center for food safety and applied nutrition 5100 paint

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.


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



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.


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.


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.


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

[email protected]

[email protected]

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

[email protected]

Caroline Cox, Research Director

Center for Environmental Health

2201 Broadway, Suite 302

Oakland, CA 94612

[email protected]

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.


[email protected]

[email protected]

Cristina Stella, Staff Attorney

Center for Food Safety

303 Sacramento Street, Second Floor

San Francisco, CA 94111

[email protected]

Urvashi Rangan, Executive Director for Food Safety and Sustainability

Consumers Union

101 Truman Avenue

Yonkers, NY 10703-1057

[email protected]

Delores E. Weis, Executive Director

Improving Kids’ Environment (IKE)

1915 W. 18th Street

Indianapolis, Indiana 46202

[email protected]

Maricel Maffini, Ph.D., Consulting Senior Scientist

[email protected]

James Huff, PhD

Formerly, Associate Director for Chemical Carcinogenesis, National Institute of Environmental

Health Sciences


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


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.


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


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


n. NTP, Report on Carcinogens, Thirteenth Edition, Methyleugenol, 2014. See

http://ntp.niehs.nih.gov/go/roc13.


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.


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.


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


v. NTP, Toxicology and Carcinogenesis Studies of Pulegone (CAS No. 89-82-7) in F344/N

Rats and B6C3F1 Mice (Gavage Studies), 2011.


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.


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



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


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


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


substances described above as flavors. As a result, no tolerance is needed.


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.


• 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


• 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


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.


• 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.


• 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


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.


• 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


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.


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.


• 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


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.


• 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.


• 7 chemicals that feature “amine” characteristics.54


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


• 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


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.


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


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.


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



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.


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.


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]


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.


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


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.


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


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



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.


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.


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.


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


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


Technology, Vol. 19, No. 2, 1965. 109 FDA, Final Rule for Synthetic Flavoring Substances and Adjuvants, 29 Fed. Reg. 14625 (October 27, 1964).


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.


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


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.


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.


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.


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.


“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.


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.


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


[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.


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


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.”


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


L5178Y Tk+/- cells

10, 15, 20, 25, 27.5,

30, 32.5, 35, 40 or 50

µg/ml

Ciaccio et al., 1998148


L5178Y Tk+/- cells

20, 25, 30 or 37.5

µm/ml

Moore et al., 1988149


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.


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


[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


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


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


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


E. Appel, Kathrin Klein, Ulrich M. Zanger and Hansruedi Glatt. Carcinogenesis

34:1025–1030


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


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)


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.


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


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


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.


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


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?”


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.


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.