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

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(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-





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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
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                                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)

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     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

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     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)

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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

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                                                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

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                                                                                                                  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

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                                                  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

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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

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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

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                                   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

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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

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                                                        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

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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

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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

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                                  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

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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

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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

-------
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.









                         REFERENCES  TO SECTION 5324









   1.  Long, E L ,  Hansen, W H , and Nelson, A.A    Fed  Proc  20, 287  (1961)




   2   Homburger, F ,  Kelley, T  Jr., Friedler, G I , and Russfield, A  B




       Med  Exp. 4, 1  (1961).




   3.  Abbott, D D., Packman, E.W ,  Wagner, B.M., and Harrison, J W.E.




       Pharmacologist  3, 62 (1961).




   4.  Hagan, E C. , Hansen, W H , Fitzhugh, O.G. , Jenner, P.M , Jones,  W  I.,




       Taylor, J M , Long, E.L , Nelson, A A., and Brouwer, J.B.   Food




       Cosmet  Toxicol ^5_, 141 (1967)




   5   Gross, M.A , Jones, W I , Cook, E L , and Boone, C C.   Proc. Am




       Assoc  Cancer Res. _9_, 24 (1967)




   6   Taylor, J M., Jones, W.I , Hagan, E C., Gross, M A , Davis, D A  ,  and




       Cook, E L    Toxicol. Appl  Pharmacol  10, 405 (1967)




   7   International Agency for Research on Cancer    IARC Monogr  10, 231




       (1976)




   8.  International Agency for Research on Cancer    IARC Monogr. 30, 183




       (1983).




   9.  International Agency for Research on Cancer    IARC Monogr  31 , 133




       (1983).




  10.  Opdyke, D.L J    Food Cosmet   Toxicol. 14, 307 (1976).




  11.  Windholz, M  (ed )   "The Merck Index," 10th edn  , Merck and Co  ,




       Rahway, New Jersey, 1983
                                      387

-------
12   Borchert, P , Wislocki, P G , Miller, J A  , and Miller,  E C    Cancer




     Res  33, 575 (1973).




13   Borchert, P., Miller, J A , Miller, E.C , and Shires, T  K    Cancer




     Res  33, 590 (1973) .




14   Drinkwater, N R., Miller, E C , Miller, J A , and Pitot, H C.   _J_




     Natl  Cancer In,st  57, 1323 (1976)




15   Wislocki, P G , Borchert, P , Miller, J A  , and Miller,  E C    Cancer




     Res. 36. 1686 (1976).




16.  Swanson, A B., Chambliss, D D , Blomquist , J C., Miller, E.C , and




     Miller, J A    Mutat  Res. 60. 143 (1979)




17   Phillips, D.H., Miller, J A , Miller, E C  , and Adams, B.   Cancer Res




     41, 176 (1981)




18   Ringk, W F    Kirk-Qthmer' s Encycloped  Chem  Technol  (3rd ed.) _6_,  142




     (1979)




19   Hawley, G G    "The Condensed Chemical Dictionary," 9th  edn., Van




     Nostrand Reinhold, New York, 1977




20   McKinney, J.D , Oswald, E., Fishbein, L. , and Walker, M.   Bull




     Environ  Contam  Toxicol. 7_> 305 (1972).




21   Eder, E., Neudecker, T , Lutz, D., and Henschler, D    Chem -Biol




     Interact  38, 303 (1982)




22.  Miller, J A., Swanson, A.B , and Miller, E C.   The Metabolic Activa-




     tion of Safrole and Related Naturally Occurring Alkenylbenzenes  in




     Relation to Careinogenesis by These Agents.  In "Naturally Occurring




     Carcinogens — Mutagens and Modulators of Carcinogenesis" (E C  Miller,




     J A  Miller, I  Hirono, T  Sugimura and S  Takayama, eds ), University




     Park Press, Baltimore, Maryland, 1979, p   111.
                                    388

-------
23   Fennell, T R , Miller, J A  , and Miller,  E  C.    Cancer  Res  44,  3231




     (1984)




24   Fennell, T R , Miller, J A  , and Miller,  E  C     Proc   Am  Assoc   Cancer




     Res  25.. 88 (1984).




