SUPPLEMENTARY INFORMATION 1: Identity for Natural Flavor Complexes as Evaluated by the Expert Panel

The Identification Description as Reviewed by the FEMA FEMA No.1 FEMA Primary Name Expert Panel Rebaudioside M ≥80%; Rebaudioside D 5-20%; Total 4895 Rebaudioside M steviol ≥95%.

Glutamic acid 35-40%; Other amino acids 1-2%; Total Corynebacterium glutamicum corn nitrogen 6-7%; Aliphatic primary alcohols, aldehydes, 4907 syrup fermentation product carboxylic acids, acetals and esters containing additional oxygenated functional groups 1-2%; Minerals 9-11%

Inosine 5´-monophosphate 20-25%; Amino acids 7-8%; Corynebacterium stationis corn 4908 Minerals 23-25%; water 28-37%; Other nucleotides 1-2%; syrup fermentation product Total nitrogen 5-8% Supraglucosylated steviol glycosides 70-80%; Rebaudioside Glucosylated steviol glycosides, 4909 A 14-20%; Steviol glycosides not further glucosylated, each 70-80% individually, not to exceed 3%; Maltodextrin 3-10% Supraglucosylated steviol glycosides 30-40%; Rebaudioside Glucosylated steviol glycosides, A 5-8%; Not more than 4% ; All other individual 4910 40% steviol glycosides not further glucosylated <3%; Maltodextrin 45-60%

Stevioside 70-80%; 13-18%; Steviobioside 1- 3%; Rebaudioside C 2-3%; Total glycosides (including 4911 extract stevioside, 70% Rebaudioside D, Rebaudioside B, Rebaudioside F, Dulcoside A, and Rubusoside) <3%

Derived from hibiscus blossom calyces (Hibiscus sabdariffa L.) , Hibiscus blossom extract is measured as water 30-60%; 4912 Hibiscus blossom extract ethanol 0-11%; ash content 5-10%; hibiscus acid 12-18%; hydroxycitric acid 2-7%; and 3-5%; phenols 1-3.5%; anthocyanins 0.3-0.6%; volatiles <0.1% Derived from refined soybean oil, refined soybean oil extract is measured as beta-Sitosterol 43-45%; Stigmatasterol 27- 4919 Refined soybean oil extract 30%; Campesterol 25-27%; Brassicasterol, trace amounts; Not to exceed Good Manufacturing Practices levels of 50 ppb 3-monochloropropylene (3-MCP) and glycidyl esters

4921 Rebaudioside D 95% Rebaudioside D ≥95%; Total other steviol glycosides <3.5%

Rebaudioside M ≥95%; Rebaudioside B <2%; 4922 Rebaudioside M 95% Rebaudioside A <1.5%; Total other steviol glycosides <1.5%

Not less than 90% of total steviol glycosides inclusive of supraglucosylated steviol glycosides; Rebaudioside A less Glucosylated steviol glycosides, 4931 than 3%; Stevioside less than 2%; other steviol glycosides not 90% further glucosylated, each individually, less than 2%; Maltodextrin less than 10%

Derived from the leaves of Chaenomeles speciosa, Chaenomeles speciosa leaf Chaenomeles speciosa leaf extract is measured as not less 4932 extract than 2% polyphenols and ≥95% non-volatile components, including carbohydrate, fat, protein and water

1 The identity descriptions for FEMA 4923-4925 are included in Cohen et al. (2020).

FEMA GRAS 29 December 2019 Derived from the leaves of Eriobotrya japonica, Eriobotrya japonica leaves extract is measured as not less than 2% citric 4933 Eriobotrya japonica leaves extract acid; Not less than 2.5% triterpene acids; Not less than 93% non-volatile components including carbohydrates, fat, protein, water.

Rebaudioside E 85-92%; Rebaudioside D 5-10%; 4936 Rebaudioside E ≥85% Steviobioside and stevioside, each individually, less than 1%

Not less than 95% Rebaudioside I; Rebaudioside B less than 4937 Rebaudioside I 95% 1%

Not less than 88% beta-bisabolene with no less than 7% 4940 beta-Bisabolene ≥88% aliphatic and aromatic hydrocarbons, typically identified as beta-sesquiphellandrene and gamma-bisabolene.

Not less than 60% nootkatone; total mono-, di- and 4941 Nootkatone complex trioxygenated sesquiterpenes up to 95%.

Derived from guaiac wood oil (Guaiacum spp.), Modified guaiac wood extract includes not less than 1.25% (3S,5R,8S)- 3,8-dimethyl-5-prop-1-en-2-yl-3,4,5,6,7,8-hexahydro-2H- 4942 Modified guaiac wood extract azulen-1-one; sesquiterpenes 9-25%; monooxygenated norsesquiterpenes and sesquiterpenes 25-40%; di- and trioxygenated sesquiterpenes 6-15%; triethyl citrate 35-50%.

FEMA GRAS 29 December 2019 Supplementary Information. Key Findings of the FEMA Expert Panel Safety Evaluations for GRAS 29

