Center for Food Safety and Applied Nutrition 5100 Paint ...
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Page 1 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
CENTER FOR SCIENCE IN THE PUBLIC INTEREST, NATURAL RESOURCES DEFENSE
COUNCIL, CENTER FOR FOOD SAFETY, CONSUMERS UNION, IMPROVING KIDS’
ENVIRONMENT, CENTER FOR ENVIRONMENTAL HEALTH, AND ENVIRONMENTAL
WORKING GROUP, JAMES HUFF
July 30, 2015
Dr. Dennis Keefe
Director of the Office of Food Additive Safety (HFS-200)
Center for Food Safety and Applied Nutrition
5100 Paint Branch Parkway
College Park, MD 20740
Re: Revised Food Additive Petition FAP 5A4810 submitted pursuant to 21 USC § 348
seeking amended food additive regulation to: 1) remove FDA’s approval at 21 CFR
§ 172.515 of seven synthetic flavors; and 2) add to that section a prohibition on use of
these seven flavors because all have been found by the National Toxicology Program to
induce cancer in man or animal. REPLACES JULY 28, 2015 SUBMISSION TO
INCLUDE A REVISED ENVIRONMENTAL REVIEW COMPONENT.
Dear Dr. Keefe:
Since 1958, federal law has stated that “no additive shall be deemed to be safe if it is found to
induce cancer when ingested by man or animal, or if it is found, after tests which are appropriate
for the evaluation of the safety of food additives, to induce cancer in man or animal . . .” (21
U.S.C. § 348(c)(3)(A)). This requirement, known as the Delaney Clause in honor of its
Congressional author, is a bright line drawn by Congress on what is not safe in food with respect
to carcinogens. This statutory requirement for food additives has not been altered in the
intervening half-century.
We hereby submit this food additive petition to the Food and Drug Administration (FDA),
pursuant to 21 USC § 348, to remove its approval of seven synthetic flavors or adjuvants from 21
CFR § 172.515 because they are not safe for use in food pursuant to the Delaney Clause. Each
has been found by the Department of Health and Human Services’ (HHS) National Toxicology
Program (NTP) to induce cancer in man or animal using tests done consistent with FDA’s
guidance1 for toxicology studies for food ingredients.2
We also petition FDA to explicitly establish a zero tolerance in 21 CFR § 172.515 for the use of
these seven flavors. The seven synthetic flavors are:
1 FDA, Guidance for Industry and Other Stakeholders: Toxicology Principles for the Safety Assessment of Food
Ingredients (Redbook 2000), 2007. Chapter IV.C.6. Accessed May 23, 2015. See
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/IngredientsAdditivesGR
ASPackaging/ucm2006826.htm. 2 See Appendix 1 and 3 for details.
Page 2 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
1. Benzophenone (also known as diphenylketone);
2. Ethyl acrylate;
3. Eugenyl methyl ether (also known as 4-allylveratrole or methyl eugenol);
4. Myrcene (also known as 7-methyl-3-methylene-1,6-octadiene);
5. Pulegone (also known as p-menth-4(8)-en-3-one);
6. Pyridine; and
7. Styrene.
For chemicals whose addition to food would violate the Delaney Clause, a zero tolerance is the
only appropriate condition of use consistent with 21 U.S.C. § 348. The explicit prohibition is
essential because their use has been allowed by FEMA3 for more than 40 years in food and
clarity is necessary to prevent their continued use. Mere revocation of approved food additive
status would be insufficient under current law and rules to assure that companies would stop
using them pursuant to a private GRAS determination regarding a particular condition of use.
For reasons elaborated upon below, a GRAS determination for carcinogens would be manifestly
an abuse of the statute. Nonetheless, it occurs as a matter of regulatory practice, as the FEMA
designations demonstrate. It is, therefore, appropriate – and even necessary – to use the food
additive petition process to set a zero tolerance for substances deemed carcinogenic to prevent
any trade association or individual food manufacturers from continuing to claim they are safe.4
FDA approved the seven food additives in 1964 and set no numerical limit on how much may be
used in food.5 The only limit is Good Manufacturing Practices at 21 CFR § 172.515(a), meaning
the additives are used in the minimum quantity required to produce their intended flavoring
effect and otherwise in accordance with all the principles of good manufacturing practice.
Shortly after FDA’s approval, FEMA designated the same flavors as GRAS, giving them a
FEMA number and describing average maximum use level in various foods considered by the
trade association to be safe. See Appendix 1 and 3.
After FDA determined that these seven synthetic flavor additives were safe, NTP found that each
induced cancer in man or animal when ingested.6 NTP’s findings were based on ingestion studies
done consistent with FDA’s Redbook.7 NTP made these findings pursuant to a Congressional
3 As noted in Appendix 1 and 3, shortly after FDA approved the seven synthetic flavors as food additives, FEMA
designated them as GRAS and set numerical limits for their use in food and beverage. 4 Nothing in the statute or regulations limits the use of a food additive petition to prohibit a substance’s specific uses
in food. The statute at 21 U.S.C. § 348(a) states that “A food additive shall, with respect to any particular use or in
intended use of such additives, be deemed unsafe for the purposes of the application of clause (2)(C) of section
402(a) . . . .” In addition, 21 CFR § 170.38(c) refers to “discontinuation of the use of the additive” as an option when
FDA determines that a substance is a food additive. It would be illogical for FDA to assert that a food additive
petition is permissible to restrict the use of a substance in food but is not allowed to establish a zero tolerance for the
same substance simply because the agency has chosen for administrative convenience to prohibit some food
additives in a different part of its regulations than those used for most food additives. FDA’s decision to identify
substances prohibited from use in human food at 21 CFR Part 189 as something other than food additives was for its
administrative convenience in the same way FDA lists prohibited substances in its Everything Added to Food in the
United States (EAFUS) database. 5 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964). 6 See Appendix 1 and 3 for details. 7 FDA, Guidance for Industry and Other Stakeholders: Toxicology Principles for the Safety Assessment of Food
Ingredients (Redbook 2000), 2007. Chapter IV.C.6. Accessed May 23, 2015. See
Page 3 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
directive at 42 U.S.C. § 241 to the HHS Secretary to conduct these types of tests. The Secretary
established NTP to perform this work.
Congress also mandated at 42 U.S.C. § 241(b)(4) that the Secretary publish a biennial report
listing substances: 1) which are known to be carcinogens or may reasonably be anticipated to be
carcinogens, and 2) to which a significant number of persons residing in the United States are
exposed. With the Secretary’s approval, NTP has designated two of these seven synthetic flavors
additives, methyl eugenol and styrene, as “reasonably anticipated to be a human carcinogen” in
its biennial report known as the Report on Carcinogens.8
Other recognized authorities, such as the International Agency for Research on Cancer (IARC)
and California Environmental Protection Agency’s Office of Environmental Health Hazard
Assessment (OEHHA), also evaluated some of the seven synthetic flavors and like NTP found
evidence to conclude that the flavors should be considered to induce cancer in man or animal.
• IARC designated five of the seven substances as Group 2B carcinogens9 based primarily
on the finding that they induce cancer in animals (see Appendix 1 and 3) and is
evaluating a seventh (beta-myrcene).10 IARC is the science program of the World Health
Organization (WHO) that was launched in 1965 to provide critical reviews and
evaluations of evidence on the carcinogenicity of a wide range of human exposures and
publishes its designations in Monographs on the Evaluation of Carcinogenic Risks to
Humans.11 The U.S. President’s Cancer Panel described IARC’s monographs on
carcinogenesis as “the ‘gold standard’ in evaluating evidence on cancer-causation . . .” 12
• OEHHA designated six of the seven flavors as carcinogens (see Appendix 1 and 3) and
required warning to consumers as part of the Safe Drinking Water and Toxic
Enforcement Act of 1986 (also known as Proposition 65).13 In 2015, OEHHA proposed
listing the seventh flavor (styrene) as a carcinogen.14
Based on the above conclusions by recognized authorities responsible for determining whether a
substance is found to induce cancer when ingested by man or animal, FDA should remove its
approval for these seven additives and prescribe a zero tolerance for them.
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/IngredientsAdditivesGR
ASPackaging/ucm2006826.htm. 8 NTP, Report on Carcinogens, Thirteenth Edition, 2014. See
http://ntp.niehs.nih.gov/pubhealth/roc/roc13/index.html. 9 See Appendix 1 and 3 for details. 10 IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, INTERNAL REPORT 14/002, Report of
the Advisory Group to Recommend Priorities for IARC Monographs during 2015–2019, 2014.
http://monographs.iarc.fr/ENG/Publications/internrep/14-002.pdf. 11 IARC, Preamble to the Monographs, accessed on May 23, 2015 at
http://monographs.iarc.fr/ENG/Preamble/currenta1background0706.php 12 The President’s Cancer Panel, Reducing Environmental Cancer Risk: What We Can Do Now, 2008-2009 Annual
Report of the President’s Cancer Panel, 2010. See http://deainfo.nci.nih.gov/advisory/pcp/annualReports/pcp08-
09rpt/PCP_Report_08-09_508.pdf. 13 OEHHA, Proposition 65, accessed on May 23, 2015 at http://www.oehha.ca.gov/prop65.html. 14 OEHHA, Proposition 65, Notice of Intent to List: Styrene, 2015. See
http://www.oehha.ca.gov/prop65/CRNR_notices/admin_listing/intent_to_list/noilstyrene2015.html.
Page 4 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
We note that, under the law, there is no reason for FDA to conduct its own hazard analysis of the
carcinogenicity of these substances given this clear body of evidence, including conclusions
from NTP, FDA’s sister program within HHS. FDA’s analysis should be limited to determining
whether the NTP study protocols were “tests appropriate for the evaluation of the safety of food
additives” under the Delaney Clause. (21 U.S.C. § 348(c)(3)(A)) Since they are consistent with
the Redbook, we maintain that this evaluation could be done quickly. Therefore, a de novo
investigation is unnecessary and could delay the agency taking action within the 90-day statutory
deadline. If FDA decides to conduct its own hazard analysis or determines that the NTP study
protocols are inappropriate, we request notification in writing from the agency. Such notice
should be made no later than in the letter sent 90 days after the agency files the petition.
Our reliance on respected recognized authorities has precedent within FDA. With the unanimous
support of FDA’s Tobacco Products Scientific Advisory Committee, the agency’s Center for
Tobacco Products classifies chemicals as Hazardous / Potentially Hazardous Constituents
(HPHC) using similar criteria. For instance, the committee recommended chemicals be
considered carcinogens if they are listed as:
• IARC Group 1 (carcinogenic to humans) or 2A (probably carcinogen to humans) or 2B
(possibly carcinogenic to humans);
• Environmental Protection Agency (EPA) known, likely, probable, or possible human
carcinogen; or
• NTP human carcinogen or reasonably anticipated to be a human carcinogen.15
FDA also relies on these authorities with respect to food additives. For example, FDA’s Office
of Food Additive Safety relied on an NTP toxicology and carcinogenesis study on ginkgo biloba
leaf extract in a warning letter issued to a food manufacturer finding that the extract was an
unapproved food additive.16
With this food additive petition, we ask that FDA amend 21 CFR § 172.515 to explicitly prohibit
the use of these seven carcinogens in food.
Appendix 1 contains the list of the seven synthetic flavor additives providing: 1) the additive
name; 2) the Chemical Abstract Service (CAS) No.; 3) FEMA No. and year of evaluation by the
FEMA Expert Panel; 4) uses and average maximum use level considered by FEMA to be safe;
and 5) authority designating the flavor as a carcinogen and year of designation sorted with most
recent first.
15 FDA, Tobacco Products Scientific Advisory Committee, Summary of minutes of August 30, 2010. See
http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/TobaccoProductsScientificAdvi
soryCommittee/UCM233611.pdf. See also FDA, Criteria developed for identifying an initial list of harmful and
potentially harmful (H/PH) constituents in tobacco products or tobacco smoke and Summary of major
recommendations of the Constituent subcommittee. See
http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/TobaccoProductsScientificAdvisoryCommi
ttee/ucm180903.htm. 16 FDA, Warning Letter SEA 13-15 to Stewart Brothers, 2013,
http://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm346316.htm.
Page 5 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Appendix 2 provides additional details on the petition required by 21 CFR Part 172; Appendix 3
supplies relevant reports on the carcinogenicity of the flavors; and Appendix 4 presents the
specific changes we seek in the regulation. This letter, all appendices, and materials provided on
a CD-ROM constitute our complete food additive petition.17 We have enclosed three copies per
21 CFR § 171.1. This petition contains no confidential information, so we ask that FDA include
it in the docket for any regulatory action it takes so the public can assess the information.
If FDA grants this petition, it will have a positive impact on the environment and public health
by reducing exposure to these seven non-essential substances. No flavor or flavor extract is
essential. With more than 2000 additional flavor additives allowed in food, the industry can
either eliminate the flavor without substitution or find a safer alternative.
If you have questions or comments, please contact Tom Neltner, our agent on this petition, at
[email protected] or 317-442-3973, and copy Erik D. Olson at [email protected], Dr. Maricel
Maffini at [email protected], and Laura MacCleery at [email protected] on all responses.
Sincerely,
Laura MacCleery, Director of Regulatory Affairs
Lisa Lefferts, Senior Scientist
Center for Science in the Public Interest
1220 L St. N.W., Suite 300
Washington, D.C. 20005
Erik D. Olson, Director, Health & Environment Program
and Senior Strategic Director for Food and Health
Natural Resources Defense Council
1152 15th St. NW, Suite 300
Washington, DC 20005
Caroline Cox, Research Director
Center for Environmental Health
2201 Broadway, Suite 302
Oakland, CA 94612
Scott Faber, Vice President for Government Relations
Renee Sharp, Director of Research
Environmental Working Group
1436 U St. NW, Suite 100
Washington, DC 20009
17 Please note that this is NOT a citizen petition.
Page 6 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Cristina Stella, Staff Attorney
Center for Food Safety
303 Sacramento Street, Second Floor
San Francisco, CA 94111
Urvashi Rangan, Executive Director for Food Safety and Sustainability
Consumers Union
101 Truman Avenue
Yonkers, NY 10703-1057
Delores E. Weis, Executive Director
Improving Kids’ Environment (IKE)
1915 W. 18th Street
Indianapolis, Indiana 46202
Maricel Maffini, Ph.D., Consulting Senior Scientist
James Huff, PhD
Formerly, Associate Director for Chemical Carcinogenesis, National Institute of Environmental
Health Sciences
Page 7 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Appendix 1: Flavors found by NTP to induce cancer in man or animal and
designated as a carcinogen by a recognized authority
Table 1: Flavors designated as a carcinogen by a recognized authority and found by NTP to induce cancer in man or animal Additive name CAS No. FEMA No. & Year
(GRAS Report No.)
Flavor uses and FEMA’s average maximum
use level considered GRAS in parenthesis
Authority and designation year (sorted with most
recent first)
Benzophenone /
Diphenylketone
119-61-9 2134 in 1965
(GRAS 3)a
Baked goods (2.4 ppm); ice cream, ices etc.
(0.61 ppm); beverages (0.5 ppm); and candy
(1.7 ppm)
Calif. Prop. 65 Carcinogen (2012)g / NTP Study
(2006)h concluded it caused cancer in two species /
IARC Possibly Carcinogenic to Humans (2B) (1999)i
Ethyl acrylate 140-88-5 2418 in 1965
(GRAS 3)a
Candy and baked goods (1.1 ppm); beverages
(0.13-0.26 ppm); ice cream, ices etc. (0.06-1
ppm); and chewing gum (0.1 ppm)
IARC Possibly Carcinogenic to Humans (2B) (1999)j
/ Calif. Prop. 65 Carcinogen (1989)k / NTP Study
(1986)l concluded it was carcinogenic in two species.
Eugenyl methyl
ether / 4-
Allylveratrole /
Methyl eugenol.
