297 918
           BENZIDINE
Ambient Water Quality Criteria
               Criteria  and  Standards Division
               Office  of Water  Planning and Standards
               U.S. Environmental  Protection Agency
               Washington, D.C.

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                       CRITERION DOCUMENT



                            BENZIDINE



CRITERIA



                          Aquatic Life



     For freshwater aquatic life, no criterion  for  benzidine can



be derived using the Guidelines, and there are  insufficient data



to estimate a criterion using other procedures.



     For saltwater aquatic life, no criterion for benzidine can



be derived using the Guidelines, and there are  insufficient data



to estimate a criterion using other procedures.



                          Human Health



     For the maximum protection of human health  from  the  poten-



tial carcinogenic effects of exposure to benzidine  through  inges-



tion of water and contaminated aquatic organisms, the  ambient



water concentration is zero.  Concentrations of  benzidine esti-



mated to result in additional lifetime cancer risks ranging from



no additional risk to an additional risk of 1 in 100,000  are pre-



sented in the Criterion Formulation section of  this document.



The Agency is considering setting criteria at an interim  target



risk level in the range of 10~5, 10"^, or 10~7 with corresponding



criteria of 1.67 x 10~3 ug/1, 1.67 x 10~4 ug/1,   and 1.67  x  10~5



ug/1/ respectively.

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Introduction
     Benzidine (4,4'-diaminobiphenyl)  is an aromatic amine.
A proven human carcinogen, its primary site of tumor induction
is the urinary bladder.  It is also mutagenic.
     The incidence of bladder tumors in humans resulting
from occupational exposure to aromatic amines (benzidine)
was first researched in Germany in 1895.  The first cases
of this condition in the United States were diagnosed in
1931 and reported in.1934.
     Several studies implicating the high risk of bladder
tumors in workers exposed to benzidine and other aromatic
amines are well documented.
     Adversary proceedings under section 307(a) of  the Federal
Water Pollution Control Act resulted in the promulgation
of a toxic pollutant effluent standard for benzidine.  The
ambient water criterion upon'which the standard was based
was 0..1 jug/1  (42 FR  2588, January 12, 1977).
     Benzidine is an aromatic amine with a molecular weight
of 184.24  (Weast, 1972).  Existing as a grayish-yellow,
white, or reddish-gray crystalline powder  (melting  point
128°C; boiling point 400°C  (Standen, 1972)),  benzidine's
solubility increases as water temperature  rises.  One gram
of benzidine will dissolve  in 2.5 liters of  cold  water.
Solubility is greatly  enhanced with dissolution  into organic
solvents  (Stecher, 1968).   Benzidine  is easily  converted
to and from  its  salt  (Morrison and Boyd, 1972).
                               A-l

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     Diazotization reactions involving benzidine will result
in colored compounds  (color will vary with molecular structure)
Because of their color, azo compounds are important as dyes
for industrial use (Morrison and Boyd, 1972).  The pKa values
for the amino groups  in benzidine were reported to be 4.66
and 3.57  (Weast, 1972).
                               A-2

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                         REFERENCES

Morrison, R.T., and R.M. Boyd.,  eds.  1972.  Organic chemistry,
2nd ed.  Allyn and Bacon, Inc.,  Boston.

Standen, A., ed. 1972.  Kirk-Othmer Encyclopedia of Chemical
Technology.  Inter science Publishers, John Wiley and Sons,
Inc., New York.

Stecher, P.G., ed. 1968.  The Merck Index. 8th ed. Merck
and Co., Inc., Rahway, N.J.

Weast, R.C., ed. 1972.  Handbook of chemistry and physics
53rd ed.  CRC Press, Cleveland, Ohio.
                               A-3

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AQUATIC LIFE TOXICOLOGY



     No appropriate data are available for freshwater or



saltwater organisms and benzidine.
                              B-l

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CRITERION FORMULATION



                          Aquatic Life



     No freshwater or  saltwater criterion  can  be derived  for



benzidine using  the Guidelines because  no  Final Chronic Value  for



either fish or invertebrate  species or  a good  substitute  for



either value  is  available, and there  are insufficient  data  to



estimate a criterion using other procedures.
                               B-2

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Mammalian Toxicology and Human Health Effects
                           EXPOSURE
Introduction
     In general, exposure to benzidine compounds occurs
in factories that synthesize benzidine and its congeners
and convert them to dyes.  It is also probable that some
exposure occurs when the closed system used in synthesis
is cleaned  (Haley, 1975).  Exposure also occurs from breath-
ing contaminated air, ingesting contaminated food, and wear-
ing contaminated clothing  (Meigs, et al. 1951).  Pointing
of brushes by Japanese kimono painters results in the inges-
tion of benzidine dyes  (Yoshida and Miyakawa, 1973), although
ingestion is not generally an important source of exposure.
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Ingestion from Water
     Water could be contaminated with benzidine and  its
derivatives and dyes if plant water  is discharged  into water
supplies serving a residential community.  However,  as of
this time no reports of such contamination have appeared
in the literature.
Ingestion from Food
     While it is possible  for food  to become contaminated
with benzidine and its derivatives  under poor  industrial
hygienic conditions, ingestion of contaminated food  is not
a real contributor to the  overall problem of benzidine toxicity,
     A bioconcentration factor  (BCF) relates the concentration
of a chemical in water to  the concentration  in aquatic organ-
isms, but BCF's are not available for the edible portions
of all four major groups of  aquatic organisms  consumed in
the United States.  Since  data  indicate  that the BCF for
lipid-soluble compounds is proportional  to percent lipids,
BCF's can be adjusted  to edible  portions using data  on percent
lipids and the amounts of  various species consumed by Americans,
A  recent  survey on  fish and  shellfish consumption  in the
United States  (Cordle, et  al.  1978}  found that the per capita
consumption  is 18.7 g/day.  From the data on the 19  major
species  identified  in  the  survey and data on the fat content
of  the  edible  portion  of  these  species  (Sidwell, et  al.
1974) ,  the  relative consumption  of  the  four  major  groups
and  the  weighted  average  percent lipids  for  each group can
be calculated:
                               C-2

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                          Consumption       Weighted Average
        Group               (Percent)          Percent Lipids

 Freshwater  fishes              12                   4.8

 Saltwater fishes               61                   2.3

 Saltwater molluscs              9                   1.2

 Saltwater decapods             18                   1.2

 Using the percentages for consumption and lipids  for each

 of  these groups, the weighted  average percent  lipids is

 2.3 for consumed fish and shellfish.

     No measured steady-state  bioconcentration factor  (BCF)

 is  available for benzidine.  A weighted average BCF of 1,150

 is  available for 3,3'-dichlorobenzidine and the calculated

 octanol-water partition coefficients for the  two  compounds

 are 35.5 and 2,190, respectively.  The proportionality  (Veith,

 et  al. Manuscript)  BCF/BCF = Antilog (0.76 log (P/P)) can

 be  used to calculate a weighted average bioconcentraticn

 factor of 50 for benzidine for  the edible portion of all

 aquatic organisms consumed by  Americans.

 Inhalation

     In the early phases of the chemical and dye  industries,

 the lack of good industrial hygienic practices and the use

of open systems made inhalation one of the principal routes

of entry of benzidine and its  derivatives into the body.

Similar inhalation exposures can occur at the  present time

unless workers wear respirators and protective clothing

while cleaning the equipment (Haley, 1975).
                               C-3

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Dermal



     Skin absorption  is the most important path of  entry



into the body.  Intact skin is readily penetrated by benzi-



dine and 3,3'-dimethylbenzidine  (Meigs, et al. 1951).   3,3'-



Dichlorobenzidine, because of its nonvolatility and large



particle size, presents less of an inhalation and skin  pene-



tration hazard than benzidine (Gerarde and Gerarde, 1974;



Rye, et al. 1970).  It is the light, fluffy, powdery nature



of benzidine base that poses the tumorigenic hazard to  benzi-



dine workers from skin absorption (Barsotti and Vigliani,



1952).  The ease of skin penetration determines the following



order of decreasing toxicity from these chemicals:  benzidine,



3,3'-dimethoxybenzidine, and 3,3'-dichlorobenzidine (Rye,



et al. 1970).



