung Bone
Marrow
+ Cloudy swelling,
fatty degeneration,
focal necrosig,
slight cirrhosis of
liver
+ Slight edema and
congestion
of lungs
+ -f Reversible non-
specific eye
changes
No adverse effects
observed
No adverse effects
observed
No adverse effects
observed
No adverse effects
observed
Reference
ii
»
Hollingsworth
et aK (1956)
n
H
n
ii

-------
                                V-27
     Hollingsworth, et al.  (1958)  also measured  the  effects  of
multiple inhalation exposures of o-DCB on  rats,  guinea pigs,
mice, rabbits and monkeys.  A range of concentrations was used,
seven hours a day, five days a week,  for six  to  seven months.  No
adverse effects were observed lit rats-, guii-fesr pig» or ia*ce> exposed:
to 49 ppm, or in rats, guinea pigs, rabbits and  monkeys exposed to
93 ppm (0.56 mg/1).
     Oral administration  of o-DCB  to  white rats  for  nine months
was conducted at doses of  0.001, 0.01 or 0.1 mg/kg/day (Varshavskaya,
1968).  Effects were observed at the  two higher  doses similar to those
described for monochlorobenzene.   The author  reported an inhibition
of mitosis in the  bone marrow, as  well as  neutropenia and abnormal
conditioned reflexes.  These changes  in the blood profile may be
important in that  they could be precursors to pancytopenia or
leukemia.  In this report,  however, Varshavskaya concluded that
no carcinogenic activity  was observable at the doses studied.  She
measured tissue DPN, TPN,  glucose-6-phosphate and alkaline phospha-
tase since it has  been claimed that an increase  in the activity of
these enzymes is indicative of carcinogenicity (Burstone, 1965).
At the 0.1 and 0.01 mg/kg  doses, o-DCB caused an increase in acid
phosphatase but a  decrease in alkaline phosphatase.
     The 0.1 mg/kg dose of o-DCB caused a  marked increase in the
amount of 17-ketosteroids  found in the urine.  This  increase is
most likely due to hyperplasia of  the adrenal cortex, since  an
increase in the weight of  the adrenals and decrease  in the ascorbic
acid concentration of the adrenals also were  observed.  The  0.001
mg/kg dose had no  observable effects  on any of the parameters  studied.

-------
                                V-28

Para-Dichlorobenzene
     Intramuscular injection of 125 or 250 mg p-DCB/kg into guinea
pigs over a 10 or 20 day period resulted in effects typical of
p-DCB toxicity (Frada and Cali, 1958; Totaro, 1961; Totaro and
Licari, 1964; Coppola, et al^. , 1963).  The observed changes included
weight loss, liver changes and blood clotting time increases.
     Oral doses of 10, 100 or 500 mg p-DCB/kg, five days a week, for
20 doses produced marked cloudy swelling and necrosis in the central
area of the liver lobules only with the highest dose (Hollings-
worth, et al_.,1956).   No effects were observed at the other doses.
Pekin ducks, fed 0.5% p-DCB in their diets for 35 days exhibited
depression of body weight gain.  Three animals died after the fourth
week.
     Oral doses of 188 or 376 mg p-DCB/kg, five days a week,
for 192 days (138 doses) in rats induced an increase in the weights
of the liver and kidneys (Holl ingsworth, et_ al_. , 1956).  At 376
mg/kg, increased splenic weight, slight cirrhosis and focal necrosis
of the liver also were observed.  There was also cloudy swelling of
the renal tubular epithelium.  No adverse effects were seen with the
18.8 mg/kg dose.  Rabbits receiving 500 mg/kg/day oral doses, 5
days a week, for a total of 263 doses showed swelling and focal
necrosis of the liver.  Other rabbits receiving a total of 92 oral
doses at 1000 mg/kg/day over a 219-day period exhibited necrosis
and cirrhosis of the liver.  CNS effects were evident as well, as
both tremors and weakness were noted.  Loss of body weight also
occurred.
     The toxicity of p-DCB also has been studied via the
inhalation route of exposure.  Zupko and Edwards (1949) exposed

-------
rats,  guinea pigs and rabbits to a high level of compound  (100 mg/1



or  16,000 ppm) 20-30 minutes/day for 5-9 days.  In all species,



adverse effects were observed on- the CNS, liver, kidney and lung.



Among  the rats (9 males and 9 females), eight animals exhibited



granulocytopenia, with three others showing a tendency toward the



same.   In the guinea pigs (9 males), granulocytopenia was observed



in five, with a tendency towards this condition in two others.  In



addition, six of the nine showed a weight loss.  Eleven of the 18



male rabbits exposed developed granulocytopenia.  Weight loss and



mucosal irritation occurred in 14 of the 18.  CNS effects manifested



as tremors and rapid and labored breathing also occurred.  Twelve of



the 18 animals died.



     Holl ingsworth, et al_. (1956) conducted a series of inhalation



studies with p-DCB, at various levels and durations of exposure,



in rats, guinea pigs, monkeys, rabbits and mice.  The concentrations



used were 96, 158, 173, 314 and 798 ppm (0.58, 0-95, 1.04, 2.05 and



4.8 mg/1, respectively).  Rats exposed to 96 ppm seven hours a day,



five days a week, for 16 days showed no abnormalities.  A 157-219



day exposure at this level in guinea pigs and mice yielded no



adverse reactons.  With the same protocol, inhalation levels of 158



and 173 ppm caused cloudy swelling or granular degeneration of the



liver, slight increase in the weight of the liver and kidneys and



some interstital edema and congestion in the lungs of rats and



guinea pigs.  Monkeys and mice showed no adverse effects at these



levels.   Rats exposed to 341 ppm for six months showed evidence of



a slight increase in the weight of the liver and kidneys.  Guinea



pigs exposed to 173 ppm for 16 days showed a slight decrease in

-------
                                V-JU
splenic weight and some lung edema and congestion.  At 314 ppm, for
six months, the liver became slightly cirrhotic with focal necrosis,
cloudy swelling and fatty degeneration.  Rabbits, exposed to an
atmosphere of 158 ppm for 157-219 days, were unaffected.  At 173
ppm for 16 days, slight edema and lung congestion were observed,
and at 798 ppm, for 1-62 exposures, reversible non-specific eye
changes were apparent.

Effects Upon Drug-Metabolizing Enzymes
     Most mammals possess a group of enzymes that specializes
in the biotransformation of foreign compounds. These enzymes are
located in the endoplasmic reticulum of the liver cells.  The
metabolic transformation of foreign compounds usually leads to the
conversion of lipophilic materials into more polar compounds, which
are eliminated more readily from the hepatocyte and excreted from
the body.  Thus, compounds which are of little nutritive value
are prevented from accumulating in cells and tissues.  In this way,
undesired effects may be avoided (see reviews in Williams, 1959;
Parke, 1968).
     The endoplasraic reticulum (ER) of the cell is the location
of many enzymes such as glucose-6-phosphatase, glucuronyl trans-
ferase, the hydroxylases and protein synthetases (Parke,  1972).
The enzymes concerned with the metabolism of drugs and other xeno-
biotics are referred to frequently as mixed function oxidases or
monooxygenases.  All of the monooxygenase drug metabolizing enzymes
require reduced nicotinamide adenine dinucleotide phosphate (NADPH2),
molecular oxygen and a cytochrome, usually P-450.  The ER is
associated not only with the oxidation of drugs, but also the

-------
                                V-31
biosynthesis of cholesterol, the catabolism of bile acids, the



oxidation of fatty acids and the oxidation of prostaglandins.  In



addition to mixed function oxygenase activities, the hepatic endo-



plasmic reticulum contains a number of reductases, some of which



utilize cytochrome P-450 and NADPH2 (Williams, 1959; Parke, 1968). *.



    The biotransformation of drugs and xenobiotics appears to



occur in two distinct phases.  The first phase includes reactions



classified as oxidations, reductions and hydrolyses.  In the second



phase, the reactions are referred to as syntheses or conjugations.



These enzyme reactions, expecially oxidations which require cyto-



chrome P-450, have been examined throughout the animal kingdom.  Con-



trary to the suggestion of an evolutionary trend for the appearance



of cytochrome P-450 in mammals, this cytochrome is apparently ubiqui-



tous (Ahokas, et al_., 1976).



     However, quantitative differences in metabolism exist,



for example, among livers from various species, among various



tissues from a single animal, and between the neonate and adult within



a species.  It also has been well documented that enzymes of



biotransformation may be regulated (stimulated or depressed)  by



xenobiotics or steroids (Parke, 1972).  While there does appear to



be a ubiquitous distribution of cytochrome P-450, the concentration



in different animal species varies greatly, as can be seen in Table



V-9.  Since these species have differing amounts of cytochrome^P-



450, they must have different abilities to manufacture toxic



intermediates.  They also have varying abilities to metabolize



benzpyrene and halogenated benzenes.

-------
                                V-33
     A number of investigators have suggested  that  a  relationship



exists between the induction of  -aminolevulinic  acid  synthetase



and the induction of drug or xenobiotic metabolizing  systems by



compounds which are known to induce liver porphyria (Rimington and



lisglery l*6*>r Psfcsnd*, et ^_., 1971r Ariyoshi,  et a^., 1975).  The



effects of a series of chlorinated benzenes  on  hepatic   -ALA



synthetase and cytochrome P-450 levels are summarized  in Table V-10.



     Poland et al. (1971) treated young female  Sherman rats by



gavage with daily doses of m-DCB in peanut oil.  Continuous daily



dosing with 800 mg/kg/day for nine days resulted  in a biphasic



pattern of urinary coproporphyrin excretion  (Figure V-6).  ALA



synthetase was measured in animals dosed daily  with 800 mg/kg/day



for 1, 3 or 5 days.  Enzyme activity measured  24 hours after the



last dose showed a steep rise after Day 1, with lesser increases



seen after Days 3 and 5 (Table V-ll).  Increases in liver size were



not sufficient to account for the changes observed.  No histological



evidence of liver damage was noted in these  animals.



     The cyclic nature of the response suggests that an adaption



or tolerance to the compound is developing,  perhaps because the



animal may be detoxifying the substance more rapidly.  This possi-



bility was tested by observing the effects of the same dosing



regimen as described above on hexobarbital (150 mg/kg i.p) sleeping



time.  Sleeping times we're significantly shortened  after one dose of



m-DCB (from 180 minutes to less than 120 minutes),  and after three



days, were only 20% of control times (180 minutes)  vs. 30 minutes



(P<0.001).  Acceleration of the rate of metabolism  of bishydroxy-



coumarin (BHC) also occurred after treatment with m-DCB.  After

-------
                                          V-34
                                       Table V-10

                 Effects of Chlorinated Benzenes on Aminol evul inic Acid
                  Synthetase  and Cytochrome P-450  Content of  Rat  Liver

        (Rimington and Ziegler,  1963;  Poland etaU, 1971; Ariyoshi et aU, 1975).
                           (Modified  from Ware  and  West,  1977)
Extent of
Conpound metabol ism
Control*
Monochlorobenzene
1 , 2-Dichlorobenzene
1 , 3-Dichl orobenzene
1 , 4-Dichlorobenzene
1,2, 3-Trichl orobenzene
1,2, 4-Trichl orobenzene
1,3, 5-Trichlorobenzene
1,2,3, 4-Tet rachl orobenzene
1,2,3, 5-Tetrachl orobenzene
1,2,4, 5-Tetrachl orobenz ene
Pentachlorobenzene
Hexachl orobenzene

High
High
High
High
High
High
Low
High
Low
Low
Very low
Very low
P-450
n moles/g
0.68
0.56
0.66
0.77
0.73
0.84
1.63
0.73
0.97
1.14
0.99
1.16
0.97
ALA
Synthetase
n moles/g/hr
22.6
49.2
36. 3+
29.8+
32.3
33.6
47.5+
34.8
33.3+
35. 4+
27.5
98. 7+
51.8
Prophyria1"

yes (1140)
yes ( 455)

yes ( 770)
yes ( 785)
yes ( 730)
—
yes ( 660)
—
-
-
yes ( 400)
*  Rats were pretreated orally with 250 mgAg body
   24 hours  after the last dose.

+  Significantly increased over control.
for three days and were sacrificec
   Numbers  in parentheses  indicate  dose  in mgAg body wt. used to produce porphyria
   in 5 days.   (Note:   glutathione  reduces porphyria produced by halogenated benzenes.)

-------
                          V-35
                       tor*
Fig. V-6. Effect of m-DCB on urinary coproporphyrin
          excretion.  Four.female rats  (90-120 g) were
          treated daily with 800 mg/kg m-DCB.  The
          24-hr urinary excretion of coproporhyrin
          (mean +_ S.D.) is plotted versus time.  The
          first dose was given on day 0, and the uri-
          nary coproporphyrin value for each day repre-
          sents a urinary collection for the previous
          24 hr.  Urinary coproporphyrin excretion was
          significantly lower on days 5 and 9  than on
          day 3.

Source:   Poland, e_t al_. (1971).

-------
                                V-36
                             Table V-ll

                 Effect of  m-Dichlorobenzene on ALA
                        Synthetase Activity*
                                      ALA synthetase (mmoles/g/hr)
Treatment             Day 1               Day 3              Day 5
Control            13.3 ± 4.4 (7)       18.8 + 4.6 (6)      16.1 + 4.8 (7)
m-DCB             52.9 + 15.3 (7)      40.5 + 6.8 (7)      30.5 + 6.2 (8)
*  Female rats were treated orally for 1,3 or 5 days with m-DCB (800 mgAg)
   or peanut oil  and sacrificed 24 hr after the last dose.  All values
   represent the  means ± S.D.  The number of animals in each group is given
   in parentheses.

   ALA synthetase activity is lower on day 5 in m-DCB-treated animals than
   on day 1 (P<0.01) or day 3 (P<0.02).

   Source:  Poland, et al. (1971)

-------
                                V-37
five days of dosing, the serum BHC levels measured  82  +_ 24  ug/ml,
as compared with control levels of 133 +_ 10 ug/ml  (P<0.025).
     Poland et. al_.  (1971) conducted further experiments  designed
to test the hypothesis that m-DCB stimulates  its own metabolism.
Liver and serum levels of m-DCB and 2,4-dichlorophenol  (DCP),  its
major metabolite, were determined after dosing daily for up to  five
days, as described  above.  The serum m-DCB concentration was higher
on Day 3 (8.89 +_ 1.61 ug/ml) than on Day 1 (3.25 +_  1.31  ug/ml), but
was significantly lower on Day 5 (5.91 +_ 2.02 ug/ml) than on Day 3
(P<0.02).  The hepatic concentration rose steeply from Day  1 (13.01
+ 6.92 ug/g tissue) to Day 3 (44.17 +_ 6.31 ug/g).   However, the dif-
ference between the concentration at Day 3 and 5 (32.1 ^ 16.18 ug/g)
was not quite statistically significant (0.10>P>0.05).   In  rats pre°
treated for four days with 40 mg/kg phenobarbital i,p.,  a known
inducer of drug metabolism, before receiving a single  800 mg/kg dose
of m-DCB, slightly lower concentrations of m-DCB in liver (8.49\+
2.37 ug/g) and serum (2.24 +_ 0.49 ug/g) were observed  as compared
with those in rats  receiving only a single dose of m-DCB.  The dif-
ferences were not significantly different, however.  The data from
this series of experiments add support to the hypothesis that m-DCB
does, in fact, stimulate its own metabolism.
     Ariyoshi et al_- (1975) studied changes in certain liver
constituents, cytochrome contents, activities of some  drug-metabo-
lizing enzymes and A-ALA synthetase in rats treated with each of
the three isomers of DCB.  Animals received oral doses of 250 mg/kg/
day for up to three days.  Activities of aminopyrine demethylase and
aniline hydroxylase were enhanced markedly by m-DCB, but cytochrome

-------
                                   V-38





content was not altered significantly after any of the three



isomers.   Delta-ALA synthetase activity was increased significantly



by treatment with o-, m- and p-DCB (63%, 32% and 42%, respectively).



However,  significant parallel changes in the cytochrome P-450



content did not occur.  All isomers increased microsomal protein



content of liver preparations.  Microsomal inorganic P content was



also increased by 36% after treatment with m-DCB.



     Carlson and Tardiff (1976) studied the effects of chlorobenzene,



1,4-dichlorobenzene, l-bromo-4-chlorobenzene, 1,2,4-trichlorobenzene



and hexachlorobenzene on adult male rats for 14 days at low doses



from 10 to 40 mg/kg body weight.  All halogenated benzenes except



monochlorobenzene decreased hexobarbital sleeping time immediately



and/or 14 days following treatment.  As can be seen in Table V-12,



cytochrome-c reductase, cytochrome P-450 content, 0-ethyl O-p-



nitrophenyl phenylphosphonothioate (EPN) detoxication, glucuronyl



transferase, benzpyrene hydroxylase and azoreductase were increased



to varying degrees.  Administration of 1,4-di- and 1,2,4-trichloro-



benzene for 90 days resulted in an increase in EPN detoxication,



benzpyrene hydroxylation and azoreductase.  The increases were still



apparent 30 days later.  1,2,4-Trichlorobenzene was the most potent



inducer of cytochrome reductase and cytochrome P-450.  Either no



change or a decrease was reported for glucose-6-phosphatase and



isocitrate dehydrogenase activities.