Since its initial publication of GRAS determinations for flavor plus) rats resulted in a no observed adverse effect level ingredients (Hall and Oser, 1965), the FEMA Expert Panel has (NOAEL) of greater than 15 mg/kg bw/day (Api et al., 2004) made available information on its determinations, including which is greater than 62,500 times the anticipated daily per conditions of intended use for individual flavor ingredients, capita intake of 1-(3,5,5,6,8,8-hexamethyl-5,6,7,8- and the scientific basis and information supporting these tetrahydronaphthalen-2-yl)ethanone from use as a flavor determinations. Included herein are the key findings for each ingredient. of the new GRAS determinations included within GRAS 29. Comprehensive monographs of the information relevant to the The Panel reviewed the GRAS application and supporting evaluations are also published as part of the FEMA Expert information regarding 2-(4-ethylphenoxy)-N-(lH-pyrazol-3-yl)- Panel’s ongoing GRAS re-evaluation program (see Hallagan N-(thiophen-2-ylmethyl)acetamide (CAS 2015168-50-8) and and Hall (2009)). For more information on the FEMA GRAS concluded that the substance is GRAS (FEMA 4880) (Smith program, please see “About the FEMA GRAS Program” on et al., 2005) for use as a flavor ingredient in the food femaflavor.org. categories and at the use levels specified in the GRAS application (see Table 2). This substance was evaluated The Panel reviewed the GRAS application and supporting individually within the context of the chemical group of information regarding 1-(3,5,5,6,8,8-hexamethyl-5,6,7,8- miscellaneous nitrogen-containing substances (JECFA, tetrahydronaphthalen-2-yl)ethanone (CAS 21145-77-7) and 2006, 2009, 2012, 2015; SLR, D19). The Panel calculated the concluded that the substance is GRAS (FEMA 4879) (Smith anticipated per capita intake (“eaters only”) of 2-(4- et al., 2005a) for use as a flavor ingredient in the food ethylphenoxy)-N-(lH-pyrazol-3-yl)-N-(thiophen-2- categories and at the use levels specified in the GRAS ylmethyl)acetamide for use as a flavor ingredient to be 290 application (see Table 2). This substance was evaluated µg/person/day, which is above the threshold of toxicological individually within the context of the chemical group of concern for structural class III (90 µg/person/day) (Munro et aromatic substituted secondary alcohols, ketones, and related al., 1996). The Panel considered the specification of the esters (Adams et al., 2007; JECFA, 2001, 2011, 2017; SLR, material to be adequately characterized by the purity assay C14). The Panel calculated the anticipated per capita intake and supporting spectral data provided for FEMA GRAS (“eaters only”) of 1-(3,5,5,6,8,8-hexamethyl-5,6,7,8- evaluation. The Panel evaluated sensory data that were tetrahydronaphthalen-2-yl)ethanone from use as a flavor included within the application and found them satisfactory ingredient to be 14 µg/person/day, which is below the with regards to intended conditions of use for the flavoring threshold of toxicological concern for structural class II (540 µg/person/day) (Munro et al., 1996). The Panel considered ingredient (Harman and Hallagan, 2013). Based on the the specification of the material to be adequately structural relative, 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)- characterized by the purity assay and supporting spectral data N-(thiophen-2-ylmethyl)acetamide (FEMA 4809), it is provided for FEMA GRAS evaluation. It is presumed that 1- presumed that 2-(4-ethylphenoxy)-N-(1H-pyrazol-3-yl)-N- (3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydronaphthalen-2- (thiophen-2-ylmethyl)acetamide will be rapidly hydrolyzed to yl)ethanone will undergo oxidation at multiple positions and yield the secondary amine product and 4-ethylphenoxyacetic the polar metabolites are excreted in the feces and urine (Api acid (Smith et al., 2018). In addition to hydrolysis, oxidation at et al., 2013; Girkin, 1998; Smith et al., 2018; Yang et al., several sites on the substance is likely, and the more polar 2015). No increases in the number of chromosome metabolites are expected to be excreted in the urine or feces aberrations in an in vitro chromosomal aberration assay in either without further reaction or after conjugation CHO cells without S9 metabolic activation were noted. (Karanewsky et al., 2015). No increases in the number of Statistically significant increases in numerical chromosomal reverse mutations were observed in the Ames assay for the aberrations were observed for this substance in the presence structurally related substance, 2-(4-methylphenoxy)-N-(1H- of S9 metabolic activation under conditions of four hours of pyrazol-3-yl)-N-(thiophen-2-ylmethyl)acetamide, in exposure with a 44-hour recovery period as well as four hours Salmonella typhimurium (S. typhimurium) strains TA98, of exposure with a 20-hour recovery period. However, TA100, TA1535 and TA1537 and Escherichia coli (E. coli) because these data were within historical control ranges and WP2uvrA in either the absence or presence of S9 metabolic gave no evidence of a dose response, the study authors activation (Karanewsky et al., 2015). For the same structural concluded that 1-(3,5,5,6,8,8-hexamethyl-5,6,7,8- relative, no genotoxic potential was observed in an in vitro tetrahydronaphthalen-2-yl)ethanone is negative for structural chromosomal aberration assay in the presence of S9 and numerical chromosomal aberrations in vitro (Api and San, metabolic activation. In the absence of S9 metabolic 1999; Curry, 1995). In an in vivo micronucleus induction activation positive results were observed. However, in a assay, no increases in the occurrence of micro-nucleated combined in vivo micronucleus/comet assay no increases in polychromatic erythrocytes were observed relative to vehicle polychromatic erythrocytes or DNA damage were observed controls in male and female ICR mice intraperitoneally (Karanewsky et al., 2015). Based on these results, as well as injected with 1-(3,5,5,6,8,8-hexamethyl-5,6,7,8- the structure of the substance and the arrangement and tetrahydronaphthalen-2-yl)ethanone (Api and San, 1999; identity of the functional groups therein, the Panel did not Gudi and Ritter, 1997). Based on these results, as well as the identify specific concerns related to the genotoxicity of 2-(4- structure of the substance and the arrangement and identity ethylphenoxy)-N-(lH-pyrazol-3-yl)-N-(thiophen-2- of the functional groups therein, the Panel did not identify ylmethyl)acetamide. A 28-day dietary study for structurally specific concerns related to the genotoxicity of 1-(3,5,5,6,8,8- related substance, 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)- hexamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethanone. A N-(thiophen-2-ylmethyl)acetamide (FEMA 4809), in male and 13-week toxicity study of 1-(3,5,5,6,8,8-hexamethyl-5,6,7,8- female rats resulted in a NOAEL of greater than 100 mg/kg tetrahydronaphthalen-2-yl)ethanone in Crl:CD(SD)BR (VAF bw/day. A 90-day dietary study for structurally related The Panel reviewed the GRAS application and supporting substance, 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N- information regarding N-(4-(cyanomethyl)phenyl)-2-isopropyl- (thiophen-2-ylmethyl)acetamide in male and female rats 5,5-dimethylcyclohexanecarboxamide (CAS 1857331-83-9) resulted in a NOAEL of greater than 100 mg/kg bw/day (Smith et al., 2005a) and concluded that the substance is (Karanewsky et al., 2015). This is greater than 20,000 times GRAS (FEMA 4882) for use as a flavor ingredient in the food the anticipated daily per capita intake of 2-(4-ethylphenoxy)- categories and at the use levels specified in the GRAS N-(lH-pyrazol-3-yl)-N-(thiophen-2-ylmethyl)acetamide from application (see Table 2). The substance was evaluated use as a flavor ingredient. individually within the context of the chemical group of aliphatic and aromatic amines and amides (JECFA, 2006, The Panel reviewed the GRAS application and supporting 2008, 2011, 2012; SLR, A7, C21). The Panel calculated the information regarding N-(3-hydroxy-4-methoxyphenyl)-2- anticipated per capita intake ("eaters only") of N-(4- isopropyl-5,5-dimethylcyclohexanecarboxamide (CAS (cyanomethyl)phenyl)-2-isopropyl-5,5- 1857331-84-0) (Smith et al., 2005a) and concluded that the dimethylcyclohexanecarboxamide from use as a flavor substance is GRAS (FEMA 4881) for use as a flavor ingredient to be 15 µg/person/day, which is below the ingredient in the food categories and at the use levels threshold of toxicological concern for structural class III (90 specified in the GRAS application (see Table 2). The µg/person/day) (Munro et al., 1996). The Panel considered substance was evaluated individually within the context of the the specification of the material to be adequately chemical group of aliphatic and aromatic amines and amides characterized by the purity assay and supporting spectral data (JECFA, 2006, 2008, 2011, 2012, 2017; SLR, A7, C21) . The provided for FEMA GRAS evaluation. Based on the lack of Panel calculated the anticipated per capita intake ("eaters amide hydrolysis observed for the structurally related only") of N-(3-hydroxy-4-methoxyphenyl)-2-isopropyl-5,5- substance, N-(3-hydroxy-4-methoxyphenyl)-2-isopropyl-5,5- dimethylcyclohexanecarboxamide from use as a flavor dimethylcyclohexanecarboxamide (FEMA 4881) in simulated ingredient to be 15 µg/person/day, which is below the gastric and intestinal matrices (Cheung, 2016a) and threshold of toxicological concern for structural class III (90 cryopreserved human hepatocytes (Cheung, 2016b), very µg/person/day) (Munro et al., 1996). The Panel considered little to no hydrolysis would be predicted for N-(4- the specification of the material to be adequately (cyanomethyl)phenyl)-2-isopropyl-5,5- characterized by the purity assay and supporting spectral data provided for FEMA GRAS evaluation. Hydrolysis of the amide dimethylcyclohexanecarboxamide. It is predicted that N-(4- bond of N-(3-hydroxy-4-methoxyphenyl)-2-isopropyl-5,5- (cyanomethyl)phenyl)-2-isopropyl-5,5- dimethylcyclohexanecarboxamide was not detected in vitro dimethylcyclohexanecarboxamide will undergo metabolism experiments in simulated gastric juice and only a very low rate primarily by hydroxylation of the alkyl substituents and ring was detected in an intestinal matrix (Cheung, 2016a). Amide positions to yield hydroxylated metabolites that are likely hydrolysis was also not detected in an in vitro study in excreted as glucuronic acid or sulfate conjugates that are cryopreserved human hepatocytes (Cheung, 2016b). Based eliminated in the urine (Smith et al., 2018). No increases in on these results, it is anticipated that N-(3-hydroxy-4- the number of reverse mutations were observed in the Ames methoxyphenyl)-2-isopropyl-5,5- assay for of structurally related substance, N-(3-hydroxy-4- dimethylcyclohexanecarboxamide will undergo metabolism methoxyphenyl)-2-isopropyl-5,5- primarily by hydroxylation of the alkyl substituents and ring dimethylcyclohexanecarboxamide (FEMA 4881), in S. positions to yield hydroxylated metabolites that are likely typhimurium strains TA98, TA100, TA1535 and TA1537 and excreted as glucuronic acid or sulfate conjugates that are E. coli strain WP2 uvrA in either the absence or presence of eliminated in the urine. Some O-dealkylation to form a S9 metabolic activation (Hashimoto, 2015). In an in vivo catechol species that may be conjugated with glucuronic acid mouse micronucleus assay, oral administration of the or sulfate, followed by excretion primarily in the urine, could structurally related substance, N-p- also occur (Smith et al., 2018). No increases in the number benzeneacetonitrilementhanecarboxamide (FEMA 4496), did of reverse mutations were observed in the Ames assay for N- not increase the frequency of micronucleated polychromatic (3-Hydroxy-4-methoxyphenyl)-2-isopropyl-5,5- erythrocytes (Pritchard, 2006). Based on these results, as well dimethylcyclohexanecarboxamide in S. typhimurium strains as the structure of the substance and the identity and TA98, TA100, TA1535 and TA1537 and E. coli stain WP2 uvrA in either the absence or presence of S9 metabolic arrangement of functional groups therein, the Panel did not activation (Hashimoto, 2015). In an in vivo mouse identify specific concerns for the genotoxic potential of N-(4- micronucleus assay, oral administration of the structurally (cyanomethyl)phenyl)-2-isopropyl-5,5- related substance, N-p-benzeneacetonitrilementhane dimethylcyclohexanecarboxamide. A 90-day male and female carboxamide (4496), did not increase the frequency of rat study for the structurally related substance, N-p- micronucleated polychromatic erythrocytes (Pritchard, 2006). benzeneacetonitrilementhanecarboxamide (FEMA 4496), Based on these results, as well as the structure of the provided in the diet resulted in a NOAEL of 100 mg/kg bw/day substance and the identity and arrangement of functional (Eapen, 2007). This is 400,000 times the anticipated daily per groups therein, the Panel did not identify specific concerns for capita intake of N-(4-(cyanomethyl)phenyl)-2-isopropyl-5,5- the genotoxic potential of N-(3-hydroxy-4-methoxyphenyl)-2- dimethylcyclohexanecarboxamide from use as a flavor isopropyl-5,5-dimethylcyclohexanecarboxamide. A 90-day ingredient. male and female rat study for the structurally related substance N-p-benzeneacetonitrilementhanecarboxamide The Panel reviewed the GRAS application and supporting (FEMA 4496) provided in the diet resulted in a NOAEL of 100 information regarding S-allyl-L-cysteine sulfoxide (CAS 556- mg/kg bw/day (Eapen, 2007). This is 400,000 times the 27-4) and concluded that the substance is GRAS (FEMA anticipated daily per capita intake of N-(3-hydroxy-4- 4883) (Smith et al., 2005a) for use as a flavor ingredient in the methoxyphenyl)-2-isopropyl-5,5- food categories and at the use levels specified in the GRAS dimethylcyclohexanecarboxamide from use as a flavor application (see Table 2). This substance was evaluated ingredient. individually within the context of the chemical group of simple aliphatic and aromatic sulfides and thiols (JECFA, 2000, 2004, 2008, 2011; SLR, B5B). The Panel calculated the anticipated per capita intake (“eaters only”) of S-allyl-L- application (see Table 2). This substance was evaluated cysteine sulfoxide from use as a flavor ingredient to be 710 individually within the context of the chemical group of linear µg/person/day, which is above the threshold of toxicological and branched-chain aliphatic, unsaturated, unconjugated concern for structural class II (540 µg/person/day) (Munro et alcohols, aldehydes, acids, and related esters (JECFA, 1999, al., 1996). This substance is known to naturally occur in fresh 2012; SLR, M1). The Panel calculated the anticipated per (Ziegler and Sticher, 1988). However, because capita intake (“eaters only”) of trans-5-dodecenal from use as only qualitative data was available, a consumption ratio could a flavor ingredient to be 0.3 µg/person/day, which is below the not be calculated. The Panel considered the specification of threshold of toxicological concern for structural class I (1800 the material to be adequately characterized by the purity µg/person/day) (Munro et al., 1996). This substance occurs assay and supporting spectral data provided for FEMA GRAS naturally in cardamom (1000 ppm) (Njissen, 2019). However, evaluation. It is presumed that S-allyl-L-cysteine sulfoxide will due to limited quantitative data, a consumption ratio could not undergo N-acetylation followed by excretion in the urine be calculated. The Panel considered the specification of the (Amano et al., 2015; Kodera et al., 2002; Smith et al., 2018). material to be adequately characterized by the purity assay Based on the structure of the substance and the arrangement and supporting spectral data provided for FEMA GRAS and identity of the functional groups therein, the Panel did not evaluation. It is presumed that trans-5-dodecenal will oxidize identify specific concerns related to the genotoxicity of S-allyl- to the corresponding unsaturated carboxylic acid. Because L-cysteine sulfoxide. A 28-day dietary study for structurally the double bond is at an odd-numbered carbon, the resulting related substance, S-allyl-L-cysteine (FEMA 4322), in male delta-3-enoyl-CoA from acetyl-CoA fragmentation must be and female Wistar rats resulted in a NOAEL of 500 mg/kg isomerized by enoyl CoA isomerase to the trans-delta-2-enoyl bw/day (Kodera et al., 2002), which is greater than 42,000 CoA before it can re-enter the fatty acid cycle for complete times the anticipated daily per capita intake of S-allyl-L- metabolism to CO2 and water (Feldman and Weiner, 1972; cysteine sulfoxide from use as a flavor ingredient. Eckfeldt and Yonetani, 1982; Nakayasu et al., 1978; Beedham, 1988; Nelson and Cox, 2008). A negative Ames The Panel reviewed the GRAS application and supporting assay was reported for the structurally related substance, 10- information regarding 6-methyl-5-hepten-2-ol (CAS 1569-60- undecenal (FEMA 3095), in S. typhimurium strains TA98, 9) (Smith et al., 2005a) and concluded that the substance is TA100, TA102, TA1535, and TA1537 in the presence and GRAS (FEMA 4884) for use as a flavor ingredient in the food absence of S9 metabolic activation (Bhatia et al., 2010; categories and at the use levels specified in the GRAS Sokolowski, 2007a). Negative results for 10-undecenal were application (see Table 2). The substance was evaluated also reported in an in vivo micronucleus test in male and individually within the context of the chemical group of female NMRI mice (Bhatia et al., 2010; Honarvar, 2007). aliphatic secondary alcohols, ketones and related substances Similar negative results were reported in the Ames assay (JECFA, 1999, 2003, 2017; SLR, A1). The Panel calculated using the same S. typhimurium strains and in vivo mouse the anticipated per capita intake ("eaters only") of 6-methyl-5- micronucleus tests using male and female NMRI mice for hepten-2-ol from anticipated use as a flavor ingredient to be another structural relative, trans-4-decen-1-al (FEMA 3264) 290 µg/person/day, which is below the threshold of (Bhatia et al., 2010; Sokolowski, 2007b; Honarvar, 2008). toxicological concern for structural class I (1800 Based on these results, as well as the structure of the µg/person/day) (Munro et al., 1996). The substance occurs substance and the arrangement and identity of the functional naturally in annatto, citrus, macadamia nuts, and tomatoes groups therein, the Panel did not identify specific concerns (Njissen, 2019). The Panel considered the specification of the related to the genotoxicity of trans-5-dodecenal. A 90-day material to be adequately characterized by the purity assay dietary study for structurally related substance, 10-undecenal and supporting spectral data provided for FEMA GRAS (FEMA 3095), in male and female Sprague-Dawley Crl:CD® evaluation. It is predicted that 6-methyl-5-hepten-2-ol will be (SD) IGS BR rats resulted in a NOAEL of 200 ppm, or metabolized primarily by conjugation with glucuronic acid approximately 14.3 mg/kg bw/day, (Liwska and Watson, followed by excretion in the urine (Kamil et al., 1953; Smith et 2012) which is greater than 2,000,000 times the anticipated al., 2018). No increases in the number of reverse mutations daily per capita intake of trans-5-dodecenal from use as a were observed in the Ames assay for structurally related flavor ingredient. substance, 6-methyl-5-hepten-2-one (FEMA 2707), in S. typhimurium strains TA98, TA100, TA1535 or TA1537 in The Panel reviewed the GRAS application and supporting either the absence or presence of S9 metabolic activation. No information regarding cis-6-dodecenal (CAS 126745-61-7) increase in micronucleated binucleated human peripheral and concluded that the substance is GRAS (FEMA 4886) blood lymphocyte cells were observed with concentrations of (Smith et al., 2005a) for use as a flavor ingredient in the food 6-methyl-5-hepten-2-ol up to 850 µg/mL in the presence or categories and at the use levels specified in the GRAS absence of S9 metabolic activation (Roy, 2015). Based on application (see Table 2). This substance was evaluated these results, the Panel did not identify any specific concerns individually within the context of the chemical group of linear related to the genotoxicity of 6-methyl-5-hepten-2-ol. A 90-day and branched-chain aliphatic, unsaturated, unconjugated oral toxicity study in male and female rat study for the alcohols, aldehydes, acids, and related esters (JECFA, 1999, structurally related substance, 6-methyl-5-hepten-2-one 2012; SLR, M1). The Panel calculated the anticipated per (FEMA 2707), resulted in a NOAEL of 50 mg/kg bw/day for capita intake (“eaters only”) of cis-6-dodecenal from use as a both sexes (Kaspers, 2002). This is greater than 10,000 times flavor ingredient to be 0.3 µg/person/day, which is below the the anticipated daily per capita intake of 6-methyl-5-hepten-2- threshold of toxicological concern for structural class I (1800 ol from use as a flavor ingredient. µg/person/day) (Munro et al., 1996). The Panel considered the specification of the material to be adequately The Panel reviewed the GRAS application and supporting characterized by the purity assay and supporting spectral data information regarding trans-5-dodecenal (CAS 68820-34-8) provided for FEMA GRAS evaluation. It is presumed that cis- and concluded that the substance is GRAS (FEMA 4885) 6-dodecenal will be oxidized to the corresponding unsaturated (Smith et al., 2005a) for use as a flavor ingredient in the food carboxylic acid. The cis double bond is isomerized to the trans categories and at the use levels specified in the GRAS double bond by 3-hydroxyacyl-CoA epimerase before re- entering the fatty acid cycle where it is converted to acetyl- the structure of the substance and the arrangement and CoA. The acetyl-CoA product then enters the citric acid cycle identity of the functional groups therein, the Panel did not where it is metabolized to yield CO2 and water (Feldman and identify specific concerns related to the genotoxicity of cis-9- Weiner, 1972; Eckfeldt and Yonetani, 1982; Nakayasu et al., dodecenal. A 90-day dietary study for structurally related 1978; Beedham, 1988; Nelson and Cox, 2008). A negative substance, 10-undecenal (FEMA 3095), in male and female Ames assay was reported for the structurally related Sprague-Dawley Crl:CD® (SD) IGS BR rats resulted in a substance, 10-undecenal (FEMA 3095), using S. typhimurium NOAEL of 200 ppm, or approximately 14.3 mg/kg bw/day strains TA98, TA100, TA102, TA1535, and TA1537 in the (Liwska and Watson, 2012), which is greater than 2,000,000 presence and absence of S9 metabolic activation (Bhatia et times the anticipated daily per capita intake of cis-9- al., 2010; Sokolowski, 2007a). Negative results for 10- dodecenal from use as a flavor ingredient. undecenal (FEMA 3095) were also reported in an in vivo micronucleus test in male and female NMRI mice (Bhatia et The Panel reviewed the GRAS application and supporting al., 2010; Honarvar, 2007). Similar negative results were information regarding mixture of 5-hydroxy-4-(4’-hydroxy-3’- reported in the Ames assay using the same S. typhimurium methoxyphenyl)-7-methylchroman-2-one and 7-hydroxy-4- strains and in vivo mouse micronucleus tests using male and (4’-hydroxy-3’-methoxyphenyl)-5-methylchroman-2-one (CAS female NMRI mice for another structural relative, trans-4- 1945993-01-0 and 828265-08-3) and concluded that the decen-1-al (FEMA 3264) (Bhatia et al., 2010; Sokolowski, substance is GRAS (FEMA 4888) (Smith et al., 2005a) for use 2007b; Honarvar, 2008). Based on these results, as well as as a flavor ingredient in the food categories and at the use the structure of the substance and the arrangement and levels specified in the GRAS application (see Table 2). This identity of the functional groups therein, the Panel did not substance was evaluated individually within the context of the identify specific concerns related to the genotoxicity of cis-6- chemical group of phenol and phenol derivatives (JECFA, dodecenal. A 90-day dietary study for structurally related 2001, 2011, 2012; SLR, C12). The Panel calculated the substance, 10-undecenal (FEMA 3095), in male and female anticipated per capita intake (“eaters only”) of from use as a Sprague-Dawley Crl:CD® (SD) IGS BR rats resulted in a flavor ingredient to be 6 µg/person/day, which is below the NOAEL of 200 ppm, or approximately 14.3 mg/kg bw/day threshold of toxicological concern for structural class III (90 (Liwska and Watson, 2012), which is greater than 2,000,000 µg/person/day) (Munro et al., 1996). The Panel considered times the anticipated daily per capita intake of cis-6- the specification of the material to be adequately dodecenal from use as a flavor ingredient. characterized by the purity assay and supporting spectral data provided for FEMA GRAS evaluation. The Panel evaluated The Panel reviewed the GRAS application and supporting sensory data included within the application and found it information regarding cis-9-dodecenal (CAS 56219-03-5) and satisfactory with regard to intended conditions of use for the concluded that the substance is GRAS (FEMA 4887) (Smith flavoring ingredient (Harman and Hallagan, 2013). It is et al., 2005a) for use as a flavor ingredient in the food presumed that the will undergo facile enzyme-catalyzed categories and at the use levels specified in the GRAS lactone ring-opening, likely followed by excretion or application (see Table 2). This substance was evaluated conjugation followed by excretion. Additional metabolic individually within the context of the chemical group of linear outcomes include hydroxylation, reduction, and conjugation and branched-chain aliphatic, unsaturated, unconjugated with sulfate or glucuronic acid (Horrigan, 2016). The mixture alcohols, aldehydes, acids, and related esters (JECFA, 1999, of 5-hydroxy-4-(4’-hydroxy-3’-methoxyphenyl)-7- 2012; SLR, M1). The Panel calculated the anticipated per methylchroman-2-one and 7-hydroxy-4-(4’-hydroxy-3’- capita intake (“eaters only”) of cis-9-dodecenal from use as a methoxyphenyl)-5-methylchroman-2-one was negative for flavor ingredient to be 0.3 µg/person/day, which is below the genotoxicity in an Ames assay with S. typhimurium strains threshold of toxicological concern for structural class I (1800 TA98, TA100, TA1535, and TA1537 as well as E. coli strain µg/person/day) (Munro et al., 1996). The Panel considered WP2 uvrA (pKM101) in the presence or absence of S9 the specification of the material to be adequately metabolic activation (Wisher, 2016). In an in vitro characterized by the purity assay and supporting spectral data micronucleus assay in CHO-K1 cells, the mixture did not provided for FEMA GRAS evaluation. It is presumed that cis- induce increases in micronucleated cells in the presence or 9-dodecenal will be oxidized to yield an unsaturated absence of S9 metabolic activation (Diaz et al., 2007; Zhao, carboxylic acid. Since the double bond is at an odd-numbered 2016). One of the constituents of the mixture, (R)-5-hydroxy- carbon, the resulting delta-3-enoyl-CoA from acetyl-CoA 4-(4’-hydroxy-3’-methoxyphenyl)-7-methylchroman-2-one fragmentation must be isomerized by enoyl-CoA isomerase to (FEMA 4834), yielded negative results in an Ames assay trans-delta-2-enoyl-CoA before it can re-enter the fatty acid conducted in S. typhimurium strains TA98, TA100, TA1535, cycle for complete metabolism to CO2 and water (Feldman and TA1537 and E. coli strain WP2 uvrA (pKM101) in the and Weiner, 1972; Eckfeldt and Yonetani, 1982; Nakayasu et presence and absence of S9 metabolic activation (Soltesova, al., 1978; Beedham, 1988; Nelson and Cox, 2008). A negative 2015). This single component of the mixture also did not Ames assay was reported for the structurally related induce increases in micronucleated cells in an in vitro substance, 10-undecenal (FEMA 3095), using S. typhimurium micronucleus assay conducted in CHO-K1 cells in the strains TA98, TA100, TA102, TA1535, and TA1537 in the presence or absence of S9 metabolic activation (Zhao, 2016). presence and absence of S9 metabolic activation (Bhatia et al., 2010; Sokolowski, 2007a). Negative results for 10- The Panel reviewed the GRAS application and supporting undecenal (FEMA 3095) were also reported in an in vivo information regarding methyl propyl sulfide (CAS 3877-15-4) micronucleus test in male and female NMRI mice (Bhatia et and concluded that the substance is GRAS (FEMA 4889) al., 2010; Honarvar, 2007). Similar negative results were (Smith et al., 2005a) for use as a flavor ingredient in the food reported in the Ames assay using the same S. typhimurium categories and at the use levels specified in the GRAS strains and in vivo mouse micronucleus tests using male and application (see Table 2). This substance was evaluated female NMRI mice for another structural relative, trans-4- individually within the context of the chemical group of simple decen-1-al (FEMA 3264) (Bhatia et al., 2010; Sokolowski, aliphatic and aromatic sulfides and thiols (SLR B4, JECFA 2007b; Honarvar, 2008). Based on these results, as well as 2000, 2004, 2008, 2011). The Panel calculated the anticipated per capita intake (“eaters only”) of m from use as (Hobbs et al., 2016). Based on these results, as well as the a flavor ingredient to be 0.03 µg/person/day, which is below structure of the substance and the arrangement and identity the threshold of toxicological concern for structural class I of the functional groups therein, the Panel did not identify (1800 µg/person/day) (Munro et al., 1996). This substance specific concerns related to the genotoxicity of 3-p-menthen- occurs naturally in beef, chive, durian, Guinea hen, kohlrabi, 7-al (Cohen et al., 2016). A 90-day dietary study of structural , and cured pork (Njissen, 2019). Due to limited relative perillyl alcohol (FEMA 2664) conducted in male and quantitative data, a consumption ratio could not be calculated. female Fischer 344 rats resulted in a NOAEL of the highest The Panel considered the specification of the material to be tested dose of 400 mg/kg bw/day (NCI, 1996) which is adequately characterized by the purity assay and supporting 20,000,000,000 times the anticipated per capita daily intake spectral data provided for FEMA GRAS evaluation. It is of 3-p-menthen-7-al from use as a flavor ingredient. In a 90- presumed that methyl propyl sulfide will undergo S-oxidation day oral gavage toxicity study in male and female beagle to the sulfoxide, and to a lesser extent the sulfone, for dogs, a NOAEL of 120 mg/kg bw/day was determined for excretion in urine (Damani, 1987). Based on these results, as perillyl alcohol (FEMA 2664) (NCI, 1996). This NOAEL is well as the structure of the substance and the arrangement 6,000,000,000 times the anticipated per capita daily intake of and identity of the functional groups therein, the Panel did not 3-p-menthen-7-al from use as a flavor ingredient. identify specific concerns related to the genotoxicity of methyl propyl sulfide.. A 14-week dietary study for structurally related The Panel reviewed the GRAS application and supporting substance, dimethyl sulfide (FEMA 2746), in male and female information regarding (E)-3-methyl-4-dodecenoic acid (CAS Wistar rats resulted in a NOAEL of 250 mg/kg bw/day 2088117-65-9) and concluded that the substance is GRAS (Butterworth et al., 1975) which is 500,000,000 times the (FEMA 4891) (Smith et al., 2005a) for use as a flavor anticipated daily per capita intake of methyl propyl sulfide from ingredient in the food categories and at the use levels use as a flavor ingredient. specified in the GRAS application (see Table 2). This substance was evaluated individually within the context of the The Panel reviewed the GRAS application and supporting chemical group of linear and branched-chain aliphatic, information regarding 3-p-menthen-7-al (CAS 27841-22-1) unsaturated, unconjugated alcohols, aldehydes, acids, and and concluded that the substance is GRAS (FEMA 4890) related esters (JECFA, 2000, 2007, 2012, 2014; SLR, M1). (Smith et al., 2005a) for use as a flavor ingredient in the food The Panel calculated the anticipated per capita intake (“eaters categories and at the use levels specified in the GRAS only”) of (E)-3-methyl-4-dodecenoic acid from use as a flavor application (see Table 2). This substance was evaluated ingredient to be 0.01 µg/person/day, which is below the individually within the context of the chemical group of alicyclic threshold of toxicological concern for structural class I (1800 primary alcohols, aldehydes, acids, and related esters (Cohen µg/person/day) (Munro et al., 1996). The Panel considered et al., 2016; JECFA, 2006, 2009, 2015; SLR, A5) . The Panel the specification of the material to be adequately calculated the anticipated per capita intake (“eaters only”) of characterized by the purity assay and supporting spectral data 3-p-menthen-7-al from use as a flavor ingredient to be 0.001 provided for FEMA GRAS evaluation. It is presumed that (E)- µg/person/day, which is below the threshold of toxicological 3-methyl-4-dodecenoic acid will undergo beta-oxidative concern for structural class I (1800 µg/person/day) (Munro et cleavage and complete metabolism to CO2 via the fatty acid al., 1996). This material naturally occurs in seeds pathway and the citric acid cycle (Williams, 1959; Nelson and (Njissen, 2019). The Panel considered the specification of the Cox, 2008). No increases in the frequency of revertant material to be adequately characterized by the purity assay colonies were observed in the Ames assay for this substance and supporting spectral data provided for FEMA GRAS in S. typhimurium strains TA98 and TA100 in the presence evaluation. It is presumed that 3-p-menthen-7-al will be and absence of S9 metabolic activation (Komai, 2016b). oxidized to yield the corresponding carboxylic acid and be Structural relative, oleic acid (FEMA 2815) did not increase excreted in urine unchanged or as a glycine conjugate (Ishida the frequency of revertant colonies in an Ames assay et al., 1989). Mutagenesis was not observed in the Ames conducted in S. typhimurium strains TA98, TA100, TA1535, assay for this substance conducted in S. typhimurium strains and TA1537 as well as in E. coli strain WP2uvrA in the TA98 and TA100 in the presence or absence of S9 metabolic presence and absence of S9 metabolic activation (Shimizu et activation (Komai, 2016a). The structural relative al., 1985). Based on these results, as well as the structure of perillaldehyde (FEMA 3557) induced reverse mutations in an the substance and the arrangement and identity of the Ames assay in the absence of S9 metabolic activation in S. functional groups therein, the Panel did not identify specific typhimurium strain TA98 but was negative in this strain in the concerns related to the genotoxicity of (E)-3-methyl-4- presence of S9 metabolic activation. This structural relative dodecenoic acid. In a 180-day study in male and female yielded negative results in S. typhimurium strains TA100, Sprague-Dawley rats, oral administration of the structural TA102, TA1535, and TA1537 in the presence and absence of relative 10-undecenoic acid (FEMA 3247) resulted in a S9 metabolic activation. In an in vitro micronucleus assay in NOAEL at the highest dose of 400 mg/kg bw/day (Tislow et human lymphocytes, perillaldehyde (FEMA 3557) did not al., 1950). This NOAEL is 2,000,000,000 times the anticipated increase the frequency of micronucleated binuclear cells in daily per capita intake of (E)-3-methyl-4-dodecenoic acid from the presence and absence of S9 metabolic activation. use as a flavor ingredient. Perillaldehyde (FEMA 3557) was negative in an hypoxanthine-guanine phosphoribosyltransferase (HPRT) The Panel reviewed the GRAS application and supporting assay in mouse lymphoma cells in the presence and absence information regarding cis-2-hexylcyclopropaneacetic acid S9 metabolic activation. Perillaldehyde (FEMA 3557) did not (CAS 4707-61-3) and concluded that the substance is GRAS increase micronucleated cells in the bone marrow of Han (FEMA 4892) (Smith et al., 2005a) for use as a flavor Wistar rats and was negative in the comet assay in the ingredient in the food categories and at the use levels duodenum in a comet/micronucleus combined assay. In the specified in the GRAS application (see Table 2). This same assay for this substance, increased comet tail intensity substance was evaluated individually within the context of the was observed in the liver at a hepatotoxic dose but these chemical group of aliphatic primary alcohols, aldehydes, values were within the laboratory’s historical control ranges esters, and acids (JECFA, 2000, 2007, 2012, 2014; SLR, M1). The Panel calculated the anticipated per capita intake (“eaters Jones, 1986; Kasamaki et al., 1982; Lawlor, 1991; Marzin, only”) of cis-2-hexylcyclopropaneacetic acid from use as a 1979a; Mortelmans et al., 1986; Nagabhushan and Bhide, flavor ingredient to be 0.001 µg/person/day, which is below 1985; Pool and Lin, 1982; Rapson et al., 1980). For the same the threshold of toxicological concern for structural class II structural relative, mixed positive and negative results were (540 µg/person/day) (Munro et al., 1996). This substance is observed in in vitro sister chromatid exchange, chromosomal reported to occurs naturally in Croton eleuteria, a medicinal aberration and micronucleus induction assays conducted in shrub, as well as the essential oil derived from it, cascarilla oil Chinese hamster cells, human lymphocytes, and human cell (Lawrence, 1977). However, these are not commonly lines (Jansson et al., 1986, 1987; Sanyal et al., 1997, Sasaki consumed as food. The Panel considered the specification of et al., 1987). (FEMA 3107) was negative in an the material to be adequately characterized by the purity unscheduled DNA synthesis assay in rat hepatocytes (Heck assay and supporting spectral data provided for FEMA GRAS et al., 1989) and was negative in bone marrow micronucleus evaluation. It is presumed that cis-2-hexylcyclopropaneacetic assays in female OF1 mice, male BDF1 mice, and in male acid will undergo conjugation to create glycine and MS/Ae mice (Inouye et al., 1988; Marzin, 1979b; Sutou et al., glucuronide conjugates. This substance could also undergo 1999). Based on these results, as well as the structure of the beta-oxidation to produce cyclopropane carboxylic acid which substance and the arrangement and identity of the functional would lead to the corresponding glycine and glucuronide groups therein, the Panel did not identify specific concerns conjugates. These conjugates can be excreted in urine (Smith related to the genotoxicity of 2-ethoxy-4- et al., 2018). No increases in the frequency of revertant (hydroxymethyl)phenol. Administration of vanillin (FEMA colonies were observed in an Ames assay for this substance 3107) to pregnant mice by oral gavage from gestation day 10 conducted in S. typhimurium strains TA98 and TA100 in the until postnatal day 30 showed no adverse effects. NOAEL presence and absence of S9 metabolic activation (Komai, values of at least 1500 mg/kg bw/day for maternal toxicity and 2016c). In an Ames assay for structural relative oleic acid teratogenicity were reported (OECD SIDS, 2002). No (FEMA 2815) in S. typhimurium strains TA98, TA100, reproductive or developmental effects were reported for Crl TA1535, and TA1537 as well as E. coli strain WP2uvrA, no CD rats administered vanillin (FEMA 3107) at concentrations increase in the frequency of revertant colonies were observed up to 500 mg/kg bw/day by gavage from 7 days prior to in the presence and absence of S9 metabolic activation cohabitation, gestation, delivery and 4 days post-parturition (Shimizu, 1985). Based on these results, as well as the (Vollmuth et al., 1990). No significant differences in weekly structure of the substance and the arrangement and identity measurements of body weight changes, food intake and of the functional groups therein, the Panel did not identify general conditions, as well as, hematology parameters were specific concerns related to the genotoxicity of cis-2- observed between groups of male Osborne-Mendel rats orally hexylcyclopropaneacetic acid. In a 180-day study in male and administered the vehicle control, 1000 or 2500 mg/kg bw/day female Sprague-Dawley rats, oral administration of the of the structural relative vanillin (FEMA 3107) for one year structural relative 10-undecenoic acid (FEMA 3247) resulted (Hagan et al., 1967). In a 13-week study in Sprague Dawley in a NOAEL of 400 mg/kg bw/day, which was the highest dose rats, oral administration of vanillin (FEMA 3107) resulted in a tested (Tislow et al., 1950). This NOAEL is 2,000,000,000 NOAEL at the highest tested dose level, 400 mg/kg bw/day times the anticipated daily per capita intake of cis-2- (Mancebo et al., 2003). In a 13-week study in male and female hexylcyclopropaneacetic acid from use as a flavor ingredient. CD Sprague Dawley rats, dietary administration of the structural relative ethyl vanillin (FEMA 2464) at doses of 500, The Panel reviewed the GRAS application and supporting 1000, and 2000 mg/kg bw/day resulted in a NOAEL of 500 information regarding 2-ethoxy-4-(hydroxymethyl)phenol mg/kg bw/day (Hooks et al., 1992) which is 1,428,000 times (CAS 4912-58-7) and concluded that the substance is GRAS the daily per capita intake of 2-ethoxy-4- (FEMA 4893) (Smith et al., 2005a) for use as a flavor (hydroxymethyl)phenol when used as a flavoring ingredient. ingredient in the food categories and at the use levels specified in the GRAS application (see Table 2). This The Panel reviewed the GRAS application and supporting substance was evaluated individually within the context of the information regarding 2-mercapto-3-methyl-1-butanol (CAS chemical group of hydroxy- and alkoxy-substituted benzyl 116229-37-9) and concluded that the substance is GRAS derivatives (JECFA, 2001, 2006, 2009; SLR, C9). The Panel (FEMA 4894) (Smith et al., 2005a) for use as a flavor calculated the anticipated per capita intake (“eaters only”) of ingredient in the food categories and at the use levels 2-ethoxy-4-(hydroxymethyl)phenol from use as a flavor specified in the GRAS application (see Table 2). This ingredient to be 21 µg/person/day, which is below the substance was evaluated individually within the context of the threshold of toxicological concern for structural class I (1800 chemical group of simple aliphatic and aromatic sulfides and µg/person/day) (Munro et al., 1996). The Panel considered thiols (JECFA, 2000, 2004, 2008, 2011; SLR, B5B). The the specification of the material to be adequately Panel calculated the anticipated per capita intake (“eaters characterized by the purity assay and supporting spectral data only”) of 2-mercapto-3-methyl-1-butanol from use as a flavor provided for FEMA GRAS evaluation. It is presumed that 2- ingredient to be 0.1 µg/person/day, which is below the ethoxy-4-(hydroxymethyl)phenol will form glucuronic acid or threshold of toxicological concern for structural class I (1800 sulfate conjugates. This substance is also presumed to µg/person/day) (Munro et al., 1996). This substance occurs undergo oxidation to ethyl vanillic acid, forming sulfate or naturally in beer (Njissen et al., 2019). However, no glucuronic acid conjugates that are expelled in urine consumption ratio could be calculated as no quantitative data (Dirscherl and Wirtzfeld, 1964; Sammons and Williams, 1941; was available. The Panel considered the specification of the Strand and Scheline, 1975; Wong and Sourkes, 1966). material to be adequately characterized by the purity assay Negative results were observed in the Ames assays for the and supporting spectral data provided for FEMA GRAS structurally related substance, vanillin (FEMA 3107), in S. evaluation. It is presumed that thiol group of 2-mercapto-3- typhimurium strains TA92, TA94, TA97, TA98, TA100, TA102, methyl-1-butanol undergoes oxidation and results in sulfenic, TA104, TA1535, TA1537, TA1538, and TA2637 using the sulfinic, and sulfonic acids products to be eliminated in the plate incorporation and pre-incubation methodologies (De urine. Alternatively, the thiol group could also undergo Flora et al., 1994; Florin et al., 1980a; Ishidate et al., 1984; methylation. Additionally, any hydroxylated products can form the corresponding carboxylic acid and/or be converted to 4601), is greater than 6,800 times the anticipated daily per sulfate or glucuronic acid conjugates and be eliminated in capita intake of the rebaudioside M from use as a flavor urine (McBain and Menn, 1969; Dutton and Illing, 1972; ingredient. Maiorino et al., 1988; Richardson et al., 1991; Renwick, 1996). Negative results were observed in an Ames assay for this substance using S. typhimurium strains TA98, TA100, The Panel reviewed the GRAS application and supporting TA1535, and TA1537 as well as E.coli strain WP2 uvrA information regarding N-(2-hydroxy-2-phenylethyl)-2- (pKM101) in the presence and absence of S9 metabolic isopropyl-5,5-dimethylcyclohexane-1-carboxamide (CAS activation (Ogura, 2014). Based on these results, as well as 2186611-08-3) and concluded that the substance is GRAS the structure of the substance and the arrangement and (FEMA 4896) (Smith et al., 2005a) for use as a flavor identity of the functional groups therein, the Panel did not ingredient in the food categories and at the use levels identify specific concerns related to the genotoxicity of 2- specified in the GRAS application (see Table 2). The mercapto-3-methyl-1-butanol. A 90-day dietary study for substance was evaluated individually within the context of the structurally related substance, 3-mercapto-2-butanol (FEMA chemical group of aliphatic and aromatic amines and amides 3502), in male and female Sprague Dawley Crl:CD® (SD) IGS (JECFA, 2006, 2008, 2011, 2012; SLR, A7, C21). The Panel BR rats resulted in a NOAEL of greater than 0.705 mg/kg calculated the anticipated per capita intake ("eaters only") of bw/day (Cox et al., 1974b) which is 352,500 times the N-(2-hydroxy-2-phenylethyl)-2-isopropyl-5,5- anticipated daily per capita intake of 2-mercapto-3-methyl-1- dimethylcyclohexane-1-carboxamide from use as a flavor butanol from use as a flavor ingredient. ingredient to be 71 µg/person/day, which is below the threshold of toxicological concern for structural class III (90 The Panel reviewed the natural flavor complex GRAS µg/person/day) (Munro et al., 1996). The Panel considered application and supporting information regarding the specification of the material to be adequately rebaudioside M (CAS 1220616-44-3) and concluded that the characterized by the purity assay and supporting spectral data material is GRAS (FEMA 4895) (Smith et al., 2005a) for use provided for FEMA GRAS evaluation. It is presumed that N- as a flavor ingredient in the food categories and at the use (2-hydroxy-2-phenylethyl)-2-isopropyl-5,5- levels specified in the GRAS application (see Table 2). This dimethylcyclohexane-1-carboxamide will undergo material was evaluated within the context of the procedure for metabolism through conjugation of the hydroxyl moiety with the FEMA GRAS evaluation of flavor ingredients produced glucuronic acid or sulfate followed by excretion in the urine. through biotechnology processes (Cohen et al., 2015; Smith Additionally, hydroxylation of the alkyl substituents and ring et al., 2005a). The Panel calculated the anticipated per capita positions would occur to yield hydroxylated metabolites likely intake ("eaters only") of rebaudioside M from use as a flavor to be excreted in the urine as glucuronic acid or sulfate ingredient to be 785 µg/person/day. Rebaudioside M is conjugates (James, 1974). No increases in the number of reverse mutations were observed in the Ames assay for N-(2- derived from the leaves of (Ohta et al., hydroxy-2-phenylethyl)-2-isopropyl-5,5- 2010). The Panel evaluated sensory data included within the dimethylcyclohexane-1-carboxamide in S. typhimurium application and found it satisfactory with regard to intended strains TA98, TA100, TA1535, and TA1537 and E. coli stain conditions of use for the flavoring ingredient (Harman and WP2uvrA in either the presence or absence of S9 metabolic Hallagan, 2013). Metabolic data exist for representative activation (Hashimoto, 2016). In an in vitro micronucleus members of each congeneric group that would predict, at the assay in Chinese hamster lung fibroma cells for N-(2-hydroxy- intake levels proposed, metabolism by well-established 2-phenylethyl)-2-isopropyl-5,5-dimethylcyclohexane-1- detoxication pathways to innocuous products (Gardana et al., carboxamide, there was a significant, concentration 2003; Geuns et al., 2003a; Geuns et al., 2007; Geuns and dependent increase in the frequency of micronucleated Pietta, 2004; Hutapea et al., 1997; Koyama et al., 2003a; polychromatic erythrocytes in the presence of S9 metabolic Koyama et al., 2003b; Nakayama et al., 1986; Renwick and activation (Furukuma, 2017). An in vivo mouse micronucleus Tarka, 2008; Roberts and Renwick, 2008; Simonetti et al., assay of N-(2-hydroxy-2-phenylethyl)-2-isopropyl-5,5- 2004; Wheeler et al., 2008; Wingard et al., 1980). The dimethylcyclohexane-1-carboxamide showed no significant genotoxicity of the major marker constituents (steviol increases of micronucleated bone marrow cells in male glycosides) has been thoroughly examined in a wide range of Crl:CD1(ICR) mice (Aoyama, 2017). Based on he overall studies. While some positive results are reported in in vitro weight of evidence based on the lack of mutagenicity in the mutagenicity assays, in vivo studies do not provide evidence Ames assay and the negative in vivo micronucleus induction of genotoxic effects (Nakajima, 2000a, b; Pezzuto et al., 1985; assay, the Panel did not identify specific concerns for the Pezzuto et al., 1986; Rumelhard et al., 2016; Suttajit et al., genotoxic potential of N-(2-hydroxy-2-phenylethyl)-2- 1993; Terai et al., 2002; Toyoda et al., 1997; Williams and isopropyl-5,5-dimethylcyclohexane-1-carboxamide. In a 90- Burdock, 2009). Based on the results for the various steviol day toxicity study in male and female Crl:CD(SD) rats, oral glycosides, the Panel did not identify specific concerns related administration of the structurally related substance, N- to the potential genotoxicity of glucosylated stevia extract. In (ethoxycarbonyl)methyl)-p-menthane-3-carboxamide (FEMA a 108-week carcinogenicity study for stevioside, no 4309), resulted in a NOAEL of 225 mg/kg bw/day (Kirkpatrick, carcinogenic effects were observed (Toyoda et al., 1997). In 2011), which is 225,000 times the anticipated daily per capita a 2-year feeding study, male and female rats were intake of N-(2-hydroxy-2-phenylethyl)-2-isopropyl-5,5- administered the equivalent of 0, 50, 150, or 550 mg/kg dimethylcyclohexane-1-carboxamide from use as a flavor bw/day of a stevia extract comprised of 74% stevioside and ingredient. A 28-day toxicity study of structurally related 16% rebaudioside A. The authors considered the NOAEL substance, N-ethyl-2-isopropyl-5-methylcyclohexane from this 2-year rat feeding study of a stevia extract to be carboxamide (FEMA 3455), in male and female Crj: CD (SD) equal to 550 mg/kg bw/day, or approximately 89.5 mg/kg rats resulted in a NOAEL of 8 mg/kg bw/day (Miyata, 1995). bw/day of rebaudioside A (Yamada et al., 1985). The NOAEL This NOAEL is 8,000 times the anticipated daily per capita for structurally related substance, rebaudioside A (FEMA intake of N-(2-hydroxy-2-phenylethyl)-2-isopropyl-5,5- dimethylcyclohexane-1-carboxamide from use as a flavor ingredient. The Panel reviewed the GRAS application and supporting information regarding trans-5-octenal (CAS 41547-29-9) and The Panel reviewed the GRAS application and supporting concluded that the substance is GRAS (FEMA 4898) (Smith information regarding allulose (CAS 551-68-8) and concluded et al., 2005a) for use as a flavor ingredient in the food that the substance is GRAS (FEMA 4897) (Smith et al., categories and at the use levels specified in the GRAS 2005a) for use as a flavor ingredient in the food categories application (see Table 2). This substance was evaluated and at the use levels specified in the GRAS application (see individually within the context of the chemical group of linear Table 2). This material was evaluated within the context of the and branch-chain aliphatic, unsaturated, unconjugated procedure for the FEMA GRAS evaluation of flavor alcohols, aldehydes, acids, and related esters (JECFA, 1999, ingredients produced through biotechnology processes 2012; SLR, M1). The Panel calculated the anticipated per (Cohen et al., 2015). This substance was evaluated capita intake (“eaters only”) of trans-5-octenal from use as a individually within the context of the chemical group of flavor ingredient to be 7 µg/person/day, which is below the aliphatic poly-hydroxy compounds and derivatives (SLR, threshold of toxicological concern for structural class I (1800 B1F). The Panel calculated the anticipated per capita intake µg/person/day) (Munro et al., 1996). This substance occurs (“eaters only”) of allulose from use as a flavor ingredient to be naturally in trace amounts in lemon peels (Cannon et al., 34,000 µg/person/day, which is above the threshold of 2015). The Panel considered the specification of the material toxicological concern for structural class 1 (1800 to be adequately characterized by the purity assay and µg/person/day) (Munro et al., 1996). This substance occurs supporting spectral data provided for FEMA GRAS evaluation. It is presumed that trans-5-octenal will undergo naturally in figs, raisins, tomato ketchup, brown sugar, and oxidation to the corresponding unsaturated carboxylic acid. caramel sauce (Oshima et al., 2006). The consumption ratio Because the double bond is at an odd-numbered carbon, the was calculated to be 0.1 based on the available quantitative resulting D-3 enoyl-CoA from acetyl-CoA fragmentation must data. The Panel considered the specification of the material be isomerized by enoyl-CoA isomerase to the trans-delta-2 to be adequately characterized by the purity assay and enoyl-CoA before it can re-enter the fatty acid cycle for supporting spectral data provided for FEMA GRAS complete metabolism to CO2 and water (Smith et al., 2018). evaluation. The Panel evaluated sensory data included within Negative results were observed in an Ames assay incubating the application and found it satisfactory with regard to S. typhimurium strains TA98, TA100, TA1535, and TA1537 intended conditions of use for the flavoring ingredient with the geometric isomer cis-5-octenal (FEMA 3749) in the 14 (Harman and Hallagan, 2013). Approcimately 98% of C- presence and absence of S9 metabolic activation (Asquith, allulose administered to rats was rapidly excreted within the 1990a). In another Ames assay conducted in S. typhimurium first 6 hours (Whistler et al., 1974). The panel also considered strains TA98 and TA100 for the constitutional isomer trans-6- a 14C radio label experiment in which eight humans were octenal (FEMA 4787), no mutagenicity was observed up to 50 administered allulose which was then eliminated through μg/plate in the presence and absence of S9 (Kawaguchi and urine. Similar rapid excretion was observed when human Komai, 2012). Based on these results, as well as the structure volunteers orally consumed allulose with the majority of the substance and the arrangement and identity of the eliminated in the urine within 24 hours of consumption. No functional groups therein, the Panel did not identify specific mutagenic potential was observed in the presence and concerns related to the genotoxicity of trans-5-octenal. A 98- absence of S9 metabolic activation in an Ames assay for this day toxicity study of structural relative cis-3-hexen-1-ol (FEMA substance conducted in S. typhimurium strains TA98, TA100, 2563) administered to male and female SPF-derived CFE TA1535, and TA1537 as well as in E. coli strain WP2 uvrA weanling rats via drinking water resulted in a No Observed (pKM101) at doses up to 5,000 μg/plate (Huntington Lab, Effect Level (NOEL) of 120 mg/kg bw/day (Gaunt et al., 1969). 2011 as cited in GRN 400). A micronucleus study of this This is 1,000,000 times the anticipated daily per capita intake substance in CD1 mice yielded no significant increases in of trans-5-octenal from use as a flavor ingredient. micronucleated polychromatic erythrocytes at concentrations up to 2,000 mg/kg/day relative to the control group The Panel reviewed the GRAS application and supporting (Huntington Lab, 2011). An in vivo chromosomal aberration information regarding N-(1-((4-amino-2,2-dioxido-1H- assay of allulose (synonym D-psicose) yielded negative benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2-methylpropan-2-yl)-2,6- results at doses up to 1,800 μg/mL (Huntington Lab, 2011). dimethylisonicotinamide (CAS 1622458-34-7; 2079034-28-7) Based on the structure of the substance and the arrangement and concluded that the substance is GRAS (FEMA 4899) and identity of the functional groups therein, the Panel did not (Smith et al., 2005a) for use as a flavor ingredient in the food identify specific concerns related to the genotoxicity of categories and at the use levels specified in the GRAS allulose. In a 90-day comparative dietary feeding study in application (see Table 2). This substance was evaluated which male Wistar rats were administered 3% allulose and 3% individually within the context of the chemical group of , or 1.67 g/kg bw/day of allulose and sucrose, miscellaneous nitrogen compounds (JECFA, 2006, 2009, 2012, 2015, 2019; SLR, D19). The Panel calculated the histopathology assessments of major organs shown no anticipated per capita intake (“eaters only”) of N-(1-((4-amino- significant toxicologic effects (Matsuo et al., 2011). A 12-18 2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2- month study of allulose and sucrose administered to male methylpropan-2-yl)-2,6-dimethylisonicotinamide from use as Wistar rats at the same doses found significantly higher mean a flavor ingredient to be 285 µg/person/day, which is above value of pathological lesions in the liver for the allulose group the threshold of toxicological concern for structural class III than the sucrose group at the conclusion of the study (Yagi (90 µg/person/day) (Munro et al., 1996). The Panel and Matsuo, 2009). The Panel determined the Lowest considered the specification of the material to be adequately Observed Adverse Effect Level (LOAEL) to be 1.67 g/kg characterized by the purity assay and supporting spectral data bw/day (Matsuo et al., 2011; Yagi and Matsuo, 2009), which provided for FEMA GRAS evaluation. The Panel evaluated is greater than 2,900 times the anticipated daily per capita sensory data included within the application and found it intake of allulose from use as a flavoring ingredient. satisfactory with regard to intended conditions of use for the flavoring ingredient (Harman and Hallagan, 2013). It is McBain and Menn, 1969; Richardson et al., 1991). An Ames presumed that N-(1-((4-amino-2,2-dioxido-1H- assay in S. typhimurium strains TA97, TA98, TA100, TA1535, benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2-methylpropan-2-yl)-2,6- and TA1537 yielded negative results in the presence and dimethylisonicotinamide is minimally metabolized and, absence of S9 at doses of up to 333 μg/plate for the structural although poorly orally bioavailable, the small percentage of relative allyl propyl disulfide (FEMA 4073) (Zeiger et al., this substance that is metabolized undergoes hydroxylation. 1988). Positive results were observed in sister chromatid Additionally, O-dealkylation followed by carbonyl reduction is exchange and chromosomal aberration assays conducted in expected to occur to a minor extent. The parent compound CHO cells for a second structural relative, allyl disulfide and its metabolites are expected to be excreted in the feces (FEMA 2028). An increase in chromosomal aberrations was (Shen, 2008, 2009). Negative results were observed for this observed in CHO cells treated with allyl disulfide (FEMA 2028) substance in Ames assays conducted in S. typhimurium in the presence of S9 and a decrease in the occurrence of strains TA98, TA100, TA1535, and TA1537 as well as in E. sister chromatid exchanges was noted for allyl disulfide coli strain WP2 uvrA in the presence and absence of S9 (FEMA 2028) in the presence of S9 (Musk et al., 1997). No metabolic activation (Bruce, 2017; Wagner, 2016). An in vitro increases in micronucleated polychromatic erythrocytes in micronucleus assay in human peripheral blood lymphocytes ICR mouse bone marrow cells were observed, when groups produced negative results for the genotoxicity of this of male ICR mice were gavage administered a 50 or 100 substance both in the presence and absence of S9 (Roy, mg/kg bw corn oil mixture of allyl disulfide (FEMA 2028), allyl 2016). Based on these results, as well as the structure of the sulfide (FEMA 2042), and diallyl trisulfide (FEMA 3265) substance and the arrangement and identity of the functional (Marks et al., 1992). Based on weight of evidence, as well as groups therein, the Panel did not identify specific concerns the structure of the substance and the arrangement and related to the genotoxicity of N-(1-((4-amino-2,2-dioxido-1H- identity of the functional groups therein, the Panel did not benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2-methylpropan-2-yl)-2,6- identify specific concerns related to the genotoxicity of hexyl dimethylisonicotinamide. A developmental toxicity study for propyl disulfide. A 90-day toxicity study of structural relative this substance in female CD® [Crl:CD® (SD)] rats resulted in propyl disulfide (FEMA 3228) in male and female Charles maternal and fetal NOAELs of the highest tested dose of River CD rats resulted in NOAELs of 7.29 and 8.17 mg/kg 1,000 mg/kg bw/day (McElroy, 2017a, b). A 28-day toxicity bw/day respectively (Posternak et al., 1969). The dose of study in male and female CD® [Crl:CD® (SD)] rats for N-(1- 7.29 mg/kg bw/day is greater than 1,400,000 times the ((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)- anticipated daily per capita intake of hexyl propyl disulfide 2-methylpropan-2-yl)-2,6-dimethylisonicotinamide resulted in from use as a flavor ingredient. a NOAEL equivalent to the highest tested dose of 100 mg/kg bw/day (Daly, 2016). A 90-day toxicity study for N-(1-((4- The Panel reviewed the GRAS application and supporting amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2- information regarding O-ethyl S-(3-methylbut-2-en-1-yl) methylpropan-2-yl)-2,6-dimethylisonicotinamide in male and thiocarbonate (CAS 2097608-89-2) and concluded that the female CD® [Crl:CD® (SD)] rats resulted in a NOAEL substance is GRAS (FEMA 4901) (Smith et al., 2005a) for use equivalent to the highest tested dose of 140 mg/kg bw/day as a flavor ingredient in the food categories and at the use (Daly, 2017), which is greater than 28,000 times the levels specified in the GRAS application (see Table 2). This anticipated daily per capita intake of N-(1-((4-amino-2,2- substance was evaluated individually within the context of the dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2- chemical group of aliphatic thiols, subgroup of acyclic sulfides methylpropan-2-yl)-2,6-dimethylisonicotinamide from use as with oxidized side-chains (JECFA, 2004, 2012; SLR, A8). The a flavor ingredient. Panel calculated the anticipated per capita intake (“eaters only”) of O-ethyl S-(3-methylbut-2-en-1-yl) thiocarbonate from The Panel reviewed the GRAS application and supporting use as a flavor ingredient to be 0.01 µg/person/day, which is information regarding hexyl propyl disulfide (CAS 64580-54- below the threshold of toxicological concern for structural 7) and concluded that the substance is GRAS (FEMA 4900) class I (1800 µg/person/day) (Munro et al., 1996). The Panel (Smith et al., 2005a) for use as a flavor ingredient in the food considered the specification of the material to be adequately categories and at the use levels specified in the GRAS characterized by the purity assay and supporting spectral data application (see Table 2). This substance was evaluated provided for FEMA GRAS evaluation. It is presumed that O- individually within the context of the chemical group of simple ethyl S-(3-methylbut-2-en-1-yl) thiocarbonate undergoes aliphatic and aromatic sulfides and thiols (JECFA, 2000, rapid hydrolysis by esterases to yield carbonothioic acid, 2004, 2008, 2011; SLR, B4). The Panel calculated the which will be excreted in the urine in its unconjugated or anticipated per capita intake (“eaters only”) of hexyl propyl conjugated form (Kurooka et al., 1976). Negative results were disulfide from use as a flavor ingredient to be 0.3 observed in an Ames assay for structurally related substance, µg/person/day, which is below the threshold of toxicological O-ethyl S-(2-furylmethyl) thiocarbonate (FEMA 4043), both in concern for structural class I (1800 µg/person/day) (Munro et the presence or absence of S9 in S. typhimurium strains al., 1996). The Panel considered the specification of the TA98, TA100, TA1535, and TA1537 as well as E. coli WP2 material to be adequately characterized by the purity assay uvrA (Verspeek-Rip, 2000). This same structural relative was and supporting spectral data provided for FEMA GRAS tested for its ability to induce chromosomal aberrations in evaluation. It is presumed that hexyl propyl disulfide will be cultured peripheral human lymphocytes under two separate metabolized to its corresponding monothiols, hexyl sulfide experiments. In the first experiment, O-ethyl S-(2-furylmethyl) and propyl sulfide, followed by enzymatic oxidation to produce thiocarbonate (FEMA 4043) tested negative at concentrations sulfenic acids, sulfinic acids, and ultimately, sulfonic acids. up to 350 μg/ml in the presence and absence of S9 when The sulfinic and sulfonic acid products are easily excreted. incubated for three hours with a 24-hour fixation time. In the The thiol products could also react with glutathione to form second experiment, a dose dependent increase of cells with mixed disulfide products that undergo reduction and oxidative chromosomal aberrations was observed at 130, 240, and 280 desulfuration or undergo oxidation to sulfonic acid through μg/ml under 24-hour exposure followed by 24-hour fixation, their corresponding thiosulfinate and sulfinic acid and at 100, 130, and 140 μg/ml after 48-hour exposure and intermediates (Dutton and Illing, 1972; Maiorino et al., 1988; 24-hour fixation, in the absence of S9. In the presence of S9, a dose dependent increase was observed at 325 and 375 presence of S9 resulted in an increase of the frequency of μg/ml (Meerts, 2001). The same structural relative tested micronuclei but no such increase was observed when the negative in an oral dose in vivo micronucleus assay in SPF lymphocytes were incubated with the test substance for 3 mice at up to the highest dose of 500 mg/kg bw/day hours followed by a 21-hour recovery period in the absence (Verspeek-Rip, 2001). Based on weight of evidence, as well of S9 or when incubated with the test substance for 24 hours as the structure of the substance and the arrangement and with no recovery period in the absence of S9 (Whitwell, 2012). identity of the functional groups therein, the Panel did not As a follow-up to these results, a combined in vivo mouse identify specific concerns related to the genotoxicity of O-ethyl micronucleus and liver comet assay for furan-2(5H)-one S-(3-methylbut-2-en-1-yl) thiocarbonate. A 28-day toxicity (FEMA 4138) was conducted in outbred male Han Wistar rats. study of O-ethyl S-(2-furylmethyl) thiocarbonate (FEMA 4043) In this assay, no increase in micronucleated polychromatic gavage administered to SPF-bred Wistar rats at 2, 8, and 32 erythrocytes was observed compared to concurrent and mg/kg bw/day resulted in a NOAEL of 8 mg/kg bw/day (van historical controls. A small increase in group mean % tail Otterdijk, 2001), which is greater than 40,000,000 times the intensity was observed at the highest tested dose of 250 anticipated daily per capita intake of O-ethyl S-(3-methylbut- mg/kg bw, but not at the mid- and low-dose treatments in the 2-en-1-yl) thiocarbonate from use as a flavor ingredient. comet assay. Coincident with the increase in group mean% tail intensity, decreased glycogen vacuolation and duodenum The Panel reviewed the GRAS application and supporting villous tip necrosis were observed for the 250 mg/kg bw group. information regarding 3-methyl-2(5H)-furanone (CAS 22122- These results were within the historical control range of the 36-7) and concluded that the substance is GRAS (FEMA testing facility, and therefore were not attributed to the 4902) (Smith et al., 2005a) for use as a flavor ingredient in the treatment with furan-2(5H)-one. The liver comet assay for food categories and at the use levels specified in the GRAS furan-2-(5H)-one yielded negative results for DNA damage in application (see Table 2). This substance was evaluated the liver (Beevers, 2014). Based on these results, as well as individually within the context of the chemical group of the structure of the substance and the arrangement and aliphatic lactones(Adams et al., 1998; JECFA, 1999, 2004, identity of the functional groups therein, the Panel did not 2011; SLR, B1C). The Panel calculated the anticipated per identify specific concerns related to the genotoxicity of 3- capita intake (“eaters only”) of 3-methyl-2(5H)-furanone from methyl-2(5H)-furanone. A 13-week toxicity study of structural use as a flavor ingredient to be 0.3 µg/person/day, which is relative 4-hydroxybutyric acid (FEMA 3291) in male and below the threshold of toxicological concern for structural female F344/N rats resulted in a NOAEL of 450 mg/kg bw/day class III (90 µg/person/day) (Munro et al., 1996). This material (NTP, 1992) which is 90,000,000 times the anticipated daily is known to occur naturally in dried bonito, parmesan cheese, per capita intake of 3-methyl-2(5H)-furanone from use as a and shoyu but no consumption ratio could be calculated as flavor ingredient. A 2-year carcinogenicity study for the same only qualitative data was available (Njissen, 2019). The Panel structural relative was administered to B6C3F1 mice and considered the specification of the material to be adequately F344/N rats by oral gavage, determined a NOAEL of 225 characterized by the purity assay and supporting spectral data mg/kg bw/day for F344/N male rats (NTP, 1992). This NOAEL provided for FEMA GRAS evaluation. It is presumed that 3- is 45,000,000 times the anticipated daily per capita intake of methyl-2(5H)-furanone will hydrolyze to its corresponding ring 3-methyl-2(5H)-furanone from use as a flavor ingredient. opened alpha,beta-unsaturated hydroxycarboxylic acid. Upon hydrolysis, condensation with acetyl CoA yields a delta2- The Panel reviewed the GRAS application and supporting enoyl product, a substrate in the fatty acid pathway. Hydration information regarding ethyl 3-methyl-2-oxopentanoate (CAS of the cis double bond in this lactone yields (R)-3-hydroxyacyl 26516-27-8) and concluded that the substance is GRAS CoA which is isomerized to (S)-3-hydroxyacyl CoA by 3- (FEMA 4903) (Smith et al., 2005a) for use as a flavor hydroxyacyl CoA epimerase before undergoing fatty acid ingredient in the food categories and at the use levels metabolism. Alternatively, the alpha,beta-unsaturated lactone specified in the GRAS application (see Table 2). This could conjugate with glutathione and be excreted as its substance was evaluated individually within the context of the cysteine or mercapturic acid derivative (Nelson and Cox, chemical group of aliphatic primary alcohols, aldehydes, 2008). In an Ames assay for 3-methyl-2(5H)-furanone in S. carboxylic acids, acetals, and esters containing additional typhimurium strains TA98, TA100, TA1535, and TA1537 as oxygenated functional groups (JECFA, 2000, 2011; SLR, well as in E. coli strain WP2 uvrA, no mutagenicity was B1B). The Panel calculated the anticipated per capita intake observed in the presence and absence of S9 at (“eaters only”) of 3-methyl-2-oxopentanoate from use as a concentrations up to 5000 μg/plate (Ogura, 2014). An in vivo flavor ingredient to be 1 µg/person/day, which is below the micronucleus assay recorded no increase in the frequency of threshold of toxicological concern for structural class I (1800 micronucleated polychromatic erythrocytes at concentrations µg/person/day) (Munro et al., 1996). The Panel considered up to 750 mg/kg bw 3-methyl-2(5H)-furanone in male the specification of the material to be adequately Crl:CD1(ICR) mice but showed significant increases in characterized by the purity assay and supporting spectral data micronucleated cells at a mid-dose level of 500 mg/kg bw in provided for FEMA GRAS evaluation. It is presumed that ethyl female Crl:CD1(ICR) mice. However, because an increase in 3-methyl-2-oxopentanoate will undergo hydrolysis to its micronucleated cells was not observed at the low and high corresponding alcohol and acid. The ethanol product would doses of 250 and 1000 mg/kg bw in the female mice, this then be excreted by exhalation or undergo further metabolism positive result was not considered significant. (Ogura, 2017). An Ames assay conducted for structural relative furan-2(5H)- in the body. The 2-oxopentanoic acid product is expected to one (FEMA 4138) in S. typhimurium strains TA98, TA100, undergo further metabolism by beta-oxidation (Leegwater and TA102, TA1535, and TA1537 yielded negative results up to van Straten, 1974). In an Ames assay for ethyl 3-methyl-2- 5000 μg/plate with and without S9 using the plate oxopentanoate conducted in S. typhimurium strains TA98, incorporation and pre-incubation methodologies (Bowen, TA100, TA1535, and TA1537 as well as in E. coli strain 2011). An in vitro micronucleus assay in human peripheral WP2uvrA, negative results were observed up to 5,000 blood lymphocytes incubated with furan-2(5H)-one (FEMA μg/plate in the presence and absence of S9 (Verspeek-Rip, 4138) for 3 hours followed by a 21-hour recovery period in the 1999). Based on these results, as well as the structure of the substance and the arrangement and identity of the functional specified in the GRAS application (see Table 2). This groups therein, the Panel did not identify specific concerns substance was evaluated individually within the context of the related to the genotoxicity of ethyl 3-methyl-2-oxopentanoate. chemical group of esters of aliphatic acyclic primary alcohols A 28-day dietary study of structurally related substance, ethyl with aliphatic linear saturated carboxylic acids (JECFA, 1999, acetoacetate (FEMA 2415), in male and female Sprague- 2012; SLR, M1). The Panel calculated the anticipated per Dawley rats resulted in a NOAEL of greater than a 1000 mg/kg capita intake (“eaters only”) of 2,6-dimethylhept-5-enyl bw/day (Cook et al., 1992). This NOAEL is 50,000,000 times formate from use as a flavor ingredient to be 0.3 the anticipated daily per capita intake of ethyl 3-methyl-2- µg/person/day, which is below the threshold of toxicological oxopentanoate from use as a flavor ingredient. concern for structural class I (1800 µg/person/day) (Munro et al., 1996). The Panel considered the specification of the The Panel reviewed the GRAS application and supporting material to be adequately characterized by the purity assay information regarding trans-tetradec-4-enal (CAS 115018-39- and supporting spectral data provided for FEMA GRAS 8) and concluded that the substance is GRAS (FEMA 4904) evaluation. It is presumed that 2,6-dimethylhept-5-enyl (Smith et al., 2005a) for use as a flavor ingredient in the food formate will undergo ester hydrolysis to yield the categories and at the use levels specified in the GRAS corresponding carboxylic acid and alcohol products, formic application (see Table 2). This substance was evaluated acid and 2,6-dimethyl heptenol. Formic acid is metabolized to individually within the context of the chemical group of linear water and CO2 while 2,6-dimethyl heptenol is expected to and branch-chain aliphatic, unsaturated, unconjugated oxidize to the corresponding unsaturated carboxylic acid, 2,6- alcohols, aldehydes, acids, and related esters (JECFA, 1999, dimethyl heptanoic acid, which would then enter the beta- 2007, 2012; SLR, M1). The Panel calculated the anticipated oxidation cycle to undergo complete metabolism to innocuous per capita intake (“eaters only”) of trans-tetradec-4-enal from products, CO2 and water (Mattia, 1999). In an Ames assay for use as a flavor ingredient to be 0.3 µg/person/day, which is structural relative 2,6-dimethyl-5-heptenal (FEMA 2389), below the threshold of toxicological concern for structural negative results were observed in S. typhimurium strains class I (1800 µg/person/day) (Munro et al., 1996). The Panel TA98, TA100, TA1535, and TA1537 both in the presence and considered the specification of the material to be adequately absence of S9. In an unscheduled DNA synthesis assay for characterized by the purity assay and supporting spectral data the same substance, negative results were observed at up to provided for FEMA GRAS evaluation. It is presumed that 1 mg/ml in rat hepatocytes (Heck et al., 1989). The same trans-tetradec-4-enal will undergo oxidation to the substance yielded no increase in incidence of micronuclei corresponding unsaturated carboxylic acid intermediate that formation in an in vivo mouse study (Wild et al., 1983). A 90- will then proceed to a single beta-oxidation cycle resulting in day toxicity study of structural relative 2,6-dimethyl-5-heptenal the corresponding delta 2-enoyl CoA product. This product (FEMA 2389) in male and female Wistar rats resulted in a then undergoes further beta-oxidation resulting in an acetyl NOAEL of 37 mg/kg bw/day (Gaunt et al., 1983) which is CoA fragment which would enter the citric acid cycle to 7,400,00 times the anticipated daily per capita intake of 2,6- dimethylhept-5-enyl formate from use as a flavor ingredient. ultimately yield CO2 and water as innocuous products (Feldman and Weiner, 1972; Eckfeldt and Yonetani, 1982; Nakayasu et al., 1978; Beedham, 1988; Nelson and Cox, The Panel reviewed the GRAS application and supporting 2008). An Ames assay for structural relative cis-5-octenal information regarding L-carnitine tartrate (CAS 36687-82-8) (FEMA 3749) conducted in S. typhimurium strains TA98, and concluded that the substance is GRAS (FEMA 4906) TA100, TA1535, and TA1537 yielded negative results up to (Smith et al., 2005a) for use as a flavor ingredient in the food cytotoxic doses in the presence and absence of S9 (Asquith, categories and at the use levels specified in the GRAS 1990). An Ames assay conducted for structural relative 10- application (see Table 2). This substance was evaluated undecenal (FEMA 3095) in S. typhimurium strains TA98, individually within the context of amino acids (JECFA, 2006, TA100, TA102, TA1535, and TA1537 resulted in negative 2012; SLR, B3). The Panel calculated the anticipated per results for mutengenicity in the presence and absence of S9 capita intake (“eaters only”) of L-carnitine tartrate from use as (Bhatia et al., 2010; Sokolowski, 2007). An in vivo a flavor ingredient to be 1510 µg/person/day, which is above micronucleus assay conducted in male and female NMRI the threshold of toxicological concern for structural class III mice gavage-administered structural relative, 10-undecenal (90 µg/person/day) (Munro et al., 1996). This substance is (FEMA 3095), resulted in no statistically significant increase naturally occurring in animal protein products and is in the frequency of micronuclei (Bhatia et al., 2010; Honarvar, endogenous to humans (Benvenga et al., 2001). However, 2007). Based on these results, as well as the structure of the since quantitative data was not available, a consumption ratio substance and the arrangement and identity of the functional could not be calculated. The Panel considered the groups therein, the Panel did not identify specific concerns specification of the material to be adequately characterized by related to the genotoxicity of trans-tetradec-4-enal. In a 90- the purity assay and supporting spectral data provided for day dietary Sprague-Dawley rats were fed an admixture FEMA GRAS evaluation. The Panel evaluated sensory data containing, 10-undecenal (FEMA 3095), a structural relative, included within the application and found it satisfactory with which resulted in no significant evidence of toxicity. A NOAEL regard to intended conditions of use for the flavoring of 14.3 mg/kg bw/day was determined for 10-undecenal ingredient (Harman and Hallagan, 2013). It is presumed that (Liwska and Watson, 2012) which is greater than 2,860,000 L-carnitine tartrate readily dissociates into L-carnitine and L- times the anticipated daily per capita intake of trans-tetradec- tartaric acid in the gastrointestinal tract. Any orally consumed 4-enal from use as a flavor ingredient. L-carnitine that is not absorbed in the gastrointestinal tract passes to the lower intestine to undergo bacterial degradation The Panel reviewed the GRAS application and supporting yielding gamma-butyrobetaine which is then eliminated in the information regarding 2,6-dimethylhept-5-enyl formate (CAS feces (EFSA, 2003; Schmidbaur et al., 1998). In an Ames 2119671-25-7) and concluded that the substance is GRAS assay for L-carnitine tartrate in S. typhimurium strains TA98, (FEMA 4905) (Smith et al., 2005a) for use as a flavor TA100, TA1535, TA1537, and TA1538, negative results were ingredient in the food categories and at the use levels observed in the presence and absence of S9 up to 5,000 μg/plate (IBR, 1991b). Based on these results, as well as the structure of the substance and the arrangement and identity GRAS (FEMA 4908) (Smith et al., 2005a) for use as a flavor of the functional groups therein, the Panel did not identify ingredient in the food categories and at the use levels specific concerns related to the genotoxicity of L-carnitine specified in the GRAS application (see Table 2). (Smith et al., tartrate. No toxicity was observed in a 150-day toxicity study 2005a). The Panel calculated the anticipated per capita intake in which New Zealand rabbits were provided structural relative of C. stationis corn syrup fermentation product from use as a sodium tartrate (FEMA 3044) as 7.7% of their diet, otherwise flavor ingredient to be 2588 µg/person/day, which is above the corresponding to an intake of 2,310 mg/kg bw/day (Packman threshold of toxicological concern for structural class III (90 et al., 1963). A 28-day study with a 28-day reversibility phase µg/person/day) (Munro et al., 1996). The Panel considered as well as a 90/93-day study of structural relative betaine the identity description of the material to be adequate for (FEMA 4223) provided in the diet to Sprague-Dawley rats FEMA GRAS evaluation. The Panel evaluated sensory data resulted in a NOAEL of 2857 mg/kg bw/day (Hayes et al., included within the application and found it satisfactory with 2003) which is greater than 114,000 times the anticipated regard to intended conditions of use for the flavoring daily per capita intake of L-carnitine tartrate from use as a ingredient (Cohen et al., 2018; Smith et al., 2005b). The flavor ingredient. metabolism of the major marker constituents, inosine 5’- monophosphate as well as other nucleotides, amino acids, The Panel reviewed the natural flavor complex GRAS minerals, water, sugars, and organic acids, are normal application and supporting information regarding components of the human diet and as such are expected to Corynebacterium glutamicum (C. glutamicum) corn syrup be digested and metabolized in a similar manner to other fermentation product and concluded that the substance is commonly consumed nutrients (Tafazoli et al., 2017). In an GRAS (FEMA 4907) (Smith et al., 2005a) for use as a flavor Ames assay for a 2:1 mixture of FEMA 4107 and this ingredient in the food categories and at the use levels substance conducted in S. typhimurium strains TA98, TA100, specified in the GRAS application (see Table 2). This material TA1535, and TA1537 and E. coli strain WP2 uvrA, negative was evaluated within the context of the procedure for the results were observed in the presence and absence of S9 FEMA GRAS evaluation of flavor ingredients produced (Tafazoli et al., 2017). An HPRT assay for the same tst through biotechnology processes (Cohen et al., 2015).The substance conducted in V79 Chinese hamster lung (CHL) Panel calculated the anticipated per capita intake of C. cells yielded negative results in the presence and absence of glutamicum corn syrup fermentation product from use as a S9 (Tafazoli et al., 2017). Based on these results, as well as flavor ingredient to be 2588 µg/person/day, which is above the the structures of the principal constituents and the identity and threshold of toxicological concern for structural class III (90 arrangement of the functional groups therein, the Panel did not identify specific concerns related to the genotoxicity of C. µg/person/day) (Munro et al., 1996). The Panel considered stationis corn syrup fermentation product. In a 90-day toxicity the identity description of the material to be adequate for study in male and female Wistar rats dietarily administered a FEMA GRAS evaluation. The Panel evaluated sensory data 2:1 mixture of C. glutamicum corn syrup fermentation product included within the application and found it satisfactory with and C. stationis corn syrup fermentation product resulted in a regard to intended conditions of use for the flavoring NOAEL of 1666 mg/kg bw/day for C. stationis corn syrup ingredient (Cohen et al., 2018; Smith et al., 2005b). The fermentation product (Tafazoli et al., 2017) which is greater metabolism of the major marker constituents, glutamic acid, than 38,000 times the anticipated daily per capita intake of C. salt, water, and other amino acids are normal components of stationis corn syrup fermentation product from use as a the human diet and as such are expected to be digested and flavoring ingredient. metabolized in a similar manner to other commonly consumed nutrients (Tafazoli et al., 2017). In an Ames assay for a 2:1 The Panel reviewed the natural flavor complex GRAS mixture of this substance with FEMA 4908 conducted in S. application and supporting information regarding glucosyl typhimurium strains TA98, TA100, TA1535, and TA1537 and steviol glycosides, 70-80% (CAS 57817-89-7) and concluded E. coli strain WP2 uvrA, negative results were observed in the that the material is GRAS (FEMA 4909) for use as a flavor presence and absence of S9 (Tafazoli et al., 2017). An HPRT ingredient in the food categories and at the use levels assay for the same test substance conducted in V79 Chinese specified in the GRAS application (see Table 2). This material hamster lung (CHL) cells yielded negative results in the was evaluated within the context of the procedure for the presence and absence of S9 (Tafazoli et al., 2017). Based on safety evaluation of natural flavor complexes (Cohen et al., these results, as well as the structures of the principal 2018; Smith et al., 2005b). The Panel calculated the constituents and the identity and arrangement of the anticipated per capita intake (“eaters only”) of glucosyl steviol functional groups therein, the Panel did not identify specific glycosides 70-80% from use as a flavor ingredient to be 1926 concerns related to the genotoxicity of C. glutamicum corn µg/person/day, which is above the threshold of toxicological syrup fermentation product. A 104-week study in Fisher 344 concern for structural class III (90 µg/person/day) (Munro et male and female rats dietarily administered monosodium al., 1996). This substance is reported to occur in Stevia glutamate (FEMA 2756) resulted in a NOAEL of 2,311 mg/kg rebaudiana leaves (Wood et al., 1955). The Panel considered bw/day, which, based on a lower bound estimate of 38.3% L- the identity description of the material to be adequate for glutamic acid content in C. glutamicum corn syrup FEMA GRAS evaluation. The Panel evaluated sensory data fermentation product (Tafazoli et al., 2017) is greater than included within the application and found it satisfactory with 140,000 times the anticipated daily per capita intake of C. regard to intended conditions of use for the flavoring glutamicum corn syrup fermentation product from use as a ingredient (Harman and Hallagan, 2013). Metabolic data exist flavor ingredient. for representative members of each congeneric group that would predict, at the intake levels proposed, metabolism by