     Environmental conditions of high air temperature and



uumidity increase skin absorption of benzidine,  3,3'-dime-



thoxybenzidine, 3, ' -d.1' chlorobenzidine,  and 3 ,3 '-dimethyl-



benzidine.   Higher amounts of benzidine are found in the



urine of workers who perspire freely and have a  wet skin



(Meigs, et  al. 1954).   Urinary benzidine measurements  indicate



that benzidine does not accumulate  in body tissues,  but



no direct human tissue determinations have been  performed



to absolutely establish this concept (Meigs,  et  al.  1951).
                              C-4

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                      PHARMACOKINETICS



Absorption and Distribution



     Benzidine is rapidly absorbed after intravenous injec-



tion into rats with maximum concentrations of free and bound



benzidine being found at two and three hours, respectively.



The highest concentrations were found in the blood, followed



by liver, kidney, spleen, heart, and lung (Soloimskaya,



1968).   Body distribution of benzidine in various tissues



and urine 4 and 12 hours after intraperitoneal injection



of 100  mg/kg was as follows:  high concentrations in the



stomach, stomach contents, and small intestine at 4 hours,



and in the small intestine and its contents at 12 hours



(Baker  and Deighton, 1953).  The amine content of the  erythro-



cytes was low at both time intervals.  Conjugated material,



indicative of metabolites, was high  in tissues and urine



at 12 hours.  Benzidine concentrations in the liver, the



target organ for toxicity in rats, were  relatively high



and constant over the 12-hour period.  When  rats were  given



20 mg of 3,3'-dimethylbenzidine subcutaneously once a  week



for eight weeks, the highest amine content was found  in



the Zymbal's gland followed by the kidney, omentum, spleen,



and liver (Pliss and Zabezhinsky, 1970) .



Metabolism and Excretion



     A pharmacokinetic study of benzidine uniformly labeled


     14           •
with   C and dichlorobenzidine labeled  in the 3,3' positions



indicated that substitution in the 3,3'  positions  of  the



benzidine molecule significantly  affects the routes of meta-



bolism and excretion.  The blood  half-life  for benzidine
                              C-5

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was 68 hours in the  rat  and 88  hours  in  the dog.   The  weekly
excretions of a dose of  0.2 mg/kg of  benzidine  in  the  rat,
dog, and monkey were 97,  96, and 83 percent respectively.
The excretion values for  the dichloro compound  were  98,
97, and 88.5 percent,  respectively.   Biliary  excretion appears
to be the main route of  excretion of  the dichloro  compound
in all three species.  The dog  and monkey excrete  free benzi-
dine with the urine, while the  rat uses  the biliary  route.
The urinary bladder  of the dog  had a  high content  of benzi-
dine, suggesting  that  this is  the reason for  urinary bladder
cancer  in this species (Kellner, et  al.  1973).
     The various  metabolites  reported for benzidine  and
its congeners  are given  in Table 1.   It  can be  seen  that
various  species  handle these  chemicals in different  ways
and  that  the  animal  metabolites differ considerably  from
those  excreted by humans.  The  improvements  in  analytical
techniques  have  made identification  of differences more
'positive.   Of  greatest interest are  the  human studies which
will  now be discussed.
      A single  oral dose of 100  mg  of benzidine  to  a  human
 resulted in the  excretion of  free  benzidine  and its  mono-
 and diacetylated conversion products in the  urine.  The
 entire dose was  not recovered indicating that fecal  excre-
 tion probably occurred.   This cannot be proven because the
 feces were not analyzed  .(Engelbertz  and Babel,  1953) .   After
 ingesting 200 mg of benzidine,  persons excreted free benzi-
 dine and N-hydroxyacetylamino benzidine in their urine (Troll,
 et al. 1963) .  In plant  workers exposed to benzidine in
                                C-6

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                                 TABLE 1

        Metabolites Formed by Biotransformation of Benzidine and
                    Benzidine Derivatives in Animals
Compound
Species
Metabolites
Reference
Benzidine
Human

Human

Human
                Human
                Human
                Monkey


                Dog

                Dog

                Dog


                Dog


                Dog

                Dog



                Guinea  pig

                Guinea  pig


                Guinea  pig

                Guinea  pig

                Guinea  pig
Acetyl N-hydroxy
   compound
N-Hydroxy acetyla-
   minobenzidine
Monoacetylbenzidine
   and diacetyl-
   benzidine
3-Hydroxybenzidine
3,3'-Dihydroxy-
   benzidine

Monoacetylbenzidin-
                 3-Hydroxybenzidine
                    and glucuronide
                 3-Hydroxybenzidine
                    hydrogen sulfate
                 3-Hydroxybenzidine
                 4,4'-Diamino-3-
                    diphenyl hydro-
                    gen sulfate
                 4-Amino-4-hydroxy-
                    biphenyl
                 Monoacetylbenzidine
                    and diacetyl-
                    bendizine

                 4'-Acetamido-4-
                    aminodiphenyl
                 4'-Acetamido-4-
                    amino-3-diphenyl
                    hydrogen sulfate
                 4'-Amino-4-diphenyl
                    sulfamic acid
                 N-Glucuronides

                 4'-Acetamido-4-
                    diphenyl sul-
                    famic  acid
Troll, et al. 1963
                                                       Haley,  1975
Haley, 1975
Haley, 1975

Haley, 1975

Rinde and
Troll, 1975

Troll and
Nelson, 1958
Sciarini and
Meigs, 1958
Bradshaw and
Clayson, 1955
Sciarini, 1957
Clayson, et al.
1959
                      Haley, 1975
                      Haley, 1975

                      Clayson, et al.
                      1959

                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959
                                 C-7

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                             TABLE 1 (Cont'd)
Compound
Species
Metabolites
 Reference
                Rabbit


                Rabbit

                Rabbit

                Rabbit


                Rabbit

                Rabbit


                Rabbit


                Rat

                Rat



                Rat


                Rat

                Rat


                Rat


                Rat

                Rat



                Mouse


                Mouse



                Mouse

                Mouse
                 3-Hydroxybenzidine
                    sulfate and
                    glucuronide
                 4'-Acetamido-4-
                    aminod iphenyl
                 3-Hydroxybenzidine

                 4'-Acetamido-4-amino-
                    3-diphenylyl
                    hydrogen sulfate
                 4'-Amino-4-diphenylyl
                    sulfamic acid
                 4'-Acetamido-4-di-
                    phenylyl sulfamic
                    acid
                 N-Glucuronides
                      Troll and
                      Nelson,
                      1958
                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959

                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959

                      Clayson, et al.
                      1959
                 3,3 '-Dihydroxy-
                    benzidine
                 N-Glucuronides
                 4'-Acetamido-4-
                    Aminod iphenyl

                 3-Hydroxybenzidine

                 4,4'-Diamino-3-di-
                    phenyl hydrogen
                    sulfate
                 4'-Acetamido-4-amino-
                    3-diphenylyl hy-
                    drogen sulfate
                 4'-Amino-4-diphenylyl
                    sulfamic  acid
                 4'-Acetamido-4-di-
                    phenylyl  sulfamic
                    acid

                 Monoacetylbenzidine
                    and diacetylben-
                    zidine
                 Monoacetylated  3-
                    hydroxybenzidine
                    glucuronide  and/or
                    ethereal  sulfate
                 N-Hydrogen sulfate
                    and/or glucuronide
                 3-Hydroxybenzidine
                    glucuronide
                      Haley,  1975
                      Elson,  et al.
                      1958
                      Clayson,  et al.
                      1959
                      Clayson,  et al.
                      1959

                      Clayson,  et al.
                      1959
                      Clayson,  et al.
                      1959

                      Clayson,  et al.
                      1959

                      Clayson,  et al.
                      1959
                      Clayson,  et al.
                      1959
                      Sciarini
                      Meigs,
                      1961a
         and
                               and
Sciarini
Meigs,
1961a
Sciarini and
Meigs, 1961a
Sciarini and
Meigs, 1961a
                                C-8

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                            TABLE 1  (Cont'd)
Compound
Species
Metabolites
Reference
                Mouse

                Mouse


                Mouse


                Mouse
3,3'-dimethyl-  Human
  benzidine     Human
(orthotolidine) Human

                Dog
3,3'-Dimethoxy- Dog
  benzidine
(dianisidine)

