"Current Awareness*
Program
Vol. H.
December 1982
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Table of Contents;
5.2.1.7 Miscellaneous Compounds
5.2.1.7.1 Aldehydes and Related Compounds 684
5.2.1.7.2 Acrylonitrile and Allylisothiocyanate 695
5.2.1.7.3 Peroxides and Peroxy Compounds 701
5.2.1.7.4 Quinones and Alloxan 712
5.2.1.7.5 C-Nitroso Compounds 71b
5.2.1.7.6 Hexamethylbenzene, Hexamethyl-Dewar-benzene and
Hexaethylidenecyclohexane 722
5.2.1.7.7 Thalidomide, Phthalate Esters and Saccharin
5.2.1.7.7.1 Thalidomide 725
5.2.1.7.7.2 Phthalate Esters and Related Compounds 732
5.2.1.7.7.3 Saccharin 7^1
5.2.1.7.8 Sulfonamides, Cyclamate and Related Compounds
5.2.1.7.8.1 Sulfonamides and Related Compounds 755
5.2.1.7.8.2 Cyclamate and Related Compounds
5.2.1.7.9 Peroxisome Proliferators
5.2.1.7.10 Bis-(Morpholino)- and Bis-(N-Methylanilino)-methane .. 778
5.2.1.7.11 Some Therapeutically-Used Agents 779
References 791
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MISCELLANEOUS COMPOUNDS
i
CARCINOGENICITY AMD STRUCTURE-ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES,
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
David Y. Lai, Ph. D.
Yin-tak Woo, Ph. D.,
Joseph C. Arcos, D. Sc., and
Mary F. Argus, Ph. D.
Prepared for the Chemical Hazard
Identification Branch "Current
Awareness" Program
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684
5.2.1.7 Miscellaneous Compounds
In addition to the agents discussed in the previous
sections., the general toxicology, 'carcinogenicity and mode of
action- of several other alkylating agents have been explored,
albeit to a limited extent. These compounds fall into widely
different chemical structure categories and probably undergo a
variety of chemical reactions with cellular constituents.
5.2.1.7.1 Aldehydes and Related Compounds. Aldehydes are
widely used in industry, agriculture and medicine (1). They have
also been detected as components of automobile and diesel exhaust
(2-4), tobacco smoke (5,6), and photochemical smog (3, 7-9). A
U.S. Environmental Protection Agency report (10) shows that some
2 to 7 billion pounds of formaldehyde are produced or imported in
the United States annually in recent years and that over 800
million pounds of this compound is released in the air every
year. In 1976, the National Institute of Occupational Safety and
Health (NIOSH) (11) estimated that 17,500,000 workers were poten-
tially exposed to formaldehyde in the workplace.
Except for some irritation and hypersensitivity reactions,
human exposure to low levels of aldehydes was thought to be
innocuous. However, because of the increasing use and ubiquitous
existence of these substances in the environment, there is
renewed concern of the potential hazard that they may represent
to human health.
Toxic Effects. Exhaustive reviews on the toxicity and the
health effects of formaldehyde and related compounds have
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685
appeared during recent years (1, 3, 10-14). The acute toxicity
data of some of these are summarized in Table CXXXI.
Acrolein (CH2=CHCHO), an olefinic unsaturated aldehyde, is
the most toxic agent of this group. In fact, acrolein was used
as a war gas during World War I. As low as 0.5-1.0 ppm of
acrolein in the air produces irritation of the respiratory and
ocular mucosa (15, 16). Higher concentrations cause death from
edema and hemorrhage in the respiratory system. The gross toxic
effects of acute and subacute exposure to acrolein in various
animal species have been described (17-23). Malonaldehyde and
formaldehyde are also highly toxic. In subacute exposure,
formaldehyde causes lung irritation (18), depression of the
central nervous system (24), and dermatitis upon contact with the
skin (25). Inhalation toxicity is manifested by lung hemorrhage
and edema, respiratory collapse and death (20). Ingestion of a
concentrated solution results in severe injury of the gastro-
intestinal tract, and liver and renal damage (26). In contrast,
hexamethylenetetramine (HMT) appears to be relatively
Insert here Text-Figure 27
innocuous. Natvig _et_ _aj^. (27) showed in a long term study that
rats fed a diet containing 0.16% HMT do not differ from control
rats fed the same diet without HMT, as regards voluntary muscular
activity, body weight, general health and relative organ
-------
Table CXXXI.
p. 1 of 2 pp.
Acute Toxicity of Some Aldehydes and HexamethylenetetramLne (HMT)
Compound
Species and route
Lethal dose or concentration
References
Acrolein
Formaldehyde
Rat, oral
Rat, s. c.
Rat, inhalation
Mouse, oral
Mouse, s. c.
Mouse, inhalation
Rat, oral
Rat, oral
Rat, s. c.
Rat, i. v.
Rat, inhalation
Rat, inhalation
Mouse, s. c.
Mouse, inhalation
Guinea pig, oral
LD
50
LC
LC
LC
50
50
50
: 46 mg/kg
= 50 mg/kg
:0. 75 mg/1 (10 min)
:0. 30 mg/1 (30 min)
'0. 02 mg/1 (4 hr)
:28 mg/kg
= 30 mg/kg
;66 ppm (6 hr)
LD^« =800 mg/kg
=600-700 mg/kg
= 420 mg/kg
= 87 mg/kg
= 820 ppm (30 min)
= 482 ppm (4 hr)
= 300 mg/kg
= 41 4 ppm (4 hr)
= 260 mg/kg
LD
LD
50
50
50
(19)
(18)
(-16)'
(16) '
(16)
(100)
(18)
(101)
(102)
(103)
(18)
(104)
(18)
(105)
(18)
(105)
(102)
-------
Table CXXXI continued
p. 2 of Z pp.
Malonaldehyde
Hexamethylene-
tetramine
Acetaldehyde
Propionaldehyde
Rat, oral
Rat, i. v.
Rat, oral
Rat, s. c.
Mouse, s. c.
Rat, oral
Rat, s. c.
Mouse, s. c.
LD5Q=632 mg/kg
LD5Q=9200 mg/kg
LD5()=1930 mg/kg
LD=640 mg/kg
5Q
mg/kg
LD =1400 mg/kg
LD5Q=820 mg/kg
LD =680 mg/kg
(106)
(104)
(19)
(18)
(18)
(19)
(18)
(18)
-------
N
H2C
CH2 CH2
N.
N
-CH2 H2C
CH2
N
Hexamethylenetetramine
Text-Figure 27
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636
weights. .However, HMT hydrolyzes to formaldehyde at acid pH and
it has been suggested that a relationship exists between the
toxicities of the two compounds ..(1, 27). The toxicity of
acetaldehyde and propionaldehyde has so far been much less
investigated. They are irritating to mucous membranes, and large
doses cause death by respiratory paralysis (18).
Mutagenic effects. The mutagenicity of aldehydes and HMT
has been studied in various test systems. The results of these
studies are summarized in Table CXXXII. ^- /^3,/fi C XX Ail
Numerous experiments have established that formaldehyde is
mutagenic in bacteria and fungi including Escherichia coli (28-
32), Pseudomonas fluorescens (28), Saccharomyces cerevisiae (33,
34), Neurospora crassa and Aspergillus nidulans (1). The
occurrence of mutations has also been reported in Drosophila (35-
38) and the grasshopper (39) fed food containing formaldehyde.
In mammalian systems, mutagenicity was observed when formaldehyde
was tested in the L5178Y mouse lymphoma assay (40), in an
unscheduled DNA synthesis test in human HeLa cells (41), and in
sister chromatid exchange assays in a Chinese hamster ovary (CHO)
cell line (42). However, formaldehyde appears to exhibit no
mutagenic activity in the Ames test, using various strains of
Salmonella typhimurium (43), or in the dominant lethal studies
conducted with Swiss mice (44). Likewise, no effect was observed
when formaldehyde was tested in the CHO/HGPRT assay using
hypoxanthinephosphoribosyltransferase (HGPRT) as a marker (45).
HMT exhibited a mutagenic effect similar to formaldehyde in
dominant lethal mutagenesis assays conducted in Drosophila
-------
Table CXXXII.
Mutagenicity of Some Aldehydes and Hexamethylenetetramine (HMT)
System
Zscherichia coli
Pseudomonas
fluorescens
Salmonella
typhLmurium
Neurospora crassa-
Aspergillus
nidulans
Saccharomyces t
cerevisiae
Drosophila
melanogaster
Mouse Lymphoma
L5178Y
CHO/HGPRT
Unscheduled DNA
Synthesis Human
Fibroblast
Chromosome
Aberrations
Dominant Lethal
in Mouse
Sister Chromatid
Exchange
Formaldehyde HMT
+(28-32)
+ (28)
+ (47)
- (43)
."''+ (1)
+ (1)
f (33, 34)
+ (35-38) + (35)
-f (40)
- (45)
+ (41)
+ (39)
- (44) + (46)
+ (42)
Compound a
Malonaldehyde Acetaldehyde Propionaldehyde Acrolein
+ (30)
- (53)
+ (48) + (55)
- (47) - (47) - (47) - (47, 52)
±(54)
+ (35) + (35) + (35)
+ (49)
f (50) - (51)
- (44)
+ (50)
a Numbers in parentheses represent the references
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687
(35). The result was confirmed in mice (46). However, the
amount of HMT used in these experiments was many times greater
than the doses used therapeutically in man.
The mutagenicity of various aldehydes in the Salmonella
typhimurium test system, using TA100 (base-pair substitutions)
and TA98 (insertions, and deletions) strains has been screened by
Sasaki and Endo (47). Only formaldehyde showed very weak
mutagenic activity within a limited range of concentrations.
Other aldehydes, namely acetaldehyde, propionaldehyde, acrolein
and malonaldehyde did not display any mutagenic effect in either
strain, with or without activation by liver microsomes. Positive
effects, however, were observed with malonaldehyde when tested in
Salmonella typhimurium strains TA1978, his D305 and his D3076
(frameshift mutants with normal excision repair) (48) as well as
in the L5178Y mouse lymphoma assay (49). Although less active
than formaldehyde, both acetaldehyde and propionaldehyde are
mutagenic in Drosophila (35). Acetaldehyde is also known to
induce sister chromatid exchange in mouse bone marrow cells, as
well as chromosomal aberrations in human lymphocytes (50) and
Vicia faba roots (51).
In agreement with the findings of Sasaki and Endo (47),
Anderson £t_ _al_. (52) could not induce point mutations by acrolein
in several strains of Salmonella typhimurium. Negative results
were also obtained with acrolein in the dominant lethal assay in
Swiss mice (44), in chromosomes of Vicia faba roots (51), in a
strain of Escherichia coli used to detect forward mutations and
reverse mutations (53), as well as in two methionine auxotroph
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683
strains of Saccharomyces cerevisiae, used to assay frameshift
mutations and base-pair substitutions (54). However, Rapoport
(35) reported that acrolein and -other aldehydes produce sex-
linked lethals in Drosophila. Mutagenicity was also observed in
a DNA polymerase-deficient strain of _E_. coli (30), a histidine
auxotroph strain of Saccharomyces cerevisiae (54) and in strains
TA1538 and TA98 (insertions and deletions) of Salmonella
typhimurium (55).
Teratogenic effects. The teratogenicity of formaldehyde,
HMT and acrolein have been studied by several investigators.
However, there is no substantive evidence that these compounds
are teratogenic.
Gofmekler (56) exposed female rats to-gaseous formaldehyde
at 0.01 and 0.8 ppm for 10-15 days before placing them with
males; immediately after the animals were further exposed for 6-
10.days to the same concentrations of formaldehyde. No gross
abnormalities were observed in the offspring, although there was
a 14-15% increase in the duration of pregnancy when compared with
controls. Also, there were no abnormalities in the offspring
when the rats were exposed to airborne formaldehyde at 4 ppm, 4
hr/day during days 1-19 of pregnancy (57). Furthermore,
following administration of formaldehyde to male rats at 0.1 ppm
in drinking water or 0.4 ppm in the air for 6 months, Guseva (58)
observed no adverse endocrine or reproductive effects in the
animals. Pregnant dogs fed diets containing either formaldehyde
or HMT on days 4-56 of pregnancy did not show any abnormality of
reproduction: no physiological or skeletal abnormalities were
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689
observed in the litters (59). Likewise, long-term studies of
rats fed HMT at 0.16% (27) or given 1% HMT in drinking water (60)
failed to show any effect on the fertility of the rats or
structural malformations of their offspring. Bouley _et_ _al_. (61)
exposed male and female rats to 1.3 mg/m acrolein vapor for 26
days and found no significant differences in the number of
pregnant animals as well as the number and mean weight of
fetuses.
No reports have been encountered in the literature on the
potential teratogenicity of malonaldehyde, acetaldehyde or
propionaldehyde.
Carcinogenicity. Since formaldehyde is a known alkylating
agent, its carcinogenic potential and that of HMT and other
aldehydes has been the subject of several studies. A summary of
the results of these studies is given in Table CXXXIII. /&£>/£
The first report of positive carcinogenic response to
formaldehyde administration was made by Watanabe ^t_ a_l_. (62) in
1954. These authors observed sarcomas at the injection site in 4
of 10 rats given weekly subcutaneous injections of 1 ml (0.4%)
formaldehyde solution over a period of 15 months. Tumors of the
liver and omentum were also observed in two of the rats bearing
the sarcomas. However, the value of this study has been in
dispute since no controls were apparent in the report. Horton et
al. (63) administered formaldehyde, by inhalation at 0.05 mg/1,
to 60 C3H mice 1 hr./day, 3 times/week for 35 weeks? after the
initial 35-week exposure the mice were treated for an additional
29 weeks at 0.15 mg/1. No pulmonary tumors or metaplasias were
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Table CXXXIII.
Carcinogenicity of Some Aldehydes and Hexamethylenetetramine
Compound
Species and strain
Principal organs affected and route
References
Formaldehyde
Hexamethylene-
tetramine
Malonaldehyde
Propionaldehyde
A cetaldehyde
A crolein
Rat,
Rat, Fisher 344
Mouse, C3H
Mouse, B6C3F1
Rat,
Rat, Wistar
Rat,
Mouse, Swiss
Mouse, Swiss
Mouse, [nbred
Rat, Hybrid
Rat,
Mouse, Albino "S"
Mouse,
Hamster, Syrian golden
Rabbit,
Local sarcomas (s. c.)
Nasal cavity (inhalation)
No significant effect (inhalation)
No significant effect (inhalation)
Local sarcomas (s. c.)
No significant effect (p. o.)
No significant effect (p. o.)
Skin, liver, kidney, lung
and rectum (topical)
Skin (topical)
No significant effect (s. c.)
Local sarcomas (a. c.)
No significant effect (p. o.)
No significant effect (topical)
No significant effect (s. c.)
No significant effect (inhalation)
No significant effect (inhalation)
(62)
(64, 65)
(63)
(71)
(67)
(68)
(69)
(70, 76. 77)
(70)
(71)
(73)
(72)
(82)
(83)
(84)
(85)
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690
found in the 15 animals surviving over 59 weeks, although
numerous changes in the lung tissue were observed. Again, this
study is considered incomplete because the animals were not.
observed for lifetime and the survival was poor. In a report of
interim results (after 18 months of a 2-year study) from a
chronic inhalation study of formaldehyde in rats, Swenberg et al.
(64) described the development of 36 squamous cell carcinomas in
the nasal cavity in 200 rats exposed to 15 ppm of formaldehyde,
6 hr. per day, 5 days per week. A similar study has been carried
out using mice by the same group of investigators (65); thus far,
there is no evidence of carcinogenicity in this species. However,
the rats, but not the mice used in the study developed an
epizootic viral infection. Therefore, it cannot be excluded that
there was a correlation between tumorigenesis and the viral
infection in the rats; the possibility of synergism will require
further investigation. Nevertheless, based on the findings of
this report and preliminary epidemiological studies, the
Carcinogen Assessment Group of U.S. Environmental Protection
Agency (66) concluded that "there is substantial evidence that
formaldehyde is likely to be a human carcinogen." Although the
study is by no means unequivocal, it appears to be corroborated
by some positive mutagenicity tests (32, 40-42) suggesting that
formaldehyde is possibly a weak carcinogen. Further studies on
the carcinogenic action of formaldehyde are needed.
The induction of sarcomas and adenomas at the site of
subcutaneous injection of HMT in 8 out of 14 rats has been
described by Watanabe and Sugimoto (67). Again, the lack of data
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691
on control animals limits the significance of the report. No
evidence of carcinogenicity of HMT was found in a large and well
controlled study conducted by Delia Porta _e_l_ _a_l_. (68) in both
rats and mice given HMT in drinking water.' Similar results were
obtained by Brendel (69) in a study of.rats given 400 mg/day HMT
orally for about a year.
Data on carcinogenicity'studies of propionaldehyde and
acetaldehyde are also quite equivocal and further investigation
seems to be desirable. Shamberger et al. (70) have shown that
38% of the mice developed skin tumors at 21 weeks after
initiation once with 0.3 mg propionaldehyde followed by daily
treatment with 0.1% croton oil. However, studies by Kiley and
Wallace (71) in 1941 showed no evidence, after 40 weeks, of
.carcinogenic activity of propionaldehyde in mice given twice
weekly subcutaneous injections of 0.5 ml propionaldehyde solution
(0.89%) for 16 weeks. Rats fed acetaldehyde in the diet over 300
days remained tumor-free (72); however, after about 1 1/2 years,
4 spindle cell sarcomas were observed in 14 of the surviving rats
subcutaneously injected with 0.1-1.0 ml, 0.5% acetaldehyde
solution 26-41 times (1-2 times/wk) (73).
The protective effects of several antioxidants against
experimental carcinogenesis (74, 75) have led to the suggestion
that malonaldehyde, an intermediary product of peroxidized
polyunsaturated fatty acids formed in animals when their diets
are low in antioxidants, may play a role in carcinogenesis.
Shamberger and coworkers (70, 76, 77) have studied the carcino-
genic potential of malonaldehyde in Swiss mice. A single dose of
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692
0.25 ml acetone containing 6 or 12 mg malonaldehyde was applied
on the back of 30 Swiss mice; after daily treatment with 0.1%
croton oil, 52% of the mice had skin tumors at 30 weeks. In
another experiment, 12 mg malonaldehyde was applied daily to
mice; 5 animals developed carcinomas of the liver, kidney, lung
and rectum. Since irradiation (78) as well as several
carcinogens, such as 7,12-dimethylbenz[a]anthracene,
benzo[a]pyrene and 3-methylcholanthrene (70, 76, 77), all bring
about increased levels of malonaldehyde following application to
mouse skin, it was suggested that malonaldehyde might be an
universal initiator of carcinogenesis (77). Considerable amounts
of malonaldehyde have been detected in beef and other meats but
not in fruits and vegetables (79). The catcinogenicity of
malonaldehyde appears to be in line with the view that
individuals who consume little or no meat and whose diets are low
in poly-unsaturated fatty acids have lower incidence of cancer
(79-81).
Ellenberger and Mohn (53) designated acrolein as a
"carcinogenic compound". However, although it has reported
mutagenicity in Drosophila (35) and in several strains of
bacterial species (30, 54, 55), no significant carcinogenic
effect attributable to acrolein has been encountered in the
literature. In a study conducted by Salaman and Roe (82),
application of 0.5% acrolein in acetone to the skin of 15 mice
for 10 weeks, followed by 0.17% croton oil treatment for 18
weeks, gave rise to 3 papillomas in 2 mice. However, a similar
incidence of papillomas was also seen in the control group
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693
treated with croton oil only. Steiner et_ _al_. (83) gave weekly
subcutaneous injections of 2.2 mg of acrolein dissolved in 0.1 ml
of sesame oil to 15 mice for 24 weeks. There were 6 survivors at
15 months, but no tumors were observed. Similar results were
obtained in inhalation studies on hamsters (84, 85) and rabbits
(86). No evidence was found that acrolein was either a .
carcinogen (84, 86) or a cocarcinogen with either diethynitros-
amine or benzo[a]pyrene in respiratory tract carcinogenesis (84,
85) .
Possible mechanisms of action. The interaction of
formaldehyde with proteins and nucleic acids (as well as their
constituents) has been amply explored (e.g., 87-90) and reviewed
extensively by Feldman (91). Some of these reactions are
believed to be involved in the production of the formaldehyde-
induced genetic effects.
Formaldehyde interacts readily with amino groups of amino
acids and proteins to form aminomethylol compounds (87).
Reactions of formaldehyde also occur with -CO-NH- groupings of
purines and pyrimidines yielding monomethylol derivatives (88).
The monomethylol derivatives of amino acids and nucleosides are
chemically reactive and can react, in turn, with amide,
guanidine, imidazole or indole groupings of other molecules to
form condensation products through methylene bridges (89, 90).
Exposure of Escherichia coli to low doses of formaldehyde induced
large numbers of interstrand cross-links in the DNA which involve
"protein bridges" between the DNA strands (31); it was suggested
that the mutagenic action of formaldehyde on bacterial DNA is
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694
actually not produced by formaldehyde itself, but is mediated by
its reaction products with proteins, which in turn react with
nucleotides (92, 93). Cross-linkage of nucleohistone and DNA by
formaldehyde has been repeatedly demonstrated (94, 95). Siomin
et al. (95) have shown that the reaction rate of formaldehyde
with nucleotides or DNA increases in the presence of amino acids
or lysine-rich histones. In addition to modification of the
bases of DNA, the reaction of formaldehyde with DNA in the
presence of amino acids is accompanied by the induction of
single-strand breaks in DNA (33, 34). The sensitivity to
formaldehyde is higher in the UV-sensitive excision-deficient
mutants of yeast (33, 34) and Escherichia coli (29) or in an
E.coli mutant defective in DNA polymerase (.92) than in their
respective wild type strains. This indicates that formaldehyde-
induced damages in DNA are susceptible to repair mechanisms. The
differences in the mutagenicity to formaldehyde among various
bacterial strains (43, 47) may be due to differences in their
repair efficiency.
The interaction of other aldehydes with cellular macro-
molecules has been much less investigated. In a study of the
action of several aldehydes to DNA, Poverennyi _et_ al_. (96)
suggested that in the presence of amino acids, aldehydes cross-
link DNA in a similar manner as formaldehyde. Malonaldehyde has
been reported to react with guanine and cytidine and form cross-
links between the strands of DNA (49, 97, 98). Cross-linking of
DNA followed by error-prone excision repair is believed to be the
mutagenic mechanism of malonaldehyde (49).
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695
Munsch et_ _al_. (99) have demonstrated that there is
substantial binding of [ nDacrolein to rat liver DNA as well as
to the sulfhydryl groups of DNA polymerase in regenerating rat
liver. Unlike other aldehydes, however, acrolein binds to DNA by
intercalating between bases. It has been hypothesized that the
critical cellular effects of acrolein involve DNA and/or enzymes
of nucleic acid synthesis (14). It is possible that the
mechanism of activation of acrolein involves conversion to
glycidol, a known carcinogen in mouse skin, through epoxidation
in vivo (14).
5.2.1.7.2 Acrylonitrile and Allylisothiocyanate. Acrylo-
nitrile (CH2=CH-CN), a highly reactive chemical agent struc-
turally resembling vinyl chloride, is widely used in industry for
the manufacture of fibers, plastics and elastomers. Over 1.5
billion pounds of acrylonitrile are produced annually in the
United States (107). Low levels of this compound enter the
environment during production, end-product manufacture and end-
product use. It is also present in cigarette smoke (108).
Recent evidence from both chronic toxicity studies in laboratory
animals and epidemiologic studies on industrial workers suggests
that acrylonitrile is a potential human carcinogen (109, 110).
In response to these findings, the U.S. Food and Drug
Administration (111) has banned the use of an acrylonitrile resin
for soft drink bottles. Recently, the National Institute for
Occupational Safety and Health (NIOSH) decreed safety measures in
the handling of acrylonitrile in the workplace, comparable to
that required for human carcinogens (109).
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696
Allylisothiocyanate (CH2=CH-CH2-N=C=S) structurally
resembles acrylonitrile; it is the product of the enzymatic
hydrolysis of sinigrin, a naturally occurring substance present
in a variety of cruciferous plants.such as cabbage, cauliflower
and horseradish (112). As a food additive, it is generally known
under the name "mustard oil". It is clinically used as a
counter-irritant. Following the discovery of its mutagenic and
carcinogenic activities in several assay systems, the wide use of
this substance as a cooking medium in certain regions of India
and some other tropical countries became suspect as a possible
causative agent in the high incidence of oesophageal cancer in
the exposed populations (113, 114). "T^le. £ X XX/1/
i
Toxic effects. The toxicity and health effects of
acrylonitrile have been reviewed (110). The acute toxicity of
acrylonitrile and allylisothiocyanate in several animal species
-si-
is summarized in Table CXXXIV. In laboratory animals, signs of
acrylonitrile intoxication vary widely in different species and
at different doses (115, 116). The effects may include damage to
the central and peripheral nervous system, and hemorrhage of the
lung, liver, kidney, adrenal or spleen (117, 118). To humans,
acrylonitrile is toxic if inhaled, ingested or in contact with
the skin. Epidemiologic studies of health impairment among
acrylonitrile workers revealed that the majority of the workers
develop functional disorders of the central nervous, the
cardiovascular and the hematopoietic systems, in addition to
other minor clinical symptoms (119, 120).
-------
Table CXXXIV.
Acute Toxicity of Acrylonitrile and A llylisothiocyanate
Compound
Acrylonitrile
A llylisothiocyanate
Species and route
Rat, oral
Rat, s. c.
Rat, inhalation
Mouse, oral
Mouse, s. c.
Mouse, inhalation
Guinea pig, oral
Guinea pig, skin
Guinea pig, inhalation
Rabbit, i. v.
Rabbit, skin
Rat, oral
Mouse, i. p.
Lethal dose or concentration
LD =62-86
LD =80-96
LC =500 ppm/4 hr
LD50=2?
LD5Q =34-35
LC =900 mg/m3/2 hr
50
LD5Q=56
LD5Q = 250
LC =576 ppm/4 hr
LD50=?2
LD5Q = 280
LD5()=148
LD5Q=38
References
(115, 117, 149)
(117, 150)
(151)
(115)
(115, 117)
(152)
(153)
(154)
(118)
(155)
(156)
(157)
(44)
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697
The toxicity of allylisothiocyanate has been much less
investigated. In animals, a lethal dose of this chemcial causes
death by respiratory and vasomot.or center paralysis (121). I2ibl. C)(XX]/
Mutagenic effects. The mutagenic action of acrylonitrile
has been demonstrated in several test systems (Table CXXXV). In
the presence of a fortified post-mitochondrial (S-9) fraction of
mouse liver, acrylonitrile is mutagenic to several strains of
Salmonella typhimurium which are sensitive to base substitution
and frame shift mutage.ns (122-124). The mutagenic activity
depends strictly on the presence of the S-9 fraction, and it has
been shown that the mutation rates vary with the animal species
from which the S-9 fraction is obtained as well as with the
pretreatment of the animals (123, 124). Acrylonitrile is also
mutagenic toward several strains of Escherichia coli? however,
the presence of an activating system is not required (125).
