Draft EFSA Scientific Opinion on the evaluation of the safety of preparations from the fruits of sweet and bitter fennel (Foeniculum vulgare Mill. and Foeniculum piperitum (Ucria) C.Presl)

References

TOX/2025/34

Last updated: 29 August 2025

This is a paper for discussion. This does not represent the views of the Committee and should not be cited.

Ackermann G, Peil M, Quarz C, Schmidt A, Halaczkiewicz M, Thomas AD, Stegmüller S, Richling E, Manolikakes G, Christmann M, Küpper JH, Schrenk D and Fahrer J, 2025. Molecular dosimetry of estragole and 1'-hydroxyestragole-induced DNA adduct formation, clastogenicity and cytotoxicity in human liver cell models. Archives of Toxicology. https://doi.org/10.1007/s00204-025-04084-2

Alajlouni AM, Al_Malahmeh AJ, Kiwamoto R, Wesseling S, Soffers AEMF, Al-Subeihi AAA, Vervoort J and Rietjens IMCM, 2016. Mode of action based risk assessment of the botanical food-borne alkenylbenzene apiol from parsley using physiologically based kinetic (PBK) modelling and read-across from safrole. Food and Chemical Toxicology, 89:138-150. https://doi.org/10.1016/j.fct.2016.01.018

Alhusainy W, Paini A, Punt A, Louisse J, Spenkelink A, Vervoort J, Delatour T, Scholz G, Schilter B, Adams T, van Bladeren PJ and Rietjens IM, 2010. Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect. Toxicology and Applied Pharmacology, 245:179-190. https://doi.org/10.1016/j.taap.2010.02.017

Alhusainy W, Paini A, van den Berg JH, Punt A, Scholz G, Schilter B, van Bladeren PJ, Taylor S, Adams TB and Rietjens IM, 2013. In vivo validation and physiologically based biokinetic modeling of the inhibition of SULT-mediated estragole DNA adduct formation in the liver of male Sprague-Dawley rats by the basil flavonoid nevadensin. Molecular Nutrition & Food Research, 57:1969-1978. https://doi.org/10.1002/mnfr.201300144

Alhusainy W, van den Berg SJ, Paini A, Campana A, Asselman M, Spenkelink A, Punt A, Scholz G, Schilter B, Adams TB, van Bladeren PJ and Rietjens IM, 2012. Matrix modulation of the bioactivation of estragole by constituents of different alkenylbenzene-containing herbs and spices and physiologically based biokinetic modeling of possible in vivo effects. Toxicological Sciences, 129:174-187. https://doi.org/10.1093/toxsci/kfs196

Al-Subeihi, A. A., Spenkelink, B., Punt, A., Boersma, M. G., van Bladeren, P. J., & Rietjens, I. M. 2012. Physiologically based kinetic modeling of bioactivation and detoxification of the alkenylbenzene methyleugenol in human as compared with rat. Toxicology and applied pharmacology, 260(3), 271–284. https://doi.org/10.1016/j.taap.2012.03.005

Anthony A, Caldwell J, Hutt AJ and Smith RL, 1987. Metabolism of estragole in rat and mouse and influence of dose size on excretion of the proximate carcinogen 1′- hydroxyestragole. Food and Chemical Toxicology, 25:799-806. https://doi.org/10.1016/0278-6915(87)90257-2

Boberg EW, Miller EC, Miller JA, Poland A and Liem A, 1983. Strong evidence from studies with brachymorphic mice and pentachlorophenol that 1'-sulfoöxysafrole is the major ultimate electrophilic and carcinogenic metabolite of 1'-hydroxysafrole in mouse liver. Cancer Research, 43:5163-5173.