3-Methoxyben-   Rat
  zidine (mono-
  substituted
  dianisidine)
                 4'-Acetamido-4-amino-
                    diphenyl
                 4,4'-Diamino-3-di-
                    phenyl hydrogen
                    sulfate
                 4'-Acetamido-4-amino-
                    3-diphenylyl hy-
                    drogen sulfate
                 N-Glucuronides
                 Diacetyl-o-tolidine
                 5-Hydroxy-o-tolidine
                 Monoacetyl-o-tolidine

                 5-Ethereal sulfate
                    of o-tolidine

                 Unidentified diamine
                   metabolite
                      Clayson, et al.
                      1959
                      Clayson, et al.
                      1959

                      Clayson, et al.
                      1959

                      Clayson, et al.
                      1959

                      Dieteren,  1966
                      Dieteren,  1966
                      Dieteren,  1966

                      Sciarini and
                      Meigs,  1961b

                      Sciarini and
                      Meigs,
                      1961b
                 4-Amino-4'-acetamido-
                   3-methoxybi-
                   phenyl
                                        Laham,  1971
                                  C-9

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unknown quantities, free  benzidine,  its roono-and  diacetylated
derivatives, and 3-hydroxybenzidine  were  identified  in  the
urine.  The latter compound comprised 78.5  to  89.7 percent
of the total  (Sciarini  and Meigs,  1961a).   This work was
a repeat of an earlier  study  by Meigs, et al.  (1954), and
confirmed the previous  findings.   It' has  been  suggested
that an 8-hour exposure to an air  concentration of 0.018
mg/m  of benzidine would  result  in a urinary excretion  of
not more than 0.026 mg/1  of diamines.  Thus an air exposure
to 0.02 mg/m  or less of  benzidine would  be safe  (Meigs,
et al. 1954).
     Dyestuff factory workers exposed to  benzidine excreted
free benzidine,  4-amino-4-oxybiphenyl, and  monoacetylbenzi-
dine  in  their urine  (Vigliani and  Barsotti, 1962).
     Exposure to 3,3'-dimethylbenzidine  results  in urinary
excretion  of  free  3,3'-dimethylbenzidine,  its  diacetyl  deri-
vative,  and 5-hydroxy-3,3'-dimethylbenzidine.  Although
the  monoacetylated  derivative was  not detected,  there is
a probability of its  formation because  3,3'-dimethylbenzidine
appears  to be metabolized similarly  to  benzidine  (Dieteren,
 1966) .
      3,3'-Dichlorobenzidine  has been identified  in  the  urine
 of workers handling benzidine yellow.   This establishes
 the weakness of the azo linkage in dyes  made  from this  compound
  (Akiyama,  1970).
      It is questionable how comparable  animal  data  are  to
 human data, and whether the former allow predictions to
 be made concerning the metabolic conversion of chemicals
  in various species.  This is taken into consideration in
                               C-10

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 the  following  discussion of  the  animal data  in Table  1 and



 their  relevance  to  the human  situation.   Intraperitoneal



 injection  of 100 mg/kg of benzidine  in mice  produced  free



 benzidine, mono-and diacetylated derivatives as well  as



 the  ethereal sulfates and glucuronates of 3-hydroxybenzidine



 (Sciarini  and  Meigs, 1961a).  -The same dose  of benzidine



 in dogs caused the  excretion  of  free benzidine and conju-



 gates  of 3-hydroxybenzidine but  no acetylated derivatives,



 because the dog  lacks this biotransformation mechanism (Scia-



 rini,  1957).   The ethereal sulfate of 3-hydroxy-benzidine



 has  been identified in dog urine and constitutes 25 to 50



 percent of the administered dose  (Sciarini and Meigs, 1958).



 The  ethereal sulfate and glucuronide were the only metabo-



 lites  found in dogs given 1 g of benzidine or rabbits given



 100  to 300 mg  of this chemical  (Troll and Nelson, 1958).



     The differences in the biotransformation of benzidine



 by the rat, mouse, rabbit, guinea pig, and dog are related



 to the presence or absence of specific enzymatic pathways.



 For  example, the dog cannot acetylate benzidine.  The rat,



 rabbit, and guinea pig can produce 4'-amino- and 4'-acetamido-



 -4-diphenylyl  sulfamic acid whereas the mouse and dog cannot.



 Other metabolites found were  4'-acetamido-4-aminodiphenyl,



 3-hydroxybenzidine,  4,4'-diamino-3-diphenylyl hydrogen sul-



 fate, and 4'-acetamido-4-amino-3-diphenylyl hydrogen  sulfate



 in the rat; and 4'-acetamido-4-aminodiphenyl, 4,4'-diamino-



 3-diphenyl hydrogen  sulfate, and 4'-acetamido-4-amino-3-



diphenylyl hydrogen  sulfate in the mouse.  4,4'-Diamino-



3-diphenylyl hydrogen sulfate was absent from rabbit  and
                              C-ll

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guinea pig urine,  although  the  other metabolites  were  present,



3-Hydroxybenzidine and  4,4'-diamino-3-diphenylyl  hydrogen



sulfate were present  in dog  urine.  In  all  cases,  N-glucuron-



ides were present  (Clayson,  et  al. 1959).   Metabolite  differ-



ences occur when different  routes of elimination  are consider-



ed.  Dogs excrete  the same  benzidine metabolites  in urine



and bile but their feces have no 3-hydroxybenzidine or N-



glucuronides (Clayson,  et al. 1959).  Comparison  of routes



of excretion of benzidine and its dichloro  derivative  in



rats, dogs, and monkeys showed  that the rat eliminated both



compounds in greater quantities in the  feces than  in the



urine; whereas the dog  eliminated the dichloro compound



to a greater extent in  the  feces.  Neither  route was decisive



in the monkey,  but more of both compounds did appear in



the urine (Kellner, et  al. 1973).  Previously it had been



shown that dog fecal excretion of dichlorobenzidine was



ten times greater  than  urinary excretion (Sciarini and Meigs,



1961b), while the opposite was true for benzidine  (Sciarini



and Meigs, 1958).



     When benzidine-based azo dyes were fed to monkeys,



benzidine and monoacetylbenzidine were found in the urine



(Rinde and Troll,  1975)  .  This shows that the monkey,  like



man, can reductively cleave the azo linkage (Akiyama,  1970).



     Intraperitoneal injection of dimethylbenzidine,  dime-



thoxybenzidine and dichlorobenzidine in dogs resulted  in



recovery of part of these chemicals in nonmetabolized  form.



The dichloro compound was not metabolized whereas the  other



two derivatives of benzidine were recovered from urine  as
                              C-12

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unidentified conjugated ethereal sulfates (Sciarini and



Meigs, 19615).



                           EFFECTS



Acute/ Subacute, and Chronic Toxicity



     lH vitro studies have shown that benzidine, 3,3'-dime-



thylbenzidine,  and 3,3'-dimethoxybenzidine are moderate



reducers of cytochrome c.  3,3'-Diaminobenzidine is a strong



reducer, whereas 3,3'-dichlorobenzidine is an ineffective



reducer.  It has been suggested that there is a relationship



between carcinogenic potential and the reduction of cyto-



chrome c (Hirai and Yasuhira,  1972; Gammer and Moore, 1973).



     There is a significant increase in urinary B -glucuro-



nidase activity in workers exposed to benzidine.  The ele-



vated activity, although decreased by removal from benzidine



exposure, does not return to normal levels (Kleinbauer,



et al. 1969; Popler, et al. 1964).



     While 3,3'-dimethylbenzidine administered  subcutaneously



to rabbits had no effect on blood phenolase activity, benzi-



dine decreased the activity of this enzyme (Nakajima, 1955).



Rats injected with benzidine showed reduced catalase and



peroxidase activity as well as a  reduction in erythrocytes



and thrombocytes and an  increase  in leucocytes  (Soloimskaya,



1968).  An intraperitoneal dose of 12.7 mg/kg of benzidine



in rats increased liver glutathione from  182 mg/100 g  to
                                t


272 mg/100 g in 24 hours  (Neish,  1967).



