CURRENT AWARENESS DOCUMENT
SAFROLE AND ITS ALKENYLBENZENE CONGENERS
SAFROLE, ESTRAGOLE, AND RELATED COMPOUNDS
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by
Yin-Tak Woo, Ph.D., D.A.B.T.
David Y. Lai, Ph.D.
Science Applications Internation Corporation
8400 Westpark Drive
McLean, Virginia 22102
EPA Contract No. 68-02-3948
SAIC Project No. 2-813-07-409
EPA Project Officer and Scientific Editor
Joseph C. Arcos, D.Sc.
Extradivisional Scientific Editor
Mary F. Argus, Ph.D.
June 1986
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5324 Safrole, Estragole and Related Compounds
53241 Introduction
Safrole (3,4-methylendioxy-1-allylbenzene), estragole (4-methoxy-l-allyl-
benzene) and related compounds constitute a relatively new class (alkenylben-
zene congeners) of carcinogens of considerable environmental importance A
variety of ring-substituted derivatives of allylbenzene* and propenylbenzene*
are found in many edible plants and spices and were or are still used as com-
ponents of pesticides, food additives and pharmaceutical preparations. The
types of edible plants or spices which contain naturally occurring alkenylben-
zene compounds include sassafras, tarragon, sweet basil, cloves, anise,
nutmeg, sweet bay, parsley, parsnip, carrots, bananas, black pepper and pro-
cessed tobacco (see Section 53245) The tendency of modern cuisine to use
spice flavors in the more concentrated form of oleoresins, extracts and essen-
tial oils (volatile oils obtained by steam distillation and/or solvent extrac-
tion) is of some concern because of the much higher concentration of alkenyl-
benzene compounds in these preparations Besides natural occurrence, there is
also evidence that treatment of oranges with several abscission agents**
(e g , eycloheximide, ^lyoxal diamine) can cause the appearance of six
alkenylbenzene compounds (e g , eugenol , elemicin) in orange juices and
essential oil (see Section 53245) Cinnamyl compounds are closely related
to alkenylbenzene compounds, they may arise from the metabolism of both
allylbenzene and propenylbenzene compounds A number of cinnamyl compounds
*In this section, the term "propenylbenzene" is used exclusively to indicate
"1-propenylbenzene " The term "allylbenzene" is used to denote
"2-propenylbenzene" in order to avoid confusion
**For explanation of the use of abscission agents, see Section 53245
340
-------
(e g , cinnamaldehyde, com feraldehyde, cinnamyl c innatnate) occur naturally in
edible plants and in wood Several synthetic cinnamyl compounds are used as
food additives and fragrances
The carcinogenicity of safrole was first discovered in 1960-1961 when
three independent studies by the U S Food and Drug Administration (1),
Homburger _et_ _al_ (2) and Abbott et al (3) presented evidence of tumor induc-
tion or preneoplastic changes in the liver of rats receiving high doses of
safrole in the diet The discovery immediately led to the banning of its use
as a flavoring agent in soft drinks (eg , root beers) and as a food addi-
tive Since these initial reports, safrole has been established to be a
relatively weak hepatocarcinogen in adult animals and a moderately active
hepatocarcinogen in preweanling male mice Several closely related compounds
(e g , dihydrosafrole, /3-asarone) have also been found to be carcinogenic by
the U S Food and Drug Administration (4-6) Since 1973, an extensive series
of systematic studies on alkenylbenzene compounds has been carried out by the
Millers and their associates at the University of Wisconsin Close to 50
metabolites, derivatives or analogs of safrole and estragole have been tested
for carcinogenic and/or mutagenic activity These studies have not only
identified several additional naturally occurring carcinogenic alkenylbenzene
compounds, but have also led to a better understanding of the structure-
activity relationships of these compounds and the role of metabolic activation
to electrophilic intermediates in the mechanism of chemical carcinogenesis
53242 Physicochemical Properties and Biological Effects
532421 PHYSICAL AND CHEMICAL PROPERTIES
The physical and chemical properties of safrole and related naturally
occurring and synthetic compounds (7-17) and cinnamyl compounds (18) have been
341
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described in various publications The structural formulas of the alkenylben-
zene and cinnamyl compounds tested for genotoxicity, and the physical proper-
ties of some of these compounds, are depicted and summarized in Tables LVII
and LVIII, respectively Derivatives of propenylbenzene exist in two
geometric isomeric forms Naturally occurring anethole and isosafrole are
predominantly in the trans isomeric form, whereas asarone is mainly in the cis
(p-) isomeric form Safrole and related naturally occurring alkenylbenzene
compounds are colorless or slightly yellow, oily liquids with the characteris-
tic odor of the plants from which they have been extracted They are prac-
tically insoluble in water, but are miscible with most organic solvents
Safrole may be chemically oxidized to yield piperonyl acrolein (3,4-methylene-
-------
0-CH2
.0
0-CH2
,0
0-CH2
,0
H2C-CH=CH2
1' 2' 3'
HC=CH-CH3
Isosafrole*
^
i 'R CHj-CH2-CH2
H2C-(!;H-CH3 CH2(OCH2CH2)2OQ,H9
Mynsticm*(R, ^
Dill apiol*(R, ^
Porsley apiol*(RrR3=CH30-,R2=H-
DihydrosafrolelR^H-l
Piperonyl sulfoxide
(R=n-C8H(7-S-)
Piperonyl butoxide
0
OCH,
H2C-CH=CH2
OCH,
H2C-CsCH
Estrogole*(RrR2-H-l trans Anethole*(R^RrH-,R2=CH3-) 2',3'-Dehydroestragole
MethyleuqenollR, =CH30-, R2--H-) a-Asarone*(R--CH30-, R,--H-,R2=CHj-)
Elemicin*lR, :R2: CH30-)
OH
OH
H3
.OCH.
H2C-CH=CH2
Eugenol*
* Naturally 0(t,uffi/iy
HOCH-CHj
Isoeuqenol*
HC=CH-CH2-R
Cmnumyl alcohol (R=HO-)
Cinnamyl anlhranilale
(R-2 arninobenzoate)
H2C-CH=CH2
l-Allyl-4 methoxynophlhalene
OH
Sesamol*
H2C-CH=CH2
1-Allylbenzene
'P
HC=CH-C^
H
Cmnamaldehyde*(R| =R2rR3rH-)
Coniferaldehyde*(R| ;CH30-, R2=HO-, R3-H-)
Smapaldehyde*(Rr R3:CH30-, R^HO-)
3,4,5-Tnmethoxycmnamaldehyde (H, =R<,-l
-------
Table LVIII
Physico-Chemieal Properties of Safrole and Related Alkenylbenzene Compounds
Compound
Safrole
Isosaf role
Dihydrosaf role
Piperonyl butoxide
Est ragole
t rans-Anet hole
3 -Asarone
Fugenol
Linndmyl alcohol
Cinnamaldehyde
t rans-Cinnamic acid
m.p b p
11°C 232-234°C
6 8°C 253°
228°
180°
( 1 mm
216°
C
C
C
Hg)
C
21 4°C 81°C
(2 3 mm
62°C 296°
-9 2°C 255°
33°C 257°
-7 5°C 252°
13J°C 300°
Hg)
C
C
C
C
C
Density
d£° = 1 096
d|° = 1 122
d|° = 1 0695
d|5 = 1 06
dj1 = 0 9645
d|° = 0 9883
d£° = 1 0664
d]l « 1 0397
d^jj = 1 1102
d£ = 1 2475
Re frac t ive
index
1
1
1
1
I
1
1
1
1
1
5383
5782
5187
50 (
5230
5614
5719
5410
5758
6195
(20°
(20°
(25°
20°C)
(17
(20°
(11°
(20°
(33°
UV absorpt ivit y
/\max * tlM
C) 236 (257), 285 (234)
C) 267 (716), 305 (329)
C)
Reference
(7)
(7
(7
)
)
(19)
5°C)
C) 259 (22,300)
C)
C)
C)
(20°C)
(1
(1
(1
(1
(1
1)
1)
1)
1)
8)
(18)
(1
8)
-------
The alkylating activity of a number of alkenylbenzene and cinnamyl com-
pounds and their derivatives has been extensively studied in view of the well
known role of alkylation in mutagenesis and carcinogenesis. Eder et al (21)
found safrole, isosafrole, eugenol and trans-cinnamyl alcohol inactive as
alkylating agent using 4-(j>-nitrobenzyDpyridine (NBP) as the nucleophilic
acceptor Miller and Miller and their associates (12, 14, 15, 17, 22-25) have
studied the in vitro reaction of various metabolites and derivatives of
safrole and related compounds with nucleophiles, such as nucleosides and sulf-
hydryl compounds Using guanosine as the nucleophile, the Millers' group
found that the relative electrophilicity of seven safrole metabolites and
derivatives follows the order I'-oxo- » I'-acetoxy- > 1'-acetoxy-2' ,3'-
epoxy- > 1'-hydroxy-21,3'-8poxy- > 2',3'-epoxy- >_ 1'-oXo-2',3'-epoxy-
> (inactive) I'-hydroxy- (see Table LIX) A similar ranking was observed for
the four estragole derivatives tested Judging from the time course of reac-
tion, the I'-oxo- and I'-acetoxy- derivatives are fast-acting with most of the
reaction completed within a few hours, whereas the epoxide derivatives react
slowly and steadily and may not reach the maximum level even after 96-97
hours Both the I'-oxo- and I'-acetoxy derivatives of safrole and estragoLe
are highly unstable, the tj/o of 1'-acetoxysafrole and 1'-acetoxyestragole in
aqueous solution is 1 8 mm. (26). A comparative study using glutathione as
the nucleophile indicates that 1'-oxosafrole is a "soft" electrophile while
1'-acetoxysafrole is a "hard" electrophile (23) 1'-Oxosafrole is expected
to prefer reacting with "soft" nucleophiles (eg, GSH and other mercapto com-
pounds) before reacting with "hard" nucleophile (e.g., oxygen and ammo groups
of purme and pyrimidme bases of DNA)
343
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Table LIX
Electrophilie Reactivities of Derivatives of Safrole
and Related Compounds with Guanosine3
Compound
1 '-Oxosafrole
1 ' -Ace toxysaf role
1 '-Ate toxysaf role- 2 ' ,3 '-oxide
l'-Hydroxysafrole-2' ,3 '-oxide
Safrole-2' ,3'-oxide
1 '-Oxosafrole-2' ,3 '-oxide
1 '-Hydroxysafrole
3 '-Acetoxyisosaf role
3 '-Hydroxyisosafrole
2 hr
-55
11 8, -20
2
2
-0 5
-0 5
<0.1
0 2
<0 1
Incubation time
17 hr 21-24 hr 96-97 hr
-80 80
20 25
15 44
10 20
58 20
48 13
1 '-Oxoestragole
1 '-Acetoxyestragole
l'-Hydroxyestragole-2' ,3'-oxide
Estragole-2' ,3'-oxide
1 '-Acetoxy-l-allyl-4-raethoxy-
naphthalene
1 '-Acetoxyallylbenzene
16 -80
20 20 -25
n.t
0.1
90
25
15, 27
20
aSummarized from the data of Borchert et_ al^. (12), Drinkwater _ejj_ _al_ (14),
Wislocki ^t__al_. (15), Miller ^^aU (22) and Phillips £t_ _al_. (17) The
numbers shown are the percentage of nucleosides (*• C-labeled) that have
reacted with the compound specified.
