SCREENING-LEVEL HAZARD CHARACTERIZATION
OF HIGH PRODUCTION VOLUME CHEMICALS
SPONSORED CHEMICALS
Anethole (isomer unspecified) (CAS No. 104-46-1)
[9th CI Name: Benzene, l-methoxy-4-(l-propen-l-yl)-]
trans-Anethole (CAS No. 4180-23-8)
[9th CI Name: Benzene, l-methoxy-4-(lE)-l-propen-l-yl-]
October 2007
INTERIM
Prepared by
High Production Volume Chemicals Branch
Risk Assessment Division
Office of Pollution Prevention and Toxics
Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, DC 20460-0001

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SCREENING-LEVEL HAZARD CHARACTERIZATION
OF HIGH PRODUCTION VOLUME CHEMICALS
The High Production Volume (HPV) Challenge Program1 is a voluntary initiative aimed at developing and making
publicly available screening-level health and environmental effects information on chemicals manufactured in or
imported into the United States in quantities greater than one million pounds per year. In the Challenge Program,
producers and importers of HPV chemicals voluntarily sponsor chemicals; sponsorship entails the identification and
initial assessment of the adequacy of existing toxicity data/information, conducting new testing if adequate data do
not exist, and making both new and existing data and information available to the public. Each complete data
submission contains data on 18 internationally agreed to "SIDS" (Screening Information Data Set1'2) endpoints that
are screening-level indicators of potential hazards (toxicity) for humans or the environment.
The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is evaluating the data
submitted in the HPV Challenge Program on approximately 1400 sponsored chemicals. OPPT is using a hazard-
based screening process to prioritize review of the submissions. The hazard-based screening process consists of two
tiers described below briefly and in more detail on the Hazard Characterization website3.
Tier 1 is a computerized sorting process whereby key elements of a submitted data set are compared to established
criteria to "bin" chemicals/categories for OPPT review. This is an automated process performed on the data as
submitted by the sponsor. It does not include evaluation of the quality or completeness of the data.
In Tier 2, a screening-level hazard characterization is developed by EPA that consists of an objective evaluation of
the quality and completeness of the data set provided in the Challenge Program submissions. The evaluation is
performed according to established EPA guidance2'4 and is based primarily on hazard data provided by sponsors.
EPA may also include additional or updated hazard information of which EPA, sponsors or other parties have
become aware. The hazard characterization may also identify data gaps that will become the basis for a subsequent
data needs assessment where deemed necessary. Under the HPV Challenge Program, chemicals that have similar
chemical structures, properties and biological activities may be grouped together and their data shared across the
resulting category. This approach often significantly reduces the need for conducting tests for all endpoints for all
category members. As part of Tier 2, evaluation of chemical category rationale and composition and data
extrapolation(s) among category members is performed in accord with established EPA2 and OECD5 guidance.
The screening-level hazard characterizations that emerge from Tier 2 are important contributors to OPPT's existing
chemicals review process. These hazard characterizations are technical documents intended to support subsequent
decisions and actions by OPPT. Accordingly, the documents are not written with the goal of informing the general
public. However, they do provide a vehicle for public access to a concise assessment of the raw technical data on
HPV chemicals and provide information previously not readily available to the public. The public, including
sponsors, may offer comments on the hazard characterization documents.
The screening-level hazard characterizations, as the name indicates, do not evaluate the potential risks of a chemical
or a chemical category, but will serve as a starting point for such reviews. In 2007, EPA received data on uses of
and exposures to high-volume TSCA existing chemicals, submitted in accordance with the requirements of the
Inventory Update Reporting (IUR) rule. For the chemicals in the HPV Challenge Program, EPA will review the
IUR data to evaluate exposure potential. The resulting exposure information will then be combined with the
screening-level hazard characterizations to develop screening-level risk characterizations4'6. The screening-level
risk characterizations will inform EPA on the need for further work on individual chemicals or categories. Efforts
are currently underway to consider how best to utilize these screening-level risk characterizations as part of a risk-
based decision-making process on HPV chemicals which applies the results of the successful U.S. High Production
Volume Challenge Program and the IUR to support judgments concerning the need, if any, for further action.