well-established detoxication pathways to innocuous products The Panel reviewed the natural flavor complex GRAS (Gardana et al., 2003; Geuns et al., 2003a; Geuns et al., application and supporting information regarding 2003b; Koyama et al., 2003a; Koyama et al., 2003b; Nikiforov Corynebacterium stationis (C. stationis) corn syrup fermentation product and concluded that the substance is et al., 2013; Purkayastha et al., 2015; Purkayastha et al., 2016; Purkayastha et al., 2014; Renwick and Tarka, 2008). feeding study of a stevia extract to be equal to 550 mg/kg The genotoxicity of the major marker constituents (steviol bw/day, or approximately 89.5 mg/kg bw/day of rebaudioside glycosides) has been thoroughly examined in a wide range of A (Yamada et al., 1985), which is greater than 4,100 times the studies. While some positive results are reported in in vitro anticipated daily per capita intake of the. anticipated daily per mutagenicity assays, in vivo studies do not provide evidence capita intake of glucosyl steviol glycosides 40% from use as a of genotoxic effects (Nakajima, 2000; Pezzuto et al., 1985, flavoring ingredient. 1986;(Rumelhard et al., 2016); Suttajit et al., 1993; Terai et al, 2002; Toyoda et al., 1997; Williams and Burdock, 2009). The Panel reviewed the natural flavor complex GRAS Based on the results for the various steviol glycosides, the application and supporting information regarding stevia Panel did not identify specific concerns related to the potential extract stevioside, 70% (CAS 91722-21-3) and concluded that genotoxicity of glucosyl steviol glycosides 70-80%. In a 108- the substance is GRAS (FEMA 4911) (Smith et al., 2005a) for week carcinogenicity study for stevioside, no carcinogenic use as a flavor ingredient in the food categories and at the use effects were observed (Toyoda et al., 1997). In a 2-year levels specified in the GRAS application (see Table 2). This feeding study, male and female rats were administered the substance was evaluated within the context of the procedure equivalent of 0, 50, 150, or 550 mg/kg bw/day of a stevia for the safety evaluation of natural flavor complexes (Cohen et al., 2018; Smith et al., 2005b). The Panel calculated the extract comprised of 74% stevioside and 16% rebaudioside anticipated per capita intake (“eaters only”) of stevia extract A. The authors considered the NOAEL from this 2-year rat stevioside, 70% from use as a flavor ingredient to be 1427 feeding study of a stevia extract to be equal to 550 mg/kg µg/person/day, which is above the threshold of toxicological bw/day, or approximately 89.5 mg/kg bw/day of rebaudioside concern for structural class III (90 µg/person/day) (Munro et A (Yamada et al., 1985), which is greater than 2,700 times the al., 1996). The Panel considered the identity description of the anticipated daily per capita intake of the. anticipated daily per material to be adequate for FEMA GRAS evaluation. The capita intake of glucosyl steviol glycosides 70-80% from use Panel evaluated sensory data included within the application as a flavoring ingredient. and found it satisfactory with regard to intended conditions of use for the flavoring ingredient (Harman and Hallagan, 2013). The Panel reviewed the natural flavor complex GRAS Metabolic data exist for representative members of each application and supporting information regarding glucosylated congeneric group that would predict, at the intake levels steviol glycosides, 40% (CAS 57817-89-7) and concluded that proposed, metabolism by well-established detoxication the substance is GRAS (FEMA 4910) (Smith et al., 2005a) for pathways to innocuous products (Gardana et al., 2003; Geuns use as a flavor ingredient in the food categories and at the use et al., 2003a; Geuns et al., 2003b; Koyama et al., 2003a; levels specified in the GRAS application (see Table 2). This Koyama et al., 2003b; Nikiforov et al., 2013; Purkayastha et substance was evaluated within the context of the procedure al., 2015; Purkayastha et al., 2016; Purkayastha et al., 2014; for the safety evaluation of natural flavor complexes (Cohen Renwick and Tarka, 2008). The genotoxicity of the major et al., 2018; Smith et al., 2005b). The Panel calculated the marker constituents (steviol glycosides) has been thoroughly anticipated per capita intake (“eaters only”) of glucosylated examined in a wide range of studies. While some positive steviol glycosides, 40% from use as a flavor ingredient to be results are reported in in vitro mutagenicity assays, in vivo 1294 µg/person/day, which is above the threshold of studies do not provide evidence of genotoxic effects toxicological concern for structural class III (90 µg/person/day) (Nakajima, 2000; Pezzuto et al., 1985, 1986;(Rumelhard et (Munro et al., 1996). The Panel considered the identity al., 2016); Suttajit et al., 1993; Terai et al, 2002; Toyoda et al., description of the material to be adequate for FEMA GRAS 1997; Williams and Burdock, 2009). Based on the results for evaluation. The Panel evaluated sensory data included within the various steviol glycosides, the Panel did not identify the application and found it satisfactory with regard to specific concerns related to the potential genotoxicity of stevia intended conditions of use for the flavoring ingredient extract stevioside, 70%. In a 108- week carcinogenicity study (Harman and Hallagan, 2013). Metabolic data exist for for stevioside, no carcinogenic effects were observed (Toyoda representative members of each congeneric group that would et al., 1997). In a 2-year feeding study, male and female rats predict, at the intake levels proposed, metabolism by well- were administered the equivalent of 0, 50, 150, or 550 mg/kg established detoxication pathways to innocuous products bw/day of a stevia extract comprised of 74% stevioside and (Gardana et al., 2003; Geuns et al., 2003a; Geuns et al., 16% rebaudioside A. The authors considered the NOAEL 2003b; Koyama et al., 2003a; Koyama et al., 2003b; Nikiforov from this 2-year rat feeding study of a stevia extract to be et al., 2013; Purkayastha et al., 2015; Purkayastha et al., equal to 550 mg/kg bw/day, or approximately 89.5 mg/kg 2016; Purkayastha et al., 2014; Renwick and Tarka, 2008). bw/day of rebaudioside A (Yamada et al., 1985), which is The genotoxicity of the major marker constituents (steviol greater than 3,700 times the anticipated daily per capita intake glycosides) has been thoroughly examined in a wide range of of the. anticipated daily per capita intake of stevia extract studies. While some positive results are reported in in vitro stevioside, 70% from use as a flavoring ingredient. mutagenicity assays, in vivo studies do not provide evidence of genotoxic effects (Nakajima, 2000; Pezzuto et al., 1985, The Panel reviewed the natural flavor complex GRAS 1986;(Rumelhard et al., 2016); Suttajit et al., 1993; Terai et al, application and supporting information regarding hibiscus 2002; Toyoda et al., 1997; Williams and Burdock, 2009). blossom extract (Hibiscus sabdariffa L.) (CAS 4775-96-2) and Based on the results for the various steviol glycosides, the concluded that the substance is GRAS (FEMA 4912) (Smith Panel did not identify specific concerns related to the potential et al., 2005a) for use as a flavor ingredient in the food genotoxicity of glucosyl steviol glycosides 40%. In a 108- categories and at the use levels specified in the GRAS week carcinogenicity study for stevioside, no carcinogenic application (see Table 2). This substance was evaluated effects were observed (Toyoda et al., 1997). In a 2-year within the context of the procedure for the safety evaluation of feeding study, male and female rats were administered the natural flavor complexes (Cohen et al., 2018; Smith et al., equivalent of 0, 50, 150, or 550 mg/kg bw/day of a stevia 2005b). The Panel calculated the anticipated per capita intake extract comprised of 74% stevioside and 16% rebaudioside (“eaters only”) of hibiscus blossom extract from use as a flavor A. The authors considered the NOAEL from this 2-year rat ingredient to be 150 µg/person/day, which is above the threshold of toxicological concern for structural class III (90 1-al (FEMA 2389) resulted in clinical signs of toxicity at the µg/person/day) (Munro et al., 1996). The Panel considered highest dose tested (3000 mg/kg bw/day). Male rats at the the identity description of the material to be adequate for high and middle dose (1500 mg/kg bw/day) showed evidence FEMA GRAS evaluation. Based on metabolic data for a of α2µ-globulin nephropathy. No such findings were found at representative member of the principal congeneric group, it is the lowest dose of 300 mg/kg bw/day (Terrill, 1990). In a 90- presumed that the group would be metabolized by well- day study in male and female Wistar rats, the same structural established pathways to yield innocuous products. An Ames relative was provided in the diet at concentrations of 9, 37, assay conducted with hibiscus blossom extract in S. and 150 mg/kg bw/day resulted in a NOAEL of 37 mg/kg typhimurium strains TA98, TA100, TA1535, and TA1537 as bw/day (Gaunt et al., 1983), which is 185,000 times versus well as in E. coli strain WP2 uvrA (pKM1010) was negative up the anticipated daily per capita intake of 3,7-dimethyl-2- to cytotoxic concentrations in the presence and absence of S9 methyleneoct-6-en-1-ol from use as a flavor ingredient. using the plate incorporation and preincubation techniques (Leuschner, 2017). Based on these assays and the The Panel reviewed the GRAS application and supporting composition of the material therein, the Panel did not identify information regarding bis-(3-methyl-2-butenyl)disulfide (CAS specific concerns related to the genotoxicity of hibiscus 24963-39-1) and concluded that it is GRAS (FEMA 4914) blossom extract. A 270-day study in which male and female (Smith et al., 2005a) for use as a flavor in the food categories Sprague-Dawley rats received hibiscus blossom extract by and at the use levels specified in the GRAS application (see water resulted in a NOAEL of 200 mg/kg bw/day (Api et al., Table 2). This substance was evaluated individually within the 2004; Lambert and Hopkins, 1996) which is 80,000 times the context of the chemical groups of simple aliphatic and anticipated daily per capita intake of hibiscus blossom extract aromatic sulfides and thiols (JECFA, 2000, 2004, 2008, 2011; from use as a flavor ingredient. SLR, B4). The Panel calculated the anticipated per capita