Similarly, mutagenic activity can be observed in Saccharomyces
cerevisiae assayed without an activating system (126).
Only very weak mutagenic effects were noted in the sex-
linlced recessive lethal tests in Drosophila melanogaster (127).
The mutation rates (0.35-0.55%) of acrylonitrile in these tests
were not considered by the authors to be different from the
spontaneous mutation rate (0.14%) (127). Acrylonitrile did not
produce chromosomal aberrations in root tips of Vicia faba (128,
129), and no mutagenic activity was found either in the L5178Y
mouse lymphoma cell assay or in the unscheduled DNA synthesis
test (130). The negative results of these studies are suspected
to be due to the high volatility and toxicity of the compound
(127, 131).
-------
Table CXXXV.
Mutagenicity of Acrylonitrile and Allylisothiocyanate
Compound
System
Acrylonitrile
Allylisothiocyanate
Salmonella
typhimariam
Escherichia coli
Saccharomyces
cerevisiae
Bacillus subtilis
Drosophila
melanogaster
Mouse Lymphoma
L5178Y
Unscheduled DNA
Synthesis
Chromosome
Aberrations
Dominant Lethal
in Mouse
+ (122-124)
+ (125)
+ (126)
(127)
(130)
(130)
(128-129)
(134)
+ (132, 133)
(44)
(134)
Numbers in parentheses represent the references.
-------
698
Data on the mutagenicity studies of allylisothiocyanate are
scarce and equivocal. The mutagenic action of allylisothio-
cyanate has been shown in Drosophila (132). There is also
evidence that it produces chromosome breakage in Triticum (133)
and in Allium (113). However, allylisothiocyanate at doses of
3.8 mg/kg and 19 mg/kg failed to induce dominant lethal effects
in mice (44). Mutagenicity assay of allylisothiocyanate in
Bacillus subtilis was also negative (134).
Teratogenic effects. Murray et al. (135) evaluated the
teratogenic potential of acrylonitrile in Sprague-Dawley rats.
Groups of 29-39 pregnant rats were given 10, 25 or 65 mg/kg
acrylonitrile by gavage on days 6-15 gestation. Adverse maternal
and fetal effects were noted at the 2 highest dose levels.
Scheufler (136) also reported acrylonitrile to be embroyotoxic
when given to pregnant mice.
No teratogenic effects in pregnant Wistar rats were observed
*
when a single dose (60 mg/kg) of allylisothiocyanate was given
orally on days 12 or 13 of gestation (137). T^.'^]e CXXX Vf
Carcinogenicity. The epidemiological data (109, 110)
implicating that acrylonitrile may be a human carcinogen are
^r
supported by several animal studies (Table CXXXVI). Maltoni et
al. (138) examined the carcinogenic effects of ingested or
inhaled acrylonitrile on Sprague-Dawley rats for about 2 years.
In the ingestion experiments, acrylonitrile was administered in
olive oil by gavage at the dose of 5 mg/kg body weight, 3 times a
week for 52 weeks. In the inhalation studies, rats were exposed
to 5, 10, 20 or 40 ppm of acrylonitrile, 4 hours daily, 5 days a
week for 52 weeks. Several types of tumors were seen after 131
-------
Table CXXXVI.
Carcinogenicity of AcryLonLtrile and Allylisothiocyanate
Compound
Species and strain
Principal organs affected and route
R eferences
Acrylonitrile
Rat, Sprague-Dawley
A llylisothiocyanate
Rat,
Mouse, albino
Mouse, C17/Icrc
Mouse, XVIIxC57Bl
Forestomach, zymbal gland,
mammary gland and brain (p. o.)
Fores tomach, zymbai gland, skin,
brain and uterus (inhalation)
Stomach, zymbal gland
and brain (p. o.)
Gastrointestinal tract, zymbal
gland, mammary gland and
brain (inhalation)
Skin (topical)
Skin (topical)
Stomach (p. o.)
None (topical)
None (s. c.)
None (i. p.)
(138)
(138)
(139)
(140)
(141)
(142)
(114)
(114)
(114)
(114)
Mouse, white
None (topical)
(144)
-------
699
weeks in both the ingestion and inhalation experiments. The most
frequent types of tumors were: mammary tumors, carcinomas of the
Zymbal gland, forestomach papillomas, acanthomas, encephalic
tumors, and carcinomas of the skin and uterus. However, some
tumors were also noted in control animals and the data indicate
only a "border-line oncogenic effect".
In another carcinogenicity bioassay, rats consuming 100 or
300 ppm acrylonitrile in drinking water for a year developed
stomach papillomas, Zymbal gland carcinomas and tumors of the
central nervous system; no such tumors were found in control
animals (139). An increase in the incidence of tumors of the
gastrointestinal tract, ear canal, mammary region and brain was
also observed in rats exposed to an atmosphere containing 80 ppm
acrylonitrile 5 hours daily, 5 days a week for 2 years (140).
The carcinogenicity of allylisothiocyanate has been tested
in mice by several investigators (114, 141-145). The data of
these studies, however, are equivocal and inconclusive. In 1938,
Visser and Ten Seldam (143) indicated that allyisothiocyanate was
not carcinogenic to the mouse skin. This was confirmed by the
findings of Larionow and Soboleva (144) who failed to induce
tumors in mice treated with allylisothiocyanate thrice weekly on
the skin over a period of 19 months. Rusch et al. (141)
reported, however, that 10% of the albino mice treated with 50%
allylisothiocyanate in acetone topically 2x/week developed skin
papillomas and carcinomas after 24 weeks. Skin tumors were also
observed by Ranadive _et_ _al_. (142) in C17/Icrc mice following
similar treatments. In another study by Ranadive and coworkers
(114), mice were given 0.05 ml allylisothiocyanate orally 6x/week
-------
700
for 12 weeks? 3 stomach papillomas were found in 14 mice examined
over 2 years. Administration of the compound by cutaneous
application (daily), s.c. injection (0.5 ml, monthly) or i.p.
injection (0.2 ml, monthly), however, did not elicit any tumors
in mice observed for their entire life span (114). Allyliso-
thiocyanate was also shown to have no co-carcinogenic effect on
mouse skin (145).
Possible mechanisms of action. The mechanisms of mutagenic
and carcinogenic action of acrylonitrile and allylisothiocyanate
are as yet unknown.
Based on the differential responses of the tester strains of
_E_. coli to the mutagenic action of acrylonitrile, Venitt and
coworkers (125) suggested that acrylonitrile might cause
nonexcisable mis-repair DNA damage thought to be associated with
the induction of DNA strand breaks (146). Cyanoethylation of
thymidine, ribothymidine and several minor t-RNA nucleosides by
acrylonitrile has been demonstrated (147). These findings lead
to the suggestion that acrylonitrile might probably react with
the thymidine residues in DNA (125).
In mammals, the metabolic fate of acrylonitrile has not been
elucidated. Hypothetically, oxidation of .acrylonitrile during
metabolism could lead to an epoxide, glycidonitrile
H C CHCN, which is structurally similar to glycidaldehyde
^o-^
H C CH-CHO, an agent demonstrated to be mutagenic and
NVOX"
carcinogenic (see Section 5.2.1.1). Recently, supporting
evidence for the formation of the suggested epoxide intermediate
-------
701
has been obtained (148). However, whether acrylonitrile has the
same mechanism of carcinogenesis as glycidaldehyde, remains to be
investigated.
Mutagenicity data imply that the effects of allylisothio-
cyanate on chromosomes probably form the basis for the carcino-
genic effect of this compound. A single application of
allylisothiocyanate to the ears of mice was noted £b increase the
mitotic activity of epithelial cells an effect observed after
the same treatment of mice with certain irritants shown to be
weak carcinogens (141). The possible correlation between the
mitotic effect and carcinogenesis of these compounds has not been
investigated. The double bonds of allylisothiocyanate are highly
reactive and should react readily with nucleophilic groups in the
genetic material. Epoxidation of the carbon-carbon double bond
could also be an important step in the bioactivation and
carcinogenesis of allylisothiocyanate.
<^3 -
5.2.1.7.3 Peroxides and Peroxy Compounds. Organic per-
oxides are compounds which contain at least two oxygen atoms
linked in the same manner as in molecular oxygen. Depending on
the chemical structure, organic peroxides may be classified
into: (i) alkyl hydroperoxides, (ii) dialkyl peroxides, (iii)
peroxyacids, (iv) peroxyesters, and (v) diacylperoxides. The term
"peroxides" and the prefix "peroxy-" are often used
interchangeably.
-------
702
ROOH
(i)
ROOR'
(ii)
0
II
RCOOH '
(iii)
0
II
RCOOR '
(iv)
0 0
II U ,
RCOOCR'
(V)
Organic peroxides "have a wide environmental occurrence and
great economic importance. They may be derived by direct air
oxidation of organic compounds or by reaction with peroxides such
as hydrogen peroxide/ alkali metal peroxides or ozone.
Peroxidation of fats and lipids may readily occur under both in
vivo and in vitro conditions (rev., 158). In vivo peroxidation
of depot fats and of lipids of nonadipose tissues is believed to
be associated with "yellow fat disease" and symptoms of vitamin E
deficiency, respectively.. Lipid-containing structures within
cell organelles may also be peroxidized. Lipid peroxidation of
biomembranes is considered to be the initial event in the toxic
action of a variety of xenobiotics (rev., 159, 160). The
relationship of such in vivo peroxidation to carcinogenesis is
unclear. .Dietary fats may be peroxidized upon heating in the
presence of air or when irradiated (158). A sample of highly
peroxidized oils and fats present in fish meal was reported to
induce hepatoms in trout (161).
The presence of peroxides in the polluted atmosphere of
several large U.S. cities has been detected. The photochemical
formation of organic peroxides from nitrogen oxides (which yield
ozone upon photoxidation) and unsaturated hydrocarbons is well
established; both reactants are present in the exhaust products
-------
703
of gasoline engines and industrial effluents. Kotin and Falk
(158) exposed mice to an atmosphere containing aerosols of
ozonized gasoline and observed substantial increase in the
induction of pulmonary tumors . A sample of aerosol condensate of
polluted air from Los Angeles was found to induce skin tumors
after repeated skin painting of mice (158); it is not known what
portion of the observed carcinogenic effect could be attributed
to peroxides.
Several organic peroxides are of great industrial import-
ance. The annual consumption or production of cumene hydroper-
Q
oxide in the U.S. was of the order of 3.06 x 10 lb. in 1977;
most of the compound was used as an intermediate in the synthesis
of phenol or acetone (162) . Other organic peroxides with annual
production greater than one million pounds in 1975 included
benzoyl peroxide, methyl ethyl ketone peroxide, di-t_-butyl
peroxide, _t_-butyl peroxybenzoate, and lauroyl peroxide; these
have been used mainly as chemical intermediates, polymerization
initiators, and bleaching and curing agents (162).
Despite their environmental importance, only about 20
peroxides have been tested for carcinogenicity, mostly by topical
or s.c. route in rodents. The following paragraphs focus on
these compounds. The structural formulas of some of. the more
complicated peroxides are depicted in Table s'^CXXX VII and CXXXVIII.
Physical and Chemical Properties and Biological Effects. The
. , , i i
physical and chemical properties of peroxides and peroxy
--------- .<-, - _ ,r3 ._ Q
compounds have been thoroughly reviewed by Mageli and Sheppard
-------
Table CXXXVII
Structural Formulas of Hydroperoxides
and Ascaridole Tested for Carcinogenicity
OOH
1-Hydroperoxycyclohex-2-ene H3C CH3
Ascaridole
:-OOH
CH3
Cumene hydroperoxide
CH=CH2
OOH
1-Hydroperoxy-l-
vinyl-cyclohexene-3
CH,
OOH
H^CH,
DiisopropyI-benzene p-Menthane hydroperoxide
hydroperoxide
p-tert-Butyl
isopropyl-benzene
hydroperoxide
H3C~C~CH3
HOO
CH-CH3
CH3
m-tert-Butyl-isopropyl-
-benzene hydroperoxide
-------
Table CXXXVIH
Structural Formulas of Some Peroxides Tested
for Carcinogenicity
0
CH,
-C-0-0-C-CH3
CH3
tert-Butyl perbenzoate
Benzoyl peroxide
H(CH2)3CH(CH3)2
. OOH
6-/3-Hydroperoxy-A4-cholestene-3-one
H3CX P~°, ,
A A
H2C 0 0 CH2
H3C H H NCH3
Methyl ethyl ketone peroxide
7,l2-Dimethyl-7,l2-
peroxybenz [o] anthracene
CH(CH2)3CH(CH3)2
A2>4-Cholestadiene peroxide
-0
i
-0
0
CH3(CH2)|0C-0--
Lauroyl peroxide
-------
704
(163). In general, the reactivity of the various classes of
peroxides roughly follows the order: peroxyacids > hydro-
peroxides > diacyl peroxides>peroxyesters > diakyl peroxides.
i
Within the individual classes, peroxides with primary alkyl
groups and lower molecular weight tend to be more unstable and
prone to explosion. Peroxides are strong oxidizing agents; they
i
may by reduced to the corresponding alcohols, ethers, anhydrides
or acids. Heterolytic decomposition of peroxides occurs in the
presence of acid or alkali yielding, in many instances, re-
arranged products. Heating or irradiation of peroxides,
particularly those with secondary or tertiary alky! groups,
I
readily cleaves the 0-0 bond homolytically to yield alkoxy (RO»),
acyl (RCO_«), hydroxyl (»OH), and after further breakdown, alkyl
(R«) radicals. The free radical formation is of great industrial
importance because of the ability of the free radical to initiate
polymerization. Free radicals are also presumed to account, at
least in part, for the biological effects of peroxides.
The literature on the toxicity of peroxides is rather
scant. The acute toxicity data of several peroxides are -r- ->-%
-^^- la-DJe
summarized in Table CXXXIX. The toxicity is apparently
dependent on the chemical structure. The hydroperoxides,
i
especially those of fatty acids, appear to be more toxic than the
relatively more stable dialkyl peroxides (^.._g_. di-t_-butyl
1 {
peroxide) or peroxyesters (.f_._g_., Jt-butyl peroxybenzoate) . The
toxicity of benzoyl peroxide has been studied quite extensively
and'has been reviewed in a NIOSH criteria document (164). The
toxic effects of human exposure to occupational levels of
-------
Table CXXXIX
Acute Toxicity of Peroxides in Rodents
Compound
Species and route
Lethal dose or concentration
References
t-Butyl hydroperoxide
Hydroperoxy methyl
oleate
Autoxidized methyl
linoleate
" ' "'". "'
Hydroperoxy methyl
linoleate
Cumene hydroperoxide
Di-t_-butyl. peroxide
Methyl ethyl ketone
peroxide
Dibenzoyl. per oxide
^t>-Butyl peroxybenzoate
Mouse, i. p.
Mouse, oral
Mouse, i. p.
Rat, i. p. : .
Rat, inhalation
Rat, s. c.
-Mouse,_i..p. _
Mouse, inhalation
Rat, oral or i. p.
Rat, inhalation
Mouse, i. p.
Rat, oral
Mouse, oral
LD =58 pimoles/mouse
(229 mg/kg)
LD .. =6 mg/mouse
(18 |jmoles/mouse)
LDc.f) = 45-60 ^.moles/mouse
50
LD50
LC
LD
LD
50
50
50
LC
LD
LC
LD
50
50
50
50
LD
= 1500-1700 p.moles/kg
; (489 mg/kg)
= 200 ppm for 4 hr
= 400 mg/kg
.=-1080 ^tmoles/mouse-
-(6780 mg/kg)
= 1 70 ppm for 4 hr
= 470-484 mg/kg
= 200 ppm for 4 hr
.= 17.1 fimoles/mouse
(167 mg/kg)
>5000 mg/kg
( '
= 2500 mg/kg ^
(182)
(183)
(182)
(184)
(185)
(186)
-(182)
(185)
: (185)
(185)
(187)
(Wazeter & Goldenthal,
cited in ref. 164)
'(186)
Contains 0. 65 moles of total peroxide/mole of starting material
-------
705
peroxides include irritation of the skin, eye, nose and throat
(163, 164).
The mutagenicity of peroxides has been virtually
unexplored. An evaluation of the mutagenicity of a preparation
of 78% benzoyl peroxide was reported (165). In in vitro tests
using Salmonella typhimurium (including strains TA-1535, 1537,
1538) and Saccharomyces cerevisiae (strain D4), benzoyl peroxide
did not exhibit any mutagenic activity even in the presence of
tissue homogenate from mice, rats or monkeys. Three peroxides
(benzoyl peroxide, cumene hydroperoxide, jt_-butyl hydroperoxide)
were tested at doses ranging from 15 to 90 mg/kg, in the dominant
lethal assay system using ICR/Ha Swiss mice; none of these
compounds was found to be mutagenic (44). The teratogenicity of
peroxides does not seem to have been studied.
Carcinogenicjty and Structure-Activity Relationships. The
carcinogenicity of 17 peroxides has been tested by Kotin and Falk
(158) and by .Van Duuren and coworkers (166-171). Table CXL
summarizes the data of Kotin and Falk (158). Nine peroxides and
one peroxide mixture were shown to induce malignant lymphomas in
C57B1 mice after subcutaneous injection. Subcutaneous sarcomas
and pulmonary adenomas were also occasionally observed. From the
available data, it appears that alkyl hydroperoxides may be more
carcinogenic than the other classes of peroxides tested. The
mixture containing _m_- and p_-isomers of jt_-butyl isopropyl benzene
hydroperoxide was the most carcinogenic, inducing malignant
lymphoma -In 50% of the treated mice. Diisopropyl benzene
hydroperoxide, was, however, much less carcinogenic. It appears
-------
Table CXL
Carcinogenicity of Peroxides in C57BL Mice after Subcutaneous Iniection'
Compound
Di-t-butyl peroxide
Methyl ethyl ketone
peroxide
Sodium peracetate
Diacetyl peroxide
Lauroyl peroxide
£-Menthane hydroperoxide
Diisopropyl benzene
hydroperoxide
m- and p_- t-Butyl isopropyl
benzene hydroperoxide
Cumene hydroperoxide
_t_-Butyl perbenzoate
Structure
(i_C4H9-0-)2
?H3
^s-f-0^
OH
O
1 ' /-x ^"V
CH C-O-O65 Na^
O
II
(CH3C-0-)2
O
1 i
(CH3^CH2\0C-°-}2
see Table CXXXVII
see Table CXXXVII
see Table CXXXVII
see Table CXXXVII
see Table CXXXVHI
Dose
(jl/moles)
100
40
80
60
40
90
50
50 C
i
50
40
s . c. sarcoma
0/35
1/34
0/37
0/38
0/43
0/^46
0/46
0/44
~\
,--'
1/30
0 0/47
Tumor incidence
lymphoma lung
7/35
3/34
3/37
4/38
2/43
6/46
2/46
22/44
1 1/30
10/47
; adenoma
1/35
1/34
0/37
1/38
0/43
2/46
3/46
3/44
0/30
0/47
""Summarized from P. Kotin and H. L. Falk [Radiation Res. Suppl. 3_, 193 (1963)].
-------
706
that the ring position and the nature of the alkyl group may
affect the carcinogenicity of the hydroperoxide, although further
analysis of the structure-activity relationship is not possible
due to the lack of data and uncertainty of the exact ring
position of the alkyl and hydroperoxy groups. Cumene hydroper-
oxide, an industrially important chemical, was found to be the
second most potent carcinogenic compound of the group, inducing
lymphomas in 29% and subcutaneous sarcomas in 3% of the treated
mice. Both di-jt_-butyl peroxide and t^-butyl peroxybenzoate
displayed some carcinogenic activity. The J>-butoxy group is
known to have a high tendency to form free radicals yielding
eventually methyl radicals. Methyl ethyl ketone peroxide,
another industrial chemical, was the only peroxide in the group
capable of inducing lymphoraas , subcutaneous sarcomas as well as
lung adenomas, although the tumor incidences were all quite
low. In addition to the above compounds, sodium peracetate,
diacetyl peroxide, lauroyl peroxide and p_-menthane hydroperoxide
were shown to be slightly carcinogenic. *73-2?J^^ C. XZ. /
The carcinogenicity data of Van Duuren and associates are
summarized in Table CX'LI. Of the 9 peroxides tested by skin
painting in mice, only three (l-hydroperoxy-l-vinyl-cyclohex-3-
ene, cyclohexene hydroperoxide and ascaridole) were considered to
be carcinogenic. Hydroperoxy methyl oleate (167), lauroyl
peroxide and benzoyl peroxide (166, 171), each induced one
papilloma among 30 mice and were not considered carcinogenic. _t_-
Butyl hydroperoxide (170), hydroperoxy methyl linoleate and
cumene hydroperoxide (167) all failed to induce tumors by topical
-------
707
route. Among the carcinogenic peroxides, 1-hydroperoxy-l-vinyl-
cyclohex-3-ene, an di, p-unsaturated hydroperoxide, was the most
potent. It induced papillomas in ,13/49 mice; 5 of these animals
developed malignant tumors. The carcinogenicity of the compound
was clearly due to the presence of the hydroperoxy group because
the corresponding hydrocarbon (l-vinyl-cyclohex-3-ene) was much
less carcinogenic, inducing only one malignant skin tumor in 30
mice. Furthermore, since l-vinyl-cyclohex-3-ene is prone to
autooxidation, the possibility that the hydrocarbon contained a
minute amount of peroxide (thus accounting for the carcino-
genicity) could not be excluded (166). The cyclohexene
hydroperoxide shown by Van Duuren _et_ _al_. (167) to be slightly
carcinogenic (inducing 1 squamous carcinoma in 30 mice) was named
l-hydroperoxy-cyclohex-3-ene but stated to be an c^, [^-unsaturated
hydroperoxide. Indeed, attack on the allylic C-H bond is the
preferred reaction in the oxidation of cyclohexene (163). The
correct name of the cyclohexene hydroperoxide should be 1-
hydroperoxy-cyclohex-2-ene or 3-hydroperoxy-cyclohexene.
Ascaridole (l,4-epidioxy-p_-menth-2-ene), a naturally occurring
endo-peroxide, is the other peroxide shown to be carcinogenic by
skin painting (167). It induced papillomas in 3/30 mice and a
squamous carcinoma in one animal. It is of interest to point out
that ascaridole may also be regarded as an o(, (o-unsaturated
peroxide. Thus, c4 ,Q-unsaturation appears to be a common
structural feature of the three carcinogenic peroxides.
The carcinogenicity of several peroxides has also been
tested by subcutaneous injection to mice and rats (168, 169). In
-------
Carcinogenicity oil-Peroxides in ICR/Ha Swiss Mice or Sprague-Dawley Rats Following Topical Application or Subcutaneous Injection'
Compound
Topical application to
mouse skin (mice
with papillomas/group size)
s. c. injection in mice
(mice with local
sarcomas/group size)
s. c. injection in rats
(rats with local
sarcomas/group size)
J>Butyl hydroperoxide
Hydroperoxy methyl oleate
Hydroperoxy methyl linoleate
6
Cyclohexene hydroperoxide
1 -Hydroperoxy-1 -vi-
nyl-cyclohex-3-ene
Cumene hydroperoxide
Lauroyl peroxide
Benzoyl peroxide
Ascaridole
0/30
1/30 (0)C
0/30
1/30 (1)
13/49 (5)
0/30
1/30 (0)'
1/30 (0)(
3/30 (1)
0/50 (low dose)
1/30 (high dose)
1/30
3/50 (low dose)
0/30 (high dose)
0/50 (low dose)
0/20 (high dose)
0/20
0/20
0/20
Summarized from the data of B. L. Van Duuren, N. Nelson, L. Orris, E. D. Palmes, and F. L. Schmitt [j. Natl. Cancer Inst. 31, 41
(1963)], L. Orris, B. L. Van Duuren, and N. Nelson CActa Un. Int. Cancrum 19, 644 (1963)], B. L. Van Duuren, L. Orris, and N.
Nelson [j. Natl. Cancer Inst. 35, 707 (1965)]," B. L. Van Duuren, L. Langseth, L. Orris, G. Teebor, N. Nelson, and M. Kuschner
[j. Natl. Cancer Inst. 37, 825 (1966)], B. L. Van Duuren, L. Langseth, L. Orris, M. Baden, and K. Kuschner [j. Natl. Cancer Inst.
3^, 1213 (1967)], B. L. Van Duuren, L. Langseth, B. M. Goldschmidt, and L. Orris [j. Natl. Cancer Inst. 39., 1217 (1967)].
See Table CXXXVII for structural formulas
The number of malignant tumors are shown in parentheses.
Not considered carcinogenic
Q
This compound was named "1-hydroperoxycyclohex-3-ene" but stated to be an a, /3 -unsaturated hydroperoxide B. L. Van Duuren, N.
Nelson, L. Orris, E. D. Palmes, and F. L. Schmitt, [j. Natl. Cancer Inst. 31, 41 (1963)3 . The correct name should be 1-hydroper-
oxycyclohex-2-ene,
-------
708
ICR/Ha Swiss mice, all three peroxides tested were found to
produce low incidences of sarcomas at the site of injection. In
agreement with skin painting experiments, cyclohexene hydroper-
oxide displayed a slight carcinogenic activity, inducing sarcomas
in 1/30 mice after weekly injections of 3.3 mg of the compound
for approximately 80 weeks. At a lower dose (0.1 mg), however,
no tumors were observed. In disagreement with the skin painting
experiments, cumene hydroperoxide and lauroyl peroxide were shown
to be slightly carcinogenic after subcutaneous injection. The
carcinogenicity of lauroyl peroxide was demonstrated by s.c.
injections of 0.1 mg of the compound but not with a higher dose
of 10 mg (168). Cumene hydroperoxide and lauroyl peroxide were
also reported to be carcinogenic after subcutaneous injection to
C57B1 mice (158). In contrast to the results in mice, none of
the four peroxides tested induced tumors after 537-542 days in
Sprague-Dawley rats (170).