1	U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm.
2	U.S. EPA. HPV Challenge Program - Information Sources; http://www.epa.gov/chemrtk/pubs/general/guidocs.htm.
3	U.S. EPA. HPV Chemicals Hazard Characterization website (http://www.epa.gov/hpvis/abouthc.html).
4	U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm.
5	OECD. Guidance on the Development and Use of Chemical Categories; http://www.oecd.org/dataoecd/60/47/1947509.pdf.
6	U.S. EPA. Risk Characterization Program; http://www.epa.gov/osa/spc/2riskchr.htm.
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SCREENING-LEVEL HAZARD CHARACTERIZATION
Anethole (isomer unspecified) (CAS No. 104-46-1)
fraws-Anethole (CAS No. 4180-23-8)
Introduction
The sponsor, the Terpene Consortium of the Flavor and Fragrance High Production Volume Consortia (FFHPVC),
submitted a Test Plan and Robust Summaries to EPA for anethole (CAS No. 104-46-1; 9th CI name: benzene, 1-
methoxy-4-(l-propen-l-yl)-) and /nmv-anctholc (CAS No. 4180-23-8; 9th CI name: benzene, l-methoxy-4-(lE)-l-
propen-l-yl-) on September 2, 2002. EPA posted the submission on the ChemRTK HPV Challenge website on
December 4, 2002 (http://www.epa.gov/chemrtk/pubs/summaries/anethole/c 14069tc.htm). EPA comments on the
original submission were posted to the website on April 8, 2003. Public comments were also received and posted to
the website. The sponsor submitted revised documents on July 19, 2005, which were posted to the ChemRTK
website on August 22, 2005.
This screening-level hazard characterization is based primarily on the review of the test plan and robust summaries
of studies submitted by the sponsor(s) under the HPV Challenge Program. In preparing the hazard characterization,
EPA considered its own comments and public comments on the original submission as well as the sponsor's
responses to comments and revisions made to the submission. A summary table of SIDS endpoint data with the
structure(s) of the sponsored chemical(s) is included in the appendix. The screening-level hazard characterization
for environmental and human health toxicity is based largely on SIDS endpoints and is described according to
established EPA or OECD effect level definitions and hazard assessment practices.
The sponsor submitted a test plan including two forms of anethole—anethole (isomer unspecified) and trans-
anethole. The unspecified isomer form of anethole contains both the cis- and /nmv-isomers. Industrially produced
anethole (isomer unspecified) consists of greater than 85% of the trans- isomer. Anethole obtained from natural
sources (e.g., anise oil or fennel oil), isolated from crude sulfate turpentine, or synthesized, is composed mainly of
the trans-isomer. Toxicity and ecotoxicity data available for both anethole (isomer unspecified) and the trans-
anethole isomer are used to address the SIDS endpoints the HPV Challenge Program. The U.S. Food and Drug
Administration (FDA) considers /nmv-anctholc to be "generally recognized as safe" (GRAS) for its intended use as
a flavoring substance [21CFR§182.60],
Sum in ;m-Conclusion
The log K of auclholc (isomer unspecified) and //¦.///>-anet hole indicates ilial their potential lo hioacciiniulalc is
e\pecled lo he low \uclholc (isomer unspecified) is rcadiK biodegradable, indicating llial il is not e\pecled In
persist in ilic cu\ iroiinicui
llie c\ alualkiu of a\ ailahlc lo\icil> dala for fish. aquatic 11 in eriehrales and aquatic plains nidicalcs ilial I lie poicuiial
acuic lia/ard of //¦.///.N-aiiclholc lo aquatic organisms is moderate
\ciitc oral lo\icil\ ofaiiclliolc (isomer unspecified) and //¦.///.N-auclholc is low Repealed diclars exposure lo ir.ni>-
auclliolc in cnlier rals or mice resulted mi hod\ weight loss and inductiou of hepatic c\ loclironic l'45<> and IM4N
acli\ it\. increased ganinia-glutaim limusfcrasc and alanine aminotransferase acli\ it\ and cholesterol lc\ els