93-15-2 2475 in 1965
(GRAS 3)a /
Reaffirmed in 2001
(GRAS 20)b /
Reaffirmed in 2002c
Jellies (52 ppm); baked goods (13 ppm);
candy (11 ppm); beverages (10 ppm); and ice
cream, ices etc. (4.8 ppm)
IARC Possibly Carcinogenic to Humans (2B) (2004)m
/ NTP Reasonably Anticipated To Be Human
Carcinogen (2002)n, o / Calif. Prop. 65 Carcinogen
(2001)p / NTP Study (2000)q found clear evidence of
carcinogenic activity in two species.
Myrcene / 7-
methyl-3-
methylene-1,6-
octadiene
123-35-3 2762 in 1965
(GRAS 3)a
Candy (0.50-13 ppm); ice cream, ices etc. (6.4
ppm); baked goods (4.9 ppm); and beverages
(4.4 ppm)
Calif. Prop. 65 Carcinogen (2015)r / NTP Study
(2010)s concluded it caused cancer in two species.
Pulegone / p-
Menth-4(8)-en-
3-one.
89-82-7 2963 in 1965
(GRAS 3)a
Reaffirmed in 2005
(GRAS 22)d
Baked goods (24-25 ppm); candy (17 ppm);
ice cream, ices etc. (5-32 ppm); and beverages
(5-8 ppm)
Calif. Prop. 65 Carcinogen (2014)t / IARC Possibly
Carcinogenic to Humans (2B) (2014)u / NTP Study
(2011)v found clear evidence of carcinogenic activity
in two species.
Pyridine 110-86-1 2966 in 1965
(GRAS 3)a
Reaffirmed in 2011
(GRAS 25)e
Beverages (1 ppm); candy and baked goods
(0.4 ppm); and ice cream, ices etc. (0.02-0.12
ppm);
Calif. Prop. 65 Carcinogen (2002)w / NTP Study
(2000)x found clear evidence of carcinogenicity in one
species.
Styrene 100-42-5 3233 in 1967
(GRAS 4)f
Ice cream, ices etc., candy and baked goods
(0.2 ppm)
NTP Reasonably Anticipated To Be Human
Carcinogen (2011)y, z / IARC Possibly Carcinogenic to
Humans (2B) (2002)aa
Page 8 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Table 1: Flavors designated as a carcinogen by a recognized authority and found by NTP to induce cancer in man or animal Additive name CAS No. FEMA No. & Year
(GRAS Report No.)
Flavor uses and FEMA’s average maximum
use level considered GRAS in parenthesis
Authority and designation year (sorted with most
recent first)
CAS = Chemical Abstract Service
FEMA = Flavor and Extract Manufacturers Association
GRAS = Generally Recognized as Safe
IARC = International Agency for Research on Cancer (part of World Health Organization)
NTP = National Toxicology Program
See next page for references.
Page 9 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
References for Table 1:
a. R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the
Consideration of Flavouring Ingredients Under the Food Additives Amendment: III. GRAS
Substances, (FEMA No. 2000-3124), Food Technology, Vol. 19, No. 2, 1965.
b. FEMA Expert Panel (R.L. Smith, J. Doull, V.J. Feron, J.I. Goodman, I.C. Munro, P.M.
Newberne, P.S. Portoghese, W.J. Waddell, B.M. Wagner, T.B. Adams, and M.M.
McGowen), GRAS Flavouring Substances 20 (FEMA No. 3964-4023), Food Technology,
Vol. 55, No. 12, December 2001.
c. R.L. Smith, T.B. Adams, J. Doull, V.J. Feron, J.I. Goodman, L.J. Marnett, P.S. Portoghese,
W.J. Waddell, B.M. Wagner, A.E. Rogers, J. Caldwell, I.G. Sipes, Safety assessment of
allylalkoxybenzene derivatives used as flavouring substances — methyl eugenol and
estragole, Food and Chemical Toxicology 40 (2002) 851–870.
d. FEMA Expert Panel (R.L. Smith, S.M. Cohen, J. Doull, V.J. Feron, J.I. Goodman, L.J.
Marnett, P.S. Portoghese, W.J. Waddell, B.M. Wagner, and T.B. Adams), GRAS Flavouring
Substances 22 (FEMA No. 4069-4253), Food Technology, Vol. 59, No. 8, August 2005.
e. FEMA Expert Panel (R.L. Smith, W.J. Waddell, S.M. Cohen, S. Fukushima, N.J..
Gooderham, S.S. Hecht, L.J. Marnett, P.S. Portoghese, I.M.C.M. Rietjens, T.B. Adams, C.L.
Gavin, M.M. McGowen, and S.V. Taylor), GRAS Flavouring Substances 25 (FEMA No.
4667-4727), Food Technology, Vol. 65, No. 7, July 2011.
f. R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the
Consideration of Flavouring Ingredients Under the Food Additives Amendment: 4. GRAS
Substances, (FEMA No. 3125-3249), Food Technology, Vol. 19, No. 2, 1965.
g. OEHHA, Chemicals Listed Effective June 22, 2012 As Known To The State Of California
To Cause Cancer: benzophenone (CAS No. 119-61-9), coconut oil diethanolamine
condensate (cocamide diethanolamine) (CAS No. 68603-42-9), diethanolamine (CAS No.
111-42-2), and 2-methylimidazole (CAS No. 693-98-1), June 22, 2012. See
http://oehha.ca.gov/prop65/prop65_list/062212list.html.
h. NTP, Technical Report on the Toxicology and Carcinogenesis Studies of Benzophenone
(CAS No 119-61-9) in F33/N Rats and B6C3F1 Mice, 2006. See
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr533/i
ndex.html.
i. IARC, Benzophenone, IARC Monograph – Volume 101-007, 2012, pp. 285-304. See
http://monographs.iarc.fr/ENG/Monographs/vol101/mono101-007.pdf.
j. IARC, Ethyl Acrylate, IARC Monograph – Volume 71-99, 1999, pp. 1447-1457. See
http://monographs.iarc.fr/ENG/Monographs/vol71/mono71-99.pdf.
k. OEHHA, Proposition 65 List of Chemicals, 2015.
http://oehha.ca.gov/prop65/prop65_list/Newlist.html.
l. NTP, Carcinogenesis Studies of Ethyl Acrylate (CAS No. 140-88-5) in F344 Rats and
B6C3F1 Mice (Gavage Studies), 1986.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr200299/abstracts/tr259/inde
x.html.
m. IARC, Methyl Eugenol, IARC Monograph – Volume 101-013, 2012, pp. 407-433. See
http://monographs.iarc.fr/ENG/Monographs/vol101/mono101-013.pdf.
n. NTP, Report on Carcinogens, Thirteenth Edition, Methyleugenol, 2014. See
http://ntp.niehs.nih.gov/go/roc13.
Page 10 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
o. NTP, Final Report on Carcinogens Background Document for Methyleugenol, 2000. See
http://ntp.niehs.nih.gov/pubhealth/roc/listings/m/methyleugenol/summary/index.html.
p. OEHHA, Chemical Listed Effective November 16, 2001 as Known to the State of California
to Cause Cancer: Methyleugenol, November 16, 2001. See
http://oehha.ca.gov/prop65/out_of_date/pdf_zip/11-16NOT.pdf.
q. NTP, Carcinogenesis Studies of Methyl Eugenol (CAS No. 93-15-2) in F344/N Rats and
B6C3F1 Mice (Gavage Studies), 2000.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr400499/abstracts/tr491/inde
x.html.
r. OEHHA, Chemical Listed Effective March 27, 2015 as Known to the State of California to
Cause Cancer: Beta-Myrcene, 2015.
http://oehha.ca.gov/prop65/CRNR_notices/list_changes/032415listbetamyrcene.html.
s. NTP, Toxicology and Carcinogenesis Studies of β-Myrcene (CAS No. 123-35-3) in F344/N
Rats and B6C3F1 Mice (Gavage Studies), 2010.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr557/i
ndex.html.
t. OEHHA, Chemicals Listed Effective April 18, 2014 as Known to the State of California to
Cause Cancer: Pentosan Polysulfate Sodium, Pioglitazone, Trimteerene, and Pulegone, 2014.
http://oehha.ca.gov/prop65/prop65_list/041814P65list.html.
u. IARC, Pulegone, IARC Monograph – Volume 108-05, 2014, pp. 1-14. See
http://monographs.iarc.fr/ENG/Monographs/vol108/mono108-05.pdf.
v. NTP, Toxicology and Carcinogenesis Studies of Pulegone (CAS No. 89-82-7) in F344/N
Rats and B6C3F1 Mice (Gavage Studies), 2011.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr563/i
ndex.html.
w. OEHHA, Chemical Listed Effective May 17, 2002 as Known to the State of California to
Cause Cancer: Pyridine, May 17, 2002. See
http://oehha.ca.gov/prop65/out_of_date/51702notice.html.
x. NTP, Toxicology and Carcinogenesis Studies of Pyridine (CAS No. 110-86-1) in F344/N
Rats, Wistar Rats, and B6C3F1 Mice (Drinking Water Studies), 2000.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr400499/abstracts/tr470/inde
x.html.
y. NTP, Report on Carcinogens, Thirteenth Edition, Styrene, 2014. See
http://ntp.niehs.nih.gov/go/roc13.
z. NTP, Final Report on Carcinogens Background Document for Styrene, 2008. See
http://ntp.niehs.nih.gov/pubhealth/roc/listings/s/styrene/summary/index.html.
aa. IARC, Styrene, IARC Monograph – Volume 82-9, 2002, pp. 437-550. See
http://monographs.iarc.fr/ENG/Monographs/vol82/mono82-9.pdf.
Page 11 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Appendix 2
Responses to elements required by 21 CFR § 171.1
Per 21 CFR § 171.1, we provide responses to the requested elements of a food additive petition
with one element per page.
Name and Pertinent Information Concerning Food Additive
The identity of the food additives are as follows:
Table 2: Description of food additives.
Additive name Chemical
formula
Formula
weight
FEMA
No.
CAS No. INS
No.
UNI No
Benzophenone / Diphenylketone C13H10O 182 2134 119-61-9 * *
Ethyl acrylate C5H8O2 100 2418 140-88-5 * *
Eugenyl methyl ether / 4-Allylveratrole /
Methyl eugenol
C11H14O2 178 2475 93-15-2 * *
Myrcene / 7-methyl-3-methylene-1,6-
octadiene
C10H16 136 2762 123-35-3 * *
Pulegone / p- Menth-4(8)-en-3-one C10H16O 152 2963 89-82-7 * *
Pyridine C3H6O 58 2966 110-86-1 * *
Styrene C8H8 104 3233 100-42-5 * *
* None found.
Directions, Recommendations, and Suggestions Regarding Proposed Use
We are asking FDA to prescribe a zero tolerance as the most appropriate condition of use of the
substances described above as flavors. We are not addressing their use as indirect additives or
food contact substances at this time. We are proposing only that they not be used as a flavor in
food.
Data establishing that food additive will have intended physical or other technical effect
We are asking FDA to prescribe a zero tolerance as the most appropriate condition of use of the
substances described above as flavors. A flavor or flavor extract is not essential to the function of
food.
Description of practicable methods to determine the amount of the food additive in the food
We are asking FDA to prescribe a zero tolerance as the most appropriate condition of use of the
substances described above as flavors. If they are not added, there need be no practical methods
to determine the amount added.
Full reports of investigations made with respect to the safety of the food additive
See Appendix 3.
Proposed tolerances for the food additive
We are asking FDA to prescribe a zero tolerance as the most appropriate condition of use of the
substances described above as flavors. As a result, no tolerance is needed.
Page 12 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Full information on each proposed change to the original regulation
See Appendix 4 for the specific changes requested to 21 CFR § 172.515. Text in strikethrough
font is to be deleted.
Environmental review component
The proposed action is categorically excluded pursuant to 21 CFR 25.32(m) as an "action to
prohibit or otherwise restrict or reduce the use of a substance in food, food packaging, or
cosmetics." It complies with the categorical exclusion criteria pursuant to 40 CFR § 1508.4. No
extraordinary circumstances as defined at 21 CFR § 25.21 exist for the action requested in this
petition which would require the submission of an Environmental Assessment.
A food manufacturer may determine that the flavor additive is not essential and choose not to
replace it. We could identify no extraordinary circumstances that would result from this removal
without replacement.18
Should the manufacturer determine that another flavor additive were needed to replace one of the
seven synthetic flavor substances covered by this petition, it would likely turn to 21 CFR §
172.515 to identify alternatives. While most of those hundreds of alternative food additives were
approved by FDA before the National Environmental Policy Act was adopted and have not been
reassessed by the agency for their current risk, we did not identify a potential for serious harm to
the environment or protected species from the marginal increase in production or use of these
alternatives.
If the manufacturer determined that these additives were also insufficient and no additives were
“generally recognized as safe” without FDA review, the manufacturers would submit a food
additive petition for agency review. In this review, the agency would consider compliance with
the National Environmental Policy Act.
For each of the seven synthetic flavors, we evaluated the alternatives more closely below:
1. Benzophenone / Diphenylketone
According to the Good Scents Company, benzophenone is reported to have “balsam rose
metallic powdery geranium” organoleptic characteristics.19 The firm identified 53 other
flavors that similar or complementary to benzophenone.
Using the firm’s search engine for organoleptic properties, we found:
• 462 chemicals that featured “balsam” characteristics,20
• 432 chemicals that featured “rose” characteristics,21
18 Note that the petitioner is not required under section 171.130(b) to provide information that only the manufacturer
would have, In re Natural Resources Defense Council, 645 F.3rd 400, 407 (DC Cir 2011). 19 Good Scents Company, Search for “benzophenone”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1016331.html. 20 Good Scents Company, Search for “balsam”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=balsam&submit.x=0&submit.y=0. 21 Good Scents Company, Search for “rose”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=rose&submit.x=6&submit.y=8.
Page 13 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
• 116 chemicals that featured “metallic” characteristics,22 and
• 72 chemicals that feature “geranium” characteristics.23
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
2. Ethyl acrylate
According to the Good Scents Company, ethyl acrylate is reported to have “harsh plastic
acrylate fruity” organoleptic characteristics.24
Using the firm’s search engine for organoleptic properties, we found:
• 11 chemicals that featured “harsh” characteristics,25
• 29 chemicals that featured “plastic” characteristics,26
• 4 chemicals that featured “acrylate” characteristics,27 and
• 1265 chemicals that feature “fruity” characteristics.28
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
3. Eugenyl methyl ether / 4-Allylveratrole / Methyl eugenol
According to the Good Scents Company, methyl eugenol is reported to have “sweet fresh
warm spicy clove carnation cinnamon” organoleptic characteristics.29 The firm identified
20 other flavors that similar or complementary to methyl eugenol.
Using the firm’s search engine for organoleptic properties, we found:
• 462 chemicals that featured “sweet” characteristics,30
• 686 chemicals that featured “fresh” characteristics,31
22 Good Scents Company, Search for “metallic”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=metallic&submit.x=0&submit.y=0. 23 Good Scents Company, Search for “geranium”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=geranium&submit.x=0&submit.y=0. 24 Good Scents Company, Search for “ethyl acrylate”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1008231.html. 25 Good Scents Company, Search for “harsh”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=harsh&submit.x=0&submit.y=0. 26 Good Scents Company, Search for “plastic”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=plastic&submit.x=0&submit.y=0. 27 Good Scents Company, Search for “acrylate”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=acrylate&submit.x=0&submit.y=0. 28 Good Scents Company, Search for “fruity”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=fruity&submit.x=0&submit.y=0. 29 Good Scents Company, Search for “methyl eugenol”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1008291.html. 30 Good Scents Company, Search for “sweet”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=sweet&submit.x=0&submit.y=0. 31 Good Scents Company, Search for “fresh”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=fresh&submit.x=0&submit.y=0.