In addition to the above studies, the potential carcino-
genicity of benzoyl peroxide was tested by Sharratt et_ _al_. (172)
and Hueper (173). In the former study, Sharratt et_ _al_. (172) fed
mice and rats diets made from flour treated with 2.8-2,800 ppm
benzoyl peroxide. Under the test conditions, there was no
evidence that benzoyl peroxide was carcinogenic. It should be
noted, however, that the above conclusion may not be considered
definitive because of the small number of animals used in the
study and the uncertainty of the actual amounts of unchanged
benzoyl peroxide present in the diet. In the latter study,
Hueper (173) investigated whether benzoyl peroxide, used as
-------
709
polymerization catalyst for silicone rubber, could account for
the local carcinogenicity of implanted silicone rubber. He
subcutaneously implanted a piece of benzoyl' peroxide-cured
silicone rubber into the neck of each of a group of 35 'rats and a
gelatin capsule containing 50 mg benzoyl peroxide into another
group of 35 rats. Ten local sarcomas occurred in the former
group; none occurred in the latter. Hueper (173) concluded that
benzoyl peroxide could not be implicated in the local carcino-
genicity of implanted silicone rubber. A number of distant
tumors did occur in some of the rats with encapsulated benzoyl
peroxide; however, it is not certain whether the effect was due
to benzoyl peroxide because of the lack of a proper control
group.
The observation that a crude preparation of oxidized
cholesterol produced tumors in mice (174) initiated an intensive
search for carcinogenic oxidation products of cholesterol (see
also page 53, Section 5.1.1.2.1, Vol. IIA). At least one
hydroperoxide of cholesterol was shown to be carcinogenic.
Fieser et al. (175) synthesized 6-|3>-hydroperoxy-A -cholestene-
3-one (see Table CXXXVIII for formula) by oxidation of A3
-cholestene-3-one. When given in three subcutaneous injections
of 5 mg each in sesame oil at distant intervals of time, to 32
Marsh-Buffalo mice, the hydroperoxide induced fibrosarcomas in 13
mice at the site of injection (average latency 9.6 months). No
fibrosarcomas were observed in litter-mates given the same amount
of hydroperoxide in aqueous colloidal solutions, nor in a control
group that received only sesame oil. It is interesting to note
-------
710
that, like many carcinogenic peroxides, 6-/3-hydroperoxy-A -
-cholestene-3-one is also a hydroperoxide with an c(, (i-unsat-
urated bond. There is some indirect, suggestive evidence that
^ -cholestene-3-one could be formed from cholesterol in the
body (175). In another earlier investigation, an unidentified
O A
peroxide of ,A ' -cholestadiene was reported to be noncar-
cinogenic after subcutaneous injection to mice (176; also quoted
in ref. 166). However, this study is inconclusive because of the
short duration (6 weeks) of the experiment. Peroxides were
believed at one time to be potential carcinogenic intermediates
of polycyclic aromatic hydrocarbons. Cook and Martin (177)
isolated several remarkably stable endoperoxides of 7,12-
dimethylbenz(a)anthracene formed by photo-oxidation. One of
these peroxides, 7,12-dimethyl-7,12-peroxybenz(a)anthracene was
tested and found to be noncarcinogenic in mice via subcutaneous
injection. The authors concluded that the formation of the
peroxide was unrelated to the carcinogenic activity of the
hydrocarbon.
Metabolism and Mechanism of Action. As may be expected from
the scanty literature on the carcinogenicity of peroxides, very
little is known concerning the role of metabolism in their
mechanism of action. Since peroxides are prone to homolytic
cleavage, the resulting free radicals are generally assumed to
account, at least in part, for the biological effects of
peroxides. It has been suggested (158) that peroxides may
directly catalyze or cause the depolymerization of DNA or RNA.
An aromatic peroxyacid (peroxyphthalic acid) was shown to react
-------
711
directly with adenosine and cytosine derivatives to form N-oxides
at the N, position (178). Treatment of native Hemophilus
influenzae DNA with succinic peroxide increases the resistance of
DNA to thermal .denaturation, suggesting possible cross-linking of
DNA strands by the peroxide (179). Jt_-Butyl peroxide produces
breakage and rearrangement of Vicia faba chromosomes (128).
These reactions could conceivably lead to mutagenesis; it remains
to be investigated whether carcinogenic peroxides act in a
similar manner.
The possibility that peroxides exert their carcinogenic
effect via the formation of epoxides has been suggested by Kotin
and Falk (158). Thus, lipoperoxides may undergo further reaction
leading to epoxidated fats. Also, peroxidic compounds may react
with unsaturated hydrocarbons to yield epoxides. Conceivably,
epoxides could be the reactive intermediates of some peroxides.
The finding that several carcinogenic peroxides have an c(/ [o
-double bond suggests the possibility of intramolecular epoxida-
tion of the double bond by the peroxy group. Alternatively, the
vicinal double bond may be epoxidized by an in vivo metabolic
pathway to yield a bifunctional carcinogenic intermediate.
The possible mechanism of carcinogenic action of ascaridole
has been explored by Melzer (180, 181). Unlike diepoxybutane
and p-butyrolactone (both carcinogenic) and hydrogen peroxide,
ascaridole failed to exert any inactivating effect on the
transforming ability of Bacillus subtilis DNA after incubating
for 2 days. In fact, ascaridole inhibits the reaction of DNA
components with hydrogen peroxide. Melzer (181) concluded that
-------
712
unlike hydrogen peroxide or peroxy acids, ascaridole probably
does not react directly with DNA. He suggested that: (a) an in
vivo metabolite of ascaridole may be the actual reactive
compound, or (b) ascaridole does not react with DNA but, rather,
primarily affects enzymes controlling nucleic acid synthesis and
metabolism (181). At least one aspect of the latter
possibilities was tested; at a concentration of 10 mM, ascaridole
was found to have no effect on deoxyribounuclease (180).
5.2.1.7.4 Quinones and Alloxan. Quinones are extensively
used as oxidizing agents, polymerization inhibitors, inter-
mediates for organic synthesis, dyes and tanning agents, etc.
Because of their xenobiotic properties, some quinones are also
£3 -
used as bactericidal agents, fungicides, insecticides and-
Pharmaceuticals (188). The U.S. Occupational Safety and Health
Administration (OSHA) requires that occupational exposure to 1,4-
benzoquinone, the simplest quinone, should not exceed an 8 hour
time-weighted average (TWA) of 0.1 ppm (0.4 mg/m ) in the working
atmosphere (189).
Quinones also occur naturally in a variety of plants and
arthropods (188, 190). The presence of insects in grains, flour
and other staple foods as a source of food contaminants have
received attention for some time. Certain insects secrete
substances containing various quinones which include 1,4-benzo-
quinone, 2-methyl-p_-benzoquinone and 2-ethyl-p_-benzoquinone (191-
193).. The toxic, allergenic, teratogenic and carcinogenic
activities-of the insect-secreted quinones have been the subject
of several studies (194-198). Alloxan, a quinone-related
compound, is a diabetogenic agent. :
-------
713
Insert here Text-Figure 28
A brief discussion' of various aspects of the toxicity and carcino'genicity of
these compounds is given below.
Toxic Effects. Ocular exposure to 1, 4-benzoquinone causes
eye irritation, conjunctivitis and injury of the cornea, and can
bring about blindness (199-201). Oral or subcutaneous adminis-
tration to animals of large doses of this compound results in
severe local irritation, convulsion, respiratory difficulties,
hypotension, toxic nephrosis and death due to paralysis of the
medullary centers, and asphyxia (202). A mixture of 2-ethyl- and /
2-methyl-p_-benzoquinone (4:1) in the diet at a level of 1000 ppm
is lethal to mice within 4 to 8 days. (195). The LD values of
1, 4-benzoquinone, 1, 4-napthoquinone and alloxan in several animal
species are summarized in Table CXLII. ^ _ "7a.^7fi C.XLII
Mutagenic Effects. Studies in several assay systems showed
that 1, 4-benzoquinone is not mutagenic. A single, i.p. injection
of 6.25 mg/kg 1, 4-benzoquinone did not induce dominant lethal
mutation in mice (46) . Mutagenic activity was not observed in
the sex-linked dominant (203) or recessive (204) lethal assays in
Drosophila melanogaster . Neither ' is there any evidence that 1,4-
benzoquinone induces chromosome breakage in human leukocytes
(204) or. roots of Vicia faba (128). Results of mutagenicity
... i
assays in Neurospora crassa (205) and Salmonella typhimurium
^--,.
(206) were also negative.
-------
Al loxan
Text-Figure 23
-------
Table CXLII.
Acute Toxicity of Quinones and Alloxan
Compound
1,
1,
4-Benzoquinone
4-Naphthoquinone
Alloxan
Species and route
Rat, oral
Rat, i. v.
Mouse, i. p.
Rat, oral
Guinea pig, oral
Rat, i. v.
LD5Q (mg/kg)
130
25
8. 5
190
400
300
References
(230)
.(230)
(231)
(232)
(232)
(233)
-------
714
Likewise, 1,2-napthoquinone was found inactive when tested
on several strains of Salmonella typhimurium in the Ames test,
regardless of the addition of a' 1'iver microsomal activation
system (206).
No data are available on the mutagenicity of alloxan and
other quinones.
Carcinogenicity. In 1940, Takizawa (207,208) reported the
induction of skin papillomas and carcinomas in mice painted with
1,4-benzoquinone and 1,4-napthoquinone, but not with 1,2-naptho-
quinone. Significantly higher incidence of lung adenocarcinoma
was also noted in mice treated with 1,4-benzoquinone compared to
control animals (207). These results were confirmed in later
studies by Takizawa and Sugishita (209) and by Sugishita (210)
although not by Tiedemann (211) in another strain. Also 1,4-
napthoquinone enhances tumorigenesis induced by 3-methylcholan-
threne (212) and 7,12-dimethylbenz(a)anthracene on the mouse skin
(145).
Inhalation studies with 1,4-benzoquinone repeatly demon-
strated the production of pulmonary adenocarcinomas in mice (213-
217). Local fibrosarcomas were observed in rats subcutaneuosly
injected with 1,4-benzoquinone (218) but not with 2-ethyl- or 2-
methyl-£-benzoquinone (198). In testing the carcinogenicity of
certain constituents of spermicidal contraceptives, Boyland e_t
al. (219) reported the induction of squamous carcinomas of the
cervix in 1 of the 20 mice injected intravaginally with 0.3% 2-
methyl-j3_-benzoquinone twice a week for 18 months. No such tumors
were seen in 30 untreated mice. The authors indicated, however,
-------
715
that this marginal carcinogenic activity of 2-methyl-p_-benzo-
quinone might have been due to nonspecific chronic inflammatory
changes resulting from the treatment.
The induction of tumors by alloxan has been studied in rats
by Petrea (220) as well as in the fish Clarias by Agrawal et al.
(221, 222). Rats subcutaneously injected with 175 mg/kg alloxan
in aqueous solution developed adenohypophyseal tumors after 1
year (220) . Leukemoid condition was observed in the fish after
repeated intramuscular injections of 50 mg/kg alloxan solution
(1% w/v) in citrate-phosphate buffer, pH 4.0 (221). In another
experiment, 80% of the alloxan-treated fish developed multiple
benign hepatomas after 30 days (222). No such tumors were found
in the controls. The carcinogenicity data of alloxan and other
quinones are summarized in Table CXLIII. .<=- /2.P./
Possible Mechanisms of Action. While the carcinogenicity of
some quinones has been established, very little is known about
their mechanisms of action.
Owing to the characteristic carbonyl groups and the two
unsaturated bonds, quinone compounds can undergo a wide variety
of reactions and interact with various chemical groups in
biological systems (188,223). Among the various cellular
constituents, proteins react the most readily with quinones
(223,224). The inactivation of enzymes as well as the inhibitory
effect of quinones on mitosis are believed to be due to cross-
linking, by interaction with free sulfhydryl and amino groups in
proteins (225,226). In vitro combination of thiols with quinones
has actually been described (227). Although there is evidence
-------
Table CXLIII.
Carcinogenicity of Quinoaes and Alloxan
Compound
Species and strain
Principal organs affected and route
References
1, 4-Benzoquinone
2-Methyl-jD-benzoquinone
2-Ethyl-£-benzoquinone
1, 4-Naphthoquinone
1, 2-Naphthoquinone
Alloxan
Mouse, s.tock
Mouse, Albino
Mouse,
Mouse, A
Rat, Wistar
Rat, Long-Evans
Mouse, BALB/c
Rat, Long-Evans
Mouse,
Mouse,
Rat, Wistar
Fish,
Skin, lung (topical)
No significant effect (topical)
Lung (inhalation)
Lung (inhalation)
Local sarcomas (s. c.)
None (s. c.)
Cervix (intravaginal)
None (s. c.)
Skin (topical)
None (topical)
Hypophysis (s. c.)
Liver, hematopoietic system (i. m.)
(207-210)
(211)
(213-215, 217)
(216)
(218)
(198)
(219)
(198)
(207, 208)
(207, 208)
(220)
(221, 222)
-------
716
that quinones interact in the cell with nuclear macromolecules or
with purified deoxyribonucleoproteins (223,228), studies with
C-labeled quinones suggest that, quinones do not bind to DNA
(229). This may provide the explanation for the negative results
of the mutagenicity tests with quinones (46,128,203-206). Thus,
it appears that cross-linking of proteins with quinones. in the
nucleus may be the critical step in the malignant transformation.
Except for 2-methyl-£_-benzoquinone (the carcinogenicity of
which in mice is equivocal), the data (Table CXLIII) suggest that
only those quinones that have an active unsubstituted hydrogen at
the 2-position will elicit tumors. With the presence of a
carbonyl (1,2-napthoquinone), a methyl (2-methyl-p_-benzoquinone)
or an ethyl (2-ethyl-£_-benzoquinone) group, at the 2-position,
quinones do not induce tumors in rats or mice. It is known that
chemical addition to quinones occurs first in the 2-position and
then in the 5-position, giving ultimately 2,5-disubstituted
quinones (188,223). Thus, it appears that cross-linking of
cellular proteins by quinones, triggering carcinogenesis,
requires the presence of an active hydrogen atom in 2-position.
5.2.1.7.5 C-Nitroso Compounds. C-Nitroso compounds contain
a nitroso group linked to a carbon atom in the molecule. There
is a considerable lack of information on the carcinogenicity of
C-nitroso compounds despite the fact that the majority of the
closely related N-nitroso compounds were found carcinogenic (see
Section 5.2.1.2). C-Nitroso compounds were studied initially for
the purpose of comparison with N-nitroso compounds (234) and 4-
-------
717
dimethylaminoazobenzene (235) for elucidating the relationships
between chemical structure and carcinogenicity. More recently,
C-nitroso compounds have attracted attention because of their
occurrence as drug metabolites, as reaction products of drugs and
nitrite, and as potential donors of nitroso group in transnitro-
sation reaction. The transnitrosation reaction has been
discussed in Section 5.2.1.2.5.1.7; conceivably, C-nitroso
compounds are also potential donors of the nitroso group.
C-Nitroso compounds have been identified as important in
vitro metabolic products of phentermine (236,237), amantadine
(238), and possibly a number of other primary amines (239),
although their in vitro significance remains obscure (240).
Reaction of nitrite with antipyrine in 10% acetic acid at room
temperature gives rise to a C-nitroso derivative, 4-nitroso-
antipyrine (241). A few industrial uses of C-nitroso compounds
have been reported (242, 243). Aromatic C-nitroso compounds have
been used in the vulcanization of synthetic rubber, as anti-
oxidant in lubricating oil, as intermediate in dye synthesis and
in the stabilization of halogenated dielectric materials. p_-
Nitroso-N,N-dimethylaniline is also a powerful germicide. To
date, only four C-nitroso compounds have been tested for
carcinogenicity; the names and structures of these compounds are
given in Table CXLIV. < /3J))e. £/Z//
Physical and Chemical Properties. The physical and chemical
properties of C-nitroso compounds have been extensively reviewed
by Gowenlock and Lutke (242). C-Nitroso compounds have
characteristic blue (aliphatic) or green (aromatic) color; they
-------
Table CXLIV
Structural Formulas of C-Nitroso
Compounds Tested "for Car.cinogeni.city
p-Nitroso-N,N-
-dimethylaniline
(NDMA)
p-Nitroso-diphenylamine
H3C .CH3
N=0
p-Nitroso-N,N-
-diethylaniline
(NDEA)
l,3-Dimethyl-4-
amino-5-nitroso
-uraci!
-------
718
(especially .those, with hydrogen atoms alpha to the nitrogen atom)
readily dimerize to form a colorless dimer or isomerize to the
oxime (RR'CH-N=O > RR'CH=N-OH). Oxidation of C-nitroso
compounds yields C-nitro compounds, whereas reduction gives rise
to N-hydroxylamines. The nitroso, group behaves in a fashion
similar. tof.the. carbonyl group-.,in. condensation reactions with a
variety.of compounds. The oral LDcn of p-nitroso-N,N-dimethyl-'
- «. + . i «j yj f ~ . 4, f (
aniline was quoted to be 65 mg/kg in the rat (244). For i;3-
dimethyl-4-amino-5-nitroso-uracil, the LD^Q was around 2 g/kg by
>
i.p. or >5 g/kg by s.c. injection'to rats (245). No information
is available on the mutagenicity or teratogenicity. of :C-nitroso
compounds. ''.:.-.
Carcinogenicity. The major findings of the carcinogenicity
studies of four C-nitroso compounds are summarized in Table /3.P/& £XL- V
CXLV. The study of p_-nitroso-N,N-dimethylaniline (NDMA) was
first reported by Kinoshita in 1940 (235). In a structure-
activity analysis of 4-dimethylaminoazobenzene, he cited.Harada1s
work which indicated that NDMA did not exert any-carcinogenic
effect in_the_rat. Only, "degeneration", of liver cells .was,-;-,
observed; however, no details of "the study were given. JD_-
Nitroso-N,N-dimeth'ylaniline was",retested by Goodall' et. al. '''(234)
in 19681' Albino rats of'undefined strain (16 of each" sex) ' were
given ad libitum drinking water containing 200 mg/liter"of 'the
compound for 365 days and kept until -natural death. "Eight-of. the
treated rats (5 females, 3 males) .developed tumors inrthe--.
esophagus ..and stomach; 3 (1. female, . 2 males) had; lymphomas. One
male bore liver tumors and one female had pulmonary tumors. The
-------
Table CXLV.
Carci.nogeni.city of C-NLtroso Compounds
Compound
Species and strain
Carcinogenicity (route)
References
rj-Nitroso-N, N-di-
methylaniline
(NDMA)
rj-Nitroso-N, N-di-
ethylaniline
(NDEA)
£-Nitroso-diphen-
y la mine
1, 3-Dimethyl-4-ami-
no-5-nitroso-uracil
Rat,
Rat, "partially
inbred albino"
Rat, NZR/Gd
Mouse, NZO/BlGd
Rat, NZR/Gd
Mouse, NZO/BlGd
Rat, F344
Mouse, B6C3F
1
Rat, Sprague-Dawley
None (route not specified)
Esophagus, stomach,
lymphatic system (oral)
(statistical significance
questionable due to lack
of proper controls)
Kidney, lung, lymphatic
system (oral)
Duodenum, lymphatic
system (oral)
No significant effect (oral)
Duodenum (oral)
Liver (oral)
Liver (oral)
Local sarcoma (s. c. )
None (i. p.)
(235)
(234)
(246)
(246)
(246)
(246)
(247)
(247)
(245)
(245)
-------
719
overall tumor incidence was 31%. No untreataed controls were
included in the study. However, based on the supplier's claim
that the rats were random-bred, the estimated "spontaneous" tumor
incidence would be less than 3%. The authors (234) concluded
that NDMA was carcinogenic in the rat and that its carcinogenic
potency was much less than that of cyclic N-nitrosamines. The
statistical analysis of the above study was, however, later
considered invalid after it was discovered that the rats used
contained some "partially inbred" rats (246). The study was
repeated using inbred male NZR/Gd rats and male NZO/BLGd mice
(246). The dose given was 300 mg/liter in the drinking water for
a total of 550 days for rats (total dose 1.2 g/rat) or 420 days
for mice (total dose 262 mg/mouse). There was a significant
increase in tumor incidence after NDMA treatment of both
species. The main sites of tumor induction were the lung (33%),
kidney (33%), and lymphatic system (13%) in the rat, and the
duodenum (55%) and lymphatic system (55%) in the mouse. The
carcinogenicity of NDMA has also been tested in the Carcino-
genesis Testing Program of the National Cancer Institute; the
results are, however, inconclusive and no final report has been
published.
The carcinogenicity of a higher homolog of NDMA, p_-nitroso-
N,N-diethylaniline (NDEA) has been studied by Goodall and
Lijinsky (246) using the same procedure as for NDMA. In the rat,
there was no significant increase in tumor incidence. In the
mouse, the only increase was in the incidence of duodenal
papillomas (46% in treated vs. 10% in control).
-------
720
p_-Nitrosodiphenylamine, another aromatic C-nitroso compound,
has recently been tested in a NCI bioassay study (247). The
compound is structurally related to NDMA and is used industrially
as rubber vulcanization accelerator and as intermediate in the
manufacture of dyes and Pharmaceuticals. Fischer 344 rats and
B6C3F, mice were used in the study. The compound was adminis-
tered in the drinXing water at two dose levels: 2,500 or 5,000
ppm for rats, and 4,254 or 9,000 ppm for mice. In the male
mouse, there was a significant increase in the incidence of
hepatocellular carcinomas in the low dose group. Male mice in
the high dose group also developed liver tumors; however, due to
the large number of early deaths in the dosed mice, no valid
statistical conclusion could be made. In the male rat, there was
a significant positive association between the dose administered
and the incidence of a combination of hepatocellular carcinomas
and neoplastic nodules. The sex of the animals apparently played
a major role; there was no evidence that the compound was
carcinogenic in either female rats or mice (247). It is
interesting to note that N-nitrosodiphenylamine, the N-nitroso
isomer of ^-nitroso diphenylamine was previously reported to be
noncarcinogenic (248, 249) but was recently found carcinogenic in
the rat (but not in mice), with the urinary bladder as the
principal carcinogenicity target organ (250).
1, 3-Dimethyl-4-amino-5-nitroso-uracil, a C-nitroso
derivative of uracil has been tested for carcinogenicity in
Sprague-Dawley rats by Schmahl (245). The compound did not exert
any carcinogenic effect after a series of intraperitoneal
-------
721
injections (15 x 0.2 g/kg) in oil suspension. After subcutaneous
injections (8x2 g/kg), however, 6 out of,the 40 treated rats
developed local sarcomas at the ..site of application (245). It is
not known whether the difference in carcinogenic activity was due
to the difference in the dose or the route of administration.
Several derivatives of uracil such as l-nitroso-5,6-dihydrouracil
(251) and uracil mustard (see Section 5.2.1.1.1) are potent
carcinogens. It appears .that the carcinogenicity of these
compounds is at least in part related to the fact that,'being
analogs of the naturally occurring pyrimidine, they may be
readily incorporated into nucleic acids and thereby initiate the
process of carcinogenesis.
In addition to the above four compounds, N,4-dinitroso-N-
methylaniline, which is both a C- and N-nitroso compound, was
reported to be "slightly carcinogenic" in female rats in a
feeding experiment (252); however, no details of the study are
available.
The role of metabolism in and the mechanism of action of
carcinogenesis by C-nitroso compounds have not been studied.
Since C-nitroso compounds lack the structural features required
for c(-C-hydroxylation, its mechanism of activation is obviously
different from that of N-nitroso compounds. j>-Nitroso-N,N-
dimethylaniline has been shown to be reduced by rat liver cytosol
in the presence of NADPH (253). The NADPH-dependent reduction of
1-nitrosoadamantane by rabbit liver microsomes has also been
demonstrated (254). In the latter study, a N-hydroxylamine
derivative has been shown to be a major end product. The
-------
722
relationship of these metabolic reactions to carcinogenesis is
not known. It is relevant, however, that N-hydroxylation has
been established as the first step in the metabolic activation of
aromatic amines to the ultimate carcinogen (see Section 5.1.4)
and it is conceivable that a similar mechanism operates here. As
-- <2 *7
depicted in Figure 37, NDMA and related compounds could be \7 '
reduced to N-hydroxylamine derivatives by C-nitroso reductase,
and acetylated? they could then generate, through the resonance
effect of the dialkylamino group, carbonium ion reactive
intermediates. Another possible mechanism of action of C-nitroso
compounds is through the formation of N-nitroso compounds by
transnitrosation. Alternatively, C-nitroso compounds may be
oxidized to C-nitro compounds, which have been shown to nitrosate
secondary amines such as morpholine (255). Realization of the
environmental significance of the C-nitroso compounds, points to
the need of further investigations in this area.
5.2.1.7.6 Hexamethylbenzene , Hexamethy 1-Dewar-benzene and
Hexaethylidenecyclohexane. These three cyclic hydrocarbons
- - - -< T^e CKL Ml
(Table CXLVI) are of interest because of their structural
similarity and chemical reactivity (256-258). Except hexa-
methylbenzene, they are not commonly encountered in the
environment and only very limited toxicological studies have been
carried out on these compounds; nonetheless, their carcinogenic
activities have been tested in mice or rats, as summarized in
Table CXLVI I. ^- -- TsCble CXLV//
Hexamethylbenzene is used in the industry as a solvent, as a
constituent of gasoline, and in organic synthesis. Some of its
-------
H
=N
=NX + OAc
Figure 37
-------
Table CXLVI
Structural Formulas of Three C/ Cyclic
^-"-.. v /--.
Hydrocarbons Tested for Carcinogenicity
CHj H,C
H3C CH3
Hexamethylbenzene Hexamethyl-Dewar- Hexaethylidene-
-benzene -cyclohexane
-------
Table CXLVII.
Carcinogenicity of Hexamethylbenzene, Hexamethyl-Dewar-benzene and Hexaethylidenecyclohexane
Compound
Species and strain Principal organs affected and route References
Hexamethylbenzene
Mouse, SaB
Skin (topical)
None (s. c.)
Mouse, Swiss Bladder (implantation)
Hexamethyl-Dewar-benzene Mouse, SaB
Liver, hematopoietic tissue (s. c.)
None (topical)
(260)
(260)
(261)
. (260)
' (260)
Hexaethy lidenecyclohexane
Rat, BD
None (s. c.)
(262)
-------
723
physico-chemical properties and toxic effects have been described
(259). Like other alkylbenzenes, direct contact of hexamethyl-
benzene with the skin causes irritation, vasodilation and
erythema. Hyperemia and hemorrhage in various tissues and organs
occur when animals are dosed with hexamethylbenzene intragas-
trically, subcutaneously or intraperitoneally. Nine of 10 rats
given orally 0.2 ml (1:1 v/v in olive oil) of the compound died
of respiratory failure due to pneumonitis with pulmonary edema
and hemorrage. (259).