     These findings demonstrate the ability of halogenated aromatic



compounds at low doses to induce enzyme systems associated with the



metabolism of foreign compounds.  This type of action may influence



the metabolism of endogenous steroids, other foreign compounds and




drugs.

-------
                                                     Table V-

                      Effect of Chlorinated Benzenes Administered Orally for 14 Days on Various
                                         Parameters of Xenobiotic Metabolism

                                             (Carlson and Tardiff, 1976)
                                         (Modified from Ware and West,  1977)

Dose
Compound (mgAg/day)
Monochloro-
benzene


1,4-Dichloro-
benzene


l-Bromo-4-
chlorobenzene


1,2,4-Tri-
chlorobenzene


Hexachloro-
benzene


0
200
400
800
0
10
20
40
0
10
20
40
0
10
20
40
0
10
20
40
Cyrochrome c EPN1"
reductase Glucuronyl- detoxication Benzpyre
(nmol Cytochrome transf erase (pg hydroxy
cytocrorne c p-450 ( E/mg (nmol/min/mg p-nitrophenol/ (nmol/m
reduced/min protein x 10) protein) 50 mg/30 min) pro
mg protein)*
100 + 25
110 + 8
109 + 9
78+5
156 + 9
151 + 6
165 4- 8
176 + 10
149 -f 22
126 -f 6
123 + 4
141 + 5
103 4- 8
139 + 15
192 + 12
183 + 9
85+7
106 + 10
109 4- 7
100 + 7

236 + 20
209 + 10
197 4- 14
133 4- 15
178 + 20
174 + 15
167 + 11
193 + 13
205 + 35
180 4- 7
202 + 7
245 + 19
72 + 13
99 4- 18
212 + 45
268 4- 15
128 + 18
204 + 18
190 + 16
150 + 27

5.8 + 0.4
9.4 4- 0.6
12.2 4- 0.3
13.0 +_ 0.5
5.9 4- 0.6
7.8 4- 0.6
11.3 4- 1.0
9.6 +_ 0.6
5.8 4- 1.4.
5.6 + 0.8
9.0 + 0.7
9.1 + 0.5
5.0 + 0.4
5.1 + 0.4
8.6 4- 1.6
8.3 +_ 0.7
6.3 + 1.0
4.5 + 0.5
6.8 + 0.3
6.6 4- 0.8

6.7 + 0.6
7.1 4- 0.5
8.4 + 0.9
7.4 + 0.7
6.7 + 0.6
7.2 4- 0.6
9.5 4- 1.1
9.0 +_ 0.5
9.5 + 0.8
11.4 + 0.9
12.4 + 1.1
14.4 + 0.6
5.8 + 0.3
10.3 + 0.9
12.0 + 0.8
15.4 + 0.4
5.3 + 0.9
15.0 + 1.1
18.4 + 0.8
18.9 + 0.7

1.39 + 0.43
0.89 + 0.05
0.92 + 0.04
0.78 + 0.16
2.36 + 0.49
1.75 + 0.13
3.53 + 0.68
2.15 +; 0.34
1.51 + 0.20
1.37 + 0.17
2.06 + 0.26
2.35 +_ 0.23
2.82 + 0.53
3.82 + 0.65
5.37 + 1.01
5.22 ± 1.14
2.79 + 0.54
4.76 + 0.58
4.08 + 0.37
4.24 + 0.82
Azoreduc-
tase
(ng/min/mg
protein)





63.9 + 2.8
67.5 + 2.5
71.4 + 4.3
79.2 +_ 6.6
59.1 + 2.5
60.9 + 2.6
69.7 + 4.2
79.4 + 2.7
72.4 + 11.1
102.0 + 2.7
91.1 + 4.6
130.7 + 5.3
122.0 + 24.9
287.7 +- 7.4
210.0 + 7.4
262.7 + 19.1
* Value is mean + S.E. for group of six rats except for benzpyrene hydroxylase group receiving 40 mgAg of I,2f4-
  trichlorobenzeTie.  In that group, there were five rats,

-------
                                V-39
Multiple  Chemical  Exposures; Actions of Combinations



     Since  many compounds, whether they are agricultural  and



industrial  chemicals or drugs, are handled similarly by  the



cytochrome  P-450 system, there would be many possibilities



for additive,  synergistic as well as antagonistic  actions.



     Experiments to determine this should be carefully designed.



For example,  in manr phenobarbital has been shown  to stimulate  drug



metabolism.  This  effect requires several days to  reach  a maximum



rate (Goodman and  Oilman, 1975).  If treatment continues, it  appears



that the  marked stimulation is lost.  This has not been  taken into



consideration in many animal studies, i.e., only the three to four



day effect  has been studied.



     From recent studies in man using identical or fraternal



twins,  the  role of the environment as an explanation for  differences



in metabolism is being deemphasized in favor of genetic differences.



It has  been postulated that people who have genetic susceptibility



to indueible drug-metabolizing enzymes may be more prone  to adverse



effects,  if there is a toxic intermediate or product formed ir\  vivo



(Goujon et  al_. , 1972; Vesell et al_. , 1976).  Goujon et aL_. (1972)



found that  hexachlorobenzene differentially inhibits aryl hydrocar-



bon hydroxylase in genetically responsive and non-responsive mice.



Similarly,  Vesell  et al_- (1976) showed that the toxicity  of chloro-



form to the kidney is different in genetically susceptible and  non-




susceptible mice.



     Goujon et aO^. (1972) and Vesell et al_. (1976) demonstrated



genetically controlled variations in the susceptibility of mice to



hexachlorobenzene  and chloroform.  Hence, exposure to a wide  range of

-------
Multiple Chemical Exposures; Actions of Combinations
     Since many compounds, whether they are  agricultural  and
industrial chemicals or drugs, are handled similarly by  the
cytochrome P-450 system, there would be many  possibilities
for additive, synergistic as well as antagonistic  actions.
     Experiments to determine this should be  carefully designed.
For example, in man, phenobarbital has been  shown  to stimulate  drug
metabolism.  This effect requires several days  to  reach  a maximum
rate (Goodman and Oilman, 1975).  If treatment  continues, it  appears
that the marked stimulation is lost.  This has  not been  taken into
consideration in many animal studies, i.e.,  only the three to four
day effect has been studied.
     From recent studies in man using identical or fraternal
twins, the role of the environment as an explanation for  differences
in metabolism is being deemphasized in favor  of genetic differences.
It has been postulated that people who have  genetic susceptibility
to inducible drug-metabolizing enzymes may be more prone  to adverse
effects, if there is a toxic intermediate or  product formed in  vivo
(Goujon e_t al_. , 1972; Vesell et_ al_. , 1976).   Goujon et al_. (1972)
found that hexachlorobenzene differentially  inhibits aryl hydrocar-
bon hydroxylase in genetically responsive and non-responsive  mice.
Similarly, Vesell et al_. (1976) showed that  the toxicity  of chloro-
form to the kidney is different in genetically  susceptible and  non-
susceptible mice.
     Goujon €st aO_. (1972) and Vesell et al_.  (1976) demonstrated
genetically controlled variations in the susceptibility  of mice to
hexachlorobenzene and chloroform.  Hence, exposure to a  wide  range  of

-------
environmental  contaminants including the halogenated benzenes could

affect  the  ability of these animals to detoxify xenobiotic substances.

Synergistic effects of the halogenated benzenes in combination or

with other  environmental  contaminants could be especially damaging

for the genetically susceptible individual.

     There  was one example of synergistic effects of halogenated

benzenes on a target organism found in the literature (Hinzer et

al., 1970).  The antifungal activity of halogenated benzenes was

synergistic with organo-tin compounds.  The mechanism of the toxicity

was not discussed.  There were no studies found on synergistic effects

in mammals  or other nonmammalian species.


Teratogenicity

     No teratogenicity studies with the dichlorobenzenes alone

were found  in the peer-reviewed literature.  Studies to assess the

embryotoxic and teratogenic potential of o-DCB and p-DCB have been

initiated and/or completed under sponsorship of the chemical indus-

try.
     Hodge, ^t al_. (1977) conducted a teratogenicity study of p-DCB

in rats.  Groups of pregnant rats (32 animals per group) were

exposed to  atmospheric concentrations of 0, 75, 200 or 500 ppm

p-DCB 6 hours/day on Days 6-15 of pregnancy.  Data were collected

only from the first 20-24 animals in each group proven to be pregnant

at the  time of sacrifice (Day 21).  Nine animals littered sponta-

eously  on Day 21 and thus were not included in the study results

(two at 75  ppmf two at 200 ppm and five at 500 ppm).

-------
                                V-42
     Maternal  weight gain was monitored over the 21 days of
                         >
pregnancy.   Exposure to p-DCB did not alter the rate of weight gain

in any group when compared with controls.

     On Day 21,  the animals were sacrificed.  The intact uteri

were examined for numbers of fetuses and resorptions.  Corpora lutea

were counted.   Upon dissection of the uteri and removal of the fe-

tuses, the resorptions which had occurred were classified as early or

late.  Resorptions are designated as late when fetal tissues are

distinguishable.  When abnormal fetuses were noted, maternal heart,

liver, lung, uterus, ovary, kidney, and adrenal were preserved for

histological examination.  Liver and lung from at least ten animals/

group also were fixed for histology.

     Each fetus was examined for viability, sex, weight and

presence of abnormalities.  Half of each litter was eviscerated,

examined for abdominal abnormalities and stained with Alizarin Red

for subsequent skeletal examination for abnormalities and degree of

ossification.

     Upon gross examination, uteri from three females exposed to 75

ppm contained excessive amounts of blood.  In one case, this appeared

to be associated with a dead fetus.  One female exposed to 200 ppm

had inflated lungs.  Upon histological examination, no lesions were

observed which were attributable to exposure to p-DCB.

     Exposure to p-DCB did not induce adverse effects on numbers

of implantations, viable fetuses, resorptions, corpora lutea or on

mean fetal  weights, mean litter weights or on implantation efficiency

(number of implantations/number of corpora lutea).  Sex ratios (male/

female) were within normal limits.  There was no increase in the

-------
                                V-43
number of runts.



     Three gross  fetal abnormalities were noted, one  in  each  exper-



imental group.  From the 75 ppm group, there was one  fetus with



gastroschisis and malrotation of the left hindlimb.   In  the 200 ppm



group, there was  a single fetus with gastroschisis and*malratation



of the right hindlimb, and in the 200 ppm group, there was one fetus



with agnathia and cleft palate.  One control fetus was found  to be



anemic.
                                i


     Upon examination for skeletal abnormalities, no  evidence was



found that maternal exposure to p-DCB resulted  in delayed ossifi-



cation of fetal bones or an increased incidence of minor abnormali-



ties.  Occasionally, 14 ribs were noted, but, in almost  all cases,



these were vestigial.



     The results  of this study suggest that maternal  exposure to



atmospheric levels of p-DCB up to 500 ppm on Days 6-15 of pregnancy



does not result in any embryotoxic, fetotoxic or teratogenic effects



in the offspring.



     Dow Chemical is completing studies in which pregnant rats



and rabbits are being exposed to o- and p-DCB via inhalation.



Final reports are not yet available to ODW.  However, when they



are, they will be subjected to review and evaluation.  Dow Chemical,



however, has submitted a review of the results of the dose range-



finding study to  determine the maximum tolerated dose of o-DCB for



the full study, as well as the final protocol for that study  (Dow,



1981).  In the probe study, groups of 10 pregnant rats and seven



pregnant rabbits  were exposed to nominal o-DCB concentrations of



0, 200, 400, or 500 ppm ( 0,^1200,^2400 or"* 3000 mg/m3).  The

-------
                                V-44
animals were exposed six hours/day on Days 6-15  (rats) or Days



6-18 (rabbits) of gestation.




     Severe maternal toxicity, as evidenced by significant decreases



in body weight, body weight gain and food consumption, increases  in



relative liver and kidney weights and signs of systemic toxicity



at gross necropsy, was observed in pregnant rats exposed to 500 ppm



of o-DCB.  Embryolethality, secondary to maternal toxicity, was ob-



served among rats in the 500 ppm exposure group.  Increased relative



liver and kidney weights and decreased food consumption were observed



among pregnant rats exposed to 400 ppm of o-DCB.  Exposure to 200



ppm of o-DCB did not produce any sign of toxicity among maternal



animals.  No statistically significant effects on reproductive



parameters were observed among rats at any exposure level.



     Among rabbits, slight maternal toxicity was observed among



pregnant animals exposed to 500 ppm of o-DCB.  Non-significant de-



creases in maternal body weight gain, and absolute and relative liver




weights were observed among rabbits exposed to 500 ppm.  Gross



observation at necropsy revealed hepatic changes indicative of mild



toxicity among pregnant rabbits exposed to 500 ppm.  No adverse ef-



fects were observed among rabbits exposed to 200 or 400 ppm of o-DCB,



and no significant effects on reproductive parameters were observed



among rabbits at any exposure level.



     Based on the results of the probe study, as summarized



above, exposure concentrations of 0, 100, 200 or 400 ppm o-DCB were



chosen as the test concentrations for the definitive teratology study




in rats and rabbits.

-------
                                V — >±O
     Recently, Dow Chemical submitted to EPA the results of the



dose range-finding study for p-DCB exposure to pregnant rabbits



(Hayes,  e_t al_.,  1982).  Pregnant females were exposed to 0, 300,



600 or 1,000 ppm (0,  1,800, 3,600 or 6,000 mg/m3).  Each was ex-



posed for 6 hrs/day on Days 6-18 of gestation.



     No maternal deaths occurred during the study and no signi-



ficant changes in gross appearance or demeanor were observed among p-



DCB-exposed rabbits.  Evidence of slight maternal toxicity was ob-



served among pregnant rabbits exposed to 1,000 ppm of DCB. 'In this



group, a decrease in body weight gain and slight decreases in absolute



and relative liver weights were observed.  In addition, histopatho-



logic examination of livers revealed decreased hepatocellular vacuoli-



zation suggestive of decreased glycogen deposition in the 1,000 ppm



group.



     No significant effects on the incidence of implantations



undergoing resorption were observed in any of the exposed groups



when compared to controls indicating that p-DCB is not embryolethal



at exposure concentrations up to 1,000 ppm.



     Based on the. results of this probe study, where evidence of



slight maternal  toxicity was observed in the 1,000 ppm group, exposure



levels of 100, 300 and 800 ppm of p-DCB were selected for the defini-



tive teratology study in rabbits.

-------
                                 V-46
Mutagenicity


   Effects on Plants


     Abnormal mitotic division  of  the  onion,  Allium cepa,  after


treatment with several halogenated benzenes has  been described by


Ostergran and Levan (1943).  Ortho-DCB produced  full  c-mitosis abnor-


malities at 300 x 10~6 mol concentration  with partial  disturbances of


mitotic division at 100 x  10~6  inol.


     Para-dichlorobenzene  also  induces abnormal  mitotic divi-


sion in higher plants (Ostergran and Levan, 1943).   Effects seen


include shortening and thickening  of chromosomes, precocious separ-


ation of chromatids, tetraploid cells,  binucleate cells and chromosome
                             /
bridges (c-mitosis).  Available studies with  p-DCB  are summarized


in Table V-13.


   Effects on Microorganisms


     Anderson _et al_. (1972) evaluated  110 herbicides for their


ability to produce point mutations  in  a number of different microbial


systems.  When tested in a culture of  histidine-requiring mutants


of Salmonella typhimurium, both o-DCB  and trichlorobenzene (isomer


not specified) gave a negative  response,  i.e., they were not


mutagenic.  Pentachlorophenol,  a metabolite of pentachlorobenzene,


also was negative in this test  system.  The metabolites of other


halogenated benzenes were not evaluated.  No liver  homogenates


containing the metabolic activating enzymes were added in order to


study the effect of conversion  of  the  test compounds to active


intermediates.

     Prasad and Pramer (1968) and  Prasad  (1970)  investigated the


mutagenic effects of the three  dichlorobenzene isomers.  The
                   »>

chemicals were evaluated for frequency of back mutation of the

-------
                                                         V-47

                                                      Table V-13

                                    Effects of p-DCB on Mitotic Division of Hants

                                         (Modified after Ware and West, 1977)
)rganism
Treatment
Fragments
Persistence
Of
Fragments
Remarks
Reference
\Lliurft
Six species
of monocoty-
ledon angio-
sperms-root
tips
Nine species
of dicotyle-
don angio-
spe rate-root
0.05, 0.1, 0.25,
0.5, 1.5 g for
five days to
seeds in Petri
dish

same doses to
four-day old
seedlings for
four hours
 4  1/2 hour soak
 in sat.  soln.
 1-4  hours
 depending on
 plant
No signs of
mitosis at
three highest
doses
Polyploidy in
root cells at
0.5 and 1.5 g
Abnormal chromo-
some numbers;
lagging chromo-
somes and dumb
bell shaped
nuclei also
occasionally seen

Frequency of frag-
ments at metaphase
tended to decrease.
Frequency high-24,
48 hrs. and de-
creased at 72, 96
hrs. 2 plants still
highly fragmented at
96 hrs. 1 complete
recovery.