intake (“eaters only”) of bis-(3-methyl-2-butenyl)disulfide from The Panel reviewed the GRAS application and supporting use as a flavor ingredient to be 0.1 µg/person/day, which is information regarding 3,7-dimethyl-2-methyleneoct-6-en-1-ol below the threshold of toxicological concern for structural (CAS 18478-46-1) and concluded that it is GRAS (FEMA class III (90 µg/person/day) (Munro et al., 1996). The Panel 4913) (Smith et al., 2005a) for use as a flavor in the food considered the specification of the material to be adequately categories and at the use levels specified in the GRAS characterized by the purity assay and supporting spectral data application (see Table 2). This substance was evaluated provided for FEMA GRAS evaluation. It is predicted that bis- individually within the context of the chemical group of linear (3-methyl-2-butenyl)disulfide will be reduced either and branched-chain aliphatic, unsaturated, unconjugated enzymatically by GSH reductase (Waring, 1996) or alcohols, aldehydes, acids, and related esters (JECFA, 1999, thioltransferases (Wells et al., 1993), or chemically by 2006, 2012; SLR, M1). The Panel calculated the anticipated exchange with GSH, thioredoxin, cysteine or other per capita intake (“eaters only”) of 3,7-dimethyl-2- endogenous thiols to yield 3-methyl-2-butenyl thiol. This methyleneoct-6-en-1-ol from use as a flavor ingredient to be simple thiol is expected to undergo enzymatic oxidation to 14 µg/person/day, which is below the threshold of yield the corresponding sulfenic acid, sulfinic acid and toxicological concern for structural class II (540 ultimately, sulfonic acid. The sulfinic and sulfonic acids are µg/person/day) (Munro et al., 1996). This substance is known water soluble and easily excreted. Alternatively, the thiol that to naturally occur in lemon eucalyptus (Eucalyptus citridora) is formed may react with glutathione and cysteine to form (Luis et al., 2017). However, because only qualitative data mixed disulfides that can then undergo reduction and was available, a consumption ratio could not be calculated. In oxidative desulfuration, or oxidation to sulfonic acid via the its evaluation the Panel noted that the major markers of intermediate thiosulfinate and sulfinic acid (Dutton and Illing, identity are ≥93%, 3,7-dimethyl-2-methyleneoct-6-en-1-ol and 1972; Maiorino et al., 1989; McBain and Menn, 1969; ≥3% 2,3,7-trimethyloct-6-en-1-ol (mixture of isomers) and Richardson et al., 1991). There are also several possible thiol- considered the specification of the material to be adequately disulfide exchange reactions that may occur (Cotgreave et al., characterized by the purity assay and supporting spectral data 1989). bis-(3-Methyl-2-butenyl)disulfide did not increase the provided for FEMA GRAS evaluation. It is anticipated that 3,7- frequency of revertant colonies in S. typhimurium strains dimethyl-2-methyleneoct-6-en-1-ol shares similar metabolic TA98, TA100, TA1535, and TA1537 as well as in E. coli strain fates as other structurally related aliphatic branched chain WP2uvrA either in the presence or absence of S9 metabolic primary alcohols. These branched-chain terpene alcohols activation by the pre-incubation method (Komai, 2017a). In a have been shown to undergo alcohol oxidation, omega- mouse micronucleus assay, gavage administration of a oxidation, hydration and selective hydrogenation to yield polar 68:20:12 mixture containing the structurally related flavor metabolites forming glucuronide or sulfate conjugates that are ingredients, allyl disulfide (FEMA 2028), allyl sulfide (FEMA rapidly eliminated in the urine (Chadha and Madyastha, 1982; 2042), and diallyl trisulfide (FEMA 3265), did not increase the Diliberto et al., 1990). The structural relative 3,7-dimethyl-2- frequency of micronucleated polychromatic erythrocytes in methyleneoct-6-en-1-al (CAS 22418-66-2) was negative for bone marrow cells. This test mixture provided estimated genotoxicity in an Ames assay in S. typhimurium strains TA98, doses of the structurally related substance, allyl disulfide TA100, TA1535, and TA1537 as well as E. coli strain WP2 (FEMA 2028), of 48 and 98 mg/kg bw/day (Marks et al., 1992). uvrA in either the presence or absence of S9 metabolic The structurally related substance, allyl propyl disulfide activation (Thompson, 2002). Based on these results, as well (FEMA 4073), did not increase the frequency of revertant as the structure and the identity and arrangement of functional colonies when tested up to 333 μg/plate in S. typhimurium groups therein, the Panel did not identify a specific concern strains TA97, TA98, TA100, TA102, TA1535, and TA1537 for genotoxic potential of 3,7-dimethyl-2-methyleneoct-6-en- either in the presence or absence of S9 metabolic activation 1-ol when used as a flavoring ingredient. A 28-day study in (Zeiger et al., 1988). In a separate experiment, both structural which male and female Sprague-Dawley rats, gavage relatives, allyl propyl disulfide (FEMA 4073) and allyl disulfide administration of the structural relative 2,6-dimethylhept-5-en- (FEMA 2028) did not increase the frequency of revertant colonies in S. typhimurium strain TA100 at doses ranging from relative 4-(4-methyl-3-penten-1-yl)-2(5H)-furanone (FEMA 0.0015-0.15 μg/mL either in the presence or absence of S9 4868) did not increase the frequency of revertant colonies in metabolic activation (Eder et al., 1982; Eder et al., 1980). the absence and presence of S9 metabolic activation at Based on the results for the substance and structural concentrations up to 500 μg/plate(Kawaguchi, 2016). Based relatives, as well as on the structure of the substance and the on the results for the substance and structural relatives, as identity and arrangement of functional groups therein, the well as on the structure of the substance and the identity and Panel did not identify a specific concern for genotoxic arrangement of functional groups therein, the Panel did not potential of bis-(3-methyl-2-butenyl) disulfide when used as a identify a specific concern for genotoxic potential of (5Z)-3,4- flavoring ingredient. In a 6-day study in female Sprague- dimethyl-5-propylidene-2(5H)-furanone when used as a Dawley rats, gavage administration of the structural relative flavoring ingredient. allyl disulfide (FEMA 2028) resulted in a NOAEL of 36 mg/kg bw/day (Munday and Manns, 1994). This is greater than The Panel reviewed the GRAS application and supporting 21,600,000 times versus the anticipated daily per capita information regarding 2-methyl-3-butene-2-thiol (CAS intake of bis-(3-methyl-2-butenyl) disulfide from use as a 124831-34-1) and concluded that it is GRAS (FEMA 4916) flavor ingredient. (Smith et al., 2005a) for use as a flavor in the food categories and at the use levels specified in the GRAS application (see The Panel reviewed the GRAS application and supporting Table 2). This substance was evaluated individually within the information regarding (5Z)-3,4-dimethyl-5-propylidene-2(5H)- context of the chemical group of simple aliphatic and aromatic furanone (CAS 2142634-65-7) and concluded that it is GRAS sulfides and thiols (JECFA, 2000, 2004, 2008, 2011; SLR, (FEMA 4915) (Smith et al., 2005a) for use as a flavor in the A8). The Panel calculated the anticipated per capita intake food categories and at the use levels specified in the GRAS (“eaters only”) of 2-methyl-3-butene-2-thiol from use as a application (see Table 2). This substance was evaluated flavor ingredient to be 0.07 µg/person/day, which is below the individually within the context of the chemical groups) of threshold of toxicological concern for structural class I (1800 furanones and related substances (JECFA, 2006, 2017; SLR, µg/person/day) (Munro et al., 1996). The Panel considered D9). The Panel calculated the anticipated per capita intake the specification of the material to be adequately (“eaters only”) of (5Z)-3,4-dimethyl-5-propylidene-2(5H)- characterized by the purity assay and supporting spectral data furanone from use as a flavor ingredient to be 0.1 provided for FEMA GRAS evaluation. It is predicted that 2- µg/person/day, which is below the threshold of toxicological methyl-3-butene-2-thiol will undergo oxidation of the thiol concern for structural class III (90 µg/person/day) (Munro et group resulting in the corresponding sulfenic acid, sulfinic al., 1996). This substance is reported to occur in kombu dashi acid, and sulfonic acid. Sulfenic acids are unstable and readily (brown algae stock) (Tomita, 2017). The Panel considered the undergo further oxidation to sulfinic and sulfonic acids or specification of the material to be adequately characterized by combine with nucleophiles. The sulfinic and sulfonic acids are the purity assay and supporting spectral data provided for water soluble and easily excreted (Dutton and Illing, 1972; FEMA GRAS evaluation. (5Z)-3,4-Dimethyl-5-propylidene- Klancnik et al., 1992; Maiorino et al., 1989; McBain and Menn, 2(5H)-furanone is predicted to be metabolized in a manner 1969; Renwick, 1996; Richardson et al., 1991). Alternatively, similar to other five-membered lactone rings. α,β-Unsaturated the thiol may react with glutathione and cysteine to form mixed lactones, such as (5Z)-3,4-dimethyl-5-propylidene-2(5H)- disulfides that can then undergo reduction and oxidative furanone, are predicted to hydrolyze to the corresponding desulfuration, or oxidation to sulfonic acid via the intermediate ring-opened α,β-unsaturated hydroxycarboxylic acids (Köppel thiosulfinate and sulfinic acids. 2-Methyl-3-butene-2-thiol is and Tenczer, 1991). Following hydrolysis, condensation with also anticipated to undergo S-methylation in mammals to acetyl CoA yields a delta-2-enoyl CoA product that is a produce the corresponding methyl thioether that can be substrate in the fatty acid pathway (Nelson and Cox, 2008). successively oxidized to the corresponding sulfoxide and Since the stereochemistry of the double bond in a lactone is sulfone (Shaw and Blagbrough, 1989; Tateishi et al., 1978; cis, hydration yields (R)-3-hydroxyacyl CoA, which is then Tateishi and Tomisawa, 1989). In an Ames assay in S. isomerized to (S)-3-hydroxyacyl CoA by 3-hydroxyacyl CoA typhimurium strains TA98, TA100, TA1535, TA1537 and E. epimerase prior to entering into fatty acid metabolism. coli strain WP2uvrA, 2-methyl-3-butene-2-thiol did not Alternately, the α,β-unsaturated lactone may conjugate with increase the frequency of revertant colonies in the absence glutathione and be excreted as the cysteine or mercapturic and presence of S9 metabolic activation (Sato, 2017). Based acid derivative (Boyland and Chasseaud, 1970; Chasseaud, on these results, as well as on the structure and the identity 1979; Fry et al., 1993). In an Ames assay in S. typhimurium and arrangement of functional groups therein, the Panel did strains TA98, TA100, TA1535, TA1537 and E. coli strain not identify a specific concern for genotoxic potential of 2- WP2uvrA, (5Z)-3,4-dimethyl-5-propylidene-2(5H)-furanone methyl-3-butene-2-thiol when used as a flavoring ingredient. did not increase the frequency of revertant colonies in the In a 14-day dietary administration study in male and female absence and presence of S9 metabolic activation at rats with the structural relative prenylthiol (FEMA 3896), an concentrations up to 5000 μg/plate (Komai, 2017b).. In an in average daily intake of 12.8 mg/kg bw/day for both sexes vitro micronucleus induction assay in human peripheral blood resulted in a significant reduction in food consumption for lymphocytes, 3,4-dimethylpentylidenefuran-2(5H)-one did males, but this is presumed to be related to palatability of the induce a statistically significant effect on micronuclei test diet since the change in food consumption was not frequency in the presence of S9 metabolic activation (Lloyd, accompanied by a significant difference in body weight gain 2014), but in a combined comet/micronucleus assay in rats, between test and control groups. Gross necropsy and the substance did not increase micronucleated cells in the histopathological examination of kidney and liver tissues bone marrow or induce DNA damage in the liver, as analyzed revealed effects related to the test article. Relative kidney and by the comet assay, when tested up to the maximum tolerated liver weights showed no significant differences between test dose of 500 mg/kg bw/day (Beevers, 2014). In an Ames assay and control groups (Wnorowski, 1997). In a 90-day study in in S. typhimurium strains TA98 and TA100, the structural albino weanling rats, dietary addition of the structural relative 2,3-butanedithiol (FEMA 3477) at a dose of 0.703 mg/kg specified in the GRAS application (see Table 2). This bw/day resulted in no hematological, biochemical and urinary substance was evaluated individually within the context of the deviations from normal ranges of tested parameter or any chemical group of linear and branched-chain aliphatic, unusual microscopic pathological observations (Morgareidge, unsaturated, unconjugated alcohols, aldehydes, acids, and 1974). In an OECD 408 compliant 90-day in male and female related esters (JECFA, 1999, 2006, 2012; SLR, M1).The Sprague-Dawley rats, gavage administration of the structural Panel calculated the anticipated per capita intake (“eaters relative 4-mercapto-4-methyl-2-pentanone (FEMA 3997) at only”) of tridec-5-enal from use as a flavor ingredient was doses of 13, 20, and 26 mg/kg/day resulted in a NOAEL of 26 calculated to be 0.3 µg/person/day, which is below the mg/kg bw/day (Bauter, 2017). This is 22,000,000 times the threshold of toxicological concern for structural class I (1800 anticipated daily per capita intake of 2-methyl-3-butene-2-thiol µg/person/day) (Munro et al., 1996). In its evaluation of tridec- from use as a flavor ingredient. 5-enal, the Panel noted that the major markers of identity are >90% cis-tridec-5-enal and >5% trans-tridec-5-enal and The Panel reviewed the GRAS application and supporting considered the specification of the material to be adequately information regarding (Z)-9-dodecenoic acid (CAS 22032-47- characterized by the purity assay and supporting spectral data 9) and concluded that it is GRAS (FEMA 4917) (Smith et al., provided for FEMA GRAS evaluation. It is predicted that 2005a) for use as a flavor in the food categories and at the tridec-5-enal will be oxidized to the corresponding use levels specified in the GRAS application (see Table 2). unsaturated carboxylic acid before proceeding into the β- This substance was evaluated individually within the context oxidation cycle (Feldman & Weiner, 1972; Eckfeldt & of the chemical group of linear and branched-chain aliphatic, Yonetani, 1982; Nakayasu et al., 1978; Beedham, 1988). The unsaturated, unconjugated alcohols, aldehydes, acids, and cis-carboxylic acid intermediate will undergo a single β- related esters (JECFA, 1999, 2006, 2012; SLR, M1). The oxidation cycle and the corresponding cis Δ-3 double bond of Panel calculated the anticipated per capita intake (“eaters the C11 intermediate will then be converted to the only”) of (Z)-9-dodecenoic acid from use as a flavor ingredient corresponding trans Δ-2 double bond (via enoyl CoA to be 0.1 µg/person/day, which is below the threshold of isomerase). It then proceeds into the next β-oxidation cycle. toxicological concern for structural class I (1800 Acetyl CoA units will continue to be cleaved off in β-oxidation µg/person/day) (Munro et al., 1996). It is anticipated that (Z)- until the final cleavage, which yields acetyl CoA and propionyl 9-dodecenoic acid will form micelle aggregates, esterify with CoA. Acetyl CoA enters the citric acid cycle directly, while glycerol in chylomicrons and low-density lipoproteins, and propionyl CoA is transformed into succinyl CoA that then then be transported via the lymphatic system for absorption enters the citric acid cycle. Based on this, it is therefore (Borgström, 1974). Alternatively, (Z)-9-dodecenoic acid could predicted that 5-tridecenal will undergo complete metabolism undergo β-oxidative cleavage and complete metabolism to to innocuous products (CO2 and water) (Nelson and Cox, carbon dioxide in the fatty acid pathway and the citric acid 2008). In an Ames assay in S. typhimurium strains TA98, cycle (Gibson et al., 1982; Masoro, 1977; Schulz, 1983). In an TA100, TA102, TA1535, and TA1537, the structurally related Ames assay conducted in S. typhimurium strains TA98 and substance, 10-undecenal (FEMA 3095), did not increase the TA100, (Z)-9-dodecenoic acid did not increase the frequency frequency of revertant colonies in the absence and presence of revertant colonies in the absence and presence of S9 of S9 metabolic activation (Bhatia et al., 2010; Sokolowski, metabolic activation. In an Ames assay in S. typhimurium 2007). In an in vivo micronucleus assay in male and female strains TA98, TA100, TA102, TA1535, and TA1537, the NMRI mice, gavage administration at doses up to 2000 mg/kg structural relative 10-undecenal (FEMA 3095) did not bw/day of the structurally related substance, 10-undecenal increase the frequency of revertant colonies in the absence (FEMA 3095), produced no statistically significant increases and presence of S9 metabolic activation using both the plate in the frequency of micronuclei (Bhatia et al., 2010; Honorvar, incorporation and pre-incubation methodologies (Bhatia et al., 2007). Based on these results, as well as the structure of the 2010; Sokolowski, 2007). In an in vivo micronucleus assay in substance and the identity and arrangement of the functional male and female NMRI mice, gavage administration at doses groups therein, the Panel did not identify a specific concern up to 2000 mg/kg bw/day of the structural relative 10- for the genotoxic potential of tridec-5-enal. A 90-day dietary undecenal (FEMA 3095) produced no statistically significant administration study for the structurally related substance, 10- increases in the frequency of micronuclei (Bhatia et al., 2010; undecenal (FEMA 3095), was conducted in male and female Honarvar, 2007). Based on these results, the very low intake Sprague-Dawley rats resulted in a NOAEL of 14.3 mg/kg of the substance and the structure and the identity and bw/day, reporting no toxicologically significant clinical arrangement of functional groups therein, the Panel did not observations or macroscopic abnormalities (Liwska, 2012; identify a specific concern for genotoxic potential of 1-ethyl- Tsang, 2018). This is 2,860,000 times the anticipated daily per (Z)-9-dodecenoic acid when used as a flavoring ingredient. A capita intake of 5-tridecenal from use as a flavor ingredient. 90-day dietary administration study for the structurally related substance, 10-undecenal (FEMA 3095), was conducted in The Panel reviewed the GRAS application and supporting male and female Sprague-Dawley rats resulted in a NOAEL information regarding refined soybean oil extract (CAS 8001- of 14.3 mg/kg bw/day due to no toxicologically significant 22-7) and concluded that it is GRAS (FEMA 4919) (Smith et clinical observations or macroscopic abnormalities (Liwska, al., 2005a) for use as a flavor in the food categories and at the 2012; Tsang, 2018). This is greater than 8,500,000 times the use levels specified in the GRAS application (see Table 2). anticipated daily per capita intake of (Z)-9-dodecenoic acid This substance was evaluated within the context of the from use as a flavor ingredient. procedure for the safety evaluation of natural flavor complexes (Cohen et al., 2018; Smith et al., 2005b). The The Panel reviewed the GRAS application and supporting Panel calculated the anticipated per capita intake (“eaters information regarding tridec-5-enal (CAS 68820-38-2) and only”) of refined soybean oil extract from use as a flavor concluded that it is GRAS (FEMA 4918) (Smith et al., 2005) ingredient was calculated to be 0.3 µg/person/day, which is for use as a flavor in the food categories and at the use levels below the threshold of toxicological concern for structural class III (90 µg/person/day) (Munro et al., 1996). The Panel considered the specification of the material to be adequately The Panel reviewed the GRAS application and supporting characterized by the purity assay and supporting spectral data information regarding 1-ethyl-2-(1-pyrrolylmethyl)pyrrole provided for FEMA GRAS evaluation. It is expected that (CAS 2204262-51-9) and concluded that it is GRAS (FEMA phytosterols, the predominant constituents of refined soybean 4920) (Smith et al., 2005a) for use as a flavor in the food oil extract, will be poorly absorbed (<5% of dietary categories and at the use levels specified in the GRAS concentrations) in the gastrointestinal tract. Excess dietary application (see Table 2). This substance was evaluated phytosterols are excreted unchanged in the feces (Maki et al., individually within the context of the chemical group of 2001; Miettinen et al., 1990; Ostlund, 2002; Rozner and Garti, pyridine, pyrrole and quinoline derivatives (JECFA, 2006, 2006; Salen et al., 1985; Sanders et al., 2000). In an Ames 2012; SLR, D3). The Panel calculated the anticipated per assay in S. typhimurium strains TA1535, TA1537, TA98, capita intake (“eaters only”) of 1-ethyl-2-(1- TA100 a phytosterol mixture (β-sitosterol, 45.5-51.0%; pyrrolylmethyl)pyrrole from use as a flavor ingredient to be campesterol, 26.7-28.1%; and stigmasterol, 17.7-18.7%) did 0.0001 µg/person/day, which is below the threshold of not increase the frequency of revertant colonies in the toxicological concern for structural class III (90 µg/person/day) absence and presence of S9 metabolic activation (Wolfreys (Munro et al., 1996). The Panel considered the specification and Hepburn, 2002). In an Ames assay in S. typhimurium of the material to be adequately characterized by the purity strains TA1535, TA1537, TA98, TA100 and E. coli strain assay and supporting spectral data provided for FEMA GRAS WP2uvrA the phytosterol mixture was esterified with fatty evaluation. 1-Ethyl-2-(1-pyrrolylmethyl)pyrrole is predicted to acids from sunflower oil, and the resulting phytosterol ester be rapidly absorbed from the gastrointestinal tract and mixture did not increase the frequency of revertant colonies in oxidized to polar metabolites that would be eliminated the absence and presence of S9 metabolic activation primarily in the urine. 1-Ethyl-2-(1-pyrrolylmethyl)pyrrole may (Wolfreys and Hepburn, 2002). Neither the phytosterol also undergo cytochrome P450 (CYP450)-induced side chain mixture nor the phytosterol ester mixture induced oxidation to yield the corresponding alcohol that is expected chromosomal aberrations in vitro in human peripheral blood to be excreted as either the glucuronic acid or sulfate lymphocytes in either the presence or absence of S9 conjugate (Gillam et al., 2000; Ruangyuttikarn et al., 1992; metabolic activation (Evans and O’Riordan, 1975 as cited in Thornton-Manning et al., 1993). 1-Ethyl-2-(1- Wolfreys and Hepburn, 2002). The phytosterol ester mixture pyrrolylmethyl)pyrrole did not increase reverse mutations in S. was negative in a mouse lymphoma forward mutation assay typhimurium strains TA98, TA100, TA1535, and TA1537 and in L5178Y tk +/- mouse lymphoma cells in the absence and E. coli strain WP2uvrA up to 500 μg/plate either in the presence of S9 metabolic activation (Wolfreys and Hepburn, presence or absence of S9 metabolic activation (Komai, 2002). No clastogenic potential was observed in the bone 2017c). The structural relative 1H-pyrrole-2-carboxaldehyde marrow of male Crl:HanWist (Glx:BRL) rats when the did not increase reverse mutations in S. typhimurium strains phytosterol ester mixture was administered via oral gavage at TA98 or TA100 either in the presence or absence of S9 doses up to 2000 mg/kg bw (Wolfreys and Hepburn, 2002). metabolic activation when tested up to 9510 μg/plate (Lee et The Panel concluded that despite the different composition of al., 1994). Based on these results and also based on the the test materials relative to the composition of refined structure and the identity and arrangement of functional soybean oil extract, these results provided sufficient evidence groups therein, the Panel did not identify a specific concern to conclude that there was a lack of genotoxic potential for for genotoxic potential of 1-ethyl-2-(1-pyrrolylmethyl)pyrrole refined soybean oil extract. The phytosterol ester (PE) when used as a flavoring ingredient. mixture, 49.4% sitosterol, 27.9% campesterol, 18.5% stigmasterol, and others was evaluated in a two-generation The Panel reviewed the GRAS application and supporting daily dietary administration reproductive study in rats. information regarding rebaudioside D 95% (CAS 63279-13-0) Administration of up to 8.1% w/w phytosterol esters, and concluded that it is GRAS (FEMA 4921) (Smith et al., equivalent to a dose of 2.5-9.1 g PE/kg bw/day dependent on 2005a) for use as a flavor in the food categories and at the the period of the study, in the diet over two generations was use levels specified in the GRAS application (see Table 2). found to have no effects on reproduction of parental F0 and This substance was evaluated within the context of the F1 generation Wistar rats nor the development of the F1 and procedure for the safety evaluation of natural flavor F2 pups or in the sexual maturation of the F1 weanlings. complexes (Cohen et al., 2018; Smith et al., 2005b). This Therefore, 8.15% w/w phytosterol esters, equivalent to 2.5 g material was evaluated within the context of the procedure for PE/kg bw/day at the most conservative period of the study, the FEMA GRAS evaluation of flavor ingredients produced the NOAEL following daily administration for PE for two through biotechnology processes (Cohen et al., 2015).The successive generations (Waalkens-Berendsen et al., 1999). A Panel calculated the anticipated per capita intake (“eaters 13-week oral gavage administration study in male and female only”) of rebaudioside D 95% from use as a flavor ingredient Sprague-Dawley rats with a mixture of plant sterol esters, to be 111 µg/person/day, which is above the threshold of isolated from soybean and esterified with oleic acid to toxicological concern for structural class III (90 µg/person/day) increase solubility, was conducted at doses of 1000, 3000 and (Munro et al., 1996). This substance is reported to occur in 9000 mg/kg bw/day. The composition of the test material was Stevia rebaudiana leaves (Chaturvedula et al., 2011; Wood et 49.4% sitosterol, 27.9% campesterol, 18.5% stigmasterol, al., 1955). The Panel considered the identity description of the and other minor components. Suppressed body weight gains material to be adequate for FEMA GRAS evaluation. The in both sexes and cardiomyopathy with mononuclear cell Panel evaluated sensory data included within the application infiltration were observed in high dose males. No other and found it satisfactory with regard to intended conditions of adverse effects were noted and a NOAEL of 3000 mg/kg use for the flavoring ingredient (Harman and Hallagan, 2013). bw/day was determined for both sexes (Kim et al., 2002). This Metabolic data exist for representative members of each is 600,000,000 times the daily per capita intake of refined congeneric group that would predict, at the intake levels soybean oil extract from use as a flavor ingredient. proposed, metabolism by well-established detoxication pathways to innocuous products (Gardana et al., 2003; Geuns the results for the various steviol glycosides, the Panel did not et al., 2003a; Geuns et al., 2003b; Koyama et al., 2003a; identify specific concerns related to the potential genotoxicity Koyama et al., 2003b; Nikiforov et al., 2013; Purkayastha et of stevia extract stevioside, 70%. In a 108- week al., 2015; Purkayastha et al., 2016; Purkayastha et al., 2014; carcinogenicity study for stevioside, no carcinogenic effects Renwick and Tarka, 2008). The genotoxicity of the major were observed (Toyoda et al., 1997). In a 2-year feeding marker constituents (steviol glycosides) has been thoroughly study, male and female rats were administered the equivalent examined in a wide range of studies. While some positive of 0, 50, 150, or 550 mg/kg bw/day of a stevia extract results are reported in in vitro mutagenicity assays, in vivo comprised of 74% stevioside and 16% rebaudioside A. The studies do not provide evidence of genotoxic effects authors considered the NOAEL from this 2-year rat feeding (Nakajima, 2000b; Pezzuto et al., 1985; Pezzuto et al., 1986; study of a stevia extract to be equal to 550 mg/kg bw/day, or Rumelhard et al., 2016; Suttajit et al., 1993; Terai et al., 2002; approximately 89.5 mg/kg bw/day of rebaudioside A (Yamada Toyoda et al., 1997; Williams and Burdock, 2009). Based on et al., 1985), This is greater than 48,000 times the anticipated the results for the various steviol glycosides, the Panel did not daily per capita intake of rebaudioside M 95% from use as a identify specific concerns related to the potential genotoxicity flavoring ingredient. of stevia extract stevioside, 70%. In a 108- week carcinogenicity study for stevioside, no carcinogenic effects were observed (Toyoda et al., 1997). In a 2-year feeding The Panel reviewed the natural flavor complex GRAS study, male and female rats were administered the equivalent applications and supporting information regarding Buchu of 0, 50, 150, or 550 mg/kg bw/day of a stevia extract leaves extract (Barosma betulina Bartl. et Wendl., B. comprised of 74% stevioside and 16% rebaudioside A. The crenulata (L.) Hook, B. serratifolia Willd.), Peppermint oil authors considered the NOAEL from this 2-year rat feeding terpeneless ( piperita L.) and Spearmint oil study of a stevia extract to be equal to 550 mg/kg bw/day, or terpeneless (Mentha spicata L.) and concluded that they are approximately 89.5 mg/kg bw/day of rebaudioside A (Yamada GRAS for use as flavoring ingredients in the food categories et al., 1985), This is greater than 48,000 times the anticipated and at the use levels specified in the GRAS applications daily per capita intake of rebaudioside D 95% from use as a (FEMA 4923-4925, respectively) (see Table 2). These flavoring ingredient. materials were evaluated within the context of the revised procedure for the safety evaluation of natural flavor complexes (Smith et al., 2005b; Cohen et al., 2018). These The Panel reviewed the GRAS application and supporting natural flavor complexes are derived from commonly information regarding rebaudioside M 95% (CAS 1220616- consumed herbs. The Panel considered the identity 44-3) and concluded that it is GRAS (FEMA 4922) (Smith et descriptions of each material to be adequate for FEMA GRAS al., 2005a) for use as a flavor in the food categories and at the evaluation. These natural flavor complexes were evaluated use levels specified in the GRAS application (see Table 2). using a rigorous procedure that considers the chemical This substance was evaluated within the context of the composition, anticipated per capita intake, metabolic fate and procedure for the safety evaluation of natural flavor toxicity of the identified constituents and potential toxicity and complexes (Cohen et al., 2018; Smith et al., 2005b). This genotoxicity of unidentified constituents (Cohen et al., 2020). material was evaluated within the context of the procedure for the FEMA GRAS evaluation of flavor ingredients produced The Panel reviewed the GRAS application and supporting through biotechnology processes (Cohen et al., 2015). The information regarding (Z)-8-pentadecenal (CAS 65398-36-9) Panel calculated the anticipated per capita intake (“eaters and concluded that it is GRAS (FEMA 4926) (Smith et al., only”) of Rebaudioside M 95% from use as a flavor ingredient 2005a) for use as a flavor in the food categories and at the to be 111 µg/person/day, which is above the threshold of use levels specified in the GRAS application (see Table 2). toxicological concern for structural class III (90 µg/person/day) This substance was evaluated individually within the context (Munro et al., 1996). This substance is reported to occur in of the chemical group of linear and branched-chain aliphatic, Stevia rebaudiana leaves (Ohta et al., 2010). The Panel unsaturated, unconjugated alcohols, aldehydes, acids, and considered the identity description of the material to be related esters (JECFA, 1999, 2006, 2012; SLR, M1). The adequate for FEMA GRAS evaluation. The Panel evaluated Panel calculated the anticipated per capita intake (“eaters sensory data included within the application and found it only”) of (Z)-8-pentadecenal from use as a flavor ingredient to satisfactory with regard to intended conditions of use for the be 0.001 µg/person/day, which is above the threshold of flavoring ingredient (Harman and Hallagan, 2013). Metabolic toxicological concern for structural class I (1800 data exist for representative members of each congeneric µg/person/day) (Munro et al., 1996). The Panel considered group that would predict, at the intake levels proposed, the specification of the material to be adequately metabolism by well-established detoxication pathways to characterized by the purity assay and supporting spectral data innocuous products (Gardana et al., 2003; Geuns et al., provided for FEMA GRAS evaluation. (Z)-8-Pentadecenal is 2003a; Geuns et al., 2003b; Koyama et al., 2003a; Koyama anticipated to be efficiently oxidized to the corresponding et al., 2003b; Nikiforov et al., 2013; Purkayastha et al., 2015; unsaturated carboxylic acid before proceeding into the beta- Purkayastha et al., 2016; Purkayastha et al., 2014; Renwick oxidation cycle for complete metabolism to innocuous and Tarka, 2008). The genotoxicity of the major marker products (Beedham, 1988; Eckfeldt and Yonetani, 1982; constituents (steviol glycosides) has been thoroughly Feldman and Weiner, 1972; Nakayasu et al., 1978; Nelson examined in a wide range of studies. While some positive and Cox, 2008). (Z)-8-Pentadecenal was negative for results are reported in in vitro mutagenicity assays, in vivo mutagenicity in an Ames assay in S. typhimurium strains studies do not provide evidence of genotoxic effects TA100, TA1535, TA98, TA1537 and E. coli strain WP2uvrA in (Nakajima, 2000b; Pezzuto et al., 1985; Pezzuto et al., 1986; either the presence or absence of S9 metabolic activation by Rumelhard et al., 2016; Suttajit et al., 1993; Terai et al., 2002; the pre-incubation method up to 5000 μg/plate (Komai, Toyoda et al., 1997; Williams and Burdock, 2009). Based on 2018a).Based on the results for the substance, as well as on the structure of the substance and the identity and mg/kg bw/day, was assigned (Gaunt et al., 1969). In a 90-day arrangement of functional groups therein, the Panel did not dietary administration study in male and female Sprague- identify any specific concerns related to the genotoxic Dawley rats, the structurally related substance, 10-undecenal potential of (Z)-8-pentadecenal. In a 98-day study in male and (FEMA 3095), was administered at 14.3, 138.6, 382.3, and female SPF-derived CFE weanling rats, the structurally 1136 mg/kg bw/day. No toxicologically significant clinical related substance, cis-3-hexen-1-ol (FEMA 2563), was observations were detected. No macroscopic abnormalities were detected. Histopathology examination showed possible provided at 310, 1250 and 5000 ppm in drinking water. Test stomach irritation associated with the route of administration. article-related changes that were observed were increased Minimal centrilobular hepatocellular hypertrophy was relative kidney and adrenal weights in males administered observed in males at 138.6, 382.3, and 1136 mg/kg bw/day, 5000 ppm. A NOAEL of 1250 ppm, or approximately 120-150 but this was not accompanied by degenerative or mg/kg bw/day, was assigned (Gaunt et al., 1969). In a 90-day inflammatory changes. The NOAEL was concluded to be 14.3 dietary administration study in male and female Sprague- mg/kg bw/day (Liwska and Watson, 2012; Tsang et al., 2018). Dawley rats, the structurally related substance, 10-undecenal This is greater than 214,000 times the anticipated daily per (FEMA 3095), was administered at 14.3, 138.6, 382.3, and capita intake of 4,7-decadienal from use as a flavor ingredient. 1136 mg/kg bw/day. No toxicologically significant clinical observations or macroscopic abnormalities were detected. The Panel reviewed the GRAS application and supporting Histopathology analysis showed possible stomach irritation information regarding 2-methylthiophene (CAS 554-14-3) and associated with the route of administration. Minimal concluded that it is GRAS (FEMA 4928) (Smith et al., 2005a) centrilobular hepatocellular hypertrophy was observed in for use as a flavor in the food categories and at the use levels males at 138.6, 382.3, and 1136 mg/kg bw/day, but this was specified in the GRAS application (see Table 2). This not accompanied by degenerative or inflammatory changes. substance was evaluated individually within the context of the The NOAEL was identified to be 14.3 mg/kg bw/day (Liwska chemical group of sulfur-containing heterocyclic compounds and Watson, 2012; Tsang et al., 2018). This NOAEL is (Cohen et al., 2017b; JECFA, 2003, 2007, 2012, 2015; SLR, 858,000,000 times the anticipated daily per capita intake of D15). The Panel calculated the anticipated per capita intake (Z)-8-pentadecenal from use as a flavor ingredient. (“eaters only”) of 2-methylthiophene from use as a flavor ingredient to be 0.01 µg/person/day, which is below the The Panel reviewed the GRAS application and supporting threshold of toxicological concern for structural class II (540 information regarding 4,7-decadienal (CAS 934534-30-2) and µg/person/day) (Munro et al., 1996). The material is known to concluded that it is GRAS (FEMA 4927) (Smith et al., 2005a) occur in nature in beef (0.0072-0.23 ppm), clams, coffee, egg, for use as a flavor in the food categories and at the use levels papaya (0.05-0.1 ppm), pork, and shrimp. Based on the specified in the GRAS application (see Table 2). This quantitative concentration data that are available, a substance was evaluated individually within the context of the consumption ratio of 23,000 could be calculated (Njissen et chemical group of linear and branched-chain aliphatic, al., 2019; Stofberg and Grunschober, 1987). The Panel unsaturated, unconjugated alcohols, aldehydes, acids, and considered the specification of the material to be adequately related esters (JECFA, 1999, 2006, 2012; SLR, M1). The characterized by the purity assay and supporting spectral data Panel calculated the anticipated per capita intake (“eaters only”) of 4,7-decadienal from use as a flavor ingredient to be provided for FEMA GRAS evaluation. 2-Methylthiophene is 4 µg/person/day, which is below the threshold of toxicological predicted to undergo ring epoxidation and S-oxidation. The concern for structural class I (1800 µg/person/day) (Munro et subsequent intermediates are expected to conjugate with al., 1996). The substance has been reported to naturally occur glutathione and then be eliminated in the urine as the in Acorus calamus L. oil (Calamus oil) (Njissen, 2019). The mercapturic acid derivatives (Cohen et al., 2017a). In an Panel considered the specification of the material to be Ames assay using S. typhimurium strains TA98 and TA100, adequately characterized by the purity assay and supporting 2-methylthiophene did not increase the frequency of revertant spectral data provided for FEMA GRAS evaluation. 4,7- colonies in either the absence or presence of S9 metabolic Decadienal is anticipated to be efficiently oxidized to the activation (Komai, 2018b). Two additional Ames assays in S. corresponding unsaturated carboxylic acid before proceeding typhimurium strains TA98, TA100 and TA102 did not show into the beta-oxidation cycle for complete metabolism to signs of mutagenic activity either in the absence or presence innocuous products (Feldman and Weiner, 1972; Eckfeldt and of S9 metabolic activation, but only limited study details were Yonetani, 1982; Nakayasu et al., 1978; Beedham, 1988; provided (Aeschbacher et al., 1989; Lee et al., 1994). Based Nelson and Cox, 2008). An Ames assay was conducted for 4,7-decadienal in S. typhimurium strains TA1535, TA1537, on these results as well as the structure of the substance and TA98, TA100 and TA102, both with and without S9 metabolic the identity and arrangement of functional groups therein, the activation using both the plate incorporation and pre- Panel did not identify a specific concern for the genotoxic incubation methodologies. No substantial increase in potential of 2-methylthiophene. In an OECD 408 compliant revertant colony numbers of any of the five tested strains was 90-day dietary administration study in male and female observed at any dose level either in the presence or absence Sprague-Dawley rats, pentylthiophene (FEMA 4387) a of S9 metabolic activation (Sokolowski, 2009).). Based on the structurally related substance, was administered at mean results for the substance, as well as on the structure of the achieved dosages of either 1.4, 7, or 33 mg/kg bw/day in substance and the identity and arrangement of functional males and 1.5, 8 or 39 mg/kg bw/day in females. No groups therein, the Panel did not identify any specific significant differences were found in mortality, body weight, concerns related to the genotoxic potential of 4,7-decadienal. food consumption or food efficiency. The clinical and In a 98-day study in male and female SPF-derived CFE ophthalmological examinations did not reveal any effects, nor weanling rats, the structurally related substance, cis-3-hexen- 1-ol (FEMA 2563), was administered at doses of 310, 1250 did histopathological evaluation. Statistically significant and 5000 ppm via drinking water. The only non-transitory test changes were observed in hematology, coagulation, and article-related changes that were observed were increased urinalysis at the two highest dose levels, but these were relative kidney and adrenal weights in males administered mostly within the historical control ranges, and did not have 5000 ppm. A NOAEL of 1250 ppm, or approximately 120-150 accompanying histological findings. Therefore, these changes were not considered to be biologically relevant. number of micronucleated polychromatic erythrocytes in the Additionally, some high dose females were observed to have high dose group slightly exceeded the vehicle and negative increased relative kidney weight, but the change was not control values but was considered not to be biologically considered to be biologically relevant since no accompanying relevant by the authors as they were not statistically significant histological effects were identified. The NOAEL of 33 and 39 (Schulz, 2012). Based on the results for the substance and mg/kg bw/day were identified for male and female rats, the structurally related substance, and also based on the respectively (Bauter, 2013). The NOAEL of 33 mg/kg bw/day structure of the substance and the identity and arrangement was greater than 198,000,000 times the anticipated daily per of functional groups therein, the Panel did not identify a capita intake of 2-methylthiophene from use as a flavor specific concern for the genotoxic potential of 4-methylidene- ingredient. 2-(2-methylprop-1-enyl)oxane.