Daniels J and and Kadlubar S, 2014. Pharmacogenetics of Sult1A1. Pharmacogenomics, 15:1823-1838. https://doi.org/10.2217/pgs.14.134

Denzer MY, Kirsch F and Buettner A, 2015. Are odorant constituents of herbal tea transferred into human milk? Journal of Agricultural and Food Chemistry, 63:104-111. https://doi.org/10.1021/jf504073d

EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2022a. Scientific Opinion on the safety and efficacy of a feed additive consisting of an essential oil from the fruit of Cuminum cyminum L. (cumin oil) for use in all animal species (FEFANAasbl). EFSA Journal 2022;20(12):7690, 26 pp. https://doi.org/10.2903/j.efsa.2022.769

EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2022b. Scientific Opinion on the safety and efficacy of a feed additive consisting of an extract of olibanum from Boswellia serrata Roxb. ex Colebr. for use in dogs and horses (FEFANA asbl). EFSA Journal 2022;20(3):7158, 24 pp. https://doi.org/10.2903/j.efsa.2022.7158

Eisenreich A, Götz ME, Sachse B, Monien BH, Herrmann K and Schäfer B, 2021. Alkenylbenzenes in foods: Aspects impeding the evaluation of adverse health effects. Foods, 10:2139. https://doi.org/10.3390/foods10092139

EMA HMPC (European Medicines Agency, Committee on Herbal Medicinal Products), 2005. Public statement on the use of herbal medicinal products containing methyleugenol. EMEA/HMPC/138363/2005. HMPC Public Statement on the use of HMP cont methyleugenol

EMA HMPC (European Medicines Agency, Committee on Herbal Medicinal Products), 2023. Public statement on the use of herbal medicinal products containing estragole. EMA/HMPC/137212/2005 Rev 1 Corr 1*. HMPC Public Statement on the use of HMP containing estragole- draft Corr 1

EMA HMPC (European Medicines Agency, Committee on Herbal Medicinal Products), 2024a. Assessment report on Foeniculum vulgare Miller subsp. vulgare var. vulgare, aetheroleum Final – Revision 1. EMA/HMPC/271394/2022. Foeniculi amari fructus aetheroleum - AR

EMA HMPC (European Medicines Agency, Committee on Herbal Medicinal Products), 2024b. European Union herbal monograph on Foeniculum vulgare Miller subsp. vulgare var. dulce (Mill.) Batt. & Trab., fructus Final – Revision 1. EMEA/HMPC/372839/2016, 10 pp. Foeniculi dulcis fructus - MO

EMA HMPC (European Medicines Agency, Committee on Herbal Medicinal Products), 2024c. European Union herbal monograph on Foeniculum vulgare Miller subsp. vulgare var. vulgare, fructus Final – Revision 1. EMA/HMPC/372841/2016, 9 pp. Foeniculi amari fructus - MO

EMA HMPC (European Medicines Agency, Committee on Herbal Medicinal Products), 2024d. Public statement on Foeniculum vulgare Miller subsp. vulgare var. vulgare, aetheroleum. EMA/HMPC/522456/2021. Foeniculi amari fructus aetheroleum - Public statement

Fennell TR, Miller JA and Miller EC, 1984. Characterization of the biliary and urinary glutathione and N-acetylcysteine metabolites of the hepatic carcinogen 1'- hydroxysafrole and its 1'-oxo metabolite in rats and mice. Cancer Research, 44:3231-3240. Characterization of the biliary and urinary glutathione and N-acetylcysteine metabolites of the hepatic carcinogen 1'-hydroxysafrole and its 1'-oxo metabolite in rats and mice - PubMed

Götz ME, Sachse B, Schäfer B and Eisenreich A, 2022. Myristicin and elemicin: Potentially toxic alkenylbenzenes in food. Foods, 11. https://doi.org/10.3390/foods11131988

Hartwig A, Arand M, Epe B, Guth S, Jahnke G, Lampen A, Martus H-J, Monien B, Rietjens IMCM, Schmitz-Spanke S, Schriever-Schwemmer G, Steinberg P and Eisenbrand G, 2020. Mode of action-based risk assessment of genotoxic carcinogens. Archives of Toxicology, 94:1787-1877. https://doi.org/10.1007/s00204-020-02733-2