2 plants recovered
before 96 hrs. 1
plant died after 48
Carey and McDonough,
1943
Sharma and Rattacharya,
1956
Sharma and Bhattacharya,
1956

-------
                                                 Table V-13 (Continued)
Organism
 Treatment
Fragments
Persistence
    Of
 Fragments
  Remarks
Reference
Nothoscordum
fragrana Kunth

Root tips
Root tips
Pollen
 (Flower  Buds)
Three species
of yicieae-
root tips
3 1/2 hrs.
soak in sat.
soln.
6 hrs. sat.
6 hours
 (3 1/2 hrs.
 sat.  soln.)
                  (6 hrs. sat,
                  sol.)
 4-6  hours  in
 soln.
 Fenugreek seeds  4-24 hours soak
                  in sat. soln.
                  not indicated
                  not indicated
                  not indicated
Erosion and frag-
mentation of chromo-
somes both at meta-
phase and anaphase

Erosion and stick-
iness increase in
fragments from 3
1/2 hour soak.

No fragmentation,
some diplochromo-
somes.  Indication
of slight disturbance
in spindle mechanism
and failure in sepa-
ration.

(Meiotic division -
only stickiness of
chromosomes noted.)

(Lagging, non-dis-
junction and stick-
iness of chromosomes.
No fragments.)

c-Mitosis abnormali-
ties.  Breaks associ-
ated with heterochro-
matic chromosome reg-
ions

No change in morph-
ology or cytology of
seed! inas
                                        Sjparma and Sarkar,
                                        1057
                                        Sjjfiarma and Sarkar,
                                                          S^iarma and Sarkar,
                                                          SJiarma and  Sarkar,
                                                          1957
                                                                              Sharma and Sarkar,
                                                                              1957
                                       Srivastava,  1966
                                                                              Qupta,  1972

-------
                                                Table V-13  (Continued)
Organism
     Treatment
Fragments
Persistence
    Of
 Fragments
Remarks
Reference
Fenugreek
seedlings
4 hours "exposure"
 (corn)
Greater than 4
hours (not speci-
fied)

3 hour soak of
0.5 cm long seed-
lings in saturated
aqueous solution,
then growth allowed
for 7 more days
Lens  escu-    4-48 hour soak of
lenta micro-  root tips in saturated
sperma        aqueous solution

              4-48 hour exposure
              of germinating
              seeds to vapors of
              25,  50, 100, 250,
              500, 800-1,000 mg
              of p-DCB crystals

              4-48 hour soak of
              germinating root
              tips in saturated
              aqueous solution
                  not            Root tip chromo-
                  indicated      somes contracted
                                 and arrested at
                                 metaphase

                                 After "longer"
                                 treatment,  mitosis
                                 appeared normal

                                 Accelerated root
                                 growth,  cell divi-
                                 sion; polypi oidy,
                                 formation of chromo-
                                 somal bridges and
                                 laggards at anaphase.
                                 Polarity of cell
                                 changed  to  an angle
                                 of 90° from normal.

                                 Germiability and
                                 growth inversely
                                 proportional to
                                 level and duration
                                 of exposure.  Mitotic
                                 and chromosomal
                                 anomalies observed:
                                 chromosome  contrac-
                                 tion and condensa-
                                 tion, fragmentation,
                                 bridges, tetraploidy,
                                 binucleate  cells
                                       Gupta, 1972
                                                                                              Gupta, 1972
                                       Sharma and Agarwal,
                                       1980
                                                                                 Sarbhoy,  1980.

-------
                                V-50
methionine-requiring (meth3) locus in the fungus Aspergillus
nidulans.  The mutagenicity of the dichlorobenzenes increased
in the following order: o-DCB-5/106 spores, m-DCB-9/106 spores
and p-DCB-11/106 spores.
     Anderson (1976) reported on a study with p-DCB designed
estimate the .mutagenic potential of this substance in the Salmonella
typhitnurium plate incorporation assay.  Mutant strains TA 1535, TA
1538, TA 98 and TA 100 were exposed to varying concentrations of
p-DCB dissolved in DMSO in two separate studies.  In the first
study, concentrations of 100, 500 and 2500 ug/plate were used.  The
experiment was run three times.  In the second study,  concentrations
of 4 and 20 ug/plate were used in addition to the three used in the
first study.  This experiment was run five times.  Exposure to
p-DCB occurred both with and without metabolic activation with S-9
mix from Alodor-treated rats.  In another series of experiments,
the tester strains were exposed to atmospheric concentrations of
p-DCB at levels of 94, 299 or 682 ppm, again with and without
metabolic activation.  This protocol was employed four separate
times.
     A greater than two-fold increase in the number of rever-
tants is the criterion by which a compound is considered to be muta-
genic in this assay system.  After atmospheric exposure to p-DCB, no
significant increases were noted in any tester strains, except in one
of the four exposures to 682 ppm in TA 1535 with metabolic activation.
This increase was not observed in three follow-up experiments.   In
the first series of experiments in which p-DCB was dissolved  in  DMSO
at three concentrations, the number of revertants in TA 1535  increased

-------
nearly  10-fold  at  500 ug/plate, with metabolic activation.  This  in-



crease  occurred in two of the three runs.  This increase was  not  ob-



served  in  the subsequent series of five runs employing five doses



including  the previously-described three.  In spite of the few posi-



tive results, the  data from all of the experiments together would sug-



gest that  p-.D.CB is not mutagenic to tester strains used in this Sal-



monella typhimurium plate incorporation assay system, either  when



dissolved  in DMSO  or in the gaseous phase.



     More  recently, Simmon et al. (1979) examined all three dichloro-



benzene isomers for mutagenic activity in both the standard Ames/



Salmonella assay and the ]2. col i WP2 system.  In the Ames assay,



tester  strains  TA  1535, 1537, 1538, 98 and 100 were employed, with



and without metabolic activation.  In the first of two experiments,



concentrations  of  0.05-1.0 ul/plate (0.065-1.3 mg/plate) of o- or



m-DCB were added to each Salmonell a strain or IS. col i culture.  No



reproducible dose-related increases in the number of revertants were



observed in either system.  In a second experiment, each compound was



retested at lower  levels, ranging from 0.0005 to 0.5 ul/plate.  Again,



no significant  changes were noted.  Para-DCB was tested in the same



manner, but at  higher dose levels:  50-1,000 ul/plate in the  first



experiment and  0.5-500 ul/plate in the second.  Again, no increases



in the  number of revertants were observed.



     Simmon, et al. (1979) also conducted tests for chromosomal



aberrations in  yeast.  All three isomers were tested for their poten-



tial  to induce  mitotic gene conversion and reciprocal recombination



in Saccharomyces cerevisiae C3, with and without metabolic activa-



tion.   At  doses ranging from 0.001-0.25%, o-DCB produced no effects.

-------
                                V-52
Toxicity was  observed at 0.05%, with and without activation.



Meta-DCB was  tested at doses ranging from 0.005 to 0.1% in two



experiments.   In both, but only after activation, enhancement of the



recombination response was observed.  Para-DCB, at dose levels rang-



ing from 0.005-5% in the first three experiments, appeared to increase



mitotic recombination, with a considerable variation in survival. In



two later  experiments, toxicity occurred, but no increase in recombi-



nation was observed.  The inconsistency of the results may be attri-



buted to the  relative insolubility of the compound.



     The differential toxicity of each of the three isomers was



evaluated  in  the DNA repair-proficient and repair-deficient strains



of E. coli (W3110 polA+/p3478 polA~) and Bacillus subtil is (H17 recV



M45 rec~)  (Simmon,  _et: al_., 1979).  In three of four experiments, 20



ug/plate o-DCB was  more toxic to the repair-deficient jE. coli (polA~)



than to  the repair-proficient strain (polA+).  There was no apparent



difference in toxicity to either strain of !3. subtil is.  At the same



concentration (20 ul/plate), m-DCB was more toxic to the repair-



deficient  strain of E. coli than to the polA+ strain in four of five



experiments.   Para-DCB, at concentrations of 1 or 5 mg/plate, had no



effect on  any of the four strains.  This result was interpreted to



mean that  either the compound was truly nontoxic under these test



conditions or that  the substance was unable to diffuse away from the



impregnated filter  paper disc in the culture dish.



     In summary, the results of these studies indicate that the



dichlorobenzenes do possess mutagencity activity in certain of the



test systems.  None were positive in the Ames/Salmonella assay system



or in the  E.  coli WP2 assay.  However, m-DCB, both with and without

-------
                                V-53
metabolic activ-atrion, increased mitotic recombination  in S_. cerevisiae.



The results with p-DCB were ambiguous.  Both o- and m-DCB were shown



to interact with and damage bacterial DNA in the E. coli W3110 polA+/



p3478 polA" differential toxicity assay system.





Effects on Animals



     There has been at least one in vitro study of the inhibitory



effect of dichlorobenzene (isomer not specified) on the number of mi-



toses in rat lung cell cultures (Guerin, _et al_., 1971).  The dose of



5 ug did not produce any significantly different number of mitoses



than the control.  In this test system, dichlorobenzene gave a nega-



tive result: it did not exert any inhibitory action on the cultures.



     Cytogenetic studies have been conducted on rat bone marrow



cells following inhalation exposures to p-DCB  (Anderson and Richard-



son, 1976).  Three series of exposures were carried out:  1) one ex-



posure at 299 or 682 ppm for two hours, 2) multiple exposures at 75



or 500 ppm, five hours/day for five days and 3) multiple exposures to



75 or 500 ppm, five hours/day, five days/week  for three months.  Ben-



zene (at 10, 750 or 7,500 ppra) was used as the positive control in



the first experiment.  Vinyl chloride (at 1,500 ppm) was used as the



positive control in the other two experiments.  Negative controls



breathed fresh air alone.  In Experiment 1, three rats were in each



treatment group, four in the negative control  group.  In the other



two experiments, there were two rats in each treatment group and two



rats in the negative control group.  In Experiment 1, 50 cells from



each animal were examined; in Experiments 2 and 3, 100 cells from



each animal were examined.

-------
                                 V-54






    Animals were  sacrificed 22 hours after termination of exposure,



following one  hour after an intraperitoneal dose of colchicine.



Bone marrow cells  from both femurs  were stained with Giemsa



and surveyed for chromosome or chromatid gaps, chromatid breaks,



fragments or other complex abnormalities.  In all three experiments,



exposure to p-DCB  failed to induce any statistically significant



effects indicative of chromosomal damage when compared to the nega-



tive controls,  whereas in all experiments, the positive controls



did produce damage at all levels employed.  Also, there was no evi-



dence  of a dose-response relationship to p-DCB exposure, whereas,



there  was with benzene in Experiment 1.  Thus, under the conditions



of these experiments, p-DCB does not appear to elicit chromosomal



damage.



    Para-dichlorobenzene also was tested in a dominant lethal



study  in the CD-I  mouse (Anderson and Hodge, 1976).  Fertile males



were exposed,  by inhalation, to plain air or to one of three levels



of p-DCB, or,  by other routes of exposure, to one of three sub-



stances known  to produce dominant lethal effects in this assay



system.  Groups of males were subdivided into the following experi-



mental groups.   Numbers of animals in each group are shown in




parentheses.

-------
                                   V-55




          Group 1:  Air (negative control)-(35)

          Group 2:  75 ppm p-DCB, 6 hr/day for 5 days  (16)

          Group 3:  225 ppm p-DCB, 6 hr/day for 5 days  (16)

          Group 4:  450 ppm p-DCB, 6 hr/day for 5 days  (16)

          Group 5:  200 mg cyclophos^h.auiLide./k.g, once by i.p.
                   injection on Day 5  (13)

          Group 6:  150 mg ethyl methanesulfone/kg,
                   orally once a day for 5 days (5)

          Group 7:  2.5 mg nitrogen mustard/kg,
                   once by i.p. injection on Day 5


     The mating protocol consisted of  placing two virgin females

in a cage containing one fertile male.  Five days later the females

were removed to a separate cage.  Two  days later, the male was

caged with two different virgin females.  This process was repeated

until the males had been mated at weekly intervals eight times.

Females were sacrificed 13 days after  the assumed date of fertili-

zation, or 15-16 days after caging with the male.  Uteri of these

females were examined for live implants, early and late deaths.

Statistically, the data were analyzed  by a simple Chi-square and a

week by week hierarchical analysis of  variance.

     Only one death occurred among males exposed to p-DCB.  This

was a male in the 75 ppm group during  Week 3.  The investigators

suggested that the death was unrelated to p-DCB exposure.  Five

males in the cyclophosamide positive control group died early,

three during Week 6 and one each in Weeks 7 and 8.

     Frequency of mating was affected  in only two of the groups.

At Week 7, in the 77 ppm-dosed males,  the frequency of mating was

100%, but significantly fewer males mated with both females (P<0.05).

-------
                                  V-56
     The  number and percentage of females becoming pregnant was

decreased significantly at Weeks 6 and 7 among those mated to

males exposed  to 75 ppm (P<0.05).  The mean total of implants per

pregnant  female in each group also was determined.  The ratio was

significantly  reduced in the 75 and 450 ppm groups at Week ff" when

compared  with  the negative control.  All positive controls showed

significant reductions at Week 1 (P<0.01), cyclophosaminde and

nitrogen  mustard at Week 2 (P<0.01) and ethyl methanesulfone at
                                   •
Week 8 (P<0.05) when compared with the negative controls.

     Early fetal deaths were analysed in three ways.  When

determining the number of females with at least one early death,

groups exposed to 225 ppm p-DCB showed an increase at Week 1

(P<0.05).  All positive controls showed significant differences at

Weeks 1 and 2  (P<0.001 or 0.05).  When comparing the mean number of

early fetal deaths/pregnancy, there were no significant differences

in any of the  p-DCB-exposed groups.  All positive control groups

exhibited differences in Week 1 and 2, with cyclophosamide also in

Week 3 (P<0.05 or less).

     Comparison of mean percentages of early deaths per total

implants  per pregnancy revealed significant differences in p-DCB

treated groups in Week 6 in the 225 ppm group (P<0.05).  Values

for the 75 ppm group in Week 1 were higher than the negative controls,

but'not significantly by Dunnett's "t" test.  No significant

differences were seen in late fetal deaths for any groups.

     Utilizing the three methods of analysis, two indicated

significant differences between p-DCB-exposed groups and negative

controls.  However, these changes were different at different times

-------
                                V-57
and they did not occur in a dose-related manner.  Therefore, the
authors suggested that these changes were not biologically signifi-
cant.  In addition, they concluded that p-DCB, at least at the
exposure levels tested, does not cause dominant lethal mutations in
germ cells, of CD-I mice.

Carcinogenicity
     Few studies have been reported which address the carcinogenic
potential of the dichlorobenzenes.  Some of these are inadequate
for judging this characteristic.
     A study by Parsons (1942) gave somewhat inconclusive
results on the carcinogenic effects of p-DCB in mice.  One group of
mice was irradiated with an unspecified source of radiation, then
given 0.2% intraperitoneal p-DCB in sesame oil in silica.  One
animal showed ascites and a sarcoma-like growth.  This animal tumor,
when grafted, gave 100% takes.  In "control" mice, i.e., those that
were not irradiated, one animal developed a sarcoma.
     Murphy and Sturm (1943) studied the effects of p-dichlo-
robenzene on induced resistance to a transplanted leukemia in the
rat.  Forty rats were immunized by intraperitoneal injection of
either defibrinated rat blood., or chopped 15 day old rat embryo.
They were exposed to saturated p-dichlorobenzene vapors for two to
three hours daily for 14 days, and then injected with 0.2 cc of
leukemia cells.
     Of the 40 animals in the immunized group exposed to p-dichloro-
benzene, 67.5% had tumors.  An immunized group not exposed to p-DCB
recorded 20.5% tumors while the control (no immunization) was 84.2%
positive.  It would appear that p-dichlorobenzene modified the

-------
                                V-58
induced resistance of the rats to the leukemia.  However, there is



not sufficient evidence to state that there are possible immuno-



suppressant effects resulting from exposure to p-dichlorobenzene.



     In the two studies by Holl ingsworth, _et al_. (1956, 1958)



described earlier, the investigators did a cursory survey for tumor



occurrence during the subchronic exposure to rats, rabbits and



guinea pigs.  No tumors were reported in any species after exposure



to either substance (o- and p-DCB).  Little confidence can be



placed in these results as the conditions of study were inadequate



for evaluating carcinogenic or mutagenic potential.



     Guerin and Curzin (1961) found that dichlorobenzene (iso-



mer not specified) gave a slight response for carcinogenic activity



in mice, as measured by the sebaceous gland and hyperplasia tests.



In both cases, dichlorobenzene (1 g/100 cc solution in acetone)



was applied to the skin of Swiss mice three times (0.1 cc solution).



The sebaceous gland test was based upon the disappearance of the



glands after application of the test compound.  The hyperplasia



test examined the thickening of the skin epithelium after applica-



tion.  On an arbitrary scale of 0 to 4, (negative to strongly  .



positive), dichlorobenzene scored 0.9 on the sebaceous gland test



and 0.7 on the hyperplasia test.



     The National Toxicology Program (NTP) recently completed



carcinogenicity bioassays in two species of rodents (rat and mouse)



for ortho- and para-dichlorobenzene.  The results of the chronic



gavage studies on the ortho isomer were presented to the NTP Board



of Scientific Counselors in a draft report last year (NTP, 1982).



However, the Board has not approved the report as yet.  Nonetheless,

-------
                                V-59
thre: .results are summarized below.  The chronic gavage studies on



p-dichlorobenzene are complete.  The results of these latter studies



on p-DCB may be presented soon.  The protocols for the chronic



studies are presented below.  In addition, the results of the



studies conducted in support of the chronic experiments with both



isomers also are discussed.