Page 14 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
• 130 chemicals that featured “warm” characteristics,32
• 549 chemicals that featured “spicy” characteristics,33
• 79 chemicals that featured “clove” characteristics,34
• 21 chemicals that featured “carnation” characteristics,35 and
• 78 chemicals that feature “cinnamon” characteristics.36
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
4. Myrcene / 7-methyl-3-methylene-1,6-octadiene
According to the Good Scents Company, myrcene is reported to have “peppery terpene
spicy balsam plastic” organoleptic characteristics.37 The firm identified 179 other flavors
that similar or complementary to myrcene.
Using the firm’s search engine for organoleptic properties, we found:
• 49 chemicals that featured “peppery” characteristics,38
• 0 chemicals that featured “terpene” characteristics,39
• 549 chemicals that featured “spicy” characteristics,40
• 462 chemicals that featured “balsam” characteristics,41 and
• 29 chemicals that feature “plastic” characteristics.42
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
32 Good Scents Company, Search for “warm”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=warm&submit.x=0&submit.y=0. 33 Good Scents Company, Search for “spicy”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=spicy&submit.x=0&submit.y=0. 34 Good Scents Company, Search for “clove”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=clove&submit.x=0&submit.y=0. 35 Good Scents Company, Search for “carnation”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=carnation&submit.x=0&submit.y=0. 36 Good Scents Company, Search for “cinnamon”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=cinnamon&submit.x=0&submit.y=0. 37 Good Scents Company, Search for “myrcene”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1016531.html. 38 Good Scents Company, Search for “peppery”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=peppery&submit.x=0&submit.y=0. 39 Good Scents Company, Search for “terpene”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=terpene&submit.x=0&submit.y=0. 40 Good Scents Company, Search for “spicy”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=spicy&submit.x=0&submit.y=0. 41 Good Scents Company, Search for “balsam”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=balsam&submit.x=0&submit.y=0. 42 Good Scents Company, Search for “plastic”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=plastic&submit.x=0&submit.y=0.
Page 15 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
5. Pulegone / p- Menth-4(8)-en-3-one
According to the Good Scents Company, pulegone is reported to have “peppermint
camphor fresh herbal buchu” organoleptic characteristics.43 The firm identified XX other
flavors that similar or complementary to pulegone.
Using the firm’s search engine for organoleptic properties, we found:
• 48 chemicals that featured “peppermint” characteristics,44
• 183 chemicals that featured “camphor” characteristics,45
• 686 chemicals that featured “fresh” characteristics,46
• 835 chemicals that featured “herbal” characteristics,47 and
• 13 chemicals that feature “buchu” characteristics.48
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
6. Pyridine
According to the Good Scents Company, pyridine is reported to have “sickening sour
putrid fishy amine” organoleptic characteristics.49 The firm identified 43 other flavors
that similar or complementary to pyridine.
Using the firm’s search engine for organoleptic properties, we found:
• 2 chemicals that featured “sickening” characteristics,50
• 35 chemicals that featured “sour” characteristics,51
• 4 chemicals that featured “putrid” characteristics,52
• 29 chemicals that featured “fishy” characteristics,53 and 43 Good Scents Company, Search for “pulegone”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1006501.html. 44 Good Scents Company, Search for “peppermint”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=peppermint&submit.x=0&submit.y=0. 45 Good Scents Company, Search for “camphor”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=camphor&submit.x=0&submit.y=0. 46 Good Scents Company, Search for “fresh”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=fresh&submit.x=0&submit.y=0. 47 Good Scents Company, Search for “herbal”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=herbal&submit.x=0&submit.y=0. 48 Good Scents Company, Search for “buchu”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=buchu&submit.x=0&submit.y=0. 49 Good Scents Company, Search for “pyridine”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1009251.html. 50 Good Scents Company, Search for “sickening”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=sickening&submit.x=0&submit.y=0. 51 Good Scents Company, Search for “sour”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=sour&submit.x=0&submit.y=0. 52 Good Scents Company, Search for “putrid”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=putrid&submit.x=0&submit.y=0. 53 Good Scents Company, Search for “fishy”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=fishy&submit.x=0&submit.y=0.
Page 16 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
• 7 chemicals that feature “amine” characteristics.54
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
7. Styrene
According to the Good Scents Company, styrene is reported to have “sweet balsam floral
plastic” organoleptic characteristics.55 The firm identified four other flavors that similar
or complementary to styrene
Using the firm’s search engine for organoleptic properties, we found:
• 1558 chemicals that featured “sweet” characteristics,56
• 462 chemicals that featured “balsam” characteristics,57
• 996 chemicals that featured “floral” characteristics,58 and
• 29 chemicals that feature “plastic” characteristics.59
Many of these flavors were approved by FDA as food additives at 21 CFR § 172.515.
54 Good Scents Company, Search for “amine”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=amine&submit.x=0&submit.y=0. 55 Good Scents Company, Search for “styrene”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/data/rw1009281.html. 56 Good Scents Company, Search for “sweet”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=sweet&submit.x=0&submit.y=0. 57 Good Scents Company, Search for “balsam”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=balsam&submit.x=0&submit.y=0. 58 Good Scents Company, Search for “floral”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=floral&submit.x=0&submit.y=0. 59 Good Scents Company, Search for “plastic”, accessed July 21, 2015 at
http://www.thegoodscentscompany.com/search3.php?qOdor=plastic&submit.x=0&submit.y=0.
Page 17 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Appendix 3
Reports on the Carcinogenicity of the Seven Flavor Additives
We are asking FDA to prescribe a zero tolerance as the most appropriate condition of the use of
the additives based on the Delaney Clause which applies to food additives and GRAS substances
such as flavors. According to 21 U.S.C. § 348(c) regarding FDA’s approval or denial of this
petition,
“(1) The Secretary shall--
(A) by order establish a regulation (whether or not in accord with that proposed by
the petitioner) prescribing, with respect to one or more proposed uses of the
food additive involved, the conditions under which such additive may be safely
used (including, but not limited to, specifications as to the particular food or
classes of food in or in which such additive may be used, the maximum
quantity which may be used or permitted to remain in or on such food, the
manner in which such additive may be added to or used in or on such food, and
any directions or other labeling or packaging requirements for such additive
deemed necessary by him to assure the safety of such use), and shall notify the
petitioner of such order and the reasons for such action; or
(B) by order deny the petition, and shall notify the petitioner of such order and of
the reasons for such action.
(2) The order required by paragraph (1)(A) or (B) of this subsection shall be issued
within ninety days after the date of filing of the petition, except that the Secretary
may (prior to such ninetieth day), by written notice to the petitioner, extend such
ninety-day period to such time (not more than one hundred and eighty days after the
date of filing of the petition) as the Secretary deems necessary to enable him to
study and investigate the petition.
(3) No such regulation shall issue if a fair evaluation of the data before the Secretary—
(A) fails to establish that the proposed use of the food additive, under the
conditions of use to be specified in the regulation, will be safe: Provided,
That no additive shall be deemed to be safe if it is found to induce cancer
when ingested by man or animal, or if it is found, after tests which are
appropriate for the evaluation of the safety of food additives, to induce
cancer in man or animal, except that this proviso shall not apply with respect
to the use of a substance as an ingredient of feed for animals which are raised
for food production, if the Secretary finds (i) that, under the conditions of use
and feeding specified in proposed labeling and reasonably certain to be
followed in practice, such additive will not adversely affect the animals for
which such feed is intended, and (ii) that no residue of the additive will be
found (by methods of examination prescribed or approved by the Secretary by
regulations, which regulations shall not be subject to subsections (f) and (g))
in any edible portion of such animal after slaughter or in any food yielded by
or derived from the living animal;
(B) shows that the proposed use of the additive would promote deception of the
consumer in violation of this Act [21 USCS §§ 301 et seq.] or would
otherwise result in adulteration or in misbranding of food within the meaning
Page 18 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
of this Act [21 USCS §§ 301 et seq.].” (21 U.S.C. § 348(c)(1)-(3)) (Emphasis
added).
Under the Delaney Clause, if an additive is found to induce cancer when ingested by man or
animal, or if it is found, after tests which are appropriate for the evaluation of the safety of food
additives, to induce cancer in man or animal, it is not safe and must not be allowed to be
intentionally added to food.
Therefore, our analysis of the safety of the seven flavors solely addresses whether the flavor
additives are prohibited based on the Delaney Clause. The extent of exposure is not a factor.
We believe this finding should rest on conclusions by recognized authorities responsible for
determining whether a substance is found to induce cancer when ingested by man or animal. We
started by looking at FDA’s evaluation. It approved the synthetic flavors as food additives in
1964 before the evidence that the flavors induced cancer became available. We have been unable
to find any evidence that FDA reassessed the safety of any of the seven flavors in light of the
cancer findings. We also looked for reassessments by FEMA.
Part I: Evaluation Organized by Recognized authority
We also looked at the recognized authorities responsible for determining whether a chemical is
found to induce cancer in man or animals. We identified three such authorities that have
evaluated the flavors listed on Table 1 for carcinogenicity. Each used a transparent process that
provided an opportunity for scientists and other stakeholders to weigh in on the decision. The
three recognized authorities are:
A. National Toxicology Program Report on Carcinogens
Since 1978, Congress has directed the program in the National Institutes of Health to publish a
report identifying carcinogens, known as the Report on Carcinogens (ROC). The most recent
ROC is the 13th edition issued in October 2014.60
NTP has designated two synthetic flavors, methyl eugenol and styrene, as “reasonably
anticipated to be human carcinogen.” This designation means there is either:
1. Limited evidence of carcinogenicity from studies in humans, which indicates that causal
interpretation is credible, but that alternative explanations, such as chance, bias, or
confounding factors, could not adequately be excluded; or
2. Sufficient evidence of carcinogenicity from studies in experimental animals, which indicates
there is an increased incidence of malignant and/or a combination of malignant and benign
tumors (1) in multiple species or at multiple tissue sites, or (2) by multiple routes of
exposure, or (3) to an unusual degree with regard to incidence, site, or type of tumor, or age
at onset; or
60 NTP, 13th Report on Carcinogens, 2014. See http://ntp.niehs.nih.gov/pubhealth/roc/roc13/index.html.
Page 19 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
3. Less than sufficient evidence of carcinogenicity in humans or laboratory animals; however,
the agent, substance, or mixture belongs to a well-defined, structurally related class of
substances whose members are listed in a previous Report on Carcinogens as either known to
be a human carcinogen or reasonably anticipated to be a human carcinogen, or there is
convincing relevant information that the agent acts through mechanisms indicating it would
likely cause cancer in humans.61
Each of NTP’s findings are discussed below.
B. National Toxicology Program Cancer Studies
NTP conducted cancer studies on five of the seven synthetic flavors: benzophenone, ethyl
acrylate, myrcene, pulegone, and pyridine.62 For each it found evidence of carcinogenicity.
Each of NTP’s findings are discussed below.
NTP’s findings were based on ingestion studies done consistent with FDA’s Redbook.63 NTP
made these findings pursuant to a Congressional directive at 42 U.S.C. § 241 to the HHS
Secretary to conduct these types of tests. The Secretary established NTP to perform this work.
C. International Agency for Research on Cancer (IARC)
IARC is the science program of the World Health Organization (WHO) launched in 1965 to
provide critical reviews and evaluations of evidence on the carcinogenicity of a wide range of
human exposures.64 It publishes Monographs on the Evaluation of Carcinogenic Risks to
Humans. The most recent monograph was Volume 108 published in 2014.65 Since it is part of
WHO, we consider IARC to be a recognized authority.
IARC designated five synthetic flavors, benzophenone, ethyl acrylate, methyl eugenol,
pulegone, and styrene, to be “possibly carcinogenic to humans” in class 2B. For five of these
flavors the agency concluded that there was sufficient evidence in experimental animals for the
carcinogenicity.
For styrene, it found limited evidence of carcinogenicity in both humans and experimental
animals but assigned it class 2B based on the cumulative evidence. IARC reached this
conclusion in 2000 and did not have the benefit of studies published after 2000 that resulted in
NTP reaching a stronger conclusion.
61 Id at p 2. 62 For the remaining two, it has designated them as reasonably anticipated to be human carcinogens in its biennial
Report on Carcinogens. 63 FDA, Guidance for Industry and Other Stakeholders: Toxicology Principles for the Safety Assessment of Food
Ingredients (Redbook 2000), 2007. Chapter IV.C.6. Accessed May 23, 2015. See
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/IngredientsAdditivesGR
ASPackaging/ucm2006826.htm. 64 IARC, Preamble to the Monographs, accessed on 2/21/15 at
http://monographs.iarc.fr/ENG/Preamble/currenta1background0706.php 65 IARC, Monographs and Supplements Available Online, accessed on 2/21/15 at
http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php.
Page 20 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
IARC listed a sixth, beta-myrcene, as priority for monograph in the 2015-2019 period.66 IARC
has not considered pyridine since NTP published its definitive study in 2000.
This designation means:
1. This category is used for agents for which there is limited evidence of carcinogenicity in
humans and less than sufficient evidence of carcinogenicity in experimental animals.
2. It may also be used when there is inadequate evidence of carcinogenicity in humans but there
is sufficient evidence of carcinogenicity in experimental animals.
3. In some instances, an agent for which there is inadequate evidence of carcinogenicity in
humans and less than sufficient evidence of carcinogenicity in experimental animals together
with supporting evidence from mechanistic and other relevant data may be placed in this
group. An agent may be classified in this category solely on the basis of strong evidence
from mechanistic and other relevant data.67
D. California’s Proposition 65 (California) OEHHA
The Safe Drinking Water and Toxic Enforcement Act of 1986, also known as Proposition 65,68 is
a regulatory program designed to protect California’s citizens and its drinking water sources
from chemicals known to cause cancer, birth defects, or other reproductive harm, and to inform
citizens about exposures to such chemicals. The Office of Environmental Health Hazard
Assessment (OEHHA) manages Proposition 65. Through a process that includes public notice
and an opportunity to comment, OEHHA has designated six of the seven synthetic flavors as
carcinogens and has proposed this designation for the seventh. A chemical is designated a
carcinogen by one of four methods:
1. A committee of independent scientists known as the Carcinogen Identification Committee
(CIC)69 is part of OEHHA's Science Advisory Board. The committee members are appointed
by the Governor and are designated as the “State's Qualified Experts” for evaluating
chemicals under Proposition 65. When determining whether a chemical should be placed on
the list, the committees base their decisions on the most current scientific information
available. OEHHA staff scientists compile all relevant scientific evidence on various
chemicals for the committees to review. The committees also consider comments from the
public before making their decisions. OEHHA designated ethyl acrylate as carcinogen
through this method.
2. An organization designated as an “authoritative body” by the CIC has identified it as causing
cancer. The following organizations have been designated as authoritative bodies: the U.S.
Environmental Protection Agency (EPA), U.S. Food and Drug Administration (U.S. FDA),
National Institute for Occupational Safety and Health, NTP and IARC. OEHHA designated
66 IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, INTERNAL REPORT 14/002, Report of
the Advisory Group to Recommend Priorities for IARC Monographs during 2015–2019, 2014.
http://monographs.iarc.fr/ENG/Publications/internrep/14-002.pdf. 67 IARC, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Preamble, (2006) p 23. See
http://monographs.iarc.fr/ENG/Preamble/CurrentPreamble.pdf. 68 OEHHA, Proposition 65, accessed on May 23, 2015 at http://www.oehha.ca.gov/prop65.html. 69 OEHHA, Proposition 65, Science Advisory Board Carcinogen Identification Committee, accessed May 23, 2015
at http://www.oehha.ca.gov/prop65/policy_procedure/CICmembers.html.
Page 21 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
three of the seven synthetic flavors, methyl eugenol, myrcene, and pyridine, as carcinogens
through this method. On February 27, 2015, it proposed listing styrene as a carcinogen; as of
July 21, 2015, the agency has not made a final decision.
