Clemson UniversityTigerPrints
All Theses Theses
5-2012
Sassafras Tea: Using a Traditional Method ofPreparation to Reduce the CarcinogenicCompound SafroleKate CummingsClemson University, [email protected]
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Recommended CitationCummings, Kate, "Sassafras Tea: Using a Traditional Method of Preparation to Reduce the Carcinogenic Compound Safrole" (2012).All Theses. 1345.https://tigerprints.clemson.edu/all_theses/1345
SASSAFRAS TEA: USING A TRADITIONAL METHOD OF PREPARATION TO
REDUCE THE CARCINOGENIC COMPOUND SAFROLE
A Thesis
Presented to
the Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Forest Resources
by
Kate Cummings
May 2012
Accepted by:
Patricia Layton, Ph.D., Committee Chair
Karen C. Hall, Ph.D
Feng Chen, Ph. D.
Christina Wells, Ph. D.
ii
ABSTRACT
The purpose of this research is to quantify the carcinogenic compound safrole in
the traditional preparation method of making sassafras tea from the root of Sassafras
albidum. The traditional method investigated was typical of preparation by members of
the Eastern Band of Cherokee Indians and other Appalachian peoples. Sassafras is a tree
common to the eastern coast of the United States, especially in the mountainous regions.
Historically and continuing until today, roots of the tree are used to prepare fragrant teas
and syrups. These traditional uses can be found across cultures throughout its range.
Products made from sassafras are banned from the market by the US Food and Drug
Administration (FDA) due to a carcinogenic compound, safrole, found in the unprocessed
root. Low levels of safrole are permitted in Europe due to the small concentration found
in common spices, including nutmeg and cinnamon. However, in sufficient doses, safrole
causes genotoxicity and cell toxicity, oxidative stress, and liver cancer in laboratory rats
after ingestion. In this study, traditionally prepared tea and the FDA method of
eliminating safrole were analyzed using high-performance liquid chromatography. These
two methods were compared for effectiveness of eliminating or reducing safrole levels.
The FDA method resulted in little to no safrole content. A smaller amount of safrole was
present in the traditionally prepared tea compared to agitate samples, used to gauge a
baseline concentration of safrole present in the root. Collaborations with the Center for
Cherokee Plants within the Cherokee community will help us to return the results of the
research and contribute resources emphasizing the cultural and historical importance of
Sassafras albidum.
iii
DEDICATION
This work is dedicated to my mother, the strongest and most loving person I
know. Also for my father, for his generosity and humor.
iv
ACKNOWLEDGMENTS
I would like to thank my major advisor, Dr. Karen Hall, first and foremost. Thank
you for your unending support and dedication to not only this project, but also for helping
me become a well-rounded individual. This project would not have been possible without
your patience, wisdom, and guidance through every step. Dr. Feng Chen, thank you for
your advice and support, as well as the generous use of your laboratory to complete the
chemical analysis. Thank you Dr. Christina Wells, for helping with the propagation trials
and planting of sassafras in North Carolina. Special thanks to Sarah McClellan-Welsh
and Kevin Welch for everything you have done, including hosting and assisting with the
tree planting project, GIS project, and sassafras tea conversations. Thank you for your
kindness, hospitality, and understanding. To Dr. Chen’s lab, especially Nina Huang and
Greg Jones, thank you for teaching me everything you knew about chemical analysis and
very basic chemistry. Thank you to Alyssa Palmer-Keriazakos, my summer intern, for
being such a cheerful, independent, and patient student of GIS and allowing me to bring
the SPRI experience full circle. I would like to acknowledge the Highlands Biological
Station for the grant which funded my summer field research, and to the Highlands-
Cashiers Land Trust, especially Kyle Pursel. Last but not least, thank you to the people
who took time out of their day to talk to me about sassafras tea.
v
TABLE OF CONTENTS
Page
TITLE PAGE .................................................................................................................... i
ABSTRACT ..................................................................................................................... ii
DEDICATION ................................................................................................................ iii
ACKNOWLEDGMENTS .............................................................................................. iv
LIST OF TABLES ......................................................................................................... vii
LIST OF FIGURES ...................................................................................................... viii
CHAPTER
I. INTRODUCTION ......................................................................................... 1
II. LITERATURE REVIEW .............................................................................. 3
Project Proposal ....................................................................................... 3
Eastern Band of Cherokee Indians ........................................................... 4
Ecology of Sassafras albidum ................................................................. 6
Safrole .................................................................................................... 10
Laboratory Studies ................................................................................. 12
Toxicity and Dosage .............................................................................. 16
Safrole in Food and Spices .................................................................... 19
Previous Studies of Foods Containing Safrole ...................................... 20
Traditional Knowledge & Implications for Chemical Analysis ............ 22
III. TRADITIONAL KNOWLEDGE OF SASSAFRAS TEA.......................... 25
Introduction ............................................................................................ 25
Traditional Harvesting, Processing, and Preparation ............................. 25
Field Methods: Harvesting ..................................................................... 27
Field Methods: Processing ..................................................................... 30
Field Methods: Preparation .................................................................... 32
Results and Discussion .......................................................................... 35
IV. CHEMICAL ANALYSIS USING HPLC ................................................... 36
vi
Table of Contents (Continued)
Page
Introduction ............................................................................................ 36
Materials and Methods ........................................................................... 37
Results .................................................................................................... 47
Observations and Discussion ................................................................. 48
Reduction of Safrole during Boiling ...................................................... 53
V. WORKING WITH NATIVE COMMUNITIES:
EDUCATION AND OUTREACH ......................................................... 55
Introduction ............................................................................................ 55
Working with Native Communities ....................................................... 55
Collaborating with the Cherokee ........................................................... 57
VI. SUMMARY ................................................................................................. 61
APPENDICES ............................................................................................................... 64
A: Propagating Sassafras albidum .................................................................... 65
B: Petitioning the Food and Drug Administration ............................................ 71
C: Reference of Previous Laboratory Studies Using Safrole ........................... 73
D: Abbreviations ............................................................................................... 98
LITERATURE CITED .................................................................................................. 99
vii
LIST OF TABLES
Table Page
3.1 Site and tree characteristics for Sassafras albidum trees harvested in
Highlands, North Carolina.. ................................................................... 31
4.1 Table of samples, extraction methods, and purpose of sample type in
preparation of HPLC analysis.. .............................................................. 41
4.2 Results of HPLC analysis in parts per million (PPM)
for 235nm UV detection. ....................................................................... 44
4.3 Milligrams and ppm of safrole per 300 mL of aqueous solution. ................ 45
4.4 Milligram of safrole hypothetically consumed by a 60 kg person............... 46
C.1 Review of studies using traditional or modern methods of preparation ...... 74
C.2 Review of studies using laboratory animals and bacterial studies
to test the toxicity of safrole and derivatives ............................................... 75
C.3 Review of studies using chemical analysis of foods containing safrole ............... 94
viii
LIST OF FIGURES
Figure Page
2.1 Heterophyllic leaves of Sassafras albidum .................................................... 9
2.2 Chemical Structure of Safrole ...................................................................... 13
3.1 Traditionally prepared sassafras tea ............................................................. 28
3.2 Segments of dried sassafras root chopped into small pieces. ...................... 33
3.3 Sassafras root being actively boiled while preparing
the traditional method samples. ............................................................. 34
4.1 Safrole standard (5,000 ppm), detected at 235 nm with an elution
time of 37.775 minutes.. ........................................................................ 42
4.2 Standard Curve for 235 nm absorption. ....................................................... 43
5.1 Stand of twenty sassafras trees planted at the Center for
Cherokee Plants in Bryson City, North Carolina. .................................. 60
5.1 Transplanted and pruned sassafras sapling .................................................. 66
1
CHAPTER ONE
INTRODUCTION
Sassafras albidum is a North American species primarily known for the tea made
from its roots and root bark. Sassafras has been in use for centuries by the Cherokee,
Chippewa, Creek, Delaware, Iroquois, Seminole, and other Native American tribes
within the East Coast range of the tree (Moerman 2011; Hamel & Chiltoskey 1975).
Many people who were raised in the Southeast remember drinking sassafras tea as a
child, and families in the mountainous regions still make root tea and syrup today. The
Eastern Band of Cherokee Indians in Western North Carolina is one such community that
have preserved and passed down the traditional knowledge needed to utilize sassafras.
The essential oil extracted from the root of sassafras contains 80-90% safrole
(Carlson & Thompson 1997). Safrole is a phenylpropanoid, a type of aromatic compound
that most likely accounts for the strong smell associated with the root (Kamdem & Gage
1995; Rasch 1998). Safrole is found in small quantities in a number of species consumed
on a daily basis, including cinnamon, ginger, nutmeg, and cocoa (Heikes 1994). On the
commercial market, sassafras extract was a common ingredient in foodstuffs, cleaning
products, and cosmetics as a flavoring agent and fragrance. Until 1960, the sale of food
containing safrole was legal up to 20 ppm (Carlson & Thompson 1997). In 1960, the
United States Food and Drug Administration (FDA) banned the presence of the
compound safrole in foodstuff. Experiments had demonstrated the hepatocarcinogenetic
(liver cancerous) effect of large doses of safrole on laboratory rats (CFR, Sec. 189). It is
2
possible to buy safrole-free extracts of sassafras tea concentrate where the safrole has
been removed according to processes set by the FDA (CFR, Sec. 172).
Many studies have demonstrated the carcinogenic effects of high doses of safrole
on laboratory rats, but all these studies have used pure safrole standard (Jin et al., 2011;
Liu et al., 1999; Ueng et al., 2005). As of yet, no research has tested the traditionally
prepared sassafras tea for carcinogenic properties. In fact, no research has investigated
the potential concentration of safrole in traditionally prepared sassafras tea. This research
is important because many people, particularly within southeastern mountain
communities, continue to prepare and consume products made from sassafras.
The purpose of this study is twofold: first, to quantify the amount of safrole
present in traditionally prepared sassafras tea. We will do this by running chemical
analysis of tea samples and samples prepared following FDA guidelines through high
performance –liquid chromatography (HPLC). The purpose of running both the FDA and
traditional samples is to quantify the effectiveness of both methods at reducing safrole
content. The second objective will focus on education and outreach projects, to return the
results of the chemical analysis to the community, with which we collaborated and
complete projects that highlight the cultural and historical importance of sassafras.
3
CHAPTER TWO
LITERATURE REVIEW
Project Proposal
The topic of this research project was suggested during a meeting with
community and tribal members connected to traditional agriculture, history, and granting
agencies of the Eastern Band. They believed researching the toxicity associated with
sassafras products would be relevant and interesting to members of the tribe, as tea and
syrup are commonly made and exchanged between Cherokee members. The products
could eventually contribute to the economy of the Cherokee if the sale of products was
achieved or desired. The banning of sassafras products on the market has raised concern
among those that consume sassafras tea, syrup, and other foodstuffs made from Sassafras
albidum. Although many continue to consume these products, there is a feeling of
hesitancy and uncertainty in conversation about the use of sassafras. For the present,
quantifying the concentration of the main carcinogenic compound in sassafras products
would be meaningful for many who have consumed sassafras their entire lives. With the
information from these results, consumers could make informed decisions on the personal
use of sassafras. Though already a culturally important plant, a study on this species
would support the continued emphasis of sassafras in the culture and honor the traditional
knowledge associated with its use.
4
Eastern Band of Cherokee Indians
The Eastern Band of Cherokee Indians (EBCI) of Western North Carolina own
more than 56,600 acres of land in five counties slightly south and adjacent to the Great
Smoky Mountains National Park (Lambert 2007). There are approximately 13,000
enrolled members, about 8,200 of which live on EBCI-owned land called the Qualla
Boundary (Hall 2006; Lambert 2007). Historically, the Cherokee territory covered about
25.6 million acres in the mountains of the southeast, primarily on the Savannah,
Hiwassee, and Tuckasegee rivers, with their principle town, Echota, on the bank of the
Little Tennessee. The Kituwah settlement, now known as the Kituwah mound, is located
along the Tuckasegee River near Bryson City, North Carolina and was possibly the
original capital of the Cherokee (Duncan and Riggs 2003; Hudson 1979).
Written history about the Cherokee began in 1540 with the exploration of De Soto
and other Spanish explorers. As the French and Spanish settled along the coast and
slightly inland in the 1500’s and 1600’s, there was only occasional contact between the
groups. Some scholars estimate these early settlers brought diseases that killed 95% of
Native Americans within the first century and a half of European contact (Duncan and
Riggs 2003). Being on the edge of the territory claimed by early European settlers, the
Cherokee were caught up in the politics and bloodshed of the Colonial Period, the French
and Indian War, and the Revolutionary War, as well as the slave trade of several ethnic
groups. Exploring settlers slowly absorbed the piedmont regions of Cherokee land and
the tribe became concentrated in the southern Appalachian Mountains (Finger 1984).
5
The “Civilization Policy” of 1789 by the new American government was a
crossroads for the Cherokee in terms of adapting to European-style schools, government,
churches and economy. The written language developed by Sequoyah in 1819, which
may have been a response to these pressures. The Treaty of New Echota of 1835, signed
without permission of the Cherokee people as a whole, gave all Cherokee land east of the
Mississippi to the United States. In 1838, the US government forced 17,000 Cherokee to
territory in Arkansas and Oklahoma on a march now known as the Trail of Tears. About
one-quarter to one-half of the ill-equipped travelers perished on the march (Finger 1984).
A group of three to four hundred Cherokee hid in the mountains of Western North
Carolina to evade capture by US soldiers. They, along with Cherokee that returned to the
territory, were the foundation for the present day Eastern Band of Cherokee Indians
(Duncan and Riggs 2003).
It is important to know this brief history of the Eastern Band when considering the
contemporary Cherokee Indians and the efforts they have made to reclaim their rights and
traditional knowledge. Many historians, ethnobotanists, and anthropologists have studied
and written about the Cherokee, including James Mooney in the 19th
century (Mooney
1992). He was followed by Frans Olbrechts, who worked with the healer Swimmer to
publish a manuscript in 1932, and then by John Witthoft, who published articles from the
1940s to 1970s. There have been a number of books written on the ethnobotany of the
Cherokee in latter half of twentieth century, including authors William Banks (1953),
Myra Jean Perry (1974), David Cozzo (2004), and Karen C. Hall (2006). The Museum of
the Cherokee Indian Press publishes the Journal of Cherokee Studies, as well as many
6
books on the history and culture of the tribe with authors like Barbara Duncan, Vernon
Crowe, and Duane H. King (MCI 2012).
Today, tribal members continue their cultural traditions including the lacrosse-like
stickball game, the Cherokee language, and traditional Cherokee arts and crafts, all of
which are integrated with contemporary festivals. They cater to tourists through
managing a casino, interpretive performances of the Trail of Tears, and selling traditional
arts and crafts. The Cherokee worldview is represented throughout the landscape where
they live, including street signs written in Cherokee language and the preservation of
culturally and spiritually important locations like the Kituwah mound. The Cherokee
language is reinforced through language immersion schools for members. Many families
continue to pass traditional ecological knowledge of plant uses through generations and
between members.
Ecology of Sassafras albidum
Sassafras is a native deciduous tree species of Eastern North America known for
its brilliant fall color and a distinct fragrance. Leaves are bright to deep green in the
summer and change to scarlet, orange, or yellow in the autumn. The tree has
heterophyllic foliage with three leaf shapes: entire, mitten (both right- and left-handed),
and trilobed, as seen in Figure 2.1. On rich sites in the Smoky Mountains, sassafras is
typically a medium-sized tree with a straight bole reaching heights of 9 to 18 m (30 to 60
ft). On the edges of the habitat range sassafras tends to be shrubby. Sassafras is a
dioecious species whose female plants produce small racemes of yellow flowers in March
7
or April (Griggs 1990). The odor of the root is very distinct; the scent has been described
as ‘sweetshop’ or spicy (Dugan 2011).
Sassafras is found along the Atlantic coast (zones 4 to 9) of the United States
from northern Florida to Canada, and west to Michigan, Illinois, and Arkansas (Cullina
2002; Rasch 1998). Sassafras is a generalist species, occupying both full sun and partial
shade sites with a variety of soil types and moistures. The tree can be found in a wide
range of ecosystems, including many species of pine dominated forests and oak-hickory,
maple-beech-birch, and aspen-birch forests. The rapid growth is achieved through a deep
taproot as well as shallow, laterally growing root system that send up root suckers
(Griggs 1990). White-tailed deer, woodchucks, black bears, and rabbits all browse the
leaves during the summer and winter. Many types of birds (wild turkeys, pileated
woodpeckers, and northern bobwhites) and some small mammals eat the ripe fruit, a
single-seeded drupe with a high energy value (Sullivan 1993). Those who wish to
propagate sassafras usually need to stake out the tree for the highly favored drupe (See
Appendix A for Propagation techniques). Sassafras is an important host plant for many
species of moths and butterflies, particularly the host-specific spicebush swallowtail
butterfly (Carter et al., 1999; Sternberg 2004).
Sassafras is typically considered shade intolerant, though it can adapt to low light
intensities of an understory with soil pH of around 5 (Bazzaz et al., 1971). S. albidum is
typically subdominant but can attain dominance by forming thickets on poor soils
through allelopathic activity, which includes the release of terpenes and
phenylpropanoids as well as other secondary defense compounds that deter pathogens
8
and herbivores (Bisset 1994; Gant et al., 1975; Sullivan 1993). These adaptations are
important when considering the chemistry of the allelopathy: the strong presence of
allelopathic compounds are found throughout the tree but mainly concentrated in the
roots. Root wood is porous with characteristic pits and oil cells (Bisset 1994). These
allelopaths include: 2-pinene, 3-phellandrene, eugenol, safrole, citrol, and s-camphor
(Griggs 1990). The sassafras root cortex contains 6-9% essential oil, which consists of
the compounds safrole (80-85% of the total oil), safrole camphor (3.25%),
methoyleugenol (1.1%), tannins (sassafrid), resin, wax, mucilage, sugar, and sitosterol.
Another one percent of the oil includes pinene, eugenol, apoil, 5-methoxyeugenol,
elemincine, estragol, and myristicin (Rasch 1998).
10
Safrole
Safrole (5-(2-propenyl)-1,3-benzodioxole) is a phenylpropene, part of the
aromatic phenylpropanoid family. Safrole has a benzene ring flanked on either side by a
dioxolane ring and a terminal, highly reactive methylene group, as seen in Figure 2.2
(Wink et al., 2008). Safrole is insoluble in water but mixes readily with chloroform,
ether, and other non-polar organic solvents (Budavaris 1989; Burdock 1997). Many of the
aromatic rings that produce smell and taste of plants are derived from the
phenylpropanoid metabolism through the shikimic acid pathway (Dewick 1997; Wink
2010). Phenylpropanoids secondary compounds associated with the taste and smell of a
plant and therefore are used as deterrents against herbivores that shy away from strong
secondary metabolites. The compounds also protect against ultraviolet radiation, fungi,
and bacteria, and can act as pollinator attractants (Hahlbrock 1989). Secondary
compound activity increases in nutrient-poor soils, which is why sassafras, known for its
ability to inhabit roadsides and abandoned fields, would have high allelopathic and
defense tendencies in these locations and therefore higher concentrations of associated
secondary compounds (Ibrahim 2001).
Due to its pleasant smell, sassafras oil used to be a common ingredient (up to 20
ppm) in foodstuffs, cleaning products, and cosmetics as a flavoring agent and fragrance
(Carlson and Thompson 1997). Then on December 3, 1960, the US FDA banned the
presence of safrole, oil of sassafras, isosafrole, and dihydrosafrole in food after
experiments showed a hepatocarcinogenic effect on laboratory rats (CFR, Sec. 189).
Many common spices, including black pepper, cocoa, mace, nutmeg, cinnamon, tarragon,
11
star anise, fennel, parsley, basil, bay laurel, dill, pimento (allspice), and cloves, contain
trace amounts of safrole (Heikes 1994; Zhou et al., 2007). Safrole is also detectable in the
range of 3-5 mg/L (ppm) in soft drinks that use the spices listed above, including the
well-known beverages Coca-Cola and Pepsi (Choong and Lin 2001). Alcoholic
beverages contain between 0.15 and 3 mg/L (ppm) (Curró et al., 1987). In a 2001
document, the European Commission allowed, “1 mg/kg in foodstuffs and beverages, 5
mg/kg for alcoholic beverages with more than 25% alcohol by volume and 15 mg/kg for
foods containing mace or nutmeg” for both safrole and isosafrole (Carlson & Thompson
1997; SCF 2002; SCF 2003). In 2005 the Council of Europe listed safrole as an Active
Principle 1, or suspected weakly carcinogenic substance; no maximum daily limit can be
set for these compounds, but they should be set as low as possible. Safrole is currently
under evaluation by the Council of Europe (CD-P-SP 2005). Sassafras was most likely
targeted because of its high concentration of safrole compared to other spices, including
the often cited nutmeg, which has 2.46 % (w/w) compared to sassafras root (89% w/w)
(Curró et al., 1987).
Pure safrole oil is still imported to Europe, Japan, and the United States from
Brazil and countries in the South Pacific. Safrole-rich species in the Cinnamomum and
Ocotea genera are harvested to be synthesized into piperonyl butoxide, used in pesticides,
and piperonal, a flavoring and perfume ingredient (Miglierini 2008; Oltramari et al.,
2004). Safrole is also an essential ingredient in the production of the illegal drug MDMA,
also known as Ecstasy (Rasch 1998). Safrole is designated as a List I Chemical by the
U.S. Drug Enforcement Administration, implying that the chemical is used in the
12
manufacture of controlled substances as well as having legitimate uses (CFR, Sec.
1310.02).
Laboratory Studies
Safrole is classified as a weak carcinogen in laboratory rodents due to the creation
of safrole intermediates that form hepatic DNA adducts (Liu et al., 1999). Conventional
genotoxicity tests, including sister chromatid exchange and micronucleus tests, tested
positive for in vitro toxicity of safrole, and many in vivo tests of safrole have established
carcinogenic doses of safrole, both through incorporating safrole into the diet and
injection (Jin et al., 2011; SCF 2002). Safrole is absorbed passively from the
gastrointestinal tract, but it is thought that safrole is non-toxic in its unaltered form.
Rather, safrole must be metabolically activated to be toxic to humans, a fact confirmed by
neutral or negative results of an Ames test of safrole (Frohne & Pfander 2004; Swanson
et al., 1979; Wink 2008). Some research has investigated utilizing the toxicity of safrole
for human advantages, including anticancer drugs, insect fumigants, contact pesticides,
and anti-fungicides (Casida et al., 1966; Catalán et al., 2010; Huang et al., 1999; Khayyat
2011; Khayyat & Al-Zahrani 2011; Kim & Park, 2008; Zhao et al., 2005).
Metabolites of safrole formed in vivo include but are not limited to
1’-hydroxysafrole (considered the most toxic), dihydrosafrole (p-n-propyl-
methylenedioxybenzene), isosafrole (1-propenyl-3,4-methylenedioxybenzene), and
eugenol (4-allyl-2-methoxyphenol) (Heikes 1994). As the body attempts to rid itself of
the non-nutritive lipophilic molecule through the liver, the defense system undergoes a
phase I reaction to convert the molecule to a nucleophilic substance (Shibamoto et al.,
14
2009). Metabolic activation of safrole to its carcinogenic derivatives can be simplified
into four [4] different transformations. The first [1] transformation involves the oxidation
of the allyl side chain in cytochrome P450 by the enzyme CYP2A6 to form 1’-
hydroxysafrole. This compound can undergo sulfation to form 1’-hydroxysafrole sulfate
(Daimon et al., 1997/8; de Vries 1997; Jeurissen et al., 2004; Zhou et al., 2007). These
electrophilic, sulfuric acid esters form safrole-DNA adducts in human hepatoma (HepG2)
cells and induce cancerous formations (Liu et al., 1999; Miller et al., 1983; Zhou et al.,
2007). Safrole-DNA adducts lead to the induction of sister chromatid exchanges and
chromosomal aberrations, which lead to mistakes in DNA replications and mutations that
has the possibility of carcinogenesis, as well as cytotoxicity (Daimon et al., 1997).
Adducts concentrate in hepatic DNA as the liver is the detoxifier and metabolizer of
drugs for the body (Nakagawa et al., 2009).
A second [2] transformation falls within a different pathway to chemical
carcinogenesis: oxidative stress, which causes faulty incorporation during DNA
replication. Safrole can undergo cleavage of the dioxolane ring to form hydroxychavicol
(4-allyl-1,2-dihydroxybenzene), shown in the pivotal Benedetti study to be the major
urinary metabolite of rodents and humans. The Benedetti et al., study is the only research
as of yet to test the results of human subjects ingesting safrole (Benedetti et al., 1977; Liu
et al., 1999). Hydroxychavicol, best known in connection to betel quid chewing, has the
potential to transform to the reactive elecrophiles ortho-quinone or para-quinone
methide. These metabolites can further transform to reactive oxygen species that can
induce oxidative damage. Hydroxychavicol is more toxic than safrole and has been
15
linked to mitochondrial dysfunction (Bolton et al., 1994; Nakagawa et al., 2009). Though,
unlike DNA-safrole adducts, oxidative stress has been shown to be reversible. The
damage was repaired within 15 days after safrole was administered, compared to stable
DNA adducts that have been detected 30 to 140 days after ingestion (Gupta et al., 1993;
Liu et al., 1999). The damage initiated by hydroxychavicol can also be prevented in vivo
by antioxidants like vitamin E (Liu et al., 1999).
The first two transformations are the main metabolic pathways in animals. A third
[3] transformation involves epoxidation of safrole by the double bond of the propenyl
group to form safrole-2’,3’-epoxide (de Vries 1997). The fourth [4] transformation is the
gamma oxidation of the allylic side chain leading to a carboxylic acid, which can
conjugate with glycine (CSF 2002). The two major safrole-DNA adducts for this
transformation are N2-(trans-isosafrol-3’-yl)2’-deoxyguanosine and N
2-(safrole-1’-yl)2’-
deoxyguanosine (Gupta et al., 1993).
The effect of safrole on laboratory rats and mice is well documented. Rodents
given large doses of pure safrole in their diet (via oral gavage, subcutaneous injection, or
mixed into food) suffered from mortality and a range of liver and kidney damages.
Physiological aliments included slight impairment such as weight loss, mild anemia, and
growth retardation. Aliments also included moderate and severe damage, including liver
tumor masses and nodules, liver enlargement, focal to chronic nephritis in the kidney and
liver, bile-duct proliferation, fatty metamorphosis, and mortality (Abbott et al., 1961;
Daimon 1998; Epstein et al., 1970). Genotoxicity assays were positive for sister
chromatid exchange (SCE), unscheduled DNA synthesis (in cultured rat hepatocytes),
16
chromosomal aberrations, gene mutation, and cell transformation (Howes et al., 1990;
Ishidate & Sufuni 1985; Mihr et al., 1985; Purchase et al., 1978). Safrole did test neutral
or negative in an Ames test (Salmonella reverse mutation assay) as mentioned before, as
well as mouse dominant lethal assay, bone-marrow micronucleus assay, and unscheduled
DNA synthesis in HeLa cells (Baker & Bonin 1985; Epstein et al., 1972; Gocke et al.,
1981; Martin et al., 1978; Mirsalis et al., 1982). Ingestion of safrole at high doses can
cause symptoms such as nephritis, unconsciousness, weakness, liver disturbance, CNS
stimulation, and skin irritation (Wink 2008). Also see Appendix C for complete chart of
laboratory studies.