Not detected using guanosine However, in another comparative test using
inosine as the nucleophile, 1'-acetoxy-l-allyl-4-raethoxynaphthalene is a more
active electrophilic reactant than 1'-acetoxysafrole and 1'-acetoxyestragole
-------
532422 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY
Toxic ity The basic acute and subacute pharmacological and pathological
effects of safrole and isosafrole in animals were first reported in 1894 and
1895 by Heffter (27, 28) Several cases of nonfatal human poisoning by
safrole and oil of sassafras (which is 80% safrole) were recorded in the lite-
rature (29, 30) The early toxicity data on safrole and related compounds in
humans and animals were reviewed by Jacob (31) and Leidy (32) in 1958
Beginning in the late 1950's and early 1960's, the U S Food and Drug
Administration undertook an extensive series of toxicity studies (4, 33-36) on
various food additives and fragrances which included many alkenylbenzene and
cinnamyl compounds The acute oral LDcQ data of some of these compounds are
summarized in Table LX There appears to be few clearly discernible struc-
ture-toxic ity relationships among these compounds One consistent observation
is that the saturated propylbenzene congeners (e g , dihydrosafrole, dihydro-
anethole, propylbenzene) are less toxic than the corresponding unsaturated
allylbenzene (e g , safrole, estragole, allylbenzene) and propenylbenzene
(e g , isosafrole, anethole, propenylbenzene) compounds (35) For propenyl-
benzene congeners (which exist in two geometric isomeric forms), there is some
evidence for a substantial positional effect on the acute toxicity of the com-
pounds The cis-isomer of anethole, for example, is about 10-30 times more
toxic than the trans-isoraer when administered intraperitoneally to rodents
(39, see, also 11, 40) The most common acute toxic sign of the alkenylbenzene
compounds is CNS depression (35) A number of alkenylbenzene compounds
appears to be capable of producing psychoactive effects (41, 42) and it is
believed that myristicin and/or elemicin may be responsible for the hallucino-
genic effect of nutmeg The liver is the most affected organ in subacute and
chronic studies, the pathological changes observed include hepatic cell enlar-
344
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Table LX
Acute Toxicity of Safrole and Related Compounds by Oral Administration
Compound
Safrole
Isosafrole
Dihydrosafrole
Piperonyl butoxide
Estragole
Anethole
Calamus oil ( g-asarone)
Eugenol
Cinnamaldehyde
Cinnarayl anthranilate
Spec IBS
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Guinea pig
Rat
Mouse
Rat
Guinea pig
Rat
Guinea pig
LD50 (rag/kg)
2,350
1,950
2,470
1,340
3,700
2,260
8,300
11,500
1,250
1,820
3,050
2,160
2,090
777
3,000
2,680
2,130
2,220
1,160
Reference
(35, 36)
(35, 36)
(36)
(36)
(36)
(36)
(37)
(37)
(35)
(35)
(35)
(35)
(35)
(35)
(35,
(35,
(35,
(35)
(35)
36)
36)
36)
Rat
>5,000
(38)
-------
gement and various degrees of focal fatty metamorphosis, bile duct prolifera-
tion and architectural irregulatory, and for some of the compounds, focal
necrosis and fibrosis, hyperplasia and neoplasia (4, 36)
Effects on microsomal mixed-function oxidases Safrole, isosafrole,
dihydrosafrole, piperonyl butoxide and several other methylenedioxyphenyl
(MDP) compounds have a dual effect on microsomal mixed-function oxidases The
immediate effect is interaction with cytochrome P-450 resulting in inhibition
of mixed function oxidases, whereas the delayed effect is the induction of new
synthesis of cytochrome P-448 or P-450 leading to an increase in mixed func-
tion oxidase activity Owing to its pharmacological and toxicological signi-
ficance, this subject has been extensively studied. Readers are referred to
reviews by Hodgson and Philpot (43) and loannides et al (44) for details
The acute inhibition of mixed function oxidases was the basis for the develop-
ment of MDP compounds (e g , piperonyl butoxide, piperonyl sulfoxide) as
synergists for insecticides (45, 46) The mechanism of inhibition is believed
to involve metabolic activation of MDP compounds to reactive intermediate(s)
which form a ligand complex with the heme group or bind covalently to the pro-
tein moiety of cytochrome P-450 Various reactive intermediates (e g ,
benzodioxolium ion, free radical, carbanion, carbene) have been postulated, at
present, the carbene intermediate (see Section 532442) is the most widely
accepted Paradoxically, the formation of metabolite-cytochrome P-450 complex
(most likely carbene ligand complex) appears to be the initiating event for
trigerring the induction of new cytochrome P-450 synthesis (47) Induction of
mixed-function oxidases by MDP compounds may occur in the liver, as well as in
extrahepatic tissues such as intestines and kidney The types of cytochrome
P-450 induced are similar to those induced by phenobarbital and 3-methylchol-
anthrene, there is also evidence for the induction of a novel type of
345
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cytochrome P-450 by isosafrole (48-50) A recent structure-activity relation-
ship study by Cook and Hodgson (51) showed that the raethyLene carbon (of the
methylenedioxy group) plays an important role in cytochrome P-450 induction,
substitution of one or both methylene hydrogen with methyl group(s) completely
abolishes metabolite-cytochrome P-450 complex formation and subsequent cyto-
chrome P-450 induction
Mutagenicity The mutagenicity of safrole and related compounds has been
studied in a variety of test organisms ranging from phages, bacteria, and
yeasts, through higher plants and drosophila, to mammalian cells Safrole was
one of the compounds selected by an International Collaborative Program for
testing in over 20 different short-term tests for careinogenicity, the results
of these studies have been published in a recent monograph (52) The
following discussion focuses only on mutagenicity studies using the Ames
Salmonella test
Close to 50 derivatives and structural analogs of safrole and estragole
have been assayed in the Ames Salmonella test The results of these studies
are summarized in Table LXI About half of these compounds were shown to be
mutagenic in one or more studies, however, for some of these compounds, con-
flicting results were reported by different investigators In some cases, the
discrepancy may be due to the use of different methods (plate incorporation
vs liquid suspension) or criteria for considering positive results Vir-
tually all the mutagenic compounds are base-pair substitution mutagens (i e.,
positive in tester strains TA100, TA1530 or TA1535), with a few compounds
(1'-acetoxysafrole, 1'-oxosafrole-2 ,'3,-oxide, 1'-acetoxyestragole and
1'-acetoxy-1-allyl-4-methoxynaphthalene) displaying weak frameshift activity
(14, 16) As the data in Table LXI indicate, compounds with saturated side
chain (e g , 2' ,3'-dihydro-2' ,3'-dihydroxysafrole, dihydrosafrole, piperonyl
346
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Table LX1
Mutagenicity of Safrole and Related Alkenylbenzene Compounds in the Ames Test
4 pages
Compound3
Safrole
1 '-Hydroxysaf role
1 '-Acetoxysaf role
1 '-Oxosafrole
Safrole-21 ,3'-oxide
r-Hydroxysafrole-21 ,3'-
oxide
l'-Acetoxysafrole-2' ,3'-
oxide
r-Oxosafrole-21 ,3'-
oxide
Safrole metabolite I
Safrole metabolite IId
Safrole metabolite IIId
2' ,3 '-Dihydrodihydroxy-
Without activation
Plate Liquid
incorporation suspension
- (16, 53-59) - (21, 56, 60)
- (14, 53, 55, 57) - (60)
+ (16)
- (57) + (60)
+ (14, 53, 55)
- (55)
+ (16, 55, 56)
+ (16, 55, 56)
+ (16, 55)
+ (16, 55)
- (56)
- (56)
- (56)
- (54)
With activation
Plate Liquid Carcino-
incorporat ion suspension geniLity
- (16, 53, 55-59) - (16, 21, 59, 60) +
+ (56, 61)
- (14, 53, 55, 57) - (60) +
+ (16)
- (55, 57) +
- (55) -, ±
* (16, 55)c -e
+ (16, 55)c +
+ (16, 55)c
+ (16, 55)c n t
- (56) - (56) n t
- (56) * (56) n t
- (56) - (56) n t
n t
sa f role
-------
Table LXI (continued)
f 4 pages
a
Compound
Myr ist ic in
Isosaf role
3'-Hydroxyisosaf role
3'-Acetoxyisosaf role
Dihydrosafrole
Piperonyl sulfoxide
Piperonyl butoxide
Estragole
1 '-Hydroxyestragole
1 '-Acetoxyest ragole
Estragole-21 ,3'-oxide
1 '-Hydroxyestragoie-
2' ,3'-oxide
Methyleugenol
Methyleugenol-21 ,3'-
Without activation
Plate Liquid
incorporation suspension
- (62)
- (54, 55, 59) - (21)
- (55)
- (55)
- (55)
- (63)
- (63, 64)
- (54, 58, 59)
w+ (16)
- (14)
+ (16)
+ (14)
+ (16, 54)
+ (16)
- (54, 59) - (65)
+ (54)
With activation
Plate Liquid Carcino-
incorporat ion suspension genicity
- (62)
- (55) - (21, 59)
- (55)
- (55) ±
- (55) +
- (63) +
- (63, 64)
- (58) +
w* (16)
- (14) *
+ (16)
n t
* (16)c -e
+ (16)c
- (59, 65) -f
n t
ox ide
-------
4 pages
Table LXI (continued)
Compound3
trans-Anethole
3'-Hydroxy-trans-
ane thole
Ot-Asarone
B-Asarone
Eugenol
Eugenol-21 ,3'-oxide
Eugenol metabolite II
Eugenol metabolite III
Isoeugenol
Allylbenzene
Allylbenzene-2' ,3'-oxide
1 '-Acetoxyal lylbenzene
1 '-Acetoxy-1-al lyl-4-
Without activation
Plate Liquid
incorporation suspension
- (58, 59)
w+ (16)
- (16)
- (66)
- (66, 67)
- (16, 54, 56, 58, 59) - (21, 64, 68)
+ (16, 54)
- (56)
- (56)
- (59, 66)
- (54) - (21)
+ (54)
w+ (14)
+ (14)
With activation
Plate Liquid Carcino-
incorporat ion suspension genicity
- (58) w+ (59, 66)
w+ (16)
+ (16)
- (66) n t
- (66) +
+ (67)
- (16, 56, 58) - (21, 56, 69, 64, 68) -
+ (16)c ±, -e
- (56) - (56) n t
- (56) - (56) n t
- (66) - (59) n.t
- (21) n t
n t .
n t
n t
methoxynaphthalene
Cinnamyl alcohol
- (21, 69)
- (21 , 69)
n t
-------
Table LXI (continued)
,1 4 pages
Compound3
Without
Plate
mcorporat ion
act ivat ion
Liquid
suspension
With
Plate
incorporat ion
act ivat ion
Liquid
suspension
Carcino-
genic it y
Coniferyl alcohol - (70)
Cinnamaldehyde
a-Chlorocinnamaldehyde
a-Bromocinnamaldehyde
d-Methylc innamaldehyde
Cinnamic acid - (70)
Cinnamyl anthranilate
Deoxypodophyllotoxin - (70)
- (70)
(21, 69, 71, 72)
(64)
(71)
(71)
(71)
(70)
(73)
(70)
(21, 69, 71, 72)
(64)c
(71)c
- (71)
- (73)
n t
n.t
n t
n t
n t
aSee Table LVII for structural formulas, w+ = weakly active
Modified Ames test Compounds are preincubated for 20 minutes with the bacteria in liquid media before plating on agar
plate.
cMutagenicity of the compound is reduced in the presence of metabolic activation system
The chemical names for these compounds are "Safrole metabolite I," 3-N,N-d imethylamino-l-O1 ,4'-methylenedioxy-
phenyl)-l-propanone, "Safrole metabolite II," 3-piperidyl-l-(3',4'-methylenedioxyphenyl)-l-propanone, "Safrole
metabolite III," 3-pyrrolidinyl-I-(3',4'-methylenedioxyphenyl)-l-propanone, "Eugenol metabolite II," 3-piper idyl-l-(3'-
methoxy-4'-hydroxyphenyl)-l-propanone , "Eugenol metabolite III," 3-pyrrolid inyl-l-(3'-methoxy-4'-hydroxyphenyl)-!-
propanone
eActive iis tumor initiator
-------
butoxide, piperonyl sulfoxide) are all inactive Most alkenylbenzene
congeners are inactive as such with some of them weakly mutagenic after
metabolic activation In contrast, all epoxides of the alkenylbenzene
congeners tested are moderately active direct-acting mutagens The relative
mutagenic potency of 2' ,3'-epoxides follows the order safrole-2',3'-oxide >
estragole-2',3'-oxide > eugenol-2',3'-oxide > methyleugenol-2',3'-oxide =
allylbenzene-2',3'-oxide (54, 55) Among the I'-oxidized derivatives of
allylbenzene congeners, only the I'-acetoxy derivatives display direct-acting
mutagenic activity, their relative mutagenic potency follows the order
1 '-acetoxy-l-allyl-4-tnethoxynaphthalene > 1 '-acetoxyestragole _>_ I'-acetoxy-
safrole » 1'-acetoxyallylbenzene (14) 3'-Acetoxyisosafrole , an isomer of
1'-acetoxysaf role , is not mutagenic 1 '-Hydroxy* derivatives of allylbenzene
congeners are either inactive or weakly mutagenic, but may be further acti-
vated by S-9 mix The only I'-oxo derivative (1'-oxosafrole) tested is
nonmutagenic A number of I'-oxidized derivatives of safrole-2' ,3'-oxide and
estragole-2',3'-oxide are also direct-acting mutagens, but their potency is
substantially lower than that of unoxidized 2',3'-oxides (16, 55) A compari-
son of the direct-acting mutagenic activity of epoxides and 1'-oxidized
derivatives of safrole, estragole and related compounds with their electro-
philic reactivity toward guanosine or inosine (see Table LIX) indicates a very
good correlation, with the notable exception of 1'-oxosafrole (which is highly
reactive but nonmutagenic) As discussed in Section 532421, I'-oxo-
safrole is a very short-lived "soft" electrophile It is probably degraded or
reacts with noncritical cellular nucleophiles before it can reach DNA
Among the various individual compounds, safrole has been consistently
shown to be nonmutagenic in the plate incorporation assay with or without
metabolic activation In modified Ames test, in which the chemical is pre-
347
-------
incubated in liquid suspension for 20 minutes before plating, two separate
studies (56, 61) showed positive results, however, these findings were not
confirmed by other investigators (see Table LXI) 3-Piperidyl-l-(3',4'-
methylenedioxyphenyl)-l-propanone ("safrole metabolite II") was also shown to
be positive in the liquid suspension assay system, but negative in the plate
incorporation system (56), no confirmatory data are available Estragole and
trans-anethole are the only two alkenylbenzene compounds that were reported to
be mutagenic without activation in one study (16), however, the weak direct-
acting activity was not detected in several other studies (54, 58, 59) With
metabolic activation, t rans-anethole is weakly or marginally active, a clear-
cut dose-response relationship was demonstrated using a liquid suspension
assay (59) (% -Asarone was shown to be inactive in one study (66) but clearly
mutagenic in TA100 after metabolic activation with S-9 in another study
(67) In the latter study, three commercial calamus-containing drugs, known
to contain p-asarone, were also mutagenic in TA100 after activation
Among cinnamyl compounds tested, cinnamaldehyde is the only compound
which displays potential mutagenic activity Cinnamaldehyde has been shown to
be a direct-acting mutagen in TA100 in one study (64), however, this activity
was not observed in several earlier studies (21, 69, 71, 72) Whereas the
mutagenicity of cinnamaldehyde may be debatable, its c\-bromo- and o(-chloro-
denvarives are potent direct-acting mutagens for TA100 (71) The greatly
enhanced mutagenicity is attributed to an increase in the electrophilicity of
the carbon at the ^-position to the aldehyde function, as a result of substi-
tution with electron-withdrawing (-1) halogen at the
-------
A comparison of the mutagenicity of safrole and related compounds with
their careinogenicity shows a notable number of discrepancies in the correla-
tion (see Table LXI) Several carcinogenic compounds (e g , safrole, dihydro-
safrole, piperonyl sulfoxide, methyleugenol, /^-asarone) are either inactive
or not consistently mutagenic On the other hand, some compounds which are
clearly mutagenic, such as safrole-2',3'-oxide , 1'-acetoxysafroie-2',3'-oxide,
estragole-2',3'-oxide, 1'-hydroxyestragole-2' ,3'-oxide and eugenol-2',3'-oxide
are either not carcinogenic or active only as tumor-initlators The lack of
correlation may be due, at least in part, to the requirement of sulfatation in
the metabolic activation of allylbenzene congeners to ultimate carcinogens
(see Section 532441) The requirement of cytosol for the metabolic acti-
vation of o(,/3-unsaturated carbonyl compounds and their corresponding allylic