Microscopic examination rc\ calcd effects on the h\ er ir.m>- \uclholc did not affect the rcproducli\ e performance
of rats in a 4-gciicraliou rcproducli\e studs \ dc\ clopnicuial lo\icil> studs indicated that //¦.///.N-aiiclholc produced
maternal and fetal lo\icil> Decreased mean hods weight and Iced consumption were seen mi dams l'cloto\icil>
was apparent as decreased I in c-horu pups, increased stillborn pups, decreased Inter si/c and decreased pup weight
mm>- \iiclholc was niiiiagcuic iu sc\cral in viini assavs using haclcrial and mammalian cells ir,m>- \iiclholc and
auclholc (isomer unspecified) did not induce chroniosonial aberrations mi in viim and in \ i\ " assa\ s. respecti\ cl>
flic poicuiial health lia/ard of auclholc (isomer uiispccilicd) and or //¦.///.N-aiielhole is niodcratc hascd on rcpcalcd-
dose and dc\ clopnicuial lo.\icil> \\ ailahlc dala indicate //¦.///.N-aiiclholc has the potential to he gcuoto\ic
\o dala gaps ha\c heen idcntilicd under the 111 J\ ( li;i I lenge I'rograni	
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1. Physical-Chemical Properties and Environmental Fate
A summary of physical-chemical properties and environmental fate data submitted is provided in the Appendix. For
the purposes of the screening-level hazard characterization, the review and summary of these data was limited to the
octanol-water partition coefficient and biodegradation endpoints as indictors of bioaccumulation and persistence,
respectively.
Octanol-Water Partition Coefficient
Anethole (isomer unspecified) (CAS No. 104-46-1)
LogKow: 3.11 (estimated)
trans-Anethole (CAS No. 4180-23-8)
LogKow: 3.39 (estimated)
Biodegradation
Anethole (isomer unspecified) (CAS No. 104-46-1)
In a ready biodegradation test using secondary effluent from an unacclimatized activated sludge as inoculum, 91%
of the test substance degraded after 28 days.
Anethole (isomer unspecified) is readily biodegradable.
Conclusion: The log Kow of anethole (isomer unspecified) and /raws-anethole indicates that their potential to
bioaccumulate is expected to be low. Anethole (isomer unspecified) is readily biodegradable, indicating that it is not
expected to persist in the environment.
2. Environmental Effects - Aquatic Toxicity
Acute Toxicity to Fish
trans-Anethole (CAS No. 4180-23-8)
Fathead minnows (Pimephalespromelas) were exposed to measured concentrations of /nmv-anctholc at 0, 0.06,
2.73, 3.96, 5.85, 10.1 and 17.2 mg/L under flow-through conditions for 96 hours.
96-h LCS0= 7.69 mg/L
Acute Toxicity to Aquatic Invertebrates
trans-Anethole (CAS No. 4180-23-8)
Daphnia magna were exposed to measured concentrations of /raws-anethole at 0, 0.06, 2.84, 5.42, 7.13, 10.9 and
14.5 mg/L under flow-through conditions for 48 hours.
48-h LCS0=6.82 mg/L
Toxicity to Aquatic Plants
trans-Anethole (CAS No. 4180-23-8)
Green algae (Pseudokirchneriella subcapitata) were exposed to /raws-anethole at stock dilutions of 0, 12.5, 25, 50
and 100% under static conditions for 96 hours.
96-h LCS0 = 9.57 mg/L
Conclusion: The evaluation of available aquatic toxicity data for fish, aquatic invertebrates and aquatic plants
indicates that the potential acute hazard of /raws-anethole to aquatic organisms is moderate.
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3. Human Health Effects
Acute Oral Toxicity
Anethole (isomer unspecified) (CAS No. 104-46-1)
(1)	Osborne-Mendel rats (5/dose) were orally administered doses (not specified) of anethole (isomer unspecified)
via gavage. Clinical signs included were depression at low doses and coma at high doses. Time of death was
between 4 hours and 4 days.
LDS0 = 2090 mg/kg-bw
(2)	Groups of mice were orally administered doses (not specified) of anethole (isomer unspecified) via gavage.
Clinical signs included were depression and coma within 15 minutes. Time of death was between 2 and 4 hours.