The carcinogenic action of hexamethylbenzene as well as
hexamethyl-Dewar-benzene has been studied in mice by Dannenberg
_et_ a_l_. (260). One skin papilloma was seen in 15 mice painted
with hexamethylbenzene (10 drops as a 0.4% acetone solution)
2x/week for over a year. However, subcutaneous injection of the
compound once a month for 4 months at doses of 5 mg (in 0.1 ml
tricaprylin) did not induce tumor development. Unlike hexa-
methylbenzene, 1 leukemia and 1 liver carcinoma were observed in
15 mice injected subcutaneously with hexamethyl-Dewar-benzene; no
tumors were seen in mice treated with hexamethyl-Dewar-benzene
via skin painting.
Tested as a potential carrier medium for pellet implantation
studies in mice, hexamethylbenzene was also found to be
associated with the development of a low incidence (11%) of
bladder carcinomas (261). It was suggested that the prolonged
presence of the chemical in the bladder, perhaps as a mechanical
irritant, had a promoting effect on the genesis of bladder
carcinomas (261).
-------
724
In the light of these preliminary findings of weak
carcinogenicity, as well as the presence of the toxic moiety of
benzene structure in the molecules, both .hexamethylbenzene and
hexamethyl-Dewar-benzene should be considered as hazardous
chemicals. The mechanism of the carcinogenic action of the two
compounds is unknown. It is tempting to speculate that cross-
linking of nuclear macromolecules with epoxides or quinones
derived from hexamethylbenzene and hexamethyl-Dewar-benzene may
be the mechanism of action. Because of the bulkiness of the
structure, epoxide or quinone formation from hexaethylidenecyclo-
hexane seems less likely. Hexaethylidenecyclohexane appears to
be devoid of carcinogenic activity as no tumors developed in rats
injected subcutaneously with the substance at doses of 25 mg/kg
or 50 mg/kg monthly for 11 months (262).
5.2.1.7.7 Thalidomide, Phthalate Esters and Saccharin.
Thalidomide, phthalate esters and saccharin are three types of
synthetic compounds that have great impact on modern society.
Thalidomide was once the favorite sleeping pill in West Germany
and the drug of choice as an antiemetic for pregnant women but
was eventually found to be a devastating teratogen. Phthalate
esters are the most commonly used plasticizers; they are widely
distributed throughout our immediate living environment and are
one of the most widespread pollutants. Saccharin is a controver-
sial artificial non-nutritive sweetener that has attracted
enormous attention in recent years because of the proposed
banning of the compound by the U. S. Food and Drug
Administration. These three types of compounds have similar
-------
725
Taife CXLV/t/
chemical structures (Table CXLVIII) and are therefore grouped
together in this Section. A vast number of articles on the
various toxic effects of these, compounds has been published in
the last three decades; a comprehensive discussion below focuses
mainly on their carcinogenicity.
5.2.1.7.7.1 Thalidomide. Thalidomide [known chemically as
N-(2,6-dioxo-3-piperidyl) phthalimide or 3-phthalimidoglutar-
imide] is a synthetic drug that brought tragedy to many families,
established the necessity of adequate testing of drugs and
delineated the field of chemical teratology. Thalidomide was
first synthesized in 1953 by a West German company and found to
be a seemingly ideal hypnotic-sedative in humans. Under the
trade name "Contergan", it was introduced to the German market in
1956 and was considered so safe that it was at one time available
without a prescription. It was approved for use in northern
Europe, England, Canada and many other countries but not in the
United States. Being effective in combating nausea due to
pregnancy, thalidomide was used by many pregnant women.
Beginning in the early 1960's, an epidemic of extremely rare form
of infant deformity known as phocomelia (Greek for "seal limb")
swept through countries where thalidomide was marketed. The
affected infants had arms so short that their hands almost
extended directly from their shoulders. It was not long before
the epidemic became linked to the drug. Before the drug could be
completely withdrawn, several thousand infants were affected.
The teratogenicity of thalidomide has subsequently been
demonstrated in some (but not all), experimental animal
-------
Table CXLVIII
Formulas of N-Arylphthalimide-related
Structures Tested for Carcinogenic Activity
Tholidomide
Phthalate ester
(R,,R2=alkyl)
Saccharin
-------
726
species. Only a very brief review of this subject is presented
in this section, while the potential carcinogenicity and
mutagenicity of thalidomide are.reviewed in detail.
Thalidomide is a solid with a melting point of 269-271°C.
It is sparingly soluble in water but soluble in dioxane,
dimethylformamide or pyridine (243). Chemically, it may be
considered as a derivative of glutamic acid or as a N-substituted
derivative of phthalimide. Thalidomide is unstable in aqueous
solution and may spontaneously hydrolyze, particularly in
alkaline solution, to breakdown products such as 4-phthalimi-
doglutaramic acid, 2-phthalimidoglutaramic acid and c(-(o-carbo-
xybenzamido) glutarimide. A small portion of these products may
be further degraded to phthalic acid, glutamine and glutamic acid
(263, 264).
As may be expected, thalidomide has a very low acute
toxicity in humans. Some would-be suicides who took large doses
of thalidomide reportedly survived without harm (265). The acute
LDcQ in mice by oral administration is of the order of ,2.0 g/kg
(266). Long-term use of the drug may lead to peripheral
neuropathy affecting mainly sensory and motor nerves (rev.,
267). These neurotoxic effects could not be consistently
reproduced in animal studies (267). Historically, the neuro-
toxicity of the drug was the main reason that alerted the
e
inadequacy of testing and prevented its distribution in the U.S.
(265, 268).
Teratogenicity. The teratogenicity of thalidomide in humans
is well documented (265, 269-271). The most susceptible period
-------
727
is between the 4th and 7th week of pregnancy. The teratogenicity
of this drug in experimental animals was first demonstrated by
Somers (272) and subsequently studied by numerous
investigators. A variety of publications (271, 273-276) have
reviewed this subject. It is important to point out that
thalidomide has a variable effect in different animal species and
strains. The teratogenic potency of the drug follows the
order: monkeys » rabbits > mice _>_ rats. In fact, the latter
two species are so insensitive that they are considered
refractory to thalidomide by many investigators. Among other
mammalian species tested, dogs (277), cats (278), armadillos
(279) and pigs (280) have also been found to be susceptible to
thalidomide although relatively few studies have been carried out
and most of the teratogenic responses in these animals do not
resemble those of humans. The monkey is the only species that
responds to thalidomide in a similar manner as humans (275, 276,
281) .
The relationship between chemical structure and the
teratogenic potency of thalidomide derivatives has been
extensively studied (264, 266, 282-284), mostly in the rabbit as
the test species. An excellent review of this topic has been
published (264). Among the various derivatives tested, only a
few are definitely teratogenic suggesting that stringent
structural rules determine this activity. Elimination,
saturation or bridging of the benzene ring of the phthalimide
moiety leads to complete loss of activity, suggesting that an
aromatic flat six-membered ring with 1, 2-dicarboximide
-------
728
(phthalimide) structure is required. In one recent study (using
mice), replacement of the benzene ring with a pyridine ring was
found to enhance the teratogenicity of the compound (285). Very
few N-substituted phthalimides are teratogenic; N-substitution
with a glutarimide ring is preferred for maximum activity.
However, the glutarimide ring is apparently not indispensable; N-
substitution with methylated isoglutamine (283) or an additional
phthalimide ring (Gillette and Schumacher, cited in ref. 264)
yields compounds with teratogenic activity. Jonsson (284)
proposed several interesting hypotheses concerning the structure-
activity relationships.
Mutagenicity. Very little information is available on the
mutagenicity of thalidomide. Kennedy et al. (286) have tested
the mutagenicity of the drug by the dominant lethal test in male
mice and by the host-mediated assay in rats using a histidine
auxotroph of Salmonella typhimurium (strain not specified) as the
test organism. In both tests, thalidomide, administered at a
dose of 0.5 or 1.0 g/"kg, failed to elicit any significant
mutagenic activity. In a recent abstract from Gordon and Blake
(287), the mutagenicity of thalidomide has been evaluated by the
Ames Salmonella test using strains TA-98, 100, 1535 and 1537. At
doses of 50-1000 g/plate, thalidomide was nonmutagenic either in
the absence or presence of a metabolic activation system which
consists of a NADPH generating system and 9000 g (S-9) liver
supernatant from Aroclor-pretreated rats. Negative results were
also obtained if the rat S-9 was replaced by: (a) maternal S-9 of
Aroclor-pretreated pregnant mice or rabbits, (b) by liver
-------
729
homogenate from fetal rabbits or (c) a human abortus. This
indicates that difference in species or developmental stage is
not a factor in the lack of activity. Preliminary cytogenetic
studies on human leukocytes also failed to demonstrate any
thalidomide-induced increase in chromosomal aberrations or sister
chromatid exchange (287). Thus, it appears that there is no
evidence that thalidomide is mutagenic.
Carcinogenicity. The potential carcinogenicity of
thalidomide has not been thoroughly investigated. Only four
studies have been described. Roe and Mitchley (288) reported
that thalidomide was weakly carcinogenic by subcutaneous
injection to mice. Among the 20 male mice of the Chester Beatty
strain, receiving 57 weekly subcutaneous injections of 15 mg
thalidomide, only two developed local sarcomas. No such
injection-site tumors were observed in arachis oil-treated
controls. In another experiment, three mice were given daily
subcutaneous injections of 7.5 mg thalidomide for 220 days; only
one of these mice developed a spindle cell sarcoma at the
injection site after 12 months. To test whether thalidomide may
have carcinogenic effect in the progeny of treated mice, Roe and
coworkers (289) gave 10 female mice daily subcutanous injections
of 7.5 mg thalidomide before mating and during gestation. The
tumor incidence of their offspring was compared to that of
offspring of untreated parents or of offspring whose fathers
received thalidomide. No significant difference was observed.
It should be noted, however, that malformations were also not
observed in this strain of mice. The lack of transplacental
-------
730
carcinogenic effect of thalidomide was also reported by DiPaolo
(cited in ref. 274); details of the study were not given. The
only other positive evidence for the carcinogenicity of
thalidomide was provided by Marin-Padilla and BenirschXe (279)
using the exotic North American 9-banded armadillo, Dasypus
Novemcinctus mexicanus. (The armadillo is a mammal that is
unique in its ability to produce single-ovum-derived multiple
offspring; it has hemochprial placenta and fetal endocrine system
closely resembling those of humans). Nineteen captured
armadillos were used in the study; they were given daily oral
administration of 100 mg/kg thalidomide at various stages. One
of these animals developed a highly unusual, metastatic
choriocarcinoma in the placenta. This anrmal also had a
malformed embryo with asymmetric phocomelia. The induction of
the tumor was attributed to thalidomide treatment because the
spontaneous occurrence of such a tumor had never been observed by
the authors (279) in their studies of over 200 pregnant
armadillos. Although this study cannot be considered as
conclusive, the coincidence of tumor induction and malformation
may be significant.
There are two recent case histories of coincidence of
thalidomide-type malformation and tumorigenesis in humans. Teppo
_et_ _al_. (290) reported that a 16-year-old Finnish male with
thalidomide-type malformation at birth subsequently developed
osteosarcoma. He was born at a time when thalidomide was in use
in Finland. The mother was treated with sedatives during early
pregnancy to combat nausea, although there was no firm record of
-------
731
the use of thalidomide. Miller et al. (291) presented a case
history of a 15-year-old male with a lymphpma of high
malignancy. This patient also .had thalidomide-induced
malformation. It is obvious that two cases are insufficient to
establish whether the association of thalidomide-induced
malformation with malignancy is purely coincidental or suggestive
of the carcinogenic effect of thalidomide in humans. However, in
view of the long latency period of chemical carcinogens in
humans, if thalidomide is carcinogenic, the beginning of the
appearance of such cases could be expected in the 1980's (290,
291).
The possible modifying effect of thalidomide on the
carcinogenicity of a known carcinogen has been investigated by
Miura et al. (292). Female Swiss mice were given topical
application of 3-methylcholanthrene (3-MC) together with oral or
intraperitoneal administration of 25 mg thalidomide 5 days a week
for 4 weeks. A significant potentiation of 3-MC carcinogenicity
by intraperitoneal administration of thalidomide was observed.
Mice given 3-MC plus thalidomide intraperitoneally developed
approximately twice as many skin papillomas as controls given 3-
MC and solvent. Oral administration of thalidomide has a slight
and statistically insignificant effect. The lack of effect by
oral route was attributed to the poor gastrointestinal absorption
of the drug. The mechanism of the potentiation of 3-MC by
thalidomide is not known. Immunosuppression was suggested as a
possible mechanism, but there is no evidence that thalidomide
suppresses serum antibody production (292).
-------
732
The role of metabolism (rev., 264) in the activation of
thalidomide remains obscure. There is no convincing evidence
that metabolism yields teratogenic (264) or mutagenic (286, 287)
intermediates. An in vivo binding study by Bakay and Nyhan (293)
revealed that the binding of thalidomide to macromolecules of rat
fetuses differs markedly from the binding of classical.chemical
carcinogens. There is no evidence of covalent binding of
thalidomide to nucleic acids. Most of the thalidomide and its
metabolites appear to bind preferentially to the highly acidic "B
class" proteins. The possible mechanisms of teratogenic action
of thalidomide have been critically reviewed by Jonsson (264) who
concluded that there is no sufficient evidence to establish that
thalidomide exerts its action by (a) acting as an acylating
agent, (b) affecting nucleic acid metabolism, (c) interfering
with folate and glutamate metabolism, (d) uncoupling oxidative
phosphorylation, or (e) suppressing .the immune system. He
hypothesized (284) that thalidomide, because of its molecular
size and structural similarity to purine bases, may initiate
teratogenic action by intercalating between base-pairs of the DNA
double helix. It should be noted that intercalation of DNA is
also a potential mechanism of initiation of mutagenesis or
carcinogenesis.
5.2.1.7.7.2 Phthalate Esters and Related Compounds.
Phthalate esters are esters of benzene dicarboxylic acid. The
term "phthalate" is generally used to denote the ortho isomer
(1,2-benzene dicarboxylate). The meta and para isomers are named
isophthalate and terephthalate, respectively. Phthalate esters
-------
733
are the most commonly used plasticizers (which Impart
flexibility, permanence and other desired .properties to plastics)
in the production of polyvinyl -chloride (PVC) resins. The annual
production of phthalate esters in the United States in 1977
amounted to 1.2 billion pounds. The most widely used phthalate
ester was di-(2-ethylhexyl)phthalate which accounted for about
1/3 of the total phthalate production. The world production of
phthalate esters is about 3-4 times the U.S. production.
Polyvinyl chloride resins, which may contain as much as 60% (by
weight) phthalate esters, are widely used in building
construction, home furnishings, appliances, automobiles, water-
proof apparels, food coverings, medical devices and numerous
other products (294). In addition, some phthalate esters are
used as solvents, fixatives, wetting agents, insect repellants,
and lubricating oil, and may be present in insecticides and
cosmetic products. It has been estimated that over 3 million
U.S. workers are occupationally exposed to phthalate esters
(295). Because of the widespread use of plastic products and the
relatively high leachability, phthalate esters have become one of
the most ubiquitous environmental pollutants (296-298).
Phthalate esters have been detected in the air (2.96, 298, 299),
water (296, 298, 300-303), soil (298, 304-306), fish (307, 308),
and various foodstuffs packed in plastic containers (296, 297,
309). The 1970 report by Jaeger and Rubin (310) of the presence
of phthalate esters in blood stored in plastic bags and in
tissues of patients who had received blood transfusions spurred
great concern about the potential health hazard of human exposure
-------
734
to phthalate esters. A conference on the subject sponsored by
the National Institute of Environmental Health Sciences was held
in 1972 (311). Many reviews on. various aspects of phthalate
esters have subsequently appeared (295-298, 312-316). In this
section, the literature of phthalate esters is focused on the.
carcinogenicity studies and recent new findings.
Physical and Chemical Properties. The physical and chemical
properties of phthalate esters have been extensively reviewed
(295, 296, 312, 317). Most phthalate esters are lipophilic,
colorless liquids of low volatility, medium viscosity and high
boiling point; they are very poorly soluble in water but are
soluble in most organic solvents. The water solubility of
phthalate esters may be enhanced by the presence of lipoprotein-
containing materials (e.g., in the blood). Phthalate esters may
be dispersed as micelles in nonionic surfactants, such as
polysorbate-80, with sonication or with heat (318).
Toxicity. Phthalate esters are generally considered to have
low acute toxicity to mammals (296, 312, 314, 316, 319-321). The
acute LD^Q values range from 10 to 49 g/kg for di-(2-
ethylhexyl)phthalate and from 3.1 to 21 g/kg for di^n.-butyl
phthalate in several animal species by various routes of
administration (319). In general, phthalate esters with shorter
alkyl side-chains are more toxic than compounds with longer side-
chains (320). Some of the acute toxic effects of phthalate
esters may be attributable to contamination with phthalic
anhydride which has an acute LD5Q of 0.8-1.6g/kg in rats (322)
and 2.2 g/kg in mice (323).
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735
Mutagenicity. The mutagenicity of various phthalate esters
has been tested in several test systems using bacteria, yeasts,
mammalian cells or mice as indicator organisms. Di-_n_-butyl
phthalate was found to be nonmutagenic in Bacillus subtilis,
Sscherichia coli (324), and Salmonella typhimurium (324-326).
Di-(2-ethylhexyl)phthalate also failed to exert any mutagenic .
effects in the Salmonella system either in the presence or
absence of metabolic activation (325, 326). Mono-(2-ethylhexyl)-
phthalate, a Xnown metabolite of di-(2-ethyl-hexyl)phthalate, was
reported to be mutagenic in _E_. coli suggesting that the monoester
may be an activated metabolite of the diester (327). In the
Salmonella system, however, there is no evidence that the
monoester is mutagenic (326). It is interesting to note that two
short-chain phthalate esters (dimethyl and diethyl) were found to
elicit a dose-dependent mutagenic effect in the absence of
metabolic activation in the Salmonella system with TA-100 (a base
substitution mutant) as the tester strain; the inclusion of liver
microsomes actually eliminated the mutagenic effect of the two
diesters (326), The mutagenicity of di-_n_-butyl phthalate has
also been tested in yeast cells (Saccharomyces cerevisiae strain
XV 185-14C); no positive response was observed even in the
presence of metabolic activation (328). In cultured Chinese
hamster cells, neither di-(2-ethylhexyl)phthalate nor di-_n_-
butylphthalate was capable of inducing chromosome aberrations
when tested without metabolic activation (324, 329, 330).
Increases in the incidence of sister chromatid exchange were
observed after exposure of Chinese hamster cells to either di-(2-
-------
736
ethylhexyl)phthlate or di-jv-butylphthalate; the effects were,
however, not dose-dependent (330). A positive response in the
dominant lethal assay was reported; treatment of male ICR mice
with single i.p. doses (equivalent to 1/3, 1/2, or 2/3 of the
acute LD5Q) of di-(2-ethylhexyl)phthalate shortly before mating
resulted in significant reduction in the number of implants/preg-
nancy, and enhancement of early fetal deaths (331).
Teratogenicity. The teratogenicity o'f phthalates has been
demonstrated in rats, mice and chickens. Singh et al. (332)
found that a variety of phthalate esters [including di-_n_-butyl
and di-(2-ethylhexyl)] were teratogenic in the rat, inducing
gross and skeletal abnormalities such as absence of tail,
anophthalmia, twisted hind legs, elongated and fused ribs, and
abnormal skull bones. In general, the teratogenic potency
correlated well with the water solubility of the compound.
Dimethoxyethylphthalate, the most soluble compound of the group,
was the most potent teratogen, whereas di-(2-ethylhexyl)-
phthalate, the least soluble compound, was very weakly
teratogenic. External and skeletal malformations were also
observed in the offspring of mice given a single dose (equivalent
to 1/4 the acute LD5Q) of di-(2-ethylhexyl)phthalate on day 8 of
gestation (327). Exposure of chick embryos to di-_n_-butyl-
phthalate and a number of other phthalates led to increased
incidence of neurological defects and other malformations (333).
Carcinogenicity. The carcinogenicity of phthalate esters
does not seem to have been adequately tested. Chronic toxicity
studies of several phthalate esters carried out prior to the 1972
-------
737
Conference of Phthalate Esters were reviewed by Krauskopf
(321). Although none of these studies gave any indication of
carcinogenicity of any of the phthalate esters, it should be
noted that many of, these studies did not have sufficiently long
duration, maximally tolerated doses or sufficient surviving
animals to critically assess the carcinogenic potential of
phthalate esters. Several phthalate esters and related compounds
have been or are being tested in the Carcinogenesis Bioassay
Program of the National Cancer Institute. At least one compound,
the widely used di-(2-ethylhexyl)phthalate, has been found to be
carcinogenic in a preliminary study. These new data are
discussed along with the earlier long-term studies in the
following paragraphs. A summary of the major findings of these
studies is presented in Table CXLIX. ^ \&b~le-
Di-n_-butyl phthalate was tested in two studies. Smith (334)
fed Sorague-Dawley rats diets containing 0.01, 0.05 and 0.25% di-
_n_-butyl phthalate for one year and observed no apparent adverse
effects. When the dietary level was increased to 1.25%,
approximately 50% of the animals died in one week but the
remainder grew normally as compared to untreated controls. A
similar finding was reported by Lefaux (cited in ref. 321).
The result of a chronic toxicity study of di(2-ethylhexyl)-
phthalate was first reported in a technical brochure of W.R.
Grace & Co. (cited in ref. 321); no adverse effects were observed
in rats fed a diet containing 0.13% of the compound for 2
years. Carpenter _et_ a_l_. (335) gave groups of 64 Sherman rats (32
of each sex) diets containing 0.04, 0.13 and 0.4% di-(2-
-------
Table CXLIX.
Carcinogenicity of Phthalate Esters and Related Compounds
Compound
Di-n-butyl phthalate
Di-(2-ethylhexyl)
phthalate
Phthalamide
Phthalic anhydride
Dimethyl terephthalate
Species and strain
Rat, Sprague-Dawley
Rat, unspecified
Mouse, B6C3FJ
Rat, unspecified
Rat, Sherman
Rat, Wistar
Rat, F344
Guinea pig
Dog
Mouse, B6C3F
Rat, F344
Mouse, B6C3F
Rat, F344
Mouse, B6C3F
Rat, F344
Organ affected
c
None
c
None
Liver
None
None
None
Liver
c
None
c
None
None
None
None
None
None
None
References
(334)
[Lefaux (1
cited in ref.
(337)
[Grace & Co.
cited in ref.
(335)
(336)
(337)
(335)
(335)
(340)
(340)
(339)
(339)
(338)
(338)
968)
321]
(1948)
321]
All tested by oral administration
Cocker spaniels and terriers
c
The duration of these experiments was one year only.
-------
738
ethylhexyl)phthalate for a maximum of 2 years. Nine of the
treated rats developed benign tumors; this tumor incidence was
not considered by the investigators to be significantly different
from that of untreated controls. About 80 first.filial
generation (F.^) rats were also maintained for 1 year on a diet
containing 0.4% of the compound; no significant increase in tumor
incidence was observed. Furthermore, Carpenter et al. (335)
tested the effect of oral administration of di-(2-ethylhexyl)-
phthalate in 93 guinea pigs and 8 dogs for 1 year and observed no
significant adverse effects. The apparent lack of carcino-
genicity was confirmed by Harris et al. (336) using Wistar-strain
rats. Groups of 86 weanling rats receiving di-(2-ethylhexyl)-
phthalate for up to 2 years at dose levels of 0.1% and 0.5% did
not develop tumors attributable to the administration of the
compound. It should be noted, however, that high mortality rates
were observed in both the control and treated groups in this
study. The carcinogenicity of di-(2-ethylhexyl)phthalate has
recently been re-tested by the National Cancer Institute (NCI).
A preliminary report (337) of the study, revealed that, in
contrast to earlier findings, the compound is a liver carcinogen
in both F344 rats and B6C3F, mice. In treated rats (receiving
1.2% of the compound in the diet), the incidence of hepato-
cellular carcinomas was 5/50 in males and 8/50 in females,
compared to 0/50 in untreated controls (significant at p=0.028
and 0.0028, respectively). In female mice receiving diets
containing 0.6% and 0.3% of the compound, the incidence of
hepatocellular carcinomas was 17/50 and 7/50, respectively (both
-------
739
significantly different from controls with 0/50). Male mice
receiving the 0.6% level also showed a significant increase of
liver tumors (19/50 treated vs. 10/50 control; p=0.037). At the
lower level of 0.3%, the incidence was not significantly
different from the control groups. Thus, the new study provides
^ c
strong evidence of the carcinogenicity of di-(2-ethylhexyl)--"'" " ^
phthalate and raises serious doubts about the safety of phthalate
esters as a group. Evaluation of the carcinogenic potential of
other phthalate esters is urgently needed.
In addition to phthalate esters, the carcinogenicity of
three related compounds (phthalamide, phthalic anhydride, and
dimethyl terephthalate) has recently been tested by the NCI
(338-340)7""
vThese compounds are of some industrial importance. Fischer 344
rats and BGCSF^ mice were used in these studies. The duration of
experiments ranged from 103 to 106 weeks. For phthalamide the
dose levels were 2.5 and 5.0% for male mice, 0.65, 1.25 and 2.5%
for female mice, 1.5 and 3.0% for male rats, and 0.5 and 1.0% for
female rats. For phthalic anhydride, the dose levels were 1.6
and 3.2% for male mice, 1.2 and 2.4% for female mice, and 0.75
and 1.5% for rats. For dimethylterephthalate, the dose levels
were 0.25 and 0.5% for mice and rats (the high dose was not the
maximajLly Jiolerated dose). None of these compounds were
carcinogenic and these negative findings shed additional light on
-------
740
the structure-activity relationships of the phthalate esters. It
is possible that the aromatic ring per se is not directly
involved in carcinogenesis, but it is the nature of the ester
groups, their molecular flexibility, and positioning on the
benzene ring which determine the carcinogenic potency.
