c/EPA
United States
Environmental Protection
Agency
Office of Water
Regulations and Standards
Criteria and Standards Division
Washington DC 20460
EPA 440, 5-80-023
October 1980
Ambient
Water Quality
Criteria for
Benzidine
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AMBIENT WATER QUALITY CRITERIA FOR
BENZIDINE
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
11
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology
William A. Brungs, ERL-Narragansett John H. Gentile, ERL-Narragansett
U.S. Environmental Protection Agency U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects:
Thomas J. Haley (author) Ahmed Ahmed
National Center For Toxicological University of Texas Medical Branch
Research
Steven D. Lutkenhoff (doc. mgr.) Roy E. Albert *
ECAO-Cin Carcinogen Assessment Group
U.S. Environmental Proteciton Agency U.S. Environmental Protection Agency
Richard A. Carchmann Herbert Cornish
Medical College of Virginia University of Michigan School of
Public Health
Patrick Durkin Karl Gabriel
Syracuse Research Corporation Medical College of Pennsylvania
Charalingayya Hiremath Roman Kuchuda
U.S. Environmental Protection Agency U.S. Environmental Protection Agency
J.F. Stara, ECAO-Cin William W. Sutton, EMSL-LV
U.S. Environmental Protection Agency U.S. Environmental Protection Agency
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, B. Gray, B. Gardiner.
*CAG Participating Members: Elizabeth L. Anderson, Larry Anderson, Dolph Arnicar,
Steven Bayard, David L. Bayliss, Chao W. Chen, John R. Fowle III, Bernard
Haberman, Charalingayya Hiremath, Chang S. Lao, Robert McGaughy, Jeffery Rosenblatt
Dharm V. Singh, and Todd W. Thorslund.
iv
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TABLE OF CONTENTS
Criteria Summary
Introduction A-l
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Acute Toxicity B-l
Residues B-l
Summary B-2
Criteria B-2
References 6-6
Mammalian Toxicology and Human Health Effects C-l
Introduction C-l
Exposure C-l
Ingestion from Water C-l
Ingestion from Food C-l
Inhalation C-3
Dermal C-3
Pharmacokinetics C-4
Absorption and Distribution C-4
Metabolism and Excretion C-4
Effects C-12
Acute, Subacute and Chronic Toxicity C-12
Synergism and/or Antagonism C-14
Teratogenicity C-14
Mutagenicity C-14
Carcinogenicity C-15
Criterion Formulation C-26
Existing Guidelines and Standards C=26
Special Groups at Risk C-28
Basis and Derivation of Criteria C-2S
References C-33
Appendix I C-48
Summary and Conclusions Regarding the Carcinogenicity
of Benzidine C-48
Summary of Pertinent Data C-53
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CRITERIA DOCUMENT
BENZIDINE
CRITERIA
Aquatic Life
The available data for benzidine indicate that acute toxicity to fresh-
water aquatic life occurs at concentrations as low as 2,500 ug/1 and would
occur at lower concentrations among species that are more sensitive than
those tested. No data are available concerning the chronic toxicity of ben-
zidine to sensitive freshwater aquatic life.
No saltwater organisms have been tested with benzidine and no statement
can be made concerning acute and chronic toxicity.
Humai
For the maximum protection of human health from the potential
carcinogenic effects due to exposure of benzidine through ingestion of
contaminated water and contaminated aquatic organisms, the ambient water
concentrations should be zero based on the non-threshold assumption for this
chemical. However, zero level may not be attainable at the present time.
Therefore, the levels which may result in incremental increase of cancer
risk over the lifetime are estimated at 10~5, 10"6, and 10~7. The
corresponding recommended criteria are 1.2 ng/1, 0.12 ng/1, and 0.01 ng/1,
respectively. If the above estimates are made for consumption of aquatic
organisms only, excluding consumption of water, the levels are 5.3 ng/1,
0.53 ng/1, and 0.05 ng/1, respectively.
VI
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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 Pollu-
tion 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 ug/1 (42 FR 2617).
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), benzi-
dine's solubility increases as water temperature rises. The solubility of
benzidine in 12°C water is 400 mg/1 (Verschueren, 1977). Solubility is
greatly enhanced with dissolution into organic solvents (Stecher, 1968).
Its log octanol/water partition coefficient is 1.81 (Radding, et al. 1977).
Benzidine is easily converted to and from its salt (Morrison and Boyd, 1972).
Diazotization reactions involving benzidine will result in colored com-
pounds (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
3.57 and 4.66 (Weast, 1972).
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Oxidation by metal cations appears to be an important route for benzi-
dine degradation in the aquatic environment (Lahav and Raziel, 1971). Ben-
zidine is not bioaccumulated to a significant extent by aquatic organisms,
and it is apparently not easily degraded by the microorganisms in sewage
plant sludge (Howard and Saxena, 1976).
A-2
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REFERENCES
Howard, P.M. and J. Saxena. 1976. Persistence and degradability testing of
benzidine and other carcinogenic compounds. EPA 560/5-76-005. Off. Toxic
Subst., U.S. Environ. Prot. Agency, Washginton, D.C.
Lahav, N. and S. Raziel. 1971. Interaction between montmorillonite and
benzidine in aqueous solutions. II. A general kinetic study. Israel Jour.
Chem. 9: 691.
Morrison, R.T. and R.M. Boyd. 1972. Organic Chemistry. 2nd ed. Allyn and
Bacon, Inc., Boston.
Radding, S.B., et al. 1977. Review of the environmental fate of selected
chemicals. EPA 560/5-77-003. Off. Toxic Subst., U.S. Environ. Prot.
Agency, Washington, D.C.
Standen, A. (ed.) 1972. Kirk-Othmer Encyclopedia of Chemical Technology.
Interscience Publishers, John Wiley and Sons, Inc., New York.
Stecher, P.G. (ed.) 1968. The Merck Index. 8th ed. Merck and Co., Inc.,
Rahway, New Jersey.
Verschueren, K. 1977. Handbook of Environmental Data on Organic Compounds.
Van Nostrand Reinhold, New York.
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*
INTRODUCTION
Static, acute tests have been conducted with five freshwater fish spe-
cies and a scud and bioconcentration factors range from 38 to 2,620. No
data are available for saltwater species.
EFFECTS
Acute Toxicity
The 96-hour LCrn values for rainbow and lake trout, red shiner, and
flagfish range from 2,500 to 16,200 ug/1 (Table 1).
Comparable tests conducted with the fathead minnow and a scud, Gammarus
pseudolimnaeus, resulted in no observed mortality at the highest test con-
centration of 20,000 yg/1 (Table 3).
Residues
The bluegill has- been exposed to C-benzidine under flow-through con-
ditions for 42 days (EG and G Bionomics, 1975). The edible portion of the
fish bioconcentrated C-residues by 38 to 44 times (Table 2). The com-
parable bioconcentration factor for the non-edible portion (viscera) of the
bluegill ranged from about 12 to about 43 times that amount present in the
edible portion. The half-life of C-residues in the fish was about 7
days.
Miscellaneous
Lu, et al. (1977) studied the behavior of benzidine in a model ecosystem
for 3 days and observed bioconcentration factors in algae, snails, mosqui-
*The reader is referred to the Guidelines for Deriving Water Quality Crite-
ria for the Protection of Aquatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation. The following tables
contain the appropriate data that were found in the literature, and at the
bottom of each table are the calculations for deriving various measures of
toxicity as described in the Guidelines.
B-J
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tos, and the mosquitofish of between 55 to 2,620 times. The result with the
mosquitofish (55x) is consistent with that discussed earlier with the
bluegill.
Summary
One freshwater invertebrate and five fish species have been exposed to
benzidine under static acute test conditions. The 96-hour LC50 values for
four fish species ranged from 2,500 to 16,200 wg/1. The LC5Q values for
the other species were greater than 20,000 ug/1. Bioconcentration factors
for the bluegill ranged from 38 to 44 and, in a model ecosystem after 3
days, bioconcentration factors ranged from 55 for fish to 2,620 for an alga.
No saltwater organism has been tested with benzidine.
CRITERIA
The available data for benzidine indicate that acute toxicity to fresh-
water aquatic life occurs at concentrations as low as 2,500 ug/1 and would
occur at lower concentrations among species that are more sensitive than
those tested. No data are available concerning the chronic toxicity of ben-
zidine to sensitive freshwater aquatic life.
No saltwater organisms have been tested with benzidine and no statement
can be made concerning acute or chronic toxicity.
8-2
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Table 1. Acute values for benzldlne (U.S. EPA, 1980)
LC50/EC50 Species Acute
Species Method* (ug/l) Value (ug/l)
FRESHWATER SPECIES
Rainbow trout, S, U 7,400
Salmo galrdner!
Lake trout. S» U 4,350
Sa 1 ve 1 1 nus namaycush
Red shiner, S, U 2,500
Notrools lutrensls
Flagflsh, S, U 16,200
Jordanel la f lorldae
7,400
4.350
2,500
16,200
* S = static, U = unmeasured
No Final Acute Value Is calculable since the minimum data base
requirements are not met.
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Table 2. Residues for banzldlne (EG i 6 Bionomics. 1975)
Bloconcentratlon Duration
Species Tissue Factor (days)
FRESHWATER SPECIES
8/ueglll, edible portion 38 to 44» 42
Leporols mncrochlrus
• Results based on 14C-resldue content.
B-4
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Table 3. Other data for beruldlne
Species
Alga,
OedogonI urn card Iacum
Snail,
Physa sp
Scud,
Gamroarus pseudolImnaeus
Mosquito (larva),
Culex pip lens
Fathead minnow,
Plmephales promelas
Mosqultofish,
Gambusla atfinis
Duration Effect
FRESHWATER SPECIES
Result
(yg/i)
3 days Model ecosystem,
bloconcentrat Ion
factor = 2,620
3 days Bloconcentrat Ion
factor = 645
96 hrs LC50
3 days Model ecosystem
b loconcentrat Ion
factor =456
96 hrs LC50
3 days Model ecosystem,
bloconcentrat Ion
factor = 55
>20,000
Reference
Lu, et al. 1977
Lu, et al. 1977
U.S. EPA, 1980
Lu, et al. 1977
>20,000 U.S. EPA, 1980
Lu, et al. 1977
B-5
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REFERENCES
14
EG and G Bionomics. 1975. Exposure of fish to C-benzidine: accumula-
tion, distribution, and elimination of 14-C residues. Res. Report to Allied
Chemical Corp.
Lu, P-Y, et al. 1977. The environmental fate of three carcinogens: ben-
zo(a)pyrene, benzidine, and vinyl chloride in laboratory model ecosystems.
Arch. Environ. Contain. Toxicol. 6: 129.
U.S. EPA. 1980. Unpublished laboratory data Environmental Research Labora-
tory, Duluth.
8-6
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Mammalian Toxicology and Human Health Effects
INTRODUCTION
In general, exposure to benzidine compounds occurs in factor-
ies 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 breathing contaminated air, ingesting contaminated
food, and wearing contaminated clothing (Meigs, et al. 1951).
Pointing of brushes by Japanese kimono painters results in the
ingestion of benzidine dyes (Yoshida and Miyakawa, 1973), although
ingestion is not generally an important source of exposure.
EXPOSURE
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 condi-
tions, 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 aquatic animals to the concentration in the water in
which they live. The steady-state BCFs for a lipid-soluble com-
pound in the tissues of various aquatic animals seem to be propor-
tional to the percent lipid in the tissue. Thus the per capita
C-l
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ingestion of a lipid-soluble chemical can be estimated from the per
capita consumption of fish and shellfish, the weighted average per-
cent lipids of consumed fish and shellfish, and a steady-state BCF
for the chemical.
Data from a recent survey on fish and shellfish consumption in
the United States was analyzed by SRI International (U.S. EPA,
1980). These data were used to estimate that the per capita con-
sumption of freshwater and estuarine fish and shellfish in the
United States is 6.5 g/day (Stephan, 1980). In addition, these
data were used with data on the fat content of the edible portion of
the same species to estimate that the weighted average percent
lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
A measured steady-state BCF of 41 was obtained for benzidine
using the edible portion of bluegills (EG & G Bionomics, 1975). A
higher BCF was found for the nonedible portion, but percent lipids
were not reported for either portion and the relative weights of
the two portions were not reported. However, for 3,3'-dichloro-
benzidine the BCF for whole body was about 3.5 times that for edi-
ble flesh (Appleton and Sikka, 1980). Thus, the steady-state bio-
concentration factor for benzidine in the whole body of the tested
bluegills can be estimated to be 140. Similar bluegills contained
an average of 4.8 percent lipids (Johnson, 1980). An adjustment
factor of 3.0/4.8 = 0.625 can be used to adjust the measured BCF
from the 4.8 percent lipids of the bluegill to the 3.0 percent
lipids that is the weighted average for consumed fish and
shellfish. Thus, the weighted average bioconcentration factor for
C-2
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benzidine and the edible portion of all freshwater and estuarine
aquatic organisms consumed by Americans is calculated to be
140 x 0.625 = 87.5.
Inhalation
In the early phases of the chemical and dye industries, the
lack of good industrial hygienic practices and the use of open sys-
tems made inhalation one of the principal routes of entry of benzi-
dine and its derivatives into the body. Similar inhalation expo-
sures can occur at the present time unless workers wear respirators
and protective clothing while cleaning the equipment (Haley, 1975).
Dermal
Skin absorption is the most important path of entry into the
body. Intact skin is readily penetrated by benzidine and 3,3'-di-
methylbenzidine (Meigs, et al. 1951). 3,3'-Dichlorobenzidine,
because of its nonvolatility and large particle size, presents less
of an inhalation and skin penetration 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 benzidine workers from skin absorption (Barsotti and
Vigliani, 1952). The ease of skin penetration determines the fol-
lowing order of decreasing toxicity from these chemicals: benzi-
dine, 3,3'-dimethoxybenzidine, and 3,3'-dichlorobenzidine (Rye, et
al. 1970).
Environmental conditions of high air temperature and humidity
increase skin absorption of benzidine, 3,3'-dimethoxybenzidine,
3,3'-dichlorobenzidine, and 3,3'-dimethylbenzidine. Higher a-
mounts of benzidine are found in the urine of workers who perspire
C-3
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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 per-
formed to absolutely establish this concept (Meigs, et al. 1951).
PHARMACOKINETICS
Absorption and Distribution
Benzidine is rapidly absorbed into rats after intravenous
injection. Maximum concentrations of free and bound benzidine are
found at two and three hours, respectively. The highest concen-
trations 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 intra-
peritoneal injection of 100 mg/kg was as follows: high concentra-
tions 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 erythrocytes
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 with
14
C and dichlorobenzidine labeled in the 3,3' positions indicated
C-4
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that substitution in the 3,3' positions of the benzidine molecule
significantly affects the routes of metabolism and excretion. The
blood half-life for benzidine was 68 hours in the rat and 88 hours
in the dog. The weekly excretions of a dose of 0.2 mg/kg of ben-
zidine in the rat, dog, and monkey were 97, 96, and 83 percent re-
spectively. 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 benzidine with the
urine, while the rat uses the biliary route. The urinary bladder
of the dog had a high content of benzidine, 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 congen-
ers are given in Table 1. It can be seen that various species han-
dle these chemicals in different ways and that the animal metabo-
lites differ considerably from those excreted by humans. The im-
provements 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 re-
covered indicating that fecal excretion probably occurred. This
cannot be proven because the feces were not analyzed (Engelbertz
and Babel, 1953). People ingesting 200 mg of benzidine excreted
free benzidine and N-hydroxyacetylamino benzidine in their urine
C-5
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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
Monoace tylbenz id i ne
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-6
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TABLE 1 (Continued)
Compound
Species
Metabolites
Reference
Benzidine
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
3-Hydroxybenzidine
sulfate and
glucuronide
4'-Acetamido-4-
aminodiphenyl
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
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
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
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 and
Meigs, 1961a
Sciarini and
Meigs, 1961a
C-7
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TABLE 1 (Continued)
Compound
Benzidine
3,3'-dimethyl-
benzidine
(orthotolidine)
3,3'-Dimethoxy-
benzidine
Species
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Human
Human
Human
Dog
Dog
Metabolites
N-Hydrogen sulfate
and/or glucuronide
3-Hydroxybenz id ine
glucuronide
4' -Acetamido-4-amino-
diphenyl
4, 4 '-Di ami no- 3-d i-
phenyl hydrogen
sulfate
41 -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
Reference
Sciarini and
Meigs, 1961a
Sciarini and
Meigs, 1961a
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
(dianisidine)
3-Methoxyben-
zidine (mono-
substituted
dianisidine)
Rat
4-Amino-4'-acetamido-
S-methoxybi-
phenyl
Laham, 1971
C-8
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(Troll, et al. 1963). In the urine of plant workers exposed to
benzidine in unknown quantities, free benzidine, its mono- and
diacetylated derivatives, and 3-hydroxybenzidine were identified.
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 concentra-
tion of 0.018 mg/m of benzidine would result in a urinary excre-
tion 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 monoacetylbenzidine in their
urine (Vigliani and Barsotti, 1962).
Exposure to 3,3'-dimethylbenzidine results in urinary excre-
tion of free 3,3'-dimethylbenzidine, its diacetyl derivative, and
5-hydroxy-3,3'-dimethylbenzidine. Although the monoacetylated
derivative was not detected, there is a probability of its forma-
tion because 3,3'-dimethylbenzidine appears to be metabolized simi-
larly 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 concern-
ing the metabolic conversion of chemicals in various species. This
is taken into consideration in the following discussion of the
C-9
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animal data in Table 1 and their relevance to the human situation.
Intraperitoneal injection of 100 mg/kg of benzidine in mice pro-
duced 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 conjugates of 3-hydroxy-
benzidine but no acetylated derivatives, because the dog lacks this
biotransformation mechanism (Sciarini, 1957). The ethereal sulfate
of 3-hydroxy-benzidine has been identified in dog urine and con-
stitutes 25 to 50 percent of the administered dose (Sciarini and
Meigs, 1958). The ethereal sulfate and glucuronide were the only
metabolites 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-di-
phenylyl hydrogen sulfate, 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 guinea pig
urine, although the other metabolites were present. 3-Hydroxyben-
zidine and 4,4'-diamino-3-diphenylyl hydrogen sulfate were present
C-10
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in dog urine. In all cases, N-glucuronides were present (Clayson,
et al. 1959). Metabolite differences occur when different routes
of elimination are considered. Dogs excrete the same benzidine
metabolites in urine and bile but their feces have no 3-hydroxy-
benzidine 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 com-
pounds in greater quantities in the feces than in the urine,
whereas the dog eliminated the dichloro compound to a greater ex-
tent 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 10 times greater than urinary excretion
(Sciarini and Meigs, 1961b), while the opposite was true for benzi-
dine (Sciarini and Meigs, 1958).
When benzidine-based azo dyes were fed to monkeys, benzidine
and monoacetylbenzidine were found in their urine (Rinde and Troll,
1975). This shows that the monkey, like man, can reductively
cleave the azo linkage (Akiyama, 1970).
Intraperitoneal injection of dimethylbenzidine, dimethoxy-
benzidine, and dichlorobenzidine in dogs resulted in recovery of
part of these chemicals in nonmetabolized form. The dichloro com-
pound was not metabolized, whereas the other two derivatives of ben-
zidine were recovered from urine as unidentified conjugated ether-
eal sulfates (Sciarini and Meigs, 1961b).
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EFFECTS
Acute, Subacute, and Chronic Toxicity
Iji vitro studies have shown that benzidine, 3,3' -dimethyl-
benzidine, 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 sug-
gested that there is a relationship between carcinogenic potential
and the reduction of cytochrorae c (Hirai and Yasuhira, 1972; Canuner
and Moore, 1973).
There is a significant increase in urinary B-glucuronidase
activity in workers exposed to benzidine. The elevated activity,
although decreased by removal from benzidine exposure, does not re-
turn 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, benzidine de-
creased the activity of this enzyme (Nakajima, 1955). Rats inject-
ed 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 272 rag/100 g in 24 hours (Neish, 1967).
Dermatitis has been reported in workers in the benzidine dye-
stuff industry, involving both benzidine and its dimethyl deriva-
tive. Individual sensitivity plays a prominent role in this condi-
tion (Schwartz, et al. 1947).
Glomerulonephritis and nephrotic syndrome have been produced
in Sprague-Dawley rats fed 0.043 percent N,N'-diacetylbenzidine.
C-12
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Both sexes developed proteinuria in 3 to 4 weeks. After two months
the females were excreting 0.1 g of protein per 24 hours. The fe-
males developed severe anemia, which was rarely seen in the males.
The former also had a hypoproteinemia, hyperlipemia, and general-
ized edema. Glomerular lesions in the females consisted of florid
epithelial crescents, progressive sclerosis, and glomerular oblit-
eration. 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 similarities
between the human nephrotic syndrome and that induced by N,N'-
diacetylbenzidine in rats, including extracapillary cell prolifer-
ation, formation of luxuriant crescents in 80 percent of the glo-
meruli, intact glomerular tufts, and the presence of normal glo-
meruli in the advanced stages of the syndrome (Barman, et al.
1952; Harman, 1971).
Rats fed N,N'-diacetylbenzidine or 4 ,4 ,4',4'-tetramethylben-
zidine 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 in rats fed benzidine (Christopher and Jairam, 1970).
Mice fed 0.01 and 0.08 percent benzidine dihydrochloride de-
veloped the following toxic symptoms: decreased carcass, 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 elements in the bone marrow and of
C-13
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the lyraphoid cells in the spleen and thymic cortex. There was a
dose-dependent body weight loss of 20 percent in males and 7 per-
cent in females. Moreover, male mice were more sensitive to ben-
zidine than female mice (Rao, et al. 1971). This disagrees with
Barman's (1971) findings in rats, but it may only be a species dif-
ference in response.
Synergism and/or Antagonism
Pertinent data could not be located in the available litera-
ture.
Teratogenicity
Embryonic mouse kidney cultures have an increased survival
time but show hyperplastic epithelial changes in the presence of
3,3'-dimethylbenzidine (Golub, 1969; Shabad, et al. 1972). Admini-
stration of 8 to 10 mg of 3,3'-dimethylbenzidine 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 transmission of the chemical or from its presence in
the milk (Golub, et al. 1974). No teratogenic effects of benzidine
derivatives in humans have been reported.
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.
C-14
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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 by
test compounds in HeLa cells (Painter and Howard, 1978). The con-
centration of a compound that is required to inhibit DNA synthesis
by 40 percent corresponds with its mutagenic effects in Salmonella
typhimurium. Benzidine has been shown to be positive in this DNA
synthesis inhibition test (Painter and Howard, 1978).
Results of a salmonella mutagenesis assay indicate that ben-
zidine 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 experi-
mental animals and humans. In the latter these chemicals have been
shown to produce bladder cancer after a long period of latency
(Clayson, 1976). Additionally, these compounds produce dermatitis,
cystitis, and hematuria in humans, indicating 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 develops
urinary bladder cancer similar to that seen in humans after expo-
sure to benzidine. The animal cancers, in general, differ signifi-
cantly in their locations. This may be related to differences in
specific target tissues or to differences in excretory pathways.
C-15
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TABLE 2
Effects of Benzidine, Its Congeners, and Metabolites
On Various Animal Species*
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 blad-
der papilloma
Cirrhosis of liver, hepato-
mas, carcinoma of Zymbal's
gland, adenomacarcinoma, de-
generation of bile ducts,
sarcoma, mammary gland car-
cinoma
Hepatoma, adenocarcinoma of
colon, carcinoma of fore-
stomach, Zymbal's gland car-
cinoma, 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 atro-
phy, bladder tumor, gall
bladder tumor
Recurrent cystitis, bladder
tumor, convulsions, liver
cirrhosis, hematuria
No pathological changes (Dura-
tion of study too short)
Bladder tumor, papilloma,
chronic cystitis, hematuria
*Source: Haley, 1975
a3,3'-Dimethoxybenzidine.
b3,3'-Dimethylbenzidine.
-------
In some cases, excessive dosage may cause death due to toxicity,
thus preventing the development of bladder cancer (Haley, 1975).
Benzidine and many other aromatic amines attack the urinary
bladder and other organs (Hueper, 1954). However, it is the met.a-
bolites of these compounds that are considered to be the proximate
carcinogens (Clayson, 1969). These aromatic amines are ring hy-
droxylated, converted to N-hydroxylated, acylated and deacylated
derivatives, and conjugated with sulfate and glucuronide (Haley,
1975). It has been suggested that the conjugated N-hydroxy com-
pounds are the active carcinogens in vivo. Bladder cancer has been
induced in rabbits and dogs fed benzidine, but these findings are
controversial (Haley, 1975). Spitz, et al. (1950) induced papil-
lary carcinoma in 1 of 7 dogs fed benzidine for five years, but the
cancer only appeared 7.5 years after the beginning of the experi-
ment. Orally administered benzidine did not produce urinary blad-
der cancer in dogs (Marhold, et al. 1967). No tumors were found in
female beagle dogs fed 1 mg/kg five days per week for three years
(Deichmann, et al. 1965). In these last two studies, the lack of a
carcinogenic effect in dogs 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).
C-17
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Benzidine administered subcutaneously to rats at a rate of 15
mg/week produced liver injury, cirrhosis, hepatomas, sebaceous
gland carcinomas, and adenocarcinomas of the rectum, but no bladder
tumors (Spitz, et al. 1950). Rats fed 0.125 percent of dihydroxy-
benzidine in the diet developed liver cirrhosis, hepatomas, adeno-
carcinomas of the colon, Zymbal's gland carcinoma, and squamous
cell carcinomas of the stomach. One sessile papilloma and two
keratinized 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 oral doses of 12 to 50 mg/rat developed mammary
gland carcinomas (Griswold, et al. 1968).
Early cirrhosis occurred in rats given benzidine by subcutane-
ous injection for six months (Pliss, 1963). Injection site sar-
comas, hepatomas, and Zymbal's gland tumors were also found and
constituted 70 percent of the tumors in 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-dicarboxylic acid
within one year (Pliss, 1969). Benzidine, in 5 mg weekly doses,
produced intestinal tumors in rats (Pliss, et al. 1973). A cumula-
tive dose of 0.75 mg/kg of benzidine for 15 days produced tumors in
20 of 22 rats, including 19 hepatomas, 18 cholangiomas, 7 intestin-
al tumors, and 4 sebaceous gland carcinomas. Subcutaneous tetra-
methylbenzidine doses of from 4.15 to 8.3 g/kg produced benign
tumors at the injection site (Holland, et al. 1974).
C-18
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Female Wistar rats given a single intraperitoneal injection of
100 or 200 rag of N,N'-diacetylbenzidine subcutaneously developed
Zymbal's gland and mammary gland tumors after 6 to 15 months. The
100 rag intraperitoneal injection produced tumors in 11 out of 18
rats while the 200 mg dose gave no tumors (Breraner and Tange,
1966).
Hepatomas, bile duct proliferation, and benign papillomas of
the urinary bladder were found in Oelph albino mice injected sub-
cutaneously with 300 mg of benzidine or dihydroxybenzidine. Only
the latter chemical caused the bladder changes (Baker, 1950).
Benzidine or 3,3-dihydroxybenzidine administered subcutan-
eously at 6 mg weekly for 52 weeks produced tumors in exposed mice
in 70 weeks. Benzidine induced hepatomas and lymphomas while the
3,3-dihydroxy derivative induced lymphomas and benign intestinal
polyps. The significance of the lymphomas is obscure because one-
third of the controls developed this condition spontaneously
(Bonser, et al. 1956). Subcutaneous administration of 3,3-dihy-
droxybenzidine in mice caused tumors of the liver and mammary
glands as well as leucosis (Pliss. 1961). Inner organ tumors de-
veloped after skin application of the chemical. Subcutaneous week-
ly 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 pulmon-
ary adenocarcinoma (Prokofjeva, 1971).
3,3-Dimethylbenzidine in a cumulative dose of 5.4 g/kg for 241
days induced 11 gastrointestinal tract tumors, 7 hepatoiaas, 7 bone
tumors, and 4 Zyntbal's gland carcinomas in rats. Total oral doses
of 500 ing in Sprague-Dawley rats produced 4 mammary carcinomas in 9
C-19
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months in 3 of 16 surviving animals (Griswold, et al. 1968). Sub-
cutaneous 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 sar-
comas were produced (Pliss and Zabezhinsky, 1970).
3,3'-Dimethoxybenzidine given subcutaneously to rats induced
Zymbal's 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 period of
latency was 293 days (Weisburger, et al. 1967).
Subcutaneous administration of 3,31-dichlorobenzidine to rats
induced tumors in 74 percent of the animals (Pliss, 1963). Tumors
appeared in the skin, sebaceous and mammary glands, intestines,
bones, and urinary bladder. Dichlorobenzidine given by ingestion
or injection into the underlying fat produced sarcomas at the
injection site, an adenocarcinoma 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 dichloro-
benzidine produced 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 dichlorobenzidine had a
significant increase in tumor incidence. Tumors developed in 13 of
C-20
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24 mice, with 4 adenocarcinomas of the mammary gland, 5 lung adeno-
mas, and 7 cases of lymphatic leukemia (Golub, et al. 1974).
The carcinogencity risk for workers exposed to benzidine 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 ben-
zidine. The latency for tumor development was 12 years (Gehrman,
1936). In England the tumor induction time averaged 16 years, but
one case occurred in two 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 associated with 14 papillomas, 7 carci-
nomas, and two cases in which the papillomas were converted to car-
cinomas (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 concentra-
tion, 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. 197Q). There is little doubt that benzidine exposure
is associated with an increase in the occurrence of bladder cancer
(International Agency for Research on Cancsr (IARC), 1972; Riches,
1972; Sax, 1975). However, there is a lack of information on the
exact concentrations of benzidine to which workers have been
exposed.
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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 nontumor 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^-naphthylamine, 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 can-
cer 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 occur-
ring prior to 1947 and 34 subsequent to 1947. Symptoms of hematur-
ia and stranguria were found in 18 cases (Billiard-Duchesne, 1960).
In Italy, 24 cancers were found in workers exposed to ben-
zidine or benzidine-^-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
C-22
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TABLE 3
Time of Discovery of Aromatic Amine
Bladder Cancer by Country*
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
*Source: Haley, 1975
C-23
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were 21 carcinomas and 16 papillomas. During the period from 1931
to 1948, 13 of 83 workers developed bladder carcinomas from ben-
zidine (Barsotti and Vigliani, 1952). The greatest exposure occur-
red in workers in filtration, pressing, drying, and milling of ben-
zidine. 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).
Studies in dyestuff plants in Japan showed 100 cases of blad-
der 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. 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
Miyakawa, 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 ^-naphthylamine 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.
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At the present time there is no evidence that 3,3'-dimethy1-
benzidine, 3,31-dimethyoxybenzidine, or 3,3'-dichlorobenzidine 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: 2 lung cancers, 1 bone marrow cancer,
6 lipomas, 3 rectal papillomas, 2 sigmoid colon carcinomas, 1
prostate carcinoma, 1 breast muscle myoblastoma, and 1 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 find-
ings (Maclntyre, 1975). It is possible that the latent period for
bladder tumors is longer for 3,31-dichlorobenzidine, since workers
exposed to benzidine plus dichlorobenzidine developed such tumors,
while those exposed to the latter compound alone did not (Gadian,
1975).
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CRITERION FORMULATION
Existing Guidelines and Standards
In 1973 the U.S. EPA proposed, but did not promulgate, a toxic
pollutant standard for benzidine (30 FR 35388).
The industrial standards instituted by the Occupational Safety
and Health Administration (OSHA) in 1974 excluded from regulation
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 proposed (41
PR 27012) based upon information on the toxicological and environ-
mental effects and the fate of benzidine. These standards, pro-
mulgated in 1977, established an ambient water criterion for ben-
zidine of 0.1 ug/1. Effluent standards were set at 10 ug/1 (daily
average) with a maximum for any single day of 50 ug/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 concen-
tration of benzidine was limited to 25 ug/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. A field survey of the Buffalo and
Niagara River areas using the chloramine-T method, with a
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sensitivity of 0.2 ug/1, showed no benzidine in the samples. How-
ever, 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 con-
verted the benzidine compounds to materials not detectable by the
analytical method used (Howard and Saxena, 1976). A Japanese sur-
vey 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 H-S or SO2 reduction (Takemura, et al. 1965).
Information on 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzi-
dine, and 3,3'-dichlorobenzidine and their dye derivatives as water
contaminants is nonexistent, and research should be instituted to
correct this deficiency.
It has been stated that benzidine resists physical and bio-
logical degradation (Lutin, et al. 1965; Malaney, et al. 1967; Rad-
ding, et al. 1975). Benzidine in water is oxidatively degraded by
free radical, enzymatic, or photochemical 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 benzidine, but the
half-lives of the two compounds are the same (Radding, et al.
1975). There is no information available 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
C-27
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intestinal bacteria reduce benzidine azo dyes to free benzidine
(Yoshida and Miyakawa, 1973), and although aquatic organisms might
also cause this same transformation, 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 ben-
zidine and its congeners and their conversion to azo dyes. There
is no maximum permissible level of contamination in the industrial
environment, although there are specific regulations governing the
manufacture of benzidine and its congeners (39 FR 3756). These
standards have reduced the risks to benzidine workers.
The use of benzidine and its congeners poses a potential risk
to workers in biochemical, chemical, and microbiological laborator-
ies where these chemicals are used as analytical reagents (Collier,
1974; Veys, 1972; Wood and Spencer, 1972). The greatest risk oc-
curs in laboratories working with known carcinogens when good lab-
oratory practices are not enforced. No epidemiological evidence is
available to determine the exact extent of the problem.
The risk to the general population from benzidine, its congen-
ers, and their dyes is unknown, but contamination of water sup-
plies, 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.
C-28
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Basis and Derivation of Criteria
The available data concerning the carcinogenicity of benzidine
in experimental animals are severely limited. It is extremely dif-
ficult to extrapolate the experimental results to man because, with
the possible exception of the dog and the rabbit, the target organs
are different. 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 suggested
criterion for benzidine was calculated using a relative risk model
described in the appendix. The calculation 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
6.5 g of benzidine-contaminated aquatic organisms. Based on the
data of Zavon, et al. (1973), a benzidine criterion of 1.2 x 10~
jug/1 is suggested to be adequate to protect the population consum-
ing the water.
Epidemiological data indicate that exposure to benzidine is
associated with an increase in bladder cancer in man. The possi-
bility that benzidine may be found in wastewater may also pose a
problem. In order to determine the extent of the potential prob-
lem, measurements must be made of wastewater, 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
C-29
-------
made 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 as-
pects 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 v. Train, criteria are to
state "recommended maximum permissible concentrations (including
where appropriate, zero) consistent with the protection of aquatic
organisms, human health, and recreational activities." Benzidine
is suspected of being a human carcinogen. Because there is no
recognized safe concentration for a human carcinogen, the recom-
mended 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 and states in the
possible future development of water quality regulations, the con-
centrations of benzidine corresponding to several incremental life-
time 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,
indicates 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.