LDS0 = 3050 mg/kg-bw
(3)	Groups of guinea pigs were orally administered doses (not specified) of anethole (isomer unspecified) via
gavage. Clinical signs included were depression. Time of death was between 1 and 7 days.
LDS0 = 2160 mg/kg-bw
trans-Anethole (CAS No. 4180-23-8)
(1)	Male rats (12/dose group) were orally administered doses (unspecified) of /raws-anethole via gavage.
LDS0 = 3200 mg/kg-bw
(2)	Male mice (12/dose group) were orally administered doses (unspecified) of /raws-anethole via gavage.
LDS0 = 5000 mg/kg-bw
Repeated-Dose Toxicity
Anethole (isomer unspecified) (CAS No. 104-46-1)
In a hepatic microsomal enzyme study, female Sprague-Dawley rats (7/dose) were orally administered anethole
(isomer unspecified) via gavage at 0, 75 or 300 mg/kg-bw/day in corn oil for 4 days. On the 5th day, body weights
were taken, rats were killed and livers were removed and homogenized. The homogenate was centrifuged and the
supernatant (S9) was used to determine cytochrome P450 and P448 activity. There were no statistically significant
differences in body weight or absolute and relative liver weight in treated rats compared to controls. At 300 mg/kg-
bw/day, ethoxyresorufin O-demethylation (EROD, cytochrome P450) and p-nitroanisole O-demethylation (PNAS,
cytochrome P448) activities were increased significantly (p < 0.05 to p < 0.005).
LOAEL = 300 mg/kg-bw/day (based on induction of hepatic cytochrome P450 and P448 activity)
NOAEL = 75 mg/kg-bw/day
trans-Anethole (CAS No. 4180-23-8)
(1) Sprague-Dawley rats (5/sex/dose) were administered /raws-anethole at 0, 150, 300, 600, 900 or 1200 mg/kg-
bw/day via the diet for 28 days. No treatment-related effects were noted at 150 or 300 mg/kg-bw/day except for
decreased feed consumption due to the poor palatability of diet. At 600 mg/kg-bw/day, decreased feed consumption
was noted in the early part of the study. Increased relative brain with stem weight (males only), increased relative
liver weight (females only), decreased relative (to brain) kidney and thymus weights (males only) and decreased
serum triglycerides in males were also noted. At 900 mg/kg-bw/day, animals exhibited decreased feed consumption
in the early part of the study. Decreases in terminal body weights (males only), absolute kidney and liver weights
(males only), absolute adrenal weight (females only), relative (to brain) adrenal weight (females only), serum
triglycerides (males), inorganic phosphorus values (males only) and cytoplasmic clearing (pallor) of hepatocytes in
centrilobular to midzonal regions were noted. Increases in relative brain with stem weight (males only), relative
liver weight (females only), gamma-glutamyltransferase and total cholesterol (females only) were noted.
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At 1200 mg/kg-bw/day, males and females showed a decrease in feed consumption, lower organ weights and
decreases in hematological parameters. Serum biochemistry changes included increased gamma-
glutamyltransferase (male and female), increased alanine aminotransferase (males), increased triglycerides (males)
and increased total cholesterol (females). Cytoplasmic clearing (pallor) of hepatocytes in centrilobular to midzonal
regions was noted in both sexes. Female rats had decreased relative (to brain) adrenal weight.
LOAEL = 600 mg/kg-bw/day (based on hepatic effects as shown in serum biochemistry results and microscopic
examination)
NOAEL = 300 mg/kg-bw/day
(2)	Mice (5/sex/dose) were administered /raws-anethole at 0, 60, 120, 240, 360 or 500 mg/kg-bw/day via the diet for
28 days. Animals were observed for clinical signs and changes in body weight and food consumption. Hematology
and serum chemistry evaluations did not reveal any changes. No treatment-related histomorphological changes were
observed in the liver at any dose. At 240 mg/kg-bw/day and above, some mice stopped eating and died. At 240
mg/kg-bw/day, body weight decreases and mortality in males were reported (mortality = 40 and 60% at 240 and 360
mg/kg-bw/day, respectively). At 500 mg/kg-bw/day, mortality in males and females was 40%. On day 29,
decreased body weights were noted (84.5% and 81% of controls for males and females). Leukocyte counts were
decreased at 360 and 500 mg/kg-bw/day.