The Panel reviewed the GRAS application and supporting The Panel reviewed the GRAS application and supporting information regarding 4-methylidene-2-(2-methylprop-1- information regarding 4-isopropoxycinnamaldehyde (CAS enyl)oxane (CAS 60857-05-8) and concluded that it is GRAS 159017-89-7) and concluded that it is GRAS (FEMA 4930) (FEMA 4929) (Smith et al., 2005a) for use as a flavor in the (Smith et al., 2005a) for use as a flavor in the food categories food categories and at the use levels specified in the GRAS and at the use levels specified in the GRAS application (see application (see Table 2). This substance was evaluated Table 2). This substance was evaluated individually within the individually within the context of the chemical group of context of the chemical group of cinnamyl alcohol and related aliphatic and aromatic ethers (JECFA, 2004, 2012; SLR, C22, substances (JECFA, 2000, 2017; SLR, C11; SLR, C16). The D12). The Panel calculated the anticipated per capita intake Panel calculated the anticipated per capita intake (“eaters (“eaters only”) of 4-methylidene-2-(2-methylprop-1- only”) of 4-isopropoxycinnamaldehyde from use as a flavor enyl)oxane from use as a flavor ingredient to be 0.03 ingredient to be 15 µg/person/day, which is below the µg/person/day, which is below the threshold of toxicological threshold of toxicological concern for structural class I (1800 concern for structural class III (90 µg/person/day) (Munro et µg/person/day) (Munro et al., 1996). The Panel considered al., 1996). The Panel considered the specification of the the specification of the material to be adequately material to be adequately characterized by the purity assay characterized by the purity assay and supporting spectral data and supporting spectral data provided for FEMA GRAS provided for FEMA GRAS evaluation. 4- evaluation. 4-Methylidene-2-(2-methylprop-1-enyl)oxane is Isopropoxycinnamaldehyde is anticipated to be metabolized predicted to be hydroxylated on the methyl groups of the by oxidation to 4-isopropoxycinnamic acid. This would then be isopropenyl moiety. The corresponding metabolite will be converted to the corresponding coenzyme A derivative, which excreted either unchanged or after conjugation (Smith et al., would then undergo glycine conjugation or beta-oxidation, 2018). In an Ames assay in S. typhimurium strains TA1535, possibly leading to the formation of the 4-isopropoxybenzoyl TA1537, TA98, TA100, and E. coli WP2 uvrA, 4-methylidene- coenzyme A derivative. This is then either conjugated with 2-(2-methylprop-1-enyl)oxane did not increase the frequency glycine, yielding 4-isopropoxyhippuric acid, or hydrolyzed to of revertant colonies either in the absence or presence of S9 yield free 4-isopropoxybenzoic acid which is then excreted metabolic activation (Komai, 2018c). In an Ames assay in S. (Nutley et al., 1994; Samuelsen et al., 1986; Snapper et al., typhimurium strains TA1535, TA1537, TA98, TA100 and 1940; Solheim and Scheline, 1973, 1976). In an Ames assay TA102 the structurally related substance, tetrahydro-4- in S. typhimurium strains TA98 and TA100, 4- methyl-2-(2-methylpropen-1-yl)pyran (FEMA 3236), did not isopropoxycinnamaldehyde did not increase the frequency of increase the frequency of revertant mutations in the absence revertant colonies in either the absence or presence of S9 or presence of S9 metabolic activation (Verspeek-Rip, 2002). metabolic activation (Kawaguchi and Komai, 2016). In an In two separate additional Ames assays in S. typhimurium OECD 471 compliant Ames assay in S. typhimurium strains strains TA1535, TA97a, TA98, TA100 and TA102 the same TA1535, TA1537, TA100, TA98, and E. coli strain WP2uvrA, structural relative did not increase the frequency of revertant 4-isopropoxycinnamaldehyde did not increase the frequency colonies in the absence or presence of S9 metabolic of revertant colonies in either the absence or presence of S9 activation (Scheerbaum, 2001). In in vitro micronucleus tests metabolic activation (Yamaguchi, 2017). In an OECD 487 in Chinese hamster V79 cells the same structural relative did compliant in vitro micronucleus induction assay in TK6 cells, not induce statistically significant increases in the frequency 4-isopropoxycinnamaldehyde did not induce statistically of micronuclei in either the absence or presence of S9 significant increases in the frequency of micronuclei in the metabolic activation in any treatment arm or treatment presence of S9 metabolic activation in a short-term treatment concentration tested (Wollny, 2012). In two OECD 474 condition. A dose-dependent increase was observed without compliant in vivo micronucleus assays, structurally related metabolic activation in the short-term treatment condition, and substance, tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran a significant, dose-dependent increase was observed in the (FEMA 3236), was administered by gavage to male and continuous treatment condition in the absence of S9 female Crl:NMRI mice at concentrations of 250, 500 or 1000 metabolic activation (Fukuda, 2017). In an OECD 474 mg/kg bw. In the first experiment, high variability in compliant rat bone marrow micronucleus study in Crl:CD(SD) micronucleated polychromatic erythrocyte incidence was rats, 4-isopropoxycinnamaldehyde was administered by noted, yielding a weak, dose-related increase in the number gavage at doses of 250, 500, or 1000 mg/kg twice at 24-hour of polychromatic erythrocytes containing micronuclei. These intervals. The test substance did not increase the frequency increases slightly exceeded the historical control range in the of micronuclei relative to controls. Clinical observations in the mid and high-dose groups but were not statistically significant dose range finder at 2000 mg/kg included decreased when compared to the controls. The authors considered the spontaneous movement and bradypnea, providing evidence increase not to be biologically relevant. In the second that the substance could achieve systemic exposure (Ishii, experiment, a weak, dose-related increase in micronucleated 2017). On the basis of a weight-of-evidence approach, polychromatic erythrocyte incidence was observed. The including the negative results observed in the Ames assays conducted on the test substance and that the positive results bw/day of rebaudioside A (Yamada et al., 1985), which is observed in the in vitro micronucleus assay were not greater than 7,500 times the anticipated daily per capita intake confirmed in the in vivo study, the Panel concluded that it did of glucosylated steviol glycosides, 90% from use as a not identify a specific concern for the genotoxic potential of 4- flavoring ingredient. isopropoxycinnamaldehyde. In a 90-day dietary study in rats, the structurally related substance, o-methoxycinnamaldehyde The Panel reviewed the GRAS application and supporting (FEMA 3181), was administered at average doses of 47.1 information regarding Chaenomeles speciosa leaf extract mg/kg bw/day and 52.2 mg/kg bw/day for males and females, (CAS 2263901-84-2) and concluded that it is GRAS (FEMA respectively. There were no biologically relevant findings in 4932) (Smith et al., 2005a) for use as a flavor in the food the study (Posternak et al., 1969). In a 90-day dietary toxicity categories and at the use levels specified in the GRAS study, structural relative, o-methoxycinnamaldehyde (FEMA application (see Table 2). This material was evaluated within 3181) was administered at concentrations of 47.1 and 52.2 the context of the procedure for the safety evaluation of mg/kg bw/day in the feed. There were no significant natural flavor complexes (Cohen et al., 2018; Smith et al., differences in growth, body weights, food intake, hematology 2005b). The Panel calculated the anticipated per capita intake or clinical chemistry measurements. Organ weights or (“eaters only”) of Chaenomeles speciosa leaf extract from use pathology observations between test and control groups as a flavor ingredient was calculated to be 71 µg/person/day, There were no differences between the exposure groups and which is below the threshold of toxicological concern for controls The NOAEL of 47.1 mg/kg bw/day is greater than structural class III (90 µg/person/day) (Cramer et al., 1984; 188,000 time the anticipated daily per capita intake of 4- Munro et al., 1996). The Panel considered the identity isopropoxycinnamaldehyde from use as a flavor ingredient. description of the material to be adequate for FEMA GRAS evaluation. The Panel concluded that the principal The Panel reviewed the natural flavor complex GRAS components of Chaenomeles speciosa leaf extract were application and supporting information regarding glucosylated carbohydrate in nature, and that these were either steviol glycosides, 90% (CAS 57817-89-7) and concluded that endogenous or would be readily metabolized in the body to the substance is GRAS (FEMA 4931) (Smith et al., 2005a) for yield innocuous products that would be excreted or use as a flavor ingredient in the food categories and at the use incorporated into the biosynthesis of other biomolecules. The levels specified in the GRAS application (see Table 2). This Panel found the metabolism data adequate for the FEMA substance was evaluated within the context of the procedure GRAS evaluation. In an Ames assay in S. typhimurium strains for the safety evaluation of natural flavor complexes (Cohen TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 et al., 2018; Smith et al., 2005b). The Panel calculated the uvrA, Chaenomeles speciosa leaf extract did not increase the anticipated per capita intake (“eaters only”) of glucosylated frequency of revertant colonies in the presence or absence of steviol glycosides, 90% from use as a flavor ingredient to be S9 in an OECD guideline 471 compliant Ames assay using 713 µg/person/day, which is above the threshold of both the plate incorporation and pre-incubation toxicological concern for structural class III (90 µg/person/day) methodologies (Roy, 2019). In an OECD guideline 487 (Munro et al., 1996). The Panel considered the identity compliant in vitro micronucleus assay, no significant increase description of the material to be adequate for FEMA GRAS in micronuclei induction was observed when Chaenomeles evaluation. The Panel evaluated sensory data included within speciosa leaf extract was incubated with human lymphocytes the application and found it satisfactory with regard to at 156.25-5,000 μg/ml for 4 hours in the presence and intended conditions of use for the flavoring ingredient absence of S9 as well as for 24 hours win the absence of S9 (Harman and Hallagan, 2013). Metabolic data exist for (Vashi, 2019). Based on these results, the Panel did not representative members of each congeneric group that would identify a specific concern for the genotoxic potential of predict, at the intake levels proposed, metabolism by well- Chaenomeles speciosa leaf extract. established detoxication pathways to innocuous products (Gardana et al., 2003; Geuns et al., 2003a; Geuns et al., The Panel reviewed the GRAS application and supporting 2003b; Koyama et al., 2003a; Koyama et al., 2003b; Nikiforov information regarding Eriobotrya japonica leaves extract (CAS et al., 2013; Purkayastha et al., 2015; Purkayastha et al., 91770-19-3) and concluded that it is GRAS (FEMA 4933) 2016; Purkayastha et al., 2014; Renwick and Tarka, 2008). (Smith et al., 2005a) for use as a flavor in the food categories The genotoxicity of the major marker constituents (steviol and at the use levels specified in the GRAS application (see glycosides) has been thoroughly examined in a wide range of Table 2). This material was evaluated within the context of the studies. While some positive results are reported in in vitro procedure for the safety evaluation of natural flavor mutagenicity assays, in vivo studies do not provide evidence complexes (Cohen et al., 2018; Smith et al., 2005b). The of genotoxic effects (Nakajima, 2000b; Pezzuto et al., 1985; Panel calculated the anticipated per capita intake (“eaters Pezzuto et al., 1986; Rumelhard et al., 2016; Suttajit et al., only”) of Eriobotrya japonica leaves extract from use as a 1993; Terai et al., 2002; Toyoda et al., 1997; Williams and flavor ingredient was calculated to be 14 µg/person/day, Burdock, 2009). Based on the results for the various steviol which is below the threshold of toxicological concern for glycosides, the Panel did not identify specific concerns related structural class III (90 µg/person/day)(Munro et al., 1996). The to the potential genotoxicity of stevia extract stevioside, 70%. Panel considered the identity description of the material to be In a 108-week carcinogenicity study for stevioside, no adequate for FEMA GRAS evaluation. The Panel concluded carcinogenic effects were observed (Toyoda et al., 1997). In that the principal components of Eriobotrya japonica leaves a 2-year feeding study, male and female rats were extract would be readily metabolized in the body to yield administered the equivalent of 0, 50, 150, or 550 mg/kg innocuous products that would be excreted or incorporated bw/day of a stevia extract comprised of 74% stevioside and into the biosynthesis of other biomolecules. The Panel found 16% rebaudioside A. The authors considered the NOAEL the metabolism data adequate for the FEMA GRAS from this 2-year rat feeding study of a stevia extract to be evaluation. No increases in reverse mutations were observed equal to 550 mg/kg bw/day, or approximately 89.5 mg/kg when 5 concentrations of Eriobotrya japonica leaves extract ranging from 15.9 to 5000 ug/plate were tested in S. presence of S9 metabolic activation. Based on the results for typhimurium strains TA98, TA100, TA97a and TA1535 and E. the substance and the structurally related substance, and also coli WP2uvrA pKM101 in the absence and presence of S9 based on the structure of the substance and the identity and (Swartz, 2018). In an in vivo micronucleus assay in male and arrangement of functional groups therein, the Panel did not female Balb/c mice, gavage administration of doses up to identify a specific concern for the genotoxic potential of 10,000 mg/kg bw/day of an ethanolic Eriobotrya japonica sodium gluconate. A 28-day gavage study of sodium leaves extract produced no statistically significant increases gluconate provided in the feed of male and female beagle in the frequency of micronuclei (Jin and Dong, 2017). Based dogs (4/sex/group) resulted in a NOAEL of 500 mg/kg bw/day on these results, the Panel did not identify a specific concern (Okamoto, 1995). In a 28-day dietary study, Crj:CD(SD) for the genotoxic potential of Eriobotrya japonica leaves Sprague-Dawley SPF rats were administered concentrations extract. In a 28-day toxicity study in male and female ICR mice equal to 0, 1.25, 2.5, or 5% w/w sodium gluconate, or 0, 1000, were administered an Eriobotrya japonica leaves extract 2000, and 4100 mg/kg bw/day (Mochizuki, 1997). The authors analogous to the test material at doses of 150, 300, or 600 concluded that the NOEL was 4100 mg/kg bw per day. In a mg/kg bw/day via gavage administration. The highest dose repeat dose 28-day toxicity study, male and female Crj:CD resulted in a statistically significant reduction in body weight (SD) rats (12/sex/group) were administered sodium gluconate in the high dose males. High dose males and females showed by gavage at doses of 500, 1000 and 2000 mg/kg bw/day statistically significant changes in several clinical biochemistry (Mochizuki, 1995). No toxicologically significant changes were parameters, but without corresponding histopathology. The observed at any dose level. Subsequent histopathological author of the study determined that the changes in clinical examinations revealed a thickening of the stomach lining in biochemistry parameters were not considered biologically male rats at the highest dose level. The Panel agreed with relevant. Based on weight reductions and biochemical the author’s conclusion that this was not of toxicological changes at the highest dose, the Panel determined a NOAEL concern to humans. The NOAEL was concluded by the Panel of 300 mg/kg bw/day. This is greater than 1,280,000 times the to be the top dose of 4100 mg/kg bw/day for both male and daily per capita intake of Eriobotrya japonica leaves extract female rats, which is greater than 229,000 times the daily per from use as a flavor ingredient (Li et al., 2017). capita intake of sodium gluconate from use as a flavor ingredient. The Panel reviewed the GRAS application and supporting information regarding sodium gluconate (CAS 527-07-1) and concluded that it is GRAS (FEMA 4934) (Smith et al., 2005a) The Panel reviewed the GRAS application and supporting for use as a flavor in the food categories and at the use levels information regarding 3-methylbutane-1,3-dithiol (CAS specified in the GRAS application (see Table 2). This material 98139-71-0) and concluded that it is GRAS (FEMA 4935) was evaluated within the context of the procedure for the (Smith et al., 2005a) for use as a flavor in the food categories FEMA GRAS evaluation of flavor ingredients produced and at the use levels specified in the GRAS application (see through biotechnology processes (Cohen et al., 2015). This Table 2). This substance was evaluated individually within the substance was evaluated individually within the context of the context of the chemical group of simple aliphatic and aromatic chemical group of aliphatic poly-hydroxy compounds and sulfides and thiols (JECFA, 2000, 2004, 2008, 2011; SLR, derivatives (JECFA, 2001; SLR, B1F). The Panel calculated D1). The Panel calculated the anticipated per capita intake the anticipated per capita intake (“eaters only”) of sodium (“eaters only”) of 3-methylbutane-1,3-dithiol from use as a gluconate from use as a flavor ingredient to be 1070 flavor ingredient to be 0.01 µg/person/day, which is below the µg/person/day, which is below the threshold of toxicological threshold of toxicological concern for structural class III (90 concern for structural class I (1800 µg/person/day) (Munro et µg/person/day) (Munro et al., 1996). The Panel considered al., 1996). The substance occurs naturally in honey the specification of the material to be adequately (Ramachandran et al., 2006). The Panel considered the characterized by the purity assay and supporting spectral data specification of the material to be adequately characterized by provided for FEMA GRAS evaluation. 3-Methylbutane-1,3- the purity assay and supporting spectral data provided for dithiol is expected to be methylated and undergo enzymatic FEMA GRAS evaluation. The Panel evaluated sensory data oxidation to yield the corresponding sulfoxide or sulfone, included within the application and found it satisfactory with followed by excretion. Alternatively, the thiols may react with regard to intended conditions of use for the flavoring glutathione to form mixed disulfides that can then undergo ingredient (Harman and Hallagan, 2013). Sodium gluconate reduction and oxidative desulfuration, or oxidation to sulfonic is predicted to be excreted unchanged in the urine and any acid via the thiosulfinate and sulfinic acid intermediates sodium gluconate absorbed in the gastrointestinal tract is (Smith et al., 2018). Negative results were obtained in Ames expected to be metabolized to innocuous products. d- assays for 3-methylbutane-1,3-dithiol in S. typhimurium TA98 Gluconic acid is an endogenously occurring metabolite of and TA100 with and without metabolic activation (Komai, glycolysis (Smith et al., 2018). In an Ames assay in S. 2018d). In an Ames assay of structurally related substance, typhimurium strains TA1535, TA1537, and TA1538 and 1,2-ethanedithiol (FEMA 3484), conducted in S. typhimurium Saccharomyces cerevisiae (strain D4), sodium gluconate did strains TA98, TA100, TA1535 and TA1537, no increase in the not increase the frequency of revertant colonies either in the frequency of revertant colonies was observed in the presence absence or presence of S9 metabolic activation (Brusick, and absence of S9 metabolic activation at up to 5000 μg/plate 1975). Sodium gluconate tested negative for genotoxicity in using the plate incorporation method both single dose and repeated oral administration (4 days) (PhillipsPetroleumCompany, 1990). In the mouse lymphoma chromosomal aberration assays in mouse bone marrow cells forward mutation assay conducted in L5178Y TK +/- mouse (Yamashita, 1974). In an Ames assay in S. typhimurium lymphoma cells, the same structurally related substance was strains TA97, TA98, TA100, and TA104 the structurally concluded by the study author to be mutagenic due to an related substance, malic acid (FEMA 2655), did not increase increase in the induction of forward mutations in the absence the frequency of revertant colonies in either the absence or of S9. No increase in forward mutations was observed in the presence of S9 metabolic activation (Pence, 1982). The Panel Toyoda et al., 1997; Williams and Burdock, 2009). Based on concluded that though the study was valid, the rates of the results for the various steviol glycosides, the Panel did not forward mutation reported in the absence of S9 metabolic identify specific concerns related to the potential genotoxicity activation were within the global historical controls and of stevia extract stevioside, 70%. In a 108- week therefore 1,2-ethanedithiol (FEMA 3484) is of no genotoxic carcinogenicity study for stevioside, no carcinogenic effects concern (Moore et al., 2003; Moore et al., 2006). The same were observed (Toyoda et al., 1997). In a 2-year feeding structurally related substance was considered by the study study, male and female rats were administered the equivalent author to be mutagenic in a sister chromatid exchange assay of 0, 50, 150, or 550 mg/kg bw/day of a stevia extract conducted in Chinese hamster ovary cells due to a positive comprised of 74% stevioside and 16% rebaudioside A. The response observed at the two highest concentrations of 16 authors considered the NOAEL from this 2-year rat feeding and 50 μg/ml without metabolic activation and at study of a stevia extract to be equal to 550 mg/kg bw/day, or concentrations greater than 1.6 μg/ml with metabolic approximately 89.5 mg/kg bw/day of rebaudioside A (Yamada activation (Pence et al., 1983). However, the Panel noted that et al., 1985). This is greater than 64,000 times the anticipated this study is of questionable relevance since the OECD daily per capita intake of rebaudioside E ≥85% from use as a withdrew its guideline for this assay (OECD, 2014). Based on flavoring ingredient. the negative Ames tests, and on the structure of the substance and the identity and arrangement of functional groups therein, the Panel did not identify a concern for the The Panel reviewed the GRAS application and supporting genotoxic potential of 3-methylbutane-1,3-dithiol. No information regarding rebaudioside I 95% (CAS 1220616-34- hematological, biochemical and urinary adverse effects were 1) and concluded that it is GRAS (FEMA 4937) (Smith et al., observed when a mean daily intake of 0.703 mg/kg bw/day of 2005a) for use as a flavor in the food categories and at the the structurally related substance, 2,3-butanedithiol (FEMA use levels specified in the GRAS application (see Table 2). 3477), was provided in the diet to male and female Sprague- This substance was evaluated within the context of the Dawley rats for 90 days (Cox et al., 1974a). The NOAEL of procedure for the safety evaluation of natural flavor 0.703 mg/kg bw/day established for the structurally related complexes (Cohen et al., 2018; Smith et al., 2005b). This substance (Cox et al., 1974a) is 4,200,000 times the material was evaluated within the context of the procedure for anticipated daily per capita intake of 3-methylbutane-1,3- the FEMA GRAS evaluation of flavor ingredients produced dithiol from use as a flavoring ingredient. through biotechnology processes (Cohen et al., 2015). The Panel calculated the anticipated per capita intake (“eaters The Panel reviewed the GRAS application and supporting only”) of rebaudioside I 95% from use as a flavor ingredient to information regarding rebaudioside E ≥85% (CAS 63279-14- be 83 µg/person/day, which is below the threshold of 1) and concluded that it is GRAS (FEMA 4936) (Smith et al., toxicological concern for structural class III (90 µg/person/day) 2005a) for use as a flavor in the food categories and at the (Munro et al., 1996). This substance is reported to occur in use levels specified in the GRAS application (see Table 2). Stevia rebaudiana leaves (Prakash et al., 2014). The Panel This substance was evaluated within the context of the considered the identity description of the material to be procedure for the safety evaluation of natural flavor adequate for FEMA GRAS evaluation. The Panel evaluated complexes (Cohen et al., 2018; Smith et al., 2005b). This sensory data included within the application and found it material was evaluated within the context of the procedure for satisfactory with regard to intended conditions of use for the the FEMA GRAS evaluation of flavor ingredients produced flavoring ingredient (Harman and Hallagan, 2013).Metabolic through biotechnology processes (Cohen et al., 2015). The data exist for representative members of each congeneric Panel calculated the anticipated per capita intake (“eaters group that would predict, at the intake levels proposed, only”) of rebaudioside E ≥85% from use as a flavor ingredient metabolism by well-established detoxication pathways to to be 83 µg/person/day, which is below the threshold of innocuous products (Gardana et al., 2003; Geuns et al., toxicological concern for structural class III (90 µg/person/day) 2003a; Geuns et al., 2003b; Koyama et al., 2003a; Koyama (Munro et al., 1996). This substance is reported to occur in et al., 2003b; Nikiforov et al., 2013; Purkayastha et al., 2015; Stevia rebaudiana leaves (Ibrahim et al., 2014). The Panel Purkayastha et al., 2016; Purkayastha et al., 2014; Renwick considered the identity description of the material to be and Tarka, 2008). The genotoxicity of the major marker adequate for FEMA GRAS evaluation. The Panel evaluated constituents (steviol glycosides) has been thoroughly sensory data included within the application and found it examined in a wide range of studies. While some positive satisfactory with regard to intended conditions of use for the results are reported in in vitro mutagenicity assays, in vivo flavoring ingredient (Harman and Hallagan, 2013). Metabolic studies do not provide evidence of genotoxic effects data exist for representative members of each congeneric (Nakajima, 2000b; Pezzuto et al., 1985; Pezzuto et al., 1986; group that would predict, at the intake levels proposed, Rumelhard et al., 2016; Suttajit et al., 1993; Terai et al., 2002; metabolism by well-established detoxication pathways to Toyoda et al., 1997; Williams and Burdock, 2009). Based on innocuous products (Gardana et al., 2003; Geuns et al., the results for the various steviol glycosides, the Panel did not 2003a; Geuns et al., 2003b; Koyama et al., 2003a; Koyama identify specific concerns related to the potential genotoxicity et al., 2003b; Nikiforov et al., 2013; Purkayastha et al., 2015; of stevia extract stevioside, 70%. In a 108- week Purkayastha et al., 2016; Purkayastha et al., 2014; Renwick carcinogenicity study for stevioside, no carcinogenic effects and Tarka, 2008). The genotoxicity of the major marker were observed (Toyoda et al., 1997). In a 2-year feeding constituents (steviol glycosides) has been thoroughly study, male and female rats were administered the equivalent examined in a wide range of studies. While some positive of 0, 50, 150, or 550 mg/kg bw/day of a stevia extract results are reported in in vitro mutagenicity assays, in vivo comprised of 74% stevioside and 16% rebaudioside A. The studies do not provide evidence of genotoxic effects authors considered the NOAEL from this 2-year rat feeding (Nakajima, 2000b; Pezzuto et al., 1985; Pezzuto et al., 1986; study of a stevia extract to be equal to 550 mg/kg bw/day, or Rumelhard et al., 2016; Suttajit et al., 1993; Terai et al., 2002; approximately 89.5 mg/kg bw/day of rebaudioside A (Yamada et al., 1985). This is greater than 64,000 times the anticipated characterized by the purity assay and supporting spectral data daily per capita intake of rebaudioside E ≥85% from use as a provided for FEMA GRAS evaluation. S-methyl 5-(1- flavoring ingredient. ethoxyethoxy) decanethioate is expected to undergo hydrolysis of the acetal or thioester groups to yield ethanol, acetaldehyde, an aliphatic 5-hydroxycarboxylic acid and The Panel reviewed the GRAS application and supporting methanethiol. Hydrolysis of the acetal group may result in information regarding S-methyl 5-(1-ethoxyethoxy) ethanol, acetaldehyde and 5-hydroxy methyl thioester and tetradecanethioate (CAS 2180135-08-2) and concluded that hydrolysis of the thioester group may result in acetal it is GRAS (FEMA 4938) (Smith et al., 2005a) for use as a carboxylic acid and methanethiol. Hydroxycarboxylic acid and flavor in the food categories and at the use levels specified in their ester and acetal derivatives are readily hydrolyzed, the GRAS application (see Table 2). This substance was absorbed and fully metabolized through the fatty acid β- evaluated individually within the context of the chemical group oxidation pathway followed by excretion. Methanethiol is of simple aliphatic and aromatic sulfides and thiols (JECFA, expected to undergo methylation and enzymatic oxidation to 2000, 2004, 2008, 2011; SLR, D1). The Panel calculated the yield the corresponding sulfoxide by sulfone, followed by anticipated per capita intake (“eaters only”) of S-methyl 5-(1- excretion. Methanethiol may also react with glutathione to ethoxyethoxy)tetradecanethioate from use as a flavor form mixed disulfides that can undergo reduction and ingredient to be 3 µg/person/day, which is below the threshold oxidative desulfuration, or oxidation to sulfonic acid via of toxicological concern for structural class I (1800 thiosulfinate and sulfinic acid intermediates. Additionally, µg/person/day) (Munro et al., 1996). The Panel considered methanethiol may be enzymatically oxidized to yield the the specification of the material to be adequately corresponding sulfenic acid, sulfinic acid and ultimately, characterized by the purity assay and supporting spectral data sulfonic acid. Sulfinic and sulfonic acids are water soluble and provided for FEMA GRAS evaluation. S-methyl 5-(1- readily excreted (Bosron and Ting-Kai, 1980; Morgareidge, ethoxyethoxy)tetradecanethioate is expected to undergo 1962; Williams, 1959). S-Methyl 5-(1-ethoxyethoxy) hydrolysis of the acetal or thioester to yield ethanol, decanethioate tested negative for mutagenicity in an Ames acetaldehyde, an aliphatic 5-hydroxycarboxylic acid and assay conducted in S. typhimurium TA98 and TA100 in the methanethiol. Hydrolysis of the acetal group may result in presence and absence of S9 metabolic activation at ethanol, acetaldehyde and 5-hydroxy methyl thioester and concentrations up to 5000 μg/plate using the pre-incubation hydrolysis of the thioester group may result in acetal method (Komai, 2018e). Based on these results as well as on carboxylic acid and methanethiol. Hydroxycarboxylic acid and the structure of the substance and the identity and their ester and acetal derivatives are readily hydrolyzed, arrangement of functional groups therein, the Panel did not absorbed and fully metabolized through the fatty acid β- identify a concern for the genotoxic potential of S-methyl 5-(1- oxidation pathway followed by excretion. Methanethiol is ethoxyethoxy)decanethioate. expected to undergo methylation and enzymatic oxidation to yield the corresponding sulfoxide by sulfone, followed by The Panel reviewed the GRAS application and supporting excretion. Methanethiol may also react with glutathione to information regarding β-bisabolene ≥88% (CAS 495-61-4) form mixed disulfides that can undergo reduction and and concluded that it is GRAS (FEMA 4940) (Smith et al., oxidative desulfuration, or oxidation to sulfonic acid via 2005a) for use as a flavor in the food categories and at the thiosulfinate and sulfinic acid intermediates. Additionally, use levels specified in the GRAS application (see Table 2). methanethiol may be enzymatically oxidized to yield the This material was evaluated within the context of the corresponding sulfenic acid, sulfinic acid and ultimately, procedure for the safety evaluation of natural flavor sulfonic acid. Sulfinic and sulfonic acids are water soluble and complexes (Cohen et al., 2018; Smith et al., 2005). This readily excreted (Bosron and Ting-Kai, 1980; Morgareidge, material was evaluated within the context of the procedure for 1962; Williams, 1959). S-Methyl 5-(1- the FEMA GRAS evaluation of flavor ingredients produced ethoxyethoxy)tetradecanethioate tested negative for through biotechnology processes (Cohen et al., 2015). The mutagenicity in an Ames assay conducted in S. typhimurium Panel calculated the anticipated per capita intake (“eaters TA98 and TA100 in the presence and absence of S9 only”) of β-bisabolene ≥88% from use as a flavor ingredient to metabolic activation at concentrations up to 5000 μg/plate be 277 µg/person/day, which is below the threshold of using the pre-incubation method (Komai, 2018e). Based on toxicological concern for structural class I (1800 these results as well as on the structure of the substance and µg/person/day) (Munro et al., 1996).). The Panel considered the identity and arrangement of functional groups therein, the the identity description of the material to be adequate for Panel did not identify a concern for the genotoxic potential of FEMA GRAS evaluation. The major marker constituent, β- S-methyl 5-(1-ethoxyethoxy)tetradecanethioate. bisabolene, is expected to undergo oxidative metabolism in a manner similar to limonene, either by side chain oxidation or The Panel reviewed the GRAS application and supporting epoxidation of the alkene groups. Alternatively, alkene groups information regarding S-methyl 5-(1- could undergo epoxidation followed by hydrolysis and ethoxyethoxy)decanethioate (CAS 2180135-09-3) and formation of glucuronic acid or sulfate conjugates, leading to concluded that it is GRAS (FEMA 4939) (Smith et al., 2005a) rapid elimination in the urine (Smith et al., 2018). The for use as a flavor in the food categories and at the use levels structurally-related substance, d-limonene (FEMA 2633), specified in the GRAS application (see Table 2). The Panel gave no evidence of mutagenicity in an Ames assay in S. calculated the anticipated per capita intake (“eaters only”) of typhimurium strains TA98, TA100, TA102, TA1535, TA1537, S-methyl 5-(1-ethoxyethoxy)decanethioate from use as a TA1538, UTH8413, and UTH8414 either in the presence or flavor ingredient to be 0.3 µg/person/day, which is below the absence of S9 metabolic activation (Connor et al., 1985; threshold of toxicological concern for structural class I (1800 Florin et al., 1980b; Haworth et al., 1983; Heck et al., 1989; µg/person/day) (Munro et al., 1996). The Panel considered Müller et al., 1993). d-Limonene was also negative in a the specification of the material to be adequately chromosomal aberration study in Chinese hamster ovary (CHO) cells at up to 500 µg/ml and a mouse lymphoma bw/day when interperitoneally injected in male NMRI mice forward mutation assay in L5178Y cells up to a maximum (Donath, 2017). Based on these results as well as on the concentration of 100 µg/ml, either in the presence or absence composition of the substance and the identity and of S9 metabolic activation (Anderson et al., 1990; Heck et al., arrangement of functional groups therein, the Panel did not 1989; Myhr et al., 1990). In an OECD 471 compliant Ames identify a specific concern for the genotoxic potential assay using S. typhimurium strains TA98, TA100, TA102, of nootkatone complex. A 28-day gavage study of major TA1535, and TA1537, the structurally related substance, marker substance, nootkatone (FEMA 3166), in male and bisabolene (a mixture of isomers) (FEMA 3331), did not female Sprague-Dawley Crl:CD (SD) IGS BR rats resulted in increase the frequency of revertant colonies in either the observations of α2u-globulin nephropathy, an effect absence or presence of S9 metabolic activation using both the determined to be specific to male rats and therefore not plate incorporation and pre-incubation methodology (OECD, relevant to human exposure.A NOAEL of 10 mg/kg bw/day 1997; Poth, 2002). Based on these results as well as on the was determined (Jones et al., 2004). This is greater than 8600 structure of the substance and the identity and arrangement times the anticipated daily per capita intake of nootkatone of functional groups therein, the Panel did not identify a complex from use as a flavoring ingredient. specific concern for the genotoxic potential of β- bisabolene ≥88%. A 90-day gavage study of d-limonene (FEMA 2633) in male and female F344/N rats (10/sex/dose) The Panel reviewed the GRAS application and supporting resulted in a NOAEL of 215 mg/kg bw/day for female rats information regarding modified guaiac wood extract (CAS (NTP, 1990) which is 46,000, times the anticipated daily per 2247239-04-7) and concluded that it is GRAS (FEMA 4942) capita intake of β-bisabolene ≥88% from use as a flavoring (Smith et al., 2005a) for use as a flavor in the food categories ingredient. and at the use levels specified in the GRAS application (see Table 2). This material was evaluated within the context of the procedure for the safety evaluation of natural flavor The Panel reviewed the GRAS application and supporting complexes (Cohen et al., 2018; Smith et al., 2005). The Panel information regarding nootkatone complex (CAS 4674-50-4) calculated the anticipated per capita intake (“eaters only”) of and concluded that it is GRAS (FEMA 4941) (Smith et al., modified guaiac wood extract from use as a flavor ingredient 2005a) for use as a flavor in the food categories and at the to be 1 µg/person/day, which is below the threshold of use levels specified in the GRAS application (see Table 2). toxicological concern for structural class III (90 µg/person/day) This material was evaluated within the context of the (Munro et al., 1996). The Panel considered the identity procedure for the safety evaluation of natural flavor description of the material to be adequate for FEMA GRAS complexes (Cohen et al., 2018; Smith et al., 2005b). This evaluation. Metabolic data of representative members of the material was evaluated within the context of the procedure for principal congeneric groups, as well as anticipated levels of the FEMA GRAS evaluation of flavor ingredients produced intake of this substance, indicate that constituents in these through biotechnology processes (Cohen et al., 2015). The congeneric groups would be metabolized to innocuous Panel calculated the anticipated per capita intake (“eaters products based on well-established metabolic pathways only”) of nootkatone complex from use as a flavor ingredient (Smith et al., 2018). In an OECD 471 compliant Ames assay to be 69 µg/person/day, which is below the threshold of of modified guaiac wood extract conducted in S. typhimurium toxicological concern for structural class III (90 µg/person/day) strains TA98, TA100, TA1535, and TA1537 as well as E. coli (Munro et al., 1996). A consumption ratio of 11 could be strain WP2uvrA, no evidence of mutagenicity was observed calculated from the quantitative data, indicating that the intake in the presence and absence of S9 metabolic activation at of nootkatone is predominantly from natural occurrence in doses up to 3160 μg/plate using both the plate incorporation foodstuffs (Njissen, 2019). The Panel considered the identity and pre-incubation methodologies (Spruth, 2017). No description of the material to be adequate for FEMA GRAS induction of micronuclei relative to controls was observed in evaluation. Nootkatone complex is expected to undergo an in vitro micronucleus study for modified guaiac wood metabolism in a manner similar to other lipophilic ketones or extract when incubated in human lymphocytes for 4 hours in those with sterically hindered functional groups, through the presence and absence of S9 at doses up to 156 μg/ml or oxidation at a ring position followed by conjugation and for 24 hours in the absence of S9 at doses up to 114 μg/ml excretion in the urine. Alternatively, reduction of the functional (Naumann, 2018). An Ames assay for triethyl citrate (FEMA ketone group could occur followed by conjugation and 3083), a major marker constituent of modified guaiac wood excretion in the urine (Smith et al., 2018). In an Ames assay extract, yielded negative results in the presence and absence for major marker constituent, nootkatone (FEMA 3166), of S9 at doses up to 5000 μg/plate (Bhalli, 2015a). No conducted in S. typhimurium strains TA98, TA100, TA102, genotoxicity was observed in an in vitro micronucleus assay TA1535, and TA1537, no increase in the frequency of for triethyl citrate (FEMA 3083) incubated in human peripheral revertant colonies was observed in either the presence or blood lymphocytes (Bhalli, 2015b). Based on these results as absence of S9 (Marzin, 1998). An in vitro micronucleus assay well as on the identity of the major marker constituents and for the same major marker substance resulted in no the identity and arrangement of functional groups therein, the statistically significant effects on micronuclei frequency in the Panel did not identify a specific concern for the genotoxic presence and absence of S9 (Stone, 2011). In a liquid potential of modified guaiac wood extract. microplate Ames assay, up to 160 μg/ml of nootkatone complex did not increase the frequency of revertant colonies when tested in S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli combined strains WP2(pKMN101) and WP2uvrA (Ziemianska, 2017). In an in vivo micronucleus assay, nootkatone complex did not increase the frequency of micronucleated bone marrow cells at up to 1000 mg/kg report provided to the FEMA Expert Panel by the International References Organization of the Flavor Industry.