Herrmann K, Engst W, Meinl W, Florian S, Cartus AT, Schrenk D, Appel KE, Nolden T, Himmelbauer H and Glatt H, 2014. Formation of hepatic DNA adducts by methyleugenol in mouse models: drastic decrease by Sult1a1 knockout and strong increase by transgenic human SULT1A1/2. Carcinogenesis, 35:935-941. https://doi.org/10.1093/carcin/bgt408

Herrmann K, Schumacher F, Engst W, Appel KE, Klein K, Zanger UM and Glatt H, 2013. Abundance of DNA adducts of methyleugenol, a rodent hepatocarcinogen, in human liver samples. Carcinogenesis, 34:1025-1030. https://doi.org/10.1093/carcin/bgt013

Jeurissen SM, Punt A, Delatour T and Rietjens IM, 2008. Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'- hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells. Food and Chemical Toxicology, 46:2296-2302.  https://doi.org/10.1016/j.fct.2008.03.010

Jeurissen SMF, Punt A, Boersma MG, Bogaards JJP, Fiamegos YC, Schilter B, van Bladeren PJ, Cnubben NHP and Rietjens IMCM, 2007b. Human cytochrome P450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes. Chemical Research in Toxicology, 20:798-806. https://doi.org/10.1021/tx700012d

Lu L-JW, Disher RM, Reddy MR and Randerath K, 1986. 32P-Postlabeling Assay in Mice of Transplacental DNA Damage Induced by the Environmental Carcinogens Safrole, 4-Aminobiphenyl, and Benzo(a)pyrene1 | Cancer Research | American Association for Cancer Research, 46:3046-3054.

Marabini L, Galli CL, Fauci PL and Marinovich M, 2019. Effect of plant extracts on the genotoxicity of 1'-hydroxy alkenylbenzenes. Regulatory toxicology and pharmacology: RTP, 105. https://doi.org/10.1016/j.yrtph.2019.03.017

Martati E, Boersma MG, Spenkelink A, Khadka DB, van Bladeren PJ, Rietjens IMCM and Punt A, 2012. Physiologically Based Biokinetic (PBBK) Modeling of safrole bioactivation and detoxification in humans as compared with rats. Toxicological Sciences, 128:301-316. https://doi.org/10.1093/toxsci/kfs174

Monien BH, Sachse B, Niederwieser B and Abraham K, 2019. Detection of N-Acetyl-S-[3'-(4-methoxyphenyl)allyl]-l-Cys (AMPAC) in human urine samples after controlled exposure to fennel tea: A new metabolite of estragole and trans-anethole. Chemical Research in Toxicology, 32:2260-2267. https://doi.org/10.1021/acs.chemrestox.9b00287

Monien BH, Schumacher F, Herrmann K, Glatt H, Turesky RJ and Chesné C, 2015. Simultaneous detection of multiple DNA adducts in human lung samples by isotope dilution UPLC-MS/MS. Analytical Chemistry, 87:641-648. https://doi.org/10.1021/ac503803m

Ning J, Louisse J, Spenkelink B, Wesseling S and Rietjens IMCM, 2017. Study on inter-ethnic human differences in bioactivation and detoxification of estragole using physiologically based kinetic modeling. Archives of Toxicology, 91:3093-3108. https://doi.org/10.1007/s00204-017-1941-x

NTP (National Toxicology Programme), 2000. Technical report on the toxicology and carcinogenesis studies of methyleugenol (CAS No. 93-15-2) in F344/N rats and B6C3F1 mice. https://ntp.niehs.nih.gov/go/tr491abs

Paini A, Punt A, Scholz G, Gremaud E, Spenkelink B, Alink G, Schilter B, Bladeren PJv and Rietjens IM, 2012. In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling. Mutagenesis, 27. https://doi.org/10.1093/mutage/ges031