LOAEL (male) = 240 mg/kg-bw/day (based on decreased body weight)
NOAEL (male) = 120 mg/kg-bw/day
LOAEL (female) = 500 mg/kg-bw/day (based on decreased body weight)
NOAEL (female) = 360 mg/kg-bw/day
(3)	CD-I mice (20/sex/dose) were administered /raws-anethole at 0, 30, 60, 120 or 240 mg/kg-bw/day via the diet
for 90 days. Severe loss of body weight and dehydration at 120 mg/kg-bw/day and higher were attributed to
inanition syndrome (starving) resulting from the poor palatability of the diet and reduced food intake. Enlarged
livers, increased liver weight and increased incidence of centrilobular hepatocellular hypertrophy were considered
adaptive physiological responses. Increased serum alkaline phosphatase was considered an adaptive response
related to reduced feed intake.
LOAEL = 120 mg/kg-bw/day (based on severe body weight loss due to non-palatability of food)
NOAEL = 60 mg/kg-bw/day
(4)	In a chronic toxicity/carcinogenicity dietary study, rats (52-78/sex/dose) were administered /raws-anethole at 0,
0.25, 0.5 or 1.0% in the diet for up to 117-121 weeks. The actual doses received were 0, 100, 200 or 400 mg/kg-
bw/day for males and 0, 120, 250 or 550 mg/kg-bw/day for females. An additional group of rats (26/sex) was
administered 1% /raws-anethole until week 54 and then received basal diet only until the end of the study. Between
weeks 42 and 45, most rats showed signs of sialodacryoadenitis (a disease of rats caused by the rat
sialodacryoadenitis virus and characterized by a severe self-limiting inflammation and necrosis of the salivary and
nasolacrimal glands) resulting in transient retardation of body weight gain. All treated groups showed lower body
weight gains. The reversal group showed no difference in body weight gain compared to controls by the end of the
study. Mortality was increased in females receiving 1% /raws-anethole (mortality results by dose and sex were not
provided in the robust summary). Reduced adiposity was reported in high-dose rats, particularly males, but was
considered an indirect effect of the poor palatability of the treated diet and decreased feed consumption. No effect
on hematological parameters was noted. Effects on the liver were sinusoidal dilatation (at 200/250 and 400/550
mg/kg-bw/day); nodular hyperplasia (at 200 and 400 mg/kg-bw/day in males and 550 mg/kg-bw/day in females) and
hepatocytic hypertrophy (at 250 and 550 mg/kg-bw/day in females).
LOAEL (male) = 200 mg/kg-bw/day (based on liver effects)
NOAEL (male) = 100 mg/kg-bw/day
LOAEL (female) = 250 mg/kg-bw/day (based on liver effects)
NOAEL (female) = 120 mg/kg-bw/day
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Reproductive Toxicity
trans-Anethole (CAS No. 4180-23-8)
(1)	In a 4-generation reproduction study, Wistar SPF rats (20/sex) were administered 0 or 1% ;ra«.v-anethole in the
diet (approximately 600 - 1500 mg/kg-bw/day) for 70 days prior to mating. Mortality, body weight, food
consumption and reproductive performance (fertility, sex ratio, date of birth, stillbirths, clinical observations, litter
size, litter viability) were monitored. /raws-Anethole did not affect the reproductive performance of rats over four
generations.
In a cross-fostering experiment, groups of six control and six treated F1 female rats (receiving 1% /nmv-anctholc in
the diet) were mated with control F1 male rats (from 4-generation portion of study). Litters born from treated
females were exchanged with litters from control females at birth and reared by the new dams. Body weight and
growth of pups were monitored. No significant difference was noted in body weights of pups nursed by mothers of
the same group and those that were exchanged and nursed by mothers from different groups. Final body weights of
pups born from treated dams but raised by control dams regained normal values by day 28. No NOAEL or LOAEL
could be assigned because only one dose was tested. No reproductive effects were noted at the tested dose.