Toxicology and Dosage
Toxicology research utilizes two models to determine the potency of compounds,
including qualitative and quantitative methods. Qualitative methods can include
observations of lesions and clinical signs, while the quantitative model examines the dose
and exposure levels. The ultimate goal is to determine a structure-activity relationship
that outlines the biological effects of a substance at physiological and biochemical levels.
To evaluate a compound for human safety, toxicology studies examine several species of
laboratory animals (rats, mice, dogs, rabbits, primates) that have similar physiology and
genetic structure to humans (Reagan-Shaw 2007). Researchers use several dose levels,
where the higher dose levels are to understand the biochemical mechanisms and a
maximum amount is established for safe consumption. The mode of ingestion or
exposure should be considered in clinical tests; for example, feeding laboratory rats the
17
food additive compounds through their diet as opposed to subcutaneous injection or oral
absorption (Dixon 1976).
As the 16th
century Swiss-German chemist/physician Paracelsus held, ‘Poison is
in everything, and no thing is without poison. The dosage makes it either a poison or a
remedy’ (“Paracelsus” 2012). In estimating toxicity and the possibility of carcinogenicity,
the dose is critical as is the perceived risk. For risk in using food additives in particular,
no amount of carcinogen is accepted as additions to food are risks than can be avoided
(Extoxnet 2012). In terms of dose, dose translation is necessary to compare doses
between different species, particularly between human and laboratory animals. The use of
body weight to compare doses between studies has been criticized as it may not
accurately correlate to volumes of blood and plasma proteins (Dixon 1976). Body surface
area (BSA) has been shown to associate these parameters, or total blood and plasma
protein volume, in addition to basal metabolism, renal functions, and oxygen and calorie
utilization. The equation used by Reagan-Shaw et al. (2007) to translate doses between
animal species and human:
Human equivalent dose (mg/kg) = Animal dose (mg/kg) * Animal Km/Human Km
(Reagan-Shaw et al., 2007). Km values (mg/m2) are listed in the paper for several species
and are based on height-weight measurements. In this thesis, mg/kg bw (body weight)
refers to mg of compound by body weight of the subject, either human or animal. The
unit listed as mg/kg refers to mg of a compound in kg of material.
Another aspect of dosage to consider is the difference in employing high versus
low dosage. The ‘Virtual Safe Dose’ for human consumption is calculated with a linear
18
model, “which assumes that cancer causation is directly proportional to dose and that
there are no unique effects of high doses” (Ames & Gold 2000). A dosage margin of
safety for humans is typically calculated by dividing a lethal dose of the compound for
laboratory rodents by a factor of 100 (Segelman et al., 1976). An exaggerated high dose
of a compound, as in experiments to understand the mechanisms of carcinogenicity, can
cause wounding and death of tissue and cells and chronic cell division of neighboring
cells. It is possible the resulting cellular necrosis and carcinogenesis is inflated in high
doses, a point discussed in literature on safrole.
The Benedetti et al. (1977) study examined the response of rats and mice to
consumption of safrole in addition to human subjects. Even at the smallest dose of 0.63
mg/kg bw (body weight), 88% of the safrole was eliminated by the rats within 24 hours.
The authors point out that when the dose of safrole for the rodents was increased to levels
common in carcinogen studies (between 500 to 1000 mg/kg bw), safrole rapidly
accumulated in the liver and kidney, and safrole was not metabolized as quickly and
therefore eliminated more slowly than rats fed smaller doses. They argue that the toxic
accumulation associated with these carcinogenic studies was due to this slow metabolism
of safrole and subsequent tissue absorption was a result of the unrealistic doses. Lower
doses (or accurate amounts of safrole in food and drinks) would not lead to safrole
accumulation in tissue muscle (Benedetti et al., 1977). Gupta (1993) found a dose of400
mg safrole/kg bw for mice to be the point of saturation of detoxification enzymes.
19
Safrole in Food and Spices
The HERP Index, or Human Exposure/Rodent Potency index lists a daily level of
safrole ingestion at 1.4 mg/kg bw per day for a 60 kg person from the various safrole-
containing spices like black pepper, star anise, cumin, cinnamon, and ginger root (HERP
2011). The European Commission estimated the daily intake of consumers (by the
quantity of safrole added to food by industry) to be 0.3 mg/day, or 0.005 mg/kg bw for a
60 kg person (SCF 2002). The WHO estimates a daily range of safrole intake as 4 to 569
μg for Americans and between 0.6 and 879 μg for those in the European Union, based on
spice content and annual volume consumed by country (WHO 2009). The median toxic
dose (TD50) of safrole, according to the HERP Index, for mice is 51 mg/kg bw per day
(HERP 2011). Segelman et al. estimate a dose of 0.66 mg/kg bw may be toxic to man,
based on a study by Epstein et al. (1970) that found 66 mg/kg bw of safrole administered
over a 21 day period to be carcinogenic to infant male mice. Studies commonly use a
margin-of-safety factor of 100 to estimate toxicity, hence the 0.66 mg/kg bw (Segelman
et al., 1976).
In the only study found with human subjects, Benedetti et al. (1977) observed the
effects of human consumption of safrole with two doses (0.163 mg and 1.655 mg). They
concluded both amounts of safrole were completely eliminated in 24 hours with little
possibility of safrole accumulating in tissue muscles. In the human subjects, safrole was
eliminated as small amounts of unchanged safrole and safrole metabolites, specifically
1,2-dihydroxy-4-allylbenzene, eugenol, and an isomer of eugenol. The metabolites 3’-
hydroxylisosafrole and 1’-hydroxysafrole (the most toxic metabolite) were not detected.
20
The authors suggest the absence of the latter metabolites could also be due to the dose or
to the differences in metabolism between man and rodent (Benedetti et al., 1977).
Safrole is present in the flowers of Piper betle, used in the betel quid chewing
tradition (similar to tobacco dip in the United States) prevalent among the male
population of Taiwan. Betel quid has been linked to the high rates of oral cancer due to
the high level (15,000 mg/kg plant material) of safrole present in the flowers, which are
used in the betel quid mixture. Exposure to safrole in the saliva during chewing is
estimated between 70 mg/L to 68120 mg/L (or 420 μmol/L) (Chen et al., 1999; Liu et al.,
2000). Research on betel quid chewing in India and Southeast Asia suggested the
presence of safrole could be causing the high rates of oral cancer and other diseases by
inhibiting the bactericidal activity and releasing reactive oxygen species (Hung et al.,
2003). A study examining betel quid chewing among pregnant women found that adverse
pregnancy outcomes, including lower birth weight, stillbirth, fetal malformation, and
premature delivery, were 2.8 times higher than women who did not chew betel quid
(Yang et al., 2001). Overall, it is difficult to compare betel quid chewing to consuming
sassafras tea due to the different paths of ingesting safrole (absorbed through the mouth
and saliva versus gastrointestinal absorption) and the range of doses.
Previous Studies on Foods Containing Safrole
A handful of studies have examined sassafras tea. Carlson & Thompson (1997)
examined several types of herbal products derived from sassafras, including leaves, tea
concentrate, herbal powder capsules, and tinctures, as well as a ‘sassafras tea’ prepared
21
from instructions from a modern herbal guide. The ‘tea’ made in this study was infused,
or the tea bag steeped in hot water for thirty minutes as opposed to the traditional method
of decocting, or boiling, the whole or roughly chopped root. They demonstrated that
herbal products of sassafras ranged from zero to a miniscule amount of safrole in tea
made from the leaves, to containing 92.4% safrole in sassafras oils. The ‘tea’ they
prepared had safrole content ranging from 0.03 to 1.37 mg per gram of powdered root
material, or only 6.9% to 17.2% of total safrole present in the root (Carlson & Thompson
1997). There were several differences between the Carlson & Thompson study and this
research, including the use of root bark powder instead of traditionally harvested and
processed root and root bark. The researchers also infused the root bark powder in hot
water instead of the decoction method that is used by all the consultants in this study.
Another study by Heikes (1994) quantified the amounts of known carcinogenic
compounds in commercial, un-brewed tea mixes, without actually heating the material.
Both of these studies (Carlson & Thompson and Heikes) focused on finding a more
efficient, cost-effective method of analysis than the official method adopted by the FDA
in the 1960’s. Kapadia et al. (1978) did not prepare tea per se, but did extract in an
aqueous solution using sassafras root bark. The researchers used petroleum ether,
methylene chloride, and ethanol to extract the oil (which would include the safrole), and
then injected 15 mg of the safrole-free ethanol extract in rats (Kapadia et al., 1978).
22
Traditional Knowledge & Implications for Chemical Analysis
Traditional knowledge, especially in Native American communities, comes from
a cumulative, inter-generational custom of passing along practices, beliefs, ideas, and
wisdom. It is transmitted through cultural behaviors and generated by close contact with
the natural environment. It encompasses every part of a society: politics, history,
anthropology, and philosophy (Huntington 2005). The inherent complexity in natural and
human social systems is recognized and categorized with the assumption that humans are
a part of the web. Balance in natural systems requires reciprocity and respect. Knowledge
is indigenous or traditional when “the meanings as well as the categories of sense making
are generated internally within a cultural community”, as well as a physical environment
(Gupta 2010; Viergever 1999).
Within the system of traditional knowledge, traditional medicine is less a remedy
or cure, and more of a way of balanced well-being. The World Health Organization
defines it as, “the sum total of knowledge, skills and practices based on the theories,
beliefs and experiences indigenous to different cultures that are used to maintain health,
as well as to prevent, diagnose, improve or treat physical and mental illnesses” (WHO
2008). When traditional medicine is taken out of context of its originating culture, it can
be termed alternative or complementary medicine. Without the history or tradition to
sustain them, medicines can be prepared or used differently from their original intention,
sometimes leading to harmful results. This is not to say that traditional knowledge
regarding plant preparation is invalid, only that if the product is of poor quality, taken
inappropriately, prepared differently, or used at the same time as other medicines, the
23
product can be ineffective or malignant (WHO 2008). The traditional knowledge and
particular species are often targeted, as in the case of sassafras tea, rather than if and how
the knowledge was taken out of cultural context.
People that use sassafras today look at the extended use of sassafras through
history as proof of its safety. Steve Brill, a wild food forager in New York, states “People
have enjoyed sassafras in moderation for thousands of years with no ill effects and
consumed it in root beer before it was replaced by artificial chemicals. I think sassafras is
safe” (Brill 1994). A home remedy book, or do-it-yourself herbal, says, “Everyone knows
that sassafras has been used for centuries as a spring tonic. Try it, it’s delicious”
(Williams 1998). Unfortunately, there are instances of injury from traditional methods
prepared incorrectly. For example, a case study published in the Journal of Postgraduate
Medicine describes an elderly woman who complained of diaphoresis. The doctor traced
the excess sweating and hot flashes to her recent habitat of consuming ten cups of
sassafras tea a day, as instructed by a family member (Haines 1991). The article does not
indicate how strongly she prepared the tea or exactly how many ounces she consumed
from ten cups. Another case study from 1987 reports a 47-year-old woman ingesting a
teaspoon, or 5mL, of sassafras oil and immediately vomiting, followed by trembling
(Grande & Dannewitz 1987).
There have been many studies highlighting the importance of traditional methods
of preparation, including several on safrole-containing species. This research closely
followed the study of Reynertson et al. on a bark tea made from Cinnamomum
carolinense, a study that specifically examined the differences in extracting the bark
24
using alcohol versus water (2005). Research in Egypt studied safrole-containing spices
collected in local markets for the degradation of safrole during common cooking methods
(Farag & Abo-Zeid 1997). A wild ginger species (Asarum spp.) used in traditional
Chinese medicine was analyzed before and after a one-hour decoction (Chen et al., 2009).
The kava-kava controversy is well known in the health food circle for multiple cases of
severe liver damage when the roots are extracted with ethanol or acetone rather than the
traditional method of maceration in water and coconut milk (Singh & Devkota 2003;
Whitton 2003).
It is important to remember as well that plant medicine is most likely derived
from secondary compounds produced for protection against herbivores, pathogens, fungi,
and other modes of attack. Plants have evolved a myriad of defense mechanism, from
neurotoxin effects that can cause confusion or sedation to physiological effecting,
including growth suppression or organ damage. These secondary compounds, often very
toxic to predators, can be harnessed by humans for beneficial effects (Briskin 2000).
The purpose of this research was not to discredit or devalue the traditional
knowledge that informed this study. Native American cultures have been using sassafras
for centuries, along with other plant species with deadly and toxic compounds. As of the
conclusion of this study, there have been no confirmed, direct linkages between drinking
sassafras tea and harm reported in any CDC Morbidity and Mortality Report or the FDA
MedWatch program (CDC 2012; MedWatch 2012). These programs are the monitoring
methods used by the FDA to follow any adverse, unexpected, or unusual results of
consuming FDA-regulated products (Love 1999).
25
CHAPTER THREE
TRADITIONAL KNOWLEDGE OF PREPARING SASSAFRAS TEA
Introduction
The methods and materials used in this study of harvesting, processing, and
preparing sassafras root are based on discussions with enrolled members of the EBCI and
non-Cherokee persons. These people have made sassafras tea for much of their lives and
many were taught as children by their parents or grandparents. They were consulted on
their methods of making tea in order to create a specific formula for tea-making to be
used as the ‘standard’ in the laboratory, a process that is replicable and uses specific
quantities. The ‘standard’ method represents a typical way to prepare tea and was a
compromise between slight differences among the processes. The harvesting, processing,
and preparing techniques varied with the consultants, including the age and habitat type
of the sassafras tree, how the root was processed for tea and storage, the proportion of
root to water in making the tea, and the length of time the root was boiled and steeped.
Traditional Harvesting, Processing, and Preparation
The processes included on harvesting, processing, and preparing sassafras root
represent one way of demonstrating how decisions were made for this research and do
not imply the totality of processes or people who use sassafras. Many consultants
harvested saplings because they are abundant, easier to dig up, and are considered weedy
by some homeowners. Some consultants dug from both young and mature trees. A few
26
indicated they dug roots in certain seasons, including only in late fall, winter, or early
spring, as these were when the plant energy was concentrated in the root. The processing
step was the most variable among root harvesters: some consultants dried the root spread
on newspaper, while others hung them in bags. The consultants either chopped the root
into chunks of various sizes and weights, whittled the root lengthwise into strips, or kept
the smaller, thinner roots whole. In making the tea, the root was boiled from 10 minutes
up to an hour, removed from the heat source, then let steep for a few hours, overnight, or
up to 24 hours.
There were several points of general agreement among the consultants. They all
emphasized the sustainable harvest of the root as to not to harm the larger trees, or only
harvesting saplings if they are considered weedy or in a site about to be bulldozed. Every
consultant indicated that they left the root bark on the roots, washed the root very
thoroughly after harvesting, and hung the root in breathable containers, most commonly a
mesh onion bag. Breathable containers are essential to keep the dried root from molding.
The consultants agreed that dried root would last many years, though typically it was
used in one-year period due to steady use.
All the consultants agreed on the proper color of the tea, though many found it
difficult to describe: light brown, almost see-through, yellowish-brown, medium honey-
colored, or the color of amber beer (See Figure 3.1). Any lighter and it would be too
weak tasting, and any darker it would overpower the senses. Many described the amount
of root to be used in a pot or single cup of tea in terms of handfuls, as in a double handful
of root per kettle of water or one chunk of root for a mug of water. The art of making
27
sassafras tea is in the experience and practice of knowing how strong the root smells as to
how strong the tea will be, and checking on the steeped tea for the color and taste. The
amount of root and water and how long it is boils and steeps is secondary to the color,
smell, and taste. The more root you use, the faster it will steep, while the less root used,
the longer you steep it.
Tea is made with either fresh root or dried root, and there seemed to be no
preference for either type. Dried root needs to be rehydrated in order to release the flavor,
which can be done by boiling the root once, letting it steep overnight, and boiling again
the next morning and let steep again, or by letting boil and steep for a longer period of
time. For the amount of tea to drink, the consultants were all in agreement. If a person
was sick, they should have about three cups of tea, otherwise one to two cups a day is
enough. Tea is typically consumed in the fall, winter, and spring, especially in the spring
months when people are liable to get sick or prepare for the changing season. Fresh tea is
best, but if a big pot is made it can be kept in the refrigerator for up to a week.
Field Methods: Harvesting
All roots were dug within two weeks during July 2011 on property managed by
either the Highlands Biological Station or the Highlands-Cashiers Land Trust. All the
land was within Highlands, North Carolina, a small town in the southern plateaus of the
Blue Ridge Mountains. Many of the harvesting sites were around 3,800 feet in elevation,
with one site at 4,100 feet. Sassafras trees grow prolifically at this elevation and habitat,
which is classified as temperate rainforest (Chamber of Commerce 2011).
29
A total of nine trees were harvested for an average of 40 grams of root material
per tree. The terrain shape index, landform index, and diameter at breast height (dbh)
were measured for each of the trees, and the habitat type was described. The roots were
removed with hand shears, and the soil samples were collected from around the
excavated roots, to a depth of six inches. Soil samples were analyzed by the Clemson
University Agricultural Services Lab for general soil properties and nutrients. With the
exception of one site (Site 7), all the primary nutrients (nitrogen, potassium, and
phosphorus) were in the low medium to low range recommended for adequate plant
growth. The soils in all the sites were moderately to extremely acidic, with an average
soil pH of 4.23 ± 0.6 (optimum soil pH for plants is 5.8 to 6.5). See Table 3.1 for
complete descriptions of harvest trees and sites.
Positive root identification was achieved by following a large surface root three to
four feet from the tree bole. The surface root sometimes dipped into the soil, where it was
followed by tunneling along the root. Identification was also attained by olfactory
identification of the excavated root, which smells characteristically of ‘sweetshop’, candy
store, or ‘spicy’ when fresh. The two young saplings that are included in the sampling
were dug up completely and the entire root system up to the main root of the colony was
collected. Only trees approved by the Highlands-Cashiers Land Trust or the Highlands
Biological Station were harvested.
The size and shape of the roots is most likely dependent on the age of the tree, site
conditions like soil type and slope, and size of the colony, if part of one. Observations
were made that the older trees had much thicker, darker roots, almost a deep reddish-
30
brown color, with potent smelling roots that smelled characteristically like a sweet-shop
and earthy. Soil surrounding older roots had a very strong sassafras smell. Young
saplings, three or four years old, had roots that were much lighter in color with a sharper,
sweeter smell then the older trees without the associated smell in the soil. Surface roots,
or roots typically acting as suckers in a colony, were very pliable and thinner compared to
non-surface roots.
Field Methods: Processing
The roots were washed thoroughly (carefully keeping the root bark) in tap water
and weighed on a Mettler H51 balance scale with 0.01 g precision. The length and
diameter were measured with a digital caliper. The roots were dried on paper for a
minimum of 48 hours to prevent mold growth, and then placed in separate brown paper
bags until prepared for samples. The bags kept in an open box in a well-ventilated room
until they were boiled for tea.
When the roots were dry, they were weighed and measured again. The roots had
an average moisture loss of 61% when air-dried, or ranging between 51% to 67%
moisture loss. One 4 g sample was dried in an oven for 6 hours, or until the dried weight
did not fluctuate, where it lost a further 9% moisture, for a total of approximately 70%
moisture difference between dried and fresh root.
The air-dried dried roots were cut into segments that each weighed from 0.1 g to 1
g approximately (See Figure 3.2). The segments were mixed together in equal parts, 10 g
from each tree, so each tree root represented one-ninth of the total weight. When root
31
Site # Height
(feet)
DBH
(inches)
Habitat Slope Soil
pH
1 17 3.6 Acidic upland with Rhododendron
and Vaccinium, part of a colony,
sandy loam soil, understory
North facing, slight
slope
3.4
2 12 1.8 Acidic upland with Rhododendron
and Vaccinium, part of a colony,
sandy loam soil, understory
Southwest facing,
slight slope
3.9
3 15.5 2.2 Acidic upland with Rhododendron
and Vaccinium, part of a colony,
sandy loam soil, understory
Southwest facing,
slight slope
4.5
4 <6 <1 Acidic upland with Kalmia,
Rhododendron, and Vaccinium,
Rocky, sandy soil, understory in a
windy, exposed mountainside
Southwest facing,
very steep
4.1
5 17 2.2 Oak-hickory forest, understory
and part of a colony
Southwest facing,
slight slope
4.3
6 57.5 28.8 Acidic soil with dense covering of
Rhododendron and Kalmia,
roadside
South facing, steep
slope.
4.1
7 <6 <1 Sapling on disturbed site, roadside South facing, slight
slope
4.9
8 49 12.5 Old-growth, acidic upland forest
with Rhododendron. Is dominant
in the canopy with no colony
West facing, some
slope
3.6
9 <6 <1 Sapling in open, disturbed site
with full sun. Part of a colony of
saplings
South facing, no slope 5.3
Table 3.1: Site and tree characteristics for Sassafras albidum trees harvested in
Highlands, North Carolina.
32
segments were selected for preparing the tea, the bag was shaken thoroughly and root
segments were chosen blindly and at random. Oven-dried root segments were not used in
tea preparation as someone would not normally dried the root in the oven before making
tea.
Field Methods: Preparing
The traditional method of preparing tea was standardized to consistently produce
a cup of sassafras tea with approximately the same color and smell each time. This was a
subjective observation, but acceptable for this study as tea is consumed with a wide range
of color and smell intensities. Just as a variety of tree age classes and habitats are
harvested, a variety of preferences exist within tea preparation. As the consultants drank
tea within this spectrum of tea intensities, we intended to cover the range of tea consumed
to reflect any differences in safrole content. Once a standard time of boiling, steeping,
and re-boiling was established, the standard method did not change to accommodate
changes in tea color.
The roots were boiled twice: once to rehydrate the roots, which were then left to
steep ‘overnight’, then boiled again to make the tea. The water and root are heated
together to boiling (when the water started visibly bubbling), which marked the
beginning of the boil time. The heat source is turned down to ‘medium’ to simmer for 15
minutes (See Figure 3.3). The tea was removed from the heat source and left to steep
‘overnight’ for 12 hours without refrigeration. The second boil was performed as the first,
or the root and water brought to a roiling boil together, with the time beginning at first
35
signs of boiling. The tea was allowed to actively boil for 20 minutes and removed from
the heat source. The root segments were used once for each replicate of each sample.
Results and Discussion
The standardized method described in this section was based on conversations
with people knowledgeable in the use of the species. This standardized method,
particularly in the preparation steps, is as similar to the traditional method as could be
replicated in a laboratory setting (using distilled water, hot plates, and laboratory
glassware instead of kitchenware). Future studies might look closely at the traditional
knowledge of sassafras, including the range of harvesting, processing, and preparation
methods.
36
CHAPTER FOUR
CHEMICAL ANALYSIS USING HPLC
Introduction
The objective of this chemical analysis was to quantify the amount of safrole
found in both the method outlined in the FDA regulation and the traditional method of
preparing sassafras tea. A third sample set, the agitate samples, was added to measure the
concentration of safrole present in the root without degrading the safrole with heat
extraction. The samples were run through a high-performance liquid chromatography
(HPLC) system using a reverse-phase C18 column method. The FDA used gas
chromatography in the 1960’s to detect and quantify safrole, but the developments in
sensitivity and reliability of HPLC lead us to use this method (CFR Sec. 189; Larry
1970). Also, many recent papers have used HPLC to quantify safrole, including a paper
by Reynertson et al., 2005 from which we adapted the methodology for the chemical
analysis. This paper was used as a model because the study was recent, so it utilized
modern analytical techniques, and because the research closely paralleled the
ethnobotanical objectives of this project.
Plant material is inherently variable, particularly in the concentration of secondary
compounds. The time of day or season of the year, soil and microclimate, herbivory and
pathogen activity, and age and history of a particular tree can determine specific chemical
quantities. The experiment was designed to encompass the spectrum of harvest and
37
preparation styles, as well as variation among young or mature trees and different habitats
and elevations.
Materials and Methods
Laboratory materials
- Safrole standard (98%) – Chem Service Inc. in West Chester, Pennsylvania.
- ACS-grade MeOH and HPLC-grade ACN – Fisher Science in Georgia.
- Water – deionized
- YMC ODS-AQ S-8 4.6 x 250mm, 5μm, 120Å HPLC column
Traditional Method
Samples 1a, 2a, and 3a (see Figure 4.1 for chart of the samples) were prepared by
boiling 2 grams of air-dried root segments in a 500 mL glass Erlenmeyer flask with 300
mL distilled water, using two hot plate/stirrers (VWR Scientific Products 370 and Fisher
Science). A small piece of aluminum foil was loosely placed over the top of the flask to
reduce water evaporation. The samples were prepared as described in Chapter 3, or
boiled for 15 minutes, let steep for 12 hours, and re-boiled for 20 minutes.
Fifty micrograms (50 μg) of safrole standard was added to the samples 2a, 3a, 2b,
and 3b, following the methods in Reynertson et al. 2005. These spiked samples would
show whether safrole is degraded without complication of root material.
The traditional method samples were run twice due to the inconsistency of the
results in the first run. Each sample set of the traditional methods has six samples
whereas the FDA method and agitate sample set contains three samples each.
38
FDA Method
The FDA samples were prepared based on the generalized methods defined by
regulations. Sassafras extract is the aqueous solution “obtained by extracting the bark
with dilute alcohol, first concentrating the alcoholic solution by vacuum distillation, then
diluting the concentration with water and discarding the oily fraction” (CFR, Sec. 172).
Samples 1b, 2b, and 3b were prepared using ACS grade methanol diluted to 80%
methanol in 500 mL glass Erlenmeyer flasks. Each of the FDA samples were stirred
using a stir plate and stir rod for 35 minutes, or the same boil time of the traditional
method samples. Following the methodology outlined by the FDA, vacuum distillation
was done using a Büchi Rotovap. The heating bath was set at 65°C, the evaporation point
of methanol. The solution was diluted back to 300 mL using distilled water. The solution
was poured into the funnel separator, allowed to settle for 10 minutes, and then run
through the separator until 5 mL remained. The remaining liquid in the funnel separator
represented any sassafras oil found in the root and was not included in the analyzed
sample. The remaining aqueous solution (after removal of the oil fraction) had no
detectable oil, characteristic sassafras scent, or color.