Paini A, Punt A, Viton F, Scholz G, Delatour T, Marin-Kuan M, Schilter B, van Bladeren PJ and Rietjens IM, 2010. A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes. Toxicology and Applied Pharmacology, 245:57-66. https://doi.org/10.1016/j.taap.2010.01.016

Phillips DH, Miller JA, Miller EC and Adams B, 1981. Structures of the DNA Adducts Formed in Mouse Liver after Administration of the Proximate Hepatocarcinogen 1′-Hydroxyestragole1 | Cancer Research | American Association for Cancer Research, 41:176-186.

Phillips DH, Reddy MV and Randerath K, 1984. 32 P-Post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally occurring alkenylbenzenes. II. Newborn male B6C3F 1 mice. Carcinogenesis, 5:1623-1628. https://doi.org/10.1093/carcin/5.12.1623

Punt A, Paini A, Boersma MG, Freidig AP, Delatour T, Scholz G, Schilter B, Bladeren PJv and Rietjens IMCM, 2009b. Use of physiologically based biokinetic (PBBK) modeling to study estragole bioactivation and detoxification in humans as compared with male rats. Toxicological Sciences, 110:255-269.  https://doi.org/10.1093/toxsci/kfp102

Punt A, Paini A, Spenkelink A, Scholz G, Schilter B, van Bladeren PJ and Rietjens IMCM, 2016. Evaluation of interindividual human variation in bioactivation and DNA adduct formation of estragole in liver predicted by physiologically based kinetic/dynamic and Monte Carlo modeling. Chemical Research in Toxicology, 29:659-668. https://doi.org/10.1021/acs.chemrestox.5b00493

Randerath K, Haglund RE, Phillips DH and Reddy MV, 1984. 32 P-Post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice. Carcinogenesis, 5:1613-1622. https://doi.org/10.1093/carcin/5.12.1613

Rietjens IMCM, Huseiny WA and Boersma MG, 2011. Flavonoids and alkenylbenzenes: New concepts in bioactivation studies. Chemico-Biological Interactions, 192:87-95. https://doi.org/10.1016/j.cbi.2010.09.016

Sangster SA, Caldwell J, Hutt AJ, Anthony A and Smith RL, 1987. The metabolic disposition of [methoxy-14C]-labelled trans-anethole, estragole and p-propylanisole in human volunteers. Xenobiotica, 17:1223-1232. https://doi.org/10.3109/00498258709167414

SCF (Scientific Committee on Food), 2001a. Opinion on estragole (1-allyl-4-methoxybenzene). SCF/CS/FLAV/FLAVOUR/6 ADD2 FINAL, Opinion on Estragole (1-Allyl-4-methoxybenzene)

SCF (Scientific Committee on Food), 2001b. Opinion on methyleugenol (4-allyl-1,2- dimethoxybenzene). SCF/CS/FLAV/FLAVOUR/4 ADD1 FINAL, Opinion on Methyleugenol (4-Allyl-1,2-dimethoxybenzene)

SCF (Scientific Committee on Food), 2002. Opinion on the safety of the presence of safrole (1-allyl-3,4-methylene dioxy benzene) in flavourings and other food ingredients with flavouring properties. SCF/CS/FLAV/FLAVOUR/6 ADD3 Final, Opinion of the Scientific Committee on Food on the safety of the presence of safrole (1-allyl-3,4-methylene dioxy benzene...

