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).
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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:
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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).
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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).
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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
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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
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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
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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
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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
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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
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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
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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
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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-
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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
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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-
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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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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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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.
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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.
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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
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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
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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.
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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.
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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.
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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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