     Dermatitis has been  reported  in workers  in the benzi-



dine dyestuff  industry,  involving  both benzidine and  its



dimethyl derivative.   Individual  sensitivity plays  a  promi-
                              C-13

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nent role in this condition  (Schwartz, et al.  1947).
     Glomerulonephritis  and  nephrotic  syndrome  have  been
produced in Sprague-Dawley  rats  fed  0.043 percent  N,N'-diace-
tylbenzidine.  Both  sexes developed  proteinuria in 3  to
4 weeks.  After  2 months the females were excreting  0.1
g of protein per 24  hours.   The  females  developed  severe
anemia which was rarely  seen in  the  males.   The former also
had a hypoproteinemia, hyperlipemia, and generalized edema.
Glomerular  lesions  in the  females  consisted  of  florid epi-
thelial  crescents,  progressive  sclerosis, and  glomerular
obliteration.   In  the males, the lesions were  slower in
developing  and less extensive,  but all males showing the
nephrotic  syndrome  also  developed  testicular atrophy. There
were  morphological  similarties  between .the  human nephrotic
 syndrome and  that  induced by N,N'-diacetylbenzidine  in rats,
 including  extracapillary cell proliferation, formation of
 luxuriant  crescents in 80 percent  of• the glomeruli,  intact
 glomerular tufts,  and the presence of  normal glomeruli in
 the advanced  stages of the syndrome (Harman, et al.  1952;
 Harman,  1971).
      Rats fed N,N'-diacetylbenzidine or  4,4,4',4'-tetramethyl-
 benzidine developed glomerular lesions with fat-filled spaces
 in the glomerular tuft  from 2 to 4.5 months of treatment
 (Dunn, et al. 1956).  Severe glomerulonephritis developed
 in rats .receiving N,N'-diacetylbenzidine by subcutaneous
 (100 mg) or intraperitoneal  (100 or 200  mg)  injections.
 These lesions were  dose related (Bremner and Tange,  1966).
 A similar  low grade glomerulonephritis has been produced
                               C-14

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 in rats  fed  benzidine  (Christopher  and  Jairam,  1970) .



      Mice  fed  0.01  and  0.08 percent benzidine dihydrochlor-



 ide developed  the following toxic symptoms:  decreased  car-



 cass,  liver, and kidney weights; increased  spleen  and thymus



 weights; cloudy swelling of the  liver;  vacuolar  degeneration



 of the renal tubules; and  hyperplasia of  the myeloid ele-



 ments  in the bone marrow and of  the lymphoid cells in the



 spleen and thymic cortex.  There was a  dose dependent body



 weight loss of 20 percent  in males  and  7  percent in females.



 Moreover, male mice were more sensitive to benzidine than



 female mice  (Rao, et al. 1971).  This disagrees  with Barman's



 (1971) findings in  rats, but it may only  be a species differ-



 ence  in response.



 Synergism.and/or Antagonism



     No available data.



 Teratogenicity



     Embryonic mouse kidney cultures have an increased  sur-



 vival time but show hyperplastic epithelial changes in  the



 presence of 3,3'-dimethylbenzidine  (Golub, 1969; Shabad,



 et al. 1972).  Administration of 8  to 10 mg of 3,3'-dimethyl-



 benzidine to mice during the last week of pregnancy resulted



 in lung adenomas and mammary gland  tumors in their  progeny.



These tumors could have resulted from transplacental transmis-



sion of the chemical or from its presence in the milk (Golub,



et al. 1974).  No teratogenic effects of benzidine  deriva-



tives in  humans have been reported.
                              015

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Mutagenicity



     The results of  the  Ames  assay on  the mutagenicity  of



benzidine are positive  (Ames, et- al. 1973; McCann,  et al.



1975; Garner, et al.  1975).   With metabolic  activation,



benzidine causes an  increase  in the recovery of  histidine



revertants in Salmonella typhimurium strain  TA 1537  and



TA 1538, both sensitive  to frameshift mutagens.  The greatest



increase was seen with TA 1538.



     Another more recently developed assay,  used to  screen



for putative mutagenic/carcinogenic compounds, has been



used to test benzidine.   This assay detects  the  inhibition



of DNA synthesis in  HeLa cells by test compounds (Painter



and Howard, 1978).   The  concentration of a compound  that



is required to inhibit DNA synthesis by 40 percent corresponds



with its mutagenic effects in Salmonella typhimurium.  Benzi-



dine has"been shown  to be positive in this DNA synthesis



inhibition test  (Painter  and Howard, 1978).



     Results of a Salmonella mutagenesis assay indicate



that benzidine causes a  significant increase in the  reversion



index of tester strains  TA 98 and TA 1538 when the compound



is activated by the  addition of human liver microsomes (U.S.



EPA, 1978).



Carcinogenicity



     Benzidine and its derivatives are carcinogenic  in both



experimental animals and  humans.  In the latter these chemi-



cals have been shown to  produce bladder cancer after a long



latent period (Clayson,  1976).  Additionally, these compounds



produce dermatitis, cystitis,  and hematuria in humans,  indi-
                               C-16

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                                 TABLE 2

          Effects of Benzidine, Its Congeners, and Metabolites
                        On Various Animal Species
                        (Adapted from Haley, 1975)
Species
Carcinogen
Effect
Mouse
Rat
Hamster
Rabbit
Dog



Monkey

Human
Benzidine

3,3'-Dihydroxybenzidine



Benzidine and its sulfate
                3,3'-Dihydroxybenzidine
Dianisidine

o-Ditoluidine


3,3'-Benzidinedioxyacetic
acid

3,3'-Dichlorobenzidine
N,N'-Diacetylbenzidine

Benzidine

o-Ditoluidine

Benzidine
Benzidine



Benzidine

Benzidine
Hepatoma, lymphoma, bile duct
  proliferation
Hepatoma, lymphoma, bile duct
  proliferation, benign bladder
  papilloma

Cirrhosis of liver, hepatomas,
  carcinoma of Zymbal's gland,   ,
  adenomacarcinoma, degeneration j
  of bile ducts, sarcoma, mammary
  gland carcinoma   .             ,
Hepatoma, adenocarcinoma of      ;
  colon, carcinoma of fore- .     '
  stomach, Zymbal's gland
  carcinoma, bladder carcinoma   ;
Zymbal's gland carcinoma,        '
  ovarian tumor
Papilloma of stomach, Zymbal's
  gland carcinoma, mammary
  tumor, leukemia
Papilloma of bladder, hepatic
  sarcoma

Extensive cancer
Chronic glomerulonephritis

Hepatoma, liver carcinoma,
  cholangiomas
Bladder cancer

Proteinuria, hematuria,  liver
  cirrhosis, myocardial  atrophy,
  bladder tumor, gall bladder
  tumor

Recurrent cystitis,  bladder
  tumor, convulsions, liver
  cirrhosis, hematuria

No  pathological changes

Bladder  tumor,  papilloma,
  chronic cystitis,  hematuria
 3,3'-Dimethoxybenzidine.
 53,3'-Dimethylbenzidine.
                                   C-17

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eating an early attack on  the  urinary bladder  and  presenting

a sign that unless exposure  is  stopped, cancer may result

(Haley, 1975) .  Table 2 gives  various animal species  and

the type of cancer induced in  them by benzidine  and its

congeners.  It should be noted  that only  the dog gets uri-
                                    /
nary bladder  cancer  similar  to  that seen  in humans after

exposure to benzidine.  The  animal cancers  in  general differ

significantly in  their  locations.  This may be related to

differences in specific target  tissues or to differences

in excretory  pathways.  In some cases, excessive dosage

may cause death due  to  toxicity,  thus preventing the  develop-

ment of bladder cancer  (Haley,  1975).

     Benzidine and many other  aromatic amines  attack  the

urinary bladder and  other  organs (Hueper, 1954).  However,

it  is  the metabolites  of  these compounds  that  are  considered

to  be  the proximate  carcinogens (Clayson, 1969).  These

aromatic  amines are  ring  hydroxylated, converted to N-hydroxy-

lated,  acylated and  deacylated derivatives, and  conjugated

with  sulfate  and  glucuronide (Haley,  1975) .   It  has been

 suggested  that the  conjugated  N-hydroxy  compounds  are the

 active carcinogens  _iii vivo.   Bladder  cancer has  been induced

 in  rabbits  and dogs  fed benzidine,  but  these  findings are

 controversial (Haley,  1975).  Spitz,  et  al.  (1950) induced

 papillary carcinoma in one of  seven  dogs  fed  benzidine for

 5 years,  but the  cancer only appeared 7.5 years  after the

 beginning of the  experiment.  Orally administered  benzidine

 did not produce urinary bladder cancer  in dogs  (Marhold,

 et al. 1967).  No tumors  were found  in  female beagle dogs

 fed 1 mg/kg  5 days a week for 3 years (Deichmann,  et al.