alcohols to mutagens (69) may also be a factor in explaining the inconsistency
between the results yielded by the plate incorporation and the liquid suspen-
sion assay systems for some of the compounds The existence of mutagenic non-
carcinogens may be related to the inability of reactive epoxides to reach
target macromolecules Alternatively, these results may suggest that somatic
cell mutation alone is insufficient to bring to completion the process of car-
cinogenesis
Teratogenicity Very little information is available on the terato-
genicity of safrole and related compounds Technical grade piperonyl butoxide
has no significant teratogenic effects in rats given daily oral doses of up to
500 mg/kg body weight of the compound from day 6 to 15 of gestation (74,
75) In a three-generation reproduction study, the progeny of rats fed diets
containing 100 or 1,000 mg/kg body weight technical grade piperonyl butoxide
showed no adverse effects However, higher doses of 10,000 and 25,000 mg/kg
caused marked reduction in pregnancies and complete infertility, respectively
349
-------
Several cinnamyl compounds used as food or fragrance additives, have been
tested in chick embryos as a screening test for teratogenicity Verrett et
al. (76) found both cinnamyl anthranilate and 0<-methylcinnamaldehyde to be
not teratogenic in developing chick embryos at doses of up to 10 mg/egg A
related compound, methyl anthranilate, was teratogenic, causing skeletal
anomalies which included micromelia and phocomeiia Abramovici and Rachmuth-
Roizman (77) tested a variety of &, /3-unsaturated aldehydes and alcohols,
including cinnamaldehyde and cinnamyl alcohol, in chick embryos. Both
cinnamyl compounds were teratogenic The optimal teratogenic dose was 0 5
innol/embryo for c innamaldehyde and 5 umol/embryo for cinnamyl alcohol, 58 2%
and 23 1% of the embryos had malformations (mainly limbs and skeleton) at the
respective dose In the same study, a number of other (X ,/3-unsaturated alde-
hydes (e g , citral, farnesal , benzaldehyde) are also teratogenic, whereas
other unsaturated alcohols and saturated aldehydes are either considerably
less active or inactive The authors (77) suggested that ex,/3-unsaturated
aldehydes, particularly those with shorter linear chains, are potential
teratogens Interaction between the liposoluble unsaturated aldehydes and
some key lipid constituents of the embryonic cell membrane was postulated to
be a possible mechanism of the teratogenic action of these compounds
532 4.3 Carcinogemcity and Structure-Activity Relationships
5 3 2 4 3.1 OVERVIEW
Since the first report in 1961 on the careinogenicity of safrole in rats,
close to 50 derivatives and related compounds have been tested for carcino-
genic activity These compounds include metabolites, synthetic derivatives
and structural analogs of safrole, estragole and eugenol The major findings
of these studies are summarized in Table LXII, the vast majority of these
350
-------
p 1 of 2
Table LXII
Summary of Comparative Careinogenesis Bioassay Data on Safrole, Related Compounds, and Their Metabolites3
Oral
Compound Rat Mouse
Safrole + (L) + (L)
l'-Hydroxysafrole + (L,F) + (L,I)
I'-Acetoxysafrole + (F) -b
1 '-Oxosaf role
1 '-Methoxysaf role
Safrole-2' ,3'-oxide
1 '-Hydroxysaf role-21 ,3 '-oxide
1 '-Acetoxysaf role-2* ,3'-oxide
Myrist ic in
Dill apiol
Parsley apiol
Isosafrole + (L)L ± (L)c
3 '-Hydroxyisosaf role
3'-Acetoxyisosafrole
3 '-Methoxyisosaf role
3'-Bromoisisafrole
Dihydrosafrole + (E)c + (F)c
1 '-Hydroxydihydrosaf role
I '-Acetoxydihydrosaf role
1 '-Met box yd ihydrosaf role
ip s c . pl „ ,
Mouse Rat Mouse initiation adenoma References
+ (L) - + (L) - - (13, 55, 78, 79)
+ (L) * (L,S) + (L) - - (13, 14, 55, 78, 79)
+ (S) + (L) - (13, 55, 79)
± (S) - - (55)
(13)
-, + - (55, 79)
* (L) * (S) * -, * (55, 79)
• (55, 79)
(79)
(79)
(79)
(13)
(13)
± (L) (13)
(13)
+ (S) (13)
(13)
(13)
(13)
(13)
-------
Table LXII (<_ontinued)
p 2 of 2
Compound
Estragole
1 '-Hydroxyest ragole
Estragole-21 ,3'-oxide
1 '-Hydroxyest ragole-21 ,3'-
oxide
l'-Hydroxy-2' ,3'-dehydro-
estragole
Methyleugenol
1 '-Hydroxyraet heugenol
El em ic in
1 '-Hydroxyelemic in
t rans-Anethole
3'-Hydroxy-t rans-ane thole
Eugenol
Eugenol-2' ,3'-oxide
I '-Hydroxyal ly I benzene
1 '-Hydroxy-l-allyl-4-methoxy-
naphthalene
Oral i p s c
Rat Mouse Mouse Rat Mouse initiation adenoma References
+ (L) + (L) + (L) - (14,
+ (L) + (L) ± (S) + (L) - (14,
+ - (79)
+ + (79)
+ (L) (79)
* (L) (79)
* (L) (79)
(79)
(79)
(79)
(79)
(79)
+ (79)
(79)
+ (L) (79)
79)
79)
aExcept where otherwise noted, most of these studies were carried out in the laboratory of J A and E C Miller under
standardized bioassay conditions Abbreviations for target organs- L, liver, F, forestomach, I, interscapuldr s c
tissue, S, local sarcoma, E, esophagus See Table LVII for structural formulas of most of the compounds
Inconclusive due to high mortality in one study and low dose in another study
CU S National Cancer Institute (NCI) or Food and Dtug Administration (FDA) data, see Table LXIII
-------
studies were carried out in the laboratory of J A and E C Miller under
standarized bioassay conditions thus permitting direct comparison of relative
potencies. These data, along with those discussed in Sections 532432,
532433 and 532434, suggest the following structure-activity
relationships
Ring substitution with methoxy groups (the raethylenedioxy group in
safrole may be considered as two methoxy groups) is an essential feature of
carcinogenic alkenylbenzene congeners The optimal number of methoxy substi-
tutions is two (e.g , safrole, methyleugenol), one of which being in the
jv-position relative to the alkenyl side chain A methoxy group in the
_p_-position may contribute to care inogenicity by stabilizing (through
resonance) the electrophilic intermediate generated during metabolic activa-
tion of the alkenyl side chain Extensive ring substitutions (e g , as in
myristicin, dill apiol, parsley apiol, elemicin) yield inactive compounds
possibly because of stenc hindrance Annelation to an additional aromatic
ring, however, appears to give rise to a potential carcinogen, l-allyl-4-
methoxynaphthalene, as indicated by the carcinogenicity of its I'-hydroxy
derivative (see Table LXII) and the potent mutagenicity of its I'-acetoxy
derivative (see Table LXI) Apparently, instead of creating steric hindrance,
the introduction of an additional aromatic ring provides a planar bicyclic
compound with a more favorable molecular geometry possibly for intercalation
into DNA 0-Demethylation of the _p_-methoxy group yields inactive compounds
(eg., eugenol) probably because of less favorable resonance stabilization as
well as easier excretion of phenolic compounds.
Modification of the alkenyl side chain can have a significant effect on
the carcinogencity of the alkenylbenzene congeners Analysis of the available
data suggests the following structure-activity relationships
351
-------
a} AlLyLbenzene congeners (e g , safrole, estragole) are
generally more carcinogenic than their propenylbenzene (A-methyl-
vinylbenzene) isomers (e g , isosafrole, trans-anetho 1e) The
lower carcinogenic activity of propenylbenzene congeners may be
related to their greater tendency to be oxidized to cinnamic
metabolites (see Section 5.3 244), which have lower genotoxic
potential
b) With the exception of 1'-oxosafrole, 1'-oxidized deriva-
tives of allylbenzene congeners are more genotoxic than their
parent compounds The I'-hydroxy derivatives of safrole,
estragole, methyleugenol (and possibly, l-allyl-4-raethoxynaph-
thalene) are all more potent as carcinogens, and they show evidence
for direct-acting carcinogenicity 1'-Acetoxysafrole is a potent
direct-acting carcinogen, whereas the I'-acetoxy derivatives of a
variety of allylbenzene congeners are all direct-acting mutagens in
the Ames test (see Table LXI) These are findings which form the
basis for the Millers' conclusion (e g , 79) that 1'-hydroxylation
followed by esterification (with suifate) represents the major
metabolic activation pathway of safrole and related compounds The
lack of genotoxicity of 1'-oxosafrole may be attributed to its high
instability and reactivity, making it unlikely to reach target DNA
molecules in a significant amount before being degraded or reacting
with noncntical cellular nucleophiles
c) In contrast to 1'-oxidized derivatives of allylbenzene
congeners, 3'-oxidized derivatives (both hydroxy and acetoxy) of
propenylbenzene congeners (e.g , isosafrole, trans-anethole) show
questionable or no carcinogenic activity As mentioned in (a),
352
-------
further oxidation to cinnamic metabolites may be a factor in limit-
ing the carcinogenic potential of these derivatives The only
carcinogenic 3'-substituted derivative is 3'-bromoisosafrole, which
is expected to be a direct-acting alkylating agent because of the
good leaving tendency of bromine group
d) The 2',3'-epoxide derivatives of a variety of allylbenzene
congeners are all direct-acting mutagens (see Table LXI) However,
they (e g , safrole-2',3'-oxide, estragole-2',3'-oxide, eugenol-
2'3,'-oxide) are inactive as "complete" carcinogens and are active
only as tumor initiators Apparently these 2',3'-epoxy derivatives
lack tumorigenesis promoting activity 2',3'-Epoxidation alone
appears to oe insufficient as a metabolic pathway to generate
electrophilic intermediates which have "complete" carcinogenic
activity
e) Saturation of the 2',3'-double bond of the allyl side
chain does not necessarily abolish the carcinogenicity of
safrole In fact, dihydrosafrole and piperonyl sulfoxide are
carcinogenic Piperonyl butoxide, another saturated derivative of
safrole (but with more extensive ring substitution), is not oar-
cinogenic.
f) Further unsaturation of the 2',3'-double bond of the allyl
side chain may have a potential to enhance the carcinogenicity of
the compound Indeed, 1'-hydroxy-2',3'-dehydroestragole is a
potent hepatic carcinogen, with a potency higher than any of the
safrole and estragole derivatives tested (see Section 532434)
353
-------
532432 CARCINOGENICITY OF SAFROLE AND RELATED COMPOUNDS
The carcinogenicity of safrole was discovered in 1960-1961 when three
separate chronic studies by the U.S Food and Drug Administration (1),
Homburger et al (2), and Abbott et al (3) all showed the induction of
hepatomas and preneoplastic changes in rats administered high concentrations
(0 5-1%) of safrole in the diet The discovery immediately led to the banning
of its use as food additive Since the initial reports, more than ten addi-
tional studies were reported confirming the carcinogenicity of safrole in
various strains of rats and mice (see Table LXIII) The compound is only
weakly hepatocarcinogenic when fed to adult rats or mice, but is a moderately
strong carcinogen when administered to preweanling male mice. Only liver
adenomas were found in male Carworth Farms CFN rats fed 1% (10,000 ppm)
safrole in the diet for 1 year, starting at the age of 6-10 weeks No tumors
were detected in rats given a dietary level of 0 1% safrole (2). In groups of
25 male and 25 female 3-week-old weanling Osborne-Mendel rats maintained on
diets containing 0, 100, 500, 1,000 and 5,000 ppm safrole for 2 years, the
respective liver adenoma incidences were 3/50, 1/50, 2/50, 8/50 and 19/50
Fourteen of the rats in the 5,000 ppm group, two in the 500-ppm group and two
in the control group bore malignant liver tumors The increase in tumor inci-
dence was significant only in the highest dose group (34) Of 18 adult
Charles River CD random-bred rats (average initial weight, 252 g) fed 5,000
ppra safrole for 22 months, only 3 developed hepatic carcinomas, no such tumors
were found in controls (55). No significant carcinogenic effects were noted
in adult CD rats 18 months after receiving 20 twice-weekly s c. injections of
18 6 iimole (3 mg) safrole (13) In adult CD-I mice, dietary administration of
0 4-0 5% safrole for 12-13 months induced liver tumors in only 12-25% of male
mice at 16-17 months after the commencement of treatment. Adult female CD-I
354
-------
Table LXIII a
Carcinogenicity of Safrole and Related Compounds
p 1 ot 2
Compound
Safrole
Myr ist icin
Dill apiol
Parsley apiol
Isosafrole
Dihydrosaf role
Species and strain
Mouse, CD-I
Mouse, BALB/c ,
B6C3F1 or BSAKFi
Mouse, B6C3F1
Mouse, A/He or A/J
Mouse, ICR/Ha
Rat, Osborne-Mendel ,
CFN or CD
Rat , CD
Dog, —
Mouse, B6C3FJ
Mouse, B6C3F1
Mouse, B6C3Fj
Mouse, B6C3F1
Mouse, B6AKFi
Rat , Osborne-Mendel
Rat , CD
Mouse, B6C3F1
or B6AKF1
Mouse, CD-I
Rat . Osborne-Mendel
Route
oral , i p
or s c.
topical*"
oral
oral , t rans-
placent al
and/or lac-
tat lonal
i P
s c
oral
s c
oral
i P
i P
i P
oral
oral
oral
s c
oral
topical0
oral
Principal
organs
affected
Liver
None
Liver
Liver
Noned
Liver, lung
Liver
None
Skin, tongue
None
None
None
Liver
(marginal)
None
Liver
None
Forestomach ,
1 iv er
None
Esophagus
References
(13, 55, 78,
79)
(55)
(80-84)
(85)
(79, 86)
(87)
(1, 2, 13,
34, 36, 55)
(13)
(Cited in 81)
(79)
(79)
(79)
(80, 81)
(80, 81)
(4, 36)
(13)
(80, 81)
(13)
(4, 33, 36)
-------
Table LXIII (continued)
2 of 2
Compound
Piperonyl
sulf oxide
Piperonyl
butoxide
SesamoL
Species and strain
Mouse, B6C3FJ
or B6AKFj
Mouse, B6C3F1
Rat, F344
Mouse, B6C3F1
or B6AKF1
Mouse, B6C3F^
Mouse, ICR/Ha
Rat , F344
Rat , —
Route
oral
oral
oral
oral
oral
s c
oral
oral
Principal
organs
affected
None
Liver
None
None
None
None
None
Various
organs
(mostly
benign)
Re ferenc.es
(80)
(88)
(88)
(80)
(89)
(90)
(89)
(91)
See also Tables LXII and LXV for careinogenesis studies of metabolites of
safrole
See Table LVII for structural formulas
cSkin painting with promotion by croton oil
Limited (24-week) bioassay only, pulmonary adenoma assay Negative results
are considered inconclusive evidence of noncarcinogenicity
eCategorized as compounds requiring additional testing due to increased, but
not significant, incidences of tumors
-------
mice appeared Co be more susceptible with 46-70% bearing hepatoraas after feed-
ing of 0.13-0.5% safrole (13, 55, 78) Similar sex difference has also been
observed in adult B6C3F^ mice given 180 twice-weekly intragastric administra-
tion of 120 mg safrole/kg body weight, the liver tumor incidences were 11% for
males and 61% for females (85). Repeated topical treatment of adult CD-I mice
with safrole (5 x 3 umol/wk for 6 wk) produced no significant carcinogenic
effects after 30 additional weeks of promotion with croton oil (13)
In contrast to adults, male infant or preweanling mice are highly suscep-
tible to the hepatocarcinogenic effect of safrole A high incidence (50-58%)
of liver tumors was observed in male Swiss albino ICR/Ha mice one year after
receiving four s c injections of safrole, totaling only 0 66 or 6 6 mg, on
days 1, 7, 14 and 21 after birth A slight increa'se in the incidence of
pulmonary tumors was also noted (87) Similar treatment of infant or pre-
weanling CD-I mice (total dose 9 45 umole or 1 5 mg) led to a significant
increase in the incidence of liver tumors in the males (14/35 treated vs 3/36
controls), however, no liver tumors developed in the 27 treated females after
16 months (13) Besides subcutaneous administration, intraperitoneal injec-
tions (total dose 9 45 uraole) and oral administration (via stomach tube, 10
twice-weekly doses of 2 5 uraole/g body weight starting at day 4 after birth)
of low doses of safrole are equally effective in inducing liver tumors (61-67%
treated vs 24-26% controls) in male CD-I mice (79).