     Two 14-day repeated dose studies with o-DCB were conducted in



B6C3F1 mice as part of the prechronic test phase of the NTP bioassay



on this substance (Battelle Columbus, 1978a, 1978b).  These studies



were designed to determine approximate doses for the three month



subchronic toxicity study.  No acute toxicity study was conducted.



In the first study, gavage doses of 0, 250, 500, 1,000, 2,000 or



4,000 mg/kg in corn oil were administered daily to six groups of



five mice of each sex (Battelle Columbus, 1978a).  By the end of



the study, 47 of the 60 mice had died, leaving 5 male and 8 female



survivors.  All mice in the top two dose groups died by Day 3.  One



female mouse in the 1,000 mg/kg group survived to the end of the



experiment.  The same was true for the 500 mg/kg group.  One male



and one female survived from the 250 mg/kg group.  In addition, one



male mouse in the control group died on Day 4.



     At gross observation, early death mice had pale livers,



beginning as early as Day 2 in the 4,000 mg/kg group and seen by



Day 7 in the 250 mg/kg females.  Other gross lesions observed in



all dose groups included reddened lung areas and yellow-green or



red small intestines.  Liver histopathology in the six male mice



receiving histopathological examination was characterized by mod-



erate to severe centrolobular necrosis at the 500 mg/kg level

-------
                                V-60
and milder (in one) or none (in two) at the 250 mg/kg level.  At


500 mg/kg, one mouse exhibited lymphoid necrosis of the spleen,


and the other animal, marked lymphoid depletion of the thymus.


Of the seven females receiving histopathological examination, the


one at 1,000 mg/kg surviving to the end of the study showed no


pathology.  Of the three examined from the 500 mg/kg group, one


of two early fatalities exhibited no pathology while the other had


moderate lymphoid depletion of the spleen.  The one survivor had no


lesions.  At 250 mg/kg, one early fatality exhibited mild centro-


lobular hepatic necrosis, the other, no histopathology.  The one


survivor examined showed no pathology.


     Since no no-effect dose level was determined from this


initial experiment, it was decided that a follow-up study would

                                                                i
be conducted, using a range of doses lower than that employed in


the first experiment.  The protocol was identical to that of the


first study; however, the doses selected were 0, 30, 60, 125, 250


or 500 mg/kg in corn oil (Battelle Columbus, 1978b).  In this


study, only two early deaths occurred:  one male in the 500 mg/kg


group on Day 3 and one female in the 125 mg/kg group Day 8.


Body weight changes in the treated animals were not markedly dif-


ferent from the controls, although males in the 30 and 60 mg/kg


groups showed a 9% lag (a 6% increase vs. a 15.7% increase in the


controls).  During the first three days of the study, all mice in


the highest dose group exhibited labored breathing, rough coats and


watery eyes.  The signs then disappeared.  Gross examination was


conducted on all animals.  The male mouse dying on Day 3 had a pale,


mottled liver, enlarged stomach and reddened small intestine.

-------
                               V-61
His-tological  examination of livers from four males and four fe-



males from the 500 mg/kg dose group revealed no changes in



two males, mild focal necrosis in one, and mild focal necrosis



as well  as cytomegaly, karyomegaly and chronic moderate multi-



focal granulomatous hepatitis in the fourth male.  Among the



four females, one exhibited moderate focal necrosis, another showed



mild focal necrosis, and the other two had mild centrolobular



degeneration with cyto- and karyomegaly.  These changes were judged



to be treatment-related.  On the basis of the results of this



second study, it was recommended that the subchronic study in



the mouse be performed at the same dose levels used in this



14-day study.



     The subchronic toxicity gavage study with o-DCB in mice was



conducted to assist in dosage selection for the 104-week chronic



study (Battelle Columbus, 1978c).  The doses used were as detailed



above (0, 30, 60, 125, 250 or 500 mg/kg/day).  Treatment groups



consisted of five animals of each sex.  Single gavage doses of the comp-



ound in corn oil were administered 5 days/week for 13 weeks.  Weekly



individual body weights and cage food consumption rates were moni-



tored.  Animals were sacrificed on Day 92 or 93, with full necrospy,



recording of organ weights and histological examination of various



tissues.  Special studies also were performed near or at the end of



the exposure period.  These included:  urinalysis in the highest



dose group and controls, hematology and clinical chemistry.  Organ/



body weight ratios were calculated and urine and liver porphy-



rin determinations made.



     During the study, lethargy and rough coats were observed

-------
in both sexes at the four highest doses.  However, by the final



week on test, only animals of both sexes at the 500 mg/kg dose



and males at 250 mg/kg showed these signs.  Body weight gain was



affected in males at 500 and 250 mg/kg  (-49% and -18%, respectively,



when compared with controls).  Female mice receiving 500 mg/kg/day



exhibited a differential weight gain of -62% when compared with



controls.  No other groups showed differential weight gains of



greater or less than 10% when compared with controls.



     Hematological parameters evaluated included hemoglobin,



hematocrit, total and differential white counts, red cell and



platelet counts, mean corpuscular volume and reticulocyte counts.



The investigators did not perform any statistical analyses on



these data, but claimed that no clinically-significant treatment-



related changes occurred.  The apparent differences in white cell



counts of treated males when compared with control was attributed



to relatively low counts among the controls.  It was suggested



that these counts were below those typically observed in that



laboratory and others (3.4 x 103/mm3 in controls vs. 5.4-6.6 x 103/



mm3 in the treated groups).  Since the individual data were not



available, the statistics cannot be done which would show whether



or not there were significant differences between the controls and



and the treated groups.  In addition, information on the "normal"



counts is' not available for evaluation.  But, since there is at



least anectodal evidence of a possible relationship between ex-



posure to ortho-dichlorobenzene and leukemias in humans, it would



be prudent to evaluate these results in greater depth.

-------
     Blood  samples were analyzed for alkaline phosphatase, SGPT and



gamma-glutamyl-transpeptidase (GGTP).  No GGTP was detected in any



sample.   Statistically significant dose-dependent changes in alkaline



phosphatase did  not occur,  although increased levels were noted in



males receiving  125 and 250 mg/kg/day.  SGPT levels in the two



surviving males  receiving 500 mg/kg/day were increased significantly



over control,  due to the high value recorded in the animal exhibiting



the hepatocellular necrosis.  The other animal showed no hepatic



histopathology.



     The following parameters were monitored during the uri-



nal ysis:  pH,  glucose, protein, bilirubin, ketones, occult blood,



specific gravity and creatinine.  The report stated that the



volume of urine  collected from treated animals, especially the



males, was  generally greater than that collected from controls.



The individual data were not available to corroborate this con-



clusion.  No record of fluid intake was kept during the study.



Decreases in specific gravity and creatinine were noted, reflect-



ing dilution due to increased urine output.  No other compound-



related effects  were observed.



     Uroporphyrin levels in treated males were generally



higher than in the controls.  In females, coproporphyrin levels were



higher in the treated animals than in controls.  Coproporphyrin levels



in treated  males and uroporphyrin levels in females were not dif-



ferent from controls.  Sex differences were seen in liver protopor-



phyrin levels, as a dose-dependent increase was observed in the



females  but not  in the males.

-------
                                V-64
     Organs weighed were: heart, lung,  kidney,  testis,  spleen,
thymus, brain, ovary and uterus.  While no statistics were done,
it appeared that relative liver weights were  increased  in the
highest dose males and females.  An  increase  of lesser  magnitude
was seen in females receiving 250" mg/kg/day.  No other  differences
were noted.
     All of the above-mentioned tissues and skeletal muscle from
control and highest dose animals were examined microscopically.
The liver, thymus, heart, spleen and thigh muscle were  examined
from animals in the 250 mg/kg group  and livers only from the 125
mg/kg group.  Lesions were observed  in  all tissues from the highest
dose animals.  These were considered to be treatment-related, since
they were absent or occurred less frequently  in the controls.
     Livers of the highest dose animals exhibited significant
centrolobular necrosis, hepatocellular  necrosis and degeneration,
and deposition of yellow-green to golden pigment considered to be
hemosiderin.  The heart showed multiple foci  of mineralization in
the myocardial fibers.  Skeletal muscle also  exhibited  mineraliza-
tion as well as necrosis and myositis.   Some  animals had lymphoid
depletion of the spleen and thymus.  One female exhibited lymphocyte
necrosis in the spleen.
     Among the animals receiving 250 mg/kg, only hepatocellu-
lar necrosis was noted in two males, with pigment deposition in
one male and hepatocellular degeneration in one male.   No lesions
were noted in the group treated with 125 mg/kg.
     The results of this study suggest  that an oral no-effect
level  can be identified in mice over 13-week  exposure period of
125 mg/kg/day.  Doses of 60 and 120 mg/kg/day were selected for use

-------
                                V-65
in the 104-week chronic study.



     A draft NTP Technical Report on the bioassay of ortho-DCB



in mice and rats was presented to the NTP Board of Scientific



Counselors on September 22, 1982.  This document does not become a



final report until it is reviewed and approved by the Technical



Reports Review Subcommittee of the NTP Board of Scientific Counsel-



ors.  Therefore, the text that follows represents only a preliminary



assessment of the data from the bioassay.



     The bioassay was conducted by administering 60 or 120 mg/kg



doses of o-DCB in corn oil, five days/week for 104 weeks.  Groups



of 50 males and 50 females comprised each dosage group and a vehicle



control group, as well.  The control group received equivalent vol-



umes of corn oil on the same schedule as the treated animals.



     No difference in survival rates were observed between the



treated and control groups of either sex.  There was no evidence



of compound-related nonneoplastic liver lesions, suggesting that a



higher dose may have been tolerated in the chronic study.



     Statistically significant positive trends in the incidence



of malignant histiocytic lymphomas occurred in mice of both sexes



(P<0.05).  However, the incidence of total lymphomas of all cell



types was not significantly increased above control.  The draft



reports states that "since the histiocytic lymphoma is a controversial



diagnosis among different pathologists and since all types of



lymphomas have the same histiogenesis, an increase in this specific



type of lyraphoma in the absence of an increase in the total incidence



of all types of lymphoma is not considered to be biologically



significant."

-------
                                V-66
     An increase in alveolar/bronchiolar carcinomas were ob-



served in male mice (control=4/5, 8% low dose=2/50, 4%, high dose=



10/50, 20%).   This increase was shown to be significant when ana-



lyzed by the  Cochran-Armitage test, but not the life-table or inci-



dental tumor  test.  Thus, this increase was discounted because the



combined incidence of males with alveolar/bronchiolar adenomas or



carcinomas was not significantly greater than controls by any of



the three tests (control=8/50, 16%, low dose=8/50, 16%, high dose=



13/50, 26%).



     Male mice also exhibited a significant decrease in hepato-



cellular adenomas at the high dose (control=8/50, 16%, low dose=



5/49, 10%, high dose=2/46, 4%)-.  This decrease was accompanied by



a negative dose-response trend using the Cochran-Armitage test.



When total incidence of adenoma or carcinoma in the high dose males



was evaluated, this decreased incidence was statistically significant



only by the life table test (control=19/50, 38%, low dose=14/49,



29%, high dose=ll/46, 24%).



     The preliminary assessment suggests that, under the con-



ditions of this bioassay, ortho-dichlorobenzene was not carcino-



genic in the  B6C3F1 mouse of either sex, but that the maximum tol-



erated dose was probably not achieved in the study.



     A 14-day repeated dose study with o-DCB also was conducted



in Fischer 344 rats as part of the prechronic .test phase of the NTP



bioassay on this substance (Battelle Columbus, 1978d).  Single oral



gavage doses  of o-DCB in corn oil of 60, 125, 500 and 1,000 mg/kg



were selected.  Five animals of each sex were placed in one of



five treatment or one control groups.  All of 10 rats in the 1,000



      group died early, the males by Day 4 and the females by Day 5.

-------
                                V-Of
No other- early  deaths occurred. The percentage body weight gain



decreased with  increasing dosage in both sexes.  Males at 250 mg/kg



showed  an -11%  deficit,  those in the 500 mg/kg group, a -16.6% dif-



ferential.   Only females at 500 mg/kg showed a weight gain deficit



exceeding 10% (-11.8%).



     No tissues from animals dying early were examined histo-



logically.   Those in the highest dose group exhibited, upon gross



examination, pale and yellow livers, yellow and/or green or red



colored contents in the  small intestine, similarly-colored fluids



in the  urinary  bladder,  red fluid in the cecum and congestion of



the vasculature of the brain.  The liver lesion was interpreted to



reflect hepatotoxicity.   No toxic lesions were observed in other



dosage  groups during gross examination.  Tissues from two males



and two females receiving 500 mg/kg/day were examined microscopi-



cally,  with no  lesions indicating toxicity.



     Based upon the results of this study, it was recommended that



the 13-week subchronic gavage study in Fischer 344 rats employ



doses of 25, 50, 100, 200 and 400 mg/kg/day, five days/week.  The



doses actually  administered in the subchronic study were 30, 60,



125, 250 and 500 mg o-DCB in corn oil (Battelle Columbus, 1978i).



Controls received corn oil.  Special studies, as described for o-



DCB in  mice and p-DCB in both species, also were conducted in this



study.   No statistical analyses were performed on the data from



these special studies, except for group means and standard devia-



tions of those  means.

-------
                                V-68
     Of  the animals used in this study, only four died early:



one male each in the 30 mg/kg (Week 11) and control  (Week 10) groups



and two  females in the highest dose group  (Weeks 6 and 9).  Only nine



males in the 250 mg/kg group completed the study; the tenth was



found to be a female during Week 10 and was removed  from the group.



     Food consumption did not vary more than 10% in  treated



groups when compared with controls (down  1.5 mg/day in the highest



dose males).  Lack of body weight gain increased with increasing



doses in both sexes.  At 250 mg/kg and 500 mg/kg, this differential



exceeded 10%.  No striking differences in hematological parameters



were noted, but without statistical analysis, it is  difficult to



determine if the changes are truly statistically non-significant.



Those parameters which may have been altered significantly in the



highest  dose males were hematocrit (down 5%) and red cell count



(down to 8.57 +^ 0.25 x 106 cells/mm3 from an average of 9.42 +_ 0.53



x 106 cells/mm3 in the control group).  There appeared to be a



trend in platelet count, directly proportional to increasing dose



in the females, with levels of  300,000 in the controls to 365,000-



600,000  as the dose increased, with the 250 mg/kg group falling out



of sequence.  This apparent change may have been due to what appeared



to be a  lower-than-normal count in the controls.



     Of  parameters measured in the clinical chemistry analyses,



cholesterol levels were increased in males in 250 and 500 mg/kg and



in females at 125, 250 and 500 mg/kg.  Triglycerides dropped in



high dose males.  The combined alpha-globulin fraction appeared to



be increased in males receiving 250 and 500 mg/kg and in females



treated  with 500 mg/kg.  Of the parameters tested in the urinaly-

-------
sis, urine volume was altered significantly.  Output  increased an



average of 157% in treated males and 187% in treated  females, when



compared with their respective controls.  Decrease  in urine creat-



inine accompanied the dilution occurring with the increased volume



output.




     Porphyrin levels in the urine showed a striking  increase in



the highest dose animals (3-6 fold), both in coproporphyrin and



uroporphyrin levels.  Liver protoporphyrin levels were not altered



significantly at any dose level.  Thus, there appeared to be ab-



normal excretion of the porphyrin, but not retention.



     Organ weights and organ/body weight ratios were  determined.



In rats of both sexes receiving 250 or 500 mg/kg, relative liver



weights were increased.  The liver/body weight ratios in the other



treatment groups did not appear to differ from the  controls.  In



addition, ratios for the other organs (spleen, kidney, testis,



ovary, uterus, thymus, brain and heart) did not appear to have been



altered.



     No consistent lesions were noted upon gross examination



at necropsy.  Microscopic examinations were performed on tissues



of all animals in the control and 500 mg/kg groups  and on the thy-



mus, liver and kidney of the animals from the 125 and 250 mg/kg



groups.  A moderate degree of centrolobular hepatocellular necrosis



was seen in the livers of the highest dose rats-which died early.



Of the survivors in that group, most showed liver lesions, either



centrolobular degeneration or necrosis of individual  hepatocytes.



This necrosis was characterized by randomly scattered cells that



were pyknotic or karyolytic, with shrunken, dark red  cytoplasm.

-------
                                V-70
Some of the 500 mg/kg group males also exhibited renal tubular



degeneration and lymphoid depletion of the thymus.  These latter



lesions were not seen in other treated groups or controls.  However,



hepatocellular necrosis was observed in the 250 mg/kg group and in one



female treated with 125 mg/kg groups was considered to be hemosiderin-



since it was PAS- and Perls positive.



     The liver lesions observed in the 250 and 500 mg/kg groups



were considered to be dose- and treatment-related, and probably



life-threatening, as were the renal and thymic lesions in the high



dose group.  The reviewing pathologist recommended that a Maximum



Tolerated Dose (MTD) of 125 mg/kg (the apparent no-effect level)



be set for both sexes of rats in the 104-week chronic study.



Ultimately, doses of 60 and 120 mg/kg were chosen for this study.



    As mentioned above, this assessment of the effects of o-



DCB in the bioassay is preliminary, pending acceptance by the NTP



Board of Scientific Counselors.



      As with the mice, groups of 50 male and 50 female rats



received 60 or 120 mg/kg doses of o-DCB in corn oil, five days/



week for 104 weeks.  Controls, also 50 of each sex, received an



equal volume of corn oil on the same schedule.