Adams, T.B., Greer, D.B., Doull, J., Munro, I.C., Newberne, Beedham, C., 1988. Molybdenum Hydroxylases, Enzyme P., Portoghese, P.S., Smith, R.L., Wagner, B.M., Weil, C.S., Systems that Metabolize Drugs and Other Xenobiotics. John Woods, L.A., Ford, R.A., 1998. The FEMA GRAS Assessment Wiley & Sons, Ltd, pp. 147-187. of Lactones Used as Flavour Ingredients. Food and Chemical Toxicology 36, 249-278. Beevers, C., 2014. 3,4-Dimethyl-5-pentylidenefuran-2(5H)- one: Combined Comet assay in the liver and a bone marrow Adams, T.B., McGowen, M.M., Williams, M.C., Cohen, S.M., micronucleus test in treated rats.Covance Laboratories Ltd., Feron, V.J., Goodman, J.I., Marnett, L.J., Munro, I.C., North Yorkshire, England. Unpublished report provided to the Portoghese, P.S., Smith, R.L., Waddell, W.J., 2007. The FEMA Expert Panel by the International Organization of the FEMA GRAS assessment of aromatic substituted secondary Flavor Industry. alcohols, ketones, and related esters used as flavor ingredients. Food and Chemical Toxicology 45, 171-201. Benvenga, S., Ruggeri, R.M., Russo, A., Lapa, D., Campenni, A., Trimarchi, F., 2001. Usefulness of L-carnitine, a naturally Aeschbacher, H.U., Wolleb, U., Löliger, J., Spadone, J.C., occurring peripheral antagonist of thyroid hormone action, in Liardon, R., 1989. Contribution of Coffee Aroma Constituents iatrogenic hyperthyroidism: a randomized, double-blind, to the Mutagenicity of Coffee. Food and Chemical Toxicology placebo-controlled clinical trial. J Clin Endocrinol Metab 86, 27, 227-232. 3579-3594.