Agitate Samples
The agitate samples were prepared with 10 g of root and 300mL of cool distilled
water. The samples were stirred with a stir plate and stir rod for 50 minutes, let sit for 20
hours, and filtered. The agitate sample went through a longer period of agitation and
steeping until the color of the resulting samples was close to color of the traditional tea
39
samples. The color was not standardized but based on the descriptions provided by the
consultants. A larger quantity of root was added for the same reason. No heat was applied
during any part of the process and the stirring was kept at a low, steady speed as to not
cause excess heat. The resulting concentration was divided by five to compensate for the
increased amount of root but no correction was taken for the increased time of agitation.
HPLC Analysis
All samples were filtered through 0.2 μm syringe filters prior to analysis. Analysis
was performed on a Shimadzu Prominence UFLC (Ultra-Fast Liquid Chromatography)
system using a reverse-phase C18 column (YMC ODS-AQ S-8 4.6 x 250mm, 5μm,
120Å). Results were monitored at 235 and 254 nm using UV detection. The mobile phase
consisted of two solutions, (solvent A) distilled water and (solvent B) acetonitrile (ACN).
The HPLC conditions were as follows: linear gradient of 5% (B) at 5 minutes, 15% (B) at
10 minutes, and 100% (B) at 45 minutes for 10 minutes, with a stop time of 55 minutes.
The injection volume was 10 μL. Retention time for safrole standard was 37.77 ± 0.23
minutes (See Figure 4.1).
The Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated
based on the blank determination method cited in Sanagi et al., 2009. LOD is the lowest
concentration of a compound that can be detected, but without the need to specifically
quantify the concentration. LOQ is the lowest quantifiable concentration identified by the
analytical procedure. LOD and LOQ are important analytical validations that define the
limits of the analytical laboratory machine (Sanagi et al., 2009). There are many accepted
40
methods for quantifying LOD and LOQ, including the blank determination method
outlined in Sanagi et al. (2009). The blank determination method was chosen for this
study as the method is applicable when a blank sample returns results with non-standard
deviations. LOD and LOQ is calculated by running at least three blank samples with no
standards or compounds added. The mean concentration and mean standard deviation of
the blank are found. LOD is the concentration of the blank sample plus three standard
deviations (xm + 3Sd) while the LOQ is the analyte concentration of the blank sample plus
ten standard deviations (xm + 10Sd), where (xm) is the mean concentration and (Sd) is the
standard deviation (Sanagi et al., 2009).
LOD and LOQ measured 1.514 ppm and 2.049 ppm for 235nm, respectively.
Triplicate samples of diluted safrole standard of 10, 50, 100, 200, and 1000 ppm were run
to configure a standard curve. Standard curves were calculated for 235 nm absorption,
yielding the following equation which with to calculate the amounts of safrole present in
the samples. For 235nm absorption: the equation was [Area = 628.2326*Concentration],
as seen in Figure 4.2. For the 235nm absorption, the p-values for the intercept variable (or
‘b’ in the equation y = mx + b) were greater than the level of significance, meaning it is
not included in calculating the sample concentrations. The R-squared value for 235nm
absorption was 0.9579. P<0.05 was considered statistically significant. Based on the
absorption spectrum of safrole, the 235 nm wavelength was determined to be a more
stable absorption time and therefore was the only wavelength reported in these results.
41
Number Sample Extraction
Method
Purpose
1a Root Traditional Quantify the presence
or absence of safrole
2a Safrole Standard Traditional No root, to ensure traditional
method eliminates safrole
3a Safrole spiked Root Traditional Quantify amount of degradation
1b Root FDA Quantify safrole to compare to #1a
2b Safrole Standard FDA No root, to compare to #2a
3b Safrole spiked Root FDA Quantify amount of degradation
Agitate Root Cold water To extract unaltered safrole using
water
Table 4.1: Table of samples, extraction methods, and purpose of sample type in
preparation of HPLC analysis. Traditional method samples are signified with the letter ‘a’
after the sample number while FDA method samples have the letter ‘b’ after the sample
number.
42
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 min
0
50
100
150
mV
Detector A Ch1:235nm
1.1
94
4.2
65
6.4
52
14.7
85
16.8
46
18.1
8018.5
10
19.0
08
19.8
26
20.5
4221.0
70
21.3
8321.5
67
22.1
35
22.4
14
22.7
5923.4
25
23.7
9324.0
12
24.3
33
24.6
25
24.8
31
25.3
08
25.5
13
26.5
1526.8
24
27.3
2527.5
70
27.8
0428.2
60
28.5
6028.8
99
29.1
06
29.4
68
30.1
7530.3
72
30.6
9230.8
25
31.0
1131.1
58
31.3
1731.4
58
31.6
7331.8
50
32.1
8832.3
42
32.4
9232.8
03
32.9
8233.1
42
33.2
7533.5
95
33.8
2034.1
74
34.5
0134.9
78
35.8
52
36.4
76
36.8
17
37.7
75
39.0
5739.4
09
39.6
7139.8
19
40.0
50
40.6
01
40.9
1941.1
96
41.7
0841.8
79
42.2
4042.7
53
43.2
67
43.4
74
43.7
8944.1
29
44.6
5645.1
26
45.2
9145.5
73
45.9
9946.1
79
46.4
2446.6
53
46.9
7847.2
08
47.3
9247.6
59
48.3
9548.6
63
48.8
66
49.1
33
49.4
6249.6
27
49.8
5350.0
20
50.2
61
54.5
39
54.8
92
61.9
15
62.7
53
Figure 4.1: Safrole standard (5,000 ppm), detected at 235 nm with an elution time of
37.775 minutes.
43
Figure 4.2: Standard Curve for 235 nm absorption. The equation generated by the linear
regression: Area = 610.03*Concentration. Triplicate runs of 10, 50, 100, 200, and 1000
ppm of safrole standard are indicated by red open circles. Upper and lower 95%
confidence intervals represented by dotted lines.
44
Sample PPM Mean
(PPM) Std Dev
1a-1 7.206
1a-2 0
1a-3 9.055
1a-4 8.917
1a-5 6.198
1a-6* 35.36 6.275¹ 3.707
2a-1 0
2a-2 1.854
2a-3 0
2a-4 0
2a-5 0
2a-6 0 0.309 0.757
3a-1 26.09
3a-2 13.80
3a-3 18.36
3a-4 5.880
3a-5 9.653
3a-6 0 12.30 9.258
1b-1 0
1b-2 0
1b-3 0 01 0
2b-1 0
2b-2 0
2b-3 0 0 0
3b-1 0
3b-2 1.93
3b-3 0 0.644 1.12
Agitate-1 21.25
Agitate-2 24.12
Agitate-3* n/a* 22.691 2.033
Table 4.2: Results of HPLC analysis in parts per million (PPM) of safrole at 235nm. The
mean and standard deviation of each of the sample sets is listed.
*Samples 1a-6 and Agitate-3, were removed from the sample sets as the results were
outliers or due to a contaminated column. The concentration was determined by the
calibration curve. 1Samples 1a are significantly greater than the samples 1b and significantly less than
Agitate 1 & 2 samples based on student t-tests at α=0.05
45
Sample Average
Safrole in mg/ 300 mL
Std. Dev.
Average Safrole in ppm or mg/L
Std. Dev.
1a 188.2 111.2 627.5 370.7
2a 9.27 22.7 30.9 75.7
3a 368.9 277.7 1229.7 925.8
1b 0 0 0 0
2b 0 0 0 0
3b 19.3 33.5 64.48 11.67
Agitate 680.6 61.0 2268.7 203.3
Table 4.3: Milligrams and ppm of safrole per 300 mL of aqueous solution.
Concentrations calculated from Table 4.2.
46
Sample Safrole in mg/ kg Std. Dev.
1a 3.14 1.85
2a 0.15 0.38
3a 6.15 4.63
1b 0 0
2b 0 0
3b 0.32 0.56
Agitate 11.34 1.02
Table 4.4: Milligram of safrole hypothetically consumed by a 60 kg person.
Concentrations calculated from Table 4.3.
47
pH of Traditionally Prepared Tea
The pH of three separately prepared, traditional tea samples was quantified using
a pH probe. The probe was rinsed with distilled water, inserted into the tea sample, and
rinsed again with distilled water. The traditionally prepared tea samples were prepared
following the methods listed above.
Results
Concentrations in ppm of the HPLC analysis of the traditional, FDA and agitate
samples can be seen in Table 4.2. One sample prepared using the traditional method with
root only (1a-6) was discarded as an outlier with a large Cook’s distance. Sample labeled
Agitate-3 in Table 4.2 was discarded due to contamination of the column.
The methods outlined in the paper by Reynertson et al., 2005 were followed in
preparing comparative samples, namely the spiked samples (2a, 3a, 2b, & 3b). The
samples 2a and 2b were prepared with 50 μg safrole standard while the samples 3a and
3b were prepared with root and 50 μg safrole standard, as outlined in Table 4.1. When
calculating the concentration of safrole standard added to the samples, the amount
suggested by Reynertson et al. (2005), it was discovered that the amount was too small to
be detected by the HPLC system. In a 300 mL solution, 50 μg contributes a concentration
of 0.67 mg/L, or 0.67 ppm. The LOD and LOQ for this system were 1.514 ppm and
2.049 ppm. Unfortunately, these samples could not be used in discussion of the results of
the chemical analysis.
The agitate samples were intended to provide a baseline amount of safrole found
in the root and extracted with water. We compared the agitate samples to the traditionally
48
prepared teas to observe how the quantity of safrole differs between samples being
actively heated and boiled and those samples simply being agitated. Although the agitate
samples were agitated longer than the traditional methods, the samples were prepared
based on color just as a person making sassafras tea will judge the readiness of the tea on
its color. The elevated amount of root in the agitate sample was compensated by dividing
the concentration by five, as five times as much root was used. It was interesting to note
that while the agitate samples did not have the characteristic sassafras smell, but they did
have a woody, earthy smell that was not unpleasant.
Based on a student t-test, the concentrations of the traditional method samples
were significantly greater than the FDA method samples (P <0.05) and significantly less
than the agitate samples (P<0.05) based on α=0.05.
Observations and Discussion
We found three important points of discussion from the chemical analysis. First,
the FDA method was extremely efficient at removing the safrole oil by agitation using
organic solvents. There was one sample with a small amount (1.878 ppm) of safrole
present, but this is probably due to imperfect laboratory procedures and not a reflection of
the methodology. We observed that the resulting liquid from the FDA method was
odorless and colorless. There was no characteristic sassafras smell associated with the
liquid and therefore, it may be unappealing for a marketable product.
The second observation related to the results of concentrations of the agitate and
traditional method samples, as listed in Table 4.3. The average safrole concentration of
49
the agitate samples was 2268.7 ppm ± 203.3, or 681 ± 61.0 mg of safrole per 300 mL of
aqueous solution. The traditional method of preparing sassafras tea (Sample 1a) resulted
in an average concentration of 627.5 ± 370.7 ppm or 188 ± 111.2 mg of safrole per 300
mL of aqueous solution. Based on these results, the traditional method samples have 3.6
times fewer grams of safrole per cup (300 mL) of tea as compared to agitate.
Since essential oil composes 6-10% of the weight of the root cortex of sassafras,
and safrole composes 80-90% of the essential oil, by taking the highest estimates the root
contains around 9% safrole (Carlson & Thompson 1997; Kamdem & Gage 1995). A cup
of tea, or around 300 mL, is typically prepared with 2 grams of root. We would then
expect the cup of tea to contain 180 mg of safrole oil. The traditional method samples
contained 188 ± 111.2 mg of safrole per cup, or close to the expected concentration. On
the other hand, roots extracted with water by agitation yielded 681 ± 61 mg of safrole per
300 mL of tea. It is difficult to compare this experiment to previous research as the
methods to agitate the sample are going to differ with each study, but it was interesting to
note the difference between the amount of safrole we expected to find and the amount of
safrole extracted by aqueous agitation. The study by Carlson & Thompson (1997)
showed an 88.9% reduction of safrole from the unbrewed teas compared to the brewed
teas. The study found ranges of 0.18 – 16.0 mg/g product in unbrewed teas that were
reduced to 0.09 – 4.12 mg/g product in the brewed teas.
Chemically, it seems unlikely that using a polar solvent (water) to extract a non-
polar solute (safrole oil) should extract more oil than a non-polar solvent (methanol or
another organic solvent). Safrole is miscible in alcohols, not water (Kamden & Gage
50
1995; Sethi et al., 1976). Though, it is difficult to compare the studies as many journal
articles do not detail extraction processes. However, it is possible to say that boiling the
root as in the traditional method aided in reducing the concentration of safrole in the
resulting aqueous solution, as compared to the agitate samples.
The third and last observation relates to the literature on safrole content and
regulations. Returning to the previous calculations, if we expect one cup of tea with 2
grams of root and root bark to have about 180 mg of safrole, a person weighing 60
kilograms would consume 3.0 mg of safrole per cup. This calculation is supported by a
similar estimate by Segelman et al. (1976) who calculated 3.0 mg/kg safrole is found in
one tea bag containing 2.5 g of sassafras bark. We found that sassafras tea has 188 ±
111.2 mg of safrole per cup (300 mL) of tea, as listed in Table 4.3. Therefore, a 60 kg
person would consume 3.13 ± 1.85 mg/kg bw of safrole per 300 mL of tea (Table 4.4). A
person consuming agitated, non-boiled tea would be consuming 11.34 ± 1.02 mg/kg bw
of safrole per 300 mL. The United States FDA does not allow the presence or addition of
safrole to any foods or beverages and the European Commission of the European Union
allows the presence of safrole in foods containing nutmeg and mace only up to 15 ppm.
Traditional tea, according to this study, has around 628 ± 370.7 ppm of safrole per 300
mL (Table 4.3).
Using the Body Surface Area (BSA) dose translation formula discussed earlier, a
60 kg person would consume 3.14 mg of safrole, which is equivalent to a dog consuming
5.8 mg/kg bw. A study by Hagan et al. (1965) conducted a six-year study that
administered safrole to dogs in doses of 5, 20, 40, and 80 mg/kg bw. After six years, with
51
the dogs receiving a tablet six days a week, the dogs experienced microscopic, minimal
focal necrosis, bile-duct proliferation, fatty metamorphosis, and hepatic cell atrophy
(Hagan 1965). Another study with dogs that spanned seven years found liver injury early
in the experiment, but hypothesized that the lack of damage found later in the study was
due to adaptation to the continuous intake of safrole (Weinberg & Sternberg 1966).
The dose equivalent for rats would be 19.4 mg/kg bw. As stated before, many
toxicity studies administer doses in the range of 100 to 1000 mg/kg bw, though Daimon
et al. (1998) tested single doses of 1, 10, 100, 250, and 500 mg/kg bw. Two DNA-safrole
adducts per 107 nucleotides were found after a dose of 10 mg/kg bw, while a 1 mg/kg bw
dose did not produce adducts and a dose of 100 mg/kg bw produced two major and two
minor adducts per 107 nucleotides. Friedman et al. (1971) found that a 10 mg/kg dose of
safrole had no effect on microsomal enzyme activity, which aid in the detoxification
process. Chang et al. (2002) found the metabolic saturation of safrole occurred between
150 and 300 mg/kg for rats, or much greater than the amount of safrole found in a cup of
tea. Saturation of safrole means the body cannot eliminate the compound quickly and the
safrole accumulates in tissue muscle, especially in the liver and kidneys, to increase the
risk of DNA adducts and carcinogenesis (Benedetti et al.,1977).
Based on these few studies, there was no indication that rats or dogs fed safrole at
the amounts listed above developed liver tumors or cancer. There are not enough studies
that have tested these particular doses on laboratory animals to conclude with certainty
that the amount of safrole found in sassafras tea will lead to carcinogenesis. These few
studies are promising, but more research needs to be conducted.
52
Plant secondary compounds, used by the plants for defense against predators and
pathogens, are utilized by humans for the same purpose, to rid the human body of disease
by way of pharmaceuticals (Bourgaud et al., 2001). Secondary compounds must be used
properly, as the same compounds that humans harness to starve off disease can harm the
body. Safrole is a proven hepatocarcinogen to laboratory animals, causing macro- and
microscopic liver lesions, liver tumors, inability to gain weight, and reduced feeding (Jin
et al. 2011; Long et al., 1963; Miller et al. 1983; Taylor et al., 1964). These symptoms
exhibited by laboratory animals make sense when we consider the traditional medicinal
qualities of sassafras tea, including ‘overfatness’ and to thin the blood during the change
of seasons, especially during the spring (Moerman 2011). Considering the laboratory
tests use high doses of safrole compared to the amount of safrole found in traditional tea,
we can hypothesize that the extreme symptoms observed in laboratory animals occur to
some degree in those drinking sassafras tea, but possibly to the human advantage. While
laboratory rats had difficulty gaining weight or had a reduced appetite while consuming
safrole, humans perhaps used small amount of safrole in sassafras tea to help shed winter
pounds. A seven year study of administering safrole to dogs found evidence of liver
injury early in the research but not late in the study. The researchers suggested the dogs
adapted to the continuous intake of safrole and regenerated the damaged liver tissue
(Weinberg & Sternberg 1966).
53
Reduction of Safrole during Boiling
A handful of studies have attempted to explain the process that occurs when
safrole-containing substances are subjected to heat. One study examined the spices star
anise, cumin, black pepper, and common cooking ginger, which contain safrole in the
essential oil, and found that washing, drying, and cooking the spices all reduced safrole to
safe levels. Boiling the spices reduced the safrole content from 955 mg safrole per kg
material in untreated seeds to 375 mg/kg in whole seeds boiled for one minute all the way
to 9 mg/kg in powdered seeds boiled for five minutes (Farag & Abo-Zeid 1997). The
study on a Chinese wild ginger reduced the 1.57 to 2.76 mg safrole per g material to 0.2
mg/g or undetected in most samples after a one-hour decoction of the root (Chen et al.,
2009). Carlson & Thompson (1997) hypothesized that the lower safrole content in their
sassafras ‘tea’ (the tea was infused not decocted) could have been due to either the
decreased solubility of sassafras oil in water or volatilization of the compounds.
Three chemical reactions could be occurring to reduce the safrole content in a cup
of boiled tea. First, the essential oil, including the safrole oil, could be still in the root.
Carlson & Thompson (1997) hypothesized that safrole content was reduced due to the
insolubility of the essential oil in the root, which agrees with their finding of high
recovery of safrole from alcohol- based sassafras oil tinctures. The second situation could
involve volatilization of safrole as it is being heated. The compound has a low vapor
pressure (0.0706 mm Hg at 25°C or 1 mmHg at 63.8°C), making safrole primarily a
vapor at room temperature and pressure (Sigma-Aldrich MDMS S9652). If the compound
was boiled at high temperatures for an extended period of time safrole would have some
54
volatility. Carlson & Thompson (1997) cited volatilization as well as reduced solubility
of safrole in water for the reason safrole content was reduced an average of 88.9% from
unbrewed to brewed tea. Many laboratory studies mention the volatilization of safrole
from safrole-spiked rodent diet, which was left in open in room temperature. One study
includes Homburger et al. (1965) that measured the evaporation rate of safrole to be
11.3% per three day period (Crampton et al., 1997; Huang et al., 1999; Long et al., 1963).
The last possibility is that safrole is being altered or degraded during the boiling
process, as described in the Reynertson et al. (2005) paper. Reynertson et al.
hypothesized that even though safrole is insoluble in water, insolubility is not the issue as
samples spiked with safrole still resulted in no detected safrole. Since there were a
number of degraded by-products after samples were boiled, it is probable that the safrole
was not volatilized. Rather, the safrole was probably degraded during the boiling process
by hydroxylation of the dioxolane ring. The hydroxylation could be due to the slightly
acidic nature of the tea, which they measured at pH 4.4 (Reynertson et al., 2005). For this
research, the average pH for the traditional method tea sample was measured at pH 5.1 ±
0.1, which is considered acidic.
55
CHAPTER FIVE
WORKING WITH NATIVE COMMUNITIES: EDUCATION AND OUTREACH
Introduction
During the research, we have collaborated with the Cherokee community, who
continue to drink sassafras tea according to their traditional method. In addition, there are
many non-Native people that grew up harvesting, processing, preparing sassafras, and
drinking sassafras tea. The Cherokee best represents a cohesive community of people that
maintain the knowledge and tradition of preparing sassafras tea. Since the Center for
Cherokee Plants has worked to maintain and continue traditional knowledge of native,
culturally signification plants, this work might not have been possible.
Working with Native Communities
This research, although not strictly participatory, is based on a model of working
with Native communities through participatory research used by many researchers. In this
model, research, education, and action are combined and research is based on the
circumstances of the community. The researchers work to connect communities with
resources, data, and scientific knowledge of benefit to them. There is an underlying
assumption that communities will benefit from the research as resources are based on an
“ecological give-and-take approach”, where knowledge, time, or mentoring by a
community is returned to them through conscience acts of reciprocity (Davis & Reid
1999; Cotton 1996).
56
Linda Tuhiwai Smith said, “The term ‘research’ is inextricably linked to
European imperialism and colonialism. The word itself, ‘research’ is probably one of the
dirtiest words in the indigenous world’s vocabulary” (1999). Unfortunately, many Native
communities around the world have been exploited by researchers. Some research has
even been harmful, especially research related to data collection and reporting on
negative issues like alcoholism or drug addiction. Such research can lead to communities
being ostracized or stigmatized, with participants feeling betrayed. In the worst cases,
participatory research has left participants feeling invaded, patronized, or inferior, which
is why many Native communities today have created strict rules to how and with whom
research can be conducted on their land (Davis & Reid 1999).
Anthropological and botanical studies in particular are in danger of being of little
use to Native communities, and unfortunately some research on traditional medicines has
been unconcerned with collaboration, effects of publication, or reciprocity. Given the
history of medical, anthropological and archeological research, contemporary research
projects with Native communities absolutely should be a collaborative effort between the
community and the researchers. Christopher (2005) outlines several recommendations for
non-Indian researchers, including involving the community from the formation of the
question to the conclusion, as well as ensuring that the community has access to the
research and results. The research should be based on values, concerns, and interests of
the community and contain culturally-appropriate methodologies (Weaver 1997). The
project should also have clear and immediate benefits for the community. Knowledge for
57
the sake of knowledge, according to activist and historian Vine Deloria Jr., should not be
tolerated by Native Americans (1969).
Collaborating with the Cherokee
Although this project was concerned with the perceived toxicity of products made
from Sassafras albidum, this research did not draw the official support of the tribe due to
the historical use of sassafras for spiritual and medicinal purposes. However, we were
able to work with the Eastern Band of Cherokee Indians Cooperative Extension to
provide a means for education and outreach about the species. The Cooperative
Extension acted as a ‘cultural consultant’ to advise us on the appropriate channels to
distribute the research results (Weaver 1997). We planted sassafras trees and created a
GIS map and pamphlets for the Center for Cherokee Plants (CCP), a project under the
Extension office. The Center for Cherokee Plants, located between Cherokee and Bryson
City, North Carolina and near the Kituwah mound, is a nursery that cultivates and
experiments with Cherokee heirloom varieties of edible crops. They collect, save, and
distribute seed within the tribe to encourage gardening with old varieties and healthy
eating. The Center emphasizes youth and elder participation by establishing community
and school gardens around the Qualla Boundary and projects including the Backyard
Ramp Patch, Native Plant Study, Cherokee Community Greens Patch, and the Cherokee
Farmers Market. They are the hub of agriculture and gardening in Cherokee as they work
with community civic clubs and are connected with youth and elders alike. The Center is
58
a medium to encourage interactions between elders, adults, and youth and the exchange
of traditional and indigenous knowledge (CCP 2011).
By working with the CCP, we were able to hold discussions about sassafras tea
and most importantly, give back to the community. Education and outreach comprise a
significant part of this project for several reasons, including to disseminate scientific
knowledge about the properties and safety of sassafras products and to highlight the
cultural and historical importance of sassafras products. We promoted the use of sassafras
in several ways: first, by planting a stand of twenty trees of sassafras on the property of
the CCP, as seen in Figure 5.1. The CCP can decide in what ways they will use trees,
either by harvesting material for demonstration purposes, allowing tribal members to
harvest material for their own use, or as a physical reminder of how sassafras is a
culturally significant species for the Cherokee. The trees also provide a basis for future
research, ranging from topics on forestry to food chemistry. We also are in the process of
creating pamphlets to be displayed with other educational material at the Cooperative
Extension office in the city of Cherokee as well as at the Center for Cherokee Plants.
Appendix A, focused on simple means of propagating sassafras, will be modified for
these pamphlets. A copy of the thesis will be given to the Museum of the Cherokee
Indian, which is also required for research under the auspices of the Tribal Research
Committee, though we did not submit this research for their approval per their request
(EBCI C.O., Sec. 70-3).
We hosted a high school intern during Summer 2011 to complete a Geographic
Information System (GIS) project to map the nursery grounds of the Center for Cherokee
59
Plants. The GIS map was made into a 3’ x 4’ laminated wall display and also into printale
sheets for record-keeping. The Center asked not for the maps to be either replicated in
print form, other than their own copies, or available on the internet, which we have
agreed not to do. This is an example of how researchers need to be flexible to the wishes
of the Native community which with they work.
The continued use of sassafras trees for traditional foods and beverages could
bring attention to land that is under development on the Qualla Boundary. Many current
stands of sassafras in use by tribal members are open fields or sites slated for bulldozing.
In the future, a non-timber forest product or products could be developed to sell if tribal
members so desired. There could be potential for members of the tribe to petition the
Food and Drug Administration based on this research, but more research and chemical
analysis would most likely be needed before individuals could market and sell sassafras
products. Appendix B contains more information on how to submit a petition to the FDA.
60
Figure 5.1: Stand of twenty sassafras trees planted at the Center for Cherokee Plants in
Bryson City, North Carolina. The three-year-old trees were planted in Spring 2011.
61
CHAPTER SIX
SUMMARY
Sassafras tea is consumed today by many communities in the Southeastern United
States, particularly in the Appalachian Mountains. Products made from the roots of
Sassafras albidum have been banned from the market by the FDA due to the presence of
the confirmed carcinogenic compound, safrole, based on tests using pure safrole standard.
Toxicological studies typically test compounds by administering large doses of the
compound to laboratory animals in order to understand the mechanisms of
carcinogenesis. These studies are needed to understand the mechanisms but should be
carefully analyzed when attempting to discuss the effects of ingesting safrole at daily
levels. Before this project, no research had quantified the concentration of safrole in
traditionally prepared sassafras tea. Additionally, no studied have used traditionally
prepared teas in laboratory rodent cancer potency tests. Even though the number of
people that consume traditional sassafras products is unknown, we can assume that many
people grew up drinking the tea continue to consume it, as well as people that recently
learned through herbal medicine guides. Due to the regular use of sassafras tea, it is
important to understand any potential dangers associated with consuming products
derived from the tree.