(2)	In a developmental/reproductive toxicity screening study, female Sprague-Dawley rats (10/dose) were
administered /raws-anethole in corn oil via gavage at 0, 35, 175 or 350 mg/kg-bw/day for 7 days prior to
cohabitation with male rats. Body weight and feed consumption were monitored. Fertility, gestation index,
implantation sites, length of gestation, number of stillborn pups, litter size, pup viability, pup weight, and clinical
observations of pups were recorded. At 350 mg/kg-bw/day, there was a significant reduction in mean body weight
and feed consumption throughout study. The number of dams with stillborn pups was markedly increased and all
pups died before postpartum day 4. At 175 mg/kg-bw/day, dams showed a marked decrease in mean body weight
on gestation days 6 and 14 and reduced feed consumption during premating days 1-8 but not during gestation.
LOAEL (systemic toxicity) = 175 mg/kg-bw/day (based on body weight effects and reduced number of live born
pups)
NOAEL (systemic toxicity) = 35 mg/kg-bw/day
LOAEL (reproductive toxicity) = 175 mg/kg-bw/day (based on reduced number of live-born pups)
NOAEL (reproductive toxicity) = 35 mg/kg-bw/day
Developmental Toxicity
trans-Anethole (CAS No. 4180-23-8)
In the developmental/reproductive toxicity screening study described previously, at 350 mg/kg-bw/day fetal toxicity
was evident as a significant decrease in number of live born pups (p < 0.01) and significant increase in number of
stillborn pups (p < 0.01) compared to controls. The pup weight/litter on postpartum day 1 was significantly (p <
0.05) decreased compared to controls. On postpartum day 4, the live litter size and the number of surviving
pups/litter were significantly decreased compared to controls (p < 0.05 and p < 0.01, respectively). The viability
index (number of live pups on postpartum day 4/number of live born pups on postpartum day 1) was significantly (p
< 0.01) decreased compared to controls. No other effects or anomalies were reported at the other doses.
NOAEL (maternal) = 35 mg/kg-bw/day
LOAEL (maternal) = 175 mg/kg-bw/day (based on decreased mean body weight and feed consumption, decreased
live-born pups)
NOAEL (developmental toxicity) = 175 mg/kg-bw/day
LOAEL (developmental toxicity) = 350 mg/kg-bw/day (based on decreased live-born pups, increased stillborn
pups, decreased litter size, decreased pup weights)
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Genetic Toxicity - Gene Mutation
In vitro
Anethole (isomer unspecified) (CAS No. 104-46-1)
In two in vitro bacterial reverse mutation assays, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537
and TA1538 were exposed to anethole (isomer unspecified) up to 200 |ig/plate. with and without S9 metabolic
activation.
Anethole (isomer unspecified) was not mutagenic in these assays.
trans-Anethole (CAS No. 4180-23-8)
(1)	In nine in vitro bacterial reverse mutation assays, /raws-anethole was tested up to 600 |ig/plate. with and without
S9 or S13 metabolic activation (one assay included cofactor PAPS), using S. typhimurium TA98, TA100, TA1535,
TA1537 and TA1538 strains. /nmv-Anctholc was not mutagenic, with and without metabolic activation in S.
typhimurium strains TA98, TA1537 and TA1538 in all nine assays. In two assays, /nmv-anctholc induced revertants
in S. typhimurium TA100 in the presence of S9 activation. /raws-Anethole also induced revertants in S. typhimurium
strain TA1535 when cofactor PAPS was added.
fraws-Anethole was mutagenic in three of the nine assays.
(2)	In two in vitro reverse mutation assays using Escherichia coli WP2 uvrA trp- strain, with and without metabolic
activation, and Saccharomyces cerevisiae, only without activation, /raws-anethole was not mutagenic.
fraws-Anthole was not mutagenic in these assays.
(3)	In two in vitro mouse lymphoma assays, /nmv-anctholc produced an increase in mutagenic activity only with S9
metabolic activation.
fraws-Anethole was mutagenic in these assays.