     There was no significant difference in the survival rates



of female rats of either treatment group or low dose males when



compared with the controls.  However, there was a significant de-



crease in the survival of the males at the high dose (P<0.001).



However, several of the males dying before the end of the study were



found to have amounts of corn oil or o-DCB in corn oil in their



lungs (3 in the control group, 8 at the low dose and 12 at the high

-------
                                V-71
dose).   Therefore, gavage trauma may have contributed to their



deaths.   The report suggests that the lower survival rate among



the dose males may not reflect that the maximum tolerated dose was



exceeded.  In fact, as was seen in the mice, there was no increase



in the incidence of nonneoplastic lesions of the liver in rats



receiving either dose of o-DCB, again suggesting that a higher dose



might well have been tolerated.



     The low dose males showed a significant increase in the



incidence of adrenal pheochromocytomas when compared with the con-



trol group by life table analysis (control=9/50, 18%, low dose=16/50,



32%, high dose=6/49, 12%).  The report concludes that since this



incidence was significant only by one of the three tests, and this



tumor expressed no dose-response trend or high dose effects, and



there were no malignant pheochromocytomas, the increase seen in the



low dose group was not related to treatment with o-DCB.



     Interstitial cell tumors of the testis in the males also



occurred with a significant positive trend when analyzed by the life



table test (control=47/50, 94%, low dose=49/50, 98%, high dose=



41/50,  82%), but with a significant negative trend when analyzed



by the Cochran-Armitage test.  The reports states that "since this



tumor is not considered to be life-threatening, this increase



detected by the life table test was discounted."



     Preliminary assessment of these data suggests that, under



the conditions of this bioassay, ortho-dichlorobenzene was not



carcinogenic in the Fischer 344 rat of either sex, but that the



maximum tolerated dose may not have been achieved.

-------
                                V-72
Para-dichlorobenzene
     A 14-day repeated dose oral gavage study was conducted in
B6C3F1 mice to determine doses for the 13-week subchronic toxicity
study (Battelle-Columbus, 1978e).  Doses of 250, 500, 1,000, 2,000
or 4,000 mg/kg in corn oil were administered to groups of five males
and five females.  Controls received corn oil only on Day 1.  All
mice receiving 4,000 mg/kg died by Day 3.  At 2,000 mg/kg, four
males died by Day 7 and two females by Day 8.  At 1,000 mg/kg, four
males and two females died by Day 8.  At 500 mg/kg, four males died
by Day 8, but all females were dead by Day 5.  At 250 mg/kg, three
males died by Day 1, three females by Day 6.  Among controls, two
males and one female died on Day 3, presumably as a result of gavage
trauma.
     Upon gross necropsy, a number of lesions were noted in
both sexes and at all dose levels.  These included:  livers of
abnormal color, either yellow or tan or reddish-chocolate, soft or
mushy small intestines from yellow-green through pink or red to
black in color, and pink to bright red lungs.  Occasionally, kidneys
were pale, and there occurred enlarged, chalk-white mandibular sali-
vary glands.  According to the investigators, no lesions were seen
microsopically in either sex or at any dose level which indicated
significant toxicity.
     Since the study did not establish a no-effect level, a second
14-day repeated dose study was performed (Battelle Columbus, 1978g).
Dose levels of 60, 125, 250, 500 and 1,000 mg/kg in corn oil were
employed.  The controls were untreated.  Only one early death, a
male in the 125 mg/kg group, occurred, purportedly due to gavage
trauma.  Only the 500 mg/kg females showed a decreased body weight

-------
gain (-10.9% vs. control).  No gross pathology was noted at necropsy.



No histology was performed.



     On the basis of these two 14-day studies, the principal



investigators recommended that the subchronic gavage studies in mice



be performed at doses of 125, 250, 500, 1,000 and 2,000 mg/kg/day,



5 days/week, for 13 weeks.  However, the final doses actually employed '



in the first subchronic study were 600, 900, 1,000, 1,500 and 1,800



mg/kg/day (Battelle Columbus, 1979a).  Ten males and ten females



were assigned to each group.  Special studies included:  urine and



liver porphyrin determination, calculation of organ/body weight



ratios, urinalysis, clinical chemistry and hematology.



     Twenty-six animals (12 males and 14 females) died before



the end of the study.  Seven males and females in the 1,800 mg/kg



died early, most within the first week.  At 1,500 mg/kg, three males



and five females died early.  One male in the 1,000 mg/kg group and



one control male died during the last week.



     No effects on food consumption were noted in any group.



Differential body weight gains in treated males of all groups were



significantly different from controls.  These differences ranged



from -50% at 1,500 mg/kg to -22.9% at 1,000 mg/kg.  The differences



did not show a dose-related trend.  Females showed a differential



weight gain of -39% at 600 mg/kg, with lesser changes as the dose



increased, but still > 10%.  Since food consumption was not altered,



the weight gain reductions were apparently unrelated to dietary intake.,



     The hematological parameters included:  hemoglobin, hema-



tocrit, total and differential white cell counts, red cell counts,



mean corpuscular volume, platelet and reticulocyte counts.  No

-------
statistical  analyses were performed on these~ data.  However, the



investigators  stated that there appeared to be no significant dif-



ferences between treated groups and controls.  However, a cursory



review of the  group data suggest that there may have been a signifi-



cant decrease  in platelet counts in the high dose males (445,000 4^



8,700 vs 691,900 ± 209,200 for controls), and a significant increase



in surviving females at the two highest doses (707,400 + 41,000 at



1,500 mg/kg, 707,500 at 1,800 mg/kg vs 492,000 + 86,631 for controls)



This latter observation may be the result of a lower than normal



platelet count among the control animals.



     The following analyses were performed on blood samples



drawn at the time of sacrifice:  serum glutamic pyruvic transami-



nase (SGPT), alkaline phosphatase, gamma-glutamyltranspeptidase



(GGTP), bilirubin, cholesterol, triglycerides, blood urea nitrogen



(BUN), glucose and total protein.  While no statistical analyses



were performed on the data, it appeared that triglycerides were



increased in males at the two highest dose levels, and that choles-



terol was increased in all males except those in the 600 mg/kg



group.  Total  protein appeared to be increased in males in the



1,800 mg/kg group.  Since there were no increases in hemoglobin



or hematocrit, this increase likely was due to an actual,  rather



than a relative, increase in blood proteins.



     Both SGPT and bilirubin may have been increased in females



in the two highest dose groups.  No control values in females were



recorded for cholesterol, triglycerides, BUN, glucose or total pro-



teins.  Thus,  it is difficult to speculate whether or not the in-



crease in cholesterol values in these groups was significantly

-------
                                V-75"
greater "than control, as would be suggested by the dose-related



upward trend,  the relationship observed in the males.



     During urinalysis, the parameters measured were:  pH, protein,



glucose, ketones, bilirubin, occult blood, specific gravity and



creatinine, as well as uroporphyrins and coproporhyrins in the



controls and two highest dose groups.



     Ketonuria was noted in one of two pooled groups of males



at the 1,500 mg/kg dose level and in both pooled groups of females



at that dose.   None was observed in urine of animals at other



doses or in the controls.  In the males, this occurrence may have



been due to the severe body weight gain differential, according to



the investigators.  However, this postulate does not hold up for



the females, since this severe weight gain differential was not



observed.  The investigators suggested that this observation was



due to contamination of the urine samples.  As was seen in the o-



DCB studies, increased urinary output occurred, especially in the



mal es.



     Urinary coproporphyrin levels in both sexes appeared to



increase significantly at the 1,500 mg/kg/dose level but only in



females at the highest dose.  No change was seen in coproporphyrin



levels in males, but appeared to be lower in the surviving female



at the highest dose.  Dose-dependent increases in liver protopor-



phyrin in both males and females occurred.  However, since no sta-



tistics were applied to the data, one cannot identify the lowest



dose level at which significant increases occurred.

-------
     Of  the  organ/body weight ratios calculated, those for the



liver were increased at all  dose levels for both males and females.



In addition,  relative uterine weights of females decreased in a



dose-related manner, with the greatest decrease in the ratio observed



in mice  receiving 1,800 mg/kg.  No differences were apparent for



lung, heart,  kidney, spleen, thymus, brain, testis or ovary.



     The only lesions noted  during necropsy were some pale and/or



yellow livers and pale kidneys in the highest dose group.  No



hemosiderin  was noted in any liver or kidney of mice from any



dosage group.



     A number of lesions were noted upon histological examina-



tion. These included lesions of the spleen, thymus, bone marrow and



lymph nodes  among animals from the two highest dose groups.  These



were considered to be treatment-related.  Hepatocellular changes



which were seen in treated animals of all groups were not seen in



the controls.  In mice from  the 1,500 mg/kg group which died before



the end  of the study, but not those who survived to the end, there



was lymphoid depletion of the spleen, lymphoid necrosis in the



thymus and hematopoietic hypoplasia in the spleen and bone marrow.



The hepatocellular changes observed in the various groups included



karyomegaly,  cytomegaly, and occasionally, large prominent nuclei



with variations in their shape along with changes in the number of



centrolobular hepatocytes.  The cytoplasm of these enlarged cells



was grainy,  sometimes hazy and amphophilic.  These changes were




dose-related.

-------
                                v-77
    Since the  hepatocellular changes were seen in animals from



all  treated dose groups,  no no-effect level could be established



from the study  results.   The changes observed at 600 mg/kg could be



characterized as minimal.  Subsequently, a second subchronic study



was  performed in the  attempt to establish the maximum tolerated



dose for the chronic  study and-a no-effect dosage level for sub-



chronic exposure  (Battelle-Columbus, 1980a).  In this second study,



target doses of 75,  150,  300, 600 or 900 mg/kg/day were chosen.  As



shown later, because  of  an error in the preparation of the doses,



those actually  administered were 84.4, 168.8, 337.5, 675 or 900



nig/kg/day, five days/week for 13 weeks.  The protocol was other-



wise identical  to  the first subchronic study, except that no special



studies were performed.   During the study, three males and seven



females died early.   These deaths were not dose-related, but rather



attributable to gavage trauma.  No significant differences were ap-



parent in  dietary  consumption or relative weight gains between



treated and control  animals of either sex.



    During gross  necropsy, no consistent observations were



made which were considered significant.  Microscopically, there



was  a significant  number of animals in the two highest dose groups



exhibiting centrolobular to midzonal hepatocytomegaly.  Few changes



of this type were  seen in animals treated at 337.5 mg/kg/day.   The



investigators determined that 337.5 mg/kg/day was the maximum tol-



erated dose for this  duration of exposure.







  ( 104-week results to be added later)

-------
     Fourteen-day repeated dose and 13-week subchronic gavage
studies  also  were conducted in Fischer 344 rats as dose-range
finding  efforts  preliminary to the 104-week chronic bioassay for
p-DCB in this species.   The first repeated dose study employed
single doses  of  60,  125, 250, 500, or 1,000 mg/kg/day p-DCB in
corn oil (Battelle Columbus, 1978f).  Doses were administered to
five males  and five females per group.  Controls were untreated.
     Only one early death was recorded, a male at the 125 mg/kg
dose level  on Day 8.  Body weight gains were slightly suppressed in
the males,  with  the greatest difference being seen at the highest
dose (-9.2%).  No gross or histological pathology was observed.
Because  of  the absence of significant signs of toxicity, it was
decided  that  the study be repeated at higher dose levels.
     A re-run of the 14-day repeated dose study was conducted
according to  the protocol of the first study.  In this study, how-
ever, doses of 500,  1,000, 2,000, 4,000 or 8,000 mg/kg/day were
administered  in  corn oil to the rats (Battelle Columbus, 1978g).
Again, controls  were untreated.  No clinical pathology studies or
histological  examinations were done.
     By  Day 3, all animals in the group receiving 2,000, 4,000 or
8,000 mg/kg had  died.  Gross pathology included pale livers,
discolored  lungs and intestines and mandibular lymph nodes
and fluid discharge from the eyes and nose.  One male rat receiving
1,000 mg/kg died on the first day, apparently due to gavage trauma.
Four females  in  that dose group died on Day 1 (1), Day 4 (1) and
Day 5 (2),  with  those dying on Day 5 showing slightly congested
livers.   One  female receiving 500 mg/kg died on Day 13 of gavage
trauma.

-------
     At  these higher doses, evidence of depressed body weight
gain appeared.   Among males, those receiving 1,000 mg/kg/day had
a 22% reduced gain;  those receiving 500 mg/kg/day showed a 24%
reduced  gain.  No significant differences were seen in the females
at any dose.
     From the results of the two repeated dose studies it was
determined initially that a subchronic gavage study in the Fischer
344 rat  be performed at dose levels of 60, 125, 250, 500 or 1,000
ntg/kg/ day.   In fact, the first subchronic study utilized doses of
300, 600, 900,  1,200 or 1,500 mg/kg/day (Battelle Columbus, 1979b).
The protocol  included use of ten animals/sex/dose or control group.
Animals  received doses of p-DCB in corn oil, five days/week for
13 weeks.  Vehicle controls received corn oil alone.  In addition
to gross necropsy and histological examination, additional special
studies  were performed as described for the subchronic studies on
o-DCB.  These included:  clinical chemistry and hematology on blood
samples  drawn the day of sacrifice, urinalysis on animals at the
1,500 and 1,200 mg/kg groups and controls, porphyrin analysis in
urine and liver of the same groups and calculations of organ/body
weight ratios.   Again, no statistical analysis of these data was
performed.
    Eight early deaths occurred in the 1,500 mg/kg males, and five
in males at 1,200 mg/kg.  One male receiving 900 mg/kg died during
Week I,  and one control male during Week 10.  Among females, 9 of
10 receiving 1,500 mg/kg died early, one of 10 at 1,200 mg/kg and
two of 10 at 900 mg/kg.  The males tended to die earlier than the
females.

-------
                                V-80
     Food consumption in the treated groups varied little



from control.   The one remaining male at 1,500 mg/kg showed a



slight increase compared with controls.  Male rats at 300 mg/kg



averaged slightly less than controls.  A dose-dependent depression



of body weight gain was seen in both sexes.  In females, a greater



than 10% difference was seen at the 900 mg/kg level and above.



All treated males exceeded the 10% differential when compared with



controls.



     Of the hematological parameters analysed (hemoglobin,



hematocrit, mean corpuscular volume, red cell, reticulocyte and



platelet counts, total and differential white cell counts), the



hemoglobin and hematocrit levels appeared to be lower in the two



surviving males at 1,500 mg/kg.  Hemoglobin levels were 17.6 jf



0.6 G/dl in controls vs. 15.3 +_ 0.2 G/dl in the treated rats.



These two males also exhibited mild anemia with average red cell



counts of 8.85 ^ 0.22 x 106 cells/mm3 compared with control levels



of 10.03 +_ 0.36 x 106 cells/mm3.  Mean corpuscular volume also was



decreased slightly (51 ^ 2 in controls vs. 48+0 in the treated



rats).  The investigators reported that no histological evidence of



bone marrow changes were apparent in these two rats, although one



other male and five females treated with this dose did exhibit bone



marrow hypoplasia.  The single surviving high dose female showed



an increase in hemoglobin, lowered white cell count, increased



red cell count and decreased mean corpuscular volume when compared



with controls.  However, one cannot establish the significance of



these observations, since they occurred in a single animal.