This study was informed by traditional knowledge of harvesting, processing, and
preparing sassafras tea, as understood from conversations with members of the Eastern
Band of Cherokee Indians (EBCI) as well as non-Cherokee well-versed in making
62
sassafras tea. The first objective of this study was to conduct chemical analysis using
HPLC. The intention was to compare the FDA method of extracting sassafras oil with a
standardized process of preparing sassafras tea to quantify the amount of safrole present.
An agitate sample was analyzed to compare the influence of heat on the quantity of
safrole. The second objective was to return the results of this study to communities that
utilize sassafras tea, namely the EBCI. We also emphasize the importance of reciprocity
for the Cherokee community, who has dedicated their own resources to this project.
The results of the chemical analysis demonstrated that the FDA method was very
effective at eliminating safrole entirely, as only one sample showed a miniscule amount
of safrole. Additionally, analysis revealed that traditionally-prepared sassafras tea
contains 188 ± 111.2 mg of safrole per 300 mL, or 0.63 ± 0.37 mg safrole per mL of
solution. This is a safrole concentration of 628 ppm in traditionally prepared sassafras
tea. Previous research examining the safrole content in root and root bark predicted a
similar quantity of safrole, or 180 mg per 300 mL. On the other hand, the agitate samples
from this research contained a higher concentration of safrole, or 680 ± 0.061 mg per 300
mL, or 2.3 ± 0.2 mg/mL of solution. This is a concentration of 2269 ± 203.3 ppm, or
more than predicted from previous estimates of safrole concentration in essential oil. As
the agitate samples in this research used water as the extraction solvent (versus organic
solvents used previously), it is difficult to conclude if safrole is reduced in the boiling
process. If we assume the agitate samples in this study are the reference point of safrole
content, then boiling does reduce the amount of safrole found in traditionally prepared
tea.
63
These three methods of extracting sassafras root resulted in important
observations. First, the traditionally made sassafras tea had a pleasant smell and color -
properties that many cultures associate with the tea. Second, the agitate samples did not
elicit the characteristic sassafras smell and it took more root and time to achieve the
desired color. Lastly, the FDA method resulted in a clear, odorless product with
undetectable sassafras odor. In hindsight, the concentration of safrole found in sassafras
tea may be serving some purpose, including simply contributing taste and scent
properties to the tea or functioning medicinally.
In conclusion, this research contributes to a growing body of knowledge, both for
those in the academic world as well as practitioners and caretakers of traditional
knowledge. This research will serve at least two purposes: first, the results of this
research will help people make informed decisions about their personal consumption of
sassafras products. Second and more broadly, this will highlight the importance of
traditional preparation methods when analyzing foods and beverages, especially those
historically used by Native communities.
65
Appendix A
Propagating Sassafras albidum
Sassafras is notoriously challenging to propagate or even find in nurseries. Not
only is it problematic to transplant (due to its deep taproot) but ripe seeds are difficult to
find in the wild (Dirr 1998). The species is dioecious so only female plants produce seed,
which are often eaten quickly by birds and wildlife. Greenish-yellow flowers produce
single-seed drupes on red pedicles that turn a dark blue-purple when mature (Bonner
2012). The species can be propagated from fresh, ripe seeds, dried seeds, cuttings, or root
scions (Rasch 1998).
Transplanting
Sassafras can be grown in a container from seed and the tree transplanted any
time of the year. A tree taken from the wild or from field plantings should be true
seedlings (not root sprouts) that are transplanted in the late winter or early spring. The
trees started by seed and grown in large pots until planting seem to transplant better than
those transplanted directly from a field (Sternberg 2004). The colonal root system of
sassafras, along with its deep taproot, makes it difficult to transplant saplings or trees
easily. Root pruning in the late fall or early winter can encourage a tree to develop a
healthy root ball before it is transplanted in the spring.
When transplanting saplings, the tree should be balled and burlapped in moist,
loamy, acidic, well-drained soil (Dirr 1998). About a third of the plant should be pruned
to encourage root growth (See Figure A.1). If the main bole or branches break, as
67
brittle wood tends to do, then the stem is cut to the ground and a new stem will sprout. A
sassafras thicket is obtained by cutting the stem back to encourage root suckers, but if a
single stemmed tree is desired, the suckers should be removed (Sternberg 2004).
Seeds
Sassafras trees produce seed around 10 years of age (minimum of 4 years old) and
reach maximum production from ages 25 to 50 with seed crops every year or every other
year. Seed viability is around 35% and the seed can remain viable for six years on the
forest floor. A study on Sassafras randaiense (Hayata) Rehder cited inhibitors in the seed
coat and cotyledon that cull germination, requiring removal or scarification of the seed
coat and followed by two to three months of cold stratification (Chen & Wang 1985).
Sassafras seeds do not store well as they are an oily seed (versus a more starchy seed),
with 47% lipid content (Bonner 2012). To break a natural dormancy of the embryo, the
seeds need a cold-wet stratification with a period of 120 days at 41°F (5°C) in moist
sandy or mineral soil (Bonner 2012; Griggs 1990; Haywood 1994). Germination can then
be tested in sand or a germination soil mix at 70° to 85° F. (21-29° C) for up to 120 days.
One method of germinating seed by the Woodlanders Nursery in Aiken, SC is to
sow seed in a ‘community pot’, or a three-gallon container with well-draining potting
soil. The seeds should be rehydrated in water for 24 hours, surface sterilized, and
scattered in a sterilized three gallon pot of soil with a half inch of soil covering the seed.
The community pot should be watered, covered with hardware cloth, and placed outside
for the entire winter until the seeds germinate in the spring. The pot is dumped out and
68
seedlings separated to be potted individually (Bob McCartney, personal communication
November 21, 2011).
Root Cuttings
A study by Sicuranza (2007) showed poor results for sassafras propagation from
stem sprouts, but worthwhile results from root cutting propagation. It has been found that
root cuttings yield plants with longer life spans, less root suckering, and better
architecture than grafted plants (Orndorff 1977). Root cuttings are collected from October
to December, when roots contain the highest percentage of carbohydrates, and placed in a
2:1:1 peat, loam, sand mixture can be planted out successfully (Del Tredici 1995, Dirr
1998). Some suggest taking root cuttings in February when the ground thaws and storing
the roots in dry sand for three weeks before planted in sandy soil. If roots are harvested in
the summer, Sicuranza (2007) showed July as the most successful month.
Evans & Blazich suggest harvesting root pieces that are 5 to 10 cm in length in
early winter. Del Tredici (1995) recommends root pieces that are 10 to 15 cm long for
roots being planted outdoors in late fall or early winter. A straight cut is made on the end
closest to the parent plant while a slanted cut is made on the opposite end (Evans &
Blazich 2011). The roots need to be washed and fibrous roots removed. The root pieces
are placed vertically in a moist medium, preferably pure sand or 1:1 peat to perlite mix
with 10-20% sand. Griggs (1990) recommends placing stem sprouts vertically or larger
roots horizontally. The straight cut, or where the root was closest to the parent plant,
should be covered by 2 to 3 inches of soil (Evans & Blazich 2011). The medium and
69
roots are stored in a cool, dark location for three weeks until put in a warm greenhouse.
There is a danger of water stress as the sudden warmth will cause shoots to develop
before roots (Sicuranza 2007).
Pests & Disease
Foliage disease is the most prominent problem among S. albidum, along with a
strong susceptibility to fire damage. Overall, sassafras is usually free from pests and
disease, probably due to the strong presence of secondary defense compounds, but it does
suffer from the occasional cankers, leaf spots, mildew, wilt, root rot, Japanese beetle, and
sassafras weevil (Dirr 1998).
Propagation Trials
Sassafras seeds were collected in August in Clemson, South Carolina and left to
air dry on newspaper. For these trials, the fleshy seed coats were allowed to dry and then
removed manually, but Bonner (2012) recommends removing the pulpy flesh before
storage or propagation by rubbing the seed over hardware cloth. Two methods of
processing and cold-wet stratifying the seeds were tested. The first method involved
removing the dried seed coat entirely, which can be done by carefully cracking open with
a small knife or fingernail and extracting the round, light brown seed. The seeds were
surface sterilized in a 10% bleach solution for 30 seconds, rinsed, and placed in a
sterilized plastic snap-lid container with damp perlite. The second method leached the
seeds of any chemicals that would inhibit germination. The seeds were placed in
70
cheesecloth like fabric and placed in a back toilet bowl for fourteen days. The seeds were
scraped with sandpaper, surface sterilized, and placed in damp perlite.
All containers were placed in the vegetable drawer of a refrigerator (at 41° F or 5° C) for
four months. In March 2012 the seeds were sown in potting soil and placed outside to
germinate. The seeds will be monitored for success rate of germination of either the
leaching method or removal of seed coat.
A third method of stratifying and germinating the seeds was a method
recommended by Woodlanders Nursery in Aiken, SC, termed ‘community pot’ (personal
communication, Nov. 21, 2011). This method (see above description) was begun for the
experiment but unfortunately had to be terminated. Future studies in sassafras
propagation should strongly consider this method.
The propagations trials are still ongoing. In Fall 2011, a dozen sassafras saplings
considered weedy by the landowner in Cherokee, NC were cut around with a sharp
shovel to encourage a root ball. Six of the trees were transplanted in March 2012 and six
more will be transplanted in Fall 2012 to the Center for Cherokee Plants. The saplings
will be monitored to measure success rate of spring and fall transplanting.
71
Appendix B
Petitioning the Food and Drug Administration
According to the FDA website, the government agency receives about 200
petitions a year (webpage last updated in 2008). The evaluation of a petition can take a
few weeks or up to a year. The majority of the petitions come from industry and
consumer groups, though individuals are allowed to submit petitions. There is a specific
format for citizen petitions that must be followed for submission, which can be found by
searching for Title 21 of the Code of Federal Regulations, Sections 10.30. It contains six
parts: action requested, statement of grounds, environmental impact, economic impact (if
applicable), certification, and identifying information.
The code that specifically targets the use of sassafras bark to make sassafras tea is
[Code of Federal Regulations: Title 21, Volume 3, Sec. 189.180 Safrole].
The full title: Title 21 – Food and Drugs, Chapter I -- Food and Drug Administration,
Department of Health and Human Services, Subchapter B – Food for Human
Consumption (Con’t), Part 189 – Substances Prohibited from Use in Human Food,
Subpart C – Substances Generally Prohibited from Direct Addition or Use as Human
Food. Sec. 189. 180 Safrole.
The code states in part (b), “Food containing any added safrole, oil of sassafras,
isosafrole, or dihydrosafrole, as such, or food containing any safrole, oil of sassafras,
isosafrole, or dihydrosafrole, e.g., sassafras bark, which is intended solely or primarily as
a vehicle for imparting such substances to another food, e.g., sassafras tea, is deemed to
72
be adulterated in violation of the act based upon an order published in the Federal
Register of December 3, 1960 (25 FR 12412)” (CFR, Sec. 189).
Specifically, the petition should outline the confirmed carcinogenic properties of
pure safrole standard and related metabolites in comparison to pivotal papers like
Benedetti et al. (1977) that found a lack of 1’-hydroxysafrole (a carcinogenic derivative
of safrole) in human subjects after ingestion of safrole. Next, the recent research on the
non-toxicity of traditionally prepared foods, beverages, and medicines should be
mentioned (Chen et al., 2009; Farag & Abo-Zeid 1997; Reynertson et al., 2005; Singh &
Devkota 2003; Whitton 2003). The results of this research should be cited as well as any
other future research on the toxicity or carcinogenicity of traditionally prepared sassafras
products.
Summaries of all the studies can be found in the following reviews:
Cropwatch.org/: Safrole
SCF. 2002. Scientific Committee on Food, European Commission: Opinion of the Scientific Committee on Food on the safety of the presence of safrole (1-allyl-3,4-
methylene dioxy benzene) in flavourings and other food ingredients with flavouring
properties. (SCF/CS/FLAV/FLAVOUR/6 ADD3 Final).
WHO 2009. World Health Organization: WHO Food Additives Series: 60. Safety
evaluation of certain food additives. The 69th
Meeting of Joint FAO/WHO Expert
Committee on Food Additives.
73
Appendix C
Reference of Previous Laboratory Studies Using Safrole
Studies on betel quid chewing were included in this chart as they gave the unique
opportunity to study the effects of safrole injection in humans. The inflorescence of Piper
betle contains 15.35 mg safrole when fresh, which metabolize to dihydroxychavicol and
eugenol (both mentioned in the chart) (Chang et al., 2002). Betel quid chewing is vastly
different from consuming sassafras products, both in the complexity and composition of
material and in the mode of safrole absorption. Both products contain safrole, so we can
observe similarities in physiological effects from a range of concentrations.
Many studies quantify the presence of carcinogen- DNA adducts as a risk for
cancer is influenced by the capability of a compound to form these covalent bonds. There
is a correlation between the incidence of carcinogen-DNA adducts and the formation of
carcinogenic tumors, but adducts do not predict the risk of cancer (Groopman & Skipper
1991).
74
Table C.1: Review of studies using traditional or modern methods of preparation. R
ed
uced
Safr
ole
Level
Not
det
ecte
d a
fter
boil
ing
0.3
75 t
o 0
.009 m
g
(in 1
g m
ater
ial)
Mai
nly
undet
ecte
d
to 0
.2 m
g (
in 1
g
mat
eria
l). A
lso
found i
n t
he
trad
itio
nal
Chin
ese
pre
par
atio
n n
o m
ore
than
0.0
2 m
g
0.0
3 t
o 1
.37 m
g (
in
1 g
mat
eria
l)
0.6
09 m
g (
in 1
mL
solu
tion)
Red
ucti
on
meth
od
Aqueo
us
dec
oct
ion
for
20
min
ute
s
Aqueo
us
dec
oct
ion
of
whole
see
ds
for
1, 5, an
d 3
0
min
ute
s an
d o
f
pow
der
ed s
eed f
or
1 a
nd 5
min
ute
s.
Aqueo
us
dec
oct
ion
for
1 h
ou
r
Aqueo
us
infu
sion
for
30
min
ute
s
Aqueo
us
dec
oct
ion
for
35
min
ute
s
Orig
inal
Safr
ole
Level
0.4
35 m
g (
in 1
g
solu
tion)
0.9
55 m
g (
in 1
g
mat
eria
l)
0.1
4 t
o 2
.76 m
g (
in 1
g m
ater
ial)
0.1
8 t
o 1
6 m
g (
in 1
g
mat
eria
l)
2.2
mg (
in 1
mL
solu
tion)
Extr
acti
on
meth
od
MeO
H
extr
acti
on
Ste
am
dis
till
atio
n
MeO
H
extr
acti
on
Aqueo
us
agit
atio
n
Pla
nt
Mate
ria
l
Cin
na
mom
u
m b
ark
Bla
ck p
epp
er
Wil
d g
ing
er
Sa
ssa
fra
s
alb
idum
-
roo
t b
ark
po
wd
er
Sa
ssa
fra
s
alb
idum
–
roo
t
Stu
dy
Rey
ner
tso
n e
t al
.,
20
05
Far
ag &
Ab
o-Z
eid
19
97
Ch
en e
t
al.,
200
9
Car
lso
n &
Th
om
pso
n 1
997
Cu
rren
t
Res
earc
h
20
12
75
Table C.2: Review of studies using laboratory animals and bacterial studies to test the
toxicity of safrole and derivatives.
Res
ult
s
No
liv
er t
um
ors
, so
me
sig
n o
f k
idn
ey
con
ges
tio
n.
At
24 m
on
ths
liv
er c
ance
rs
was
fo
un
d,
& c
ellu
lar
chan
ges
in
th
e
kid
ney
s, a
dre
nal
s, t
hy
roid
, p
itu
itar
y,
and
rep
rod
uct
ive
org
ans.
Req
uir
es p
rolo
ng
ed
exp
osu
re b
efo
re c
ellu
lar
chan
ges
.
Saf
role
was
neu
tral
or
wea
kly
po
siti
ve
Fo
r ra
ts,
wit
hin
24
ho
urs
88
% o
f th
e
low
est
do
se o
f 0
.63 m
g/k
g w
as
elim
inat
ed,
78%
of
the
60 m
g/k
g d
ose
was
eli
min
ated
, an
d 2
5%
was
eli
min
ated
at a
do
se o
f 7
45
mg
/kg.
Fo
r hu
man
s, t
he
do
se l
evel
s w
ere
abso
rbed
an
d
elim
inat
ed w
ith
in 2
4 h
ou
rs.
Un
able
to
det
ect
met
abo
lite
s in
man
.
Ex
amin
ed a
dif
fere
nt
met
abo
lic
pat
hw
ay
safr
ole
co
uld
un
der
go,
nam
ely
th
roug
h
ox
idat
ion
to
qu
ino
id m
etab
oli
tes
in l
iver
mic
roso
mes
in
a n
on
-en
zym
atic
pro
cess
.
Co
nc.
39
0 p
pm
fo
r 2
4
mo
nth
s an
d
11
70
pp
m f
or
22
mo
nth
s
Rat
s: O
ne
do
se,
fro
m 0
.63
to
75
0 m
g/k
g;
Hu
man
s: 0
.163
to 1
.655
mg
(1.6
55
mg
is
0.0
24
mg
/kg
fo
r
a 6
8 k
g p
erso
n)
Cm
pd
.
sou
rce
Nat
ura
l
oil
of
sass
afra
s
and
safr
ole
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Tri
al
Mo
del
Ro
den
t
In v
itro
Sal
mo
nel
la
Am
es t
est
Mal
e
Sp
rag
ue-
Daw
ley
rats
(10
0-
20
0 g
) an
d
hu
man
(5
0-
85
kg
)
Mal
e
Sp
rag
ue-
Daw
ley
rats
(1
80
-
20
0 g
)
Art
icle
Ab
bo
tt,
D. D
., E
.W.
Pac
km
an,
B.M
. W
agn
er a
nd
J.W
.E.
Har
riss
on
. C
hro
nic
ora
l
tox
icit
y o
f o
il o
f sa
ssaf
ras
and
safr
ole
. P
har
mac
olo
gis
t 3
: 6
2.
Bak
er,
R.S
.U.
and
A.M
.
Bo
nin
. T
ests
wit
h S
alm
on
ella
pla
te i
nco
rpo
rati
on
ass
ay.
In:
Ash
by a
nd
de
Ser
res
(ed
s.)
Ev
alu
atio
n o
f S
ho
rt-t
erm
Tes
ts f
or
Car
cin
og
ens.
Els
evie
r, N
.Y.
Ben
edet
ti,
M.
S.,
A.
Mal
on
e,
A.
L. B
roil
let.
Ab
sorp
tion
,
met
abo
lism
an
d e
xcr
etio
n o
f
safr
ole
in
th
e ra
t an
d m
an.
To
xic
olo
gy 7
(1):
69
-83
.
Bo
lto
n,
J.L
., e
t al
. E
vid
ence
that
4-a
lly
l-O
-qu
ino
nes
spo
nta
neo
usl
y r
earr
ang
e to
thei
r m
ore
ele
ctro
ph
ilic
qu
ino
ne
met
hid
es:
po
ten
tial
bio
acti
vat
ion
mec
han
ism
fo
r
the
hep
ato
carc
ino
gen
saf
role
.
Ch
em R
es.
To
xic
ol.
7:
44
3-
45
0.
Yea
r
19
61
19
85
19
77
19
94
76
Tes
tin
g t
he
theo
ry t
hat
saf
role
un
der
go
es i
n v
ivo
con
ver
sio
n t
o e
lect
rop
hy
llic
all
yli
c an
d b
enzy
lic
este
rs t
hat
are
th
e tr
ue
carc
ino
gen
s. 1
'-
hy
dro
xy
safr
ole
is
the
pri
mar
y m
etab
oli
te o
f
safr
ole
an
d a
pro
xim
ate
carc
ino
gen
, m
ore
so
th
an
safr
ole
.
Sh
ow
ed 1
'-h
yd
roxy
safr
ole
is
mo
re c
arci
no
gen
ic
than
saf
role
in
ad
ult
mal
e ra
ts,
alm
ost
hal
f o
f ra
ts
giv
en 1
'-h
yd
rox
ysa
fro
le d
evel
op
ed l
iver
carc
ino
mas
by
8 m
on
ths
ver
sus
rats
giv
en s
afro
le
0.0
4%
dev
elo
pin
g l
iver
car
cin
om
as b
y 1
6
mo
nth
s. T
he
rod
ents
gai
ned
wei
gh
t m
ore
slo
wly
,
bu
t un
til
they
sta
rted
dev
elo
pin
g t
um
ors
,
surv
ivo
rsh
ip w
as h
igh
.
Saf
role
tes
ted
po
siti
ve
for
Sis
ter
Ch
rom
atid
Ex
chan
ge
assa
y.
Sh
ow
ed t
hat
man
y o
f th
e co
mpo
un
ds
vo
lati
lize
d
du
rin
g t
he
incu
bat
ion
per
iod.
Tes
ted
th
e re
cov
ery
of
the
com
po
un
ds
fro
m h
ou
sefl
ies
and
mic
e.
90
mg
was
inje
cted
in
to
rats
or
30
mg
/100
g b
ody
wei
gh
t
Rat
s an
d m
ice:
fed
via
die
t
con
c. o
f 0
.5%
,
0.4
%,
or
0.3
%
safr
ole
, 0
.55
%
or
0.4
4%
1'-
hy
dro
xy
safr
ole
for
8.5
to
11
mo
nth
s
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Sta
nd
ard
Rat
s (3
00
g),
ham
ster
s,
and
gu
inea
pig
s.
CD
rat
s
(23
0-2
60
g)
and
CD
-
1 m
ice
(27
-
29
g)
In v
ivo
mam
mal
ia
n c
ell
gen
oto
xic
it
y a
ssay
Ho
use
fly
and
mal
e
alb
ino
mic
e
Bo
rch
ert,
P.,
et
al.
Th
e
met
abo
lism
of
the
nat
ura
lly
occ
urr
ing
hep
ato
carc
ino
gen
safr
ole
to
1'-
hyd
rox
ysa
fro
le
and
th
e el
ectr
op
hil
ic
reac
tiv
ity
of
1'-
acet
ox
ysa
fro
le.
Can
cer
Res
earc
h 3
3:
57
5-5
89.
Bo
rch
est,
P.,
et
al.
1'-
hy
dro
xy
safr
ole
, a
pro
xim
ate
carc
ino
gen
ic m
etab
oli
te o
f
safr
ole
in
th
e ra
t an
d m
ou
se.
Can
cer
Res
. 3
3:
590
- 60
0.
Bra
dle
y,
M.O
. M
easu
rem
ent
of
DN
A s
ing
le-s
tran
d b
reak
s
by
alk
alin
e el
uti
on
in
rat
hep
ato
cyte
s. I
n:
Ash
by a
nd
de
Ser
res
(ed
s).
Ev
alu
atio
n
of
Sho
rt-t
erm
Tes
ts f
or
Car
cin
og
ens.
Els
evie
r, N
.Y.,
35
3-3
57
.
Cas
ida,
J.
E.,
et
al.
Met
hy
len
e-C
14-d
iox
yp
hen
yl
com
pou
nd
s:
Met
abo
lism
in
rela
tio
n t
o t
hei
r sy
ner
gis
tic
acti
on
. S
cien
ce 1
53
(37
40
):
11
30
-11
33
19
73
19
73
19
85
19
66
77
Saf
role
an
d s
ix d
eriv
ativ
es w
ere
test
ed-
the
der
ivat
ives
pro
ved
to
be
mo
re c
yto
tox
ic t
o
hu
man
can
cer
cell
s th
an s
afro
le.
Rat
s w
ere
use
d t
o e
stab
lish
a l
inea
r d
ose
-
resp
on
se r
elat
ion
ship
- fo
und
th
at t
her
e w
as a
met
abo
lic
satu
rati
on
bet
wee
n 1
50 a
nd
300
mg
/kg i
n r
ats.
Th
e sa
fro
le m
etab
oli
tes
dih
yd
roxy
chav
ico
l an
d e
ug
eno
l w
ere
fou
nd
in t
he
uri
ne
of
non
-bet
el q
uid
ch
ewer
s, a
t th
e
lev
els
of
no
t d
etec
ted
to
5.4
mic
rog
ram
s/m
g
crea
tin
ine,
pro
bab
ly d
ue
to s
pic
es i
n t
he
die
t.
Liv
er w
eig
ht
incr
ease
d t
o 2
0%
aft
er w
eek
1.
Liv
er w
eig
ht
con
tinu
ed t
o i
ncr
ease
to
60
%
mo
re t
han
th
e co
ntr
ol
gro
up
by w
eek
8 w
ith
a d
ecre
ase
in d
rug
met
abo
lizi
ng
en
zym
e
acti
vit
y.
Cy
toch
rom
e P
-450
was
in
itia
lly
80
% h
igh
er,
bu
t lo
wer
ed t
o 2
0-3
0%
hig
her
afte
r 1
6 w
eek
s. N
oti
ced
en
larg
ed
cen
tril
ob
ula
r h
epat
ocy
tes,
in
div
idu
al c
ell
nec
rosi
s, a
nd
nec
rosi
s o
f h
epat
ocy
tes.
Use
d 3
2P
-po
stla
bel
ing
ass
ay t
hat
saf
role
form
s D
NA
add
uct
s, i
n t
urn
in
du
ces
sist
er
chro
mat
id e
xch
ang
es a
nd
ch
rom
oso
mal
aber
rati
on
s as
say
s.
Rat
s: 0
, 3
0, 75
,
15
0,
30
0 m
g/k
g.
Tes
ted
uri
ne
of
rats
an
d h
um
an
wh
o c
hew
ed
qu
id b
etw
een
2
- 1
00 t
imes
a
day
Die
t co
nta
inin
g
0.2
5%
(w
/w)
safr
ole
fo
r 8
5
wee
ks,
10
%
mo
re s
afro
le
was
in
itia
lly
add
ed d
ue
to
vo
lati
lity
0.0
25
to
0.2
mg
/ml
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Bre
ast
and
colo
rect
al
can
cer
cell
lin
es
Ad
ult
mal
e
Wis
tar
rats
an
d
hu
man
uri
ne
Fem
ale
Wis
tar
alb
ino
rat
s
(80
-100
g)
Ch
ines
e
ham
ster
lun
g c
ells
Cat
alán
, L
. E
., e
t al
. S
yn
thes
is o
f
Nin
e S
afro
le D
eriv
ativ
es a
nd
Th
eir
An
tip
roli
fera
tiv
e A
ctiv
ity
To
war
ds
Hu
man
Can
cer
Cel
ls.
Jou
rnal
of
the
Ch
ilea
n C
hem
ical
So
ciet
y 5
5:
2, 2
19
-222
.
Ch
ang,
M.J
.W., C
.Y. K
o, R
.F.
Lin
& L
.L.
Hsi
eh.
Bio
log
ical
mo
nit
ori
ng
of
env
iro
nm
ent
exp
osu
re t
o s
afro
le a
nd
th
e
Tai
wan
ese
bet
el q
uid
ch
ewin
g.
Arc
h.
Env
iro
n.