3. An agency of the state or federal government requires that it be labeled or identified as
causing cancer or birth defects or other reproductive harm. Most chemicals listed in this
manner are prescription drugs that are required by the U.S. FDA to contain warnings relating
to cancer or birth defects or other reproductive harm. None of the seven flavors were
designated as carcinogens through this method.
4. The California Labor Code list of chemicals meeting certain scientific criteria and identified
as causing cancer or birth defects or other reproductive harm. This method established the
initial chemical list following voter approval of Proposition 65 in 1986 and continues to be
used as a basis for listing as appropriate.70 OEHHA designated two of the seven synthetic
flavors, benzophenone and pulegone, through this method.
Part II: Evaluations Organized by Carcinogenic Flavor
We incorporate the referenced findings of the recognized authorities as well as the FEMA Expert
Panel’s analysis by reference and summarize them below for each of the seven flavor additives.
A. Benzophenone / Diphenylketone (CAS No. 119-61-9)
FEMA’s Expert Panel determined benzophenone to be GRAS at average maximum use levels of
0.5 to 2.4 ppm, assigned it FEMA No. 2134, and published its conclusion in 1965 as part of
FEMA’s GRAS 3 report.71 The specific average maximum use levels are: baked goods at 2.4
ppm; ice cream, ices etc. at 0.61 ppm; beverages at 0.5 ppm; and candy at 1.7 ppm. A year
earlier, FDA approved the flavor as a food additive without establishing numerical maximum
levels at 21 CFR §121.1164.72 In 1977, FDA recodified this section without altering the
requirements for the flavor to 21 CFR §172.515.
In 2012, California’s OEHHA designated benzophenone as a carcinogen73 based on IARC’s
article in Lancet designating the chemical as a 2B carcinogen.74
70 OEHHA, Proposition 65 in Plain Language, accessed on 2/21/15 at
http://www.oehha.ca.gov/prop65/background/p65plain.html. 71 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: III. GRAS Substances, (FEMA No. 2000-3124), Food
Technology, Vol. 19, No. 2, 1965. 72 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964). 73 OEHHA, Chemicals Listed Effective June 22, 2012 As Known To The State Of California To Cause Cancer:
benzophenone (CAS No. 119-61-9), coconut oil diethanolamine condensate (cocamide diethanolamine) (CAS No.
68603-42-9), diethanolamine (CAS No. 111-42-2), and 2-methylimidazole (CAS No. 693-98-1), June 22, 2012. See
http://oehha.ca.gov/prop65/prop65_list/062212list.html. 74 Grosse Y, Baan R, Secretan-Lauby B, El Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Islami F,
Galichet L, Straif K, on behalf of the WHO International Agency for Research on Cancer Monograph Working
Group (2011). Carcinogenicity of chemicals in industrial and consumer products, food contaminants and
flavourings, and water chlorination byproducts. Lancet Oncology 12(4):328-9. [URL:
http://www.thelancet.com/journals/lanonc/article/PIIS1470-2045%2811%2970088-2/fulltext]
Page 22 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
In 2006, in response to a study it conducted, NTP stated that “We conclude that benzophenone
caused kidney cancer in male rats, liver tumors in male mice, and histiocytic sarcomas in female
mice. Benzophenone may also have been associated with development of leukemia in male and
female rats and with liver tumors in female mice.”75
NTP went on to explain that:
Under the conditions of these 2-year studies, there was some evidence of carcinogenic
activity* of benzophenone in male F344/N rats based on increased incidences of renal
tubule adenoma; mononuclear cell leukemia in male F344/N rats may have been related to
benzophenone exposure. There was equivocal evidence of carcinogenic activity of
benzophenone in female F344/N rats based on the marginally increased incidences of
mononuclear cell leukemia and histiocytic sarcoma. There was some evidence of
carcinogenic activity of benzophenone in male B6C3F1 mice based on increased incidences
of hepatocellular neoplasms, primarily adenoma. There was some evidence of carcinogenic
activity of benzophenone in female B6C3F1
mice based on increased incidences of
histiocytic sarcoma; the incidences of hepatocellular adenoma in female B6C3F1 mice may
have been related to benzophenone exposure.76
Administration of benzophenone in feed resulted in increased incidences and/or severities
of nonneoplastic lesions in the kidney and liver of male and female rats and in the liver,
kidney, nose, and spleen of male andfemale mice. 77
In 1999, IARC designated benzophenone as “Possibly Carcinogenic to Humans (2B)” based on
its analysis of mouse and rat studies.78 It stated that:
Benzophenone was tested for carcinogenicity by oral administration in the diet in one
study in mice and rats and by dermal application in one study in mice. Oral
administration of benzophenone significantly increased the incidence of hepatocellular
adenoma, and hepatocellular adenoma, hepatocellular carcinoma and hepatoblastoma
(combined) in male mice and histiocytic sarcoma in female mice. It increased the
incidence of mononuclear-cell leukaemia in male and female rats (not statistically
significant in females), renal tubule adenoma in male rats and histiocytic sarcoma in
female rats (not statistically significant). Dermal application of benzophenone did not
induce tumours in mice. Tumours of the kidney, histiocytic sarcomas and
hepatoblastomas are rare spontaneous neoplasms in experimental animals.79
75 NTP, Technical Report on the Toxicology and Carcinogenesis Studies of Benzophenone (CAS No 119-61-9) in
F33/N Rats and B6C3F1 Mice (Feed Studies), 2006. See http://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr533.pdf. 76 Id. 77 Id. 78 IARC, Benzophenone, IARC Monograph – Volume 101-007, 2012, pp. 285-304. See
http://monographs.iarc.fr/ENG/Monographs/vol101/mono101-007.pdf. 79 Id.
Page 23 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
IARC concluded that “There is sufficient evidence in experimental animals for the
carcinogenicity of benzophenone.” 80
B. Ethyl Acrylate (CAS No. 140-88-5)
A FEMA Expert Panel determined ethyl acrylate to be GRAS at average maximum use levels of
0.06 to 1.1 ppm, assigned it FEMA No. 2418, and published its conclusion in 1965 as part of
FEMA’s GRAS 3 report.81 The specific average maximum use levels are: candy and baked
goods at 1.1 ppm; beverages at 0.13-0.26 ppm; ice cream, ices etc. at 0.06-1 ppm; and chewing
gum at 0.1 ppm. A year earlier, FDA approved the flavor as a food additive without establishing
numerical maximum levels at 21 CFR §121.1164.82 In 1977, FDA recodified this section without
altering the requirements for the flavor to 21 CFR §172.515.
In 1999, IARC designated ethyl acrylate as “Possibly Carcinogenic to Humans (2B)”83 It stated
that:
Ethyl acrylate was tested for carcinogenicity by oral gavage in mice and rats. Dose-
related increases in the incidence of squamous-cell papillomas and carcinomas of the
forestomach were observed in both species. Ethyl acrylate was tested by inhalation in the
same strains of mice and rats; no treatment-related neoplastic lesion was observed. No
treatment-related tumour was observed following skin application of ethyl acrylate for
lifespan to male mice (IARC, 1986).84
Regarding the rat studies, IARC stated that:
Three groups of 25 male Fischer 344 rats, two months of age, were treated with 200
mg/kg bw ethyl acrylate (purity, 99%) by gavage in corn oil on five days per week for six
or 12 months. Control rats received 5 mL corn oil/kg bw per day on five days per week
for 12 months. Five rats from each treatment group were killed 24 h after the last dose.
The remaining rats were killed at 24 months of age. All animals were examined for gross
lesions and the stomachs were collected and fixed in formalin. Microscopic examination
was restricted to three or four sections of the stomach. No treatment-related neoplastic
lesions were observed in the forestomach of rats exposed to ethyl acrylate for six months
and autopsied at 24 months of age. After 12 months of ethyl acrylate administration, all
rats showed hyperplastic lesions but no neoplastic lesions were detected. However, when
rats received ethyl acrylate for 12 months and were killed after nine months of recovery,
they developed squamous-cell carcinomas (3/13) and papillomas (1/13) (Ghanayem et
80 Id. 81 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: III. GRAS Substances, (FEMA No. 2000-3124), Food
Technology, Vol. 19, No. 2, 1965. 82 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964). 83 IARC, Ethyl Acrylate, IARC Monograph – Volume 71-99, 1999, pp. 1447-1457. See
http://monographs.iarc.fr/ENG/Monographs/vol71/mono71-99.pdf. 84 Id.
Page 24 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
al., 1993). [The Working Group noted that histopathological evaluation was limited to
the stomach.]85
IARC concluded that “There is sufficient evidence in experimental animals for the carcinogenicity of
ethyl acrylate.” 86
In 1989, California’s OEHHA designated ethyl acrylate as a carcinogen based on the analysis of
its independent committee of cancer expert.87
In 1986, in response to a study it conducted, NTP stated “Under the conditions of these studies,
ethyl acrylate was carcinogenic for the forestomach of F344/ N rats and B6C3F1 mice, causing
squamous cell carcinomas in male rats and male mice, squamous cell papillomas in male and
female rats and male mice, and squamous cell papillomas or carcinomas (combined) in male and
female rats and mice. Evidence for carcinogenicity was greater in males than in females. Ethyl
acrylate also caused irritation of the forestomach mucosa in male and female rats and mice.”88
C. Eugenyl methyl ether / 4-Allylveratrole / Eugenyl Methyl Ether (CAS No. 93-15-2)
FEMA Expert Panel determined methyl eugenol (also known as eugenyl methyl ether) to be
GRAS at average maximum use levels of 4.8 to 52 ppm, assigned it FEMA No. 2476, and
published its conclusion in 1965 as part of FEMA’s GRAS 3 report.89 The specific average
maximum use levels are: jellies at 52 ppm; baked goods at 13 ppm; candy at 11 ppm; beverages
at 10 ppm; and ice cream, ices etc. at 4.8 ppm. A year earlier, FDA approved the flavor as a food
additive without establishing numerical maximum levels at 21 CFR §121.1164.90 In 1977, FDA
recodified this section without altering the requirements for the flavor to 21 CFR §172.515.
In 2004, IARC designated methyl eugenol as “Possibly Carcinogenic to Humans (2B).”91 It
stated that:
Methyl eugenol was tested for carcinogenicity by oral administration by gavage in one
study in mice and one study in rats and by intraperitoneal administration to mice in one
study. In mice, oral administration of methyl eugenol caused a significantly increased
incidence of liver tumours (hepatocellular adenoma, hepatocellular carcinoma and
hepatoblastoma) in both sexes. In rats, oral administration of methyl eugenol caused a
significantly increased incidence of liver tumours (hepatocellular adenoma,
85 Id. 86 Id. 87 OEHHA, Chemicals Listed Effective November 4, 2011 as Known to the State of California to Cause Cancer:
Five Chemicals, November 4, 2011. See http://oehha.ca.gov/prop65/docs_admin/110411LClist.html. 88 NTP, Carcinogenesis Studies of Ethyl Acrylate (CAS No. 140-88-5) in F344 Rats and B6C3F1 Mice (Gavage
Studies), 1986. http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr200299/abstracts/tr259/index.html. 89 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: III. GRAS Substances, (FEMA No. 2000-3124), Food
Technology, Vol. 19, No. 2, 1965. 90 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964). 91 IARC, Methyl Eugenol, IARC Monograph – Volume 101-013, 2012, pp. 407-433. See
http://monographs.iarc.fr/ENG/Monographs/vol101/mono101-013.pdf.
Page 25 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
hepatocellular carcinoma, hepatocholangioma and hepatocholangiocarcinoma) and
benign and malignant neuroendocrine tumours of the glandular stomach in males and
females, and renal tubule adenoma of the kidney, mammary gland fibroadenoma, skin
fibroma, skin fibroma or fibrosarcoma (combined) and mesothelioma in males in the
main and stop-exposure experiments. Tumours of the kidney, fibromas and fibrosarcomas
of the skin, mesotheliomas, hepatoblastomas and hepatocholangiocarcinomas are rare
spontaneous neoplasms, and neuroendocrine tumours of the glandular stomach are
extremely rare spontaneous neoplasms in experimental animals. In the main and stop-
exposure experiments in rats, there was consistency in the tumour response for cancers of
the liver and glandular stomach in males and females, and of the kidney in males.92
IARC also stated that “Intraperitoneal injection of methyl eugenol caused a significantly
increased incidence of hepatocellular adenoma in male mice. 1′-Hydroxymethyleugenol, a
metabolite of methyl eugenol, was tested for carcinogenicity by intraperitoneal injection in one
study in mice, and caused a significantly increased incidence of hepatocellular adenoma in
males.”93
IARC concluded that “There is sufficient evidence in experimental animals for the
carcinogenicity of methyleugenol.”94
In 2002, NTP designated methyl eugenol to be “Reasonably Anticipated To Be Human
Carcinogen.”95 In its 13th Report on Carcinogens published in 2014, NTP stated that:
Methyl eugenol is reasonably anticipated to be a human carcinogen based on sufficient
evidence of carcinogenicity from studies in experimental animals.” Regarding cancer
studies in experimental animals it said “Oral exposure to methyl eugenol caused tumors
in two rodent species and at several different tissue sites. Methyl eugenol administered by
stomach tube caused benign or malignant liver tumors (hepatocellular adenoma or
carcinoma) in rats and mice of both sexes. In rats, methyl eugenol also caused benign or
malignant stomach tumors (neuroendocrine tumors) in both sexes and tumors of the
kidney (renal-tubule adenoma), mammary gland (fibroadenoma), and skin (fibroma or
fibrosarcoma) in males. Malignant neuroendocrine tumors of the stomach in male mice
also were considered to be related to methyl eugenol exposure (NTP 2000). Earlier
studies found that methyl eugenol and two structurally related allylbenzenes, safrole and
estragole, caused liver tumors in mice when administered by intraperitoneal injection
(IARC 1976, Miller et al. 1983). Safrole is listed in the Report on Carcinogens as
reasonably anticipated to be a human carcinogen and by the International Agency for
Research on Cancer as possibly carcinogenic to humans.96
NTP evaluated studies on the mechanisms of carcinogenesis and stated:
92 Id. 93 Id. 94 Id. 95 NTP, Report on Carcinogens, Thirteenth Edition, Methyleugenol, 2014. See http://ntp.niehs.nih.gov/go/roc13. 96 Id.
Page 26 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Mechanistic studies indicate that liver tumors induced by methyl eugenol and structurally
related allylbenzenes result from metabolism of these compounds to DNA-reactive
intermediates. Methyl eugenol may be bioactivated by three different pathways: (1)
hydroxylation at the 1′ position of the allylic side chain to yield
1′-hydroxymethyleugenol, followed by sulfation of this intermediate to form
1′-hydroxymethyleugenol sulfate, (2) oxidation of the 2′,3′-double bond of the allylic side
chain to form methyleugenol-2,3-oxide, and (3) O-demethylation followed by
spontaneous rearrangement to form eugenol quinone methide. Formation of protein
adducts and DNA adducts in the livers of animals (and in cultured human hepatocytes)
exposed to allylbenzenes and induction of liver tumors by these compounds in animals
have been attributed to activation via the hydroxylation pathway, because similar effects
were produced by the 1′-hydroxy metabolites and because these effects were inhibited by
pretreatment with sulfotransferase inhibitors (Boberg et al. 1983, Miller et al. 1983,
Randerath et al. 1984, Gardner et al. 1996, NTP 2000).97
NTP further said:
Methyl eugenol, safrole, and estragole caused unscheduled DNA synthesis in rat
hepatocytes, and their corresponding 1′-hydroxy metabolites were more potent genotoxic
agents than were the parent compounds (Howes et al. 1990, Chan and Caldwell 1992).