In other studies, B6C3Fi and B6AKF^ mice were given 464 rag/kg body weight
safrole by gavage from days 7-28 after birth and subsequently 1,112 ppm in the
diet for up to 82 weeks Liver tumors occurred in 11/17 male and 16/16 female
mice of the first strain, 3/17 male and 16/17 female mice of the second
strain, these incidences were significantly higher than those of the controls
when the data in both sexes were combined (80, 81) Lipsky et_ _al_ (82-84)
355
-------
described the sequential histopathoiogicai and biochemical changes during
safrole-induced carcinogenesis in BALB/c tnioe Vesselinovitch gt al (85)
studied transplacental and lactational carcinogenesis by safrole in B6C3F,
mice Pregnant mice, nursing mothers or offspring were given oral doses of
120 mg safrole/kg body weight via gavage No significant increases in liver
tumors were observed in offspring exposed in utero on days 12, 14, 16 and 18
of gestation, however, renal epithelial tumors (not seen in controls)
developed in 77, of females Male (but not female) offspring nursed by
safrole-treated mothers had a significantly higher incidence of liver tumors
(34% treated vs 3% controls) A combination of transplacental, lactational
and post-weanling exposure brought about high incidences of liver tumors (51%
males, 80% females) in mice of both sexes. Besides rodents, dogs were
reported to be susceptible, carcinomas of the skin and tongue were seen in
dogs receiving safrole for 6 years (unpublished FDA data, cited in 81)
Besides safrole, a variety of related compounds have been tested for car-
cinogenic activity Myristicin, dill apiol and parsley apiol, three naturally
occurring substances that are closely related to safrole (differing only by
one or two additional methoxy group(s) on the ring), have been found to be
noncarcinogenic when injected into preweanling male B6C3Fi mice on days 1, 8,
15 and 22 after birth at a total dose of 4 75 jumoles (79). It appears that
extensive ring substitution reduces or abolishes the carcinogenicity of
safrole, possibly because of steric hindrance
Isosafrole, an isomer of safrole, has marginal or no carcinogenic activ-
ity in mice and is hepatocarcinogenic in rats, with a potency substantially
lower than that of safrole In B6C3Fi mice, oral administration of isosafrole
(215 mg/kg body weight by i p injection on days 7-28 after birth followed by
517 ppm in the diet for up to 82 weeks) led to a small, marginally significant
356
-------
increase (5/18 treated males and 1/16 treated females vs 8/79 and 0/87 con-
trols, P = 0 05 with combined data) in the incidence of liver tumors The
same treatment had no carcinogenic effect in B6AKF^ mice (80, 81) In young
adult Osborne-Mendel rats, dietary administration of 5,000 ppm for 2 years
induced liver tumors in 5/50 rats (including 3 hepatocellular carcinomas) com-
pared to 19/50 (14 malignant) safrole-treated rats A higher dose (10,000
ppm) was acutely toxic whereas lower doses (2,000 and 1,000 ppm) were not car-
cinogenic (4, 36) No significant careinogenicity was noted in adult CD rats,
18 months after receiving 20 twice weekly s c injections of 18 6 jumole iso-
safrole (13). Thus, a change of the side chain from allyl to propenyl reduces
the carcinogenic activity of the compound.
Of the three side chain-saturated derivatives of safroie that have been
tested for carcinogenic activity, two (dihydrosafrole and piperonyl sulfoxide)
are carcinogenic in at least one animal species suggesting that saturation of
the side chain does not necessarily abolish the carcinogenicity of the com-
pound Dihydrosafrole has been shown to induce tumors of the forestomach and
the liver in mice and tumors of the esophagus in rats Oral administration of
dihydrosafrole (464 mg/kg body weight by i p injection on days 7-28 after
birth followed by 1,400 ppm in the diet for up to 82 weeks) to B6C3F^ and
B6AKFi mice brought about significant increase in the incidences of hyper-
plasia and tumors of the forestomach Fifteen of 17 (88%) treated female
B6C3F^ mice and 14 of 18 (78%) treated female B6AKF^ mice had forestomach
tumors (including 3 malignant) compared to 5/17 (28%)* and 0/15 female
*The incidence of stomach tumors in strain B6C3F^ control mice (23% males, 28%
females) reported in this study were substantially higher than historical
controls No explanation was given for this unusual finding
357
-------
controls Male B6AKF1 mice also had a higher incidence (7/17 treated ^s_ 0/18
controls) of stomach tumors (81) Besides stomach tumors, carcinomas of the
liver developed in 10/17 (60%) male BSCSFj mice and 7/17 (41%) male B6AKFj
mice in significantly higher incidences than controls No such increases were
seen in female mice (80, 81) A clear-cut dose-response relationship in the
induction of esophageal tumors was seen in Osborne-Mendel rats maintained on
diets containing 2,500, 5,000 or 10,000 ppm dihydrosafrole for 2 years The
respective tumor incidences were 20%, 74% and 75% Of these esophageal
tumors, 5%, 32% and 50% were malignant No tumors were observed in rats fed
1,000 ppm of the compound (4, 33, 36) Besides rodents, there is some evi-
dence that dogs given dihydrosafrole for 2 years had hyperplasia of the
esophagus (FDA unpublished data, cited in 81)
The potential carcinogenicity of piperonyl sulfoxide was first investi-
gated by Innes et al (80) as a part of a large-scale test of industrial and
agricultural chemicals Two strains (B6C3Fi and B6AKFi) of mice were given
46 4 mg/kg body weight of the compound by stomach tube on days 7-28 after
birth and 111 ppm in the diet for up to 82 weeks The compound was classified
as suspect carcinogen requiring additional testing because an increased,
though not significant incidence of tumors (reticulum cell sarcoma) was
observed (80) A subsequent study by the U S National Cancer Insitute (88)
showed that technical grade piperonyl sulfoxide (88% pure with 12% related
compounds) is carcinogenic toward male B6C3F^ mice producing an increased
incidence of hepatocellular carcinoma The compound is not carcinogenic
toward female mice and Fischer 344 rats of both sexes The compound was
administered for 105 weeks in the diet at doses of 1,500 or 3,000 ppra for male
mice, 3,000 or 6,000 ppm for female mice, 350 or 700 ppm for male rats and 700
or 1,400 ppm for female mice with the high doses being the maximum tolerated
358
-------
dose The incidences of hepatocelluLar carcinoma in male mice were 6/18,
31/50 and 46/50 for control, low-dose and high-dose groups, respectively
(88). Technical grade piperonyl butoxide was also categorized as a compound
requiring additional testing in the study of Innes et al (80) However, a
subsequent National Cancer Institute bioassay (89) showed no evidence of
carcinogenicity of the compound in either B6C3Fi mice or Fischer 344 rats, fed
maximum tolerated doses in the diet for 2 years Piperonyl butoxide is also
noncarcinogenic in Swiss albino ICR/Ha mice one year after receiving repeated
s c injections (totaling 30 mg) 1-19 days after birth (90) There is evi-
dence, however, that the compound acts synergistically with Freon 112 or 113
(see Section 532436)
Besides the above-mentioned compounds, there is indication that sesamol
(l-hydroxy-3,4-methylenedioxybenzene), a naturally occurring minor constituent
of sesame oil, may be weakly carcinogenic Ambrose et al (91) found a total
of 20 "proliferative lesions" in 134 rats fed diets containing 0 016 to 1 0%
sesamol for 400-634 days Sixteen of these lesions (which include adenomas,
papillomatous foci, polyps and nodules) were benign while four were either
malignant (1 fibrosarcoma of the ovary) or undetermined Most of the lesions
were distributed in the mammary and adrenal glands, bladder, uterus and
ovary No such lesions were found in the controls, nor in rats receiving the
lowest doses (0 008%) of the compounds
532433 CARCINOGENICITY OF ESTRAGOLE AND RELATED COMPOUNDS
Estragole, a naturally occurring substance stucturally related to
safrole, is hepatocarcinogenic in CD-I or B6C3F, mice by three different
routes of administration (see Table LXIV). Repeated subcutaneous injections
of estragole totaling 4 4 or 5 2 umoles between the days 1 and 22 after birth
359
-------
Table LXIV
Careinogenicity of Estragole and Related Compounds3
Compound
Estragole
Methyleugenol
Elemic in
Eugenol
Species
Mouse,
Mouse ,
Mouse ,
Mouse ,
Mouse ,
Mouse ,
Mouse ,
and strain
CD-I or B6C3F1
A/J
B6C3F1
B6C3Fj
B6C3F1
CD-I
ICR/Ha
Rat, F344
t rans-Anethole
( isoestragole)
Calamus oil
( B "Asarone)
Mouse ,
Mouse ,
Rat , —
CD-I or B6C3F1
A/ He or A/J
Route
oral , i p
or s c
i P
i P
i.p
oral
oral or i.p
skin
painting
oral
oral or i p
i P
oral
Principal
organs
affected
Liver
Nonec
Liver
None
Liver
(equivocal)
None
None
None
None
Nonec
Intest ines
References
U4,
(79)
(79)
(79)
(92)
(79)
(93)
(92)
(79)
(79,
(5, 6
79)
,
86)
)
aSee also Tables LXII and LXV for carcinogenesis studies of metabolites and
derivatives of estragole.
See Table LVII for structural formulas
cLimited (24-week) bioassay only, pulmonary adenoma assay Negative results
are considered inconclusive evidence of noncarcinogenicity
Oil of calamus obtained from Jamrau, India It consists of the following
compounds; g -asarone, 75 8%, calamene, 3.842, calaraol, 3 2%, a -asarone,
1.322, camphene, 0 92%, @ -pinene, 0 56%, and asaronaldehyde, 0 2%
-------
induced, after 15 months, hepatocellular carcinomas in 23% and 39% of male
CD-I mice, respectively (14) Some 73% of male CD-I mice developed hepatomas
14 months after receiving 10 twice-weekly intragastric administrations of 2 5
umole/g body weight of the compound starting on day 4 after birth Like with
safrole, preweanling female mice are refractory to the carcinogenic effect of
estragole Repeated intraperitoneal injections to preweanling male CD-I mice
(total dose, 9 45 jumole) or B6C3F^ mice (total dose, 4 75 umole) led to
hepatoma incidences of 65% (after 12 months) and 83% (after 18 months),
respectively (79) The carcinogenic potency of estragole is approximately
equal to that of safrole in preweanling male mice
Two ring-substituted higher homologs of estragole have been tested for
carcinogenic activity in male preweanling B6C3Fi mice by Miller et al (79)
The introduction of one additional methoxy group enhances tne carcinogenicity
of the compound as indicated by the higher tumor incidence and higher average
number of hepatomas per mouse in preweanling male mice given methyleugenol
(96%, 3 2) than in those given equimolar doses of estragole (83%, 2 4) How-
ever, further substitution with a methoxy group (yielding elemicin) abolishes
the carcinogenic activity of the compound Interestingly, these structure-
activity relationships parallel those of safrole in which the optimal number
of methoxy substitutions (considering methylenedioxy group as two methoxy
groups) appears to be two (see Section 532432)
Ring hydroxylation and shift of the methoxy group from the para to the
meta position (yielding eugenol) , together, reduce or virtually abolish the
carcinogenic activity of estragole Thus, eugenol was found completely non-
carcinogenic in the study of Miller et al (79), in which a total dose of 25
imiole/g body weight was administered orally or a total dose of 9 45yUmole/
mouse was injected intraperitoneally to preweanling male CD-I mice As dis-
360
-------
cussed in Section 522532 (Vol IIIA), there is some evidence that eugenol
increases the incidence of hepatocellular carcinomas in B6C3F^ mice but the
evidence is equivocal due to the absence of dose-dependency In Fischer 344
rats, eugenol is clearly noncarcinogenic (92) The lack of (or lower) car-
cinogenicity of eugenol may be due to poorer absorption, to the generally
easier excretion of phenolic compounds, and to shift of the methoxy group from
para to meta which is a less effective position for resonance stabilization of
raetabolically generated electrophilic intermediate(s)
Also in accord with the greater carcinogenic potency of safrole than iso-
safrole, trans-anethole (isoestragole) is inactive under conditions in which
estragole was carcinogenic In fact, trans-anethole showed no significant
carcinogenicity in preweanling male CD-I mice given a total dose (50 xmole/g
body weight) twice that of estragole by gavage or an equimolar dose by
intrapentoenal injection (79). trans-Anethole is also inactive in the pul-
monary adenoma assay using strain A/He mice (79, 86)
In contrast to the lack of carcinogenicity of trans-anethole, there is
some evidence that its close analog, p-asarone, may be carcinogenic Oil of
calamus, a volatile oil present in the rhizomes (about 1%) of the plant Acorus
calamus (commonly called sweet flag in U S ), was found carcinogenic in rats
(5, 6). In this study, groups of rats were fed diets containing 0, 500,
1,000, 2,500 or 5,000 ppm oil of calamus Malignant mesenchymal tumors of the
small intestines were found in treated rats at all levels, and the incidence
was dose-related The tumors were first detected after 59 weeks of feeding
The particular variety of oil of calamus (from Jammu, India) used in this
study consists of 75 8% /9-asarone, 3 84% calamene, 3 2% calamol , 1 32%
cy'-asarone, 0 92% camphene, 0 56% (3-pinene and 0 2% asaronaldehyde Owing to
its abundance in oil of calamus, the carcinogenic activity of the oil is
generally attributed to /3-asarone
-------
532434 CARCINOGENICITY OF METABOLITES OF SAFROLE, ESTRAGOLE AND
RELATED COMPOUNDS
In an attempt to elucidate the metabolic activation pathways of safrole,
estragole and related compounds, Millers and associates have synthesized a
variety of metabolites and derivatives" and tested their carcinogenic activ-
ity The major findings of these studies are summarized in Table LXII For
comparison of relative potency, the careinogenicity data (by i p administra-
tion to preweaniing mice) of some of these compounds are summarized in Table
LXV
Six metabolites of safrole have been tested for carcinogenic activity by
various routes of administration By oral administration, 1'-hydroxysafrole
(5,500 ppm in diet) induces a high incidence (89-92%) of hepatocellular car-