Co
nta
m. T
ox
ico
l.
43
: 43
2–
43
7.
Cra
mp
ton
, R
.F.,
et
al.
Lo
ng
-ter
m
stu
die
s o
n c
hem
ical
ly i
nd
uce
d
liv
er e
nla
rgem
ent
in t
he
rat.
II.
Tra
nsi
ent
ind
uct
ion
of
mic
roso
mal
en
zym
es l
ead
ing
to
liv
er d
amag
e an
d
no
du
larh
yp
erp
lasi
a p
rod
uce
d b
y
safr
ole
an
d P
on
ceau
MX
.
To
xic
olo
gy 7
: 3
07
-326
.
Dai
mo
n H
., S
. S
awad
a, S
.
Asa
ku
ra &
F. S
agam
i. A
nal
ysi
s
of
cyto
gen
etic
eff
ects
an
d D
NA
add
uct
fo
rmat
ion
ind
uce
d b
y
safr
ole
in
Ch
ines
e h
amst
er l
un
g
cell
s. T
erat
og
enes
is
Car
cin
og
enes
is M
uta
gen
esis
17
(1):
7-1
8.
20
10
20
02
19
77
19
97
78
No
mo
rtal
ity
. T
he
sin
gle
do
ses
did
no
t in
du
ce
chro
mo
som
e ab
erra
tio
n,
bu
t th
e re
pea
ted
do
ses
incr
ease
d a
ber
ran
t ce
lls
in t
he
liv
er.
Th
e
sin
gle
do
ses
at 1
00
, 2
50
and
500
mg
/kg
an
d
the
rep
eate
d d
ose
s in
du
ced
sis
ter
chro
mat
id
exch
ang
es.
DN
A a
ddu
cts
wer
e fo
un
d i
n t
he
sin
gle
do
ses
of
10 (
2 a
dd
uct
s) a
nd
10
0,
25
0,
& 5
00
(4 a
ddu
cts)
mg
/kg
wh
ile
no a
ddu
cts
wer
e o
bse
rved
in
1 m
g/k
g
Pu
rpo
se o
f th
is s
tud
y w
as t
o d
isco
ver
th
e
acti
vat
ion
or
enh
ance
men
t o
f sa
fro
le t
hat
tes
ts
po
siti
ves
fo
r th
e A
mes
tes
t. S
afro
le t
este
d
po
siti
ve
usi
ng
str
ain
TA
153
5, ac
tiv
ated
by
rat
liv
er i
nje
cted
wit
h s
afro
le i
n v
ivo
. P
rev
iou
s
stu
die
s te
sted
saf
role
wit
ho
ut
bei
ng
mod
ifie
d
and
sh
ow
ed s
afro
le t
o b
e n
egat
ive
in a
n A
mes
test
.
Th
e 1
'-h
yd
roxy
met
abo
lite
s o
f b
oth
est
rag
ole
and
saf
role
wer
e fo
un
d t
o b
e m
ore
carc
ino
gen
ic t
han
th
e p
aren
t co
mp
ou
nd
s. 5
9%
of
the
mic
e g
iven
1'-
hyd
roxy
safr
ole
dev
elo
ped
liv
er t
um
ors
.
Fiv
e d
ose
s o
f
62
.5, 1
25
or
25
0 m
g/k
g,
sin
gle
do
ses
of
1,
10
, 1
00
, 2
50
,
or
500
mg
/kg
via
sto
mac
h
tub
e
42
mg
/rat
/day
via
i.p
.
inje
ctio
n f
or
thre
e d
ays
New
bo
rn m
ice:
tota
l o
f 4
.43
mic
rom
ole
s; 9
-
12
wee
k o
ld
mic
e: t
ota
l o
f
5.1
9
mic
rom
ole
s,
via
se
inje
ctio
n
Saf
role
Sta
nd
ard
Saf
role
stan
dar
d,
syn
thes
iz
ed t
o 1
'-
hy
dro
xy
safr
ole
Rat
(F3
44
)
mo
del
Ad
ult
rats
CD
-1
mic
e,
new
bo
rn
(16
g)
and
9-1
2
wee
k o
ld
(35
g)
Dai
mo
n, H
., S
. S
awad
a, S
.
Asa
ku
ra,
and
F.
Sag
ani.
In
viv
o
gen
oto
xic
ity
an
d D
NA
add
uct
lev
els
in t
he
liv
er o
f ra
ts t
reat
ed
wit
h s
afro
le.
Car
cin
og
enes
is 1
9:
14
1-1
46
.
Do
ran
ge,
J. L
. et
al.
Co
mp
arat
ive
surv
ey o
f m
icro
som
al a
ctiv
atio
n
syst
ems
for
mu
tag
enic
stu
die
s o
f
safr
ole
, M
uta
tio
n R
esea
rch
5
3:
17
9-1
80
.
Dri
nk
wat
er,
N.
R. et
al.
Hep
ato
carc
ino
gen
icit
y o
f es
trag
ole
(1 a
lly
l-4
-met
ho
xyb
enze
ne)
an
d
1'-
hyd
roxy
estr
ago
le i
n t
he
mo
use
and
mu
tag
enic
ity
of
1'-
acet
ox
yes
trag
ole
in
bac
teri
a. J
.
Nat
l. C
ance
r In
st.
57
: 1
32
3-1
331
.
19
98
19
78
19
76
79
S
afro
le r
eact
s to
fo
rm a
saf
role
met
abo
lite
- cy
toch
rom
e P
-45
0
com
ple
x.
Do
min
ant
leth
al a
ssay
: sa
fro
le t
este
d
neg
ativ
e
Th
e b
ile
and
uri
ne
sam
ple
s sh
ow
ed
safr
ole
met
abo
lize
d i
nto
iso
safr
ole
and
dih
yd
rosa
fro
le.
Th
e sa
fro
le a
nd
met
abo
lite
s w
ere
slo
wly
eli
min
ated
in
the
bil
e.
2%
(w
/w)
via
die
t fo
r tw
o
wee
ks.
0.0
4 m
l v
ia
intr
aven
ou
s
inje
ctio
n
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Mal
e
Sp
rag
ue-
Daw
ley
rats
(2
00
g)
Sw
iss
mic
e 8
-10
wee
ks
old
Sp
rag
ue-
Daw
ley
mic
e (3
50
g)
Elc
om
be,
C.
R. et
al.
Stu
die
s o
n
the
inte
ract
ion
of
safr
ole
wit
h r
at
hep
atic
mic
roso
mes
. B
ioch
em.
Ph
arm
aco
l. 2
4:
142
7-1
433
.
Ep
stei
n,
S.S
., e
t al
. D
etec
tio
n o
f
chem
ical
mu
tag
ens
by
th
e
do
min
ant
leth
al a
ssay
in
th
e
mo
use
. T
ox
ico
log
y a
nd
App
lied
Ph
arm
aco
log
y 2
3:
28
8-
32
5.
Fis
hb
ein
, L
. et
al.
Th
in-l
ayer
chro
mat
og
rap
hy
of
rat
bil
e an
d
uri
ne
foll
ow
ing i
ntr
aven
ou
s
adm
inis
trat
ion
of
safr
ole
,
iso
safr
ole
, an
d d
ihy
dro
safr
ole
. J.
Ch
rom
atog
. 2
9:
26
7-2
73
.
19
75
19
72
19
67
80
Th
e sa
fro
le h
ad n
o e
ffec
t o
n
mic
roso
mal
en
zym
e ac
tiv
ity
.
Saf
role
tes
ted
neg
ativ
e
Saf
role
was
neu
tral
or
wea
kly
po
siti
ve
Ex
amin
ed D
NA
usi
ng
32P
-po
stla
bel
ing
assa
y a
t 0
.5, 1
, 2
, 3
, 7
, 1
5,
and
30
day
s.
Sh
ow
ed a
lin
ear
resp
on
se o
f sa
fro
le-
DN
A a
dd
uct
s, r
each
ing
a t
hre
sho
ld a
t
the
10
mg
do
se,
wh
ere
the
tiss
ue
cou
ld
be
satu
rate
d a
nd
th
e el
imin
atio
n o
f th
e
com
pou
nd
wo
uld
occ
ur
mo
re s
low
ly.
10
mg
/kg
on
e
tim
e d
ose
of
safr
ole
via
i.p
.
inje
ctio
n
On
e do
se o
f
0.0
01
, 0
.01,
0.1
,
1.0
, an
d 1
0.0
mg
/mou
se o
r
0.0
4, 0
.4,
4,
40
,
or
400
mg
/kg
bw
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Mal
e
Sw
iss
Alb
ino
mic
e (2
0-
25
g)
Bo
ne-
mar
row
mic
ron
ucl
eus
assa
y
In v
itro
Sal
mo
nel
l
a re
ver
se
mu
tati
on
assa
y
(Am
es
test
)
Fem
ale
CD
-1
mic
e (2
5
g)
Fri
edm
an,
M. A
., E
. A
rno
ld,
Y.
Bis
ho
p, an
d S
.S.
Ep
stei
n.
Ad
dit
ive
and s
yn
erg
isti
c
inh
ibit
ion
of
mam
mal
ian
mic
roso
mal
en
zym
e fu
nct
ion
s
by
pip
ero
ny
l bu
tox
ide,
saf
role
and
oth
er m
eth
yle
ned
ioxy
ph
eny
l
der
ivat
ives
. E
xp
erie
nti
a 2
7:
10
52
-10
54
.
Go
cke,
E.,
M.T
. K
ing,
K.
Eck
ard
t, a
nd
D.W
ild
.
Mu
tag
enic
ity
of
cosm
etic
s
ing
red
ien
ts l
icen
sed
by
th
e
Eu
rop
ean
Com
mun
ity
. M
uta
t.
Res
. 9
0:
91
-10
9.
Gre
en,
N.R
. an
d S
avag
e, J
.R.,.
Scr
een
ing
of
safr
ole
, eu
gen
ol,
thei
r n
inh
yd
rin
po
siti
ve
met
abo
lite
s an
d s
elec
ted
seco
nd
ary
am
ines
fo
r p
ote
nti
al
mu
tag
enic
ity
. M
uta
tio
n
Res
earc
h,
57
, 1
15
- 12
1.
Gu
pta
, K
.P., K
.L. v
an G
ole
n,
K.L
. P
utm
an &
K.
Ran
der
ath
.
Fo
rmat
ion
and
per
sist
ence
of
safr
ole
-DN
A a
dd
uct
s o
ver
a
10
,000
-fo
ld d
ose
ran
ge
in m
ou
se
liv
er.
Car
cin
og
enes
is 1
4 (
8):
15
17
–1
52
1.
19
71
19
81
19
78
19
93
81
T
he
rats
fee
d 1
0,0
00
pp
m d
ied
aft
er
62
wee
ks,
and
th
e re
st e
xp
erie
nce
d g
row
th r
etar
dat
ion
,
incr
ease
d m
ort
alit
y i
n t
he
mal
es (
at 5
00
0pp
m),
liv
er e
nla
rgem
ent
wit
h t
um
or
mas
ses
and
no
du
les,
mil
d h
yp
erp
lasi
a o
f th
yro
id, an
d
incr
ease
of
chro
nic
nep
hri
tis
in t
he
kid
ney
. T
he
do
gs
exp
erie
nce
d l
iver
en
larg
emen
t an
d n
odu
les
at t
he
20
mg
/kg d
ose
an
d l
iver
dam
age
by
fo
cal
nec
rosi
s, b
ile-d
uct
pro
life
rati
on
, fa
tty
met
amo
rph
osi
s, a
nd
oth
er c
han
ges
at
the
5
mg
/kg d
ose
.
At
750
mg
/kg,
9 o
ut
of
10
rat
s d
ied
at
19 d
ays,
at 5
00
mg
/kg
, 1
of
10 r
ats
die
d a
fter
46
day
s,
and
at
250
mg
/kg
, n
o r
ats
die
d b
ut
they
exp
erie
nce
d l
iver
en
larg
emen
t an
d f
oca
l
nec
rosi
s. N
o e
vid
ence
of
tox
icit
y f
or
iso
safr
ole
.
Dih
yd
rosa
fro
le w
as f
ou
nd
to
be
a es
op
hag
eal
carc
ino
gen
. M
ice
sho
wed
sim
ilar
liv
er c
han
ges
as f
ou
nd
in
th
e ra
ts.
Th
e ra
ts r
ecei
vin
g h
igh p
rote
in d
iets
(3
0%
pro
tein
), t
he
liv
er a
pp
eare
d n
orm
al c
om
par
ed t
o
the
con
tro
l w
hil
e o
ther
die
ts w
ith
lo
wer
pro
tein
had
sm
alle
r li
ver
s w
hen
fed
wit
h s
afro
le.
Th
e
safr
ole
sp
iked
die
ts r
esu
lted
in
liv
ers
wit
h n
o f
at
pre
sen
t. R
ats
giv
en s
afro
le l
ived
lo
ng
er t
han
th
e
con
tro
l, b
ut
ther
efo
re h
ad a
hig
her
lev
el o
f
hep
ato
mas
.
Rat
s, a
du
lt:
10
00
, 2
50
0,
50
00
, an
d
10
,000
pp
m
for
two
yea
rs;
Do
gs
(2M
,
2F
): 5
and
20
mg
/kg f
or
6
yea
rs
Rat
s, a
du
lt:
25
0,
50
0,
75
0
mg
/kg
/day
for
105
day
s;
Mic
e: 2
50
and
50
0
mg
/kg f
or
60
day
s
0.5
% s
afro
le
via
die
t fo
r
fiv
e d
iets
wit
h v
ary
ing
lev
els
of
pro
tein
, fa
t,
carb
oh
yd
rate
s, a
nd
sal
ts.
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Osb
ou
rne-
Men
del
rats
an
d
pu
re-
bre
ed
bea
gle
do
gs
Osb
ou
rne-
Men
del
rats
an
d
Sw
iss
mic
e
Osb
ou
rne-
Men
del
mal
e ra
ts
(11
4 g
)
Hag
an,
E.C
., W
.H.
Han
sen
, O
.G.
Fit
zhug
h,
P.M
. Je
nn
er,
W.I
. Jo
nes
,
J.M
, T
aylo
r, E
.L.
Lo
ng
,
A.A
. N
elso
n,
and
J.B
.
Bro
uw
er.
Fo
od
flav
ou
rin
gs
and
com
pou
nd
s o
f re
late
d
stru
ctu
re.
II.
Su
bac
ute
an
d
chro
nic
to
xic
ity
. F
ood
Co
sm. T
ox
ico
l. 5
: 1
41
-
15
7.
Hag
an,
E.C
., P
. M
. Je
nn
er,
W.
I. J
on
es,
O.
G.
Fit
zhu
gh,
E.
L. L
ong
, J.
G.
Bro
uw
er,
W. W
elfa
re.
To
xic
pro
per
ties
of
com
pou
nd
s re
late
d t
o
safr
ole
. T
ox
ico
log
y a
nd
Ap
pli
ed P
har
mac
olo
gy
7(1
): 1
8-2
4.
Ho
mbu
rger
, F
., P
.D.
Bo
gd
on
off
and
T.F
.
Kel
ley
. In
flu
ence
of
die
t
on
ch
ron
ic o
ral
tox
icit
y o
f
safr
ole
an
d b
utt
er y
ello
w
in r
ats.
P
roce
edin
gs
of
the
So
ciet
y f
or
Exp
erim
enta
l
Bio
logy
and
Med
icin
e 1
19
(4):
11
06
-11
10
.
19
67
19
65
19
65
82
S
afro
le t
este
d p
osi
tiv
e fo
r
gen
oto
xic
ity
act
ivit
y,
at a
hig
her
do
se
than
pre
vio
us
stu
die
s (P
rob
st e
t al
.
19
81
) th
at t
este
d s
mal
l d
ose
s.
Bo
th i
nse
cts
sho
wed
su
scep
tib
ilit
y t
o
the
fum
igan
t to
xic
ity
of
safr
ole
. T
her
e
was
sli
gh
t fe
edin
g d
eter
ren
ce a
gai
nst
S.
zea
ma
is d
ue
to r
edu
ced
fo
od
con
sum
pti
on
and
no
det
erre
nce
fo
r T
.
cast
an
eum
.
Saf
role
did
no
t ef
fect
gro
wth
of
A.
act
ino
myc
etem
com
ita
ns
or
S. m
uta
ns,
bu
t d
id d
ecre
ase
the
gro
wth
of
E. co
li.
Saf
role
red
uce
s th
e re
leas
e o
f
sup
ero
xid
e an
ion
, b
ut
no
t th
roug
h a
dir
ect
kil
lin
g o
f n
eutr
oph
ils,
or
a h
ost
def
ense
mec
han
ism
. S
afro
le r
edu
ced
anti
mic
rob
ial
acti
vit
y b
ut
did
no
t
dem
on
stra
te c
yto
tox
icit
y.
10
^-3
M s
afro
le
for
a
un
sch
edu
led
DN
A s
yn
thes
is
assa
ys
in
cult
ure
d r
at
hep
ato
cyte
s.
Fu
mig
ant
stud
y
at 0
.9 m
g/c
m^3
and
co
nta
ct
tox
icit
y s
tud
y a
t
2.0
4-1
6.2
mg
/g
of
food
.
5 m
M a
nd
10
mM
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Mal
e F
isch
er
34
4 r
ats.
Sit
op
hil
us
zea
ma
is a
du
lts
and
Tri
bo
liu
m
cast
an
eum
adu
lts
and
larv
ae
Ora
l
pat
ho
gen
s
incl
ud
ing
Act
ino
ba
cill
us
act
ino
myc
etem
com
ita
ns,
Str
epto
cocc
us
mu
tan
s,
and
Po
ryp
hyr
om
on
as
gin
giv
ali
s
Ho
wes
, J.
A., V
.S.W
. C
han
,
and
J.
Cal
dw
ell.
Str
uct
ure
-
spec
ific
ity
of
the
gen
oto
xic
ity
of
som
e n
atu
rall
y o
ccu
rrin
g
alk
eny
l-b
enze
nes
det
erm
ined
by
th
e u
nsc
hed
ule
d D
NA
syn
thes
is a
ssay
s in
rat
hep
ato
cyte
s. F
ood
Ch
em.
To
xic
ol.
28
: 5
37
-54
2.
Hu
ang,
Y. H
o S
H,
Kin
i R
M.
Bio
acti
vit
ies
of
safr
ole
an
d
iso
safr
ole
on
Sit
op
hil
us
zea
ma
is (
Co
leo
pte
ra:
Cu
rcu
lio
nid
ae)
and
Tri
bo
liu
m
cast
an
eum
(C
ole
op
tera
:
Ten
ebri
on
idae
). J
ou
rnal
of
Eco
nom
ic E
nto
mo
log
y 9
2:
67
6-6
83
.
Hu
ng
, S
.-L
., Y
.-L
. C
hen
, an
d
Y.-
T.
Ch
en. E
ffec
ts o
f sa
fro
le
on
th
e d
efen
siv
e fu
nct
ion
s o
f
hu
man
neu
trop
hil
s. J
ou
rnal
of
Per
iod
on
tal
Res
earc
h 3
8:
130
–
13
4.
19
90
19
99
20
03
83
Liv
er-c
ell
tum
ors
fo
und
in
14 o
f
34
mal
es a
nd
32
of
33
fem
ales
.
Saf
role
tes
ted
po
siti
ve
Est
abli
shed
acu
te t
ox
icit
y a
t
thes
e co
nce
ntr
atio
ns.
Hy
bri
d m
ice,
7
day
s o
ld:
464
mg
/kg o
ver
21
day
s v
ia
sto
mac
h t
ub
e,
then
12
65
mg
/kg o
ver
82
wee
ks
via
die
t
19
50
mg
/kg
bw
for
rats
an
d
23
50
mg
/kg
fo
r
mic
e
Saf
role
stan
dar
d
Hy
bri
d m
ice
fro
m C
57
BL
/6
(fem
ale)
an
d
C3
H/A
nf
or
AK
R (
mal
e)
stra
ins
In v
ivo
mam
mal
ian
cell
gen
oto
xic
ity
assa
y:
chro
mo
som
al
aber
rati
on
s
Rat
an
d m
ice
Inn
es,
J.R
., B
.M. U
llan
d,
M.G
. V
aler
io,
L.
Pet
ruce
lli,
L.
Fis
hb
ein
, E
.R.
Has
t,
A.J
. P
allo
ta,
R.R
. B
ates
, H
.L. F
alk
, L
.L.
Gar
t, M
. K
lein
, I.
Mit
chel
l, a
nd
J.
Pet
er.
Bio
assa
y o
f p
esti
cid
es a
nd
in
du
stri
al
chem
ical
s fo
r tu
mo
rig
enic
ity
in
mic
e: a
pre
lim
inar
y n
ote
. J.
Nat
l. C
ance
r In
st.
42
: 11
01
-11
14
.
Ish
idat
e, M
. an
d T
. S
ofu
ni.
Th
e in
vit
ro
chro
mo
som
al a
ber
rati
on
tes
t u
sin
g
Ch
ines
e h
amst
er l
un
g (
CH
L)
fib
rob
last
cell
s in
cu
ltu
re.
In:
Ash
by a
nd
de
Ser
res
(ed
s).
Ev
alu
atio
n o
f S
ho
rt-t
erm
Tes
ts
for
carc
ino
gen
s. E
lsev
ier,
N.Y
.: 4
27
-
43
2.
Jen
ner
, P
.M.,
E.C
. H
agan
, J.
M. T
aylo
r,
E.L
. C
oo
k,
and O
.G.
Fit
zhug
h.
Fo
od
flav
ou
rin
gs
and c
om
po
un
ds
of
rela
ted
stru
ctu
res.
I.
Acu
te O
ral
To
xic
ity
. F
oo
d
Co
sm. T
ox
ico
l. 2
: 3
27
-343
.
19
69
19
85
19
64
84
Fo
un
d t
he
form
atio
n o
f 1
'-hy
dro
xy
safr
ole
in t
he
hu
man
liv
er m
icro
som
es.
Var
iou
s
hu
man
cy
toch
rom
e P
45
0 e
nzy
mes
met
abo
lize
saf
role
, in
clu
din
g e
nzy
mes
P4
50
2C
9*
1,
P4
50
2A
6,
P4
50
2D
6*
1,
and
P4
50
2E
1.
Bo
th d
ose
s ex
per
ien
ced
dec
reas
ed b
od
y
wei
gh
t g
ain
, li
ver
en
larg
emen
t in
th
e m
ales
and
fem
ales
(0
.5%
gro
up
on
ly),
an
d s
ing
le
cell
nec
rosi
s. M
ale
rats
ex
per
ien
ced
tub
ula
r h
yal
ine
dro
ple
ts,
tub
ula
r
reg
ener
atio
n,
gra
nu
lar
cast
, p
elv
ic
calc
ific
atio
n,
and i
nte
rsti
tial
cel
l
infi
ltra
tio
n i
n t
he
kid
ney
s.
Th
e m
ajo
r m
etab
oli
c p
ath
way
fo
r sa
fro
le i
s
clea
vag
e o
f th
e m
eth
yle
ned
iox
yp
hen
yl
mo
iety
as
wel
l as
ex
pir
atio
n o
f ca
rbo
n
dio
xid
e fr
om
th
e m
eth
yle
ne
carb
on
.
Met
abo
lism
of
safr
ole
, (w
hic
h i
s v
ola
tile
)
resu
lted
in
les
s v
ola
tile
, m
ore
po
lar,
an
d
eth
er-s
olu
ble
met
abo
lite
s. F
ou
nd
th
e
met
abo
lite
s o
f sa
fro
le w
ere
mo
re v
ola
tile
.
Th
e sa
fro
le-f
ree
sass
afra
s ex
trac
t p
rod
uce
d
loca
l tu
mo
rs i
n 6
6%
of
the
rats
, th
ou
gh
stu
dy
do
es n
ot
ind
icat
ed i
f th
e sa
mp
les
wer
e te
sted
fo
r sa
fro
le l
evel
s p
rio
r to
inje
ctio
n.
Rat
s: 6
9.1
(0.1
% i
n
die
t) a
nd
27
5.6
(0.5
%)
mg
/kg
/day
for
13
wee
ks
Mic
e: 5
mic
rom
ole
s/k
g b
w;
rats
&
ham
ster
s:
10
mic
rom
ole
s/k
g b
w
Rat
s: 1
5
mg
via
s.c
.
inje
ctio
n
for
78
wee
ks
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Sas
safr
as r
oo
t
bar
k,
extr
acte
d w
ith
pet
role
um
eth
er,
met
hy
len
e
chlo
rid
e, a
nd
eth
ano
l
Hu
man
cyto
chro
me
P4
50
en
zym
es
and
mic
roso
mes
fro
m l
iver
cell
s li
nes
Mal
e an
d
Fem
ale
F3
44
gp
t d
elta
rat
s
Mal
e S
wis
s-
Web
ster
mic
e
(18
-20 g
),
mal
e S
pra
gu
e-
Daw
ley
rat
s
(15
0-1
70 g
),
mal
e h
amst
er
(18
0-2
00 g
)
NIH
Bla
ck
rats
, 1
-2
mo
nth
old
Jeu
riss
en,
S.
M. F
. et
al.
Hum
an
Cy
toch
rom
e P
45
0 E
nzy
me
Sp
ecif
icit
y f
or
Bio
acti
vat
ion
of
Saf
role
to
th
e P
rox
imat
e
Car
cin
og
en 1
-Hy
dro
xy
safr
ole
.
Ch
emic
al R
esea
rch
in
To
xic
olo
gy.
17
(9
): 1
24
5-1
250
.
Jin
, M
., A
. K
ijm
a, Y
. S
uzu
ki,
D.
Hib
i, T
. In
ou
e, Y
. Is
hii
, T
.
No
hm
i, A
. N
ish
ikaw
a, K
.
Og
awa,
T. U
men
ura
.
Co
mp
reh
ensi
ve
tox
icit
y s
tud
y o
f
safr
ole
usi
ng
a m
ediu
m-t
erm
anim
al m
od
el w
ith
gp
t d
elta
rat
s.
To
xic
olo
gy 2
90
: 3
12
-321
.
Kam
ien
ski,
F.X
. an
d J
.E.
Cas
ida.
Im
po
rtan
ce o
f
dem
eth
yle
nat
ion
in
th
e
met
abo
lism
in
viv
o a
nd
in
vit
ro
of
met
hy
len
e-d
ioxy
ph
eny
l
syn
erg
ists
an
d r
elat
ed
com
pou
nd
s in
mam
mal
s.
Bio
chem
ical
Ph
arm
aco
lolg
y 1
9:
91
-11
2.
Kap
adia
, G
. J.
, E
. B
. C
hun
g,
B.
Gh
osh
, Y
. N
. S
hu
kla
, S
. P
.
Bas
ak,
J. F
. M
ort
on
, S
. N
.