Methyl eugenol caused morphological transformation of Syrian hamster embryo cells
(Kerckaert et al. 1996), sister chromatid exchange in Chinese hamster ovary (CHO) cells
(NTP 2000), intrachromosomal recombination in yeast (Schiestl et al. 1989), and DNA
repair in Bacillus subtilis (Sekizawa and Shibamoto 1982). It did not cause mutations in
Salmonella typhimurium (NTP 2000) or Escherichia coli (Sekizawa and Shibamoto
1982), chromosomal aberrations in CHO cells (NTP 2000), or micronucleus formation in
the peripheral-blood erythrocytes of mice (NTP 2000). A higher frequency of b-catenin
mutations was observed in liver tumors from mice exposed to methyl eugenol than in
spontaneous liver tumors from unexposed mice (Devereux et al. 1999). Methyl eugenol’s
lack of mutagenicity in bacteria may be due to the need for sulfation in the metabolic
activation of methyl eugenol to its ultimate mutagenic or carcinogenic form.98
In 2001, California’s OEHHA designated methyl eugenol as a carcinogen based on NTP’s study
saying that NTP “concluded that there is clear evidence of the carcinogenic activity of
methyleugenol in male and female F344/N rats and in male and female B6C3F1 mice.”99
In 2000, in response to a study it conducted, NTP stated:
Under the conditions of these 2-year gavage studies, there was clear evidence of
carcinogenic activity* of methyleugenol in male and female F344/N rats based on the
increased incidences of liver neoplasms and neuroendocrine tumors of the glandular
stomach in male and female rats and the increased incidences of kidney neoplasms,
97 Id. 98 Id. 99 OEHHA, Chemical Listed Effective November 16, 2001 as Known to the State of California to Cause Cancer:
Methyleugenol, November 16, 2001. See http://oehha.ca.gov/prop65/out_of_date/pdf_zip/11-16NOT.pdf.
Page 27 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
malignant mesothelioma, mammary gland fibroadenoma, and subcutaneous fibroma and
fibroma or fibrosarcoma (combined) in male rats. A marginal increase in the incidence of
squamous cell neoplasms of the forestomach may have been related to methyleugenol
administration in female rats. There was clear evidence of carcinogenic activity of
methyleugenol in male and female B6C3F1 mice based on the increased incidences of
liver neoplasms. Neuroendocrine tumors of the glandular stomach in male mice were also
considered related to methyleugenol administration.100
NTP further found, “[i]n male and female rats and mice, methyleugenol administration caused
significant increases in the incidences of nonneoplastic lesions of the liver and glandular
stomach.”101
In 2002, the FEMA Expert Panel reaffirmed that methyl eugenol was generally recognized as
safe.102 While not disputing OEHHA’s conclusion that it was a carcinogen, the Panel stated that
the:
hazard determination uses a mechanism-based approach in which production of the
hepatotoxic sulfate conjugate of the 10-hydroxy metabolite is used to interpret the
pathological changes observed in different species of laboratory rodents in chronic and
subchronic studies. In the risk evaluation, the effect of dose and metabolic activation on
the production of the 10-hydroxy metabolite in humans and laboratory animals is
compared to assess the risk to humans from use of methyl eugenol and estragole as
naturally occurring components of a traditional diet and as added flavouring substances.
Both the qualitative and quantitative aspects of the molecular disposition of methyl
eugenol and estragole and their associated toxicological sequelae have been relatively
well defined from mammalian studies. Several studies have clearly established that the
profiles of metabolism, metabolic activation, and covalent binding are dose dependent
and that the relative importance diminishes markedly at low levels of exposure (i.e. these
events are not linear with respect to dose). In particular, rodent studies show that these
events are minimal probably in the dose range of 1-10 mg/kg body weight, which is
approximately 100–1000 times the anticipated human exposure to these substances. For
these reasons it is concluded that present exposure to methyl eugenol and estragole
resulting from consumption of food, mainly spices and added as such, does not pose a
significant cancer risk. Nevertheless, further studies are needed to define both the nature
and implications of the dose–response curve in rats at low levels of exposure to methyl
eugenol and estragole.103
In essence, FEMA says that despite methyl eugenol causing cancer in an animal, the cancer risk
is not significant enough. This analysis is inconsistent with the Delaney Clause.
100 NTP, Carcinogenesis Studies of Methyl Eugenol (CAS No. 93-15-2) in F344/N Rats and B6C3F1 Mice (Gavage
Studies), 2000. http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr400499/abstracts/tr491/index.html. 101 Id. 102 R.L. Smith, T.B. Adams, J. Doull, V.J. Feron, J.I. Goodman, L.J. Marnett, P.S. Portoghese, W.J. Waddell, B.M.
Wagner, A.E. Rogers, J. Caldwell, I.G. Sipes, Safety assessment of allylalkoxybenzene derivatives used as
flavouring substances — methyl eugenol and estragole, Food and Chemical Toxicology 40 (2002) 851–870. 103 Id.
Page 28 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
D. Myrcene / 7-methyl-3-methylene-1,6-octadiene (CAS No. 123-35-3)
A FEMA Expert Panel determined myrcene to be GRAS at an average maximum use level of 13
ppm in candy, 6.4 ppm in ice cream, ices, etc., 4.9 ppm in baked goods, and 4.4 ppm in
beverages, assigned it FEMA No. 2762, and published its conclusion in 1965 as part of FEMA’s
GRAS 3 report. 104
In 2014, California’s OEHHA designated beta-myrene to be a carcinogen based on the NTP
study conducted in 2010.105
In 2011, in response to a study it conducted, NTP stated “We conclude that β-myrcene caused
kidney cancers in male rats and liver cancer in male mice, and the occurrence of kidney tumors
in female rats and liver tumors in female rats may have been related to β-myrcene administration.
In addition β-myrcene was associated with other lesions of the kidney in rats, the liver in mice,
and the nose in male rats.”106
NTP concluded that there was clear evidence of carcinogenic activity of beta-myrcene in male
F344/N rats and male B6C3F1 mice.107
E. Pulegone / p- Menth-4(8)-en-3-one (CAS No. 89-82-7)
A FEMA Expert Panel determined pulegone to be GRAS at average maximum use levels of 5 to
25 ppm, assigned it FEMA No. 2731, and published its conclusion in 1965 as part of FEMA’s
GRAS 3 report.108 The specific average maximum use levels are: baked goods at 24-25 ppm;
candy at 17 ppm; ice cream, ices etc. at 5-32 ppm; and beverages at 5-8 ppm. A year earlier,
FDA approved the flavor as a food additive without establishing numerical maximum levels at
21 CFR §121.1164.109 In 1977, FDA recodified this section without altering the requirements for
the flavor to 21 CFR §172.515.
104 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: III. GRAS Substances, (FEMA No. 2000-3124), Food
Technology, Vol. 19, No. 2, 1965. 105 OEHHA, Chemical Listed Effective March 27, 2015 as Known to the State of California to Cause Cancer: Beta-
Myrcene, 2015. See http://oehha.ca.gov/prop65/CRNR_notices/list_changes/032415listbetamyrcene.html. 106 NTP, Toxicology and Carcinogenesis Studies of β-Myrcene (CAS No. 123-35-3) in F344/N Rats and B6C3F1
Mice (Gavage Studies), 2010.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr557/index.html. 107 NTP, Toxicology and Carcinogenesis Studies of β-Myrcene (CAS No. 123-35-3) in F344/N Rats and B6C3F1
Mice (Gavage Studies), 2010.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr557/index.html. 108 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: III. GRAS Substances, (FEMA No. 2000-3124), Food
Technology, Vol. 19, No. 2, 1965. 109 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964).
Page 29 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
In 2014, IARC designated pulegone as “Possibly Carcinogenic to Humans (2B)” saying “There is
sufficient evidence in experimental animals for the carcinogenicity of pulegone.”110 Regarding the
animal carcinogenicity data, IARC stated that
Pulegone was tested for carcinogenicity after oral administration in one study in mice and
one study in rats. In male and female mice given pulegone by gavage, there was a
significant increase in the incidences of hepatocellular adenoma, and hepatocellular
adenoma and carcinoma (combined) in males and females, and of hepatoblastoma in males.
In female mice, the incidence of osteoma or osteosarcoma (combined) was higher than that
in historical controls. In female rats given pulegone by gavage, there was an increase in the
incidence of urinary bladder papilloma and of urinary bladder papilloma or carcinoma
(combined). In males, there were no treatment-related increases in tumour incidences.111
Regarding the mechanistic data, IARC stated that:
Pulegone is readily absorbed in humans. It is metabolized in humans and rodents to
isomers of hydroxypulegone, predominantly by hepatic oxidation at the 5-, 9-, and 10-
positions. In rodents, 9-hydroxypulegone is further oxidized to menthofuran, which is
converted to a reactive epoxide and a reactive aldehyde (γ-ketoenal). 5-Hydroxypulegone
is converted to piperitenone, which is then hydroxylated at the 9-position and further
converted to an analogous furan metabolite and to the γ-ketoenal. Further metabolism of
the γ-ketoenal produces 4-methyl-2-cyclohexenone and p-cresol. Pulegone was not
mutagenic in standard bacterial assays, either with or without exogenous metabolic
activation. Studies in humans and rodents indicated that some of the pulegone
metabolites deplete hepatic levels of glutathione and can bind to cellular proteins. This
may result in chronic regenerative cell proliferation, which may be related to the
carcinogenicity observed in the liver and urinary bladder in experimental animals.112
In 2014, California’s OEHHA designated pulegone to be a carcinogen based on the IARC
determination.113
In 2011, in response to a study it conducted, NTP stated “We conclude that pulegone caused
cancer of the urinary bladder in female rats and cancer of the liver in male and female mice.
Neoplasms of the bone in female mice were also possibly associated with administration of
pulegone. There were no increases in cancers in male rats receiving pulegone. Pulegone also
caused an unusual kidney lesion, hyaline glomerulopathy, in male and female rats and mice.”114
110 IARC, Pulegone, IARC Monograph – Volume 108-05, 2014, pp. 1-14. See
http://monographs.iarc.fr/ENG/Monographs/vol108/mono108-05.pdf. 111 Id. 112 Id. 113 OEHHA, NOTICE OF INTENT TO LIST PULEGONE BY THE LABOR CODE MECHANISM, February 7,
2014. See http://oehha.ca.gov/prop65/CRNR_notices/admin_listing/intent_to_list/noilLCset20pulegone.html. 114 NTP, Toxicology and Carcinogenesis Studies of Pulegone (CAS No. 89-82-7) in F344/N Rats and B6C3F1 Mice
(Gavage Studies), 2011.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr563/index.html.
Page 30 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
NTP considered that there was clear evidence of carcinogenic activity of pulegone in female
F344/N rats and male and female B6C3F1 mice.115
In 2005, the FEMA Expert Panel reaffirmed that pulegone was generally recognized as safe in its
22nd report on flavoring substances.116 However, this analysis was done well before the 2011
studies upon which IARC based its conclusions.
F. Pyridine (CAS No. 110-86-1)
A FEMA Expert Panel determined pyridine to be GRAS at an average maximum use level of
0.02 to 1 ppm, assigned it FEMA No. 2731, and published its conclusion in 1965 as part of
FEMA’s GRAS 3 report.117 The specific average maximum use levels are: beverages at 1 ppm;
candy and baked goods at 0.4 ppm; and ice cream, ices etc. at 0.02-0.12 ppm. A year earlier,
FDA approved the flavor as a food additive without establishing numerical maximum levels at
21 CFR §121.1164.118 In 1977, FDA recodified this section without altering the requirements for
the flavor to 21 CFR §172.515.
In 2002, California’s OEHHA designated pyridine as a carcinogen finding that there was an
“Increased incidence of malignant tumors in male and female mice” based on an NTP study
published in 2000.119 It stated that NTP “concluded that there is clear evidence of carcinogenic
activity of pyridine in male and female B6C3F1 mice.”120
In 2000, IARC designated pyridine was “Not Classifiable as to its Carcinogenicity to Humans
(Group 3)” based on NTP studies done in 1997 not the 2000 studies considered by OEHHA.121
In 2011, despite the conclusions of OEHHA, the FEMA Expert Panel reaffirmed that pyridine
was generally recognized as safe in its 25nd report on flavoring substances.122 The Panel based its
decision upon pyridine’s:
115 NTP, Toxicology and Carcinogenesis Studies of Pulegone (CAS No. 89-82-7) in F344/N Rats and B6C3F1 Mice
(Gavage Studies), 2011.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr563/index.html. 116 FEMA Expert Panel (R.L. Smith, S.M. Cohen, J. Doull, V.J. Feron, J.I. Goodman, L.J. Marnett, P.S. Portoghese,
W.J. Waddell, B.M. Wagner, and T.B. Adams), GRAS Flavouring Substances 22 (FEMA No. 4069-4253), Food
Technology, Vol. 59, No. 8, August 2005. 117 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: III. GRAS Substances, (FEMA No. 2000-3124), Food
Technology, Vol. 19, No. 2, 1965. 118 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964). 119 OEHHA, Chemical Listed Effective May 17, 2002 as Known to the State of California to Cause Cancer:
Pyridine, May 17, 2002. See http://oehha.ca.gov/prop65/out_of_date/51702notice.html. 120 Id. 121 IARC, Pyridine, IARC Monograph – Volume 77-16, 22000, pp. 503.
http://www.inchem.org/documents/iarc/vol77/77-16.html. 122 FEMA Expert Panel (R.L. Smith, W.J. Waddell, S.M. Cohen, S. Fukushima, N.J. Gooderham, S.S. Hecht, L.J.
Marnett, P.S. Portoghese, I.M.C.M. Rietjens, T.B. Adams, C.L. Gavin, M.M. McGowen, and S.V. Taylor), GRAS
Flavouring Substances 25 (FEMA No. 4667-4727), Food Technology, Vol. 65, No. 7, July 2011.
Page 31 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
efficient detoxification in humans; its low level of flavor use; the lack of genotoxic and
mutagenic potential; the safety factor calculated from results of subchronic studies (NTP,
2000) indicating a margin of safety of at least 1,000; the conclusion that the statistically
significant findings in the NTP mouse bioassay, of an increased incidence of
hepatocellular neoplasms in male and female B6C3F1 mice were secondary to
pronounced hepatotoxicity at high dose levels; the conclusion that the increased
incidence of renal neoplasms in male F344/N rats occurs via a dose-dependent non-
genotoxic mode of action; and the conclusion that the increased incidence of
mononuclear cell leukemia in female F344/N rats is likely species- and sex-specific, the
biological significance of which remains uncertain. Based on these conclusions, the use
of pyridine as a flavor ingredient is not considered to produce any significant risk to
human health.123
In essence, FEMA says that despite methyl eugenol causing cancer in an animal, the cancer risk
is not significant enough. This analysis is inconsistent with the Delaney Clause.
In 2000, in response to a study it conducted, NTP stated:
Under the conditions of these 2-year drinking water studies, there was some evidence of
carcinogenic activity* of pyridine in male F344/N rats based on increased incidences of
renal tubule neoplasms. There was equivocal evidence of carcinogenic activity of
pyridine in female F344/N rats based on increased incidences of mononuclear cell
leukemia. There was equivocal evidence of carcinogenic activity in male Wistar rats
based on an increased incidence of interstitial cell adenoma of the testis. There was clear
evidence of carcinogenic activity of pyridine in male and female B6C3F1 mice based on
increased incidences of malignant hepatocellular neoplasms.124
In F344/N rats, exposure to pyridine resulted in increased incidences of centrilobular
cytomegaly and degeneration, cytoplasmic vacuolization, and pigmentation in the liver of
males and females; periportal fibrosis, fibrosis, and centrilobular necrosis in the liver of
males; and bile duct hyperplasia in females. In male Wistar rats, pyridine exposure
resulted in increased incidences of centrilobular degeneration and necrosis, fibrosis,
periportal fibrosis, and pigmentation in the liver, and, secondary to kidney disease,
mineralization in the glandular stomach and parathyroid gland hyperplasia.125
G. Styrene (CAS No. 100-42-5)
A FEMA Expert Panel determined styrene to be GRAS at an average maximum use level of 0.2
ppm in ice cream, ices, candy, and baked goods, assigned it FEMA No. 3233, and published its
123 Id. 124 NTP, Toxicology and Carcinogenesis Studies of Pyridine (CAS No. 110-86-1) in F344/N Rats, Wistar Rats, and
B6C3F1 Mice (Drinking Water Studies), 2000.
http://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr400499/abstracts/tr470/index.html. 125 Id.