                               C-18

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 1965).   The  lack  of  a  carcinogenic  effect  in  dogs  in  these



 latter  two studies is  probably  related  to  the known long



 latency for  benzidine  cancer  induction  and  the shortness



 of  both studies.



      Extensive  bile  duct proliferations and cysts  appeared



 along with cholangiofibrosis, hepatomas, and  liver cell



 carcinoma but no  urinary bladder  tumors were  found in  hamsters



 fed benzidine at  0.1 percent  of the diet throughout their



 life  spans (Saffiotti, et al. 1967).



      Benzidine  administered subcutaneously  to rats at  a



 rate  of  15 mg/week produced liver injury, cirrhosis, hepa-



 tomas,  sebaceous  gland carcinomas, and  adenocarcinomas of



 the rectum but  no bladder tumors  (Spitz, et al.  1950).



 Rats  fed 0.125  percent of dihydroxybenzidine  in  the diet



 developed liver cirrhosis, hepatomas, adenocarcinomas  of



 the colon, Zymbal's gland carcinoma, and squamous  cell car-



 cinomas of the  stomach.  One  sessile papilloma and two kerati-



 nized squamous  cell carcinomas were found  in  the bladder



 wall  (Baker,  1953).  Intraperitoneal or subcutaneous  injection



 of N,N'-diacetylbenzidine in Wistar rats induced tumors



 of Zymbal's gland and of the mammary glands 6  to 15 months



 later.  Glomerulonephritis was also reported  and appeared



 to be dose related.  Female Sprague-Dawley rats given  12



 to 50 mg/rat  orally developed mammary gland carcinomas (Griswold,



 et al. 1968) .



     Early cirrhosis occurred in rats given benzidine  by



 subcutaneous  injection for 6 months (Pliss, 1963).  Injec-



 tion site sarcomas, hepatomas, and Zymbal gland tumors were



also found,  and constituted 70 percent of the  tumors in





                              C-19

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these rats  (Pliss,  1964).   Benzidine was more toxic to the



females.  Tumors  of Zymbal's gland and the liver were induced



by 3,3 '-benzidine dicarb&xylLc aeg.idw4t.h4.ji 1 year (Pliss,



13b9; .  Benzidine,  i-n  5  mg  weekly. doses, produced..intestinal



v.i,.i-..ors  in rats  (Pliss,  et al.  1973)-.   A cumulative dos.e



c:~ 0.75 me/kg of  benzidine  for 15  days produced tumors in



"0 of 27 rats,  including 19 hepatomas, 18 cholangiomas,



7 intestinal tumors and  4 sebaceous gland carcinomas.   Subcu-



taneous tetramethylbenzidine doses of  from 4.15 to 8.3 g/kg



produced benign tumors at the  injection site (Holland, et



al. 1974).



     Female Wistar  rats  given  a  single intraperitoneal injec-



tion of 100 or  200  mg of N,N.'^diacetylbenzidine subcutan-



eously developed  Zymbal  gland  and  mammary gland, tumors after



6 to 15 months.   The 100 mg intraperitoneal injection  pro-



duced tumors in 11  out of 18 rats  while the 200 mg  dose



gave no tumors  (Bremner  and Tange,  1966).



     Hepatomas, bile duct proliferation,  and benign  papillomas



of the urinary  bladder *'ei.e found  in Delphi  albino mice inject-



ed subcutaneously with 300  mg  of benzidine  or dihydroxybenzi-



dine»  Only the latter chemical caused  the  bladder changes



(Baker,  1950).



     Benzidine or 3,3-dihydroxybenzidine  administered  subcu-



taneously at 6 mg weekly for 52 weeks  produced  tumors  in



exposed mice ia 70  weeks.   Benzidine induced  hepatomas and



lymphomas whi]_° the  3 ,.1-dihydroxy  derivative  induced lymphomas



and benign intestinal polyps.  The  significance  of the lympho-



mas i.=- obscure because one-third of the controls  developed



this condition  spontaneously  (Bonser,  et  al.  1956).  Subcuta-






                               C-20

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neous administration of 3,3-dihydroxybenzidine in mice caused
tumors of the liver and mammary glands as well as leucosis
(Pliss, 1961).   Inner organ tumors developed after skin
application of  the chemical.   Subcutaneous weekly doses
of 6 mg of benzidine to C3HA mice induced hepatomas in 31
of 46 animals after 15 to  16 months.   One animal developed
a pulmonary adenocarcinoma (Prokofjeva, 1971).
     3,3-Dimethylbenzidine in a cumulative dose of 5.4 g/kg
for 241 days induced 11 gastrointestinal tract tumors, 7
hepatomas, 7 bone tumors and 4 Zymbal's gland carcinomas
in rats.  Total oral doses of 500 mg in Sprague-Dawley rats
produced 4 mammary carcinomas in 9 months in 3 of 16 surviving
animals  (Griswold, et al.  1968).  Subcutaneous injection
of 3,3'-dimethylbenzidine in rats caused skin tumors, large
sebaceous gland tumors, and mammary tumors in 60  to 70 percent
of the animals.  When 20 mg of the chemical was  implanted
subcutaneously, hepatocellular carcinomas and subcutaneous
sarcomas were produced  (Pliss and Zabezhinsky, 1970).
     3,3'-Dimethoxybertzidine given subcutaneously  to  rats
induced  Zymbal gland tumors in two animals and an  ovarian
tumor and a  fibroadenoma of the mammary gland in  another
one  (Pliss,  1963) .  Both male and female Fischer  strain
rats developed tumors of the gastrointestinal tract,  skin,
breast,  and  ear duct after receiving  260 oral 10  mg  doses
of 3,3'-dimethoxybenzidine.  The'  latency was  293  days  (Weis-
burger,  et al.  1967).
     Subcutaneous administration  of 3,3'-dichlorobenzidine
to rats  induced tumors  in 74 percent  of  the animals  (Pliss,
1963).   Tumors appeared in the skin,  sebaceous  and mammary
                               C-21

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glands/ intestines, bones,  and  urinary bladder.  Dichloroben-
zidine given by ingestion or  injection into  the underlying
fat produced sarcomas  at the  injection site,  an adenocarci-
noma in the intestine,  papillomas  in  the  urinary bladder,
and tumors in the  sebaceous and mammary glands  (Pliss,  1959) .
Total doses of 300  mg/rat orally of dichlorobenzidine pro-
duced no  tumors  (Griswold,  et al.  1968).   Rats  fed  1,000
mg/kg in  the diet  developed mammary gland tumors in both
sexes and  Zymbal's  gland and  hematopoietic tumors  in males
 (Stula, et al. 1971,  Stula, et al. 1975).  Progeny  of BALB/c
mice given total  subcutaneous doses of 8  to  10  mg  of dichloro-
benzidine  had a  significant increase  in  tumor incidence.
Tumors  developed  in 13 of  24  mice; with  4 adenocarcinomas
of  the  mammary gland,  5 lung  adenomas and 7  cases  of lymphatic
 leukemia  (Golub,  et al. 1974) .
      The  carcinogencity risk  for  workers  exposed to benzi-
 dine  is 14 times  higher than  for  the  unexposed  population
 (Case,  et al.  1954).   In the  American dyestuff  industry,
 24  cases  of  bladder carcinomas were  found in workers exposed
 to  aromatic  amines including  benzidine.   The latency for
 tumor  development was 12 years (Gehrman,  1936).  In England
 the tumor induction time averaged 16  years but  one case
 occurred in 2 years (Case,  et al.  1954).   In 30 cases of
 bladder tumors the induction period  varied from 8  to 32
 years, with an average of  15.9 years.  The concentration
 of benzidine in the exposure appeared to be the main factor
 in early tumor induction.   Benzidine manufacturing was  asso-
 ciated with 14 papillomas, 7 carcinomas and 2 cases in  which
                                C-22

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 the  papillomas  were converted  to  carcinomas  (Scott,  1952).