25   Wiseman, R W , Miller, J A  , Miller,  E  C  ,  Drinkwater,  N R ,  and




     Blomquist, J C    Proc  Am  Assoc   Cancer Res   25,  85  (1984)




26   Phillips, D H , and Hanawalt, P C     Careinogenesis 3,  929 (1982)




27   Heffter, A    Versuche uber die Wirkungen des  Safrols  und Isosafrols




     sowie einiger anderer Substanzen, die Dioxyraethylen-Gruppe




     enthaltend   In "Proceedings of XI  Congress o  Medico  Internazionale,"




     Vol  3, Rome, 1894, p  32




28   Heffter, A    Arch  Exp. Path  Pharmacol  35.  342  (1895)




29   Albright, LM    Cine in .Lancet-Clinic  21.  631  (1888)




30   Gang, J 0    Arch  Pis  Child  28, 475 (1953).




31   Jacobs, M B    Amer  Perfom  Aromat   71,  57 (1958)




32   Leidy, W P    Amer  Perfurn  Aromat  71, 61  (1958)




33   Long, E L., and Jenner, P M    Fed  Proc. 22,  275  (1963)




34   Long, E L , Nelson, A A , Fitzhugh, 0 G., and  Hansen,  W.H    Arch




     Pathoi. 75, 595 (1963)




35   Jenner, P M , Hagan, E.C , Taylor,  J.M.,  Cook,  E L ,  and Fitzhugh,




     0 G    Food Cosmet  Toxicol  _2_, 327 (1964)




36.  Hagan, E C , Jenner, P M , Jones, W I , Fitzhugh,  0 G.,  Long, E  L  ,




     Brouwer, J G , and Webb, W K    Toxicol.  Appl.  Pharmacol  _7_>  18  ^965)




37   Draize, J H , Alvarez, E , Whitesell, M.F , Woodward,  G , Hagan, E  C  ,




     and Nelson, A A    J  Pharmacol  Exp  Therap   93,  26  (1948)




38   Opdyke, D L J.   Food Cosmet  Toxicol   13,  751  (1975)
                                    389

-------
39   Caujolle, F , and Meynier, D    C R  Lebd   Seanc   Aiad   Sci   Paris  246,




     1465 (1958)




40   LeBourhis, B.   "Les Proprietes Biologiques de  L'Anethole," MaLoine,




     Paris, 1973




41   Shulgin, AT..  Nature, (London) 210, 380 (1966)




42.  Cesano de Mello, A , Carlini, E A., Dressier,  K  ,  Green,  J P ,  Kang,




     S ,  and MargoLis, S    Psychoparmacologia  Berl . 31 ,  349  (1973)




43   Hodgson, E , and Philpot, RM    Drug Me tab   Rev  _3_,  231  (1974)




44.  loannides, C., Delaforge, M , and Parke, D V    Food  Cosmet   Toxicol




     19.  657 (1981)




45   Sun, Y P , and Johnson, E R    J  Ag r ic . Food Chem. J_,  261  (1960)




46.  Hodgson, E , and Casida, J E    Biochim. Blophys.  Acta  42,  184  (1960)




47   Fennel1, T R., Sweatman, B C , and Bridges, J.W    Chem  -Biol




     Interact  31, 189 (1980).




48   Dickens, M , Bridges, J.W , Elcombe, C.R ,  and  Netter,  K J    Biochem




     Biophys  Res  Commun. 80, 89 (1978).




49   Ryan, D E., Thomas, P E , and Levin, W     Properties  of  Liver Micro-




     somal Cytochrome P-450 from Rats Treated with Isosafrole    In "Micro-




     soraes and Drug Oxidation" (M.J  Coon, A H   Conney,  R.W   Estabrook,  H V




     Gelboin, J.R. Gillette and P.J  O'Brien, eds  ), Academic  Press,  New




     York, 1980, p. 167




50   Fisher, G.J., Fukushima, H , and Gaylor, J.L    J   Biol   Chem  256,




     4388 (1981).




51   Cook, J C , and Hodgson, E    Toxicol. Appl.  Pharmacol.  68, 131  (1983)




52   de Serres, F J , and Ashby, J  (eds )   "Evaluation of  Short-term Tests




     for Carcinogens   Report of the International Collaborative Program,"




     Progress in Mutation Research, Vol. I, Elsevier/North Holland,




     Amsterdam, The Netherlands, 1981.
                                    390

-------
53   MuCann, J , Choi, E , Yamasaki, E, and Ames, B N     Proc   Nat   Ac ad




     Sci  U.S A  72, 5135 (1975)




54   Derange, J -L  , Delaforge, M , Janiaud, P  , and Padieu,  P    C  R   Soc




     Biol  171, 1041 (1977)




55   Wislocki, P G  , Miller, E C., Miller, J A  , McCoy,  E C  , and




     Rosenkranz, H  S    Cancer Res. 37, 1883 (1977)




56   Green, N R ,  and Savage, J R    Mutat  Res  57. 115  (1978)




57   Rosenkranz, H  S , and Potrier, L A    J. Natl. Cancer Inst  62, 873




     (1979).