cinomas in adult male rats A few papillomas of the forestomach have also
been observed Under similar conditions, safrole (5,000 ppm in diet) induces
only a low incidence (6-16%) of liver tumors Fed at a lower dietary level
(4,100 ppm), 1'-acetoxysafrole is not hepatocarcinogenic but induces a high
incidence (64%) of multiple papilloma (with a few squamous cell carcinomas) of
the forestomach (13, 55) At an even lower dietary level (2,500 ppra), I'-oxo-
safrole is not carcinogenic (55) Subcutaneous injections of 1'-aeetoxysaf-
role consistently led to the induction of injection-site sarcomas in 20-30% of
the rats tested (13, 79) 1'-Hydroxysafrole-21,3'-oxide is approximately
equipotent to 1'-acetoxysafrole in the induction of local sarcomas (79)
1'-Hydroxysafrole induces much fewer local sarcomas (13) or no local sarcomas
(79), but is hepatocarcinogenic 1'-Oxosafrole has little or no local car-
cinogenic activity (55) whereas safrole and safrole-21,3'-oxide are both inac-
tive by the s c route (79) These results, along with mutagenicity data and
metabolic studies, led Millers and associates to conclude that I'-hydroxy-
362
-------
Table LXV
Relative Carcinogenicity of Metabolites of Safrole, Estragole and Related Compounds
in the Induction of Liver Tumors in Male Infant Mice by Inttaperitoneal Administrationa
CD-I roiceb
Total dose % Incidence Av no hepatoma
Compound (uraol) at 12 months per mouse
Safrole 9 45 67 19
I'-Hydroxysafrole 4 72 65 27
I'-Hydroxysafrole- 9 45 55 10
2',3'-oxide
Estragole 9 45 65 17
1 '-Hydroxyest ragole
l'-Hydroxy-2' ,3'-de-
hydroest ragole
Methyleugenol
1 '-Hydroxymethyl-
eugenol
r-Hydroxy-l-allyl-4-
met hoxynaphthalene
aSummarized from E C Miller, A B Swanson, D H Phillips, T L
Total dose
( umol)
3 75
4 75
1 87
1 9
1.86
4 75
2 85
3.75
Fletcher, A
B6C3Fj micec
% Indicence at Av no hepatoma
12 months 13-18 months per mouse
92 27
83 24
93 2 7
98 56
97 94
96 32
93 35
65 1 I
Lein and J A Miller [Cancer Res 43, 1124
(1983)
Compound given in 10 doses during the first 4 or 5 weeks after birth
cCompound given in 4 doses prior to weaning at 22 days of age
-------
safrole is a proximate carcinogen of safrole 1'-Acetoxysafrole is a poten-
tial ultimate carcinogen, however, there is no evidence that ester ification of
1'-hydroxysafrole with acetic acid occurs to any significant extent Both
1'-hydroxysafrole-2',3'-oxide and 1'-oxosafrole possibly also represent ulti-
mate carcinogens The evaluation of the degree of carcinogenic activity of
the latter is limited by its instability and high toxicity
Various metabolites of safrole have also been assayed in mice The
results support the conclusion that 1'-hydroxysafrole is a proximate carcino-
gen of safrole 1'-Hydroxysafrole is more potent than the parent compound in
male preweanling mice given i.p. (see Table LXV) or s.c. (55) injections of
the compounds In adult female mice, 1'-hydroxysafrole induces slightly fewer
hepatomas than does the parent compound, but the decrease may be due to the
induction, by 1'-hydroxysafrole, of a large number of angiosarcomas in inter-
scapular subcutaneous tissue, shortening the lifespan of the animals (79)
1'-Acetoxysafrole is more hepatocarcmogenic than safrole after s c injec-
tion, however, the compound lacks local carcinogenic activity in mice (55)
As in rats, 1'-oxosafrole and safrole-2' ,3'-oxide are inactive whereas
1'-hydroxysafrole-2',3'-oxide is carcinogenic in mice Another putative elec-
trophilic metabolite, 1'-acetoxysafrole-2' ,3'-oxide, also fails to induce any
significant carcinogenic effect in mice. There is strong evidence that
1'-sulfooxysafrole (the sulfate ester of 1'-hydroxysafrole) is the major ulti-
mate electrophilic and carcinogenic metabolite of 1'-hydroxysafrole Boberg
%t_ jil_ (78) recently showed that the carcinogenic effect of 1'-hydroxysafrole
in female adult CD-I mice can be virtually eliminated by treating the animals
with pentachlorophenol, a sulfotransferase inhibitor. Brachymorphic mice,
which lack the enzyme system for the synthesis of 3'-phosphoadenosine-5'-
phosophosulfate (PAPS, activated sulfate), are much less responsive than their
363
-------
phenotypically normal Littermates to the induction of liver tumors by
1'-hydroxysafrole Attempts to synthesize 1'-sulfooxysafrole have thus far
been unsuccessful because of its high instability and reactivity
In contrast to enhancement of carcinogenicity of safroie by 1'-oxidation
of the allyl side chain, there is little or no evidence that oxidation at the
corresponding position of isosafrole brings about activation of the compound
(13) By subcutaneous injection to adult male rats, 3'-hydroxyisosafrole
fails to show any carcinogenic effect 3'-Acetoxyisosafrole is also inactive
for inducing local tumors at the injection site, but may have a marginal or
weak hepatocarcinogenic effect. Repeated s.c. injections (2 times/week for 10
weeks) of 18 6 jumoles of the compound led to the induction of one liver car-
cinoma among 18 rats, the effect was considered to be probably a direct con-
sequence of the treatment because of the extremely low spontaneous incidence
of liver carcinomas in Fischer rats Of the two other tested 3'-substituted
derivatives of isosafrole, 3'-methoxyisosafrole is inactive while 3'-bromoiso-
safrole displays local carcinogenic activity, inducing sarcomas at the injec-
tion site of 2/18 rats These results suggest that the role of 3'-oxidation
in the metabolic activation of isosafrole is questionable and remains to be
elucidated Further studies by oral administration are needed The local
carcinogenic effect of 3'-bromoisosafrole is probably due to the good leaving
tendency of the bromine group (see Section 5.2 1 1 2 1, Vol IIIA) generating
the electrophilic carbonium ion on the allyl moiety.
As with safroie, the I'-hydroxy derivative of estragole is substantially
more potent than its parent compound by i p. injection (see Table LXV) to pre-
weanling male mice. 1'-Hydroxyestragole is approximately equipotent to estra-
gole by dietary administration to adule female CD-I mice. The compound
exhibits marginal local carcinogenic activity after s c. injection, inducing
364
-------
local sarcomas in only 3/20 rats (79) Like safrole-2' ,3'-oxide, estragole-
2',3'-oxide is also noncarcinogenic after i p or s c injection to pre-
weanling male mice, and only shows some tumor-initiatory activity The
results indicate that 2",3'-epoxidation is less important than 1'-hydroxyla-
tion and subsequent ester ification in the metabolic activation of estragole
This conclusion is further supported by the finding that 1'-hydroxy-2',3'-
dehydroestragole is a strong hepatocarcinogen In fact, the compound is more
potent than any of the estragole and safrole derivatives thus far tested (see
Table LXV) The replacement of the double bond by a triple bond is expected
to eliminate the possibility of epoxidation, leaving 1'-hydroxylation as the
sole metabolic pathway of side chain oxidation. The mechanism of enhancement
of carcinogenic activity by further unsaturation of the 2',3'-bond is not
clear It is possible that the acetylenic bond may contribute to carcinogenic
activity by stabilizing the electrophilic intermediate (such as carbonium ion)
giving it a greater chance to reach target DNA molecules.
Among other carcinogenic congeners, 1'-hydroxyraethyleugenol is at least
as carcinogenic or possibly more potent than its parent compound (see Table
LXV) 1'-Hydroxy-l-allyl-4-methoxynaphthalene is an active hepatocarcinogen
when administered intraperitoneally to preweanling male mice (see Tab 1e LXV),
while its I'-acetoxy derivative is a direct-acting mutagen (see section
5.3 2.4.2 2). These results indicate that 1'-hydroxylation followed by ester-
ification is a common metabolic activation for all carcinogenic allylarene
congeners
Several metabolites of noncarcinogenic analogs or of trans-anethole (a
noncarcinogenic isomer) of estragole have been tested for carcinogenic
activity. 1'-Hydroxyelemicin is inactive after i p injection to preweanling
mice (see Table LXV), indicating that the lack of carcinogenicity of elemicin
365
-------
LS not due to inability to undergo side chain oxidation but possibly to
excessive substitution of the aromatic ring Like 3'-hydroxyisosafrole,
3'-hydroxy-trans-anethole (3'-hydroxyisoestragole) is also inactive In
agreement with the results of safrole-21,3'-oxide and estragole-21,3'-oxide,
eugenol-2',3'-oxide has little or no "complete" carcinogenic activity but is
active as an initiator in skin tumorigenesis
532435 CARCINOGENICITY OF CINNAMYL (CINNAMIC) COMPOUNDS
Cinnamyl (cinnamic) compounds have been used widely as food additives,
fragrances or flavoring agents Some cinnamyl compounds are found in wood
products or smoke They are structurally related to safrole, estragole and
their isomers, 3'-hydroxyisosafroLe, for example, is actually a ring substi-
tuted derivative of cinnamyl alcohol. Several cinnamyl compounds have been
tested for carcinogenic activity (see Table LXVI). In the pulmonary adenoma
assay by Stoner et al (86), both cinnamyl alcohol and cinnamaldehyde are
inactive in A/He mice given i.p injections of maximally tolerated doses of
the compounds, 3 times a week for 8 weeks and observed for another 16 weeks
It should be noted, however, that owing to the nature of the study (short
duration of only 24 weeks, insensitivity to many hepatocarcinogens), the
negative results are suggestive but not conclusive evidence for the lack of
carcinogenicity of the compounds
Schoental and Gibbard (95) showed that 3,4,5-trimethoxycinnamaldehyde is
carcinogenic in rats. In a small scale experiment in which two doses of the
compound (150 mg/kg body weight, i p , as a 20% suspension in aqueous ethanol,
followed within one week by a s c dose of 100 mg/kg in dimethylformamide)
were given to 6 young adult rats, 4 of 4 rats which survived longer than 17
months developed tumors These consisted of a sarcoma in the peritoneal
366
-------
Table LXVI
Carcmogenicity of Cinnamyl Compounds
Species
Compound3 and strain
Cinnamyl alcohol Mouse, A/He
Cinnamyl aldehyde Mouse, A/ He
Rat , —
3,4,5-Tr imethoxy- Rat, —
cinnamaldehyde
Cinnamyl anthranilate Mouse, B6C3Fj
Mouse , A/ He
Rat, F344
Methyl anthranilate Mouse, A/He
Anthranilic acid Mouse, B6C3F1
Rat, F344
Route
i P
i P
i P /
s c
1 P /
s c
oral
i P
oral
1 P
oral
oral
Principal
organs
affected
Nonec
Nonec
None
Nasal cavity,
peritoneum,
testis
Liver
Lung
Pancreas ,
kidney
Nonec
None
None
References
(86)
(86)
(Schoental and
Gibbard, cited
in 94)
(95)
(96)
(86)
(96)
(86)
(97)
(97)
aSee Table LVII for structural formula
-------
cavity, a mesothelioraa of the tunica albuginea of the testis, and two nasal
squamous carcinomas. The latent period for these tumors ranged from 20 to 25
months In view of the extremely low spontaneous incidence of nasal carcinoma
in rats, the results are of particular importance in the light of several
reports of increased incidences of nasal tumors among woodworkers and popula-
tion groups exposed to wood smoke (see Section 5 3 2.4 5) Whereas it is not
known whether 3,4,5-trimethoxycinnamaldehyde as such is present in wood
lignins, several closely related compounds have been found in wood (see
Section 53245). In contrast to its 3,4,5-trimethoxy derivative, unsubsti-
tuted cinnamaldehyde is not carcinogenic (unpublished data by Schoental and
Gibbard, cited in 94) Apparently, the raethoxy groups, particularly the _p_-
methoxy group, confer carcinogenic activity to the compound.
Of relevance to the above finding is an interesting report by Sab me et
_al_. (98) that C3H Avy mice, which have a high "spontaneous" incidence of
mammary and hepatic tumors in the laboratories of the National Cancer
Institute in United States, developed much fewer "spontaneous" tumors when
tested in Australia. The discrepancy has been attributed to the use in the
NCI laboratory of red cedar (Juniperus virginiana) wood bedding which is known
to contain methylenedioxy- and polymethoxylignins such as podophyllotoxin It
has been suggested that podophyllotoxin may account for some of the carcino-
genic effects of red cedar wood bedding From the structural point of view,
podophyllotoxin may be considered as consisting of one molecule of a safrole
derivative and one molecule of 3,4, 5-trunethoxy-
phenol derivative and it is possible that the
compound may in fact yield these derivatives
in the course of its metabolic or microbial
degradation (94, 95). CH30'
Podophyllotoxin
367
-------
CLtinamyL anthranilate, a synthetic flavoring agent, was first found to be
carcinogenic in the pulmonary adenoma assay by Stoner et al. (86) The com-
pound produced a statistically significant increase in the incidence and
multiplicity (average number/mouse) of lung adenomas in strain A/He mice which
received i p injections of maximally tolerated doses (up to 500 rag/kg body
weight) three times a week for 8 weeks and survived for an additional 16
weeks The carcinogenicity of the compound has been confirmed in a carcino-
genesis bioassay by U S National Cancer Institute and National Toxicology
Program (96) Groups of 50 Fischer 344 rats and B6C3Fi mice of each sex were
fed diets containing 15,000 or 30,000 ppm (maximally tolerated dose) cinnamyl
anthranilate for 103 weeks and then observed for an additional 2 or 3 weeks
Significant, dose-related increases in the incidences of liver tumors
(carcinomas or adenomas) were observed in dosed mice (males control 47%, low
dose 60%, high dose 79%, females control 14%, low dose 41%, high dose
67%) The chemical is also carcinogenic in male (but not female) rats, induc-
ing low incidences of acinar-cell carcinomas or adenomas of the pancreas (a
rare type of tumor) and tumors of the renal cortex It is particularly
interesting to note that when tested separately, neither the cinnamyl alcohol
moiety nor the anthranilic acid moiety of cinnamyl anthranilate are active in
the pulmonary adenoma assay. Anthranilic acid (2-aminobenzoic acid) is also
noncarcinogenic in B6C3F^ mice and Fischer 344 rats in a 2-year feeding study
using maximally tolerated doses (97).