-------
     Clinical  chemistry analyses on blood drawn at sacrifice
included:   gamma-glutamyl-transpeptidase, alkaline phosphatase,
bilirubin,  cholesterol, triglycerides, total protein, blood urea
nitrogen, glucose and globulin fractions.  Of these parameters,
there was evidence of a possible dose-related increase in alkaline
phosphatase in females, with a substantial increase in the lone
survivor at" the highest dose.  Trends also appeared in both sexes
as an increase in cholesterol levels with increasing dose and an
increase in albumin levels at the highest doses.  The total pro-
tein increases seen at the higher doses likely are due to the
increase in the albumin levels.
     The investigators reported no significant changes in the
parameters  measured in the urinalysis:  pH, protein, glucose,
creatinine, ketones, bilirubin, occult blood and specific gravity.
As previously  described for o-DCB, urine volume of rats treated at
the higher  dose levels was significantly increased:  246% in males
at 1,200 mg/kg, 142% in females at this dose.  The few survivors
from the 1,500 mg/kg groups had urine volumes 6-10 times greater
than control levels.  Since drinking water intake was not moni-
tored,  one  cannot speculate on the consequences of this alteration
in output.
     Porphyrin levels in urine and liver also were monitored
in the two  higher dose group and control males_  Significant in-
creases in  both coproporphyrin and uroporphyrin levels in the
urine were  observed.  A similar increase was seen in the urine of
the surviving  highest dose female.  No data for survivors at 1,200
mg/kg were  presented.  Liver protoporphyrin levels apparently were

-------
                                v-ai
not increased  in either sex.   On the contrary, the levels in the
survivors  at the highest dose were depressed somewhat.  Therefore,
there  was  no retention of porphyrins in treated animals, just as
was reported for o-DCB.
    Several organs  were weighed at the time of sacrifice.  These
included:   heart, liver, kidney, uterus, ovary, testis, brain,
thymus and spleen.   The relative liver weights were increased clearly
in animals of  both sexes at all  doses except the lowest (300 mg/kg).
An increase at this  dose level was seen in females, but it may not
have been  statistically significant.  Dose-related changes in brain/
and uterus/body weight ratios also were observed.  The investigators
suggested  that these differences might be due to the significant
lack of body weight  gain in the affected groups at the higher dose
levels.
    Histological examination was performed on tissues from
animals in the control and three highest dose groups.  The kidneys
and lungs  from males treated at 300 and 600 mg/kg also were examined.
    Retinal atrophy was seen in almost all of the animals, the
degree of  severity of which was inversely related to the time of
death.  Thus,  those dying early showed few signs; those dying later
had severe bilateral atrophy.
    Pulmonary lesions related to the gavage procedure were
seen in animals of all tested groups.  Presumably, aspiration of
the test material occurred.
    Of significance was the degeneration and necrosis of the
hepatocytes, hypoplasia of bone marrow, lymphoid depletion of the
spleen and thymus,  epithelial necrosis and villar bridging of the

-------
                                V-83
mucosa of  the small intestine and epithelial necrosis of the nasal
turbinates that occurred in animals in the two highest dose groups.
None of the lesions were observed in animals receiving doses of 900
mg/kg or less.  Thus,  these changes were considered to be treatment-
and dose-related.
     Most  of the males surviving beyond the halfway point of
the study  exhibited renal lesions characterized by multifocal
degeneration or necrosis of the cortical tubular epithelium.  An
amorphous  eosinophilic material was often present in the lumen of
thes'e tubules.  Some thickening of the basement membrane of these
cells was  visible.  Occasionally, a dilatation of some tubules
could be observed at the corticomedullary junction.  These tubules
also had degenerated epithelia and were filled with material simi-
lar to that described above.
     The investigator describing the pathology stated that the
renal lesions are not unusual for male rats of this strain.  How-
ever, he would have expected to have seen lesions of some magnitude
in the control animals, and to a lesser degree in treated ones.
Thus, he concluded that while the lesion was not clearly dose-related,
it might be at least partially treatment-related and for this reason,
no no-effect dose level could be established from the results of
this study.  Thus, it was decided that the subchronic gavage study
in rats would be repeated, employing lower dose levels of 37.5, 75,
150, 300 or 600 mg/kg/day (Battelle Columbus, 1980b) .  The protocol
employed was identical to that described above for the first sub-
chronic study, except that no special studies were done.  A few
early deaths occurred during the second study.  All were attributed
to gavage  trauma.

-------
                                V-84
     Na significant differences in food consumption were ob-
served.   No dose group had a greater than -10% differential weight
gain when compared with controls.  Histological examination was
performed on all control and 600 mg/kg dose animals, as well as
all early death animals, and kidney from males at the two lower
doses.
     Microscopically, some lung pathology was noted which was
attributed to aspiration of the gavaged material.  There also was
an increased incidence and severity of renal cortical degeneration
in males receiving the two higher doses, as had been observed in
the earlier study.  Females showed no significant changes at any
dose level.  Some rats also exhibited myocardial degeneration and
lymphoid hypoplasia in lung tissue.  These changes were character-
ized as being normal for this strain and age of rat.  The reviewing
pathologist suggested that the MTD for males be set at 150 mg/kg
and for females at 600 mg/kg for the chronic study.  Therefore,
Battelle Columbus proposed doses for the chronic study of 75 and
150 mg/kg in males, 300 and 600 mg/kg in females.  Tracor Jitco
suggested 150 and 300 in males, 300 and 600 mg/kg in females.

      (104-week study to be added later.)

Other Carcinoqenicity Studies
     Long term inhalation studies with p-DCB have been conducted
in mice and rats.  Groups of Swiss strain mice (75 males and 75 fe-
males per group) were exposed to airborne concentrations of 0, 75
or 500 ppm p-DCB five hours/day, five days/week for 57 weeks for all
female groups and the 500 ppm males and for 61 weeks for the 0 and

-------
                                V-85
75-ppro males.   Males were sacrificed at these times.  Females were
held unexposed until sacrifice after 75 or 76 weeks.
     The original objective of this study was to assess the
chronic toxicity and carcinogenic potential of p-DCB in the mouse.
However, because of fighting among the males during the early
stages of the study and a high background incidence of respiratory
disease in both sexes resulting in high mortality rates, the investi-
gators felt that this objective was not attained satisfactorily.
The following information was gathered from the study, however.
     At termination, blood samples were drawn from at least ten
males in each group to determine the blood levels of urea and glucose
and serum alanine and aspartate transaminase activities.  Because of
insufficient or clotted samples, no urea or glucose levels were ob-
tained from the 500 ppm group.  No significant changes in blood
glucose or plasma alanine transaminase activity were observed in
the 75 ppm group.  Blood urea concentrations appeared to be slightly
reduced in the 75 ppm group, but this likely was due to the rela-
tively high control levels observed.  There was some evidence of
an increase in the plasma aspartate transaminase activities in both
treatment groups, but this increase was not statistically signifi-
cant.
     Urinalysis was performed for pH, glucose, bilirubin, specific
gravity, protein and coproporphyrin, in males only.  No significant
differences were observed in any of the parameters.  Several hemato-
logical parameters were studied in the males:  hemoglobin levels,
packed cell volume, total white cell count and differential,
platelet count and red cell morphology, red cell count, mean red

-------
cell  count,  mean hemoglobin concentration and raethemoglobin content.
Femoral bone marrow smears also were examined.  Slight reductions
in hemoglobin concentration and packed cell volume were observed in
isolated males in all three groups.  These animals also showed
slight increases in methemoglobin.  Among the 500 ppm-dosed animals,
there was a slight, but not statistically significant, decrease in
the mean total white cell count.  No bone marrow changes were
observed.
     While tissues from animals of both sexes and all treatment
groups were examined grossly, only those from females sacrificed
when moribund or at the end of the study were examined histologi-
cally.  The "epithelial repair" observed in the nasal sinuses and
the "resolving pneumonia" seen in the lungs of both test and con-
trol animals were said to be related to the high incidence of
respiratory disease in the colony thought to be caused by a Sendai
virus.  Lesions in the liver (hepatitis) and the kidney (inflamma-
tion) were seen in similar quantities in both control and test
animals.  No significant increases in numbers of neoplastic lesions
were observed in either treatment group when compared with the con-
trol group run concurrently or with historical controls from this
laboratory.
     From the data available, it was concluded that the admini-
stration of p-DCB by inhalation at levels up to 500 ppm for longer
periods of exposure, followed by a period of recovery, did not pro-
duce any significant non-neoplastic lesions or increase the number
or types of neoplastic lesions in female mice.

-------
                                V-87
     A similar long term inhalation study was conducted  in



Alderly Park  Wistar-derived albino rats (Riley, et al_.,  1980a) .



Groups of  animals (76-79 animals/sex/group) were exposed to airborne



concentrations of 0,  75 or 500 ppm p-DCB five hours/day, five days/



week for 76 weeks.  Survivors at this time were left unexposed for



36 additional weeks.   Interim sacrifices (5 animals/sex/group) were



conducted at  26, 52 and 76 weeks.  Body weights were determined



weekly until  Week 13  and then monthly thereafter.  Food  and water



consumption was monitored biweekly until Week 15, then every six



weeks thereafter.  Urinalysis, clinical chemistry and histopatholo-



gical parameters as described above for the mouse study  also were



analyzed in the rat study.  Hepatic aminopyrine demethylase acti-



vity also was measured at the 52 week sacrifice.  Organ weights



were measured at each sacrifice date.  •



     No consistently  significant effects on mortality rates, food



and water consumption, body weight gain, clinical chemistry, hema-



tology or histopathology were observed in either sex, at either



exposure dose or at any of the sacrifice times when compared with



controls at the same  times.  Group mean body weights in  the high



dose females  were significantly depressed from Weeks 4-38 except



at Week 10, but were  not significantly different at Week 50 or



later.  Increase in liver and kidney weights (both sexes at Weeks



76 and 112),  heart and lung (both sexes at Week 112) and urinary



protein and coproporphyrin output (in males) were noted  in animals



exposed at 500 ppm.  No increases in tumor incidence or  types



were produced at either dose in either sex.  The investigators



concluded that, under the conditions of this study, p-DCB was not

-------
carcinogenic  to rats at doses up to 500 ppm.  An increase in hepa-



tic  hyperplasia was seen in treated females, but not in controls



or treated males.   Hemosiderosis was increased in treated males at



both doses, but not in females.  Focal chronic hepatitis with



infiltration  was increased in all treated animals, but signifi-



cantly so only in  the animals exposed at 500 ppm.  A slight in-



crease in myocardial calcification was seen in high dose males.



Adrenal hyperplasia was increased only in the low dose males.  The



investigators concluded that non-neoplastic changes of a minor



nature occurred in the high dose animals, but were absent or insig-



nificant in the low dose animals.

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                                VI-1
VI.   HEALTH EFFECTS IN HUMANS
                                      •
     Most  of the poisoning incidents reported in the literature

resulted  from inhalation.  Accidental inhalation can occur either

at home or at work.  There are several cases, however, when a

chlorinated benzene was accidentally or deliberately ingested.

Table VI-1 summarizes the available literature for ortho-

and para-dichlorobenzene.  Again, no reports appear in the litera-

ture concerning m-DCB.

o-Dichlorobenzene

     One  case of sensitization to o-dichlorobenzene was re-

ported for a man who regularly handled window sashes dipped in the

compound  (Downing,  1939).  When applied to the skin, there was a

burning sensation after 15 minutes and for the duration of expo-

sure.  The site of  application showed a reddish hue which increased

up to 24  hours later, when blisters formed.  A brown pigmentation

formed later and persisted for three months.  The man was forced to

seek alternative employment.

     Dupont (1938)  described the reactions of a group of sewage

workers performing  cleaning operations in the sewer at a point di-

rectly below a pipe discharging sewage from a dry-cleaning establish-

ment.  Inhalation of the fumes caused irritation of the eyes and

upper respiratory tract and vomiting.  Apparently, no deaths occur-

red, but  no clinical follow-up was described.

     An 18-year old female employee of a dry-cleaning shop was

admitted  to the hospital with severe acute anemia (Gadrat, et al.,

1962).  She had been employed as an ironer in that shop for about

6  months  prior to admission.  During her employment, she was

-------
                                                          Vi—i!
                                                       Table VI-1

                                  Report of Human Exposure to o- and p-Dichlorobenzene
                                            (Modified from Ware, West, 1977)
Compound   Subject
 Exposure
    Symptoms
Clinical Report
Follow-up Studies    Reference
OrDCB      Sewage       o-DCB effluents
           workers      from dry-clean-
                        ing establish-
                        ment above sewer
                   Eye and upper res-
                   piratory tract ir-
                   ritation, vomiting
                      o-DCB intoxication    Not indicated
                                         Dupont,  1938
           47 year
           old male
            18 year
            old fanale
            40 year
            old male
            53 year
            old male
o-DCB in dipping
solution for
window sashes,
occupational

Dry-cleaning
and dyeing shop
1940-1950 occu-
pational expo-
sure to solvent:
80% o-DCB, 15%
p-DCB

1932-1961, glue
containing 2%
o-DCB, methyl
ethyl ketone
& cyclohexane,
(no benzene or
homologues)
Water blisters on
face, hands, arms
Pallor, tiredness,
headaches, vomit-
ing, violent gas-
tric pains
Weakness, fatigue
Weakness, fatigue
Eczematoid derma-
titis due to o-DCB
Severe hemolytic
anemia: 1.5 x
erythocytes/mm-*
 Not indicated
Downing, 1939
Chronic lymphoid
leukemia
Chronic lymphoid
leukemia, periph-
eral and abdominal
adenopathy, spleno-
megaly
 10 months later    Gadrat et al.,
 erthyrocytes       1962
"excellent" but
 leucocyte equil-
 ibrium showed
 tendency to
 neutropenia

 Treatment ongoing  Girard, et al.,
                    1969
 Died 1968
Girard et al.,
1969

-------
                                                 Table VI-1 (Continued)
Compound   Subject
              Exposure
                     Symptoms
                    Clinical Report
                     Follow-up Studies
                          Reference
o-DCB
p-DCB
15 year
old
female
           60 year
           old male
62 year
old male
            19 year
           old
            female

            60 year
            old male
           Wife of
           male
           above
            36 year
            old
            female
Cleaned clothes
with products
containing 37%
o-DCB, (no ben-
zene or toluene)

1930 to 1960
shipping mono,
o-di- and tri-
chlorobenzene

p-DCB in bath-
room
           Preparation of
           p-DCB for 18
           months

           Heavy p-DCB moth
           ball vapor in
           house for 3 to
           4 months
           As  above
            p-DCB moth killer
            in house
Initially hos-
pitalized with
ret reel av icul a r
adenopathy
                              Weakness, tired-
                              ness
Asthenia, dizzi-
ness
                   Asthenia, dizzi-
                   ness, weight loss
                   Weight loss,
                   loose bowels,
                   tarry stools,
                   numbness, clumsi-
                   ness

                   Weight and
                   strength loss,
                   abdominal swell-
                   ing, jaundice
                   Periorbital
                   swelling, intense
                   headaches, profuse
                   rhinitis
Acute myeloblastic
leukemia
                    Anemia: 3 x 106
                    erythrocytes/mm-*
Light hyperchromic
anemia, after 1
month, increase in
anemia, hypogran-
ulocytosis

Slight anemia,
reactional hyper-
leucocytosis

Acute yellow
atrophy of the
liver (confirmed
by autopsy)
                    Acute yellow
                    atrophy of the
                    liver (confirmed
                    by autopsy), spleno-
                    megaly

                    Exposure to p-DCB
Died 10 months later
of 100% peripheral
1eukoblastosis
                     Not indicated
Girard  et  al.,
1969
General hematologi-
cal improvement but
increase in hypogra-
nulocytosis at 6
months

Not indicated
                       Girard et al.,
                       1969
Perrin, 1941
                                         Developed ascites
                                         and died
Petit and
Champeix,
1948

Cotter, 1953
                     Died 1 year after
                     initial exposure
                                                                                              Cotter, 1953
                     Symptoms subsided
                     within 24 hours
                       Cotter, 1953

-------
                                      Table VI-1 (Continued)
Compound
p-DCB
>
Subject
34 year
old
fanale
52 year
old male
Exposure
Demonstrating
p-DCB containing
products
p-DCB exposure
in fur storage
plant
Symptoms
Tiredness, nausea
headache, vomiting
Weakness, nausea,
blood, vomiting,
jaundice.
Clinical Report
Subacute yellow
atrophy and cirrho-
sis of the liver
Subacute yellow
atrophy of the
liver
Follow-up Studies
Not indicated
4 years later
reported "in good
health-
Reference
Cotter, 1953
Cotter, 1953
20 year    p-DCB manufacture Loss of weight
old male   1 to 7 months     exhaustion, de-
(+ 26      exposure          crease of appetite
workmates)
53 year
old
fanale
3 year
old
male

19 year
old
female
12 to 15 year     Cough, progressive
exposure to p-DCB dyspnea, fatigue,
moth balls in     mucoid sputum
house
                                       Methemoglobinemia
                                       and other blood
                                       pathologies
                     Pulmonary granulo-
                     matosis? focal
                     necrosis of liver
Played with p-DCB Cough, listlessness.  Acute hemolytic
crystals          black urine          anemia
4-5 p-DCB pel-
lets ingested
daily for 2-
1/2 years
Increased patchy
pigmentation of
skin
 21 year
 old
 pregnant
 female
Pica for p-DCB
toilet blocks
first trimester
General tiredness,
mild anorexia, diz-
ziness, edema of
ankles
Due to p-DCB inges-
tion.  Unsteadiness
and tremors on ceas-
ing consumption
thought to be psycho-
logical, not physio-
logical

Hemolytic anemia
                       All workers trans-
                       ferred to other
                       working environ-
                       ment

                       Not indicated
                                            Complete recovery
Pigment returned to
normal
                                                                  Wallgren,
                                                                  1953
                      Weller and
                      Crellin,
                      1953
                                             Hallowell,
                                             1959
Frank and
Cohen, 1961
Healthy child
delivered several
months later
Campbell and
Davidson,
1970

-------
                                VI-5
continuously  exposed to fumes of cleaning solution containing 95%



o-DCB and 5%  p-DCB.   She exhibited pallor, weakness, headaches,



vomiting and  severe  pains.   She was diagnosed as having a severe



hemolytic anemia  (1,500,000 RBCs/mm3), accompanied by leukocytosis and



polynucleosis,  and the presence of some immature elements belonging



to the granulocytic  and erythrocytic series.  Vigorous treatment



and a change  of employment  resulted in essentially a full recovery.



    Girard,  e_t al_.  (1969)  reported three cases of leukemia which



they attributed to chronic  exposure to o-dichlorobenzene (o-DCB).



One man hospitalized for chronic lymphoid leukemia worked with a



solvent containing 80% o-DCB and 15% p-DCB for 10 years.  A girl



hospitalized  with acute myeloblastic leukemia died 10 months later



of peripheral leukoblastosis.  She reportedly had a neurotic compulsion



to remove dirt and grease stains from her clothes, which she did



repeatedly  with a product containing 37% o-DCB (no benzene or



toluene).   Another man exposed to a glue containing 2% o-dichloro-



benzene, methyl ethyl ketone and cyclohexane for a period of 29



years died  of chronic lymphoid leukemia.  No further details of



these incidents were given.