58   To, L.P , Hunt, T P , and Andersen, M E.   Bull. Environ   Contain




     Toxicol. 28,  647 (1982).




59   Sekizawa, J ,  and Shibamoto, T    Mutat  Res  101,  127  (1982)




60   Simmon, V F    J. Natl. Cancer Inst  62, 893 (1979)




61   Dorange, J -L  , Janiaud, P , Delaforge, M., Levi, P   ,and  Padieu,  P




     Mutat  Res  53, 179 (1978)




62   Buchanan, R L  , Goldstein, S., and Budroe, J D    J  Food  Sci   47, 330




     (1981).




63   NTP   "NTP Technical Bulletin No  5," National Toxicology  Program,




     Research Triangle Park, North Carolina, 1981.




64   Ishidate, M  Jr., Sofuni, T , Yoshikawa, K , Hayashi, M.,  Nohmi, T ,




     Sawada, M , and Matsuoka, A    Food Chem   Toxicol    22,  623 (1984)




65   NTP-  "NTP Technical Bulletin No  9," National Toxicology  Program,




     Research Triangle Park, North Carolina, 1983.




66   Hsia, M T S ,  Adamovics, J A , and Kreamer, B L     Chemosphere  8,  521




     (1979).




67   Goggelmann, W  , and Schimmer, 0    Mutat   Res. 121 ,  191  (1983)




68   Pool, B.L , and Lin, P Z    Food Chem. Toxicol. 20,  383  (1982)
                                    391

-------
69   Lutz, D ,  Eder, E , Neudecker, T  , and Hensuhler, D    Mutat   Res   93,




     305 (1982)




70   Mohtashamipur, E , and Norpoth, K    Int  Ann  Ociup  Environ  Health




     54, 83 (1984)




71   Neudecker, T , Ohrlein, K , Eder, E , and Henschler, D    Mat at  Res




     110. 1 (1983)




72   Sasaki, Y ,  and Endo, R    Mutat  Res  54, 251  (1978)




73   Dunkel, V C  , and Simmon, V F     IARC Sci. Publ. 27, 283  (1980)




74   Kennedy, G L  Jr , Smith, S H., Kinoshita, F.K  , Keplinger, M.L , and




     Calandra,  J C    Food Cosmet. Toxicol. 15, 337  (1977).




75   Khera, K S , Whalen C., Angers, G , and Trtvett, G    Toxicol  Appl




     Pharmacol. 47, 353 (1979).




76   Verrett, M J , Scott, W.F , Reynaldo, E F , Alterraan, E K., and Thomas,




     C A    Toxicol  Appl  Pharmacol  56, 265 (1980)




77   Abramovici,  A , and Rachmuth-Roizman, P    Toxicology 29,  143  (1983)




78.  Boberg, E W  , Miller, E C , Miller, J A , Poland, A., and  Liem, A




     Cancer Res  43. 5163 (1983).




79   Miller, E C  , Swanson, A.B , Phillips, D H , Fletcher, T  L  , Liem,  A  ,




     and Miller,  J A    Cancer Res  43, 1124 (1983)




80   Innes, J RM , Ulland, BM , Valerio, M G , Petrucelli, L , Fishbein,




     L., Hart,  E R , Pallotta, A.J., Bates, R R., Falk, H L  ,  Gart, J.J  ,




     Klein, M , Mitchell, I., and Peters, J    J  Natl  Cancer  Inst  42,




     1101 (1969)




81   Reuber, M D    Digestion 19, 42 (1979)




82   Lipsky, M M  , Hinton, D E , Klaunig, J E , and  Trump, B.F   J  Natl




     Cancer Inst. 67, 365 (1981).
                                    392

-------
83   Lipsky, M M , Hinton, D E  , Klaunig, J  E  ,  GoLdblatt,  P J ,  and  Trump,




     B F    J  Nat!  Cancer Inst  67, 377 (1981)