532436 MODIFICATION OF CARCINOGENESIS BY SAFROLE AND RELATED
COMPOUNDS
As for virtually all carcinogens, the carcinogenicity of safrole and
related compounds is modified by a number of host and environmental factors
The modification studies provided important clues to the understanding of the
368
-------
activating metabolic pathways of the carcinogens Wislocki et al (55) showed
that phenobarbital (PB, 0 1% in drinking water), a well known inducer of
microsomal mixed-function oxidases, enhances the hepatocarcinogenicity of
safrole (0 5% in diet) in CD rats The incidence of hepatocellular carcinomas
in rats receiving both PB and safrole was 67%, substantially higher than that
of rats given safrole (17%) or PB (6%) alone The enhancing effect was attri-
buted to the increased oxidation of safrole to the proximate carcinogen,
1'-hydroxysafrole As much as 10 times more 1'-hydroxysafrole was detected in
the urine of rats pretreated with PB before safrole administration (12)
Boberg et al (78) demonstrated that chronic administration of a nontoxic
level (0 05%) of pentachiorophenol, a potent inhibitor of cytosolic sulfo-
transferase (100, 101), in the diet of adult female CD-I mice strongly
inhibits (to the extent of 82-100%) the hepatocarcinogenic effect of both
safrole (0 13 or 0 25% in diet) and 1'-hydroxysafrole (0.14 or 0 27% in
diet) Induction of hepatic tumors by a single i.p injection of I'-hydroxy-
safrole to preweanling B6C3Fi mice is also inhibited by prior treatment with
pentachiorophenol Brachymorphic* mice, which are deficient in enzymes for
synthesis of 3'-phosphoadenosme-5'-phosphosulfate (PAPS, the sulfated
coenzyme also known as "activated sulfate," needed for the sulfotransferase),
develop much fewer liver tumors than their phenotypically normal littermates
when exposed to 1'-hydroxysafrole (dietary administration to adult females or
i.p. to preweanling males). These results, along with metabolism and DNA
*Brachymorphism, a recessive trait in mice, is phenotypically characterized by
disproportionately short stature as a result of undersulfation of the glycos-
aminoglycans in the cartilage (102) Homozygotic brachyraorphic (bm/bm) mice
have reduced capacity to synthesize PAPS because of defect in one or both of
the enzymes involved in the synthesis of PAPS from ATP and sulfate ion (103,
104) The heterozygotic (+/bm) and wild type ( + /+) mice are phenotypically
indistinguishable
369
-------
binding studies (see Section 5 3 244), provide convincing evidence that
1'-hydroxylation by cytochrome P-450-dependent microsomal mixed-function
oxidase followed by sulfation by cytosolic sulfotransferase represents the
principal metabolic pathway for activation of safrole to its ultimate carcino-
genic form, 1'-suifooxysafrole
An unusual case of syncarcinogenesis was noted by Epstein et al (90) in
a study involving piperonyl butoxide and Freon 112 or 113 (see Section
5 2.1 38, Vol IIIB) When administered singly, neither fluoroalkane nor
piperonyl butoxide is carcinogenic in neonatal mice However, combined treat-
ment of piperonyl butoxide with either Freon 112 or 113 induce liver tumors in
male mice The mechanism of synergism is unclear and is hypothesized to
involve modification of metabolism of Freon by piperonyl butoxide. In this
respect, it is relevant to note that safrole, isosafrole and related
methylenedioxybenzene compounds (including piperonyl butoxide) are inducers of
microsomal mixed-function oxidases (see Section 5 3 2.4 2 2), have been shown
to modify the metabolism of carcinogens (e g , 105), and may be expected to be
possible modifiers of careinogenesis
5 3 2 4 4 Metabolism and Mechanism of Action
5 3 2 4.4 1 METABOLISM
Metabolism of Safrole The metabolism of safrole has been extensively
studied and was the subject of several comprehensive reviews between 1977 and
1983 (44, 106-109) The initial metabolism of safrole involves three prin-
cipal types of reactions (a) 1'-hydroxylation of the allyl side chain, (b)
epoxidation of the 2',3'-double bond of the allyl side chain, and (c) oxida-
tion ("demethylenation") of the methylenedioxy group. The resulting metabo-
lites are further metabolized by a variety of pathways, giving rise to a large
370
-------
number of metabolites (see Fig 13) The relative importance of each of these
pathways which contribute, to some extent, to the careinogenicity of safrole,
is discussed below
There is ample evidence that 1'-hydroxylation is the predominant meta-
bolic activation pathway of safrole. 1'-Hydroxysafrole (compound II in Fig
13) has been detected in the liver, plasma, urine (mainly conjugated as
glucuronide) and bile of several species of animals given safrole (12, 13, 78,
110-113). Human volunteers given low doses of safrole did not excrete
1'-hydroxysafrole in their urine (112). Pretreatment with typical inducers of
microsomal mixed-function oxidases (e.g., phenobarbital, 3-methylcholanthrene)
increases the urinary excretion of conjugated 1'-hydroxysafrole by rats or
mice, but not by hamsters and guinea pigs (12). 1'-Hydroxysafrole is regarded
to be the proximate carcinogen of safrole because (a) it is more carcinogenic
(see Section 5.3 2.4 3.4) and mutagenic (see Section 5.3.2.4 2.2) than the
parent compound, (b) pretreatment of rats with phenobarbital enhances the
careinogenicity of safrole (see Section 5 3 2.4.3.6), and (c) it can be
further metabolized to electrophilic, ultimate carcinogens. At least three of
these metabolites — 1'-sulfooxysafrole (compound III in Fig 13), I'-oxo-
safrole (VI) and 1'-hydroxysafrole-21,3'-oxide (IV) — have been considered to
be possible candidates for ultimate carcinogen
The evidence that 1'-sulfooxysafrole may be an ultimate carcinogen of
safrole was first obtained by Wislocki et al. (15) who demonstrated that in
vitro incubation of 1'-hydroxysafrole with mouse or rat liver cytosol in the
presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS, the sulfated coenzyme
also known as "activated sulfate," needed for sulfotransferase) generates
reactive intermediates which bind covalently to nucleic acids. I'-Acetoxy-
371
-------
LEGEND TO FIGURE 13
Fig. 13. The principal metabolic pathways of safrole (I). The chemical
names of the metabolites are II, 1'-hydroxysafrole, III, 1'-sulfooxysafrole,
IV, r-hydroxysafrole-2' ,3'-oxide; V, 1' ,2' ,3'-trihydroxy-2' ,3'-dihydrosaf-
role; VI, I'-oxosafrole; Vila, 3f-(glutathion-£-yl)-l'-oxo-21,3'-dihydro-
safrole (SR= glutathionyl group), Vllb, S'-O^-acetylcystein-Jr-yD-l'-oxosaf-
role (SR « 2^-acetylcysteinyl group); Villa, S-JJ^-dimethylaraino-l-O' ,4 '-
methylenedioxyphenyl)-l-propanone (R » methyl group), VHIb, 3-pipendyl-l-
(31,4'-methylenedioxyphenyl)-l-propanone (NR2 * piperidyl group), VIIIc,
3-pyrrolidinyl-l-(3',4'-methylenedioxyphenyl)-l-propanone (NR2 = pyrrolidinyl
group), IX, 3'-hydroxyisosafrole; X, 3,4-methylenedioxybenzoylglycine, XI,
safrole-2',3'-oxide, XII, 2',3'-dihydroxy-2',3'-dihydrosafrole; XIII, 2-
hydroxy-3-(3' ,4'-methylenedioxyphenyDpropionic acid; XIV, 3,4-dihydroxy-l-
allylbenzene (allylcatechol), XVa, eugenol; XVb, 3-hydroxy-4-methoxy-l-allyl-
benzene, XVI, 2',3'-epoxypropylcatechol, XVII, 2*,3'-dihydroxypropylcatechol.
Virtually all hydroxylated metabolites can be further conjugated with
glucuronic acid whereas carboxylated metabolites can be conjugated with
glycine. PAPS = 3'-phosphoadenosine-5'-phosphosulfate ("activated sulfate")
-------
OR
CH-CH=CH2
OS03H
PAPS
CH-CH-CH2
OH V
t
W.
,
[0]
CH2-CH=CH2
I
OH
CH2-CH-CH2
xo
H0
I
- OH
H
CH2~CH=CH2
ZZa,b
OH
I5F
CH2-CH=CH2
OH
CH2-CH-CH
C-CH2-CH?-NR2
„ 2 2 2
0
2nTa,b,c
o
C-NH?CH2COOH
II 2 2
0
CH-CH-CH2OH
I I
OH OH
31
o-\
CHa-CH-COOH
OH
2HT
CH2-CH-CH20H
OH
TVTT
Fig. 13
-------
safrole*, a chemically synthesized model 1'-ester derivative of 1'-hydroxysaf-
role, is a reactive electrophiie (see Section 532421) and a direct-acting
carcinogen (see Section 532 4.3.4) and mutagen (see section 5324 2.2)
Strong evidence for an important role of 1'-sulfooxysafrole in the hepatocar-
cinogenesis by safrole or 1'-hydroxysafrole in the mouse has recently been
provided by Boberg et al (78) They showed that chronic administration of a
nontoxic level of pentachiorophenol, a potent inhibitor of sulfotransferase,
strongly inhibits (by as much as 82-100%) the hepatocarcinogenic activity of
safrole and 1'-hydroxysafrole. Brachymorphic mice, which lack competent
enzymes for synthesis of PAPS, develop much fewer liver tumors than normal
mice in response to 1'-hydroxysafrole treatment (see Section 5.3.2.4.3.6). In
both cases, reduced levels of covalent binding of 1'-hydroxysafrole to DNA and
RNA were observed These results, together with the finding that the major
DNA adducts of 1'-hydroxysafrole in the mouse liver are formed via an ester of
1'-hydroxysafrole (114, see also Section 5 3.2.4.4.2), led to the conclusion
(78, 109) that 1'-sulfooxysafrole is the major ultimate electrophiliu
metabolite, responsible for the DNA binding and carcinogenicity of I'-hydroxy-
safroie (and safrole) in the mouse liver.
The presence of 1'-oxosafrole as a metabolite of safrole was first
deduced from the study of Oswald et al (115) who detected three nitrogen-
containing metabolites in the urine of rats and guinea pigs given safrole
These metabolites were identified as 3-N,N-dunethylamino-l-(3',4'-methylene-
dioxyphenyl)-l-propanone (compound Villa in Fig. 13), 3-pipendyl-l-(3' ,4'-
methylenedioxyphenyl)-l-propanone (compound VHIb) and 3-pyrrolidinyl-l-
(31 ,4'-methylenedioxyphenyl)-l-propanone (compound VIIIc). All three of these
*Attempts to demonstrate in vitro formation of 1'-acetoxysafrole (by substi-
tuting PAPS with acetyl-CoA) were unsuccessful (15).
372
-------
A-ammoketones ("Mannich base") may decompose to yield 1'-oxosafrole upon
heating and are believed to be the condensation (Michael addition) products of
the vinyiketone (1'-oxosafrole) with the secondary amines (dimethyiamine,
piperidine, pyrrolidine) present in body fluids (20, 115) The possibility
that 1'-oxosafrole may be a potential ultimate carcinogen of safrole was
raised by Wislocki et al (15) who demonstrated the strong electrophilic reac-
tivity of the compound (see Section 532421) However, attempts to demon-
strate the carcinogenic or mutagenic activity of synthetic 1'-oxosafrole have
thus far been unsuccessful (55). The lack of genotoxicity of 1'-oxosafrole
has been attributed to its extremely high reactivity and instability (55) and
to the "soft" nature of its electrophilicity (23) In rats and mice given a
single i p dose of 1'-oxosafrole, the two major biliary and urinary
metabolites detected were the GSH-conjugates, 3'-(glutathion-S-yI)-i'-oxo-
2',3'-dihydrosafrole (compound Vila) and 3'-(N-acetylcystein-S-yl)-l'-oxo-
2' ,3'-dihydrosafrole (compound Vllb) Apparently, in contrast to I'-sulfooxy-
safrole (a "hard" electrophile), 1'-oxosafrole (a "soft" electrophile) is
extensively detoxified by glutathione (a "soft" nucleophile) and must first
deplete cellular GSH before it can react with the oxygen atoms or ammo groups
("hard" nucleophiles) of nucleic acid bases (23)
1'-Hydroxysafrole-21,3'-oxide is an in vitro metabolite of 1'-hydroxysaf-
role (15) The reaction is catalyzed by rat or mouse liver raicrosomes and is
dependent on NADPH. The yield is substantially higher when an inhibitor of
epoxide hydrase is included in the incubation medium (55). Trace amounts of
1'-hydroxysafrole-21,3'-oxide (as glucuronide) and 1',2',3'-trihydroxy-2',3'-
dihydrosafrole (compound V) have also been found in the urine of rats given
safrole (110, 111). 1'-Hydroxysafrole-21,3'-oxide is a relatively long-lived
electrophilic metabolite (15, see also Section 5 3 2 4 2.1) It is a direct-
373
-------
acting mutagen and carcinogen and is also active as a tumor initiator (55,
79) The metabolite is considered to be a possible ultimate carcinogen of
safrole, although its contribution to overall DNA binding and the carcino-
genicity of safrole still remains to be investigated
A fourth metabolic pathway of 1'-hydroxysafrole is isoraerization to
3'-hydroxyisosafrole (or 3,4-methylenedioxycinnamyl alcohol, compound IX in
Fig 13) This pathway is called "cinnaraoyl pathway" by some investigators
The mechanism has been postulated to involve protonation of the hydroxy group
of l'-hydrox,ysafrole, loss of H20 to form allylic cation, isomerization of the
allylic cation and rehydration to form 3'-hydroxyisosafrole (116) The equi-
librium strongly favors the formation of 3'-hydroxyisosafrole (116)
3'-Hydroxyisosafrole is further metabolized by oxidation and conjugation with
glycine to yield the hippuric acid derivative, 3,4-methylenedioxybenzoyl-
glycine (compound X) as the major urinary metabolite (110, 117). Owing to the
lack of careinogenicity and mutagenicity of 3'-hydroxyisosafrole and 3'-acet-
oxyisosafrole, the cinnamoyl pathway is generally considered to represent the
detoxication of safrole It should be noted, however, that one of the pos-
sible intermediates in this pathway, 3,4-methylenedioxycinnamaldehyde, is
potentially carcinogenic because a closely related compound, 3,4, 5-tnmethoxy-
cinnamaldehyde, is in fact carcinogenic (see Section 5.3 24 3 5).