    Girard,  et al .  (1969)  also reported the case of a 60-



year old male,  who for 30 years had worked in a job during which



he had been in contact with mono-, o-di- and trichlorobenzene.



At the time he was seen by  the authors, he exhibited symptoms



of weakness and tiredness.   Clinical studies revealed that he



suffered from anemia, with  an erythrocyte count of 3 million/mm3.



No follow-up of this individual was described.

-------
                                VI-6
     In cases  where moderate exposures to p-dichlorobenzene



were  documented,  patients complained of severe headaches, profuse



rhinitis  and periorbital  swelling for approximately 24 hours after



exposure  (Cotter,  1953;  Campbell  and Davidson, 1970).   Anorexia,



nausea, vomiting,  weight loss and yellow atrophy of the liver were



reported  for high exposure concentrations (Petit and Champeix,



1948; Cotter,  1953; Hallowell ,  1959).



     Wallgren  (1953)  reported loss of weight, exhaustion, decrease



of appetite and blood dyscrasias  in 27 men who manufactured p-di-



chlorobenzene  for 1 to 7 months.   Cotter (1953) described the



case  of  a woman who demonstrated  products containing p-DCB and



who complained of tiredness, nausea, headache and vomiting.  Clinical



studies  showed that she had subacute yellow atrophy and cirrhosis



of the liver.



     Heavy use of p-dichlorobenzene as either a moth-repellent



or a  deodorizer apparently resulted in weakness, nausea, vomiting



of blood  and jaundice (Perrin,  1941; Cotter, 1953; Weller and Crellin,



1953).  One man and his wife died within months of each other of



acute yellow atrophy of the liver (confirmed by autopsy).  Their



house was apparently saturated with p-DCB moth ball vapor for a



period of at least three to four  months (Cotter, 1953).



     There are at least two reports of deliberate ingestion of



p-dichlorobenzene.  One woman who developed a pica for p-DCB during



the first trimester of her pregnancy complained of general tired-



ness, mild anorexia,  dizziness  and edema of the ankles.  She was



hospitalized with hemolytic anemia and delivered a healthy child



several months later (Campbell  and Davidson, 1970).  Another

-------
                                VI-7
wontanr who  ingested  4  to 5 p-DCB pellets (size not indicated) daily



for  2 1/2  years  complained about increased patchy pigmentation.



Unsteadiness  and tremors occurred when she stopped taking the



pellets, but  these  symptoms were thought to be due to psychological



rather  than physiological withdrawal (Frank and Cohen, 1961).

-------
                               VII-1
VII.  MECHANISM(S) OF TOXICITY






     Of the various toxic effects occurring after exposures



to the dichlorobenzenes, information on the mechanisms of toxi-



city is available only for the necrosis noted in the liver, which



perhaps also can be applied to similar changes in the kidney and



lung, and for the induction of porphyria via acceleration of



synthesis in the heme pathway.



     Many workers have studied the possibility that cellular



damage caused by many drugs and xenobiotics is mediated via chemi-



cally reactive metabolites.  Many of the metabolites formed are



chemically inactive, but certain of the metabolites such as the



arene oxides or epoxides may interact with physiological or bio-



chemical processes, causing either pharmacological or toxicologi-



cal effects.



     Studies of halogenated benzenes, including the dichloro-



benzenes, demonstrate that hepatic necrosis produced on exposure



to these compounds results from their conversion to reactive toxic



intermediates.  Reid and co-workers have shown that an increase in



toxicity can be correlated with an increase in covalent binding of



metabolites to proteins within liver cells.  This relationship can



be seen both in. vivo and ijn vitro.  In the presence of the result-



ing hepatic necrosis, an increase in mercapturic acid excretion can



be measured, as well as a decrease in available glutathione levels



(see Table VII-1).  This suggests that with sufficient depletion of



glutathione (greater than 20-25%), the liver loses its ability to



"detoxify" the chemical by complexing with the substance to form a



less reactive substance, and, thus, proportionately more reactive

-------
                                              Table VII-1

                  Covelent Binding,  Hepatotoxicity and Mercapturic Acid Excretion oE
                                    Halogenated Benzene  Derivatives

                     (Reid et aU,  1971j  Reid  et aU, 1973; Reid and Krishna, 1973)
                                      (After Ware and West, 1977)
Compound

Monobronobenzene
Monochl orobenzene
Monoiodobenzene
Monofl uorobenzene
o-Dichlorobenzene
p-Dichl orobenzene


(1 nMAg)
(1 nMAg)
(1 nMAg)
(1 nMAg)
(0.5 nMAg)
(0.5 nMAg)
Covalent binding
(nM/mg protein
+ S.E.)
(N=6)
0.534 + 0.050+
0.604 + 0.044+
0.323 + 0.054
0.060 + 0.004+
0.234 + 0.015S
0,021 + 0.002S
Hepatic
necrosis

Yes
Yes
Yes
No
Yes
No
Mercapturic Gtutathione
acid excretion concentration
(% of control)*

3 +
3 +
3 +
+
2 +
+

67
Not detennined
66f
82
48f
101
*  GLutathione concentration determined 3 hours  after administration of the hydrocarbon.

+  Killed at 24 hourg

t  P<0.01 compared with control.

S  Killed at 6 hours.

-------
                               VII-3
metabol'ite is available to  interact  with  tissue proteins,  resulting



in cellular and tissue damage.  A  threshold  does seems  to  exist  for



the halobenzene-induced necrosis.   (Reid,  et al_.,  1971;  Reid,  et



al_.,  1971; Reid and Krishna,  1973).  The  stimulation  of  metabolism



by pretreatment with phenobarbital  potentiates  hepatic  damage  in



rats, as can be seen in Table VII-2.   Conversely,  blocking  metabolism



by SKF-525A (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride)



or piperonyl butoxide (a pesticide  synergist) prevents  their hepa-



toxicity  (Reid et al_., 1973;  Reid  and  Krishna,  1973).



     Just as there are species difference  in metabolism  there



are also differences in the rate and degree  of  metabolism  of a



halogenated benzene in different organs,  i.e.,  lung may  differ from



kidney which differs from liver, etc.   Phenobarbital  does  not



increase covalent binding in  the lung  as  shown  for the liver if



administered before exposure  to o-DCB  (Reid,  e_t al_.,  1973;  see



Table VII-3).  Nevertheless, both  the  lung and  kidney exhibit



pathology following exposure  to the  dichlorobenzenes  (Hollingsworth,



_et al_., 1956; Battell e-Columbus, 1979b).  The primary mechanism  of



toxicity in these tissues is likely  to be  the same as that  described



in the liver:  necrosis due to binding of  the reactive metabolite



to cellular and tissue proteins, thereby  interfering  with  the



normal physiological and biochemical processes.



     The halobenzenes, the dichlorobenzenes  among  them,  also



have  been si-town to i-nditee perptiyria  (Rimington  and Ziegler, 1963;



Carlson, 1977, Battelle-Columbus,  1978c,  19781,  1979a,  1979b).



This  condition, a disturbance in porphyrin metabolism,  is  character-



ized  by increased formation and excretion of porphyrin  precursors,

-------
                                              Table VI1-2

         Effect of Phenobarbital and SKF-525A Administration on Cbvalent Binding of Halogenated
             Benzene Derivatives to Rat Liver Protein Ir± Vivo 6 Hours After Administration

                              (Reid et aU, 1973; Reid and Krishna, 1973)
                                      (After Ware and West, 1977)
                                         Control
                                         (nM/mg protein
                                            + S.E.)
                                            (N=6)
                       Phenobarbital
                       (nM/mg protein
                         +_ S.E.)
                            (N=6)
                       Phenobarbital +
                       SKF-525A3
                       (rm/mg protein
                           _+ S.E.)
                           (N=6)
Monobromobenzene-l^C (1 mM/kg)

Monochlorobenzene-l^C (1 mM/kg)

Monoiodobenzene-14C (1 mMAg)

Monofluorobenzene-^C (1 mM/kg)

o-Dichlorobenzene-^c (Q.5

p-Dichlorobenzene-14C (0.5
0.267 +_ 0.034

0.364 jf 0.053

0.090 j- 0.015

0.085 HH 0.015

0.234 +_ 0.015

0.021 + 0.002
0.550 4; 0.031+

1.268 + 0.278^

0.545 ± 0.129+

0.054 + 0.008

0.308 + 0.038

0.012 + 0.001+
0.036 +_ 0.024f

0.438 + 0.144§

0.666 4 0.304

0.052 +_ 0.005

0.186 +_ 0.014§

0.006 + 0.001f
a Diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) (75 mg/kg i.p.) was given 1 hour
  before the hepatotoxin.

+ P<0.01 compared with controls.
* P<0.01 compared with phenobarbital alone.
§ P<0.02 compared with phenobarbital alone.

-------
                                       Table VI1-3

                      Binding of Aromatic Hydrocarbons in Rat Lung:
                                 Effect of Phenobarbital*

                                   (Reid et aK, 1973)
                           (Modified from Ware and West, 1977)
Compound
o-Dichlorobenzene-^C ,
0.5 mM/kg
p-Dichlorobenzene-l^C,
0.5 mMAg
Time of
Sacrifice
(hour)
6
24
6
24
Binding of Hydrocarbon in Lung
(mumole/mg protein)
Control
27.3 + 1.4
20.9 + 2.4
4.6 -f 0.2
3.4 + 0.4
Phenobarbital
18.9 +
15.3 +
3.4 4-
1.8 +
2.7 (p<0.05)
2.2
0.5
0.2
Values are the means -f SE of 6 animals.

-------
                              vii-b
cutaneous photosensitivity, frequent hemolytic  anemia  and  spleno-



megaly.   Acute abdominal and nervous system manifestations may  also



occur.   As was described in some detail earlier,  exposure  to  the



dichlorobenzenes leads to the induction of the  mitochondrial



enzyme,  A -aminolevulinic acid synthetase, resulting in  an increased



production from heme of aminolevulinic acid and then other constit-



uents of the heme synthetic pathway  (Rimington  and  Ziegler,  1963;



Poland,  ejt al_., 1971; Ariyoshi, £t al_., 1975).   When these levels



exceed those needed for maintenance  of the system,  clinical



symptoms may be manifested.

-------
                            viii-i                          OCT  3 I S83



VIII. QUANTIFICATION £F TOXICOLOGICAL EFFECTS


     The quantification of toxicological effects of a chemical

consists of an assessment of the non-carcinogenic and carcino-

genic effects.  In the quantification of non-carcinogenic

effects, an Adjusted Acceptable Daily Intake  (AADI) for the

chemical is determined.  For ingestion data,  this approach

is illustrated as follows:

     Adjusted ADI =    (NOAEL or MEL in mg/kg)(70 kg)
                     (Uncertainty  factor)(2 liters/day)

The 70 kg adult consuming 2 liters of water per day is used

as the basis for the calculations.  A "no-observed-adverse-effect-

level" or a "minimal-effect-level" is determined from animal

toxicity data or human effects data.  This level is divided

by an uncertainty factor because,  for these numbers which are

derived from animal studies, there is no universally acceptable

quantitative method to extrapolate from animals to humans,

and the possibility must be considered that humans are more

sensitive to the toxic effects of  chemicals than are animals.

For human toxicity data, an uncertainty factor is used to

account for the heterogeneity of  the human population in

which persons exhibit differing sensitivity to toxins.  The

guidelines set' forth by  the National Academy  of Sciences

(Drinking Water and Health, Vol.  1, 1977) are used in estab-

lishing uncertainty factors.  These guidelines are as follows:

an uncertainty factor of 10 is used if there  exist valid

experimental  results on  ingestion  by humans,  an uncertainty

factor of 100 is used  if there exist valid results on long-

-------
                            VIII-2
term feeding  studies on experimental animals, and an uncertainty
factor of 1000 is used if only limited data are available.
     In the quantification of carcinogenic effects, mathematical
models are used to calculate the estimated excess cancer
risks associated with the consumption of a chemical through
the drinking  water.  EPA's Carcinogen Assessment Group has
used the multistage model, which is linear at low doses and
does not exhibit a threshold, to extrapolate from high dose
animal studies to low doses of the chemical expected in the
environment.   This model estimates the upper bound (95%
confidence limit) of the incremental excess cancer rate that
would be projected at a specific exposure level for a 70 kg
adult, consuming 2 liters of water per day, over a 70 year
lifespan.  Excess cancer risk rates also can be estimated
using other models such as the one-hit model, the Weibull
model, the log it model and the probit model.  Current
understanding of the biological mechanisms involved in cancer
do not allow for choosing among the models.  The estimates
of incremental risks associated with exposure to low doses
of potential carcinogens can differ by several orders of
magnitude when these models are applied. The linear, non-
threshold multi-stage model often gives one of the highest
risk estimates per dose and thus would usually be the one
most consistent with a regulatory philosophy which would
avoid underestimating potential risk.
     The scientific data base, which is used to support the
estimating of risk rate levels as well as other scientific

-------
                            VI11-3







endeavors,  has an inherent uncertainty.  in addition, in



many areas, there exists only limited knowledge concerning



the health  effects of contaminants at levels found in drinking



water.  Thus, the dose-response data gathered at high levels of



exposure are used for extrapolation to estimate responses at



levels  of exposure nearer to the range in which a standard



might be set. In most cases, data exist only for animals; thus,



uncertainty exists when the data are extrapolated to humans.



When estimating risk rate levels, several other areas of



uncertainty exist such as the effect of age, sex, species



and target organ of the test animals used in the experiment,



as well as  the exposure mode and dosing rates.  Additional



uncertainty exists when there is exposure to more than one



contaminant due to the lack of information about possible



additive, synergistic or antagonistic interactions.





Non-Carcinogenic Effects



     The principal toxic effects of the dichlorobenzenes in



humans and other animals from both acute and longer-term



exposures include central nervous system (CNS) depression, blood



dyscrasias  (granulocytopenia, hemolytic anemia and leukemias),



lung, kidney and liver damage.  In addition to liver



necrosis, the dichlorobenzenes also can produce porphyria.



The appearance and intensity of these and other adverse



effects are dependent upon dose and duration of exposure.



Death following high level acute exposure usually results



from the CNS effects (primarily, respiratory failure).  Deaths



in humans have been reported following accidental exposure.

-------
                            VIII-4

     Several investigators have determined  the acute  lethal
dose levels after exposure to ortho- and para-dichlorobenzene
in several species.  These data are summarized in Table VIII-1
(same as Table V-3).
     Varshavskaya (1968), in her comparative studies  on the
adverse effects of the lower chlorinated benzenes, showed that,
when determining the LD50S, o-DCB was slightly less toxic in mice
and rats than was monochlorobenzene (MCB),  and that p-DCB was
even less toxic than either MCB or o-DCB. o-DCB was slightly
more toxic than MCB and p-DCB in rabbits and guinea pigs.  Thus,
in general, one may conclude that o-DCB is  acutely more toxic
than is p-DCB.
     A number of studies with o-dichlarobenzene and p-dichloro-
benzene are available in which dose-response data are described
and which allow the identification of no-observed-adverse-effect-
levels (NOAELs) following longer-term or lifetime periods of
exposure.  Animals were exposed both by gavage and by inhalation
for periods of time constituting a subchronic or chronic
exposure.  Since several gavage studies are available for
evaluation, only these, and not the inhalation studies, will
be used in the quantification of toxicological effects, as
this route of exposure is more appropriate  for the development
of allowable exposure levels in drinking water.  Comparable
-steadies with the meta- isomer of dichlorobenzene have not
been reported.  Therefore, it will be assumed that ADIs
developed for o-dichlorobenzene also will be appropriate for
m-dichlorobenzene.  This assumption is defensible for several

-------
                                      VI I I — ±
                                  Table  VIII-1

                Acute Toxicity Data  for  o-  and  p-Dichlorobenzene
Animal
        Route
                              LCLp
o-Dichlorobenzene

Rat            Oral
Rat            Oral
Mouse          Oral
Rabbit         Oral
Guinea Pig     Oral
Guinea Pig     Oral
Rat
Guinea Pig
Guinea Pig
Inhal
Inhal
Inhal
                       500 mg/kg
                      2138 mg/1
                      2000 mg/1
                      1875 mg/1
                      3375 mg/1
                      2000 mg/kg
                                            821  ppm/7  hr
                                            800  ppm/7  hr
                                            800  ppm/24 hr
                             Reference
                                           NIOSH, 1978
                                           Varshavskaya,
                                           Varshavskaya,
                                           Varshavskaya,
                                           Varshavskaya,
                                           Hollingsworth,
                                           1958

                                           Hollingsworth,
                                           Hollingsworth,
                                           Cameron, et al
                                           1968
                                           1968
                                           1968
                                           1968
                                            et al
                                                                  195,8
                                                                  1958
                                                                  ., 1937
p-Dichlorobenzene
Rat
Rat
Rat
Mouse
Rabbit
Guinea
Guinea
Mouse
Pig
Pig
Oral
Oral
Oral
Oral
Oral
Oral
Oral
SC
 500 mg/kg
2500 mg/kg
2138 mg/1
3220 mg/1
2812 mg/1
7593 mg/1
2800 mg/kg
5145 mg/kg

-------
                            VIII-6




reasons: 1) in general, in mutagenicity and other short-term

tests,  the meta isomer behaved more like the ortho isomer

than like the para isomer, and 2) short- and longer-term

studies with o- and p-DCB suggest that the ortho isomer is

somewhat more toxic than the para isomer on a mg/kg basis.