Genetic Toxicity - Chromosomal Aberrations
In vitro
trans-Anethole (CAS No. 4180-23-8)
/nmv-anctholc. when tested in Chinese hamster cells up to 0,2|iL/mL. did not produce a significant increase in the
percentage of cells with chromosomal aberrations with or without metabolic activation. Positive controls were used
in the assay but the responses were not provided in the robust summary.
fraws-Anethole did not induce chromosomal aberrations in this assay.
In vivo
Anethole (isomer unspecified) (CAS No. 104-46-1)
In an in vivo micronucleus assay, groups of male Swiss albino mice were gavaged with anethole (isomer
unspecified) at 0, 250, 500 or 1000 mg/kg-bw/day for 7 days. Anethole (isomer unspecified) did not increase the
polychromatic erythrocyte (PCE)/normochromatic erythrocyte (NCE) ratio.
Anethole (isomer unspecified) did not increase micronuclei in this assay.
Genetic Toxicity - Other
DNA Effects
In vitro
Anethole (isomer unspecified) (CAS No. 104-46-1)
(1)	In two in vitro unscheduled DNA synthesis (UDS) assays using Fisher 344 male rat hepatocytes, with and
without metabolic activation, anethole (isomer unspecified) did not induce unscheduled DNA effects when tested at
concentrations ranging from 10"6 to 10"2M. Cytotoxic concentration was 10"3 M.
Anethole (isomer unspecified) was not mutagenic in these assays.
(2)	In an in vitro bacterial DNA repair assay using Bacillus subtilis, with and without metabolic activation, anethole
(isomer unspecified) was not mutagenic.
Anethole (isomer unspecified) was not mutagenic in this assay.
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trans-Anethole (CAS No. 4180-23-8)
In four in vitro UDS assays using Fisher 344 or Sprague-Dawley rat hepatocytes, with and without metabolic
activation, /ram-anctholc was not mutagenic.
fraws-Anethole was not mutagenic in these assays.
In vivo
trans-Anethole (CAS No. 4180-23-8)
In an in vivo UDS assay, groups of female SD-CD rats were gavaged with /nmv-anctholc at 0, 1, 125 or 500 mg/kg-
bw in trioctanoin. Sixteen hours following treatment, rats were anaesthetized and hepatocytes were isolated and
seeded. No UDS response was observed at any dose level.
fraws-Anethole was not mutagenic in this assay.
Additional Information
Carcinogenicity
trans-Anethole (CAS No. 4180-23-8)
In the chronic toxicity/carcinogenicity dietary study described previously, (under Repeated-Dose Toxicity), the only
statistically significant finding in neoplastic lesions was an increase in the incidence of liver tumors in 550 mg/kg-
bw/day dosed females. The authors noted that the increased incidence of hepatocellular carcinomas reported in
high-dose females were "late onset", had no effect on longevity and was still within the range of historical controls.
Conclusion: Acute oral toxicity of anethole (isomer unspecified) and /nmv-anctholc is low. Repeated dietary
exposure to /raws-anethole in either rats or mice resulted in body weight loss and induction of hepatic cytochrome
P450 and P448 activity, increased gamma-glutamyltransferase and alanine aminotransferase activity and cholesterol
levels. Microscopic examination revealed effects on the liver. /raws-Anethole did not affect the reproductive
performance of rats in a 4-generation reproductive study. A developmental toxicity study indicated that trans-
anethole produced maternal and fetal toxicity. Decreased mean body weight and feed consumption were seen in
dams. Fetotoxicity was apparent as decreased live-born pups, increased stillborn pups, decreased litter size and
decreased pup weight. /nmv-Anctholc was mutagenic in several in vitro assays using bacterial and mammalian
cells. /raws-Anethole and anethole (isomer unspecified) did not induce chromosomal aberrations in in vitro and in
vivo assays, respectively.
The potential health hazard of anethole (isomer unspecified) and/or /nmv-anctholc is moderate based on repeated-
dose and developmental toxicity. Available data indicate /nmv-anctholc has the potential to be genotoxic.
4. Hazard Characterization
The log Kow of anethole (isomer unspecified) and /ram-anctholc indicates that their potential to bioaccumulate is
expected to be low. Anethole (isomer unspecified) is readily biodegradable, indicating that it is not expected to
persist in the environment.