Pra
dh
an. C
arci
no
gen
icit
y o
f
som
e fo
lk m
edic
inal
her
bs
in
rats
. Jo
urn
al o
f N
atio
nal
Can
cer
Inst
itu
te 6
0 (
3):
683
-686
.
20
04
20
11
19
70
19
78
85
B.
sub
tili
s w
as n
ot
inh
ibit
ed b
y
safr
ole
or
safr
ole
ep
ox
ide,
bu
t it
was
inh
ibit
ed b
y s
afro
le h
yd
rop
ero
xid
e.
S.
au
reu
s w
as a
lso
no
t in
hib
ited
by
safr
ole
, b
ut
it w
as b
y s
afro
le e
po
xid
e
and
saf
role
hy
dro
per
ox
ide.
E.
coli
was
in
hib
ited
by a
ll t
hre
e
com
pou
nd
s.
Th
e ph
oto
syn
thes
ized
co
mp
ou
nd
s
der
ived
fro
m s
afro
le i
nh
ibit
ed
gro
wth
of
Ca
nd
ida a
lbic
an
s.
Saf
role
was
eff
ecti
ve
agai
nst
th
e ri
ce
wee
vil
via
fu
mig
atio
n,
lik
ely
th
rou
gh
vap
or
acti
on
th
rou
gh
th
e re
spir
ato
ry
syst
em.
Fo
un
d t
hat
th
e es
sen
tial
oil
dir
ectl
y f
rom
th
e p
lan
t w
as m
ore
effe
ctiv
e th
an p
uri
fied
co
mp
oun
ds,
as t
he
com
po
un
ds
in t
he
esse
nti
al o
il
fro
m t
he
pla
nt
cou
ld b
e ac
tin
g
syn
erg
isti
call
y.
25
mic
rog
ram
/m
L o
f es
sen
tial
oil
on
fil
ter
pap
er f
or
the
fum
igan
t
bio
assa
y
Saf
role
stan
dar
d
Saf
role
stan
dar
d,
ph
oto
syn
th
esiz
ed t
o
com
pou
nd
Asi
asa
rum
sieb
old
i
esse
nti
al
oil
Gra
m-p
osi
tiv
e
bac
teri
a B
aci
llu
s
sub
till
is,
Sta
ph
ylo
cocc
us
au
reu
s, a
nd
gra
m-n
egat
ive
bac
teri
a E
. co
li
Ca
nd
ida
alb
ica
ns,
fun
gal
pat
ho
gen
Fu
mig
atio
n
bio
assa
y a
gai
nst
the
rice
wee
vil
Sit
op
hil
us
ory
zae
Kh
ayy
at, S
. A
an
d S
. H
. A
l-Z
ahra
ni.
Th
erm
al,
pho
tosy
nth
esis
an
d
anti
bac
teri
al s
tud
ies
of
bio
acti
ve
safr
ole
der
ivat
ive
as p
recu
rso
r fo
r
nat
ura
l fl
avo
r an
d f
rag
ran
ce.
Ara
bia
n J
ou
rnal
of
Ch
emis
try
,
do
i:1
0.1
016
/j.a
rab
jc.2
01
1.0
9.0
14.
Kh
ayy
at, S
. A
. P
ho
tosy
nth
esis
of
dim
eric
cin
nam
ald
ehy
de,
eu
gen
ol,
and
saf
role
as
anti
mic
rob
ial
agen
ts.
Jou
rnal
of
Sau
di
Ch
emic
al S
oci
ety
,
do
i: 1
0.1
016
/j.j
scs.
20
11.0
7.0
14.
Kim
, J.
an
d I
-K.
Par
k. F
um
igan
t
tox
icit
y o
f K
ore
an m
edic
inal
pla
nt
esse
nti
al o
ils
and
co
mpo
nen
ts f
rom
Asi
asa
rum
sie
bo
ldi
roo
t ag
ain
st
Sit
op
hil
us
ory
zae
L.
Fla
vou
r an
d
Fra
gra
nce
Jo
urn
al 2
3:
79
-83.
20
11
20
11
20
08
86
Ind
uct
ion
of
ben
zopy
ren
e h
yd
roxy
lase
, w
ith
max
imu
m e
nzy
me
acti
vit
y w
ith
in 1
4 d
ays.
Incr
ease
s w
her
e o
bse
rved
in
hep
atic
bip
hen
yl
2-
hy
dro
xy
lase
an
d 4
-hyd
rox
yla
se a
ctiv
ity
,
mic
roso
mal
pro
tein
, li
ver
wei
gh
t, c
yto
chro
me
P-4
50
co
nte
nt.
Hep
atic
an
ilin
e 4
-hy
dro
xy
lase
acti
vit
y w
as i
nh
ibit
ed.
Saf
role
in
du
ces
a d
ose
-dep
end
ent
incr
ease
of
hep
atic
lip
id h
yd
rop
ero
xid
es a
nd
8-h
yd
roxy
-2'-
deo
xy
gu
ano
sin
e. T
he
hig
her
th
e do
se,
the
earl
ier
the
lip
id h
yd
rop
erod
ixe
lev
els
reac
hed
max
imu
m. O
xid
ativ
e d
amag
e is
rep
aire
d w
ith
in
15
day
s. V
itam
in E
pre
ven
ted
lip
id p
ero
xid
atio
n
bu
t no
t 8
-hyd
roxy
-2'-
deo
xyg
uan
osi
ne
form
atio
n.
Th
e h
igh
est
do
se -
red
uce
d b
od
y w
eig
ht
gai
n,
mil
d a
nem
ia,
and
leu
ko
cyto
sis.
Liv
er i
nju
ry -
mo
der
ate
to s
ever
e at
th
e h
igh
est
do
se,
to v
ery
slig
ht
at t
he
10
0 m
g/k
g d
ose
. M
agli
gn
ant
and
ben
ign t
um
ors
at
hig
hes
t d
ose
, d
ecre
asin
g
amo
un
ts o
f b
enig
n t
um
ors
.
An
imal
s w
ere
pre
trea
ted
wit
h s
afro
le a
nd
iso
safr
ole
bef
ore
in
ject
ed w
ith
2-
acet
amid
ofl
uo
ren
e in
crea
sed
th
e ac
tiv
ity
of
liv
er m
icro
som
al m
ater
ial
for
hy
dro
xy
lati
on
of
2-a
ceta
mid
ofl
uo
ren
e, w
hic
h c
an i
nh
ibit
liv
er
carc
ino
gen
esis
.
Die
t o
f 0
.25
%
iso
safr
ole
fo
r 1
4
day
s
Rat
s: s
ing
le 0
,
25
0,
50
0,1
000
mg
/kg v
ia i
p
inje
ctio
n,
wit
h
on
e g
roup
giv
en
30
0 m
g/k
g
Vit
amin
E
Rat
s, a
du
lt:
10
0,
50
0,
10
00
, 2
00
0
mg
/kg o
ver
tw
o
yea
rs,
or
5,
25
,
50
, an
d 1
00
mg
/kg
/day
.
Rat
s, i
n v
ivo
stu
dy
: 10
0
mg
/kg b
w,
In
vit
ro s
tud
y:
100
mg
/kg b
w,
and
30
0 m
g/k
g b
w;
Ham
ster
s: 2
00
mg
/kg b
w
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Mal
e W
ista
r
alb
ino
rat
s
Mal
e
Sp
rag
ue-
Daw
ley
rat
s
(20
0 g
)
Osb
orn
e-
Men
del
rat
s
Mal
e al
bin
o
Wis
tar
rats
(60
-120
g)
and
Sy
rian
go
lden
ham
ster
s
(10
0 g
)
Lak
e, B
. G
. &
D. V
. P
ark
e.
Ind
uct
ion
of
ary
l
hy
dro
carb
on
hyd
roxy
lase
in
var
iou
s ti
ssu
es o
f th
e ra
t b
y
met
hy
len
edio
xy
ph
eny
l
com
pou
nd
s. B
ioch
em.
J.
13
0:
86.
Liu
, T
. Y
., C
. C
. C
hen
, C
. L
.
Ch
en,
and
C.
W. C
hi.
Saf
role
-in
du
ced
Ox
idat
ive
Dam
age
in t
he
Liv
er o
f
Sp
rag
ue-
Daw
ley
Rat
s. F
oo
d
and
Ch
emic
al T
ox
ico
logy
37
: 69
7-7
02
.
Lo
ng
, E
.L.,
A.A
. N
elso
n,
O.G
. F
itzh
ugh
and
W.H
.
Han
sen
. L
iver
tu
mo
urs
pro
du
ced
in
rat
s b
y f
eed
ing
safr
ole
. A
rch
ives
of
Pat
ho
logy
75
: 5
95
-604
.
Lo
tlik
ar,
P. D
. &
Was
serm
an,
M.
B. E
ffec
ts
of
safr
ole
an
d i
sosa
fro
le
pre
trea
tmen
t o
n N
- an
d r
ing
-
hy
dro
xy
lati
on
of
2-
acet
amid
ofl
uo
ren
e b
y t
he
rat
and
ham
ster
. B
ioch
em.
J.
12
9:
937
-94
3.
19
72
19
99
19
63
19
72
87
Pre
gn
ancy
alt
ered
th
e b
ind
ing
of
safr
ole
to
DN
A b
y i
ncr
easi
ng
th
e
add
uct
bin
din
g t
o l
iver
an
d k
idn
ey
DN
A b
y 2
.3-3
.5 t
imes
.
2 m
ajo
r ad
du
cts
form
ed i
n t
he
N2
po
siti
on
of
gu
anin
e w
ith
saf
role
an
d
1'-
hyd
roxy
safr
ole
Saf
role
tes
ted
neg
ativ
e fo
r
un
sch
edu
led
DN
A s
yn
thes
is a
nd
is
no
t m
uta
gen
etic
in
bac
teri
a. 1
'-
hy
dro
xy
safr
ole
was
als
o
no
nm
uta
gen
etic
.
Saf
role
id
enti
fied
as
a ca
rcin
og
en,
no
n-m
uta
gen
ic,
wit
h l
ess
than
70
rev
erta
nt
colo
nie
s o
n a
pet
ri d
ish
per
10
00
mic
rog
ram
s o
f ch
emic
al
inco
rpo
rate
d i
n t
he
pla
te a
nd
<0
.01
rev
erta
nts
per
nm
ol
per
pla
te.
Use
d
stra
ins
TA
100
, T
A1
53
5, T
A1
53
7,
and
TA
98
.
On
e ti
me
do
se o
f
97
mg
/kg
to
pre
gn
ant
and
no
n-p
reg
nan
t
mic
e
Saf
role
Sta
nd
ard
Saf
role
stan
dar
d
ICR
mic
e
Mic
e
Un
sch
edu
le
d D
NA
syn
thes
is
assa
ys
in
HeL
a ce
lls
Sa
lmon
ella
/
mic
roso
me
test
Lu
, L
.J.,
et
al.
Dif
fere
nce
s in
th
e
cov
alen
t b
ind
ing
of
ben
zo(a
)py
ren
e,
safr
ole
, 1
’-h
yd
rox
ysa
fro
le a
nd
4-
amin
ob
iph
eny
l to
DN
A o
f p
reg
nan
t
and
no
n-p
reg
nan
t m
ice.
Can
cer
Let
ters
31
: 43
-52
.
Lu
, L
.J.,
et
al.
32
P-P
ost
-lab
elli
ng
assa
y i
n m
ice
of
tran
spla
cen
tal
DN
A
dam
age
ind
uce
d b
y t
he
env
iron
men
tal
carc
ino
gen
s sa
fro
le,
4-
amin
ob
iph
eny
l, a
nd
ben
zo(a
)py
ren
e.
Can
cer
Res
. 4
6:
304
6-3
05
4.
Mar
tin
, C
.N.,
A.C
. M
c D
erm
id a
nd
R.L
. G
arn
er.
Tes
tin
g o
f k
no
wn
carc
ino
gen
s an
d n
on
carc
ino
gen
s fo
r
thei
r ab
ilit
y t
o i
ndu
ce u
nsc
hed
ule
d
DN
A s
yn
thes
is i
n H
eLa
cell
s. C
ance
r
Res
earc
h 3
8:
26
21
-262
7.
McC
ann
, J.
et
al.
Det
ecti
on
of
carc
ino
gen
s as
mu
tag
ens
in t
he
Sal
mo
nel
la/m
icro
som
e te
st:
Ass
ay o
f
30
0 c
hem
ical
s. P
roce
edin
gs
of
the
Nat
ion
al A
cad
emy
of
Sci
ence
of
the
US
A 7
2 (
12
): 5
135
-513
9.
19
86
19
86
19
78
19
75
88
Saf
role
tes
ted
po
siti
ve
for
gen
e
mu
tati
on
ass
ay.
Fo
un
d l
iver
can
cer
in 6
1%
(v
. co
ntr
ol
of
24
%)
in m
ales
an
d 1
3%
in
fem
ales
.
Liv
er c
ance
r in
67
% (
v. 26
% c
on
tro
l),
lun
g a
den
om
as i
n 1
4%
fo
r bo
th
com
pou
nd
s. A
t b
oth
do
ses,
liv
er c
ance
r
in 6
8%
of
the
mic
e. L
ung
ad
eno
mas
wer
e fo
un
d i
n 5
% f
or
safr
ole
an
d 1
0%
in 1
'-hy
dro
xy
safr
ole
. L
iver
ca
nce
r in
55
% by
1'-
hyd
roxy
safr
ole
.
Saf
role
did
no
t in
du
ce u
nsc
hed
ule
d
DN
A s
yn
thes
is,
may
be
fro
m t
he
low
met
abo
liza
tio
n o
f 1
'-h
yd
roxy
safr
ole
.
Hy
dro
xy
chav
ico
l is
an
in
term
edia
te
met
abo
lite
of
safr
ole
an
d m
ore
to
xic
than
saf
role
as
it c
ause
s li
ver
cel
lula
r
mit
och
ond
ria
to d
ysf
un
ctio
n a
nd
cau
ses
gen
erat
ion
of
reac
tiv
e o
xyg
en s
pec
ies.
Mic
e: s
afro
le a
t
40
0 m
g/k
g t
en
tim
es &
23
00
or
46
00
mg
/kg
;
1'h
yd
rox
ysa
fro
le
at 8
1 m
g/k
g, 45
mg
/kg,
& 3
90
0
mg
/kg.
Rat
s:
10
00
, 1
10
0,
12
00
mg
/kg
A s
ing
le d
ose
of
20
0 o
r 1
00
0
mg
/kg
1 m
M o
f sa
fro
le
in 1
x 1
0^6
cell
s/m
L
Sta
nd
ard
Saf
role
stan
dar
d
Saf
role
stan
dar
d
In v
ivo
mam
mal
ia
n c
ell
gen
oto
xic
i
ty a
ssay
:
gen
e
mu
tati
on
CD
-1
mic
e (1
.4
g:
Day
1;
3.5
g:
Day
8;
7 g
:
Day
15
;
13
g:
Day
22
)
Mal
e
Fis
cher
-
34
4 r
ats
Mal
e
F3
44
/Jcl
(20
0-2
40
g)-
hep
ato
cyt
es w
ere
iso
late
d
Mih
r, B
., L
. B
ow
ers,
an
d W
.J.
Cas
par
y.
Ass
ays
for
the
ind
uct
ion
of
gen
e m
uta
tio
ns
at t
he
thy
mid
ine
kin
ase
locu
s in
L51
78
Y m
ou
se
lym
pho
ma
cell
s in
cu
ltu
re.
In:
Ash
by
and
de
Ser
res
(ed
s.).
Ev
alu
atio
n o
f
Sh
ort
-ter
m T
ests
fo
r C
arci
no
gen
s.
Els
evie
r, N
.Y.:
555
-56
8.
Mil
ler,
C.,
A.B
. S
wan
son
, D
.H.
Ph
illi
ps,
T.L
. F
letc
her
, A
. L
iem
, an
d
J.A
. M
ille
r. S
tru
ctu
re-a
ctiv
ity
stu
die
s
of
the
carc
ino
gen
icit
ies
in t
he
mo
use
and
rat
of
som
e n
atu
rall
y o
ccu
rrin
g
and
sy
nth
etic
alk
eny
lben
zen
e
der
ivat
ives
rel
ated
to
saf
role
an
d
estr
ago
le.
Can
cer
Res
. 4
3:
112
4-
11
34
.
Mir
sali
s, J
.C.,
K.
Ty
son,
and B
.E.
Bu
tter
wo
rth
. D
etec
tio
n o
f g
eno
tox
ic
carc
ino
gen
s in
th
e in
viv
o –
in
vit
ro
hep
ato
cyte
DN
A r
epai
r as
say
.
En
vir
on
. M
uta
gen
4:
55
3-5
62
.
Nak
agaw
a Y
., T
. S
uzu
ki,
K.
Nak
ajim
a, H
. Is
hii
, A
. O
gat
a.
Bio
tran
sfo
rmat
ion
an
d c
yto
tox
ic
effe
cts
of
hyd
roxy
chav
ico
l, a
n
inte
rmed
iate
of
safr
ole
met
abo
lism
, in
iso
late
d r
at h
epat
ocy
tes.
Ch
emic
o-
Bio
log
ical
In
tera
ctio
ns
18
0:
89
-97
.
19
85
19
83
19
82
20
09
89
Nin
hyd
rin
-po
siti
ve
bas
ic m
ater
ials
wer
e
excr
eted
in
th
e u
rin
e.
Saf
role
dec
reas
ed t
he
amo
un
t o
f g
lyco
gen
in t
he
liv
er.
Ad
eny
late
cy
clas
e an
d 3
:5-
cycl
ic A
MP
may
co
ntr
ibu
te, b
ut
no
rad
ren
alin
e d
id n
ot
incr
ease
bip
hen
yl
2-
or
4-h
yd
rox
yla
ses.
Incr
ease
d c
yto
chro
me
b5
an
d c
han
ged
th
e
abso
rpti
on
sp
ectr
um
, w
hic
h c
ou
ld r
esu
lt
fro
m b
iosy
nth
esis
of
a h
epat
ic
mic
roso
mal
hae
mo
pro
tein
. T
he
new
abso
rpti
on
sp
ectr
um
was
no
t o
bse
rved
wh
en t
hio
acet
amin
e w
as a
dd
ed t
o t
he
safr
ole
in
du
ctio
n.
2 m
ajo
r ad
du
cts
form
ed i
n t
he
N2 p
osi
tio
n
of
gu
anin
e w
ith
saf
role
an
d 1
'-
hy
dro
xy
safr
ole
Sin
gle
do
se
of
75
-300
mg
/kg b
w
12
5 m
g/k
g
per
day
Saf
role
stan
dar
d
Mal
e
Sp
rag
ue
Daw
ley
rats
(2
00
-
50
0 g
)
Rat
mo
del
Rat
mo
del
Mic
e
mo
del
Osw
ald
, E
. O
., L
. F
ish
bei
n &
B.J
.
Co
rbet
t. M
etab
oli
sm o
f n
atu
rall
y
occ
urr
ing
pro
pen
ylb
enze
ne
der
ivat
ives
.
I. C
hro
mat
og
rap
hic
sep
arat
ion
of
nin
hy
dri
n-p
osi
tiv
e m
ater
ials
of
rat
uri
ne,
Jou
rnal
of
Ch
rom
ato
gra
ph
y 4
5:
437
-445
.
Par
ke,
D.
V.
& H
. R
ahm
an. T
he
ind
uct
ion
of
hep
atic
mic
roso
mal
enzy
mes
by
saf
role
. B
ioch
em.
J. 1
19
:
53
P.
Par
ke,
D.
V.
& H
. R
ahm
an.
Ind
uct
ion
of
a n
ew h
epat
ic m
icro
som
al h
aem
op
rote
in
by
saf
role
an
d i
sosa
fro
le.
Bio
chem
. J.
12
3:
9P
.
Ph
illi
ps,
D.,
M.V
. R
eddy
and
K.
Ran
der
ath
. 3
2P
- P
ost
lab
elli
ng
an
aly
sis
of
DN
A a
dd
uct
s fo
rmed
in
th
e li
ver
s o
f
anim
als
trea
ted
wit
h s
afro
le,
estr
ago
le
and
oth
er n
atu
rall
yo
ccu
rin
g
alk
eny
lben
zen
es.
II.
New
bo
rn m
ale
B6
C3
F1
mic
e. C
arci
no
gen
esis
5:
162
3-
16
28
.
19
69
19
70
19
71
19
84
90
2 m
ino
r ad
du
cts
form
ed i
n t
he
N6
po
siti
on
of
aden
ine
wit
h s
afro
le
and
1'-h
yd
roxy
safr
ole
Saf
role
tes
ted
po
siti
ve
in b
oth
Saf
role
tes
ted
po
siti
ve
for
the
Am
es t
est,
cel
l tr
ansf
orm
atio
n,
deg
ran
ula
tio
n,
and
seb
aceo
us-
gla
nd
sup
pre
ssio
n a
nd
tes
ted
neg
ativ
e fo
r te
traz
oli
um
red
uct
ion
and
th
e im
pla
nt
test
s.
2 m
ajo
r ad
du
cts
form
ed i
n t
he
N2
po
siti
on
of
gu
anin
e w
ith
saf
role
and
1'-h
yd
roxy
safr
ole
. A
fter
a
sin
gle
do
se o
f 10
mg
of
safr
ole
,
on
e ad
du
ct f
orm
ed i
n 1
40
,000
nu
cleo
tid
es.
RA
L x
10
^7
val
ues
for
safr
ole
wer
e 4
91
+/-
12
9 f
or
10
mg
and
11
4 +
/- 5
0 f
or
the
2 m
g
do
se.
12
mic
rom
ole
s/
mo
use
of
1'-
[2',3
'-3H
]-
hy
dro
xy
safr
ole
via
i.p
.
inje
ctio
n.
2 m
g f
or
DN
A
bin
din
g s
tud
ies
or
10 m
g f
or
add
uct
per
sist
ence
stu
die
s. M
ice:
80
mg
/kg
or
40
0 m
g/k
g.
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Fem
ale
CD
-1
mic
e, 8
-10
wee
ks
old
(30
g)
E.
coli
an
d
Sa
cch
aro
myc
es c
erev
isia
e
in v
itro
assa
ys
Am
es t
est
wit
h
Sa
lmon
ella
typ
him
uri
um
;
Rab
in's
tes
t
and
oth
ers
Fem
ale
CD
-1
mic
e (2
5 g
)
Ph
illi
ps,
D.H
., J
.A. M
ille
r, E
.C.
Mil
ler,
and
B. A
dam
s. N
2 a
tom
of
gu
anin
e an
d
N6
of
aden
ine
resi
du
es a
t si
tes
for
cov
alen
t b
ind
ing
of
met
abo
lica
lly
acti
vat
ed 1
’- h
yd
roxy
safr
ole
to
mo
use
liv
er D
NA
in
viv
o.
Can
cer
Res
. 4
1:
26
24
-26
71
.
Po
irie
r, L
.A.
and
F.
J. d
e S
erre
s. I
nit
ial
Nat
ion
al C
ance
r In
stit
ute
stu
die
s o
n
mu
tag
enes
is a
s a
pre
-scr
een
fo
r
chem
ical
car
cin
og
ens:
an
ap
pra
isal
. J.
Nat
l. C
ance
r In
st.
62
: 9
19
-926
.
Pu
rch
ase,
I.
F.
H.
et a
l. A
n e
val
uat
ion
of
6 s
ho
rt-t
erm
tes
ts f
or
det
ecti
ng
org
anic
ch
emic
al c
arci
no
gen
s, B
r. J
.
Can
cer
37
: 8
73
-90
3.
Ran
der
ath
, K
., R
.E.
Hag
lund
, D
.H.
Ph
illi
ps
and
M.V
. R
eddy
. 3
2P
-
Po
stla
bel
lin
g a
nal
ysi
s o
f D
NA
ad
du
cts
form
ed i
n t
he
liv
ers
of
anim
als
trea
ted
wit
h s
afro
le,
estr
ago
le a
nd
oth
er
nat
ura
lly
-occ
urr
ing
alk
eny
lben
zen
es.
I.
Ad
ult
fem
ale
CD
-1 m
ice.
Car
cin
og
enes
is 5
: 1
613
-162
2.
19
81
19
79
19
78
19
84
91
Mic
e th
at o
nly
dra
nk c
ola
-dri
nk
s
dev
elo
ped
100
-200
DN
A a
dd
uct
s in
10
^9
DN
A n
ucl
eoti
des
, w
ith
saf
role
5-6
% o
f
the
add
uct
s an
d m
yri
stic
in 5
0-8
0%
. A
t 8
wee
ks,
saf
role
re
pre
sen
ted
5-6
% o
f th
e
tota
l ad
du
cts.
Fo
r m
yri
stic
in,
nu
tmeg
,
and
mac
e, s
afro
le a
dd
uct
s re
pre
sen
ted
3.5
-8.5
% o
f th
e to
tal
addu
cts.
Dih
yd
rosa
fro
le i
nje
stio
n r
esu
lted
in
neo
pla
sms
of
the
fore
sto
mac
h.
Saf
role
and
iso
safr
ole
did
no
t h
ave
sto
mac
h
tum
ors
, bu
t sa
fro
le d
id i
ncr
ease
th
e
inci
den
ces
of
liv
er c
arci
no
mas
.
A d
ose
of
20 m
g/k
g o
f sa
fro
le d
ou
ble
d t
he
mea
n s
leep
ing
tim
e co
mp
ared
to
th
e
con
tro
l w
hen
ad
ded
to
th
e sl
eep
ing
tab
let
com
pou
nd
sod
ium
pen
tob
arb
ital
. S
afro
le
did
no
t si
gn
ific
antl
y a
ffec
t th
e sl
eep
ing
tim
e in
ass
oci
atio
n w
ith
eth
ano
l.
In t
he
mic
e th
at r
ecei
ved
4.5
0 g
/kg
bw
, 1
0
ou
t o
f 3
0 d
ied
and
th
ree
mic
e h
ad l
un
g
tum
ors
. T
he
mic
e th
e re
ceiv
ed 0
.90
g/k
g
bw
, 3
ou
t o
f 3
0 d
ied
and
6 m
ice
had
lun
g
tum
ors
.
Mic
e :c
ola
dri
nk
s
inst
ead
of
wat
er (
for
4 o
r 8
wee
ks)
, m
ice
giv
en a
sin
gle
10
mg
do
se o
f m
yri
stic
in,
nu
tmeg
, o
r m
ace
via
gas
tric
in
tub
atio
n.
46
4 m
g/k
g o
f sa
fro
le
& d
ihy
dro
safr
ole
:
21
5 m
g/k
g o
f
iso
safr
ole
; 1
,11
2
pp
m s
afro
le,
1,4
00
pp
m d
ihy
dro
safr
ole
,
& 5
17
pp
m
iso
safr
ole
.