Page 32 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
conclusion in 1967 as part of FEMA’s GRAS 4 report.126 A year earlier, FDA approved the
flavor as a food additive without establishing numerical maximum levels at 21 CFR
§121.1164.127 In 1977, FDA recodified this section without altering the requirements for the
flavor to 21 CFR §172.515.
In 2011, NTP designated styrene as “Reasonably Anticipated To Be Human Carcinogen” saying
the chemical “is reasonably anticipated to be a human carcinogen based on limited evidence of
carcinogenicity from studies in humans, sufficient evidence of carcinogenicity from studies in
experimental animals, and supporting data on mechanisms of carcinogenesis.”128 In its 13th
Report on Carcinogens published in 2014, NTP evaluated the limited evidence of carcinogenicity
in humans stating that it is:
based on studies of workers exposed to styrene that showed (1) increased mortality from
or incidence of cancer of the lymphohematopoietic system and (2) increased levels of
DNA adducts and genetic damage in lymphocytes from exposed workers. Elevated risks
of lymphohematopoietic cancer were found among workers with higher exposure to
styrene after an appropriate elapsed time since first exposure. In some studies, the risks
increased with increasing measures of exposure, such as average exposure, cumulative
exposure, or number of years since first exposure. However, the types of lymphohe-
matopoietic cancer observed in excess varied across different cohort studies, and excess
risks were not found in all cohorts. There is also some evidence for increased risks of
esophageal and pancreatic cancer among styrene-exposed workers. Causality is not
established, as the possibility that the results were due to chance or to confounding by
exposure to other carcinogenic chemicals cannot be completely ruled out. However, a
causal relationship between styrene exposure and cancer in humans is credible and is
supported by the finding of DNA adducts and chromosomal aberrations in lymphocytes
from styrene-exposed workers.129
Regarding animal studies, NTP stated:
The evidence from studies in rats is insufficient for reaching a conclusion concerning the
carcinogenicity of styrene. Lung tumors were not observed in rats (IARC 2002);
however, findings for mammary-gland tumors were equivocal. The incidence of
mammary-gland tumors was increased in female Sprague-Dawley rats exposed to styrene
in the drinking water (mammary fibroadenoma; Huff 1984) or by inhalation (malignant
tumors; Conti et al. 1988), but decreased incidences of mammary-gland tumors
(adenocarcinoma) were reported in another inhalation-exposure study of rats of the same
strain (Cruzan et al. 1998).130
Regarding the mechanisms of carcinogenesis, NTP stated that:
126 R.L. Hall and B.L. Oser on behalf of FEMA Expert Panel, Recent Progress in the Consideration of Flavouring
Ingredients Under the Food Additives Amendment: 4. GRAS Substances, (FEMA No. 3125-3249), Food
Technology, Vol. 19, No. 2, 1965. 127 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964). 128 NTP, Report on Carcinogens, Thirteenth Edition, Styrene, 2014. See http://ntp.niehs.nih.gov/go/roc13. 129 Id. 130 Id.
Page 33 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Although styrene disposition differs quantitatively among species, no qualitative
differences between humans and experimental animals have been demonstrated that
contradict the relevance of cancer studies in rodents for evaluation of human hazard.
Detection of styrene-7,8-oxide–DNA adducts at base-pairing sites and chromosomal
aberrations in lymphocytes of styrene-exposed workers supports the potential human
cancer hazard from styrene through a genotoxic mode of action.131
In 2013, the U.S. District Court for the District of Columbia affirmed NTP’s decision in the face
of an industry challenge to the agency’s determination. The court said “In short, the Report
provides a rational explanation for the Secretary’s decision to list styrene as a reasonably
anticipated human carcinogen, and this explanation is adequately supported by the administrative
record.”132
In 2014, the National Research Council, at Congress’ request,133 reviewed NTP's assessment of
styrene for the Report on Carcinogens. The NRC committee concluded that:
NTP correctly determined that styrene should be considered for listing [as a carcinogen]
in the RoC. There is sufficient evidence of exposure to a significant number of persons
residing in the United States to warrant such consideration. NTP adequately documented
that exposure to styrene occurs in occupational settings and in the general public
regardless of smoking status.134
After conducting a scientific review of the styrene assessment presented in the NTP 12th
RoC, the committee finds that the overall conclusion reached by NTP in 2011, that
styrene is “reasonably anticipated to be a human carcinogen,” was appropriate. The
following points of the listing criteria support NTP’s conclusion:135
• “There is limited evidence of carcinogenicity from studies in humans.”
Publications available to NTP as of June 10, 2011, provided limited but credible
evidence that exposure to styrene is associated with lymphohematopoietic,
pancreatic, and esophageal cancers. The most informative human epidemiologic
studies that support that conclusion are those by Ruder et al. (2004), Wong et al.
(1994), Kolstad et al. (1994), and Kogevinas et al. (1994). The evidence is limited
in that chance, bias, or confounding factors could not be adequately excluded.136
• “There is sufficient evidence of carcinogenicity from studies in experimental
animals.” Literature published by June 10, 2011, provided sufficient evidence that
131 Id. 132 U.S. District Court for the District of Columbia, Styrene Information and Research Center v.
Sebelius, Civil Action 11-1079, 2013. Seehttps://ecf.dcd.uscourts.gov/cgi-
bin/show_public_doc?2011cv1079-56. 133 2012 Consolidated Appropriations Act (112th Congress, 1st Session; Public Law 112-74). 134 National Research Council, Review of the Styrene Assessment in the National Toxicology Program 12th Report
on Carcinogens, 2014. See http://www.nap.edu/catalog/18725/review-of-the-styrene-assessment-in-the-national-
toxicology-program-12th-report-on-carcinogens. 135 Id. 136 Id.
Page 34 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
“there is an increased incidence of . . . a combination of malignant and benign
tumors” in experimental animals induced by styrene administered by multiple
routes of exposure (inhalation and oral gavage). The most informative
experimental animal studies that support that conclusion are studies in mice (NCI
1979; Cruzan et al. 2001).137
• “There is convincing relevant information that the agent acts through mechanisms
indicating it would likely cause cancer in humans.” Literature published by June
10, 2011, provided convincing evidence that genotoxicity is observed in cells
from humans who were exposed to styrene. That evidence is derived from a large
body of publications. In addition, styrene-7,8-oxide “was listed in a previous
Report on Carcinogens as . . . reasonably anticipated as a human carcinogen.”
Styrene-7,8-oxide, a compound that is structurally related to styrene, is a major
metabolite of styrene in both experimental animals and humans; it was first listed
in the 10th RoC as reasonably anticipated to be a human carcinogen.138
In 2002, IARC designated styrene as “Possibly Carcinogenic to Humans (2B).”139 Regarding the
human studies, IARC stated:
The increased risks for lymphatic and haematopoietic neoplasms observed in some of the
studies are generally small, statistically unstable and often based on subgroup analyses.
These findings are not very robust and the possibility that the observations are the results
of chance, bias or confounding by other occupational exposures cannot be ruled out.140
Regarding animal carcinogenicity data, IARC stated:
Styrene was tested for carcinogenicity in mice in one inhalation study and four oral
gavage studies. In the inhalation study, in male mice there was an increase in the
incidence of pulmonary adenomas and in female mice, there was an increase in the
incidence of pulmonary adenomas, and only an increase in that of carcinomas in the high-
dose group. Two of the gavage studies were negative. The other two were considered
inadequate for an evaluation of the carcinogenicity of styrene. A screening study by
intraperitoneal administration also did not find an increase in tumour incidence or
multiplicity in mice. Styrene was tested for carcinogenicity in rats in four gavage studies,
one drinking water study and two inhalation studies. Overall, there was no reliable
evidence for an increase in tumour incidence in rats. Styrene 7,8-oxide is a major
metabolite of styrene and has been evaluated previously (IARC, 1994b). The evaluation
at that time was that there was sufficient evidence in experimental animals for the
carcinogenicity of styrene 7,8-oxide.141
137 Id. 138 Id. 139 IARC, Styrene, IARC Monograph – Volume 82-9, 2002, pp. 437-550. See
http://monographs.iarc.fr/ENG/Monographs/vol82/mono82-9.pdf. 140 Id. 141 Id.
Page 35 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
IARC concluded that “There is limited evidence in humans for the carcinogenicity of styrene”
and “There is limited evidence in experimental animals for the carcinogenicity of styrene.”142
However, IARC’s analysis, published in 2000 did not consider later studies that NTP relied upon
to reach its determination.
Part III: Expanded Literature Search
For each of the seven flavors, we conducted an updated literature search for studies conducted
after the publication of their respective NTP reports. We limited the searches to studies
conducted between the NTP report publication date and July 1, 2015.
We searched the following databases: PubMed; TOXNET; Google Scholar; European database
for flavouring substances;143 European Food Safety Authority (EFSA) Register of Questions.144
When available, we searched the references cited in IARC monographs and EFSA’s Scientific
Opinion on Flavouring Groups.
We did not include the reports from OEHHA, IARC, or FEMA described in the previous section.
The search included the following terms:
• Chemical names: benzophenone, diphenylketone, ethyl acrylate, eugenyl methyl ether, 4-
allylveratrole, methyl eugenol, myrcene, 7-methyl-3-methylene-1,6-octadiene, pulegone, p-
menth-4(8)-en-3-one, pyridine, styrene, cancer, carcinogenesis, carcinogenic, mutagenicity,
mutagenic, genotoxicity, gene toxicity, DNA damage, DNA adducts
• CAS Register numbers: 119-61-9, 140-88-5, 93-15-3, 123-35-3, 89-82-7, 110-86-1, 100-42-5
• FL No.: 07.032, 09.037.
Table 3 Summary of Results of Expanded Literature Search.
Additive name Results
Benzophenone / Diphenylketone No study found in the publicly available scientific literature that
does not support our claim that benzophenone is a carcinogenic
chemical.
Ethyl acrylate 3 in vivo and 6 in vitro studies found and described below.
Eugenyl methyl ether / 4-
Allylveratrole / Methyl eugenol.
7 in vivo, 3 in vitro studies, and 4 studies analyzing the available
data found. They are described below.
Myrcene / 7-methyl-3-
methylene-1,6-octadiene
No study found in the publicly available scientific literature that
does not support our claim that myrcene is a carcinogenic
chemical.
Pulegone / p- Menth-4(8)-en-3-
one.
1 in vivo study found and described below.
142 Id. 143 EFSA, Fl@vouring substances, accessed July 16, 2015 at
http://ec.europa.eu/food/food/chemicalsafety/flavouring/database/dsp_search.cfm. 144 EFSA, Register of Questions, accessed July 16, 2015 at http://registerofquestions.efsa.europa.eu/raw-
war/ListOfQuestionsNoLogin?0.
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Pyridine No study found in the publicly available scientific literature that
does not support our claim that pyridine is a carcinogenic
chemical.
Styrene 2 studies analyzing the available data found and described below.
A. Ethyl acrylate:
We found three in vivo and seven in vitro studies related to cancer. Each is described below.
1. In vivo studies:
a. Relationship between the Time of Sustained Ethyl Acrylate Forestomach Hyperplasia
and Carcinogenicity. (1993) B.I. Ghanayem, I.M. Sanchez, R.R. Maronpot, M. R.
Elwell, and H. B. Matthews. Environmental Health Perspectives 101:277-280
The report stated that:
Data presented in this report demonstrated that EA administration to male F344
rats resulted in the development of FS hyperplasia, which was sustained for as
long as EA administration continued. FS hyperplasia induced by continued
administration of EA for 6 months was largely reversible after cessation of dosing
(Table 1). No treatment-related neoplastic lesions were observed in the FS of rats
exposed to EA for 6 months and sacrificed at 24 months of age (Table 1).
After 12 months of EA administration, forestomachs of all treated rats showed
evidence of hyperplastic lesions, but there was no evidence of gross or
microscopic tumors. However, when animals receiving EA for 12 months were
sacrificed after 9 months of recovery, gross examination showed
papillomas/carcinomas arising from the FS mucosa of some chemical-treated
animals. Microscopic diagnosis in H&E-stained sections further confirmed the
development of FS tumors in these EAtreated rats (Table 1). The incidence of FS
squamous cell carcinomas and papillomas in EA-treated rats was 3/13 and 1/13,
respectively, (combined incidence of 4/13).
b. Demonstration of a temporal relationship between ethyl acrylate-induced
forestomach cell proliferation and carcinogenicity. (1994) Burhan I. Ghanayem,
Idalia M. Sanchez, H. B. Matthews, and Michael R. Elwell. Toxicologic Pathology
22:497-509
The report stated that:
[F]orestomach hyperplasia/cell proliferation was sustained as long as EA
administration continued. Reversal of such effects was time-dependent and cell
proliferation remained enhanced after cessation of EA dosing in the 12-mo study
but not in the 6-mo dosing protocol.
Page 37 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Present work also demonstrated that EA-induced lesions progressed in the
absence of chemical administration. While hyperplasia/cell proliferation was
detected at the end of the 12-mo dosing regimen, squamous cell papillomas and
carcinomas were seen during the recovery period. In conclusion, these data
indicate that the time of sustained enhancement of epithelial cell proliferation
plays a critical role in EA-induced forestomach carcinogenicity.
c. The histopathological and biochemical response of the stomach of male F344/N rats
following two weeks of oral dosing with ethyl acrylate. (1990) Clay B. Frederick,
George A. Hazelton, and Jerry D. Frant. Toxicologic Pathology 18:247-256
The report stated that:
Ethyl acrylate administered by gavage: Primary compound-related
histopathological changes were noted only in the forestomach of treated animals.
None of the effects observed in treated animals were observed in the gastric
epithelium of control animals (Fig. 1; Table I). The treatment-related changes
consisted of diffuse and focal epithelial hyperplasia, hyperkeratosis, subacute to
chronic submucosal inflammation, submucosal edema, and ulcers/erosions.
Ethyl acrylate administered in the drinking water: As in gavage dosing,
compound-related histopathological findings occurred only in the forestomach.
These findings, consisting of epithelial hyperplasia, hyperkeratosis, submucosal
inflammation, and focal epithelial hemorrhage, were generally much less severe
than those noted with comparable doses by gavage.
The authors discussed that “The severe depletion of NPSH [non-protein sulfhydryl]
content in the rat forestomach following repeated gavage dosing of EA appears to be
causally related to the histopathology observed at relatively high doses. This
conclusion is mechanistically plausible and is supported by correlation of the
biochemical and histopathology data with regard to target tissue specificity, dose
response, and the effect of a change in the rate of dose delivery in going from gavage
to drinking water dosing at a comparable daily body burden.”
They concluded that “The metabolic results in the present study support the
histopathological observations and tumor results by suggesting that spreading the
daily body burden of EA throughout a 24 hr period (as opposed to a single bolus
dose) allows effective detoxification.
No NPSH depletion was observed in animals that were dosed in the drinking water,
even at doses that exceeded the highest gavage dose studied when evaluated on a
daily body burden basis. This observation is consistent with the conclusion that
continual exposure to low oral doses of EA is not associated with severe tissue
toxicity or carcinogenicity.”