The role of metabolism in the activation of phthalate esters
is not clearly understood. Metabolic studies of di-(2-ethyl
hexyDphthalate in the rat have established that the diester is
substantially hydrolyzed after ingestion to the monoester, mono-
(2-ethylhexyl)phthalate, by nonspecific lipases in the pancreatic
juice (341-343). Side-chain oxidation of the monoester then
occurs with the formation of CJ- and (ol-l)-oxidation products
(alcohols, ketones or acids) as the principal urinary metabolites
(342, 344). Biochemical and histochemical studies indicate that
the hepatic effects of di(2-ethylhexyl)phthalate are reproducible
by mono-(2-ethylhexyl) phthalate (but not by phthalic acid or 2-
ethylhexanol) suggesting that the partial hydrolysis of the
diester to the monoester may be a necessary step for the
manifestation of the effects of di-(2-ethylhexyl)phthalate
(345). In vitro hydrolysis studies of a variety of phthalate
esters by hepatic and intestinal preparations from various animal
species (including humans) reveal that monohydrolysis of
phthalate esters appears to be a common metabolic step (346).
The relevance of monohydrolysis to the potential carcinbgenicity
of phthalate esters is not known. Yagi et_ _al_. (327) have
reported that mono-(2-ethylhexyl)phthalate is mutagenic in _E_.
coli; however, this could not be confirmed in the Salmonella
-------
741
system (326). On the other hand, Rubin _et_ al_. (326) have
demonstrated that dimethyl and diethyl phthalates are mutagenic
in the Salmonella system; the inclusion of liver microsomes
actually abolishes the mutagenicity of the diesters. Obviously,
further studies are needed. The chemical structure of phthalate
esters could allow the formation of arene oxide as reactive
intermediate; however, there is no experimental evidence that the
epoxidation of the aromatic ring actually occurs.
5.2.1.7.7.3 Saccharin. Saccharin [1,2-benzisothiazol-3(2H)-one
1,1-dioxide; see Table CXLVIII for chemical structure] is a
controversial artificial nonnutritive sweetener that has been
used in the U.S. for more than 70 years. It was first syn-
thesized by Remsen and Fahlberg in 1879 for use as an anti-
septic. Being 200-700 times sweeter than sugar, saccharin was
recognized as a potential artificial sweetener. The reduced
sugar supply in World War I first brought about a demand for this
artificial sweetener. The demand has since been escalating, as
increasing concern for personal health and appearance propels
many people toward weight reduction or maintainance programs.
Since the banning of cyclamate in 1969, saccharin has become the
only artificial sweetener allowed in the U.S. It is now present
in dietetic soft drinks, tabletop sweeteners, processed foods,
cosmetics (toothpaste, mouthwash, lipstick), Pharmaceuticals
(pill coating), smokeless tobacco products and cattle feeds. The
total U.S. production and import amounted to 7.6 million pounds
in 1977. The committee for a Study on Saccharin and Food Safety
Policy of the National Academy of Sciences (347) has recently
-------
742
estimated that approximately 50-70 million Americans (including
about 4/5 of the approximately 5 million diabetics and 1/3 of
children under 10) consume products containing saccharin. .The
estimated average daily intake is 25-155 mg for nondiabetics and
54-173 mg for diabetics. On a weight basis, children under 10
consume the highest amount of saccharin of any age group. It
should be noted that although saccharin is widely claimed to be
beneficial in the prevention or treatment of obesity, diabetes
and dental caries, the Committee (347) could find no unequivocal
scientific evidence to accept (or rule out) these claims.
Saccharin may, however, provide possible benefits in making
dentrifices and Pharmaceuticals more palatable (to promote proper
use) and in fulfilling the psychological need or reliance of some
consumers on the nonnutritive sweetener.
The safety of saccharin has been a question of controversy
since its introduction into the market. It was first banned from
foods in the U.S. in 1912. The ban was lifted during World War
I. In 1958, saccharin was approved for use as a food additive in
the U.S. and was included in the GRAS (generally recognized as
safe) list. Since the banning of cyclamate, the safety of
saccharin has been questioned. In 1972, with new evidence of
possible carcinogenicity, the U.S. Food and Drug Administration
(FDA) removed saccharin from the GRAS list. In 1977, prompted by
the results of three independent two-generation studies which all
showed significantly higher incidences of bladder tumors in male
rats exposed to high doses of saccharin jLn_ utero, from milk and
through ingestion, the Canadian government banned the use of the
-------
743
sweetener in foods. The U.S. FDA also announced its intention to
ban saccharin but the proposed ban has been suspended due to
public pressure and awaits further evaluation. Saccharin has
been the subject of many reviews and comments in recent years
(e.g., 348-350). The National Academy of Sciences has reviewed
the safety of saccharin several times (347, 351-354). In this
Section, a brief review on saccharin is presented with emphasis
on carcinogenicity.
Saccharin is mainly manufactured by two different pro-
cesses: the older Remsen-Fahlberg process which uses toluene or
£_-toluenesulfonamide as the starting material and the newer
Maumee process which starts with either phthalic anhydride or
anthranilic acid and yields purer product. Saccharin is
generally prepared as the sodium salt although calcium and
ammonium salts are also available. Saccharin may be hydroloyzed
to £_-sulfamoylbenzoic acid or ammonium salt of c>_-sulfobenzoic
acid in alkaline or acidic medium, respectively.
Toxicity, Teratogenicity, Mutagenicity. Saccharin has a low
order of acute toxicity. The LE>CQ values by oral administration
are approximately 17 g/Tcg for mice and rats (355, 356), 7.4 - 8.4
g/kg for hamsters (357), and 5-8 g/kg for rabbits (355, 356).
There is no evidence of teratogenicity of saccharin, given either
alone or in combination with cyclamate, in mice (358, 359), rats
(360, 361), or rabbits (360). The mutagenicity of saccharin has
been extensively tested in bacterial systems (362-365; revs.,
347, 356, 366), Drosophila melanogaster (367; rev., 356),
cultured mammalian cells (330, 364, 368, 369; revs., 347, 366),
-------
744
and rodents (370, 371). Saccharin was found to be slightly
mutagenic in some studies but inactive or marginally active in
others. The apparent contradiction is believed to be due, at
least in part, to the differences in the amount and type of
impurities present in different batches of saccharin used (347,
356, 363, 367). In the Ames Salmonella assay, it appears that
highly purified saccharin is not mutagenic (362-365). Some
commercially produced impure samples may, however, exhibit weak
mutagenic activity in in vitro assays or host-mediated assays
(362). Urine from mice given oral doses of saccharin also
displayed some mutagenic activity (362). Stoltz et_ al_. (363)
found that the mutagenic activity of commercial saccharin was
mainly associated with impurities extractible by organic
solvents. They further compared the mutagenicity and
carcinogenicity (in two-generation studies) of several samples of
saccharin and concluded that the two parameters do not
correlate. Rao et al., (365) have recently investigated the
possibility of using the Ames assay to test the co-mutagenicity
of promoters. Saccharin was found to be ineffective in modifying
the mutagenic activity of a wide variety of known mutagens. The
authors (365) concluded that the assay was incapable of detecting
saccharin as a promoter of mutagenesis. Seven mutagenicity
studies of saccharin in Drosophila were reviewed by Kramers
(356); only one was considered positive, three were suggestive,
two could not be evaluated and one was not statistically
significant. Impurities appear to play a determining role in the
mutagenicity in Drosophila, but two of the major impurities,
-------
745
_o_- and p_-toluenesulfonamides, were not considered responsible
(367). Mutagenicity studies of saccharin in cultured mammalian
cells have also been equivocal (revs., 347, 366). Most
chromosome aberration tests appear to be negative (366). At
least three studies, however, showed increased incidences of
sister chromatid exchange in Chinese hamster cells after exposure
to high doses of saccharin (330, 368, 369). Cell transformation
assay using BHK 21/C1 13.cells was negative (364) but using 10T
1/2 cells, Mondal et al. (372) demonstrated that saccharin acted
as a promoting agent (this study will be further discussed in the
following paragraphs). In dominant lethal tests, saccharin was
considered positive in male mice (370) but negative in female
mice (371).
Carcinogenicity. The carcinogenicity studies, including
some unpublished or preliminary reports, of saccharin have been
extensively reviewed by the NAS Committee on Saccharin (347) and
by Rueber (348) in 1978. The final reports of some of these
studies have since been published. These studies may be divided
into three categories: (a) one-generation studies, (b) two-
generation studies, and (c) studies of saccharin in combination
with other carcinogens or promoters. A comprehensive summary of
each of these categories is presented in Tables CL, CLI and CLII,
respectively.
The carcinogenicity of saccharin was first tested by
Fitzhugh e_t_ jal_. (373). Osborne-Mendel rats were fed diets
containing 0.01-5.0% sodium saccharin for 2 years. No adverse
effects were observed among rats receiving 1% or less saccharin
-------
746
in the diet. In the 5% group, there was an increase (7/17) of
lymphosarcoma. Four of these 7 rats had both thoracic as well as
abdominal lymphosarcoma; such an-occurrence is very uncommon
among untreated rats of this strain. The data of Fitzhugh et al.
(373) were re-examined by Long and Haberman (cited in ref. 348)
who affirmed the statistical significance of increased incidence
of lymphosarcoma but pointed out that the study was inconclusive
with respect to the urinary bladder as a potential target,
because of the lack of histologic examination of the organ. At
least nine other feeding studies have since been carried out.-- ^
^fir- iSib
using various strains of rats (Table CL). With one exception,
none of these studies showed any unequivocal evidence of carcino-
genicity of saccharin. Although increases in the incidence of
tumors at some sites were observed, none of these increases was
statistically significant. The only exception was from part of a
two-generation study by the Canadian Health Protection Branch
(374) in which a significantly increased incidence of tumors of
the urinary bladder was observed among male parents. Seven of
the 38 male rats fed a diet containing 5% saccharin developed
bladder tumors (4 benign, 3 malignant) compared to only 1 in 36
control rats. No such carcinogenic effect was observed in female
rats or in rats fed lower doses of saccharin.
The carcinogenicity of saccharin in the mouse was first
tested by Allen _et_ _al_. (375) in 1957 by the pellet implantation
technique (see Section 4.3.3.5). Pellets weighing 9-11 mg and
containing 20% saccharin and 80% cholesterol were surgically
implanted into the urinary bladder of mice. After 52 weeks, 4/13
-------
Table CL.
p. 1 of 2 pp.
. a
Carcinogenicity of Saccharin
Species, strain and sex
Dose and route
Carcinogenicity
References
Rat, Osborne-Mendel,
M & F
Rat, Boots-Wistar, M & F
Rat, Sprague-Dawley,
M & F
Rat, Charles River
CD, M & F
Rat, Charles River
Sprague-Dawley, M
Rat, Charles River
Sprague-Dawley, M & F
Rat, Wistar, M & F
0. 01 -5% in diet
0. 005-5% in diet
0. 2%, 0. 5% in diet
1%, 5% in diet
0.09-2.7 g/kg/day in diet
1%, 5% in diet
5% in diet
2. 5 g/kg/day in diet
2. 0 or 4. 0 g/kg/day
in diet or water
Lymphocytic tissue
None
None
No significant effect
No significant effect
No significant effect
Urinary bladder (male)
None
No significant effect
[373 ; data re-examined by
Long and Haberman, 1969
(cited in refs. 347, 348)]
[Lessel, 1959 (cited
in refs. 347, 348)]
(383)
[Litton Bionetics, 1972
(cited in ref. 348)]
(413)
[BioResearch Consultants, 1973
(cited in refs. 347, 348)]
(374)
(384)
(387)
-------
Table CL, continued
p. 2 of 2 pp.
Rat, Fischer, M
Mouse, unspecified
Mouse, Swiss, F
Mouse, Swiss SPF, M & F
Mouse, HaM/ICR, M & F
Mousej dde, M fc F
Hamster, Syrian golden
Monkey, Rhesus
5% in diet
9-11 mg pellet0
surgical implantation
Q
20-24 mg pellet
surgical implantation
5% in diet
0. 2%, 0. 5% in diet
1%, 5% in diet
0. 2-5% in diet
0. 1 56-1. 25% in drinking water
0.02-0.5 g/kg/day in
aqueous solution
None
Urinary bladder
Urinary bladder
None
No significant effect
Vascular system,
lung (male)
Ovary (female)
None
No evidence of
carcinogenicity (study
still in progress)
(388)
(375)
(376)
(377)
(359)
[BioResearch Consultants,
1973 (cited in ref. 348)]
[Natl. Inst. Hygienic Sciences
Japan (cited in ref. 348)]
(357)
(378)
Does not include two-generation studies
Urinary bladders were not examined his to logically in this study.
Containing 20% saccharin and 80% cholesterol
-------
747
mice developed bladder tumors (3 malignant, 1 benign) compared to
only one bladder carcinoma among 24 controls (significant at
p<0.01). In 1970, Bryan et_ a_l_. (376) re-tested saccharin by the
same procedure, using a much larger number of mice and confirmed
the carcinogenicity of the sweetener toward the bladder. The
tumor incidence was 47-52% in experimental and 12-13% in
controls. It should be noted that there is a lack of consensus
regarding the significance of pellet implantation studies; some
investigators (e.g., 349) are of the opinion that the study
demonstrated the promoting, rather than the carcinogenic, effect
of saccharin.
Saccharin has a variable effect by oral administration to
various strains of mice (Table CL). In female Swiss mice, Roe et
al. (377) found no evidence of carcinogenicity after feeding the
animals a diet containing 5% saccharin for 18 months. A similar
finding was reported by Kroes et al. (359) using lower doses
(0.2% or 0.5%) of saccharin. An unpublished study by BioResearch
Consultants (cited in ref. 348), however, reported that male
HaM/ICR mice fed diets containing 1% or 5% saccharin had
significant increases in the incidences of tumors in the lung or
in the vascular system. A brief report by the National Institute
of Hygienic Sciences of Japan (cited in ref. 348) indicated a
significant increase in the incidence of ovarian tumors in female
strain dde mice.
The carcinogenicity of saccharin has also been tested in
Syrian golden hamsters and Rhesus monkeys. In the hamster, oral
administration of 0.156, 0.312, 0.625 and 1.25% saccharin in the
-------
748
drinking water for life did not bring about any increase in tumor
incidence, compared to the controls (357). In the monkey, a
preliminary report of an ongoing -study showed no evidence of
carcinogenicity after 5.4 years of daily oral administration of
0.02-0.5 g/kg saccharin (378).
Thus far, the most consistent evidence of carcinogenicty of _
-
-------
Table CLI.
Urinary Bladder Tumor Incidences in Rats After Successive Prenatal, Lactational
and Postnatal Exposures to Saccharin (Two-generation Studies)
Study
W. A. R. F.a
F.D.A.b
Canadian
Dose
(% diet)
0%
5%
0%
5%
7. 5%
0%
5%
Bladder tumor incidence
Male
O/ 1 6
'?e/l6 (p=0. 003)
lf/25
lf /21 (n. s.)
7g/23 (p=0. 018)
0/42
12k/45 (p =0. 0002)
Female
0/17
0/20 (n. a.)
0/24
0/28 (n. s.)
2h/31 (n. s.)
0/45
2m/49 (n. s.)
References
(379, 380; see
also 347, 348)
(381)
(374)
I
a
Wisconsin Alumni Research Foundation. Sprague-Dawley rats and saccharin manufactured by the
Remsen-Fahlberg process were used in this study.
U.S. Food and Drug Administration. Charles River Sprague-Dawley rats and saccharin by the
Remsen-Fahlberg process were used.
c
Canadian Health Protection Branch. Charles River Sprague-Dawley rats and saccharin by the Maumee
process were used.
p-Values for statistical analyses are shown in parentheses; n. s. = not significantly different from controls.
Tumor pathology: all transitional cell carcinomas; transitional cell polyp- *4 transitional cell car-
cinomas, 2 papillomas, 1 polyp; 1 transitional cell carcinoma, 1 polyp; 4 benign, 8 malignant; both
malignant.
-------
749
An analysis of the combined data of the three studies by the NAS
Committee on Saccharin (347) led the Committee to suggest that
"ingestion of saccharin at the 5%. or 7.5% dietary level may have
contributed to an increase in benign uterine tumors and ovarian
lesions in female rats." .
The major findings of studies of saccharin in combination
with other carcinogens or promoters are summarized in Table [3.bj£.
CLII. In the only available initiation-promotion study (82),
saccharin was found active as an initiator of skin tumorigenesis
in mice. Skin painting of a total dose of 0.24g saccharin (given
in 10 thrice weekly applications) followed by 18 weekly
applications of 0.25% croton oil led to the induction of 14 skin
tumors in 7 of the 20 mice; for comparison, only 7 tumors were
found in 5 of the 53 mice given croton oil alone.
Before the banning of cyclamate in 1969, cyclamate and
saccharin were widely used as tabletop sweeteners as a 10:1
mixture. The carcinogenicity of such a mixture in rodents has
been tested by several groups of investigators. Price et al.
(382) first released a preliminary report showing the induction
of transitional cell tumors of the urinary bladder in 8 (7 male,
1 female) out of 80 rats given 2.5 or 2.6 g/kg/day of the mixture
for up to 105 weeks. No such tumors were observed in control
rats or rats treated with lower doses (1.12 or 0.5 g/kg/day) of
the mixture. It should be noted that this study was somewhat
complicated by the addition of cyclohexylamine to the diet of
some rats starting at the 79th week. This report precipitated
the banning of cyclamate. A final report of this study was
-------
Table CLII.
p. 1 of 2 pp.
Carcinogenicity of Saccharin in Combination with Other Carcinogens or Promoters
Intended test of
saccharin activity
Other carcinogen
a
or promoter used
Test system and method
Results
References
As initiator
As carcinogen
or promoter
As promoter
As promoter
Croton oil (known
promotor)
Cyclamate (as carcinogen
or promoter)
MNU (known bladder
carcinogen)
FANFT (known bladder
carcinogen)
"S" strain mice; skin painting
of saccharin followed by
by croton oil
Wistar-FDRL rats; feeding
of a 10:1 mixture of
cyclamate and saccharin
Sprague-Dawley rats; same
treatment as above
Wistar rats (males only);
same treatment as' above
SPF Swiss mice; same
treatment as above
Wistar rats; instillation of a
"subcarcinogenic" dose of
MNU into bladder followed
by feeding saccharin
Fischer rats (males only);
feeding of FANFT
followed by saccharin
Increased incidence
of skin tumor
Increased incidence
of bladder tumor
No evidence of
carcinogenicity
No evidence of
carcinogenicity
No evidence of
carcinogenicity
Increased incidence
of bladder tumor
Increased incidence
of bladder tumor
(82)
(361, 382)
(383)
(384)
(359)
(385-38-7)
(388)
-------
TabJt; CLII continued
p. Z of Z pp.
A s promoter
As promoter
As inhibitor
3-MC (known carcinogen)
B(a)P (known carcinogen)
Z-AAF (known carcinogen)
C3H10T1/2 mouse embryo
cells; exposure to
"sub-transforming" dose of
3-MC followed by continuous
treatment with saccharin
Swiss mice (females only);
intragastric instillation
of B(aJP followed by
saccharin feeding
Horton Sprague-Dawley
rats (females only);
simultaneous feeding of
2-AAF and saccharin
Increased incidence
of transformation
"(372)
No evidence of synergism
or promotion
Decreased incidence of
mammary and ear duct tumors
(377)
(389)
''Abbreviations used: MNU = N-methyl-N-nitrosourea; FANFT = N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide; 3-MC'= 3-methylcholanthrene;
B(a)P = benzo(a)pyrene; Z-AAF = 2-acetylaminofluorene
-------
750
recently published (361). A total of 12 rats (9 male, 3 female)
were found to have malignant bladder tumors in the 2.5 g/kg
group. No bladder tumors were found among the controls or in the
1.12 g/kg group. Two rats in the 0.5 g/kg group had benign
bladder tumors. In contrast to the above finding, however, three
other studies failed to confirm the carcinogenicity of the
cyclamate-saccharin mixture.. Schmahl (383) could not observe any
carcinogenic effects after feeding Sprague-Dawley rats diets
containing cyclamate (5 or 2%) and saccharin (0.5 or 0.2%) for
lifetime. Furuya _et_ _al_. (384) reported that none of the 54-56
male Wistar rats developed bladder tumors after ingesting 2.5
g/kg/day cyclamate-saccharin mixture for up to 28 months. Kroes
et al. (359) were also unable to find any evidence of carcino-
genicity of cyclamate-saccharin mixture in SPF Swiss mice.
The possibility that saccharin may act as a promotor or
cocarcinogen in bladder carcinogenesis has been tested by Hicks
and coworkers (385-387). Wistar rats were first administered a
single "sub-carcinogenic" or "initiating" dose (1.5 or 2.0 mg) of
a strong bladder carcinogen, N-methyl-N-nitrosourea (NMU) by
intravesicular instillation through a catheter into the urinary
bladder; they were then given daily oral administration of 2 or 4
g/kg saccharin in the drinking water or diet. In one such study
(387), the incidences of bladder tumors were 0% for the untreated
controls, 0% for MNU only, 1% for saccharin only (not signifi-
cantly different from controls), and 52% for the MNU plus
saccharin group. The time of appearance of the first tumor was
reduced from 95 weeks in the saccharin group to just 8 weeks in
-------
751
the MNU plus saccharin group. The results can be interpreted to
indicate either a promotion by sacccharin pf the carcinogenic
effect of MNU or a synergism between MNU and saccharin as a
potential weak carcinogen. The effect of saccharin in
combination with another bladder carcinogen, N-[4-(5-nitro-2-
furyl)-2-thiazolyl3 formamide (FANFT), has recently been
investigated by Cohen et_ a_l_. (388). Male Fischer rats given for
6 weeks a diet containing 0.2% FANFT had a bladder tumor
incidence of 25% (5/20 rats with 4 malignant tumors). The tumor
incidence was increased to 94% (18/19 rats; all tumors malignant)
if the rats were given a 5% saccharin diet immediately following
FANFT treatment. No bladder tumors were observed if the rats
were given saccharin only, suggesting that saccharin acted as a
promoter in the potentiation of FANFT carcinogenesis. The
promoting effect of saccharin could still be readily observed if
saccharin was given 6 weeks after the cessation of FANFT
treatment. The promoting effect of saccharin may also be
demonstrated by in vitro carcinogenesis (cell transformation)
study. Mondal _et_ _al_. (372) showed that incubation of 10T 1/2
mouse embryo fibroblasts in a medium containing 2 mg/ml saccharin
for 24 hours did not produce any oncogenic transformation of the
cells. However, if the cells were pretreated with a "sub-
transforming" or "initiating" dose (0.1/ig/ml) of 3-methyl-
cholanthrene, subsequent treatment with 100/tg/ml saccharin led
to significant transformation. It was concluded that saccharin
probably acted as a promoter and that its promoting activity was
about 1000-fold less than that of the classical promoter, 12-0-
tetradecanoyl-phorbol-13-acetate.
-------
752
In contrast to the above findings, an earlier report by Roe
_et_ _a_l_. (377) did not indicate any co-carcinogenic or promoting
effect of saccharin with benzo(a)pyrene in Swiss mice.
Intragastric instillation of a single dose of 50/M/g benzo(a)-
pyrene increased the incidence of tumors of the forestomach;
however, feeding the mice a diet containing 5% saccharin for 18
months had no significant effect on the carcinogenic effect of
the hydrocarbon. In a study by Ershoff and Bajwa (389),
saccharin was found to inhibit, rather than to promote, the
carcinogenic effect of 2-acetylaminofluorene (2-AAF). Female
Horton Sprague-Dawley rats fed a diet containing 300 ppm 2-AAF
had a tumor incidence of 91.7% with the mammary gland and ear
duct as the principal targets; simultaneous feeding of 2-AAF and
5% saccharin decreased the tumor incidence to 50%. The mechanism
of the apparent inhibition is not known. The authors were
cautious to point out the possibility that the decreased tumor
incidence may have been due, at least in part, to a decreased
intake of calories.
Since commercial saccharin is produced by two different
manufacturing processes yielding products with different
impurities, and since conflicting results were obtained in
saccharin studies, it was suspected (353) that impurities may
play a role in affecting the potential carcinogenicity of
saccharin. This possibility has been investigated, but is now
considered highly unlikely (347). The carcinogenicity of _o_-
toluenesulfonamide (see Section 5.2.1.7.8), the principal
impurity of Remsen-Fahlerg saccharin, has recently been
-------
753
studied. It is noncarcinogenic in one study (374) and is so very
weakly carcinogenic in another study (390), that it is unlikely to
exert any significant effect as,,an impurity of saccharin.
Furthermore, as shown in Table CLI, saccharin produced by the two
/
different processes had nearly the same carcinogenic effects in
the two-generation studies. The impurities present in.the Maumee
saccharin are so low that if any of them were responsible for the
observed carcinogenic effect of saccharin it would have to be an
extremely potent carcinogen (347, 374).
The potential carcinogenicity risk of human consumption of
saccharin has been a subject of great concern and controversy.
The NAS Committee on Saccharin (347, 354) has recently investi-
gated the risk and benefits of saccharin use; the readers are
referred to their reports for details. Three types of epidem-
iologic studies have been carried out: (a) time-trend studies
which provide crude measurements relating the change pattern of
bladder tumor incidence in a general population against the use
of saccharin (391, 392), (b) studies on diabetics which
investigate the risk of bladder tumorigenesis among diabetics who
are heavy users of saccharin (393-395), and (c) case-control
studies in which the extent of saccharin use among patients with
bladder tumors is compared with that of a group without bladder
tumors (396-400). Thus far, no sufficiently unequivocal evidence
is available to support or to refute an association between
saccharin use and bladder tumor induction (347).
The pharmacokinetics of saccharin has been extensively
studied (rev., 347, 353); saccharin is readily absorbed and
-------
754
rapidly excreted, mainly in the urine, as unmetabolized parent
compound. Several investigators (401-403) detected trace amounts
(<1% of the administered dose) -of 2-sulfamoylbenzoic acid, 2-
sulfobenzoic .acid and/or carbonate as urinary metabolites in the
rat. However, these findings could not be confirmed by others
(405-407). In a recent study by Sweatman and Renwick (408), the
metabolic study was carried out under conditions known to cause
induction of bladder tumors. Rats were exposed to saccharin in
utero and throughout weaning, and then maintained on a 5%
saccharin diet. Even under these conditions, no metabolism was
detected strongly suggesting that the carcinogenic effect of
saccharin is due to the unmetabolized parent compound.