10
or
20
mg
/kg v
ia
i.p
. in
ject
ion
alo
ng
wit
h s
od
ium
pen
tob
arb
ital
an
d
10
0 m
g/k
g v
ia i
.p.
inje
ctio
n o
f et
han
ol
Ov
er 2
4 w
eek
s,
mic
e re
ceiv
ed a
to
tal
of
4.5
0 o
r 0.9
0 g
/kg
bw
of
safr
ole
ov
er
12
in
ject
ion
s.
Saf
role
Sta
nd
ard
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Fem
ale
ICR
mic
e, 6
-7
wee
ks
Hy
bri
d
mic
e
Fem
ale
wh
ite
mic
e
(22
-40 g
)
A/H
e
mic
e, 6
-8
wee
ks
old
(18
-
20
g)
Ran
der
ath
, K
.P.,
K.L
. P
utm
an,
and
E. R
and
erat
h.
Fla
vo
r
con
stit
uen
ts i
n c
ola
dri
nk
s
ind
uce
hep
atic
DN
A a
dd
uct
s in
adu
lt a
nd
fet
al m
ice.
Bio
chem
ical
Bio
ph
ysi
cal
Res
earc
h C
om
mun
icat
ion
s 1
92
(1):
61
-68
.
Reu
ber
, M
. D
. N
eop
lasm
s o
f th
e
fore
sto
mac
h i
n m
ice
ing
esti
ng
dih
yd
rosa
fro
le,
Dig
esti
on
, 1
9,
42
-47
Set
o,
T. A
. &
W. K
eup.
Eff
ects
of
alk
ylm
eth
oxy
ben
zen
e an
d
alk
ylm
eth
yle
ned
ioxy
ben
zen
e
esse
nti
al o
ils
on
pen
tob
arb
ital
and
eth
ano
l sl
eep
ing
tim
e. A
rch
Inte
rnd
e P
har
mac
od
yn
amie
et
de
ther
apie
18
0:
23
2-2
40.
Sto
ner
, G
. D
. et
al.
Tes
t fo
r
carc
ino
gen
icit
y o
f fo
od
add
itiv
es
and
ch
emo
ther
apeu
tic
agen
ts b
y
the
pu
lmon
ary
tu
mo
r re
spo
nse
in
stra
in A
mic
e, C
ance
r R
esea
rch
33
: 30
69
-30
85
.
19
93
19
79
19
69
19
73
92
Saf
role
was
neu
tral
or
wea
kly
po
siti
ve
in
the
Am
es t
est.
1'-
hy
dro
xy
safr
ole
was
mu
tag
enic
fo
r st
rain
TA
100
. T
ox
icit
y
incr
ease
d w
ith
ad
dit
ion
of
NA
DP
H-
fort
ifie
d r
at l
iver
mic
roso
mes
an
d
cyto
sol.
Pre
vio
us
stu
die
s w
ith
po
siti
ve
mu
tag
enic
pro
per
ties
use
d a
pre
incu
bat
ion
per
iod,
wh
ich
may
be
hav
e
crea
ted
met
abo
lite
s.
No
rat
s d
ied
ou
t o
f si
x t
este
d f
or
safr
ole
,
bu
t an
av
erag
e o
f 2
.5 m
acro
sco
pic
liv
er
lesi
on
s w
ere
ob
serv
ed.
Th
e ra
ts l
ost
wei
gh
t an
d w
ere
in p
oo
r co
nd
itio
n a
t th
e
end
of
the
exp
erim
ent.
Saf
role
in
hib
its
CY
P1
A2
, C
YP
2A
6,
CY
P2
D6,
CY
P2E
1, an
d C
YP
3A
4
Saf
role
an
d/o
r m
etab
oli
tes
cro
ssed
th
e
pla
cen
ta a
nd
was
del
iver
ed t
o i
nfa
nts
via
lact
atio
n,
bu
t y
ou
ng
mic
e h
ad h
ad m
uch
hig
her
su
rviv
al r
ate
(>9
0%
) w
hen
in
ute
ro a
nd
nu
rsin
g f
rom
mo
ther
s
rece
ivin
g s
afro
le c
om
par
ed t
o w
ean
ed
rats
th
at r
ecei
ved
dir
ect
safr
ole
do
ses.
Sa
lmo
nel
la
typ
him
uri
um
stra
ins
TA
100
,
TA
15
35
, an
d
TA
98
65
0 m
g/k
g b
w
via
gav
age,
or
1/3
of
the
LD
50
of
safr
ole
12
0 m
icro
gra
m
per
g o
f b
w t
o
pre
gn
ant
mic
e,
lact
atin
g
mo
ther
s, a
nd
4
wee
k o
ld
off
spri
ng
Saf
role
stan
dar
d
Saf
role
stan
dar
d
Saf
role
stan
dar
d
In v
itro
Sal
mo
nel
l
a re
ver
se
mu
tati
on
assa
y
(Am
es
test
)
Osb
orn
e-
Men
del
or
Sh
erm
an
rats
(1
80
-
35
0 g
)
Esc
her
ich
ia c
oli
-
exp
ress
ed
hu
man
P4
50
Fem
ale
C5
7B
L/6
J
and
mal
e
C3
HeB
/F
eJ m
ice,
8
wee
ks
Sw
anso
n,
A.B
., D
.D.
Ch
amb
liss
, J.
C.
Bla
nq
uis
t, E
.C.
Mil
ler
and
J.A
.
Mil
ler.
Th
e m
uta
gen
icit
ies
of
safr
ole
,
estr
ago
le,
tran
s-an
eth
ole
an
d s
om
e o
f
thei
r k
no
wn
or
po
ssib
le m
etab
oli
tes
for
Sal
mo
nel
la t
hy
ph
imu
riu
m
mu
tan
ts.
Mu
tat.
Res
. 6
0:
143
-15
3.
Tay
lor,
J.M
.,P
.M. Je
nn
er a
nd
W.I
.
Jon
es.
A c
om
par
iso
n o
f th
e to
xic
ity
of
som
e al
lyl,
pro
pen
yl
and
pro
py
l
com
pou
nd
s in
th
e ra
ts.
Tox
ico
logy
and
Ap
pli
ed P
har
mac
olo
gy
6:
37
8-
38
7.
Uen
g, Y
-F.,
et
al.
Inh
ibit
ion
of
hu
man
cy
toch
rom
e P
45
0 e
nzy
mes
by
the
nat
ura
l h
epat
oto
xin
saf
role
. F
ood
and
Ch
emic
al T
ox
ico
logy
43
: 70
2-
71
2.
Ves
seli
no
vit
ch,
S.D
., K
.V. R
ao, an
d
N.
Mih
ailo
vic
h. T
ran
spla
cen
tal
and
lact
atio
nal
car
cin
og
enes
is b
y s
afro
le.
Can
cer
Res
. 3
9:
437
8-4
38
0.
19
79
19
64
20
05
19
79
93
Ex
amin
ed t
he
ov
er 7
yea
rs.
Fo
un
d s
om
e ti
ssu
e in
jury
in
the
beg
inn
ing
of
the
exp
erim
ent,
bu
t co
ncl
ud
ed t
he
do
gs
may
hav
e ad
apte
d t
o t
he
con
tin
uo
us
inta
ke
of
safr
ole
.
2 m
ajo
r ad
du
cts
form
ed i
n t
he
N2 p
osi
tio
n o
f g
uan
ine
wit
h
safr
ole
an
d 1
'-hy
dro
xy
safr
ole
.
20
% h
ad h
epat
om
as.
Am
es
test
: 1
'-ac
eto
xy
safr
ole
, sa
fro
le-
2',3
'-o
xid
e, 1
'-ac
eto
xy
safr
ole
,
and
1'-o
xy
safr
ole
are
dir
ectl
y
mu
tag
enic
in
mu
tag
enic
in
Sal
mo
nel
la s
trai
n T
A15
35
.
Saf
role
ox
ide
inh
ibit
s
ang
iog
enes
is i
nd
uce
d b
y
can
cer
cell
s b
oth
in
vit
ro a
nd
in v
ivo
.
Bro
msu
lfal
ein
excr
etio
n c
urv
e,
solu
ble
en
zym
e, t
he
lip
id a
nd
gly
cog
en
of
the
liv
er,
and
tnit
ro-r
edu
ctas
e
0.1
mic
rom
ole
s o
f
1'-
hyd
roxy
safr
ole
per
g o
f b
od
y
wei
gh
t
10
or
2 m
g p
er 1
00
g o
f b
od
y w
eig
ht
via
i.p
. in
ject
ion
;
Sa
lmon
ella
str
ain
TA
10
0
Sy
nth
esi
zed
fro
m
stan
dar
ds
Sy
nth
esi
zed
fro
m
stan
dar
ds
Saf
role
stan
dar
d
syn
thes
iz
ed t
o
safr
ole
ox
ide
Do
g m
od
el
Mal
e h
yb
rid
mic
e
B6
C3
F1
Mal
e C
D
rats
; C
D-1
mic
e, a
nd
mal
e
ham
ster
s;
Am
es t
est
Vas
cula
r
end
oth
elia
l
cell
s, A
54
9
lun
g c
ance
r
cell
s, r
at
aort
a fr
om
mal
e
Sp
rag
ue-
Daw
ley
rat
s
Wei
nb
erg
, M
. S
. &
S. S
. S
tern
ber
g.
Eff
ect
of
chro
nic
saf
role
ad
min
istr
atio
n
on
hep
atic
en
zym
es a
nd
fu
nct
ion
al
acti
vit
y i
n d
og
s. T
ox
ico
logy
and
Ap
pli
ed
Ph
arm
aco
log
y 8
: 2
.
Wis
eman
, R
.W.,
et
al.
Fu
rth
er
char
acte
riza
tio
n o
f th
e D
NA
ad
du
cts
form
ed b
y e
lect
rop
hil
ic e
ster
s o
f th
e
hep
ato
carc
ino
gen
s 1
'-h
yd
roxy
safr
ole
an
d
1'-
hyd
roxy
estr
ago
le i
n v
itro
an
d i
n t
he
mo
use
liv
er i
n v
ivo
, in
clu
din
g n
ew
add
uct
s at
C-8
an
d N
-7 o
f gu
anin
e
resi
du
es.
Can
cer
Res
. 4
5:
30
96
-310
5.
Wis
lock
i, P
.G., E
.C.
Mil
ler,
J.A
. M
ille
r,
E.C
. M
cCo
y,
and
H.S
. R
ose
nk
ran
z.
Car
cin
og
enic
an
d m
uta
gen
ic a
ctiv
itie
s o
f
safr
ole
, 1
'-h
yd
roxy
safr
ole
an
d s
om
e
kn
ow
n o
r po
ssib
le m
etab
oli
tes.
Can
cer
Res
. 3
7:
18
83
-18
91
.
Zh
ao,
J, J
. M
iao
, B
. Z
hao
, S
. Z
han
g, D
.
Yin
. S
afro
le o
xid
e in
hib
its
ang
iog
enes
is
by
ind
uci
ng
apo
pto
sis.
Vas
cula
r
Ph
arm
aco
log
y 4
3:
69
-74
.
19
66
19
85
19
77
20
05
94
Table C.3: Review of studies using chemical analysis of foods containing safrole.
R
esu
lts
Ex
amin
ed a
dif
fere
nt
met
abo
lic
pat
hw
ay
safr
ole
co
uld
un
der
go,
nam
ely
th
roug
h
ox
idat
ion
to
qu
ino
id m
etab
oli
tes
in l
iver
mic
roso
mes
in
a n
on
enzy
mat
ic p
roce
ss.
Th
e ‘t
ea’
mad
e in
th
is s
tud
y w
as i
nfu
sed
or
the
tea
bag
ste
eped
in
ho
t w
ater
fo
r
thir
ty m
inu
tes
as o
ppo
sed
to
th
e
trad
itio
nal
met
ho
d o
f d
eco
ctin
g, o
r
bo
ilin
g, th
e w
ho
le o
r ro
ugh
ly c
ho
pp
ed
roo
t. T
hey
dem
on
stra
ted
th
at h
erb
al
pre
par
atio
ns
of
sass
afra
s ra
ng
ed f
rom
har
mle
ss f
or
tea
mad
e fr
om
th
e le
aves
, to
con
tain
ing
92
.4%
saf
role
in
sas
safr
as o
ils.
Th
e ‘t
ea’
they
pre
par
ed h
ad s
afro
le
con
ten
t ra
ng
ing f
rom
0.0
9 t
o 4
.12
mg
per
cup
, o
r on
ly 6
.9 t
o 1
7.2
% o
f to
tal
safr
ole
pre
sen
t in
ro
ot.
Th
e sa
fro
le m
etab
oli
tes
dih
yd
roxy
chav
ico
l an
d e
ug
eno
l w
ere
fou
nd
in
th
e u
rin
e o
f n
on
-bet
el q
uid
chew
ers,
at
the
lev
els
of
no
t d
etec
ted
to
5.4
mic
rog
ram
s/m
g c
reat
inin
e, p
rob
ably
du
e to
sp
ices
in
th
e d
iet.
Co
nce
ntr
ati
on
s
0,
30
, 7
5,
15
0,
30
0
mg
/kg o
ne
tim
e.
Tes
ted
th
e u
rin
e
of
the
rats
an
d o
f
hu
man
su
bje
cts
wh
o c
hew
ed b
etel
qu
id b
etw
een
2 -
10
0 t
imes
a d
ay.
Ch
emic
al
An
aly
sis
HP
LC
,
UV
spec
tro
ph
oto
met
er
Liq
uid
Ch
rom
ato
gra
ph
y
HP
LC
So
urc
e
Saf
role
stan
dar
d
Po
wd
ere
d l
eav
es,
gu
mb
o
file
, ro
ot
bar
k,
roo
t
po
wd
er,
her
bal
cap
sule
mat
eria
l,
un
bre
we
d t
ea.
Saf
role
stan
dar
d
Art
icle
Bo
lto
n,
J.L
., e
t a.
Ev
iden
ce
that
4-a
lly
l-O
-qu
ino
nes
spo
nta
neo
usl
y r
earr
ang
e to
thei
r m
ore
ele
ctro
ph
ilic
qu
ino
ne
met
hid
es:
po
ten
tial
bio
acti
vat
ion
mec
han
ism
for
the
hep
ato
carc
ino
gen
safr
ole
. C
hem
Res
. T
ox
ico
l.
7:
443
-45
0.
Car
lso
n,
M.
and
R. D
.
Th
om
pso
n. L
iqu
id
Ch
rom
atog
rap
hic
Det
erm
inat
ion
of
Saf
role
in
Sas
safr
as-D
eriv
ed H
erb
al
Pro
du
cts.
Jo
urn
al o
f A
OA
C
Inte
rnat
ion
al 8
0 (
5):
102
3-
8.
Ch
ang,
M.J
.W., C
.Y. K
o,
R.F
. L
in &
L.L
. H
sieh
.
Bio
log
ical
mo
nit
ori
ng
of
env
iro
nm
ent
exp
osu
re t
o
safr
ole
an
d t
he
Tai
wan
ese
bet
el q
uid
ch
ewin
g.
Arc
h.
En
vir
on
. C
on
tam
. T
ox
ico
l.
43
: 43
2–
43
7.
Yea
r
19
94
19
97
20
02
95
S
afro
le c
on
ten
t o
f th
e h
erb
al d
rug
extr
acte
d b
y M
eOH
ran
ged
bet
wee
n
1.5
7 t
o 2
.76
mg
/g.
Aft
er a
on
e ho
ur
dec
oct
ion
, sa
fro
le c
on
ten
t w
as
bet
wee
n 0
.20
mg
/g a
nd
no
t
det
ecta
ble
. T
he
med
icin
e fo
rmu
las
con
tain
ed 0
.06
mg
/g o
f sa
fro
le t
o n
ot
det
ecta
ble
.
Dev
elo
ped
sim
ple
met
hod
to
an
aly
ze
soft
dri
nk
s; f
ou
nd
20
ou
t o
f 25
so
ft
dri
nk
s h
ad l
evel
s o
f sa
fro
le a
nd
/or
cis-
iso
safr
ole
ex
ceed
ed t
he
lim
it o
f 1
mic
rog
ram
/mL
set
by
reg
ula
tio
ns,
tho
ug
h t
he
safr
ole
all
cam
e fr
om
'nat
ura
l se
aso
nin
g e
xtr
acts
'.
Fo
un
d s
afro
le p
rese
nt
in a
lco
ho
lic
dri
nk
s w
ith
lev
els
fro
m 6
.6 m
g/l
to
abse
nt.
Det
erm
ined
HP
LC
had
a
hig
h r
eco
ver
y r
ate
of
esse
nti
al o
ils-
for
safr
ole
rec
ov
ery
ran
ged
fro
m
95
.6 t
o 1
00
%.
3 g
of
po
wd
ered
her
bal
dru
g
dec
oct
ed i
n w
ater
or
extr
acte
d w
ith
MeO
H.
A
trad
itio
nal
Ch
ines
e
med
icin
e fo
rmu
la
that
co
nta
ined
3 g
of
Asa
ri r
adix
was
dec
oct
ed.
So
ft d
rin
ks
con
tain
ed 3
-5 m
g/L
(pp
m)
of
safr
ole
.
HP
LC
Gas
Ch
rom
atog
rap
hy
HP
LC
wit
h
flu
ori
met
ri
c d
etec
tio
n
and
GC
-
MS
Asa
ri r
adix
et
rhiz
om
a
Co
mm
erci
al
soft
dri
nk
s,
app
le c
ider
,
and
roo
t b
eer
Co
mm
erci
al
alco
ho
lic
bev
erag
es
and
ess
enti
al
oil
s o
f
nu
tmeg
,
sass
afra
s,
cin
nam
on
,
and
an
ise
Ch
en, C
., D
. S
pri
ano
, T
.
Leh
man
n,
and B
. M
eier
.
Red
uct
ion
of
Saf
role
an
d
Met
hy
leu
gen
ol
in A
sari
rad
ix
et r
hiz
om
a b
y D
eco
ctio
n.
Fo
rsch
Kom
ple
men
tmed
16
:
16
2-1
66
.
Ch
oo
ng
, Y
.-M
. &
H.-
J. L
in.
A R
apid
and
Sim
ple
Gas
Ch
rom
atog
rap
hic
Met
ho
d f
or
Dir
ect
Det
erm
inat
ion
of
Saf
role
in
So
ft D
rin
ks.
”
Jou
rnal
of
Fo
od
and
Dru
g
An
aly
sis
9(1
): 2
7-3
2.
Cu
rró
, P
., G
. M
ical
i, a
nd
F.
Lan
uzz
a. D
eter
min
atio
n o
f β
-
asar
on
e, s
afro
le,
iso
safr
ole
,
and
an
eth
ole
in
alc
hoh
oli
c
dri
nk
s by
hig
h-p
erfo
rman
ce
liq
uid
ch
rom
atog
rap
hy.
Jou
rnal
of
Ch
rom
oto
gra
phy
40
4:
273
-27
8.
20
09
20
01
19
87
96
Was
hin
g a
nd d
ryin
g r
edu
ced
th
e sa
fro
le c
on
ten
t
by
any
wh
ere
fro
m 9
4 t
o 7
4%
. G
amm
a
irra
dia
tio
n r
edu
ced
th
e sa
fro
le c
on
ten
t b
y 9
7 t
o
86
%, o
r o
nly
to
saf
e le
vel
s o
f sa
fro
le a
t th
e h
igh
lev
els.
Mic
row
ave
rad
iati
on
red
uce
d t
he
safr
ole
con
ten
t 9
2 t
o 5
7%
, bu
t on
ly t
he
lon
ges
t ti
me
red
uce
d t
o s
afe
lev
els
and
th
e sa
mp
les
bu
rned
.
Bo
ilin
g t
he
seed
s fo
un
d b
oil
ing w
ho
le s
eed
fo
r
5 m
inu
tes
and
bo
ilin
g p
ow
der
ed s
eed
fo
r 1
min
ute
was
eff
ecti
ve
at r
edu
cin
g s
afro
le t
o s
afe
lev
els.
New
met
ho
d o
f d
eter
min
ing
saf
role
, eu
gen
ol,
and
4-a
lly
l-1
,2-d
imet
ho
xy
ben
zen
e. T
este
d
un
bre
wed
tea
an
d f
oun
d s
afro
le l
evel
s b
etw
een
29
9 a
nd 1
7,4
00 m
g/k
g.
Tes
ted
th
e re
cov
ery
rat
es o
f co
mp
oun
ds
usi
ng
GC
an
d r
eco
mm
end
s ad
op
tin
g t
he
met
hod
fo
r
qu
anti
tati
ve
det
erm
inat
ion
of
safr
ole
, w
hic
h h
as
bee
n a
dop
ted
by
th
e F
DA
.
12
0 g
of
bla
ck
pep
per
see
ds
wer
e w
ash
ed &
dri
ed,
bo
iled
,
exp
ose
d t
o
gam
ma
irra
dia
tio
n,
and
mic
row
ave
irra
dia
tio
n b
oth
in w
ho
le s
eed
and
po
wd
ered
seed
fo
rm.
Gas
Ch
rom
atog
rap
hy
SF
E w
ith
GC
-MS
Gas
Ch
rom
atog
rap
hy
Sp
ices
:
Bla
ck
pep
per
,
gin
ger
roo
t,
cum
in,
star
an
ise.
Un
bre
wed
sass
afra
s
tea,
com
pou
nd
stan
dar
ds
Saf
role
stan
dar
d
Far
ag,
S.E
.A. &
M. A
bo
-
Zei
d.
Deg
red
atio
n o
f th
e
nat
ura
l m
uta
gen
ic
com
pou
nd
saf
role
in
sp
ices
by
coo
kin
g a
nd
irr
adia
tio
n.
Nah
rung
41
(6
): 3
59
-361
.
Hei
kes
, D
.L. S
FE
wit
h G
C
and
MS
det
erm
inat
ion
of
safr
ole
an
d r
elat
ed
ally
lben
zen
es i
n s
assa
fras
teas
. Jo
urn
al o
f
Ch
rom
atog
rap
hic
Sci
ence
32
(7
): 2
53
-258
.
Lar
ry,
D.
Gas
Ch
rom
atog
rap
hic
Det
erm
inat
ion
of
Saf
role
and
Rel
ated
Co
mp
ou
nd
s in
No
nal
coho
lic
Bev
erag
es:
Co
llab
ora
tiv
e S
tudy
.
Jou
rnal
of
the
AO
AC
54
(4):
90
0-9
02
.
19
97
19
94
19
70
97
E
xtr
acts
by
MeO
H s
how
ed
safr
ole
pre
sen
t in
th
e b
ark
by
0.4
35
% (
w/w
). S
afro
le
mea
sure
d 0
.000
1%
(w
/w)
in
the
tea
mad
e b
y b
oil
ing
in
wat
er,
wh
ich
is
abo
ut
30
0
tim
es m
ore
saf
role
pre
sen
t in
the
met
han
ol
extr
acte
d
sam
ple
s.
Ex
trac
ted
sas
safr
as o
il u
sin
g
pet
rol,
wh
ich
was
evap
ora
ted
an
d e
xtr
acte
d
agai
n w
ith
MeO
H. O
ily
frac
tio
n w
as a
nal
yze
d t
o f
ind
19
com
po
nen
ts o
f sa
ssaf
ras
oil
.
2 m
ajo
r ad
du
cts
form
ed i
n
the
N2 p
osi
tio
n o
f gu
anin
e
wit
h s
afro
le a
nd
1'-
hy
dro
xy
safr
ole
.
Ex
trac
ted
2.0
1 g
of
bar
k
wit
h M
eOH
. E
xtr
acti
ng
5.0
1 g
of
bar
k w
ith
wat
er
by
bo
ilin
g,
incl
ud
ing
on
e
sam
ple
set
th
at w
as a
lso
extr
acte
d w
ith
MeO
H.
An
oth
er s
amp
le s
et w
as
spik
ed w
ith
50
mic
rog
ram
s o
f sa
fro
le
stan
dar
d a
nd
bo
iled
in
wat
er
0.1
mic
rom
ole
s o
f 1
'-
hy
dro
xy
safr
ole
per
g o
f
bo
dy
wei
gh
t
HP
LC
GC
-MS
HP
LC
Cin
na
mo
mu
m
caro
lin
ens
e tr
un
k
bar
k
Po
wd
ered
roo
t b
ark
Sy
nth
esiz
ed f
rom
stan
dar
ds
Rey
ner
tso
n,
K.
A., M
. J.
Bal
ick
,
R.
Lee
, W
. R
ayno
r, Y
. P
elep
, an
d
E.
J. K
enn
elly
. A
Tra
dit
ion
al
Met
ho
d o
f C
inn
am
om
um
caro
lin
ense
Pre
par
atio
n
Eli
min
ates
Saf
role
fro
m a
Th
erap
euti
c P
ohn
pea
n T
ea.
Jou
rnal
of
Eth
nop
har
mac
olo
gy
10
2 (
2):
26
9-2
74.
Set
hi,
M.
L.,
G.
Su
bb
a R
ao, B
. K
.
Ch
ow
dh
ury
, J.
F.
Mo
rton
, &
G. J.
Kap
adia
. Id
enti
fica
tio
n o
f vo
lati
le
con
stit
uen
ts o
f S
ass
afr
as
alb
idu
m
roo
t o
il. P
hy
toch
emis
try
15
: 1
77
3-
17
75
.
Wis
eman
, R
.W.,
T.R
F
enn
ell,
J.A
Mil
ler,
an
d E
.C.
Mil
ler.
Fu
rth
er
char
acte
riza
tio
n o
f th
e D
NA
add
uct
s fo
rmed
by
ele
ctro
ph
ilic
este
rs o
f th
e h
epat
oca
rcin
og
ens
1'-
hy
dro
xy
safr
ole
an
d 1
'-
hy
dro
xy
estr
ago
le i
n v
itro
an
d i
n
the
mo
use
liv
er i
n v
ivo
, in
clu
din
g
new
ad
du
cts
at C
-8 a
nd
N-7
of
gu
anin
e re
sid
ues
. C
ance
r R
es.
45
:
30
96
-31
05
.
20
05
19
76
19
85
98
Appendix D
Abbreviations
Abbreviation Meaning
Bw Body Weight
CCP Center for Cherokee Plants
CDC Center for Disease Control and Prevention
CFR Code of Federal Regulations
EBCI Eastern Band of Cherokee Indians
FDA Food and Drug Administration
GIS Geographic Information System
HPLC High Performance-Liquid Chromatography
MCI Museum of the Cherokee Indian
mg/kg bw milligram/kilogram of body weight
ppm Parts Per Million
SCF Scientific Committee on Food (European Commission)
WHO World Health Organization
99
LITERATURE CITED
Abbott, D.D., E.W. Packman, B.M. Wagner and J.W.E. Harrisson. 1961. Chronic oral toxicity of oil of
sassafras and safrole. Pharmacologist 3: 62.
Ames, B.N. & L.S. Gold. 2000. Paracelsus to parascience: the environmental cancer distraction. Mutation
Research Frontiers 477: 3-13.