Page 38 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
2. In vitro studies: The following six studies for ethyl acrylate showed positive genotoxicity data. They
were evaluated by JECFA and referenced by EFSA in its 2010 Flavouring Group
Evaluation 71:145
Endpoint Test system Concentration Reference
Mitotic recombination S. cerevisiae D61.M ≤1 095 µg/ml Zimmermann &
Mohr, 1992146
Forward mutation Mouse lymphoma
L5178Y Tk+/- cells
20 µg/ml Tennant et al.,
1987147
Forward mutation Mouse lymphoma
L5178Y Tk+/- cells
10, 15, 20, 25, 27.5,
30, 32.5, 35, 40 or 50
µg/ml
Ciaccio et al., 1998148
Forward mutation Mouse lymphoma
L5178Y Tk+/- cells
20, 25, 30 or 37.5
µm/ml
Moore et al., 1988149
Forward mutation Mouse lymphoma
L5178Y Tk+/- cells
2.5 – 40 µg/ml McGregor et al.,
1988150
Chromosomal aberration Chinese hamster ovary
cells
299 µg/ml Loveday et al.,
1990151
Chromosomal aberration Mouse lymphoma
L5178Y Tk+/- cells
20, 25, 30 or 37.5
µm/ml
Moore et al., 1988
Chromosomal aberration Chinese hamster ovary
cells
299 µg/ml Tennant et al., 1987
Sister chromatid exchange Chinese hamster ovary
cells
150 µg/ml Tennant et al., 1987
Sister chromatid exchange Chinese hamster ovary
cells
150 µg/ml Loveday et al., 1990
Sister chromatid exchange Mouse lymphoma
L5178Y Tk+/- cells
20.0 – 37.5 µg/ml Moore et al., 1988
145 Table 2.1, page 14. Scientific Opinion on Flavouring Group Evaluation 71 (FGE.71): Consideration of aliphatic,
linear, alpha,beta-unsaturated carboxylic acids and related esters evaluated by JECFA (63rd meeting) structurally
related to esters of branched- and straight-chain unsaturated carboxylic acids. Esters of these and straight-chain
aliphatic saturated alcohols evaluated by in FGE.05Rev2 (2009). EFSA Panel on Food Contact Materials, Enzymes,
Flavourings and Processing Aids (CEF). EFSA Journal 2010; 8(2):1401 146 Zimmermann, F.K., Mohr A., 1992. Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione,
2,3-hexanedione, ethyl actylate, dibromoacetonitrile, 2-hydroxypropionitrile induce chromosome loss in
saccharomyces cerevisiae. Mutat. Res. 270, 151-166. 147 Tennant, R.W., Margolin, B.H., Shelby, M.D., Zeiger, E., Haseman, J.K., Spalding, J., Caspary, W., Resnick,
M., Stasiewicz, S., Anderson, B., Minor, R., 1987. Prediction of chemical carcinogenicity in rodents from in vitro
genetic toxicity assays. Science 236, 933-941. 148 Ciaccio, P.J., Gicquel, E., O´Neill, P.J., Scribner, H.E., Vandenberghe, Y.L., 1998. Investigation of the positive
response of ethyl acrylate in the mouse lymphoma genotoxicity assay. Toxicol. Sci. 46, 324-332. 149 Moore, M.M., Amtower, A., Doerr, C.L., Brock, K.H., Dearfield, K.L., 1988. Genotoxicity of acrylic acid,
methyl acrylate, ethyl acrylate, methyl methacrylate, and ethyl methacrylate in L5178Y mouse lymphoma cells.
Environ. Mol. Mutag. 11, 49-63. 150 McGregor, D.B., Brown, A., Cattanach, P., Edwards, I., McBride, D., Riach, C., Caspary, W.J., 1988. Responses
of the L5178Y tk+/tk- mouse lymphona cell forward mutation assay: III. 72 coded chemicals. Environ. Mol. Mutag.
12, 85-153. 151 Loveday, K.S., Anderson, B.E., Resnick, M.A., Zeiger, E., 1990. Chromosome aberration and sister chromatid
exchange tests in chinese hamster overy cells in vitro. V. Results with 46 chemicals. Environ. Mol. Mutag. 16, 272-
303.
Page 39 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
B. Eugenyl methyl ether / 4-Allylveratrole / Methyl eugenol:
We found seven in vivo and three in vitro studies related to cancer as well as four studies
analyzing the available data. Each is described below.
1. In vivo studies:
a. Histopathological and Immunohistochemical Characterization of Methyl Eugenol–
induced Nonneoplastic and Neoplastic Neuroendocrine Cell Lesions in Glandular
Stomach of Rats. (2015) Kyathanahalli S. Janardhan, Yvette Rebolloso, Geoffrey
Hurlbur, David Olson, Otis Lyght, Natasha P. Clayton, Margarita Gruebbel,
Catherine Picut, Cynthia Shackelford, and Ronald A. Herbert. Toxicologic
Pathology, 43: 681-693
The report stated that:
[M]ethyl eugenol induced NE [neuroendocrine] cell hyperplasias and benign and
malignant NE tumors in the fundic stomach of male and female F344/N rats.
These lesions appear to be of ECL [enterochromaffin-like] cell origin.
Immunohistochemically, the lesions consistently expressed chromogranin A and
synaptophysin and not gastrin or somatostatin. Therefore, chromogranin A or
synaptophysin can be used as a marker in suspected NE cell lesions in the
glandular stomach of rats. Metastatic lesions resembled the primary neoplasms in
both histology features and IHC staining characteristics.
b. The natural basil flavonoid nevadensin protects against a methyleugenol-induced
marker of hepatocarcinogenicity in male F344 rat. (2014) Wasma Alhusainy, Gary
M. Williams, Alan M. Jeffrey, Michael J. Iatropoulos, Sean Taylor, Timothy B.
Adams, Ivonne M.C.M. Rietjens. Food and Chemical Toxicology 74:28–34
The authors concluded that the “tumor formation could be lower in rodent bioassays
when methyleugenol would be dosed in a matrix containing SULT inhibitors [in other
words, basil] such as nevadensin compared to experiments using the pure
methyleugenol.” The report stated that:
The alkenylbenzene methyleugenol occurs naturally in a variety of spices and
herbs, including basil, and their essential oils. At high dose levels methyleugenol
induces hepatocarcinogenicity in rodents following bioactivation to 1′-
sulfooxymethyleugenol which forms DNA adducts. This study investigated
whether the inhibitory effect of the basil flavonoid nevadensin on sulfotransferase
(SULT)-mediated bioactivation of methyleugenol observed in vitro would also be
reflected in a reduction of DNA adduct formation and a reduction in an early
marker for liver carcinogenesis in an 8-week rat study. Co-exposure to
methyleugenol and nevadensin orally resulted in a significant inhibition of liver
methyleugenol DNA adduct formation and in inhibition of hepatocellular altered
Page 40 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
foci induction, representing indicators for initiation of neoplasia. These results
suggest that tumor formation could be lower in rodent bioassays when
methyleugenol would be dosed in a matrix containing SULT inhibitors such as
nevadensin compared to experiments using the pure methyleugenol.
c. Formation of hepatic DNA adducts by methyleugenol in mouse models: drastic
decrease by Sult1a1 knockout and strong increase by transgenic human SULT1A1/2.
(2014) Kristin Herrmann, Wolfram Engst, Walter Meinl, Simone Florian, Alexander
T. Cartus, Dieter Schrenk, Klaus Erich Appel, Tobias Nolden, Heinz Himmelbauer
and Hansruedi Glatt. Carcinogenesis 35:935–941
The report stated that:
A single dose of 0.05 mg/kg (0.28 µmol/kg) methyleugenol was sufficient to form
detectable levels of DNA adducts in the liver of ko-tg mice. This dose is 4-fold
below the estimated mean daily intake of methyleugenol by humans from foods.
In agreement with this efficiency in adduct formation even at low doses, we were
able to clearly demonstrate the presence of N2-MIE-dG adducts, and often also
N6-MIE-dA adducts, in 29 out of 30 human liver biopsy specimens studied (25).
Based on the results in the mouse lines with varying SULT1A status, it is obvious
to expect that individual variation in the SULT1A1 level, e.g. due to copy number
polymorphism, may affect the susceptibility.
d. Methyleugenol hepatocellular cancer initiating effects in rat liver. (2013) Gary M.
Williams, Michael J. Iatropoulos, Alan M. Jeffrey, Jian-Dong Duan. Food and
Chemical Toxicology 53:187–196
The report stated that:
In the present study, the LD [low dose] of 62 mg/kg body weight/d of MEG,
resulting in a cumulative dose at 16 weeks of 3000 mg/kg body weight, induced
HAF [foci of altered hepatocytes], but no hepatocellular neoplasia, even with PB
[phenobarbital] promotion. Thus, this was a dosage which was not capable of
producing a detectable induction of hepatocellular neoplasia, under the
experimental conditions studied here, in spite of producing formation of DNA
adducts, increased hepatocellular proliferation and histopathological changes. The
LD used in the NTP bioassay at 105 weeks, which was hepatocarcinogenic, was
19,000 mg/kg (Table 9). Thus, identification of a cumulative dose of <3000
mg/kg body weight that does not induce preneoplastic hepatocellular effects,
would contribute to defining a no-observed-adverse-effect level for MEG
initiation and consequent carcinogenicity. The relationship of such a dose to
human exposure would assist in risk assessment.
e. Abundance of DNA adducts of methyleugenol, a rodent hepatocarcinogen, in human
liver samples. (2013) Kristin Herrmann, Fabian Schumacher, Wolfram Engst, Klaus
Page 41 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
E. Appel, Kathrin Klein, Ulrich M. Zanger and Hansruedi Glatt. Carcinogenesis
34:1025–1030
The report stated that:
We analyzed surgical human liver samples from 30 subjects for the presence of
DNA adducts originating from methyleugenol using isotope-dilution ultra-
performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS).
Twenty-nine samples unambiguously contained the N2-(trans-methylisoeugenol-
3′-yl)-2′-deoxyguanosine adduct. A second adduct, N6-(trans-methylisoeugenol-
3′-yl)-2′-deoxyadenosine, was also found in most samples, but at much lower
levels, in agreement with the results from experimental models. The maximal and
median levels of both adducts combined were 37 and 13 per 108 nucleosides
(corresponding to 4700 and 1700, respectively, adducts per diploid genome). This
is the first demonstration of DNA adducts formed by a xenobiotic in human liver
using UPLC-MS/MS, the most reliable method available. It has been estimated
for diverse rat and mouse hepatocarcinogens that 50–5500 adducts per 108
nucleosides are present after repeated treatment at the TD 50 (daily dose that
halves the probability to stay tumor-free in long-term studies). We conclude that
the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts
and, therefore, may pose a significant carcinogenic risk.
f. In Vivo Genotoxicity of Methyleugenol in gpt Delta Transgenic Rats Following
Medium-Term Exposure. (2012) Meilan Jin, Aki Kijima, Daisuke Hibi, Yuji Ishii,
Shinji Takasu, Kohei Matsushita, Ken Kuroda, Takehiko Nohmi, Akiyoshi Nishikawa,
and Takasi Umemura. Toxicological Sciences 131:387–394
The authors concluded that “the MEG dose that induces preneoplastic lesions in the
liver resulted in in vivo genotoxicity in the reporter gene mutation assay. The data
presented in this study provide valuable information regarding the development of
risk assessments for the flavoring agents classified as alkoxysubstituted
allylbenzenes.” The report stated that:
[T]he role of genotoxicity as a possible mechanism of action is not fully
understood even though the DNAreactive metabolite of MEG has been identified.
In this study, a gpt delta transgenic rat model was used to clarify whether
genotoxic mechanisms are involved in MEG-induced hepatocarcinogenesis
following medium-term exposure. F344 gpt delta rats were subjected to repeated
oral administration of MEG at dosages of 0, 10, 30, or 100 mg/kg (a carcinogenic
dose) for 13 weeks. The relative weight of the liver of the male and female rats
that were administered 100mg/kg MEG and the absolute weight of the liver of the
male rats that were administered 100 mg/kg MEG were significantly increased. In
addition, the number and area of glutathione S-transferase placental form (GST-P)
positive foci and proliferating cell nuclear antigen (PCNA) positive cell ratios in
the hepatocytes were significantly increased in the male and female rats that were
administered 100 mg/kg MEG compared with the control animals. In the in vivo
Page 42 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
mutation assays, a significant increase in the gpt and Spi− mutant frequencies was
observed in both sexes at the carcinogenic dose. These results suggest the possible
participation of genotoxic mechanisms in MEG-induced hepatocarcinogenesis.
g. Methyleugenol Genotoxicity in the Fischer 344 Rat Using the Comet Assay and
Pathway-Focused Gene Expression Profiling. (2011) Wei Ding, Dan D. Levy,
Michelle E. Bishop, E. Lyn-Cook Lascelles, Rohan Kulkarni, Ching-We Chang,
Anane Aidoo, and Mugimane G. Manjanatha. Toxicological Sciences 123:103–112
This study, conducted by FDA scientists, including from CFSAN, acknowledges
methyl eugenol’s “carcinogenicity” and aimed at clarifying “inconclusive results”
from existing studies on methyl eugenol genotoxicity. The report stated that:
In summary, these results indicate that exposure of MEG to male F344 rats at
doses that produce tumors in rodents failed to induce DNA damage beyond
control levels as measured by the standard alkaline Comet assay. On the other
hand, Endo III–modified in vivo Comet assay designed for the detection of
oxidative DNA damage at pyrimidines showed a significant increase in DNA
damage 6 and 8 h following MEG exposure.
Additionally, analysis of gene expression indicated that genes associated with
DNA damage and repair in the liver were suppressed following exposure to MEG.
Taken together, the results indicate that MEG exposure is associated with
suppression of DNA repair gene expression and the induction of oxidative DNA
damage which may be at least partially responsible for MEG-induced
genotoxicity in rodents. Further studies specifically analyzing a free radical
pathway are necessary to confirm the genotoxic mode of action of MEG in rodent
carcinogenesis.
2. In vitro studies:
a. Physiologically based kinetic modeling of bioactivation and detoxification of the
alkenylbenzene methyleugenol in human as compared with rat. (2012) Al-Subeihi AA,
Spenkelink B, Punt A, Boersma MG, van Bladeren PJ, Rietjens IM. Toxicology and
Applied Pharmacology 260:271-284
The authors’ model predicted that the formation of 1'-sulfooxymethyleugenol
(1'HMES), which readily undergoes desulfonation to a reactive carbonium ion and
can form DNA or protein adducts, is the same in the liver of both human and male
rat. It also predicted a linear rate of formation of the metabolite in both species,
concluding that “kinetic data do not provide a reason to argue against linear
extrapolation from the rat tumor data to the human situation.” The report stated that:
This study defines a physiologically based kinetic (PBK) model for
methyleugenol (ME) in human based on in vitro and in silico derived parameters.
With the model obtained, bioactivation and detoxification of methyleugenol (ME)
Page 43 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
at different doses levels could be investigated. The outcomes of the current model
were compared with those of a previously developed PBK model for
methyleugenol (ME) in male rat. The results obtained reveal that formation of
1'-hydroxymethyleugenol glucuronide (1'HMEG), a major metabolic pathway in
male rat liver, appears to represent a minor metabolic pathway in human liver
whereas in human liver a significantly higher formation of 1'-oxomethyleugenol
(1'OME) compared with male rat liver is observed. Furthermore, formation of
1'-sulfooxymethyleugenol (1'HMES), which readily undergoes desulfonation to a
reactive carbonium ion (CA) that can form DNA or protein adducts (DA), is
predicted to be the same in the liver of both human and male rat at oral doses of
0.0034 and 300 mg/kg bw. Altogether despite a significant difference in
especially the metabolic pathways of the proximate carcinogenic metabolite
1'-hydroxymethyleugenol (1'HME) between human and male rat, the influence of
species differences on the ultimate overall bioactivation of methyleugenol (ME)
to 1'-sulfooxymethyleugenol (1'HMES) appears to be negligible. Moreover, the
PBK model predicted the formation of 1'-sulfooxymethyleugenol (1'HMES) in the
liver of human and rat to be linear from doses as high as the benchmark dose
(BMD10) down to as low as the virtual safe dose (VSD). This study shows that
kinetic data do not provide a reason to argue against linear extrapolation from the
rat tumor data to the human situation.
b. Genotoxic potential of methyleugenol and selected methyleugenol metabolites in
cultured Chinese hamster V79 cells. (2012) Groh IA, Cartus AT, Vallicotti S, Kajzar
J, Merz KH, Schrenk D, Esselen M. Food Funct 3:428-436
The authors showed that methyleugenol metabolites may be causing even more DNA
damage than the parent compound. The report stated that:
Methyleugenol is a substituted alkenylbenzene classified by the European Union's
Scientific Committee on Food as a genotoxic carcinogen. We addressed
cytotoxicity, genotoxicity and mutagenicity caused by methyleugenol and
selected oxidative methyleugenol metabolites in Chinese hamster lung fibroblasts
V79 cells. Cytotoxicity was measured by two cell proliferation assays, water
soluble tetrazolium salt (WST) 1 and sulforhodamine B (SRB) assays.