Amano, H., Kazamori, D., Itoh, K., Kodera, Y., 2015. Bhalli, J., 2014a. cis-3-hexenol (CAS # 928-96-1): Bacterial Metabolism, excretion, and pharmacokinetics of S-allyl-L- reverse mutation assay: plate incorporation method with a cysteine in rats and dogs. Drug Metab Dispos 43, 749-755. confirmatory assay, Covance Laboratories Inc., Greenfield, IN, USA. Unpublished report to the FEMA Expert Panel. Anderson, B.E., Zeiger, E., Shelby, M.D., Resnick, M.A., Provided by the Research Institute for Fragrance Materials. Gulati, D.K., Ivett, J.L., Loveday, K.S., 1990. Chromosome aberration and sister chromatid exchange test results with 42 Bhalli, J., 2014b. cis-3-hexenol (CAS # 928-96-1): In vitro chemicals. Environmental and Molecular Mutagenesis 16, 55- micronucleus assay in human peripheral blood lymphocytes, 137. Covance Laboratories Inc., Greenfield, IN, USA. Unpublished report to the FEMA Expert Panel. Provided by the Research Aoyama, N., 2017. Micronucleus test of CA-047 ITO in mice, Institute for Fragrance Materials. UBE Scientific Analysis Laboratory, Inc., Yamaguchi Prefecture, Japan. Unpublished report provided to the FEMA Bhalli, J., 2015a. Triethyl citrate (CAS # 77-93-0): Bacterial Expert Panel. Reverse Mutation Assay: Plate Incorporation Method with a Confirmatory Assay. Covance Laboratories, Greenfield, Api, A., Smith, R.L., Pipino, S., Marczylo, T., de Matteis, F., Indiana, USA., Unpublished report to the FEMA Expert Panel. 2004. Evaluation of the oral subchronic toxicity of AHTN (7- Provided by the Research Institute for Fragrance Materials. Acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4- tetrahydronaphthalene) in the rat. Food and Chemical Bhalli, J., 2015b. Triethyl citrate (CAS # 77-93-0): In Vitro Toxicology 42, 791-801. Micronucleus Assay in Human Peripheral Blood Lymphocytes. Covance Laboratories, Greenfield, Indiana, Api, A.M., Ritacco, G., Sipes, I.G., 2013. Disposition and USA., Unpublished report to the FEMA Expert Panel. excretion of 14C-AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl- Provided by the Research Institute for Fragrance Materials. 1,2,3,4-tetrahydronaphthalene) and 14c-hhcb (1,3,4,6,7,8- hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-gamma-2- Bhatia, S., Politani, V.T., Api, A., 2010. Genotoxicity Tests benzopyran) after intravenous administration to Sprague- Conducted on a Group of Structurally Related Aldehydes, Dawley rats and domestic pigs. International journal of Unpublished report to the FEMA Expert Panel. Provided by toxicology 32, 288-295. the Research Institute for Fragrance Materials.