Direct epoxidation of the 2',3'-double bond of the allyl side chain is
the second principal initial metabolic pathway of safrole This pathway is
called the "epoxide-diol pathway" by some investigators. Trace amounts of
safrole-21,3'-oxide (compound XI in Fig. 13) have been found in the urine of
t
rats and guinea pigs given safrole (110) and in in vitro studies using rat
hepatocytes (118) Its presence can also be deduced from the detection of its
dihydrodiol, 2',3'-dihydroxy-2',3'-dihdyrosafrole (compound XII) and further
374
-------
oxidation product, 2-hydroxy-3-(3 ,4-methylenedioxyphenyL) propionic ac id (com-
pound XIII), as urinary metabolites (110, 119) Safrole-2',3'-oxide is a
relatively long-lived electrophile It is capable of directly reacting with
nucleosides (15, see also Section 532421) and is a direct-acting mutagen
(Section 532422) However, safrole-2',3'-oxide appears to play a limited
role in careinogenesis by safrole It is inactive as a "complete" carcinogen
and is active only as a tumongenesis initiator (see Sections 532431 and
5 3 2.4 3 4)
Oxidation of the raethylenedioxy group of safrole is the third and the
major initial metabolic pathway of the compound This pathway is called the
"demethylenation pathway" by some investigators The predominant urinary
metabolite in animals (110, 112, 113) or humans (112) exposed to safrole is
allylcatechol (3,4-dihydroxy-l-allylbenzene, compound XIV in Fig 13) Small
amounts of eugenol (XVa) and its isomer, 3-hydroxy-4-raethoxy-l-allylbenzene
(XVb) and some raonohydroxy metabolites have also been detected The intact
allyl side chain of the above metabolites may be further oxidized to yield
epoxides (e g , 2',3'-epoxypropylcatechol, compound XVI) which in turn can be
hydrated to diol metabolites (e g , 2',3'-dihydroxypropylcatechol, compound
XVII) and further oxidized to corresponding propanoic acids (110, 120) The
demethylenation pathway is generally considered to represent detoxification
because the resulting metabolites lack the resonance-stabilizing _p_-methoxy
group, are much more hydrophilic, and are expected to be more readily
excreted However, there is some suggestive evidence that reactive interme-
diate(s) (e g , carbene) may be generated during demethylenation of the
methylenedioxy group and interact with cytochrome P-450 and the endoplasraic
reticulura to contribute to carcinogenesis by epigenetic mechanism(s) (see
further discussion in Section 532 4.4 2) Formaldehyde is a possible
375
-------
metabolite in the demethylation pathway, however, there is no experimental
evidence for this so far
Metabolism of Myristicin, Isosafrole and Dihydrosafrole Very little
information is available on the metabolism of myristicin. /3-Aminopropiophe-
nones (compounds Villa,b,c in Fig 13) corresponding to those reported for
safrole have been detected in the urine of animals given myristicin (121)
suggesting that 1'-oxomyristicin may be a metabolic intermediate of
myristicin Myristicin binds covalently to DNA following metabolic activation
(see Section 532442), the nature of the DNA adduct is not known The
metabolism of isosafrole and dihydrosafrole has recently been studied by
Klungs^yr and Scheline (117) Demethylenation of the methylenedioxy group is
by far the most predominant metabolic pathway, accounting for 92% and 95% of
the total metabolism of isosafrole and dihydrosafrole, with 4-propenylcatechol
(1,2-dihydroxy-4-propenylbenzene) and 4-(l-propyl)-catechol as the major
metabolite, respectively For isosafrole, the other metabolites (e g ,
3'-hydroxyisosafrole, 3,4-methylenedioxycinnamic acid, 1',2'-dihydroxydihydro-
safrole, 3,4-methylenedioxybenzoyl glycine) are attributable to metabolism via
the epoxide-diol and the cinnamoyl pathways Trace amounts of 1'-hydroxysaf-
role have been detected in the urine of rats given 3'-hydroxyisosafrole
(116). Besides demethylenation, dihydrosafrole is also metabolized by ring
hydroxylation and I1- as well as 2'-hydroxylation Interestingly,
1',2'-dihydroxydihydrosafrole is also a metabolite (albeit very minor,
accounting for 0 2% of the total dose) of dihydrosafrole, despite the lack of
double bond in the side chain
Metabolism of Estragole and Related Compounds. The metabolism of estra-
gole and related compounds bears a close resemblance to safrole and related
compounds, with the exception that the demethylenation step is replaced by the
376
-------
0-demethyLation step Solheira and Scheline (122) showed that estragole is
metabolized in the rat by (a) the 0-demethylation pathway to yield
4-hydroxy-l-allylbenzene (approx 39-46% of the dose), (b) the epoxide-diol
pathway to yield 2",3'-epoxide, 2',3'-dihydrodiol and eventually 2-hydroxy-3-
(4-methoxyphenyl)propionic acid and 4-methoxybenzoyl glycine (approx 17-31%
of the dose), and (c) 1'-hydroxylation to yield 1'-hydroxyestragole (approx
5-10% of the dose) which in turn undergoes isomerization to 3'-hydroxyiso-
estragole and further metabolism by the cinnamoyl pathway A metabolic study
by Drinkwater et^ ^1_ (14) found that 23% of an intraperitoneal dose (1 85
mmol/kg body weight) of estragole may be recovered as 1'-hydroxyestragole
(mostly as conjugates) in the urine of preweanling mice (which are highly
susceptible to the hepatocarcinogenic effect of estragole) Zangouras et al
(123) showed thai the proportion of the dose converted to 1'-hydroxyestragole
in rodents is nonlinearly dose-dependent increasing from 1% at 0 05 rag/kg to
12% at 1,500 rag/kg. DNA binding studies (see Section 532442) led the
Millers and their associates (109, 114) to suggest that, as with safrole,
1'-sulfooxyestragole may be the principal electrophilic metabolite responsible
for the DNA binding and carcinogenicity of estragole The metabolism of two
higher homologs (methyleugenol and elemicin) of estragole has also been
studied by Solheira and Scheline (124, 125) The most significant change is
the substantial decrease in the importance of the 0-demethylation pathway with
the increase of ring substitution with methoxy group(s). For both compounds,
the cinnamoyl and the epoxide-diol pathways are prominent. Large amounts of
I'-hydroxy derivatives of methyleugenol and elemicin have been found in the
bile (124, 125). Some of the I'-hydroxy metabolites appear to be oxidized to
I'-oxo metabolites, as suggested by the detection of /3-ammopropiophenones as
urinary metabolites (124, 126).
377
-------
As with the methoxyallylbenzene congeners, the extent of ring substitu-
tion can have a dramatic effect on the metabolism of methoxypropenylbenzene
congeners 0-Demethylation is the predominant metabolic pathway in the
metabolism of trans-anethole (127, 128), but this pathway is only a very minor
one for its dimethoxy and trimethoxy homologs, isoraethyleugenol and iso-
elemicin (124, 125) The cinnamoyl pathway and, to a lesser extent, the
epoxide-diol pathway account for most of the metabolism of isomethyleugenol
and isoelemicin in the rat (124, 125). The metabolism of trans-anethole
displays significant dose-dependence and species difference At low doses, as
much as 56-72% of the compound is metabolized by the 0-demethylation path-
way. At high doses, however, the 0-demethylation pathway appears to be
saturated and the cinnamoyl and epoxide-diol pathways dominate A species
comparison study indicates that rats favor the epoxide-diol pathway while mice
favor the cinnamoyl pathway (128)
5 3.2 4 4 2 MECHANISM OF ACTION
Safrole and several of its congeners are genotoxic carcinogens Covalent
binding to DNA of safrole and its proximate carcinogen, 1'-hydroxysafrole has
been convincingly demonstrated by Boberg et al (78) Modifying factors
(eg., pentachlorophenol, brachymorphism), which inhibit the hepatocarcino-
genicity of these compounds in mice by 82-100% (see Section 5.3 2 4.3 6), also
reduce their covalent binding to liver DNA by 85-89% There is, moreover, a
reasonably good correlation between DNA-binding activity and carcinogenicity
of congeners of safrole Randerath j2t__al_ (129) studied the in vivo covalent
binding of 10 alkenylbenzene compounds to mouse liver DNA The "covalent
binding indexes" of the compounds follow the order methyleugenol (36 0) >
safrole (28 4) = estragole (28 4) > myristicm (10 7) > dill apiol (7 7) >
parsley apiol (3 0) > elemicin (2.3) > anethole (0.16) > allylbenzene (0 16) >
378
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eugenoL (no binding) The three compounds (methyleugenoL , safrole and
estragole) that exhibit the highest DMA-binding activities are carcinogenic,
their relative carcinogenic potencies (see Table LXV) correlate with their
covalent binding indexes The seven compounds that show a lower level or lack
of DNA-binding activities are all noncarcinogenic (see Section 53243)
The covalent binding indexes of some of these compounds (e g , mynsticin,
dill apiol) appear to suggest a greater genotoxic potential than the
carcinogenicity data would indicate It would be interesting to investigate
whether the nature of DNA adducts and the repair efficiency of these adducts
of carcinogenic compounds differ from those of noncarc inogenic compounds
Consistent with carcinogenicity data, the covalent binding index of estragole
is substantially higher than that of anethole (isoestragole) Another in vivo
DNA binding study by Fennell et al (24) showed high levels of covalent
binding of 1'-hydroxy-2',3'-dehydroestragole, a very potent hepatocarcinogen
(see Table LXV), to mouse liver DNA
The nature of DNA-adducts formed in mouse liver following administration
of the I'-hydroxy derivative (the proximate carcinogen) of safrole, estragole
and 2' ,3'-dehydroestragole has been studied by the Millers and associates (17,
24, 25) Four or five major nucleoside adducts have been found in the hepatic
DNA of mice given 1'-hydroxyestragole (25, 114). They were identified as
N -(estragol-1'-yOdeoxyguanosine (two diastereomers) , N2-( trans- isoestragol-
9 A
3'-yl)deoxyguanosine, N -(cis-isoestragol-3'-yl)deoxyguanosine and N -(trans-
isoestragol-3'-yDdeoxyadenosine These adducts arise as the result of the
reaction of an ester of 1'-hydroxyestragole with purine bases in DNA by either
SN1, SN2 or a modified SN2 (SN2f) mechanisms (see Fig 14). Five analogous
nucleoside adducts have been found in mice given 1'-hydroxysafrole (25, 114,
130) These results, coupled with the demonstration of the critical role of
379
-------
OCH
CH-CH=CH2
DNA(dGuo)
Adduct I -*
DNA(dGuoordAdo)-
Adducts nj
OCH,
CH-H
II
CH2
dR
Adduct I
OCH,
H-Ci
o
Adduct E
dR
QCH3
ll
Adduct IE
OCH3
I)
I
CH2-NH
dR
Adduct JZ
Fig. 14. Proposed mechanisms by *rtiich an ester of 1'-hydroxyestragole
can react with purine bases in DNA to yield the adducts found in mouse liver
DMA in vivo. In the formulas, X » -OSOj or -OCOCH^, dR » deoxyribose. The
chemical names of the adducts -are: Adduct I, _N^-(estragol-l '-yDdeoxyguano-
sine (two diastereomers), Adduct II, j^-(tran3-isoestragol-3'-yl)deoxy-
guanosine, Adduct III, _N -(cis-isoestragol-S'-yDdeoxyguanosine. Adduct IV,
jjf6-(tran8-i8oe3tragol-3'-yl)deoxyadenosine [Modified from D.H. Phillips, J. A
Miller, E. C. Miller and B. Adams. Cancer Res. 41. 176 (1981)].
-------
suifation in caremogenesis by safrole or 1'-hydroxysafrole (78), suggest that
1'-hydroxylation followed by suifation is the predominant metabolic activation
pathway for both safrole and estragole Only a single nucleoside adduct,
which comigrates on high performance liquid chromatography (HPLC) with
r\
N -(2',3'-dehydroestragol-1'-yl)deoxyguanosine (obtained by in vitro reaction
of 1 '-acetoxy-21 ,3 '-dehydroestragole with dGMP) , has been detected in hepatic.
DNA of mice given 1'-hydroxy-2',3'-dehydroestragole (24) suggesting that an
electrophilic 1'-ester is the ultimate carcinogen of the compound
The molecular mechanism of carcinogenesis after the initial covalent
binding of the carcinogen to DNA is not clearly understood. Most of the
7 ft
N -guanine and N -adenine adducts are removed quite rapidly from mouse liver
DNA through the repair mechanism(s) Nonetheless, a significant fraction of
each adduct persists for up to 20 days after treatment (17) It has been
postulated that the formation of adduct at the N^-position of adenine may lead
to mutation by causing raispairing, between deoxycytidine and the imino tauto-
meric form of deoxyadenosine, during DNA replication (131). Adduct formation
can facilitate the cleavage of the sugar-phosphate backbone in DNA leading to
apurinic/apyrimidinic sites which could, under certain special conditions (see
Section 531142), lead to mutation. Alternatively, chemically induced DNA
repair is often error prone and can lead to infidelity of DNA replication.