84   Lipsky, M M , Hinton, D E  , Klaunig, J  E.,  and  Trump,  B F    J  Natl




     Cancer Inst  67. 393 (1981)




85   Vesselinovitch, S D  , Rao, K V N., and  Mihailovich,  N     Cancer  Res




     3j, 4378 (1979)




86   Stoner, G D , Shimkin, M B , Kniazeff,  A  J  ,  Weisburger,  J H ,




     Weisburger, E K , and Gori, G.B    Cancer Res   33,  3069 (1973)




87.  Epstein, S S , Fujn, K , Andrea, J  , and Mantel, N.    Toxicol   Appl




     Pharmacol  16, 321 (1970)




88   NCI   "Bioassay of Piperonyl Sulfoxide  for  Possible  Carcinogenicity,"




     NCI Tech  Rep  No  124, U.S  National Cancer  Institute, Bethesda,




     Maryland, 1979




89   NCI   "Bioassay of Piperonyl Butoxide for Possible  Carcinogenicity,"




     NCI Tech  Rep. No  120, U  S  National Cancer  Institute, Bethesda,




     Maryland, 1979




90   Epstein, S S , Joshi, S., Andrea, J  , Clapp,  P  , Falk,  J  ,  and Mantel,




     N.   Nature (London) 214, 526 (1967)




91   Ambrose, A M , Cox, A J  Jr , and De Eds, F     J  Agric  Food  Chem  j6_,




     600 (1958)




92.  NTP   "Carcinogenesis Studies of Eugenol  in F344/N  Rats and  B6C3F1 Mice




     (Feed Studies)," NTP Tech. Rep. No   223,  National Toxicology Program,




     Research Triangle Park, North Carolina, 1983




93.  Van Duuren, B L , and Goldschmidt, B M    J   Natl   Cancer Inst   56,




     1237 (1976)




94   Schoental, R    Carcinogens in Plants and Microorganisms    In  "Chemical




     Carcinogens" (C E  Searle, ed.), ACS Monogr.  No. 173,  American Chemical




     Society, Washington, D C , 1976, p  626

-------
 95   Schoental, R ,  and Gibbard, S    Br  J  Cancer 26,  504  (1972)




 96   NCI   "Bioassay of Cinnamyl Anthranilate for Possible Care mogenicity,"




      NCI Tech  Rep  No  196, U S  National Cancer Institute,  Bethesda,




      Maryland, 1980.




 97   NCI   "Bioassay of Anthranilic Acid for Possible Careinogenicity,"  NCI




      Tech  Rep  No  36, U S  National Cancer Institute,  Bethesda, Maryland,




      1978




 98   Sab me, J R , Horton, B J , and Wicks, M B.   J. Natl   Cancer  Inst   50,




      1237 (1973)




 99   Petcher, T J ,  Weber, H P , Kuhn, M , and von Wartburg,  A    ^J  Chem




      Soc. Per km Trains. 2_< 288 (1973).




100   Meerman, J H N , van Doom, A B D , and Mulder, G.J    Cancer  Res   40,




      3772 (1980)




101   Meerman, J H N , Beland, F A , and Mulder, G J    Careinogenesis 2,  413




      (1981)




102   Orkin,  R W ,  Pratt, RM , and Martin, G R.   Dev. Biol   50, 82 (1976)




103   Sugahara, K , and Schwartz, N B    Prpc  Nat  Acad   Sci  USA  76,




      6615 (1979)




104   Sugahara, K , and Schwartz, N.B.   Arch  Biochem. Biophys   214,  589




      (1982)




105   Mehta,  R., Labuc, G E , and Archer, M C    J  Natl   Cancer  Inst  71,




      1443 (1984)




106   Rostron, C    Food Cosmet  Toxicol  15, 645 (1977)




107.   Schelme, R R.    "Mammalian Metabolism of Plant Xenobiotics,"  Academic




      Press,  New York, 1978, p  108.
                                     394

-------
108   Miller, J A , Miller, E C , and Phillips, D H    The Metabolic  Activa-




      tion and Carcinogenicity of Alkenylbenzenes that Occur  Naturally  in




      Many Spices   In "Carcinogens and Mutagens in the Environment"  (H F




      Stich, ed ), Vol  I , CRC Press, Boca Raton, Florida, 1982, p   83




109   Miller, E A , and Miller, J A    Br. J  Cancer  48, 1 (1983)




110   Stillwell, W G , Carman, M J , Bell, L  , and Horning, M.G.   Drug^




      Metab  Dispos. _2_, 489 (1974).