The mechanism of action of saccharin is not known. In the
apparent absence of detectable metabolism, this anionic compound
is not expected to act as a classical, electrophilic carcin-
ogen. This view is supported by the study of Lutz and Schlatter
(406) who showed that saccharin did not bind covalently to DNA of
liver or bladder in the rat. The limits of detection in this
o 7
study were 1 x 10 and 1 x 10 mole saccharin per mole DNA
phosphate for the liver and bladder, respectively. Compared to
dimethylnitrosamine binding to liver DNA, the "covalent binding
index" of dimethylnitrosamine is at least 5 to 6 orders of
magnitude greater than that of saccharin. The authors (406)
suggested that the carcinogenicity of saccharin is probably due
to an indirect mechanism involving damage to the bladder
epithelium. Miyata et_ a_l_. (409) have investigated,by alkaline
sucrose density gradient analysis, the ability of saccharin to
-------
755
cause damage, of bladder DNA. Under conditions that led to
extensive DNA damage by the bladder carcinogen, N-butyl-N-(3-
carboxypropyl)-nitrosamine, saccharin had no significant ability
to cause DNA damage. Ashby et al. (364) have proposed that
saccharin may exert carcinogenic action by epigenetic mechanisms,
e.g., inhibition of DNA repair enzymes, interference with
cellular hormonal function, or disturbance of cellular control
mechanisms. Boyland (349) is of the opinion that saccharin
should be considered a promotor rather than a complete
carcinogen. He postulates that the induction of bladder tumors
by saccharin in two-generation studies could be due to the
promoting action of saccharin together with endogenous initiators
of carcinogenesis. It should be noted that the concept of
endogeneous carcinogens is highly controversial. Fukushima and
Cohen (410). have recently demonstrated the ability of saccharin
to induce hyperplasia of bladder epithelium, a well known
property of promoters (411, 412). However, they pointed out that
the possibility that saccharin may also have weak initiating
activity and, therefore, a weak "complete" carcinogenic activity
can still not be excluded.
5.2.1.7.8 Sulfonamides, Cyclamate and Related Compounds.
5.2.1.7.8.1 Sulfonamides. The sulfonamides occupy a prominent
position as chemotherapeutic agents. About 150 sulfonamides have
been marketed, mostly as antimicrobials, at one time or another
(414). In spite of the advent of the antibiotics, sulfonamides
are still widely used because of their low cost and high
efficacy. Most sulfonamides are structural analogs of jp_-
-------
756
aminobenzoic acid and exert their antimicrobial action by
preventing the enzymatic condensation of the latter with
glutamylpteridine compounds to form folic acid, an essential
precursor of purine synthesis.
Relatively few sulfonamides have been tested for carcino-
genicity. Among these, four are of particular interest and have
*£-
been studied more thoroughly (see Table CLIII for formulas) . _o_-
Toluene-sulfonamide (not shown) is the major contaminant of
saccharin and was, at one time, suspected to play a role in the
alleged carcinogenic action of the controversial sweetener (see
Section 5.2.1.7.7). Sulfanilamide is the simplest member of the
antibacterial sulfonamide series; it was first tested for
carcinogenicity in 1938 (415) because of its structural
resemblance to certain carcinogenic dyes. 2-p_-Methoxybenzene-
sulfonamido-5-isobutyl-l,3,4-thiadiazole is a sulfonamido
derivative of thiadiazole; thiadiazole compounds are effective
hypoglycemic agents. 4-Ethylsulfonylnapthalene-l-sulfonamide was
developed as an anti-convulsant and diuretic drug, but was never
introduced into the market after it was found to have hyper-
plastic activity toward the urinary tract epithelium in several
animal species and carcinogenicicity toward the bladder in
mice. This Section focuses on the carcinogenicity studies of
these four compounds.
The acute toxicity of the sulfonamides discussed here is
quite low. The LD^Q values by oral administration.are 4.87 g/kg
for £_-toluene-sulfonamide in rats (416); 3.9 and 2.0 g/kg for
sulfanilamide in rats and dogs, respectively (416); and 0.47 and
-------
Table CLIII
Structural Formulas of Cyclamate and
Some Sulfonamides Tested for Carcinogenicity
NHS03"
M
J2
Cyclamate
(M++= bivalent
metal cation)
S02NH2
S02C2H5
4-Ethylsulfonylnaphthalene-
-l-sulfonamide
CH.O
2-p-Methoxybenzenesulfonamido-
-5-isobutyl-l,2,3-thiadiazole
-------
757
0.53 g/kg for 2-p_-methoxybenzenesulfonamido-5-isobutyl-l,3,4-
thiadiazole in mice and rats, respectively (417). 4-Ethyl-
sulfonylnapthalene-1-sulfonamide brings about hperplasia in the
urinary tract epithelium of mice, rats and possibly rabbits, but
not guinea pigs (418, 419). The hyperplastic activity is
reversible and is dependent on the continous administration of
the compound. Structure-activity studies (418-420) revealed that
(a) the sulfonamide group is essential for the hyperplastic
activity, (b) modification of the alkylsulfonyl group does not
necessarily abolish activity, but the change of the position of
the group on the naphthalene ring does bring about loss of
activity. Very little information is available on the tera-
togenicity and mutagenicity of these compounds. As a group,
antibacterial sulfonamides display little teratogenic activity in
experimental animals (421). In particular, sulfanilamide has
been found to be nonteratogenic in rats and rabbits and
marginally active in mice (rev., 421). Despite extensive use,
there is no epidemiologic evidence to implicate antibacterial
sulfonamides, used as drugs, as human teratogens (421). Only &-
toluene-sulfonamide has been extensively tested for mutagenicity;
it was found to be nonmutagenic in (a) the Ames Salmonella test
either in the presence or absence of the S-9 activation system
(363, 364), (b) a Drosophila test for sex-linked recessive lethal
mutations (367), and (c) a cell transformation assay using
cultured mammalian cells (364).
The major findings of the carcinogenicity studies of the
four sulfonamides are summarized in Table CLIV. o-Toluenesulfon-
-------
Table CLIV.
Carcinogenicity of Sulfonamide and Sulfonamido Compounds
Compound
Species and strain
Carcinogenicity and route
References
o-Toluenesulfonamide
Sulfanilamide
(jD-aminobenzene
sulfonamide)
2-j3-Methoxybenzene-5-iso-
butyl-1, 3, 4-thiadiazole
4-Ethyls ulfonylnaphtha-
lene-1 -sulfonamide
(ENS; "HPA";
hyperplastic agent)
Rat, Sprague-Dawley
Rat, Charles River
Sprague-Dawley
Mouse, C57, C3H
or dba
Mouse, Swiss albino
Mouse, C57, C3H, A
or Swiss albino
Rat, Wistar
Dog, mongrel
Mouse, (AbxIF)F
or (C57xIF)F
1
1
Mouse, (C57xIF)F
1
Mouse, (AxIF)F
Rat, unspecified
1
Urinary bladder (oral) (390)
None (oral) (374)
(two-generation study)
None (s. c.) (415, 422)
Local sarcoma (s. c.) , (423)
None (oral) (423, 424)
Urinary bladder (oral) (417)
) ,
None (oral) (41-7)
Urinary bladder (oral) (419, 425,
426, 429)
Urinary bladder (pellet (425)
implantation)
Urinary bladder, mammary (427, 431)
gland (oral)
No evidence of Carcinogenicity (428)
in bladder
-------
758
amide was first tested for carcinogenicity by Schmahl (390);
Sprague-Dawley rats were fed daily doses of 20 or 200 mg/kg c>_-
toluenesulfonamide in the diet-from the age of 3 months to
death. Among the 76 animals in the high-dose group one had a
carcinoma and four developed papillomas in the urinary bladder,
while in the low-dose group 3 of the 75 rats bore bladder
papillomas. Almost all these tumors occurred after the rats were
beyond two years of age.. None of the 71 controls had bladder
tumors. In contrast to the above finding, Arnold et_ jal_. (374)
were unable to confirm the carcinogenic effect of the compound in
Charles River Sprague-Dawley rats in a two generation study. In
this study, the parent (FQ) generation animals were given daily
oral administration of 2.5, 25 or 250 mg/kg ^-toluenesulfonamide
for 3 months before mating. The females continued to receive _p_-
toluenesulfonamide during gestation and lactation. The filial
(F^) generation animals, upon weaning, were placed for lifetime
on diets containing the same levels of _o_-toluenesulfonamide as
their parents. None of these treatments brought about any
significant increase in tumor induction in either the FQ or F,
generation. Thus, even a combination of in utero exposure,
exposure via mother's milk and lifetime feeding failed to
demonstrate the carcinogenic effect of _p_-toluenesulfonamide.
Sulfanilamide (p_-aminobenzenesulfonamide) was first tested
for carcinogenicity by Lewis (415) because of its structural
similarity to some known carcinogens. An unspecified number of
2- to 3-month-old C57 and C3H mice were given a single s.c.
injection of 12.5-25 mg sulfanilamide in oil suspension. Twenty-
-------
759
five of these mice survived one month and were given a second
dose of 12 mg sulfanilaraide. After 219 days, 10 of these mice
were sacrificed; none of them had any turner. The remaining mice
were kept for 300 days and still found to be free from tumors.
The lack of carcinogenicity of sulfanilamide was confirmed by
Zamecnik and Koletsky (422). Fifty-two Bar Harbor dba strain
mice were given repeated s.c. injections of 40-60 mg sulfanil-
amide, over a 4 1/2 month period, for a total of 320 mg for each
mouse. At the end of one year the 23 mice still alive were
sacrificed; no evidence of tumors was found in any of these
mice. Thus, both studies showed lack of carcinogenicity of
sulfanilamide; however, it should be pointed out that neither
test was of sufficiently long duration to exclude carcinogenic
effect at older age. In contrast to the above, Haerem (423)
demonstrated a weak carcinogenic effect of sulfanilamide by s.c.
injection to Swiss albino mice. Among the 20 mice that received
2 injections of 15 mg sulfanilamide in lard, 2 developed spindle
cell sarcomas at the site of injection and died after 222 and 261
days, respectively. The significance of this study could not be
evaluated because matched controls were not included in this
study. Sulfanilamide has also been tested by oral administration
with no evidence of carcinogenicity. Haerem (423) fed 20 Swiss
albino mice a diet containing 2.5% sulfanilamide; none of these
animals developed tumors. In a short communication, Figge et al.
(424) reported a multi-generation study in which sulfanilamide
was fed to mice of various strains (C3H, C57, A) for 8 genera-
tions over a period of 5 years. No carcinogenic effects of the
compound were observed. 'No details of the study were given. .
-------
760
The carcinogenicity of 2-£-methoxybenzenesulfonamido-5-
isobutyl-1, 3,4-thiadiazole was tested in two animal species by
Rosen _et_ _al_. (417). In two separate experiments, the drug was
found to induce tumors in the urinary bladder of Wistar rats. In
the first experiment, rats were fed diets containing 0.05, 0.1,
0.2, 0.4, 0.6 or 0.8% of the compound for 12-18 months. Three of
the 4 females in the 0.8% group and 2 of the 5 females in the
0.6% group developed bladder tumors within 18 months. No bladder
neoplasms were detected in controls or rats fed lower doses of
the drug. In a confirmatory experiment, 60 rats were fed a diet
containing 0.8% of the drug for 16 months. Among the 44
survivors, 9 males and 4 females had bladder tumors; none of the
31 controls had such tumors. Histopathologically, the tumors
were classified as transitional cell papillomas or papillary
carcinomas of the bladder epithelium. The chronic toxicity of
the drug was also investigated using mongrel dogs. Groups of 4
dogs (2 of each sex) were respectively treated with 25, 50, 100,
or 200 mg/kg of the drug in capsule, once a day, 5 days a week.
After 56 weeks, half of the dogs were sacrificed and their
various organs examined histopathologically; the remaining dogs
were sacrificed after 3 years and only their urinary bladders
were examined. None of these examinations revealed any evidence
of tumor induction by the drug. It should be noted, however,
that even a 3-year-period may not be regarded to be of
sufficiently long duration for carcinogenicity testing involving
dogs because of their relatively long life span.
-------
761
4-Ethylsulfonylnapthalene-l-sulfonamide (ENS) is the most
extensively studied sulfonamide, due mainly to the efforts of
Clayson and his associates. The.carcinogenicity of this drug was
first reported by Bonser and Clayson (419). Sixty-five (Ab x IF)
hybrid mice were fed a diet containing 0.01% of the drug for 65
weeks. Among the 28 mice (12 males, 16 females) that survived
more than 30 weeks, 10 (corresponding to 36%) were found to have
carcinomas of the urinary bladder. Of these 10 mice only one was
male indicating that the females were considerably more
susceptible to the carcinogenic action of ENS. This -finding was
confirmed in a more extensive study using the same strain of mice
(425). The bladder tumor incidences were 46% for females and 15%
for males. The carcinogenicity of ENS was also demonstrated in
two other strains of mice. The (C57 x IF)?^ mice responded to
ENS in a similar manner as (Ab x IF)F, mice, with bladder tumor
incidences of 44% for females and 16% for males (426). Castrated
males and females had bladder tumor incidences of 21% and 28%,
respectively, indicating the importance of hormonal involvement
in ENS carcinogenesis. The closely related (A x IF)F, mice were
also susceptible to ENS; however, their susceptibility to bladder
carcinogenesis was considerably less than that of (Ab x IF)F^
mice (427). Among the (A x IF)F, mice that survived more than 40
weeks of oral administration of 0.005 or 0.01% ENS in the diet,
there were only 6 bladder carcinomas and one papilloma in 74
females (9% incidence) and no bladder tumors in 77 males. In
contrast to the lower susceptibility to bladder carcinogenesis,
female (A x IF)F, mice developed more mammary carcinomas in
-------
762
response to ENS treatment. However, since one of the parent
strains (strain A) carries mammary tumor virus, it is not known
whether the increase in the incidence of mammary tumors was due
'--,. "
to a direct carcinogenic action of ENS or was the result of a
synergism with or a potentiation of the action of the tumor virus
(427). In addition to oral administration, ENS was also tested
by bladder implantation in (C57 x IF)F, mice (425). A signif-
icant increase in the incidence of bladder carcinomas (22% in
experimental vs. 4.5% in control) was observed. The authors
concluded that ENS was a locally acting carcinogen to the urinary
bladder epithelium. In contrast to mice, rats appear to be
resistant to the bladder carcinogenic action of ENS (428).
The carcinogenic action of ENS may be modified by a variety
of exogenous factors. Of great importance is the recent finding
that the carcinogenic action of ENS is inhibited by simultaneous
treatment with ammonium chloride (429, 430). This observation
may shed light on the possible mechanism of action of ENS which
will be further discussed below.
The effect of pretreatment of mice with strong carcinogens
on the carcinogenicity of ENS has been investigated. Both 7,12-
dimethylbenz(a)anthracene (419) and 2-acetylaminofluorene (425)
slightly reduce rather than enhance the carcinogenic action of
ENS in females. Their modifying effect in male mice cannot be
evaluated because of the relatively low tumor incidences. While
whole body irradiation of mice has no significant effect on the
carcinogenicity of ENS, combined treatment results in a decrease
in the induction of mammary tumors (431). Oral administration of
-------
763
ENS to mice increases the induction of bladder tumors by
implantation of pellets containing the bladder carcinogens, 2-
amino-1-naphthol hydrochloride (432) or ,2-amino-l-phenylazo-2-
naphthol (425). Since ENS is also an effective hyperplastic
agent, it is not known whether the enhancement is due to
promotion or synergism.
The role of metabolism'in the carcinogenic action of
sulfonamides is unclear. As a group, sulfonamides undergo
metabolic alterations involving mainly acetylation and oxida-
tion. The acetylated forms of some sulfonamides are less soluble
and may contribute to crystalluria (433). The metabolism of ENS
in the mouse has been investigated by Bradshaw (434). Orally
administrated ENS is excreted mostly unchanged in the urine. The
major metabolites are 4-(2'-hydroxyethylsulfonyl)-naphthalene-l-
sulfonamide and its glucuronide; other minor unidentified
metabolites and a compound tentatively identified as 4-ethyl-
sulfonyl-naphthalene-1-sulfonic acid are also excreted. It is
not known whether any of these metabolites may represent the
proximate carcinogen of ENS.
The possible mechanism of action of ENS has been extensively
studied. Lawson et al. (435) demonstrated that acute doses of
ENS cause specific increase in DNA synthesis in the mouse
bladder; however, the increase appears to be mainly a response to
chemically induced cellular injury. This response diminished or
disappeared as ENS feeding continued, suggesting that a sustained
stimulation by ENS was not needed for bladder carcinogenesis.
Levi et_ _a_l_. (436) found that ENS caused cell proliferative
-------
764
changes not shared by strong carcinogens, such as 2-acetyl-
aminofluorene. At least part of the ENS effect was secondary to
the increase in the alkalinity of the urine (as a result of the
inhibition of -carbonic anhydrase by ENS) and the subsequent
increase in the production of bladder.calculi (crystalluria).
The critical question is whether ENS itself, or alkalinuria, or
crystalluria is responsible for the induction of the tumors.
FlaXs and coworkers (429, 430) have recently shown that
simultaneous administration of ammonium chloride with ENS to
female mice completly abolished the bladder carcinogenic action
of the latter. Apparently, the correction of alkalinuria by
ammonium chloride treatment greatly reduces the formation of
bladder calculi and prevents the induction of tumors. Further
studies are needed to elucidate whether the sex difference in
susceptibility to bladder carcinogenesis by ENS could be
accounted for by any difference in the production of alkalinuria
and crystalluria. Also, it remains to be elucidated whether ENS
is a complete carcinogen or a co-carcinogen.
5.2.1.7.8.2 Cyclamate and Related Compounds. Cyclamate
(cyclohexylsulfamate, see Table CLIII for structure) is another
controversial, artificial nonnutritive sweetener. It was widely
used in the U.S. and many other countries until 1969. Sodium
cyclamate was first discovered to have sweetening properties by
Michael Sveda in 1937, during studies of organic sulfamates.
Although cyclamate (30-40 times sweeter than sugar) is not as
sweet as saccharin, it has less bitter aftertaste and is more
resistant to boiling in acidic or alkaline solutions. Based on
-------
765
negative findings in earlier toxicity studies, cyclamate was
included in the 1959 GRAS (generally recognized as safe) list.
The popularity of cyclamate grew rapidly and soon replaced
saccharin as the major sugar substitute. 3y 1968, an estimated
17 million pounds of cyclamate were manufactured in the U.S.; 69%
of which were used in beverages, 19% as tabletop sweetener
(mostly as 10:1 cyclamate: ,saccharin mixture), 6% in foods, 4%
in non-food items and 2% is exported (437). The discovery of
cyclohexylamine as an important metabolite of cyclamate raised
doubts about the safety of the sweetener. Cyclamate was first
reported to induce bladder tumors in mice by pellet implantation
(438). Shortly afterwards, the induction of bladder tumors in
rats fed high doses of a 10:1 mixture of cyclamate and saccharin
was observed (382). Oral administration of calcium cyclamate
alone was also demonstrated to be carcinogenic in rats (439).
These findings led to the banning of the use of cyclamate in
foods by the U.S. Food and Drug Administration in 1969. The
banning has drawn much criticism and stimulated new
investigations. A number of recent studies failed to confirm the
carcinogenicity of cyclamate. A Temporary Committee for Review
of Data on the Carcinogenicity of Cyclamate (NCI, cited in ref.
381) recently concluded that the carcinogenicity of cyclamate in
experimental animals is not definitively established.
Cyclamate is generally used as the sodium or calcium salt.
The free acid is fairly strongly acidic and is sparingly soluble
in water. Sodium or calcium cyclamate is freely soluble in
water. Solutions of sodium cyclamate are practically neutral,
-------
766
and are resistant to boiling under mildly acidic or alkaline
conditions. The acute toxicity of cyclamates is very low. The
LD^Q values of sodium cyclamate in mice are 10-17 g/kg by oral
administration, 7 g/kg by i.p. and 4-5 g/kg by i.v. injection;
the corresponding values for rats are 12-17, 6, and 3.5 g/kg
(440, 441). Cyclohexylamine, the major metabolite of cyclamates,
is considerably more toxic with an acute LD^Q (oral) of 0.2-0.7
g/kg in rodents (416). In humans, cyclamate produces stool
softening and diarrhea if ingested in relatively large amounts.
Cyclamate and cyclohexylamine may influence the therapeutic
effects of a number of commonly used drugs by affecting the
absorption, binding or metabolism of drugs (437).
Cyclamate and cyclohexylamine have been reported to induce a
variety of teratogenic effects in chick embryos (442, 443);
however, there are no convincing data to indicate that these
compounds may be teratogenic in mammalian species. The
occurrence of eye abnormalities in rat embryos following
treatment of pregnant rats with cyclamate was observed in one
study (444), but a number of other investigators (359, 361, 381,
445-448) failed to detect any significant teratogenic effects of
either cyclamate or cyclohexylamine in rats, mice or rabbits.
Extensive investigations on the mutagenicity of cyclamate
and cyclohexylamine yielded contradictory results. A compre-
hensive review of the mutagenicity literature has recently been
presented by Cattanach (449). The indicator test organisms used
in the mutagenicity tests included bacteria, plant cells,
Drosophila, cultured mammalian cells arid whole animals. In
-------
767
eleven in vitro studies with mammalian cells, nine investigators
reported the occurrence of a small but consistent increase in
minor cytogenetic damages (chromatid breaks and gaps but no
exchanges) following exposure of cell cultures to sodium or
calcium cyclamate; two reported negative findings. Similar
cytogenetic damage was detected in three of the four in vitro
studies in which cyclohexylamine was used. In vivo cytogenetic
studies were even more conflicting. Three of the five papers on
cyclamate and two of the six papers on cyclohexylamine reported
positive findings? the rest were all negative. In some cases
(e.g., 450, 451), the experimental procedures and .animals used
were identical but the results were contradictory. In dominant
lethal tests, sodium cyclamate was consistently found to be
inactive in mice (371, 452, 453) and rats (439). There is also
no evidence that cyclohexylamine or its N-hydroxy derivative are
mutagenic in dominant lethal tests (rev., 449).
The carcinogenicity of cyclamate has been tested in several
animal species; the major findings of these studies are {S.bjG, C£, y
summarized in Table CLV. Fitzhugh et al. (373) and Richards et
al., (440) were the first to report independently the lack of
carcinogenicity of cyclamate in the rat. In the study of
Fitzhugh et al. (373), Osborne-Mendel rats were fed diets
containing 0.01-5% sodium cyclamate for two years. No adverse
effects were observed among rats receiving 1% or less cyclamate
in the diet. In the 5% group, the animals had a moderate amount
of diarrhea throughout the experimental period; the only gross
and microscopic effects were those resulting from mild inani-
-------
Table CLV.
Carcinogenicity of Cyclamate
p. 1 of 2 pp.
Species, strain and sex
Rat,
Rat,
M
Rat,
Rat,
Rat,
Rat,
unspecified, M &c F
Os borne -Mend el,
& F
Holtzman, M
Sprague-Dawley
Wistar, M & F
Charles River
Dose and route
0. 05-1. 0% in diet
0. 01 -5% in diet
0. 4-10% in diet
1%, 2% in diet
2%, 5% in diet
1.0 or 2.0 g/kg/day in
drinking water or diet
2, 5 g/kg/day in diet
5% in diet
Carcinogenicity
None
None
Bladder
Bladder (questionable
s ignificance)
None
No significant effect
None
None
References
(440)
(373)
(439)
(439)
(383)
(387)
(384)
(381)
Sprague-Dawley, M & F
Mouse, Swiss
(two-generation study)
20-24 mg pellets3
surgically implanted
into bladder
Bladder
(438)
-------
Table CLV continued
p. 2 of 2 pp.
Mouse, C3H, M & F
RIII, M & F
XVII/G, F
C3HxRIII, M
Mouse, Swiss, F
Mouse, unspecified
Mouse, ASN-CSI, M & F
Mouse, Swiss SPF, M & F
Hamster, Syrian
golden, M & F
Dog, unspecified
Monkey, Rhesus
20-25 mg/day, oral
20-25 mg/day, oral
20-25 mg/day, oral
20-25 mg/day, oral
5% in diet
unspecified, oral
0. 7-7% in diet
2%, 5% in diet
0. 156-1. 25% in
drinking water, oral
0.5 or 1.0 g/day, oral
0. 2 g/kg/day, oral
None
None
Lung
Liver, lung
None
x
No significant effect
(preliminary report)
None
None
None
None (only 4 animals;
insufficient duration)
No evidence of carcinogenicity
after 6. 4 yr (study
still in progress)
(454)
(454)
(454)
(454)
(377)
(456)
(457)
(359)
(357)
(440)
(378)
Containing 20% cyclamate and 80% cholesterol
-------
763
tion. In the study of Richards et_ a_l_. (440), rats of an
unspecified strain were placed on a diet containing 0.05, 0.1 or
1% sodium cyclamate for 18-30 months. Histologic examination of
the heart, lung, liver, kidney, intestinal tract, spleen and sex
organs did not show pathological changes attributable to the
feeding of the sweetener.
Evidence that cyclamate alone may be carcinogenic in the rat
was provided by the study of Friedman 'et al. (439). In one
experiment, Osborne-Mendel rats were fed 0.4, 2 or 10% sodium or
calcium cyclamate in commerical chow diets for 101 weeks.
Transitional cell carcinomas with various degrees of invasiveness
were detected in the urinary bladder of 3 of the 23 rats
receiving calcium cyclamate; two of these carcinomas v/ere in the
low-dose (0.4%) group. In 15 rats receiving sodium cyclamate, 3
developed papillomas in the urinary bladder. No such tumors were
detected in 38 control rats. Although a dose-response relation-
ship was not established in this study, the results were highly
suggestive of the carcinogenicity of cyclamate toward the urinary
bladder of the rat. It should be noted, however, that some of
the rats in this study were infected with parasites which could
enhance the carcinogenicity of some bladder carcinogens. In
another experiment reported by Friedman et_ _al_. (439), male
Holtzman rats were fed 1 or 2% calcium cyclamate in semisynthetic
diets containing 10 or 20% casein for 75 weeks. Only one out of
the 31 treated rats developed a transitional cell papillomatosis
in the urinary bladder; this rat also had stones or calcium
deposits in the kidney and bladder. This finding was probably of
-------
769
questionable significance because of the low incidence and the
presence of bladder calculi.
In contrast to the positive" findings of Friedman et al.
(439), four recent chronic toxicity studies of cyclamate in rats
all failed to confirm the carcinogenicity of the sweetener.