 Only a  few weeks of exposure followed by a latent  period



 of several years can produce bladder tumors  (Deichmann  and



 Gerarde,  1969).  A latent period  of 18.6 years  has also



 been reported  (Hamblin, 1963).  Initial exposure concentration,



 exposure  duration and years of  survival following  exposure



 as well as work habits and personal hygiene  are  involved



 in the development of carcinomas  where benzidine appears



 to be implicated (Rye, et al.  1970).  There  is  little doubt



 that benzidine  exposure is associated with an increase  in



 the  occurrence  of bladder cancer  (Int. Agency Res. Cancer,



 1972; Riches, 1972; Sax, 1975).   However, there  is a lack



 of information on the exact concentrations of benzidine



 to which  workers have been exposed.



     Long exposure to benzidine produced bladder tumors



 in 13 out of 25 men (Zavon, et al. 1973).  Comparison of



 the  two groups showed that the tumor group was exposed  to



 benzidine for an average of 13 years while the non-tumor



 group was exposed for an average  of less than 9 years.



 Observations were carried out for approximately 12 years



 following exposure.   Ambient air  benzidine in the  plant



 varied from 0.005 to 0.415 mg/m   with one area giving a



 value of 17.6 mg/m  (Wendel, et al. 1974).   Death  records



of 171 workers showed that 18 were due to bladder  and kidney



cancers  and that there was a higher rate of  neoplasms of



 the digestive system.   It appeared that there could have



been  a synergistic  effect between benzidine  and ^-naphthyla-
                               C-23

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mine, since these workers  were  exposed  to  both  chemicals



(Mancuso and El-Attar,  1966,  1967).



     When benzidine dyestuff  manufacturing  begins  in  any



country the incidence of bladder  tumors among exposed workers



increases.  Table 3 shows  the times of discovery of aromatic



amine bladder cancer in a  number  of countries.  Urinary



system tumors occurred  in  17  percent of the workers in one



benzidine plant.  The highest rate of tumors was in the



group exposed for 6 to  10  years  (Kuzelova,  et al.  1969).



Men working in a French aromatic  amine plant developed bladder



tumors.  One Normandy factory had 54 cases, with 17 occurring



prior to 1947 and 34 subsequent to 1947.  Symptoms of hema-



turia and stranguria were  found in 18 cases (Billiard-Duchesne,



1960) .



     In Italy, 24 cancers  were  found in workers exposed



to benzidine or benzidine-xB-naphthylamine  (Vigliani  and



Barsotti, 1962).  Italian  benzidine workers were found to



have developed 47 cases of bladder cancer during the period



from 1931 to 1960.  There  were 21 carcinomas and 16 papil-



lomas.  During the period  from 1931 to 1948, 13 of 83 workers



developed bladder carcinomas from benzidine (Barsotti and



Vigliani, 1952).  The greatest exposure occurred in workers



in filtration, pressing, drying, and milling of benzidine.



Maximum latency for benzidine tumors was 16 years from the



cessation of exposure.   Ten papillomas and seven carcinomas



were found in a cohort of 858 benzidine dyestuff workers



(Forni, et al. 1972).
                              C-24

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                   TABLE 3
Time of Discovery of Aromatic Amine Bladder
       Cancer by Country (Haley, 1975)
 Country                             Year
 Germany                             1895
 Switzerland                         1905
 United Kingdom                      1918
 U.S.S.R.                            1926
 United States                       1931
 Austria                             1932
 Italy           '                    1936
 Japan                               1940
 France                              1946
                  C-25

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     Studies in dyestuff plants in Japan showed 100 cases
of bladder cancer during the period 1949 to 1970.  Benzidine
production workers accounted for 11.25 percent of  the cases
and benzidine users for 1.45 percent.  Eight cases developed
cancer of the upper urinary tract and not the bladder.
                                     t
There was a long latent period of 16.25 years  (Tsuchiya,
et al. 1975).  The silk kimono painters are the highest
risk bladder cancer group  in Japan because they point their
brushes,  thereby ingesting benzidine dyes  (Yoshida and Miya-
kawa, 1973).
     There  was a high  incidence of bladder tumors,  (21.3
percent)  in benzidine  workers  in a coal tar dye factory.
The  latent  period was  18.4 years for papillomas and 18.7
for  carcinomas  (Goldwater, et  al. 1965).  A further study
showed  that the combined  exposure to benzidine plus ^-naph-
thylamine increased  the bladder cancer rate to 45.5 percent
 (Kleinfeld, et al.  1966).  Occupational bladder cancers
are  morphologically  similar  to spontaneous bladder tumors
found  in the  general  population.  Both have a  tendency  for
high recurrence after  treatment.
      At the present  time  there is no evidence  that 3,3'-
dimethylbenzidine,  3,3'-dimethyoxybenzidine, or 3,3'dichloro-
benzidine are human  bladder  carcinogens  (Rye,  et  al.  1970).
However, future  epidemiological study  may  show them to  be
 carcinogenic  agents.   No  bladder  neoplasms related to exposure
 to 3,3'-dichlorobenzidine over a  35-year period were  found.
 However, the following neoplasms  were  reported  in 17  workers:
 two lung cancers,  one bone marrow  cancer,  six  lipomas,  three
                               C-26

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rectal papillomas, two sigmoid colon carcinomas, one prostate
carcinoma, one breast muscle myoblastoma, and one basal
cell epithelioma  (Gerarde and Gerarde, 1974).  No bladder
tumors were found in British workers handling this chemical
but the worker exposure time of less than 16 years could
account for these findings (Maclntyre, 1975).  It is possible
that the latent period for bladder tumors is longer for
3,3'-dichlorobenzidine since workers exposed to benzidine
plus dichlorobenzidine developed such tumors while those
exposed to the latter compound alone did not (Gadian, 1975).
                              C-27

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                     CRITERION FORMULATION
Existing  Guidelines and Standards
      In 1973  the  Environmental Protection Agency proposed
but did not promulgate  a toxic pollutant standard for benzi-
dine  (30  FR 35388).
      The  industrial standards instituted by the Occupational
Safety and Health Administration  in  1974 excluded from regula-
tion  any  compounds containing less than  0.1 percent  benzidine,
These standards did  not recognize a  safe level  of water
contamination and provided  no provisions for  environmental
monitoring.
      New  standards for  benzidine  discharges have  been pro-
posed (41 FR  27012)  based upon  information  on the toxico-
logical and environmental effects and the  fate  of benzidine.
These standards,  promulgated  in 1977, established an  ambient
water criterion for  benzidine of  O.ljug/1.  Effluent  stan-
^ards were set at  10'jug/l (daily  average) with  a  maximum
for any single day  c 50 jug/1.  Based on a  monthly average,
daily loading was  limited to  0.13 kg/1000 kg of benzidine
produced.  The standards set  for  users of benzidine-based
dyes were the same except that the maximum  daily  effluent
concentration of  benzidine was limited to 25 jug/1  (42 FR
2617).
Current Levels of  Exposure
     It is essential that consideration be given  to the
manner in which benzidine and its congeners and the dyes
derived from them  contaminate water  supplies.  In most cases
these chemicals are a hazard only in  the vicinity of  dye
and pigment plants where wastes escape or are discharged.
                              C-28

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A field survey of the Buffalo and Niagara river areas using
the chloramine-T method, with a sensitivity of 0.2 ug/1,
showed no benzidine in the samples.  However, this method
of analysis is photosensitive and leads to low estimates
of benzidine.  Moreover, the samples may have been below
the level of detectability or oxidative degradation may
have converted the benzidine compounds to materials not
detectable by the analytical method used  (Howard and Saxena,
1976).  A Japanese survey of the Sumida River area detected
0.082, 0.140, and 0.233 mg/1 of benzidine in the water.
The authors believed that the benzidine came from azo dyes
by H2S or S02 reduction (Takemura, et al. 1965).
     Information on 3,3'-dimethylbenzidine,  3,3'-dimethoxy-
benzidine, and 3,3'-dichlorobenzidine and their dye deriva-
tives as water contaminants  is non-existent  and research
should be instituted to correct this deficiency.
     It has been stated that benzidine resists  physical
and biological degradation  (Lutin, et al.  1965; Malaney,
et al. 1967; Radding, et al. 1975).  Benzidine  in water
is oxidatively degraded by  free radical,  enzymatic or photo-
chemical processes  (Radding, et al. 1975).   Its half-life
in water has been estimated  to be  100 days.  Air oxidation
of benzidine in  water seems  to occur readily (Howard  and
Saxena, 1976).
     Humic material seems to bind  3,3'-dichlorobenzidine
tightly and  its  degradation  appears to be slower  than benzi-
dine, but the half-lives of  the  two compounds  are  the same
(Radding, et al. 1975).  There is  no  information  available
                               C-29