Thus, to assume that the meta isomer is more similar to the

ortho isomer would be consistent with a regulatory philosophy

that seeks to avoid underestimating the potential risk to

human health.



o-Dichlorobenzene (and/or m-Dichlorobenzene)

     Hollingsworth, e_t aJL. (1958) gave rats a series of 138

doses of o-DCB over a period of 192 days ( 18.8, 188 or 376

rag/kg/day, five days a week) by intubation.  No adverse effects

were observed at the lowest dose.  With the intermediate dose,

a slight increase in liver and kidney weight was noted.  At the

highest dose, there was a slight decrease in the weight of the

spleen and a modest increase in the weight of the liver accompanied

by cloudy swelling.

     If one were to assume that the results of this study were

appropriate for use in developing an acceptable daily intake

(ADI),  it would be derived thusly:


       18.8 mg/kg/day x 70 kg x 1.0 x 5 =  0.94 mg/day
         100  x.  10                   7
                                               (for a 70 kg adult)

-------
                            VIII-7



     Where:    18.8 mg/kg/day = NOAEL

                       70 kg = weight of protected individual

                         1.0 = ratio of administered dose absorbed

                         5/7 = conversion of 5 day/week dosing
                               regimen to 7 day/week

                         100 = uncertainty factor, appropriate
                               for use with NOAEL from animal
                               studies with no comparable human
                               data ( longer-term exposure duration)

                          10 = uncertainty factor, appropriate
                               for use with data from exposure
                               duration significantly less than
                               1 ifetime


     Varshavskaya (1968) administered o-DCB orally to white

rats for nine months at doses of 0.001, 0.01 or 0.1 mg/kg/day.

Effects were observed at the two higher doses.  The author

reported an inhibition of mitosis in the bone marrow, as well

as neutropenia and abnormal conditioned reflexes.  These changes

in the blood profile can be important in that they could be

precursors to pancytopenia or leukemia.  In this study, however,

no carcinogenic activity was observed.  Also, at the two

higher doses, there was an increase in acid phosphatase and

a decrease in alkaline phosphatase.  At the highest dose, a marked

increase in the amount of 17-ketosteroids in the urine occurred.

This was attributed to hyperplasia of the adrenal cortex, as an

increase in adrenal weight and a decrease in ascorbic acid content

of the adrenals also were observed.   The O.OTTL mgAg/Say

dose had no observable effects on any of the parameters studied.

-------
                            VIII-8
     If one were to assume that the results of the Varshavskaya

study were appropriate for use in developing an ADI for o-DCB,

it could be derived thusly:


     0.001 mg/kg/day x 70 kg x 1.0 =  0.00007 mg/dav
          100 x 10                                 *
                                                (for a 70 kg adult)


     Where:  0.001 mg/kg/day = NOAEL

                       70 kg = weight of protected individual

                         1.0 = ratio of administered dose absorbed

                         100 = uncertainty factor, appropriate
                               for use with NOAEL from animal
                               studies with no comparable human
                               data ( longer-term exposure duration)

                          10 = uncertainty factor, appropriate
                               for use with data from exposure
                               duration significantly less than
                               lifetime

     Subchronic gavage studies with o-DCP in mice and rats

were conducted to assist in dosage selection for the NTP 104-

week carcinogenicity study (Battelle-Columbus, 1978c,i).

Single doses in corn oil were administered 5 days/week for

13 weeks.  Treated mice received doses of 30, 60, 125, 250 or 500

nig/kg/day; treated rats received 30, 60, 125, 250 or 500 mg/kg/day.

Controls received corn oil.  Other protocol details are

described in Chapter V.

     In the mice, body weight gain was decreased significantly

in animals of both sexes at the 500 mg/kg dose level and in males

at the 250 mg/kg dose.  Of the hematological parameters tested,

white cell counts of treated males were lower than those of

control males.  It was suggested that this was due to lower

-------
than normal control values, as observed typically in that



laboratory.  Since individual data were not available for



statistical analysis, it cannot be shown whether or not



the differences between the controls and the treated groups



were statistically significant.  But, since there is at least



anecdotal evidence to suggest a possible relationship between



exposure to o-DCB and leukemia in humans, it would be prudent



to evaluate these results in greater depth.



     Increased, but apparently not statistically-significant,



blood alkaline phosphatase levels were observed in males



receiving 125 and 250 mg o-DCB/kg/day.  SGPT levels were



increased significantly in the two surviving males receiving



500 mg/kg/day, due to the high value for the one animal



exhibiting hepatocellular necrosis.  Urine volume was greater



in the treated animals than in controls, but no record of



fluid intake was kept.  The significance of this observation



is unknown, since no parameters of the urinalysis measured



were altered, except for the decreases in specific gravity and



creatinine levels.



     Males receving 500 mg/kg  exhibited higher uroporphyrin levels



than did male controls; females at that dose showed higher coproporphyin



levels.   These parameters were not measured in the other dose



groups.   In addition, a dose-dependent increase in liver proto-



porphyrin was observed in the females, but not in the males.



Even without statistical evaluation, one could observe that



liver weights in the highest dose group of both sexes were



increased significantly.  An increase of lesser magnitude

-------
                           VIII-10



was" observed in the females at the 250 mg/kg/day dose level.

Histopathological examination of several tissues revealed

that no lesions were apparent in animals treated with 125 mg/kg/day

or lower doses of o-DCB.

     Interpretation of the results of the study suggests that

an oral NOAEL of 125 mg/kg/day could be identified in mice, if

one discounts the observations concerning the white cell counts.

In the absence of the raw data, at this time, it will be

assumed that the investigators have interpreted this finding

correctly.   Under these circumstances, if one were to assume

that the results of the mouse 90-day study were appropriate

for use in  developing an ADI, it could be derived thusly:


     125 mg/kg/day x 70 kg x 1.0 x 5  -  6<25 ma/day
         100 x 10 x                7
                                            (for a 70 kg adult)

     Where:   125 mg/kg/day = NOAEL

                      70 kg = weight of protected individual

                        1.0 = ratio of administered dose absorbed

                        5/7 = conversion of 5 day/week dosing
                              regimen to 7 day/week

                        100 = uncertainty factor, appropriate for
                              use with NOAEL from animal studies
                              with no comparable human data
                              (longer-term exposure duration

                         10 = uncertainty factor, appropriate for
                              use with data from exposure duration
                              of significantly less than lifetime

     In the rats, body weight gain was decreased significantly

at the two  higher doses (250 and 500 mg/kg/day).  Cholesterol

levels were increased in males at the two higher doses and in

females at  the three higher doses.  The combined alpha-globulin

-------
                           VHI-li               	"    '      "



fraction  appeared to be increased in females at the highest dose

and males at  the  two highest doses.  As in the mice, urinary

output was increased substantially, with concomitant decreases

in specific gravity and creatinine-

     Both uro-  and coproporphyrin levels in the urine increased

significantly in  aninmals receiving 500 mg/kg/day.  No measurments

of these  parameters were made in other dose groups.  However,

liver protoporphyrin levels were not changed.  Absolute and

relative  liver  weights were increased in the 250 and 500

mg/kg/day groups. Histopathological examination of tissues

revealed  liver  and kidney changes in the highest dose group,

and  liver changes in the 250 mg/kg/day groups.  As for the

mice, 125 mg/kg/day was identified as the NOAEL for the rats.

     If one were  to assume that the results of the study in which

rats  were exposed to o-DCB subchronically were appropriate for

use  in  developing an ADI, it could be derived thusly:
     125 mg/kg/day x 70 kg x 1.0 x 5  =  6.25 mg/day
         100 x 10 x                7
                                            (for a 70 kg adult)
     Where:    125 mg/kg/day = NOAEL

                      70 kg = weight of protected individual

                        1.0 = ratio of administered dose absorbed

                        5/7 = conversion of 5 day/week dosing
                              regimen to 7 day/week

                        100 = uncertainty factor, appropriate for
                              use with NOAEL from animal studies
                              with no comparable human data
                              (longer-term exposure duration)

-------
                           VIII-16



     Where:   337.5 mg/kg/day = NOAEL

                       70 kg = weight of protected individual

                         1.0 = ratio of administered dose absorbed

                         5/7 = conversion of 5 day/week dosing
                               regimen to 7 day/week

                         100 = uncertainty factor, appropriate for
                               use with NOAEL from animal studies
                               with no comparable human data
                               (longer-term exposure duration)

                          10 = uncertainty factor, appropriate
                               for use with data from exposure
                               duration significantly less
                               than lifetime

     In the  rats, no significant differences in food consumption

or body weight gain were observed between treated and control

animals of either sex at any dose.  Microscopically, there

was an increased incidence and severity of-renal cortical

degeneration in males receiving the two highest doses, as had

been observed in the first subchronic rat study.  Females showed

no significant changes at any dose level.  The NOAEL for this

study was established at 150 mg/kg/day.

     If the  results of this rat subchronic study were considered

to be appropriate for use in developing an ADI, it could be

derived thusly:

     150 mg/kg/day x 70 kg x 1.0 x 5  =  7.5 mg/day
          100 x 10                 7
                                           (for a 70 kg adult)

-------
                            VIII-17
     Where:    150 mg/kg/day  =  NOAEL

                       70  kg  =  weight  of  protected  individual

                        1.0  =  ratio of administered  dose  absorbed

                        5/7  =  conversion of  5  day/week  dosing
                                regimen to 7  day/week

                        100  =  uncertainty factor,  appropriate  for
                                use with  NOAEL  from animal studies
                                with no comparable  human data
                                (longer-term  exposure duration)

                          10  =  uncertainty factor,  appropriate
                                for use with  data from exposure
                                duration  significantly less
                                than lifetime
Quantification of Non-carcinogenic  Effects


     Table VIIl-2 summarizes the ADIs derived  from the

available gavage studies on o- and  p-dichlorobenzene which

contain adequate dose-response data  identifying NOAELs.  As

can be seen, a wide range of numbers was derived.  For o-DCR

(and m-DCB), the ADIs range from 0.00007 mg/day (Varshavskaya,

1968) to 60 mg/day (NTP, 1982; preliminary report).  For

p-DCB, the ADIs range from 0.94 mg/day  (Hollingsworth, et al.,

1956) to 16.9 mg/day (Battelle-Columbus, 1980a).  However, a

rationale can be presented by which certain of these numbers

can be eliminated and others supported.

   Ortho-dichlorobenzene (and, meta-dichlorobenzene)

     While the Varshavskaya study suggests that effects can

be seen at very low doses when compared with the other studies,

little in the way of quantitative experimental detail was

presented in her publication.  Therefore, it is difficult to

-------
                           VIII-19

assess  fully the results presented, and one cannot conclude
that  this  paper should be the basis for the development of an
Adjusted Acceptable Daily Intake (AADI).
     The fact that the ADIs generated from the chronic studies
in the  NTP bioassay are larger than those derived from the
subchronic studies preceding them would suggest that the 10-
fold  uncertainty factor used to estimate a chronic ADI from
subchronic data may be unnecessarily large for this compound.
However, the NTP Board of Scientific Counselors has not yet
approved the report prepared on the bioassay of o-DCB.  Therefore,
it is prudent to reserve judgment on its validity until such
time as the report is approved.
     The results of Hollingsworth, et al. (1958) suggest
an ADI  of 0.94 mg/day while those of the subchronic studies
preceding the NTP bioassay suggest an ADI of 6.25 mg/day.
Each ADI was derived from an NOAEL ( 18.8 mg/kg vs. 125 mg/kg,
respectively).  Since the highest NOAEL should be used to
derive an ADI, it is more appropriate to use the NOAEL established
in the NTP subchronic studies, than the NOAEL from the Hollingsworth
study.   In addition, the NTP subchronic studies employed
additional doses {5 vs 3), thereby allowing for a more precise
identification of a NOAEL.  It also should be noted that the
minimal effect dose identified in the Hollingsworth study
(188.8  mg/kg) is somewhat higher than the NOAEL established
in the NTP subchronic studies.
     For o-DCB ( and, m-DCB), then, if one were to use the ADI
from the NTP subchronic studies to determine the AADI, it would
be derived thusly:

-------
      AADI „ ADI = 6.25 mq/day  =  3.125 mg/l/day
             21     21
     This AADI assumes that the protected individual  (.a 70 kg

adult)  drinks 2 liters of water/day and that the sole source

of exposure to o- or m-dichlorobenzene is via that drinking

water.   [It is important to note that the odor threshold for

o-DCB and m-DCB in water has been identified as 0.01 and 0.02

ppm, respectively (Kolle, 1972).  Therefore, any MCL for

these compounds may have to consider the asthetic, as well as

the toxic, consequences of exposure to these compounds in

drinking water].


p-Dichlorobenzene

     The results of the Hollingsworth, et al. (1956) study

suggest an ADI of 0.94 mg/day while those from the subchronic

studies preceding the NTP bioassay suggest ADIs of 7.5 mg/day

(rats)  and 16.9 mg/day (mice).  It is apparent from these

three studies, as well as the acute toxicity studies described

earlier, that the rat is more sensitive to p-DCB toxicity

than is the mouse.  Therefore, to be consistent with the

philosophy that one uses data from the most sensitive

animal  species when estimating the potential risk to the

human,  the data from the experiments in the rat should be

used in deriving an AADI.

     The ADI derived from the Hollingsworth study was based

upon an NOAEL of 18.8 mg/kg; the ADI from the NTP subchronic

study in the rat was derived from an NOAEL of 150 mg/kg.

-------
Since the highest NOAEL should be used to derive an ADI, it

is more appropriate to use the NOAEL established in the NTP

subchronic study than the NOAEL from the Hollingsworth study.

The NTP subchronic study employed additional treatment doses

(5 vs. 3), thereby allowing for a more precise identification

of an NOAEL.  In addition, it should be noted that the minimal

effect level identified in the Hollingsworth study (188 mg/kg)

was somewhat higher than the NOAEL established in the NTP

subchronic study.

     As with o-DCB (and m-DCB), it may be that any ADIs derived

from the as yet unreported NTP chronic studies would be

larger than those derived from the subchronic studies preceding

them because the 10-fold uncertainty factor applied to accommodate for

the difference in duration of exposure may be unnecessarily large.

However, until the report has been approved by the NTP Board of

Scientific Counselors and published as final, it is appropriate

to develop an ADI and AADI based upon the subchronic data.  The

AADI can be reevaluated at a later date.

     For p-DCB, then, if one were to use the ADI from the NTP

subchronic study in rats to determine the AADI, it would be

derived thusly:

        AADI _ ADI = 7.5 mq/day  =  3.75 mg/l/day
             ~ 2 1     21

-------
                          VIII-18


                        Table VIII-2

           Possible ADIs for the Dichlorobenzenes
Compound
 Experiment
Possible ADI
o-DCB/m-DCB
Hollingsworth,  et  al
     (1958)
  subchronic  rat

Varshavskaya (1968)
   subchronic rat

Battelle-Columbus
    (1978c)
  subchronic  mouse

Battelle-Columbus
    (19781)
  subchronic  rat

   NTP (1982)
  chronic mouse
(preliminary  report)

   NTP (1982)
   chronic rat
(preliminary  report)
   0.94 mg/day



   0.07 ug/day


   6.25 mg/day



   6.25 mg/day



     60 mg/day



     60 mg/day
 p-DCB
 Hollingsworth, et al .
      (1956)
  subchronic rat

 Battelle-Columbus
     (1980a)
 subchronic mouse

 Batt el1e-Columbus
     (1980b)
  subchronic rat
   0.94 mg/day
                                                 16.9  mg/day

                                                  7.5  mg/day

-------
                             VIII -22








     i'M.;..<3  A ADI ^ssv.i-sos  that <:ho p r: '".' '- •- ' ' I: e '1 i ; id L v i. ^I'er




the asthotic,  as~ we 1.1 as the  toxic, ociu-.3q.K- . cos of exL'0*ure




to p-dichlorobenxene in drinking water].






C_a i-^if. og o n ic_ E f_f -^c 1; s




     Poth o-nCB  ^-!id p-DCB  have bo<-,-n tnstod by fja\',-ge




for their carcinogenic  potential in F344  rats a iid  Mfi(j3Fl  i;'ii;o




in Lhe NT? Pioassay Prog can.   A draft  report of ti-.o ro-:-nl!:s




of the studios -vith o-DCB  is  a vr. i lab lo  ( ^TP , 1^82).  A  •..•opo'.'t




ol: the results of the studies with p--DCB  has not boen ;nade




a v.i i lab 1 e o s yet.




     The preliminary assessment of the data from the studies




on o-DCB suggests that, under the tost conditions, o-DCB  does




not possess carcinogenic potential-  Kov.'cvoc,  until the N;TP




Board of Scientific Counselors approves  the draft  no port ,




this asnosc^icnt  nust remain preliminary.



     Since no  report, preliminary or oth-'i c.v/i. se ,  is available




on the results of the studies v/ith p-DCB,  no asscsr.nont of




its carcinogenic potential can be made at  this time.






Quantification of Carcinoge^nic^ !•: f_0 ^c t s_
     Preliminary assessment  of i:ha \'TP  ^io.-.say on o-DCB




suggests that  it was not carcinogenic u.-.dor  the con-.! i i-.ions

-------
                            VTTI-23








o f. i-'"> '5 o x p e c i. 
-------
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-------
                               IX-3
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                               IX-6
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                               IX-7
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