Baker, R.S.U. and Bonin, A.M., 1985. Tests with Salmonella plate incorporation assay. In: Ashby and de
Serres (eds.) Evaluation of Short-term Tests for Carcinogens. Elsevier, N.Y.
Bazzaz, F. A., V. Paape, and W. R. Boggess. 1971. Photosynthetic and respiratory rates of Sassafras
albidum. Forest Science 18: 218-222.
Benedetti, M.S., A. Malone, A.L. Broillet. 1977. Absorption, metabolism and excretion of safrole in the rat
and man. Toxicology 7(1): 69-83.
Bisset N. 1994. Sassafras lignum. In: Herbal Drugs and Phytopharmaceuticals. CRC Press, Germany,455–
56.
Bolton, J.L., N.M. Acay, and V. Vukomanovic. 1994. Evidence that 4-allyl-O-quinones spontaneously
rearrange to their more electrophilic quinone methides: potential bioactivation mechanism for the
hepatocarcinogen safrole. Chemical Research in Toxicology 7: 443-450.
Bonner, F.T. 2012. Sassafras albidum (Nutt.) Nees. In: Woody Plant Seed Manual. p. 1027-1028. U.S.
Department of Agriculture, Agriculture Handbook 727. Washington, DC.
Brill, S. 1994. Identifying and Harvesting Edible and Medicinal Plants in Wild (and Not So Wild) Places.
HarperCollins Publishers, New York.
Briskin, D.P. 2000. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to
human health. Plant Physiology 124 (2): 507-514.
Bourgaud, F., A. Gravot, S. Milesi, and E. Gontier. 2001. Production of plant secondary metabolites: a
historical perspective. Plant Science 161 (5): 839-851.
Budavaris, S. 1989. The Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals. 11th ed.
Merck & Co. Inc., Rahway, NJ.
Burdock, G. A. 1997. Encyclopedia of Food and Color Additives. Vol. 3. CRC Press, Florida.
Carlson, M. and R. D. Thompson. 1997. Liquid chromatographic determination of safrole in sassafras-
derived herbal products. Journal of AOAC International. Vol. 80, No. 5, p. 1023-8.
Carter, M., P. Feeny, and M. Haribal. 1999. An oviposition stimulant for spicebush swallowtail butterfly,
Papilio troilus, from leaves of Sassafras albidum. Journal of Chemical Ecology 25 (6):1233-1245.
Casida, J.E., J.L. Engel, E.G. Essac, F.X. Kamienski, L. Kuwatsuka. 1966. Methylene-C14
-dioxyphenyl
compounds: metabolism in relation to their synergistic action. Science 153 (3740): 1130-1133
100
Catalán, L.E., A.M. Villegas, L.T. Liber, J.V. García, M.C. Fritis, and H.C. Altamirano. 2010. Synthesis of
nine safrole derivatives and their antiproliferative activity towards human cancer cells. Journal of
the Chilean Chemical Society 55 (2) 219-222.
CCP. 2011. Center for Cherokee Plants. http://www.indiancountryextension.org/extension/program/center-
cherokee-plants. Accessed 5 November 2011.
CDC 2012. Center for Disease Control and Prevention, Morbidity and Mortality Weekly Report (MMWR).
http://www.cdc.gov/mmwr/. Accessed 23 February 2012.
CD-P-SP 2005. Council of Experts on Flavouring Substances (P-SP-AR), 53rd
Meeting. Strasbourg, 12-14
April 2005. RD 53 REC.
CFR, Sec. 172: Code of Federal Regulations, Title 21, Vol. 3, Ch. 1, Pt. 172.580 Safrole. Accessed 4
March 2011.
CFR, Sec. 189: Code of Federal Regulations, Title 21, Vol. 3, Ch. 1, Pt. 189.180 Safrole. Accessed 4
March 2011.
CFR, Sec. 1310.02: Code of Federal Regulations, List of Chemicals from 21 CFR Section 1310.02(a). U.S.
Department of Justice, Drug Enforcement Administration, Office of Diversion Control. Accessed
3 February 2012.
Chamber of Commerce, Highlands, North Carolina. 2011. http://www.highlandschamber.org/. Accessed 27
August 2011.
Chang, M.J.W., C.Y. Ko, R.F. Lin & L.L. Hsieh. 2002. Biological monitoring of environment exposure to
safrole and the Taiwanese betel quid chewing. Arch. Environ. Contam. Toxicol. 43: 432–437.
Chen, C., D. Spriano, T. Lehmann, and B. Meier. 2009. Reduction of safrole and methyleugenol in Asari
radix et rhizoma by decoction. Forsch Komplementmed 16: 162-166.
Chen, C.-L. C.-W. Chi, K.-W. Chang, and T.-Y. Liu. 1999. Safrole-like DNA adducts in oral tissue from
oral cancer patients with a betel quid chewing history. Carcinogenesis 20 (12): 2331-2334.
Chen, W., and P. Wang. 1985. Somatic embroyogenesis and plant regeneration on Sassafras randaiense
(Hay.) Rehd. Bot. Bull. Academia Sinica 26: 1-12.
Choong, Y-M., and H.-J. Lin. 2001. A rapid and simple gas chromatographic method for direct
determination of safrole in soft drinks. Journal of Food and Drug Analysis 9 (1): 27-32.
Christopher, S. 2005. Recommendations for conducting successful research with Native Americans.
Journal of Cancer Research 20 (51): 47-51.
Cotton, C.M. 1996. Ethnobotany: Principles and Applications. John Wiley & Sons, New York.
Cozzo, D.N. 2004. Ethnobotanical classification system and medical ethnobotany of the Eastern Band of
Cherokee Indians (Doctoral Dissertation). University of Georgia, Athens, GA.
Crampton, R.F., T.J.B. Gray, P. Grasso, and D.V. Parke. 1977. Long-term studies on chemically induced
liver enlargement in the rat. II. Transient induction of microsomal enzymes leading to liver
damage and nodularhyperplasia produced by safrole and Ponceau MX. Toxicology 7: 307-326.
101
Cullina, W. 2002. Native Trees, Shrubs, and Vines: A Guide to Using, Growing, and Propagating North
American Woody Plants. Houghton Mifflin Co., Boston, MA.
Curró, P., G. Micali, and F. Lanuzza. 1987. Determination of β-asarone, safrole, isosafrole, and anethole in
alchoholic drinks by high-performance liquid chromatography. Journal of Chromotography 404:
273-278.
Daimon H., S. Sawada, S. Asakura & F. Sagami. 1997. Analysis of cytogenetic effects and DNA adduct
formation induced by safrole in Chinese hamster lung cells. Teratogenesis Carcinogenesis
Mutagenesis 17(1): 7-18.
Daimon H., S. Sawada, S. Asakura, & F. Sagami 1997-1998. Inhibition of sulfotransferase affecting in vivo
genotoxicity and DNA adducts induced by safrole in rat liver. Teratogenesis Carcinogenesis
Mutagenesis 17(6): 327-337.
Davis, S. M. and R. Reid. 1999. Practicing participatory research in American Indian communities.
American Journal of Clinical Nutrition 69: 755S-759S.
De Vries, J. 1997. Food Safety and Toxicity. CRC Press, New York.
Del Tredici, P. 1995. Shoots from roots: a horticulture review. Arnoldia 55(3): 11-19.
Deloria, V. Jr. 1969. Custer Died for Your Sins. University of Oklahoma Press, Norman, OK.
Dewick, P. M. 1997. Medicinal Natural Products: A Biosynthetic Approach. John Wiley & Sons, New
York.
Dirr, M. 1998. Manual of Woody Landscape Plants: Their Identification, Ornamental Characteristics,
Culture, Propagation and Uses. Stipes Publication, Champaign, IL.
Dixon, R.L. 1976. Problems in extrapolating toxicology data for laboratory animals to man. Environmental
Health Perspectives 13: 43-50.
Dugan, H. 2011. The Ephemeral History of Perfume: Scent and Sense in Early Modern England. The John
Hopkins University Press, Baltimore.
Duncan, B. R. and B. H. Riggs. 2003. Cherokee Heritage Trails Guidebook. The University of North
Carolina Press, NC.
EBCI C.O., Sec. 70-3. Eastern Band of Cherokee Indians, Code of Ordinances: Part II Code of Ordinances,
Chapter 70 – Skeletal Remains and Burial Site Preservation, Article 1. – In General, Section 70-3:
Historic preservation of skeletal remains. http://library.municode.com/index.aspx?clientId=13359.
Accessed 12 February 2012.
Epstein, S.S., K. Fujii, J. Andrea, and N. Mantel. 1970. Carcinogenicity testing of selected food additives
by parenteral administration to infant Swiss mice. Toxicology and Applied Pharmacology 16 (2):
321-334.
Epstein, S.S., E. Arnold, J. Andrea, W. Bass and Y. Bishop. 1972. Detection of chemical mutagens by the
dominant lethal assay in the mouse. Toxicology and Applied Pharmacology 23: 288- 325.
102
Evans, E. & F.A. Blazich. Plant propagation by leaf, cane, and root cutting: instructions for the home
gardener. NC State University, Horticulture Information Leaflets: 1/99 HIL-8700. Accessed 1
November 2011.
Extoxnet 2012: Extension Toxicology Network. Oregon State University Cooperative Extension.
“Carcinogenicity”. Accessed 1 March 2012.
Farag, S.E.A. and M. Abo-Zeid. 1997. Degradation of the natural mutagenic compound safrole in spices by
cooking and irradiation. Nahrung 41 (6): 359-361.
Finger, J. R. 1984. Eastern Band of Cherokees: 1819 -1900. University of Tennessee Press, Knoxville, TN.
Friedman, M.A., E. Arnold, Y. Bishop, and S.S. Epstein. 1971. Additive and synergistic inhibition of
mammalian microsomal enzyme functions by piperonyl butoxide, safrole and other
methylenedioxyphenyl derivatives. Experientia 27: 1052-1054.
Frohne, D., and H.J. Pfander. 2004. Poisonous Plants: A Handbook for Doctors, Pharmacists,
Toxicologists, Biologists, and Veterinarians. Second Ed. Timber Press, Portland.
Gant, R.E. and E.C. Clebsch. 1975. The allelopathic influences of Sassafras albidum in old-field succession
in Tennessee. Ecology 56: 604-615.
Gocke, E., M.T. King, K. Eckardt, and D. Wild. 1981. Mutagenicity of cosmetics ingredients licensed by
the European Community. Mutat. Res. 90: 91-109.
Grande, G.A. and S.R. Dannewitz. 1987. Symptomatic sassafras oil ingestion. Veterinary and Human
Toxicology 29 (6) 447.
Griggs, M.M. 1990. Sassafras albidum (Nutt.) Nees. In: Burns, R.M.; Honkala, B.H., Technical
coordinators. Silvics of North America. Volume 2. Hardwoods. Agricultural Handbook 654.
Washington, DC: U.S. Department of Agriculture, Forest Service: 773-777.
Groopman, J.D. and P.L. Skipper. 1991. Molecular Dosimetry and Human Cancer: Analytical,
Epidemiological, and Social Considerations. Florida: CRC Press.
Gupta, A.K. 2010. Indigenous knowledge: ways of knowing, feeling, and doing. In: Ashok Jain (ed.),
History of Science, Philosophy and Culture in Indian Civilization: Science and the Public. New
Delhi: Centre for Studies in Civilizations.
Gupta, K.P., K.L. van Golen, K.L. Putman and K. Randerath. 1993. Formation and persistence of safrole-
DNA adducts over a 10,000-fold dose range in mouse liver. Carcinogenesis 14 (8): 1517–1521.
Hagan, E.C., P. M. Jenner, W. I. Jones, O. G. Fitzhugh, E. L. Long, J. G. Brouwer, and W. Welfare. 1965.
Toxic properties of compounds related to safrole. Toxicology and Applied Pharmacology 7(1): 18-
24.
Hahlbrock K., and D. Scheel. 1989. Physiology and molecular biology of phenylpropanoid metabolism.
Annual Review of Plant Physiology and Plant Molecular Biology 40: 347–69.
Haines, J.D. 1991. Sassafras tea and diaphoresis. Postgraduate Medicine 90 (4): 75-76.
103
Hall, K. C. 2006. Ethnobotany of the Eastern Band of Cherokee Indians: A Path to Sustaining Traditional
Identity with an Emphasis on Medicinal Plant Use (Doctoral Dissertation). Clemson University,
Clemson, SC.
Hamel, P. B. and M. U. Chiltoskey. 1975. Cherokee Plants: Their Uses- a 400 year History. Herald
Publishing Co., Sylva, NC.
Haywood, J. 1994. Seed viability of selected tree, shrub, and vine species stored in the field. New Forests 8
(2): 143-154.
Heikes, D.L. 1994. SFE with GC and MS determination of safrole and related allylbenzenes in sassafras
teas. Journal of Chromatographic Science 32 (7): 253-258.
HERP 2011. Ranking Possible Cancer Hazards from Rodent Carcinogens, Using the Human
Exposure/Rodent Potency Index (HERP). http://potency.berkeley.edu/pdfs/herp.pdf. Accessed 4
December 2011.
Homburger, F., P.D. Bogdonoff and T.F. Kelley. 1965. Influence of diet on chronic oral toxicity of safrole
and butter yellow in rats. Proceedings of the Society for Experimental Biology and Medicine 119
(4): 1106-1110.
Howes, J.A., V.S.W. Chan, and J. Caldwell. 1990. Structure-specificity of the genotoxicity of some
naturally occurring alkenyl-benzenes determined by the unscheduled DNA synthesis assays in rat
hepatocytes. Food Chem. Toxicol. 28: 537-542.
Huang, Y., S.H. Ho, R.M. Kini. 1999. Bioactivities of safrole and isosafrole on Sitophilus zeamais
(Coleoptera: Curculionidae) and Tribolium castaneum (Coleoptera: Tenebrionidae). Journal of
Economic Entomology 92: 676-683.
Hudson, C. 1979. The Southeastern Indians. University of Tennessee Press, Knoxville.
Hung, S.-L., Y.-L. Chen, and Y.-T. Chen. 2003. Effects of safrole on the defensive functions of human
neutrophils. Journal of Periodontal Research 38: 130–134.
Huntington, H.P. 2005. ‘We Dance Around a Ring and Suppose’: Academic Engagement with Traditional
Knowledge. Arctic Anthropology 42 (1): 29-32.
Ibrahim, R. K. 2001. Phenylpropanoid Metabolism. In Encyclopedia of Life Sciences, John Wiley & Sons,
NY.
Ishidate, M. and T. Sofuni. 1985. The in vitro chromosomal aberration test using Chinese hamster lung
(CHL) fibroblast cells in culture. In: Ashby and de Serres (eds). Evaluation of Short-term Tests for
carcinogens. Elsevier, N.Y.
Jeurissen, S.M.F., J.J.P. Bogaards, H.M. Awad, M.G. Boersma, W. Brand, Y.C. Fiamegos, T.A. van Beek,
G.M. Alink, E.J.R. Sudholter, N.H.P. Cnubben, IM.C.M. Rietjens. 2004. Human cytochrome P450
enzyme specificity for bioactivation of safrole to the proximate carcinogen 1-hydroxysafrole.
Chemical Research in Toxicology. 17 (9): 1245-1250.
Jin, M., A. Kijma, Y. Suzuki, D. Hibi, T. Inoue, Y. Ishii, T. Nohmi, A. Nishikawa, K. Ogawa, and T.
Umenura. 2011. Comprehensive toxicity study of safrole using a medium-term animal model with
gpt delta rats. Toxicology 290: 312-321.
104
Kamdem, D.P., and D.A. Gage. 1995. Chemical composition of essential oil from the root bark of
Sassafras albidum. Planta Medica 61 (6): 574-5.
Kapadia, G.J., E.B. Chung, B. Ghosh, Y.N. Shukla, S.P. Basak, J.F. Morton, S.N. Pradhan. 1978.
Carcinogenicity of some folk medicinal herbs in rats. Journal of National Cancer Institute 60 (3):
683-686.
Khayyat, S.A. 2011. Photosynthesis of dimeric cinnamaldehyde, eugenol, and safrole as antimicrobial
agents. Journal of Saudi Chemical Society, doi: 10.1016/j.jscs.2011.07.014.
Khayyat, S.A and S.H. Al-Zahrani. 2011. Thermal, photosynthesis and antibacterial studies of bioactive
safrole derivative as precursor for natural flavor and fragrance. Arabian Journal of Chemistry,
doi:10.1016/j.arabjc.2011.09.014.
Kim, J. and I-K. Park. 2008. Fumigant toxicity of Korean medicinal plant essential oils and components
from Asiasarum sieboldi root against Sitophilus oryzae L. Flavour and Fragrance Journal 23: 79-
83.
Lambert, D. 2007. Eastern Band of Cherokee Indians First Steps Project. Tribal Energy Program, Program
Review Meeting. http://apps1.eere.energy.gov/tribalenergy/pdfs/0711review_lambert.pdf.
Accessed 20 May 2011.
Larry, D. 1970. Gas chromatographic determination of safrole and related compounds in nonalcoholic
beverages: collaborative study. Journal of the AOAC 54 (4): 900-902.
Liu, C.J., C.L. Chen, K.W. Chang, C.H. Chu, and T.Y. Liu. 2000. Safrole in betel quid may be a risk factor
for hepatocellular carcinoma: case report. CMAJ 162(3): 359–360.
Liu, T.Y., C.C. Chen, C.L. Chen, and C.W. Chi. 1999. Safrole-induced oxidative damage in the liver of
Sprague-Dawley rats. Food and Chemical Toxicology 37: 697-702.
Long, E.L., A.A. Nelson, O.G. Fitzhugh and W.H. Hansen. 1963. Liver tumours produced in rats by
feeding safrole. Archives of Pathology 75: 595-604.
Love, L.A. 1999. Safety monitoring of botanicals by government agencies. In: D. Eskinazi (ed.), Botanical
Medicine: Efficacy, Quality Assurance, and Regulation. Mary Ann Liebert, Inc. Publishers, NY.
Martin, C.N., A.C. Mc Dermid and R.L. Garner. 1978. Testing of known carcinogens and noncarcinogens
for their ability to induce unscheduled DNA synthesis in HeLa cells. Cancer Research 38: 2621-
2627.
MedWatch 2012. MedWatch search ‘sassafras’. http://www.fda.gov/Safety/MedWatch/default.htm.
Accessed 10 March 2012.
Miglierini, G. 2008. Sassafras oil, a key raw material now largely banned from the market. Chemistry
Today 26 (5): 59-62.
Mihr, B., L. Bowers, and W.J. Caspary. 1985. Assays for the induction of gene mutations at the thymidine
kinase locus in L5178Y mouse lymphoma cells in culture. In: Ashby and de Serres (eds.).
Evaluation of Short-term Tests for Carcinogens. Elsevier, N.Y.
105
Miller, C., A.B. Swanson, D.H. Phillips, T.L. Fletcher, A. Liem, and J.A. Miller. 1983. Structure-activity
studies of the carcinogenicities in the mouse and rat of some naturally occurring and synthetic
alkenylbenzene derivatives related to safrole and estragole. Cancer Res. 43: 1124-1134.
Mirsalis, J.C., K. Tyson, and B.E. Butterworth. 1982. Detection of genotoxic carcinogens in the in vivo – in
vitro hepatocyte DNA repair assay. Environ. Mutagen. 4: 553-562.
Moerman, D. 2011. “Sassafras albidum”. Native American Ethnobotany Database. 5 November 2011.
http://herb.umd.umich.edu/
Mooney, J. 1992. History, Myths, and Sacred Formulas of the Cherokees. Historical Images: Asheville,
North Carolina.
MCI: Museum of the Cherokee Indian. “Publications”. http://www.cherokeemuseum.org/education-
publications.htm. Accessed 15 February 2012.
Nakagawa Y., T. Suzuki, K. Nakajima, H. Ishii, A. Ogata. 2009. Biotransformation and cytotoxic effects of
hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes. Chemico-
Biological Interactions 180: 89-97.
Oltramari, A.C., K.V. Wood, C. Bonham, R. Verpoorte, M.S.B. Caro, A.M. Viana, E.L. Pedrotti, R.P.
Maraschin, and M. Maraschin. 2004. Safrole analysis by GC-MS of prototrophic (Ocotea
odorifera (Vell.) Rohwer) cell cultures. Plant Cell, Tissue, and Organ Culture 4500: 1-5.
Orndorff, C. 1977. Propagation of woody plants by root cuttings. Combined Proceedings, International
Plant Propagators’ Society 27: 402-406.
"Paracelsus." Encyclopædia Britannica Online. Accessed 19 February 2012.
http://www.britannica.com/EBchecked/topic/442424/Paracelsus.
Purchase, I.F., E. Longstaff, J. Ashby, J.A. Styles, D. Anderson, P.A. Lefevre, and F.R. Westwood. 1978.
An evaluation of 6 short-term tests for detecting organic chemical carcinogens, Br. J. Cancer
37(6): 873-903.
Ratsch, C. 1998. The Encyclopedia of Psychoactive Plants. Parkstreet Press, Vermont.
Reagan-Shaw, S., M. Nihal, and N. Ahmad. 2007. Dose translation from animal to human studies revisited.
The FASEB Journal 22: 659-661.
Reynertson, K.A., M.J. Balick, R. Lee, W. Raynor, Y. Pelep, and E.J. Kennelly. 2005. A traditional method
of Cinnamomum carolinense preparation eliminates safrole from a therapeutic Pohnpean tea.
Journal of Ethnopharmacology 102 (2): 269-274.
Sanagi, M.M., S.L. Ling, Z. Nasir, D. Hermawan, W.A. Ibrahim, and A. Abu Naim. 2009. Comparison of
signal-to-noise, blank determination, and linear regression methods for the estimation of detection
and quantification limits for volatile organic compounds by gas chromatography. Journal of
AOAC International 92(6): 1833-1838.
SCF. 2002. Scientific Committee on Food, European Commission: Opinion of the Scientific Committee on
Food on the safety of the presence of safrole (1-allyl-3,4-methylene dioxy benzene) in flavourings
and other food ingredients with flavouring properties. (SCF/CS/FLAV/FLAVOUR/6 ADD3
Final).
106
SCF. 2003. Scientific Committee on Food, European Commission: Opinion of the Scientific Committee on
Food on Isosafrole. (SCF/CS/FLAV/FLAVOUR/30 Final).
Segelman, A.B., F.P. Segelman, J. Karliner, and D. Sofia. 1976. Sassafras and herb tea: potential health
hazards. Journal of American Medical Association 236 (5): 477.
Sethi, M.L., G. Subba Rao, B.K. Chowdhury, J.F. Morton, & G.J. Kapadia. 1976. Identification of volatile
constituents of Sassafras albidum root oil. Phytochemistry 15: 1773-1775.
Shibamoto, T. and L.E. Bjeldanes. 2009. Introduction to Food Toxicology. Elsevier Academic Press, New
York. Second ed.
Sicuranza, J. 2007. Development of Native alternatives to invasive horticultural plants: vegetative
propagation of Linder benzoin (L.) Bl. and Sassafras albidum (Nutt.) Nees (Masters Thesis).
University of Rhode Island, Kingston, RI.
Sigma-Aldrich MDMS S9652. “Safrole”. Version 4.1, Accessed 28 February 2012.
Singh, Y.N, and A.K. Devkota. 2003. Aqueous kava extracts do not affect liver function tests in rats. Planta
Medica 69: 496-499.
Smith, L.T. 1999. Decolonizing Methodologies: Research and Indigenous Peoples. Zen Books Ltd, New
York, NY.
Sternberg, G. with J. Wilson. 2004. Native Trees for North American Landscape: From the Atlantic to the
Rockies. Timber Press, Portland, OR.
Sullivan, J. 1993. “Sassafras albidum”. Fire Effects Information System [online], U.S. Department of
Agriculture, Forest Service. Rocky Mountain Research Station.
http://www.fs.fed.us/database/feis/.
Swanson, A.B., D.D. Chambliss, J.C. Blanquist, E.C. Miller and J.A. Miller. 1979. The mutagenicities of
safrole, estragole, trans-anethole and some of their known or possible metabolites for Salmonella
thyphimurium mutants. Mutat. Res. 60: 143-153.
Taylor, J.M., P.M. Jenner and W.I. Jones. 1964. A comparison of the toxicity of some allyl, propenyl and
propyl compounds in the rats. Toxicology and Applied Pharmacology 6: 378-387.
Ueng, Y-F., C-H. Hsieh, and M-J Don. 2005. Inhibition of human cytochrome P450 enzymes by the natural
hepatotoxin safrole. Food and Chemical Toxicology 43: 702-712.
Viergever, M. 1999. Indigenous knowledge: an interpretation of views from indigenous peoples. In: What
is Indigenous Knowledge? Voices from the Academy. Ed. L. M. Semali and J. L. Kincheloe.
Falmer Press: New York, NY. 333-343.
Weaver, H.N. 1997. The challenges of research in Native American communities: incorporating principles
of cultural competence. Journal of Social Services Research 23 (2): 1-15.
Weinberg, M.S. and S.S. Sternberg. 1966. Effect of chronic safrole administration on hepatic enzymes and
functional activity in dogs. Toxicology and Applied Pharmacology 8: 2.
107
Williams, J.C. 1998. Jude’s Herbal Home Remedies: Natural Health, Beauty & Home-Care Secrets.
Llewellyn Publications, St. Paul, Minnesota.
Wink, M. and B.-E. van Wyk. 2008. Mind-Altering and Poisonous Plants of the World. Timber Press,
Portland.
Wink, M. 2010. Biochemistry of Plant Secondary Metabolism. Wiley-Blackwell, Iowa. Annual Plant
Reviews, Vol. 40.
Whitton, P.A., A. Lau, A. Salisbury, J. Whitehouse, and C S. Evans. 2003. Kava lactones and the kava-
kava controversy. Phytochemistry 64: 67-679.
WHO 2008. World Health Organization. Fact sheet N*134: “Traditional Medicine”.
http://www.who.int/mediacentre/factsheets/fs134/en/. Accessed 14 February 2012.
WHO 2009. World Health Organization: WHO Food Additives Series: 60. Safety evaluation of certain
food additives. The 69th Meeting of Joint FAO/WHO Expert Committee on Food Additives.
Yang, M.-S., T.-C. Chung, M.-J. Yang, T.-Y. Hsu, and Y.-C. Ko. 2001. Betel quid chewing and risk of
adverse birth outcomes among aborigines in Eastern Taiwan. Journal of Toxicology and
Environmental Health 64 (6): 465-472.
Zhao, J, J. Miao, B. Zhao, S. Zhang, and D. Yin. 2005. Safrole oxide inhibits angiogenesis by inducing
apoptosis. Vascular Pharmacology 43: 69-74.
Zhou, G.D., B. Moorthy, J. Bi, K.C. Donnelly and K. Randerath. 2007. DNA adducts from
alkoxyallylbenzene herb and spice constituents in cultured human (HepG2) cells. Environmental
and Molecular Mutagenesis 48 (9): 715-21.