Ill   Levi, P , Janiaud, P ,  Delaforge, M , Morizot , J.P  , Maume, B F , and




      Padieu, P.   C R. Soc  Biol  171, 1034  (1977)




112   Benedetti, M S , Malnoe, A., and Broillet, A.L    Toxicology 7, 69




      (1977)




113.  Janiaud, P , Delaforge, M , Levi, P., Maume, B F ,  and  Padieu,  P




      Coll  Int  CMS 286, 431 (1977)




114   Phillips, D H , Miller, J.A., Miller, E C , and Adams,  B.   Cancer Res




      41. 2664 (1981)




115   Oswald, E 0 , Fishbein, L , Corbett, B J , and Walker,  M P    Biochim




      Biophys  Acta 230, 237  (1971)




116.  Peele, J D  Jr , and Oswald, E.O    Bull  Environ   Contain  Toxicol   19,




      396 (1978)




117   Klungs^yr, J , and Scheline, R R.   Biomed  Mass Spectrometry 9, 323




      (1982).




118.  Delaforge, M , Janiaud, P., Maume, B F., and Padieu, P.   Direct Evi-




      dence of Epoxide Metabolic Pathways for Natural Ailylbenzene Compounds




      in Adult Rat Liver Cell Culture.  In "Recent Developments in Mass




      Spectrometry in Biochemistry and Medicine" (A.  Frigerio, ed ), Vol.  I,




      Plenum Press, New York, 1978, p  521.
                                     395

-------
119   Delaforge, M ,  Janiaud, P ,  Levi, P ,  and Morizot, J P    Xenoblot ica




      10, 737 (1980).




120   Delaforge, M ,  Janiaud, P ,  Chessebeuf, M , Padieu, P , and Maume,




      B F    Possible Occurrence of the Epoxide-diol Pathway  for Hepatoear-




      cinogenic Safrole in Cultured Rat Liver Cells, as Compared with  Whole




      Animal   A Metabolic Study by Mass Spectrometry   In "Advances  in Mass




      Spectrometry in Biochemistry and Medicine" (A  Frigerio, ed ), Vol   II,




      Spectrum Publ , New York, 1976, p  65




121.   Oswald, E 0 , Fishbein, L ,  Corbett, B J , and Walker, M P    Biochim




      Biophys  Acta 244, 322 (1971).




122   Solheim, E., and Scheline, R. R    Xenoblotica 3,, 493 (1973)




123   Zangouras, A ,  Caldwell, J , Hutt, A J., and Smith, R.L.   Biochem




      Pharamcol  30,  1383 (1981)




124   Solheim, E , and Scheline, R.R    Xenob10tic a 6, 137 (1976)




125   Solheira, E , and Scheline, R R    Xenobiotica 10, 371 (1980)




126   Oswald, E 0 , Fishbein, L ,  Corbett, B.J , and Walker, M P    J




      Chromatogr  73, 43 (1972)




127   Sangster, S A , Caldwell, J., Smith, R L , and Farmer,  P B    Food




      Chem  Toxicol  22, 695 (1984).




128   Sangster, S A , Caldwell, J , and Smith, R L    Food Chem  Toxicol   22,




      707 (1984)




129   Randerath, K ,  Haglund, R E , Phillips, D.H , and Reddy, M V     Proc




      Am  Assoc  Cancer Res  25, 85 (1984)




130   Phillips, D H , Hanawalt, P C., Miller, J.A., and Miller, EC.   _J_




      Supramol. Struct  Cell Biochem. 16, 83 (1981)




131   Kadlubar, F.F , Unruh, L E., Beland, F A , Straub, KM  , and  Evans,




      F.E    Carcinogenesis  1, 139 (1980)
                                     396

-------
132   Phillips, D H ,  and Hanawalt, P C    Caremogenesis 3, 935  (1982)




133   Drinkwater, N R , Miller, E C , and Miller, J.A    Biochemistry  19,




      5087 (1980)