Api, A.M., San, R.H.C., 1999. Genotoxicity tests with 6-acetyl- Borgström, B., 1974. Fat Digestion and Absorption, Intestinal 1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro- Absorption. Springer Science + Business Media, pp. 555-620. 4,6,6,7,8,8-hexamethylcyclopenta-y-2-benzopyran. Mutation research 446, 67-81. Bosron, W.F., Ting-Kai, L., 1980. Alcohol dehydrogenase. , in: W.B. Jacoby (Ed.), Ezymatic Basis of Detoxification. Asquith, J.C., 1990a. Bacterial reverse mutation assay ST Academic Press, New York, pp. 231-248. 14C 89, Toxicol Laboratories Limited, Herefordshire, England. Unpublished report provided to the FEMA Expert Panel. Bowen, R., 2011. Reverse mutation in five histidine-requiring strains of Salmonella typhimurium: 3,4-Dimethyl-5- Asquith, J.C., 1990b. Bacterial reverse mutation assay ST pentylidenefuran-2(5H)-one, Study no. 8233097 ed. 13C 89. Toxicol Laboratories Limited, Herefordshire, England. Unpublished report provided to the FEMA Expert Panel, Unpublished report provided to the FEMA Expert Panel. Covance Laboratories Ltd., North Yorkshire, England.

Bauter, M., 2017. 2-Methyl-4-oxopentane-2-thiol: A 90-day Boyland, E., Chasseaud, L.F., 1970. The Effect of Some oral gavage study in rats. Product Safety Labs, Dayton, New Carbonyl Compounds on Rat Liver Glutathione Levels. Jersey. Unpublished report provided to the FEMA Expert Biochemical Pharmacology 19, 1526-1528. Panel by the International Organization of the Flavor Industry. Bruce, S., 2017. Bacterial Reverse Mutation Assay of Bauter, M.R., 2013. 2-Pentylthiophene: a 90-day dietary study Synthetic Intermediate of FEMA 4899. BioReliance in rats, Product Safety Labs, Dayton, NJ, USA. Unpublished Corporation, Rockville, MD, USA. Unpublished report provided to the FEMA Expert Panel. Cohen, S.M., Eisenbrand, G., Fukushima, S., Gooderham, N.J., Guengerich, F.P., Hecht, S.S., Rietjens, I., Bastaki, M., Brusick, D., 1975. Mutagenic evaluation of compound FDA Davidsen, J.M., Harman, C.L., McGowen, M.M., Taylor, S.V., 75-5 000527-07-1 sodium gluconate, FCC, fine granular. 2020. FEMA GRAS assessment of natural flavor complexes: Unpublished report provided to the FEMA Expert Panel, Litton Mint, buchu, dill and caraway derived flavoring ingredients. Bionetics, Inc., Kensington, Maryland. Food and Chemical Toxicology https://doi.org/10.1016/j.fct.2019.110870. Butterworth, K.R., Carpanini, F.M.B., Gaunt, I.F., Kiss, I.S., Gangolli, S.D., 1975. Short-term toxicity of dimethyl sulphide Connor, T., Theiss, J., Hanna, H., Monteith, D., Matney, T., in the rat. Food and Cosmetics Toxicology 13, 15-22. 1985. Genotoxicity of organic chemicals frequently found in the air of mobile homes. Toxicology Letters 25, 33-40. Cannon, R.J., Kazimierski, A., Curto, N.L., Li, J., Trinnaman, L., Janczuk, A.J., Agyemang, D., Da Costa, N.C., Chen, M.Z., Cook, W.M., Purchase, R., Ford, G.P., Creasy, D.M., 2015. Identification, synthesis, and characterization of novel Brantom, P.G., Gangolli, S.D., 1992. A 28-day feeding study sulfur-containing volatile compounds from the in-depth with ethyl acetoacetate in rats. Food and Chemical Toxicology analysis of Lisbon lemon peels (Citrus limon L. Burm. f. cv. 30, 567-573. Lisbon). Journal of agricultural and food chemistry 63, 1915- 1931. Cotgreave, I.A., Atzori, L., Moldeus, P., 1989. Thiol-disulphide exchange: physiological and toxicological aspects. Wiley- Chadha, A., Madyastha, M., 1982. Omega-hydroxylation of Blackwell, New York. acyclic monoterpene alcohols by rat lung microsomes. Biochemical and Biophysical Research Communications 108, Cox, G.E., Bailey, D.E., Morganreidge, K., 1974a. 90-day 6. feeding study in rats with compound 14865 (2,3- butanedithiol). Food and Drug Research Laboratories, Inc. , Chasseaud, L.F., 1979. The role of glutathione and Unpublished report provided to the FEMA Expert Panel. glutathione S-transferases in the metabolism of chemical . carcinogens and other electrophilic agents. Advances in Cox, G.E., Bailey, D.E., Morganreidge, K., 1974b. 90-Day cancer research 29, 175-274. Feeding Study in Rats with Compound 14935. Food and Drug Laboratories Inc. Unpublished report provided to the FEMA Chaturvedula, V.S., Upreti, M., Prakash, I., 2011. Diterpene Expert Panel. glycosides from Stevia rebaudiana. Molecules 16, 3552-3562. Cramer, G.M., Ford, R.A., Hall, R.L., 1978. Estimation of toxic Cheung, Y.-L., 2016a. CA-016: Stability in simulated gastric hazard-A decision tree approach. Food and Cosmetics and intestinal fluids, Envigo CRS Limited, Cambridgeshire, Toxicology 16, 255-276. UK. Curry, P.T., 1995. In vitro mammalian cytogenetic test with 6- Cheung, Y.-L., 2016b. CA-016: Human hepatocyte Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN), Study No. metabolism, Envigo CRS Limited, Cambridgeshire, UK. G94AP80.337005 ed. Microbiological Associantes Inc, Rockville, Maryland, USA. Unpublished report to the FEMA Cohen, S.M., Fukushima, S., Gooderham, N.J., Hecht, S.S., Expert Panel provided by the Research Institute for Fragrance Marnett, L.J., Rietjens, I.M.C.M., Smith, R.L., Bastaki, M., Materials. McGowen, M.M., Harman, C., and Taylor, S.V. 2015. GRAS flavoring substances 27. Food Technol. 69(8), 40-59. Daly, J., 2016. FEMA 4899: A 28-day dietary administration toxicity study in rats. MPI Research, Mattawan, Michigan, Cohen, S.M., Eisenbrand, G., Fukushima, S., Gooderham, USA. Unpublished report to the FEMA Expert Panel N.J., Guengerich, F.P., Hecht, S.S., Rietjens, I., Davidsen, J.M., Harman, C.L., Taylor, S.V., 2018. Updated procedure for the safety evaluation of natural flavor complexes used as Daly, J., 2017. FEMA 4899: A 13-week dietary administration ingredients in food. Food Chem Toxicol 113, 171-178. toxicity study in rats. Unpublished report provided to the FEMA Expert Panel., MPI Research, Mattawan, Michigan, Cohen, S.M., Fukushima, S., Gooderham, N.J., Guengerich, USA. F.P., Hecht, F.M., Rietjens, I.M., Smith, L.M., Bastaki, M., Harman, C.G., McGowen, M.M., Valerio, L.G., Taylor, S.V., Damani, L.A., 1987. Metabolism of sulphur-containing drugs, 2017a. Safety evaluation of substituted thiophenes used as in: D.J. Benford, J.W. Bridges, G.G. Gibson (Eds.), Drug flavoring ingredients. Food and Chemical Toxicology, 40-59. metabolism -- from molecules to man. Taylor & Francis, London, New York, Philadelphia, pp. 581-603. Cohen, S.M., Fukushima, S., Gooderham, N.J., Guengerich, F.P., Hecht, S.S., Rietjens, I.M.C.M., Smith, R.L., Bastaki, M., De Flora, S., Bennicelli, C., Rovida, A., Scatolini, L., Harman, C.L., McGowen, M.M., Taylor, S.V., 2016. FEMA Camoirano, A., 1994. Inhibition of the "spontaneous" expert panel review of p-mentha-1,8-dien-7-al genotoxicity mutagenicity in Salmonella typhimurium TA102 and TA104. testing results. Food and Chemical Toxicology 98, Part B, Mutation research 307, 157-167. 201-209. Dhinsa, N.K., 2006. FEMA 4928 Twenty-eight day repeated Cohen, S.M., Fukushima, S., Gooderham, N.J., Guengerich, dose oral (gavage) toxicity study in the rat. Unpublished report F.P., Hecht, S.S., Rietjens, I.M.C.M., Smith, R.L., Bastaki, M., provided to the FEMA Expert Panel, Safepharm Laboratories Harman, C.L., McGowen, M.M., Valerio, L.G., Taylor, S.V., Limited, Derbyshire, UK. Unpublished report to the FEMA 2017b. Safety evaluation of substituted thiophenes used as Expert Panel. flavoring ingredients. Food and Chemical Toxicology 99, 40- 59. Diaz, D., Scott, A., Carmichael, P., Shi, W., Costales, C., FEMA 1978. SLR C11. Scientific literature review of cinnamyl 2007. Evaluation of an automated in vitro micronucleus assay alcohol and related substances. PB284-961/AS. National in CHO-K1 cells. Mutation research 630, 1-13. Technical Information Service.

Diliberto, J.J., Srinivas, P., Overstreet, D., Usha, G., Burka, FEMA 1978. SLR C14. Scientific literature review of aryl L.T., Birnbaum, L.S., 1990. Metabolism of citral, an alpha- substituted secondary alcohols, ketones and derivatives. beta-unsaturated aldehyde, in male F344 rats. American PB284-963/AS. National Technical Information Service. , Society for Pharmacology and Experimental Therapeutics 18, 1978. 866-875. FEMA 1978. SLR C16. Scientific literature review of alpha- Dirscherl, W., Wirtzfeld, A., 1964. Vanillinsaure im alkylcinnamaldehydes and related substances. PB283- menschlichen Urin Ihre Isolierung, Bestimmung und Herkunft 505/AS. National Technical Information Service. Acta endocr., Copenh 47. FEMA 1978. SLR C21. Scientific literature review of Donath, C., 2017. Mammalian micronucleus test in murine and related compounds. PB284-868/AS. National Technical peripheral blood cells with nootkatone 70 natural. l, Eurofins Information Service. BioPharma, Planegg, Germany. Unpublished report to the FEMA Expert Panel. FEMA 1978. SLR C22. Scientific literature review of aromatic ethers. PB284-867/AS. National Technical Information Service. Dutton, G.J., Illing, H.P.A., 1972. Mechanism of biosynthesis of thio-β-d-glucuronides and thio-β-d-glucosides. Biochem. J. FEMA 1978. SLR D1. Scientific literature review of dithiols 129, 539-550. and related substances. PB291-113/AS. National Technical Information Service. Eapen, A., 2007. A 90-day oral (dietary) toxicity study of GR- 72-0180 in rats with a 28-day recovery period, WIL Research FEMA 1978. SLR D12. Scientific literature review of aliphatic Laboratories, LLC, Ashland, Ohio, United States. ethers. PB141166/AS. National Technical Information Unpublished report to the FEMA Expert Panel. Service.

Eckfeldt, J.H., Yonetani, T., 1982. [79] Isozymes of aldehyde FEMA 1978. SLR D15. Scientific literature review of dehydrogenase from horse liver, Methods in Enzymology. thiophene derivatives. PB291-102/AS. National Technical Elsevier BV, pp. 474-479. Information Service. 1978.

Eder, E., Henschler, D., Neudecker, T., 1982. Mutagenic FEMA 1979. SLR D3. Scientific literature review of pyrrole and properties of allylic and α,β-unsaturated compounds: related substances. . PB296-007/AS. National Technical Consideration of alkylating mechanisms. Xenobiotica 12, 831- Information Service. 848. FEMA 1979. SLR D9. Scientific literature review of furanones Eder, E., Neudecker, T., Lutz, D., Henschler, D., 1980. and related substances. PB85141240/AS. National Technical Mutagenic potential of allyl and allylic compounds. Information Service. 1978, 1979. Biochemical Pharmacology 29, 993-998. FEMA 1979. SLR D19. Scientific literature review of EFSA, 2003. Opinion of the Scientific Panel on Food miscellaneous substances. PB85141299/LL. National Additives, Flavourings, Processing Aids and Materials in Technical Information Service. 1979, 1983. Contact with Food (AFC) on a request from the Commission related to L-carnitine-L-tartrate for use in foods for particular FEMA 1979. SLR B1F. Scientific literature review of aliphatic nutritional uses. poly-hydroxy compounds and derivatives. PB85-141117/LL. National Technical Information Service. 1976, 1979. Feldman, R.I., Weiner, H., 1972. Horse liver aldehyde dehydrogenase. I. Purification and characterization. J Biol FEMA 1984. SLR B1B. Scientific literature review of aliphatic Chem 247, 260-266. keto- and hydroxy-acids with oxygen functions and related compounds. PB85141091/LL. National Technical Information FEMA 1975. SLR A7. Scientific literature review of aliphatic Service. 1976, 1979, 1984. . amines. PB85141075/LL. National Technical Information Service. FEMA 1984. SLR B1C. Scientific literature review of aliphatic lactones. PB86-155850/LL. National Technical Information FEMA 1975. SLR A8. Scientific literature review of aliphatic Service. 1979, 1984. thiols. PB85141075/LL. National Technical Information Service. FEMA 1984 SLR B3. Scientific literature review of amino acids. PB85141125/LL. National Technical Information FEMA 1976. SLR B4. Scientific literature review of aliphatic Service. mono-, di- and trisulfides. . PB85141133/LL. National Technical Information Service. FEMA 1984. SLR C9. Scientific literature review of Benzyl alcohol, , benzoic acid and related compounds. FEMA 1976. SLR, B5B. Scientific literature review of selected . PB85141216/LL. National Technical Information Service. oxygenated derivatives of mercaptans and sulfides. 1978, 1984. PB85141158/LL. National Technical Information Service. FEMA 1984. SLR C12. Scientific literature review of phenols. PB86-155868/AS. National Technical Information Service. 1979, 1984. Istituto di Tecnologie Biomediche, Segrate, Italy. Unpublished FEMA,1984.SLR A1. Scientific literature review of aliphatic report provided to the FEMA Expert Panel. ketones, secondary alcohols and related esters. PB85141059/LL. National Technical Information Service. Geuns, J.M.C., Malheiros, R.D., Moraes, V.M.B., Decuypere, E.M.P., Compernolle, F., Buyse, J.G., 2003b. Metabolism of FEMA 1985. SLR A5. Scientific literature review of alicyclic Stevioside by Chickens. Journal of agricultural and food compounds of carbon, hydrogen and oxygen. PB86- chemistry 51, 1095-1101. 155835/LL. National Technical Information Service. 1976, 1978, 1985. Gibson, G.G., Orton, T.C., Tamburini, P.P., 1982. Cytochrome P -450 induction by clofibrate. Purification and FEMA 1985. SLR M1. Scientific literature review of aliphatic properties of a hepatic cytochrome P -450 relatively specific primary alcohols, aldehydes, esters and acids. PB86-155926. for the 12- and 11-hydroxylation of dodecanoic acid (lauric National Technical Information Service. 1974, 1978, 1979, acid). Biochem. J. 203, 161-168. 1984, 1985. Gillam, E.M., Notley, L.M., Cai, H., De Voss, J.J., Guengerich, Flanders, L., 2017. Micronucleus Test in the Mouse., Envigo F.P., 2000. Oxidation of indole by cytochrome P450 enzymes. Research Limited, Derbyshire, England. Unpublished report Biochemistry 39, 13817-13824. provided to the FEMA Expert Panel. Girkin, R., 1998. Metabolite profiles in rat and pig urine. Florin, I., Rutberg, L., Curvall, M., Enzell, C.R., 1980a. Huntington Life Sciences Ltd. Cambridgeshire, England. Screening of smoke constituents for mutagenicity Unpublished report to the FEMA Expert Panel. using Ame's test. Toxciology 18, 219-232. Gomes-Carneiro, M.R., Felzenszwalb, I., Paumgartten, Florin, I., Rutberg, L., Curvall, M., Enzell, C.R., 1980b. F.J.R., 1998. Mutagenicity testing (+/-)-, 1,8-cineole, