EPA 560/5-77-005
POTENTIAL INDUSTRIAL
CARCINOGENS AND MUTAGENS
Environmental Protection Agency
Prepared for the Office of Toxic Substances
Washington, D.C.
May 5,1977
-------
This document is available to the public through the
National Technical Information Service
Springfield, VIrginia 22151
-------
EPA 560/5-77-005
POTENTIAL INDUSTRIAL CARCINOGENS AND MUTAGENS
Lawrence Fishbein
Project Officer
Carl R. Morris
Prepared for the Office of Toxic Substances
Environmental Protection Agency
Washington, D.C.
Contract #EPA-IAG-D4-0472
May 5, 1977
-------
This draft report has been reviewed by the Office
of Toxic Substances, EPA, and approved for publi-
cation. Approval does not signify that the con-
tents necessarily reflect the views and policies
of the Environmental Protection Agency, nor does
mention of trade names or coimnercial products
constitute endorsement or recommendation for use.
-------
TABLE OF CONTENTS
Page
Infroduction 1-28
II. Tabular Summaries 29-42
III. Alkylating Agents 43-117
A. Epoxides 43-55
B. Lactones 56-60
C. Aziridines 61-65
D. Alkyl Sulfates 66-69
E. Sultones 70-72
F. Aryldialkyltriazenes 73-75
G. Diazoalkanes 7 6-79
H. Phosphoric Acid Esters 80-86
I. Alkane Halides 87-93
J. Halogenated Alkanols 94-95
K. Halogenated Ethers 96-104
L. Aldehydes 105-117
IV. Acylating Agents 118-121
V. Peroxides 122-129
VI. Halogenated Unsaturated and Saturated
Hydrocarbons and Aromatic Derivatives 130-198
A. Unsaturated Hydrocarbons 13 0-162
B. Saturated Hydrocarbons 163-170
C. Alkanols 94-95
D. Ethers 96—104
E. Aryl Derivatives 171-172
F. Polyaromatics 173-197
-------
VII. Hydrazines Hydroxylamines, Carbamates 198-224
A. Hydrazines 198-211
B. Hydroxylarnines 212-219
C. Carbamates 220—224
Tm. Nitrosamines 225-244
IX. Aromatic Amines 245-262
X. Azo Dyes 263-269
XI. Heterocyclic Amines 270-278
XII. Nitrofurans 279-285
,WI. Anthraquinones 286-295
XIV. Aromatic Hydrocarbons 296-302
XV. Cyclic Ethers 303—307
XVI. Phosphoramides 308-310
XVII. Nitroalkanes 311-312
XVIII. Azides 313—314
Summary 315-316
-------
Introduction
There is a continuing need to assess the status of existing potentially hazardous
chemicals and strategies as well as those that may be introduced in the future that
may impact on man and the environment, primarily from a predictive view of avoiding
or ameliorating catastrophic episodes similar to those which have occurred in the past
involving agents such as methyl mercury, cadmium, PCBs, PBBs, vinyl chloride,
chlorinated dioxins and a number of chlorinated pesticides Ce. g., Kepone, Mirex).
It is generally acknowledged that the past few decades has witnessed an unparalled
expansion of chemical industry with the concomittant development of many new organic
chemical products as well as enhanced product application. Hence, the number and
amounts of chemicals and end products that man is potentially exposed to is staggering.
There are approximately 3,500,000 known chemicals with about 25,000 chemicals in
significant production in the United States alone, increasing at the rate of about 700
new industrial chemicals per year 1 . In 1975, the world production of plastics, rubber
products and fiber products was 40, 10 and 30 million tons respectively 2 . It is esti-
mated that of all the chemicals on the market, approximately 6,000 have been tested
to determine whether they cause cancer 3 ’ 4 , of these approximately 1,000 compounds
have thus far been found to be tumorigenic in animals 4 .
We do not know with precision what percentage of existing chemicals as well as
those which enter the environment annually may be hazardous, primarily in terms
of their potential carcinogenicity, mutagenicity, and teratogenicity. For example,
although the etiology of human neoplasia, with rare exceptions is unknown, it has
been stated repeatedly that a large number of cancers can be attributed to environ-
mental factors in proportions that can vary from 75%5, to 80%617 to 9Q%8 Although
these percentages can be disputed, the fact remains that a certain proportion of human
1
-------
cancers can be attributed to environmental factors 911 , (e.g., exposure to toxic
chemicals, including: benzidine, 2-naphthylamine, 4-aniinobiphenyl, 4-nitrobiphenyl,
bis(chlorornethyl)ether. vinyl chloride, auramine chromium and inorganic arsenic).
If estimates are correct that 60 percent or more of all human cancers are due to
environmental agents, then about 500, 000 cases per year may be involved’ 2 . Exposure
to chemical agents is known to cause a range of occupational cancers, mainly of the
skin, bladder, lungs and nasal sinuses 9 Recently, increased bladder cancer
rates have been found in certain counties where chemical industries were concen-
12,12a .
trated . It is also pro)ected that as the 20—30 year lag period for chemical carcino—
genesis is almost over, a steep increase in the human cancer rate from suspect
chemicals may soon occur 13 .
Concomittant with the potential cancer risk of environmental agents, is the growing
concern over the possibility that future generations may suffer from genetic damage
by mutation-inducing chemical substances to which large segments of the population
may unwittingly be exposed 418 .
Few can dispute the desirability and sense of urgency in controlling the number
of carcinogenic and/or rnutagenic agents that are already in the environment or that
which may be introduced in the future.
The major objectives of this review are to: (1) consider and collate a number of
industrially significant compounds encompassing a spectrum of structural categories
that have b en reported to be carcinogenic and/or mutagenic in order to better assess
the nature of the present potential risk; and (2) to determine whether there are
structural and biological similarities amongst these agents which would better permit
a measure of predictability and prioritization in both the screening of new or untested
2
-------
compounds and the determination of which of the existing potential chemical car-
cinogens to investigate in long-term bioassays.
The cost of examining for carcinogenicity (by existing long-term testing procedures)
of large numbers of suspect chemicals already in the environment as well as those
that will be introduced, is expensive and time consuming. For example,
animal tests of a substance can take as much as 2 to 3 years and cost upward of
sioo, ooo 19 .
Bartsch 21 estimated that the world capacity for testing carcinogenicity (in long-term
tests) is only about 500 compounds per year 21 .
Currently, there are about 450 chemicals now being screened for carcinogenicity
at 28 different U . S. laboratories under the sponsorship of the National Cancer
Institutes Carcinogenesis Program 20 .
Chemical carcinogens and mutagens represent a spectrum of agents varying
in quantitative requirements by a factor of at least io , with strikingly different
biological activities, ranging from highly reactive molecules that can alkylate
macromolecules and cause mutations in many organisms to compounds that are
hormonally active and have neither of these actions 2224 .
Approximately 100 chemicals have been shown to be definitely carcinogenic in
experimental animals 9 ’ 10 In many carcinogenesis studies, the type of cancer observed
is the same as that found in human studied (e. g., bladder cancer is produced in
man, monkey, dog and hamster by 2-naphthylamine, while in other instances,
species variations can exist resulting in the induction of different types of neo-
plasms at different locations by the same carcinogen (e.g., benzidine causes
liver cell carcinoma in the rat and bladder carcinoma in dog and man) 9 ’ 10•
3
-------
A program aimed at identifying and eliminating exposure to potential car-
cinogens and/or mutagens, undoubtedly requires the development of rapid,
inexpensive screening methods to augment long-term animal tests (for potential
carcinogens) in order to focus on the hazardous chemicals among the many
thousands to which humans are exposed.
Mutagenicity screening is now apparently both feasible and necessary for chemicals
now in, and those which will enter the environment. The mutagenic activity of certain
reactive chemicals can be detected in prokaryotic and enkaryotic cells. Short-term
microbial tests (in Salmonella typhixnurium, Escherichia coli and Bacillus subtilus
in combination with in vitro metabolic activation) for mutation-induction include
assays for both forward and reverse mutation at specific loci, as well as tests for
inhibition of DNA repair 416,21,23,25 28
The mutagenic activity of some chemicals have also been detected in Saccharo-
mycesl 4 ]S1 29 3 O, Neurospora 14 ’ 15 ’ 31 and Drosophila 14 ’ 15 ’ 32 .
Chemically-induced stable phenotypic changes have been induced in mammalian
cell culture systems that include Chinese hamster cells 15 ’ 3236, L5178Y mouse
lymphoma cells 15 ’ 37-40, human skin fibroblasts 15 ’ 41 ’ 42 and a human lymphoblastoid
cell line 43 .
Unscheduled DNA synthesis (a measure of excision repair) in human fibroblasts
has been used as a prescreen for chemical carcinogens and mutagens, both with and
without metabolic alteration 4448 .
Although at present there are many test systems available that involve different
genetic indicators and metabolic activation systems for detecting mutagenic activity,
all appear to possess individual advantages and limitations ‘
4
-------
Hence, the belief is generally held that a battery of test systems is needed to
detect the genetic hazards caused by chemicals ’ 416 ’ 21 ’ 28 ’ 4854 . The utilization
of a battery of tests should provide confirmation of positive test data as well as re-
ducing the possibility of false negative tests.
Tier systems (hierarchical) approaches to mutagenicity testing and potential
regulatory control of environmental chemicals that have been proposed by Dean 53 ,
Bartsch 21 , Bridges 50 ’ 51 and Bora 52 are illustrated in Tables 1 and 2 and Figures
1 and 2 respectively. Flamm 49 stressed that “the genetic effects of concern to man
would include the entire myriad of mutational events known to occur in man such as
base—pair substitutions, base additions or del,etions, which comprise the category
referred to as point mutations, as well as the other category of mutations that are
chromosomal in nature and are represented by chromosome deletions, rearrangements
or non-disjunctions”.
Aspects of the evaluation of environmental mutag ens have been described in
regard to the estimation of human risk’ 4 ’ 15,21,49-60 Of all the test systems currently
employed, the Ames test using a rat-liver rnicrosome activation has been evaluated
13,23,25,26,48,61—64
in the greatest detail
Results accumulated up to the present time using a rat-liver rnicrosome test in
vitro with Salmonella typhiinurium strains developed by Ames 23 ’ 25 ’ 26 ’ 6164 have
shown that about 8 0-90% of the carcinogens tested were also mutagens, while the
number of false positives and false negatives was much lower, ranging from 10 to
5%23 2527,4816164 For example, the assay of 300 chemicals utilizing Salmonella/
23,25,26,61—63
microsome test in Ames laboratory included almost all of the known
human carcinogens (e . g., 4-aminobiphenyl, -naphthylamine, benzidine, bischloro-
methylether and vinyl chloride) and hence demonstrated a definite correlation between
5
-------
carcinogenicity and mutagenicity in the testing. A tabulation of these data 25 ’ 48 ’ 62
as a function of chemical class (Table 3) showed a high level of ascertainment in classes
such as aromatic amines , alkyl halides , polycyclic aromatic , nitroaro-
matics and heterocyclics (E), nitrosamines (G), fungal toxins and antibiotics (H),
mixtures (I), and azo dyes and diazo dyes CL) and lower positive response for esters,
epoxides and carbamates (D, 76%), miscellaneous heterocycles (J, 25%) and iniscelia-
neous nitrogen compounds (K, 78%). It should be stressed that for all of the classes
tested, the number of compound within each class is small, and does not permit, at
persent, the distinction that the level of ascertainment varies markedly as a function
of chemical class 48 . Similarly, the data on 63 non-carcinogenic chemicals tested
which show that 22.5% (14/62) are mutagens do not indicate compelling data that the
positive test data occur in any particular chemical class.
The recent evaluation of 6 short-term tests for detecting organic chemical carcinogens
by Purchase et al 64 using 120 organic chemicals, 58 of which are known human or
animal carcinogens, disclosed a 93% of accurate predictions employing the Ames test 61
(S. typhimuriuin strains TA 1535, 1538, 98 and 100 with rat liver microsomal preparation
S-9 fraction: cofactor 1: 3). In the study of Purchase et al 58 , a cell transformation
assay with neonatal Syrian hamster kidney fibroblasts (BHK 21/C 13) and either
human diploid lung fibroblasts (W1-38) or human liver cells (Chang) were treated
with the above test compounds in liquid tissue culture medium (without serum) and
the S-9 mix of the Ames test 6 ’ to aid in the metabolism, yielded a 94% of accurate
predictions. When the responses of the Ames test and cell transformation assay were
compared, it was found that they agree with each other in correctly predicting the
activity of 106 of the 120 compound (88%), while in contrast, they both disagree in
64
only 2 cases those of diethylstilbestrol and vinyl chloride
6
-------
The prospect is widely held that short term tests will offer a method of rapidly
searching a group of compounds for potential carcinogens in order that priorities
may be set for conventional long term studies’°’ 14-16,21,49-64 It has been stressed
that in tests used for preliminary screening, a small proportion of false positive and
false negative results may be acceptable, but for a final test, no false negative results
can be accepted 21 . However, it is also conceded that despite the extent to which
short—term test systems might be improved, there will always remain a finite level
of false positives and negatives. Hence, a number of limitations of mutagenicity test
systems are acknowledged, e . g., some of the factors that determine the processes of
cancer development in vivo cannot be duplicated by mutagenicity systems in vitro .
Other determining factors are: biological absorption and distribution; the concen-
tration of ultimate reactive metabolites available for reaction in organs and animal
species with cellular macromolecules; the biological half-life of metabolites; DNA
repair mechanisms between the test system and the whole animal (excision, strand
break, post-replication and photoreactivation); immuno-surveillance; and organ-
specific release of proximate or ultimate carcinogens by enzymic deconjugation 21 ’ 28 .
Criticisms of submammallian testing have been reported and range from the
current limitations of microbial assays to assess the carcinogenic potential of metals,
organometallics, hormones and particulates 65 and the lack of correlation (in potency
or activity) between microbial mutagenicity and rodent carcinogenicity in a group
of direct-acting and metabolically activated agents (polycyclic hydrocarbons) 66 .
Additionally, although it is conceded that many chemical carcinogens may exhibit
mutagenic activity in certain assay procedures, there are exceptions such as the
nucleic acid base analogs and the acridines which are excellent mutagens but are
not known to be carcinogenic in vivo . The view is also held by some, that the mutational
7
-------
origin of cancer remains an unproven hypothesis with evidence in support of other
66—70
mechanisms
It is useful to briefly summarize several key considerations of reactivity of chemical
carcinogens that are particularly germane for the predictive value of mutagenicity
tests in chemical carcinogenesis. Many chemical carcinogens are reactive electrophiles
per Se, e.g., alkylating agent, acylating en, and other electrophiles 2 ’ 71-78
Despite the diversity in chemical structures of known carcinogens and mutagens,
such as alkylating agents, N-nitrosamines and N-thtrosamides, rutro aryl- and furan
derivatives, aromatic and heterocyclic amines and azo dyes, carbainates, polycyclic
aromatic hydrocarbons, chlorinated hydrocarbons and naturally occurring compounds
(e.g., pyrolizidine alkaloids, aflatoxin), recognition of a common element in chemical
carcinogens and mutagens has rapidly progressed since it was understood that the
majority of carcinogens (procarcinogens) and many mutagens need metabolic activation
in the host for transformation to their so-called ultimate reactive forms 21 ’ 7 78 . Some
procarcinogens are often chemically or spontaneously converted to ultimate carcino-
gens by hydrolytic reactions and often exhibit a broad spectrum of activity in many
species and target organs 71 ,80-83 Other procarcinogens which require host-controlled
biochemical activation (dependent on specific enzyme systerns) 8286 may exhibit more
specific and/or restricted carcinogenic tity 587 . It should also be noted that
the procarcinogen (and its derivatives) are subject to deactivation reactions which
can lead to compounds possessing either no carcinogenic activity or less carcinogenic
potential than the parent compound 73 .
The common denominator of these ultimate reactive metabolites of carcinogens is
their electrophilicity (electron-deficient reactants). They are compounds which react
8
-------
with electron-rich sites in cellular nucleic acids and proteins causing mutagenic effects
frequently paralled by the onset of DNA repair processes 21 ’ 71-73•
Figures 3 and 4-7 illustrates a generalized scheme of the metabolic activation of
chemical carcinogens, possible mechanisms of action of these agents, and steps in the
carcinogenic process respectively 73 ’ 75 . Typical activation reactions (procarcinogens
- proximate carcinogen - ultimate carcinogen) for a variety of agents are shown in
77
Figure 8 . An illustration of some of the factors influencing the formation of reactive
metabolites and their interaction with biological functions in liver cells has been provided
by Arrhenius 78 for the case of aromatic amines (Figure 9). Figure 10 illustrates the
site of interaction of a number of chemical carcinogens with DNA in vivo and in vitro 76 ,
although all four bases of DNA and in some instances the phosphodiester backbone
are targets for one or more carcinogens under some circumstances, by far the most
reactive groups are the purine nitrogens. The N-7 of guanine appears to be the most
reactive site, followed by the N-3 and N-7 postions of adenine 76 .
It is recognized that although many bioassays and safety assessments have con-
sidered single agents (principally purified materials), the role of trace contaminants,
continuous exposure to low levels of multiple agents, co-carcinogens and other factors
are of importance in the evaluation of the sequence of chemical carcinogenesis and the
73,75,87,88
etiology of human cancer
Despite the converging tendency of chemicals to be both carcinogenic and mutagenic
it cannot be known at present whether all carcinogens will be found to be mutagens
and all mutagens, carcinogens, e . g., for classes of compounds such as base analogs
which do not act via electrophilic intermediates and steroidal sex hormones which are
carcinogenic in animals and not yet been shown to be mutagens, different cancer-
inducing mechanisms may be implied.
9
-------
The industrial chemicals considered in this report were limited to organic com-
pounds and selected on factors including: their reported carcinogenicity and/or
mutagenicity, their chemical structures and relationships to known chemical carcino-
gens and mutagens, their volume or use characteristics and suggested or estimated
potential populations at risk.
It should also be noted that while the numbers of individuals directly involved
in the preparation of these chemicals and their byproducts (e. g., plastics, polymers,
etc.) are relatively small in number compared to many industrial segments and
processes, the degree of exposure to potentially hazardous (carcinogenic and muta-
genic) substances can be very substantial indeed. Substantially greater numbers of
individuals may be indirectly exposed to these potential carcinogens and inutagens
vIa (1) use applications which may contain entrained materials, (2) inhalation,
ingestion or absorption of these agent va air, water and food sources resulting from
escape into the atmosphere, leaching into water and food, etc.
In terms of worker exposure, the predominant rats of exposure are via inhalation
and dermal absorption and secondarily from ingestion of food and water.
It should also be stressed that there are instances where the volume produced of
a potentially hazardous chemical is not the overriding consideration. This would
pertain to materials that also have broad utility as laboratory and analytical reagents
(e.g., sodium azide 1 semicarbazide, hydz-oxylamine, hydrazine, diazomethane, etc.)
and hence the individuals in contact with these substances may be relatively larger
than a high-volume hazardous monomer handled in a closed system.
This assessment of potentially hazardous industrial chemicals cannot be complete,
but it will attempt to focus on the possible correlative features (e. g., structural) of
10
-------
a number of significant industrial chemicals that have been reported to be carcinogenic
and/or mutagenic and hence enable a more facile prediction of potential chemical
hazards in the future. It is also hoped that the enclosed tabular compilation will also
provide a more rational basis for the prioritization of those compounds shown to be
mutagenic in individual and/or tier systems and hence are potential candidates for
long-term animal studies to more definitively ascertain their carcinogenicity.
11
-------
TABLE 1
A PREDICTIVE TESTING SCHEME FOR CARCINOGENICITY OR MUTAGENICITY
OF INDUSTRIAL CHEMICALS 53
Phase 1: initial screen
(a) Screening test with sensitive micro—organisms
(i) Salmonella tyhpimurlum TA 1538 (frame shift)
(ii) Escherichia coil WP2 (base—pair substitution)
(iii) Saccharomyces cerevlsiae (mitotic gene conversion)
(b) Nicrosomal assay using rat liver homogenate with the above four micro-
organisms
Cc) Cytotoxicity study with HeLa cells and cultured rat liver (RL 1 ) cells
Cd) Chromosome study in cultured rat liver cells
(e) Short—term exposure of rats by a relevant route to the highest tolerated
dose followed by histological examination and analysis of chromosome damag
Phase 2:
(a) Microsomal assay using liver homogenates from mice and other species
(b) Dominant lethal assay in male mice
Cc) Assay of gene mutation in cultured mammalian cells
Cd) Assay of malignant transformation in cultured cells or by a host—mediated
approach
Phase 3:
(a) An in vivo assay of gene mutation
(b) Dominant lethal assay in male rats
(c) Dominant lethal assay in female rats
(d) In vivo chromosome study in Chinese hamsters or mice or both
(e) Long—term carcinogenicity studies in one or two species
(f) Pharmacokjnetjc studies and biochemical studies at the sub—cellular level
-------
TABLE 2
FRANEWORK OF CARCINOGENICITY TEST PROCEDIJRES 21
VALID DATA ON
TEST SYSTEM
NO DATA ON
Target organ in man;
high risk groups
Carcinogenic in man
Epidetniological
studies
Positive
Threshold dose;
individual risk
Level A
Predictive value for
estrapolation (at
present limited);
target organ; threshold
dose
Species and organ speci-
ficity; dose response in
animals
Carcinogenicity test
in animals
Positive
Level B
Species and/or organ
specificity; correlation
between mutagenic and
carcinogenic potency
Mechanism of metabolic
activation in animals
and man; type of genetic
damage
Mutagenicity tests
Microbial, mammalian,
human cells/activation
in vivo and in vitro
Chemicals
Level C
13
-------
___________ Q i.ttt IL,. tu a t . ..e ..etos .1
. tses 1c rtlk to . &‘ s1u.tto.. ot
TIER 3 rt.i
___________ to r dtgttI l. potttal bso.ftts
1.j.c%
Fig 1 Three-tier framework for mutagenicity crcening. 50, 51
p s.
14
-------
O.f’.C MIO 44
5.- 0’Y t$C F ’ . s.. w o.
4 ( . .i’CA4 .S S 5 4.LC1 4U, C4OlN?*4. £OS4 ’ C CT
4 1
P1 SE
St
I
++
) s;e. ‘ .-————— RL4CT
— +
____ -
++ T V
.E ,ICY sE C
1
I
.4
Ssheme 2 Phu. 8. 1. in vitro bad 1ImlcrosomaL urut. assey for detection of gene mutations. 2. Host-
mediated assay (HIWA) detection at gene mutations in bacteria. 3. in vitro (direct and znicro,omat) and in
vivo (HMA) detection of gene mutation, gene conversion and rnitotic reeombination in eukaryotes. 4. In
- vitro detection of DNA damag. and repair In human arid mammalian cells. 5. In vitro detection of
c romnsomeaberTatjonsInhumaa and masnmaliancells. 6. In vitro detection of gene mut tionain cultured
human and mammalian cell lines. Phase C. 7. Zn vivo (bone marrow and penpherai blood) detection of chro-
mosome abeer-stionsandlor micronuctei in experimental maznmaZs. 8. In vivo (HMA) detection of chromo-
some aberrations an human cells. 9. Detection of dominant letitaJa in erjmentaI mammals. 10. In vivo
detection of heritable tran,locstions In mammalian germ cells. 11. In vivo detection of chromosonie aber-
rations in exposed human populations. Phase D. 12. In vivo detection of gen. mutations in mammals
(sPecific locui test). 13. In vtvo detection of genetic defects in exposed human populations. Phase E. (a)
Establishment of dose response relationship,. (b) Risk benefit considerations and evaluation of genetic
risk to human population. (c) Estimation of acceptable safe dose levels. Phase F. (d) itecominendations.
(e) Regu!atcirl actions. (+4) Positive — substitute available — reject; (a-) positive — flew chemical—has
considerable industrial potential, — chemical iii use — considerable social and economic be-nefit: (2)
results inconclusive or negative but other considerations e.g structural similarity makes it a suspect chemi-
cal — further test: (—) negative In all tests — no reasonable doubt — accept.
PF44SE p
Lcd” to.
•jjT ItO uS 5
Bora, K. C., Mutation Rns., 41 (1976) 73—82
PHA$E F
15
-------
PRECARCI NOCENS
4,
PROXIPV TE CARCI NOGENS
1 ’
CARCINOGENIC CLECTRW’IIIUC R(ACTANTS
CULT IWITE CARCINOGENS)
NUCLEOPHILES TN CRITICAL
CELLULAR TARGt TS:
I -8i SES IN NUCLEIC ACIDS
-AMINO ACIDS IN IlOTEINS
4, —OTHER CELL COMPONENTS
ALTERED NUCLEIC ACIDS OR PROTEINS OR BOTH
GENETIC EFPECTS EPTCENETIC EFFECTS
DIRECT: MUTATIONS CHANGES IN GENOME
‘1’ , \
INDIRECT: ACTIVATION OF I ACTIVATION OF
VIRAL INFORMATION\ j, 1 , ,VIRAL INFORMATION
SELECTION OF LATENT
NEOPLASIA
Figure 3 Metabo& acsiva ion of chemical carcinoge z, eni possible mech.
onisms of eden oJ these ngcnts’
16
-------
Chemical Cnrclnocjon
Metabolic
{ Activation
Ultimate Carcinogen
I Specific
4 Receptor
Altered Receptor
Expression
I’i , .ure 4. Sequence 0/
—.- Latent Tumor Cell
I Growth
‘ r Development
Differentiated Tumor
Progression
Undifferentiated Cancer
complex rven ..s during chemical carcino-
genesis
POSITIVE EVENTS
Altered Receptors
Expression
Latent tumor cell
Growth,
Development
Differentiated tumor
MODIREk1S
Macromoiccular synthesis
Control of differentiation
Gene Action systems
Cocarcinogens
Growth stimulants or
inhibitors
Nutritional status
Endocrine status
Immunologic compctencC
l ’iguri’ 6 Laser slaps in carcinogenic proce.cs involving eta.
imleuls uflecling I/ I a derrlopmne,,t arul gro,,’ilt o/ carcinogen.
mu umlifie ! ills and eon slit urn k’s
Positive Events
Chemical Carcinogen
Met i.’bolic
Activation
Ultimate Cnreinoge
# Specific
Receptors
Altered Receptors
Negative Events
Metabolic Detoxification
and Excretion
f blocks
4’ repaIr
Repair
POSITIVE EVENTS
Modifiers
Species, strain, age, sex, diet,
Intestinal flora,
Mixture of agents
Nucleophltic trapping agents,
Availability of Receptors,
Modifier of growth and ropeir
Repair systems
C—
‘—I
MODIFiERS
Differentiated Tumor
Progression
Undifferentiated Cancer
Fige e .5. P;rst steps of chemical ccrcinogenesls, involving activation of pro.
carc: ”ogcr. -‘ reaction of resulting ut’tirrzate or primary ccrci.- .cgen wit/i specific
cellular receptors, including DNA. These reactions are controlled end modified
by nume e 5ac ors, some of which are noted.
immunologic Status
Gene and chromatin
stability
Surgory
Radiation
Chemotherapy
Immunotherapy
Figure 7., Last steps In carcinogenic process leading to
malignancy, including spread by metastasis. Much more
fundamental inlormation is required to understand fully
these steps. It is these terminal steps which often are
responsible for the jatal outcome If not controlled by the
modifiers listed.
-------
TYPICAL ACTIVATION REACTIONS 77
PROCARCINOGEN —PROXIMATE CARCINOGEN—.ULTIMATE CARCINOGEN
POLYCYCLIC AROMATIC IIYOROCAR8 S
rgigq
B(NZO(a)ANTHRACENE 5 6 - EPOXIDE
(BENZO(a)PYRENE WITH
ADDITIONAL RING)
LRYLAMINES oa ARYLAMIDIS
HETEROCYCIJC AMINES
c1T r
0 0 OCH 3
00
çç
AFLATOXIN 8 2,3—EPOXIOE
ESTER
C-C•CH 2 C—C.CH 2
Q j
Hz 1 12
I ’-HYDROXY 0 ESTER
DERIVATIVE
9’
9H
HNCOOC 2 H 5 HNCO+
N-IIYDROXY CARBONIUM
DERIVATIVE RESIDUE
OR’ CK2OR OR’ CH OR OR’ CH2
uJ
PYRROLIZIQ INE PYRROLIC CARDONIUM
ALKALOID DERIVATIVE RESIDUE
OH 0 ESTER
S I
NHCOCH 3 NCOCH 3
cx zx
N-2—FLUDRENYLACETAMIDE N-HYOROXY
(or 2-ACETYLAMIN0Fw0RENE) OfRIVATIVE
CH 3 _________
“N-NO
CH ’
DIMETI4’Vl.N ITROSAMINE
________ HYDROXYMETHYL
CH 3 N—CONH 2 ) r INTERMEDIATE
NO
METHYLNITROSOUREA
CIt 3 NHNH C I t 3 —
HYDROXYNETHYL
I, 2 DIMETHYLHYDRAZ(NE DERIVATIVE
VINYL CHLORIDE EPOXIDE
‘ -I.
CCJ 4 •,
CAR OP1 TETRACHLORIDE
N 2 C.CHCI H 2 C” HCI
and on the species, the strain, and certain environmental t ciors such asensyme Inducers or inhibiio,
Formation of the “ t ’imale carcinogen ii lso under enzymatic control. The exact nature of the LII5,,II I
carcinogen has been fully documented in all cases. lirca ,iw of the high reactIvity of thc
ht’mkaIt, they cannot oIecn be Isolated. Their nature U usually clucitlared on the basis vi tIn
pr.cur,orx or their rOducis of Interaction,
HC—C’ CIt 2
SAFROLE
H 2 N COOC 2 H 5
ETHYL
CA RBAM ATE
ACTIVE ESTER
(SULFATE, ACETATE)
R H OR —COCH 3
HITROARYL OR HETEROCYCUC
COMPOUNDS
J J NHOH NHO ESTER
-+ c
METHYL
CARBONIUM
ION
2—NITRONAPHTHALENE 2- HYDROXYLAMIPIO
NAPHTHALE IIE
OH H 0 14
— .
OH 0 ESTER
3— IIYDROXYXANTHINE
METHYLAZOX’VMETHAN OI.
FU.L 5 5 8. Schematic biochemical activation of typical procarcinogens. lit some Instances, several
reactions arc in’ oh ed where a proczrciIIOVCfl is converted C c , a provin’ arcinogen, an intermediat e,
more SCtist molecule which. PIowevcr,docs nothaveastructisresuce stitcaninleractdirectlywith
crucUl macrnmolecular receptors In the cill. Nonetheless, in some instances, such as with the
.u’!.,rnin.s. tI, ’, sin, is, eontroII ,ngcvsn; since it Is highly dependent on thestructureofthechemlcal
-------
FIG. 9. FACTORS INFLUENCING THE FOR TION OF REACTIVE 1ETABOLIT
AND THEIR INTERACTION WITH BIOLOGICAL FUNCTIONS IU LIVER CELLS
MEDIAN EMINENCE
stress
diet
ç geQt
The liver cell and its organelles are represented by the outer cell
esthrane (outer irregular line), the endoplascriic reticulum continuc us with
the outer nuclear membrane (inner irregular line) with attached poly—
ribosomes, and the nucleus. At the top is shown the brain median eminence,
which rticipates in stress reactions. Straight arrows represent metabolic
;athways, curvilinear thin arrows stimulatory effects, and curvilinear thick
arrows inhibitory effects. Electrophilic intermediates released from
eta olic steps connected with N-oxygenation interact with metabolic
functions involved in detoxication of the amines themselves and cortico—
steroids, thereby giving rise to self-perpetuating increased production of
reactive metabolites. The electrophilic metabolites also interact with
genetic functions associated with cell structures in close vicinity to the
site of nascence of these metabolites, i.e., ribosomes, messenger RNA and
r. cl us. Endoqanous and exogenous factors shown to increase the produc—
ti n of reactive metabolites in viva or in vi;ro are shown on the right
rrhenjus,
urincry met.
19
-------
3 4 UNZP V4t 1
OMNDEN, $U ENU;MN)SS;
IRIFUICTIONAI. M.KYLATIN
AftNTS;3 l.
ZAAF*MAS?;4N00? 1 71rM*
FIG*U.] .3). Sites of inleracuon of cheincal carcinogens with DNA in vivo znd in vu , ..
bNA C1*Ue
- * 7&rMI*
A
a.AuI
MMU;N MM$ OMS MIUIS
I
IIN%DMS
B
76
20
-------
TAUT..E1.3 48
MUTAGENICITY OP CHEMICAL CAItCINOGENS a
Class
Type
Carcinogens
Non-carcinogens
Total No.
Positive
ll.esponsc
Total No.
Positive
Responte
tested
number
percentage
tested
number
I’orcentai e
A.
Aromiitie amines
25
23
92
11
2
18
0.
Aikyl haikles
20
18
90
3
2
(37
C.
Polycyciic aromatles
26
26
100
8
2
12.5
0.
E.
Esters. epoxide and carbaniates
Nitrc, ari,matlcs ann heterocycles
17
28
13
28
76
100
8
4
2
3
25
75
F.
Miscellaneous all phatics and aromatlcs
5
20
12
0
0
0.
Nitroanmines
21
20
95
0
0
0
II.
Fungal toxing and antibiotics
8
8
100
2
0
0
I.
Mixtures
1
1
100
J.
MisceUanoou* heterocyclus
4
1
25
7
0
0
K.
Miscellaneous nitrogen compounds
9
7
78
4
2
50
L.
Azodyesisnddiazodyes
11
11
100
3
1
33
a Adapto
175
d from McCann et al. 25 • McCann and Ames 6
157
89.7
02
14
22.5
-------
References for Introduction
1. National Cancer Institute, 4th Annual Collaborative Conference, Carcinogenesis
Program, Orlando, Fla., Feb. 22-26 (1976)
2. Vouk, V. B., WHO-NIEHS Symposium on Potential Health Hazards from Technolo-
gical Developments in Plastic and Synthetic Rubber Industries, Research Triangle
Park, NC, March 1-3 (1976)
3. Anon, The spector of cancer, Env. Sci. Technol. , 9 (1975) 116
4. Toth, B., Synthetic and naturally occurring hydrazines as possible causative
agents, Cancer Res. , 35(1975) 3693
5. World Health Organization, Prevention of Cancer, Tech. Rept. Ser. No. 276 ,
Geneva, Switzerland (1964)
6. Haddow, A., Proceedings of the 9th mt. Cancer Congr. Tokyo, Japan, 1966,
R. J. C. Harris, ed., UICC Monograph Series Vol. 9: 111-115, Springer,
Verlag, New York, NY (1967)
7. Boyland, E., The correlation of experimental carcinogenesis and cancer in man,
Progr. Exp. Tumor Res. , 11(1969) 222-234
8. Higginson, J., Present Trends in Cancer Epidemiology, Proc. Eighth Canadian
Cancer Conf. Honey Harbour, 1968: 40-75, Pergamon Press, NY (1968)
9. Toniatis, L., The IARC Program on the Evaluation of the Carcinogenic Risk of
Chemicals to Man, Ann. NY Acad. Sci. , 271 (1976) 296
10. WHO, “Health Hazard of the Human Environment”, World Health Organization,
Geneva (1972) 213—323
11. Hueper, W. C., Occupational and Environmental Cancer of the Urinary System,
Yale University Press, New Haven (1969)
12. Train, R. E., Environmental cancer, Science , 195 (1977) 443
12a. Hoover, , Mason, T. J., McKay, F. W., and Fraumens, J. F., Geographical
patterns of cancer mortality in the United States, In “Persons at High Risk of
Ca ncer: An Approach to Cancer Etiology and Control” Academic Press, New
York, San Francisco, London (1975) pp. 343-360
13. Blu.m, A., and Ames, B. N., Flame-retardant additives as possible cancer hazards,
Science , 195 (1977) 17—22
14. Committee 17, Environmental mutagenic hazards, Science , 187 (1975) 503-514
15. DHEW, Approaches to Determining the Mutagenic Properties of Chemicals: Risk
to Future Generations, DHEW Subcommittee on Environmerrtal Mutagenesis,
Washington,D.C. April, (1977)
22
-------
16. DeSerres, F. J., Prospects for a revolution in the methods of toxicological
evaluation, Mutation Res. , 38(1976) 165-176
17. Crow, J. F., Chemical risk to future generations, Scientist and Citizen , 10
(1968) 113—117
18. Auerbach, C., The chemical production of mutations, Science , 158 (1967) 1141
19. Anon, Application for Ames test expanded, Chem. Eng. News , 54 (1976) 18
20. Maugh, T. H., Can potential carcinogens be detected more quickly? Science ,
183 (1974) 94
21. Bartsch, H., Predictive value for mutagenicity tests for chemical carcinogenesis,
Mutation Res. , 38(1976) 177—190
22. Shubik, P., and Clayson, D. B., Environmental cancer and chemical agents,
Ann. Intern. Med. , 85(1976) 120—121
23. McCann, J., Ames, B. N., The Salmonella/Microsome Mutageriicity Test:
Predictive Value for Animal Carcinogenicity, In Proceedings of Conference on
Origins of Human Cancer, Cold Spring Harbor Laboratory, New York, Sept.
7—14 (1976)
24. Russell, K., and Meselson, M., Quantitative Measures of Carcinogenic and
Mutagenic Potency, In Proceedings of Conference on Origins of Human Cancer,
Cold Spring Harbor Laboratory, New York, Sept. 7-14 (1976)
25. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N., Detection of Carcinogens
as mutag ens in the Salmonella/microsonie test: Assay of 300 chemicals, Proc.
Nati. Acad. Sci. , 72(1975) 5135—5139
26. Ames, B. N., and McCann, J., Carcinogens are mutagens: A simple test system,
in R. Montesano, H. Bartsch and L. Tomatis (eds) Screening Tests in Chemical
Carcinogenesis, Lyon (IARC Scientific Publications No. 12) 1976
27. Sigimura, T., Yahagi, T., Nagao, M., Takeuchi, M., Kawachi, T., Hara, K.,
Yamasaki, E., Mtsushima, T., Hashixnoto, Y., and Okada, M., Validity of
Mutagenicity Tests Using Microbes as a Rapid Screening Method for Environ-
mental Chemicals, In: R. Montesano, H. Bartsch, and L. Tomatis (eds) Screening
Tests in Chemical Carcinogenesis, Lyon, IARC Scientific Publications No. 12
1976
28. Stoltz, D. R., Poirier, L. A., lying, C. C., Strich, H. F., Weisburger, J. H.,
and Grice, H. C., Evaluation of short-term tests for carcinogenicity, Toxicol.
Appl. Pharmacol. , 29 (1974) 157—180
29. Mortimer, R. K. and Manny, T. R., Mutation induction in yeast, in A. Hollaender
(ed) Chemical Mutagens: Principles and Methods for their Detection, Plenum
Press, New York, (1976) pp. 289—310
23
-------
30. Brusick, D., and Andrews, H., Comparison of the genetic activity of dimethyl-
nitrosainines, ethyl methanesulfonate, 2-acetylaminofluorene and ICR- 170 in
Saccharomyces cerevisiae strains D 3 . D 4 and D 5 using in vitro assays with and
without metabolic activation, Mutation Res. , 26 (1974) 491-500
31. Ong, T., and De Serres, F. J., Mutagenicity of chemical carcinogens in
Neurosporacrassa, Cancer Res. , 32(1972) 1890-1893
32. Duncan, M. E., and Brookes, P., The induction of azaguanine-resistant mutants
in cultured Chinese hamster cells by reactive derivatives of carcinogenic hydro-
carbons, Mutation Res. , 21 (1973) 107-118
33. Huberman, E., Aspiras, L., Heidelberger, C., Grover, P. L., and Sims, P.,
Mutagenicity to mammalian cells of epoxides and other derivatives of polycyclic
hydrocarbons, Proc. Nail. Acad. Sd . (US) 68 (1971) 3195-3 199
34. Huberinan, E., Donovan, P. J., and DiPaolo, J. A., Mutation and transformation
of cultured mammalian cells by N-acetoxy-N-2-fluorenylacetamide, J. Nail.
Cancer Inst. , 48(1972)837—840
35. Kao, F. T., and Puck, T. T., Genetics of somatic mammalian cells, VII. Induction
and isolation of nutritional mutants in Chinese hamster cells, Proc. Nail. Acad.
Sd . (US) 60 (1968) 1275—1281
36. Kao, F. T., Cell mutagenesis studies in vitro using auxotrophic markers, in
P.O.P. TsO and J. A. DiPaolo (eds) Chemical Carcinogenesis, Part B, Marcel
Dekker, inc., New York (1974) pp. 565—573
37. Clive D., Flamnm, W. G., Machesko, M. R., and Bernheim, N. J., A mutational
assay system using the thymidine kinase locus in mouse lyrnphoma cells,
Mutation Res. , 16(1972) 77-87
38. Capizzi, R. L., Papirineister, B., Mullins, J. M., and Cheng, E., The detection
of chemical mutagens using the L5178Y/Asn- rnurine leukemia in vitro and in a
hos-mediated assay, Cancer Res. , 34(1974) 3073—3082
39. Fischer, G. A., Predictive tests in culture of drug-resistant mutants selected in
vivo, Nail. Cancer Inst. Monograph , 34(1971) 131-134
40. Summers, W. P., Chemically induced mutation of L5178Y mouse leukemia cells
from asparagine-dependence to asparagine-independence, Mutation Res. , 20
(1973) 377—385
41. Rappaport, H., and DeMars, R., Diaminopurine-resistant mutants of cultured,
diploid human fibroblasts, Genetics , 75 (1973) 335—345
42. Maher, V. M., and Wessel, 3. E., Mutations to azaguanine resistance induced in
cultured diploid human fibrob lasts by the carcinogen, N-acetoxy-2-acetylamino-
fluorene, Mutation Res. , 28 (1975) 277-284
24
-------
43. Sato, K., Slesinski, R. S., and Littlefield, 3. W., Chemical mutagenesis at the
phosphoribosyltransterase locus in cultured human lymphoblasts, Proc. Nati.
Acad. Sci . (US) 69 (1972) 1244—1248
44. San, R. H., and Stich, H. F., DNA repair synthesis of cultured human cells
as a rapid bioassay for chemical carcinogens, mt. 3. Cancer , 16 (1975) 284-291
45. Stich, H. F., San, R.H., and Kawazoe, Y., DNA repair synthesis in mammalian
cells exposed to a series of oncogenic and non-oncogenic derivatives of 4-nitro-
quinoline—l-oxide, Nature , 229 (1971) 416—419
46. Stich, H. F., and San, R. H., DNA repair and chromatin anomalies in mammalian
cells exposed to 4-nitroquinoline-1-oxide, Mutation Res. , 10 (1970) 389
47. Ahmed, F. E., Hart, R. W., and Lewis, N. 3., Pesticide induced DNA damage
and its repair in cultured human cells, Mutation Res. , 42 (1977) 161-174
48. DeSerres, F. 3., Mutagenicity of chemical carcinogens, Mutation Res. , (1976)
41 43—50
49. Flamm, W. G., Atier system approach to mutagen testing, Mutation Res. , 26
(1974) 329—333
50. Bridges, B. A., The three-tier approach to mutagenicity screening and the concept
of radiation-equivalent dose, Mutation Res. , 26 (1974) 335—340
51. Bridges, B. A., Use of a three-tier protocol for evaluation of long-term toxic
hazards particularly mutagenicity and carcinogenicity, In Screening Tests in
Chemical Carcinogenesis, WHO/IARC Pubi. No. 12, eds Montesano, R., Bartsch,
H., and Tomatis, L., Lyon (1976) pp. 549-568
52. Bora, K. C., A hierarchical approach to mutagenicity testing and regulatory
control of environmental control chemicals, Mutation Res. , 41 (1976) 73-82
53. Dean, B. 3., A predictive testing scheme for carcinogenicity and mutagenicity
of industrial chemicals, Mutation Res. , 41 (1976) 83-88
54. Bochkov, N. P., Sram, R. J., Kuleshov, N. P. and Zhurkov, V. S., System
for the evaluation of the risk from chemical mutagens for man: Basic principles
and practical recommendations, Mutation Res. , 38 (1976) 191-202
55. Ehling, U. H., Mutagenicity testing and risk estimation with mammals, Mutation
Res. , 41(1976) 113—122
56. Drake, 3. W., Environmental mutagenesis: Evolving strategies in the USA,
Mutation Res. , 33(1975) 6572
57. Breweri, J. G., Practical evaluation of mutagenicity data in mammals for estimating
human risk, Mutation Res. , 41 (1976) 15-24
25
-------
58. Purchase, I. F. H., Longstaff, E., Asby, J., Styles, J. A., Anderson, D.,
LeFevre, P. A., and Westwood, Evaluation of s ix short term tests-for detecting
organic chemical carcinogens and recommendations for their use, Nature , 264
(1976) 624—627
59. Sobels, F. H., Some thoughts on the evaluation of environmental mutagens,
Mutation Res. , 38(1976) 361—366 -
60. Srazn, R. J., Genetic risk from chemicals: Mutagenicity studies and evaluation,
Rev. Czeck. Med. , 21(1975) 186—193
61. Ames, S. N., McCann, J., and Yarnasaki, E., Methods for detecting carcinogens
and mutagens with the Salmonella/mammalian rnicrosome rnutagenicity test,
Mutation Res. , 31 (1975) 347—364
62. McCann, J., and Ames, B. N., Detection of carcinogens as mutagens in the
Salrnonella/microsome test: Assay of 300 chemicals, Part II, Proc. Natl. Acad.
Sci. , 73(1976)950—954
63. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N., Detection of car-
cinogens as mutagens: Bacterial tester strains with R factor plasmids, Proc.
Nat. Acad. Sci. , 72(1975) 979—983
64. Purchase, I. F. H., Longstaff, E., Ashby, J., Styles, J. A., Anderson, ID.,
Lefevre, P. A., and Westwood, F. R., Evaluation of six short-term tests for
detecting organic chemical carcinogens and recommendations for their use,
Nature , 264 (1976) 624—627
65. Legator, M. S., The inadequacies of sub-mammalian tests for mutagenicity, In
“Molecular and Environmental Aspects of Mutagenesis’ t , Thomas, Springfield
(1974)
66. Sivak, A., The Ames Assay, Science , 193(1976) 271
67. Pitot, H. C., Neoplasia: A somatic mutation or a heritable change in cytoplasmic
membranes? J. Nati. Cancer Inst. , 53(1974) 905-911
68. Pierce, G. B., Neoplasms, differentiation and mutations, Am’. J. Pathol. , 77
(1974) 103—118
69. Mintz, B., and flhinensee, K., Normal genetically mosaic mice produced from
malignant teratocarcinoma cells,
Proc. Nat!. Acad. Sd. , 72(1975) 3585-3589
70. Nery, R., Carcinogenic mechanisms: A critical review and a suggestion that
oncogenesis may be adaptive on togenesis, Chem. Biol. Interactions , 12 (1976)
145—169
26
-------
71. Miller, J. A., and Miller, E. C., Metabolic and reactivity of chemical carcinogens,
Mutation Res. , 33(1975)25
72. Miller, J. A., Carcinogenesis by chemicals: An overview, G.H.A. Cowes
Memorial Lecture, Cancer Res. , 30(1970) 559-576
73. , E. C., and Miller, 3. A., The metabolism of chemical carcinogens to
reactive electrophiles and their possible mechanisms of action in carcinogenesis,
In “Chemical Carcinogens” ed. Searle, C. E., ACS Monograph No. 173, American
Chemical Society, Washington, D.C. (1976) 737-762
74. Lawley, P. D., Carcinogenesis by alkylating agents, In “Chemical Carcinogenesis”
ed. Searle, C. E., ACS Monograph No. 173, American Chemical Society, Washing-
ton, D.C. (1976) pp. 83—244
75. Weisburger, J. H., Bioassays and tests for chemical carcinogens, In “Chemical
Carcinogens” ed. Searle, C. E., ACS Monograph No. 173, American Chemical
Society, Washington, D.C. (1976) pp. 1-23
76. Sarma, D. S. R., Rajalakshmi, S., and Farber, E., Chemical carcinogenesis:
Interactions of carcinogens with nucleic acids, In “Cancer” ed. Becker, t .; ‘ “fl 4 d
(1975) pp. 235—287
77. Weisburger, J. H., and Williams, G. M., Metabolism of chemical carcinogens,
In “Cancer” ed. Becker (1975) pp. 185-234
78. Arrhenius, E., Comparative metabolism of aromatic amines, In ”Chemical Car-
cinogenesis Assays”, eds. Montesano, R., and Tomatis, L., IARC Pubi. No. 10,
International Agency for Research on Cancer, Lyon (1974) pp. 25-37
79. Selkirk, J. K., Huberman, E., and Heidelberger, C., Epoxide is an intermediate
in the microsomal metabolism of the chemical carcinogen dibenz(a,h)-anthracene
Biochem. Biophys. Rest Coxnmun. , 43 (1971) 1010
80. Kriek, E., Carcinogenesis by aromatic amines, Biochim. Biophys. Acta. , 355
(1974) 177
81. Boutwell, R. K., Colburn, N. H., Muckerman, C. S., In vivo reactions of
-propiolactone, Ann. N.Y. Acad. Sci. , 163 (1969) 751
82. Clayson, D. B., and, R. C., Carcinogenic aromatic amines and related
compounds, In “Chemical Carcinogens”,. ed. Searle, C. E., ACS Monograph NO.
173, American Chemical Society, Washington, D.C. (1976) 366-461
83. Sugiznura, T., Kawachi, T.,
Experimental stomach cancer,
Methods Cancer Res. , 7(1973)245-308
27
-------
84. Oesch, F., Mammalian epoxide hydrases: Inducible enzymes catalysing the inaction
of carcinogenic and cytotoxic metabolites derived from aromatic nd olefinic compounds,
Xenobiotica , 3(1972) 305
85. Weisburger, J. H., Weisburger, E. K., Biochemical formation and pharmacological,
toxicological and pathological properties of hydroxylarnines and hydroxamic acids,
Pharrn. Rev. , 25(1973) 1
86. Grover, P. L., Hewer, A., Sims, P., Metabolism of polycyck hydrocarbons by
rat—lung preparations, Biochem. Pharmacol. , 23 (1974) 323
87. Falk, H. L., Possible mechanisms of combination effects in chemical carcinogenesis,
Oncology , 33 (1976) 77—85
88. Nashed, N., Evaluation of a possible role for antiniutagens, antiteratogens-, and
anticarcinogens in reducing environmental health hazards, Env. Filth. Persp .
1.4 (1976) 193—200
28
-------
LI. Tabular Summaries of Potential Industrial Carcinogens and Mutag ens
Table 1 is a summary of 90 compounds examined, structurally tabulated in terms
of 16 major classes and 19 structural sub—sets, chemical name or synonym, chemical
abstract number (CAS#). reported carcinogenicity and/or mutagenicity, and production
quantities where known.
The compounds are arranged in the order in which they appear in the subsequent
chapters and are divided into 14 tabular headings as follows: 1. Alkylating Agents,
2. Acry ting Agents, 3. Peroxides, 4. Halogenated Unsaturated and Saturated Hydro-
carbons and Aromatic Derivatives, 5. Hydra zines, hydroxylamines and carbamates,
6. Nitrosamines, 7. Aromatic Amines, 8. Azo Dyes. 9. Heterocyclic Amines, 10. Nitro-
furans, 11. Anthraquinones, 12. Aromatic Hydrocarbons, 13. Cyclic Ethers, 14. Phos-
phoramides, 15. Nitroalkanes, and 16. Azides.
It is well recognized that there are overlaps in these categorical arrangements.
For example, three classes of halogenated derivatives (e.g., alkane halides, halo-
genated alkanols, and halogenated ethers) are categorized as to their reactivity and
hence are included under alkylating agents but are also tabulated under halogenated
derivatives (4) above for the sake of categorical completeness.
In some instances, classes were listed that had only one iUustrative example
(e.g., lactones, diazoalkanes, carbamates, aromatic hydrocarbons, cyclic ethers.,
phosphoramides and azides). This was felt warranted as the future may weU reveal
additional examples within these classes.
No attempt has been made to present an exhaustive critique and review of the
literature for each of the chemical classes considered in succeeding chapters. Rather
a more balanced review was sought containing germane elements ( where known ) of
synthesis (and identification of important trace contaminants) use categories, potential
29
-------
populations at risk, biological and physical properties, chemical reactivity and
stability, metabolic fate, test systems employed for mutagenicity assay, and test
species and site of tumors in carcinogenicity assays.
There is an acknowledged paucity in many instances of definitive information
regarding domestic production levels (as well as the amount of imported substances),
dissipative levels, aspects of environmental persistence, degradation, transformation,
migration into environmental sinks and material balance.
There are instances of conflicting information as to the carcinogenicity ani/or
mutagenicity of the agents described. Clearly additional data will have to be obtained
in the case of potential carcinogenic substances (as disclosed by mutagenic assay).
Hence long-term bioassay will be required where desired in selected warranted cases,
to definitively establish the assessment of carcinogenicity.
30
-------
Mt,cugt iiici.cy (Il
TM I.I•: 1. Ptitcnt [ nl Industrial Carcinogens ‘I
I I I 1 °‘
- U
find Nut igcns 0
U) k) W.-4
o 1 .I Productior
( 4I U) W0.I4. U)rl W OWOQJ
Class Chemical Name & Synonym CASh
I. Alkylating Agents
A. Epoxides
H 2 ç,—p1 2 Ethylene Oxideb 75—21—8 + 0 + + + 0 + 0 4,870 x 1o 6
0 lbs (1975)
H 2 c SHCH 3 Propylene Oxideb 75—26—9 + 0 0 + + 0 0 0 0 2,315 106
lbs (1975)
H 2 q J1cH 2 cl Epichlorohydrinb 106—89—8 + + 0 + + + + — 0 450 x lO 6 lbs
(1976)
H2c PH-CH2°H GlycIdol 55—65—25 0 + 0 + + 0 0 0 0 >1000 lbs
(1974)
H 2 c —çHCH0 Glycidaldehyde 1 ’ 765344 + + + 0 0 0 0 0 0 <1000 lbs
0 (1976)
:: _:; —c pi 2 tyrene Oxide 1 ’ 96—09-3 ± + 0 0 + 0 0 0
B. Lactones
d11 2 —çH 2 B—Propiolactone (BPL) ’ 57—57—8 + + 0 + 0 0 0 0 0 <1000 lbs
0—C = 0 (1974)
C. Aziridines
b
Aziridine (Ethyleneimine) 151—56—4 + .0 + + + + + 0 0 <4.8 , io 6
H 2 lbs (1974)
-------
Msitarcuiclty ,
•1
TAi E I Potential Industrial Ca rcio cns .—l s-i
S
I I tO
and Mutagen$ Cu J 0 Cu 0 rd Cu a
I•du) Productlon
Cu OQ I Cu i
Class Chemical Name & Synonym CAS# Care in Cu S S Cu 14 5 0 5 0 ) Quantities
_____________ ___________________ _______ ____
C. Azirldines (coat)
/C11 3 b
2—Methylaziridine 75—55—8 + + 0 0 0 0 0 0 0 >1000 lbs
(Propyleneimine) (1974)
4
+ 0 00 + 0 0 0 OlxlOlbs
2 )N_CH 2 CH 2 0H 2_(1_Aziridinyl)_Ethanolb 1072—52—2 - (1974)
D. lkylsulfates
11 3 C0 b
- —OC R 3 Dimethylsulfate (DMS) 77—78—1 + + 0 + + 0 0 0 0 >1000 lbs
(1974)
64 57 5 + + + + + 0 0 0 0 >1000 lbs
H 5 C 2 0— 0C 2 R 5 Diethylsulfate (DES) b — — (1974)
N
S. Sultones
1120714 + 0 + 0 0 0 0 + 0 <1000 lbs
1,3—Propane Suitone ’ 1633—83—6 (1973)
b
_cH 1,4—Butane Sultone + 0 + 0 0 0 0 0 0 <1000 lbs
— — 2 (1974)
F. Aryldialkyltriazenes
N 1mN N(CR 3 ) 2 3,3-Dimethyl-1—phenyl— 7227910 + •+ + + + 0 0 0 0 <1000 lbs
triazene (DMPT) (1974)
Cl@_NN_N(CH 3 ) 2 1—(4--Chlorophenyl)—3,3— 7203909 + + + + + 0 0 0 0 <1000 lba
dimethyltriazene (1974)
-------
Mutagen.ieity
‘—I
TABLE 1. Potential Industrial Carcinogens
S U
I I IC) e.-i i
and Mutagens
I 0 - • - ‘ l’roduction
e oo•-i B .— i.im o
cuss
Class Chemical Name & Synonym CAS# Carcin. Quantities a
G. Diazoalkanes
b 34—88—3 + 0 + + + 0 0 0 0 <1000 lbs
CH 2 =N N Diazomethane
(1974)
H. Phosphoric Acid Estei
(CH 3 O) 3 P0 Trimethyl Phosphate (TMP) 512—.56—1 0 + 0 + + + + 0 0 io 6 lbs
(1974)
(C 2 H 5 0) 3 P=0 Triethyl Phosphate (TEP) 78—40—0 0 0 0 0 + 0 0 0 0 <1000 lbs
(1974)
(BrCH 2 CH(Br)CH 2 O) 3 P=0 Tris(2,3—dibromopropyl)— 126—72—7 + + 0 0 + 0 + 0 0 10 , lbs
phosphate (Tris—BP) (1975)
rn
I. Alkane Halides
C1CH 2 CH 2 C1 1,2—Ethylene Dichioride 107—06—2 0 + 0 0 + 0 0 0 0 9165 x 10 6 Th
(1974)
BrCH 2 CH 2 Br 1,2—Ethylene Dibromide ’ 106-934 + + 0 + + 0 - - 332 x lO 6 lbs
(1974)
BrCH 2 ç HCH 2 C1 1,2—Dibromo—3—chloro— 96-12-8 + + 0 0 o 0 0 0 0
Br propane
J. Halogenated Alkanols
C1CH 2 CH 2 OH 2—Chioroethanol (Ethylene— 107—07—3 + 0 0 0 0 0 0 0 >1000 lbs
chiorohydrin) (1974)
-------
Mutágenicity
TABLE 1. Potential Industrial Carcinogens
and Mut igens U
— Class CIi inical Name & Synonym C4SI CarCiL . J L Quantiti.c
K. Halogenated Ethers
C 1CH 2 OCH 3 Methyichioromethylether 107—30—2 + + 0 0 0 0 0 0 0 >1000 lbs
(CMME) (1974)
*b
C1CH 2 OCH 2 C1 . Bis(chloromethyl)ether .32-88—1 + + 0 0 0 0 0 0 0 <1000 lbs
(BalE) (1974)
Cl(CH 2 ) 2 0(dH 2 ) 2 cl Bis(2—chloroethyl)ethe ’ 111444 ± 0 0 + 0 0 0 O <1000 lbs
(1974)
L. Aldehydes
HQ1O Formaldehyde 50—00—0 ± + 0 + + 0 0 0 0 5765 x 106
lbs (1974
CH 3 CHO Acetaldehyde 75—07—0 0 0 0 0 + 0 0 0 0 1670 x 10
lbs (19 6)
CR 2 —CHCH O Acro lejxt 10202—8 0 0 0 0 + 0 0 0 0 61 x 10 lbs
(1974)
II.Acylating Agents
b
Diinethylcarban yl Chloride 79—44—7 + + 0 0 0 0 0 0 0 <1000 lbs
(DMMC (1974)
c 2 H Diethylcarbamoyl Chloride 88—10—8 0 + 0 0 0 0 0 0 0 1.5 10 4 Tha
(1974)
—c—cl Bensoyl Chloride 98—88—4 + + 0 0 0 0 0 0 0 15 x lO 6 lba
(1972)
CH 2 C-0 Ketene 46-35—14 0 0 0 + 0 0 0 0
-------
Ilutagwiicity U,
TABLE 1. Potential Industrial Carcinogens
andMutagens
H Production
Class Chemical Name & Synonym CASh Carcin. j °- ‘ 5 Quantities
.11. Peroxides
(CH 3 ) 3 COOC(CH 3 ) 3 Di—tert.butylperoxide 110—05—4 0 0 0 + 0 0 0 0 0 3 x IO 6 lbs
(1974)
(CH 3 ) 3 C0011 Tert.butylperoxide 75—91—2 0 + 0 + + 0 0 0 0 >1000 lbs
C 6 H 5 C(CH 3 ) 2 00H Cumene Hydroperoxide 80—15-9 0 + 0 + 0 0 0 0 0 3062 x i0 6
lbs (1974)
HOOC(CH 2 ) 2 C OOH Succinic Acid Peroxide 3504130 0 + 0 0 0 0 0 0 0 >1000 lbs
(1974)
H 2 0 2 Hydrogen Peroxide 772—28—41 0 ± 0 + 0 0 0 0 1.9 X 1O 5
lbs (1974)
CH 3 C000H Peracetic Acid (Peroxy 79—21—0 0 + 0 0 0 0 0 0 0
acetic acid; acetyihydro—
peroxide)
IV.Halogenated Unsat’d and
Sat’d Hydrocarbons and
Aromatic Derivatives
A. Unsat’d Hydrocarbons
*b 6
CH 2 CHC 1 Vinyl Chloride (VCM) 75—01—4 + + + + 0 + - + 5621 x 10
lbs (1974)
CH 2 CC1 2 Vinylidene Chlorid (1,1— 75—35—4 + + 0 0 0 0 0 0 0 0 x 106 lbs
dichloro—ethylene) (1974)
ClCH CCl Trichioroethyleneb 79—01—4 + + + 0 0 0 0 o 0 610 x lO 6 lbs
2 — (1976)
cH 2 =ç—cH H 2 Chloroprene (2—chloro—1,3— 126—99—8 + + 0 0 0 6 + o o 349 x lO 6 lbs
butadiene)*b (1975)
çl i
H —ç —c=ç —ç —H Trans_1,4_dichlOrObutefleb 764—41—0 + + + 0 0 0 0 0 0
R H 01 (1,4—dichloro—2—butene)
C1 2 C=CC1 2 ‘Tetrachioroethylene 127-18-4 + 0 0 0 0 0 0 0 1210 x io 6
(perchioroethylene) (1976)
-------
Nutagenicity tn
‘ -4
TAliLE 1. Potential Industrial Carcinogens —4 i
ii
and Mutagens I I u
n$U o4 O j 5t t
1..l U) )- 5 -4C .Ci.ij)
o - roduction
C]a ; Chemical Name & Synonym CASII Carcin. € 42) 0. 4 r1 5 0 (2) 0 ‘ Quant [ ties
_______________ ______ ____
B. Sat’d Hydrocarbons
b
cuci 3 Chloroform 7—66—3 + — 0 0 0 0 0 0 0 300 t i0 6
lbs (1974)
Cd 4 Carbon Tetrachlorid 6—23—5 + — 0 0 0 0 0 0 0 1000 x i 6
lbs (197k)
CH 3 Cl Methyl Chloride 74—87—3 0 + 0 0 0 0 0 0 0 493 X 10 lbs
(1974)
CH 3 I Methyl Iodide 74—88—4 + + 0 0 0 0 0 0 0 19,000 ] bs
(1975) 6
C1CH 2 CH 2 C 1 1,2—ethylene dichloride 107—06—2 0 + 0 0 + 0 0 0 0 9165 x 10
lbs (197k)
BrCH 2 CH 2 Br 1,2—ethylene dibromide 106—93—4 + + 0 + + 0 0 0 0 332 X 10 lbs
(1974)
C. Halogenated Alkanols
C1CH 2 CH 2 OH 2—Chloroethanol (Ethylene— 107—07—3 + 0 0 0 0 0 0 0 >1000 lbs
chlorohydrin) (1974)
D. Halogenated Ethers
C 1CH 2 OCH 3 MethylchlorOlflethYlether 107—30—2 + + 0 0 0 0 0 0 0 >1000 lbs
(1974)
C1CH 2 OCH 2 C 1 Bis(chloromethYl)ether 432—88—1 + + 0 0 0 0 0 0 0 <1000 lbs
(BcME) (1974)
Cl(CR 2 ) 2 0(CH 2 ) 2 C 1 Bis(2 —chlOroethYl)ether” 542881 ± ± 0 0 + 0 0 0 0 <1000 lbs
(1974)
-------
Mutagenicity U,
TABLE 1. Potential Industrial Carcinogens
01 0 )
and Mutagens I I I U 10 r4 4 1
10k’ O10O€0I lfl 10 60
I rI tfl
U ‘ 10 o o roduction
Class Chemical Name & Synonym CASt! Carcin. 01 01 01 0. 1-. 10 -I 0) 0 0 ) 0 W Quantities
__________________ _____________________ _______ _____ ±: ?:!. ‘ _2 __ ________
E. Aryl Derivatives
CH 2 C 1 Benzyl Chloride 100—44—7 + + + 0 0 0 0 0 0 80 x lO 6 lbs
(1972)
F. Polyaromatics
Polychlorinated Biphenyls ’ 1336363 + + 0 0 0 0 0 40 x lO 6 lbs
(Cl)-’ __f — . cl) — — — — (1974)
V. Hydrazines, Hydroxylamii
Carbamates
N
A. Hydrazines
H 2 N—NH 2 Hydrazineb 302—01—2 + + 0 0 + 0 0 — + 3.1 X lO 6 lbs
b (1971) 6
CE 1,1—Dimethylhydrazine 57—14—7 + 0 0 0 0 0 0 0 0 <1.1 x 10
CH NNH2 (UDMH) lbs (1973)
CH 3 —NH—NHCH 3 1,2_Dimethylhydrazineb 54—07—3 + 0 0 0 0 0 0 0 0 <1000 lbs
(SDMH) (1974)
b
Hydrazine Carboxamide 57—56—7 0 0 0 0 0 + 0 0 0 >1000 lbs
0 (semicarbazide) (1971)
-------
?lutagenicity m
r 1
TAB1.) I. Potential. industrial C . rci un eus
I I lu I I U
•iiid Mulagens o o
I 4 S . Production
cUQI S S
Class Chemical Name & Synonym cASII Carcin. ., ___ Quantities B
B. Hydroxylamines
Hydroxylamine p803498 o + + + o + + 0 o lbs
(1974)
cH 3 — —0H N—Methylhydroxylamine 93771 o + 0 0 + 0 0 0 0 <1000 lbs
H (1974)
0—Methylhydroxylamine p7-62—9 0 + 0 + 0 + 0 0 0 <1000 lbs
(1974)
C. Carbamates
11 2 N_g_0C 2 11 5 Ethyl Carbamate (Urethan)b 51—79—6 + + + + + 0 - 0 1 x 1O 5 lbs
(1972)
00
VI . Nitrosamines
‘N NO Dimethy lnitrosoainineb 62—75—9 + ± ± ± + + 0 o a <1000 lbs
— (DMN) (1976)
2 N—NO Diethylnitrosainine (DEN)” 55—18—5 + ± ± ± + + 0 o o < ® lbs
(1974)
II.Aromatic Amines
.6
N H 2 Bensidine 92—87—5 + + o 0 0 0 0 0 0 1.5 x 10 lbs
(1972)
H 2 N ®NH 2 3,3’—Dichlorobenzidine 1 ’ 91—94—1 + (1972)
+ 0 0 0 0 0 0 0 4.6 x IO 6 lbs
2_Aminoblphenyib 90415 + + 0 - 0 0 0 0 0 0 <1000 lbs
________________ _______ ‘to,’.’
-------
tiutagenicity 0
—I
. 4 J •u
TABLE 1. Potential Industrial Carcinogens
I I IU t .-4 U
andflutagens
I . u, i H Production
U i c EUtn o
C d S S Cd o. I.i .i ..-i w o w o w antities a
Class Chemical Name & Synonym CAS# Carcin. u -____
‘ II.Aromatic /unines (cont)
4 —Aininobiphenyl 92—67—1 + + 0 0 0 0 0 0 0 <1000 lbs
(1974)
1—Naphthyla nine (C( —naph— 134—32—7 ± + 0 0 0 0 0 0 0 7 x io6 lbs
thylainine) (1974)
NH
2 2_NaPhthY] Ibmine ( —naph- 1—59—8 + + 0 0 0 0 0 0 0 <1000 lbs
thylamine) (1974)
Cl Cl
H 2 N 3CH 2 / NH 2 4,4’—Methylene is(2—chlorI —
aniline) (MOCA) 101—14—4 ± + 0 0 0 0 0 0 0 7.7 x lO 6 lbs
(1972)
CH CH3
H 2 N CH NH 2 4,4’—Me hylene Bis(2—methy: —
Os
aniline 1807552 + 0 0 0 0 0 0 0 0 <1000 lbs
(1974)
III.Azo Dyes
Azobenzeneb 103—33-3 ± + 0 0 0 0 0 0 <1000 lbs
(1974)
® .N N_®NH 2 paraAmino Azo Benzene ’ 60—09-3 + + 0 0 0 0 0 0 0 3.3 x lO 5 lbs
(1974)
p—Dime 1 hylamino Azo Benzen 60—11-7 + + 0 0 0 0 0 0 0 1 x 1O 4 lbs
(DAB) (1971)
CII H 3
ortho—Ainino Azo Toluene 97- 56—3 + + 0 0 0 0 0 0 0 4.5 x lO 5 lbs
(o-AT?’ (1973)
-------
IL. ti.J. LLy .,
TABLE 1. Potential. Industrial Carcinogens
S S
and Mutagens I I I Q i I IJ
cuu oi oi
I n I -i 0 Productian
J 4
Class Cla’inicai Name & Synonym cAS# Carcin ( U S S . 5 5 0 5 0 5 Quanttti s a
___________ _________________ ______ ____ e _u - _J _______
IX.Heterocyclic Aromatic
Mines
Quinoline 91-22—5 + + 0 0 0 0 0 0 0
OH N 8—Hydroxyquinoline 184243 ± + 0 0 0 0 0 0 0
X. Nitrofurans
Nitrofuran 609392 0, + 0 0 0 0 0 0 0 <1000 lbs
0 2 N f [ TIr’ 1 NHCCH 3 N—(4—(5-nitro-2-furyl)—2- (1974)
0 thiazolyl)acetamide) (NFTA 531—82—8 0 + 0 0 0 0 0 0 0 <1000 lbs
0
O 2 NE JL__lUIN N- (4— (5-nit ro-2 --furyl) —2- (1974)
‘0 thiazolyl)formamide (FANFT 24554265 + + 0 0 0 0 0 0 0 <1000 lbs
(1974)
.1. Anthraguinones
0 01! 1,2—dlhydroxy—9,10—anthra— 74-48-0
X X j0H quinone (alizarin) 0 + 0 0 , 0 0 0 0
0
fi J 1,4—dlhydroxy—9,10—anthra— 81—64—1 0 + 0 0 0 0 0 0 0
quinone (Quinizarin)
OH
-------
flutagLnlclty n
. -1
TABLE 1. Potential Industrial Carcinogens
a a
I I I U 5 -4
and Mutagens o o a a
I ‘ U) Produ
U I -i C d 0 0 0 Cd E -I E J n • ction
Class Chemical Name & Synonym CAS# Carcin. W Ow 050 UQ fltjLjesa
____ 4 C 4
U. Anthraguinones (cont)
0
OH 1,2,3—Trihydroxy—9,1O— 602-64-2 0 + o 0 0 0 0 0 0
O H anthraquinone (Anthragallol
0
OH
J OH 1,2,4—Trihydroxy—9,1O—
anthraquinone (Purpurin) 81—54—9 0 + 0 0 0 0 0 0 0
o OH
O
1,4—Dfamino—9,1O—anthra 128—95—0 0 + 0 0 0 0 0 0 0
qu inone
O
iII. Aromatic Hydrocarbons
Benzen ’ 74—43—2 + 0 0 0 0 + + 0 0 1.4 billion
gallons
(1976)
:111. Cyclic Ethers
,CH 2 -CH
1,4_Dioxaneb 123911 + 0 o 0 0 0 0 0 0
CH 2 —CH 2
-------
Mtlt3gunle; [ ty u,
•TABLl 1. 1’otcntI ii Industrial Cart im cn
and Hutagens t U
Product1an
Class Chemical Name & SyLlonym CAS# Carcin. “ Quantities
XIV. Phosphoramides
N H 3 )2
(CH 3 ) 2 N 7 P O Hexamethyl phosphoramide + 0 0 0 0 ± 0 0 0
N(CH ) (Tris(dimethylamino)phos- 680-31-9
3 2 phine oxide, HMPA)
XV. Nitroalkanes
CH 3 -CH-CH 3 2-Nitropropane 79-46-9 + 0 0 0 0 0 0 0 0
NO 2
XVI.Azides
+-
Na NNN Sodium azide 266—28—22 0 + 0 0 0 0 0 <1000 lbs
(1974)
+ Reported positive in the literature
— Reported negative in the literature
0 Not tested, unreported, or unknown
* IluiTlan carcinogen
a Data from Stanford Research Institute (SRI) CheMical Producers Index; Chemical Marketing Rsports
Chemical Week; Chest. Eng. News
b Reviewed in IARC (International Agency for Research on Cancer) Monographs on the Evaluation of Carcinogenic
Risk of Chemicals to Man
-------
III. ALKYLATING AGENTS
A. Epoxides
The epoxides (oxiranes, cyclic ethers) include a number of very reactive reagents
that are exceptions to the generalization that most ethers are resistant to cleavage.
Because of the strain energy of oxiranes, they react with acidic reagents even
more rapidly than acyclic ethers do, producing n-substituted alcohols. The direction
of opening of an oxirane in the SN 2 and acid-catalyzed processes differs. The less
highly substituted carbon (sterically more accessible) is the site of the attack in the
SN 2 process, whereas the more highly substituted carbon (more stable carbonium ion)
is the site of attack in the acid-catalyzed process (Figure 1). Due to their reactivity,
a number of epoxides have broad general utility.
1. Ethylene Oxide (H 2 ç H 2 ; 1 ,2-epoxyethane) possesses a three-membered
ring which is highly strained and readily opens under mild conditions; e. g., even
in the unprotonated form it reacts with nucleophiles to undergo SN 2 reactions. The
ease of this reaction is ascribed to the bond angle strain of the 3-membered ring
(estimated to be about 27k cal/mode) a strain that is relieved in the course of the
ring-opening displacement reaction the bond angle strain providing a driving force
for the reaction 1 . The commercial importance of ethylene oxide which is used in enor-
mous quantities, lies in its readiness to form other important compounds, e . g.,
ethylene glycol, diethylene glycol, the cellosolves and carbitols, dioxane, ethylene
chlorohydrin and polymers (carbowax) (Figure 1). Ethylene oxide is also widely
employed in the production of non-ionic surface-active agents, triethylene glycol,
ethanolamines, choline, and, a wide variety of organic chemicals, as well as broadly
used in fumigation and sterilization 2 and as an intermediate for polyethylene tereph-
thalate polyester fibre.
43
-------
In limited studies, no carcinogenic effect was found when ethylene oxide was
tested in ICR/Ha Swiss mice by skin application and in rats by subcutaneous injection 2 .
Ethylene oxide reacts with DNA, primarily at the N-7 position of guano sine, forming
N-7-hydroxyethylguanine 3 . Ethylene (1, 2- 3 H) oxide alkylated protein fractions taken
from different organs of mice exposed to air containing 1.15 ppm of the labelled agent 4 .
The highest activity was found in lung followed by liver, kidney, spleen and testis 4 .
Ethylene oxide produces reverse mutations in S. typhiinurium TA 1535 strains
(without activation) 5 and in Neurospora crassa at the adenine locus 6 , induces recessive
lethals 7 ’ 8 ’ ‘ , translocations °’ U, and minute mutations’ 2 in Drosophila melanogaster .
Exposure of male Long-Evans rats for 4 hours to 1.83 g/m 3 (1000 ppm) ethylene
oxide produced dominant lethal mutations 5 , while chromosome aberrations were found
in bone-marrow cells of male rats of the same strain exposed to 0.45 g/m 3 (250 ppm)
ethylene oxide for 7 hours/day for 3 days 5 . Ethylene oxide induces chromosome
aberrations in mammalian somatic cells ’ 3 ” 4 .
2. Propylene Oxide (1, 2-epoxypropane; H 2 q - -CH 3 ) (less reactive than ethylene
oxide) is used largely as an intermediate in the production of polyether polyols which
are used to make polyurethane foams; other major uses include the production of pro-
pylene glycol for the manufacture of unsaturated polyester resins; conversion to
dipropylene glycol, glycol ethers and synthetic glycerin 2 . Propylene oxide has also
been us d as a fumigant for a spectrum of materials ranging from foodstuffs to plastic
2
medical indstruments
Propylene oxide is produced by two processes, viz., (1) chiorohydrin process
from 1-chloro-2-propanol and Ca(OH) 2 , and (2) a peroxidation process based on the
oxidation of isobutane to tert.butyl alcohol and tert.butyl hydroperoxide, the latter
after separation, is used to oxidize propylene to propylene oxide 15 .
44
-------
Propylene oxide is carcinogenic in rats producing local sarcomas following sub-
cutaneous injection 2 ’ 16• reacts with DNA at neutral pH to yield N-7-(2-hydroxy-
propyl)guanine and N-3-(2-hydroxypropyl)adenine as the major products 17 . Pro-
pylene oxide induces reverse mutations in Neurospora crassa 18 and recessive mutations
in Drosophila melanogaster 8 ’ 9 ’ 19 .
3. Epichiorohydrin (1 -chlqro -2,3 -epoxypropane; c bloropropylene oxide;
CH 2 -FH-CH 2 C1) is produced by chiorohydrination of allyl chloride (obtained by
chlorination of propylene) 20 , and is extensively used as an intermediate for the
manufacture of synthetic glycerins, epoxy resins, (e.g., via reaction with Bisphenol-
A), elastomers, and in the preparation of pharmaceuticals, textile coatings, cleaning
agents, glycidyl ethers, paper sizing agents, ion-exchange resins, surface active
agents, corrosion inhibitors, inks and dyes 2 ° and as a solvent for resins, gums,
cellulose and paints.
Epichiorohydrin has recently been reported to produce squamous cell carcinomas
of the nasal epithelium in rats following inhalation at levels of 100 ppm for 6 hours!
day 21 . Epichiorohydrin has been previously shown to induce local sarcomas in mice
following subcutaneous injection 22 .
Epichlorohydrin (without metabolic activation) at concentrations of 1-50 mM per
1 hr, induced reverse mutations in S. typhimurium G46 and TA 100 tester strains 23 .
The mutagenic activity with TA 1535 tester strain was markedly reduced in the pre-
sence of liver homogenates 24 . Epichlorohydrin produced reverse mutations in E.
coli 25 and in Neurospora crassa’ 8 , recessive lethal mutations in Drosophila melano-
gaster 8 , was mutagenic in Kiebsiella pneumorLiae 26 .
45
-------
Doses of 50 and 100 mg/kg of epichiorohydrin after 3 hours increased the fre-
cjuency of reverse mutations using S. typhimurium strains G46, TA 100 and TA 1950
in ICR female mice in a host-mediated assay 23 .
Mutagenic activity (as determined with the TA 1535 strain of S. typhimurium )
was detected in the urine of mice after oral administration of 200-400 mg/kg epich-
lorohydrui 24 . Although an initial evaluation of mutagenic activity (utilizing the above
system) in the urine of 2 industrial workers exposed to a concentration in excess
of 25 ppm was regarded as borderline, additional mutagenic testing revealed more
definitive evidence of activity, with the active compound appearing as a conjugate 24 .
Epichlorohydrin induced dose-dependent chromosome abnormalities in bone
marrow of ICR mice injected i.p. to a single dose of 1-50 mg/kg or repeated doses
of 5 x 5-20 mg/kg, or given p.o. in a single dose of 5-100 mg/kg or repeated doses
of 5 x 20 mg/kg 23 . Epichlorohydrin did not induce any dominant lethal mutation in
ICR mice when given i.p. in a single dose of 5-40 mg/kg 23 , 150 mg/kg 27 , repeated
doses of 5 x 1-10 mg/kg, p.o. in a single dose of 20 or 40 mg/kg or by repeated doses
at 5 x 4-20 mg/kg 23 . Human peripheral lymphocytes exposed to i0 - i - epichioro-
hydrin in vitro during the last 24 hours of cultivation showed chromosomal aberrations 23 .
It was 4-5 times less mutagenic than the polyfunctional mutagenic agent TEPA when
tested analogously 28 ; the epichlorohydrin induced changes were mainly classified
23
as chromatid and isochroinatid breaks and exchanges . These results demonstrated
the ability of epichlorohydrin to induce gene and chromosome mutations in somatic
cells. The finding of no changes in gametic cells was suggested to be the result of
biotransformation changes of epichiorohydrin into forms which then cannot reach
gametic cells in a concentration capable of inducing dominant lethal effects’ 9 .
46
-------
NIOSH estimated that approximately 50, 000 U.S. workers are occupationally
exposed to epichiorohydrin. Approximately 550 million pounds of epichiorohydrin
29
wereproducedintheU.S. in 1975
4. Glycidol (2, 3-epoxy-1-propanol; CH 2 -,pH-CH 2 OH) is widely employed in
textile finishings as water repellant finishes, and as intermediate in production of
glycerol and glycidyl esters, esters and amines of industrial utility. It is mutagenic
in Drosophila 8 , Neurospora 8 , and S. typhimurium tester strains TA 98 (for frame-
shift mutagens) and TA 100 (for base pair substitution mutagens) 30 , both with and
without rat liver microsomal extract (RME) (although less effective in the presence
of RME) and in Kiebsiella penumomae auxotroph
5. Glycidaldehyde (2,3-epoxy--1-propanol; CH 2 -pHCHO) is prepared from acrolein
by the action of hydrogen peroxide or sodium hypochlorite 31 . It has been used as a
cross-linking agent, vapor-phase disinfectant and suggested synthetic intermediate 3 ’.
Glycidaldehyde is carcinogenic in ICR/Ha Swiss mice following skin application 32
or subcutaneous injection 33 and in Sprague-Dawley rats following its subcutaneous
33,34
admlrustratLon
Glycidaldehyde produces base-pair mutations in S. typhimurium TA 153 535,
TA 10OO 35 and TA ioo30 tester strains (on a molar basis glycidaldehyde was about
20 to 50 times more potent in producing mutations than glycidol in TA 10030).
Glycidaldehyde induces reverse base-pair mutations in Saccharomyces cerevisiae
strain S211 36 and petite cytoplasmic mutations by strain N123 of S. cerevisiae 36 .
It produces base-pair transitions (primarily A-T to G-C), frame-shift mutations
and some deletions in bacteriophage T4 37 ’ 38 and is mutagenic in Kiebsiella peneumoniae 26 .
47
-------
6. Styrene Oxide (1, 2-epoxyethylbenzene; epoxystyrene; phenyl oxirane;
phenylethylene oxide; -C -/H 2 ) is produced either via the epoxidation of styrene
with peroxyacetic acid or by the chiorohydrin route from x-phenyl-(3-iodoethanol and
K0H 39 . It is used as a reactive diluent in epoxy resins and as an intermediate in the
preparation of agricultural chemicals, cosmetics, surface coatings and in the treatment
of textiles and fibers 39 .
Styrene oxide has been tested by skin application in C 3 H and Swiss ICR/Ha mice
with no significant increase in the incidence of skin tumors observed 40 ’ 41 .
Styrene oxide induces reverse mutations in S. typhimurium strains TA 1535
and TA ioo42.43 without metabolic activation, (producing base-pair substitutions).
Previous recent mutagenic analyses with styrene oxide on strains of S. typhimurium
44
(TA 1537 and TA 1538) sensItive to frame-shift producing agents were negative
Styrene oxide also induces forward mutations in Schizosaccharomyces pombe , mitotic
gene conversions in strain 1)4 of Saccharomyces cerevisiae and is a potent mutagen
in the production of forward mutations in mammalian somatic cells in culture (aza-
guanine-resistantmutants in V79 Chinese hamster cells) 45 . In this latter case, it
was more active tK2n ethylmethane suiionate 46
It should be stressed that styrene oxide is a metabolite in the proposed transfor-
mation of styrene to hippuric acid in man and animals’ 1T , viz.,
9H
R-CH = CH 2 - R-C i-çH 2 -‘ R-çH-cH 2 - R-ÔH-COOH
OHOH /
Styrene Styrene Styrene / Mandelic
R C 6 H 5 oxide glyco ,/ I acid
R-C-NHCH 2 COOH - R-COOH - R-CH 2 OH R-ç-COOH
0
Hippuric acid Benzoic Benzyl Phenyl glyoxylic
acid alcohol acid
48
-------
For example, as postulated by Leibman 49 , styrene is metabolically converted
to styrene oxide, and subsequently to styrene glycol by microsomal mixed function
oxidases and microsomal epoxide hydrase from the liver, kidneys, intestine, lungs,
and skin from several mammals 50 . The principal metabolites which have been
detected in the urine of factory workers exposed to styrene vapor on the job, or
volunteers exposed under controlled conditions for 4-60 ppm styrene vapor for 2
47,48
hours are mandelic acid and phenyiglyoxylic acid
Styrene is produced in quantities in excess of 1 million tons per year in the
United States as well as in considerable quantities in Europe and Japan. The prin-
cipal areas of application of styrene is in the production of plastics and resins (e.g.,
polystyrene resins; styr ene-acrylnitrile copolyiners; styrene-butadiene copolymer
resins; styrerre-butadiene rubber and acrylonitrile-butadiene-styrene (ABS) ter-
polymer).
In addition to styrene vapors detected in the air of vulcanization plants producing
butadiene—styrene rubber soles, it has been found as a constituent of coal gas, coal
45
tar, and of gasoline produced by cracking processes and recently as a contaminant
in samples of drinking water in the U.S . 51 .
Information as to the mutagenicity is somewhat conflicting. Thus while Milvy
and Garro 42 reported styrene to be rion-mutagenic when tested (without activation)
with S. typhimuriurn TA 1535, TA 100, TA 1537, TA 1538, and TA 98 on agar overlay
plates, styrene was reported by Vaino et a1 43 to be xnutagenic toward TA 1535 and
TA 100 only after metabolic activation. This would suggest that styrene seems to be
an indirectly acting mutagen to TA 1535 and TA 100, the same strains which are also
sensitive to styrene oxide 35 ’ 42,43• In the presence of liver homogenate, styrene
49
-------
appears to be even more mutagenic to strain TA 1535 than styrene oxide which may
be due in part to the complex factors involved in chemical-homogenate association 43 .
Styrene was non-mutagernc when tested on forward mutation and gene-conversion
systems of yeast (S. pombe and S. cerevisiae respectively). However, it was
mutagenic only in a host-mediated assay with yeast (S. cerevisiae ) when tested at
very high doses (1000 mg/kg) 45 .
50
-------
Ack1-cata yzed ring opening
SN2nucIeophhc rng op nng
H .
RCHCH 2 OH — RC —,CH 2
Br
( ) RMgX
(2) H O
RCHCH ’
OH
RCHCH 3
OH
Fig. 1. Acid-caialvz d and S%2 nucleophilic Tiflf openirl ction of
o irane .
H C ‘
NOCH CH OH H-
Ethylene g)ycol
(HOCH 2 CH 2 ) 0
Diethylene glycol
________ H:C’
CH,...CH CH OH }{OCH 2 CH.OCH, Cft O(CH 3 ).O(CU 2 ) 2 O}I
0 M:thylc*c’oiiol
,CH CH
CH 3 CH?’
Dne
C!CH 2 CH OH
HO
Ethylene chIorohy rin
H...-C(CH:CH O).H
Carbow3
Fio. 2. . Some comrr.erci3 ;y irnport3u deriva:iv s of ethv1cc. o :.
\c
5].
-------
References for Epoxides
1. Gutsche, C. D., and Pasto, 0. J., Fundamentals of Organic Chemistry, Prentice—
Hall, Englewood Cliffs, NJ (1975) p. 296
2. IARC, Vol. 11, Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial
Chemicals and General Considerations on Volatile Anaesthetics, International
Agency for Research on Cancer, Lyon, 1976, pp. 157-167, 191-196
3. Brookes, P., and Lawley, P. D., The alkylation of guanosine and guanylic acid,
J. Chem. Soc . (1961) 3923-3928
4. Ehrenberg, L., Hiesche, K. D., Osterman-Golkar, S., and Wennberg, I., Effects
of genetic risks of alkylating agents: Tissue doses in the mouse from air con-
taminated with ethylene oxide, Mutation Res. , 24 (1974) 83-103
5. Embree, J. W., and Hine, C. H., Mutagenicity of ethylene oxide, Toxicol. Appi.
Pharmacol. , 33(1975) 172—173
6. Kolmark, G., and Westergaard, M., Further studies on chemically induced
reversions at the adenine locus of Neurospora, Hereditas , 39 (1953) 209—224
7. Bird, M. J., Chemical production of mutations in Drosophila : Comparison of
techniques. J. Genet. , 50(1952) 480—485
8. Rapoport, I. A., Dej stvie okisi etilena, glitsida i glikoles na gennye mutatsii
(Action of ethylene oxide glycides and glycols on genetic mutations), DokI.
Acad. Nauk. SSR , 60 (1948) 469-472
9. Rapoport, I. A., Alkylation of gene molecule, Doki. Acad. Nauk. SSR , 59 (1948)
1183—1186
10. Watson, W. A. F., Further evidence on an essential difference between the genetic
effects of mono- and bifunctional alkylating agents, Mutation Res. , 3 (1966) 455-457
11. Nakao, Y, and Auerbach, C., Test of possible correlation between cross-linking
and chromosome breaking abilities of chemical mutagens, Z. Vererbungsl , 92
(1961) 457—461
12. Fahmy, 0. G., and Fahny, M. J., Gene elimination in carcinogenesis: Reinter-
pretation of the somatic mutation theory, Cancer Res. , 30 (1970) 195-205
13. Strekalova, E. E., Mutagenic action of ethylene oxide on mammals, Toksikol.
Nov. Prom. Khimschesk. Veshchestv., , 12 (1971) 72-78
14. Fomenko, V. N., and Strekalova, E. E., Mutagenic action of some industrial
poisons as a function of concentration and exposure time, Toksikol. Nov.
Promyscien. Khirnschesk. Veshchestv. , 13 (1973) 51—57
52
-------
15. Lowenheim, F. A., and Moran, M. K., Propylene oxide, In Faith, Keyes and
Clark’s Industrial Chemicals, 4th ed., John Wiley & Sons, New York (1975)
pp. 692—697
16. Walpole, A. L., Carcinogenic action of alkylating agents, Ann. NY Acad. Sci. ,
68 (1958) 750—761
17. Lawley, P. D., and Jarman, M., Alkylation by propylene oxide of deoxyribo-
nucleic acid, adenine, guanosine and deoxyguanylic , Biochem. J. , 126
(1972) 893—900
18. K$lmark, G., and Giles, N. H., Comparative studies on monoepoxides as inducers
of reverse mutations in Neurospora, Genetics , 40 (1955) 890-902
19. Schalet, A., Drosophila Info. Service, 28(2) (1954) 155
20. Lowenheim, F. A., and Moran, M. K., Epichiorohydrin, In Faith, Keyes and
Clark’s Industrial Chemicals, 4th ed., John Wiley & Sons, New York (1975)
pp. 335—338
21. Anon, Epichiorohydrin causes nose cancers in rats, NYU’s Nelson Reports,
Pesticide & Toxic News , 5 (19) (1977) 27—28
22. Van Duuren, B. L., Goldschrnidt, B. N., Katz, C., Siedman, I., and Paul,
J. S., Carcinogenic activity of aklylating agents, J. Nati. Cancer Inst. , 53
(1974) 695—700
23. Sram, Cerna, M., and Kucerova, M., The genetic risk of epichlorohydrin as
related to the occupational exposure, Biol. Zbl. , 95 (1976) 451-462
24. Kilian, D. J., PuJ.lin, T. G., Connor, T. H., Legator, M. S., and Edwards, H. N.,
Mutagenicity of epichiorohydrin in the bacterial assay system: Evaluation by
direct in vitro activity and in vivo activity of urine from exposed humans and
mice, Presented at 8th Annual Meeting of Environmental Mutagen Society, Colorad,
Springs, Cob. Feb. 13—17 (1977) p. 35
25. Strauss, B., and Okubo, S., Protein synthesis and the induction of mutations by
E.colibyalkylatirtg agents, 3. Bact. , 79(1960) 464-473
26. Voogd, C. E., Mutageic action of epoxy compounds and several alcohols, Mutation
Res. , 21(1973) 52-53
27. Epstein, S. S., Arnold, E., Andrea, 3., Bass, W., and Bishop, Y., Detection of
chemical mutagens by the dominant lethal assay in the mouse, Toxicol. Appl.
Pharmacol. , 23(1972) 288—325
28. Kucerova, M., Polivakova, Z., Sram, R., and Matousek, V., Mutagenic effect
of epichiorohydrin. I. Testing on human lymphocytes in vitro in comparison with
TEPA, Mutation Res. , 34(1976) 271-278
53
-------
29. Anon, NIOSH recommends new epichlorohydrin standard, revision on allyl
chloride, Toxic Materials News , 3 (1976) 154
30. Wade, M., and Moyer, J.., The mutagenicity of epoxides, Fed. Proc. , 35 (1976)
404
31. IARC, Vol. 11, Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial
Chemicals and General Considerations on Volatile Anesthetics, International
Agency for Research on Cancer, Lyon (1976) PP. 176-181
32. Van Duuren, B. L., Orris, L., and Nelson, N., Carcinogenicity of epoxides,
lactones, and peroxy compounds, II, J. Nati. Cancer Inst. , 35 (1965) 707-717
33. Van Duuren, B. L., Langseth, L., Orris, L., Teebor, G., Nelson, N., and
Kuschner, M., Carcinogenicity of epoxides, lac tones and peroxy compounds,
N. Tumor response in epithelial and connective tissue in mice and rats, J.
Nati. Cancer Inst. , 37(1966)825—834
34. Van Duuren, B. L., Langseth, L., Orris, L., Baden, M., and Kushner, M.,
Carcinogenicity of epoxides, lactones and peroxy compounds, V. Subcutaneous
injection inrats, J. Nati. Cancer Inst. , 39(1967) 1213-1216
35. McCann, J., Choi, E., Yainasaki, E., and Ames, B. N., Detection of carcinogens
as mutagens in Salmonella/xnicrosome test: Assay of 300 chemicals, Proc. Nati.
Acad. Sd. , 72(1975) 5135—5139
36. Izard, M. C., Recherches sur les effects mutagenes de l’acroleine et de ses deu.x
epoxydes le glycidol et le glycidal sur S. cerevisiae , C .R. Acad. Sci. Ser. D.,
27.6(1973) 3037—3040
37. Corbett, T. H., Heidelberger, C., and Dove, W. F., Determination of the muta-
genie activity to bacteriophage T4 of carcinogenic and noncarcinogenic compounds,
Mol. Pharmacol. , 6(1970) 667—679
38. Corbett, T. H., Dove, W. F., and Heidelberger, C., Attempts to correlate car-
cmogenic with mutagenic activity using bacteriophage, mt. Cancer Congr.
Abstr. , 10 (1970) 61-62; 10th mt. Cancer Congr., Houston, TX May 22-29 (1970)
39. IARC, Vol. 11, Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial
Chemicals and General Considerations on Volatile Anesthetics, International
Agency for Research on Cancer, Lyon (1976) pp. 201-208
40. Weil, C. S., Condra, N., Huhn, C., and Striegel, .1. A., Experimental carcino-
genicity and acute toxicity of representative epoxides, J. Am. md. Hyg. Assoc. ,
24 (1963) 305—325
41. Van Duuren, B. L., Nelson, N., Orris, L., Palmes, E. D., and Schmidt, F. L.,
Carcinogenicity of epoxides, lactones and peroxy compounds, J. Nati. Cancer
Inst. , 31 (1963) 41-55
54
-------
42. Milvy, P., and Garro, A. J., Mutagenic activity of styrene oxide (1,2-epoxy ethyl
benzene) a presumed styrene metabolite, Mutation Res. , 40 (1976) 15-18
43. Vai.nio, H., Paakkonen, R., Ronnholm, K., Raunio, V., and Pelkonen, 0., A
study on the mutagenic activity of styrene and styrene oxide, Scand. 3. Work .
Env. Hlth. , 3(1976) 147—151
44. Glatt, H. R., Oesch, F., Frigerio, A., and Garattini, S., Epoxides metabolically
produced from some known carcinogens and from some clinically used drugs,
I. Difference in mutagenicity, mt. J. Cancer , 16 (1975) 787—797
45. Loprieno, N., Abbondandolo, A., Barale, R., Baroncelli, S., Bonatti, S.,
Bronzetti, G., Cammellini, A., Corsi, C., Corti, G., Frezza, D., Leporini, C.,
Mazzaccaro, A., Nieri, R., Rosellini, D., and Rossi, A., Mutagenicity of
industrial compounds: Styrene and it possible metabolite styrene oxide,
Mutation Res. , 40(1976) 317—324
46. Abbondandolo, A., Bonatti, S., Colella, C., Corti, G., Matteucci, F., Mazzac-
caro, A., and Rainaldi, G., A comparative study of different protocols for
mutagenesis assays using the 8-azaguanine resistance system in Chinese hamster
cultured cells, Mutation Res. , 37 (1976) 293—306
47. Ohtsuji, H., and Ikeda, M., The metabolism of styrene in the rat and the stimu-
latory effect of phenobarbital, Toxicol. Appi. Pharmacol. , 18 (1971) 321-328
48. Ikeda, M., and Imamura, Evaluation of hippuric, phenylglyoxylic and mandelic
acids on urine as indicators of styrene exposure, mt. Arch. Arbeitsmed. , 32
(1974) 93—101
49. Leibman, K. C., Metabolism and toxicity of styrene, Env. Hith. Persp. , 11
(1975) 115—119
50. Oesch, F., Mammalian epoxide hydrase: Inducible enzymes catalyzing the
inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and
olefinic compounds, Xenobiotica , 3 (1973) 305-340
51. Dowty, B. J., Carlisle, D. R., and Laseter, J. L., New Orlean’s drinking water
sources tested by gas-chromatography-mass spectrometry, Env. Sci. Technol .
9 (1975) 762—765
55
-------
3. Lactones
Lactones constitute a class of highly reactive compounds that possess a broad
spectrum of current and suggested industrial uses including: wood processing,
protective coatings and impregnation of textiles, modification of flax cellulose,
urethan foam manufacture, intermediates in the preparation of insecticides, plasti-
cizers and medicinals 1 .
p-Propiolactone (CH 2 —t H 2 ; BPL; -hydroxypropionic acid lactone;
0—Co
hydracryclic acid, -lactone) is by far the most important lactone produced commer-
cially. It should be noted that BPL is produced form formaldehyde and ketene which
have been found to be inutagenic 14 . Commercial grade BPL (97%) can contain
trace quantities of the reactants•. Samples of the common commercial product have
also been found to contain impurities including: acrylic acid, acrylic anhydride,
acetic acid arid acetic anhydride 5 . Industrially, BPL is used mainly as an inter-
mediate in the production of acrylic acid and esters. The very high chemical reac-
tivity of BPL is due to the presence of a strained four-membered lactone ring. It
is a nucleophilic alkylating agent which reacts readily with acetate, halogen, thio-
1,5—7
cyanate, thiosuiphate, hydroxyl and suiphydryl ions . 7-(2-Carboxyethyl)-
guanine (in the enol form) has been suggested to be the major binding product
of -propiolactone with both DNA and RNA in vivo 6 .
P: 0pi01acto1 is carcinogenic in the mouse by skin application, subcutaneous
or intraperitoneal application and in the rat by subcutaneous injection 5 ’ 8 ’ 9 , while
oral administration in the rat gave some indication of carcinogenicity 5 ’ 10
f3—Propiolactone has been shown to be mutagemc in Vicia faba 11 ’ 12 , Neurospora ,
E. coli 13 and Serratia marcescens’ 3 ’ 14, in the Salmonella/microsome test 15 , causes
chrornosomal aberrations in Vicia faba , Allium’ 6 and Neurospora and effects a
decline in the transforming activity of DNA from Bacillus subtillis 17
56
-------
-Propio1actone is mutagenic in bacteriophage T4 inducing primarily guanine//
cytosine to adenine//thymine base pair transitions 18 . This type of misparing was
suggested to be the most probable cause of BPL induced mutagenesis 18 .
57
-------
o C}4 2 CH 2 C00
CHO
N44
I
C’4
f9)! 5) 4:: 5:r°°’
o cFtcH 2 coo,4
U
• I
Fi’. 1. J at•ft of 3-propit.! c1o triih g’ta,io-
Si,W and (1’ riraiL $
7-(2•Carh..xv thv1)gua&ii e is he prirn irr
producc obtaiixrd irs’rn t,rIr’, ,&s ‘4 mr, t e ik1,i
LJNA tre 1Ted £4 1 1ir’, w rh J- p .iIz ct. ne
An inere d hPltiz.t .?n t N-I “i -(2nrb .
ethvI)x - uu 1 .x,ine u v c:u i: L psiring wilh
thvmid ,tt. dunn r jiIiettio - Orh r u,ist-
((flenee of the react iaI r,f with
d,,.’gti .jsi: iitcIitd dep.I! :r,n, which is
favored under ;widie cor,,li?i,,L s , od ring
. peiuiti . which i I:tv(,rett iittde bn ic Cuflhlitton5
58
-------
References for Lactones
1. Fishbein, L., Flamm, W. G., and Falk, H. L., Chemical Mutagens, Academic
Press, New York, (1970) pp. 21S—216
2. Rapoport, I. A., Acetylation of Gene Proteins and Mutations, Doki. Akad. Nauk.
SSSR , 58 (1947) 119
3. Auerbach, C., Drosophila Tests in Pharmacology, Nature , 210 (1966) 104
4. Khishin, A. F. E., The Requirement of Adenylic Acid for Formaldehyde M’L.itagenesis,
Mutation Res. , 1(1964) 202
5. IARC, -Propiolactone, In Vol. 4, International Agency for Research on Cancer
Lyon, France (1974) 259—269
6. Boutwell, R. L., Colburn, N. H., and Muckerman, C . C., In Vivo Reactions of
3-Propiolactone, Ann. NY Acad. Sd. , 163(1969)751
7. Fishbein, L., Degradation and Residues of Alkylating Agents, Ann. NY Acad.
Sci. , 163(1969)869
8. Van Duuren, B. L., Carcinogenic Epoxides, Lactones, and Halo-ethers and
Their Mode of Action, Ann. NY Acad. Sci. , 163 (1969) 633
9. Dickens, F., and Jones, H. E. H., Further Studies on the Carcinogenic Action
of Certain Lactones and Related Substances in the Rat and Mouse, Brit. 3. Cancer ,
19 (1965) 392
10. Van Duuren, B. L., Langseth, L., Orris, L., Teebor, G., Nelson, N., and
Kuschner, M., Carcinogenicity of Epoxides, Lactones and Peroxy Compounds
IV. Tumor Response in Epithelial and Connective Tissue in Mice and Rats,
3. Nati. Cancer Inst. , 37(1966)825
11. Smith, H. H., and Srb, A. M., Induction of Mutations with 3-Propio1actone,
Science , 114 (1951) 490
12. Swanson, C. P., and Merz, T., Factors Influencing the Effect of J3-Propiolactone
on Chromosomes of Vicia Faba, Science , 129 (1959) 1364
13. Mukai, F., Belman, S., Troll, W., and Hawryluk, I., The Mutagenicity of Aryl-
hydroxylamines, Proc. Am. Assoc. Cancer Res. , 8 (1967) 49
59
-------
14. Kaplan, R. W., Probleme Der Pri Lfung Von Pharmaka, Zusatzstoffen Und Chemikallen
Auf Ihre Mutationsauslösende Wirkung, Naturwiss , 49 (1962) 457
15. MeCann, J., Choi, E. Yamasaki, E., and Ames, B. N., Detection of Carcinogens
as Mutagens in Salmonella/Microsome Test: Assay of 300 Chemicals, Proc. Nail.
Acad. Sci. , 72(1975) 5135
16. Sin:i.th, H. H., and Lofty, T. A., Effects of -Propiolactone and Ceepryn on Chromo-
soines of Vicia and Allum, Am. J. Botany , 42 (1955) 750
17. Kubinski, H., and Kubinski, Z. 0., Effects of Monoalkylating Carcinogens and
Mutagens on Transforming DNA, Fed. Proc. , 35 (1976) 1551
18. Corbett, T. H., Heidelberger, C., and Dove, W. F., Determination of the
mutagenic activity to bacteriophage T4 of carcinogenic and noncarcinogenic
compounds, Mol. Pharmacol. , 6(1970) 667-679
-------
C. Aziridines
Aziridines are extremely reactive alkylating agents which can undergo two
major types of reactions” 2: (1) ring-preserving reactions in which an aziridine
(e. g., ethyleneimine) acts as a secondary amine reacting with many organic
functional groups containing an active hydrogen, undergo replacement reactions
of the hydrogen atom by nucleophilic attack at one of the methylene groups and
(2) ring-opening reactions similar to those undergone by ethylene oxide. Aziri-
dines, because of their dual functionality and high degree of reactivity, exhibit
actual or potential utility in a broad range of applications 2 including: (1) textiles:
crease proofing, dyeing and printing, flame proffing, water-proffing, shrink
proffing, form stabilization and stiffening; (2) adhesives and binders; (3) petro-
leum products and synthetic fuels; (4) coatings; (5) agricultural chemicals;
(6) ion-exchange resins; (7) curing and vulcanizing polymers; (8) surfactants;
(9) paper and printing; (10) antimicrobials; (11) flocculants and (12) chemothera-
peutics.
1. Aziridine (H 2 ç\ ; ethyleneimine; azacyclopropane; dihydro-IH-aziridine;
H .NH
dimethyleniinine) has been used principally in the polymerization to polyethylene-
imine (which can contain less than 1 mg/kg of residual monomer) 1 . Principal
uses for polyethyleneimine are as a flocculant in water treatment and in the textile
and paper industries where it is used as a wet-strength additive (due to its cationic
nature resulting in adhesion to cellulose compounds) 1 . Other areas of utility of
aziridine include its use as an adhesion promoter in various coating applications
and as an intermediate in drug, cosmetic and dye manufacture; in the production
of 2-aziridinyl ethanol and triethylenemelamine and as an intermediate and monomer
61
-------
for oil additive compounds, ion-exchange resins, coating resins, adhesives, polymer
stabilizers and surfactants
Aziridine is carcinogenic in two strains of mice following its oral administration
producing an increased incidence of liver-cell and pulmonary tumors” 3 .
Aziridine induces both transmissible translocations and sex-linked recessive
lethal mutations in Drosophila melanogaster 4 ’ 5; specific locus mutations in silkworms
( Bombyx Mon) 6 . Aziridine also produces leaky mutants, mutants with polarized
and non-polarized complementation patterns, and non-complementing mutants and
multilocus deletions in Neurospora crassa 7 and induces mitotic recombination 8 and
gene conversion in Saccharomyces cerevisial 9 .
Azinidine induces chromosome aberrations in cultured human cells 10 , mouse
embryonic skin cultures 11 and Crocker mouse Sarcoma l 88 . When rabbits were
inseminated with spermatozoa which had been treated with azinidine in vitro , only
40% of embryos were found to be viable relative to the number of corpora lutea in
12
comparison to 78% in controls . Azinidine has been reported to posses teratogenic
13
activity
CH 3
2. 2-Methyla zinidine (H d. .. propyleneimine; 2-methylazacyclopropane)
H 2 è
is a highly reactive chemical intermediate mainly used in the modification of latex
surface coating resins to improve adhesion 14 . Polymers modified with 2-methyl-
azinidine or its derivatives have been used in the adhesive, textile and paper
industries, because of the enhanced bonding of imines to cellulose derivatives 14 .
2-Methylazinidine has also been employed to modify dyes for specific adhesion to
celiulose. Derivatives of 2-methylazinidine have been used in photography, gelatins,
synthetic resins, as modifiers for viscosity control in the oil additive industry and
as flocculants in petroleum refining 14 .
62
-------
2—Methylaziridine has been reported to be a powerful carcinogen affecting a
wide range of organs in the rat when administered orally 15 . For example, brain
tumors (gliomas) and squamous cell carcinomas of the ear duct have been found
in both sexes; disseminated granulocytic leukemia in males and a number of multiple
mammary tumors (some metastasizing to the lung) were found in females at the end
of 60 weeks following twice weekly 10 and 20 mg/kg oral administrations.
2-Methylaziridine (as well as aziridine) have been shown to be mutagenic in
the Salmonella/microsome test system’ 6 .
3. 2- ( 1-A ziridinyl) - ethanol (H 2 C 13 -hydroxy- 1 -ethylaziridine;
H 2 C,NCH 2 CH 2 OH;
N- (2-hydroxyethyl ) a ziridine; 1- (2 -hydroxyethyl) ethylenimine; a ziridine ethanol)
can be prepared by the addition of aziridine to ethylene oxide’ 7 . (It should be
noted that both reactive intermediates are carcinogenic and mutagenic). It is
reported to be used commercially in the modification of latex polymers for coatings,
textile resins and starches, as well as in the preparation of modified cellulose pro-
ducts such as paper, wood fibers and fabrics 17 . 2-(1-Aziridinyl)-ethanol is car-
cinogenic in mice producing malignant tumors at the site of its injection 18 and has
been reported to induce sex-linked recessive lethals in Drosophila melanogaster ’ 9 .
63
-------
References for Aziridines (Imines )
1. IARC, Azirithne, In Vol. 9, International Agency for Research on Cancer, Lyon.(tV/c)
2. Fishbein, L., Flamm, W. 0., and Falk, H. L., 11 Chemical Mutagens”, Academic
Press, New York (1970) pp. 412-152
3. limes, J. R. , Ulland, B. M., Valerio, M. , Petrucelli, L., Fishbein, L.,
Hart, E. R., Pallotta, A. J., Bates, R. R., Falk, H. L., Gart, J. J., Klein, M.,
Mitchell, I., and Peters, J., Bioassay of Pesticides and Industrial Chemicals
for Tumorigenicity in Mice: A Preliminary Note; J. Natl. Cancer Inst. , 42
(1969) 1101
4. Alexander, M. L., and Glanges, E., Genetic Damage Induced by Ethylenimine,
Proc. Nat. Acad. Sci. , 53(1965) 282
5. Sram, R. J., The Effect of Storage on the Frequency of Translocations in
Drosophila Mutation Res. , 9 (1970) 243
6. Inagaki, E., and Oster, I. I., Changes in the Mutational Response of Silkworm
Spermatozoa Exposed to Mono- and Polyfunctional Alkylating Agents Following
Storage, Mutation Res. , 7 (1969) 425
7. Ong, T. M., and deSerres, F. J., Mutagenic Activity of Ethylenixnine in Neuro-
sporaCrassa, Mutation Res. , 18(1973)251
8. Zimmermann, F. K., and VonLaer, U., Induction of Mitotic Recombination with
Ethylenimine in Saccharomyces Cerevisiae, Mutation Res. , 4 (1967) 377
9. Zimmermann, F. K., Induction of Mitotic Gene Conversion by Mutagens, Mutation
Res. , 11(1971) 327
10. Chang, T. H., and Elequin, F. T., Induction of Chromosome Aberrations in Cul-
tured Human Cells by Ethyleniinine and its Relation to Cell Cycle,
Mutation Res. , 4 (1967) 83
11. Biesele, J. J., Philips, F. S., Thiersch, J. B., Burchenal, J. H., Buckley,
S. M., and Stock, C. C., Nature , 166(1950) 1112
12. Nuzhdin, N. I., and Nizhnik, G. V., Fertilization and Embryonic Development
of Rabbits After Treatment of Spermatozoa In Vitro With Chemical Mutagens,
Dokl. Akad. Nauk. SSSR Otd. Biol. , 181(1968) 419
13. Murphy, M. L., DelMoro, A., and Lacon, C., The Comparative Effects of Five
Polyfunctional Alkylating Agents on the Rat Fetus with Additional Notes on the Chick
Ann. N.Y. Acad. Sci. , 68(1958) 762 Embryo,
64
-------
14. IARC, 2-Methylaziridine, In Vol. 9, International Agency for Research on Cancer,
Lyon, (1975) pp 61—65( )
15. Ulland, B., Finkeistein, M , Weisburger, E. K., Rice, J. M., and Weisburger,
J. H., Carcinogenicity of Industrial Chemical Propylene linine and Propane
Sultone, Nature , 230 (1971) 460
16. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N., Detection of Carcinogens
as Mutagens in the Salmoneila/microsome Test: Assay of 300 Chemicals, Proc.
Nat. Acad. Sci. , 72(1975) 5135
17. IARC, 2-(1-Aziridinyl)-ethanol, In Vol. 9, International Research on Cancer
Lyon, (1975) pp. 47-50
18. Van Duuren, B. L., Meichionne, S., Blair, R., Goldschmidt, B. M., and
Katz, C., Carcinogenicity of Isoesters and Epoxides and Lactones: Aziridine
Ethanol, Propane Sultone and Related Compounds, J. Natl. Cancer Inst. ,
46 (1971) 143
19. Filippova, L. M., Pan shin, 0. A., and Kostyankovskii, R. G., Chemical
Mutagens, IV. Mutagenic Activity of Germinal Systems, Genetika , 3 (1967) 134
65
-------
D. Alkyl Sulfates
Alkyl sulfates such as dimethyl- and diethyl sulfates are very reactive alkylating
agents that have been extensively employed both in industry and the laboratory for
converting active-hydrogen compounds such as phenols, amines and thiols to the
corresponding methyl and ethyl derivatives” 2
0
1. Dirnethyl sulfate (H 3 COI-OCH 3 ; dimethyl monosulfate; methyl sulfate, sulfuric
0
acid, dimethyl ester; DMS) has been extensively used as a rnethylating agent both in
industry and the laboratory. Its utility includes the methylation of cellulose, pre-
paration of alkyl lead compounds, preparation of alkyl ethers of starch, solvent for
the extraction of aromatic hydrocarbons, curing agent for furyl alcohol resins and
the polymerization of olefins 3 . DMS has been employed commercially for the pre-
paration of quaternary ammonium methosulfate salts (via its reaction with the res-
pective tertiary amine) 1 . Included in this group are six cationic surfactants:
dimethyl dioctadecylaxnrnonium methosulfate; (3 —lauramidopropyl) -trimethyl ammonium
xnethosulfate; (3-olearnideopropyl) -trirnethyl amrnonium rnethosulfate; the metho-
sulfate of a stearic acid-diethanolamine condensate; the methosulfate of N- (2-hydroxy-
ethyl) -N, N’, t -tris (2-hydroxypropyl) -ethylenediainenl di stearate; and the metho-
sulfate of N, N 2 , N’ , N -tetrakis (2 -hydroxypropyl) ethylenediaminedioleate. DMS
has also been used for the preparation of anticholinergic agents (e. g., diphemanil
methyl sulfate and hexocycliuxn methyl sulfate, and the parasympathomimetic agent,
neostigmine methyl sulfate’.
Dimethyl sulfate has been shown to be carcinogenic in the rat (the only species
tested) by inhalation 1 ’ 4, subcutaneous injection 4 and following pre-natal exposure 4 .
It is carcinogenic to the rat in a single-dose exposure’ ‘ 4. The possibility of
carcinogenicity of dimethyl sulfate in man occupationally exposed for 11 years has
66
-------
been raised 5 , however good epidemiological evidence is unavailable to confirm
this” 5 .
Dimethyl sulfate is mutagenic in Drosophila 6 ’ 7 , E. coli 6 and Neurospora 8 ’
10
and induces chromosome breakage in plant material
p
2. Diethyl sulfate (C 2 H 5 O- --OC 2 H 5 ; diethyl monosulfate; ethyl sulfate; sulfuric
0
acid, diethyl ester; DES) has been employed in a variety of ethylation processes
in a number of commercial areas and organic synthesis including 2 ’ : finishing of
cellulosic yarns, etherification of starch, stabilization of organophosphorus insec-
ticides, as a catalyst in olefin polymerization and acrolein-pentaerythritol resin
formation. DES has been used as the ethylating agent for the commercial preparation
of a number of cationicsurfactants including: (2-aminoethyl) ethyl (hydrogenated
tallow alkyl) (2-hydroxyethyl) ammoniuin ethosuiphate; 1 -ethyl-2- (8-heptadec enyl) -
1- (2-hydroxyethyl) -2 -imida zolinium ethosulphate; N-ethyl-N-hexadecyl-morpho-
linium ethosulphate; N-ethyl-N- (soybean oil alkyl) morpholinium ethosulphate;
ethyl dimethyl (mixed alkyl) ammonium ethosulphate; and triethyl octadecyl ammo-
mum ethosulphate.
Diethyl sulfate is carcinogenic in the rat (the only species tested) following
subcutaneous administration and pre-natal exposure 2 ’ 4 . The evidence for carcino-
genicity of diethyl sulfate in the rat following oral administration is inconclusive 2 ’ 4 .
6,7,11,12 13—15
Diethyl sulfate has been found xnutagenic in Drosophila , E. coli
bacteriophage T-2 ’ 6 , Neurospora 8 , S. pombe 17 , and Aspergillus nidulans 18 .
67
-------
References for Sulfates
1. IARC, Dimethyl Sulfates, In Vol. 4, International Agency for Research on Cancer,
Lyon, France (1974) pp. 271-276
2. IARC, Diethyl Sulfate, In Vol. 4, International Agency for Research on Cancer,
Lyon, France (1974), 277-281
3. Fishbein, L., Flamm, W. G., and Falk, H. L., “Chemical Mutagens”, Academic
Press, New York, (1970) PP. 216—217
4. Druckrey, H., Kruse, H., Preussmann, R., Ivankovic, S., and Landschi!itz,
C., Cancerogene Aikylierende Substanzen. m. Alkylhalogenide, -Sulfate-,
Su]fonate Und Ringgespannte Heterocyclen, Z. Krebsforsch. , 74 (1970) 241
5. Druckrey, H., Preussmann, R., Nashed, N., and Ivankovic, S., Carcinogenic
Alkylierende Substanzen I. Dimethylsulfate, Carcinogene Wirkung an Ratten
Und Wahrscheinliche Ursache Von Berufskrebs., Z. Krebsforsch. , 68 (1966) 103
6. Alderson, T., Ethylation Versus Methylation in Mutation of E. Coli and Drosophila
Nature , 203 (1964) 1404
7. Pelecanos, M., and Alderson, T., The Mutagenic Action of Diethylsulfate in Droso-
phiia Melanogaster I. The Dose-Mutagenic Response to Larval and Adult Feeding
Mutation Res. , 1(1964) 173
8. Mailing, H. V., Identification of the Genetic Alterations in Nitrous Acid-Induced
AD-3 Mutants of Neurospora Crassa, Mutation Res. , 2 (1965) 320
9. Kolmark, G., Mutagenic Properties Of Esters Of Inorganic Acids Investigated by the
Neurospora BackMutation Test, Ser. Physiol. , 26(1956) (1956) 205-220
10. Loveless, A., and Ross, W. C. J., Chromosome Alteration and Tumour Inhibition
by Nitrogen Mustards: The Hypothesis of Cross-linking Alkylation, Nature , 166
(1950) 1113
11. Alderson, T., and Pelecarios, M., The Mutagenic Activity of Diethylsulfate in
Drqsophila Melanogaster, II. The Sensitivity of the Immature (Larval) and Adult
Testis, Mutation Res. , 1(1964) 182
12. Pelacanos, M., Induction of Cross-Overs, Autosotnal Recessive Lethal Mutations,
and Reciprocal Transloctions in Drosophila After Treatment with Diemthyl, Nature ,
210 (1965) 1294
13. Alderson, T., Brit. Empire Cancer Campaign Ann. Rept., (1963) p. 416
14. Strauss, B., and Okubo, S., Protein Synthesis and the Induction of Mutations by
E. Coli by Alkylating Agents, J. Bact. , 79 (1960) 464
68
-------
15. Zamenhof, S., Leidy, G., Hahn, E., and Alexander, H. E., Inactivation and
Unstabilization of the Transforming Principle by Mutagenic Agents, J. Bact. , 72
(1956) 1
16. Loveless, A., The Influence of Radiomimetic Substances on DNA Synthesis and
Function Studied in E. Coil Phage Systems, III. Mutation of T2-Bacteriophage as
a Consequence of Alkylation In Vitro : The Uniqueness of Ethylation, Proc. Roy.
Soc., B150 (1959) 497
17. Heslot, H., S. Pombe: A New Organism for the Study of Chemical Mutagenesis,
Abhandl. Deut. Akad. Wiss. Berlin. Ki. Med. , 1(1960) 98-105
18. Alderson, T., and Clark, A. M., Interlocus Specificity for Chemical Mutagens in
Aspergilus Nidulans, Nature , 210 (1966) 593
69
-------
E. Sultones
Su.ltones such as the 1,3-propane-, and 1,4-butane derivatives are
being increasingly employed industrially to introduce the sulphopropyl and sul-
phobutyl groups (-CH 2 CH 2 CH 2 SO 3 and -CH 2 CH 2 CH 2 CH 2 SO 3 respectively) into
polymer chains containing nucleophilic centers in order to enhance water solu-
bility and confer an anionic character 1 . For example, the simplest sultone,
1,3-propane sultone (3-hydroxy-1-propanesulphonic acid sultone; 1, 2 -oxathiolane-
2,2-dioxide) is a xnonofunctional alkylating agent and reacts with nucleophiles,
Y along the general pathway (1) or (2) as follows 2 :
y +P 2 3>so 2 - Y-CH 2 CH 2 CH 2 SO 2 O (1)
+ P z- 2 SO - Y -CH CH CH SO 0 (2)
CH—O 2 2 2 2 2
A large number of suiphopropylated products and their potential uses 1 ’ 3
include: (a) derivatives of amines, alcohols, phenols, met-captans, suiphides
and amides useful as detergents, wetting agents, lathering agents and bacterio-
stats; (b) soluble starches used in the textile industry; Cc) solubilized cellulose,
which was reported to have soil-suspending properties; Cd) dyes; (e) an anti-
static additive for polyamide fibers; (f) cation-exchange resins (prepared by
condensing the sulphonic acid product derived from phenol and propane sultone
with formaldehyde); and (g) phosphorus-containing sulphonic acids (produced
from organic phosphines, neutral esters of trivalent phosphorous acids, and
phosphorous and phosphoric triainides), useful as insecticides, fungicides,
surfactants and vulcanization accelerators.
1,3-Propane sultone is carcinogenic in the rat when administered orally,
intravenously or by pre-natal exposure, and exhibits a local carcinogenic effect
-------
in the mouse and the rat when given subcutaneously 1 ’ 47 . Propane sultone acts as
a complete carcinogen having both initiating and promoting activity when given as
a single application of a 25% W/V solution in toluene or after repeated applications of
a 2.5%W/V solution for up to 58 weeks in two strains of Mice, CF1 and C3H 8 . In addi-
tion to a high incidence of skin tumors, a statistically significant increase in systemic
neoplasia was found. The exposed CF1 mice had a higher incidence of neoplasia of
lymphoreticular and lung origin, while female C 3 H mice showed a higher incidence of
mammary gland and uterine tumors 8 . 1,4-Butane sultone is chemically far lessi-eactive 9 ”°
1,3-Propane sultone has been classified as a potent mutagen toward Schizesaccharo-
10
myces pombe while the mutagenic effectiveness of 1,4-butane sultone towards S.p be
was found to be much lower 9 ’ 10
In an evaluation of the dependence of mutagenic effectiveness of chemical reactivity
it was found that the mutagenic effectiveness of 1, 3-propane and 1,4-butane sultone,
if expressed per alkylating event at a certain low nucleophilicity was the sanie as that
10
of methyl- and ethyl methanesulfonate . This then indicates that alkylation of certain
groups of DNA with a low nucleophilic strength has approximately the same mutagenic
effect independent of the structure of the alkyl (e. g., including the 3-sulfopropyl- and
9
4-sulfobutyl- groups despite the realtive bulkiness and negative charge of these groups
Rapid decline in the transforming activity of DNA from Bacillus subtilis exposed to
either 1,3-propane or -propiolactone has been noted indicating that large complexes
of DNA induced by these carcinogens following initial fragmentation of the polymer may
be inactive se and that their residual transforming activity may be due to the relatively
unaltered DNA chains on the periphery of the complex 11 .
71
-------
References for Sultones
1. IARC, Vol. 4, International Agency for Research on Cancer, Lyon (1974),
pp. 253—258
2. Goldschmidt, B. M., Frenkel, K., and Van Duuren, B. L., The Reaction
of Propane Sultone with Guanosine, Adenosi.ne and Related Compounds,
3. Heterocyclic Chem. , 11 (1974) 719
3. Fischer, F. F., Propane Sultone, mt. Eng. Chem. , 56 (1964) 41
4. Druckrey, H., Kruse, H., Preussmann, R., Ivankovic, S., Landschi itz, C.,
and Gimmy, J., Cancerogene Allylierende Substanzen, IV, 1, 3-Propanesulton
und 1,4-Butansulton, Z. Krebsforsch. , 75 (1970) 69
5. Druckrey, H., Kruse, H., and Preussmann, R., Propane Sultone, A Potent
Carcinogen, Naturwiss , 55 (1968) 449
6. Ulland, B., Finkeistein, M., Weisburger, E. K., Rice, J. M., and Weisbur-
ger, J. H., Carcinogenicity of the Industrial Chemicals Propylene Imine and
Propane Su].tone, Nature , 230 (1971) 460
7. Van Duuren, B. L., Meichionne, S., Blair, R., Goldschmidt, B. M., and
Katz, C., Carcinogenicity of Isoesters of Epoxides and Lactones: Aziridine-
ethanol, Propane Sultone and Related Compounds, 3. Natl. Cancer Inst. , 46
(1971) 143
8. Doak, S. M. A., Simpson, B. 3. E., Hunt, P. F., and Stevenson, D. E., The
Carcinogenic Response in Mice to the Topical Application of Propane Sultone
to the Skin, Toxicology , 6 (1976) 139=154
9. Nilsson, T., “Nagra Sultoner Deras Framst .11ninoch Hydrolys’ t , Ph. D. Thesis,
Lund University, Lund, Sweden, 1946
10. Osterman-Golkar,. S., and Wachtmeister, C. A., On the Reaction Kinetics in
Water of 1,3-Propane Su.ltone and 1,4-Butane Sultone: A Comparison of
Reaction Rates and Mutagenic Activity of Some Alkylating Agents, Chern.
Biol. Interactions , 14 (1976) 195
11. Heslot, H., Etude Quantitative De Reversions Biochimiques Induites Chez La
Levure Schizosaccharomyces Pombe Par Des Radiations Et Des Substances
Radiomitnetiques, Abbandi. Deut. Akad. Wiss., Berlinki. Med. , (1962) 193
12. Kubinski, H., and Kubinski, Z. 0., Effects of Monoalkylating Carcinogens
and Mutagens on Transforming DNA, Fed. Proc. , 35 (1976) 1551
72
-------
F. Aryldialkyltriazenes
Triazenes of the general formula X- -NN-N(CH 3 ) 2 (x= substituents);
phenyl or a heterocyclic residue are industrial intermediates, as well as anti-neo-
plastic agents and have been patented as rodent repeflents and herbicides. Their
biological effects such as carcinogenicity” 2 mutagenicity in Neurospora crassa 3 ,
Drosophila melanogaster 4 ’ 5 , a yeast (S. cerevisiae) 4 ’ 6 ’ 7 and S. typhimurium 8
(metabolically activated) and toxicity 9 are suggested to be dependent on at least
two molecular mechanisms 8 . (Figure 1) One mechanism involves non-en zymic
cleavage of the diazoarnino side chain liberating arenediazonium cations. In the
other mechanism, the major metabolic pathway is an enzymic oxidative mono-dealkyl-
lation yielding the corresponding monoalkyltriazenes, with subsequent hydrolysis
yielding alkylating reactants 8 ’ 10 (e.g., methylating species similar to those formed
from alkylnitrosoureas). A more recent report 11 has suggested a common 3,4-epoxy
intermediate to account for the formation of modified anilines during the catabolic
degradation of the carcinogen l-(4-chlorophenyl)-3,3-dimethyl triazene into 3-
chloro-4-hydroxyaniline.
73
-------
N N
— 1 • N_CH3
x ‘ CH 1
I -N
ac -HYDROXYLATION
+
o- CH
N N..CH 3
x
I
* O—NN—CH J
frn.-i .. n mt .. int.rt.i,•di. Ls.
r
74
-------
References for Arylalkyltriazenes
1. Preussmann, R., Ivankovic, S., Landschdtz, C., et a., Carcinogene Wirkung
Von 13 Aryldialkyllriazenen an BD-Ratten, Z. Krebsforsch. , 81 (1974) 285
2. Druckrey, H.,lvankovic, S., and Preussinann, R., NeurotropeCarcinogene
Wirkung Von Phenyl—Dimethyltriazene an Ratten, Naturwiss , 54 (1967) 171
3. Ong. T. M., and DeSerres, F. J., The Mutagenicity of 1-Phenyl-3,3-dimethyl-
triazene and 1-Phenyl-3-monornethyltriazene in Neurospora Crassa, Mutation Res .
13 (1971) 276
4. Kolar, G. F., Fahrig, R., and Vogel, E., Structure Activity Dependence In Some
Novel Ring-Substituted 3, 3-Dimethyl-1--phenyl Triazenes. Genetic Effect in D.
Melanogaster and S. Cerevisiae, Chem. Biol. Interactions , 9 (1974) 365
5. Vogel, E., Chemische Konstitution Und Mutagene Wirkung VI. Induction Dominanter
Und Rezessiv-Geschlechts gebundener Lethal Mutationen Durc h Aryldialkyltria zene
Bei Drosophila Melanogaster, Mutation Res. , 11 (1971) 379
6. Fahrig, R., Metabolic Activation of Aryldialkyltriazenes in the Mouse: Induction of
Mitotic Gene Conversion in Saccharomyces Cerevisiae in the Host Mediate Assay,
Mutation Res. , 13(1971) 436-439
7. Siebert, D., and Kolar, G. F., Induction of Mitotic Gene Conversion by 3, 3-Dimethyl-
Phenyltriazene, 1, (3-Hydroxyphenyl) -3, 3-Dimethyltriazene in Saccharomyces
Cerevisiae, Mutation Res. , 18 (1973) 267
8. Malaveille, C., Kolar, G. F., and Bartsch, H., Rat and Mouse Tissue-Mediated
Mutagenicity of Ring-Substituted 3,3-Dimethyl-1-Phenyltriazenes in Salmonella
Typhimuriurn, Mutation Res. , 36 (1976) 1
9. Andrysova, A., Rambousek, V., Jirasek, J., Zverina, V., Matrka, M., and
Marhold, J., 1 -Aryl-3, 3-Dialkyltriazene Compounds. Toxicity of Parasubstituted
1-Phenyl-3, 3-Dialkyltriazene Compounds Physiol. Bohenoslov. , (1972) 63
10. Preussmanrt, R., Von Hodenberg, A., and Hengy, A., Mechanisms of Carcinogens
with 1-Aryl-3, 3-dialkyltriazenes. Enzymatic Dealkylation by Rat Liver Microsomal
Fraction In Vitro, Biochem. Pharmcol. , 18 (1969) 1
11. Kolar, G. F., and Schiesiger, J., Biotransformation of 1-(4-Chlorophenyl)-3,3-
Dimethyltriazene Into 3-Chloro-4-hydroxyaniline, Cancer Letters , 1 (1975) 43
75
-------
G. Diazoalkanes
Diazoalkanes represent an extremely reactive class of alkylating agents. Dia-
zoalkanes are sufficiently basic to abstract protons from many compounds containing
acidic hydrogens, the rate of protonation increasing as the acidity of the proton
donor increases. The protonated diazoalkane (an alkyldiazonium ion) is exceedingly
unstable, losing molecular nitrogen yielding a carbonium ion which then becomes
affixed to whatever nucleophile is available. Hence the overall reaction is a replace-
ment of the nitrogen of the diazoalkane by the hydrogen and accompanying nucleo-
philic portion of the protic compound 1 .
Diazomethane (CH 2 = N = N) is a powerful methylating agent for acidic compounds
such as carboxylic acids, phenols, and enols, and, as a consequence, is both an
important laboratory reagent and has industrial utility (with acids, diazomethane
yields esters and with enols it gives 0- alkylation). When heated, irradiated with
light of the appropriate wavelength, or exposed to certain copper-containing catalysts,
diazomethane loses molecular nitrogen and forms carbene, via., CH 2 N 2 - CH 2 : + N 2
Carbenes are exceedingly reactive species which, for example, can add to alkenes
to form cyclo ropanes. Carbenes can react with the electrons of a carbon-hydrogen
bond to “insert” the carbon of the carbene between carbon and hydrogen, e.g.,
transforming -CH to -C-CH 3 1 .
Diazomethane can react with many biological molecules, especially nucleic acids
and their constituents. For example, its action on DNA includes methylation at
several positions on the bases and the deoxyribose moiety as well as structural
alterations that result in lower resistance to alkaline hydrolysis and altered hyper-
chromicity.
76
-------
Diazomethane has been found to be carcinogenic in rats and mice 6 and its role
as the active agent responsible for the carcinogenic action of many compounds (e . g.,
nitroso derivatives) noted 7 . The mutagenicity of diazomethane in Drosophila ’ 0 ,
Neurospora 10 12 and Saccharomyces cerevisiae ’ 3 ’ 14 have been described. The
implication of diazoinethane in the inutagenesis of riitroso compounds has also been
15—18
cited
77
-------
References for Diazoalkanes
1. G utsche, C. D., and Pasta, D. J., “Fundamentals of Organic Chemistry”, Prentice-
Hall, Englewood Cliffs, NJ (1975) p. 693
2. Friedman. 0. M., On a Chemical Degradqtion of Deoxyribosenucleic Acid,
lBiochem. Biophys; Acta. , 23(1957) 215
3. Friedman, 0. M., Mahapatra, G. N., Dash, B., and Stevenson, R., Studies on
the Action of Diazomethane on Deoxyribonucleic Acid, Biochim. Biophys. Acta. ,
103 (1965) 286
4. Kriek, E., and Emmelot, P., Methylation of Deoxyribonucleic Acid by Diazomethane
Biochim. Biophys. Acta. , 91, (1964) 59
S . Holy, A., and Scheit, K. H., On the Methylation of Dinucleoside Phosphates with
Diazomethane, Biochim. Biophys. Acta , 123 (1966) 430
6. Schoental, R., Carcinogenic Action of Diazomethane and of Nitroso-N--Methyl Urethane
N ture , 188 (1960) 420
7. Rose, F. L., in “The Evaluation of Drug Toxicity” (ed) Walpole, A. L., and
Spinks, A., Little, Brown, Boston, Mass. (1958) p. 116
8. Mizrahi, I. J., and Emmelot, P., The Effect of Cysteine on the Metabolic Changes
Produced by Two Carcinogenic N-Nitroso dialkylamines in Rat Liver, Cancer Res .
22 (1962) 339
9. Druckrey, H., Carcinogenicity and Chemical Structure of Nitrosamines,
Acta. Unio Intern. Contra Cancrurn , 19 (1963) 510
10. Rapoport, I. A., Alkylation of Gene Molecule, Doki. Akad. Nauk. SSSR , 59 (1948)
1183
11. Jensen, K. A., Kolmark, G. , and Westergaard, M., Back-Mutations in Neurospora
Crassa Adenine Locus Induced by Diazomethane,
Hereditas , 35(1949) 521 1
12. Jensen, K. A., Kirk, I., Kolinark, G., and Westergaard, M., Chemically Induced
Mutations in Nuerospora, Cold Spring Harbor Symp Quant. Biol. , 16 (1951) 245-261
13. Marquardt, H., Zimmermann, F. K., and Schwaier, R., Nitrosamide Als Mutagene
Agentien, Naturwissenschaften , 50 (1963) 625
14. Marquardt, H., Schwaier, R., and Zimmermann, F., Nicht-Mutagenit t Von Nitro-
sarninen Bei Neurospora Crassa, Naturwissenschaften , 50 (1963) 135
78
-------
15. Zirnmermann, F. K., Schwaier, R.. and Von Laer, U., The Influence of pH on the
Mutagenicity in Yeast of N-Methylnitrosamides and Nitrous Acid, Z. Vererbungslehre ,
97 (1965) 68—71
16. Schwaier, R., Vergleichende Mutations Versuche Mit Sieben Nitrosamiden Im
Ruckmutationstest an Hefen, Z. Vererbungslehre , 97 (1965) 55-67
17. Schwaier, R., Zirrimermann, F. K., and Von Laer, U., The Effect of Temperature
on the Mutation Induction in Yeast by N-Alkylnitrosamides and Nitrous Acid, Z.
Vererbungslehre , 97 (1965) 72-74
18. Marquardt, H., Zimmermann, F. K., and Schwaier, R., The Action of Carcinogenic
• Nitrosamines and rosamides on the Adenine-G-45 Reverse Mutations System of
S. Cerevisiae, Z. Vererbungslehre , 95 (1964) 82-96
79
-------
H. Phosphoric Acid Esters
Phosphoric acid (HO- -OH), as a tribasic acid, can form mono-, di-, and triesters
with a broad spectrum of alcohols, thiols and phenols, a number of the resultant
reactive organophosphates have utility as alkylating agents, intermediates in chemical
synthesis and as organophosphorus insecticides 1 (e.g., Dichiorvos, Parathion,
Malathion, Diazinori).
,
The common structural element of all organophosphates is P-O-C-, with both
I
phosphorus and carbon being electrophilic sites. Alkylation (e. g., methylation or
ethylation) can occur as a result of nucleophilic attack on the carbon atom with sub-
sequent cleavage of the C-O bond. Alternatively, a nucleophile can preferentially
attack the phosphorus atom and undergo phosphorylation. The type and rate of
reaction with a given nucleophile depends to a major extent on its nature, as in the
presence of several nucleophiles such as occur in competitive reactions in a living
cell 1 .
1. Trimethylphosphate [ (CH 3 O) 3 P0; phosphoric acid-trimethyl ester, TMP I is
the simplest trialkyl ester of phosphoric acid and has been mainly employed as a
methylating agent, in the preparation of organophosphorus insecticides [ e.g.,
Dichlorvos (DDVP) (CH3O)2R H via reaction with chloral] 2 , as a low-
cost gasoline diti 34 and as a catalyst for polyester uftur 56 .
7
Trimethylphosphate is known to alkylate E. coh, phage T 4 B , to cause chromo-
some breaks in bone marrow cells of rats 8 ’ 9 , or cultured human lymphocytes 10 ,
11—14
and to produce mutations in bacteria , including Salmonella typhiniurium tester
14 15 16 17—19
strains with R factor plasmids , Neurospora , Drosophila and mice
A number of additional phosphate esters have suggested utility in diverse industrial
purposes including: catalysts for curing resins, chemical intermediates, solvents,
80
-------
gasoline and lubricant additives, anti-foaming antioxidants and flotation agents 20 .
2. Triethyiphosphate [ (C 2 H 5 0) 3 P0, phosphoric acid, -iethyl ester, TEP] is
used to impart flame-resistance in polyesters 21 , as a heat stabilizer for neoprene
rubber 22 and as a plasticizer for injection moldable bisphenol-based polyesters 23 .
Triethylphosphate has been shown to be mutagenic in Drosophila 13 .
81
-------
3. TRIS (2, 3-DIBROMOPROPYL)PHOSPHATE
Currently, about 300 million pounds of flame-retardant chemicals are being
produced mainly for use in fabrics, plastics and carpets. Approximately two-thirds
of this amount are inorganic derivatives such s alumina trihydrate and antimo iy
oxide while the remaining one-third are large numbers of brominated and chlorinated
organic derivatives 2428 .
Tris (2, 3-dibromopropyl)phosphate (tris-BP) [ (BrCH 2 CH (Br)CH 2 O) 3 P0 I is
the most widely used flame-retardant additive for childrens sleepwear. Commercial
preparations of tris-BP can be obtained in two grades, viz. HV (High in volatiles)
and LV (low in volatiles). A typical LV sample has been reported to contain the
following impurities 29 : 0.05% 1, 2-dibromo-3-chloropropane (BrCH 2 CHBrCH 2 C1) (I);
0.05% 1 2 ,3-tribromopropane (BrCH 2 CHBrCH 2 Br) (IU .; and 0.20% 2, 3-dibrornopro-
panol (BrCH 2 CHBrCH 2 OH) (III).
About 65% of the 10 million pounds of tris-BP produced annually in the United
States by 6 manufacturers are applied to fabrics used for childrens fabrics, with
the remainder used as a flame retardant in other materials such as urethane foams 30 .
A significant portion of the total (approximately 10%) is estimated to reach the
environment from textile finishing plants and launderies while most of the remainder
is postulated to eventually end up on solid wastes (e.g., manufacturing waste and
used clothing) 30 .
Tris-BP is added to fabrics used for children’s garments to the extent of 5-10%
by weight.
Tris (2, 3-dibromopropyl)phosphate is n utagenic 29 ’ 31 ’ to histidine-requiring strains
of Salmonella typhimuriuin (Ame& Salmonella/microsome test 33 ). For example,
Prival et a1 3 ’ reported tris-BP mutagenic to S. typhimurium sti ains TA 1535 and
82
-------
TA 100, but not TA 1537 indicating that tris-BP induces mutations of the base-pair
substitution type. On a quantitative basis, no significant activity was found among
9 different commercial samples, including high and low volatile materials from 5
different supplies. Highly purified samples of tris—BP containing 0.029% 1,2,3—tn-
bromopropane and less than 0.002% each of 1, 2-dibromo-3-chloropropane had
approximately the same mutagenic activity as the commercial samples 31 . Each of
the 3 contaminants (I, II, and HI) displayed some mutagenic activity, but insufficient
to account for the mutagenicity of tnis-BP when the level of these compounds in
31
tnis-BP was taken into account
Extracts of fabrics treated with tris-BP were also found capable of inducing
31
mutations in TA 1535 and TA 100 strains of S. typhimurium
Tnis-BP has been found to induce heritable mutations (sex-linked recessive
lethals) in Drosophila melanogaster 34 as well as unscheduled DNA synthesis 35 and
repairable breaks in DNA in human cells in culture 36 .
The carcinogenicity of an impurity of tris-BP, e. g., dibromochioropropane (I)
should also be noted. This compound caused a high incidence of squamous carcinoma
of the stomach in both rats and mice as early as 10 weeks after initiation of feeding
37,38
by oral intubution
83
-------
References for Phosphoric Acid Esters
1. Wild, D., Mutagenicity Studies on Organophosphorus Insecticides, Mutation
Res. , 32(1975) 133
2. British Crop Protection Council, “Pesticide Manual”, (ed) Martin, H., Worcester,
(1968) p. 152
3. Ehrenberg, L., Osterman-Golkar, S., Singh, D., and Lundquist, U., On the
Reaction Kinetics and Mutagenic Activity of Methylating and 3-Halogenoethy-
lating Gasoline Additives, Radiation Botany , 15 (1974) 185
4. Kerley, R. V., and Flet, A. E., Fuels for Automotive Engines, U . S. Patent
3,807,974, April 30, (1974), Chem. Abstr. , 81(1974) 172852T
5. Watrnabe, T., Ichikawa, H., Yokouch, R., Manufacture of Polyesters for Film—
molding Use, Japan Pat. 73 42, 712, Dec. 7 (1973) Chem. Abstr. , 81 (1974)
136948 V
6. Murayama, K., and Yamadera, R., Heat-Stable Polyesters, Japan Kokai, 73,
102, 194, Dec. 22(1973), Chem. Abstr. , 81(1974) 14004P
7. Gunianov, G. D., and Gumanov, L. L., Mutagenic Action of Alkyl Phosphates
on T 4 B Phage and Alkylation of Phage DNA, Doki. Akad. Nauk., SSSR , 198,
(1972) 1442—1444
8. Adler, I. D., Ramarao, G., and Epstein, S. S., InVivo Cytogenetic Effects of
Trimethyiphosphate and of Tepa on Bone Marrow Cells of Male Rats, Mutation
Res. , 13(1971) 263
9. Legator, M. S., Palmer, K. A., and Adler, I. D., A Collaborative Study of
In Vivo Cytogentic Analysis. I. Interpretation of Slide Preparations, Toxicol.
Appi. Pharmacol. , 24(1973) 337
10. Söderman, G., Chromosome Breaking Effect of Gasoline Additive in Cultured
Human Lymphocytes, Hereditas , 71 (1972) 335
11. YacPhee, D. G., Salmonella Typhimurium His G46 (R-Utrecht): Possible Use
in Screening Mutagens and Carcinogens, Appi. Microbiol. , 26 (1973) 1004
12. Voogd, C. E., Jacobs, J. J. J. A. A., Vanderstel, J. J., On the Mutagenic
Action of Dichiorvos, Mutation Res. , 16 (1972) 413
13. Dyer, K. F., and Hanna, Comparative Mutagenic Activity and Toxicity of
Triethylphosphate and Dichiorvos in Bacteria and Drosophila, Mutation Res .
21 (1973) 175
84
-------
14. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N., Detection of Car-
cinogens as Mutagens: Bacterial Tester Strains with R Factor-Plasmids, Proc.
Nati. Acad. Sci., , 72(1975) 979
15. Kolmark, G., quoted in Epstein, S. S., Base, W., Arnold, E., and Bishop, Y.,
Mutagenicity of Trimethyiphosphate in Mice, Science , 168 (1970) 584
16. Dyer, K. F., and Hanna, P. J., Mutagenic and Antifertility Activity of Trirnethyl-
phosphate in Drosophila Melanogaster, Mutation Res. , 16 (1972) 327
17. Dean, B. J., and Thorpe, E., STudies with Dichiorvos Vapor in Dominant Lethal
Mutation Tests on Mice, Arch. Toxicol. , 30 (1972) 51
18. Epstein, S. S., Trimethyiphosphate (TMP), EMS Newsletter , 2 (1969) 33
19. Epstein, S. S., Bass, W., Arnold, E., and Bishop Y., Mutagenicity of Trimethyl-
phosphate in Mice, Science (1970) 584
20. Hanna, P. J., and Dyer, K. F., Mutagenicity of Organophosphorus Compounds
in Bacteria and Drosophila, Mutation Res. , 28 (1975) 405-420
21. Byrd, S. M., Jr., Flame-Retardant Polyesters: Two Approaches, Proc. Annual
Conf., Reinf. Plast. Compos. Inst. Soc. Plast. md., 29 (1974) 23D, 9 pp.,
Chem. Abstr. , 81(1974) 1064993G
22. Takahashi, H., Taketsume, M., and Nakata, M., Ch1or’ prene Rubber Composition,
Japan Patent, 74 00, 981, Jan 10 (1974) Chem. Abstr. , 81 (1974) 79138E
23. Sakata, H., Okamoto, T., Hasegawa, H., and Nagata, K., Polyester Compositions
With Improved Workability, Japan Kokai, 74 34, 546, April 2 (1974), Chem.
Abstr. , 81(1974) 137062
24. Pearce, E. M., and Liepins, R., Flame Retardants, Env. Hith. Persp , 11 (1975)
59—69
25. Hutzinger, 0., Sundstrorn, G., and Safe, S., Environmental Chemistry of Flame
Retardants I. Introduction and Principles, Chemosphere , 1 (1976) 3-10
26. Anderson, E., V.,
Chem. Eng. News. , March 22 page 12
27, WHO, Health Hazards from New Environmental Pollutants, Tech. Rept. Series
No. 586 (1976) 52-63 World Health Organization, Geneva
28. LeBlanc, R. B., Textile Industries, Feb. 1976, P. 28
29. Kerst, A. F., Toxicology of Tris(2,3-Dibromopropyl)phosphate , J. Fire Flamm.,
Fire Retardant Chem . (Suppi), 1 (1974) 205
85
-------
30. EPA, Summary Characterizations of Selected Chemicals of Near-Term Interest,
EPA Rept. 560/ 4—76-004, April, 1976, p. 53—54, Office of Toxic Substances,
Environmental Protection Agency, Washington, D.C.
31. Prival, M. J., McCoy, E. C., Gutter, B., and Rosenkranz, H. S., Tris(2,3-
dibromopropyl)phosphate: Mutagenicity of a Widely Used Flame Retardant,
Science , 195 (1977) 76—78
32. Ames, B. N., and Blum, A., Flame-Retardant Additives as Possible Cancer
Hazards, Science , 195 (1977) 17—23
33. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N., Detection of Carcinogens
as Mutagens in Salmone]la/Microsome Test: Assay of 300 Chemicals, Proc. Natl.
Acad. Sci . (USA) 72 (1975) 5135
34. Valencia, R., personal communication cited in reference 30
35. Stich, H. F., cited by Blum, A., and Ames, B. N., in reference 31
36. Gutter, B., and Rosenkranz, H. S., cited by Prival et al in reference 30
37. Olson, W. A., Haberman, R. T., Weisburger, E. K., Ward, J. M., and
Weisburger, J. H., Induction of Stomach Cancer in Rats and Mice by Halogenated
Aliphatic Fumigants, J. Nail. Cancer Inst. , 51 (1973) 1993
38. Powers, M. B., Voelker, W., Page, N. P., Weisburger, E. K., and Kraybiil,
H. F., Carcinogerncity of Ethylene Dibromode (EDB) and 1, 2-Dibromo-3-
Chioropropane (DBCP) Toxicol. Appl. Pharmacol. , 33 (1975) 171-172
86
-------
I. ALKANE HALIDES
1. Ethylene Dichloride (1, 2-dichioroethane; C1CH 2 CH 2 C1) is produced in large
quantities by the oxychiorination of ethylene and is used mainly for the production
of vinyl chloride monomer (VCM) with lesser utility for the manufacture of 1, 1 ,l-trich-
loroethane (methyl chloroform), trichioroethylene, ethyleneamines and perchloroethy-
lene, ethyleneamines and perchloroethylene. Ethylene dichloride se and in com—
bination with ethylene dibromide is also used in large amounts as a lead scavenging
agent in gasoline, as a component of fumigants (with ethylene dibromide) for grain,
upholstery and carpets and in various solvent applications.
Ethylene dichioride has been found in 11 raw water locations at levels of < 0.2—
3.]. g/l and 26 finished water locations (32.0% of total) at levels of 0.2-6
No apparent carcinogenicity data concerning ethylene dichloride exists although
a carcinogenicity study of the compound in rodents is currently in progress at the NCI *
Ethylene dichloride, without activation, is a weak mutagen in S. typhirnurium
TA 1530, TA 1535, and TA 100 tester strains 2 ’ 3 . It should be noted that when chloro-
acetaldehyde was compared directly to reversion of TA 100 with two other metabolites
of ethylene dichloride, e. g., chioroethanol and chloroacetic acid, on a molar basis,
.2
chloroacetaldehyde was hundreds of tunes more effective . Ethylene dichloride is also
mutagenic in E. coli (DNA polymerase deficient p 01 A strain) 4 and increased the
frequency of recessive lethals and induced chromosome disjunction in Drosophila 5 ’ 6
2. Ethylene Dibromide (1,2-dibromoethane; DBE; BrCH 2 CH 2 Br) prepared by
the reaction of ethylene and bromine, is used principally as an additive (scavenger)
in leaded gasoline. Relatively smaller amounts are used as pesticides in soil furnigants
(as a nematocide) and in grain and commodity fumigants, industrial solvents and as
87
-------
a chemical intermediate. Over 300 million pounds of ethylene dibromide are produced
annually in 5 companies in the U.S The numbers of workers engaged in the pro-
duction and use applications of ethylene dibromide (as well as ethylene dichioride)
as well as their levels of exposure is not currently known.
The concentration of ethylene dibromide in gasoline is variable, but is in the order
of 0.025% (wt/vol) 7 . (Ethylene dichioride is also used in admixture with DBE). The
chief sources of ethylene dibromide and dichioride emissions are from automotive
sources via evaporation from the fuel tank and carburetor of cars operated on leaded
fuel. Emissions from these sources have been estimated to range from 2 to 25 mg/day
for 1972 through 1974 model-year cars in the U.S. 7 .
Very limited and preliminary air monitoring data for ethylene dibromide, show air
concentration values of 0.07-0.11 pg/rn 3 (about 0.01 ppb) in the vicinity of gasoline
stations along traffic arteries in 3 major cities; 0.2-1.7 pg/rn 3 (about 0.1 ppb) at an
oil refinery and 90-115 pg/rn 3 (10-15 ppb) AT DBE manufacturing sites in the U.S.,
suggesting that DBE is present in ambient air at very low concentrations 7 .
It should be noted that the increased use of unleaded gasoline should result in
lower ambient air levels of ethylene dibrornide from its major sources of emissions 7 ’ 8 .
Ethylene dibromide has also been found in concentrations of 96 pg/m 3 , up to a
mile away from a U.S. Dept. of Agriculture’s fumigation center 8 .
Concentrations of ethylene dibromide on the order of 1 ppg have been found in
samples from streams of water on industrial sites. Limited information suggests that
ethylene dibroinide degrades at moderate rates in both water and soil 7 .
The use of ethylene dichloride and dibrornide in fumigant mixtures of disinfecting
fruits, vegetables, foodgrains, tobacco, seeds, seedbeds, mills and warehouses,
suggests the possibility that their residues se or that of their respective hydrolytic
88
-------
products (e . g., ethylene chiorohydrin or bromohydrin) may be present in fumigated
materials 912 .
Although materials such as ethylene dichloride and dibromide are volatile, and
their actual occurrence in processed or cooked foodscan possibly be considered negli-
gible, more significant exposure is considered more likely among agricultural workers
or those fumigating grain and crops in storage facilities and the field, than among
consumers of the food products 13 .
Ethylene dibromide induced squamous cell carcinomas in the stomachs of both
Osborne-Mendel rats and (C56B1XC3H)fl mice when administered via chronic oral
intubation at maximum tolerated doses (MTD) and at half MTD’s 13 .
Ethylene dibromide, without metabolic activation induces base-pair substitution
reverse mutations in S. typhiznuriuxn TA 1530, TA 1535, TA 100, and G 46 plate
assays 4 ’ 14-16• When tested in polymerase assays which are believed to be indicators
of repairable DNA damage, ethylene dibromide was more toxic to E. colip34 7 8 (pol A)
than to E. coliW3llO (po]. A ) hence suggesting that it can damage DNA 4 .
Ethylene dibromide was not mutagenic in plate assays with Serratia marcescens 21
or in the host-mediated assay in mice 15 . Ethylene dibromide induced recessive lethal
mutations in the ad-3 region of a two-component heterokaryon of Neurospora crassa 17 ’ 18
as well as X-chromosomal recessive lethals in Drosophila 19 and visible mutations in
20,21
mutable clones in the Tradescantia stamen hair somatic test system where it
exhibited good dose-response relationships with surface exposures as low as 3 .6 ppm
20
compared to 5 ppm with ethyl methane sulfonate (EMS)
Ethylene dibromide did not cause dominant lethal mutations in mice when admini-
stered orally (5 doses totalling 50 or 100 mg/kg) or by i.p. injections (18 or 90 mg/kg) 22 .
QO
-------
It did not cause chromosome breaking effects in human lymphocytes or in Alluirri
23
roots
The mutagenicity of the vicinal 1, 2-dibromides was suggested to be a consequence
of their ability to react to form highly unstable bromonium ions in solution, via.,
H 2 C-çH 2 - Br + H 2 q 5H 2 , “biological alkylating agents” which can alkylate cellular
nucleophiles including DNA. The initial product of alkylation of a hetero-atom such as
0, N, or S would be the 2-brornoethyl derivatives, which would be a “half-mustard”
type reagent capable of another alkylation reaction. Hence 1, 2-dibromoethane (as
well as 1, 2-dichioroethane) could be considered as bi-functional alkylating agents,
capable of introducing cross-links into biological molecules 19 .
Nauman et a1 19 and Ehrenberg et a1 24 also designated ethylene dibromide as an
alkylating agent suggesting that it reacts via an SN 1 mechanism.
Antifertility effects of ethylene dibromide have been attributed by Edwards et a1 25
to a direct alkylating effect of its primary metabolite, the glutathione conjugate of
bromoethane, which is a more reactive alkylating agent than ethylene bromide.
3. 1, 2-Dibromo-3-chloropropane (DBCP; BrCH 2 -çH-CH 2 C1) is used as a fumigant
Br
and as an intermediate in organic synthesis. DBCP has been detected in the order
of 0.05% in LV grade (low in volatiles) commercial preparations of the flame-retardant
26
tris [ (2,3-dibromopropyl)phosphatej . Residues of DBCP have also been found on
grains as a result of fumigation.
1,2-Dibromo-3-chloropropane has been shown to induce a high incidence of squa-
mous cell carcinomas of the stomach in both rats and mice treated via chronic oral
intubation with MTD and half MTD doses of the agent 13 . In addition, DBCP induced
mammary adenocarcinomas in the female rats 13 . Analogously to ethylene dibromide,
90
-------
in DBCP the bromine atoms are activated so that the compounds probably act as
alkylating agents 13 .
DBCP is mutagenic for S. typhimurium tester strains TA 100 (with S-9 microsomal
activation) 27 and TA 1535 with and without activation 26 .
91
-------
References for Alkane Halides
1. Environmental Protection Agency, Draft Report for Congress: Preliminary
Assessment of Suspected Carcinogens in Drinking Water, Office of Toxic Sub-
stances, Washington, D.C., October 17 (1975)
2. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N., Detection of Car-
cinogens as mutagens: Bacterial tester strains with G factor plasmids, Proc.
Nati. Acad. Sci . (USA) 72 (1975) 979—983
3. McCann, J., Simon, V., Streitweiser, D., and Ames, B. N., Proc. Nati. Acad.
Sci. , 72(1975) 3190—3193
4. Brem, H., Stein, A. B., and Rosenkranz, H. S., The rnutagenicity and DNA-
modifying effect of haloalkanes, Cancer Res. , 34 (1974) 2576-2579
5. Shakarnis, V. F., 1, 2-Dichloroethane-induced chromosome non-disjunction and
recessive sex-linked lethal mutation in Drosophila melanogaster, Genetika , 5
(12) (1969) 89—95
6. Rapoport, I. A. Reaction of gene proteins with ethylene chloride, Akad. Nauk.
SSSR Doki. Biol. Sci. , 134 (1960) 745—747
7. Environmental Protection Agency, Sampling and Analysis of Selected Toxic Sub-
stances, Task 11-Ethylene Dibromide, Final Report, Office of Toxic Substances,
Environmental Protection Agency, Washington, D.C., Sept. (1975)
8. Anon, Ethylene dibromide “ubiquitous” in air, EPA report says, Toxic Materials
News , 3 (1976) 132
9. Berck, B., Fumigant residues of carbon tetrachioride, ethylene dichioride, and
ethylene dibromide in wheat, flour, bran, middlings and bread, J. Agr. Food
Chern. , 22(1974) 977-984
10. Wit, S. L., Besemer, A. F. H., Das, H., Goedkoop, W., Loostes, F. E., and
Meppelink, Rept. No. 36/39, Toxicology (1969) National Institute of Public
Health, Bilthoven, Netherlands
1].. Fishbein, L., Potential hazards of fumigant residues, Env. Hith. Persp. , 14
(1976) 39—45
12. Olomucki, E., and Bondi, A., Ethylene dibromide fumigation of cereals I. Sorption
of ethylene dibromide by grain, J. Sci. Food Agr. , 6 (1955) 592
13. Olson, W. A., Haberman, R. T., Weisburger E. K., Ward, J. M., and Weisburger,
J. H., Induction of stomach cancer in rats and mice by halogenated aliphatic fumi-
gants, J. Nail. Cancer Inst. , 51 (1973)
92
-------
14. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N., Detection of carcino-
gens as mutagens in the Salmoneila/microsome test: Assay of 300 chemicals,
Proc. Nati. Acad. Sci. , 72(1975) 5135-5139
15. Buselxnaier, V. W., Rohrborn, G., and Propping, P., Mutagenit ts-untersuchungen
mit pestiziden im hos-mediated assay und mit dem dominanten-letal test an der
maus, Biol. Zbl. , 91(1972) 311—325
16. Buselmaier, W., Robrborn, G., and Propping, P., Comparative investigation of
the mutagenicity of pesticides in mammalian test systems, Mutation Res. , 21
(1973) 25—26
17. Mailing, H. V., Ethylene dibromide: A potent pesticide with high mutagenic ac-
tivity, Genetics , 61 (1969) s 39
18. DeSerres, F. J., and Mailing, H. V., Genetic analysis of ad-3 mutants of Neuro-
spora crassa induced by ethylene dibromide, A commonly used pesticide, EMS
Newsletter , 3 (1970) 36—37
19. Vogel, E., and Chandler, J. L. R., Mutagenicity testing of cyclamate and Some
pesticides in Drosophila melanogaster, Experientia , 30 (1974) 621-623
20. Sparrow, A. H., Schairer, L. A., and Villalobos-Pietrini, R., Comparison of
somatic mutation rates induced in tradescantia by chemical and physical mutagens,
Mutation Res. , 26(1974) 265—276
21. Nauman, C. H., Sparrow, A. H., and Schairer, L. A., Comparative effects of
ionizing radiation and two gaseous chemical mutagens on somatic mutation induction
in one mutable and two non-mutable clones of tradescantia, Mutation Res. , 38
(1976) 53—70
22. Epstein, S. S., Arnold, E., Andrea, J., Bass, W., and Bishop, Y., Detection
of chemical mutagens by the dominant lethal assay in the mouse, Toxicol. Appi.
Pharmacol. , 23(1972) 288—325
23. Kristoffersson, U., Genetic effects of some gasoline additives, Hereditas , 78 (1974) 319
24. Ehrenberg, L., Osterman-Holkar, S., Singh, D., Lundquist, U., On the reaction
kinetics and mutagenic activity of methylating and -ha.iogenoethylating gasoline
derivatives, Radiat. Biol. , 15 (1974) 185—194
25. Edwards, K., Jackson, H., and Jones, A. R., Studies with alkylating esters, II.
A chemical interpretation through metabolic studies of the infertility effects of
ethylene dimethanesulfonate and ethylene dibromide, Biochem. Pharmacol . 19
(1970) 1783—1789
26. Prival, M. J., McCoy, E. C., Gutter, B., and Rosenkranz, H. S., Tris(2,3-
dibromopropyl)phosphate: Mutagenicity of a widely used flame retardant, Science
195 (1977) 76—78
27. Blum, A., and Ames, B. N., Flame-retardant additives as possible cancer hazards,
Science , 195 (1977) 7—23
93
-------
Halogenated Alkanols
2-Chloroethanol (ethylene chlorhydrin, C1CH 2 CH 2 OH) has been used as an inter-
mediate for the preparation of ethylene oxide, indigo, acrylic and methacrylic esters,
thiodiethylene glycol and 2, 2-dichiorodiethyl ether; lesser uses are as solvent, and
in the preparation of insecticides, herbicides, and growth modifiers. Additional sources
of 2-chioroethanol (as well as 2-bromoethanol) can arise from the use of fumigants
such as ethylene oxide, ethylene dichloride and ethylene thbromide 3 .
Ethylene oxide in the presence of chloride ion forms 2—chioroethanol in vitro and
probably in vivo 4 . 2-Chioroethanol is known to be metabolized n vivo 5 and in vitro
to chioracetaldehyde by rat 5 or human 6 liver alcohol dehydrogenase. Chloroacetal-
4.
dehyde (which is chemically very reactive binding with glutathione) is further meta-
bolized to chioroacetic acid 4 . 2-Chloroethanol is also considered a likely metabolic
product of vinyl chloride which is carcinogenic and mutagenic 79 as well as a precursor
of chioroacetaldehyde from the metabolism of 1, 2-dichloroethane 10 .
2-Chioroethanol when tested for carcinogenicity in rats at 10 mg/kg and lower
doses by sub-cutaneous administration twice/week for 1 year did no increase the
tumor incidence comparable with those of controls 4 .
2-Chloroethanol is mutagenic in S. typhimurium TA 153011, TA 153510,12 and
TA 10010 with and without metabolic activation. 2-Chioroethanol is weakly mutagenic
in S. typhimurium TA 1530 when incorporated in an agar overlay in a Petri dish’ 3 . In
addition, it preferentially inhibited the growth of E. coli pol A strain, but was found
to be the least active when compared to the other haloethanols in regard to the diameters
of the zones of growth inhibition (e.g., bromoethanol > iodoethanol > chloroethanol) 13 .
The mutagenicity order of activity when tested in Klebsiella penumoniae was iodoethanol
> bromoethanol > chioroethanol This order of activity correlated with the decrease
of bond dissociation energies between the halogens and carborr atoms
94
-------
References for Halogenated Alkanols
1. Fishbein, L., Potential hazards of fumigant residues, Env. Hith. Persp. , 14
(1976) 39—45
2. Fishbein, L., Degradation and residues of alkylating agents, Ann. N.Y. Acad.
Sci. , 163 (1969) 869—894
3. Wesley, F., Rouke, B., and Darbishire, 0., The formation of persistent toxic
chiorohydrins in foodstuffs by fumigation with ethylene oxide and propylene oxide,
J. Food Sci. , 30(1965) 1037
4. Balazs, T., Toxicity of ethylene oxide and chioroethanol, FDA By-Lines , 3 (1976)
15 0—155
5. Johnson, M. K., Metabolism of chioroethano]. in the rat, Biochem. Pharmacol . 16
(1967) 185—199
6. Blair, A. H., and Vallee, B. L., Some catalytic properties of human liver dehy-
drogenase, Biochemistry , 5 (1966) 2026-2034
7. Bartsch, H., and Montesano, R., Mutagenic and carcinogenic effects of vinyl
chloride, Mutation Res. , 32 (1975) 93-114
8. Watanabe, R. E., Hefner, R. E., Jr., and Gehring, P. G., Preliminary studies
of the fate of inhaled vinyl chloride monomer in rats, Ann. NY Acad. Sci. , 246
(1975) 135—148
9. Loprieno, N., et al., Evaluation of the genetic effects of vinyl chloride monomer
(VCM) under the influence of liver microsomes, Mutation Res. , 40 (1976) 85—96
10. McCann, J., Simon, V., Streitsweiser, D., and Ames, B. N., Mutagenicity of
chloroacetaldehyde, a possible metabolic product of 1, 2-dichioroethane (ethylene
dichioride), chioroethanol, and cyclophosphamide, Proc. Nat].. Acad. Sci. , 72
(1975) 3190—3193
11. Malaveille, C., et al., Mutagenicity of vinyl chloride, chloroethyleneoxide, chioro-
acetaldehyde and chioroethanol, Biochem. Biophys. Res. Communs. , 63 (1975)
363—370
12. Rannug, U., Gôthe, R., and Wachtmeister, C. A., The mutagenicity of chioro-
ethylene oxide, c hioroac etaldehyd e, 2-c hioroethanol and c hioroac etic acid,
conceivable metabolites of vinyl chloride, Chem. Biol. Interactions , 12 (1976)
251—263
13. Rosenkranz, S., Carr, H. S., and Rosenkranz, H. S., 2-Haloethanols: Mutagenicity
and reactivity with DNA, Mutation Res. , 26 (1974) 367-370
14. Voogd, C. E., and Van Der Vet, P., Mutagenic action of ethylene halogenhydrins,
Experientia , 25 (1969) 85-86
95
-------
K. Haloethers
Haloethers, primarily alpha chloromethyl ethers, represent a category of alkylating
agents of increasing concern 1 3 due to the establishment of a causal relationship
between occupational exposure to two agents of this class and lung cancer in the U.S.
and abroad 313 . These haloethers are bis (chioromethyl) ether (BCME, C1CH 2 QCH 2 C1).
and chioromethyl methyl ether (methyl chloroznethyl ether, CMME, CICH 2 OCH 3 ). These
agents are widely used in industry as chioromethylation agents in organic synthesis
for preparation of anion-exchange resins, formation of water repellants and other
textile-treating agents; manufacture of polymers and as solvents for polymerization
reactions.
BCME can be produced from paraformaldehyde, sulfuric acid and hydrogen chloride
while CMME can be produced via the reaction of methanol, formaldehyde, and anhydrous
hydrogen chloride. It should be noted that commercial grades of CMME can be con-
taminated with 1% to 8% BCME 6 ’ 7 .
The potential for BCME formation increases with available formaldehyde and
chloride 14 6 (in both gaseous and liquid phases), viz., 2Cl+2HCHO+2H - C1CH 2 -
OCH 2 C1+H 2 0.
The reaction is believed to be an equilibrium much in favor of the reactants. The
extent of hazard from the combination of formaldehyde and HCI to form BCME is tin-
nown at present, and to date, the results appear scanty and disparate 10 .
The hydrolytic reactions of BCME and CMME can be depicted as follows:
H 2 0
Cl-CH 2 -OCH 3 — CH OH +HC1 + CH 2 OJ
CMME
H 2 0 ‘1 ’
Cl-CH 2 -O-CH 2 C1 __________________
BCME
96
-------
Potential sources of human exposure to BCME appear to exist primarily in areas
including (1) its use, in chiorornethylafing (cross-linking) reaction mixtures in anion-
exchange resin production 14 ; (2) segments of the textile industry using formaldehyde-
containing reactants and resins in the finishing of fabric and as adhesives in the
laminating and flocking of fabrics’ 6 and (3) in non-woven industry which uses as
binders, thermosetting acrylic emulsion polymers comprising rnethylol acrylamide.
since a finite amount of formaldehyde is liberated on the drying and curing of these
bor. ding agents’ 6 .
NIOSH has confirmed the spontaneous formation of BCME from the reaction of
formaldehyde and hydrochloric acid in some textile plants and is now investigating
the extent of possible worker exposure to the carcinogen’ 7 . However, this finding
has recently been disputed by industrial tests in which BCME was not formed in air
by the reaction of textile systems employing hydrochloric acid and formaldehyde 18 .
Evidence of the human carcinogenicity of BCME and CMME have been cited 3 3 .
Regulations published recently by OSHA in U.S., specifically list both BCME and CMME
as human carcinogens 5 . Epidemiological studies on an industry—wide basis in the
United States, have disclosed some 30 cases of lung cancer in association with BCME
and CMME 11 .
The carcinogenicity of BCME and CMME by skin application to mice and by sub-
cutaneous administration to mice and rats 1 ’ 10, the induction of lung denomas by
intrapentoneal injection of BCME in newborn mice’ 9 and by inhalation of CMME and
BCME 20 , and the induction of squamous carcinomas of the lung and esthesioneuro—
epitheliomas in rats by inhalation exposure 21 ’ 22 of 0.1 ppm BCME 5 hr/day, 5 days!
week through their lifetime as well as in groups of rats given 10, 20, 40, 60, 80 and
100 exposures to 0.1 ppm BCME and then held until death, ha.re all been reported.
97
-------
Van Duuren et al 1 ’ 2 ’ 23 suggested that the 0-haioethers be classified with the
biologically active alkylating agents (e.g., nitrogen mustards, epoxides, ç3 -lactones,
etc.). The high chemical reactivity of the -ha1oethers is attributed to the reactivity
of the halogen atom in displacement reactions. In comparing the carcinogenicity of
11 chloroethers 2 ’ 10 (Figure 1, Table 1), in general, bifunctional cc-chloroethers are
more active than their zuonofurtctional analogs. As the chain length increases, activity
decreases, and as chlorine moves further away from the ether oxygen, carcinogenic
activity also decreases. It was also noted that in a general way, the more carcino-
genicafly active compounds are the most labile; as stability increases, carcinogenicity
also decreases 0 .
While BCME and CMME have received the most attention of the haloethers because
of their human carcinogenic activity, it is important to note that other haloethers have
industrial utility or have been found as industrial by-products. For example, bis-
(2—chioroethyl) ether (C1CH 2 CH 2 OCH 2 CH 2 C1) (BCE) has been extensively used as
a solvent in paint and varnish industry and in textile industry for grease spotting
and removal of paint and tar brand marks from raw wool; other uses include its utility
as an extractive for lubricating oils in the petroleum industry and its application as
a soil insecticide.
ci ci
Bis (2-chioroethyl) ether and bis (2 -chloroisoprop yl) ether (CH -ç -0-c -CH 3 )
CH CH 3
are by-products of the chlorohydrin process for making ethylene and propylene
oxides. Both have been found in U.S. rivers as a result of industrial outfall 24 ’ 25
and also identified in waterways in the Netherlands 26 . Both bis(2-chloroethyl)ether
and bis(2-chloroisopropyl)ether have also been found in samples of finished drinking
water in concentrations somewhat higher than those found in the raw water’ 0 ’ 24 .
The induction of hepatomas by bis(2-chloroethyl)ether ir lifetime feeding experi-
ments in mice has been reported 27 .
98
-------
A comparison of the carcinogenic and mutagenic activity (in E. coli and S. _____
murium microbial systems) of a number of haloethers has been described (Table 2)10,28,29.
The agents shown are direct-acting and do not require metabolism for mutagenic activity.
Bis(2-chloroethyl)ether has also been reported to be mutagenic in Drosophila 30 .
However, while it was not mutagenic in the standard Salmonella/microsome assay or
towards E. coli WP2 using a siniilar procedure 31 it was mutagenic when tested with
S. typhirnurium strains TA 1535 and TA 100 and was weakly mutagenic in strains
TA 1538 and TA 98 and E. coil WP2 when tested in desiccators to contain the volatile
fumes 31 . In suspension assays BCE was rnutagethc when assayed with S. typhimurium
TA 1535 and TA 100 and with S. cerevisiae D3. BCE was not mutagexuc in host-
mediated assays when given as a single oral dose or when administered for 2 weeks
prior to the injection of the S. typhimurium into the peutoneal cavity 31 . BCE as well
as bis(2-chloroisopropyl)ether did not induce heritable translocations when tested
in mice 32 . BCME is rnutagenic in S. typhimui-ium 33 .
99
-------
Cl—CH 2 —O—CH 3 I 0
C1—CH --O—CH 2 —CI
,H ,H - S
H 3 C—C—O—C—cH 3 3
‘c i L Vr_Cl
H—C—O—CH 4
‘ci Ckr O
O-CH 2 O-04 2 —c 14-cI
C1,H ,O-CH 2 04 5
Cl—CH 2 —C—H 21
a ci ‘ci ci O—CI4 2 —cH 3
Fiouia 1. Structure of 11 chloroethers tested for carcinogenic activity. 2
T atz 1
CAScisOGEsICTTY or HALOGEIr4ATED CoMPoUNDS’
Subcuta.
Subcuta-
Carcinogenicizy of
Halogenated Compounds
‘( .
zOUSC
VhoIe Initiati ng
Carcinogen Agent
‘ °
jection
in Mice:
Sarcom
injec.
lion Site/
ze0
Injection
in Rats:
Sarcomas
at Injec-
lion Site/
Mice with Papiltoanas/
Group
Group
Compound
Total Mice/Group t
Size
Size
CMME, 1
0 12/40 (5)
10/30
1/20
BCME.2
13/20 (12) 5/20 (2)
—
7/20
Bs(a.chIo4oethyl)ether, 3
— 7/20 (0)
4/30
—
a. iz-Dichloromethyl methyl ether,
4
0/20 (0) 3/20 (1)
—
—
Bis(3-chloroethyl)ether, 5
— 3/20 (0)
0/20 (0) 3/20 (1)
2/30
—
—
—
O.zachloro.di-n-propyl ether, 6
2,3-Dichlorotetrahydrofuran, 7
— 5/20 (1)
1/30
—
L2-Thchloroethylene carbonate, 8
— 3/20 (0)
2130
—
Epichlorohydrin, 9
Perchlorocyclobut-2-enoon, 10
Monochloroacetaklehyde diethyl
acetal, Il
— 0/20 (0)
— —
0/20 1/20 (0)
2/50
1/30
—
—
—
—
• From Reference Z
t Number of mice with carcinomas given in parentheses.
100
-------
TABLE
COMPAaLSON OF CARCI OGFNIC AND MtJ I GENIC
(is MIQOBIAL SYSTEMS) AcTwrr? *
No.
Biological Activity of Halog
Compound
enated Compounds
Mutagenic
Activity
Carcinogenic
Activity
1
Chloromethyl methyl ether
+
+
2
Bis(chloromethyl)ether
+
- -
3
Bis(cL-chloroethyl)ether
+
+
4
,rz-dichtoromethyl ether
. -
+
5
Bis-($-chloroethyi) ether
—
6
Octachloro-di-n-propyl ether
not tested
+
7
2,3-dichlorotetrahydroiuran
—
+
8
1,2-dichloroethylene carbonate
—
—
9
Epichiorohydrin
+
+
10
Perchlorocylclobutenenone
+
—
11
Chloroacetaldehyde diethyl acetal
- I-
—
12
Dimethyl carbamyl chloride
. -
+
* F
rom References 3 d 34
101
-------
References for Halogenated Ethers
1. Van Duuren, B. L., Goldschmidt, B. M., Katz, C., Langseth, L., Mercado, C.,
and Sivak, A., Alpha—baloethers: A new type of alkylating carcinogen, Arch.
Env. HIth. , 16(1968) 472—476
2. Van Duuyen, B. L., Katz, C., Goldschmidt, B. M., Frenkel, K., and Sivak, A.,
Carcinogenicity of haloethers. II. Structure-activity relationships of analogs of
bis(chloromethyl)ether, J. Nati. Cancer Inst. , 48 (1972) 1431-1439
3. IARC, Bis(chlorornethyl)ether, In Monograph No. 4., Lyon (1974), pp. 231-238
4. IARC, Chiorornethylmethylether, In Monograph No. 4, Lyon (1974) pp. 239-245
5. OSHA, Occupational Safety & Health Standards: Carcinogens, Fed. Reg. , 39
(20) (1974) 3768—3773; 3773—3776
6. Albert, R. E., et al. , Mortality patterns among workers exposed to chloroxnethyl
ethers, Env. Hith. Persp. , 11(1975) 209—214
7. Figueroa, W. G. • Raszkowski, R., and Weiss, W., Lung cancer in chioromethyl
methyl ether workers, New Engi. J. Med. , 288(1973)1094-1096
8. Weiss, W., and Figueroa, W. G., The characteristics of lung cancer due to chloro—
methyl ethers, J. Occup. Med. , 18(1976) 623-627
9. Weiss, W., Chioromethyl ethers, cigarettes, cough and cancer, J. Occup. Med. ,
18 (1976) 194—199
10. Nelson, N., The chloroethers-occupational carcinogens: A summary of laboratory
and epidemiology studies, Ann. NY Acad. Sci. , 271 (1976) 81-90
ii. Nelson, N., The carcinogenicity of chloroethers and related compounds, Meeting
on Origins of Human Cancer, Cold Springs Harbor Laboratory, New York Sept.
7—14 (1976) p. 8
12. Sakabe, H., Lung cancer due to exposure to bis(chloromethyl) ether, md. Hlth. ,
1]. (1973) 145
13. Thiess, A. M., Hey, W., and Zefler, H., Zue Toxikologie von dichlorodiznethyl
ther-verdacht auf kanzerogene wirkung auch beim menschen, Zbl. Arbeits
Med. , 23(1973) 97
14. Rohm & Hass Co., News release: Reaction of formaldehyde and HC1 forms
bis-CME, Rohm & Hass, Phila., PA, Dec. 27 (1972)
102
-------
15. Kallos, G. 3., anrid Solomon, R. A., Investigation of the formation of bis-
(chioromethyl) ether in simulaated hydra gen chloride-formaldehyde atmos-
pheric environments, Amer. md. Hyg. Assoc. T. , 34(1973)469-473
16. I-iurwitz, M. D., Assessing the hazard from BCME in formaldehyde-containing
acrylic emulsions, Amer. Dyestuff Reptr. , 63 (1974) 62—64, 77
17. Anon, Indusfry s problems with cancer aired, Chem. Eng. News , 53 (1975) 4
18. Anon, Dow says bis(chloromethyl)ether does not form during textile operations,
Toxic Materials News , 3 (20) (1976) 157
19. Gargus, J. L., Reese, W. H., Jr., and Rutter, H. A., Induction of lung adenoinas
in new born mice by bis(chloromethyl)ether, Toxicol. Appi. Pharmacol. , 15
(1969) 92—96
20. Leong, B. K., MacFarland, H. N., and Reese, W. H., Jr., Induction of lung
adenomas by chronic inhalation of bis(chloromethyl)ethers, Arch. Env. Hlth. ,
22 (1971) 663—666
21. Laskin, S., Kuschner, M., Drew, R. T., Capiello, V. P., and Nelson, N.,
Tumors of the respiratory tact inducted by inhalation of bis (chloromethyl) ether,
Arch. Env. Hlth. , 23(1971)125—176
22. Kuschner, M., Laskin , S., Drew, R. T., Cappiello, V., and Nelson, N.,
Inhalation carcinogenicity of aipha-haloethers, UI. Lifetime and limited period
inhalation studies with BCME at 0.1 ppm, Arch. Env. Hlth. , 30 (1975) 73-77
23. Van Duuren, B. L., Carcinogenic epoxides, lactones and halo-ethers and their
mode of action, Ann. NY Acad. Sd. , 163 (1969) 633-651
24. Kleupfer, R. I D., and Fairless, B. J., Characterization of organic components
in municipal water supply, Env. Sci. Technol. , 6 (1972) 1062—1063
25. Rosen, A. A., Skeel, R. T., and Ettinger, M. B., Relationship of river water
to specific organic contaminants, 3. Water Pollut. Con -ol Fed. , 35 (1963) 777-782
26. Zoetman, B. C. J., Rijks Instituut Voor Drink Water Voorziening, Personal
communication cited in reference number 10.
27. Innes, 3. R., Ulland, B. M., Valerio, M. G., et al., Bioassay of pesticides and
indus ial chemicals for tumorigenicity in mice: A preliminary note, J. Nati.
Cancer Inst. , 42 (1969) 1101—1114
28. Mukai, F., and Troll, W., The mutagenicity and initiating activity of some aromatic
amine metabolites, Ann. NY Acad. ScL , 163 (1969) 828
29. Mukai, F., and Hawryluk, I., The mutageriicity of some halo-thers and haloketones,
Mutation Res. , 21 (1973) 228
103
-------
30. Auerbach, C., Robson, J. M., and Carr, J. G., The chemical production of
mutations, Science , 105 (1947) 243
3].. Sixnmon, V. F., Kauhanen, K., and Tardiff, R. G., Mutagenicity assays with
bis(2-chloroethyl)ether, Abstracts of Meeting of International Congress of
Toxicology, Toronto, March 3 - April 2 (1977) P. 31
32. Jorgenson, T. A., Rushbrook, C. J., Newell, G. W., and Tardiff, R. G., Study
of the rnutagenic potential of bis(2-chloroethyl) and bis(2-chloroisopropyl) ethers
in mice by heritable translocation test, Abstracts of 8th Annual Meeting Environ-
mental Mutagen Society, Colorado Springs, Colorado, Feb. 13-17 (1977), p. 76
33. McCann J., Choi, E., Yamasaki, E., and Ames, B.N., Detection of Carcinogens
as mutagens in the Salmonefla/rnicrosome test: Assay of 300 chemicals, Proc.
Natl. Acad. Sci. , 72(1975) 5135—5139
104
-------
L. Aldehydes
The carbonyl group (as typified in aldehydes and ketones) occurs in many sub-
stances of both biological and commercial importance. Compounds such as aldehydes
have been employed in a large number of organic syntheses and reactions. For example,
as a result of the polarization of the carbonyl group, = 0 ( ) C - 0, aldehydes
(and ketones) have a marked tendency to add nucleophilic species (Lewis bases) to the
carbonyl carbon, followed by the addition of an electrophilic species (Lewis acids) to
the carbonyl oxygen, the reactions are classified as 1,2 nucleophilic additions, via.:
0 + Nu E S c O E which can involve carbon or nitrogen
I Nu / Nu
nucleophilies.
1. F.91 ma4dehyde (H 2 CzO), the simplest of the aldehydes is considerably more reactive
than its higher homologs. The chemical stability associated with enol-keto tautomerism
in the higher aldehydes is lacking in formaldehyde where the carbonyl groups is
attacked directly to two hydrogens.
Formaldehyde is used extensively as a reactant in a broad spectrum of commercial
processes because of its high chemical reactivity and good thermal stability 1 ’ 2 These
reactions can be arranged in three major categories, via., 1) self-polymerization
reactions; 2) oxidatio -reducdon reactions and addition or condensation reactions
with a large number of organic and inorganic compounds (Figure 1).
Since pure formaldehyde is a gas at ordinary temperatures and hence cannot be
readily handled, it is marketed chiefly in the form of aqueous solutions containing
37% to 50% formaldehyde be weight. In aqueous solution formaldehyde exists entirely
,OH
in the hydrated form H 2 C
OH
Formaldehyde has a marked tendency to react with itself to form linear polymers
(designated as paraformaldehyde) or a cyclic trimer (designated as trioxane); i.e.,
105
-------
H
HO \ HC CH 2
HO(CHzO) H — - ,.c=o 2
H 0 0
\/
CH 2
Parafornialdehyde Trioxane
In general, the major chemical reactions of formaldehyde with other compounds
involve the formation of methylol (---CH 2 OH) or methylene derivatives. Other typical
reactions include alkoxy-, amido—, amino—, cyano—, halo—, sulfo—, and thiocyano—
rnethylations. For example, reactions with amides and carbamates yield rnethylol
derivatives, e . g. 1 methylolureas and methylol carbamates which are used in the treat-
ment of textiles (for crease—resistance; crush proof, flame resistance and shrinkproof
fabrics).
Aldol condensations are important in the synthesis of -hydroxycarbonyl compounds
which can be used in further synthesis, e . g., pentaerythritol production. Methylol
derivatives are highly reactive species which can be polymerized to yield methylene or
ether bridges, e . g., phenolic resins. Condensation of formaldehyde with ammonia
yields hexamethylenetetramine which undergoes many reactions including decomposition
into formaldehyde and ammonia, and nitramine formation upon nitration.
The major uses of formaldehyde and its polymers are in the synthetic resin industry
(e.g., in the production of urea-formaldehyde-, phenolic—, polyacetal—, and melamine-
formaldehyde resins) and in the manufacture of peritaerythritol and hexamethylene—
tetraruine. Production levels are currently approximately 6000 million pounds
annually on a 37% basis) 2 . Over 50% of the formaldehyde produced is used in the
manufacture of resins.
Pentaerythritol is used mainly in alkyd surface coating resins, rosin and tall oil
resins, varnishes, pharmaceuticals, plasticizers and insectic des 2 .
106
-------
Formaldehyde is employed in a number of minor applications in agriculture, paper.
textile and dyestuffs manufacture, medicine, analysis, etc. (Table 1).
The mutagenicity of formaldehyde has been described most extensively for Droso-
phila 3 (with hydrogenperoxide) 12 and established for Neurospora cassida (also with
13—15 .15—18
hydrogen peroxide) and E. coli
Formaldehyde effects on E. coli B/r in a special mutant lacking a DNA polyrnerase
(pol A) and, therefore, a repair deficient strain were elaborated by Rosenkranz 17 .
Formaldehyde treatment of poi A and pol A strains showed differential toxicity,
determined by the “zone of inhibition” surrounding a formaldehyde-soaked disc placed
on the surface of the growth agar. There was a preferential inhibition of growth in
the poi A strain, indicating that some repair capability may affect the survival of
formaldehyde treated bacteria.
In the above studies, Rosenkranz 17 also described the interaction of known car-
cinogens Ce . g. methyl inethanesulfonate and N-hydroxylaminofluorene) with both
the pol A and P 0I A strains of E. coli and concluded that “in view of the present
findings and because the procedure used seems to be quite reliable for detecting car-
cinogens, it would seem that continued use of formaldehyde requires reevaluation and
monitoring as exposure to even low levels of this substance might be deleterious
especially if it occurs over prolonged periods of time, a situation which probably
increases the chance of carcinogenesis”.
Formaldehyde is also rnutagenic in E. coli. B/r strains which were altered in another
repair function, Hcr. (This strain lacks the ability to reactivate phage containing
UV-induced thymine dimers because it lacks an excision function.) Strains of E.
B/r which were Hcr showed more mutation to streptomycin resistance or to trypto han
+
independence than did the repair competent Hcr strain. Ultraviolet inactivation of
107
-------
Hcr strains was enhanced by treatment with formaldehyde, possibly indicating some
effect of formaldehyde on the repair function 18 .
Formaldehyde has been found to combine with RNA or its constituent nucleotides 1921
with the formation of hydroxymethylamide (HOCH 2 CONH 2 ) and hydroxyrnethylamine
(NH 2 CH 2 OH) by hydroxymethylation of arnido (-CONH 2 ) and amino groups respectively.
Formaldehyde has been found to combine more readily with single stranded poly-
nucleotides such as replicating DNA 22 or synthetic poly A 23 . The reaction products
may also include condensation products of adenosine such as methylene bis AMP. The
possibility of formation of these compounds in vivo has led to the postulation that
adenine dirners may be found in polynucleotides in situ or may be erroneously incor-
porated into polynucleotides 21 ’ 22 .
An alternative mechanism of action for formaldehyde involving the formation of
peroxidation products by autooxidation of formaldehyde or by its reaction with other
molecules to form free radicals has been proposed 15 . The synergism between hydrogen
peroxide and formaldehyde in producing mutations in Neurospora has been described 15 .
The combination of formaldehyde and H 2 0 2 was found to be differentially mutagenic
at two loci, adenine and inositol utilization. These two loci showed divergent dose
response curves when similarly treated with formaldehyde and H 2 0 2 . This was taken
as evidence for a mutagenic peroxidation product.
A number of carcinogenic studies of formaldehyde se 2 ’ 26 as well as that of
hexamethylene te amine 27 ’ 28 (an agent known to release formaldehyde) have been
reported. To date the assessment of the carcinogenicity based on these studies would
appear to be equivocal.
108
-------
FigtLre I Ge iera1 Rea:ti s oi Fornaldehyde
Oxidatjo — Reduction
A C ,D + 3f 11 , —HCO..f + 2H 2 0 + 2e
3• C ..O + 2. ( 3)2 + 30H - 2Az + RCOO 4 4MM 3 + 2H,C 2e Tollins Rea tjo
c. z: _o _____ c 3 o1 Cannizzarc, Reaction
+ + + Crossed Cannizzaro Reacti
Addition
E. + + Cyanohydrin Formation
It
4 - I - +
F. c:-. + i so —H—C—SO 3 a Additjo of Bisulfite
O 4
G• “ 2 — — c1c 2 cC i 2 c1 . . H 2 0 Bis(chloronethyl)ethor for ati on
H. C 6 N H 12 + 5H 2 0 Nexanethyjenetetramjne for atiori
I. + _‘iL —R-NBCH 2 OM Condensation ajth a ines
.i. + — Conden at±on itch a incs
x. +F: : P.CO C 2 O.4 RCOM(G1 2 O 4) 2 Condcn tio t cd.th anid::
L. C :O :t h RO—C1 OH ROCR —OR eta1 Formation
M eo
‘ . C -- — .“ --C-3 -ç —a Aldol Cond n otion
Da . .
+ R cH 2 —c-c-a”’ + H 2 0 Mannich Reaction
?eact cn wttn Active H
Q•g(
-. £CLd O _ CH OH MeChylol Formation
2 nase ._ 2
?. 20 — - RCH 2 (O X) H 2 0 RCH 2 OH + )C gC’4 Crignard
- - — Formation of po1yox’. athy1ene
- r .( H,O) •H + (fl—1)H 2 0
— - — n
-------
TABLE 1
Minor Uses of Formaldehyde and Its Products
Agriculture
1. Treatment of bulbs, seeds and roots to destroy microorganisms.
2. Soil disinfectant.
3. Prevention of rot and infections during crop storage.
4. Treatment of animal feed grains.
5. Chemotherapeutic agent for fish.
Dyes
1. Manufacture of intermediate for production of rosaniline dyes.
2. Preparation of phenyl glycine, an intermediate in the manufacture of indigo
dyes.
3. Used to prepare formaldehydesulfoxylates which are stripping agents.
Metals Industries
1. Pickling agent additive to prevent corrosion of metals by H 2 S.
2. Preparation of silver mirrors.
3. Hexamethylenetetramine is used to produce nitriotriacetic acid and formal-
dehyde to produce ethylenediaminetetracetic acid. These compounds are
excellent metal sequestering agents.
Paper
Formaldehyde is used to improve the wet-strength, water shrink, and grease
resistance of paper, coated papers and paper products.
Photography
1. Used in film to harden and insolubilize the gelatin and reduce silver salts.
2. Photographic development.
Rubber
1. Prevent putrefaction of latex rubber.
2. Vulcanize and modify natural and synthetic rubber.
3. Hexamethylenetetramine is used as a rubber accelerator.
4. Synthesis of tetraphenylmethylenediarnine. a rubber antioxidant.
Hydrocarbon Products
1. Prevent bacterial action from destroying drilling fluids or muds.
2. Remove sulfur compounds from hydrocarbons.
3. Stabilize gasoline fuels to prevent gum formation.
4. Modify fuel characteristics of hydrocarbons.
110
-------
TABLE 1 (continued)
Leather
Tanning agent for white washable leathers.
Solvents and Plasticizers, Surface Active Compound
1. Synthesis of ethylene glycol.
2. Synthesis of formals.
3. Synthesis of inethylene derivatives.
4. Synthesis of surface active compounds.
Starch
Formaldehyde is used to modify the properties of starch, by formation of acetals
and hemiacetals.
Textiles
Modification of natural and synthetic fibers to make them crease, crush and flame
resistant and shrink-proof.
Wood
Used as an ingredient in wood preservatives.
Concrete and Plaster
Formaldehyde is used as an additive agent to concrete to render it impermeable
to liquids and grease.
Cosmetics and Deodorants
Formaldehyde is utilized in deodorants, foot antiperspirants and germicidal soaps.
Disinfectants and Fumigants
Formaldehyde is employed to destroy bacteria, fungi, molds, and yeasts in houses,
barns, chicken coops, hospitals, etc.
Medicine
1. Treatment of athletes foots and ring worm.
2. Hexarnethylenetetramine is used as a urinary antiseptic.
3. Conversion of toxins to toxoids.
4. Synthesis of Vitamin A.
5. Urea-formaldehyde is used as a mechanical ion exchange resin.
Analysis
Small quantities are used in various analytical techniques.
111
-------
2. Acrolein
Acrolein (CH 2 CHCHO; acrylic anhydride; 2-propenal) as well as its dimer (3,4-
dihydro-2-forniyl-2H-pyran) are used commercially for the synthesis of a variety of
chemicals useful in textile mi hi , paper treating, and the manufacture of rubber
chemicals, pharmaceuticals, plasticizers and synthetic resins. The extreme reactivity
of acrolein is attributed to the conjugation of a carbonylic group with the vinyl group
within its structure.
Relatively large quantities of acrolein are consumed in the manufacture of derivatives
such as 1,2, 6-hexanefriol, hydroxyadipaldehyde, and glutaidehyde via the intermediate
acrolein dimer (3, 4-dihydro-2-formyl-2H-pyran), via.:
/cHZ CH HC’ CH
+112
HC CHCHO I - IC CHCHO
0 0
H,H 2 0
2H 2
HOCH 2 CHOH (CH 2 ) 4 0H 4 OCH—CH—OH (CH 2 ) 3 CHO
1,2 ,6-Hexanetriol 2-Hydroxyadipaldehyde
Other important reactions of acrolein involve its ability to undergo a variety of
polymerization reactions (homo-, co-, and graft polymerization) as well as to undergo
reactions with ammonia and formaldehyde, respectively, to yield the industrially
important derivatives acrylonitrile and pentaerythritol. One of the largest uses is
in the production of methionine which is used in supplementing fowl, swine, and
ruminant feeds. Epoxidation of acrolein with hydrogen peroxide yields glycidaldehyde
which is extensively used as a cross-linking agent for textile treatment and leather
tanning.
112
-------
Acrolein has also been suggested for the stabilization of photographic baths 29
and for the removal of odors from waste liquids containing deodorants 30 .
Acrolein (as well as formaldehyde) have been found to be among the most cyto-
toxic aldehydes (or ketones) of a large number of organic solvents examined in short-
term in vitro incubations with Ehrlich-Landschuetz diploid (EL])) ascites tumor cells 31 .
In a comparison of the cytotoxicity of short-chain alcohols and aldehydes in
cultured neuroblastoma cells it was found that the cytotoxicity of the alcohols increased
as the number of carbons in the compound increased, whereas toxicity of the aldehydes
33
increased with decreasing chain length . The marked cytotoxicity of acrolein was
ascribed to the presence of both the carbonyl and the carbon-carbon double bond
since propion aldehyde, having only the carbortyl group, and allyl alcohol, having
only the CC double bond, were less toxic 32 .
Acrolein has been found mutagenic in Drosophila 33 .
3. - Acetaldehyde (ethanol; acetic aldehyde, CH 3 CHO) is employed primarily as an
intermediate in the production of paraldehydes, acetic acid, acetic anhydride, penta-
erythritol, butyl alcohol, butyraldehyde, chioral, 2-ethyl hexanol and other aldol
products, peroxy acetic acid, cellulose acetate, vinyl acetate resins and pyridine deri-
vatives 34 . Lesser uses of acetaldehyde include the production of aniline dyes; thermo-
setting resins from the condensation products with phenol and urea; the preparation
of Schiff bases (via reaction with aliphatic and aromatic amines) which are used as
accelerators and antioxidants in the rubber industry. Acetaldehyde has been used as
a preservative for fruit and fish, as a denaturant for alcohol, in fuel compositions, for
hardening gelatin, glue and casein products, for the prevention of mold growth on
leather, and as a solvent in the rubber, tanning and paper industries 34 ’ 35 .
Aldehyde is manufactured via the hydration of acetylene, the oxidation or dehydro-
genation of ethanol, or the oxidation of saturated hydrocarbons or ethylene 34 . Ace—
taldehyde is a highly reactive compound exhibiting the general reactions of aldehydes,
113
-------
e. g., undergoing numerous condensation addition and polymerization reactions. In
addition, acetaldehyde is readily oxidized to peroxy compounds such as peroxy
acetic acid, acetic anhydride, or acetic acid, the principal product(s) dependent
on the specific oxidation conditions employed.
It is the product of most hydrocarbon oxidations; it is a normal intermediate
product in the respiration of higher plants; it occurs in traces in all ripe fruits and
may form in wine and other alcoholic beverages after exposure to air. Acetaldehyde
is an intermediate product in the metabolism of sugars in the body and hence occurs
in traces in blood. It has been reported in fresh leaf tobacco 36 as well as in tobacco
snioke 37 ’ 38 and in automobile and diesel exhaust 39 ’ 40 .
Information as to the mutagenicity of aldehydes (with the exception of formaldehyde)
33
is scant. Acetaldehyde has been found mutagenic in Drosophila
114
-------
References for Aldehydes
1. Kirk-Othiier, “Encyclopedia of Chemical Technology” 2nd ed., Vol. 10, Wiley-
Interscience, New York (1966) p. 77.
2. Environmental Protection Agency, “Investigation of Selected Potential Environmental
Contaminants-Formaldehyde, Final Report”, Office of Toxic Substances, Environ-
mental Protection Agency, August, 1976
3. Alderson, T., Chemically Induced Delayed Germinal Mutation in Drosophila,
Nature . 207(1965) 164
4. Auerbach, C., Mutation Tests on Drosophila Melanogaster with Aqueous Solutions
of Formaldehyde, Am. Naturalist , 86 (1952) 330-332
5. Khishin, A. F. E., The Requirement of Adenylic Acid for Formaldehyde Mutagenesis
Mutation Res. , 1(1964) 202
6. Burdette, W. J., Tumor Incidence and Lethal Mutation Rate in a Tumor Strain of
Drosophila Treated with Formaldehyde, Cancer Res. , 11 (1951) 555
7. Auerbach, C., Analysis of the Mutagenic Action of Formaldehyde Food. III Con-
ditions influencing the effectiveness of the treatment, Z. Vererbungslehre , 81
(1956) 627—647
8. Auerbach, C., Drosophila Tests in Pharmacology, Nature , 210 (1966) 104
9. Rapoport, I. A., Carbonyl Compounds and the Chemical Mechanisms of Mutations,
C.R. Acad. Sci. USSR , 54 (1946) 65
10. Kaplan, W. D., Formaldehyde as a Mutagen in Drosophila, Science , 108 (1948)
11. Auerbach, C., The Mutagenic Mode of Action of Formalin, Science , 110 (1949) 119
12. Sobels, F. H., Mutation Tests with a Formaldehyde-Hydrogen Peroxide Mixture
in Drosophila, Am. Naturalist , 88 (1954) 109-112
13. Dickey, F. H., Cleland, G. H., and Lotz, C., The Role of Organic Peroxides in
the Induction of Mutations, Proc. Nati. Acad. Sci. , 35 (1949) 581
14. Jensen, K. A., Kirk, I., Kolmark, G., and Westergaard, M., Cold Spring Symp.
Quant. Biol., 16 (1951) 245 Chemically Induced Mutations in Neurospora
15. Auerbach, C., and Ramsey, D., Analysis of a Case of Mutagen Specificity in
NeurosporaCrassa, Mol. Gen. Genetics , 103(1968)72
115
-------
16. Demerec, M., Bertani, G., and Flint, J., Chemicals for Mutagenic Action on
E.coli,Am. Naturalist , 85(1951) 119
17. Rosenkranz, H. S., Formaldehyde as a Possible Carcinogen, Bull. Env. Contam.
Toxicol. , 8(1972) 242
18. Nishioka, H., Lethal and Mutagenic Action of Formaldehyde, Hcr and Hcr Strains.
E. Call, Mutation Res. , 17 (1973) 261
19. , D. E., The Applicability of Formol Titration to the Problem of End-Group
Determinations in Polynucleotides. A Preliminary Investigation, Biochem. Biophys.
Acta. , 40 (1960) 62
20. Haselkorn, R., and Doty, P., The Reaction of Formaldehyde with Polynucleotides,
J. Biol. Chem. , 236(1961) 2730
21. Alderson, T., Significance of Ribonucleic Acid in the Mechanism of Formaldehyde
Induced Mutagenesis, Nature , 185 (1960) 904
22. Voronina, E. N., Study of the Spectrum of Mutations Caused by Formaldehyde in
E.Coli K-12, In Different Periods of a Synchronized Lag Period, Soy. Genet. , 7
(1971) 788
23. Fillippova, L. M., Pan’ shin, 0. A., and Koslyankovskii, F. K., Chemical Mutagens,
Genetika , 3 (1967) 135
. Watanabe, F., Matsunaga, T., Soejima, T., and Iwata, Y., Study on the Carcino-
genicity of Aldehyde, 1st. Report, Experimentally Produced Rat Sarcomas by
Repeated Injections of Aqueous Solution of Formaldehyde, Gann. , 45 (1954) 451
25. Watanabe, F., and Sugimoto, S., Study on the Carcinogenicity of Aldehyde 2nd
Report , Seven Cases of Transplantable Sarcomas of Rats Appearing in the Area
of Repeated Subcutaneous Injections of Urotropin, Gann. , 46 (1955) 365
26. Horton, A. W., Tye, R., and Stemmer, K. L., Experimental Carcinogenesis of
the Lung, Inhalation of Gaseous Formaldehyde on an Aerosol Tar By C3H Mice,
J. Nati. Cancer Inst. , 30 C 1963) 31
27. Della orta, G., Colnagi, M. I., and Parxniani, G., Non-Carcinogenicity of Hex-
amethylenetetramine in Mice and Rats, Food Cosmet. Toxicol. , 6 (1968) 707
28. Brendel, R., Untersuchungen an Ratten Zur Vertraglickeit Von Hexamethylene
Tetramin, Arznei-Forsch. , 14 (1964) 51
29. Shirasu, K., Iwano, H., and Hatano, T., Stabilizing Baths for Color-Photographic
Materials, German Offen., 2, 361, 668, June 20 (1974), Chem. Abstr. , 81 (1974)
P84404T
116
-------
30. Nomura, K., Ito, H., Futai, Y., and Watarai, S., Deodorization of Bad Smelling
Substances with Alkaline Deodorizers Containing Glycine-System Surfactants
Chem. Abstr. , 81(1974) P53906D
31. Holmberg, B., and Malrnfors, T., Cytotoxicity of Some Organic Solvents, Env.
Res. , 7(1974) 183
32. Koerker, R. L., Berlin, A. J., and Schneider, F. H., The Cytotoxicity of Short-
Chain Alcohols and Aldehydes in Cultured Neuroblastoma Cells, Toxicol. Appl.
Pharmacol. , 37(1976) 281—288
33. Rapoport, I. A., Mutations Under the Infliience of Unsaturated Aldehydes, Dokl.
Akad. Nauk. SSSR , 61 (1948) 713
34. Hayes, E. R., Acetaldehyde, In Kirk-Othmer Encyclopedia of Chemical Technology,
2nd ed., Vol. 1., Interscience Publishers, New York (1963) pp. 75-95
35. Merck & Co., “The Merck Index”, Ninth ed., Merck & Co., Inc. Rahway, NJ
(1976) p. 35
36. Shaw, W. G. J., Stephens, R. L., and Weybrew, 3. A., Carbonyl constituents
in the volatile oils from flue-cured tobacco,
Tobacco Sci. , 4(1960) 179
37. Irby, R. M., Harlow, E.S., Cigarette smoke. I. Determination of vapor constituents,
Tobacco , 148 (1959) 21
38. Johnstone, R. A. W., and Plimmer, 3. R., The chemical constituents of tobacco
and tobacco smoke,
Chem. Revs. , 59(1959) 885
39. Linnel, R. H., and Scott, W. E., Diesel exhaust analysis,
Arch. Environ. Hith. , 5(1962) 616
40. Ellis, C. F., Kendall, R. F., and Eccleston, B. H., Identification of oxygenates
in automobile exhausts by combined gas-liquid chromatography and I.R. techniques,
Anal. Chem. , 37(1965) 511
117
-------
IV. Acylating Agents
Acylating agents Ce . g., acid chlorides or anhydrides) are another class of
highly reactive electrophiic agents (via the initial formation of a complex with a
Lewis acid catalyst such as aluminum chloride). Acylation and alkylation processes
are closely related, for example, intheir activity toward arenes. The reaction
effectively introduces an acyl, RCO-, into an aromatic ring and the product
is an aryl ketone (or an aldehyde if the acid chloride is formylchloride). It can
be assumed that these substances can interfere with normal biological reactions
because of their high chemical reactivity to biochemical substances.
1. Dirnethylcarbarnovichioride (CH 3 .. DMCC) is prepared via the reaction
CH( N-C-Cl,
of phosgene and trimethylamine and is used primarily inthe preparation of pharma-
ceuticals , e. g. , neostigrnine bromide, neostigmine inethylsulfate and pyridostigmine
bromide, agents used in the trea nent of myasthenia gravis and secondarily as an
intermediate in the synthesis of carbamates which are used as pesticides, drugs
and industrial intermediates in the synthesis of dyes and unsymmetrical dimethyl-
hydrazine (a rocket l 2• Dimethylcarbamaylchloride has also been found at
levels of up to 6 ppm during production of phthaloylch1orides 3 .
The carcinogenic potential (high incidence and short latency period) of dimethyl-
carbamoyl chloride by inhalation in rats has recently been reported 2 . Eighty-nine
of 93 rats exposed by inhalation to 1 ppm DMCC developed squamous cell carcinomac
of the nose within 200 days. The carcinogenic potential of DMCC was first reported
in l972 in a preliminary note, and in 1974 describing a high incidence of skin
tumors and subcutaneous sarcomas, along with some papillary tumors of the lung
in ICR/Ha Swiss mice following applications of DMCC to skin by both subcutaneous
and intraperitoneal injection.
118
-------
Dimethylcarbamoyl chloride has been shown to be mutagenic in Salmonella
typhimurium TA 100 and TA 98 containing an R factor (plasmids carrying antibiotic
6 2
resistance genes) and two E. coil strains (WP2 and WP25)
2. Diethylcarbamoyl chloride DECC) is used commercially pri-
c Hç
marily in the synthesis of anthelmintic diethylcarbamazine citrate 2 . DECC has
recently been found to be less mutagenic than DMCC in E. coil strains WP2 and WP25 2 .
It should be noted that other acylating agents, e.g., benzoyl chloride, phtha-
loyl chloride are widely employed as chemical intermediates. A recent report
has cited the incidence of lung cancer among benzoyl chloride manufacturing
workers in Japan The manufacturing process involved the initial chlorination
of toluene to benzotrichloride with subsequent hydrolysis or reaction of the inter-
mediate benzotrichloride with benzoic acid to yield benzoyl chloride. The se-
quence of reactions is as follows: c 3 Cd 3 + 3J- c l
H 2 0
C 6 1-1 5 C001-I \ J
Minor reaction products in the original chlorination step were found to be
benzyl chloride (C 6 H 5 CH 2 C1) and benzal chloride (C 6 H 5 CHC1 2 ). Benzyl chloride
has been shown to be carcinogenic in rats 8 and mutagenic in Salmonella typhi-
murium TA 100 and TA 98 tester strains with R-factor plasmids 6 . The high
reactivity of both benzoyl chloride and benzotrichloride used as reagents to in-
troduce benzoyl (C 6 H 5 CO ) and benzenyl (C 6 H 5 C ) radicals respectively suggest
the potential of these reagents for carcinogenic and/or mutagenic activity.
119
-------
3. Ketene (CH 2 C=O; ethenone) is a highly reactive acylating agent formed by pyro-
lysis of virtually any compound containing an acetyl group, e.g., acetone. Ketene
is widely used in organic synthesis for the acylation of acids, hydroxy compounds,
aromatic hydrocarbons (Friedel-Crafts acylation), amines etc . The major areas of
utility of ketene include: the manufacture of acetic anhydride and the dimerization to
diketene which is an important intermediate for the preparation of dihydroacetic acid,
ac etoacetic esters, acetoac etanilide, N, N, -dialkylacetoac etamides, and cellulose esters
which are used in the manufacture of fine chçmicals, drugs, dyes, and insecticides.
Ketene has utility as a rodenticide 10 , in textile finishing 11 , in the acetylatiort of
viscose rayon fiber’ 2 , as an additive for noncorrosive hydrocarbon fuels’ 3 , and in
the acetylation of wood for improved water resistance 14
Ketene has been found mutagenic in Drosophila 15 , but non-mutagenic in
Neurospora 16 .
1—,
I’.
-------
References for Acylating Agents
1. Anon, Dupont Takes Steps to Protect Workers Against DMCC, Occup. Hith.
Safety Letter, 6 (1976) 7
2. Flnklea, 3. F., Dimethylcarbamoyl Chloride (DMCC), National Institute of
Occupational Safety and Health, Current Intelligence Bulletin, July 7, 1976
3. E .1. dupont de Nemours & Co., Dixnethylcarbaxnoyl Chloride, Advisory Letter
to NIOSH, June 23, 1976
4. Van Duuren, B. J., Dimethyl Carbamyl Chloride, A Multi Potential Carcinogen,
J. Natl. Cancer Inst. , 48(1972) 1539
5. Van Duuren, B. J., Goldschmidt, B. M., Katz, C., Seidman, I., and Paul, T .S
Carcinogenic Activity of Alkylating Agents, J. Natl. Cancer Inst. , 53 (1974) 695
6. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N., Detection of
Carcinogens as Mutagens: Bacterial Tester Strains with R Factor Plasznids,
Proc. Nati. Acad. Sci. , 72(1975) 979
7. Sakabe, H., Matsushita, H., Koshi, S., Cancer Among Benzoyl Chloride Manu-
facturing Workers, Ann. N.Y. Acad. Sci. , 271 (1976) 67
8. Druckrey, H., Kruse, H., Preussmann, R., Ivankovic, S., and Landscht Itz,
C., Cancerogene Alkylierende Substanzen, U I. Alkylhalogenide,—Sulfate-, Sul-
fonate Urid Ringgespante Heterocyclen, Z. Krebsforsch. , 74 (1970) 241
9. Lacey, R. N., In “Advances in Organic Chem. Methods Results”, (eds, Raphael,
R. A., Taylor, E. C., Wynberg, H.) Wiley Interscience, New York(1960), p. 213
10. Farbenfabriken Bayer Akt., British Patent 862,866 (1961), Chem. Abstr. , 55,
15823b (1961)
11. Gagliardi, D. D., Symposium on Textile Finishing. I. Chemical Finishing of
Cellulose, Am. DyestuffRepr. , 50(1961)34-36
12. Takigawa, M., and Kanda, I., Toho Reiyon Kenkyu Hokoku 3, 30 (1956); Chem.
Abstr. , 53(1959) 7602
13. Fields, J. E., and Zopf , Jr., G. W., U.S. Patent 2,291,843 (1960)
14. Karlsons , I., Svalbe, K., Ozolina, I.,, and Sterni, S., USSR Patent 391,924,
27 July (1973), Chem. Abstr. , 81(1974) 39278tJ
15. Rapoport, I. A., Dokl. Akad. Nauk, SSSR , 58 (1947) 119
16. Jensen, K. A., Kirk, I., Kolmark, G., and Westergaard, M., Coldspring Harbor
Symp. Quant. Biol . 16(1951) 245
121
-------
V. Peroxides
The utility of a variety of organic peroxides in a broad spectrum of commercial
and laboratory polymerization reactions is well established. The commercial organic
peroxides are highly reactive sources of free radicals: RO: OR - R0 + OR.
Organic peroxides are employed predominantly in the polymer industry (e.g., plas-
tics, resins, rubbers, elastomers, etc.) and are used in applications including the
following: (a) initiators for the free-radical polymerizations and/or co-polymeri-
zations of vinyl and diene monomers, (b) curing agents for resins and elastomers
and (c) cross-linking agents for polyolefins. Miscellaneous uses of organic peroxides
in the polymer industry include: vulcanization of natural and butadiene rubbers;
curring polyurethanes and adhesives; preparation of groft copolymers; cross-linking
polyethylenes and ethylene-containing co-polymers; and as flame retardant synergists
for polystyrene 1 .
1. Di-tert. butyl peroxide [ (CH 3 ) 3 COOC (CH 3 ) 3; bis (1, 1-dimethyl ethyl )peroxide]
is used extensively in organic synthesis as a free radical catalyst, as a source of
reactive methyl radicals; as an initiator for vinyl monomer polymerizations and co-
polymerizations of, styrene, vinyl acetate, and acrylics and as a curing
agent for thermoset polyesters, styrenated alkyds and oils, and silicone rubbers,
as a vulcanization agent for rubber 2 , in lubricating oil manufacture 3 , for cross-
linking of Lire-resistant polybutadiene moldings 4 and for cross-linking of high devsity
polyethylene 5 .
Di-tert-butyl peroxide is mutagenic in Neurospora 6 but has been reported to be
inactive toward transforming-DNA 7 .
2. tert-Butyl hydroperoxide [ (CH 3 ) 3 COOH; 1, 1-dimethyl ethyl hydroperoxide] is
used as an initiator for vinyl monomer polymerizations and copolymerizations with
122
-------
styi-ene, vinyl acetate, acrylics, acrylarnide, unsaturated polyesters 4 ’ 8 ’ 9 and as
a curing agent for thermoset polyesters.
The mutagenic effect of tert-bu*yl hydroperoxide in DrosophilalO)U, E. coli’ 2 ,
and Neurowora 13 and its induction of chromosome aberrations in Vicia faba1 4 lS
and Oenothera 16 has been described.
3. Cumene hydroperoxide [ C 6 H 5 C (CH 3) 2 00H; 1—methyl- 1-phenyl ethyl hydro-
peroxide) is employed as an initiator for vinyl monomer polymerizations and co—
polymerizations with styrene, acrylics, butadiene—styrene, cross-leaked foamed
1,17,18
polyesters , as a curing agent for therrnoset polyesters, styrenated alkyds
and oils, acrylic monomers, ‘ and as a promoter for oxidation of hydrocarbons
Curneme hydroperoxide is mutagenic in E. coil 12 and Neurospora 6 ’ 7 .
0
4. Succinic acid peroxide [ HOOC-CH 2 -CH 2 -C-O-OH} is used as an inidicator for
vinyl monomer polymerizations and copolymerizations with ethylene and fluorolefins.
It is mutagenic in E. coil’ 2 ’ 20 , and has been found to inactivate T2 phage 21 , and
transforming DNA of H. influenzae 22 .
5. Peracetic acid (peroxyacetic acid; acetyl hydroperoxide; ethaneperoxic acid;
CH 3 C000H) is generally prepared from acetaldehyde via autoxidation or from
23
acetic acid and hydrogen peroxide . Organic peroxyacids are the most powerful
oxidizing agents of all organic peroxides 23 .
The commercial form of peroxyacetic acid is usually in 40% acetic acid, and
is primarily employed as a bleaching and epoxidizing agent. The range of utility
of peracetic acid includes: (1) the bleaching of textiles and paper-pulp; (2) as
a catalyst for polymerization of aminopropronitrile; (3) as a co-catalyst for a
sterospecific polymerization of aldehydes; (4) as a fungicide; (5) disinfectant
123
-------
for rooms arid medical machines; (6) sterilizing agent for blood serum for tissue
culture; (7) as an oxidizing and hydroxylating reagent in organic synthesis,
and (3) as a bactericide in food industries.
Peracetic acid (as well as hydrogen peroxide) have been found mutagenic
in S. typhimurium inducing mainly deletions with H 2 0 2 being the more effluent
agent 24 . For lower concentrations, cells are protected against these peroxides
by superoxide dismutase and catalase. A 500 fold ratio between the concentrations
of H 2 0 2 and peracetic acid producing a similar biological effect indicated that
cells were more efficiently protected against hydrogen peroxide. This was
believed to result from the decomposition of peracetic acid via °2 radical liberation;
thence subsequently converted by superoxide dismutase into H 2 0 2 which is
further decomposed by catalase. Once the cell protection is overcome (e.g.,
at levels of Sg/l H 2 0 2 and 20 mg/i peracetic acid) the differences in survival
suggest that induced genetic damage by H 2 0 2 would be partly repaired. whereas
that induced by peracetic acid would not 24 .
It is also of importance to consider hydrogen peroxide se in terms of its
utility, sources, and mutagenic activity.
6.. Hydrogen peroxide undergoes a variety of reactions:
Decomposition 2 H 2 O -+ 2 H 2 0 ÷ O (1)
Molecular addition H 2 0 2 + Y - YH 2 0 2 (2)
Substitution H 2 0 2 + RX - ROOH ÷ HX (3)
or H 2 0 2 + 2 RX -, ROOR ÷ 2 HX (4)
H 7 0, as oxidizing agent H 2 0 2 + Z ZO + H 2 0
H 2 O- as reducing agent H 2 0 2 ÷ W -+ WH 2 O (6)
124
-------
In entering into these reactions, hydrogen peroxide may either react as a molecule
or it may first ionize or be dissociated into free radicals. The largest commercial
use for H 2 0 2 is in the bleaching of cotton textiles and wood and chemical (Kraft and
sulfite) puips. (The rate of bleaching appears directly related to alkalinity, with
the active species assumed to be the perhydroxyl anion 00H.) The next largest
use of H,0 2 is in the oxidation of a variety of important organic compounds. For
example, soubean oil, linseed oil, and related unsaturated esters are converted to
the epoxides for use as plasticizers and stabilizers for polyvinyl chloride. Other
important commercial processes include the hydroxylations of olefinic compounds,
the synthesis of glycerol from propylene, the conversion of tertiary amines to corres-
ponding amine oxides, and the conversion of thiols to disulfides and sulfides to
sulfoxides and sulfones. In the textile field, I-I O is used to oxidize vat and sulfur
dye.
In addition, many organic peroxides are made from hydrogen peroxide. These
include peroxy acids such as peroxyacetic acid; hydroperoxides such as tert-butyl
hydroperoxides; diacyl peroxides such as benzoyl peroxide; ketone derivatives
such as r ethylethylketone peroxide. Other uses of hydrogen peroxide include its
use as a blowing agent for the preparation of foam rubber, plastics, and elastorners,
bleaching, conditioning, or sterilization of starch, flour, tobacco, paper, and fabric 25
and as a component in hypergolic fuels 26 . Solutions of 3% to 6% H 2 0 2 are employed
for germicidal and cosmetic (bleaching) use, although concentrations as high as
30% H 2 0, have been used in dentistry 27 . Concentrations of 35% and 50% H 2 0 2 are
used for most industrial applications.
Hydrogen peroxide has been found mutagenic in . coli 28 ’ 29 , Staphylococcus
6 33,34
aurcus 3032 , and Neurospora (including mixtures of Hydrogen peroxide
125
-------
and acetone, and hydrogen peroxide and formaldehyde) 33 . The inactivation of
transforming DNA by hydrogen peroxide 7 ’ 3537 , as well as by peroxide-producing
agents (e.g., compounds which contain a free N-OH group as hydroxylamirte, N-
xnethylhydroxylamine, hydroxyurea, hydroxyurethan arid hydroazines on exposure
to oxygen) has been described 1 ’ 3 ’. Hydrogen peroxide has induced chromosome
aberrations in strains of ascites tumors in mice 38 and in Vicia faba 39 .
40 . t1
Hydrogen peroxide has also been found to be non-mutagenic in bacteria
4’ 43
and Drosophila -. Recent studies by Thacker and Parker on the induction of
mutation in yeast by hydrogen peroxide suggest that it is inefiluent in the induction
of nuclear gene mutation. This could be because radical action produces certain
types of lesions, leading to inactivation and mitochondrial. genome mutation, but
not to point mutational changes.
There is general agreement that the effects of hydrogen peroxide, (as veU as
hydrogen peroxide-producing agents) on DNA are caused by the free radicals they
generate.
Hydrogen peroxide decomposes into two OH radicals in response to
UV irradiation or spontaneously at elevated temperatures. It also gives rise to
H00 radicals in the presence of reduced transition metals (e.g., Fe+±, Cu+).
These radicals can then react with organic molecules to produce relatively more
stable organic peroxy radicals and organic peroxides which may later decompose
again into free radicals. This process of ‘ t radical-exchang&’ sustains the effective-
ness of short-lived radicals such as .O}i, H00, and H• and gives them an oppor-
tunity to reach the genome where they can exert their effect 1 .
126
-------
References for Peroxides
1. Fishbejn, L., Flamm, W. G., and Falk, H. L., “Chemical Mutagens”, Academic
Press, New York, (1970) PP. 269—273
2. Maeda, I., Aoshima, M., Low-Temperature Vulcanization of Chlorinated Rubbers,
Ger. Offen., 2,401,375, August14 (1974), Chem. Abstr. , 82(1975) P183704
3. Bitter, J. G. A., Ladeur, P., Maas, R. J., and Bernard, J. ID. J., Lubricating
Oils with High Viscosity Index, Ger. Qffen. 2,361,653, June20 (1974), Chem.
Abstr. , 82(1975) P46240Z
4. Musashi, A., Yama zaki, M., and Hiruta, M., Hardenable Flame Proof Polybuta-
diene Composition Ger. Offen. 2,403,639, August 15 (1974), Chem. Abstr. , 82
(1975) P179950A
5. Proskurnina, N. G., Akutin, M. S., and Budnitskii, Y. M., Cross-Linkmg of
High-Density Polyethylene by Peroxides on Zeolites, Plast. Massy , 12 (1974) 50,
Chexn. Abstr. , 82 (1975) 140928B
6. Jensen, K. A., Kirk, I., Kolmark, G., and Westergaard, M., Chemically Induced
Mutations in Neurospora, Cold Springs Harbor Symp. Quant. Biol. , 16 (1951)
245—261
7. Latarjet, R., Rebeyrotte, N., and Demerseman, P., In “Organic Peroxides in
Radiobiology”, (ed. Haissinsky, M.) Pergamon Press, Oxford (1958) p. 61
8. Meyer, H., Schmid, ID., Schwarzer, H., and Twittenhoff, H. J., Setting of
Unsaturated Polyester Resins, U.S. Patent 3,787,527, Jan. 22 (1974), Chem.
Abstr. , 81(1974) 50578N
9. Ulbricht, J., Thanh, V. N., Effect of REdox Systems on the Degree of Polymeri-
zation and in Homogeneity of PVC, Plaste Kaut. , 21 (1974) 186, Chem. Abstr. ,
81 (1974) 37902
10. Altenberg, L. S., The Production of Mutations in Drosophila by Tert.Butyl Hydro-
peroxide, Proc. Nati. Acad. Sci. , 40(1940) 1037
11. Altenberg, L. S., The Effect of Photoreactivating Light on the Mutation Rate Induced
in Drosophila by Tert.Butyl Hydroperoxide, Genetics , 43 (1958) 662
12. Chevallier, M. R., and Luzatti, ID., The Specific Mutagertic Action of 3 Organic
Peroxides on Reversal Mutations of 2 Loci in E. Coli, Compt. Rend. , 250 (1960)
1572—1574
13. Dickey, F. H., Cleland, G. H., and Lotz, C., The Role of Organic Peroxides in
the Induction of Mutations, Proc. Nati. Acad. Sci. , 35 (1949) 581
127
-------
14. Revell, S. H. • Chromosome Breakage by X-rays and Radiomimetic Substances
in Vicia, Heredity , 6 (1953) Suppi., 107-124
15. Loveless, A., Qualitative Aspects of the Chemistry and Biology of Radiomirnetic
(Mutagenic) Substances, Nature , 167 (1951) 338
16. Oehlkers, F.. Chromosome Breaks Induced by Chemicals, Heredity Suppi. , 6
(1953) 95—105
17. Doyle, E. N., Thermosetting Unsaturated Polyester Foam Products, U. S. Patent
3,823,099, July 9 (1974) Chem. Abstr. , 81 (1974) P137087A
18. Gruzbarg, K. A., and Barboi, W. M., Copolytnerization for Cross linking of
Oligomeric Polyest rs with Vinyl Monomers in Presence of Various Initiators,
Teknol. Svoistva Polim. Mater Radiats. Otverzhdeniya. (1971) pp. 34-46, Chem.
Abstr. , 81(1974) 14061E
19. Toi, Y ., Shinguryo, H., Nakano, S., and Tsuchida, R., Polymerizing Acrylic
Monomers Absorbed in Organic Materials, Japan Kokai, 74 16,791, Feb. 14 (1974)
Chem. Abstr. , 81 (1974) P50704A
20. Luzzati, D., and Chevallier, M. R., Comparison of the Lethal and Mutagenic Action
of an Organic Peroxide and of Radiations on E. Coli, Ann. Inst. Pasteur. , 93 (1957)
366
21. Latarjet, R., Ciba Foundation. Symp. Ionizing Radiations Cell Metab. (1957)
p. 275
22. Wieland, H., and Wingler, A., Mechanism of oxidation processes. V. Oxidation
of aid ehydes,
Ann. Chem. , 431(1923) 301—322
25. Young, J. H., U. S. Patent 2,777,749, Jan. 15, Bleaching and Conditioning of
Solids with Hydrogen Peroxide, Chem. Abstr. , 51 (1957) 5442g
26. Ayers, A. L., and Scott, C. R., U.S. Patent 2,874,535 (Feb. 24), Substituted
Furans as Hypergolic Fuels, Chem. Abstr. , 53 (1959) 9621B
27. Lude’ ”ig , R., Z. Deut. Zahnaerztl. , 55, (1960) 444; The Use of High Concentrations
of Hydrogen Peroxide in Dentistry
28. Deinerec, M., Bertani, G., and Flint, J., Chemicals for Mutagenic Action on
E. Coil. Am. Naturalist , 85 (1951) 119
128
-------
29. Iyer , V. N., and Szyba].ski, W., Two Simple Methods for the Detection of Chemical
Mutagens, Appi. Microbiol., , 6 (1958) 23
30. Wyss, 0., Stone, W. S., and Clark, 3. B., The Production of Mutations in Staphyl-
ococcus Audreus by Chemical Trea nent of the Substrate, 3. Bacteriol. , 54 (1947) 767
31. Wyss, 0., Clark, J. B., Haas, F., and Stone, W. S., The Role of Peroxide in the
Biological Effects of Irradiated Broth, j. Bacteriol. , 56 (1948) 51
32. Haas, F. L., Clark, J. B., Wyss, 0., and Stone, W. S., Mutations and Mutagenic
Agents in Bacteria, Am. Naturalist. , 74 (1950) 261
33. Dickey, F. H., Cleland, G. H., and Lotz, C., The Role of Peroxides in the Induction.
of Mutations, Proc. Nati. Acad. Sci. US , 35 (1949) 581
34. Wagner, R. P., Haddox, C. R., Fuerst, R., and Stone, W. S., The Effect of
Irradiated Medium, Cyanide and Peroxide on the Mutation Rate in Neurospora,
Genetics , 35 (1950) 237
35. Luzzati, D., Schweitz, H., Bach, M. L., and Chevallier, M. R., Action of
Succin.ic Peroxide on Deoxyribonucleic Acid (DNA), J. Chirn. Phys. , 58 (1961),
1021
36. Zamenhof, A., Alexander, H. E., and Leidy, G., Studies on the Chemistry of the
Transforming Activity, I. Resistance to Physical and Chemical Agents, 3. Expti.
Med. , 98(1953) 373
37. Freese, E. B., Gerson, 3., Taber, H., Rhaese, H. 3., and Freese, E. E.,
Inactivating DNA Alterations by Peroxides and Peroxide Inducing Agents,
Mutation Res. , 4(1967) 517
38. Schöneich, J., The Induction of Chromosomal Aberrations by Hydrogen Peroxide
in Strains of Ascites Tumors in Mice, Mutation Res. , 4 (1967) 385
39. Lilly, L. J., and Thoday, 3. M., Effects of Cyanide on the Roots of Vicia Faba,
Nature . 177 (1956) 338
40. Doudney, C. 0., Peroxide Effects on Survival and Mutation Induction in UV
Light Exposed and Photo-reactivated Bacteria, Mutation Res. , 6 (1968) 345-353
41. Kimball, R. F., Hearon, 3. Z., and Gaither, N., Tests for a Role of H 2 0, in
X-ray Mutagenesis. II. Attempts to Induce Mutation by Peroxide, Radiatio n Res. ,
3 (1955) 435—443
42. Sobels, F. H., Peroxides and the Induction of Mutations by X-Rays, Ultraviolet,
and Formaldehyde, Radiaon Res., Suppi. 3 (1963) 171—183
43. Thacker, 3., Parker, W. F., The Induction of Mutation in Yeast by Hydrogen
Peroxide, Mutation Res. , 38 (1976) 43—52
129
-------
VI. HALOGENATED UNSAT’D AND SAT’D HYDROCARBONS AND RELATED DERIVATiVES
The halogenated aliphatic hydrocarbons represent one of the most important cate-
gories of industrial chemicals from a consideration of volume, use categories, envir-
onmental and toxicological considerations and hence most importantly, potential popu-
lation risk.
In recent years, there has been recognized concern over the environmental and
toxicological effects of a spectrum of halogenated hydrocarbons, primarily the organo
chlorine insecticides and related derivatives, e.g., DDT, dieldrin, Mirex, Kepone,
polychiorinated biphenyls (PCBs) and chlorinated dioxins. This concern has now
been extended to practically all of the major commercial halogenated hydrocarbons,
numerous members of which have extensive utility as solvents, aerosol propeflants,
degreasing agents, dry-cleaning fluids, refrigerants, flame-retardants, synthetic
feedstocks, cutting fluids and in the production of textiles and plastics, etc., and
hence are manufactured on a large scale.
A. Unsat d Hydrocarbons-Vinyl and Vinylidene Derivatives
1. Vinyl chloride (chloroethylene; VCM; CH 2 CHC1) is used in enormous
quantities primarily (97%) for the production of homo-polymer (for PVC production)
and co-polymer resins (e.g., Saran and other plastics). Lesser quantities of vinyl
chloride are used in the production of 1,1, l-trichioroethane (methyl chloroform), as
an additive in specialty coatings and as a component of certain propellant mixtures.
It is important to note the VCM production processes because of the halocarbon pre-
cursors and intermediates as well as the nature of the potential carcinogenic and muta
genic trace impurities. VCM monomer production processes employ one of the following:
(1) the acetylene plus hydrogen reaction; (2) the direct chlorination of ethylene and
dehydrochiorination and (3) the balanced direct and oxychlorination of ethylene and
130
-------
dehydrochiorination. The overall processes differ primarily in the manner in which
the intermediate ethylene dichioride is produced. The bulk of VCM is produced by
process (3) above 1 ’ 2 and a typical commercial product can contain the following
impurities in mg/kg 2 : unsaturated hydrocarbons, 10; acetaldehyde, 2; dichioro
compounds, 16; water, 15; HC1, 2; non volatiles, 200; iron, 0.4; phenol as a sta-
bilizer, 25-50; and trace amounts of organic impurities including: acetylene, 1,3-
butadiene, methyl chloride, vinylidene chloride and vinylacetate 1 .
As has been previously noted in this report, ethylene dichloride is mutagenic in
3,4
Drosophila , in S. typhimuriurn TA 1530, TA 1535 and TA 100 tester strains
(without metabolic activation) 5 ’ 6 and in E. coli (DNA polymerase deficient pol A
7
strain
The growth patterns of vinyl chloride monomer (VCM) per se as well as that of
its primary end product polyvinyl chloride (PVC) plastic resin have been well docu-
mented 1 ’ 8-10 Vinyl chloride production in the United States exceeded 2.6 billion
kg in 1974 (about one-third of the Western World’s output) with the annual growth
rate expected to exceed 10% per year through the 1980’s 1 . The world production of
PVC in 1975 is estimated to be 9-10 million tons. The total world-wide employment
in the VCM and PVC producing industries is over 70, 000 workers. Those employed
in industries using PVC as a basic element are believed to number in the millions 9 .
PVC is produced (in U.S.) via 4 major processes (in % total production) as
follows: (1) suspension polymerization, 78; (2) emulsion polymerization, 12;
(3) bulk polymerization, 6; and (4) solution 4.
The hazard of vinyl chloride was originally belived to primarily concern workers
employed in the conversion of VCM to PVC who may receive a particularly high
exposure of VCM in certain operations (e.g., cleaning of polymerization kettles) or
131
-------
a long-term exposure to relatively low concentrations in air of VCM at different
factory sites. Much larger populations are now believed to be potentially at risk
including: (1) producers of VCM, (2) people living in close proximity to VCM- or
PVC producing industries, (3) users of VCM as propellant in aerosol sprays; (4)
persons in contact with resins made from VCM; (5) consumers of food and beverage
products containing leachable amounts of unreacted VCM from PVC packaged materials
and (6) ingestion of water containing utireacted VCM leached from PVC pipes.
Gaseous vinyl chloride is emitted at both vinyl chloride and PVC resin plants
and is distributed into the atmosphere surrounding the emissions source in patterns
that depend on the amount of vinyl chloride released, the nature of the plant area
from which it is released and the meterological conditions’. It is estimated that the
total vinyl chloride escaping to the atmosphere in the United States exceeds 100 million
1 .
kg per year . Vinyl chloride loss from the average VC plant is estimated to be about
0.45 kg/l00 kg of VCM produced, and from the average PVC plant, approximately
4 kg/l00 kg of PVC produced’. Based on limited data, ambient concentrations of vinyl
chloride exceeded 1 ppm (2560 .Lg/m 3 ) less than 10% of the time in residential areas
located in the vicinity of plants producing VC or PVC. The maximum concentration
of vinyl chloride found in ambient air was 33.0 ppm (84,480 .Lg/m 3 ) at a distance of
0.5 km from the center of the plant 1 .
The concentration of residual VCM monomer in PVC powder that is fabricated into
final products is also an important determinant of VCM in the ppm range. The entrapped
concentration is dependent upon the production process and can range from 0.1 to 5.8
thousand ppm, which can be liberated during fabrication, particularly when heated’.
PVC leaving certain plants may contain 200-400 ppm VCM, on delivery to the customer,
132
-------
the level of VCM is about 250 ppm, and after processing, levels of 0.5-20 ppm are
reached, depending on the method of fabrication.
Early occupational exposure studies have revealed a wide range of VCM concen-
trations dependent on the manufacturing processes involved 11 . The air concentration
of VCM in a polymerization reaction prior to ventilation has been reported 11 to be
7800 mg/rn 3 and range from 1560-2600 mg/rn 3 in a polymerization reactor after washing 12 .
Concentrations of VCM in the working atmospheres in some plants producing PVC
have been reported in the ranges of 50-800 mg/rn 3 (20-312 ppm) 13 and 100-800 mg/m 3
(40—312 ppm) with peaks up to 87,300 mg/rn 3 (34,000 ppm) 13 .
Vinyl chloride has been found in municipal water supplies in the United States” 4
in representative samples of the nation s community drinking water supplies that
chlorinate their water and represent a wide variety of raw water sources, treatment
techniques and geographical locations.
The sources of the vinyl chloride found in the Miami and Philadelphia water
supplies (5.6 and 0.27 .tg/1 respectively) have not been identified 1 .
Available results indicate that migration of vinyl chloride from rigid PVC water
pipes does occur, and that it is a linear function of the residual vinyl chloride level
in the pipe itself 1 . Only limited data are available on vinyl chloride emissions from
the incineration of plastics. The quantities of vinyl chloride and combustion products
1
varied as a function of temperature as well as with the type of plastics and their polymers
It is believed that vinyl chloride should disappear significantly in its transport
over long distances, however, in the immediate vicinity of emission sources, vinyl
chloride can be considered a stable pollutant 1 . While no mechanism is presently known
for the removal of vinyl chloride from the air at night, biological sinks such as
microbial removal in soil may be of significance in depletion of vinyl chloride over
133
-------
long time periods. However, such sinks would not be expected to be important in
terms of urban scale transport of vinyl chloride’.
Vinyl chloride has been shown to produce tumors of different types, (especially
angiosarcomas of the liver) as well as lung adenomas, brain neuroblastonia
lymphomas in mice, rats and l amsters 15 20 Extensive world wide epidemiological
studies to date have indicated about 50 cases of angiosarcomas of the liver associated
with VCM exposure among workers employed in the manufacture of PVC resins 10 ’ 2 24 .
24 . .
Infante et al cited a significant excess of mortality from cancer of the lung and brain
in addition to cancer of the, among workers occupationally exposed to VCM.
The risk of dying from cancer of the lymphatic and hematopoietic system also appears
to increase with an increase in latency. A study of cancer mortality among populations
residing proximate to VCM polymerization facilities also demonstrated an increased
risk of dying from CNS and lymphatic cancer 24 . However, it was noted by Infante
et .i24 that although these findings raise cause for concern about out-plant emissions
of VCM, without further study these cancers cannot be unequivocally interpreted as
being related to out-plant exposure to VCM 24 .
Vinyl chloride has been shown to produce chromosome breaks in exposed wor-
kers 2528 . The mode of mutagenic action of VCM in special tester strains of SalmoneUa
23, 29—32
typhimurium appears to -be multifaceted. For example, it could be mutagenic
3l, or could be active via its microsomal metabolites such as chioroethylene
oxide and 2-chloroacetaldehyde 23 ’ 29 ’ produced in the presence of liver microsomes
from mice 23 ’ 29 , rats 23 ’ 29 ’ 3 ° and humans 23 ’ 29 . It was also reported that the stiinu-
latory effect of hepatic extracts is not due to microsomal activation but rather it is
due to a nonenzymatic reaction and that mutagenic activity of VCM in Salmonella might
involve a free radical mechanism 33 . VCM is mutagenic in Drosophila melanogaster 34 ,
134
-------
and produced gene mutations in S. pombe (forward mutations) and gene conversions
in two loci of a diploid S. cerevisiae in the presence of liver xnicrosomes 35 and in
the host-mediated assay when mice were treated with an oral dose of 700 mg/kg 35 .
In the absence of metabolic activation, vinyl chloride was not mutagenic or recombino-
genetic in S. cerevisiae strains D5 and XV185-14C 36 . VCM (in gaseous form or in
ethanol solution) was also non-mutagenic in two strains of Neurospora crassa 37 and
not xnutagenic in male CD-i mice at inhalation levels of 3,000, 10,000, and 30,000 ppm
for 6 hours/day for 5 days as measured by the dominant lethal test 38 .
Elmore et a1 39 screened without exogenous activation seven potential metabolites
of vinyl chloride in their pure forms as well as the related epichlorohydrin in tester
strains of Bacillus and Salmonella . Chlorooxirane (chloroethylene oxide), chioro-
ac etaldehyde, c hioroac etaldehyde monomer hydrate, chloroac etaldehyde dimer hydrate,
chioroacetaldehyde timer and epichiorohydrin produced significant mutagenic activity
in S. typhimurium strains sensitive to base-pair mutation. A recombination repair
deficient strain of B. subtilis was inhibited in growth by these compounds, whereas
excision repair deficient and wild type strains of B. subtilis were relatively unaffected.
Table 1 illustrates the bacteria tester strains used and Table 2 is a summary of mutagen
activity in the above microbial systems.
Vinyl chloride at a concentration of 0. 0106 M (723 ppm) in nutrient broth was
negative in both the Salmonella and Bacillus cultures. (High concentrations of VCM
(20% V/V in air-200, 000 ppm) produced rnutagenic action in previous assays with
Salmonella tester strains) ‘
Chioroethanol and chloroacetic acid which probably are metabolic intermediates
as shown in Scheme 1 were non-mutageniC at 1 mM concentrations in the above muta-
genicity assays of Elmore et al 39 . Scheme 2 illustrates the metabolic pathways of vinyl
135
-------
chloride as proposed by Hefner et a1 40 . Among the compounds tested by Elmore et
a1 39 in this scheme, chioroacetaldehyde and chiorooxirane (compounds 5 and 4
respectively-Table 2) were the most mutagenic with the lowest toxic side effects.
Hence, it was suggested that they may be the active carcinogenic derivatives of vinyl
chloride 39 . However, chioroacetaldehyde monomer hydrate (compound 6-Table 2)
was considered a more realistic choice as the ultimate carcinogen than the monomer
compound 5 which reacts immediately with water. The lower mutagenic activity of
chlorooxirane (compound 4) compared to chioroacetaldehyde monomer hydrate may
reflect the unstable nature of chlorooxirane as an X-chloroether 30 . One mode of
action of chiorooxirane is a rearrangement to chloroaceta]dehyde 41 via the NIH shift 42 .
Another is a homolytic ring cleavage to yield a stabilized diradical intermediate
C1CH—CH 2 O with both being capable of reacting with DNA to account for the muta-
genicity of chiorooxirane (compound 4)30•
Bartsch and Montesano 8 proposed a possible biotransformation of VCM in rats
and an alternative biotransformation of VCM involving mixed function oxidase
(Schemes 3 and 4). Products obtained by reaction of 2-chioroacetaldehyde with
adenosine or cytidene are illustrated in Scheme 5. Base alterations of this type
(Scheme 5) in the bacterial DNA may explain the activity of 2-chioroacetaldehyde as
a bacterial rnutagen since this compound induced base—pair substitutions in S.
8
typhunurium TA 1530
136
-------
ABLE I
BACTERIA TESTER STRAINS 39
A) Sulmoneila lyphimurium LT-2 tester strains
\Il tester strains contain uvrB repair mutations hjch eliminat.e the excision repair system; mutations in
hc histidin operon; and rfa mutations which alter the cell wait by increasing permcab lity and elimi-
• . ting pathogenicity. The resistance tr nss- factor. “R” factor, enhances the error pro;i.’ recombination
. .pair system thus making the stratns more susceptible to mutation (151. The strains ausceptible to base-
nz .ir substitution contaigl mutations in the histjdjne G46 operon and those susceptible to frai seshift muta-
!on contain mutations in the histidivie operon C 3076 (TA 1537) or D3052 (TA 1538. TA 98).
ttrains
R” factor
? Iutation detected
TA 1535
—
base-pair substitution
TA 100
+
base-pair substitution
TA 1537
—
frameshitt
EA 1538
—
frameshift
TA 98
+
frameshift
(3) J3ø I1lzas subiilis tester strains
Tr ’ denotes a requirement for tryptophan; Mit-S denotes sensitivity to mitomycin C. hcr denotes a
host-cell reactivation DNA repair capacity; hcr ischs a host-cell reactivation DNA repair capacity; rcc de-
notes a recombination DNA repair capaciLy: rcc 1acl s a recombination DNA repair capacity; uur is sen-
.itjse to ultraviolet-induced DNA damage.
Strains
Phenotype
DNA repair
68 M
Prototroph (wild type)
hcr . rcc
Ftcx-9
Trp
hcr. rr.c
rB-13
Trp
uur , rec
MC-1
Trp, Mit-S
hcr , rec
TABLE.2 3
SUMMARY OF MUTAGEN ACTIVITY IN MICROBIAL SYSTEMS
NI. no inhibition of growth detected in B. sub(iUs MC-1; NR, no increase of revertantS inS. typhiriuriurs
TA 100 compared to control; +. active; ++. very active. AcetaldehYde. a potential metabols e of corn-
pound 1. and allyl chloride, the parent olefin of compound 9, were nagative in these two systems.
Compounds tested
B. subtil s
Repair assay
S. typhirnuriurn
Reversion assay
I H 2 C CHCl
NI
NR
2 CIH 2 C-CfI 2 OH
NI
NR
3 CIH 2 C-COOH
NI
NR
4 ciHç -d11 2 -Q
+
5 Clll C.CItO
++
6 CIH 2 C-CH(O}i)2
++
++
7 Clll 2 C.CHOit.O -CHOH-Cti Cl
S (Cl1I 2 C- HO-)3
++
++
++
+
9 CIH;C-CFI-Ci1:9
NI
++
Control: 4.nitcoquirsoline-NOXide
++
137
-------
CICH 2 —CH 2 OH (2) ce Jar —O C—CH(NH 3 )— H 2
SH
CH 2 = CHCI (1) C1CH 2 —C0 2 H H CH 2 H 2 —S
, cell Jar
( ) S(CH 2 —CO ,H) 2
Scn me 1. Vinyl thiorlde metabolites,
SCHEI%IE I
. C1—CH=CH 2 (1) C1—CH 2 —CH 2 —CH ( 2 )i u ne
talcohol (5)
C ICH 2 —CHO
dehydrogenaaa
C1—CH 2 —CHO (5)- ’ C1—CH 2 —CO 2 H (3) SCHEMEI
urine
11202
II. C1CH 2 CH OH (2) C1CH CH 2 OQH CICH 2 CHO (5)
catalase
IlL CI—CH=CH (1) oxidase C1—CH—-CH 2 O (4) C1—CH 2 —CHO (5)
0
Sebem, II. The metabolic pathways of vinyl chloride
138
-------
SCHEME 3
• • • 8
A possible biotransformation of VCM in rats
alcohol
C1HC = CH 2 - C1H 2 C-CH 2 OH ) C1H 2 C-CHO - C1H 2 C-COOH
dehydrogenase
I UI IV V
SCHEME 4
An alternative biotransformation of VCM, involving microsomal mixed-function
oxidase 8 .
- C1H 2 C-CHO - C1H 2 C-COOH
IV V
Products obtained by reaction of 2-chioracetaldehyde with adenosine (VI) or
cytidine (VU) 8 .
a
E.R. 2
I
r water soluble I
su1ph00njug te5J *.
SCHEME 5
HC1
HOOH
(VI)
HOOH
(VII)
139
-------
2. Vinylidene Chloride
Vinylidene chloride (1, 1-dichioroethylene, CH 2 =CC1 2 ) (DCE) is used prin-
cipally as an intermediate for the copolyrnerization with other monomers such as
acrylonitrile, vinyl chloride, styrene, vinyl acetate, etc. These copolymers (in latex,
fiber, film and resin forms) are referred to as “Saran” and have wide utility mainly
for film wraps for food. Saran production is estimated at about 150 million pounds
43
per year
Specific impurities in vinylidene chloride monomer depend upon its method
of manufacture and isolation. A typical commercial grade of vinylidene chloride,
99.7% by weight as prepared by dehydrochiorination of 1,1, 2-trichioroethane with
lime or caustic 44 contains the foilowing impurities (in ppm): vinyl chloride, 850;
cis-i, 2-dichloroethylene, 500; trans-i, 2-dichloroethylene, 1500; 1, 1-dichioroethane,
10; ethylene dichioride, 10; 1,1, 1—trichloroethane, 150, and ii, 2-trichloroethylene,
10. Phenol at levels of 0.6-0.8% or 200 ppm of monomethyl ether or hydroquinone
(MEHG) are added to prevent polymerization during shipment and storage. A typical
analysis of vinylidene chloride monomer (unstabilized) largely produced in the United
States 45 includes as impurities (in ppm): vinyl chloride, 28; vinyl bromide; trans-
1,2-dichloroethylene, 1000; and cis-i,2-dichloroethylene, 410. The annual produc-
tion of vinylidene chloride in the U . S. is about 60 million lbs/year.
Vinylidene chloride is not known to occur in nature. However, it could occur as
a decomposition product of 1,1, i-trichloroethane 46 . Rigorous quantitative data is
lacking as to its occurrence in occupational exposure environments as well as in air,
water and food. Vinylidene chloride has been reported to be a trace impurity in vinyl
chloride monomer 47 . Workers involved in manufacturing facilities using VCM
monomer in polymerization processes (e.g., PVC) can be exposed to vinylidene chloride
47,48
concentrations in amounts of less than 5 ppm and more frequently to trace amounts
140
-------
The number of workers engaged in the production of vinylidene chloride monomer
per se in the United States (compared to vinyl chloride monomer) appears to be small
e.g. 75 and 12-15 at two major vinylidene chloride production facilities 50 . Estimates
of the number of workers engaged in the preparation of polymers and co-polymers
of vinylidene chloride and vinyl chloride (e.g., the preparation of Saran wrap) are
not available, nor are data available at present on workers exposed to only vinylidene
chloride during their working lifetimes.
Although the widespread use of vinylidene polymers as food wraps could result
in the release of unreacted monomer into the food chain 43 ’ 51, and vinylidene chloride
copolymers containing a minimum of 85% vinylidene chloride have been approved for
use with irradiated foods 52 , information is scant as to the migration of unreacted
monomers from these sources either into food or via disposal of the polymeric material
a . se. One report states that no more than 10 ppm of unreacted vinylidene chloride
is contained in Dow’s product Saran Wrap and that within detectable limits, no more
53
than 10 ppb could get into food, even under severe conditions of use
Aspects of the reported carcinogenicity of vinylidene chloride appear conflicting
and indicate sex, species, and strain specificity. Maltoai 54 reported that Swiss male
mice exposed to 25 ppm of vinylidene chloride in air for 4 hours daily, 4-5 weeks,
for 52 weeks, developed adenocarcinoma of the kidney no effects were found in Balb/C;
C56B1 or C 3 H mice or Sprague-Dawley rats and hamsters similarly exposed to vinylidene
chloride 54 . However, ViolaDS reported that male and female Wistar rats exposed to 100
ppm of vinylidene chloride by inhalation developed abdominal lymphomas and sub-
cutaneous fibromas.
141
-------
Two year studies at Dow Chemical Co., involving both vinylidene chloride
administered in the drinking water (60, 100 and 120 ppm) and repeated inhalation
(10 or 40 ppm 6 hours/day; 5 days week; after 5 weeks, 75 ppm for up to 18 months)
to male and female Sprague-Dawley rats have been carried out 56 ’ and indicated
no dose-related clinical differences or cumulative mortality differences or findings
of neoplasia. Reproduction studies with vinylidene chloride administered to Sprague
Dawley rats by inhalation or ingestion in the drinking water showed the compound to
be neither a teratogen or mutagen or one adversely affecting reproductivity 57 . The
vinylidene chloride (99.5%) tested in the Dow studies contained trace amounts (ppm)
of the following impurities: vinyl bromide, 4; vinyl chloride, 3-50; trans-i, 2-dich-
loroethylene, 138-1300; cis-1,2-dichloroethylene, 0.013-0.16%; 1,1, l-trichloroethane,
0.03; and 1 1,2-trichloroethane 56 .
Winston et a1 58 and Lee 59 reported the only tumor in rats exposed to 55 ppm
vinylidene chloride for 9 months was a subcutaneous hemangiosarcoma of the skin
in one of the 9 rats. Exposure of mice to 55 ppm of vinylidene chloride for up to 9
months resulted in the development of one hepatic hemangiosarcoma. Bronchiolar
adenoma and/or acinar proliferation in the lung also occurred in 5 of 42 mice.
Vinylidene chloride induces point mutation in the histidine-auxotroph strains of
Salmonella tyhpimurium TA 1530 and TA 100 when tested in the presence of rat- or
mouse liver in vitro 60 (its mutagenic activity was higher than that of VCM). The
mutagenic response, which was greater in TA 100 strain increased in both strains
after exposure of 2% vinylidene chloride in air. The lower mutagenic response
observed with a concentration of 20% vinylidene chloride may have resulted from an
inhibitory action of vinylidene chloride and or its metabolite(s) on the microsomal
enzymes responsible for its metabolic activation. It was postulated by Bartsch et a1 6 °
that 1, 1-dichioroethylene oxide (in analogy with chioroethylene oxide the
142
-------
suggested primary metabolite of vinyl chloride 8 ) may be a primary reactive meta-
bolite of vinylidene chloride. It is also considered possible that partial dechlorination
of vinylidene chloride by microsomal enzymes results in vinyl chloride and its meta-
bolic products 60 .
Vinylidene chloride has also been found to be mutagenic when tested in a meta-
61
bolizing in vitro system with E. coli K12 . In contrast to the results in S. typhi-
murium 60 , the mutagenicity of VCM was several times higher than that of vinylidene
chloride when tested in E. coil (back-mutation system, arg ) 61 .
No chromosomal aberrations have been found in Sprague-Dawley rats exposed
to 75 ppm vinyliderie chloride 6 hrs/day, 5 days/week for 26 weeks
Hathaway 62 recently proposed the in vivo metabolism of vinylidene chloride in
rats to proceed as shown in scheme 6. Of the compounds shown, chioroacetic acid,
thiodiglycollic acid, thioglycollic acid, dithioglycollic acid and lactam compounds
have been isolated.
143
-------
SCHEME 6 Proposed mammalian metabolism of vinylidene chloride 63 .
çi
H 2 C = Cd 2 - H 2 S-. d1 - C1CH 2 ç-Cl - C1CH 2 COOH
/‘GSH 0 + GSH
ri-cH-cH 2 scH 2 cooH I
LONH
HOOCCHCH SCH COOH
• 2 2
I
‘ hf ‘1
HOOC-çHCH 2 SCH 2 COOH
S(CH 2 COOH) 2
*
HSCH 2 COOH
*
[ SCH 2 COOHZ]
I
‘H
0
AC
144
-------
3. Trichioroethylene
Trichioroethylene (1-chloro-2, 2 -dichloroethylene; 1, 1. 2-trichloroethylene
C1CHCC1 2 ) is prepared primarily by the chlorination and dehydrochioririation of
ethylene dichloride 63 . Approximately 90% is used in the U . S. for vapor degreasing
of fabricated metal parts and 6% is used as a chain terminator for polyvinyl chloride
production 63 . Additional areas of utility include: as an extract in food processing
Ce. g., for decaffeinated coffee), as a chemical intermediate; as a solvent in the textile
industry and research laboratories; as an ingredient in printing inks, lacquers,
varnishes and adhesives, and in the dry—cleaning of fabrics. A pharmaceutical grade
of trichloroethylene is used as a general anesthetic.
The number of workers exposed to trichioroethylene has been estimated to be
approximately 283 thousand 64 .
Because of its implications in smog production in the U.S. and resultant legis-
lation restricting its use, it is expected that during the next 5 years consumption of
trichloroethylene for metal cleaning will decline at an average rate of 3% and be most
probably replaced by 1, 1, 1-trichloroethane and perchloroethylene 65 .
Emissions of commercial organic solvent vapors into the atmosphere have been
increasing dramatically in the last decade 46 . The loss of trichloroethylene and per-
chloroethylene to the global environment in 1973 was estimated to be each over 1
46
million tons
Concentrations of trichloroethylene vapor in degreasing units have been reported
to range from 20-500 ppm (at head height above the bath) with the highest levels being
over the baths which relied on manual removal of articles 66 , and between 150 and 250
ppm in degreasing rooms 2 se 67 . Levels of 1076-43,000 mg/rn 3 (200-8000 ppm) of
trichioroethylene have been reported in a small U.S. factory 68 .
145
-------
Concentrations of anesthetics (including trichioroethylene) in operating rooms
to which surgeons and nurses were exposed varied from 0.3 to 103 ppm 69 . It is
estimated that about 5000 medical, dental, and hospital personnel are routinely ex-
posed to tri.chloroethylene 64 .
Trichloroethylene has been detected in air samples at 8 locations in 5 U . S. states
in 1974, with typical concentrations ranging from 0. 18 ppb in urban areas to less
than 0.02 ppb in rural areas 70 , and has been more recently detected in air over 3
New Jersey industrial cities 71 .
Slightly enhanced levels of trichioroethylene following chlorination of water at
72,73
sewage treatment plants have been found in the U . S. . Trichioroethylene has
been found in the organic constituents of Mississippi River water (before and after
treatment) and in the organic constituents of commercial deionized charcoal filtered
water 74 . Trichioroethylene concentrations of 54 kg/day of 1.2 ng/l in average raw
wastewater flow have resulted from a decaffeination process used in the manufacture
of soluble (instant) coffee in California 75 .
The National Cancer Institute (NCI) in the U.S. has recently issued a “state of
concern” alert, warning producers, users, and regulatory agencies that trichloro-
ethylene administered by gastric intubation to B6C3F mice induced predominantly
hepatocellular carcinomas with some metastases to the , e. g.. 30 of 98 (30. 6%)
of the mice given the low dose (1200 mg/kg and 900 mg/kg for male and female
respectively) and 41 or 95 (43.2%) of the mice given the higher dose (2400 mg/kg
and 1800 mg/kg for male and female respectively). Only one of 40 (2. 5%) control
64,76
mice developed these carcinomas
146
-------
No hepatocellular carcinomas were observed in both sexes of Osborne-Mendel
rats administered trichioroethylene at levels of 1 .0 or 0. 5 g/kg by gastric intubation
5 times weekly for an unspecified period 64 .
No liver lesions or hepatomas were found in NLC mice given oral doses by gavage
of 0 1 ml of a 40% solution of trichloroethylene in oil twice weekly for an unspecified
77
period
Trichloroethylene (3.3 mM) in the presence of a metabolic activating rnicrosomal
system induced reverse mutations in E. coli strain K12 61 . It has also been shown
to induce frameshift as well as base substitution mutation in S. cerevisiae strain
78
XV185—14C in the presence of mice liver homogenate
Trichioroethylene is metabolized in rats to trichloroacetic acid and trichloro-
ethanol which are proposed to have been derived from a primary metabolite, trich-
loroethylene oxide 79 81 Trichioroethylene is also metabolized to trichloroethanol and
tz-ichloroacetic acid in dogs 82 .
Epoxides are now recognized as obligatory intermediates in the metabolism of
olefins by hepatic microsomal mixed-function oxidases 43 ’ 83 . The formation of meta-
bolites such as trichioroethanol and trichloroacetic acid implies rearrangement of the
ansient trichioroethylene oxide intermediate into chioral. This has been confirmed
in studies involving: (1) the rearrangement of the oxides belonging to a series of
chlorinated ethylenes 84 , (2) and the identification of chloral as a trichioroethylene
85 86
metabolite in vitro and in vivo . Chloralhydrate has been shown to be mutagenic
87
in Antirrhinum
147
-------
4. Tetrachioroethylene
Tetrachloroethylene (perchioroethylene; C1 2 C=CC1 2 ) is prepared primarily via
two processes: (1) the Huels method whereby direct chlorination of ethylene yields
70% perchioroethylene, 20% carbon tetrachioride and 10% other chlorinated products
and (2) hydrocarbons such as methane, ethane or propane are simultaneously chlori-
nated and pyrolyzed to yield over 95% perchioroethylene plus CC1 4 and HC1.
The world-wide production of perchioroethylene in 1972 was 680 million kg; its
growth rate estimated at 7%/year with a total world production estimated at 1100
million kg for 1980. The consumption pattern for perchioroethylene in the United
States in 1974 is estimated to have been as follows: textile and dry cleaning industries,
69%; metal cleaning, 16%; chemical intermediate Ce. g. , preparation of trichioroacetic
acid in some fluorocarbons), 12%; and miscellaneous uses, 3%. Perchioroethylene
is used as a solvent in the manufacture of rubber solutions, paint removers, priiting
inks, and solvent soaps, as a solvent for fats, oils, silicones and sulfur and as a
heat-transfer medium 89 .
The number of workers engaged in the production and applications of perchioro-
ethylene is not known at present nor are data available as to the levels of occupational
exposure.
Depending on its source strength, meteorological dilution, sunlight intensity, and
the presence of other trace constituents, perchioroethylene or its predominant product,
70
phosgene, accordingly may or may not be observed in non-urban areas . It was
estimated that an ambient concentration of 10 ppb perchioroethylene observed in New
York City should lead to the formation of 12 ppb phosgene 70 (TLV100 ppb). In 1974,
at 8 locations in 5 industrial states, the concentrations of perchioroethylene ranged
from 1 .2 ppb in the urban areas to less than 0. 02 ppb in rural areas. Perchioroethylene
148
-------
was measured at concentrations exceeding 0. 06 ppb at least 50% of the time at all
locations 70 .
Chlorination at sewage treatment plants has resulted in slightly enhanced levels
of perchloroethylene in water 72 ’ 73• Similarly to trichioroethylene, perchioroethylene
(5 g/l) has been found in the organic constituents of Mississippi River water and in
the organic constituents of commercial deionized charcoal-filtered water 74 .
Perchioroethylene has very recently reported to be carcinogenic in NCI studies,
producing liver hepatocellular tumors in B 6 C 3 F 1 hybrid male and female mice when
tested at MTD and MTD dose levels in corn oil solution by gavage 90 ’ 91• No carcino-
genic activity was observed in analogously treated Osborne-Mendel rats of both sexes 90 .
Perchloroethylene has not been found to be carcinogenic in inhalation studies
with rabbits, mice 92 , rats, guinea pigs and monkeys.
Perchioroethylene, as well as the cis- and trans-isoniers of l,2-dichloroethylene
were found to be non-mutagenic when tested in the metabolizing in vitro system with
E. coli K12 61 . The mutagenicity of vinyl chloride, vinylidene chloride, trichioroethy-
lene, in the above test system was attributed to their initially forming unstable oxiranes,
whereas halocarbons such as perchloro ethylene and cis- and trans - 1, 2-dichloro-
ethylene which form much more stable oxiranes were non-mutagenic 61 ’ 93 .
Figure 7 illustrates the metabolic pathways of tetrachloroethylene as proposed
by Bonse and Henschler 93 . The metabolic formation of trichioroacetic acid can be
explained by the primary formation of the oxirane and subsequent rearrangement to
trichioroacetyl chloride and its subsequent hydrolysis.
149
-------
Figure 7. Metabolism of Tetrachioroethylene (Perchloroethylene) 93 .
ci a ci c i
/ ___ \/ / ___ 0 ___
c=c
/ N CC CCI,C — CCI,—C
/ \ N N
Ci C i C I C l CL
H o
/ thermal
C=C C — C CHCL, —C
/ N / N N
Cl
C I Ci Cl Ci
metabolic
‘9
Ccl, —C
N
/
CCI , — CH ,OH CC !, —c
OH
150
-------
5. Chloroprene
Chloroprene (2-c hiorobutadiene; 2-chioro- 1, 3 -butadiene; CH 2 -CH=CH 2 )
is the monomer for neoprene, the specialty rubber. It is prepared by two major
routes: (1) the addition of hydrogen chloride to vinyl acetylene and (2) the chlori-
nation of butadiene to a mixture of dichiorobutenes; from which 3, 4-dichloro-1-
butene is isolated and then is subj ected to dehydrochlorination 94 . The latter method
is believed to be the basis of the current U . S. production of chioroprene. A typical
specification for chloroprene made from butadiene is as follows: chioroprene, 98.5%
mm., 1-chiorobutadiene, 1 .0% max., aldehydes (as acetaldehydes), 0.2% max.,
3,4-dichlorobutene-1, 0.01% max., dimers, 0.01% max., peroxides, 1 ppm max.,
and rio detectable amount of vinyl acetylene 95 .
Chioroprene is extremely reactive, e.g., it can polymerize spontaneously at
room temperatures, the process being catalyzed by light, peroxides and other free
radical initiators 96• It can also react with oxygen to form polymeric peroxides and
because of its instability, flammability and toxicity, chioroprene has no end product
96 96
uses . An estimated 2, 500 workers are exposed to chioroprene in the United States
Neoprene is obtained by emulsion polymerization of chloroprene consists mainly
of transpolychioroprene. There are two main classes the sulfur modified type and the
96,97
non-sulfur modified type, indicating the differences in polymerization techniques
The main areas of utility of neoprene are in automative applications, cable sheaths,
hoses, adhesives, fabrics and a large number of technical rubber articles 96 .
Chioroprene which has been used since 1930 in the manufacture of synthetic rubber,
has recently been suggested to be responsible for an increased incidence of skin and
lung cancer in exposed workers in the USSR 98 ’ 99 . During the period 1956-70,
151
-------
epidemiological studies of industrial workers in the Yerevan region revealed 137
cases of skin cancer among 24,989 persons over 25 years of age. Three precent of
the workers exposed to chioroprene and 1.6% of those working in industries using
chioroprene derivatives developed skin cancer, compared to only 0.4% for persons
working in non-chemical industries. The chioroprene workers who developed skin
cancer had an average age of 59.6 years and an average duration of employment of
98
9.5 years
During the same period, 87 cases of lung cancer were identified among 19, 979
workers in the same region. The group exposed to chloroprene or its derivatives
had the highest incidence of lung cancer (1.16%). These workers’ average age was
44.5 years with an average duration of employment of 8 .7 years. Of the 34 cases of
lung cancer in this group, 18 were among persons having a direct and prolonged
exposure to chioroprene monomer, the remaining 16 involved individuals exposed
to chloroprene latexes 99 . High rates of skin and lung cancer have also recently been
reported among workers in a U.S. plant who had handled chioroprene and its
derivatives 100 .
Chromosome aberi ations in lymphocytes from peripheral blood of workers exposed
101
to chioroprene has been reported . A significant rise in the number of chromosome
aberrations in blood cultures of those exposed to an average chioroprene concentration
of 18 pprxk for 2 to more than 10 years was noted 101 .
Exposure of S. typhimuriuni TA 100 strain to 0.5-8% of chioroprene vapor in air
in the absence of any metabolic activation system caused a linear increasing mutagenic
response, (reaching 3 times the spontaneous mutation rate at a concentration of 8%)60.
Exposure to a higher concentration (2 0%) caused a strong toxicity in the bacteria.
152
-------
This mutagenic and/or toxic effect could be caused by a direct action of chioroprene
or more likely, by one of its enzymic (bacteria) or non enzymic breakdown products
Up to a 3-fold increased mutagenic response was found when a fortified 9000 g liver
supernatant from either phenobarbitone-treated or untreated mice was added to such
assays supporting an enzymic formation of mutagenic metabolite(s) from chloroprene 6 °
(probably an oxirane (epoxide) in analogy with vinyl chloride, vinylidene chloride,
and trichioroethylene)
153
-------
Cl H
6. , 4—dichlorobutene (1, 4-dIchloro-2-butene; HC -C=C--C -H) is employed in
HHH l
the U . S. mainly as an intermediate in the manufacture of hexamethylenediamine and
chloroprene. Hexamethylenediamine is further used as a chemical intermediate in
the production of nylon 66 and 612 polyamide resins, while chloroprene is used in
the production of polychioroprene rubber. While the U .5. production of hexamethylene-
diamine and polychioroprene rubber in 1975 was 340 and i43 .9 million kilograms
respectively, the percentage originally derived from tr ,4-dichlorobutene is
102
not known
Trans-i, 4-dichiorobutene has been shown to be weakly carcinogenic by sub-
cutaneous and intraperitoneal administration in ICR/HA Swiss mice but no carcino-
genic in mice via skin application 103 .
Trans-i, 4-dichiorobutene produced mutations in S. typhimurium
TA 100 strains’ 04 with the mutagenic effect enhanced by liver microsomal fractions
from mouse or humans. It has also been reported rnutagenic in E. coli ’ 05 and S. cerevisiae 106
It has been suggested that trans-i,4-dichlorobutene-2 could conceivably be
metabolized to an epoxide intermediate which is analogous in structure to open-chain
p-ch1oroethers 03 .
154
-------
References for Halogenated Hydrocarbons and Related Derivatives
1. EPA, Scientific and technical assessment report on vinyl chloride and polyvinyl
chloride, Office of Research and Development, Washington, D.C., June, 1975
2. IARC, Some anti-thyroid and related substances, nitrofurans and industrial che-
micals, Monograph No. 7, International Agency for Research on Cancer, Lyon, 1974
3. Sakarriis, V. F., 1, 2-Dichioroethane-induced chromosome non-divergence of the
X-chromosome and recessive sex-linked lethal mutation in Drosophila melanogaster
Genetika , 5 (1969) 89-95
4. Rapoport, I. A., The reaction of genic proteins with 1,2-dichloroethane, Akad.
Nauk. SSR Doki. Biol. Sci. , 134 (1960) 745-747
5. McCann, J., pingarn, N. E., Kobori, J., and Ames, B. N., Carcinogens a
mutagens: Bacterial tester strains with R factor plasmids, Proc. Natl. Acad.
Sci. , 72(1975) 979—983
6. McCann, J., Simon, V., Streitweiser, D., and Ames, B. N., Mutagenicity of
chloroacetaldehyde, a possible metabolic product of 1, 2-dichioroethane, chlorO-
ethanol, and cyclophosphamide, Proc. Nat!. Acad. Sci. , 72(1975) 3190-3193
7. Brem, H., Stein, A. B., and Rosenkranz, H. S., The mutagenicity and DNA-
modifying effect of haloalkanes, Cancer Res. , 34 (1974) 2576-2579
8. Bartsch, H., and Montesano, R., Mutagenic and carcinogenic effects of vinyl
chloride, Mutation Res. , 32(1975) 93-114
9. Levinson, C., Vinyl chloride: A case study of the new occupational health hazard,
ICF (Pub!.) Geneva, 1974
10. IARC , Report of a working group on vinyl chloride, Lyon, IARC Internal Tech-
nical Report No. 74/005, 1974
11. Cook, W. A., Giever, P. M., Dinman, B. D., and Magnuson, H. J., Occupational
acroosteolysis, II. An industrial hygiene study, Arch. Env. Hith. , 22 (1971) 74-82
12. Lange, C. E., Juhe, S., Stein, G., and Veltman, G., Die sogenannte Vinyl
Chloride krankheit, Eirie Berufsbeding Systemkierose? mt. Arch. Arbeits, Med .
32 (1974) 1—32
13. Filatova, V. S., and Gronsberg, E. S., Sanitary hygienic conditions of work in
the production of po1ych1orvin Tl tar and measures of improvement, Gig. Sanit .
22 (1957) 38—42
14. Environmental Protection Agency, Preliminary Assessment of Suspected Carcinogens
in Drinking Water, An Interim Report to Congress, Office of Toxic Substances, EPA
Washington, D.C., June (1975)
155
-------
15. Maltoni, C., The value of predictive experimental bioassays in occupational and
environmental carcinogenesis, Ambio , 4 (1975) 18
16. Maltoni, C., and Lefemine, G., Carcinogenicitybioassays of vinyl chloride, I.
Research plans and early results, Environ. Res. , 7 (1974) 387-405
17. Viola, P. L., Bigotti, A., and Caputo, A., Oncogenic response of rat skin, lungs,
and bones to vinyl chloride, Cancer Res. , 31 (1971) 516-519
18. Maltoni, C., La ricerca sperimentale ei suoa risultati, Sapere , 776 (1974) 42-45
19. Maltoni, C., and Lefemine, G., La potenzialita dei saggi sperimentali nella predi—
zione dei rischi oncogeni ainbientali. Un esempio: Ii cloruro di vinile, Rend. Sci.
Fis. Mat. Nat. , 66(1974) 1—11
20. Maltoni, C., and Lefemine, G., Carcinogenicity bioassays of vinyl chloride,
currentresults, Ann. NY Acad. Sci. , 246(1975) 195-218
21. Creech, J. L., and Johnson, M. N., Angiosarcoma of liver in the manufacture of
polyvinyl chloride, J. Occup. Med. , 16 (1974) 150
22. Holmberg, B., and Molina, M. J., The industrial toxicology of vinyl chloride:
A review, Work Environ. Health , 11 (1974) 138-144
23. Bartsch, H., and Montesano, R., Mutagenic and carcinogenic effects of vinyl
chloride, Mutation Res. , 32 (1975) 93—114
24. Infante, P. F., Wagoner, J. K., and Waxweiler, R. J., Carcinogenic, mutagenic
and teratogenic risks associated with vinyl chloride, Mutation Res. , 41 (1976)
131—142
5. Purchase, I. F. H., Richardson, C. R., and Anderson, D., Chromosornal and
dominant lethal effects of vinyl chloride, Lancet , 11 (1976) 410-411
26. Hansteen, I. L., Hillestad, L., and Thiis-Evensei , E., Chromosome studies on
workers exposedto vinyl chloride, Mutation Res. , 38 (1976) 112
27. Funes-Cravioto, F., Lambert, B., et al., Chromosome aberrations in workers
exposed to vinyl chloride, Lancet , 1 (1975) 459
28. Ducatnan, V., Hirschhorn, K., and Selikoff, I. J., Vinyl chloride exposure and
human chromosome aberrations, Mutation Res. , 31 (1975) 163-169
29. Bartsch, H., Malaveille, C., and Montesano, R. , Human, rat and mouse liver-
mediated mutagenicity of vinyl chloride in S. typhimurium strains, mt. 3. Cancer
15 (1975) 429—437
30. Rannug, U., Johansson, A., Ramel, C., and Wachtrneister, C. A., The mutagenicity
of vinyl chloride after metabolic activation, Ambio , 3 (1974) 194-197
156
-------
31. McCann, J., Simon, V., Streitswejser, D., and Ames, B. N., Mutagenicity of
chioroacetaldehyde, A possible metabolic product of 1,2-dichioroethane, chioro-
ethanol, and cyclophosphamide, Proc. Nail. Acad. Sci . 7?. (1975) 3190-3193
32. Malaveille, C., Bartsch, H., Barbiri, A., Camus, A. M., and Montesano, R.,
Mutagenicity of vinyl chloride, chioroethylene oxide, ch1 roacetaldehyde and
chioroethanol, Biochem. Biophys. Res. Comm. , 63 (1975) 363-370
33. Garro, A. J., Guttenpian, J. B., and Milvy, P., Vinyl chloride dependent muta-
genesis: Effects of liver extracts and free radicals, Mutation Res. , 38 (1976) 81-88
34. Magnusson, J., and Ramel, C., Mutagenic effects of vinyl chloride in Drosophila
melanogaster, Mutation Res. , 38 (1976) 115
35. Loprieno, N., Barale, R., Baroncelli, S., Bauer, C., Bronzetti, G., Cammellirii,
A., et al., Evaluation of the genetic effects induced by vinyl chloride monomer
(VCM) under mammalian metabolic activation: studies in vitro and in vovo,
Mutation Res. , 40(1976) 85—96
36. Shahin, M. M., The non-mutagenicity and recombinogenicity of vinyl chloride
in the absence of metabolic activation, Mutation Res. , 40 (1976) 269—272
37. Drozdowicz, B. Z., and Huang, P. C., Lack of mutagenicity of vinyl chloride in
two strains of Neurospora crassa, Mutation Res. , 48 (1977) 43-50
38. Anderson, D., Hodge, M. F. E., and Purchase, I. F. H., Vinyl chloride: Dominant
lethal studies in male CD-i mice, Mutation Res. , 40 (1976) 359—370
39. Elmore, J. D., Wong, J. L., Laumbach, A. D., and Streips, U. N., Vinyl chloride
mutagenicity via the metabolites chiorooxirane and chloroacetaldehyde monomer
hydrate, Biochim. Biophys. Acta. , 442 (1976) 405—419
40. Hefner, J. R. E., Watanabe, P. G., and Gehring, P. J., Preliminary studies on
the fate of inhaled vinyl chloride monomer in rats,
Ann. NY Acad. Sci. , 246(1975) 135—148
41. Walling, C., and Fredericks, P. S., Positive halogen Compounds, IV. Radical
reactions of chlorine and t.butyl hypochiorite with some small ring compounds,
3. Am. Chem. Soc. , 84 (1962) 3326—3331
42. Daly, J. W., Jermna, D. M., and Witkop, B., Arene oxides and the NIH shift.
The metabolism, toxicity and carcinogenicity of aromatic compounds, Experientia
28 (1972) 1129
43. Anon, Vinylidene chloride linked to cancer, Chem. Eng. News , Feb. 28 (1977) p. 6-7
44. Wessing, R., and Edwards, F. G., Poly(vinylidene chloride) in Mark, H. F.,
McKetta, 3. J., and Othmer, D. F. (eds) Encyclopedia of Chemical Technology,
2nd ed., Vol. 21, Interscience, New York, pp. 275—303
157
-------
45. Dow Chemical Co., Published references and literature review pertaining to
toxicological properties of vinylidene chloride monomer, 1976
46. McConnell, G., Ferguson, 0. M.., and Pearson, C. R., Chlorinated hydrocarbons
in the environment, Endeavor , 34 (1975) 13-27
47. Ott, M. G., Langner, R. R., and Holder, B. B., Vinyl chloride exposure ma
controlled industrial environment. Arch. Env. Hith. , 30 (1975) 333-339
48. Jaeger, R. J., Conolly, R. B., Reynolds, E. S., and Murphy, S. D., Biochemical
toxicology of unsaturated halogenated monomers, Env. Hith Persp. , 11 (1975) 121-123
49. Kramer, C. D., and Mutchier, J. E., The correlation of clinical and environmental
measurement for woz kers exposed to vinyl chloride, Am. md. Hyg. Assoc. J .
33 (1975) 19-30
50. Anon, Rap for film wrap, Chem. Week , Oct. 16 (1974) p. 20
51. Lehman, A. J., Chemicals in foods: a report to the Association of Food and Drug
Officials on Current Developments, Assoc. Food and Drug Officials, U. S. Quart.
Bull., 15 (1951) 82—89
52. Anon, Food additives, packaging material for use during irradiation of pre-packaged
foods, Fed. Register , 33 (1968) 4659
53. , Vinylidene chloride: No trace of cancer at DOW, Chem. Eng. News , March
14 (1977) pp. 21-22
54. Maltoni, C., Recent findings on the carcinogenicity of chlorinated olefins, NIEHS
Conference on Comparative Metabolism and Toxicity of Vinyl Chloride and Related
Compounds, Bethesda, MD May 2-4 (1977)
55. Viola, P. L., Carcinogenicity studies on vinylidene chloride (VDC), NIEHS
Conference on Comparative Metabolism and Toxicity of Vinyl Chloride and Related
Compounds, Bethesda, MD, May 2-4 (1977)
56. Raxnpy, L. W. ., Toxicity and carcinogenicity studies on vinylidene chloride, NIEHS
Conference on Comparative Metabolism and Toxicity of VInyl Chloride and Related
Compo iids , Bethesda, MD, May 2-4 (1977)
57. Norris, J. M., The MCA-Toxicology Program for Vinylidene Chloride, Presented
at 1977 European Tappi Meeting, Hamburg, Germany, Jan. 26 (1977)
58. Winston, J. M., Lee, C. C., Bhandari, J. C., Dixon, R. C., and Woods, 3. S.,
A study of the carcinogenicity of inhaled vinyl chloride and vinylidene chloride
(VDC) in rats and mice, Abstracts mt. Congress of Toxicology, Toronto, March 3-
April 2 (1977) p. 32
158
-------
59. Lee, C. C., Toxicity and carcinogenicity of vinyl chloride compared to vinyli-
dene chloride, NIEHS Conference on Comparative Metabolism arid Toxicity of Vinyl
Chloride and Related Compounds, Bethesda, MD, May 2-4 (1977)
60. Bartsch, H., Malaveille, C., Moritesano, R., and Tomatis, L., Tissue-mediated
mutagenicity of vinylidene chloride and 2-chiorobutadiene in Salmonella
typhimurium, Nature , 255 (1975) 641-643
61. Greim, H., Bonse, G., , Z., Reichert, D., and Henschler, ID., Mutagenicity
in vitro and potential carcinogenicity of chlorinated ethylenes as a function of
metabolic oxirane formation, Biochem. Pharmacol. , 24 (1975) 2013
62. Hathaway, ID. E., Comparative mammalian metabolism of vinylidene and vinyl
chloride in relation to outstanding oncogenic potential. NIEHS Conference on
Comparative Metabolism and Toxicity of Vinyl Chloride Related Compounds,
Bethesda, MD, May 2—4 (1977)
63. IARC, Trichioroethylene, In. Vol. 11, ‘Cadmium, Nickel, and Some Epoxides,
Miscellaneous Industrial Chemicals and General Considerations on Volatile
Anaesthetics”, International Agency for Research on Cancer, Lyon (1976) pp.
263—27 6
64. Lloyd, 3. W., Moore, R. M., and Breslin, P., Background information on trich-
loroethylene, 3. Occup. Med. , 17(1975)603-605
65. Anon, Trichioroethylene-report abstract, Chem. Econ. Newsletter (SRI Inst.)
Nov.-Dec. (1975)
66. Glass, W. I., A survey of trichloroethylene degreasing baths in Auckland,
Occup. Hlth. Bull. , December 1961
67. Takamatsu, M., Health hazards in workers exposed to trichioroethylene vapour,
Kumanoto Med. 3. , 15(1962) 4354
68. Kleinfeld, M., and Tabershaw, I., Trichloroethylene toxicity. Report of five
fatal cases, Arch. Ind. Hyg. , 10(1954) 134—141
69. Corbett, T. H., Retention of anesthetic agents following occupational exposure,
Anesth. Analg. Current Res. , 52(1973)614-618
70. Lillian, D., Sign, H. B., Appleby, A., Lobban, L. Arnts, R., Gumpert, R.,
et al, Atmospheric fates of halogeriated compounds, Env. Sci. Technol. , 9
(1975) 1042—1048
71. Anon, Carcinogenic and other suspect substances, Toxic Materials News , 3 (17)
(1976) 15
72. Environmental Protection Agency, Draft Report for Congress: Preliminary
Assessment of Suspected Carcinogens in Drinking Water, Office of Toxic Sub-
stances, Washington, D.C., October 17, 1975
159
-------
73. Bellar, T. A., Lichtenberg, J. J., and Kroner, R. C., The occurrence of
organohalides in chlorinated drinking waters, J. Am. Waterworks Assoc. ,
66 (1974) 703—706
74. Dowty, B. 3., Carlisle, D. R., and Laseter, J. L., New Orleans drinking water
sources tested by gas chromatography-mass spectrornetry, Env. Sci. Technol. ,
9 (1975) 762—765
75. Camisa, A. G., Analysis and characteristics of trichloroethylene wastes, J.
Water Pollut. Control Fed. , 47(1975) 1021—1031
76. National Institutes of Health, HEW News, June 14 (1976)
77. Rudali, G., A propos de 1 ’activit ’oncog ne de quelquea hydrocarbures halo-
gen s uti1is s en therapeutique, 111CC Monograph , 7 (1967) 138-143
78. Shahin, M. M., and Von Borstel, R. C., Mutagenic and lethal effects of a:—benzene
hexachioride, dibutyl phthalate and trichioroethylene in Saccharomyces Cerevisiae,
Mutation Res. , 48(1977) 173—180
79. Daniel, J. W., Metabolism of 36 C1-labelled trichloroethylene and tetrachioro-
ethylene in the rat, Biochem. Pharmacol. , 12 (1963) 795
80. Parke, D. V., In “The Biochemistry of Foreign Compounds” Vol. 5 (eds) Campbell,
P. N., Datta, S. D., and Engel, L. L., Pergamon Press, Oxford (1968)
81. Powell, J. F., Trichioroethylene: Absorption, elimination and metabolism,
Brit. J. md. Med. , 2(1945) 142—145
82. Butler, T. C., Metabolic transformation of trichloroethylene, 3. Pharmacol. Exp.
Ther. , 97(1949)84—92
83. Maynert, E. W., Foreman, R. L., and Watanabe, T., Epoxides as obligatory
intermediates in the metabolism of olefins to glycols, 3. Biol. Chem. , 245 (1970)
5234
84. Bonse, G., Urban, T., Reichert, D., and Henschler, D., Chemical reactivity,
metabolic oxirane formation and biological reactivity of chlorinated ethylenes in
the isolated perfused rat liver preparation, Biochein. Pharmacol. , 24 (1975)
18 29—1834
85. Byington, K. H., and Leibman, K. C., Metabolism of trichioroethylene by liver
microsomes. II. Identification of the reaction product as chioral hydrate, Molec.
Pharmacol. , 1(1965)247-254
86. Scansetti, G., Rubino, G. F., and Trompeo, G., Studio sully intossicazione
cronica da trielina II I. Metabolismo del trichioroetilene, Med. Lavoro. , 50 (1959)
743-7 54
160
-------
87. Barthelmess, A., Mutagene arzneimittel, Arzneimittelforsch. , 6 (1956) 157-168
88. Anon, Chem. Marketing Reptr., May 8 (1972) pp. 3, 47
89. Hardie, ID. W. F., Chlorocarbons and chlorohydrocarbons, tetrachioroethylene,
In R. E. Kirk and D.F. Othmer (eds) Encyclopedia of Chemical Technology, 2nd
ed., Vol. 5, John Wiley and Sons, New York, pp. 195-203
90. Weisburger, E. K., Carcinogenicity studies of halogenated hydrocarbons, NIEHS
Conference on Comparative Metabolism and Toxicity of Vinyl Chloride and Related
Compounds, Bethesda, MD May 2-4 (1977)
91. Anon, NCI Clearinghouse Subgroup Finds Tris Tetrachioroethylene Carcinogenic,
Toxic Materials News , 4 (9) (1977) p. 60
92. Kylin, B., S imegi, I., and Yliner, S., Hepatotoxicity of inhaled trichioroethylene,
and tetrachioroethylene, long-term exposure, Acta. Pharmacol. Toxicol. , 22
(1965) 379—385
93. Bonse, G., Henschler, D., Chemical reactivity, biotransformation, and toxicity
of polychiorinated aliphatic compounds, CRC Crit. Revs Toxicology q (1976)
395-409
94. Bauchwitz, P. S., Chioroprene, In R. E. Kirk arid ID. F. Othmer (eds) Encyclo-
pedia of Chemical Technology, 2nd ed., Vol. 5, John Wiley and Sons, New York,
1964, pp. 215—231
95. Beliringer, F. J., and Hollis, C. E., Make chioroprene from butadiene, Hydro-
carbon Processing , 47 (1968) 127-130
96. Lloyd, J. W., Decoufle, P., and Moore, R. M., Background information on
chioroprene, J. Occup. Med. , 17(1975)263—265
97. VanOss, J. F., ‘Chemical Technology: An Encyclopedic Treatment”, Barnes and
Noble Books, Vol. 5, New York (1972) pp. 482-483
98. Khachatryan , E. A., The role of ch] .oroprene in the process of skin neoplasm
formation, Gig. Tr. Prof. Zab. , 18(1972) 54-55
99. Khachatryan, E. A., The occurrence of lung cancer among people working with
chioroprene, Vop. Oncol. , 18 (1972) 85-86
100. Anon, Industry’s problems with cancer aired, Chem. Eng. News , 53 (1975) 4
101. Katosova, L. D., Cytogerietic analysis of peripheral blood of workers engaged in
the production of chloroprene, Gig. Tr. Prof. Zab. , 10 (1973) 30-33
102. U.S. International Trade Commission, Synthetic Organic Chemicals United States
Production and Sales of Elastomers, 1975 Preliminary, June, Washington, D.C.
U.S. Government Printing Office (1976) p. 2
161
-------
103. Van Duuren, B. L., Goldschmidt, B. M., and Siedman, I., Carcinogenic activity
of di- and tn-functional cx-chloroethers and of 1,4-dichlorobutene-2 in ICR/HA
Swiss Mice, Cancer Res. , 35(1975) 2553-2557
104. Bartsch, H., et al., Malaveille, C., Barbin, A., Planche, G., and Montesano, R.,
Alkylating and mutagenic metabolites of halogenated olefins produced by human and
Proc. Am. Assoc. Cancer Res. , (1976) P. 17 Toronto, animal tissues.
May 4-8 Proc. 67th Ann. Meeting of Amer. Assoc. Cancer Res.
105. Mukai, F. H., and Hawryluk, I., The mutagenicity of some haloethers and halo-
ketones, Mutation Res. , 21(1973) 228
106. Loprieno, N., Mutagenicity assays using yeasts with carcinogenic compounds,
Second Meeting of Scientific Committee of the Carlo Erba Foundation, Dec. 12
(1975) pp. 129—140
162
-------
B. Saturated Halogenated Hydrocarbons
Alkyl halides can enter into a variety of nucleophilic substitution reactions and
hence are exceedingly useful for the synthesis of other compounds. A variety of
compounds containing hetero atoms are capable of acting as nucleophiles toward
alkyl halides, e.g., H 2 0, H 2 S, ROH and RSH. More reactive nucleophiles, the corres-
ponding anions, HO , HS, R0, and RS can also react with a variety of alkyl halides.
1. Methyl Chloride (CH 3 CI, monochioromethane) is produced by the action of
hydrogen chloride on methanol, with the aid of a catalyst, in either the vapor or the
liquid phase. Originally methyl chloride was used almost entirely as a refrigerant,
but this use has been largely preempted by the chlorofluoromethanes. The current
major uses of methyl chloride are: (1) as a catalyst carrier in the low temperature
polymerization production of butyl rubber; (2) in the production of silicones and
1
(3) tetramethyl lead . Other uses of methyl chloride include: as a paint remover,
in solvent degreasing operations, in the formation of carbonated quaternized acrolein-
copolymer anion exchanger 2 , and quaterization of tertiary amines 3 .
Methyl chloride has also been shown to be present in tobacco smoke 4 suggesting
an additional potential portion of the population that may be exposed to this agent.
Methyl chloride has recently been reported to be highly mutagenic in S. typhimurium
tester strain TA 1535 (which can detect mutagens causing base pair substitutions).
It should be noted that metabolic activation was not required to detect mutagenesis.
2. Chloroform (trichloromethane, CHCI 3 ) is made principally via the chlorination
of methane with lesser amounts produced by the limited reduction of carbon tetrachloride.
It is used in extensive quantities principally in the manufacture of chiorodifluoromethane
(C1F 2 HC) for use as a refrigerant and an aerosol propellant and as a raw material for
163
-------
the manufacture of fluorinated resins (e. g., Teflon, polytetrafluoroethylene, PTFE).
Other uses of chloroform include: extractant and industrial solvent in the prepara-
tion of dyes, drugs, pesticides, essential oils, alkaloids, photographic processing,
industrial dry cleaning, as a fumigant, in pharmaceuticals and toiletries (until recently
in mouthwashes, dentrifices), hair tinting and permanent-waving formulations, and
in fire extinguishers (with carbon tetrachioride).
NIOSH estimates that 40, 000 people in the United States may be exposed to chloroform
in their working environxnent 6 .
Chloroform is widely distributed in the atmosphere 7 ’ 8 and water 9 ’ 10 (including
municipal drinking water primarily as a consequence of chlorination) 9 ’ 10 A survey
of 80 American cities by EPA found chloroform in every water system in levels ranging
from <0.3-311 ppb 9 .
Chloroform is carcinogenic in Osborne-Mendel rats and G6C3F1 mice following
long-term oral intubation at maximum tolerated and half maximum tolerated doses” 12•
In rats, malignant and benign primary kidney tumors were found while chloroform
treated mice showed significant incidences of hepatocellular carcinomas” 12
Chloroform and other halogenated hydrocarbons produce pathological effects by
localizing in target tissues and binding covalently to cellular macromolecules’ 315 .
Information as to the mutagerucity of chloroform is scant. Chloroform (as well as
carbon tetrachioride) gave negative results when tested with microsomal incubates
with S. typhimurium TA 1535 and E. coli K-12 for base pair substitution and S.
typhimurium TA 1538 for frame-shift mutations’ 6 .
3. Carbon Tetrachloride (tetrachioromethane, CC1 4 ) is manufactured primarily
via the chlorination of methane and to a limited extent by the chlorination of carbon
disulfide 17 . It is produced in extensive quantities and employed largely for the
164
-------
production of fluorocarbons, e . g., dichiorofluoromethane (CF 2 C1 2 ) and trichioro-
fluoromethane (CFC1 3 ). Other areas of utility include: in grain fumigants (alone,
or mixed with ethylene bromide or chloride); fire extinguishers (with 10% CHC1 3
or trichloroethylene); solvent for oils, fats, resins and rubber cements; cleaning
agent for machinery and electrical equipment; in synthesis of nylon-7 and other organic
chlorination processes.
While losses of carbon tetrachioride to the global environment were estimated to
be in the order of 1 million tons in 19748, the occurrence of CC1 4 in the atmosphere
cannot be accounted for from direct production emission data.
Carbon tetrachioride is found in many sample waters (rain, surface, portable
and sea) in the sub-ppb range 8 , Cd 4 has been found in 10% of the U.S. drinking
water supplies at levels of < 2-3 ppb in a recent EPA survey of 80 cities 9 .
Thirteen halogenated hydrocarbons have been identified recently in samples of
New Orleans drinking water and 5 halogenated hydrocarbons were found in the pooled
plasma from 8 subjects in that area. Carbon tetrachioride and tetrachioroethylene
were found in both the plasma and drinking water. Considerable variation in the
relative concentrations of the halogenated hydrocarbons was noted from day to day
in the drinking water. In view of the lipophilic nature of CC1 4 , it was suggested
that a bioaccumulation mechanism may be operative, if drinking water was the only
source of such materials 18
Carbon tetrachioride has produced liver tumors in the mouse, hamster, and rat
following several routes of administration including inhalation and oral 19 ’ 20 A
number of cases of hepatomas appearing in men several years after carbon tetrachioride
poisoning have also been described 21 ’ ” ’.
.s.
-------
The chemical pathology of Cd 4 liver injury is generally viewed as an example
of lethal cleavage 23 , e.g., the splitting of the CC1 3 -Cl bond which takes place in the
mixed function oxidase system of enzymes located in the hepatocellular endoplasmic
reticulum. While two major views of the consequences of this cleavage have been
suggested, bith views take into account the high reactivity of presumptive free radical
products of a homolytic cleavage of the CCI 3 -Cl bond 21 . One possibility is the direct
attack (via alkylation) by toxic free radical metabolites of CC1 4 metabolism on cellular
constituents, especially protein sulfhydryl groups 24 . In homolytic fission, the two
odd-electron fragments formed would be trichioromethyl and monatomic chlorine free
radicals (e.g., CC ] 4 + e - CC ] 3 + C1). Fowler 25 detected hexachioroethane
(CC1 3 CC1 3 ) in tissues of rabbits following Cd 4 intoxication.
An alternative view has emphasized peroxidative decomposition of lipids of the
endoplasmic reticulum as a key link between the initial bond cleavage and pathological
phenomena characteristic of CC ] 4 liver injury 21 .
Similarly to chloroform, information as to the mutagenicity of carbon tetrachloride
is scant. Carbon tetrachioride gave negative results when tested in E coli and
Salmonella typhiniurium’ 6 ’ 26 . The synergistic effect of Cd 4 on the mutagenic effecti-
vity of cyclophosphamide in the host-mediated assay with S. typhimurium has been
reported 27 . CC ] 4 did not effect the mutagnicity of cyclophosphamide when tested
in vitro with S. typhimurium strains G46 and TA1950. Cd 4 was non-mutagenic when
assayed in a spot-test with the above strains of S. typhimurium .
4. Miscellaneous Halogenated Derivatives-Although the emphasis abovehas
been on saturated chlorinated hydrocarbons, it should be noted that other halo genated
related derivatives (e . g., brominated and iodinated) are potentially hazardous
compounds.
166
-------
Methyl iodide (lodomethane, CH 3 I) is used primarily as a methylating agent in
the preparation of pharmaceutical intermediates and in organic synthesis 28 . It also
is used to a limited extent in microscopy due to its high refractive index, and as a
reagent in testing for pyridine.
Methyl iodide is carcinogenic in BD strain rats by subcutaneous administration
inducing local sarcomas after single or repeated injections 29 ’ 30 . In a limited study
in A/He mice, methyl iodide caused an increased incidence of lung tumors after
inti-aperitoneal injection 31 .
Methyl iodide has been reported to be mutagenic in S. typhimurium TA 100 when
plates were exposed to its vapors. The mutagenic activity was slightly enhanced in
the presence of rat liver microsoinal fraction (900 ox g) 26 . Methyl iodide did not
increase the back mutation frequency in Aspergillus nidulans to methionine in
dependence at concentrations of 0.01 - 0.1 M for 5—15 minutes 32 .
It should also be noted that methyl iodide can be formed in nuclear reactor
environments 33 . An ambient concentration of 80 x io12 by volume (0.08 ppb) of
methyl iodide has been reported in the air of New Brunswick, NJ 3 .
Methyl bromide (bromomethane, CH 3 Br) an intermediate in organic synthesis
and a grain fumigant, apparantly has not been reported to have been tested for
carcinogenicity or mutageniCity.
167
-------
References for Saturated Halogenated Hydrocarbons
1. Cowenheim, F. A., and Moran, M. K., “Industrial Chemicals” 4th ed., John
Wiley, New York (1975) PP. 530-538
2. Clemens, D. H., and Lange, R. J., Ion-exchange resins from acrolein copolymers,
US Patent 3,813,353, May28 (1974) Chem. Abstr. , 81 (1974) P121747E
3. Mitsuyasu, T., and Tsuji, J., Quaternary arnmonium salts, Japan Kokai, 7,426,209
Chern. Abstr. , 81 (1974) P33374
4. Chopra, N. M., and Sherman, L. R., Systematic studies on the breakdown of
p , p’-DDT in tobacco smokes. Investigations of methyl chloride, dichloromethane
and chloroform in tobacco smoke, Anal. Chem. , 44 (1972) 1036
5. Andrews, A. W., Zawistowski, E. S., and Valentine, C. R., A comparison of
the mutagenic properties of vinyl chloride and methyl chloride, Mutation Res. ,
40 (1976) 273
6. Anon, Chloroform causes cancer in laboratory animals, NCI Study Report, Toxic
Materials News , 3(1976) 36
7. Yung, Y. L., McElroy, M. B., and Wofsy, S. C., Atmospheric halocarbons: A
discussion with emphasis on chloroform, Geophys. Res. Letters , 2 (1975) 397-399
8. McConnell, G., Ferguson, 0. M., and Pearson, C. R., Chlorinated hydrocarbons
in the environment, Endeavor , 34 (1975) 13-27
9. Environmental Protection Agency, Draft Report for Congress: Preliminary Assess-
ment of Suspected Carcinogens in Drinking Water, Office of Toxic Substances,
Washington, D.C., October 17 (1975)
10. Bellar, T. A., Lichtenberg, J. 3., and Kroner, R. C., The occurrenceof organo-
halides in chlorinated drinking waters, J. Am. Waterworks Assoc. , 66 (1974)
703—706
11. Powers, M. B., and Welker, R. W., Evaluation of the oncogenic potential of
chloroform long term oral administration in rodents, 15th Annual Meeting Society
of Toxicology, Atlanta, GA, March 14-18 (1976)
12. National Cancer Institute, Report on the Carcinogenesis Bioassay of Chloroform,
National Cancer Institute, Bethesda, MD, March (1976)
13. Ilett, K. F., Reid, W. D., Sipes, I. G., and Krishna, G., Chloroform toxicity
in mice: Correlations of renal and hepatic necrosis with covalent bonding of
metabolites to tissue macromolecules, Exp. Mol. Pathol. , 19 (1973) 215
168
-------
14. Reid, W. D., and Krishna, G., Centrolobular hepatic necrosis related to covalent
binding of metabolites of halogenatéd aromatic hydrocarbons, Exp. Mol. Pathol. ,
18 (1973) 80—95
15. Brodie, B. B., Reid, W. D., Cho, A. K., Sipes, G., Krishna, G., and Gillette,
J. R., Possible mechanism of liver necrosis caused by aromatic organic compounds,
Proc. Natl. Acad. Sci. , 68(1971) 160
16. Uehleke, H., Greim, Fl., Kramer, M., and Werner, T., Covalent binding of halo-
alkanes to liver constituents, but absence of mutagenicity on bacteria in a meta-
bolizing test system, Mutation Res. , 38 (1976) 114
17. Hardie, D. W. F., Carbon tetrachioride, In Kirk-Othmer (ed.) Encyclopedia of
Chemical Technology, 2nd ed, Vol. 5, lnterscience, New York, (1964) p. 128-139
18. Dowty, B., Carlisle, D., Laseter, J. L,, and Storer, J., Halogenated hydrocarbons
in New Orleans drinking water and blood plasma, Science , 187 (1975) 75-77
19. IARC, Vol. 1, International Agency for Research on Cancer, Lyon (1972) pp. 53-60
20. Warwick, G. P., In “Liver Cancer”, International Agency for Research on Cancer,
Lyon (1971), pp. 121—157
21. Tracey, 3. P., and Sherlock, P., Hepatoma following carbon tetrachioride poisoning,
New York St. 3. Med. , 68(1968) 2202
22. Rubin, E., and Popper, H., The evolution of human cirrhosis deduced from
observations in experimental animals, chiorofluorocarbons in the atmosphere,
Medicine , 46 (1967) 163
23. Rechnagel, R. 0., and Glende, E. A., Jr., Carbon tetrachloride: An example
of lethal cleavage, CRC Crit. Revs. In Toxicology (1973) 263-297
24. Butler, T. C., Reduction of carbon tetrachioride in vivo and reduction of carbon
tetrachioride and chloroform in vth o by tissues and tissue constituents, 3. Pharmacol.
Exp. Therap. , 134(1961) 311
25. Fowler, S. S. L., Carbon tetrachioride metabolism in the rabbit, Brit. J. Pharmacol .
37 (1969) 773
26. McCann, 3., Choi, E., Yamasaki, E, and Ames, B. N., Detection of Carcinogens
as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc.
Nail. Acad. Sd. , 72(1975) 5135—5139
27. Braun, R., and Schôneich, 3., The influence of ethanol and carbon tetrachioride
in the host-mediated assay with Salmonella typhimurium, Mutation Res. , 31 (1975)
191—194
169
-------
28. Hart, A. W., Gergel, M. G., and Clarke, J., Iodine, In Kirk, R. E., and
Othxner, D. F. (eds) Encyclopedia of Chemical Technology, 2nd ed., Vol. 11,
John Wiley and Sons, New York (1966) pp. 862-863
29. Preussmann, R., Direct alkylating agents as carcinogens, Food Cosmet. Toxicol .
6 (1968) 576—577
30. Druckrey, H., Kruse, H., Preussmann, R., Invankovic, S.. and Lanschi ltz, C.,
Canc erogene alkylierende substan zen. III. Alkyl-halogenide, -sulfate, sulfonate
und ringespannte heterocyclen, Z. Krebsforsch. , 74 (1970) 241-270
31. Poirier, L. A., Stoner, G. D., and Shimkin, M. B., Bioassay of alkyl halides
and nucleotide base analogs by pulmonary tumor response in strain A mice,
Cancer Res. , 35(1975) 1411—1415
32. Moura Duarte, F. A., Efeitos mutagenicos de alguns esteres de acidos inorganicos
em aspergillus nidulans (EIDAM) winter, Ciencia e Cultura , 24 (1971) 42-52
33. Barnes, R. H., Kircher, 3. F., and Townly, C. W., Chemical-equilibrium studies
or organic-iodide formation under nuclear-reactor-accident conditions, AEC
Accession No. 43166 Rept. No. BMI-1781, Chern. Abstr. , 66 (1966) 71587V
34. Lillian, D., and BirSingh, H., Absolute determination of atmospheric halocarbons
by gas phase coulometry, Anal. Chem. , 46 (1974) 1060-1063
170
-------
C. Aryl Halogen Compounds
Most aryl halides are usually much less reactive than alkyl or allyl halides
toward nucleophilic reagents in either SN 1 - or SN 2 -type reactions. However,
in contrast to phenyl halides, benzyl halides are quite reactive, are analogous in
reactivity to allyl halides and are hence readily attacked by nucleophilic reactants
in both SN 1 - and SN2-displacement reactions. This reactivity is related to the
stability of the benzylcation, the positive charge of which is expected to be exten-
sively delocalized .
Benzyl chloride ( 2) -CH 2 C1; chloromethylbenzene; -chlorotoluene) is
used principally (65-70%) in the U . S. as an intermediate in the manufacture of butyl-
benzylphthalate, a vinyl resin plasticizer, while the remaining 30-35% is employed
as an intermediate in the production of benzyl alcohol, quaternary ammonium
chlorides and benzyl derivatives such as benzyl acetate, cyanide, salicylate and
2
cinnamate
Suggested uses of benzyl chloride include: in the vulcanization of fluororubbers 3
and in the benzylation of phenol and its derivatives for the production of possible
4
disinfectants
Benzyl chloride has been shown to induce local sarcomas in rats treated by
5,6
subcutaneous injection
Benzyl chloride was reported to be weakly mutagenic in S. typhimurium TA 100
strain 7 .
171
-------
References for Aryl Halogen Compounds
1. Roberts, 3. D., Caserio, M. C., “Modern Organic Chemistry” W. A. Benjamin,
Inc. New York (1967) p. 571
2. IARC, Vol. 11, International Agency for Research on Cancer, Lyon (1976) 217-223
3. Okada, S., and Iwa, R., Fluorolefin elastomer stocks, Japan Kokai 7, 414, 560,
Feb. 8 (1974) Chem. Absti-. , 81 (1974) P50806K
4. Janata, V., Simek, A., and Nemeck, 0., Benzyl phenols, Czeck Patent 152, 190,
Feb. 15 (1974) Chem. Abstr. , 81(1974) P25347D
5. Druckrey, H., Kruse, H., Preussmann, R., Wankovic, S., and Lanschi jtz, C.,
Cancerogene alkylierende substanzen. III. Alkyl-halogenide-sulfate sulfonate
und ringgesparinte heterocyclen, Z. Krebsforsch. , 74 (1970) 241
6. Preussmann, R., Direct alkylating agents as carcinogens, Food Cosmet. Toxicol .
6 (1968) 576
7. McCann, 3., Springarn, N. E., Kobori, 3., and Ames, B. N., Carcinogens as
mutagens: Bacterial tester strains with R factor plasmids, Proc. Nati. Acad.
Sci. , 72 (1975) 979—983
172
-------
D. Halogenated Polyaromatics
Halogenated biphenyls,as aretypical of aryl halides, generally are quite stable
to chemical alteration. Polychiorinated biphenyls (PCB’s) (first introduced into com-
mercial use more than 45 years ago) are one member of a class of chlorinated aromatic
organic compounds which are of increasing concern because of their apparent ubiqui-
tous dispersal, persistence in the environment, and tendency to accumulate in food
chains, with possible adverse effects on animals at the top of food webs, including
1—7
man
Polychiorinated biphenyls are prepared by the chlorination of biphenyl and hence
are complex mixtures containing isomers of chiorobiphenyls with different chlorine
contents 8 . It should be noted that there are 209 possible compounds obtainable by
substituting chlorine for hydrogen or from one to ten different positions on the biphenyi
ring system. An estimated 40-70 different chlorinated biphenyl compounds can be
present in each of the higher chlorinated commercial mixture 9 ’ o. For example,
Arochior 1254 contains 69 different molecules, which differ in the number and position
of chlorine atoms’°.
It should also be noted that certain PCB commercial mixtures produced in the U.S.
and elsewhere (e.g., France, Germany, and Japan) have been shown to contain other
classes of chlorinated derivatives, e. g., chlorinated naphthalenes and chlorinated
dibenzofurans 7 ’ 1114• The possibility that naphthalene and dibenzofuran contaminate
the technical biphenyl feedstock used in the preparation of the commercial PCB mixtures
cannot be excluded. Table 1 illustrates the structures of the chlorinated biphenyls;
chlorinated naphthalenes; chlorinated dibenzofurans and lists the extent of chlorination
as well as the number of chlorinated derivatives possible.
173
-------
The PCBs have been employed in a broad spectrum of applications because of their
chemical stability, low volatility, high dielectric constants, nonflammability, and
general compatability with chlorinated hydrocarbons. The major areas of utility have
included: heat exchangers and dielectric fluids, in transformers and capacitors,
hydraulic and lubricating fi, diffusion pump oils, plasticizers for plastics and
coatings, ingredients of caulking compounds, printing inks, paints, adhesives and
carbonless duplicating, flame retardants, extender for pesticides, and electrical
circuitry and component.
The rats and routes of transport of the PCBs in the environment” 2, 5, 6 and
1,2 ,15—20 1,3,5,7,21—23
their accumulation in ecosystems , and toxicity have
been reviewed.
It is generally acknowledged that the toxicological assessment of commercially
available PCBs has been complicated by the heterogeneity of the isomeric chiorobiphenyls
and by marked differences in physical and chemical properties that influence the rates
of absorption, distribution, biotransformation and excretion” 7. 21-29
The lower chlorine homologs of PCBs (either examined individually se or
in Aroclor mixtures) are reported to be more rapidly metabolized in the rat then the
higher homologs 3034 . Sex-linked differences were also disclosed (e.g., the
biological half-life of Aroclor 1254 in adipose tissue of rats fed 500 ppm was 8 and
12 weeks in males and females respectively) 3 ”.
The lowest PCB homolog found from Aroclor 1254 in human fat was pentachloro-
biphenyl 3 ’.
The metabolism of many PCB isomers have consistently shown the formation of
various hydroxylated urinary excretion products. For example, the metabolism of
4,4’-dichlorobiphenyl in the rat yielded four monohydroxy-, four dehydroxy- and
two trihydroxy inetabolites (Scheme 35• The structures of the major metabolites
174
-------
in the rat are consistent with epoxidation of the biphenyl nucleus followed by epoxide
ring opening accompanied by a 1, 2-chlorine shift (NIH shift). The formation of
minor rat metabolites, 4-chloro-3 ’-biphenylol appeared to occur via reductive de-
chlorination 35 .
Urinary metabolites of 2, 5, 2’, 5 ‘-tetrachiorobiphenyl (TCB) in the non-human
primate included: TCB; monohydroxy-TCB; dehydroxy-TCB; hydroxy-3 , 4-dihydro-
37
3, 4-di hydroxy-TCB; and trans - 3,4 ‘-dihydro -3 ,4-dihydro-TCB
In studies involving the metabolism of 2,2 l ,4,4 , 5, 5 ‘-hexachlorobiphenyl by
38,39
rabbits rats and mice , it was shown that the rabbit excreted hexachiorobiphenylol-,
39,40
pentachlorobiphenylol and methoxypentachlorobiphenylol compounds (Scheme
while rats and mice excreted only a hexachlorobiphenylol 38 .
41
These results and previous studies by Gardner and co-workers as well as
in vitro metabolism studies with 4-chlorobiphenyl 42 indicate that PCBs are metabolized
via metabolically activated arene oxide intermediates.
It is also of potential importance to note the presence of methyl sulfone metabolites
43
of PCB (as well as DDE) recently found in seal blubber . The toxicological signi-
ficance of these metabolites have not been elucidated to date.
Cl-C
Cly ClK 7 Cl
0= S0 0=5=0
CH 3 CH 3
Methyl Sulphone of PCB Methyl Sulfone of DDE
(X + y = 3 - 7)
Increasing evidence indicates that not all chioribiphenyl congeners produce the
7,29,49-51 Morphological alterations in both acute and
same pharmacologic effects
chronic toxicity have been studied in rats, monkeys, mice and cows 7 ’ 5257 , the
organ consistently affected was the liver. For example, when male Sprague-Dawley
175
-------
rats were fed a diet containing mixtures of PCB isomers (Aroclor 1248, 1254 and
1262) at a concentration of 100 ppm in the diet for 52 weeks, there was a decided
increase in their total serum lipids and cholesterol and a transient increase in tri-
glycerides accompanied by distinct morphological changes in the liver 57 . Genera-
lized liver hyperfrophy and focal areas of hepatocellular degeneration were followed
by a wide spectrum of repair processes. The tissue levels of PCB were greater
in the animals receivuig the high chlorine mixtures and high levels persisted in
these tissues even after the PCB treath ient had been discontinued.
Indirect effects of PCB exposure are related to increased rnicrosomal enzyme
activity and include alteration in metabolism of drugs, hormones, and pesticides 7 ’ 44 ’ 58-60
A large portion of the human population has detectable levels of PCB in adipose tissue 2 ’ 6
and recent preliminary reports have revealed that PCBs have been found in 48 of 50
samples of mothers’ milk in 10 states 6 63 (the average levels in the 48 samples was
2.1 ppm). The 1971-74 adipose levels of PCB in the U.S. showed that of 6500 samples
examined, 77% contained PCB (e.g., 26% contained <1 ppm; 44% contained 1-2 ppm
63
and 7% contained > 2 ppm . There were no sex differences and the levels of PCB
increased with age 63 .
The 1971-1974 PCB ambient water levels were as follows: of 4472 water samples,
130 were in the range of 0.1 to 4.0 ppb (detection limit: 0.1-1.0 ppb) 63 ; of 1544
sediment samples, 1157 were in the range of 0.1 to 13, 000 ppb (limit of detection 0.1
1.0 ppb) 63 . In a recent accidental plant discharge episode, Hudson River sediments
64
near Fort Edward, New York were found to contain 540-2980 ppm PCBs . Soil levels
of PCBs in the 1971-1974 period ranged from 0.001-3.33 ppm (average 0.02 ppm) in
1,434 samples taken from 12 of 19 metropolitan areas 63 . In limited surveys in ambient
air an average of 100 ng/cni of PCB was found for each of 3-24 hr samples from
176
-------
Miami, Florida, Jacksonville, Florida and Fort Collins, Colorado 63 . Where PCBs
have been found in food, it has generally been in fish samples from the Great Lakes
area 64 with contamination arising mainly through the environment. Where as in the
past, milk and dairy products, eggs, poultry, animal feeds, infant foods as well as
paper food packaging received PCBs principally from agricultural and industrial
64
applications
Although there has been a sharp curtailment of PCB production and dispersive
use applications from a record high of 70 million lbs in 1969, it is believed that it
will take several years for ecosystems such as Lake Michigan to cleanse itself of
the compounds even if no new input is made 65 . Due to its inertness and high adsorption
coefficient, the PCBs have accumulated in the bottom sediments. The final sink for
PCB is predicted to be degradation in the atmosphere, with some fraction being
buried in underlying sediments of lakes 65 . Figure 3 illustrates the possible routes
of loss of PCBs into the environment.
It is important to note that even with the cessation of PCB production L
other environmental sources of PCB may exist. For example, it has been reported
that some PCBs are products of DDT photolysis 4 ’ 66 ’ 67 (Figure 4). Uyeta et a1 68
recently reported the photoformation of PCBs from the sunlight irradiation of mono-,
di-, tn-, tetra-, and hexachlorobenzenes.
Gaffniey 68 a recently reported the formation of various mono-, di-, and tnichioro-
biphenyls resulting from the final chlorination of municipal wastes containing biphenyl.
Laboratory chlorination of influent and effluent from a municipal waste treatment
facility also resulted in the formation of these and other chloroorganic substances
such as di- and trichlorobenzenes.
177
-------
Previous clinical aspects of human poisoning in Japan (“Yusho” disease) involving
at least 1000 people consuming rice bran oil contaminated with Kanechlor 400 (a PCB
containing48%chlorinewith2,4,3’,4’—,2,5,3’,4’—, 2 ,3,S,4 ’—and3,4,3,4’—tetra—
chlorobiphenyl, and 2,3, 5,3’ , 4’ —pentachlorobiphenyl) 69 are well documented” ‘ ‘ 70, 71
It has also been claimed that there are an estimated 15, 000 victims of TtYusho disease
72
although only 1081 persons have been officially diagnosed as such
It has been very recently reported by Hirayama 73 that five of the Yusho victims
died of liver cancer within 5 years after consuming the contaminated cooking oil.
Recent reports of high cancer rates among Mobil Oil employees at its Paulsboro,
N.J. refinery exposed to PCBs (Aroclor 1254) have suggested a possible link between
PCB exposure and skin (melanoma) or pancreatic cancer 7476 . The Mobil study
indicated that 8 cancers developed between 1957 and 1975 among 92 research arid
development and refinery workers exposed for 5 or 6 years in the late 1940’s and early
1950’s to varying levels of Aroclor 1254. Of the 8 cancers, 3 were malignant melanomas
and two were cancers of the pancreas. NIOSH said “this is significantly more skin
cancer (melanoma) arid pancreatic cancer than would be expected in a population of
this size, based on the Third National Cancer Survey” 75 .
It should be noted that Monsanto Co. could find no casual relationship between
cancer and PCB exposure at its plant in Sauget, Ill. The Monsanto study was based
on a review of the records of more than 300 current and former employees at the illinois
plant which had been engaged in PCB production since. 1936 .
Earlier indications of the carcinogenicity of PCBs were reported in 1972 by Naga-
saki et al 78 who cited the hepatocarcinogenicity of Kaneclor-500 in male dd mice fed
500 ppm of the PCB. The hepatomas appeared similar to those induced by the gamma-
isomer of benzene hexachloride (BHC) 79 ’ 80 , whereas Kaneclor-400 and Kaneclor-
300 had no carcinogenicity activity in the liver of mice. The Kaneclor-500 sample
178
-------
contained 55.0% pentachlorobiphenyl, 25.5% tetrachiorobiphenyl, 12.8% hexachioro-
biphenyl and 5. 0% trichlorobiphenyl. Later studies by Ito et a1 8 ’ also demonstrated
that Kaneclor-500 not only induced hepatic neoplasms in mice when fed at levels of
500 ppm in the diet for 32 weeks but also promoted the induction of tumors by alpha-
PHC and beta-BHC. Kimbrough et a1 82 reported the induction of liver tumors in
Sherman strain female rats fed 100 ppm of Aroclor 1260 in their diet for approximately
21 months. Recent studies also suggest that PCBs exert a potent promoting action
in experimental azo dye hepato carcinogenesis 83 .
Conflicting evidence to date exists concerning mutagenic effects of mixtures of
63, 42,84—89
PCBs. Tests on Drosophila with PCB of mixed degrees of chlorination did
not indicate ary chromosome-breaking effects 85 . However, it was suggested by
Ramel 84 , that PCBs may have an indirect bearing on mutagenicity and carcinogenicity
since they induce enzymatic detoxification enzymes in liver micro somes.
No chromosomal aberrations have been observed in human lymphocyte cultures
86 87
exposed to Aroclor 1254 at 100 ppm levels . Keplinger et al employing a dominant
88,89
lethal assay, reported no evidence of mutagenic effects of Aroclors. Green et al
reported a lack of mutagenic activity as measured by dominant lethal test for male
Osborne-Mendel rats subjected to 4 different regimens of Aroclor 1242 and 1254.
While the above studies of Green et al 88 ’ 89 were negative in regard to chromosomal
mutations, they do not entirely rule out the possibility that PCBs may induce point
mutations. However, to date there are no known reports in the literature concerning
the induction of point mutations by PCBs in laboratory model systems.
A recent comparison of the mutagenic activity of Aroclor 1254 (average chlorine
content, 4. 96% Cl/molecule), 2, 2’, 5, 5 ‘-tetrachiorobiphenyl (4 Cl/molecule), Aroclor
1268 (average chlorine content, 9.7 Cl/molecule), Aroclor 1221 (average chlorine
179
-------
content, 1 .15 Cl/molecule) and 4-chiorobiphenyl showed that as the degree of chlori-
nation decreased, the mutagenicity to Salmonella typhimurium TA 1538 strain (in the
QO 42
presence of liver homogenate’ ) increased . This strain is sensitive to frameshift
mutagens 90 . The influence of the degree of chlorination on the mutagenicity to the
mutant strain TA 1538 also complements the observations that as the chlorine content
of the PCB substrate increases, the metabolic rate decreases 31 ’ 91
It is also important to note the recent report that the in vitro metabolism of 4-
chlorobiphenyl proceeds via an arene oxide intermediate and is accompanied by
binding to the endogenous microsomal RNA and protein 42 . Preliminary results also
suggest binding of PCB to exogenous DNA 42 which confirms an earlier report of
Allen and Norback 92 . Covalent binding of the 2, 5, 2’ , 5’-tetrachlorobiphenyl metabolites
(e.g., trans dihydrodihydroxy) to cellular macromolecules was suggested by Allen
and Norback 93 to be a possible pathway for the carcinogenic action of the PCBs.
Teratogenic studies appear to be thus far nondefinitive 7 . However, while the
PCBs have not exhibited known or clearly defined teratogenic effects in mammals,
their easy passage across the plucenta suggests the potential for some form of fetal
toxicity 7 ’ 72, 9’4 Placental transport of PCBs have been reported for the rabbit, rat 95 ,
mouse and cow as well as observed among “Yusho” patients 7 ’ 96 ’ 7 .
No account of the toxicity of the polychlorinated biphenyls can be complete
without stressing the possible role of trace contaminants 7 ’ 11-14, e.g., the chlorinated
dibenzofurans. For example, embryotoxicity of the PCBs Clophen A069 and Phenoclor
DP-6 has been attributed to chlorinated dibenzofurans present as trace contaminants in
the commercial preparations 11 ’ 98 Subsequently, tetra-, penta-, and hexachioro—
dibenzofurans were detected in a number of American preparations of PCBs (e.g.,
180
-------
Aroclor 1248, 1254, 1260), concentrations of the individual chlorodibenzofurans
were in the order of 0 . 1 gig/kg of the PCB. Chlorinated dibenzofurans have been
considered as possible causes of embryonic mortality and birth-defects observed
in PCB-feeding experiments in birds 99 ’ 100 The chlorinated dibenzofurans are
structurally related to the chlorinated dibenzo-p-dioxin (Table 1) some of which
101
are both highly toxic and teratogenic
A number of possibilities exist to account for the presence of chlorodibenzofurans
in commercial PCB mixtures. One explanation considers the presence of the parent
compound (dibenzofuran) in the technical grade biphenyl subjected to the chlori-
nation process. It is also conceivable that chlorinated dibenzofurans may be produced
from PCBs in the environment. Two possible mechanisms for such a transformation
are illustrated in Figure 5, both of which involve hydroxy derivatives.
As cited earlier, hydroxylation is a route of metabolism of the PCBs. Polar
oxygenated compounds have also been found as photolytic products of the PCBs.
It should be stressed that the transformation of only 0.002% of a major constituent
of an Aroclor mixture to the corresponding chlorinated dibenzofurans would produce
concentrations in the mixture corresponding to the values reported by Vos et al
as toxicologically significant 102 ’ 103
To date, there have been no published findings of chlorinated dibenzofurans
in aquatic samples or in foods. The extremely low levels of these trace contaminants
in the original organic chemicals and/or complex mixtures (e. g., PCBs, chlori-
nated phenols) would stress the requirement for analytical procedures permitting
the sampling, concentration and detection in the parts-per-billion to parts-per-
trillion range.
Although the overwhelming stress thus far in a consideration of the halogenated
polyaromatics has focused on the polychlorinated biphenyls, it must be noted that
181
-------
structurally related derivatives such as the polybrominated biphenyls (PBBs) have
105,106
been increasmgly employed, primarily as fire retardants . For
example, the PBBs are incorporated into thermoplastics at a concentration of about
15% to increase the heat stability of the plastic to which it is added. About 50% of the
PBBs manufactured are used in typewriter, calculator, microfilm reader and business
machine housings. One-third is used in radio and television parts, thermostats
and electrical showers and hand tools and the remainder is used in a variety of other
types of electrical equipment 7 ’ 1O5
The recent accidental contamination in 1973 of animal feed and livestock through-
out Michigan of polybrorninated biphenyl flame retardants (Firemaster BP-6) 104 ’ 105
has stimulated extensive studies of the potential for water contamination, transport,
bioaccurnulation, biological and toxicological nature of this class of environmental
agent.
While no immediate adverse health effects were noted in several thousand Michigan
farm families which consumed milk and dairy products contaminated with PBB s, it
is not possible to determine at this date any chronic or delayed effects that might be
105
attributed to the PBBs or the potential ability of this chemical to cause birth defects
Fireniaster BP-6, the PBB responsible for the adverse effects in livestock and
poultry in the Michigan contamination eposides was found to consist (to the extent
of 70%).of a mixture principally of hexabromo-, and heptabromobiphenyl 105 . Fire-
master BP-6 fed at doses up to 2000 ppm in diets to pregnant rats and mice resulted
in exencephaly in the offspring of mice receiving both 100 and 2000 ppm dosages;
cleft polate and defective kidneys were found in the offspring of mice fed at the
1000 ppm level ’ 05 . Firemaster BP-6 has also been shown to be an inducer of rat
hepatic microsomal mixed function oxidase (analogous to the PCBs) and hence it
182
-------
was predicted that environmental exposure to BP-6 may affect the rate of metabolism
of a wide range of both endogenous and exogenous substrates in mammals 106 .
PBBs are produced by direct bromination of biphenyl and it could be anticipated
that very complex mixtures of compounds differing from each other both in number
of bromine atoms per molecule and by positional isomersion are formed 105 . The
possibility also exists (analogous to the PCBs) that halogenated dibenzofurans (e.g.,
brominated dibenzofurans) may be trace contaminants in certain PBB formulations.
There is a paucity of data concerning the toxicity of individual brominated
biphenyl isomers. No terata were observed in offspring from rats fed 100 mg/kg
107
hexabromobiphenyl on days 6,8,10,12,14 and 16 of pregnancy . Rats treated
with PBB and coichicine were found to have higher metaphase and mitotic indices
(when bone-marrow was studied cytogenetically) than non-treated animals; no
107
chromosome aberrations were noted
193
-------
rat metabohsrn of
SCHEME 1
4 4’—c1 ch1O1ob PheflY’-
c
C
dr€ Ct
hydrOXy Rt on
HO
b
6 09
decb or natiOfl
C -4 --Q
-ol
184
-------
CL v’-”
O }c cI
LJ
H3 - Ct
A C [
Suggested met bc ic p2ff ay ot n rabbts.
‘U
V
II
‘V
7
U
CL
C t
N
V I
185
-------
Fig.3 Pd sihIe routes of oss of PCB into the en ronrnent.
nQ_• Qafl
—I : , °$t .n1et$.tr (a )
186
-------
Cr
L )
I.i— —CC 1
C L
—‘p’
-
D E
C l 4
c
0
C t)
FC3
3 6- d.chIa fLucrenone
Fig. / Proposed scheme ibr th Qegradati. -1 of DDT in sunk ht
r.
a!
C:
DD:
187
-------
2.4,5.Z’.3 5-he*4corob pP .’y
C,
CL—” /f ’ \
.. j/ \ /
_Ø .:
2,4 S 34’- pntachtorobiphe yI
C .. C i
.‘E - eorOd,De’ZC’ afl
Fig. I’ Possible routes of trar.sforria:ior of PCRs t. c orinat d dib ’z.,fur n .
188
-------
TABLE 1
STRUCTURES, EXTENT OF POSSIBLE CHLORINATION AND
NUMBER OF CHLORINATED DERIVATIVES OF CHLORINATED
BIPHENYLS, NAPHTHALENES AND DIBENZO-p-DIOXINS
Structure
Chlorinated
D eriva .v es
Possible
Chlorinated
biphenyls
Chlorinated
nap hthalenes
Chlorinated
diben zofurans
Chlorinated
dib en zo -p -dioxins
C
x1—1O
209
xn=l—8
75
m1—8
135
m1—8
75
Name
Extent of Chlori-
nation Possible
Number of
189
-------
D. References for Halogenated Polyaromatics
1. Panel on Hazardous Substances, Polychiorinated Biphenyls-Environmental Impact,
Environ. Res. , (1972) 249
2. Hammond, A. L., Chemical Pollution: Polychiorinated Biphenyls, Science , 175
(1972) 155 —
3. National Swedish Environment Protection Board, PCB Conference, Wenner-Gren
Center, Stockholm, Sweden, Sept. 29 (1970)
4. Peakall, D., and Lincer, 3., Polychiorinated Biphenyls: Another Long-Life Wide-
spread Chemical in the Environment, Bioscience , 20 (1970) 958
5. Ahmed A. H., PCBs in the Environment, Environment , 18 (1976) 6
6. Nisbet, I. C. T., and Sarofim, A. F., Rates and Routes of Transport of PCBs in
the Environment, Env. Hith. Persp. , 1(1972)21
7. Dept. HEW, Final Report of the Subcommittee on the Health Effects of Polychiorinated
Biphenyls and Polybrominated Biphenyls, Washington, D.C. July (1976).
8. Hubbard, H. L., Chlorinated Biphenyl and Related Compounds, InKirk-Othmer’s
“Encyclopedia of Chemical Technology”, 2nd ed. 5 (1964) 289
9. Hutzinger. 0., Safe, S., and Zitko, V., “The Chemistry of PCBs ”, CRC Press,
Cleveland, Ohio (1974)
10. Zitko, V., Hutzinger, 0., and Safe, S., Retention Times and Electron-Capture
Responses of Some Individual Chiorobiphenyls, Bull. Env. Contam. Toxicol. ,
6 (1971) 160
11. Vos, 3. G., Koeman, 3. 3., Vandermaas, H. L., TenNoever de Brauw, M. C.,
and DeVos, Identification and Toxicological Evaluation of Chlorinated Dibenzo-
furan and Chlorinated Naphthalene in Two Commercial Polychlorinated Biphenyls,
Food Cosmet. Toxicol. , 8 (1970) 625
12. Bowes, G. W., Identification of Chlorinated Dibenzofurans in American Polychlori-
nated Biphenyls, Nature , 256 (1975) 305
13. Roach, 3. A. G., and Pomerantz, I. H., The Findings of Chlorinated Dibenzo-
furans in Aroclor PCB’s of Recent Manufacture, Paper No. 53, Presented at 88th
Annual Meeting of Assn. of Official Analytical Chemists, Washington, D.C.
October 14-17 (1974)
15. Risebrough, R. W., and deLappe, R. W., Polychlorinated Biphenyls In Ecosystems,
Env. Hith. Persp. , 1(1972) 39
1 C”
j.
-------
16. Gustafson, C. G , PCBs-Prevalent and Persistent, Env. Sci. Technol. , 4 (1970)
814
17. Ruopp, D. J., and deCarlo, V. J., Environmental Levels of PCBs, mt. Conf. on
Environ. Sensing and Assessment, Las Vegas, Nev., Sept. 14-19 (1975)
18. Maugh, T. H., Chemical Pollutants: Polychlorinated Biphenyls Still a Threat,
Science , 190 (1975) 1189
19. Kolbye, A. C., Food Exposures to Polychlorinated Biphenyls, Environ. Hlth.
Persp. , 1(1972) 85-88
20. Anon, The Rising Clamor About PCBs, Env. Sci. Technol. , 10 (1976) 122
21. Fishbein, L., Toxicity of Chlorinated Biphenyls, Ann. Rev. Pharmacol. , 14
(1974) 139
22. Kimbrough, R. D., The Toxicity of Polychiorinated Polycyclic Compounds and
Related Compounds, Crit. Revs. Toxicol. , 2 (1974) 445
23. Allen, J. R., Response of the Non Human Primate to Polychlorinated Biphenyl
Exposure, Fed. Proc. , 34(1975) 1675
24. Grant, D. L., Phillips, W. E. J., and Villeneuve, D., Metabolism of Polychlorinated
Biphenyl (Aroclor 1254) Mixture in the Rat, Bull. Env. Contam. Toxicol. , 6
(1971) 102
25. Bush, B., Turnasonis, C. F., and Baker, F. D., Toxicity and Persistence f
PCB Homologs and Isomers in the Avian System, Arch. Env. Con. Tox. , 2 (1974)
195
26. Sissons, D., and Welti, D., Structural Identification of Polychiorinated Biphenyls
in Commercial Mixtures by Gas-Liquid Chromatography, Nuclear Magnetic Reson-
ance and Mass Spectrometry, J. Chromatog. , 60 (1971) 15
27. Webb, R. G., and McCall, A. C., Identities of Polychlorinated Biphenyl Isomers
inAroclors, 3. Assoc. Off. Anal. Chem. , 55(1972)746
28. Ecobichon, D. J., and Comeau, A. M., Comparative Effects of Commercial Aroclors
on Rat Liver Enzyme Activities, Chem. Biol. Interactions , 9 (1974) 314
29. Ecobichon, ID. 3., and Comeau, A. M., Isomerically Pure Chlorobiphenyl Congeners
and Hepatic Function in the Rat: Influence of Position and Degree of Chlorination,
Toxicol. Appi. Pharmacol. , 33 (1975) 94
30. Matthews, H. B., and, M. W., The Distribution and Excretion of 2, 4,
5,2’,5-Pentachlorobiphenyl in the Rat, Drug Metab. Dispos. , 3 (1975) 211-219
191
-------
31. Matthews, H. B., and Anderson, M. W., Effect of Chlorination on the Distribution
and Excretion of Polychlorinated Biphenyls, Drug Metab. Dispos. , 3 (1975) 371-380
32. Braunberg, R. C., Dailey, R. E., Brouwer, E. A., Kasza, L., and Blaschka,
A. M., Acute, Subacute, and Residual Effects of Polychiorinated Biphenyl (PCB)
in Rats. Biological Half-life in Adipose Tissue, J. Toxicol. Env. Hith, , 1 (1976)
683—688 —
33. Mehendale, H. M., Uptake and Disposition of Chlorinated Biphenyls by Isolated
Perfused Rat Liver, Drug Metab. Dispos. , 4(1976) 124-132
34. Peterson, R. E., Seymour, J. L., and Allen, 3. R., Distribution and Biiary
Excretion of Polychlorinated Biphenyls in Rats, Tox. Appi. Pharmacol. , 38
(1976) 609—619
35. Biros, F. 3., Walker, A. C., and Medbery, A., Polychiorinated Biphenyls in
Human Adipose Tissue, Bull. Env. Contam. Toxicol. , 5 (1970) 317-323
36. TuIp, M. T. M., Sundsträm, G., and Hutzinger, 0., The Metabolism of 4,4’-
Dichiorobiphenyl in Rats and Frogs, Chemosphere , 6 (1976) 425-432
37. Hsu, I. C.. Van Miller, 3. P., Seymour, 3. L., and Allen, J. R., Urinary
Metabolites of 2,5,2’, 5’-Tetrachlorobiphenyl in the Nonhuman Primate, Proc.
Soc. Exp. Biol. Med. , 150(1975) 185—188
38. Jensen, S., and Sundstrorn, G., Metabolic Hydroxylation of a Chiorobiphenyl
Containing only Isolated Unsubstituted Positions -2, 2’4,4 5, 5’ -Hexachiorobiphenyl ,
Nature , 25]. (1974) 219-220
39. Hutzinger, 0., Jamieson, W. 1)., Safe, S., Paulmann, L., and Ammon, R.,
Identification of Metabolic Dechlorination of Highly Chlorinated Biphenyl in Rabbit,
Nature , 252(1974) 698 -699
40. Sundström, G., Hutzinger , 0., and Safe, S., The Metabolism of 2,2’ , 4, 4’, 5, 5 -
Hexachiorobiphenyl by Rabbits, Rats and Mice, Chemosphere , 4 (1976) 249-253
41. Gardner, A. M., Chen, J. T., Roach, 3. A. G., and Ragelis, E. P.,
Biochem. Biophys. Res. Comm. , 55 (1973) 1377
42. Wyndham , C., Devenish, J., and Safe, S., The In Vitro Metabolism, Macro-
molecular Binding and Bacterial Mutagenicity of 4-Chiorobiphenyl, A Model
PCB Substrate, Res. Communs. Chem. Pathol. Pharrnacol. , 15 (1976) 563-570
43. Jensen, S., and Jansson, B., Methyl Sulfone Metabolites of PCB and DDE,
Ambio , 5—6 (1976) 257—260
44. Vos, 3. G., and Notenboom-Ram, E., Comparative Toxicity Study of 2,4,5,2’,4’,5’-
Hexachiorobiphenyl and a Polychiroinated Biphenyl Mixtuie in Rabbits, Toxicol.
Appl. Pharmacol. , 23(1972) 563
192
-------
45. Figita, S., Tsuji, Kato, K., Saeki, S., and Tsukamoto, H., Effects of Biphenyl
Chlorides on Rat Liver Microsomes, Fukuoka Acta. Med. , 62 (1971) 30
46. Ecobichon, D. J., and MacKenzie, D. 0., The Uterotrophic Activity of Commercial
and Isomerically Pure Chlorobiphenyls in the Rat, Res. Commun. Chem. Pathol.
Pharmcol. , 9(1974)85
47. Chen, P. R., Mehendale, H. M., and Fishbein, L., Effect of Two Isomeric Tetra-
chlorobiphenyls on Rat and Their Hepatic Microsomes, Arch. Env. Contam.
Tox. , 1(1973)36
48. Johnstone, G. J., Ecobichon, D. J., and Hut zinger, 0., The Influence of Pure
Polychiorinated Biphenyl Compounds on Hepatic Function in the Rat, Toxicol.
Appi. Pharmacol. , 28 (1974) 66
49. Jao, L. T., Hass, J. R., and Mathews, H. B., In Vivo Metabolism of Radioactive
4-Chloro- and 4,4’-Dichlorobiphenyl in Rats, Toxicol. Appl. Pharmacol. , 37
(1976) 147
50. Mathews, H. B., and Tuey, D. B., The Distribution and Excretion of 3, 5,3 , 5’-
Tetrachiorobiphenyl in the Male Rat, Toxicol. Appi. Pharmacol. , 37 (1976) 148
51. Seymour, J. L., Peterson, R. E., and Allen, J. R., Tissue Distribution and
Biliary Excretion of 2,5,2’, 5 ’-Tetrachlorobiphenyl (TCB) and 2,4,5,2’, 4’, 5’-
Hexachiorobiphenyl (HCB) in Male and Female Rats, Toxicol. AppI. Pharmacol. ,
37 (1976) 171
52. Kimbrough, R. D., Linder, R. E., and Gaines, T. B., Morphological Changes in
Livers of Rats Fed Polychiorinated Biphenyls: Light Microscopy and Ultrastructure,
Arch. Env. Hlth. , 25(1972) 354
53. Nagasaki, H., Tomii, S., Mega, T., Maruganii, M., and Ito, N., Hepatocar-
cinogenicity of Polychiorinated Biphenyls in Mice, Garin , 63 (1972) 805
54. Nishizumi, M., Light and Electron Microscope Study of Chlorobiphenyl Poisoning
in Mouse and Monkey Liver, Arch. Env. Hith. , 21 (1970) 620
55. Platonow, N. S., and Chen, N. Y., Transplacental Transfer of Polychiorinated
Biphenyls (Aroclor 1254) in Cow, Vet. Rec. , 92 (1973) 69
56. Kasza, L., Weinberger, M. A., Carter, C., Hinton, D. E., Trump, B. F., and
Brouwer, E. A., Acute, Subacute and Residual Effects of Polychlorinated Bi-
phenyl (PCB) in Rats. II. Pathology and Electron Microscopy of LIver and Serum
Enzyme Study, J. Toxicol. Env. Hith. , 1 (1976) 689
57. Allen, 3. R., Carstens, L. A., and Abrahamson, L. 3., Responses of Rats Exposed
to Polychlorinated Biphenyls for 52 Weeks, 1. Comparison of Tissue Levels of
PCB and Biological Changes, Arch. Env. Contam. , 4 (1976) 404-419
193
-------
58. Grant, D. L., Moodie, C. A., and Phillips, W. E. J., Toxicodyanamics of
Aroclor 1254 in the Male Rat, Env. Physiol. Biochem. , 4 (1974) 214
59. Fuhremann, T. W., and Lichtenstein, E. P., Increase in the Toxicity of Organo-
phosphorus Insecticides to House Flies Due to Polychiorinated Biphenyl Com-
pounds, Toxicol. Appl. Pharmacol. , 22 (1972) 628
60. Villeneuve, D. C., Grant, D. L., and Phillips, W. E. J., Modification of Pento-
barbital Sleeping Tunes in Rats Following Chronic PCB Ingestion, Bull. Env.
Contam. Toxicol. , 7(1972) 264
61. Anon Worrisome PCB Levels Found in Mothers’ Milk in 10 States, Toxic Mat.
News , 3(1976) 141
62. Anon, PCB Contamination of Mothers’ Milk Spurs Inquiry, Eriv. Hith. Letter ,
15 (1976) 4
63. DHEW/EPA, Meeting to Review and Evaluate EPA’s Sampling of PCBs in the
Fat of Mothers’ Milk, DHEW Committee to Coordinate Toxicology and Related
Programs in Cooperation with Environmental Protection Agency, Bethesda,
MD, Sept. 23, 1976
64. Anon, The Rising Clamor About PCBs, Eriv. Sci. Technol. , 10 (1976) 122-123
65. Neeley, W. B., A Material Balance Study of Polychiorinated Biphenyls in Lake
Michigan, Sci. Total Environ. , 7 (1977) 117-129
66. Plimmer, J. R., and Kligebiel, U. L., PCB Formation, Science , 181 (1973)
994-995
67. Moilanen, K. W., and Crosby, Amer. Chem. Soc. Meeting, 165th, Dallas, TX
April (1973)
68. Uyeta, M., Taue, S., Chikasawa, K., and Mazaki, M., Photoformation of Poly-
chlorinated Biphenyls from Chlorinated Benzenes, Nature , 264 (1976) 583-584
68a. Gaffney, P. E., Chiorobiphenyls and PCBs: Formation During Chlorination,
J. Water Pollut. Control , 49 (1977) 401-404
69. Saeki, S., Tsutsui, A., Oguri, K., Yoshimura, H., and Hamana, M., Isolation
and Structure Elucidation of the Amin Component of KC-400 (Chiorobiphenyls),
Fukuoka Acta Med. , 62(1971)20
70. Kuratsune, M., et al., Morikawa, Y., Hirohata, T., Nishizumi, M., Kochi, S.,
An Epidemiologic Study on “Yusho” or Chlorobiphenyls Poisoning, Fukuka
Acta Med. , 60(1969) 513—532
71. Kuratsune, M., Yoshimura, T., Matsuzaka, 3., et al., Yusho, A Poisoning
Caused by Rice Oil Contaminated with Chlorobiphenyls, Env. Hlth. Persp. ,
1 (1972) 119
72. Umeda, G., Kanemi Yusho: PCB Poisoning in Japan, Amb o (1972) 132-134
194
-------
73. Hirayma, T., Meeting on Origins of Human Cancer, Cold Spring Harbor, NY
Sept. 7—14 (1976)
74. Balm, A. K., Rosenwaike, I., Herrmann, N., et al., Melanoma After Exposure
to PCBs, New Engi. 3. Med. , 295 (1976) 450
75. Anon, Mobil Study Suggests Possible Link Between PCBs and Human Cancer,
Occup. Hith. Safety Letter , 6 (1976) 4
76. Anon, High Cancer Rates Among Workers Exposed to PCBs, Chem. Erig. News ,
54 (1976) 26
77. Anon, Monsanto Finds No Link Between Cancer and PCBs in Workers, Occup.
Hith. Safety Letter , 5 (1976) pp. 5-6
78. Nagasaki, H., Tomii, S., and Mega, T., Hepatocarcinogenicity of Polychiorinated
Biphenyls in Mice, Gann , 63 (1972) 805
79. Nagasaki, H., Tomii, S., Mega, T., Marugami, M., and Ito, N., Development
of Hepatomas in Mice Treated with Benzene Hexachloride, Gann , 64 (1971) 431
80. Kimura, N. T., Baba, T., Neoplastic Changes in the Rat Liver Induced by
Polychiorinated Biphenyl, Gann , 64 (1973) 105
81. Ito, N., Nagasaki, H., Arai, M., Makiura, S., Sugihara, S., and Hirao, K.,
Histopathologic Studies on Liver Tumorigenesis Induced by Mice by Technical
Polychiorinated Biphenyls and Its Promoting Effect on Liver Tumors Induced
byBenzeneHexachloride, 3. Nati. Cancer Inst. , 51(1973) 1637
82. Kimbrough, R. D., Squire, R. A., Lmder, R. E., Strandberg, 3. D., Montali,
R. 3., and Burse, V. W., Induction of Liver Tumors in Sherman Female Rats
by Polychiorinated Biphenyl Aroclor 1260, 3. Nati. Cancer Inst. , 55 (1975)
1453—1459
83. Kimura, N. T., Kanematsu, I., and Baba, T., Polychlorinated Biphenyl(s) as
a Promotor in Experimental Hepatocarcinogenesis in Rats, Z. Krebsforsch. Kim.
Onkol. , 87(1976) 257—266
84. Ramel, C., Mutagenicity Research and Testing in Sweden, Mutation Res. , 33,
(1975) 79—86
85. Nilsson, B., and Ramel, C., Genetic Tests on Drosophila Melanogaster with
Polychiorinated Biphenyls (PCB), Hereditas , 77 (1974) 319
86. Hoopingarner, R., Samule, A., and Krause, D., Polychiorinated Biphenyl
Interactions with Tissue Culture Cells, Env. Hith. Persp. , 1 (1972) 155
87. Keplinger, M. L., et al., Toxicological Studies with Polyclorinated Biphenyls
PCB Conference, Quail. Roost. Conf. Center, Rougemorit, NC, Dec. (1971)
195
-------
88. Green, S., Sauro, F. M., and Friemann, L., Lack of Dominant Lethality in Rats
Treated with Polychlorinated Biphenyls, Food Cosmet. Toxicol. , 13 (1975) 507
89. Green, S., Carr, J. V., Palmer, K. A., and Oswald, E. J., Lack of Cytogenetic
Effects in Bone Marrow Spermatogomal Cells in Rats Treated with Polychlorinated
Biphenyls, Bull. Env. Contam. Toxicol. , 13 (1975) 14
90. Ames, B. N., Darston, W. E., Yamasaki, E., and Lee, F. D., Carcinogens are
Mutagens: A Simple Test for Combining Liver Homogenates for Activation and
Bacteria for Detection, Proc. Nail. Acad. Sci. , 70 (1973) 2281-2285
91. Ghiasuddin, S. M., Nenzer, R. E., and Nelson, J. 0., Metabolism of 2,5,2’ -
Trichioro-, 2,5,2’, 5’-Tetrachloro- and 2,4,5, 2’, 5’-Pentachlorobiphenyl in
Rat Hepatic Microsomal Systems, Toxicol. Appi. Pharmacol. , 36 (1976) 187-194
92. Allan, J. R. and Norback, D. H., Pathobiological Responses of Primates to
Polychlorinated Biphenyl Exposure, In National Conference on Polychlorinated
Biphenyls, Proceedings, 43-49 (1975)
93. Allan, J. R. and Norback, D. H., Carcinogenic Potential of the Polychlorinated
Biphenyls, In Origins of Human Cancer Meeting, Cold Spring Harbor, New York,
Sept. 7-14 (1976) Abstract p. 75
94. Grant, D. L., Viileneuve, D. C., McCully, K. A., and , W. E. J.,
Placental Transfer of Polychiorinated Biphenyls in the Rabbit, Env. Physiol. ,
1 (1971) 61
95. Takagi, Y., Otake, T., Kataoka, M., Murata, Y., Aburada, S., Akasaka, S.,
Hashirnoto, K., Uda, H., and Kitaura, T., Studies on the Transfer and Distri-
bution of 4 C-Polych1orinated Biphenyls from Maternal to Fetal and Suckling
Rats, Toxicol. Appi. Pharmacol. , 38 (1976) 549-558
96. Kojima, T., Fukumoto, H., Makisumi, S., ChlorQ-Biphenyl Poisonings, Gas-
Chromatographic Detection of Ch.loro-Biphenyls in the Rice Oil and Biological
Materials, Jap. J. Legal Med. , 23 (1969) 415
97. Inagaini, K., Koga, T., Tomita, Y., Poisoning of Chlorobiphenyls, Shokuhin.
Eisei Gaku Zasshi , 10 (1969) 415
98. Vos, J. G., and Koeman, J. H., Comparative Toxicologic Study with Polychlori-
nated Biphenyls in Chickens with Special Reference to Porphyria, Edema Forma-
tion, Liver Necrosis, and Tissue Residues, Toxicol. Appi. Pharm. , 17 (1970) 565
99. Tumasonis, C. F., Bush, B., and Baker, F. D., PCB Levels in Egg Yolks
Associated with Embryonic Mortality and Deformity of Hatched Chicks, Arch.
Env. Contarn. Toxicol. , 1 (1973) 312
100. Gilbertson, M., and Hale, R., Early Embryonic Mortality in a Herring Gull
Colony in Lake Ontario, Canad. Field-Naturalist , 88 (197 ) 354
196
-------
101. Schwetz, B. A., Norris, J. M., Sparschu, G. L., Rowe, V. K., Gehring, P.
J., Emerson, J. L., and Gerbig, C. G., Toxicology of Chlorinated Dibenzo-
p-dioxins, Env. Hith. Persp. , 5(1973)87
102. Vos, J. G., Koeman, 3. H., Vandermaas, H. S., Debraun, M. C. and DeVos,
H., Identification and Toxicological Evaluation of Chlorinated Dibenzofuran and
Chlorinated Naphthalene in Two Commercial Polychiorinated Biphenyls, Food
Cosmet. Toxicol. , 8(1970) 625—633
103. Vos, 3. G., and Koeman, 3. H., Dermal Toxicity Studies of Technical Polychiori-
nated Biphenyls and Fractions Thereof in Rabbits,
Toxicol. Appi. Pharmacol. , 19 (1971) 617
104. Sundstrom, G., Hutzinger, 0., and Safe, S., Identification of 2,2’ ,4,4’, 5, 5’-
Hexabromobiphenyl as the Major Component of Flame Retardant, Firemaster
BP-6, Chemosphere , 1 (1976) 11
105. Carter, L. 3., Michigan’s PBB Incident: Chemical Mix-up Leads to Disaster,
Science , 192 (1976) 240
106. Dent, 3. G., Netter, K. 3., and Gibson, 3. E., Characterization of Various
Parameters of Rat Hepatic Microsomal Mixed Function Oxidase after Induction
by Polybrominated Biphenyls, Toxicol. Appi. Pharmacol. , 37 (1976) 147
107. Ficsor, G., and Wertz, G. F., Polybrominated Biphenyl Nonteratogenic C-
rnitosis Synergist in the Rat, Presented at 7th Annual Meeting of Environmental
Mutagen Society, Atlanta (1976)
197
-------
VU. Hydra zines, Hydroxylamines, Carbamates
A. Hydra zine and Derivatives
Hydrazine and its derivatives have many properties similar to those of amines
in forming gaits and acyl derivatives, as well as undergoing alkylation and conden-
sations with carbonyl compounds.
Hydrazine and a number of its derivatives possess a broad spectrum of utility
in the preparation of agricultural chemicals,. medicinals, textile agents, explosives,
fuels, plastics, preservatives, blowing agents, and in metal processing (Table 1).
1. Hydrazine (H 2 N-NH 2 ; diamide, diamine) is used primarily in the manufacture
of the herbicides maleic hydrazide and 3-amino-l,2,4-triazole; blowing agents such
as azodicarbonamide, benzenesulfonylhydrazide and 4,4’ -oxybis (ben zene-sulfonyl-
hydrazide); the manufacture of medicinals such as isoniazid; in rocket fuels; as
hydrazine hydrate in boiler feedwater treatment; and as a chemical intermediate in
the production of the hydrazine salts used in soldering fluxes’.
Hydrazine or hydrazine salts (e.g., sulphate) have been shown to be carcino-
genic in mice after oral and intraperitoneal administration and in rats following oral
athninistration ’ 6 .
Hydrazine has been shown to be an effective agent in producing mutations in
phage 7 , bacteria 8 1 , higher plants 12 ’ 13 , and Drosophila 14 ’ l5• Hydrazine did not
produce detectable levels of dominant lethals in mice 16 ’ 17 though it did produce
mutations in S. typhiinurium used in the host-mediated assay in mice 18 . It should
be noted that the dominant lethal assay tests primarily for chromosomal aberrations,
where as most of the successful tests with hydrazine have been for mutations more
likely to be single locus events 10 .
198
-------
2. 1,1-Ditnethyl hydrazine (CH 3 \ ; UDMH; unsymmetrical dimethyl hydra-
CM/N NH 2
zine) is made by the reduction of N-nitrosodimethylamine’ (a potent carcinogen ’ 9 ’ 20
21
and mutagen ) is used principally as a storable high-energy propellant for liquid-
fueled rockets, and in the manufacture of N-dimethylaminesuccinamic acid, a plant-
growth regulator. Potential uses of 1, 1-dimethyihydrazine include its use as a chemical
intermediate in the manufacture of aminimides 1 , and as a cross-linking catalyst for the
22,23
production of polymethacrylate anaerobic adhesives
UDMH has been reported to induce abnormalities in the morphology of sperm in
the Cauda epidymides of mice which reached maximum levels < 3 weeks after exposure
of the animals to their agent 24 . UDMH is carcinogenic in mice after oral administration 1 ’ 25 ’ 26
3. 1, 2-Dimethylhydrazine (CH 3 -NH-NH-CH 3 ; SDMH; symmetrical-dimethyihydrazine)
is carcinogenic in mice, rats and hamsters following oral, subcutaneous or intramuscular
administration 27 . SDMH is considered not to be carcinogenic se, but is activated in
vivo by metabolic processes to form the ultimate carcinogen 27 ’ 28 The postulated
activation pathway of this carcinogen proceeds by a series of oxidations through azo-
methane, azoxymethane and methylazoxymethanolto form the proximate carcinogen
28,29
methyldiazonium hydroxide
It should be noted that although small quantities of SDMH are offered, there is no
aDparent commercial quantity produced nor are there now known commercial uses for
DMH 5 .
4. Hydrazine carboxamide (I-I 2 NCONHNH 2 ; semicarbazide; aminourea; carbamyl
hydrazine) is a nitrogen nucleophilic reagent that is used extensively for the prepara-
tion of semicarba zones, viz., R 2 C0 + NH 2 NHç -NH 2 - R 2 C NNHCONH 2 . Usually
0
these derivatives are solids and are excellent for the isolation and characterization of
aldehydes and ketones in synthesis and analysis. Other suggested areas of utility of
199
-------
semicarbazide include: in the manufacture of Thiokol rubber foam 30 ; as a cross-linking
agent for oxidized ethylene polymers 31 ; and acrylic fibers 32 ; as a stabilizer for ethylene-
vinylacetate polymers 33 ; in the synthesis of plant-growth regulators 34 ; in the pre-
paration of anion-exchangers from polyethylene-polyamines 35 ; and in phosphors 36 .
Semicarbazide is teratogenic in chick embryo 37 and has recently been reported to
exhibit mutagenic action on the spermatocyte chromosomes of the grasshopper, Spatho-
sternum prasiniferum 38 . Aberrations such as chromatid and chromosome breaks,
translocations, fragments and bridges were found with the sex chromosome and the
long antosonies being affected. It was postulated that semicarbazide reacts with DNA
and the chromosome in a manner analogous to that of hydroxylamine and hydra zine.
Hydroxlamine and semicarbazide have a common chemical affinity for the carboxyl group.
Hydroxylamine liberates all the four base pairs from DNA which, in turn, results in
the breakage of the sugar-phosphate backbone 38 . Mitra 39 previously reported the
induction of chromosome aberrations on mouse marrow chromosomes while Rieger and
Michaelis 40 observed no effect on Vicia faba chromosomes.
It is also of importance to consider major compounds which yield hydrazine and
acetyl hydrazine as metabolic products. Recent clinical studies have suggested that
the hydrazine moiety of the widely used antituberculosis, isoniazid (N CONHNH 2
isonicotinyihydrazide; INH) may be responsible for the serious hepatitis
that has been observed 41 ’ 42 . Acetyl hydrazine is also a metabolite of iproniazid
(isonicotinyl acid; 2-isopropyihydrazine; N -CONHNHCH(CH 3 ) 2 ) an anti-
43,44
depressant removed from chrucal use because of high incidence in liver injury
The similar fates of acetylisoniazid and isopropylisoniazid support the view that hepatic
injury caused by hydrazide drugs may be due to the metabolic activation of their
hydra zine moieties 4 :). Nelson et a1 45 recently demonstrated that acetyihydrazine and
200
-------
isopropylhydrazine were oxidized by cytochrom P-450 enzymes in human and rat liver
microsomes to highly reactive acylating and alkylating agents covalent binding of
these metabolites to liver macromolecules paralled hepatic cellular necrosis. The
metabolites formed from these and probably other monosubstituted hydrazines are
reactive electrophiles.
A reaction scheme which was consistent with the experimental findings was proposed
45
as follows : OH
Microsomal H
R--N--NH 2 - > R--N--NH
oxidation
Tissue acylation
R--N NH - or alkylation
4 f
R+, R - RH
R=CH 3 -C=O,CH 3 -CH-CH 3
Another possible mechanism involves a second oxidation of the diazene to form a
diazohydroxide, a reactive intermediate similar to that envisioned by Magee and Barnes 46
for carcinogenic nitrosamines and by Druckrey 47 for 1, 2-diakyihydrazines.
The mutagenicity of isonicotinyihydrazine in E. coli 4850 has been reported.
Substituted hydrazine derivatives are receiving considerable attention in pharma-
cology and toxicology because of their widespread use as herbicides and rocket fuels,
as intermediates in chemical synthesis, and as therapeutic agents for the treatment of
tuberculosis, depression, and cancer. Besides the liver necrosis found in therapy
with isoniazid and iproniazid, hydrazines are known to produce many other toxic res-
ponses including methemoglobinemia, hemolysis, fatty liver, mutagenesis, and carcino-
genesis 5153 .
Table 2 lists 20 tumorigenic hydrazine compounds in terms of their structures,
species tested, effected organs and route of administration 3 . It should be restressed
that a number of these hydrazines are used in industry, agriculture and medicine.
201
-------
They include, in addition to hydrazine, 1, 1-dimethyihydrazine, monomethylhydrazine,
carbamylhydrazine, isonicotinylhydrazide discussed above, derivatives such as 2-
hydroxyethylhydrazine, a ripener for pineapple and other plants 54 and N-isopropyl-
(2-methyihydra zino )p-toluamide , an antineoplastic drug 55 .
To what extent humans are exposed to the industrial hydra zines is presently not
known, nor are there sufficient data relating to the extent of residues in food of the
ripening agent 2-hydroxyethylhydrazine, or maleic hydrazidide (a widely used plant
growth retardant which can contain traces of free precursor hydra zine).
The mutagenicity of hydrazine and some of its derivatives has recently been
reviewed by Kimball 56 . Hydrazine can react with the pyrimidiner in DNA to saturate
the 5,6 double bond (especially of thymine) to form N 4 -aminocytosine. It can also
open up the pyrimidine ring with consequent loss of pyrimidines from DNA. Hydrazine
can react either directly with DNA or through intermediate radical reactions including
the formation of H 2 0 2 . A number of substituted hydrazines can also act in much the
same way. Other hydrazines, especially the methyl derivatives, can act as alkylating
56
agents to alkylate purines, primarily
Although hydrazine E se has riot been reported to produce chromosomal aberrations,
several of its derivatives including isoriiazid (which is believed to produce hydrazine
in vivo ) have been reported to produce chromosomal aberrations and other nuclear
anomalies. Table 3 lists these compounds and their reported effects. It should be noted
that most of the compounds producing chromosomal effects are methylated derivatives
of hydrazine which might be acting as alkylating agents 56 .
The metabolic fate of hydrazines 57 ’ 58 and have been recently reviewed.
iU humans and most other mammalian species, one of the most important biotransformation
reactions affecting hydrazine-hydrazide compounds is acetylaticri of the terminal nitrogen
202
-------
group. Biotransformation appears to be essential to the capacity of many hydra zine
derivatives to inhibit monoamine oxidase. Other reactions of the hydrazines, e . g.,
hydrolysis, oxidation, and reduction frequently results in the formation of metabolites
57,58
with potent biological effects
203
-------
TABLE 1
Utility of Hydrazine and Its Derivatives
Application Form
1. As a reducing agent
a. Corrosion inhibitor (oxygen Hydra zine
scavenger)
b. Silver plating of glass and plastics Hydrazine hydrate
c. Soldering fluxes Hydrazine hydrochloride
e. Inhibitor of color and odor forma- Stearic hydra zide
tion in soaps
2. Reactive chemicals
a. Extender for urethan polymers Hydrazine
b. Terminator of emulsion polyxneri- Hydrazine with dialkyl dithiocarbamates
zation
c. Curing of epoxy resins Dihydrazides
d. Rocket fuel Hydrazine; unsym. dimethyihydrazine
e. Blowing agent 2 ,2’ zoisobutyronitrile a zodicarboamide
3. Agricultural chemicals
a. Plant growth regulators Maleic hydra ide
b. Defoliants 3—Amino-i, 3, 4—triazole
c. Plant growth stimulator -Hydroxyethylhydrazine
4. Medicinals
a. Antitubercular agents Isonicotinic acid hydrazide
b. Psychic energizers 3 -Phenylisopropyihydra zine
c. Hypotensive agents l-Hydrazinophthalazine
d. Topical antiseptic Nitrofura zone
e. Polycythemia vera Phenyihydrazine
204
-------
Tjhk 2
Tumorigenic h,dra:ine compounds
Compound Species Organ Treatment References
Mice Lungs p.o. 39
l ’kit.t-t. , ,t,’t—’n.
CH ,—CH ,—CH 3 —CH 1 —CH,—NH—NH 3 • HO \Iice Lungs, blood cssds p.o. 33
N-Arnylhydrazine HO
Slice Lungs. l mphoreticular po. 2,34
tissue
k.s,vsa,ni.
NH 5 —NH ---CH ,—CH ,—CH ,—CH,HCI Slice Lungs p.o. 44
N Butylhydrazine HCI
NH 2 —NH---CO—NH 1 .HC I Mice Lungs.bloodvessels p.o. 43
Carbamyihydrazine. HCI
o
Mice Lungs p.o.
CH,—CH 2 ——NH—NH—CH ,—CH,2HC I Rats Lvmphoreiicuiar and nerve s.c. 5
l,2-Oiethylhvdrazinc 2HC I tissucs. liver. eihmotur.
binal
(CH ,) 1 N_NH 1 Mice Lungs, blood vessds. kidney. p.o. 2 . 37
I. l-Dimeth)lhydrazlne liver
CH,—NH— Il—C H, 211€ I SI ice Colon, lunes. h!,nid . seI’s s p 0. 46, 52
l,2-Dime thyihydrazine . 2HCI Hamsters Liver. ioniach. intestine. m., p.o. 27. 36
blood vcs cls
Rats Intestine s.c., p.o. 6
NH ,—NH—CH,—CH , HC1 Mice Lungs. blood vcsscls p.o.