Schmahl (383) did not observe any carcinogenic or toxic effects
after lifetime feeding of 208 Sprague-Dawley rats diets
.containing 2 or 5% sodium cyclamate (equivalent to daily doses of
1.0 or 2.5 g/kg or total doses of 0.88 or 2.2 leg/kg). Furuya et
al. (384) administered to a group of 54-56 Wistar rats daily oral
doses of 2.5 g/kg sodium cyclamate for 12-28 months; there was no
animal that developed any urinary bladder tumors. Hicks and
Chowaniec (387) administered sodium cyclamate in the diet or
drinking water to 228 Wistar rats at dose levels of 1.0 or 2.0
g/kg. At the end of two years, 3 of the 228 rats developed
tumors in the urinary bladder. Although the tumor incidence was
greater than that in control rats (0/96), the statistical
significance of the increases could not be established. In the
most recent study of Taylor _et_ _al_. (381), calcium cyclamate was
fed at a dietary level of 5% to Cnarles River Sprague-Dawley rats
for two generations. No urinary bladder neoplasms were
observed. Thus, even after in utero, lactational and lifetime
exposure, the rats did not develop any bladder tumors.
The carcinogenicity of cyclamate in experimental animals was
first shown by Bryan and Ertu'rk (438) using the pellet implanta-
tion technique. These investigators surgically implanted
cholesterol pellets containing cyclamate into the lumen of the
-------
770
urinary bladder of mice; pellets of cholesterol alone were used
in control mice. At the end of the study, mice exposed to
cyclamate exhibited a significantly higher incidence of bladder
carcinomas (78% and 61% in two experiments) than did the controls
(12-13%). Furthermore, the carcinomas in the exposed mice were
frequently multiple and often more invasive. In contrast to the
above, cyclamate does not seem to have any significant carcino-
genic activity by oral administration. Rudali et al. (454) were
the only group to demonstrate a weak carcinogenic activity of
cyclamate in some strains of mice. They fed 20-25 mg sodium
cyclamate daily to 4 different strains of mice from the age of 30
days to death. No carcinogenic effects were observed in strains
C3H and RIII. In the female XVII/G mice, there was an increase
in the incidence of lung tumors (16/20 experimental vs. 3/16
control). In the F^ hybrid of CSHxRIII, treated males had
significantly more tumors of the liver and lung than did the
controls (65% vs. 43%). Rudali _et_ _al_. (454) suggested that
cyclamate may, in some strains, have a weak carcinogenic activity
possibly "in the form of an acceleration or an accentuation". In
his review, Price (455) has pointed out that many investigators
do not consider the above data as conclusive evidence for
carcinogenicity. At least four different studies, indicating the
lack of carcinogenicity of cyclamate by oral administration to
mice, have been reported. Roe _et_ _al_. (377) fed 50 female Swiss
mice a commercial diet containing 5% sodium cyclamate for 18
months. No urinary bladder tumors were detected and there was no
evidence that cyclamate induced tumors in any other organ. In a
-------
771
preliminary communication, Crampton (456) reported that only 2 of
248 mice had bladder tumors in their cyclamate study? the data
were insufficient to indicate any significant carcinogenic
activity. Brantom et al. (457) observed no carcinogenic effect
attributable to cyclamate after feeding the sweetener to groups
of 60 ASN-CSI mice at dietary levels of 0.7, 1.75, 3.5 or 7.0%
for 80 weeks. Finally, in a multi-generation study by Kroes et
al. (359), a dietary level of 2 or 5% sodium cyclamate was found
to have no carcinogenic effect in Swiss SPF mice.
In addition to the rat and the mouse, the carcinogenicity of
cyclamate has been tested in Syrian golden hamsters, dogs, and
Rhesus monkeys. Althoff et al. (357) observed no significant
increase in tumor incidence in hamsters receiving drinking water
containing 0.156 to 1.25% sodium or calcium cyclamate for
lifetime. The tumor incidences were 10.1% in 168 controls, 14.2%
in 247 animals given sodium cyclamate, and 11.9% in 210 animals
given calcium cyclamate; the highest dose corresponded to the
maximum tolerated dose. No neoplasms of the urinary tract were
found. Only four dogs were used in the study by Richards et al.
(440); the animals received 0.5 or 1.0 gram sodium cyclamate
daily in the diet. One dog was accidentally killed after 3
months; the other three were sacrificed after 15 months. None of
these animals had any tumors. The results are suggestive but by
no means conclusive of the lack of the carcinogenicity of
cyclamate because of the short duration of the experiment and the
small number of animals. In a study on monkeys, Coulston et al.
(413) reported that there was no evidence of carcinogenicity
-------
772
after administration of a solution of sodium cyclaraate at the
dose of 0.2 g/kg/day. The study was still in progress at the
time of their report.
The discoverey of cyclohexylamine as a major metabolite of
cyclamate has raised questions about its role in carcino-
genesis. The carcinogenicity of cyclohexylamine has been tested
by several groups of investigators (359, 382, 383, 445). Three
of these studies found no evidence for the carcinogenicity of
cyclohexylamine in rats (383, 445) or mice (359). Only one study
(382) reported the occurrence of a tumor of the urinary bladder
in one male rat out of 8 male and 9 female rats that survived
daily oral administration of 15 mg/kg cyclohexylamine sulfate for
2 years. The statistical significance of this study was not
analyzed.
The ability of cyclamate to act synergistically with, or to
promote the carcinogenesis of, other carcinogens has received
some attention. The well-publicized study of Price _e_t a_l_. (382)
that a 10:1 mixture of cyclamate and saccharin induced bladder
tumor in rats has been discussed in Section 5.2.1.7.7. This
finding could not be confirmed in several recent studies (359,
383, 384); [the readers are referred to Section 5.2.1.7.7 for
details of these studies]. Like saccharin, cyclamate appears to
have a variable modifying effect on the action of other
carcinogens. Hicks and Chowaniec (387) demonstrated synergistic
(or promoting) effect in bladder carcinogenesis in the rat
.between a "sub-carcinogenic" dose of the strong bladder
carcinogen, N-methyl-N-nitrosourea, and a cyclamate-containing
-------
773
diet. The tumor incidences were 0, 1.3 and 49% in groups
receiving 1.5 mg N-methyl-N-nitrosourea alone, cyclamate alone
and N-methyl-N-nitrosourea plus cyclamate, respectively.
Moreover, the time of appearance of the first tumor was reduced
from 87 weeks (cyclamate alone) to 8.5 weeks by the combination
*
treatment. In sharp contrast to the above finding, Schmahl and
Kru'ger (458) found that cyclamate (2.5 g/kg/day) did not enhance
the bladder carcinogenic effect of 4-hydroxybutylbutylnitrosamine
(another typical bladder carcinogen) in male Sprague-Dawley
rats. Similarly, Roe et al. (377) observed that sodium cyclamate
(5% in diet) had no significant modifying effect on the induction
of gastric tumors by benzo(a)pyrene in Swiss mice. At the other
end of the spectrum, inhibition of tumor induction by 2-acetyl-
aminofluorene in rats by sodium cyclamate was noted by Ershoff
and Bajwa (389). The tumor incidence (mainly mammary gland and
ear duct tumors) was 91.7% in rats receiving a diet containing
300 ppm 2-acetylaminofluorene alone for 40 weeks, but decreased
to 16.7% if sodium cyclamate was also included in the diet. The
possibility that the lower tumor incidence might be due to the
lower intake of calories and/or 2-acetylaminofluorene was
considered by the authors.
The metabolism of cyclamate has been extensively studied.
At the time of its introduction, cyclamate was believed to be
metabolically inert and almost totally excreted in the urine
unchanged (440). With the refinement of analytical methodology,
various investigators have since found that metabolism of
syclamate does occur, albeit to a minor extent, and cyclohexyl-
-------
774
amine is the major metabolite (rev., 449, 459, 460). The
metabolism of cyclamate has been demonstrated to occur in rats,
guinea pigs, rabbits, dogs and monkeys, as well as in human
volunteers. The metabolic capability exhibits a great varia-
bility among various subjects or animals depending on .the route
of administration, the sugar content of the diet, and the extent
of prior feeding with cyclamate. Pretreatment with antibiotics
greatly diminishes cyclamate metabolism. These, along with
several other lines of evidence, have led many investigators to
conclude that the bacterial flora in the intestinal tract is
mainly, if not entirely, responsible for the metabolism of
cyclamate (rev., 460). Cyclohexylamine may be further metaboli-
zed in some species. Cyclohexanol, cyclohexanone and N-hydroxy-
cyclohexylamine have been detected as minor urinary metabolites
in humans and monkeys (461-463); however, in rats and dogs,
cyclohexylamine appears to be the only metabolite. The role of
metabolism in the generation of potential carcinogenic inter-
mediates from cyclamate has not been estabished, since none of
the known metabolites appears to have any demonstrated mutagenic
or carcinogenic activity. lB.ble. L/. V/
5.2.1.7.9 Peroxisome Proliferators. Recently a new class
of carcinogens involving a number of structurally diverse
* ^->~
chemicals (Table CLVI), which induce peroxisome proliferation in
*Peroxisomes are cytoplasmic organelles present in almost all
eukaryotes. They are characterized by their content of catalase
and other oxidases. While their exact function(s) in cells are
unknown, it has been suggested that peroxisomes may play a role
in gluconeogenesis, lipid metabolism, and the production and
degradation of H000 (464, 465).
£ /*
-------
Table CLVI
Structural Formulas of Peroxisome
Proliferators Tested for Carcinogenic Activity
CH3
I
0-C-COOC2H5
CH3
Clofibrate
Nafenopin
N-S02
Tibric acid
Cl
COOH
\
N
CH,
.Cl
S-CH2COOH
Wy-14,643
' /=
N
Cl
0
CH,
S-CH2-C-NH-CH20H
BR-931
-------
775
the liver, has been identified (466-471). Although these
compounds induce hepatomegaly, they possess the therapeutically
exploitable property of bringing-about hypolipidemia (468-470,
472). For example, one of them, clofibrate (ethylchlorophenoxy-
isobutyrate, known as Atromid-S), has been used for more than 15
years for the treatment of hyperlipoproteinemia in the United
States and Europe (472, 473). Many patients have received 2 g
clofibrate daily for periods up to 6 years (473). In an epidem-
iological study of the potential hazard of the long-term use of
clofibrate, it was found that there were more deaths from
malignant tumors of various types among the 5,000 males given
clofibrate than among the control group (474). This observation
is in accord with the results of long-term studies in laboratory
animals. Notably higher incidences of liver and pancreatic
tumors were seen in rats fed 0.5% (v/w) clofibrate for 18-23
months than in the controls (475, 476). Moreover, tumors of the
stomach, kidney, urinary bladder, lung, and parotid gland were
found in the clofibrate treated animals (476). The carcinogenic
action of other peroxisome proliferators in mice and rats has
also been reported. These studies are summarized in Table CLVII
and discussed below. ^» iSlbje. CL Y'f
Reddy et al. (477) observed first in 1976, the development
of hepatocellular carcinomas in 9 of 9 male and 12 of 12 female
acatalasemic mice fed 0.1% nafenopin (2-methyl-2 [p_-(l, 2, 3,4-
tetrahydro-1-naphthyl) phenoxy] propionic acid) in the diet for
12 months and then 0.05% for 8 more months. No liver tumors were
found in the 15 male and 15 female acatalasemic control mice.
-------
Table CLVII
Carcinogenicity of Peroxisome Proliferators
a
Compound
Clofibrate
Nafenopin
Wy-14, 643
BR-931
Tibric acid
Species and strain
Rat, F344
Rat, F344
Rat, F344
Mouse, Cs
Rat, F344
Mouse, Cs
Rat, F344
Mouse, Cs
Rat, F344
Principal organs affected
Liver, pancreas
Liver, pancreas, stomach, kidney,
urinary bladder, lung
and parotid gland
Liver, pancreas
Liver
Liver
Liver
Liver
Liver
Liver
References
(475)
(476)
(478, 479)
(477)
(466)
(466)
(467)
(467)
(467)
All tested by oral administration.
-------
776
Similar observations were made in rats given 0.1% nafenopin in
the diet for 25 months. Eleven of 15 rats (73%) developed
hepatocellular carcinomas, and 3 of 15 rats (20%) developed
pancreatic acinar cell tumors after 18 months (478). The
metastatic growth and transplantability of these nafenopin-
induced tumors were established (478, 479).
The compound, Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-
pyrimidinylthio] acetic acid), given at 0.1% level in the diet
for 6 months and then at 0.5% level for 8 1/2 more months,
induced hepatocellular carcinomas in 18 of 18 acatalasemic mice
(466). Rats fed this compound at 0.1% level in the diet for 16
months also developed hepatocellular carcinomas with an incidence
of 15/15 (466). Some of these Wy-14,643-induced liver tumors
observed in mice and rats metastasized to the lungs.
Recent evidence indicates that two other potent peroxisome
proliferators, BR-931 [4-chloro-6-(2,3-xylidino)-2-pyrimid-
inylthio(N-|3-hydroxyethyl)-acetamide] and tibric acid [2-chloro-
5-(3,5-dimethylpiperidinosulphonyl) benzoic acid], are also
carcinogenic when fed to rats and/or mice (467). Hepatocellular
carcinomas were found in 20 of 20 rats reciving 0.2% and in 7 of
10 rats receiving 0.05% BR-931 in the diet for 16 to 19 months.
Eleven of 12 mice fed 0.2% BR-931 for 19 months also developed
liver tumors, histologically resembling those found in rats.
Tibric acid was given to rats at 0.2% dietary level for 7 1/2
months, 0.1% for 4 months and then 0.5% for 5 months. Thirty of
31 animals that survived the chronic treatment developed
hepatocellular carcinomas. No liver tumors were observed in the
controls.
-------
777
It was suggested that clofibrate may promote the growth of
preexistent cancers in humans (474). In F344 rats, Wy-14,643 and
to a lesser extent clofibrate have actually been found to
potentiate diethylnitrosamine hepatocarcinogenesis (480).
The mechanism whereby the peroxisome proliferators initiate
and/or promote tumorigenesis is unknown. Recently, Warren et al.
(481) suggested that the peroxisome proliferators or their
metabolites neither interact with nor damage cellular DNA. Their
conclusion was based on the findings that: (i) no mutagenic
activity was detected in the Ames test using strains TA98, TA100,
TA1535, TA1537 and TA1538 of Salmonella typhimurium with
clofibrate, nafenopin, Wy-14,643 and BR-931 either in the absence
or presence of the liver microsomal S-9 fraction; and (ii) in
contrast to other known DNA-damaging carcinogens (methylni-
trosourea, benzo[a]pyrene, etc.), the carcinogenic peroxisome
proliferators fail to irreversibly suppress the rate of DNA
replication of lymphocytes, regardless of the presence of a liver
microsomal activating system. The authors indicated the
possibility that peroxisome proliferators might exert their
carcinogenic effect via the epigenetic mechanism suggested for
dioxane (482) and saccharin (364).
The possible relationship between peroxisome proliferation
and liver tumorigenesis induced by these chemicals has been
investigated. It was suggested that carcinogenesis may be
induced by the high intracellular level of H202, generated by
peroxisomal oxidases (431). Feinstein _et_ _al_. (483) noted the
enhancement of hepatocarcinogenesis in acatalasemic mice fed
-------
773
aminotriazole, an inhibitor of catalase. They ascribed this to
the decreased rate of degradation of H202/ which has been shown
to induce chromosome aberrations and DNA repair synthesis in
cultured mammalian cells (484). It is of interest in this
connection that the activity of catalase, a marker enzyme for
peroxisomes, is also decreased in many tumors induced by
peroxisome proliferators (466). Furthermore, increased mitotic
activity has been shown in livers of mice and rats fed Wy-14,643
or nafenopin (466, 477). Reddy et_ _al_. (477) suggested that the
persistently high mitotic activity in liver cells, due to
peroxisome proliferators, may predispose to chromosomal
abnormalities leading eventually to carcinogenesis.
5.2.1.7.10 bis-(Morpholino)- and bis-(N-Methylanilino)-
methane. Owing to their relatively low toxicity and their
capacity to amino-alkylate nucleic acids in cells, many
methylene-bis compounds with a secondary amine of the type
\ /
N-CH2~N have been used as chemotherapeutic agents in the
experimental treatment of tumors (485-487). For instance, the
LD^Q value of bis-(morpholino)-methane in rats is 1,000 mg/kg
and, under acidic condition, it can be easily degraded to produce
the alkylating carbonium-imonium ions (488). In contrast to
compounds that require metabolic activation to form alkylating
intermediates, most direct-acting alkylating agents are generally
regarded as non-carcinogens or only weak carcinogens (489). Bis-
(morpholino)-methane, however, was found to be highly carcino-
genic in rats (488, 489). Upon subcutaneous injection (once
-------
0 N-CH2-N 0
bis-(Morpholino)-methane
N-CHo-N-
I 2 I
CH3 H3C
bis-(N-Methylanilino)-methane
Text-Figure 29
-------
779
weekly at the dose of 50 rag/kg for 40 weeks) to BD strain rats,
the compound gave close to 100% incidence (11/12 and 9/12) of
local sarcomas in 392 to 587 days. One neurosarcoma of the
sciatic nerve and some lung metastases were also observed. The
vegetable oil used as solvent was not carcinogenic in control
experiments.
Insert here Text-Figure 29
Another compound with amino-alkylating activity, bis-(N-
methylanilino)-methane has also been tested for carcinogenic
activity (488). One BD strain rat died 495 days after receiving
a single subcutaneous dose (850 mg/kg) of the compound. Upon
autopsy, a polymorphocellular fibrosarcoma at the injection site,
2 inguinal solid tumors (histologically shown to be squamous cell
carcinomas) and a mammary adenocarcinoma were found. Table
CLVIII summarizes the carcinogenicity data of the above two
compounds. ^ "JSible C-L V///
The induction of tumors in animals administered cancer
chemotherapeutic drugs has been repeatedly demonstrated (490,
491). It is likely that bis-(morpholino)- and bis-(N-methyl-
anilino)-methane induce tumors through a mechanism similar to
that accounting for their cytostatic effect (485-487), that is
amino-alkylation of DNA.
5.2.1.7.11 Some Therapeutically-Used Agents. The long
suspected association between certain pathological conditions and
cancer has led to the concern that drugs used in the treatment of
-------
Table CLVIII. .
Carcinogenicity of bis-(Morpholino) - and bis -(N-Methylanilino)-methane
Compound Species and strain Principal organs affected and route References
bis-(Morpholino)-methane Rat, BD Local sarcoma, brain (s. c.) (488, 489)
bis-(N-Methylanilino)-methane Rat, BD Local sarcoma, mammary (488)
adenocarcinoma (s. c.)
-------
780
cancer may be carcinogenic. Indeed, a number of pharmaceutical
agents have been shown to date to be weak carcinogens in various
animal species (492, 493) and the justification of the use of
'""" "'
these drugs in human therapy has been discussed (493). Below are
briefly reviewed the therapeutically-used agents (Table CLIX)
which have been documented to be carcinogenic. The LD^Q values
and the carcinogenicity studies of these substances are
summarized in Tables CLX and CLXI, respectively. /&pJeS CL
Methotrexate. Methotrexate (4-amino-N -methylpteroyl-
glutamic acid) has been used in the treatment of psoriasis and
various neoplastic diseases including acute leukemia, chorio-
carcinoma, breast cancer, lung cancer, and epidermoid cancer of
the head and neck (494-498). However, because of its adverse
reactions and high toxicity, methotrexate should be used only if
the therapeutic indications are strong, and then with extreme
caution (498-502). Fetal death and/or congenital anomalies have
been reported in women administered methotrexate during pregnancy
(503, 504) as well as in animal studies (505-508).
Rustia and Shubik (509) tested the carcinogenic potential of
methotrexate in a lifetime study in Swiss mice and Syrian golden
hamsters. Seven-week-old mice or hamsters of both sexes were
given methotrexate (10, 8, 5 or 3 ppm for mice and 20, 10 or 5
ppm for hamsters) in the diet until the animals died. No
significant increase in the incidence of tumors was observed in
either species when compared with the controls. Also, Schmahl
and Osswald (510), who administered 1 mg/kg methotrexate
intravenously weekly for 1 year to BR46 strain rats, did not
-------
Table CLIX
Structural Formulas of Therapeutically-Used
Agents Tested for Carcinogenicity
QV-CHz-CHz-Nv
I '
1 -
,N
Chloropromozine
Oxolomine
Pyrimethamine
COOH
I
Methotrexate
/CH3
,CH2-CH2-N
CH3
Methapyrilene
-------
Table CLX.
Acute Toxicity of Some Therapeutically Used Agents
Compound
Methotrexate
Pyrimethamine
Phenytoin
Phenobarbital
Oxolamine
Methapyrilene
Chlorpromazine
Species and route
Rat, i. p.
Mouse, i. p.
Mouse, oral
Mouse, i. p.
Rat, i. p.
Rat, i. v.
Mouse, oral
Mouse, s. c.
Rat, oral
Rat, i. v.
Mouse, oral
Mouse, i. p.
Mouse, s. c.
Mouse, i. p.
Rat, oral
Rat, s. c.
Guinea pig, oral
Rat, oral
Mouse, oral
JLD _ (mg/kg)
50
1 7
94
128
74
280
141
200
400
240
209
350
250
300
351
521
150
375
225
792
References
(501)
(502)
(604)
(604)
(605)
(606)
(607,)
(607)
(608)
(609)
(610)
(611)
(610)
(586)
(612)
(612)
(592)
(608)
(613)
-------
Table CLXI.
Carcinogenicity of Some Therapeutically Used Drugs
Compound
Species and strain
Principal organ affected and route
References
Methotrexate
Pyrimethamine
Phenytoin
Phenobarbital
Oxolamine
Methapyrilene
Chlorpromazine
Mouse, Swiss
Hamster, Syrian golden
Rat, BR46
Mouse, XVII/Bln, AWD
Mouse, A/He
Rat, Sprague-Dawley
Rat, Holtzman
Mouse, C57B1,
C3H/F, SJL/J
Mouse,
Mouse, CF1
Mouse, C3H
Rat, Charles River CD
Rat, Fischer 344
Rat, Sprague-Dawley
Rat, Wistar,
Sprague-Dawley
None (p. o.)
None (p. o.)
None (i. v.)
Lung, liver and skin (p. o.)
Lung (i. p.)
Mammary gland (p. o.)
Hematopoietic system (p. o.)
Hematopoietic system (i. p.)
Hematopoietic system (i. p.)
Liver (p. o.)
Liver (p. o.)
Urinary bladder (p. o.)
Liver (p. o.)
None (p. o.)
Liver? (p. o.)
(509)
(509)
(510)
(491)
(530)
(549, 550)
(552)
(553)
(554)
(563, 564)
(565)
(589)
(593)
(549, 594)
. (598)
-------
731
observe any carcinogenic effect of the drug. However, when given
to XVII/Bln and AWD strain mice of both sexes in the drinking
water, methotrexate (0.1 mg/kg/day) showed definite carcino-
genicity in 18-24 months (491). Predominantly lung adenomas and
carcinomas and, to a lesser extent, hepatomas, skin and
subcutaneous tumors were observed. A high incidence of mammary
adenocarcinomas was also found in a group of 32 female Sprague-
Dawley rats injected intraperitoneally with a combination of
various levels of several anti-cancer drugs including metho-
trexate (490). Furthermore, the enhancing effect of methotrexate
on 7,12-dimethylbenz(a)anthracene induction of malignant
carcinomas of the buccal pouch in Syrian golden hamsters has been
reported (511).
Both the therapeutic and toxic effects of methotrexate are
known to be due to its inhibition of dihydrofolate reductase, an
enzyme required to reduce folic acid in the process of DNA.
synthesis and cellular replication (495, 512). The mechanism by
which this antifol exerts the carcinogenic action, however,
remains unclear. Studies on the ability of methotrexate to
damage DNA in vivo yielded conflicting results (513-520). In a
comparative study of the cytogenetic effects of methotrexate in
human bone-marrow cells and in lymphocytes, Jensen and Nyfors
(518) found that methotrexate has a chromosome-breaking effect on
the former but not the latter, and attributed previous negative
results (514, 515) to the use of lymphocytes which may not be as
sensitive indicators as bone-rnarrow cells. Moreover, they
indicated that the micronucleus test was more sensitive than
-------
782
chromosome analysis for the study of the clastogenic effect of
methotrexate. Their views were supported by Melnyk' _et_ _al_. (519)
who also found chromosome aberrations in bone-marrow cells but
not in cultured lymphocytes or fibroblasts treated with
methotrexate. Moreover, that methotrexate induces micronuclei in
murine bone-marrow erythrocytes has been shown (520).
Pyrimethamine. As with methotrexate, pyrimethamine inhibits
dihydrofolate reductase and suppresses the growth of malaria
parasites (521, 522). Since 1949 this antipyrimidine has been
widely used as a antimalarial agent in Africa and other parts of
the world (522). Recently, the use of pyrimethamine as an
immunosuppressive agent has been reported (523). Although
current views do not support the hypothesis that pyrimethamine
may be implicated in the etiology of Burkitt's lymphoma, there
appears to be some linkage between malarial infection and
neoplastic diseases in African population (522, 524, 525). Early
studies showed that pyrimethamine easily crosses the placental
barrier and is also excreted in mothers' milk (526). Evidence
that this antifol has teratogenic properties has been established
in rats (527, 528) and chick embryos (529).
Stoner et al. (530) conducted a large-scale bioassay to
study the carcinogenicity of food additives and chemotherapeutic
agents in strain A/He mice. They found, after 24 weeks, a
significant increase in the incidence of lung tumors among
animals which received a total dose of 125 mg/kg body weight of
pyrimethamine distributed in 5 intraperitoneal injections over 3
weeks.
-------
733
Chromosomal abnormalities have been observed in vitro in
human peripheral lymphocytes (531) as well as in vivo in bone-
marrow cells of humans (532) or -rats (533) treated with pyri-
methamine. The drug was also found to induce sex-linked
recessive lethals in Drosophila melanogaster (531).
Phenytoin. Phenytoin (hydantoin), an important anti-
epileptic drug, is often used in the management of generalized
tonic-clonic (grand mal.) or complex partial (psychomotor)
seizures (534). Although it is generally considered to be a
relatively safe anticonvulsant, various undersired adverse
reactions have been reported (534-537). Recent observations and
epidemiological studies in the U.S. and European countries
suggest a possible connection between the- use of phenytoin and
the significantly increased incidence of various types of cancers
in patients or birth defects in children (538, 539). Voluminous
reports have appeared confirming the teratogenicity of phenytoin
in animal studies (540, 541-546). The teratogenic effect of
phenytoin is now recognized as the "fetal hydantoin syndrome"
(547, 548).