Cultured human cells exposed to 1'-acetoxysafrole or 1'-acetoxyestragole
undergo DNA repair replication shortly (4-11 hours) after the treatment
(26) DNA damage, consistent with the presence of apurinic/apyrimidinic
sites, have been demonstrated in a small fraction of these cells (132) How-
ever, a comparative study by Drinkwater et al. (133) show that the capacity of
a variety of structurally different types of carcinogens (including
1'-acetoxyestragole) to produce apurinic/apyrimidinic sites in supercoiled DNA
380
-------
does not correlate well with their rautagenic activity in the Ames test Also,
a number of mutagenic, electrophilic metabolites of alkenylbenzene compounds
(e g , safrole-2',3'-oxide, estragole-2' ,3'-oxide) are inactive as "complete"
carcinogens (see Sections 532431 and 5 3 2.4 3 4) suggesting that somatic
cell mutation alone may not be sufficient to explain the complete process of
carcinogenesis The elucidation of the mechanism of careinogenesis by
safrole, estragole and related compounds awaits further studies
In addition to the genotoxic mechanisms described above, there is sugges-
tive evidence that the methylenedioxy group may contribute to the overall car-
cinogenicity of safrole and related compounds through epigenetic mechanisms
Studies by various investigators (rev in 44) show ligand complexing and
oovalent binding of reactive intermediates (most likely carbene intermediate,
see Fig. 15) of safrole with the heme and protein moieties, respectively, of
cytochrome P-450. Such binding may cause structural and functional changes in
the endoplasmic reticulum resulting in loss of ribosomes ("degranulation")
known indeed to occur following treatment with safrole or other carcinogens
(134) It has been suggested (134) that loss of ribosomes could lead to
impairment of glycoprotein synthesis and contribute to the process of
malignant transformation by epigenetic mechanisms The simultaneous loss of
cytoohrome P-450 activities through ligand complexing and eovalent binding
and, on the other hand, increase in cytochrome P-448 activities through enzyme
induction by safrole may also be contributory factors because such changes
have been found to be often conducive to chemical carcinogenesis (134)
Another theoretically possible way by which the methylenedioxy group may
contribute to careinogenicity is through the release of formaldehyde Being a
cyclic acetal, the methylenedioxy group may, under acidic conditions, be
degraded to diol and formaldehyde (which is reactive and carcinogenic, see
381
-------
OH
CH2CH=CH2
Safrole
CH2CH=CH2
CH2CH=CH;
Safrole carbene
Cytochrome
P-450
CH2CH=CH2
Safrole carbene
cytochrome P-450
complex
Fig. 15. Proposed possible mechanism of formation of a safrole carbene
cytochrome P-450 complex [Adapted from C. loannides, M. Delaforge and D. V.
Parke: Food Cosmet. Toxicol. 19, 657 (1981)].
-------
Section 52171) The induction of forestomach tumors in mice by dihydro-
safrole lends some support to this hypothesis The in situ release of reac-
tive compound at or near the target site is expected to be more hazardous than
the administration of the compound at a distant site However, this hypo-
thesis does not explain why other methylenedioxy derivatives discussed in
Section 5 3 2 4.1 2 are incapable of inducing stomach tumors.
532 4 5 Environmental Significance
Safrole and its congeners including the cinnamyl compounds may occur in
the environment as naturally occurring constituents of food materials of plant
origin, of spices and herbal medicines, as synthetic food additives, as cos-
metics and toiletry ingredients, or as pesticide residues. The natural occur-
rence, economic production and uses of a number of these compounds are sum-
marized in Table LXVII In particular, the essential oils, extracted by steam
distillation or solvent extraction from parts of plants (e g., root, rhizome,
seed, bark), contain high concentrations of alkenylbenzene and related com-
pounds. For example, as much as 93% of a sample of Brazilian oil of sassafras
is safrole (149) Plants from different parts of the world may contain widely
different quantities of alkenylbenzene compounds. The oil of calamus
extracted from Indian Acorus plants contains about 80% /3-asarone (5, 145),
whereas that of European variety contains only 5% /3-asarone (146) An
analysis of myristicin content in essential oils extracted from roots of 24
different varieties of cultivated parsnip showed a wide range of 18.3 to 66.2%
(138) A number of essential oils were or still are extensively used as food
flavoring agents or as cosmetics ingredients Besides natural occurrence,
there is some evidence that treatment of oranges with certain abscission
agents* (such as cycloheximide, 5-chloro-3-methyl-4-nitro-lH-pyrazol, glyoxal
diamine) may cause the appearance of eugenol, methyleugenol, cis- and trans-
* for footnote, see p 3B3 332
-------
methylisoeugenol , eLeraicin and isoelemicin in orange juices at levels of 4-40
ppb and in essential oils (150) The mechanism of enhancement of this
chemically induced environmental formation of alkenylbenzene compounds is
unknown Several methylenedloxy compounds structurally related to safrole
(e g., piperonyl butoxide and sulfoxide), are used as insecticides or insecti-
cide synergists. There is little information on the extent of food contamina-
tion by residues of these pesticide synergists A study of the environmental
fate of methyleugenol, an insect attractant useful in control of fruit fly,
showed tj/2 of 16 hours in soil and 24 hours in water at 22°C When topically
applied to the surface of tomatoes, about 3.8% of the dose was still present
after 24 hours, none was detected after 5 days (151).
Human exposure to alkenylbenzne and cinnamyl compounds occurs mainly
through ingestion of foodstuffs or drugs to which naturally occurring or
synthetic flavor additives are added. Safrole, isosafrole and dihydrosafrole
were used as flavoring agent in root beer prior to the banning of their use in
foods by the U S. Food and Drug Administration (FDA) in 1961 To some extent,
sassafras bark is still being used as herbal tea or as an ingredient thereof,
and in folk medicine (152). According to Segelman £t__al_ (153), a single
herbal tea bag sold by some producers may contain 2 5 mg sassafras bark equi-
valent to 200 mg safrole. If completely absorbed, the consumption of such tea
could lead to exposure to a potentially hazardous dose of 3.0 mg/kg body
*Abscission agents are synthetic plant-growth regulating chemicals which
promote the separation or shedding of a plant part (e g , leaf, flower, fruit
or stem) from the parent plant They are used (a) to thin (shed) fruits in
trees with too many fruits, so that the size and quality of the remaining
fruits may be improved, (b) to shed leaves just before mechanical harvesting
of crops such as cotton, and (c) to promote separation of mature fruits from
tree branches for easier picking.
383
-------
weight (a total subcutaneous dose of approximately 66 mg/kg body weight is
carcinogenic in infant mice). It is interesting to note that safrole is
detected in the expired air of a group of 62 nonsmoking volunteers with no
known exposure to safrole suggesting that the compound is being bioaccumulated
involuntarily in the general population (154) /3 -Asarone was at one time
used as a flavoring agent (confering bitter flavor) in liqueurs and vermouth
at levels of up to 10-30 ppra (147) before it was banned by FDA in 1967 Its
use is still permitted in some countries (137). Calamus drugs containing
A-asarone are being used in Europe Several commercial calamus drugs were
shown to be mutagenic in the Ames test (67), long-term use of these drugs may
represent a carcinogenicity risk. Estragole, the major constituent of oils of
tarragon and basil, has been used as a flavoring agent in gourmet types of
vinegar (155) as well as in a variety of food products (candy, chewing gum,
ice cream) at levels of 2-50 ppm (135) Cinnamyl anthranilate has been used
as a synthetic flavoring agent (to imitate grape or cherry flavor) in the
United States and in Europe. It is added to a variety of food products (e g ,
chewing gums, ice cream, baked goods, gelatin, beverages) at levels of 1 7 to
730 ppm (135). The banning of this compound was reported to be under consi-
deration by FDA (156). The World Health Organization (157) recommended that
no acceptable daily intake (ADI) should be allocated to any of the above food
additives
Among alkenylbenzene and cinnamyl compounds with equivocal or no evidence
for carcinogenicity, trans-anethole is the most widely used food additive It
is reported to be used in a variety of food products such as nonalcoholic
beverages (11 ppm), alcoholic beverages (1,400 ppm), ice cream (26 ppm), candy
(340 ppm), baked goods (150 ppm), and chewing gums (1,500 ppm) to impart the
popular aniseed flavor (135). The estimated annual consumption of the com-
384
-------
pound as a food additive in the United States is 70 tons, which represents an
average daily intake of 60 ug per person (144) In France, as much as 200
tons of the compound is used annually because of the popularity of alcoholic
and non-alcoholic aniseed beverages (40). Cinnamaldehyde is another popular
food flavoring (cinnamon) agent used in food products at levels ranging from
7 7 to 4,900 ppm (135) The World Health Organization (157) recommended that
the acceptable daily intake for humans should not exceed 0.7 mg/kg body
weight. Myristicin is the principal physiologically important ingredient of
oils of nutmeg and mace, which enjoyed high esteem in the early Middle Ages in
the Arab world and in India as almost a panacea for treatment of a wide
variety of ailments such as toothache, dysentery, cholera, rheumatism, hali-
tosis and skin diseases (137) The medicinal use of these spices declined
sharply during the 19th century when their narcotic, toxic and hallucinogenic
properties were discovered Myristicin is also found in many edible plants
and in black pepper (see Table LXVII), its concentration in parsnip root is
particularly high (138)
The literature on the potential careinogenicity risk of human exposure to
naturally occurring alkenylbenzene compounds is rather scanty Morton (152,
158, 159) reported that cancer of the esophagus was the most common type of
tumor among male cancer patients in South Carolina (U S.A ). She suggested
that induction of esophageal cancer may be associated with the common use of
sassafras tea (which contains safrole) as a folk medicine for cure of fever,
pneumonia, bronchitis and mumps among the native residents, particularly by
the black population. In this respect, it is important to note that dihydro-
safrole, a synthetic derivative of safrole, induces indeed esophageal cancer
in rats. Although safrole itself is predominantly a hepatocarcinogen in
rodents, its potential to induce esophageal cancer should not be discounted
385
-------
p 1
Table LXVII
Environmental Occurrence, Production and Economic Uses of Safrole and Related Compounds
Compound
Natural Occurrence/
Economic Production
Uses
References
Safrole3
Myristicin
Dill apiol
Parsley apiol
Isosafrole3
Dihydrosafrole3
Piperonyl butoxide
Piperonyl sulfoxide
Estragole3
Sassafras , sweet basil,
cinnamon, nutmeg, mace,
ginger, black pepper
Nutmeg , mace , parsnip ,
black pepper, carrot,
parsley, celery, dill
Dill, Indian dill
Parsley, fennel, sassafras
Ylang ylang
Synthetic, U.S production/
import volume in 1977
4 million Ib
Synthetic, U S production
volume in 1978 334,000 kg
Synthetic
Tarragon , sweet basil ,
anise, U S production
volume in 1977 >1,360 kg
Flavoring agent in root
beerc , tea, folk medicine
Flavoring agent , herbal
medic me
Flavoring agent
Flavoring agent
Flavoring agent0, fragrance,
production of piperonyl
butoxide
Flavoring agent0, production
of ptperonyl butoxide
Insecticide synergist
Insecticide synergist
Flavoring agent, fragrance
(7, 135)
(136-140)
(79)
(79)
(7, 10, 135)
(7, 141)
(8, 142)
(88, 142)
(135, 142)
-------
Table LXVII (continued)
p. 2
Compound
Natural Occurrence/
Economic Production
Uses
References
Methyleugenol'
El eraic in
trans-Anethole
3 -Asaronea
Eugenol
Cinnamaldehyde
Cinnamyl
anthranilate3
Sweet bay, cloves, lemon-
grass, black pepper, U S
production volume in 1977
>1,800 kg
Flavoring agent, fragrance,
insect attractant
Nutmeg, elemi gum, sassafras Flavoring agent
Anise , fennel , coriander,
U S production in 1977
1 1 million kg
Calamus (Acornus calamus)
plants , plants of Asarum
and Asiasarum genera
Cloves , allspice, arti-
choke, pimenta, bayleaf,
cinnamon
Cinnamon , also produced
synthetically, U S pro-
duction volumes in 1977
910 kg
Synthetic, U S production/
import volume in 1977
454 kg
Flavoring (aniseed) agent,
fragrance
Flavoring agent0 in bitters,
liqueurs and vermouths,
herbal medicine
Flavoring agent, fragrance,
ingredient in temporary
denture fillings
Flavoring (cinnamon) agent
for foods, beverages,
Pharmaceuticals and
liqueurs
Flavoring (grape, cherry)
agent , cosmetics
fragrance
(79, 136, 143)
(79)
(10, 135, 143)
(5, 137, 145-148)
(135)
(18, 135)
(9, 135)
aClear evidence of careinogenicity demonstrated, World Health Organization recommends that no
acceptable daily intake should be allocated
The compound is a (the) major constituent in essential oil derived from the plant
cUse now banned in U S by the Food and Drug Administration
Under consideration for banning
-------
Besides safrole, sassafras and other herbal teas may contain other carcino-
genic substances, such as tannin (see Section 5 3.2 6 3) The possibility of
syncarcinogenesis among these food components should be investigated
In contrast to the scanty epidemiologic literature on alkenylbenzene com-
pounds, there is ample epideraiologic evidence to indicate excess cancer (par-
ticularly nasal adenocarcinoma) risk among woodworkers exposed to wood dust
(especially furniture and cabinet makers) throughout the world (rev. in
160). In addition, the high incidence of nasopharyngeal cancer among the
Southern Chinese and the Highland Kenyans was postulated to be associated, in
part, with chronic exposure to wood smoke (161) Although most of the above
mentioned population groups at risk are often simultaneously exposed to other
industrial or environmental chemicals or agents (e.g , formaldehyde, benzene,
herbicides, paint solvents, nitrosamines, Epstein-Barr virus) with carcino-
genic potential, it is believed that at least part (if not most) of the car-
cinogenic effects is contributed by naturally occurring chemicals present in
wood dust or wood smoke Despite some early work, the search for naturally
occurring carcinogens in woods is still in the exploration stage (94, 162)
The potential carcinogens include phenolic and flavonoid compounds (see
Sections 53262 and 532 6.3), substituted cinnamyl compounds and podo-
phyllotoxin. Smapaldehyde (3,5-dimethoxy-4-hydroxycinnamaldehyde) and
related compounds have been detected in the smoke of Chinese incense derived
from sandal wood, in eucalyptus wood abundant in Kenya and in other angio-
spermous woods (94, 163-165). A closely related compounds, 3,4,5-trimethoxy-
cinnamaldehyde, has been shown to be a nasal carcinogen in rats (see Section
5.3.2 4.3.5) Podophyllotoxin is suspected to account for the apparent car-
cinogenic effect of red cedar (Juniper virginiana) wood bedding in the induc-
tion of "spontaneous" tumors in C3HAvy mice (see Section 5 3 2.4.3 5)
386
-------
Whether these compounds could contribute to the apparent human carcinogenicity
of wood dust or wood smoke remains to be elucidated.
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SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 532.4
1 loannides, C., Delaforge, M , and Parke, D.V Food Cosmet Toxnoi
19, 657-666 (1981)
2 Ringk, W.F. Kirk-Qthmer' s EncycLoped Chem Techno1 (34d edn ) _6_,
142-149 (1979)
3 Miller, J A , and Miller, E C. Br J. Cancer 48, 1-15 (1983)
4 Miller, J A , Miller, E.C , and Philips, D.H The Metabolic
Activation of Alkenylbenzenes that Occur Naturally in Many Spices In
"Carcinogens and Mutagens in the Environment, Vol. I, Food Products"
(H.F. Stich, ed.), CRC Press, Boca Raton, Florida, 1982, pp. 83-96
5. International Agency for Research on Cancer "Some Naturally Occurring
Substances," IARC Monographs on the Evaluation of Carcinogenic Risks of
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399
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