Etnylhydraztne. HCI
NH 3 —NH,. 11250, Mice Lungs. liver p.o
Hydrazine sulrate Rats Liver, lungs p.o. 32
Nil ,—NH.—-CH ,—CH,OH Mice Liver p.o.
2-Hydroxyethy Ihydrazine
Slice Lungs p o. 2
Mice Lungs. l’.niphoreticular p.o.. i.p. 13
0 0 ’ tissue, kidne%
Rats Breast. lur’gc. blood p.o.. p. 14
,,
Slice Lungs p a. 2
• - r_..N.i,.t_ .LIn
‘3 Slice Lungs p o.
I —
C1l,—\H—\ 11 3 Mice Lung’s po. 35
Methylhvdraiir ie Hamsters kupifer cells. cecum p.o. 40
‘i Rats Central and peripheral ncr- s.c.. p.o. 4
vous sysicrils. huibu, ol-
factorius
(if,—NH—\H—CH ,—(il ,—Cf!,—Cli, 2HCI R is Large ir.tcstine. hu hus p0. 4
l__%lcus.l_2_buis .1r.i I le lit. oll acirius
C (‘ -—-—.
\1ce lungs p .o.
205
-------
TABLE 3 56
CHItOMOSOMAL ABEft AT!ONS AND ANOMALIES PRODUCED BY HYDRAZXNE DE IVATXVES
Compound
Test object
Effect
Methvlhytirarmnc
Human Icukocytci in vitro
from treated patients
Chromutid aberrations
I .2.Dimcthylhydrazine
Sacchurnmyces cereuisiae
Mitotic crossing-over
1-((N-Methvuiydyazjno)methyll-N- tsopropyl benzamide
Ehrhch ascites cells
Chromatid aberrations
(sometimes cafled ‘ethylhydrzine’)
Eh:lich ascites cells
I I m;itopi wtii, eiIl f T I L
Karyotype changes, mitotic inhibition
ChroI?I a t d iberra tions
2-1 e,i,.v(—t—iuethy Ihydrarinc
Ehrlidi ascites cells
. sciLcs tumor cells in vitro
Ehrlich ascites and ucLa
cells in vitro
Bone marrow. spleen. nar-
cissus root tips in vivo
Human lymphocytes and
mouse spicea cells in
vitro
Cliruniatid aberratiøns. mitotie itilill, itiuii
Transtucations
No aberrations
No abcrration
No aberrations
S jccinic acid. mono(2,2-dimethylhydraiide)
Root tips from soaked barley
seeds, pollen mother cells
from plants germinated
from soaked seeds
Various chromosome abnormalities
X-(Methylhydrazinomethyl) nicotir.amide
Lep d um satiuum
Mitotic inhibition
2-Methvthydrazide-3-r .itroquirtoiine4-ca boxy1ic aced
Lep dium saiiuum
Mitotic inhibition
3-Thiosemicarbazide
Meiotic cells in Vicja (aba
plants sprayed with
compound
Various abnormalities
Isonicotinic hydrazitle (isoniazid)
}himan Icukocytes in vitro
Rat bone niarrow
Chromatid aberrations. achromatic letions
Gaps. breaks, deletions. fragments
lson cotinic 2-isopropylhydrazidu (iproniazide)
Vicia lobe root tips
Normal karyotype. mitotic inhibition
206
-------
References for Hydrazine and Derivatives
1. IARC, Vol . ‘, International Agency for Research on Cancer, Lyon, France,
pp. 127—136; 137—143(,i )
2. Biancjfjorj, C., and Seven, L., The Relation of Isoniazid (INH) and Allied
Compounds to Carcinogenesis in Some Species of Small Laboratory Animals:
A Review, Brit. 3. Cancer , 20 (1966) 528
3. Kelly, M. G., OGara, R. W., Yancey, S. T., Gadekar, K., Botkin, C., and
Oliverio, V. T., Comparative Carcinogenicity of N-isopropyl-cc-(2-methyl-
hydra zino) -p-toluamide HC1 (Procarbazine Hydrochloride), its Degradation
Products, Other Hydrazines, and Isonicotinic Acid Hydrazide, J. Nati. Cancer
Inst. , 42(1969) 337
4. Toth, B., Investigation on the Relationship Between Chemical Structure and
Carcinogenic Activity of Substituted Hydrazines, Proc. Am. Ass. Cancer
Res. , 12(1971)55
5. Toth, B., Lung Tumor Induction and Inhibition of Breast Adenocarcinomas by
Hydrazine Sulfate in Mice, 3. Nati. Cancer Inst. , 42 (1969) 469
6. Seven, L., and Biancifiori, C., Hepatic Carcinogenesis in CBA/Cb/Se Mice
and Cb/Se Rats by Isonicotini Acid Hydrazide and Hydrazine Sulfate, 3. Natl.
Cancer Inst. , 41(1968) 331
7. Chandra, S. V. S. G., and Reddy, G. M., Specific Locus Mutations in Maize
by Chemical Mutagens , Curr. Sci. , 40(1971)136-137
8. Lingens, F., Mutagene Wirkung Von Hydrazin Aug E. Coli-Zellen, Naturwiss ,
48 (1961) 480
9. Lingens, F., Erzeugung Biochemischer Mangel Mutanten Von E. Coli Mit Hilie
Von Hydrazin Und Hydrazin Derivaten, Z. Naturforsch. , 193 (1964) 151-156
10. Kimball, R. F., and Hirsch, B. F., Tests for the Mutagenic Action of a Number
of Chemicals on Haemophilus Influenzae with Special Emphasis on Hydrazine,
Mutation Res. , 30(1975) 9—20
11. Kimball, R. F., Reversions of Proline-Requiring Auxotrophs of Haemophilus
Infiuenzae by N-methyl-N’ -nitro -nitrosoguanidine and Hydra zine, Mutation
Res. , 36(1976) 29—38
12. Jam, H. K., Raut, R. N., and Khamanker, Y. G., Base-Specific Chemicals
and Mutation Analysis in Lycopersicon, Heredity , 23 (1968) 247
13. Chu, B. C. F., Brown, D. M., and Burdon, M. G., Effect of Nitrogen and
Catalase on Hydroxylamine and Hydrazine Mutagenesis, Mutation Res. , 20
(1973) 265—270
207
-------
14. Shukla, P. T., Analysis of Mutagen Specificity in Drosophila Melanogaster,
Mutation Res. , 16(1972) 363—371
15. Jam, H. K., and Shukla, P. T., Locus Specificity of Mutagens in Drosophila,
Mutation Res. , 14 (1972) 440—442
16. Epstein, S. S., Arnold, E., Andrea, J., Bass, W., and Bishop, Y., Detection
of Chemical Mutagens by the Dominant Lethal Assay in the Mouse, Toxicol.
Appl. Pharmacol. , 23(1972)288
17. Epstein, S. S., and Shafner, H., Chemical Mutagens in the Human Environment,
Nature , 219 (1968) 385
18. Rôhrborn, G., Propping, P., and Buselmaier, W., Mutagenic Activity of
Isoniazid and Hydrazmne in Mammalian Test Systems, Mutation Res. , 16 (1972)
189
19. Magee, P. D., and Barnes, J. M., Carcinogenic Nitroso Compounds,
Adv. Cancer Res. , 10(1967) 163
20. Druckrey, H., Preussmann, R., and Schm hl, D., Carcinogenicity and Chemical
Structure of Nitrosamines,
Acta. Unio. Intern. Contra. Cancrum. , 19 (1963) 510
21. Fishbein, L., Flamm, W. G., and Falk, H. L., “Chemical Mutagens”, Academic
Press, New York, (1970) pp. 165-167
22. Tsuji, I., Azuxna, K., Kato, H., Tachimichi, H., Motegi, A., and Suzuki, 0.,
Cross-linking Accelerators for Anaerobic Adhesives, Japan Kokai., 74, 120,889,
Nov. 19 (1974), Chem. Abstr. , 82, (1975) P126144M
23. Azuina, K., Tsuji, I., Kato, H., Tatemichi, H., Motegi, A., Suzuki, 0., and
Kondo, K., Anaerobic-Hardenable Adhesives, Ger. Offen., 2,402, 427, August 8
(1974), Chem. Abstr. , 82, (1975) 59020C
24. Wyrobek, A. J., and London, S. A., Effect d Hydrazines on Mouse Sperm Cells,
Proc. Ann. Conf. Environ. Toxicol. 4th, (1973), AD-781031, pp. 417-432,
Chem. Absti- , 82, (1975) 150111U
25. Toth, B., Comparative Studies with Hydrazine Derivatives. Carcinogenicity
of 1. 1-Dirnethylhydrazine, Unsymmetrical (1, 1—DMH) in the Blood Vessels,
Lung, Kidneys, and Liver of Swiss Mice, Proc. Am. Assoc. Cancer Res. , 13
(1972) 34
26. Toth, B., 1,1-Dimethyl Hydrazmne (Unsymmetrical) Carcinogenesis in Mice.
Light Microscopk and Ultrastructural Studies on Neoplastic Blood Vessels,
J. Nati. Cancer Inst. , 50 (1973) 181
208
-------
27. IARC, 1 ,2-Dimethylhydrazine, In Vol. a/ , International Agency for Research
on Cancer, Lyon, France, pp. 145-152(i 2q)
28. Druckrey, H., Production of Colonic Carcinomas by 1,2-Dialkyihydrazines and
Azoxyalkanes, in “Carcinoma of the Colon and Antecedent Epithelium’ T , ed.
W. J. Burdette, Thomas Pubi., Springfield, Ill. (1970) pp. 267-279
29. Fiala, E. S., Kulakis, C., Bobotas, G., and Weisburger, J. H., Detection and
Estimation of Azomethane in Expired Air of 1,2-Dimethyihydrazine-Treated Rats,
J. Nati. Cancer Inst. , 56(1976) 1271
30. United States Rubber Co., Foamed, Vulcanized Polysulfide Rubber, Ger. Patent
1,229,717, Dec. 1, (1966), Chem. Abstr. , 67 (1967) P12366V
31. Carbazate Cross-linking Agent and Thermosetting Resins, Chem. Abstr. , 73 (1970)
P26293E
32. Zharkova, M. A., Kudryavtsev, G. I., Khudoshev, I. F., and Romanova, T. A.,
Khim. Volokna. , 2(1969)49
33. Meinc e, E. R., Color-stabilized Copolymers of Ethylene and Vinylacetate, Ger.
Offen., 1,953,693, June 18(1970) Chem. Abstr. , 73(1970) PJ6821T
34. Roechling, H., Hartz, P., and Hoerlein, G., Plant-Growth Regulators, Ger. Offen.,
2,3 2,000, Jan. 16 (1975), Chem. Abstr. , 82 (1975) 156326Q
35. Samborskii, I. V., Vakulenko, V. A., Chetverikov, A. F., Pedikova, L. N.,
and Nekrasova, L. G., Anion-Exchangers, USSR Patent 398,570, Sept. 27 (1973)
Chem. Abstr. , 81(1974) P106565A
36. Isojima, T., Phosphor, Japan Kokai, 73102,780, Dec. 24 (1973), Chern. Abstr. ,
81 (1974) P19153W
37. Bodit, F., Stoll, R., and Maraud, R., Effects of Hydroxyurea, Semicarbazide and
Related Compounds on Development of Chick Embryo, C.R. Soc. Biol. , 160 (1960)
960
38. Bhattacharya, A. K., Chromosome Damage Induced by Semicarbazide in Sperma-
tocytes of a Grasshopper, Mutation Res. , 40 (1976) 237
39. Mitra, A. B., Effect of pH on Hydroxylamine (HA) Phenyl Hydrazine (PH) and
Semicarbazide (SC) Induced Chromosome Aberration Frequency in Mice, J. C ’to1.
Genet. , 6 (1971) 123—127
40. Rieger, R., and Michaelis, A., Die Ausl sung Von Chromosomen Aberrations Bei
Vicia Faba Durch Chemische Agenzien, Die Kulturpflante , 10 (1962) 212
41. Mitchell, 3. R., Long, M. W., Thorgeirsson, U. P., and Jailow, D. 3., Acetylaticn
Rats and Monthly Liver Function Tests During One Year of Isoniazid Preventive Therapy
Chest , 68 (1975) 181
209
-------
42. Mitchell, J. R. , Thorgeirsson, U. P., Black, M., Timbrell, 3. A., etal., Increased
Incidence of Isoniazid Hepatitis in Rapid Acetylators: Possible Relation to Hydrazine
Metabolites, Clin. Pharmacol. Ther. , 18 (1975) 70
43. Mitchell, J. R,, Jollow, D. J., and Gillette, J. R., Relationship Between Metabolism
of Fo ign Compounds and Liver Injury, Israel 3. Med. Sci. , 10 (1974) 339
44. Snodgrass, W. R., Potter, W. Z., Timbrell, J. A., and Mitchell, 3. R., Possible
Mechanism of Isoniazid —Related Hepatic Injury, Clin. Res. , 22 (1974) 323A
45. Nelson, S. D., Mitchell, J. R., Timbrell, 3. A., Snodgrass, W. R., and Cororan,
G. B., Isonazid and Ipronazid: Activation of Metabolites to Toxic Intermediates in
Man and Rat, Science , 193 (1976)
46. Magee, P. N., and Barnes, 3. M., Carcinogenic Nitroso Compounds, Adv. Cancer
Res. , 10(1967) 163
47. Druckrey, H., Specific Carcinogenic and Teratogenic Effects of “Indirect T ’ Alkylating
Methyl and Ethyl Compounds and their Dependency on Stages of Ontogeriic Developments,
Xenobiotica , 3 (1973) 271
48. Freese, E., Bautz, E., and Freese, E. B., The Chemical and Mutagenic Specificity
of Hydroxylamine, Proc. Nail. Acad. Sci. US , 47 (1961) 845-855
49. Jam, H. K., and Raut, R. N., Differential Response of Some Tomato Genes To Base
Specific Mutagens, Nature , 211 (1966) 652
50. Lingens, F., Mutagenic Effect of Hydrazine on E.Coli, Z. Naturforsch. , 19b,
(1964) 151
51. Back, K. C., and Thomas, A. A., Aerospace Problems in Pharmacology and Toxi-
cology, Annual Rev. Pharmacol. , 10 (1970) 395
52. Juchau, M. R., and Herita, A., Metabolism of Hydrazine Derivatives of Interest,
Drug Metabi. Rev. , 1(1972) 71
53. Toth, B., Synthetic and Naturally Occurring Hydra zines as Possible Causative
Agents, Cancer Res. , 35(1975) 3693
210
-------
54. Gowing, D. P., and Leeper, R. W., Induction of Flowering in Pineapple by Beta-
Hydroxyethyl Hydrazine, Science , 122 (1955) 1267
55. Mathe, G., Schweisguth, 0., Schneider, M., Amiel, 3. L., Berumen, L., Brule, G.,
Cattan, A., and Schwarzenberg, L., Methyihydrazirie in Treatment of Hodgkin’s
Disease, Lancet , 2 (1963) 1077-1080
56. Kimball, R. F., The mutagenicity of hydrazine and some of its derivatives,
Mutation Res. , 39(1977) 111—126
57. Colvin, L. B., Metabolic fate of hydrazines and hydrazides, J. Pharm Sci. ,
58 (1969) 1433—1443
58. Juchau, M. R., and Horita, A., Metabolism of hydrazine derivatives of pharma-
cologic interest, Drug Metabolism Reviews , 1 (1973) 71-100
211
-------
B. Hydroxylamines
Hydroxylamine and its salts as well as certain of its derivatives are widely
employed (as nitrogen nucleophiles) in the transformation of organic compounds to
derivatives which in turn may be intermediates (e. g., oximes) in pharmaceutical
or other industrial syntheses of complex molecules. A major area of utility of hydro-
xylamine is in the synthesis of caprolactam, the raw material for nylon 6.
The oxidation-reduction capabilities of hydroxylamine make it useful in many
applications, e . g., as a reducing agent for many metal ions, and for the termination
of peroxide-catalyzed polymerizations.
Hydroxylamine (NH 2 OH) and its hydrochloride or sulfate salts are used in appli-
cations including: prevention of discoloration of rayon and cellulose products 1 ,
vinylidene chloride polymers 2 and paper-pulp 3 ; as bleaching agents for phenol
resin fibers 4 ; modification of acrylic fibers 5 ; fire-proofing of acrylic fibers 6 ; in
multicolor dyeing of acrylic fibers 7 ; as catalysts for polymerization of acrylamide 8 ;
conjugated diolefins 9 ; in electroplating 10 ; in soldering fluxes for radiators 13 ; in
photographic color developers ’ 2 and emulsions 13 ; in the stabilization of water solu-
tions of fertilizers 14 ; as antishining agents in paints; and complexing agents for metals
and as a laboratory reagent for the preparation and determination of oximes.
Hydroxylamine and certain hydroxylamine derivatives (e. g., CH 3 NHOH and
NH 2 OCH 3 ) (as well as the closely related hydrazine) are mutagenic to different
degrees in bacteria and to transforming DNA 14 . Each have in common the ability
to interact specifically with pyrimidines under specific conditions including pH,
concentration of reagent, and oxygen tension. Their mutagenicity also depends
markedly upon the above conditions, although, in general, hydroxylamine and its
analogs are appreciably more mutagenic than hydrazine.
212
-------
At high concentrations and high pH (approximately pH 9), hydroxylamine
(0.1 M to 1.0 M) reacts exclusively with the uracil moieties of nucleic acids.
At low pH (approximately pH 6) and high concentration (0. 1 M to 1. 0 M),
hydroxylamine reacts exclusively with cytosine moieties of DNA, aminating only
the C-4 atom’ 6 . Paradoxically, at lower concentrations of hydroxylamine, the
reaction involves all four bases’ 6 ’ 17 analogous to that reported for higher pH’s
(see above). Furthermore, it is well known that hydroxylamine is highly toxic
at low concentrations’ 8 , where it is only weakly mutagenic 19 , and yet, at the
higher concentrations at which it is mutagenic, little or no cytotoxicity is observed.
The explanation may relate to the degradation products of hydroxylamine (e.g.,
hyponitrous acid) rather than the compound per se 20 .
The reaction of hydroxylamine with cytosine and related compounds 21 its
effects and induction of mutations in transforming DNA 2225 , bacteriophage
26 27,28 29 30 .31
(S13 and X174 , T4 ), Neurospora , Saccharomyces pombe , E.coh
and induction of chromosome aberrations in human chromosomes 32 , cultured Chinese
33 34,35 36
hamster cells , mouse embryo cells and in Vicia faba . have been described.
N- and 0-derivatives of hydroxylamine, e.g., N—methyl- and 0-methyl-
hydroxylamine, have also been found to be mutagenic in transforming DNA of
B. subtillis 22 and Neurospora 37 , and to induce chromosome aberrations in Chinese
hamster cells 34 . The selective reaction of 0-methylhydroxylamine with the cytidine
nucleus has been reported by Kochetov et a1 38 .
Among the known chemical mutagens, hydroxylamine as well as its 0-
methyl and N-methyl analogs are of particular interest because of their apparent
specificity and ability to induce point mutations. The mutagenic activity is due
39—42
largely to its reactions with cytosine residues in DNA, or RINA , optimal in
2 3
-------
slightly acid medium. Under these conditions uracil reacts to a minor extent with
subsequent ring opening, so that in RNA this reaction is inactivating rather than
43
mutagenic . Adenine also reacts to a small extent with hydroxylamine. The
mechanism of reaction of hydroxylamine with 1-substituted cytosine residues is
illustrated in scheme i 13. The adduct I is a presumed intermediate, the instability
of which has prevented its detection or isolation. The final products are compounds
II and/or III, which are interconvertible under the conditions of the reaction 44 as
shown in scheme 1.
Evidence for compound III as the product responsible for hydroxylamine muta-
genesis is based largely on the observation that hydroxylamine is highly mutagenic
27,45,46
against the T-even bacteriophages , the DNA of which contains, in place
of cytosi.ne, free and/or glucosylated 5-hydroxymethylcytosine, and the demonstra-
tion that such 5-substituted cytosine residues react with hydroxylamine by only one
pathway to give uniquely compound IV, the 5-substituted analogue of compound III
47,48 4
(scheme 2) . In addition N -hydroxycytidine (i . e., compound III) has been
49, 50
shown to be highly mutagenic in two selected bacterial systems
43
In a recent study of Shugar et al involving the molecular mechanism of by-
droxylamine mutagenesis. it was reported that at least in the case of 5-substituted
cytosine residues in essentialDNA (such as found in the T-even and other bacterio-
phages), hydroxylamine mutations are unlikely to be due exclusively to simple
Watson-Crick base-pair transitions. Hence, hydroxylarnine, in a number of instances,
can affect transitions other than the normally expected C- U(T). Similar consi-
derations are believed to apply to mutations possibly resulting from the reaction
of hydroxylamine with adenine residues 43 .
214
-------
..r 3]
.,‘ J
II
C
215
-------
References for Nydroxylamines
1. Smith, F. R., and Scharppel, 3. W., Hydroxylamine-Treated Hemicellulose-
Containing Regenerated Cellulose Product, U. S. Patent 3,832,277. August 27
(1974) Chem. Abstr. , 81(1974) p. 154499D
2. Moore, C., Inhibiting Discoloration of Vinylidene Chloride Polymers, U. S.
Patent 3,817,895, June 18(1974), Chem. Abstr. , 81(1974) P1219303
3. Andrews, D. H., Canadian Patent 611,510 (1960), Chem. Abstr. , 55 (1961)
10888H
4. Yano, M., Mon. F., Ohno, K., Bleaching of Phenolic Resin Fibers, Japan
Kokai 74 36, 972, April.5 (1974), Chem. Abstr. , 81 (1974) P92968V
5. Mikhailova, L. P., Antonov, A. N., Pichkhadze, S. V., and Soshina, S. M.,
Carbon Fibers, U.S.S.R. Patent, 389,184, July 5 (1973), Chem. Abstr. , 81
(1974) P50908V
6. Ono, M., Sahara, H., and Akasaka, M., Continuous Nonflammable Treathient
of Acrylic Fibers, Japan Kokai, 74 54, 631, May28 (1974), Chem. Abstr. , 81
(1974) 154443F
7. Muroya, K., Ono, M., Sahara, H., and Akasaka, M., Acrylic Fiber Goods
Having Multicolor Effects, Japan 73 28, 990. Sept. 6 (1973), Chem. Abstr. ,
81 (1974) P27100S
8. Das, S., Kar, K. K., Palit, S. R., Catalysts for Polymerization of Acrylamide,
J. Indian. Chem. Soc. , 51(1974) 3931
9. Rakhmanku lov, D. L., Makismova, N. E., Melikyan, V. R., and Isagulyants,
V. I., Conjugated Diolefin, USSR Patent 434,075, June 30 (1974), Chem.
Abstr. , 81 (1974) P77447F
10. Rosenberg, W. E., Aqueous Acid Electroplating Baths, U. S. Patent 3,808,110,
April30 (1974), Chein. Abstr. , 81 (1974) P32620R
11. Zobkiv, B. A., Kovalysko, Y. M., Egorov, G. Y. A., and Ishchenko, V. 0.,
Flux for Soldering Radiators, USSR Patent 399,330, October 3 (1973), Chem.
Abstr. , 81(1974) P531974
12. Fisch, R. S., Photographic Developer Replenisher Concentrates, U. S. Patent
3,785,824, Jan. 15 (1974), Chem. Abstr. , 81 (1974) P56593K
13. Mason, L. F. A., German Patent 1,057,875 (1959), Chem. Abstr. , 55(1961) 8137C
14. Nabiev, M. N., An irova, A. M., Badalova, E. K., and Saibova, M., Fertilizers
with Manganese Trace Element, USSR Patent 242,850, April 25 (1974), Chem.
Abstr. , 81(1974) P103952P
216
-------
15. Fishbein, L., Flamm, W. G., and Falk, H. L., “Chemical Mutagens” Academic
Press, New York (1970) PP. 27-28
16. Freese, E., Bautz, E., and Freese, E. B., The Chemical and Mutagenic Specificity
of Hydroxylamine,
Proc. Nati. Acad. Sci. , 47, (1961) 844
17. Tessman, I., Ishiwa, H., and Kumar, S., Mutagenic Effects of Hydroxylamine In
Vivo, Science , 148 (1965) 507
18. Gray, J. D. A., Lambert, R. A., Bacteriostatic Action of Oximes,
Nature , 162 (1948) 733
19. Schuster, H., The Reaction of Tobacco Mosaic Virus Ribonucleic Acid With Hydroxy—
lamine, 3. Mol. Biol. , 3 (1961) 447
20. Bendich, A., Borenfreund, E., Korgold, G., Kirm, M., and Bolis, M., in ‘Acidi
Nucleic e. Loro Fungione Biologica”, pg. 214 Fondazione Basselli, Instituto Lombardo
Pavia, Italy, (1964)
21. Brown, D. M., and Schell, P., The Reaction of Hydroxylamine with Cytosine and
Related Compounds, 3. Mol. Biol. , 3 (1961) 709
22. Freese, E. B., and Freese, E., Two Seperaple Effects of Hydroxylamine on Trans-
forming DNA, Proc. Nail. Acad. Sci. U.S. , 52 (1964) 1289
23. Freese, E., and Sh-ack, H. B., Induction of Mutations in Transforming DNA by
Hydroxylamine, Proc. Nati. Acad. Sci. , 48 (1962) 1796
24. Kapadia, R. T. ,and Srogl, M., Inactivation of B. Subtilis Transforming DNA by
Mutagenic Agents,
Folio Mircobiol. (Prague) , 14 (1968) 51
25. Freese, E., and Freese, E. B., Mutagenic and Inactivating DNA Alterations,
Radiation Res . Suppl., 6 (1966) 97-140
26. Tessman, I., Ishiwa, H., and Kumar, S., Mutagenic Effects of Hydroxylamino In
Vivo, Science , 148 (1965) 507
27. Freese, E., Freeze, E. B., and Bautz, E., Hydroxylamine as a Mutagenic and
Inactivating Reagent, 3. Mol. Biol. , 3 (1961) 133
28. Dhillio, E. K. S., and Dhillio, T. S., N-methyl-N’-nitro-N-nitrosoguanidien and
Hydroxylamine Induced Mutants of the rh-Region of Phage T4, Mutation Res. , 22
(1974) 223—233
217
-------
29. Mailing, H. V., Hydroxylamine as a Mutagenic Agent for Neurospora Crassa,
Mutation Res. , 3(1966) 470
30. Loprieno, N., Guglielminetti, R., Bonatil, S., and Abbondanoalo, A., Evaluation
of the Genetic Alterations Induced by Chemical Mutagens in S. Pombe Mutation
Res., 8 (1969) 65
31. Androsov, V. V., Molecular Mechanisms of Mutations in E. Coli K-12 Under the
Action of N-Nitroso-N-Methyl Urea, Doki. Akad. Nauk. SSSR , 215 (1974) 1481
32. Engel, W., Krorxe, W., and Wold, U., Die Wirkung Von Thioguanin, Hydroxyl-
aminunds-Broxnodesoxyuridin auf Menschliche Chromosomen In Vitro, Mutation
Res. , 4(1967) 353
33. Somers, C. F., and Hsu, T. C., Chromosome Damage induced by Hydroxylainine
in Mammalian Cells, Proc. Nati. Acad. Sci. , 48 (1962) 937
34. Borenfreund, E., Krim, M., and Bendich, A., Chromosomal Aberrations Induced
by Hyponitrite and Hydroxylamine Derivatives, 3. Nati. Cancer Inst. , 32 (1964) 667
35. Bendich, A., Borenfreund, E., Korngoid, G. C., and Krim, M., Action of
Hydroxylarnine on DNA and Chromosomes, Federation Proc. , 22 (1963) 582
36. Natarajan, A. T., and Upadhya, M. D., Localized Chromosome Breakage Induced
by Ethyl Methane Sulfonate and Hydroxylamine in Vicia Faba, Chromosoma , 15
(1964) 156—169
37. Mailing, H. V., Mutagenicity of Methyihydroxylarnines in Neurospora Crassa,
Mutation Res. , 4(1967) 559
38. Kochetkov, N. K., Budowsky, E. 3., and Shibaeva, R. P., The Selective Reaction
of 0-Methyihydroxylamine with the Cytidine Nucleus, Biochixn. Biophys. Acta .
68 (1963) 493—496
39. Kochetkov, N. K., and Budowsky, E. 3., The Chemical Modification of Nucleic Acids
Prog. Nucleic Acid Res. Mol. Biol. , 9(1969) 403-438
40. Singer, B., and Fraenkel-Conrat, H., The Role of Confirmation in Chemical
Mutagenesis, Prog. Nucleic Acid Res. Mol. Biol. , 9 (1969) 1-29
41. Phillips, J. H., and Brown, D. M., The Mutagenic Action of Hydroxylamine,
Prog. Nucleic Acid Res. Mol. Biol. , 7 (1967) 349-368
42. Banks, G. R., Mutagenesis: A Review of Some Molecular Aspects, Sci. Prog. ,
59 (1971) 475—503
43. Shugar, D., Huber, C. P., and Birnbaum, G. I., Mechanism of Hydroxylamine
Mutagenesis. Crystal Structure and Confirmation of 1, 5-Dimethyl-N 4 -hvdroxy-
cytosine, Biochim. Biophys. Acta. , 447 (1976) 274-284
2 iS
-------
44. Janion, C., and Shugar, D., Preparation and Properties of Some 4-Substituted
Analogs of Cytosine and IDihydrocytosine, Acta, Biochim. Pol. , 15 (1968) 261-272
45. Champe, S. P., and Benzer, S., Reversal of Mutant Phenotypes by 5—Fluorouracil:
An Approach to Nucleotide Sequences in Messenger-RNA, Proc. Nail. Acad. Sci .
U.S., 48(1962) 532—546
46. Schuster, H., and Vielmetter, W., Studies on the inactivating and mutagenic
effects of nitrous acid arid hydroxylamines on viruses,
J. Chim. Phys. , 58(1961) 1005—1010
47. Janion, C., and Shugar, D., Reaction of Hydroxylamine with 5-Substituted Cytosines
Biochem. Biophys. Res. Commun. , 18(1965)617-622
48. Janion, C., and Shugar D., Mutagenicity of Hydroxylamirie: Reaction with
Analogs of Cytosine, 5(6)Substituted Cytosines and Some 2-Keto-4-Ethoxy Pyrimidines,
Acta. Biochim. Pol. , 12(1965) 337—355
49. Salganik, R. I., Vasjunina, E. A., Poslovina, A. S., and Andreeva, I. S.,
Mutagenic Action of N 4 -Hydroxycytidine on Escherichia Coli B cyt, Mutation
Res. , 20 (1973) 1—5
50. Popowska, E., and Janion, C., N 4 -Hydroxycytidine-A New Mutagen of a Base
Analog Type, Biochem. Biophys. Res. Commun. , 56 (1974) 459-466
-------
C. Urethans
Urethanes (carbamic acid esters) are widely utilized in a variety of applications
including: in the plastics industry as monomers, co-monomers, plasticizers, and
fiber and molding resins, in textile finishing, in agricultural chemicals as herbicides’,
insecticides, and in insect repellants 2 , fungicides 3 and molluskicides, in pharma—
ceutical chemicals as psychotropic drugs, hypnotics, and sedatives, anticonvulsants,
miotics, anesthetics, and antiseptics. Urethans are also used as surface-active agents,
selective solvents, dye intermediates, and corrosion inhibitors.
Urethan (H 2 N- -C 2 H 5 ; ethyl carbamate) is a pulmonary carcinogen in mice 46
and in rats’, induces carcinomata of the forestomach in hamsters, and is teratogenic
in mice 8 , hamsters 9 , fish 10 , and amphibia 11 .
The mutagenic action of urethane has been recently reviewed by Bateman 12 .
Urethan has been reported to induce mutations in plants 1214 , bacteria 11 ’ 15-18,
11,19—1 . . .
Drosophila and transforming DNA • is non-mutagenic in Neurospora
No evidence has been found for the production of dominant lethal mutations in mice
and rats at anesthetic levels of urethan (lg/kgY 1 ’ 25 28 Chromosome aberrations
have been induced in somatic cells 11 ’ 29 34• Congeners of urethan, e.g., methyl-,
propyl, and butyl carbamate are also active as mutagens in bacteria 17 .
Urethan is metabolized by mammals (rat, rabbit, man) to n-hydroxyurethan and
35,36
N-acetyl-N-hydroxyurethan, and according to Boyland and co-workers , the
carcinogenic and aritileukemic effects attributed to urethan are probably caused by
the hydroxyurethan metabolites which act as alkylating agents toward mercaptoamino
acids and react with cytosine residues of RNA. The mechanism is similar to, but
distinct from, that of the action of alkylating agents which react mainly with guanine
of nucleic acid; however, in both cases, the same base pairs, guaxiine-cytosine, are
modified.
‘.4-
-------
The induction of chromosome aberrations by N-hydroxyurethan in Vicia faba 36 ,
mammalian cells in culture 37 ’ 38 , as well as its inactivation of transforming DNA
have also been reported 22 ’ 39 .
Freese et a1 4 ° suggested that even N-hydroxyurethan does not itself react with
DNA but reacts with oxygen yielding peroxy radicals and other derivatives, some
of which are apparently the active reagents.
Nery 4 ’ reported that hydroxyurethan and its esters act directly on cytosine under
physiological conditions: Carboxyethylating it (hence showing that it could act
directly on DNA). It has been shown 42 that labelled urethan injected into mice appeared
as labelled carboxyethylated cytosiLne in the DNA and RNA of their livers and lungs
(the two organs most susceptible to urethan induced rzeoplasia) 12 ’ 42 .
The possibility of a direct link between mutagenicity and carcinogenicity is the
observation by Colnaghi et a1 43 that chromosome aberrations are found in the thymus
of mice within days of freathient with urethan; these mice subsequently developed
aneuploid thymic lymphosarcomata.
221
-------
References for Urethans
1. George, D. K., Moore, D. H., Brian, W. P., and Garman, 3. A., Relative Her-
bicidal and Growth Modifying Activity of Several Esters of N-Phenylcarbamic Acid,
3. Agr. Food Chem. , 2(1954) 353
2. Ferguson, G. R., and Alexander, C. C., Heterocyclic Carbamates Having Systemic
Insecticidal Action, J. Agr. Food Chem. , 1(1953)888
3. Mowry, D. T., andPiesbergew, N. R., U.S. Patent 2,537,690 (1951), Chem.
Abstr. , 45 (1951) 2142d
4. Henshaw, P. S., Minimal Number of Anesthetic Treatments with Urethane Required
to Induce Pulmonary Tumors, 3. Nati. Cancer Inst. , 4 (1943) 523
5. Cowen, P. N., Some Studies on the Action of Urethane On Mice, Brit. 3. Cancer ,
1, (1947) 401
6. Orr, 3. W., The Induction of Pulmonary Adenomata in Mice by Urethane, Brit.
3. Cancer , 1 (1947) 311
7. Jaffe, W. G., Carcinogenic Action of Ethyl Urethan on Rats, Cancer Res. , 7 (1947)
107
8. Sinclair, 3. G., A specific transpiacental effect of urethan in mice, Texas Rept.
Biol. Med. Med. , 8(1950) 623—632
9. Ferm, V. H., Severe Developmental Malformations: Malformations Induced by
Urethane and Hydroxyurea in the Hamster, Arch. Pathol. , 81 (1966) 174
10. Battle, H. I., and Hisaoka, K. K., Effects of Ethyl Carbamate (Urethan) on the
Early Development of the Teleost (Brachydariio Rerio), Cancer Res. , 12 (1952)
334
11. McMillan, D. B., and Battle, H. I., Effects of Ethyl Carbamate (Urethan) and
Related Compounds on Early Developmental Processes of the Leopard Frog. Rana
Pipiens, Cancer Res. , 14(1954) 319
12. Bateman, A. 3., The mutagenic action of urethane, Mutation Res. , 39 (1976) 75-96
13. Oehlkers, F., Chromosome breaks induced by chemicals, Heredity Suppl. , 6
(1953) 95—105
14. Oehlkers, F., Die auslosung von chromosomen mutationeri in der meiosis durch
em wirkung von chemikalein, Z. Vererbungslehre , 81 (1943) 313
-------
15. Bryson, V., Carbamate induced phage resistant mutants of E. coli, Proc. 8th
mt. Congress Genetics, (1948) 545
16. Latarget, R., Buu-Hoi, N. P., and Elias, C. A., Induction of a specific mutation
in a bacterium by a water-soluble cancerigen, Pubbi. Staz, Zool. Napoli. , 22
Suppi., (1950) 78—83
17. Bryson, V., Carbamate-induced phage-resistant mutants of E. coli
Hereditas Suppl . 545 (1949)
18. Demerec, M., Bertani, G., and Flint, J., A survey of chemicals for mutagenic
action on E. coli, Am. Naturalist , 85 (1951) 119-136
19. Vogt, M., Mutations ai islosung bei Drosophila durch athylurethan, Experientia ,
4 (1948) 68
20. Vogt, M., Urethan induced mutations in Drosophila, Pubbl. Staz. Zool. Napoli ,
22, Suppi. (1950) 114
21. Oster, I. I., The induction of mutations in Drosophila melariogaster by orally
administered ethyl carbamate (urethane), Genetics , 40 (1955) 588-589
22. Freese, E. B., The effects of urethan and hydroxyurethan on transforming DNA,
Genetics , 51 (1965) 953—960
23. Jensen, K. A., Kirk, I., Kolmark, G., and Westergaard, M., Chemically induced
mutations in Neurospora, Cold Spring Harbor Symp. Quant. Biol. , 16 (1951)
245—261
24. Guglieminetti, R., Bonatti, S., and Loprieno, The mutageriic activity of N-nitro-
N-methyl urethane and N-nitroso-N-ethyl urethane in S. pombe, Mutation Res. ,
3 (1966) 152—157
25. Bateman, A., A failure to detect any mutagenic action of urethane in the mouse,
Mutation Res. , 4 (1967) 710—712
26. Jackson, H., Fox, B. W., and Craig, A. W., The effect of alkylating agents on
male rat fertility, Brit. 3. Pharmacol. Chemotherapy , 14 (1959) 149-157
27. Kennedy, G. L., Jr., Arnold, D. W., and Keplinger, M. L., Mutagenic response
of known carcinogens, Mutation Res. , 21 (1973) 224-225
28. Yumkawa, K., Lack of effect of urethane on the induction of dominant lethal mutations
in male mice, Nat. Inst. Genet. Mishima. Ann. Rep. , 19 (1968) 69-70
29. Boyland, E., and Koehier, P. C., Effects of urethane on mitosis in the Walker rat
carcinoma, Brit. J. Cancer , 8 (1964) 677-684
223
-------
30. Platonova, G. M., Comparative study of the mutagenic action of urethane and
N-hydroxyurethane on the chromosomes of embryonic lung cells of A/SN and
C57blackmice, Soy. Genet. , 5(1969) 262-263 (Engl. transi.)
31. Pogosyants, E. E., Platonova, G. M., Toikacheva, E. N., and Ganz enko, L. F.,
Effect of urethane on mammalian chromosomes in vitro, Soy. Geriet. , 4 (1968)
902—911 (Engi. ti-ansi.)
32. Tolkacheva, E. N., Piatonova, G. M., and Vishenkova, N. S., Mechanism of the
mutagenic action of urethane in mammalian cells in vitro, Soy. Genet. , 6 (1970)
1077—1082 (Engi. Ti-ansi.)
33. Rapoport, I. A., Derivatives of carbamic acid and mutations, Bull. Exp. Biol.
Med. , 23(1974) 198—201
34. Dean, B. J., ChemicaUy induced chromosome damage, Lab. Animal , 3 (1969) 157-174
35. Boyland, E., and Nery, R., The metabolism of urethane and i-elated compounds,
Biochem. 3. , 94(1965) 198—208
36. Boyland, E., Nery, R., and Peggie, K. S., The induction of chromosome aberrations
in Vicia Faba root meristems by N-hydroxyurethan and related compounds, Brit.
J. Cancer , 19 (1965) 878
37. Borenfreund, E., Krim, M., and Bendich, A., Chromosomal aberrations induced
by hyponitrite and hydroxylamirie derivatives, J. Nati. Cancer Inst. , 32 (1964)
667—679
38. Bendich, A., Borenfreund, E., Korngold, G. C., and Krim, M., Action of hy-
droxylamine on DNA and chromosomes, Federation Proc. , 22 (1963) 582
39. Freese, E. B., Gerson, 3., Taber, H., Rhaese, H. J., and Freese, E., Inactivating
DNA alterations induced by peroxides and peroxide-producing agents, Mut. Res .
4 (1967) 517
40. Freese, E., Skiarow, S., and Freese, E. B., DNA damage caused by antidepressant
hydrazines and related drugs, Mutation Res. , 5 (1968) 343
41. Nery, ‘R., Aqrlation of cytosine by ethyl N-hydroxycarbamate and its acyl deri-
vatives and the binding of these agents to nucleic acids and proteins, 3. Chem. Soc .
(C), (1969) 1860—1865
42. Boyland, E., and Williams, K., Reaction of urethane with nucleic acids in vivo,
Biocheni. 3. , 111(1969) 121-127
43. Colnaghi, M. I., Defla Porta, G. D., Parmiani, G., and Caprio, G., Chromosornal
changes associated with urethan leukemogenesis in mice, Int. J. Cancer , 4 (1969)
327—333
224
-------
VIII. NITROSAMINES
The category of N-nitroso compounds comprise N-nitrosamines (I), N-nitrosamides
(IIa,b) and N-nitrosamjdjnes (III). The N-nitrosamines (e.g., dimethylnitrosoamine)
are chemically stable under physiological conditions and exert their adverse biological
effects via metabolic activation to reactive intermediates, predominantly by mixed-
function oxidases.
In contrast, the N-nitrosamides (e.g., nitrosomethylurea) are unstable at physio-
logical pH and decompose non-enzymatically to reactive derivatives (in most cases
alkylating species).
R N(R C 2 H 5 O C N R NH 2 C N R
N0 O N=O O N=O
Dialkyl-N-Nitrosoamine N-Alkyl-N-Nitrosourethane N-Alkyl-N -Nitrosour ea
(I) (ha) Cub)
- N-ç-N
NO 2 NH W=O
N-Alkyl-N’ -Nitro guanidine (III)
Nitrosamines possess considerable diversity of action, especially as carcinogens 5
Their occurrence, whether as direct emissions of N-nitroso compounds or via localized
release of large amounts of precursor compounds (e.g., secondary amines, nitrogen
oxides, nitrate, nitrites) effluent discharges from sewage treatment plants or run&ff
from feedlots or croplands treated with amine pesticides, ammonium fertilizers or nitro-
genous organic materials 6 6 or accidental products in food processing and use,
tobacco smoke 1722 , or via the body burden contributed by in vivo nitrosation 2326
reactions, has sparked ever increasing intensive investigations as to the overall scope
of the potential sources, mechanism of in vitro and in vivo formation, body burdens
as well as to the need to develop a proper scientific foundation for a human health risk
assessment’ 7 ’ 22,23,27—36
225
-------
A number of nitrosamines have been patented for use as gasoline and lubricant
additives, antioxidants, and pesticides. Dimethylnitrosamine [ (CH 3 ) 2 N-NO; DMNI
is used primarily in the electrolytic production of the hypergolic rocket fuel 1, 1-
dirriethylhydrazine 37 ’ 38 . Other areas of utility include the control of nematodes 39 ,
the inhibition of niti-ification in soil 40 , use as plasticizer for acrylonitrile polymers 41 ,
use in active metal anode—electrolyte systems (high-energy batteries) 42 , in the
preparation of thiocarbonyl fluoride polymers 43 , in the plasticization of rubber 44 ,
and in rocket fuels 45 . Some N-nitroso compounds have been used as organic accelera-
tors and anti-oxidants in the production of rubber, including N-nitrosodiphenylamine,
N, Nt -nitrosopentamethylenetetramine, polyrnerized N-nitroso-2, 2,4-trim ethyl- 1-2 -
dihydroquinolene, and N—methyl-N- 4 —dinitrosoaniline 4 6
It is of importance to cite several aspects of potential nitrosamine exposure and
contamination that have recently been brought to light . Synthetic cutting fluids,
semi-synthetic cutting oils and soluble cutting oils may contain nitrosamines, either
45—50
as contaminants in anunes, or as products from reactions between amines and nitrite
Concentrations of nitrosamines have been found in certain synthetic cutting oils at
levels ranging from 1 ppm to 1000 ppm. It is believed that there are 8 to 12 additives
49
that could be responsible for nitrosamine formation in cutting oils and that approxi-
mately 750,000 to 780,000 persons employed by more than 1,000 cutting fluid manu-
facturing firms are endangered, in addition to an undetermined number of machine
shop workers who use the fluids 49 ’ 50 .
N-nitroso compounds, primarily dimethylnitrosamine (DMN) have been found to
be present as air pollutants in the ambient air of residential areas of Baltimore 51
with DMN levels varying from 16 to 760 ng/m 3 while on an industrial site in Baltimore,
226
-------
DMN levels reached 32,000 ng/m 3 (10.67 ppb) of ambient air in close proximity to
a chemical factory which manufactured unsymmetrical dimethyihydrazine for which
DMN was used as an intermediate 52 . In Belle, West Virginia, DMN has been found
near chemical factories which handle dimethylamine 53 .
Another area of recent concern involves the finding of nitrosamines in a variety
of herbicides 54 . These ranged from less than 50 micrograms to 640,000 micrograms
per liter of nitrosamines (e.g., N-nitrosodimethylamine and N-nitrosodipropylamine) 4 .
It has been estimated that 950 to 1, 000 pesticide products may contain nitrosamines,
and a sizeable number of these are available for use by homeowners 55 . High levels
of nitrosamines in soils (believed to arise from the use of triazine herbicide which
can combine with nitrogen fertilizer) have been previously reported 58 as well as
plant uptake and leaching of dimethylnitrosamine 59 .
N-Nitrosodiethanolamine has been found in amounts ranging from 1 ng/g (1 ppb)
to a high of about 48,000 ppb in about 30 toiletry products (e.g., cosmetics, hand and
body lotions and shampoos. The N-nitroso compound found probably results from
nitrosation of di and/or triethanolamine emulsiliers by a nitrite compoundS 9 a.
With regard to the mutagenicity of nitroso compounds, it is of note that the
nitrosamines which are belived to require enzymatic decomposition before becoming
active carcinogens (e . g., dixnethyl- and diethylnitrosamines) are mutagenic in Droso-
6 —64 65 66,67
phila 0 and Arabidopsis thaliana and inactive in microorganisms such as E coil
Serratia marcesens 66 , Saccharomyces cerivisiae 68 ’ 69 and Neurospora 70 (but active
71 72
in Neurospora in the hydroxylating model system of Udenfriend , or in the presence
of oxygen). Eleven carcinogenic N-nitrosamines have been found mutagenic on S.
typhimurium TA 100 (but not TA 98) when the bacteria, test substance were pre-incubated
7,
with rat liver S-9 mix, and then poured on a plate
Methylvinyl -, methylbenzyl-, and N -methylpipera zine nitrosamine have all been
found mutagenic in Drosophila0O . However, ethyl tert-butylnitrosamine (which has
227
-------
no carcinogenic action) was found also to be nonmutagenic in Drosophila . Presumably,
this substance is not degraded in vivo .
N-dealkylation of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) by
tissue specific microsomal mixed-function oxidases is believed to generate alkylating
73—77 3,5 78
intermediates that are responsible for the mutagenic , toxic and carcinogenic
effects of the parent compound in vivo and in vitro . Possible mechanisms for the
metabolic activation and deactivation of dialkylnitrosamines are illustrated in Figures
1 and 2.
Optimal cofactor and reaction conditions for an in vitro mutagenicity assay using
G-46 and TA 1530 strains of S. typhimurium where the latter was specifically reverted
to histidine protrophy by both DMN and DEN following biotransformation through base
pair substitutions have been described 74 ’ 9. The relationship between the site of
metabolic activation, mutagenicity and carcinogenicity and the effect of enzyme inducers
has been further delineated by Bartsch et a1 77 . The enzymatic conversion of DMN and
DEN into alkylating intermediates is paralleled by the formation of the corresponding
alcohols and/or aldehydes, which are further metabolyzed to CO 2 in vitro and in vivo .
To elucidate further the mechanism of action of DMN, a stable derivative (N, C-
acetoxy methyl-N--methylnitrosamine, ADMN) of the postulated primary product of
metabolism, hydroxy-methyl-methyl nitrosamine has been studied. The mutagenic
activity of ADMN in Drosophila , compared to DMN was found to be 10 times as effective,
on a dose basis 79 .
The higher carcinogenicity to rats of the acetoxy derivative ADMN compared with
the parent DMN has also been observed 80 ’ 8 ’. This strongly suggests that ADMN could
be regarded as DMN’s proximate metabolite. A comparative genetic study on the
testicular tissue of Drosophila with ADMN and DMN to assess the role of intracellular
metabolism via an elaboration of the dose effect on the metabolically inert sperm and
228
-------
the metaboli:ing early germ cells (spermatocytes and spermatozonia) with respect to
the induction of the non-specific X-chromosome recessives (lethals and visibles and the
specific effects on representatives of the RNA genes (especially DNA) was recently
reported by Fahmy and Fahmy 82 .
The correlation between mutagenicity and carcinogenicity of a large number of
N-nitroso compounds has recently been reviewed by Montesano and Bartsch 83 while
Neale has reviewed the mutagenicity of nitrosamides and nitrosamidines in micro-
organisms and plants 84 . Table 1 summarizes data that have accumulated up to 1975
concerning the mu:ageriicity of a number of representative N—nitroso compounds in
various systems involving different genetic indications. Mutagenicity data from direct
mutagenicity assays, tissue- and host-mediated assays, dominant lethal tests, data
on chrornosomal aberrations and tests in Drosophila melanogaster are listed 83 .
The increasing evidence of an empirical correlation between mutagenicity and
carcinogenicity is shown. in Figure 3 in which the biological activities of 23 N—
ni osamides and 24 N-nitrosamines are plotted. Carcinogenicity data are taken from
Table 1; positive mutagenicity results were obtained from one of the test systems
considered, and negative mutagenic results were plotted only if they were negative
in all the test systems. Of the 47 N-nitrosamines and N-niu-osamides plotted, 38
compounds were found to be carcinogenic and i utagenic, five carcinogens were
not detected as mutagens, three non-carcinogens were non-mutagenic and only one
compound reported to be non-carcinogenic exhibited a mutagenic effect 83 .
229
-------
R—CH.
Nitrosamine
R—CIV
Enz ’ -’c - :‘
V
R—CH
i-Hydroxynitrosarnine
R—CH
R. CH.) Mor ’aIk) Initresarnine.
H
-RCHO
R—CH -—N= N—-OH Diazo ydroxide
Ge
RCH—N N — }40 Diazoalkanc
R—CH —N N Dia:cniurn s Jt
V
R—CH 1 - Czr o i& rn on
Fic / Possible rnechani rn tcr ti-e i :-irc mciabc srn ar.d re cx on of di Iky!r i:ros-
al ines. Druckrey. H., SchiL b h. .A.. S ’ hI, D., P e smann. R., and Jvznkcvic. S.,
Ar:qt imgrtd-Frjrsch. 33, S41 (I
230
-------
DNA :RNA & PROTEIN ALKYLAUON
9J
F . 2. Met.a’oot c ac: .ation (left side) and d toxicati3n ( i:h side) .V,N-d.i iky1ni rosamjnea.
231
N. N — DIALKYL—NITROSAMINES
Activation Deactivation
t RCHO -
CH 2 R
RCHO
t
[ HO. ;E ; CH R ] RCH?OH
-------
CAICINOGEN
30
10
4JtA$f
A r . _________
‘V
NO$CA CINOO1N
Fjg.3. Cortelation b.tw••n mutagenic and caith ojenic effects of N-nittoso compounds. Data on 47 N-
nltroso compounds wars .xttacted fzoin Table I -
232
-------
TABLE 1
CARCINOGENICITY AND MUTAGENICITY Of N -NZTROSO COMPOUNDS
Compound. Genetic Indicator
Carcinogenielty a: species and
Gen.tjc changes Muta
A
genial
ty b
B
C
D
principal taSsel organs
-
F
(1) N-Nitrosodlm .thylam ga
S. typhi,nurlum
Rat: liver, kidney, human, mouse, rat, baa- Reverse mut. — +
nasal cavitiei ster. liver MS C
Mouse: Lung, liver, kidney rat. mouse, hamster Reverse mut.
S.G. Hamster: liver, nasal cavities lung MS
European hamster: liver, kidney mouse and rat Reverse mut. +
Rabbit; mastomys; guinea pig;
trout: newt; aquarium fish; E.coli
mink: rat liver MS Reverse or forward — +
liver mut. and preferen-
tial growth jnhibuLio
ret kidney MS Reverse mut.
mouse Rever c mut. +
fl. subi Ills
mouse livct MS Reverse mut. — +
Saccharam yee , c.,evisia .
Udenfnersd hydroxyla’ Beck mut,, gene recom. +
tion system, mouse bination and conversion,
liver MS petit. mut, canavanine
resistant mist.
mouse Gene recombination +
and conversion
Serratia maree$ccns a Mot.
mouse Reverse rout. +
Neumap,rn crease Reverse mut.
Udi-niriclid hydroxyla. Forward mut, +
tion system, mouse
liyrr MS
mouse l”urward rout, +
Drosophila melenogaslcr Recessive lethal rout, +
Chinese hanisler cells V.79
rat liver MS Th logoanine. — +
reaistunt mist.
Chinese hainsier c cl i , CflO .Ki Nutritional auxo- + +
— tropic mutant
Mtgrine l.ukaemic calls
L5 178 YIA,,n
mouse Aspareglneindepes s- +
dent mist.
Mouse Dominant lethal rout.
Carelnog.nicity da.a wer, extracted from the article by Magee at al. [ . unless otherwise specified.
b u.agenjcfty assays considered were: A, direct test; B, in vitro tissuezszediated assay; C, host-mediated cssay; 0, dominant lethal test: F:, chromosomal tierra-
lions; F, test in Drosophila melarogaster. The mutagerijc response, esprcsaed as positive or negative is as evaluated by the authors of this article.
1$. microsomal iystcrn consisting either of a post-rnitochondrial fraction orof pursfied microsomes, fortified with cofactora of mixed-function oxidases.
1’. Matsusatma. M. Xagao and T. Suglmura personal communication.
233
-------
Genetic (ndic4ur Genetie changes Mutjge,,kjty assays b
C,srcmogenicity U; ,ecics and A 13 C D E F
pttncipal tatgc$
(2) .‘J -Nltromdlethylan%iflt
S. typhimunum
Rat: liver. oesopbagus, nasel cavities, rat, mouse and ham- Reverse mut. — +
kidney 4cr MS
Mouse: liver, hang. orestomach, rat, mouse and ham- Reverse mut,
oeiopbagua. nasel cavities star lung
S.C. hamster: t *cbea. larynx neml mouse, rat Reverse mut.
caa4ties, brag. liver
Chines. bam.der: oesophagus, Laze- Z. coil
stom.ch, liver ret liver MS Reverse snut. — +
European hamster: nasal cavities, rat kidney MS Reverse mut. —
trachea, bronchi, larynx mouse Forward mul,
Guinea-pig; rabbit; dog: pig; trout;
flrnclydanio re t - jo; grass parakeet; Sacchammyces esreuisla. Oack end reverse
t onkey: mut., gene conversinfl
liver Udenfrlcnd hydroxyla- Forward and pc(iic — +
tion system, mouse liver mut., gene tecoinbin-
MS tints
mouse Gene recomblnation 4
ud ennvcrsl’t,i
.ierrnlh, mseeePecnl us Mut,
mouse Reverse mul,
-— N uru p ,rnt cruem UaCII mul. —
Udenlricnd hydvvxyLt- Forward inul, — +
tion system
mouse Forward snut. 4
L)ro ophi1a m.tanogasler Recessive lethal mut. +
Mouse Dominant lethal
Rat liver cells in vivo Chromosome aberrations +
( l) X-NitrosomethyMnylamln.
Drosophila ,nelnnotaster Recessive lethal emit. +
Ral: Orsophagu., pharyn.r, tongue,
nasal c gveties
(4) N’Niirosode-n-propylamizse
S. typhimurium
R 5 ; .ver, oe,oplugu,, tongue rat and hamster liver, Reverse muC — +
S.C. amster: rsaajl Cavities, hamster lung MS
;rschea [ 2021 rat lung, hamster and Reverse mist.
rat kidney MS
E. coil
-rat liver MS Reverse mut. — +
rat kidney MS lteverse mist. —
Succliarnsn ye t - a ceret,isinc
Udeitiriend hydruxyla. Gene rvcomb natinmt — +
tion system
Caratisogenicity data were extracted from the aritele by Magee et al. unless otherwise specified.
The mutagenicl:y assays considered were A, direct test; B, In vitro tiss’me mediated assay; C, hosi rnediated assay; D, dominant lethal test; S. chroino orflal aberra-
.ios: F. test n ‘)‘ opoils vme!anogaater. The mutagenic response, expressed as positive or negative is as evaluated by the authors of this article.
MS. n,icosortal system consisting either of a post-mitochondrjai fraction oi of purified microsorncs, fortified with cofactors of mixed-function oxidases.
1 ‘ ‘, ).Iatsushims, ‘4. Nigao and T. Suvimura: penonal communication.
234
-------
TALILI. l(couUnucd)
Cooipound CCUCLIC Indicator Genetic changes Mutagenicity b
CarcIno en(cigy 5; and A U C D R r--
pt1flC pji target organs
(21) N-Nltrosomorpi olinc
S. typhimurzum
RaL: Ltver, nasal cavities, kidney, human and rat Iivex MS Revesas awL — +
oesophagus, ovary mouse Rcvcsse muL +
Mouse: liver, lung -
S.C. hamster: trachea, Larynx, bronchi E coil BIOChCInICM mut.
rat liver MS Reverse Taut. +
kidney MS Reverse mut. —
Mouse Dominant lethal mug.
Drosophila melanogostej- Recessive lethal mug. +
and translocation
(22) N-Nitrosopyrrolidine
S. typhirncjrlum
Rat: liver, nasal cavities, testis human and Sat liver MS Reverse mut. — +
Mouse: lung
S.G. hamster: tx*chea, lung
(23) N-Nitrosopiperidine
S. typhirnuriwn
flat: oesophagus, liver, nasal c viUe,. human and rat liver MS Reverse mut. — +
hsrynx, trachea
Mouse: forestomach, liver, lung. R. coli Biochemical mug.
oesophagus rat liver MS Reverse mut. +
S.C. hamster: trachea, lung, larynx rat kidney MS Reverse mug. —
Monkey: liver
(4) N-Nitrosopiperazirse
S. typhimursunm
Rat: nasal cavi ties mouse Reverse mug. - +
( ) .V -Nitroso-N -methyIpiperazine
S. typhimurium
R .. : nasal cavities human and r t liver MS Reverse mut. — +
mouse Reverse mug. +
Drosophila metanogastep Recessive lethal owl. - +
(26) N,N .DinitrosOPiperazifle
S. typhimurlwn
Rat: oe,ophagus. liver, nasal cavitle , mouse Reverse met. +
furestomach
M ,use lung, liver J . enti Bmochemieal mut.
Drojophita melanogas let Recessive lethal mug. +
- -—•. - — ______ -
Carcj msngenje lt, d ts were extracted (corn the article by Magee ci al. unless otherwise specified.
The mutimgestiejgy ttiays considered were: A, direct test; IS. in vitro tissue-mediated assay: C, host-mediated assay: D, dominant lethal test; R, chromosomal aocyra-
lions: F, test in flrnsnpItjt melanngcr!er. Tic motagenic ns onse , ex7resscd as positive Or flcgatiVO Is as ev Iuated by the authors of this article.
MS. micyo nrnai qvsLcnm c(.fl.slsjlng either of a post-mitochondrial frsctinn or ot purified mimcrosomnes. fortified with CofaCtors of ntlxcd—iunctii,n Gxid;ises.
:1 T. .Msisushims, .t. Natt.,o 50( 1 T. Sufimura: ;crsonsI communication,
235
-------
r. ili E jfcontinue*l)
Compound G netie indicator Genetic changes Mutagcnlcity y 1 b
Carcinogemciiy : specks and A B C 0 B F
princi a3 target organs . - -
(27) N-Niisoao-WmethYlIfle*
S. typhin.uriwfl
Rat: ce ttal and peripheral nervou, mouse Reverse inuL. + +
system. intestine, kidney. for&
stomach. glandular stomach. skin H, colt Revene mid., preferS +
and a n.xrg Jaw, bladds.. atasus , entlal growth Inhibition
mouse Forward mut.
Mouse: luog. baemapoiselc ayetem.
forastomach. iddn.v, sida. User Sacchasomycrl cs.ssiala. Gene conversion and +
(only nsw’boen), osMal nvous reverse mat.
system
S.G. hamsts. latedlas. pharyan.
oe ,qpbagus. trachea. haunch!. end mouse R.vma. mat. + +
cav y. shin and annexea and s.c. dt.
of InJection D,o.ophll. moienogc.t.r Recessive lethal miii.
European hamst s.c. sit. ol InJac-
tion Chinese hamster cells V79 8-azaguani s- eslstaxtt + 4
Guinea.plg: *omaeh. pancreas. ear mist.
duct
Rabbit: cuitist nervous sydses, Chineja hamster celia Nutritional auxotrophic + +
Iniesilne. thin CIlO .IC I miii.
Dog: central and pedoheral narveus
aynasn Mouse Dominant lethal mist. +
coliphag. ?2 ,-mui +
Aap.rtflla.a .,id,aiaaa Forward and reverse +
• miii.
if. ,abf ills (transforming Fluorescent ind mist.. +
DNA) inactivation
—
(28) y.Njtrose4.3 ,digi 5ethyhirea -
B. 5 ubtilis (trsnforndflg Fluorescent md rnut.. +
Rat: central and peripheral nervous DNA> ina ct lv at lOfi
sygems, kidney
Mouse: ba.inapoi tic system Sacchcromyc .s c.revisIae Reverse inut
(29) N-Ni soishnethylurta
Saecherom yes, esrevi a les Revers. mist.
Rat: central and peripheral nervous
sysmans, kidney. asin
(30) .V -NItrose-N..thvlure*
H. coil Reverse mi i i., prefer- +
Rat: central and peripheral nervous entt*l growth Inhibition
systems, kidney, hsemapoiedc
system, skin, intestine, ovary. coUpling. T 2 r-mut +
uterus
Mouse: hsamapoletlc sydese, lung,
c .gtral and p.riphe,al nervous sy
tem. kidney
(n) X .Nitro.Y.n -ProPYluseS
S.ccheromycel c.revusics Gene conversion
5 Carciaogsnlcit, dst* wer* extract.d from the ax cIe by Mage. eta!. ‘1 unless otherwis, specified.
b , mutageniclty ways cons*deysd wers A, direct test; B. in vitro tIssue-mediated assay: C. ho.t.mediated assay; 0. dominant lethal test E. chromosomal aberra.
tions: F. test In Drosophila ,‘wlsnogestsr. Toe mutagenic response, expressed as ponuve or negative is as evaluated by the authors of this article.
C MS. mjcro.omai System consisting either of a post .m ltochunddal fraction o -ot purified microsomca. totttfied with cotact .nr of mixed.(unciiori oxldases.
d ‘, Maiaushima, M. agao and T. Susimura: personal communication.
236
-------
T \BL.t l(continued)
Cwnp nu d Genetic Indicator Genetic etuinges — hlutaganicity b
Cagcwe genicity species .snd A B C I) E P
i.HinclPal t.afl:et O5 3US
( 4) Y-Nitro,o?J-methylurethane
E. coli Preferential growth
Rat: forestomach. lung. oesophaaus. inhibition; forward
intestine, kidney, ovary and reverse mul.
Mouse: lung, forestomach
S.G. hamster: oeaophag is. (ore- B. s btiIia Preferential growth +
stomach inhibition
Guinea-pig: pancreas, s .c. sit, of
imection Saceharom yeas care visiac Gene con version. back +
tnut., respiratory de-
Ilcient mut.
$erratia ,narerseena Back mut. +
Drosophila meianogaaier Recessive lethal mut.,
t ran aloc*L lon
Chinese hamster el3.s Nutritional auxoiropic +
(CIlO- ICI) xnut.
Schtrosaccherornycs porn be Reverse and forward +
n%Ut.
Collectntrichum cocodes Nutritional auxoirophic + -
tout.
lIae,nophilua influenzae Novobtocin-rcslst.ant +
tout.
S. iyphimununl Reverse mut. +
,Venro pum emesa Reverse tSIIIL. +
CBS) N.n:noso N-ethylurcthane
E. coil Prc(ercnti.4 growth +
.it: (orestcim ch. intcattne inhibition
Sacchcrurnyces cere isiae Back and reverse tout., +
gene conversion
Neurospore crease Reverse tout. +
Schizo acchatOrnycEs po rtbe Reverse and forward +
rnut.
Coliectobichum cocodEs Nutritional auxotrophic +
mut.
Drosophila rneinnogasfrr Recessive lethal mut.
C 6) .V Nitroso.N’.D.gluCOSyI.2-methY1Ure3 (StreptozO (Ocin)
S. (yphirntsrf urn
? at: kidney mouse Reverse tout. + +
Chinese hamster: liver
Mouse Dominant lethal tout. +
( 7) Ni;rocO N.mctiiYLICetatoide
E. coil Forward mut. +
Rtt: (,testornach
SocclearOmyCeS cere;isice Reverse tout. +
Drosophila meianogciler Recessive lethal tout., +
translocation
* Carein enlcity data were extracted from the article by Magee et at. [ ..aaIJ unless otherwise specified.
b The mulagm,teity assays considered were: A. direct test: 3. In vitro tissue-mediated assay; C. host-mediated assay; D. dominant lethal test; R. chromosomat aberra
tions; test in r)rn.ophita ,,sslanogesle?. The niutaSenic response. e-s ressed as nsitive or negative is as evaluatcd by the authors of thiS article.
C MS. mierocomal system onsist(ng either of a post-mitochondrlai traetion or of purified rnkrosonics. fortified wiTh cofactors of mixcd-f ;.ncUon OxidaseS.
d T. : iatsushI Th%. M. :; 3o arid T. Su rnur personal communicatiOn.
237
-------
T ’ i1L l(continuc j)
Gcnetic lndkalor Genetic chuntes Mut .n enicjty b
C ,-ir rn.: . s Cies and A U C II K F
iiiic;’.il taret orijns
(47) v.Njt , --me;nyl.N-nitroguani4ine
S. typhiinurium Reverie mut. +
Rat iand 1 zsto&’.iich Lorestomach. mouse Reverse rnuL +
‘ .e 5tine. s .c. s ie of injection
!oi . e. inl.e tulc. f0re31.urnaCh. s.un t . cuii Revur-jo and forward +
(s.fle of UWiC iOn) . mut
S.C. tamster: gandta at stomach, mouse Forward mut. +
j n e 5tiflC
Rabbit: (ung Sccchozoni).ces cite uisic Forward and rev.,,. +
t )og: stomach. intesti ne mut.
mouse Geis. converoon + +
Neurospora crass. Forwards recess ye +
lethal mut. deletions
Chinese hamster cells Nutritional auaotropbk • +
(CHO-KI) mut.
Chinese hamster cells V79 8-Azaguauins-resistant +
inut.
Murin. leukaemla cells Asparagjn.-mdepen. +
L5 178Y/Asn dent mut.
Haemophilua inf l uenza. Novobiocln-r .sis +
tent Inut.
Aap .rgiliui nidul ‘as Forward ind severs. +
inut.
— - Ta.owaa (transforming Pluoreseenl ind muL. ÷
DNA) Inactivation
Drosophila melanogaster Recessive and domi- + +
nant tethzl mut..
chromosome aberra-
tions
B. sublills Reverse muL. preferen- +
tial growth inlilbilloit
SernUia na,cescen,
mouse Reverse anuS, 4 +
Mouse t)ominant lethal rnut. +
rh $e 11 rc iasa inut. +
Humon skin fibrobla its Chromosome aberra- +
from Xrrn.frrmn pig- tions
fltrnh,su,pt patients
(I 3) .V’ sO- h.V-n.t,oZuani(line
5, lypi.imUP Uin Revcrse mu).. +
flat: for—stomach, intestine
M ose: ; iit (site of injection). F. Coli flCvcrse mu)..
oesophacus. intestine
S . harmstcr: gandular ttornach. Saccharonsyees ccreris oc Forward 4 reverse +
duodenum rout.
)og: stomach
a (:s. o r ucn, ’ data i 5 estrscted from the article by Magee Ct al. .2i unless otherwise specified.
b , mutageniesty ama considered were: A, direct test; B, in vitro tlsauemed aed assay; C. host-mediated assay; D, dominant lethal test; E, chromosomal aberra.
tiona: F, :esl in Dro.OPna!a .‘ efanogasfzr. ‘The mutageuic response, expressed as positive or negative is as evaluated by the authors of this article.
C MS. mnicrosomai system consisting either ot a post-mjto hondcjal fraction or of purified microsoanca. fortified with cofactori of mixed-function oxidsac,.
T Mate on.. r.i. Nag o and T. Sutimura: personal communication.