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on the dimethyl- and dimethoxy  derivatives.   This  deficiency
must be corrected.
     Benzidine  is converted  to  a  chloramine  type compound
during water chlorination  processes  (Jenkins and Baird,
1975) .  Soil and intestinal  bacteria  reduce  benzidine  azo
dyes to free benzidine  (Yoshida and Miyakawa,  1973), and
although  aquatic organisms might  also cause  this same  trans-
formation,  no data  are  available  to prove  this point.   It
should be remembered  that  the  hydrochlorides of benzidine
are  much  more soluble  in water  than  the  free amines  and
are  more  resistant  to  degradation than the latter  (Bowman,
et al. 1976).
Special Groups  at  Risk
     A potential  health hazard exists in the production
of benzidine and  its  congeners and their  conversion  to azo
dyes.  There is no maximum permissible level of contamina-
tion in  the industrial environment although  there  are  spe-
cific  regulations  governing  the manufacture  of benzidine
and  its  congeners  (39 FR 3756)  .  These standards have  re-
duced  the risks to benzidine workers.
      The use of benzidine and  its congeners  poses  a  poten-
 tial risk to workers in biochemical,  chemical, and microbio-
 logical   laboratories where these chemicals are used  as ana-
 lytical   reagents (Collier, 1974;  Veys, 1972; Wood  and  Spen-
 cer, 1972).  The greatest risk occurs in laboratories  working
 with known carcinogens when good laboratory practices  are
 not enforced.  No epidemiological evidence is available
 to determine the exact extent  of the problem.
                               C-30

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      The risk  to the  general  population  from benzidine,



 its congeners,  and  their  dyes is  unknown,  but contamination



 of water supplies,  which  is known to  occur in Japan  (Takemura,



 et al.  1965),  poses a yet to  be determined risk.   There



 also is a potential risk  for  workers  in  the garment,  leather,



 and homecraft  industries  where the  benzidine dyes  are used.



 Basis  and Derivation  of Criteria



      The available  data concerning  the carcinogenicity of



 benzidine in experimental animals are severely limited.



 It is  extremely  difficult to  extrapolate the experimental



 results to man  because, with  the  possible  exception of the



 dog and the rabbit, the target organs are  diffe* >nt.   Moreover,



 the metabolites  produced  by the various  species, in general,



 differ  significantly  from those produced by man  (Haley,



 1975) ,  although  3-hydroxybenzidine  and its conjugation products



 are common to both  man and animals.



     Despite the limitations  of the available  data, a suggest-



 ed  criterion for benzidine was calculated  using the linear



 non-threshold model described in  the appendix.  The calcula-



 tion assumes a risk of 1  in 100,000 of developing  cancer



 as  a result of daily  consumption  of 2 liters  of benzidine



 contaminated water  and the daily  consumption  of 18.7  g of



 benzidine contaminated aquatic organisms.   Based on the



data of Zavon,  et al.   (1973),  a benzidine  criterion of 1.67



 x 10    lwg/1)  is suggested to be  adequate  to protect  the



population consuming the water.



     Epidemiological data indicate that exposure to benzidine



is associated  with an  increase in bladder  cancer in man.
                              C-31

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The possibility  that  benzidine may be  found  in  wastewater
may also pose a  problem.   In order to  determine  the  extent
of the potential problem,  measurements must  be  made  of  waste-
water not only for 'benzidine but -also  for  its congeners.
Moreover, further evaluation must be made  on these chemicals
and their azo dye derivatives to determine their stability
to microbiological degradation.  It is essential that studies
of their carcinogenicity  in experimental animals be  ma,de
at doses which produce a  bare minimum  of liver  pathology.
A detailed pharmacokinetic study should be undertaken to
establish routes of absorption, body transport, storage
and excretion of benzidine, its congeners, and  the azo dyes
synthetized from them.  Programs covering both  industrial
hygienic and epidemiologic aspects of  exposure  to benzidine
and its congeners to  establish the degree of dermal  and
pulmonary absorption  are  a necessity if we are to prevent
this chemically  induced cancer from occurring.
     Under the Consent Decree in NRDC  vs. Train, criteria
are to state "recommended  maximum permissible concentrations
(including where appropriate, zero)  consistent with  the
protection of aquatic organisms, human health, and recreation-
al activities."  Benzidine is suspected of being a human
carcinogen.  Because  there is no recognized safe concentration
for a human carcinogen, the recommended concentration of
benzidine in water for maximum protection of human health
is zero.
     Because attaining a  zero concentration level may be
infeasible in some cases  and in order  to assist the Agency
                               C-32

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     and  States  in  the  possible  future  development  of  water  quality
     regulations, the concentrations  of benzidine corresponding
     to several  incremental  lifetime  cancer  risk  levels have
     been estimated.  A cancer  risk  level provides  an  estimate
     of the additional  incidence of  cancer that may be expected
     in an exposed  population.   A risk  of 10   for  example,  indi-
     cates a probability of  one additional case of  cancer for
     every 100,000  people exposed, a risk of 10   indicates  one
     additional  case of cancer  for every million  people exposed,
     and so forth.
          In the Federal Register notice of availability of  draft
     ambient water  quality criteria,  EPA stated that it is con-
     sidering setting criteria at an interim target risk level
     of 10" , 10   or 10"  as shown in the table  below.
Exposure Assumptions           Risk Levels and Corresponding Criteria^
                               0   10~7         1£~6        1£~5
2 liters of drinking water     0   1.67 x 10"5  1.67 x 10~4 1.67 x 10~3
                                      jug/1         ;ug/l        /ig/1
and consumption of 18.7
grams of fish and shellfish (2)
                               0   5.:
                                      jug/1         /ag/1        ;ug/l
Consumption of fish            0   5.24 x 10~5  5.24 x 10~4 5.24 x  10~3
and shellfish only.
     (1)  Calculated by applying a modified  "one hit"  extrapolation
     model described in the FR 15926, 1979.  Appropriate  bioassay
     data used in  the calculation of the model are  presented
     in Appendix I.  Since the extrapolation model  is  linear
     to low doses, the additional lifetime  risk  is  directly propor-
     tional to the water concentration.  Therefore,  water concen-
                                    C-33

-------
trations corresponding to other risk levels can be derived
by multiplying or dividing one of the risk levels and corres-
ponding water concentrations shown in the table by factors
such as 10, 100, 1,000, and so forth.
(2)  Thirty-two percent of benzidine exposure results from
                                    /
the consumption of aquatic organisms which exhibit an average
bioconcentration potential of 50 fold.  The remaining 68
percent of benzidine  exposure results from drinking water.
     Concentration levels were derived assuming a lifetime
exposure to various amounts of benzidine,  (1) occurring
from the consumption  of both drinking water and aquatic
life grown in water containing the corresponding benzidine
concentrations  and,  (2) occurring solely from the consumption
of  aquatic life grown in  the waters containing the correspond-
ing benzidine concentrations.
     Although total exposure information for benzidine is
discussed  and an  estimate of the contributions from other
sources  of exposure can  be made, this data will not be factored
into the ambient  water quality criteria formulation because
of  the tenuous  estimates.  The criteria presented, therefore,
assume an  incremental risk  from  ambient water exposure only.
                               034

-------
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                               C-48

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                         APPENDIX I
              Summary and Conclusions Regarding
              the Carcinogenicity of  Benzidine*
     Benzidine ((1,I'-Biphenyl)-4,4'-diamine)  is used in
the manufacture of dyes,  as a reagent for  detection of H202
in milk and as a reagent  for hemoglobin.
     It appears that the  greatest  hazard  to exposure from
benzidine occurs during its manufacture.   Absorption through
the skin is the primary route of entry into the body, although
other routes of exposure  such as inhalation and ingestion
also exist.  Exposure to  benzidine,  its derivatives, and
other chemicals involved  in the manufacture of dyes has
long been known to be associated with an elevated incidence
of bladder cancer in workers in Germany,  England, Italy,
France, Switzerland, Japan, and the United States  (see Haley,
et al. 1975, Zavon, et al. 1973, Clayson,  1976).
     Epidemiological data clearly demonstrate that benzidine
is a bladder carcinogen in humans and experimental evidence
indicates that it can induce cancer in a variety of organs
in several species of animals.  Several animal  studies have
reported carcinogenic effects of benzidine in hamsters  (liver),
rats  (liver and Zymbal glands), and mice  (liver).  Dogs
have been reported to develop urinary bladder tumors  following
chronic exposure to large doses of benzidine  (Spitz,  et
*This summary has been prepared and approved by  the Carcino-
gens Assessment Group, EPA, on July 15, 1979.
                               C-49

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al. 1950; Bonser, et al. 1956).  However, the small numbers


of animals involved make the significance of these findings


questionable.