C-30
-------
In the Federal Register notice of availability of draft am-
bient water quality criteria, the U.S. 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 ,,.
(daily intake) and Corresponding Criteria^
10"7 10"6 10"5
2 1 of drinking 1.2 x 10"5 1.2 x 10"4 1.2 x 10"3
water and consumption jug/1 wg/1 jug/1
of 6.5 g of fish
and shellfish (2)
Consumption of fish 5.3 x 10"5 5.3 x 10~4 5.3 x 10"3
and shellfish only. ug/1 jug/1 ug/1
(1) Calculated from the relative risk model for epidemiology stud-
ies as described in the Human Health Methodology Appendices to
the October 1980 Federal Register notice which announced the
availability of this document and in Appendix 1. Appropriate
data used in the calculation of the model are also presented
in Appendix I. Since the extrapolation model is linear at low
doses, the additional lifetime risk is directly proportional
to the water concentration. Therefore, water concentrations
corresponding to other risk levels can be derived by multiply-
ing or dividing one of the risk levels and corresponding water
concentrations shown in the table by factors such as 10, 100,
1,000, and so forth.
(2) Approximately 22 percent of benzidine exposure results from
the consumption of aquatic organisms which exhibit an average
C-31
-------
bioconcentration potential of 87.5-fold. The remaining 78
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 sole-
ly from the consumption of aquatic life grown in the waters con-
taining the corresponding 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 am-
bient water exposure only.
C-32
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APPENDIX I
Summary and Conclusions Regarding
the Carcinogenicity of Benzidine*
Benzidine ((1,1'-biphenyl)-4,4'-diamine) is used in the manu-
facture of dyes, as a reagent for detection of H-O- 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 ef-
fects of benzidine in hamsters (liver), rats (liver and Zymbal's
gland), and mice (liver). Dogs have been reported to develop urin-
ary bladder tumors following chronic exposure to large doses of
benzidine (Spitz, et al. 1950; Bonser, et al. 1956). However, the
*This summary has been prepared and approved by the Carcinogens
Assessment Group, EPA, on July 15, 1979.
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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 route of excre-
tion 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 benzidine is excreted in the urine in the form of 3-
hydroxybenzidine. In rats, it is questionable whether this metabo-
lite is even formed, but it is formed in the dog and rabbit.
Three studies have reported mutagenic activity of benzidine
towards Salmonella typhimurium (TA 1537 and TA 1538) in the pres-
ence 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 Sciences (NAS, 1975) calculated an
estimate of the total benzidine exposure of occupationally exposed
humans on the basis of the urinary levels. The NAS report present-
ed 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. 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
C-49
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had a fibroadenoma. Based on these data/ the concentration of ben-
zidine in water, calculated to keep the lifetime cancer risk below
10~5, is 8.5 x 10"4 jug/1.
Although the criterion value derived from human exposure data
is higher than that calculated from the most sensitive animal sys-
tem, it seems reasonable that human epidemiological data are most
appropriate for estimating human risks. The 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 1.2
x 10"3 iig/1.
C-52
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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 benzidine in water
calculated to keep the lifetime cancer risk below 10" . In this
study 25 workers in a benzidine manufacturing 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 of exposure was 43 years and at
the end of observation 56 years. The estimated total accumulated
dose of 130 mg/kg was estimated from average urinary levels of ben-
zidine in these workers at the end of a workshift (see Table 1 and
Zavon, et al. 1973). The criterion was calculated from the follow-
ing parameters:
Average weight of man = 70 kg
Observed incidence of bladder cancer = 13/25 (52 percent)
Bioconcentration factor of benzidine = 87.5
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 ,
ters of water and 0.0065 kg fish.
a lifetime risk of 10~ , assuming a daily ingestion of 2 li-
C-53
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The average duration of benzidine exposure for this cohort was
11.46 years. The average urine level of benzidine at the end of a
workshift was approximately 0.04 mg/1 of urine* Assuming the aver-
age urine output per day for a man is 1.2 I/day the expected urine
concentration would be
0.04 nig/1 x 1.2 I/day
= 0.0480 mg/day worked
Assuming a recovery factor of 1.45% (i.e., 1.45% of the actual
exposure concentration was found in the urine according to Rhinde
and Troll, 1974 ) and assuming an average human body weight of 70
kg, the estimated exposure is
-r 70 = 0.0473 mg/kg/day worked
Assuming that an average of 240 days are worked in a year, the re-
sulting average lifetime exposure would be
0.0473 x x —=— « 0.0063 mg/kg/day,
JV-I -»U . J
where 11.46 is the average duration of exposure and 56.5 years is
the average age of the cohort at the end of study.
The carcinogneic potency of benzidine is estimated, using the model
P = 1 - exp £-Bdt~ j, as
B »[- In (1 - 13/25JJ -j- [0.0063 x (56. 5/71. 3 ) 3 J
= 234.13 (mg/kg/day)""1,
where 71.3 years represents the average life span in the U.S. and
56.5 years is the average age of the cohort at the end of the study.
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Therefore, the water concentration is calculated as:
70 x 10"5
234.13 (2 + 0.0065 x 87.5)
1.16 x 10~6 mg/1
1.16 ng/1.
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