238
-------
References for Nitrosamines
1. Magee, P. N., Barnes, J. M., The Production of Malignant Primary Hepatic
Tumors in the Rat by Feeding Dimethylnitrosamine, Br. 3. Cancer (1956)
10 114—122
2. Heath, D. F., Magee, P. N., Toxic Properties of Dialkylnitrosamines and Some
Related Compounds, Br. 3. hid. Med. , 19 (1962) 276-282
3. Magee, P. N., Barnes, 3. M., Carcinogenic Nitroso Compound, in Haddow, A.,
Weinhouse, S., (eds): Advances In Cancer REsearch , 10(1967) 163-246
4. Druckrey, H, et al., Erzeugung von Krebs durch eine einmalige Dosis van
M ethylnitroso-Harnstoff und verschiedenen Dialkynitrosaminen an Ratten,
Z. Krebsforsch , 66 (1964) 1-10
5. Druckrey, H., et al., Organotrope Carcinogene Wirkungen bei 65 Verschiedenen
N-nitroso—Verbindungen an BD-Ratten, Z. Krebsforsch , 69 (1967) 103-201
6. Magee, P. N., Possibilities of Hazard from Nitrosamines in Industry, Ann.
Occup. Hyg. , 15(1972) 19—22
7. Feuer, H. (ed), ‘ T The Chemistry of the Nitro and Nitroso Groups’, New York,
Interscience Publishers Inc., (1969)
8. Lijinsky, W., Epstein, S. E., Nitrosamines as Environmental Carcinogens,
Nature , 225 (1970) 21—23
9. Shapley, D., Nitrosamines: Scientists on the Trail of Prime Suspect in Urban
Cancer, Science , 191 (1976) 270
10. Wolfe, N. L., Zepp, R. G., Gordon, 3. A., and Fincher, R. C., N-nitrosamine
Formation From Atrazine, Bull. Env. Contam. Toxicol. , 15 (1976) 342
11. Bogovski, P., and Walker, E. A., N-Nitroso Compounds in the Environment,
IARC Scientific Publication No. 9, International Agency for Research on Cancer,
Lyon, (1974)
12. Ayanaba, A., and Alexander, M., Transformation of Methylamines and Formation
of a Hazardous Product, Dimethylnitrosamine, in Samples of Treated Sewage
and Lake Water, 3. Environ. Qual. , 3 (1974) 83-89
13. Ayanaba, A., Verstraete, W., and Alexander, M., Formation of Dimethylnitrc
samine, a Carcinogen and Mutagen, in Soils Treated with Nitrogen Compounds,
Soil Sci. Soc. Am. Proc. , 37(1973) 565—568
14. Tate, R. L., and Alexander, .M., Formation of Dimethylamine and Diethylamine
in Soil Treated with Pesticides, Soil Sci , 118 91974) 317—321
239
-------
15. Tate, R. L., and Alexander, M., Stability of Nitrosamines in Samples of Lake
Water, Soil, and Sewage, J. Nati. Cancer Inst. , 54 (1975) 327-330
16. Elespuru, R., Lijinsky, W., The Formation of Carcinogenic Nitroso Compounds
From Nitrite and Some Types of Agricultural Chemicals, Food Cosmet. Toxicol. ,
11 (1973) 807—817
17. Issenberg, P., Nitrite, Nitrosamines, and Cancer, Fed. Proc. , 35(1976)1322
18. Fiddler, W., et al., Formation of N-Nitrosodimethylamine from Naturally Occurring
Quaternary Ammonium Compounds and Tertiary Amines, Nature , 236 (1972) 307
19. Scientific Status Summary, Nitrites, Nitrates, and Nitrosamines in Food: A
Dilemma, J. Food Sd. , 37(1972) 989—991
20. Serfontein, W. J., Smit, J. J., Evidence for th Occurrence of N-nitrosamines
in Tobacco, Nature , 214 (1967) 169—170
21. Neurath, G., Zur Frage des Vorkommens von N-nitroso-vergindungen in
Tabakrauch, Experientia , 23 (1967) 400-404
22. Wolff, I. A., Wasserman, A. E., Nitrates, Nitrites, and Nitrosamines, Science
177 (1972) 15—19
23. Low, H., Nitroso Compounds-Safety and Public Health, Arch. Env. HIth. , 29
(1974) 256
24. Sander, J., Kann Nitrit in der Menschlichen Nahrung Ursache Einer Krebsent-
stehung durch Nitrosaminebildung sein? Arch. Hyg. Bakt. , 151 (1967) 22-24
25. Sander, J., Self, F., Bakterielle Reduktion van Nitrat im Magen des Menschen
als Ursache einer Nitrosamin-Bildung, Arzr.eim Forsch , 19 (1969) 1091-1093
26. Hawksworth, G., Hill, M. J. , The Formation of Nitrosamines by Human Intestinal
Bacteria, Biochem. 1. , 122 (1971) 28P—29P
27. Magee, P. N.,
Food Cosrnet. Toxicol. , 9 (1971) 207
28. Wogan, G. N., and Tannenbaum, S. R., Environmental N-Nitroso Compounds:
Implications for Public Health,
Toxicol. Appi. Pharmacol. , 3]. (1975) 373
29. Scanlan. R. A., In: “Critical Reviews in Food Technology” Cleveland, CRC
Press, (1975) V 0 1. 5, No. 4
240
-------
30. Sen, N. P., In: “Toxic Constituents of Animal Foodstuffs “, edited by I. F. Liener,
New York, Academic, (1974)
31. Shank, R. C., Toxicology of N-Nitrosocompounds, Toxicol. Appl. Pharmacol .
31 (1975) 361
32. Mirvish, S. S., Formation of N—Nitroso Compounds: Chemistry, Kinetics and
In Vitro Occurrence, Toxicol. Appi. Pharmacol. , 31 (1975) 325
33. Fiddler, W., The Occurrence and Determination of N-Nitroso Compounds,
Toxicol. Appi. Pharmacol. , 31 (1975) 352
34. Anon, Dupont to Limit Nitrosamine Emissions: EPA Studies Nitrosamine Problem,
Toxic Materials News , 3 (1976) 111
35. Anon, EPA Advisory Unit Recommends No Immediate Regulatory Action on Nitro-
samines, Toxic Materials News , 3 (1975) 139
36. Anon, Science Advisory Board Endorses Closer Look at Nitrosamines, Env. Hith.
Letter , 15 (1976) 1
37. Horvitz, D., and Cerwonka, E., U.S. Patent 2,916,426 (1959), Chem. Abstr .
54, (1960) 6370c
38. National Distillers and Chem. Corp., British Patent 817,523 (1959), Chem. Abstr .
54 (1960) lO6Olc
39. Maitlen, E. G., U. S. Patent 2,970,939 (1961), Chem. Abstr. , 56 (1961) 11752f
40. Goring, C. A. I., U.S. Patent 3,256,083 (1966), Chem. Abstr. , 65 (1966) 6253d
41. Lytton, M. R., Wielicki, E. A., and Lewis, E., U. S. Patent 2,776,946 (1957);
Chem. Abstr. , 51(1957) 5466e
42. Elliot, W. E., Huff, J. R., Adler, R. W., and Towle, W. L., Proc. Ann. Power
Sources Conf. , 20(1966) 67-70; Chern. Abstr. , 66(1967) 100955w
43. Middieton, W. J., U. S. Patent 3,240,765 (1966), Chem. Abstr. , 64 (1966) 19826f
44. Lel’Chuck, Sh. L., and Sedlis, V.1., Zh. Priki. Khim. , 31(1958)128; Chem.
Abstr. , 52(1958) l778 7 g
45. Kiager, K., U. S. Patent 3,192,707 (1965).
46. Bcyland, E., Carter, R. L., Gorrod, 3. W., and Roe, F. 3. C., Carcinogenic
properties of certain rubber additives, Europ. 3. Cancer , 4 (1968) 233
241
-------
47. Anon, Nitrosamines Warning, Am. md. Hyg. Assoc. J. , 37 (1976) A—9
48. Anon, Nitrosamines Reported in Industrial Cutting Oils, Occup. 111th. Safety
Letter , 5(1976) 34
49. Anon, Nitrosamines Found in Cutting Oils, Toxic Materials News , 3 (1976) 156-157
50. Anon, NIOSH Issues Nitrosamine Alert, Toxic Materials News , 3 (1967) 167
51. Fine, D. H., Roundbebler, D. P., Sawicki, E., Krost, K., and DeMarrais, G. A.,
N-Nitroso Compounds in the Ambient Community Air of Baltimore, Maryland,
Anal. Letters , 9(1976) 595—604
52. Pellizzari, E. D., Bunch, J. E., Bursey, J. T., Berkley, R. E., Sawicki, E.,
and Krost, K., Estimation of N-Nitrosodimethylamine Levels in Ambient Air by
Capillary Gas-Liquid Chromatography/Mass Spectrometry, Anal. Letters , 9
(1976) 579=594
53. Fine, D. H., Roundehier, D. P., Belcher, N. M., and Epstein, S. S.,
Science , (1976) in press, cited in ref. 51
54. Anon, Nitrosamines Found in Herbicides: In Vivo Formation Documented, Toxic
Materials News , 3 (1976) 148
55. Anon, Nitrosanünes: EPA Refuses Suspension Request; NIOSH Promises Alert,
Toxic Materials News , 3 (1976) 156
56. Anon, Nitrosamines Found in Commercial Pesticides, Chem. Eng. News , 54
(1976) 33
57. Anon, EPA Investigating Nitrosaxnine Impurities in 24 Herbicides, Chem. Eng.
News , 54 (1976) 12
58. Anon, High Levels of Nitrosamines in Soils Lead to Government Task Force,
Pesticide Chem. News , 4 (1976) 56
59. Dean-Raymond, D., and Alexander, M ., Plant Uptake and Leaching of Dimethyl-
nitrosatnine, Nature , 262 (1976) 394—395
59a.Anon, N-nin-osamines Found in Toiletry Products, Chem. Eng. News , 55(1977) 7,8
60. Pasternak, L., Untersuchung Uber Die Mutagene Wirkung Verschiedner Nitrosamin
Und Mitrosamid-Verbindungen. Arzneimittel-Forsch. , 14 (1964) 802
61. Pasternak, L., Mutagene Wirkung Von Dimethyl Nitrosamin Bei Drosophila
Melanogaster, Naturwissenschaften , 49 (1962) 381
62. Pasternak, L., The Mutagenic Effect of Nitrosamines and Nitroso Methyl Urea,
Untersuchungen Uber Die Autagene Wirkung von Nitrosaminen Und Nitrosomethyl
Harnstoff, Acta. Biol. Med. Ger. , 10 (1963) 436-438
242
-------
63. Fahn y, 0. G., and Fahmy, M. J.., Mutational Mosaicismin Relation to Dose With
the Amine and Amide Derivatives of Nitroso Compounds in Drosophila, Mutation
Res. , 6(1968) 139
64. Fahniy, 0. G., Fahmy, M. 3., Massasso, 3., and Ondrej, M., Differential Muta-
genicity of the Amine and Amide Derivatives of Nitroso Compounds in Drosophila
Mutation Res. , 3(1966) 201
65. Veleminsky, 3., and Gichner, T., The Mutagenic Activity of Nitrosamines in
Arabidops{s Thaliana, Mutation Res. , 5 (1968) 429
66. Geisler, E., Uber Die Wirkung Von Nifrosaminen Auf Mikroorganisrnen, Natur-
wissenschaften , 49 (1962) 380
67. Pogodina, 0. N., 0 Mutagennoy Aktionosti Kancerogenoviz Gruppy Ni -osarninov,
Cvtolo ia , 8 (1966) 503
68. Marquardt, H., Zimrnermann, F. K., and Schwaier, R., Die Wirkung Krebsauslô-
sender Nitrosamine und Nitrosamide Auf Den Adenin-6-45-Ri ck Mutationssystern
Von S. Cerevisiae, Z. V -ebungslehre , 95 (1964) 82
69. Marcuardt, H., Zimmermann, F. K., and Schwaier, R., NitrosamideAls Mutagene
Agentien, Naturwiss. , 50(1963) 625
70. Marcuardt, H., Schwaier, R., and Zimmermann, F., Nicht-Mutagenit .t Von
N osaminen Bei Neurospora Crassa, Naturwiss. , 50 (1963) 135
71. Malling, H. V., Mutagenicity of Two Potent Carcinogents, Dimethylnitrosamine
and Diethylnitrosamir.e in Neurospora Crassa, Mutation Res. , 3 (1966) 537
72. Yahagi, I., Nagao, M., Seino, Y., Matsushima, T.;Sugimura, T., and Okada, M.,
Mutagenicities of N-nitrosamines on Salmonella, Mutation Res. , 48 (1977) 121-130
73. a1 .ing, H. V., Dimethyl Nitrosarnine: Formation of Mutagenic Compounds by
Interaction with Mouse Liver Microsornes, Mutation Res. , 13 (1971) 425
74. MaLing, H. V., and Frant, C. N., In Vitro Versus In Vivo Metabolic Activation
of Mi tagens, Env. Filth. Persp. , 6(1973)71
75. Czygan, P., Greim, H., Garro, A. J., Hutterer, F., Schaffner, F., Popper, H.,
Rcsenthal, 0., and Cooper, D. Y., Microsomal Metabolism of Dimethylrdtrosamine
and :he Cyrochrom P-450 Dependency of its Activation to a Mutagen, Cancer Res. ,
33 (1973) 2983
76. Bartsch, H., Macaveille, C., and Montesano, R., The Predictive Value of Tissue-
Mediated Mutagenicity Assays to -tssess tne Carcinogenic Risk or Chemicals In
‘Screening Tests in Chemical CarcinogeneSis’ (eds) Montesano, R., Dartsch, H.,
and Tomads, L., WHO/IARC Pubi. No. 1, International Agency for Research on
Cancer, Lyon (1976) pp. 467491
243
-------
77. Bartsch, H., Ia1aveille, C., and Montesario, R., In Vitro Metabolism and Microsome-
mediated Mutagenicity of Dialkynitrosainines in Rat, Hamster, and Mouse Tissues,
Cancer Res. , 35(1975) 644
78. Heath, D. F., The Decomposition and T3xicity of Dialkylniuosamines in Rats,
Biochem. J. , 85(1962)72
79. Fahmy, 0. G., Fahrny, M. J., and Wiessler, M.,-Acetoxy-Dimethvl-Nitrosarnin:
Approximate Metabolite of the Carcinogenic Amine; Biochem. Pharmacol. , 24
(1975) 1145
80. Rice, J. M., Joshi, S. R., Roller, P. P., and Wenk, M. L., Methyl (Acetoxymethyl)
Nitrosamine: A New Carcinogen Highly Specific for Colon and Small Intestine
(Abstract); 66th Meeting. Proc. Am. Assoc. Cancer Res. . 16 (1975) 32
81. Wiessler, M., and Schmahl, D., Zur Carcinogenen Wirkling Von N-Nitroso-
Verbindur.gen. V Acetoxymethyl-Methyl-Ni -osamin; Z. FZrebsiorsch . (1975) in press
82. Fahmy, 0. C., and Fahmy, M. J., Mutagenic Selectivity of Carcinogenic Nitroso
Compounds: III. N-Acetoxymethyl-N-?. e:hyl Ni osamine, Chem. Biol. Int. , 14
.9io) 21
83. Montesano, R., and Bar:sch, H., Mutagenic And Carcinogenic N-Ninoso Compounds
Possible Environmental Hazards, Muta cn Res . 32 (1976) 179
84. Neale, S., Mutagenicity of Nitrosamides and Nin-osainidines in Microorganisms
and Plants, Mutation Res. , 32(1976) 22?
244
-------
IX. AROMATIC AMINES
Aromatic Arnines
1. Benzidine and Analogs-Benzidine and related diaminobiphenyls, homologous
tolidines and other derivatives are of considerable importance as organic inter-
mediates for the manufacture of a wide variety of organic chemicals and intermediates
for azo dyes. For examples, the major uses of benzidine are: (1) based on the
conversion of the amino groups to dyestuffs via facile diazotization with nitrite ion
and thence coupling with aromatic acceptors (e.g., naphthols) and (2) the high-
temperature reaction of the amino groups with polyurethanes to effect cross-linking
to yield products with enhanced physical properties’. Benzidine and related
diaminobiphenyls have also been extensively employed in analytical chemistry for
the detection of a large number of inorganic ions and compounds 2 , (e.g., HCN,
sulfate, nicotine, and sugar e). A solution of benzidirte in 50% acetic acid has been
used widely for the determination of the presence of human Nood.
Benzidine (H 2 N-®-®NH 2 ) , 3, 3’-dichlorobenzidine (H 2 N® - NH 2 )
3, 3 1 -dimethylbenzidine o—toluidine; H Ne - -NH 2 ), 3, 3 ‘-dimethoxyben zidine
(o-dianisidine; H 2 N_@- NH 2 ) have been used since 1930 principally in the
manufacture of dyestuffs and pigments. 3,3’-Dichlorobenzidine is also used alone
and in blends with 4,4’-methylenebis(2-chloroanlline) as a curing agent for liquid-
castable polyurethane elastom ers.
Additional important uses of 3,3’—dimethoxybenzidine include its use as an
intermediate in the production of o-dianisidinediisocvar.ate, and the detection of
the presence of a number ef metals, thiocyanates and nitrites.
Estimates of the number of people exposed to benzidir .e and 3, 3’-dichlorobenzidine
are difficult to obtain. It l as been suggested that 62 pecpie in the U.S. are exposed
245
-------
to the former and between 250 and 2500 to the latter 3 . It is possible that exposures
could be exceeded since 1.5 million pounds of benzidine were produced in the U.S.
in 1972 while 3.5 million pounds of 3, 3 1 -dichlorobenzidine were produced domes-
tically in addition to another 1.4 million pounds imported in l971 ’ .
2 3,5—10
Although benzidine is a recognized bladder carcinogen in exposed workers
analogous to other aromatic arnines, the nature of the precise mechanisms responsible
for the induction of neoplasia following exposure to diverse aromatic amines is not
known.
Evidence exists that the metabolism of these compounds is analogous to that
observed with other aromatic antines, via., ring hydroxylation, N-hydroxylation,
of the monoacetyl derivative, and conjugation with sulfate and glucuroriic acidUlS.
It has also been suggested that the sulfate and glucuronide conjugates of the aromatic
amines might be the carcinogenically active forms in vivo 3 .
Tables 1 and 2 summarize the bio-transformation of benzidine and the physiologic
changes induced by benzidine and congeners in various species respectively. A
summary of the metabolic pathways by which aromatic amines may modify nucleic
acids and proteins is shown in Figure 1 using 4-aminobiphenyl as an illustrative example.
It should be noted that the metabolites listed in Table 1 are in the main postulated , with
confirmatory evidence largely lacking.
Benzidine and its analogs Ce. g., 3,3’ -dichlorobenzidirie; 3, 3 , 5, 5’ -tetrafluoro-
beazidine) have been shown to be frameshift mutagens in a liver mixed function
oxidase system with S. typhimuriurn TA l53816 17 Other compounds tested in
this study 17 , 3, 3 ’-dianisidine (3,3 ‘-dimethoxybenzidine) and 3,3’, 5, 5 ’-tetramethyl-
18 10
benzidine which were either weak carcinogens or noncarcinogenic respectively
were found to have slight mutagenic activity (in the activated system only) and no
mutagenic activity respectively indicating a good correlation between animal
246
-------
car:inogenicity experiments and the bacterial mutagenicity assay. Benzidine has
also been shown to be rnutagenic in the micronucleus test in rats 2 ° inducing high
incidences of micronucleated erythrocytes following both dermal application and
subcutaneous injection. 2-Amino-, and 4-aminobiphenyl have been found mutagenic
in the Salrnonella/niicrosome test 21 . 4-Aminobiphenyl is carcinogenic in the mouse,
rat, rabbit, and dog 2 . A high incidence of bladder carcinomas has been reported
in one series of workers occupationally exposed to commercial 4-aminobiphenyl 2 .
247
-------
TABLE 1
Summary of Benzidine Biotransformation in Various Species 3
Species Metabolites
Mouse Monoacetylated 3—OH ethereal sulfate
Monoacetylated 3-OH glucu.ronide
N-Hydrogen sulfate and/or glucuronide
3-OH-Benzidine glucuronide
Rat 3,3’-Dihydroxybenzidine (?)
4 ‘-Acetarnido-4-amino -3 -diphenylyl hydrog en sulfate
4’—Amino-4-diphenylyl sulfamnic acid
4’-Acetarnido-4-diphenylyl sulfamic acid
Guinea pig 4 t -Acetamido -4-aminodiphenyl N-glucuronide
4’-Acetamido-4-amino-3-diphenylyl hydrogen sulfate
Rabbit 3’-OH-Benzidine sulfate and glucuronide
4’-Acetamido-4-arnino-3-diphenylyl hydrogen sulfate
4’-Amino—4-diphenylyl sulfamic acid
4 t -Acetamido-4-diphenyiyl sulfamic acid
N -Glucuronides
4! —Acetamido-4-aminodiphenyl
3-OH-Benzidine
Dog 3-OH-Benzidine
3-OH-Benzidir.e hydrogen sulfate
4-Amino-4-hydroxybiphenyl
4,4 -Diamino-3-diphenyl sulfate and glucuronide
Monkey Monoacetylbenzidine
Man 3,3’—Dihydroxvbenzidine (?)
Mono- and diacetylbenzidine
3-ON-Berizidine
N-Hydroxy acety laminobenzidine
248
-------
TABLE 2
Types of Physiologic Changes in Various Species 2 ’ 3
Species Carcinogen Physiologic change
Mouse Benzidine Hepatoma, lymphoma, bile duct
proliferation
3,3 ‘-Dihydroxybenzidine Hepatoma, lymphoma, bile duct
proliferation, benign bladder papilloma
Rat Benzidine and its sulfate Cirrhosis of liver, hepatomas,
carcinoma of Zymbal’s gland, adeno-
carcinoma, degeneration of bile ducts,
sarcoma, mammary gland carcinoma
3 3 t.Dichlorobenzidine Extensive cancer
3,3 ‘-Dimethyoxybenzidine Intestinal, skin, Zymbal, gland
carcinoma, bladder papilloma
3, 3 —Dihydroxybenzidine Hepatoma, adenocarcinoma of colon,
carcinoma of fore stomach, Zymbal’s
gland carcinoma, bladder carcinoma
Dianisidine Zyrnbal’s gland carcinoma, ovarian
tumor
o-Ditoluidine Papilloma of stomach, Zymbal’s gland
carcinoma, mammary tumor leucoses
3, 3 ‘-Benziniedioxyacetic Papilloma of bladder, hepatic sarcoma
N, N’ -Diac etylbenzidine Chronic glomerulonephritis
Hamster Benzidine Hepatoma, liver carcinoma, cholangiomas
3,3’-Dichlorobenzidirie Transitional cell carcinomas of the
bladder, liver cell tumors
3,3’ -Dimethoxybenzidine Fore stomach papilloma, urinary
bladder tumors
o-Ditoluidine Bladder cancer
Rabbit Benzidine Proteinuria hematuria, liver cirrhosis,
myocardial atrophy, bladder tumor,
gall bladder tumor
Do Benzidine Recurrent cystitis, bladder tumor,
convulsions, liver cirrhosis, heniaturia
Monkey Benzidine o pathology
Man Benzidine Bladder tumor, papilloma, chronic
cystitis, hematuria
249
-------
FIGURE 1
PATHWAYS BY WHICH 4-AMINOBIPHENYL MAY BE ACTIVATED METABOLICALLY *
K I>.—K NH CH 3
Amine
Ti
Ilydroxamic acid
>
--——- ( ( NH 2
Amine
, >
JOH
011
Ilydroxylanilne
11 NN
- g-cCH3
Hydroxami.c acid
esters
1
Arylacetamide—nucleic acid
& protein adducts
- )
U i
o—Aininophenol
@-® -N02
Nitro
1
NH
o—Quinone lime
;::
Nitroso
I
Azoxy
Ilydroxylamine es
Arylamine—nucleic acid
& protein adducts
Arylamine—prote in
adducts
*Baetke, K., Aromatic Amines Program, Mechanistic Approaches to Carcinogenics; NCTR Report, Nov. 6 (1976)
-------
2. Naphthylamine and Analogs-1-Naphthylamine (alpha-naphthylamine) is used
as a intermediate in the preparation of a large number of compounds with the major
uses including the manufacture of dyes, herbicides (e.g., N-1-naphthylphthalamic
acid) and antioxidants. Occupational exposure to commercial 1-naphthylamine
containing 4-10% 2-naphthylamine is strongly associated with bladder cancer in
22
man . However, it is not possible at present to determine unequivocally whether
22
l-naphthylamine free from the 2 -isomer is carcinogenic to man
The carcinogenicity of l—naphthylamine in animals is equivocal. For example,
no carcinogenic effect of 1-naphtylamine was found in the hamster following oral
administration; inconclusive results were obtained in mice after oral and subcutaneous
administration and in dogs. 1-Naphthylamine, if carcinogenic at all, was less so to
the bladder than was the 2-isomer 22 . The carcinogenicity of rnetabolites of
1-naphthylamine (e.g., N-(1-naphthyl)--hydroxylamine) in rodents has been re-
22—24
ported . N—Hydroxy-1-naphthylamine is a much more potent carcinogen than
N-hydroxy--2-naphthylamine.
21
1-Naphthylamine is mutagenic in the Salmonella/microsome test
2-Naphthylamine (beta -naphthylamine) has been used as an intermediate in the
manufacture of dyes and antioxidants. Earlier uses of 2-naphthylamine included
its utility in the manufacture of 5-acid (2-amino-5-naphthoi7sulfonic acid), gamma-
acid (7-amino-1-naphthol3SUlfOfliC acid), in the synthesis of N-alkyl-2naphthyl
amines used as dye intermediates and the anti-oxidant N-phenyl-2-naphthylamine’ .
2-Naphthylamine is present as an impurity in commercial l-naphthylamine (e. g.,
22
in U . S. produced product the content has been present at levels of 0. 5% or less)
2-Maphthylainine has been found in cigarette smoke (e. g., 0. 02 pg/cigarette which
25
is equivalent to 1 tg/SO cigarettes) the daily exposure of a heavy cigarette smoker
251
-------
2-Naphthylamine has also been found in coal-tar 26 and in gas retort houses at
atmospheric levels sufficient to give a human exposure of 0.2 tg/day 27 .
Epidemiological studies have shown that occupational exposure to 2-naphthyl-
amine, either alone, or when present as an impurity in other compounds, is strongly
associated with the occurrence of bladder cancer 22 ’ 28-34
2-Naphthylamine, administered orally, produced bladder carcinomas in the
23,35,36 22,27 38,39
dog and monkey and at high dosage levels, in the hamster
Although oral administration of 2-naphthylamine increased the incidence of hepatomas
in the mouse 4 ° it demonstrated little, if any, carcinogenic activity in the rat and
22,40, 41
rabbit
Evidence to date suggests that several carcinogenic metabolites, rather than
a single proximate carcinogen are responsible for the carcinogenic activity demon-
strated by 2-naphthylamine 22 ’ 42 ’ 43 . These include: 2-naphthylhydroxylamine
and/or an 0-ester therof; bis (2hydroxylamino 1-nap hthyl )phosphate; bis (2-amino-
1. -naphthyl)phosphate; 2-amino -1 -naphthol free and! or conjugated and 2-hydroxyl-
aininolnaphthol.
The demonstrated carcinogenicity spectrum of the metabolites is from highly
active to weakly active in some species and testing system(s) 22 ’ 4247 .
Metabolic pathways leading to probable proximate carcinogens of 2-naphthylamine
42,43
are iUustrated in Figure 2
Twenty-four metabolites of 2—naphthylamine have been identified in the urine of
rats, rabbits, dogs or monkeys by Boyland 44 and Boyland and Manson 45 . The
following mechanisms were suggested to represent the metabolic pathways 23 ’ 44 ’ 45 :
252
-------
(1) N-hydroxylation followed by conversion to 2-amino-i-naphthyl
mercapturic acid, 2 -nitrosonaphthalene and rearrangement to
2-amino-i -nap hthol
(2) Oxidation at C 5 and C 6 to an arene oxide which rearranges to
5-hydroxy-2-naphthylamine, reacts with water to form a 5,6-
hydroxy dihydro derivative and forms a 5-hydroxy-6-mercapturic
acid
(3) Conjugation of the amino group with acetic, sulphuric, or
glucosiduronic acid
(4) Secondary conjugation of the hydroxyl group with phosphate
sulfuric or glucosiduronic acid.
Recent reports have highlighted the potential problem of the metabolic conversion
of industrial chemical precursors to 2-naphthylamine 48 ’ 49 . For example, phenyl-
beta-naphthylamine (PBNA) which is not currently regulated by OSHA, is widely
used as a antioxidant in the rubber industry, as an antioxidant for grease and oils
in the petroleum industry, as a stabilizer for the manufacture of synthetic rubber
and as an intermediate in the synthesis of dyes as well as other antioxidants
In a recent study in the U.S. with volunteers, 3-4 micrograms of 2-naphthylamine
were found in the urine of individuals who had 50 mg of PBNA (contaminated with
0.7 g 2-naphthylamine) and from workers estimated to have inhaled 30 mg PBNA 49 .
These findings indicated that PBNA is at least partially metabolized by man to 2-
48 50
naphthylamine and confirmed an earlier study in the Netherlands - ‘ where volun-
teers who consumed 10 mg PBNA (containing 0.032 micrograms of 2—naphthylamine
as an impurity) were found to have 3 to 8 micrograms of 2-naphthylamine in their
urine samples.
253
-------
It should be noted that 15,000 workers are at potential risk of exposure to
phenyl-beta-naphthylainine during its manufacture and use 49 .
The carcinogenic potential of the metabolism of 2-nitronaphthalene (an uninarketed
byproduct produced during the commercial preparation of l-naphthylamine) has
also recently been stressed 48 ’ 49 . 2-Nitronaphthalene (analogous to PENA) is meta-
bolized by beagle dogs to 2-naphthylamine. In earlier studies, female dogs fed 100
mg of 2-nitronaphthalene daily for 8 months, after 10.5 years, bladder papillomas
were observed in various stages of maligtiancy of 3 of 4 dogs 48 .
2-Naphthylamine as well as 2-naphthyl- and l-naphthylhydroxylamines are
mutagenic in the Salmonella/microsome test 21 . However, N-hydroxy-1-, and N-
hydroxy-2-iiaphthylamines although toxic, were not mutageriic to intracellular T4
51 . . 52
phage . Earlier studies indicated that N-hydroxy-l-naphthylamine and N-hydroxy-
2—naphthylaxnine caused mutations in bacteria 52 ’ However, the significance of
the mutagenesis data reported in these studies 2 ’ was suggested 51 to be marginal
(e.g., less than a 10 fold increase in the frequency of revertants in back-mutation
experiments).
254
-------
Figure 2. o ic P.Lthw }s LCadtng to Probabk Prox ma e Ca cinc. nc o1 -Na 7 hth}1arninc
CQmu d in parenth s rcprcsertt hy othe ic& r aboIi es an Io ’ s to tho Iouid
ot r aromatic amines and azo dyes. Solid fines rre d rnoi zrat d o tes of rneta-
ar d the broken lines are hypothetical pathways . [ From J. C. Arco a d M. F. ArguS.
A. crt. C -’cer R ’s.II, 305 (I968J )
NO
ccxcv:ii
255
-------
3. Methylene Bis-Aniline Analogs-4 , 4’ -Methylenedianiline (bis- (4-aminophenyl) -
methane; DDM; MDA; DAPM; H 2 N CH 2 - -NH 2 ) is used (unisolated) princi-
pally in the manufacture of polymethylene polyphenyl isocyariate (used in rigid
polyurethane foam), and 4, 4’-methylenediphenyl isocyanate (mostly used in the
production of Spandex fibers). The major usage for isolated DAPM is in the produc-
tion of the corresponding halogenated diamine 4,4 ‘-rnethylenebis (cyclohexylamine)
which is employed in polyurethane coatings. Isolated DAPM is also used as an
intermediate in the production of polyarnide-imide resins and fibers; in the synthesis
of pararosaniline dyes and as a curing agent for liquid-castable polyurethane
elastomers and epoxy resins 54 .
Over 200-million pounds of DDM are manufactured annually in the U.S. by
condensation of aniline with formaldehyde in the presence of an acid catalyst 55 ,
NIOSH estimates that 2,500 workers are exposed to DDM. Approximately 90% of DDM
produced is consumed in crude form at its production site by reaction with phosgene
in the preparation of the intermediates such as isocyanates and polyisocyanates
which are used in the manufacture of rigid polyurethane 55 .
Extremely limited carcinogenicity studies have been reported (in the rat) which
to date do not permit a definite conclusion regarding the carcinogenicity of DAPM
in this species 54 . Severe hepatotoxic effects of exposure to DAPM in man have been
reported 55 ’ 56; however, no significant epidemiological data have been reported 54 .
44 -Methylene bis (2-chloroaiuline) (MOCA; DACPM; H 2 N 3 CH 2 NH 2 )
is used primarily as a curing agent for isocyanate-containing polymers and as an
agent for curing liquid-castable polyurethane elastomers suitable for molded mechanical
articles and for potting and encapsulating purposes 57 . It is frequently formulated
256
-------
with other aromatic diamines (e . g., 3,3 ‘-dichlorobenzjdjne or 4,41 -methyle -ie-
dianiline) to prepare curing agents. Very small quantities (e. g., approximately
1% of the total consumed) of MOCA are believed to be used as a curing agent for epoxy
and epoxy-urethane resin blends 57 . Commercial production of MOCA is believed
to involve the reaction of formaldehyde with orthochloroaniline, with U.S. produc-
tion in 1972 amounting to approximately 7.7 million pounds 57 .
MOCA when administered at levels of 0.2% and 0.1% in the diet of mice produced
vascular tumors at the higher dose level and hepatomas at both levels 58 and hepa-
tornas and lung tumors in rats maintained throughout their lifespan on a low protein
diet containing 0.1% MOCA 59 . MOCA is mutagenic in the Salmonella/microsome
21
test
4• 4, 4’ — Methylene bis ( 2—methylaniline ) (4,4 ‘-methylenediortho toluidine; 4, 4’-
diamino-3, 3’-dimethyldiphenylmethane; H 2 N? -( -NH 2 ) has been produced
commercially in the past probably via the reaction of formaldehyde with ortho-
toluidine 60 . It has been used (as an unisolated intermediate) in the manufacture
of the corresponding diisocyanate, 4,4’-methylene bis(ortho-tolylisocyanate) for
use in the production of polyurethanes 60 . 4,4 -Methylene bis(2-methylaniline)
has al%o been used in the synthesis of the dye Cd. Basic Violet 2. 4 ,4 ’-Methylene
bis(2-methylaniline) is carcinogenic in the rat after oral administration, the only
60
species and route tested
257
-------
References for Aromatic Amities
1. Radding, S. B., Holt, B. R., Jones, J. L., Liu, D. H., Mill, T., and Hendry,
D. G., Review of the Environmental Fate of Selected Chemicals, Stanford
Research Inst. Rept. Jan. 10 (1975)
2. IARC, Monograph No. 1, International Agency for Research on Cancer, Lyon
(1972) pp. 80—86; 89—91; 74—79
3. Haley, T. 3., Benzidine revisited: A review of the literature and problems
associated with the use of benzidine and its congeners, Clin. Toxicol. , 8, (1975)
13—42
4. Anon, Final Rules Set for Exposure to Carcinogens, Chem. E g. News , Feb. 11
(1974) p. 12
5. Hueper, W. C., Occupational and Environmental Cancers of the Urinary System,
Yale University Press, New Haven and London (1969)
6. Case, R. A. M., Hosker, M. E., McDonald, D. B., and Pearson, 3. T. (1954)
Tumors of the urinary bladder in workmen engaged in the manufacture and use
of certain dyestuff intermediates in the British Chemical Industry. I. The role
of aniline, benziduie, OC.. -naphthylamine and -naphthylamine, Brit. J. md.
Med. , 11(1954) 75
7. Goldwater, L. J., Rosso, A. 3., and Kleinfeld, M., Bladder tumors in a coal
tar dye plant, Arch. Env. Hlth. , II (1965) 814
8. Ubelin, F. Von., and Pletscher, A., Atiologie und prophylaxe gewerblicher
tumoren in der farbstoff-industrie, Schweiz. Med. Wschr. , 84 (1954) 917
9. Anon, Federal Register , 39 (1974) 3756 (Jan. 29, 1974)
10. Zavon, M. R., Hoegg, U., and Bingham, E., Benzidine Exposure as a Cause
of Bladder Tumors, Arch. Env. Filth. , 27 (1973) 1
11. Miller, 3. A., and Miller, E. C., The metabolic activation of carcinogenic aromatic
amines and amides, Progr. Exptl. Tumor Res. , 11(1969) 273
12. Miller, 3. A., and Miller E. C., Chemical carcinogenesis: Mechanisms and
approaches to its control, 3. Nati. Cancer Inst. , 47 (1971) V-XIV
13. Miller, 3. A., Carcinogenesis by Chemicals: An Overview-G.H.A. Clowes
Memorial Lecture, Cancer Res. , 30 (1970) 559—576
14. Gu ann, H. R., Malejka-Giganti, D., Barry, E. 3., and Rydell, R. F., On the
correlation between the hepato carcinogenicity of the carcinogen N-2-fluoreny
lacetamide and its metabolite activation by the rat, Cancer Res. , 32 (1972) 15541560
2 5S
-------
15. Arcos, J. C., arid Argus, M. F., ‘ T Chernjcal Induction of Cancer , Vol. IIB,
Academic Press, New York (1974) PP. 23-37
16. Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D., Carcinogens are
mutagens. Simple test system combining liver homogenates for activation and
bacteria for detection, Proc. Nati. Acad. Sci . (USA) 70 (1973) 2281-2285
17. Garner, R. C., Walpole, A. L., and Rose, F. L., Testing of some benzidine
analogs for microsomal activation to bacterial mutagens, Cancer Letters , 1
(1975) 39—42
18. Hadidian, Z., Fredrickson, T. N., et al., Tests for chemical carcinogens.
Report on the activity of derivatives of aromatic amines, nitrosamines, quino-
lines, nitroalkanes, amides, epoxides, aziridines and purine antimetabolites,
3. Nati. Can. Inst. , 41(1968) 985
19. Holland, V. R., Saunders, B. C., Rose, F. L., and Walpole, A. L., Safer
substitute for benzidine in the detection of blood, Tetrahedron. , 30 (1974) 3299
20. Urwin, C., Richardson, J. C., and Palmer, A. K., An evaluation of the
mutagenicity of the cutting oil preservative Grotan BK, Mutation Res. , 40,
(1976) 43
21. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N., Detection of carcino-
gens as mutagens in the Sairnonella/microsome test: Assay of 300 chemicals,
Proc. Nat. Acad. Sci. , 72(1975) 5135
22. IARC, Monograph No. 4, International Agency, Lyon (1974) Pp. 87-96
23. Boyland, E., Busby, E. R., Dukes, C. E., Grover, P. L., and Manson, D.,
Further experiments on implantation of materials into the urinary bladder of
mice, Brit. 3. Cancer , 18 (1964) 575
24. Radomski, 3. L., Brill, E., Deichmann, W. B., and Glass, E. M., Carcino-
genicity testing of N-hydroxy and other oxidation and decomposition products
of 1— and 2-naphthylamine, Cancer Res.. , 31 (1971) 1461
25. Hoffmann. D., Masuda, Y., and Wynder, E. L., ‘ < -Nap hthylamine and fi
naphthylamine in cigarette smoke, Nature , 221 (1969) 254
26. Treibl, H. G., Naphthalene derivatives. In Kirk, R. E. and Othmer, D. F., eds,
‘Encyclopedia of Chemical Technology” 2nd ed., John Wiley & Sons, Vol. 13,
New York, p. 708
27. Battye, R., Bladder carcinogens occurring during the production of “fown”
gas by coal carbonisation. In ‘ Transcripts of the XV International Congress
on Occupational Health, Vienna , Vol. VI-2 (1966) p. 153
259
-------
28. Veys, C. A., Two epidemiological inquiries into the incidence of bladder tumors
in industrial workers, J. Nat. Cancer Inst. , 43 (1969) 219
29. Vigliani, E. D., and Barsotti, M., Environmental tumors of the bladder in some
Italian dye-stuff factories, Acta. Un. mt. Cancer , 18 (1961) 669
30. Temkin, I. S., “Industrial Bladder Carcinogenesis” Pergarnon Press, Oxford,
London, New York, Paris (1963)
31. Tsugi, I., Environmental arid industrial cancer of the bladder in Japan, Acta.
Un. mt. Cancer , 18 (1963) 662
32. Gehrmann, G. H., Foulger, J. H., and Fleming, A. J., Occupational carcinoma
of the bladder, In Proceedings of the North International Congress of Industrial
Medicine, London, (1948), Bristol, Wright, p. 427
33. Hueper, W. C., ‘Occupational Tumors and Allied Diseases” Thomas Pubi.,
Springfield, Di.
34. Billiard-Duchesne, J. L., Cas francais de tumeurs professionnelles de la vessie,
Acta. Un.Int. Cancr. , 16(1960) 204
35. Conzelman, G. M., Jr., and Moulton, J. E., Dose-response relationships of the
bladder tumorigen 2-naphthylamine: A study in beagle dogs, J. Nat. Cancer
Inst. , 49 (1972) 193
36. Bonser, G. M., Clayson, D. B., Jull, J. W., and Pyrah, L. N., The carcino-
genic activity of 2-naphthylamine, Brit. J. Cancer , 10 (1956) 533
37. Conzelman, G. M., Jr., Moulton, J. E., Flanders, L. E. III, Springer, K.,
and Crout, D. W., Induction of transitional cell carcinomas of the urinary
bladder in monkeys fed 2-naphthylamine, 3. Nat. Cancer Inst. , 42 (1969) 825
38. Saffiotti, U., Cefis, F., Montesano, R., and Sellakumar, A. R., Induction of
bladder cancer in hamsters fed aromatic ainines. In “Bladder Cancer. A Symposium”
eds Deichmann, W., and La.mpe, K. F., Aesculapius Press, Birmingham, Ala.
(1967) p. 129
39. Sellakumar, A. R., Montesano, R., and Saffiotti, U., Aromatic Amines carcino-
genicity in hamsters, Proc. Amer. Ass. Cancer Res. , 10 (1969) 78
40. Bonser, G. M., Clayson, D. B., Jull, 3. W., and Pyrah, L. N., The carcinogenis
properties of 2-amino-1-naphthol hydrochloride and its parent amine 2-naphthyl-
amine, Brit. 3. Cancer , 6(1952) 412
41. Hadidian, Z., Fredrickson, T. N., Weisburger, E. K., Weisburger, J. H., Glass,
R. M., and Mantel, N., Tests for chemical carcinogens. Report on the activity
of derivatives of aromatic amines, nitrosamines, quinolines, nitroalkanes, amides,
epoxides, aziridines and purine anti-rnetabolites, 3. Nat. Can. In. , 41 (1968) 985
260
-------
42. Arcos, J. C. and Argus, M. F., ‘Chemical Induction of Cancer”, Vol. IIB,
Academic Press, NY (1974) 247-253
43. Arcos, J. C., and Argus, M. F., Molecular Geometry and Carcinogenic Activity
of Aromatic Compounds. New Perspectives, Adv. Cancer Res. , 11 (1968) 305
44. Boyland, E., The biochemistry of cancer of the bladder, Brit. Med. Bull. , 14
(1958) 153
45. Boyland, E., and Manson, D., The metabolism of 2-naphthylamine and 2-naph-
thyihydroxylamine derivatives, Biochem. J. , 101 (1966) 84
46. Radomski, 3. L., and Brill, E., The role of N-oxidation products of aromatic
amines in the induction of bladder cancer in the dog, Arch. Toxicol. , 28 (1971)
159
47. R domski, 3. L., Brill, E., Deichmann, W. B., and Glass, E. M., Carcinogenicity
te3tng of N-hydroxy and other oxidation and decomposition products of 1-, and
2-naphthylamine, Cancer Res. , 31(1971) 1461
48. Anon, NIOSH Issues alert on precursors of beta-naphthylamine, Occup. Hith.
Safety Letter , 6 (24) (1976) 4—5
49. Moore, R. M., Jr., Woolf, B. S., Stein, H. P., Thomas, A. W., and Finklea,
3. F., Metabolk precursors of a known human carcinogen, Science , 195 (1977)
344
50. Kummer, R., and Tordoir, W. F.,
Tijdschr. Soc. Geneesk. , 53(1975)415
51. Corbett, T. H., Heidelberger, C., Dove, W. F., Determination of the mutagenic
activity to bacteriophage of T4 of carcinogenic and non-carcinogenic compounds,
Mol. Pharmacol. , 6 (1970) 667—679
52. Perez, G., and Radomski, 3. L., The mutagenicity of the N-hydroxy naphthyl-
amines in relation to their carcinogenicity, md. Med. Surg. . 34 (1965) 714-716
53. Bellman, S., Troll, W., Teebor, G., and Mukai, F., The carcinogenic and muta-
genic properties of N-hydroxyaminonaphthalenes, Cancer Res. , 28 (1968) 535-542
54. IARC, Monograph No. 4, International Agency for Research on Cancer, Lyon
(1974) pp. 79—85
55. Anon, NIOSH warns that DDM may cause toxic hepatitis in workers, Toxic
Materials News , 3 (4) (1976) 26
261
-------
56. Kopelman, H., Robertson, M. H., Sanders, P. G., and Ash. I., The epping
jaundice, Brit. Med. J.,i(1966 ) 514
57. IARC. Monograph No. 4., International Agency for Research on Cancer, Lyon
(1974) pp. 65—71
58. Russfield, A. B., Homberger, F.., Boger, E., Weisburger, E. K., and Weis-
burger, J. H., The carcinogenic effect of 4,4-methylene-bis-(2-chloroaniline)
in mice and rats, Toxicol. Appi. Pharmacol. , (1973)
59. Grundmann. E., and Steinhoff, D., Leber und lungentumoren nach 3,3’-dichlor-
4,4’-diaminodiphenylmethan bei ratten, Z. Krebsforsch. , 74 (1970) 28
60. IARC, Vol. 4, International Agency for Research on Cancer, Lyon (1974) p. 73-77
262
-------
X. Azo Dyes
1. Azobenzene-Azo dyes form the largest and most versatile class of all dyes.
They are a well defined group of compounds characterized by the presence of one or
more azo groups (-NN-). Chemically, the azo class is subdivided according to the
number of azo groups present, into mono-, di-, tris-, tetrakis-, and higher azo deri-
vatives.
Azo dyes have a multitude of uses depending on their chemical structures
and method of application. Their areas of utility include: dyeing of wool, silk, leather,
cotton, paper and the synthetic fibers (e.g., acetate, acrylics, polyamides, polyesters,
viscose rayon); for the coloring of paints, plastics, varnishes, printing inks, rubber,
cosmetics, food, drugs; for color photography; diazotypy and for staining polish as
well as absorbing surfaces 1 .
Azobenzerie ( -N = N-s) has been used for the production of benzidine
and its salts and as an intermediate in the manufacture of insecticides, dyes, rubber
accelerators and pyrazolone derivatives 2 . Limited carcinogenicity studies of azo-
benzene in mice and rats have been reported with inconclusive results 2 . Azobenzene
(of undefined purity) has been reported to be zion-mutagenic when tested in Drosophila
for the production of X-linked recessive iethals 3 , and mutagenic in the Salmonella
4
TA 100/microsome test
2. para-Aminoazobenzene-para-Aminoazobenzene [ 4-(Phenylazo)-benzenamine;
-N N-®NH 2 1 is used as an intermediate in the production of acid yellow,
diazo dyes and indulines; as a dye for lacquers, varnishes, wax products, oil stains
and styrene resins. p—Aminoazobenzerie is carcinogenic in rats following its oral
administration producing liver tumors, and by application to the skin producing epi-
2
dermal tumors . p-Aminoazobenzene is mutagenic in the Salmonella TA1538/microsome
test 4 ’
263
-------
3. ortho-Aminoazotoluene-ortho-Aininoazotoluene [ 4-methyl-4- (2-methyiphenyl) -
azo-benzenamine] is used to color oils, fats and waxes 2 , and is carcinogenic in mice,
rats, hamsters and dogs following its oral administration, producing mainly tumors of
the liver, gall-bladder, lung, and urinary bladder. ortho-Aminoazotoluene is mutagenic
in Salmonella TA 1538/microsome test 4 ’ .
4. para-Dimethylaminoazobenzene-para-Dimethylaminoazoben zene [ N, N-
dirnethyl-4-aminoazobenzene; DAB; N, N-dimethyl -4- (phenylazo) -ben zenamide;
-N = N@N CH is used for coloring polishes and other wax products, poly-
CR 3 2
styrene, gasoline, soap, and as an indicator
Para-dimethylaminoazobenzene and its derivatives have been extensively
employed in modern experimental studies on amino azo dye carcinogenesis 6 . A
great range of activity has been displayed employing a broad spectrum of derivatives
of DAB. Tables 1 and 2 illustrate the synoptic tabulation of structural requirements
for hepato carcinogenicity of para-dimethylaminobenzene in the rat for substitution
in the 4 1,4L., 4 1,4L. and uniform substitutions in the 2—, and 61_,
positions respectively. Table 3 depicts the synoptic tabulation of structural require-
ments for hepato carcinogenicity of DAB demonstrating the effect of simultaneous
substitution by different substituents.
DAB is carcinogenic in rats, producing liver tumors after its oral administration
by several routes, and in dogs, producing bladder tumors following its oral
administration 2 .
DAB as well as N-methyl-4-aminoazobenzene (MAE) ® NN®-N-CH 3 )
are mutagenic in S. tv’ohimurium TA 100 and TA 98 strains metabolically activated
by rat liver microsomal enzymes (S-9 mix) 4 ’ 6 . N-acetoxy-N-methyl-4-arninoazo-
264
-------
ben zene and N-ben zoyloxy-N-methyl -4-aminoazoben zene and their 4’ -methoxycarbonyl
derivatives were also mutagenic in TA 100 and TA 98 tester strains and did not require
metabolic activation by S-9 mix 6 . The correlation of the mutagenicity of DAB and its
derivatives with carcinogenicity can be tabulated as follows 6 :
Compound Mutag eriicity Carcinog enicity
TA100 TA98
S-9 mix (-)+
(-) +
p—Dimethylaminoazobenzene (DAB) - + - + +
3 ’-methy l-DAB - + - + +
N-methyl-4-aminoazobenzene (MAB) - + - + +
2-methyl-DAB - + - + +
4-aminobenzene (AB) - + - + +
o-aminoazotoluene Co-AT) - + - + +
3-methoxy-AB - + - + +
N-hydroxy-AB - + - + +
N-acetoxy-MAB + + + + -
N-benzoyloxy-MAB + + + + +
4’-methoxycarbony lNa cetoXyMAB + + + +
4’-methoxycarbonylNbenZOYlOXYMAB + + + +
N-hydroxy-MAB - + + +
4-methoxycarbony l-N-hydroXyMAB - + + +
4’-methoxycarbonylMAB - - - -
7
All the carcinogenic azo dyes and their derivatives tested were mutagenic
These findings (as well as those reported by others 2 ) suggest that azo dyes are
metabolized to ultimate carcinogens which modify bases in DNA. This alteration in
DNA bases probably results in mutation as well as carcinogenesis. N-acetoxy-MAB
7
would appear to be a likely ultimate carcinogen
265
-------
Tabk 1
Synoptic Tabulation of Struczur 1 Rqu re nts for
Hepatocarcinogcnicity of 4-Dimeth zobr zeae in the Rat.
Substitution in the 4 -.4-. I -. a 1- o t on
W kl active if
—CH
—c
‘ • active if
—F
—, 0 by
;: Iacing tr.e
priIne ring
.ith r id& e-
4-X-o :dc
Moderately active if
—C 3 H.
—C.H 9
—SCH,
ha::i e if
—0:-i
0C.H
— 3r
—CF
—COOH
—NH OC -CH 3
N CH,i.
—C H
—SO H
—S Ot0H’
Inactive if
—N=CH—
—CH=N—
—C0 .NH—
—CO--CHCH—
—NH H 2 N—
Active mainly it sites
other than liver if
—CH=CH--
Transition to
4-aminostilbene
—S—
Tran :tion to
4-aminodiphenyl
sulfides
Transit,on to
4-arninobipbenyl
.-..
-N=Ni-- < —N
‘S. \J ‘ ci-i ,‘
2 3 ‘ .
.Aczz e if
—NH-CH 3
—N’CH 3 )C H,
N!C:H,):. prodded that
4 -e:hvl substituted.b or ‘prime’
ri p!aced b pyridinc-
4- V-oxide
Vea iy active if
—NCH CH0
hacthe if
— CH iCH 2 C 4 H,
N CH,lCH:CHj0H
—N CH -CH 0H) 5
—NH-C-H,
—NtC .I1 l 2
—“ .C H4
—Nt CH ),
—NH•CHO
—NH.
—OCH 3
—OH
—H
Cc’m ed from: J. A. Miller and E. C. Milkr [ Adran. C nc?rR ’j. 1.339 (l95 : E. C. Miller
I M’llerJ.Nat. Cancer l ast. I . 1571 (1955)]: J. A. Mr. E. C. Mi !er. aid 0. C. Finger
:c . : r Re;. 17. 5S7(l957 : K. Kino itzi (I9 , 1937 ) i 1. 1. rO t:!e a I94O)asquotcd in
J. L. H rweI: Strvey of Compounds \Vhich Have T : d fo Carcinogenic Activity.”
266
-------
i’ubk’ 2
Sviit’piie l.tlHII.IIitHI of Stru ttur ,tl ltc iuir mcnts for I Icpai ’ ’ ’. sreiiio c ieily oF 4.I)ii,ietliyIainii Zoben/ .eiic
inih in Stibstitul ins in the 2—. 3—. 5—. 6—. 2—. 3’’. 5’—, and 6’—povilions
RcLU iv
act ivit I
cv (4 -dim
ethyLi in
inoazob
‘nzenc 6)
I’ositionv
—CIl ,
—C 2 Il
—1’
—Cl
—Dr
—N()
—CF
——Oil
—0C 1 1 3
—OC,H
—SCIIf
—COOlI’
2
0’
>10
0
0
0
2
0
Weakly active
2’
23
o
7
4
2
0
3
6
\Vcakly active
3’
1012
6
10-12
0
2.6
2Y
0
2,4’
0’
3.4’
0
2’.V
40
2’,)’
0
II )
‘.: ‘
( I
..lt)
( I
tI’
5 6’
4
> 10
(1
3 ’,S ’
(1
>10
2’,4’.6’
0
>10
0
2’.4 ’.S’
0
2’.3 ’.4’
0
2,3.5’
0
3 ’.4 ,5 ’
9
2’.3 ’,6’
I.)
2.6.Y.5
0
2,5.2’,S’
2.6,2 ’.4 ’.6’
4
0
Compiled From: J. A. Miller and 13. C. Miller (J. Exp. Med. 87, 139 (l94 1)]: i. A. Miller and U. C. Miller [ #lih’an. Cuncer Rev. 1. 339 (1953)]: .1. A.
Miller, 13. C. Miller, and C. C. Finger [ Cwa’r Rt’s, Il, 3 7 (1957)); N. Nagao (1940, 1941) and 1. Sasaki c i uL( 1940) asquolcd in J. L. liariwell:
“Survey of Compounds Which I Live Ileen Testcd for Carcinogenic Activity.” U.S. Public I lealth Service Pubi. No. 149, \Vashington, D.C., 1951.
p. 366 ( # 99 ): J. C. Areos and J. Simon (Ar:neimiiiel.I.’orsrh. 12, 270 (1962)); IL V. Drown 1J. Medicinal Chetn. II, 1234 (1965)]; 0. M. Bcbawi.
V. S. Kim, and J. P. t.ambooy [ Cancer Res. 30. 1520(1970)) : fl.V. l3rown and A. Kiuegel (.1. Alt’dic’isial (‘ht’,ii. IS. 212 (1972)] .
a’
-------
Tab t 3-
S roptic Tabulaton of Struczural R 4ui: : i r H of 4—
Dtl arnin azob nzene. EFect 01 Si .:e _s 5i: z by D T r nt
Subst :
PositiOnS
P alative
a.tivity’
2
3
.
—CH
—C-H.
16
—CH 3
—C H 5
—CH 3
—cn
—CH)
—C l
—F
—CH,
CH.
—o: -i
—C
—CH 3
—2
6-7
>>>1O
0
>ID
3-S
C0r?::cd f:o n: K. Su iura, M. L. Cro; : - a:d C. J.K ;! J. .\ at. Cai;c r
I—a;. 15. 7 (1954 : I. C. .rcos and i. :.4-: . r-F ’ . 12 2 0 (I952 :
G.M. tb wi. V. S. Kim, and 1. P. La boo -. :c - - l5: o,.
R l the to an arWtr ry sca: dard activi c f 6 o di ethamiaoazo-
b nzenef e Voun I, S ction 4.3.6.2 4, çp.
268
-------
References for Azo Dyes
1. Johnson, R. F., Zenhausern, A., and Zollinger, H., Kirk-Othmer “Encyclopetha
of Chemical Technology” Vol. 2, 2nd ed,, Interscierxce, New York (1963) p. 863-
910
2. IARC, Vol. 8, International Agency for Research on Cancer, Lyon (1975); pp. E3-
60; 61—74; 127—146
3. Demerec, M., Wallace, B., Witkin, E. M., and Bertani, C., In: ‘Carnegie Insti -
tution of Washington Yearbook, l948 l949” No. 48, Washington, D.C. (1949)
pp. 154—166
4. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N., Detection of Carcinogens
as Mutagens in the Salmonella/microsome Test: Assay of 300 Chemicals, Proc.
Nati. Acad. Sci. , 72(1975) 5135
5. Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D., Carcinogens are
Mutagens: A Simple Test System Combining Liver Homogenates for Activation
and Bacteria for Detection, Proc. Nati. Acad. Sci. , 70 (1973) 2281-2285
6. Arcos, 3. C., and Argus, M. F., Chemical induction of cancer, Vol. IIB, Academic
Press, New York and London (1974) pp. 142-175
7. Yahagi, T., Degawa, M., Seino, Y., Matsushima, T., Nagao, M., Sugimura, T.,
and Hashimoto, Y., Mutagenicity of Carcinogenic Azo Dyes and Their Derivatives
Cancer Letters , 1(1975) 91—96
269
-------
XI. Heterocvclic Amines
1. Quinoline Ebenzo(b)pyridine; 1-benzazine; leucoline( occurs in small
an .ount in coal, tar and petroleum, and is a volatile component in roasted cocoa’.
It. is produced by many synthetic procedures 2 including: a) the Skraup synthesis by
heating; aniline with glycerine and nitrobenzene in presence of sulfuric acid and b) via
the mt eraction of aniline with acetyladehyde and a formaldehyde hemiacetal 3 . Quino-
line is a weak tertiary heterocyclic base and it and its derivatives exhibit reactions
which are familiar in the benzene and pyridine series 4 . For example, electrophilic
substi.tution occurs almost exclusively in the benzene ring (partly because of the
deactivation of the pyridine ring by the hetero atom), while nucleophilic substitution
occux ‘ S in the pyridine ring.
‘ uinoline is used in the manufacture of dyes 4 , deodorants (e.g., 8-hydroxyquino-
line sulfate aluminum salt); local anesthetics (dibucaine); anti-malarials (4- and 8-
ami noquinoline derivatives; 4-quinolineinethanols) 4 . Quinoline derivatives are the
parent substances of quinine and other plant alkaloids. Quinoline is also used as a
so lvent for resins and terpenes and as a preservative for anatomical specimens .
Patented areas of suggested utility of auinoline include: anti-knock additive
for gasoline 6 , catalyst for hardening of epoxy resins 7 , dimerizadon of isoprene 8 ,
catalyst for the dehydrohalogenation of ihaloalkanes to dihaloalkenes 9 and in petro-
10
leum recovery
2. 8-Hydroxyquinoline (8-quinolinol; oxyquinoline; hydroxybenzopyridine (I))
is used as a fungistat and in the analysis of and separation of metallic ions, The citrate
salt of 8-hydroxyquinoline is employed as a dissirifectant while the copper derivative
(copper 8-quinolinolate; cupric-8-hydroxyquinolate) is used as a mildew proofing
agent, as a fungicide in the treatment of textiles, as an ingredient of paints, in wood
—‘
-------
11
paper and plastics preservation, in agriculture and in miscellaneous other uses.
In the U.S., an estimated 75% of approximately 132, 000 lbs (domestic and imported
12,13
copper 8 —quinolinolate) is used in the treathient of textiles (e.g., in fabric, rope,
thread, webbing and cordage).
Copper 8 -quinolinoate (II) can be prepared by mixing solutions of copper salts with
8—hydroxyquinoline 14 , and is available in many forms including: as a 5.0% liquid
concentrate in combination with 17 .6% pentachiorophenol and 2. 4% tetrachiorophenol
and in combination with zinc petroleum sulfonate’ 5 . The technical grade of copper
8-quinolinate can contain both free copper and free 8—hydroxyquinoline as impurities.
041 W0dt NHOH
I
- .c
Quinoline was recently reported to be carcinogenic in Sprague—Dawley rats
inducing hepatocellular carcinomas and hemangioendotheliamas in the livers of animals
16
fed a basal diet containing 0.05, 0.10 or 0.25% quinoline for about 16 to 40 weeks
2—Chioroquinoline did not induce any nodular hyperplasia or other neoplastic changes
when rats were treated analogouslylO. While there are no data on the possible formation
of quinoline metabolites in the liver, it is belived that quir.oline may be activated only
in the liver, perhaps by the formation of the N-oxide. It is also possible that quinoline
derivatives may be the proximal carcinogen(s) of cuinoline. The lack of carcinogeni-
city of 2—chloroquinoline is suggested to possibly be related to its more difficult
conversion to an N-oxide 16 .
The carcinogenicity of 8-hydroxyquinoline appears to be conflicting. While it
has been reported to induce tumors when implanted into mouse bladder as a pellet
17 18
with cholesterol , or instilled into rat vagina , it has also been shown to be non-
- P71
‘ -I
-------
carcinogenic in mice or intraveginal administration’ 9 ’ 20, in hamsters by intratesticular
injection 21 or when fed to mice or rats under various experimental conditions 2225 .
While no increase in the incidence of tumors was noted in two strains of mice
following oral administration of copper 8-hydroxyquinoline 25 ,26, a significantly in-
creased incidence of reticulum cell sarcomas was observed in males of one strain of
mice following single subcutaneous injection of copper-8-hydroxyquinoline 25 ’ 26 .
4-Nitroquinoline-1-oxide (4NQO) (UI) and its related compounds have been
shown to be carcinogenic in rats, mice, quinea pigs, hamsters and rabbits, producing
such tumors as papillomas of the skin, lung carcinomas, and fiberosarcomas 2732 .
4-Nitroquinoline-1-oxide has been the most intensively studied of the quinolines,
in regard to rnutagenic activity. It is mutagenic for both prokaryotic organisms such
.33 . 34,35 • 36 37
as E. coli and its phages , Salmonella typhimurium ‘ and Streptomyces
griseoflavus 38 , and for eukaryotic microorganisms such as Aspergillus niger 39 ,
Neurospora crassa 40 , and Saccharomyces cerevisiae 41 ’ 42 .
Base-pair substitutions arise at G . C base pairs which are the site of 4NQO attack.
Hence, 4NQO induces GC - AT transitions. G C - TA transversions and possibly
GC - C.Gtransversions 43 .
4NQO and its reduced rnetabolite 4-hydroxyaininoquinoline-1-oxide (4HAQO) (IV)
bind covalently to cellular macromolecules such as nucleic acid and protein 4448 . It
has recently been reported that seryl-tRNA synthetase is an 4HAQO-activating enzyme 49
and that 4HAQO may be activated by both seryl- and prolyl tRNA synthetases which are
capable of 4—HAQO-activation may possess a unique conformation enabling them to
- . 49
aminoacylate in vivo the N-hydroxyl group of the carcinogen (e. g., carcinogenic
aromatic amines and nitro compounds).
272
-------
The mutagenic activity of quinoline and a number of its derivatives have been
recently determined utilizing Salmonella typhimurium tester strains TA 100 and TA 98
in the presence or absence of an S-9 metabolic activation system 42 (Table 1). Quino-
line was mutagenic to both TA 100 and TA 98 only when activated with the S-9 mix.
While isoquinoline was non-mutagenic with or without metabolic activation the isomeric
methyl quinolines (e.g., 4, 6, 7, or 8 methyl derivatives) were all mutagenic to both
strains, only when activated. 6-Nitro quinoline was the only derivative that was
mutagenic to both strains with or without metabolic activation. This suggests that the
nitro group at the 6 position (analogous to 4NQO) can be reduced to a hydroxyamino
group by an enzyme(s) of S. typhimurium producing an active metabolite.
273
-------
TA3L 1
sTa;CTCIUSA .3 UT . Z C1T!ES OP QIUNOL! AND iTS DEkIVATIvES
C — ourd a ne Structt.re Mutag.-nicity
With Witbaul
S.9 mix S.9 mix
1 Quinotii ie +
2 ±n c
3 4. i thy lqumo1ine +
4 6-Methy lquinoline ÷
5 7 -M thy iqu inoIine ÷
6 8-MethytquuioUDe +
7 3-Methylisoquinollne — —
-
8 1-Nltroquinoline + +
9 6.Nitroquiziohne ‘- -• -I .
It ’) 2•Ch1oroquinoI ne
11 4 ,7-Dichloroquinoline +
12 8-Hydroxyquinoline
13 4-Dihydroxyquino line 4
14 2- ’ k th l-8-hydroxy- +
— -I - ’ .-
qUlflU iIflC ‘ -
15 8-}{yd roxyquinoiine 5- _# _•_ +
suifunic acid
5 -CbIuro-8-hy 3r xy- +
qt .hrn l&ne
1 5.7 -i)ich loro-S-hydruxy.
quinuime
5 ,7 -D b o .n -S- i ydcox-
q inc ’ Irne
C-
Qu± 1it e-S-sut onyI
c Th ritIe
2 ) Q ino!ine-2 -tarb , xyiic
acid
274
-------
References for Quinoljnes
1. Vitzhum, 0. G., Werkhoff, P., and Hubert, P., Volatile components of roasted
cocoa, Basic Fraction, J. Food Sd. , 40 (1975) 911-916
2. Manske, R. H. F., The chemistry of guinolines, Chem. Revs. , 30 (1942) 113
3. Cislak, F. E., and Wheeler, W. R., Quinoline, U. S. Patent 3, 020, 281, Feb. 6,
1962, Cherri. Abstr. , 56 (1962) Pl4248e
4. Kulka, M., ‘Quinoline and Isoquinolin&’, in Kirk-Othmer, Encyclopedia of
Chemical Technology, 2nd eds,, Vol. 16, Interscience Publishers, New York,
Pp. 865—886 (1968)
5. Merck&Co.,
Merck Index, 9th ed., Rahway, NJ (1976) p. 7682
6. Friberg, S. E., and Lundgren, L. E. G., Motor Fuel with High Octane Number,
Ger. Offen. 2,440,521, March 6, 1975, Chem. Abstr. , 83(1975) 63261V
7. Wynstra, J., and Stevens, J. J., Jr., Hardenable Resin Composition, Ger. Offen.
2,065,701, May 22, 1975, Chem. Abstr. , 83 (1975) 148382K
8. Mon. H., Imaizuini, F., and Hirayanagi, S., Isoprene Dimers, Japan Patent
7,511,884, May 7, 1975, Chem. Abstr. , 83(1975) 148079S
9. Tomio, A., Fukui, E., Yokoyama, 1., loka, M., and Ohkoshi, Y., Dihaloalkenes,
Japan Patent 7,433,166, Sept. 5, 1974, Chem. Abstr. , 83(1975) 6164C
10. Brown, A., Chichakli, M., and Fontaine, M. F., Improved Vertical Conformance
via a Stream Drive, Canadian Patent 963,803, March, 4, 1975, Chem. Abstr .
83 (1975) 134786H
11. Turner, N. J., Industrial Fungicides, In Kirk-Othmer, Encyclopedia of Chemical
Technology, 2nd ed., Vol. 16, Interscience Publishers, New York, (1966)
pp. 231—236
12. U.S. International Trade Commission, Synthetic Organic Chemicals, U. S.
Production and Sales, Pesticides and Related Products, 1975 Preliminary, U.S.
Government Printing Office, Washington, D.C., (1976) p. 4
13. U.S. International Trade Commission, Imports of Benzenoid Chemicals and
Products, 1974, USITC Publication No. 762, U.S. Government Piinting Office,
Washington, D.C., (1976) p. 26
14. Spencer, E. Y., Guide to the Chemicals Used in Crop Protection, 6th eds.,
University of Western Ontario, Research Branch, Agriculture Canada, Univ.
of Western Ontario, London, Ontario, Pubi. No. 1093 (1973)
275
-------
15. Environmental Protection Agency, EPA Compendium of Registered Pesticides,
Vol. II. Fungicides and Nematicides, U. S. Govt. Printing Office, Washington,
D.C. (1973) pp. C-54—00.01-Z —07’-OO.Ol
16. Hirao, K., Shinohara, Y., Tsuda, H., Fukushlina, S., Takahashi, M., and
Ito, N., Carcinogenic activity of quirioline on rat liver, Cancer Research , 36
(1976) 329—335
17. Allan, M. J., Boylarid, E., Dukes, C. E., Horning, E. S., and Watson, J. G.,
Cancer of the urinary bladder induced in mice with metabolites of aromatic
amines and tryptophan, Brit. 3. Cancer , 11(1957) 212-218
18. Hoch-Ligeti, C., Effect of prolonged administration of spermicidal contraceptives
on rats kept on low protein or on full diet, 3. Natl. Cancer Inst. , 18 (1957)
661—685
19. Boyland, E., Charles, R. T., and Gowing, N. F. C., The induction of tumors
in mice by intraveginal application of chemical compounds, Brit. 3. Cancer ,
15 (1961) 252—256
20. Boyland, E., Roe, F. J. C., and Mitchley, B. C. V., Test of certain constituents
of spermicides for carcinogenicity in genital tract of female mice, Brit. 3. Cancer
20 (1966) 184—187
21. Umeda, M., Screening tests of various chemical substances as carcinogenic
hazards (Report 1), Gann , 48 (1957) 57—64
22. Hadidian, Z., Fredrickson, T. N., Weisburger, E. K., Weisburger, 3. H.,
Glass, R. M., and Mantel, N., Tests for chemical carcinogens. Report on
the activity of aromatic amines, zutrosamir.es, quinoithes, nitroalkanes, amides,
epoxides, aziridines and purine antimetabolites, 3. Nail. Cancer Inst. , 41
(1968) 985—1036
23. Trihaut, R. C., Researchers sur les risques de riociuite a long terme de l’hydroxy-
8-quinoleine au cours de son utilisation comme coriservateur alimeritaire, Ann.
Pharm. Fr. , 21(1963) 266
24. Yainamoto, R. S., Williams, G. M., Frankel, H. H., and Weisburger, J. M.,
8-Hydroxyquinoline: Chronic toxicity and inhibitory effect on the carcinogenicity
of N-2-fluorenylacetamide, Toxicol. Appi. Pharmacol. , 19 (1971) 687-698
25. lanes, J. R. M., Ulland, B. M., Valerio, M. G., Petruc eli, L., Fishbein, L.,
Hart, E. R., Pallotta, A. 3., Bates, R. R., Falk, H. L., Gart, 3. J., Klein,
M., Mitchell, I., and Peters, 3., Bioassay of pesticides and industrial chemicals
for tumorigenicity in mice: A preliminary note. 3. Natl. Cancer Inst. , 42
(1969) 1101—1114
276
-------
26. National Technical Information Service, Evaluation of Carcinogenic, Teratogenic
and Mutagenic Activities of Selected Pesticides and Industrial Chemicals, Vol. I,
Carcinogenic Study, U. S. Dept. of Commerce, Washington, D.C.
27. Mori, K., Induction of pulmonary and uterine cancers, and leukemia in mice by
injection of 4 -nitroquinoline, Gann , 56 (1965) 513-518
28. Mori, K., Kondo, M., Koibuchi, E., and Hashimoto, A., Induction of lung cancer
in mice by injection of 4 -hydroxyaminoquinoline-i-oxide, Gann , 58 (1961) 105-106
29. Takahashi, M., and Sato, H., Effect of 4-nitroquinoline-l-oxide with alkyl
benzerie sulfonate on gastric carcinogertesis in rats, Gann Monograph 8 (1969)
241—246
30. Magao, M., and Sugimura, T., Molecular biology of the carcinogen, 4-nitro-
quinolirie—1-oxide, Adv. Cancer Res. , 23(1976)131-169
31. Endo, H., Carcinogenicity, In ‘Results in Cancer Research: Chemistry and
Biological Actions of 4-Nitroquinoline-1-oxide ” (eds) Endo, H., Ono, T., and
Sugimura, T., Vol. 34, Springer-Verlag, Berlin, Heidelberg, New York (1971)
pp. 32—52
32. Takayama, S., Effect of 4-riitroquinoline-n-oxide painting on azo dye hepato
carcinogenesis in rats, with note on induction of skin fibro sarcoma, Gann ,
52 (1961) 165—171
33. Kondo, S., Ichikawa, H., Iwo, K., and Kato, T., Base-c Aangemutagenesis and
prophage induction in strains of E. coil with different DNA repair capacities,
Genetics , 66 (1970) 187—217
34. Ishizawa, M., and Endo, H., Mutagenesis of bacterio phage T4 by a carcinogen
4-nitroquinoline-1-oxide Mutation Res. , 12 (1971) 1-8
35. Ishizawa, M., and Endo, H., Mutagenic effect of a carcinogen, 4-nitroquinoline-
1-oxide, in bacteriophage T4, Mutation Res. , 9 (1970) 134-137
36. Ames, B. N., Lee, F. D., and Durston, W. E., An improved bacterial test system
for the detection and classification of mutageris and carcinogens, Proc. Natl. Acad.
Sd. , USA 70 (1973) 782—786
37. Hartxnan, P. E., Levine, K., Hartman, Z., and Berger, H., Hycanthone: A frame-
shift mutagens, Science , 172 (1971) 1058—1060
38. Mashima, S., and Ikeda, Y., Selection of rnutagenic agents by the Streptomyces
reverse mutation test, Appi. Microbiol . ,“ 6 (1958) 45—49
39. Okabayashi, T., Studies on antifungal substances, VIII. Mutagenic action of
4-nitroquinoline—loxide , J.Ferment. Technol. , 33(1955) 513-516
277
-------
40. Matter, B. E., Ong, T., and DeSerres, F., Mutageriic activity of 4 —nitroquino-
line- 1-oxide and 4- hydroxyaminoquinoline- 1 -oxide in Neurospora cras sa ,
Gann,63 (1972) 265—267
41. Epstein, S. S., and St. Pierre, J. A., Mutagenicity in yeast of nitroquinolines
and related compounds. Toxicol. Appi. Pharmacol . 15, (1969) 451-460
42. Nagai, S., Production of respiration-deficient mutants in yeast by a carcinogen,
4-nitroquinoline-1-oxide, Mutation Res. , 7 (1969) 33 3-337
43. Prakash, L., Stewart, J. W., and Sherman, F., Specific induction of transition
and transversions of G C base pairs by 4-nitroquinoline-1-oxide in iso-1-cyto-
chrome C mutants of yeast, J. Mol. Biol. , 85 (1974) 51—65
44. Sugimura, T., Okabe, K., and Endo, K., The metabolism of 4-nitroquinoline-1-
oxide. I. Conversion of 4-nitroquinoline- 1-oxide to 4 -aminoquinoline- 1-oxide by
rat liver enzymes, Gann , 56 (1965) 489-501
45. Tada, M., and Tada, M., Interaction of a carcinogen, 4-nitroquinoline-1-oxide,
with nucleic acids: Chemical degradation of the adducts, Chemico-Biol.