     The difference in organotropic properties of benzidine


among the different species is probably due to both its
                                    i

route of excretion and metabolism.  For example, in humans


and dogs, benzidine or its metabolites are largely excreted


through  the  urine, whereas, in mice and rats, excretion  is


largely  through  the bile.  In man, 70 to 90 percent of benzi-


dine is  excreted in the  urine in the form of 3-hydroxybenzi-


dine; in rats, it is questionable whether this metabolite


is even  formed,  but it is  formed in the dog and  rabbit.


     Three studies have  reported mutagenic activity of benzi-


dine towards Salmonella  typhimurium  (TA 1537 and TA 1538)


in the presence  of a rat liver mixed function oxidase system


(Ames, et al.  1973; McCann, et al. 1975).


     The carcinogenic and  mutagenic activities of benzidine


in animal systems clearly  substantiate  the epidemiological


findings that show benzidine  to be carcinogenic  in humans.


In a recent  report, The  National Academy of Science  (NAS,


1976) calculated an estimate  of the total benzidine exposure


of occupationally exposed  humans on the  basis of the  urinary


levels.  The NAS report  presented  a table comparing tumor


incidence  and total accumulated dose  in  humans and  two species


of laboratory animals.


      Table  1 contains  data from  the NAS  (1975) report


as well  as  additional  animal  data.
                               C-50

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                                     TABLE 1

            Degree of Exposure and Reported Cancer Frequencies for Agents
                     Carcinogenic  to Man  and  Laboratory  Animals
        Conditions
        of Exposure
Total Accumulated
  dose (mg/kg)
Cancer
Reference
Man     13.6 yr;  occupational

Mouse   1/wk; 32  - 52 wks
        S.C. injection

Mouse   1/wk; 52  wks
        S.C. injection

Rat     1/wk; 64  wks
        S.C. injection

Rat     1/3 days  for 30 days
        gastric intubation
          200   52% bladder (13/25)  Zavon, et al. 197:

       10,000   67% liver (31/46)    Prokofjevea, 1971
       10,400   12% liver (7/60)
                 Bonser, et al. 19-1
        3,200    4% liver (6/152)    Spitz, et al. 195(
          100   78% mammary (7/9)    Gr
           50   50% mammary (5/10)     1968
       control   4% mammary (5/132)
                   iswold, et al,
*dose calculated on basis of an average rat we'ght of .25 kg, from NAS, 1975,


     On the basis of the data presented in this table, it is

     apparent that in animal studies where benzidine was injected

     and where liver tumors were induced,  much higher doses of

     benzidine were required than in the sensitive mammary tumor

     rat model system and in the doses estimated to give a

     high bladder cancer incidence in man.

          The data from the human epidemiology study of Zavon,
t
     et al.  1973 was used to estimate the  concentration of benzi-

     dine in water calculated to keep the  lifetime cancer risk

     below 10~ .   In this study 25 workers in a benzidine manufac-

     ture plant,  were observed for the appearance of bladder

     cancer  over  a period of 13 years.  In this series 13 of

     25 men  developed bladder tumors after a mean exposure period
                                   C-51

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of 13.61 years, their  average  age  at  the  end  of  exposure



was 44 years and at  the  end of a 13 year  observation  was



57 years.  The men not showing evidence of cancer  had a



mean exposure period of  8.91 years, their average  age at



the end of exposure  was  43 years and  at the end  of  observation



56 years.  The estimated total accumulated dose  of  200 mg/kg



was estimated from average urinary levels of  benzidine in



these workers at the end of a  workshift (see  Table  1  and



Zavon, et al. 1973).   From this data  the concentration of



benzidine in water calculated  to keep lifetime cancer risk



below 10~5 is 1.67 x 10~3 jug/1.



     Four animal studies shown  in Table 1 were considered



for possible use in  the  calculation of the water quality



criterion.  The most sensitive  response occurred in the



Griswold study, where  10  to 20  female Sprague-Dawley  rats



per treatment group were  administered benzidine  by gastric



-ntubation in ten equal  doses  at three-day intervals over



a 30-day period and  jbserved for nine months.   Total doses



of 25 and 12 mg/1 benzidine/rat induced carcinomas in 7/9



and 5/10 animals, respectively, compared to 5/132 animals



in the control group.  All tumor-bearing rats  had multiple



carcinomas and one had a  fibroadenoma.  Based  on these data,



the concentration of benzidine  in water, calculated to keep


                                 — 5             —4
the lifetime cancer risk  below 10  ,  is 8.5 x  10   jug/1.



     Although the criterion value derived  from human exposure



data is higher than that  calculated from the most sensitive



animal system, it seems  reasonable that human  epidemiological



data are most appropriate for estimating human risks.   The
                               C-52

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study of Zavon, et al.  (1973) was selected  as  the  data  base
for deriving the water quality criterion.   Based on  these
data, the concentration of benzidine  in  water  calculated
to keep the lifetime cancer  risk below 10    is 1.67  x  10
jug/1.
                              C-53

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National Academy of Science. 1975. Pest control: An assessment
of present and alternative technologies. Vol. 1: Contemporary
pest control practices and prospects:  The report of the
Executive Committee. Washington, D.C. 20418.
                               C-54

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                   Summary  of  Pertinent  Data



      The  data  from the  human  epidemiology  study  of  Zavon,

 et  al.  1973  was  used  to estimate  the  concentration  of  benzi-

 dine  in water  calculated to keep  the  lifetime  cancer risk

 below 10~  .  In  this  study 25 workers in a benzidine manufac-

 turing  plant were  observed for  the  appearance  of  bladder

 tumors  after a mean exposure period of  13.61 years, their

 average age  at the end  of  exposure  was  44  years  and at the

 end of  a 13  year observation was  57 years.  The men not

 showing evidence of cancer had  a  mean exposure period  of

 8.91  years,  their  average  age at  the  end or exposure was

 43 years and at  the end  of observation  56  years.  The  estimat-

 ed total accumulated dose  of 200  mg/kg  was estimated from

 average urinary  levels  of  benzidine in  these workers at

 the end of a workshift  (see Table 1 and Zavon, et al.  1973).

 The criterion was  calculated from the following parameters:

 Average weight of  man =  70 kg

 Observed incidence of bladder cancer  =  13/25 (52 percent)

 Accumulated  dose = 200 mg/kg

 Bioconcentration factor of benzidine  =  50

 X = average  daily  exposure producing  lifetime risk of  10"

B* = potency factor, which is an estimate  of the linear depen-
    dency of cancer rates on lifetime average dose

C = concentration of benzidine  in water, calculated to produce
    a lifetime  risk of 10  ,  assuming a daily ingestion
    of 2 liters of water and 0.0187 kg  fish.
                              C-55

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Workers were assumed  to  have  received  200  mg/kg  of benzidine

in a lifetime.  At  the end of  a  13-year  observation period,

the average age of  the workers was  57  years.   Therefore,

benzidine exposure  on a  ing/day basis amounts  to:

                          200  x 70
                          365 x 57
                                  =  .673 mg/day
This gives a response at  57 years of 52% so  that



                          .52 - 1 - e-B<'673>
                          B = .734 = 1.091
                             7STJ
                         B* = B(tf)3 = 1.091  (7_2)3 =  2.021
                                (tf)           (57)
                          (2.021)  (X) fg10"
                          X = 4.9  x 10   mg/day to obtain a rate

                              of  10~5 or 4.9 x 10   pg/day

Therefore:

                          C(2 -l- 50 x .0187) = 4.9 x 10~3

                          C = 1.67 x 10~3 pg/1

From this data the concentration  of benzidine in water calculated

to keep lifetime cancer risk below 10   is 1.67 x 10   ug/1.
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