The evaluation of available toxicity data for fish, aquatic invertebrates and aquatic plants indicates that the potential
acute hazard of /nmv-anctholc to aquatic organisms is moderate.
Acute oral toxicity of anethole (isomer unspecified) and /raws-anethole is low. Repeated dietary exposure to trans-
anethole in either rats or mice resulted in body weight loss and induction of hepatic cytochrome P450 and P448
activity, increased gamma-glutamyltransferase and alanine aminotransferase activity and cholesterol levels.
Microscopic examination revealed effects on the liver. /nmv-Anctholc did not affect the reproductive performance
of rats in a 4-generation reproductive study. A developmental toxicity study indicated that /raws-anethole produced
maternal and fetal toxicity. Decreased mean body weight and feed consumption were seen in dams. Fetotoxicity
was apparent as decreased live-born pups, increased stillborn pups, decreased litter size and decreased pup weight.
/nmv-Anctholc was mutagenic in several in vitro assays using bacterial and mammalian cells. /raws-Anethole and
anethole (isomer unspecified) did not induce chromosomal aberrations in in vitro and in vivo assays, respectively.
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The potential health hazard of anethole (isomer unspecified) and/or /nmv-anctholc is moderate based on repeated-
dose and developmental toxicity. Available data indicate /nmv-anctholc has the potential to be genotoxic.
5. Data Gaps
No data gaps have been identified under the HPV Challenge Program.
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APPENDIX
Summary Tabic of the Screening Information Data Set
as Submitted under the U.S. HPV Challenge Program
Endpoints
SPONSORED CHEMICAL
Anctholc (isomer unspecified)
(104-46-1)
SPONSORED CHEMICAL
frw/.v-Anctholc
(4180-23-8)
Structure



ch3
°T"1
CH3
°
W CH3
Melting Point (°C)
21.3
21.4
Boiling Point (°C)
234
236
Vapor Pressure
(hPa at 25°C)
0.041 (at21°C)
0.0634
Log K„w
3.11
3.39
Water Solubility
(mg/L at 25°C)
111 (estimated)
139.8 (estimated)
Indirect (OH) Photodegradation
Half-life (t1/2)

2.015 hours
Stability in Water (Hydrolysis) (ti/2)
Anethole (isomer unspecified) is
expected to be stable in aqueous
solution.
/rcw/.v-Anctholc is expected to be
stable in aqueous solution.
Fugacity
(Level III Model)
Air (%)
Water (%)
Soil (%)
Sediment (%)

0.53
29.8
69.1
0.60
Biodegradation at 28 days (%)
91
Readily biodegradable
—
Fish
96-h LCS0 (mg/L)

7.69
Aquatic Invertebrates
48-h ECS0 (mg/L)

6.82
Aquatic Plants
72-h ECS0 (mg/L)

9.571 (96-h)
Acute Oral Toxicity
LDS0 (mg/kg-bw)
2090 - 3050
2900 - 5900
Repeated-Dose Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)

NOAEL = 60
1 .OA F.I. = 120
NOAEL = 100 - 120 (chronic)
LOAEL = 200 - 250 (chronic)
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Summarv Tabic of the Screening Information Data Set
as Submitted under the U.S. HPV Challenge Program
Endpoints
SPONSORED CHEMICAL
Anctholc (isomer unspecified)
(104-46-1)
SPONSORED CHEMICAL
frw/.v-Anetholc
(4180-23-8)
Reproductive Toxicity
NOAEL/LOAEL
(mg/kg-bw/day)
—
NOAEL = 35
1 .OA F.I. = 175
Developmental Toxicity
NOAEL/LOAEL
(mg/kg-bw/day)
Maternal and Developmental toxicity
—
NOAEL = 35
1 .OA F.I. = 175
Genetic Toxicity - Gene Mutation
In vitro
Negative
Positive
Genetic Toxicity - Chromosomal
Aberrations
In vitro
—
Negative
Genetic Toxicity - Chromosomal
Aberrations
In vivo
Negative
—
Genetic Toxicity - Other
DNA Effects
In vitro
Negative
Negative
Genetic Toxicity - Other
DNA Effects
In vivo
—
Negative
— indicates that endpoint was not addressed for this chemical.
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