Interactions , 3 (1971) 225-229
46. Tada, M., Tada, M., and Takahashi, T., Interaction of a carcinogen, 4-hydroxy-
aminoquinoline-1-oxide with nucleic acids, Biochem. Biophys. Res. Commun. ,
29 (1967) 469—477
47. Ikegami, 5., Neinoto, N., Sato, W., and Sugirnura, J., Binding of ‘ 4 C-labeled
4-nitroquinoline-1-oxide to DNA in vivo, Chernico-Biol. Interactions , 1 (1969/
1970) 321—330
48. Andoh, T., Kato, K.. Takaoka, T., and Katsuta, H., Carcinogenesis in tissue
culture XIII Binding of 4-nitroquinoline-1-oxide-3H to nucleic acids and proteins
ofLP3andJTC—25•P3cells, Tnt. J. Cancer , 7(1971) 455—467
49. Tada, M., and Tada, M., Seryl-t-RNA synthetase and activation of the carcinogen
4-nitroquinoline --1-oxide, Nature , 255 (1977) 510-512
50. Nagao, M., Yahagi, T., Seino, Y., Sugimura, T., and Ito, N., Mutagenicities
of quinoline and its derivatives, Mutation Res. , 42 (1977) 335-342
278
-------
x i i. Nitrofurans
Nitrofuran derivatives constitute a large category of important heterocyclic
compounds many of which have been widely used as food additives, feed additives,
human medicines and veterinary drugs 13 . Many of the nitrofuran derivatives which
have been studied for their mutagenic and/or carcinogenic activities are chiefly
classified into four groups 2 : (1) 5-thiazole derivatives, (2) and (3) consist of those
compounds which have vinyl or acryl residues and azomethin residues, respectively,
next to position 2 of the 5-nitrofuran (NO 2 - E 7J ) and (4) other derivatives not
0
classified in groups (1), (2) and (3). The potential carcinogenicity of nitrofurans
4
has been known since 1966 . Some nitrofuran 57 , nitrothiophene 8 , nitroimi- 1
azole
and nitrothiazole 6 derivatives were found to be carcinogenic recently.
Nitrofurazone (NO 2 - t.!J i -CHNNHCONH 2 ; 5-nitro-2-furaldehyde semicarbazone)
which as been used extensively in human and veterinary medicine has been known
as a mutagen in E. coli since 196411. However, the mutagenicity of nitrofurans in
2,12—17
microorganisms has received extensive attention only recently and has been
2
recently reviewed by Tazima et al
Data compiled on structure and activity relationship strongly suggested that
the nitro group was responsible for the carcinogenicity and mutagenicity of nitro-
furans. Hydroxylaminofuran was proposed as the active intermediate. Nitroreduction
9,23
19—21 22 1
of nitrofuran , nitrothiophene , and nitrothiazole by animal tissues has
24
been shown. Nitroreduction of nitrofuran was also observed in bacteria
Nitrofurantoin (1- [ (5-nitrofurylidene)arfliflO] -hydantoin, furantoin) the nitro-
furan utilized most in antimicrobial chemotherapy has recently been shown to be
a mutagen in S. typhimurium TA 100 and TA-FR 1 strains following its metabolism
by rat liver nitroreductaSe 25 . Recently, the presence of mutagenic activity was
279
-------
demonstrated (utilizing S. typhimurium TA 100) in the urine of patients given
metronida zole [ 1- (2-hydrozyethyl) -2--methyl-5--nitroimadazolej a drug commonly
used in treatment of amebiases. Trichomonas vaginitis and infections caused by
anerobic microbes 26 . This activity was due to unmodified metronidazole and a
least four of its metabolites (including a hydroxyamino derivative). Metronidazole
has been shown previously to induce lung tumors and malignant lyinphomas in mice 27 .
The list of nitrofurans tested for carcinogenicity is shown in Table 1 , while
Table 2 lists the thtrofuran derivatives whose mutagenicity and/or DNA damaging
capacity in bacteria have been studied 2 . Table 3 illustrates the correlation between
prophage inducibiity, inutagenicity and carcinogenicity of nitrofurans and related
compounds tn E. coli
280
-------
a --
x.(2 Hydroxyethy))-3-5-IUt
furfurylidine)_amiflO 2 -imidar01idifle
Nifuridene
Mammarr
Kidney
Mouse + Leukemia
Stowaclt
Lung
Mammart
Mammary
Forestomacb
3lammarr
Bladder
Renal
Mammary
Hamster ÷ Bladder
Mouse + Bladder
Leukemia
flog + Bladder
Ureter
Renal
Gal! bladder
Mammary
Mammary
Leukemia
Bladder
Mammart
Gal: bladder
Forestomach
Forestomach
Not desctthed
Not descnbed
Not described
Breast
Intestine
Uterus
4- ‘ Not described ..
UST OF 1TROFtRA)S TESTED FOR CARCINOGENIC1TY — - -- -______ -
Compound Group Test Carcino- Principal
auin al geniticy tumour site
FXT +
I Rat
liNT
DMNT
FAN
I Rat ±
Mouse +
I Rat +
I Rat +
NFrA I Rat +
Mouse +
Hamster +
Dog ±
2 .2.2-Tn UorO-X-4-(3-fli O.2-furv1 - I Mouse +
thiazolyl} .acetainide
ANF I Mouse +
4 -(5-N3trO..2-iu rvl th1azole I Not +
described
2 -Methyl - 4 .(5.nztro -z -furvl)th azole I Not +
described
2- (Dimethy1ammo)methylimzno- II Not +
5.2 -(5.nltro2 -urvI)vlnyl.s.3.4- described
oxadiazole
2-(2-Furvl) -3-(5-mtrO-2-fnrvl) II Mouse +
acrylamide (AFZ)
Nitrofurazone III Rat +
Nitrofurantoin III Rat —
-Nitro-z-furamidoxime III Rat —
4 Meryl _( nicroIuryllUrylidifle)-. III Rat
amiao].n-imidazolidinone
Furmethonol III Rat
TABLE
281
Foreatomach
Mammary
+ Mammary
Breast
Mammary
Breast
Ovary
Kidney pelvis
Lymphoma
+ Mammary
III Rat
Breast
Kidney
Uterus
III Rat ± Mammary
Breast
Salivary gland
Lyrnphoma
Adenoma
3 A. - .amido3 -(5 -nitO -.2-fUryI) IV Rat
6-H- , , 4 -o,cadiazine
5 Nitro z (uraametbaftdi0l diacetate I V Rat
4 .6_Diamino 2 -(5-uit O - -i’iIY’}5 IV Rat
triazine
3 Acetamtd .) -3 -(3 -nitr 0 20 3t) IV Rat
6-Y-x .2. 4 -oxad azofle
X_ S (S XitrO iUtYi)T.3.4 IV Mouse
thiadiazoi-3 v1:aceramide
IV Rat
triazine 2. 4 diyl:bi s acetam te
± Hemangioendo-
thelial sarcoma
± Mammary
± Hemangioendo-
thelial sarcoma
Forestomach
± Mammary
I ’ Not
oxadiazol.5 ylthY l)3cet described
Forrn:c acid 2 : 4 (2.iurYI -2- I Rat —
th iazolyt:hyd?aZ e
Formic acid -( 4 -meth l- I Rat —
rh a o [ yl3hYdraZt. e
F iraldeh%’dC semicarbarone I ( Rat — - . s. I
-- ---
-------
TABLE U
• F T
HNT
DMNT
FA FT
NFTA
z -Trinuoro-N-r 4 -(5-nitro-2-
i -li-z-tha 3olyl:- etamide I
i
th iazo v l:phenyIa&uin* I
2-ChiorO- 4 43-nltro-.z-
furyIjthi azo e I
4 -( - i. o-a-fury1)thiazote I
-Mac1w -4-{ -ni o-z-iuzy1)
th a ok I
hvdrane-. .(z.fury1)
R.3- hi*ZOZC I
- .;.phe iy1 thiazol. I
YFT II
F- 4 x6 I X
5 - .i -3- 2z) -L ac lic acid II
3 .(5- ro.z4uryI)acrylamid. It
II
--P svi -3- 5 -aitro.2 -fury1)
ac - amide It
-For IwIaaiino-4 - -(5-nitro -z-furyl)
y J -r.3-th.iazo II
-Acev!ami o.. 4 - 2-(5 -nitco -2 -fury))
t. 3 -tb azole Ii
-(D hyIarz ino}methy1i noj-
5-
oxadia.zo le II
U
it’
Fu ethono 111
itroi rancoin It t
F zQlidone III
X f roxi e III
: -.5 -Ni ro2 -ftzrfur tidzae).3*N.N
d nyI-propby1-aminourea HCI III
III
2- 3-Y . Iro-2-iurfur31iden.)
aziao tthaool
2- 5 - L o-2-iurfurvhdeae)
a i o ethanol X-oxjd
4 Meth: 1.t :(3 nitro4uth1rl.
elammo-:-imidazolidinone
5-A :etado-3-(5-n.trO . !-furyl)
z-(5- itr a-furyI)-3-p ipendino -
propan.z -on -semicarba.zone HCI
5-l5- itr o -2-fu1) - 4 -J i -x.t, 4 .
trizole
5- flTO . -2-furanmethandiol
diacetate
5-Nitro -2 -fnroic acid
4.6-Diamino- -(5-nitTo-2-furvl)- -
triazine
5-Acet amido -3-(3-nitro-2 -furvl).
6-K-z,2.4-oxadiazine IV
X 3-(3-Nitro-2-fury i-I.3. 4 -
thiadiazol-2-vlacCtamjde IV
N-(3-(5-nitro- -iuryh-I . .4 -
oxadiazol-5-vl)merh-l;at raittjde IV
$-Nitro-2 -methyl furan IV
I ±++
I
I ++±+
I +++±
I ++++
+4+ ± +4+
+++ + 4+4
+4+ +
±44+ ±4
+4
+4+ +
MWaiio,. ,.Ub
E. coli WPz
y: uar
audfov uvv&
(her)
++
++
+4 +4
+4
+
+
+
+
4. .
+4+ 4 - I -
+4 ÷
+
+4
++ +4
4-I-
±
+÷
4÷
+
÷
+
LIST 0? S1taOYVS I DaalvAttvas Waosa TAGe.!CLTY AND(OR D (A DAMAGING cAPAcrry ZN BACTZRI HAVE
NEaN aTvDLEr t
C.).y 1fl4L Group Ripeir ks ’
E. coti
Y c 1 /r.c4
S. Eyp i.
mMn. N
TA 19 7 81
TAz 3 8
8. suàit
rsc 4 (r.c S
,ec’JncB
±
4+
+
4 -1-
+4+
+4+
±4+
+4+
++++ +
+÷++ +
4±
+4+ ++ 4÷
+4+
+4
+
4+
+
4-4
+±+
÷
+44+ +4
+4+4 ±
+4
+
+4
+
+
+
+
+4
++
+4
4+-I-
+4
‘Ii
‘U
‘U
U I
.Iv
±4
±
±4
l v
IV
lv
TV
+4
IV
— — -r
—
±
±4
±+
+4 ±
a Difference in diameters of inhibition zones between wild and mutant s:rains:
iomrn; ±±. imm: —. 3rTUn: —. <3mm.
• Strongly muta enie; .-. moderatdv or weakly muta enic; —. non muta enic.
282
-------
TABLE 3-
CO ELAT ON I3ETWEEN PROPHAGE INOCCIRIUIY , IUTAG N1C TY AND CARCINOGENJCITY OR ITR0-
FCR’ S A P ZLATED COMPOCNDS IN E. Coil”
c i.,otuid
Group
Inducibili v
of props &
( ,igini)°
Mutagenicity
(by— I,y -)
per pla.teC
Carcino-
•i
Nitroluran derivatives
YET
II
0.!
300
±
FAYPT
I
o.i
20!
±
HNT
I
o.i
S3
±
FNT
I
o. j
S9
±
2.Trr o-Y- 4-(5-nttro-
2 r;. l-2-t a Iazo1yIacetamide
I
0.5
.
- -
5 -Ac t3rnido-3-(3-tro-2 furVt)-
6-1-:.:. 4 -oxad:azme
I V
0.5
27
±
D iYT
1
1.0
I II
±
N :rofu ra.zc’ ne
I [
i.o
12
±
Furaa derivatives
F0TIT :C acici-2- 4-(2-fUr/!)
2 -tazo .ihvdrazide
I
io.o
o
—
Formic acid-2( 4 -methyi-2-
tbzc,Iv 1vU1 ’aZI(tC
1
10.0
0
—
—
Stra;n u cd: E. coli T .
h 5t ’:r cnacen ration of compound required to induce r as5 in E. rolL
C S ai’ .i d: F. c li \VP2 and its derivative uvrA.
283
-------
References for Nitrofurans
1. Grunb ierg, E. and Titsworth, E. H., Chemotherapeutic Properties of Hetero-
cyclic Compounds: Monocyclic Compounds With Five-Membered Rings, Ann.
Rev. r ficrobiol. , 27(1973) 317—346
2. Tazima, Y., Kada, T., and Murakami, A., Mutagenicity of Nitrofuran Derivatives,
Includin g Furyl Furamide, A Food Preservative, Mutation Res. , 32 (1975) 55-80
3. Miura, K., and Reckendorf, H. K., The Nitrofurans, Prog. Med. Chem. , 5
(1967) 32 0—381
4. Stein, R. J., Yost, D., Petroliunas, F., and Von Esch, A., Carcinogenic Activity
of Nitrofur,ans. A Histologic Evaluation, Federation Proc. , 25 (1966) 291
5. Cohen, S. !‘.L, and Bryan, 0. T., Carcinogenesis Caused by Nitrofuran Deri-
vatives, In: ttPharmacology and the Future of Man.” Proceeding of the 5th
mt. Cortgre ;s on Pharmacology, Vol. 2., pp. 164-170, Basel, Switzerland:
S. Karger, As.. G., (1973)
6. Cohen, S. M., Ert lrk, E., Von Esch, A. M., Crovetti, A. 3., and Bryan, G. T.,
Carcinogenici ty of 5 -Nitrofurans and Related Compounds with Amino-Heterocyclic
Substituents, J. Natl. Cancer Inst. , 54 (1975) 841-850
7. Morris, 3. E., Price, J. M., Lalich, J. J., and Stein, R. J., The Carcinogenic
Activity of Some 5-Nitrofuran Derivatives in the Rat, Cancer Res. , 29 (1969) 2145-
2156
8. Cohen, S. M., and Bryan, G. T., Carcinogenicity of 5-Nitrothiophene Chemicals
in Rats, Federation Proc. , 32 (1973), 825
9. Cohen, S. M., Ertifirk, E., Von Esch, A. M., Crovetti, A. 3., and Bryan, G. T.,
Carcinogenicity o.f 5-Nitrofurans, 5-Nitroimidazoles, 4-Nitrobenzenes, and
Related Compounds, 3. Nail. Cancer Inst. , 51 (1973) 403-417
10. Rustia, M., and Shubik, P., Induction of Lung Tumors and Malignant Lymphotnas
inMicebyMetronidazole, 3. Nail. Cancer Inst. , 48(1972)721—729
11. Zampieri. A., and Greenberg, 3., Nitrofurazone as a Mutagen in E.Coli,
Biochem. Biophys. Res. Commun. , 14(1964) 172-176
12. McCalla, D. R., and Voutsinos, D., On the Mutagenicity of Nitrofurans, Mutation
Res. , 26(1974) 3—16
13. McCalla, D. R., Voutsinos, D., and Olive, P. L., Mutagen Screening with
Bacteria: Niridazole and Nitrofurans, Mutation Res. , 31 (1975) 31-37
14. Yahagi, T., Matsushima, T., Nagao, M., Seino, Y., Sugimura, T., and Bryan,
0. T., Mutagenicity of Nitrofuran Deivatives on a Bacterial Tester Strain with
an R Factor Plasmid, Mutation Res. , 40 (1976) 9
2g4
-------
15. Yahagi, T., Nagao, M., Bara, K., Matsushima, T., Sugimura, T., and Bryan,
G. T., Relationships Between the Carcinogenic and Mutagenic or DNA-modifying
Effects of Nitrofuran Derivatives, Including 2- (2-Furyl) -3- (5-nitro-2-furyi) -
acrylamide, AFood Additive, Cancei- Res. , 34(1974) 2266-2273
16. Wang, C. Y., Muraoka, K., and Bryan, G. T., Mutagenicity of Nitrofurans,
Nitro thiophenes, Nitropyrroles, Nitroimida zole, Aminothiop henes, and Amino-
thiazoles in Salmonella Typhimuriuxn, Cancer Res. , 35 (1975) 3611
17. Rosenkranz, H. S., Jr., Speck, W. T., Stambaugh, 3. E., Mutagenicity of
Metronidazole: Structure Activity Relationships, Mutation Res. , 38 (1976) 203
18. Kada, T., E. Coil Mutagenicity of Furyifuramide,
Jap. 3. Genet. , 48(1973) 301
19. Morita, M., Feller, D. R., and Gillette, 3. R., Reduction of Niridazole by Rat
Liver Xanthine Oxidase, Biochem. Pharmacol. , 20 (1971) 217-226
20. Wang, C. Y., Behrens, B. C., Ichikawa, M., and Bryan, G. T., Nitro—reduction
of 5-Nitrofuran Derivatives by Rat Liver Xanthine Oxidase and Reduced Nico-
tinamide Adenine Dinucleotide Phosphate-Cytochrome c Reductase, Biochem.
Pharmacol. , 23(1974) 3395—3904
21. Wang, C. Y., Chiu, C. W., and Bryan, G. T., Nitroreduction of Carcinogenic
5-Nitrothiophenes by Rat Tissues, Biochem. Pharmacol. , 24 (1975) 1563-1568
22. Wang, C. Y., Chiu, C. W., and Bryan, G. T., Nitroreduction of Carcinogenic
5—Nitrothiophenes by Rat Tissues, Biochem. Pharmacol., , 24 (1975) 1563
23. Anon, Niridazole by Rat Liver Microsomes, Biochem. Pharmacol. , 20 (1971)
203—215
24. Asnis, R. E., The Reduction of Furacin by Cell-free Extracts of Furacin-resistant
and Parent-susceptible Strains of E. Coli, Arch. Biochem. Biophy , 66 (1957)
203—216
25. Rosenkranz, H. S., and Speck, W. T., Activation of Nitrofurantoin to a Mutagen
by Rat Liver Nitroreductase, Biochem. Pharmacol. , 25 (1976) 1555
26. Speck, W. T., Stein, A. B., and Rosenki-anz, H. S., Mutagenicity of Metroni-
dazole: Presence of Several Active Metabolites in Human Urine, 3. Nati. Cancer
Inst. , 56(1976) 283
27. Rustia, M., Shubik, P., Induction of Lung Tumors and Malignant Lymphomas
in Mice by Metronidazole, 3. Nati. Cancer Inst. , 48 (1972) 721
285
-------
XIII. 9, 10-Anthraguinones
A large number of naturally occurring and synthetic anthraquinones have been
widely employed as dyestuffs and coloring agents i i textiles, foods, drugs, cosmetics,
and hair dyes 1 . Table 1 lists the mono- to hexa- hydroxyanthraquinones and their
respective common names.
Alizarin (1, 2-dihydroxy-) (I); quinizarin (1, 4-dihydroxy-) (II); anthragallol
(1,2, 3-trihydroxy-) (III) and purpurin (1,2, 4-trihydroxy-) (IV) anthr aquinones
are among the best known of the hydroxylated derivatives and have achieved impor-
tance as mordant dyestuffs and as intermediates for the manufacture of a number of
important anthraquinone intermediates and as intermediates for the production of
dyes for wool and synthetic fibers 1 . Because of their rather unique ability to form
lakes with metallic ions, many hydroxyanthraquinones are also used for the detection
and estimation of metals.
Other areas of utility of the 1,4- and 1, 5-dihydroxyanthraquinones and 1, 2,4-
trihydroxyanthraquinones are in the production of acrylate-ethylerie polymers for
hot-melt adhesives and laminates 2 as light stabilizers for polystyrene 3 and in the
case of the 1, 4-dihydroxy derivative (quinizarin), as lubricants for pneumatic tools 4 .
(I) (II) (III) (IV)
Among the nitroartthraquinones that have utility in the preparation of amino-
anthraquinones for dyestuffs are the l-riitro-, 1,5-dinitro-, and 1,8-dinitro anthra-
quinones.
In general, anthraquinone 2 se is a relatively inert compound. In spite of its
quinone structure, many reactions characteristic of quinone compounds, either do
286
-------
not occur, or if so, only with difficulty. However, it is the base material for the
manufacture of a group of dyes.
The carcinogenic activity of th arithraquinones have been sparsely examined.
1-Amino anthraquinone has been reported to be carcinogenic in rats 5 . 1-Methyl
amino anthraquinone fed intragastrically was carcinogenic in rats, while 2-amino
anthraquinone induced cystic changes in the kidneys 6 . 2, 6-Diamino anthraquinone
was also tested in this study and found negative 6 .
Brown and Brown 7 recently described the screening of ninety 9,10-anthraquinone
derivatives and related anthracene derivatives for mutagenicity with 5 S. typhimurium
tester strains, TA 1535, TA 100, TA 1537, TA 1538, and TA 98, with and without
mammalian microsomal activation. Three patterns of mutagenesis were apparent in
the approximately 35% of the compounds considered to be mutagenic. These are:
(1) direct frameshift mutagenesis by certain derivatives bearing free hydroxyl groups.
The most potent were anthragallol (1, 2, 3-trihydroxy-); purpurin (1, 2, 4-trihydroxy-)
and anthrarufin (1, 5-dihydroxy-) anthraquinones. While some hydroxy anthraquinones
particularly at lower concentrations, exhibited activation by mammalian microsomal
preparations, the majority of mutagenic hydroxy anthraquinories appeared to revert
strain TA 1538 ( his 3076) specifically. (2) Frameshift mutagenesis by certain derivatives
with primary amino groups, and, in a few cases, with secondary amino groups.
Frameshift mutagenesis was poteritiated with mammalian microsomes, and activity
with strain TA 100 (sensitive to base-pair substitution) was observed in a few cases,
e.g. 1, 2-diamirio anthraquinone. (3) AnthraquiriOnes with one or more nitro groups
exhibited the least specificity with regard to tester strain reverted and to rnicrosomal
activation; all 7 nitro anthraquinories tested were mutagenic. In anthraquinones
287
-------
containing mixed ttmutagenicll functional groups, the type of mutagenesis observed
was usually NO 2 > OH> NH 2 . Table 2 illustrates the screening of a number of
anthraquinone derivatives and related compounds with . typhimurium tester strains
TA 1535, TA 100, TA 1537, TA 1538, and TA 98/mammalian microsomal test.
At present, it is not known whether hydroxy anthraquinones revert TA 1537 by
simple intercalation or a more reactive process 7 . It was suggested that possible
oxidative metabolites or chemical oxidation products involved in the latter process
might include cyclic peroxides as precursors to cis-dihydrodiol-arithraquinones 8 ’ 9
or phenoxide free radicals 10 ’ ”
288
-------
Table 1 Hydzo’ yanthr .tquinone /
Poeition of
Name OH group
ehohydro ryan braquioone 1-
2-
alizarin 1,2..
purpuroxanthin, xanthopurpurin 1,3-.
quinizasin
anthraruñn 1,5-
1,6—
chrysazin is..
hyetaza rin 2,3-
aatbraf avin 2,6-
isoanthraflavin 2,7-
4nthracene Brown; anthragnUol 1,2,3-
pwpurin 1,2,4-
Alizarine Brilliant Bordesu R 1,2,5-
fl3vOpu rpu rin 1,2,6-
anthrapurpurin 1,2,7-
• Alizarne Cyanine It 1,2,4,5,5-
Anthracene Blue YR 1,2,4,3,6,S..
289
-------
rAsL 2
SCREENING OF ANTHRAQUINONE DERIVATIVES AND RELATED COMPOUNDS 0R MUTA-
ENICITY WITH SALMONELLA TYPH!MURWMIMAMMALIAN MICROSOMAL TEST
Test compound b C Number
TA1535
of Hja rev
ettants/pla
t., C
TAIOO
TA1 537
TA1538 TA9&
(a) Bvdrozylated anthx%-
qu aon.s and related
compounds
1,8.9-Thhydroxyaa thaac.n.
(Anthaslin) (1) C
1.2-D hydro*yztthra-
q unon. (Ahaartn) (0)
1 .4-Dihydroxyanthraquinon.
(Quirtiza.rin) (1)
1.5 -dihydzoxy sn±hzaqumone
(Auth fin) (1)
1. 8-D nydroxy i th.aqutnon.
(C)uysazin. Danthron) (2)
100 — — — a
+ — — ± t
500 — * $ a a *
+ — — a a
2000 — * a a a
4 — — — — *
100 — — +4 — *
+ — ++
500 — — + —
+ — — + — t
1000 — — — + —
+ — —
2000 — a a — — —
+ a — —
100 — — — 4+4+
+ — — ±
500 — — —
+ — — +
2000 — — — +44+
+ — — +
50 — — — —
+ — — ++
100 — — — +4
+ — — +4
2000 — — ± +44+
+ — — ++
100 — — — #4-4.4
+ — — ++
500 — — — +4+4
+ — — +
1000 — — —
+ — — +
2000 — a
+ a — +
±
+
4.
* $ t
+4+
ControlX± SD(N)d
—
—
21±15
98±28
10±5
13± 6
27±17
(114)
(114)
(121)
(122)
(132)
—
4.
22±24
(100)
92*25
(106)
12±4
(112)
36±16
(113)
41±23
(112)
10 —
+
20 -
+
4+ - -
+
+4+ - -
+4 - -
— t
—
— ±
4.
a a
290
-------
TABLE 2 (continued)
Test compound gzg b S-9 C Number o
TA1535
I His re
vertants/pla
te a
TA100
TA1537
TA1538 TA9S
2,6-Dthydroxyanthzaquu on. 100 — — — — — —
(AnthraflavIc Acid) (0) + — — ± — —
500 — — — — — —
+ — — ±
1000 — — — ± — —
+ — — ±
1 S-Dthydro ,cy-3-m etby1- 100 — — — ± — —
anthraquinon. + — — + — —
(Chrysophanic Acid) 500 — — — — — —
+ — — +
2000 — — — — — —
+ — — +++ —
Leuco 1.4.dihyd rOXy1fltht* 100 — — — +4 ± —
quu on. (Leucoquixuzazifl> + — — + 4 — —
(2) 500 — — — +4 4 — —
+ — — +4 — —
2000 — — — 4+ — —
+ — — +4 — —
1.2.3-Tri ydroxyanIhra 20 — — — — +4+4. i-i .
quutone (Antbraia11o ) + — — ± +4 +
(1) 100 — — — +4 I I-
+ — — +4+ ++, -I +444
500 — — ft 4+4 -i-tii i ti-
+ — ± 4+4 +++ - I-i-I-I- -f
1.2.DihydroxY.gaflthl ofle 100 — — — ++ — ft
(Anthraobifl) (2) + — — + — —
500 — — — + — —
+ —
3 .Metby1.1,8.94rihYdZOXY 50 — — — — —
anthracene (ChrysaxObin) + — — + — —
(1) 100 — — — ++ — —
+ — — +
500 — — — ft — —
+ — +4+ — —
1.2.5.8 TetrahYdrOXYanth 50 — — +4. ± —
quinone (QuiflJ3 Z*ñfl) (0) + — $ 4+4 — —
100 — — $ + ± ft
4 — $ +4+ — —
500 — — — +4+ — —
+ — — +4+ — —
l.3,8 TrhYdXOXY m0tbY 50 — — — — — —
anthraquinOftl (Emodin) + — — 44+ . , . — —
250 — — — — —
4 . — — +4 — —
2000 — — — — —
+ — —
-------
50 — — —
+ — — +4+
100 — — — +
+ — —
500 — — 4+ 4 .
+ — + #4-4—t-S’#
Lsuco.1.4.5.8 .t.tr,hy4z0Z1’
anthraquinons (2)
100 — — — +4+ t —
+ — — +4 — —
500 — — — 4+ — —
+ — —
1.Xydroxy4.anOS nthr
qulnon. (thap.ra. Red 15) (2)
1.N.Ac.t)i-4.hydzozyanthra-
qutt ons (2)
10 — — — +44+
+ — — +
50 — — * +44-4
+ — — 4+
100 — — — +4+
4- — — 4-4
500 — * * S
+ 5 . *
100 — — — +4+
+ — — 4-4
500 — — — +4+
+ — — 4-I•
2000 — — — +4+
+ — — +
50 — — — 4-4
+
100 — — — +44
+
500 — — — +4+
+ — — ±
100 — — — +44+ + ++
+ — ± +4+
500 — — — +4+
+ — 4+4+
2000 - - - + 4 4
+ — +4. +44+
(1) 100 —
+
500 —
4
1000 —
+
2000 —
+
- - +4+
- - 4+
— — 4+
— +4+
— — +
- - 4+
- - +4.
L\BLE 2cenUnu d)
T .stcomPo and S-9 Number
TA1S3 S
of Hi
rev.rtants!pIat.
TA100
TA1531
TA1538 TA9S
1 5 -DLbydxoxy-4.S diamino
enthraquinons (1)
+
4+
+4+
+44+
+4+
+44+
4+
+4
+4+
4+4+
+4+
lilt ’ -
4 S
(Purpurln) (1)
— ±
±
* —
4 +
• S
• C
± *
±
(b) Arainated anthiaqwnone$
1.2.Dlamoantbz*quinOAS (3)
1 -uaamuioanuiraouinonu
+4
4+
+4
+
+4+4
+4+
4+
+4+
+44+
+4
+44+
4+
4+ 4 .4 4+
4+4+
±
4+
+4+
+44.
292
-------
TABLE 2 (cont ued)
100 — — — — — —
+ — — — ±
500 - - - - - -
+ — —
2000 — — — — — —
+ — — 4 +4 ++
100 — — — — —
+ — — +
500 — — — ± —
+ — —
2000 — — — + — —
+ — —
- - +4
— + 44 +
* S $
444 $ *
+
a S
4.4
1-Benzarnido-5-chloro-
anlbzaquLnone
1-X.Aceiyl-4 0-acety1-
anthraquinorte (4)
1.4-Dia .in no 2.3 dthyd.ro-
a.nth.raquinon. (4)
1-.- riA1ino-2-methyl-
anthraqt none
50 — — — — ±
+ —
100 — — — — —
+ — — 2 — + 4
500 — •- — ± +
+ — 2 +4 = +4 -4
500 — — — +++ -.-+ — —
+ — — ++++ —
50 — — — — — —
+ — —
100 — — — + — —
+ — —
500 — a * — — S
+ * S + — a
500 — — ± — — —
+ — +
1 5 -Dtarnmo-anthtaquinoni
Cc ) Nitrated anthrsquinone
den aUves
(0) 100 — — — — — —
+ — —
.500 — — — — — —
+ — — ± 2 2
2000 — — — — — —
+ — — — + 4-
1 .8-Dihydzo y -4.5-din2tro-
anthraquinone (0)
± ±
+4- - . 44444
4.,. +4+
+4+4 •44..# +4+
4+4-4 +1-p +4+
Test compound b S-9 C Number
TA1535
of H i s 4
revert n S/Ptate
a
TA100
TA1537
1A1538 TA9a
2,6-DzamLnoanthraquinone (0)
1.4.5 .8-Teraazrnnoanthxa-
qumone (Disperse B%ue 1)
(0)
Anthraq ui non.-1-dia2onium
chionde (Fast Red A Salt)
(0)
10 —
+
200 —
+
400 —
+
100 — — +
+ — +4+ +
500 — — ++ +4-444.4
+ — +4 +4 -4+4
2000 — —
+ ±
293
-------
T. BLE 2(conunued
1 .Niro-5eulfonalo-AQ
+
+
+4 +
+4
+ t t
500 — — — — + 4 +
+ — — — t
1000 — — — — — —
+ + =
1.4•Diamino5-nitro-
anthrsquinon. (0)
1-Am no-2-carbozyl ate-4-
mtro-anthraquinon. (0)
50 — — — 4+
+
100 — — — 4+4
+ — —
500 — — S 4 .
+
50 — —
+
100 — —
+
500 — —
+
+4
+
4+4.
*
#4+
+4
50 — — — — +
+ — — — 4
100 — — -#4.
+ — — + +4
500 — — +4 +4+4
+ — — + .4.
a Number of His revertan:/plate expressed as multiples of the mean background reversion fre-
quency for a given assay (X), thus an n(+) score indicates a number exceeding 2 X; ± indi-
cates a marginal effect i.e. <2X; asterisks indicate microbial toxicity at concentration em-
ployed; —, negative.
b Quantity of material/plate, i.e. incorporated into top agar.
C Indicates plates without (—) or with (4) Aroclor 1254 stimulated rat liver microsomal
preparation ( ‘ S-9 mix”).
d SDN) grand mean and standard deviation for control plates of all assays (N plates)
rounded to nearest integer.
Number of minor (usually fluorescent) impurities detected by TLC.
- ino-2 .rnetbyI...nthra-
quinone (0)
re,: compound b S.pC Number
TA1535
of H j
re ertantsfpIate
a
TA100
TA1537
TA1538 TA98
100 — — +
+ — +4+
500 — — +
+ — 4-4-4
1270 — — —
+ ± 4 4+
+
+4
+
4+4
4+
74 8+
54. • 6+
7+ 3.
74 8+
4.4+4+
+ 4 +44+
+
+4
+4
8+
5+
8+
8+
+4+
+4+
+4
4+
+
+44+4
+4
1-Ni tro-6(7)sulfonato-
anthraQuinone (0)
(d) Miscelianeous taduding
known znutag.ns
1-Methoxy-anthzaquinon.
1 Arninoanthracen.
2-Aminoanthracene
1-A ruinopyrene--
3-Arninopyrene
500 —
-
4+
—
4
—
—
-p4.
—
—
20 —
—
—
+
+4
4+4-
1 —
—
—
—
—
—
+
+4-4
6+
-I---
tIII
71
10 —
—
—
+4.4
4
4 +
.4
- I-1-+t-4.
10 —
—
++
:
+4+
+
44-4.
4.4.
4.4 -4.4
294
-------
References for Anthraguinones
1. Cofranesco, A., Anthraquinone derivatives, In Kirk-Othmer Encyclopedia of
Chemical Technology, 2nd eds., Vol. 2, Interscience Publishers, New York
(1966) pp. 465—477; 478—489; 501-533
2. Baumann, H., Bauer, P., and Glaser, R., Ger. Offen., 2,335, 141, Feb. 28
(1974) Chem. Abstr. , 81(1974) 38559Z
3. Nakamura, K., and Honda, K., Photodegradable polystyrenes, Kobunshi Ron-
bunshu, 31 (1974) 373—376; Chem. Abstr. , 81 (1974) 136580iJ
4. Hartmann, L. M., Pneumatic tool lubricant, U.S. Patent 3,801,503, April 2 (1974)
Chem. Abstr. , 81(1974) 52113F
5. Laham, S., Grice, H. C., and Sinclair, J. W., Studies in chemical carcinogenesis
III. —Aminoanthraquinone , Toxicol. Appl. Pharmacol. , 8 (1966) 346
6. Griswold, D. P., Casey, A. E., Weisburger, E. K., and Weisburger, J. H.,
The carcinogenicity of multiple intragastric doses of aromatic and heterocyclic
nitro or amino derivatives in young female Sprague-Dawley rats, Cancer Res .
28 (1968) 924—933
7. Brown, J. P., and Brown, R. J., Mutagenesis by 9, 10-anthraquinone derivatives
and related compounds in SalmoneUa typhimurium, Mutation Res. , 40 (1976) 203-224
8. Daly, J. W., Jernia, D. M., and Witkop, B., Arene oxides and the NIH shift:
The metabolism toxicity and carcinogenicity of aromatic compounds, Experientia ,
28 (1972) 1129—1149
9. Double, J. C., and Brown, J. R., The interaction of aminoalkylaminoanthra-
quinones with deoxyribonucleic acid, 3. Pharm. Pharmacol. , 27 (1975) 5 02-507
3. Nagata, C., Inomata, M., Kadoma, M., and Tagashira, Y., Electron spin response
study on the interaction between the chemical carcinogens and tissue components.
II I. Determination of the structure of the free radical produced either by stirring
3,4 benzopyrene with albumin or incubating it with liver homogenates, Gann ,
59 (1968) 289—298
11. Nagata, C., Tagashira, Y., Kodama, M., and Imamura, A., Free radical produced
by interraction of aromatic hydrocarbons with tissue components, Gann , 57 (1966)
437—444
295
-------
XW. AROMATIC HYDROCARBONS
1. Benzene (C 6 H 6 ), the parent hydrocarbon of the aromatic group, is produced
in enormous amounts principally from coal tar distillation and from petroleum by
catalytic reforming of light naphthas from which it is isolated by distillation or solvent
extraction. The broad spectrum of utility of benzene (commercially sometimes called
“Benzol”) includes the following 1 : extraction and rectification; intermediate for
synthesis in the chemical and pharmaceutical industries; the preparation and use
of inks in the graphic arts industries ; as a thinner for paints; as a degreasing and
cleaning agent; as a solvent in the rubber industry; as an anti-knock fuel additive;
and as a general solvent in various laboratories. Industrial processes involving
the production of benzene and chemical synthesis usually are performed in sealed
and protected systems.
Currently benzene is consumed by the chemical industry in the
U.S. at the rate of 1.4 billion gallons annually and is expected to increase when
additional production facilities become available’. NIOSH estimates that approximately
2 million workers in the U . S. are potentially exposed to benzene ‘ . Increased
concern for benzene as a significant environmental pollutant arises from public
exposure to the presence of benzene in gasoline and the possibility of its increased
content in gasoline
NIOSH considers “that benzene is leukemogenic” and recommends that “for
regulatory purposes It be considered carcinogenic in man” 3 and hence recommends
that OSHA revise its standard to limit exposure to 1 ppm 3 .
1,4-7
A number of recent reviews on benzene toxicity have appeared . Benzene
has long been suggested as a leukemogenic agent based on many individual cases
of leukemia which have been linked to leukemia 1 ,412• It should be noted that although
2%
-------
in the majority of cases the individuals were subjected to mixed exposures, benzene
was the agent common to all cases’. It has been also suggested that it is possible
that all cases reported as “leukemia associated with benzene exposure” have resulted
from exposure to rather high concentrations of benzene and other chemicals’. Dose-
response relationships in chronic exposure of humans to benzene and details of the
1
extent of exposures are generally considered to be either lacking of inadequate
Conflicting epidemiological surveys relating to a correlation between leukemia
and benzene exposure should be cited. For example, in the first major epidemilo-
gical survey 9 , a study of 28,500 shoe workers showed an annual incidence of
leukemia of 13/100,000 compared to 6/100,000 in the general population. However,
an epidemiologic study 13 on 38,000 petroleum workers who had potential exposures
to benzene failed to indicate an increase of leukemia.
The role of benzene-induced chromosome aberrations is not currently definitive 1 .
14—16
Chromosomal aberrations of both the stable and unstable type have been noted
In general, the chromosomal aberrations were higher in peripheral blood lymphocytes
of workers exposed to benzene than in the controls even in the absence of avert signs
of bone-marrow damage. The stable type of chromosomal aberrations persisted
several years after recovery from benzene hemopathy. It was suggested that benzerxe
might induce various types of chromosomal aberrations and that leukemia may develop
in cases when potentially leukemia alone with selective advantage is produced as a
14
toxic response to benzene exposure
Numerous studies involving benzene-induced lymphocyte chromosome damage
and hemopothies have been cited’ 724 . It should be stressed that no quantitative
data on total benzene exposure were available on all of the above studies on chromosome
aberration on human, with all indications suggesting very high levels (e.g., several
297
-------
1
hundred ppm) of benzene . In general, no correlation was found between the
persistence of chroinosornal changes and the degree of benzene poisoning ” 24
Exposure of cultured human leukocytes and Heba cells to 2.2 x 10 3 M benzene
has resulted in a decrease in DNA synthesis. Cultured human leukocytes exposed
to dose levels of 1.1 x 10 3 M and 2.2 x 10 3 M exhibited chromosome aberrations
consisting of breaks and gaps 25 .
Chromosornal aberrations have been noted in rats exposed to 0.2g/kg/day of
benzene, O.8g/kg/day of toluene and a mixture of benzene and toluene at levels
of 0.2 and 0.8g/kg/day respectively
Rabbits injected subcutaneously with a dose of 0.2mg/kg/day of pure benzene
showed a normal karyotype in 15 of 16 test animals. However, the frequency of
mitoses with chromosomal aberrations which was initially in the range of 5.9% increased
to 57.8% after an average of 18 weeks 27 .
It should be noted that animal experiments have not been supportive of the view
that benzene is a leukexnogenic agent 1 ’ 28 ’ 29 . However, aco-leukornogenic role for
benzene could explain the failure to induce leukemia in benzene—exposed animals’.
The role of benzene metabolism in its toxicity as well as the significance of
benzene-induced chromosome aberrations appears to be undefined’. Urinary excretion
products following benzene exposure include phenol, hydroquinone, catechol, hy-
droxyhydroquinone, trans-trans muconic acid, and L-phenylmercapturic acid 30 .
The major route of metabolism in all species tested was conjugation which included
both ethereal sulfate and glucuronide conjugates.
The rate of benzene metabolism depends on the dose administered as well as
the presence of compounds which either stimulate or inhibit benzene metalDolism ’
2q8
-------
Although the mechanism of benzene hydroxylation has not been definitively
determined, it has been suggested that the reactions occurr via an arene oxide
intermediate 30 . While benzene oxide has not been found in liver rnicrosomes
(probably due to its extreme lability) it should be noted that incubation of benzene
oxide with inicrosomes yields the metabolic products of benzene and that naphtha-
lene oxide has been isolated from the incubation of naphthalene with microsomes 30 .
In summary, it has been established that exposure to commercial benzene or
benzene-containing mixtures may result in damage to the haematopoietic system 1 ’ 30 ’ 31
although the mechanism by which benzene acts is now known.
In advanced stages, the result can be pancytopenia due to bone marrow aplasia.
DNA synthesis is reduced in bone marrow of benzene—treated animals either because
of inhibition of enzymes involved in DNA synthesis or because of lesion revealed as
reduced incorporation of tritiated thymidine in DNA occurs at some point in the cell
cycle.
A relationship between exposure to benzene or benzene-containing mixtures and
the development of leukemia is suggested by many case reports 1 ’ 30 ’ 31 . However,
it would appear that more definitive data are required to enable a more accurate
assessment of the myelotoxic , leukemogenic and chromosome-damaging effects of
1, 30, 31
benzene
299
-------
References for Benzene
1. National Academy of Sciences, A Review of Health Effects of Benzene, National
Academy of Sciences, Washington, D.C., June (1976)
2. Kay, K., Toxicologic and cancerogenic evaluation of chemicals used in the
graphic arts industries, Clin. Toxicol. , 9 (1976) 359-390
3. Anon, NIOSH links benzene to leukemia, Chemecology (Oct. 1976) p. 5
4. Berlin, M., Gage, J., and Johnson, E., Increased arornatics in motor fuels:
A review of the environmental and health effects, Work Env. Hith. , 11 (1974) 1-20
5. Saita, G., Benzene induced hypoplastic anaemias and leukaemias. In Girdwood,
R. H., ed. tt Blood Disorders Due to Drugs and Other Agents Amsterdan,
Excerpta Medica. (1973) p. 127—146
6. U.S. Dept. of Health, Education and Welfare, Public Health Service, National
Institute for Occupational Safety and Health. Criteria for a Recommended Standard-
Occupational Exposure to Benzene. HEW Pubi. No. (NIOSH) 74-137. Washington,
D.C., U. S. Government Printing Office (1974)
7. Benzene in the Work Environment. Considerations bearing on the question of
safe concentrations of benzene in the work environment (MAK-Wert). Commu-
nication of the Working Group Establishment of MAK_Werteu of the Senate
Commission for the Examination of Hazardous Industrial Materials, prepared in
cooperation with Dr. Gertrud Buttner. Boppard, Germany, Harald Boldt
Verlag (1974)
8. Eckardt, R. E., Recent development in industrial carcinogens, 3. Occup. Med. ,
15 (1973) 904—907
9. Aksoy, M., Erdein, S., and Dincol, G., Leukemia in shoe-workers exposed
chronically to benzene, Blood 44 (1974) 837-841
10. Aksoy, M., Erdem, S., Dincol, K., Hepyuksel, T., and Dincol, G., Chronic
exposure to benzene as a possible contributary etiologic factor in Hodgkin s
disease, Blut , 28 (1974) 293-298
11. Aksoy, M., Dincol, K., Erdem, S., and Dincol, G., Acute leukemia due to
chronic exposureto benzene, Am. 3. Med. , 52(1972) 160—166
12. Vjadana, E., and Bross, I. D. J., Leukemia and occupations, Prey. Med. , 1
(1972) 513
13. Thorpe, J. 3., Epidemiologic survey of leukemia in persons potentially exposed
tobenzene, 3. Occup. Med. , 16(1974) 375-382
14. Vigliani, E. C., and Forni, A., Benzene, chromosome changes and leukemia,
J. Occup. Med . 11 (1969) 148—149
300
-------
15. Forni, A. M., Capellini, A., Pacifico, E., and Vigliani, E. C., Chromosome
changes and their evolution in subjects with past exposure to benzene, Arch.
Environ. Health , 23 (1974) 385—391
16. Forni, A., Pacifico, E., and Limox-xta, A., Chromosome studies in workers
exposed to benzene or toluene or both, Arch. Environ. Hith. , 22 (1971) 373-378
17. Forni, A., and Moreo, L., Cytogenetic studies in a case of benzene leukaemia,
Eur. 3. Cancer , 3(1967) 251-255
18. Forni, A., and Moreo, L., Chromosome studies in a case of berizene-induced
erythroleukaemia, Eur. 3. Cancer , 5 (1969) 459-463
19. Hartwich, G., Schwanitz, G., and Becker, J., Chromosome anomalies in a case
ofbenzeneleukaemia, Ger. Med. Monthly , 14(1969)449—450
20. Khan, H., and Khan, M. H., Cytogenetic studies following chronic exposure
to benzene, Arch. Toxiko].. , 31 (1973) 39-49
21. Sellyei, M., and Kelemen, E., Chromosome study in a case of granulocytic
leukaemia with Pelgerisation” 7 years after benzene pancytopenia, Eur. J.
Cancer , 7 (1971) 83—85
22. Tough, I. M., and Court Brown, W. M., Chromosome aberrations and exposure
to ambient benzene, Lancet , 1 (1965) 684
23. Pollini, G., and Colombi, R., Lymphocyte chromosome damage in benzene blood
dyscrasia, Med. Lay. , 55 (1964) 641-654
24. Tough, I. M., Smith, P. G., Court Brown, W. M., and Harnden, D. G., Chromo-
some studies on workers exposed to atmospheric benzene. The possible influence
of age, Eur. J. Cancer , 6(1970)49-55
25. Koizumi, A., Dobashi, Y., Tachibana, Y., Tsuda, K., and Katsunuma, H.,
Cytokinetic and cytogenetic changes in cultured human leucocytes and HeLa cells
induced by benzene, md. Health (Japan) 12 (1974) 23-29
26. Dobrokhotov, V. B., The mutagenic influence of benzene and toluene under
experimental conditions, Gig Sanit. , 37 (10) (1972) 36-39 (Translated by Air
Pollution Technical Information C enter, Environmental Protection Agency,
Research Triangle Park, NC. Translation No. HS-138)
27. Kissling, M., and Speck, B., Chromosome aberrations in experimental benzene
intoxication, Helv. Med. Acta. , 36 (1971) 59-66
28. Laerum, 0. D., Reticulum cell neoplasms in normal and benzene treated hairless
mice, Acta. Pathol. Microbiol. Scand. Sect. A , 81 (1973) 57-63
301
-------
29. Ward, J. M., Weisburger, J. H., Yamamoto, R. S., Benjamin, T., Brown, C. A.,
and Weisburger, E. K., Long-term effect of benzene in C57BL/6N mice, Arch.
Environ. Health , 30 (1975) 22-25
30. Snyder, R., and Kocsis, J. J., Current concepts of chronic benzene toxicity,
CRC Crit. Rev. Toxicol.. , 3 (1975) 265-288
31. IARCI Vol. 7, International Agency for Research on Cancer, Lyon (1974)
pp. 203—221
302
-------
XV. Cyclic Ethers
1, 4-Dioxane
,CH CH 2 \
1,4-Dioxane (diethylene—l,4--dioxide; dioxan) (0 2 0) is made by
CH CH(
polymerizing ethylene oxide with caustic soda, or by deh drating ethylene glycol.
It is commercially widely employed since 1930 specially as a solvent for lacquers,
varnishes, paints, dyes, fats, waxes, resins and plastics; lesser amounts are
employed as a solvent in laboratory synthesis, and in the preparation of tissues
for histology.
The pharmacokinetic and metabolic fate of 1,4-dioxane has been shown to be
dose dependent in rats due to a limited capacity to metabolize dioxane to 3—hydroxy-
ethoxyacetic acid (I-IEAA) 2 , the major urinary metabolite.
1,4-Dioxane and HEAA were found in the urine of dioxarie plant personnel
exposed to a time-weighted average concentration of 1 .6 ppm dioxane for 7 .5 hrs.
The average concentrations of dioxane and HEAA in samples of urine collected at
the end of each workday were 3 . 5 and 414 pmol/liter respectively 3 . The high ratio
of HEAA to dioxane (118 to 1) suggests that low-exposure concentrations, dioxane
is rapidly metabolized to HEAA.
The principle toxic effects of dioxane have long been known to be centrilobular,
hepatoceflular and renal tubular, epithelial degeneration and necrosis 4 ‘.
More recent reports by Argus et a1 8 and Hoch-Ligeti et a1 9 described nasal and
hepatic carcinomas in rats ingesting water containing large doses of diexane (up to
1.8% of dioxane in the drinking water for over 13 months). For example, Argus et
a1 8 reported hepatomas in Wistar rats maintained in drinking water containing 1%
0
dioxane for 63 weeks while Hoch-Ligeti et al described the induction ot nasal cavity
carcinomas in Wistar rats maintained on drinking water containing from 0.75 to 1.8%
dioxane for over 13 months.
303
-------
Studies by Kociba et al 10 in 1974 indicated a dose response for the toxicity of
dioxane in Sherman strain rats. Daily administration of 1% dioxane (in drinking
water to male and female rats 1015 and 1599 mg/kg/day respectively) for up to 2
years caused pronounced toxic effects including the occurrence of hepatic and nasal
tumors. There was an induction of untoward effects, liver and kidney damage, but
not tumor induction in male and female rats receiving 0.1% dioxane (equivalent to
approximately 94 and 148 mg/kg/day respectively) in the drinking water, and female
rats receiving 0.01% dioxane in the drinking water (equivalent to approximately 9.6
and 19.0 mg/kg/day, respectively) showed no evidence of tumor formation or other
toxic effects considered to be related to treatment.
Gebririg et a l” postulated that the toxicity and carcinogenicity of 1,4—dioxane
(as weU as vinyichioride) are expressed only when doses are sufficient to overwhelm
their detoxification mechanisms. When such doses are given, there is a dispropor-
tionate retention of the compound se and/or its metablites in the body. Also
observed are changes in the biochemical status of the animals consistent with accepted
mechanisms for cancer induction 11 .
Although dioxane is considered to be a weak to moderate hepatic carcinogen 8 ’ 12,
the mechanism of its carcinogenic action is not understood. Earlier suggestions were
advanced that by virtue of the potent hydrogen bond breaking 13 and protein denaturing
action 14 of dioxane, the molecular basis for carcinogenic action lies in the inactivation
of key cellular macro-molecules involved in metabolic control. Although acute toxicity
studies suggested involvement of microsomal mixed function oxidases, pre-treatment
of rats with enzyme inducers had little or no effect on covalent binding 12 . No
microsome-catalyzed dioxane binding to exogenous DNA was observed under conditions
that allowed significant binding of benzo (a)pyrene. Incubation of isolated microsomes
304
-------
or nuclei also showed no enzyme-catalyzed binding of dioxane 12 . It had been earlier
postulated by Hoch-Ligeti et a1 9 that a reactive free radical or a carbonium ion may
arise in the metabolism of dioxane and may represent a proximate carcinogen. Another
possibility was that a peroxide of dioxane may account for its carcinogenicity 9 .
305
-------
REFERENCES FOR 1 , 4-DIOXANE
1. Young, J. D., and Gehring, P. J., The dose-dependent fate of 14-dioxane
in male rats, Toxicol. Appi. Pharmacol. , 33 (1975) 183
2. Young, 3. ID., Braun, W. H., LeBeau, 3. E., and Gehring, P. 3., Saturated
metabolism as the mechanism for the dose-dependent fate of 1,4-dioxane in rats,
Toxicol. Appi. Pharmacol. , 37 (1976) 138
3. Young, 3. ID., Braun, W. H., Gebring, P. 3., Horvath, B. S. & Daniel, R. L.,
1,4-Dioxane and -hydroxyethoxyacetic acid excretion in urine of humans
exposed to dioxane vapors, Toxicol. Appi. Pharmacol. , 38 (1976) 643-646
4. IDe Navasquex, A., Experimental tubular necrosis of the kidney accompanied
by liver changes due to dioxan poisoning, 3. Hyg. , 35 (1935) 540-548
5. Fairley, A., Linton, E. C., and Ford-Moore, A. H., (1934) The toxicity to
animals of 1,4-dioxane, 3. Hyg. , 34(1934) 486-501
6. Schrenk, H. H., and Yant, W. P., Toxicity of dioxane, 3. md. Hvg. Toxicol. ,
18 (1936) 448—460
7. Kesten, H. D., Mulinos, M. G., and Pomerantz, L., Pathologic effects of certain
glycols and related compounds, Arch. Pathol. , 27 (1939) 447-465
8. Argus, M. F., Sohal, R. S., Bryant, G. M., Hoch-Ligeti, C., and Arcos, 3. C.,
Dose-response and ultrastructural alterations in dioxane carcinogenesis, Eur.
3. Cancer , 9(1973) 237—243
9. Hoch-Ligeti, C., Argus, M. F., and Arcos, 3. C., Induction of carcinomas in
the nasal cavity of rats by dioxane, Brit. J. Cancer , 24 (1970) 164-170
10. Kociba, R. 3., McCollister, S. B., Park, C., Torkelson, T. R., and Gehring,
P. 3., 1, 4-Dioxarie. I. Results of a 2-year ingestion study in rats, Toxicol.
ppl. Pharmacol. , 30 (1974) 275—286
11. Gehring, P. 3.. Watanabe, P. G., and Young, 3. D., The relevance of dose-
dependent pharmacokinetics and biochemical alterations in the assessment of
carcinogenic hazard of chemicals, Presented at Meeting of Origins of Human
Cancer, Cold Spring Harbor, NY, Sept. 7-14 (1976)
12. Woo, Y. T., Argus, M. F., and Arcos, 3. C., Dioxanecarciriogenesis apparent
lack of enzyme-catalyzed covalent binding to macromolecules, The Pharmacologist
(1976) 158
13. Argus, M. F., Arcos, 3. C., Alam, A., and Mathison, 3. H., A Viscornetric
Study of Hydrogen-Bonding Properties of Carcinogenic Nitrosamines and Related
Compounds, 3. Med. Pharm. Chem. , 7 (1964) 460-465
306
-------
14. Bemis, J. A., Argus, M. F., and Arcos, 3. C., Studies on the Denaturation of
Biological Macromolecules by Chemical Carcinogens. III. Optical Rotary Dispersion
and Light Scattering Changes in Oval Bumin During Denaturation and Aggregation
by Water Soluble Carcinogens, Biochim. Biophys. Acta. , 126 (1966) 275-286
307
-------
XVI. HEXAMETHYL PHOSPHOBAMIDE
Hexamethyl phosphoramide (hexamethyl phosphoric-acid, triamide, fris-
(dimethylamino)-phosphine oxide, HMPA) ((CH 3 ) 2 N- O ) is used primarily
N(CH 3 ) 2
in the following areas: (1) as a solvent for polymers, (2) polymerization catalyst,
(3) stabilizer for polystyrene against thermal degradation, (4) as a selective solvent
for gases, (5) as an additive for polyvinyl and polyolefine resins to protect against
degradation by U .V. light and (6) as a solvent in organic and organometallic reactions
in research laboratories 2. In the U.S., hexamethyl phosphoramide is used by its
major producer as a processing solvent for Aramid (aromatic polyamide fiber) 3 .
The use of hexamethyl phosphoramide as a solvent in research laboratories has been
reported to account for more than 90% of the estimated 5000 people who are occupa-
tionally exposed to thie chemical in the u. 2• Hexamethyl phosphoramide has also
been evaluated as a chemosterilant for insects (e.g., houseflies, Musca domestica 4 )
and to a lesser extent as an antistatic agent, flame retardant and as a de-icing additive
for jet fuels 2 .
Recent studies in 1975 have indicated that inhalation of HMPA vapor (400 or 4000
ppb by volume) in air 6 hrs/day for 5 days each week for 6 to 8 months produces
squamous cell carcinomas of the nasal cavity in Charles River CD rats 5 .
In an earlier study in 1973 involving a small number of 6 week old Sherman rats
fed diets containing HMPA at concentrations of 6.25, 3.12, 1.56 and 0.78 mg/kg by
weight for 2 years, a low incidence of tumors (mainly reticulum-cell or lymphosar-
comas of the lungs) were noted in all groups 6 .
Although chromosome aberrations have been noted in insects treated with HMPA’’ 8 ,
no increase in chromosomal aberrations in Chinese hamster lung cells treated with
308
-------
5 x io 3 vi HMPA resulted in chromosomal aberrations in 11% of the cells, while only
6 o of the control cells contained abnormal chromosomes. These values are not
significantly different at the 5% level of statistical significance’°. HMPA induced
a high frequency of ressive lethal mutations in the sperm of Bracon hebetor 11 , testi-
cular atrophy in rats 12 and a marked antispermatogenic effect in rats’ 2 ” 3 and mice’ 3 ,
309
-------
REFERENCES FOR HEXAMETHYL PHOSPHORAMIDE
1. Robert, L. Properties and uses of hexamethyl phosphorotriamide, Chimie Indus . -
Genie Chimigue , 97 (3) (1967) 337—345
2. U.S. Dept. Health, Education, & Welfare, Background Information on Hexamethyl-
phosphoric Triamide, National Institute for Occupational Safety & Health, Rockville,
MD (1975) pp. 1—4
3. Anon, Hexamethyl phosphoramide causes cancer in laboratory animals, Chem.
Eng. News , 53 (1975) 17
4. Borkovec, A. B. Physiological effects of insect chemosterilants,
Adv. Pest. Control Res. , 7 (1966) 43-54
5. Zapp, J. A., Jr., HMPA: A possible carcinogen, Science . 190(1975) 422
6. Kimbrough, R. D., and Gaines, T. B., The chronic toxicity of hexamethyl phos-
phoramide in rats, Bull. Env. Contam. Toxicol. , 10 (1973) 225-226
7. Grover, K. K., Pillai, M. K. K., and Dass, C. M. S., Cytogenetic basis of
chemically induced sterility in Culex Pipiensfatigans, Wiedemann III. Chemo-
sterilant-induced damage in the somatic chi omosomes, Cytologia , 38 (1973) 21-28
8. LeChance, L. E., and Leopold, R. A., Cytogenic effect of chemosterilants in
housefly sperm: Incidence of polysptermy and expression of dominant lethal
mutations in early cleavage divisions, Canad. J. Genet. Cytol. , 11 (1969)
648—659
9. Sturelid, S., Chromosome-breaking capacity of TEPA and analogs in Vicia
Faba and Chinese hamster cells, Hereditas , 68 (1971) 255-276
10. Chang, T. H., and Klassen, W., Comparative effects of tretarnine, TEPA, Apholate,
and their structural analogs on human chromosomes in vitro, Chromosorna , 24
(1968) 314—323
11. Palmquist, J., and LaChance, L. E., Comparative inutagenicity of two chemo-
sterilants, tepa and hempa in sperm of Bi-acon Hebetor, Science , 154 (1966) 915-917
12. Kimbrough, R. M., and Gaines, T. B., Toxicity of hexarnethyl phosphoramide
in rats, Nature , 211 (1966) 146-147
13. Jackson, H., and Craig, A. W.,
Proc. 5thI.P.P.F. Conf. (l967)p. 49
310
-------
XVIII.. NITROALKANES
2-Nitropropane
Nitroalkanes (nitroparaffins) are derivatives of the alkanes in which the nitro
+ ._O
group may be represented as a resonsance hybrid, e.g., R-N R-N
Primary and secondary mononitroalkanes are acidic substances which exist in tan-
tomeric equilibria with their nitronic acids These nitroalkanes undergo aldol-type
condensations with aldehydes and ketories to yield nitroalcohols, reactions of nitro-
alkanes and primary or secondary arnines yield Mannich bases 1 .
2-Nitropropane (isonitropropane; CH 3 CHCH 3 ) is prepared by reacting nitric acid
1
with an excess of propane ; the process also yields nitromethane, nitroethane and
l-nitropropane.
2-Nitropropane is used as an industrial solvent for vinyl, epoxy, nitrocellulose,
chlorinated rubber coatings and adhesives 2 . Other areas of utility include: the
production of derivatives such as nitro alcohols, alkanol amines and polynitro com-
pounds as a vehicle for other miscellaneous resins for printing on plasticized polyvinyl
chloride films 1 , as a stabilizer for chlorohydrocarbons 3 ’ 4 and as a corrosion inhibitor 5 .
It is estimated that 100,000 workers in the U.S. are exposed to 2-nitropropane 2 .
2-Nitropropane is not known to occur naturally but has been detected in tobacco
smoke with other riitroalkanes, the levels were found to correlate with tobacco nitrate
contents 5 . The smoke content of a filterless 85mm U.S. blend cigarette was found to
contain (rig): 2-nitropropane, 1 . 1; l-nitropropane, 0. 13; 1-nitrobutane, 0.71; nitro-
ethane, 1.1; and nitrornethane, 0.53.
NIOSH has recently reported that 2-nitropropane produced liver cancer in rats
after 6 minths exposure at about 200 ppm and suggested that “it would be prudent to
handle 2-nitropropane as if it were a human carcinogen .
Information as to the mutagenicity of 2-nitropropanes appears to be lacking.
311
-------
References for Nitroalkanes
1. Martin, J. C., and Baker, P. J., Jr., Nitroparaffins In Kirk-Othmer Encyclo-
pedia of Chemical Technology, 2nd ed., Vol. 13, Interscience, New York, pp.
8 64—88 5
2. Anon, 2-Nitropropane causes cancer in rats, Chem. Eng. News , May 2 (1977) p. 10
3. Hara, K., Stabilized methyl chloroform, Japan Kokai, 7455, 606, 30 May (1974)
Chem. Abstr. , 82(1975) 3756E
4. Sawabe, S., Genda, G., and Yarnamoto, T., Stabilizing ch1oi ohydrocarbons by
addition of aliphatic alcohols, polyalkyl ethers and nitroalkanes, Japan Patent
7403,963, 29 Jan (1974), Chem. Abstr. , 81(1974) 151517X
5. Hoffman, D., and Rathkamp, G., Chemical studies on tobacco smoke. III. Primary
and secondary nitroalkanes in cigaret smoke, Beitr. Tabakforsch. , 4 (1968) 124-
134
312
-------
XVIII. Azides
Azides (both inorganic and organic) are highly reactive nucleophilic agents
that have been widely employed in the preparation of a variety of intermediates.
The action of hydrazoic acid (HN 3 ) on a carboxylic acid, the action of sodium azide
(NaN 3 ) on an acid chloride, or the action of hydrazine on an ester followed by
treatment of the resulting hydrazide (RCONHNH 2 ) with nitrous acid, all produce
the acyl or aryl azide (RCON 3 or ArCON 3 ). Acyl and aroyl azides rearrange by
thermal or photochemical processes to yield isocyanates via nitrene intermediates.
Isocyanates are of importance in pharmaceutical, pesticide and polymer synthesis.
Areas of utility of sodium azide include: in fungicidal 1 and nematocidal corn-
2 . . . . 3,4
pounds , as a gas-generating agent for inflating protective bags , as a preservative
in diluents used with automatic blood cell counters and as a common reagent in
hospitals and chemical laboratoriesD.
Sodium azide effectively reverts Salmonella typhirnurium strain TA 1530 indi-
cating that it is a base-substitution mutagen. Sodium azide is ineffective on strains
TA 1531, TA 1532, and TA 1534 which are frameshift mutants 6 . Sodium azide has
6
been reported to be a powerful and efficient mutagen when used on barley seeds
and has been suggested as a very useful rnutagen for practical plant breeding
applications 6 . The mutagenic action of sodium azide was not associated with chromo-
some aberrations 6 ’ 7 . Azide treatment has been shown to slightly increase the fre-
quency of penicillin- and streptomycin- resistant mutants in Staphylococcus
aureus ( Micrococcus pvogenes var. aureus) 8 . However, in Drosophila , azide
treatment alone induced no sex—linked recessive lethals 9 ’ 10, although in combination
with carbon monoxide a slight increase in lethal mutations occurred 1 .
313
-------
References for Azides
1. McConnell, W. D., Control of Pythium SPP and Scierotium SPP Using Azides,
U. S. Patent 3,812,254, May21 (1974), Chem. Abstr. , 81(1974) 73408W
2. Wilner, W. D., Nematocidal Mixtures, Ger. Offen. , 2,359 ,226, June 6 (1974),
Chem. Abstr. , 81(1974) P164782E
3. Klager, K., and Dekker, A. 0., Non—toxic Gas Generation, U. S. Patent,
3,814,694, June 4 (1974) Chem. Abstr. , 81 (1974) 172482X
4. Harada, I., Harada, T., Shiki, T., and Shiga, Y., Gas-generating Agent for
Inflating Protective Bags for Passengers in Case of Automobile Accidents. Japan
Kokai, 74 10, 887 (1972), Chem. Abstr. , 81 (1974) 39546E
5. Anon, Explosion Warning Issued for Azides, Chem. Eng. News , 54 (1976) 6
6. Nilan, R. A., Sideris, E. G., Kleinhofs, A., Sander, C., and Konzak, C, F.,
Azide-A Potent Mutagen. Mutation Res. , 17 (1973) 142
7. Sideris, E. G., and Argyrakis, The Effect of the Potent Mutagen Azide on
Deoxyribonucleic Acid, Mutation Res. , 29 (1975) 239
8. Berger, H., Haas, F. L., Wyss, 0., and Stone, W. S., Effect of Sodium A zide
on Radiation Damage and Photoreaction, J. Bact. , 65 (1953) 538
9. Sobels, F. H., The Effect of Pretreatment with Cyanide and Azide on the Rate
of X-ray Induced Mutations in Drosophila, Z. Vererbungslehre , 86 (1955)
399—404
10. Sobels, F. H., The Influence of Catalase Inhibitors on the Rate of X-ray Induced
Mutations in Drosophila Melanogaster, Proc. 1st Intern. Photobiol. Congr.,
Amsterdam , (1954) 332—335
11. Clark, A. M., Genetic Effects of Carbon Monoxide, Cyanide and Azide on
Drosophila, Nature , 181 (1958) 500-501
314
-------
Summary
Ninety industrial chemicals, illustrative of 16 major classes (e.g., 1) aikyl-
lating agents, 2) acylating agents, 3) peroxides, 4) halogenated derivatives, 5) by-
drazines, hydroxylamines and carbamates, 6) nitrosamines, 7) aromatic amines,
8) azo dyes, 9) heterocyclic aromatic amines, 10) nitrofurans, 11) anthraquinones,
12) aromatic hydrocarbons, 13) CyCliC ethers, 14) phosphoramides, 15) nitroalkanes,
and 16) azides) and 19 structural sub-categories have been reviewed primarily in
terms of their reported carcinogenicity and/or mutagenicity. The compounds were
selected based on factors including: their reported carcinogenicity and/or mutagenicity,
their chemical structures and relationships to known carcinogens or mutagens, their
volume or use characteristics, and suggested or estimated potential populations at risk.
Additionally, germane aspects (where known) of their synthesis (primarily in
terms of the nature of the possible hazardous trace impurities), use patterns, chemical
and biological reactivity and stability, environmental occurrence and metabolic fate
have been included for cohesiveness of eatrnent.
It is important to note that in 52 of the above cases, both carcinogenicity and muta-
genicity of individual compounds were reported. Thirty-one compounds have been
reported to be mutagenic and non-carcinogenic and seven compounds are carcinogenic
and non-rnutagenic. In a number of cases, there are no reports of a compound having
been tested for carcinogenicity or mutagenicity or they are currently on test. In some
cases, conflicting carcinogenicity and/or mutagenicity results for the same compound
were reported.
The largest number of industrial agents that have been reported to be carcinogenic
and/or mutagenic are alkylating and acylating agents classified under 12 structural
headings, viz., epoxides lactones, aziridines, alkylsulfates, sultones, aryidiakyl
triazenes, diazoalkanes, phosphoric acid esters, alkane halides, halogenated alkanols,
315
-------
halogenated ethers and aldehydes. The major industrial class of demonstrated carcino-
genic and/or mutagenic activity are the halogenated hydrocarbons comprising saturated
and unsaturated derivatives including: alkanes, alkanols, ethers, vinyl and vinylidene
analogs, alkyl, aryl and polyaromatic derivatives.
Although the industrial organic potential chemical carcinogens and mutagens
were considered as discrete entities, it is recognized that man is exposed to a
broad galaxy of environmental agents and hence considerations relative to possible
synergistic, potentiating, co-carcinogenic, co-mutagenic and/or antagonistic inter-
actions of carcinogenic and mutagenic and non-carcinogenic and non—mutagenic
chemicals are of vital importance.
It is also important to restress that the mutagenicity of a compound is important
se and suggestive to a degree of the compounds potential carcinogenicity. However,
it is recognized that more definitive elaboration of a compounds carcinogenicity can
only be obtained at present by long-term bioassay.
It should also be noted that although a relatively small number of industrial organic
compounds were reviewed in this report many structurally related agents are currently
in use. It would appear prudent to consider their potential toxicity as well in the
event that they have been untested in regard to carcinogenicity and/or mutagenicity.
We cannot be absolutely certain of the significance to man of findings of neopiasia
in test animals or positive mutagenic effects in a variety of test systems. However, it
would also appear prudent to minimize the burden and risk of potentially carcinogenic
and mutagenic agents in the environment, lest we bequeth o subsequent generations
the risk of catastrophic exposures for which redress would be either difficult or
impossible.
316
U.S. VER 4ENT PRINTING OPTICS : 1977 0—241—037/45
-------
TECHNICAL REPORT DATA
(Please read Ji .stj ucf ions on the reverse befoee completing)
1. REPORT NO. 2.
3. RECIPIENTSACCESSIOIIPNO.
I. TITLE AND SUBTITLE
Potential Industrial Carcinogens and Mutagens
5. REPORT DATE
May . 1977
6.PERFORMINGORGANIZAT’ONCODE
. AUTHOR(S)
Lawrence Fishbein
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
National Center for Toxicological Research
Jefferson, Arkansas 72079
10. PROGRAM ELEMENT NO.
11.CONTRACTIGRANTNO.
EPA-IAG-D4-0472
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final RPport
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ana. nal.. i
Ninety industrial chemicals, illustrative of 16 majdr classes and 19 structural
sub-categories have been reviewed primarily in terms of their reported carcinogenicity
and/or mutagenicity. The compounds were selected based on factors including: their
reported carcinogenicity and/or mutagenicity, their chemical structures and relation-
ships to known carcinogens or mutagens, their volume or use characteristics, and
suggested or estimated potential populations at risk.
Additionally, germane aspects (where known) of their synthesis (primarily in term
of the nature of the possible hazardous trace impurities), use patterns, chemical and
biological reactivity and stability, environmental occurrence and metabolic fate have
been included for cohesiveness of treatment.
It is impor tant to note that in 52 of the above cases, both carcinogenicity and
mutagenicity of individual compounds were reported. Thirty-one compounds have been
reported to be mutagenic and noncarcinogenic and seven compounds are carcinogenic and
nonmutagenic. In a number of cases, there are no reports of a compound having been
tested for carcinogenicity or mutagenicity or they are currently on test. In some
cases, conflicting carcinogenicity and/or mutagenicity results for the same compound
were reported. The largest number of industrial agents that have been reported to be
carcinogenic and/or mutagenic are alkylating and acylating agents classified under 12
structural ka ilj flQç -
KEY WORDS AND DOCUMENT ANALYSIS
—
— b.IDENTIFIERS/OPEN ENDED TERMS
C. COSATI Field/Group
19. SECURITY CLASS (This Report)
to public through the
nformati on Service, 20. SECURITY CLASS (This page)
22151.
21. NO. OF PAGSS
319
22. PRICE
EPA Form 2220-1 (9-73)
------- |