Schulte-Hubbert R, Küpper J-H, Thomas AD and Schrenk D, 2020. Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells. Toxicology, 444:152566. https://doi.org/10.1016/j.tox.2020.152566

Smith RL, Adams TB, Doull J, Feron VJ, Goodman JI, Marnett LJ, Portoghese PS, Waddell WJ, Wagner BM, Rogers AE, Caldwell J and Sipes IG, 2002. Safety assessment of allylalkoxybenzene derivatives used as flavouring substances — methyl eugenol and estragole. Food and Chemical Toxicology, 40:851-870. https://doi.org/10.1016/s0278-6915(02)00012-1

Solheim E and Scheline RR, 1973. Metabolism of alkenebenzene derivatives in the rat. I. p-Methoxyallylbenzene (Estragole) and p-methoxypropenylbenzene (Anethole). Xenobiotica: The Fate of Foreign Compounds in Biological Systems, 3. https://doi.org/10.3109/00498257309151538

Suparmi S, Ginting AJ, Mariyam S, Wesseling S and Rietjens I, 2019. Levels of methyleugenol and eugenol in instant herbal beverages available on the Indonesian market and related risk assessment. Food and Chemical Toxicology, 125:467-478. https://doi.org/10.1016/j.fct.2019.02.001

Suzuki Y, Umemura T, Ishii Y, Hibi D, Inoue T, Jin M, Sakai H, Kodama Y, Nohmi T, Yanai T, Nishikawa A and Ogawa K, 2012. Possible involvement of sulfotransferase 1A1 in estragole-induced DNA modification and carcinogenesis in the livers of female mice. Mutation Research, 749:23-28. https://doi.org/10.1016/j.mrgentox.2012.07.002

Tremmel R, Herrmann K, Engst W, Meinl W, Klein K, Glatt H and Zanger UM, 2017. Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels. Archives of Toxicology, 91:3329-3339.  https://doi.org/10.1007/s00204-017-1955-4

Vesselinovitch SD, Rao KV and Mihailovich N, 1979. Transplacental and Lactational Carcinogenesis by Safrole1 | Cancer Research | American Association for Cancer Research, 39:4378-4380.

Wiseman RW, Miller EC, Miller JA and Liem A, 1987. Structure-Activity Studies of the Hepatocarcinogenicities of Alkenylbenzene Derivatives Related to Estragole and Safrole on Administration to Preweanling Male C57BL/6J × C3H/HeJ F1 Mice1 | Cancer Research | American Association for Cancer Research, 47:2275-2283

Yang S, Diem M, Liu JDH, Wesseling S, Vervoort J, Oostenbrink C and Rietjens I, 2020. Cellular levels and molecular dynamics simulations of estragole DNA adducts point at inefficient repair resulting from limited distortion of the double-stranded DNA helix. Archives of Toxicology, 94:1349-1365. https://doi.org/10.1007/s00204-020-02695-5

Yang S, Kawai T, Wesseling S and Rietjens IMCM, 2022. In vitro and in silico study on consequences of combined exposure to the food-borne alkenylbenzenes estragole and safrole. Toxicology In Vitro, 79:105290. https://doi.org/10.1016/j.tiv.2021.105290

Yang S, Meng Z, Li Y, Chen R, Yang Y and Zhao Z, 2021. Evaluation of physiological characteristics, soluble sugars, organic acids and volatile compounds in ‘Orin’ apples (Malus domestica) at different ripening stages. Molecules, 26:807. https://doi.org/10.3390/molecules26040807

Zangouras A, Caldwell J, Hutt AJ and Smith RL, 1981. Dose dependent conversion of estragole in the rat and mouse to the carcinogenic metabolite, 1'-hydroxyestragole. Biochemical Pharmacology, 30. https://doi.org/10.1016/0006-2952(81)90329-4

Zeller A, Horst K and Rychlik M, 2009. Study of the metabolism of estragole in humans consuming fennel tea. Chemical Research in Toxicology, 22:1929-1937. https://doi.org/10.1021/tx900236g

Zhou G-D, Moorthy B, Bi J, Donnelly KC and Randerath K, 2007. DNA adducts from alkoxyallylbenzene herb and spice constituents in cultured human (HepG2) cells. Environmental and Molecular Mutagenesis, 48:715-721. https://doi.org/10.1002/em.20348