134   Parke, D V    The Endoplasmic Reticulum   Its Role in Physiological




      Functions and Pathological Situations.  In "Concepts in Drug




      Metabolism   Part B" (P. Jenner and B  Testa, eds ), Marcel Dekker, New




      York, 1981, p  1




135   Furia, T E ,  and Bellanca, N  (eds )   "Fenaroli's Handbook of Flavor




      Ingredients," Chemical Rubber Company, Cleveland, Ohio, 1971




136.  Russell, G F ,  and Jennings, W G    J. Agr  Food Chem. 17,  1107  (1969)




137   Hall, R L    Toxicants Occurring Naturally in Spices and Flavors,   In




      "Toxicants Occurring Naturally in Foods," 2nd edn , National Academy of




      Sciences, Washington, D.C , 1973, p  448




138.  Stahl, E    J  Agr  Food Chem  29, 890 (1981)




139   Buttery, R G ,  Seifert, RM , Guadagni, D G , Black, D R ,  and Ling,




      L C.   J  Agr  Food Chem. 16, 1009 (1968)




140   Lichtenstein, E P , and Casida, J.E    J. Agric. Food Chem  11,  410




      (1963).




141.  Karstadt, M , and Bobal, R    Teratog  Carcinog  Mutag  _2_,  151 (1982)




142   Metcalf, R L    Kirk-Othmer's Encycloped. Chem  Tech. (3rd  edn ) 13,




      425 (1981)




143   Helmes,  C.T , Sigman, C C., Atkinson, D L , Papa, P A , Thompson,  K.L  ,




      Valentini, M.A , McCaleb, K E , Bulian, E.S , and Rich, P A   J




      Eviron  Sci  Health A18, 797 (1983).




144   Flavor and Extract Manufacturers' Association (FEMA)   "Scientific




      Literature Review of Anisole and Derivatives in Flavor Usage," FEMA,




      Washington, D.C , 1978
                                     397

-------
145   Guenther, E    "The Essential Oils," Vol  VII, Van Nostrand,  New  York,




      1952




146   Larry, D    J  Assoc  Off  Anal  Chem  56, 1281 (1973)




147   Miller, J A    Naturally Occurring Substances that can  Induce Tumors




      In "Toxicants Occurring Naturally in Foods," 2nd ed  , National Academy




      of Sciences, Washington, D C ,  1973, p  530




148   Stahl, E., and Keller, K    Pharmazie 36, 53 (1981)




149   Gembella, G    Sci  Pharm. (Wien) 26, 8 (1958)




150   Moshonas, M G ,  and Shaw, P E    J  Agric  Food Chem  26,  1288 (1978)




151   Shaver, T N , and Bull, D L    Bull  Environ  Contam  Toxicol  24, 619




      (1980)




152   Kapadia, G J , Rao, G S , and Morton, J.F.   Herbal  Tea Consumption  and




      Esophageal Cancer   Jto "Carcinogens and Mutagens in  the Environment




      (H F  Stich, ed  ), Vol  III, CRC Press, Boca Raton,  Florida,  1983,




      P  3




153   Segelman, A B ,  Segelman, F.P , Karliner, J., and Sofia, R D    J  Am




      Med. Assoc  236, 477 (1976)




154   Krotoszynski, B K , and O'Neill, H J    J  Environ   Sci  Health A17 ,




      855 (1982)




155   Milker, DM    Nutr. Cancer 2,  217 (1981)




156   U.S  Food and Drug Administration   Fed  Register 47, 22545 (1982)




157.   World Health Organization   WHO Tech. Rep  Ser. 669, 41 (1981)




158   Morton, J F    "Folk Remedies of the Low Country," Seeman  Publishing,




      Miami, Florida,  1974




159   Morton, J F    "Atlas of Medicinal Plants of Middle  America," C C




      Thomas Publ , Springfield, Illinois, 1981
                                     398

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160   IARC   "Wood, Leather and Some Associated Industries,"  1ARC Monographs




      on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol




      25, International Agency for Research on Cancer, Lyon,  France,  1981,




      412 pp




161   Shanmugaratnam, K    Int. Rev  Exp  Pathol. 10, 361 (1971)




162.   Sigman, C C , Helraes, C T ,  Fay, J R., Lundquist, P L  , and Perry,




      L R    J  Environ  Sci  Health A19, 533 (1984)




163   Schoental, R., and Gibbard,  S    Nature (London) 216,  612 (1967)




164   Gibbard, S ,  and Schoental,  R.   J  Chromatog  44, 396  (1969)




165.   Schoental, R    Cancer Res.  30, 252 (1970).









             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




      Chemicals to  Man, Vol  10,  Int. Agency Res. Cancer, Lyon, France, 1976,




      353 pp.
                                     399

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