Genotoxicity was determined by using single cell gel electrophoreses (comet
assay) and the in vitro micronuclei test, while mutagenicity was investigated with
the hypoxanthinephosphoribosyl transferase (hprt) assay. Methyleugenol and
1'-hydroxymethyleugenol showed no or marginal cytotoxic effects, but caused
DNA strand breaks at concentrations ≥10 µM. The metabolites
methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol exhibited growth
inhibitory properties with IC(50)-values of 70-90 µM after 48 h or 72 h of
incubation. These metabolites significantly enhanced cytotoxicity and DNA
damage after 1 h of incubation. Overall, no increase in formamidopyrimidine
DNA glycosylase sensitive sites were detected with the comet assay. The
metabolites 1'-hydroxymethyleugenol and methyleugenol-2',3'-epoxide exceeded
the DNA strand breaking properties of the parent compound methyleugenol.
Page 44 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
However, only 3'-oxomethylisoeugenol and methyleugenol-2',3'-epoxide induced
the formation of micronucleated cells in comparison to the negative control.
These compounds were found to be not or rather weakly mutagenic at the hprt
locus. In summary, phase I metabolites exceeded the cytotoxic and genotoxic
properties of the parent compound methyleugenol.
c. DNA adducts from alkoxyallylbenzene herb and spice constituents in cultured human
(HepG2) cells. (2007) Zhou GD , Moorthy B, Bi J, Donnelly KC, Randerath K.
Environ Mol Mutagen 48:715-721
The authors demonstrated methyl eugenol formed DNA adducts in human liver cells.
The liver was reported by NTP to be the target organ for methyl eugenol
carcinogenicity. The report stated that:
Alkoxy derivatives of allylbenzene, including safrole, estragole, methyleugenol,
myristicin, dill apiol, and parsley apiol, are important herb and spice constituents.
Human exposure occurs mainly through consumption of food and drinks. Safrole,
estragole, and methyleugenol are weak animal carcinogens. Experimental data
reveal the genotoxicity and/or carcinogenicity of some allylbenzenes; however,
except for safrole, the potential capacity of allylbenzenes for forming adducts in
human cellular DNA has not been investigated. In the present study, we have
exposed metabolically competent human hepatoma (HepG2) cells to three
concentrations (50, 150, and 450 muM) of each of the six aforementioned
allylbenzenes and shown by the monophosphate (32)P postlabeling assay that
each compound formed DNA adducts. With the exception of methyleugenol,
DNA adduction was dose dependent, decreasing in the order, estragole
>methyleugenol > safrole approximately myristicin > dill apiol > parsley apiol.
These results demonstrate that safrole, estragole, methyleugenol, myristicin, dill
apiol, and parsley apiol are capable of altering the DNA in these cells and thus
may contribute to human carcinogenesis.
3. Analysis using available data:
a. Quantitative comparison between in vivo DNA adduct formation from exposure to
selected DNA-reactive carcinogens, natural background levels of DNA adduct
formation and tumour incidence in rodent bioassays. (2011) Paini A, Scholz G,
Marin-Kuan M, Schilter B, O'Brien J, van Bladeren PJ, Rietjens IM. Mutagenesis
26:605-618
The report stated that:
Dose-response data on both carcinogenicity and in vivo DNA adduct formation
were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1,
methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and
tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the
US Environmental Protection Agency BMD software. DNA adduct levels at this
Page 45 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
dose were extrapolated assuming linearity of the DNA adduct dose response. In
addition, the levels of DNA adducts at the BMD(10) were compared to available
data on endogenous background DNA damage in the target organ. Although for
an individual carcinogen the tumour response increases when adduct levels
increase, our results demonstrate that when comparing different carcinogens, no
quantitative correlation exists between the level of DNA adduct formation and
carcinogenicity. These data confirm that the quantity of DNA adducts formed by
a DNA-reactive compound is not a carcinogenicity predictor but that other factors
such as type of adduct and mutagenic potential may be equally relevant.
Moreover, comparison to background DNA damage supports the notion that the
mere occurrence of DNA adducts above or below the level of endogenous DNA
damage is neither correlated to development of cancer. These data strongly
emphasise the need to apply the mode of action framework to understand the
contribution of other biological effect markers playing a role in carcinogenicity.
b. Application of the margin of exposure (MoE) approach to substances in food that are
genotoxic and carcinogenic Example: Methyleugenol, CASRN: 93-15-2. (2010)
Benjamin Smith, Peter Cadby, Jean-Charles Leblanc, R. Woodrow Setzer. Food and
Chemical Toxicology 48: S89–S97.
The authors acknowledge that methyl eugenol causes cancer in animals and has
“weak genotoxic activity”. However, they also state that “Because of the evidence of
hepatotoxicity of methyleugenol in all groups of rats and mice, the study cannot be
recognised as conclusive for carcinogenicity at lower, non-toxic doses.”
The main point of their review is to show that there is sufficient data to calculate a
margin of exposure which ranged from 100 to 800 depending on the assumptions
used in the exposure estimation.
c. Correlation of Tumors with DNA Adducts from Methyl Eugenol and Tamoxifen in
Rats. (2004) William J. Waddell, Neil H. Crooks, and Paul L. Carmichael.
Toxicological Sciences 79, 38–40
This report evaluates the dose response for DNA adduct formation from methyl
eugenol and compares that threshold with the carcinogenicity threshold and also with
doses to which humans are exposed. The report stated that:
It appears that adduct formation begins at a dose to rats of about 1019.3 molecules
of methyl eugenol/kg/day. This is about one order of magnitude below the dose
that produces tumors in rats. [T]umor formation from methyl eugenol did not
begin until there were about 30 adducts/108 nucleotides.
The safety factor for methyl eugenol is several orders of magnitude; therefore,
there should be no cause for concern for humans at current levels of exposure.
Page 46 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
d. Safety assessment of allylalkoxybenzene derivatives used as flavouring substances —
methyl eugenol and estragole. (2002) R.L. Smith, T.B. Adams, J. Doull, V.J. Feron,
J.I. Goodman, L.J. Marnett, P.S. Portoghese, W.J. Waddell, B.M. Wagner, A.E.
Rogers, J. Caldwell, I.G. Sipes. Food and Chemical Toxicology 40:851–870
This is a report from the FEMA panel. They stated that
“[H]azard determination uses a mechanism-based approach in which production
of the hepatotoxic sulfate conjugate of the 10-hydroxy metabolite is used to
interpret the pathological changes observed in different species of laboratory
rodents in chronic and subchronic studies. In the risk evaluation, the effect of dose
and metabolic activation on the production of the 10-hydroxy metabolite in
humans and laboratory animals is compared to assess the risk to humans from use
of methyl eugenol and estragole as naturally occurring components of a
traditional diet and as added flavouring substances. Both the qualitative and
quantitative aspects of the molecular disposition of methyl eugenol and estragole
and their associated toxicological sequelae have been relatively well defined from
mammalian studies. Several studies have clearly established that the profiles of
metabolism, metabolic activation, and covalent binding are dose dependent and
that the relative importance diminishes markedly at low levels of exposure (i.e.
these events are not linear with respect to dose). In particular, rodent studies show
that these events are minimal probably in the dose range of 1-10 mg/kg body
weight, which is approximately 100–1000 times the anticipated human exposure
to these substances. For these reasons it is concluded that present exposure to
methyl eugenol and estragole resulting from consumption of food, mainly spices
and added as such, does not pose a significant cancer risk. Nevertheless, further
studies are needed to define both the nature and implications of the dose–response
curve in rats at low levels of exposure to methyl eugenol and estragole.
C. Pulegone / p-Menth-4(8)-en-3-one:
We found the following in vivo study published in 2012:
Mode of Action of Pulegone on the Urinary Bladder of F344 Rats. (2012) Mitscheli S. Da Rocha,
Puttappa R. Dodmane, Lora L. Arnold, Karen L. Pennington, Muhammad M. Anwar, Bret R.
Adams, Sean V. Taylor, Clint Wermes, Timothy B. Adams, and Samuel M. Cohen. Toxicological
Sciences 128:1-8
The authors concluded that “the present studies support the hypothesis that the MOA [mode of
action] for pulegone-induced tumorigenicity in female rats involves urothelial cytotoxicity
followed by regenerative cell proliferation, ultimately leading to tumors.” The report stated that:
Essential oils from mint plants, including peppermint and pennyroyal oils, are used at low
levels as flavoring agents in various foods and beverages. Pulegone is a component of
these oils. In a 2-year bioassay, oral administration of pulegone slightly increased the
Page 47 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
urothelial tumor incidence in female rats. We hypothesized that its mode of action
(MOA) involved urothelial cytotoxicity and increased cell proliferation, ultimately
leading to tumors. Pulegone was administered by gavage at 0, 75, or 150 mg/kg body
weight to female rats for 4 and 6 weeks. Fresh void urine and 18-h urine were collected
for crystal and metabolite analyses. Urinary bladders were evaluated by light microscopy
and scanning electron microscopy (SEM) and bromodeoxyuridine (BrdU) labeling index.
Pulegone and its metabolites, piperitenone, piperitone, menthofuran, and menthone, were
tested for cytotoxicity in rat (MYP3) and human (1T1) urothelial cells by the 3-(4,5-
dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. No abnormal urinary
crystals were observed by light microscopy. Urine samples (18-h) showed the presence of
pulegone, piperitone, piperitenone, and menthofuran in both treated groups. By SEM,
bladders from treated rats showed superficial necrosis and exfoliation. There was a
significant increase in the BrdU labeling index in the high-dose group. In vitro studies
indicated that pulegone and its metabolites, especially piperitenone, are excreted and
concentrated in the urine at cytotoxic levels when pulegone is administered at high doses
to female rats. The present study supports the hypothesis that cytotoxicity followed by
regenerative cell proliferation is the MOA for pulegone-induced urothelial tumors in
female rats.
D. Styrene:
We found two studies that reviewed the NTP finding in its 12th Report on Carcinogens. Each is
summarized below:
1. Review of the Styrene Assessment in the National Toxicology Program 12th Report on
Carcinogens: Workshop Summary. (2014) Committee to Review the Styrene Assessment
in the National Toxicology Program 12th Report on Carcinogens; Board on
Environmental Studies and Toxicology; Division on Earth and Life Studies; National
Research Council. See http://www.ncbi.nlm.nih.gov/books/NBK241556/
The committee concluded that “[b]ased on credible but limited evidence of
carcinogenicity in traditional epidemiologic studies, on sufficient evidence of
carcinogenicity in animals, and on convincing evidence that styrene is genotoxic in
exposed humans, this report finds that compelling evidence exists to support a listing of
styrene as, at a minimum, "reasonably anticipated to be a human carcinogen."”
2. The Weight of Evidence Does Not Support the Listing of Styrene as "Reasonably
Anticipated to be a Human Carcinogen" in NTP's Twelfth Report on Carcinogens. (2013)
Rhomberg LR, Goodman JE, Prueitt RL. Human and Ecological Risk Assessment 19:4-27
The authors stated that “[t]here are no consistent responses among the human data of the
kind that one would expect if there were true biological causation. The diversity of
proposed tumor endpoints in human studies raises more questions than it answers—why
is it that styrene would affect some tumor responses in some studies, and other responses
(requiring other modes of action) in other studies?”
Page 48 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
They also argue that “[f]or styrene, however, it is clear that the processes responsible for
the tumorigenesis observed in mice do not occur in rats. It is not only that rats do not
show a tumor impact of styrene inhalation (the hypothesized generality of which across
mammals is the basis for inferring its relevance to humans), but also that the specific
mode of action—the tissue-specific metabolic activation—is not present. In short, the
proposed generalization of effects across mammals is contradicted. Moreover, there is no
indication that the mode of action would be present in humans, either.”
Styrene was listed as “reasonably anticipated to be a human carcinogen” in the twelfth
edition of the National Toxicology Program’s Report on Carcinogens based on what we
contend are erroneous findings of limited evidence of carcinogenicity in humans,
sufficient evidence of carcinogenicity in experimental animals, and supporting
mechanistic data. The epidemiology studies show no consistent increased incidence of, or
mortality from, any type of cancer. In animal studies, increased incidence rates of mostly
benign tumors have been observed only in certain strains of one species (mice) and at one
tissue site (lung). The lack of concordance of tumor incidence and tumor type among
animals (even within the same species) and humans indicates that there has been no
particular cancer consistently observed among all available studies. The only plausible
mechanism for styrene-induced carcinogenesis—a non-genotoxic mode of action that is
specific to the mouse lung—is not relevant to humans. As a whole, the evidence does not
support the characterization of styrene as “reasonably anticipated to be a human
carcinogen,” and styrene should not be listed in the Report on Carcinogens.
Page 49 Carcinogenic Flavors Food Additive Petition – FAP 5A4810
Appendix 4
Requested changes to 21 CFR § 172.515
Note that we have removed chemicals from the table unaffected by this petition.
TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION
DEPARTMENT OF HEALTH AND HUMAN SERVICES SUBCHAPTER B--FOOD FOR HUMAN CONSUMPTION (CONTINUED)
PART 172 -- FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR
HUMAN CONSUMPTION
Subpart F--Flavoring Agents and Related Substances
Sec. 172.515 Synthetic flavoring substances and adjuvants.
Synthetic flavoring substances and adjuvants may be safely used in food in accordance with the
following conditions.
(a) They are used in the minimum quantity required to produce their intended effect, and
otherwise in accordance with all the principles of good manufacturing practice.
(b) They consist of one or more of the following, used alone or in combination with flavoring
substances and adjuvants generally recognized as safe in food, prior-sanctioned for such use, or
regulated by an appropriate section in this part.
Benzophenone; diphenylketone.
Ethyl acrylate.
Eugenyl methyl ether; 4-allylveratrole; methyl eugenol.
Myrcene; 7-methyl-3-methylene-1,6-octadiene
Pulegone; p- menth-4(8)-en-3-one.
Pyridine.
Styrene.
(c) [Delta]-Decalactone and [Delta]-dodecalactone when used separately or in combination in
oleomargarine are used at levels not to exceed 10 parts per million and 20 parts per million,
respectively, in accordance with 166.110 of this chapter.
(d) BHA (butylated hydroxyanisole) may be used as an antioxidant in flavoring substances
whereby the additive does not exceed 0.5 percent of the essential (volatile) oil content of the
flavoring substance.
(e) The following flavoring substances shall not be used as a flavor in food.
Benzophenone; diphenylketone.
Ethyl acrylate.
Eugenyl methyl ether; 4-allylveratrole; methyl eugenol.
Myrcene; 7-methyl-3-methylene-1,6-octadiene.
Pulegone; p- menth-4(8)-en-3-one.
Pyridine.
Trans,trans-2,4-hexadienal.
Styrene.