Phenytoin displayed marginal to moderate carcinogenicity
when bioassayed in rats and mice. Griswold et al. (549, 550)
administered a single dose of 150 mg phenytoin to 20 female
Sprague-Dawley rats by gavage and observed, after 6 months, a
mammary carcinoma in one of the animals. No tumors were found in
the controls. In occasional patients, phenytoin induces
lymphadenopathy which pathologically resembles malignant
lymphomas (551). It is interesting to note that malignant
-------
784
lymphoma appeared in one of the 19 female Holtzman rats which
survived after administration of 0.2% phenytoin in the diet for
44 1/2 weeks (552). Similarly, lymphomas were observed in 12.5%
"--..._'
C57B1, 12.0% .C3H/F and 14.3% SJL/J strain mice fed phenytoin
sodium at the dose of 60 mg/kg body weight daily for .168 days
(553). No tumors were seen in the controls. In a study in which
0.6 mg/animal phenytoin was given intraperitoneally daily, to 50
random bred albino mice for 66 days, 6 animals developed
lymphomas and 4 developed leukemias after 9 months. In the 50
untreated controls, 1 lymphoma and 1 lung adenoma were observed
after 11 months (554).
Marquez-Monter _et_ _a_l_. (555) noted a significant increase in
the incidence of chromosome aberrations in the peripheral blood
leukocytes of 20 epileptic patients treated with phenytoin
sodium. This observation is in accord with the findings obtained
in rats. Abnormal metaphases were found in 26% of bone-marrow
cells of the animals given phenytoin orally at the dose of 250
mg/kg body weight daily for 5 days whereas only 8% was observed
in the controls (556, 557). MacKinney _et_ _al_. (558) reported that
the hydroxylated metabolite of phenytoin, 5-[4-hydroxphenyl]-5-
phenyl-hydantoin, is more potent to inhibit the polymerization of
microtubules and the completion of metaphase of cultured human
lymphoctes; however, hydroxylation does not significantly affect
inhibition of DNA or protein systhesis by phenytoin.
Phenytoin (115 or 145 mg/kg body weight) exhibited no
mutagenic effect in dominant lethal studies conducted in mice
(44). Other systems should be investigated to explore the
possible mutagenicity of this drug.
-------
785
Phenobarbital. Phenobarbital (PB) and its sodium salt are
commonly used as an anticonvulsant, hypnotic and sedative. In
combination with phenytoin or other anticonvulsants, they .are
also, used in the treatment of epilepsy (534). Epidemiological
surveys in various parts of the world have indicated possible
association between treatment with antiepileptic drugs and the
increased cancer incidence or congenital malformations in
offspring (559). Evidence that PB is teratogenic in mice (540,
560), rats (561) and rabbits (562) has been presented.
Previous discussion (Section 5.1.2.7 in Vol. IIB) has
indicated that amytal (5-ethyl-5-isoamylbarbituric acid), a
hypnotic drug closely related to PB, is carcinogenic in mice.
The carcinogenic potential of PB has also been investigated in
rodents. In a life-time study, CF1 mice beginning age 4 weeks
were fed a dietary level of 500 ppm phenobarbital sodium. After
26 months, liver tumors were found in 80% males and 75% females,
campared with 24% and 23%, respectively, in the control animals
(563). Lung metastases were observed in another experiment when
the mice were exposed to 1000 or 3000 ppm PB (563). Similar
findings were made by Ponomarkov et al. (564) who administered
phenobarbital sodium (0.05%) to 4-week-old CF1 mice in the
drinking water. The incidence of liver tumors after 120 weeks
was 78% in treated males and 62% in treated females, compared
with 27% in male and 0% in female controls. Peraino _et_ _al_. (565)
studied the enhancement of spontaneous hepatocarcinogenesis in
mice by dietary PB. Beginning with one- or three-month-old
animals, C3H mice were fed a control diet or a diet containing
-------
786
0.05% PB. After 12 months, a significant increase in the liver
tumor incidence was observed in mice treated with PB as compared
to the controls, regardless of-population density or sex. Based
on the observation that PB did not decrease the degree of
differentiation of the tumors, however, the investigators
suggested that PB only accelerated the expression of spontaneous
liver tumorigenesis.
The above hypothesis was further investigated by the same
laboratory studying the effects of dietary PB on hepatocar-
cinogenesis induced in rats by 2-acetylaminofluorene (AAF) (566-
568). It was observed that simultaneous feeding of AAF and PB
repressed, whereas sequential feeding of the two compounds
enhanced, the hepatocarcinogenic effects of AAF. The protective
effect of PB against hepotocarcinogenesis of other chemicals such
as diethylnitrosamine (569, 570), 4-dimethylaminoazobenzene (571)
or aflatoxin (572) have also been reported. The effects are
ascribed to the induction of microsomal enzymes that detoxify the
carcinogens (573, 574). The enhancement of the tumorigenesis by
PB involved both an increase of the rate at which new tumor loci
appeared and of the growth rate of the tumors. Since stimulatory
effects of PB on macromolecular synthesis and stabilization in
the liver are well established (575-577), the authors speculated
that anabolic changes in liver metabolism by PB treatment may
magnify the consequences of molecular changes produced by prior
exposure to AAF (567). Evidence of promoter effect of PB on
hepatocarcinogenesis by other agents has also been provided (578-
580) .
-------
737
Although PB was reported to be a weak mutagen in Drosophila
melanogaster (581), no chromosome abnormalities were noted in
human leukocytes (582) or fibroblasts (583) exposed in vitro to
high concentrations of PB. There is also no indication that PB .
is mutagenic in the Salmonella.typhimurium strains TA98, TA100,
TA1535 or TA1537 (584, 585).
Oxolamine. Oxolamine (3-phenyl-5-(3-diethylaminoethyl-l, 2, 4-
oxadiazole) is an active antitussive agent. It is also an anti-
inflammatory, analgesic, local anesthetic and antispasmodic agent
(586). Both acute and chronic experiments in mice, rats, and
dogs have indicated that Oxolamine has low toxicity and few side
effects (586, 587). Bladder irritation is, however, a conse-
quence of the administration of high dose.s of the drug (587,
588).
The carcinogenic potential of oxolamine has been extensively
investigated by Barren (589) and evaluated by Price (455). In
1963, Barron (589) observed after 12 months the development of
urinary bladder carcinomas in 32 of 36 rats given by gavage (250
mg/kg body weight), the citrate salt of oxolamine 5 days/week for
15 weeks, and then 350 mg/kg for the following 37 weeks. Tumors
in 22 of the 32 rats were invasive. Diffusive precancerous
lesions in the urinary bladder were also found after 1 year in
dogs administered the drug at the same dose levels and by the
same route. No such lesions were seen during the course of the
studies in the controls of both species. These observations led
to the conclusion that prolonged oral administration of the
citrate salt of oxolamine has a carcinogenic effect on the
-------
788
urinary bladder of the rat and probably of the .dog. Metabolic
studies with oxolamine showed that diethylamine is the metabolite
responsible for the bladder irritation (588, 590, 591). Whether
there is a correlation between the carcinogenic effect and the
irritation of the bladder epithelium has yet to be established.
Methapyrilene. This antihistaminic agent, a common
ingredient in many over-the-counter cold remedies and sleeping
aids, had been used until recently by millions of people in the
United States (592, 593). In a study exploring the possible
formation of carcinogenic N-nitroso compounds in the mammalian
stomach by the reaction of secondary and tertiary amines with
nitrite, Lijinsky and Taylor (594) observed that 9 of 30 Sprague-
Dawley rats developed liver neoplasms after receiving 0.1%
luethapyrilene hydrochloride together with 0.2% sodium nitrite in
the drinking water for 18 months. Control rats treated .with
nitrite only, or untreated, did not develop liver tumors. In
further studies (593) approximately 100% of Fischer rats of both
sexes given 0.1% methapyrilene in the diet with or without 0.2%
sodium nitrite developed hepatocellular carcinomas and
cholangiocarcinomas after 43 to 64 weeks. More than 50% of the
tumors also metastasized to distal organs. Following these
reports, methapyrilene was removed from the market.
Despite the strong carcinogenicity of methapyrilene in the
liver of rats, mutagenic activity was not observed in the Ames
test with or without activation (595).
Chlorpromazine. Because of its psychotropic properties,
this phenothiazine derivative has been extensively used as a
-------
789
tranquil|zer for the management of psychotic disorders and manic-
depressive states. It is also often used as a sedative and
antiemetic. The pharmacology and clinical use of chlorpromazine
have been described (.?_._2_« 498, 596). The drug is not recommended
for pregnant women, since epidemiological studies have indicated
a possible association between maternal drug intake arid
congenital malformations of the infants (597). Fetotoxicity and
decreased performance of the offspring have been demonstrated in
teratogenicity testing of chlorpromazine in rodents (498).
Significant data on the carcinogenicity of chlorpromazine
are scanty. In 1959, a French report (598) described liver
tumorigenesis in rats administered chlorpromazine in the diet.
Nonetheless, the carcinogenicity of the drug is difficult to
evaluate since it is not clear from the report whether p_-
dimethylaminoazobenzene (DAB) was also in the diet. The authors
indicated a slight acceleration of DAB tumorigenesis in the rat
liver by chlorpromazine. Salyamon (493) erroneously quoted the
French report (598) and claimed that: "chlorpromazine when
chronically administered to mice produces hepatomas in almost
100% of the animals". Recent studies showed no tumorigenesis. in
female Sprague-Dawley rats, which received 0.2% chlorpromazine in
the drinking water (594) or 20 mg/rat chlorpromazine
hydrochloride by gavage (549) and were observed for 6 months or
longer. Chlorpromazine did not induce micronuclei (599) nor
chromosome aberrations in vitro (600-602). The compound also has
no mutagenic activity in the Ames test using several Salmonella
strains, in the presence or absence of liver S-9 fraction
-------
790
activation system; mutagenic products, presumably nitrosamines
(603), are formed, however, when chlorpromazine is reacted with
sodium nitrite in acidic solutfoii (595).
-------
791
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606. Leuschner, F., Neumann, W. and Reith, H.: Arzneim-Forsch.
27, 811 (1977). *
607. Balsamo, A., Barili, P.L., Crotti, P., Macchia, B., Macchia,
F., Cuttica, A. and Passerini, N.: J. Med. Chem. 20, 48
(1977).
608. Goldenthal, E.I.: Toxicol. Appl. Pharmacol. 18, 185 (1971).
609. Vohland, H.W., Schirop, Th., Barckow, D., Kreutz, G., and
Streichert, B.: Arch. Toxicol. 40, 211 (1978).
610. Squires, R.F. and Lassen, J.B.: Biochem. Pharmacol. 17, 369
(1968).
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854
611. McColl, J.D., Chubb, F.L., L.ee, C.F., Hajdu, A. and
Komlossy, J. : J. Med. Chem. 6, 584 (1963).
612. Sunshine, I., "Handbook of Analytical Toxicology," Chemical
Rubber Co. Cleveland, Ohio, 1969. p.73.
613. Dehaen, P.: "Drugs in Research," Academic Press, New York,
1964, p. 397.
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855
SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.2.1.7
1. Auerbach, C., Moutschen-Dahmen, M., and Moutschen, J.: Genetic and
Cytogenetical Effects of Formaldehyde and Related Compounds. Mutation
Res. 39, 317-362 (1977).
2. U.S. Environmental Protection Agency: "Investigation of Selected
Potential Environmental Contaminants: Formaldehyde" EPA-560/2-76-009,
Office of Toxic Substances, U.S. Environmental Protection Agency,
Washington, B.C., 1976, 204 pp.
3. U.S. Environmental Protection Agency: "Investigation of Selected
Potential Environmental Contaminants: Acrylonitrile" EPA 560/2-78-003,
Office of Toxic Substances, U.S. Environmental Protection Agency,
Washington, D.C. 1978, 234 pp.
4. International Agency for Research on Cancer: "Some Miscellaneous
Pharmaceutical Substances" IARC Monographs on Evaluation of Carcinogenic
Risk of Chemicals to Man, Vol. 13, Lyon, France, 1977, 255 pp.
5. Schm'ahl, D., Thomas, C. and Auer, R. : "latrogenic Carcinogenesis"
Springer-Verlag, New York, 1977, 120 pp.
6. Mageli, O.L., and Sheppard, C.S.: Peroxides and Peroxy Compounds,
Organic. Kirk-Othmer Encycl. Chem. Tech. 14. 769-820 (1967).
7. J'onsson, N.A. : Chemical Structure and Teratogenic Properties III. A
Review of Available Data on Structure-Activity Relationships and
Mechanism of Action of Thalidomide Analogues. Acta Pharm. Suecica 9,
521-542 (1972).
8. Price, J.M.: Etiology of Bladder Cancer. In "Benign and Malignant
Tumors of the Urinary Bladder" (E. Maltry, Jr., ed.), Chapter 7, Medical
Exam. Publ., Flushing, N.Y., 1971, p. 189-264.
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856
9. National Academy of Sciences: "Saccharin: Technical Assessment of Risk
and Benefits, Part I" PB-292695, National Technical Information Service,
. Springfield, Va., 1978.
10. Thomas, J.A., Darby, T.D., Wallin, R.F., Garvin, P.J., and Martia, L.:
A Review of the Biological Effects of Di-(2-ethylhexyl) Phthalate.
Toxicol. Appl. Pharmacol. 45. 1-27 (1978).
11. Lee, D.H.K., and Flak, H.L. (eds.): "Conference on Phthalic Acid Ester",
Environmental Health Perspectives Experimental Issue No. 3, 1973, 182 pp.
12. Peakall, D.B.: Phthalate Esters: Occurrence and Biological Effects.
Residue Rev. 54. 1-42 (1975).
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NOTES ADDED AFTER COMPLETION OF SECTION 5.2.1.7
Aldehydes and Related Compounds. According to a report by Selikoff and
Hammond (1), the final results (24 months) of a CIIT-sponsored (Chemical
Industry Institute of Toxicology) inhalation study of formaldehyde indicated a
significantly higher incidence (43%) of squamous cell carcinomas of the nasal
cavity in rats exposed to 15 ppra than the incidence (18%) reported at 18
months (see Section 5.2.1.7 ref. 64). Moreover, nasal carcinomas were also
found in 3 rats exposed to 6 ppm and 2 mice exposed to 15 ppm formaldehyde.
Tumors of this type were not observed in matched controls and in 7,000
historic controls. After evaluating the final data, the
authors (1) concluded that the epizootic viral infection seen in rats in this
study did not seem to play a significant role in the development of the nasal
carcinomas. The carcinogenic effect was attributable to formaldehyde expo-
sure. Further evidence for the carcinogenicity of formaldehyde has been
provided by another chronic study recently completed at the Institute of
Environinetnal Medicine, New York University Medical Center (la). Squanous
cell carcinomas of the nasal cavity were previously observed in 25/100 male
Sprague-Dawley rats exposed (544 times, 6 hr/exposure) to formaldehyde (14.6
ppm) and hydrochloric acid (10.6 ppm) over a period of 27 months. Subsequent
studies showed that formaldehyde alone produced about the same incidence of
nasal carcinomas as the combined exposure of formaldehyde and hydrochloride
(which may produce bis-(chloromethyl)-ether; see Section 5.2.1.1.2) indicating
that the carcinogenic effect was due to fornaldehyde.
The rautagenicity of halogenated acroleins has been investigated by Rosen
_et_ _al_. (2). In contrast to the weak or lack of mutagenicity of acrolein in
Ames test, the halogenated derivatives are potent mutagens. The number of
revertants (TA 100) induced per nmole of 2-bromo-acrolein was 700. Metabolic
activation was not required for full activity (which actually decreased in the
presence of microsoines). Crosslinking involving Schiff base formation at the
38
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carbonyl group and Michael addition at the double bond was postulated to be a
possible mechanism of action. The potential carcinogenic risk of acrolein to
humans has been evaluated by an International Agency for Research on Cancer
study group (3), which concluded that inadequate information is available.
Acrylonitrile. The in vitro metabolism of acrylonitrile has recently been
studied by Duverger-Van Bogaert et^ jtl. (4). Four metabolites: cyanoacetic
acid, cyanoethanol, acetic acid and glycolaldehyde were identified. These
findings along with mutagenicity studies led the authors to postulate an in
vitro metabolic scheme involving the formation of a radical species and an
epoxide, as intermediates. Peter and Bolt (5) have demonstrated irreversible
binding of acrylonitrile to rat liver microsomal protein. This reaction does
not require metabolic activation indicating that acrylonitrile may directly
alkylate nucleophiles.
C-Hitroso-compoundi p-Nitrosophenol, a C-nitroso compound, has recently
been found to be mutagenic towards Salmonella typhimurium strain TA 1538 (a
frameshift mutant). No metabolic activation was required. Since most
N-nitroso compounds are mutogenic toward base-pair substitution mutants, it
appears that p-nitrosophenol may exert its genotoxic effect by mechanisms
different from those described for N-nitroso compounds (6).
Phthalate Esters. A number of phthalate esters and related compounds have
been tested for mutagenicity under the National Toxicology Program. Thus far,
the following compounds: butyl benzyl phthalate, di-(2-ethylhexyl) adipate,
di-(2-ethylhexyl) phthalate, diethyl phthalate, phthalic anhydride,
terephthalic acid, and tetrachlorophthalic anhydride have all been found to be
nonmutagenic in Salmonella typhimurium (7). In a paper presented at the
National Toxicology Program/Interagency Regulatory Liaison Group Conference on
Phthalate in June, 1981 (in Washington, D.C.), Kozumbo and Rubin (Johns
Hopkins University) reported that dimethyl and diethyl phthalates were muta-
genic toward Salmonella typhimurium strain TA 100. Inclusion of metabolic
activation system (S9 mix) abolished the activity. Monoraethyl phythalate was ^x
not mutagenic. The ortho-posi^tion of the two methyl groups was crucial for "2/7
^~*"^ N>>~^-/
mutagenic ativity of dimethyl phthalate; both meta- and para- derivatives were
inactive. Shiota et_jLU (8) demonstrated that di-(2-ethylhexyl) phthalate and
di-ji-butyl phthalate, both at high dose levels, were embryotoxic and possibly
teratogenic (borderline level significance) to ICR-JCL mice. The major mal-
formations observed were neural tube defects (exencephaly and epina bifida).
39
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The final results of the NCI/NTP carcinogenic!ty study of di-(2-ethyl-
hexyl)-phthalate (DEHP) are now available (9). The compound was found to be a
liver carcinogen for F344 rats and B6C3F, mice of either sex. The incidences
of hepatocellular carcinomas or neoplastic nodules in the control, low dose
(1/2 high dose) and high dose (maximum tolerated dose) groups were 6, 12, 24%
for male and 0, 12, 26% for female rats. For the mice, the corresponding
incidences of hepatocellular carcinomas or adenomas were 28, 52, 58% for males
and 2, 24, 36% for females. Two related compounds were tested concurrently.
Butyl benzyl phthalate (10) was not carcinogenic toward B6C3F, mice of either
sex but may have been responsible for the increased incidence of leukemia in
female F-344 rats (male rats were inadequately tested due to compound-related
mortality). Di-(2-ethylhexyl) adipate (11) was not carcinogenic toward F344
rats of either sex but was carcinogenic for female and possibly male B6C3F,
mice, causing increased incidences of hepatocellular adenomas or carcinomas
(the incidences were: 26, 41, and 55% for control, low dose, and high dose
males; 6, 38, and 37% for females).
The mechanism of action of phthalate esters remains to be elucidated.
Some suggestive evidence emerged recently that the hepatocarcinogenic action
of certain phthalate esters may be related to their ability to induce peroxi-
some proliferation. This topic is further discussed below.
Peroxisome Proliferators. An International Agency for Research on Cancer
Study Group (12) has recently reviewed the literature on Clofibrate and
Nafenopin. An epidemiological study by Bergstrom _££_£!. (13) presented
several case histories of possible induction of human cancer by Clofibrate;
however, it could not be ascertained at this stage whether the carcinogenic
effect was attributable to Clofibrate treatment. Von Daniken _£t__al. (14) have
recently shown the lack of covalent binding of Clofibrate to rat liver DNA
supporting the view that the drug may exert its carcinogenic action by some
epigenetic mechanism.
Certain phthalate esters and related compounds have been shown to cause a
spectrum of pathological and biochemical changes similar to that produced by
hepatocarcinogenic peroxisome proliferators. Significant induction of hepatic
peroxisome proliferation, hepatomegaly and hypolipidemia was observed in rats
(15-18) and mice (16) treated with di-(2-ethylhexyl)-phthalate (DEHP).
Similar effects were also noted in rats treated with di-(2-ethylhexyl)-adipate
40
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(DEHA), di-(2-ethylhexyl)-sebacate (DEHS), mono-(2-ethylhexyl)-phthalate,
2-ethylhexyl alcohol, 2-ethylhexyl aldehyde or 2-ethylhexanoic acid but not
with diethyl phthalate, phthalic acid, adipic acid, hexyl alcohol, hexyl
aldehyde or hexanoic acid (15, 17). These observations led Moody and Reddy
(17) to suggest that the structural moiety responsible for perosisome proli-
feration may be the branched 2-ethylhexyl side-chain. Considering the diverse
chemical structure of various hepatocarcinogenic peroxisome proliferators,
Reddy (19) postulates that potent hepatic peroxisome proliferators as a class
are carcinogenic. Further studies are needed to test whether this hypothesis
is applicable to all potent peroxisome proliferators.
The mechanism by which peroxisome proliferators trigger carcinogenesis
remains to be elucidated. As discussed in Section 5.2.1.7.9, one possibility
is that carcinogenesis may be related to the elevated intracellular level of
H-Oo generated by peroxisomal oxidases. In this respect, it is interesting to
note that an increase in active oxygen species has also been proposed to be
associated with patients with "Bloom's Syndrom" (BS), an autosomal recessive
human disease characterized by high cancer incidence (as much as 10,000 times
higher cancer risk than normal individuals), growth retardation, immunodefi-
ciency and skin sensitivity to sunlight. Recent studies by Cerutti and asso-
ciates (20-22) revealed that skin fibroblasts from BS patients are deficient
in enzymes (e.g., superoxide dismutase) responsible for detoxifying active
oxygen species and that superoxide radicals generated by UV irradiation are
involved in the chromosomal damages of BS fibroblasts. Since active oxygen
species are continuously generated in most tissues by various pathways, it is
conceivable that a defect in the detoxifying mechanism may lead to an accumu-
lation of active oxygen species which may account for the abnormally high
cancer incidence.
Therapeutically-Used Agents- An International Agency for Research on
Cancer Study Group (12) has recently evaluated the carcinogenic risk of 16
pharmaceutical drugs to humans. Clayson (23) has presented an excellent
overview of drugs and therapeutic procedures investigated as possible human
carcinogens and drugs demonstrated to be carcinogenic in laboratory animals.
41
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References for Section 5.2.1.7 Update
1. Selikoff, I. J., and Hammond, E. C., "Carcinogen!city of Formaldehyde.
Final Report" Report to the American Cancer Society by the Environmental
Cancer Information Unit, Environmental Sciences Laboratory, Mount Sinai
School of Medicine of the City Univeristy of New York, February 25, 1981.
la. Albert, R. E., Sellakumar, A.R., Laskin, S., Kuschner, M., Nelson, N., and
Snyder, C.A.: J. Natl. Cancer Inst. (in press).
2. Rosen, J. D., Segall, Y., and Casida, J. E.: Mutat. Res. 78, 113 (1980).
3. International Agency for Research on Cancer: IARC Monog. 19, 479 (1979).
4. Duverger-Van Bogaert, M., Lambotte-Vandepar, M., DeMeester, C., Rollmann,
B., Poncelet, F., and Mercier, M.: Toxicol. Letters 7, 311 (1981).
5. Peter, H., and Bolt, H. M.: Xenobiotica 11, 51 (1981).
6. Gilbert, P., Rondelet, J., Poncelet, F., and Mercier, M.: Food Cosmet.
Toxicol. 18, 523 (1980).
7. National Toxicology Program: NTP Technical Bulletin, Issue Number 4,
p. 7, April, 1981.
8. Shiota, K., Chou, M. J., and Nishimura, H.: Environ. Res. 22, 245
(1980).
9. NCI/NTP: "Carcinogenesis Bioassay of Di-(2-ethylhexyl) phthalate" DHHS
(NIH) publ. No. 81-1773, National Cancer institute/National Toxicology
Program, Bethesda, Maryland/Research Triangle Park, North Carolina, 1981.
10. NCI/NTP: "Bioassay of Butyl Benzyl Phthalate for Possible Carcino-
genicity" DHHS (NIH) publ. No. 80-1769, National Cancer
Institute/National Toxicology Program, Bethesda, Maryland/Research
Triangle Park, North Carolina, 1981.
11. NCI/NTP: "Bioassay of Di-(2-ethylhexyl) adipate for Possible
Carcinogenic!ty" DHHS (NIH) Publ. No. 80-1768, National Cancer
Institute/National Toxicology Program, Bethesda, Maryland/Research
Triangle Park, North Carolina.
12. IARC: IARC Monographs on the Evaluation of the Carcinogenic Risk of
Chemical to Humans, Vol. 24, "Some Pharmaceutical Drugs," International
Agency for Research on Cancer, Lyon, 1980, 337 pp.
13. Bergstrom, I., Boethius, G., and Rydstrom, P. 0.: Lakartidningen 76,
2538 (1979).
14. Von Daniken, A., Lutz, W. K., and Schlatter, C.: Toxicol. Letters 7, 311
(1981).
15. Lake, B. G., Gangolli, D., Grasso, P., and Lloyd, A. G. : Toxicol. Appl.
Pharmacol. 32, 355 (1975).
42
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16. Reddy, J. K., Moody, D. E. , Azarnoff, D. L., and Rao, M. S.: Life Sci.
_1^, 941 (1976).
17. Moody, D. E., and Reddy, J. K.: Toxicol. Appl. Pharraacol. 45, 497
(1978).
18. Canning, A. E., and Dallner, G.: FEES Letters 130, 77 (1981).
19. Reddy, J. K.: Hepatic Peroxisome Proliferative and Carcinogenic Effects
of Hypolipidemic Drugs, jn "Drugs Affecting Lipid Metabolism" (R.
Fumagalli, D. Kritchevsky and R. Paoletti, eds.) Elsevier/North Holland,
The Netherlands, 1980, p. 301.
20. Zbinden, I., and Cerutti, P.: Biochem. Biophys. Res. Commun. 98, 579
(1981).
21. Eraerit, I., and Cerutti, P.: Proc. Natl. Ac ad. ScjL. USA _78_, 1868 (1981).
22. Hirschi, M., Netrawali, M. S., Reiasen, J. F., and Cerutti, P. A.: Cancer
Res. 41, 2003 (1981).
23. Clayson, D. B.: Therapeutic Agents and Procedures in Cancer Induction,
In "Carcinogens in Industry and the Environment" (J. M. Sontag, ed.)
Chapter 12, Dekker, New York, 1981, p. 535.
43
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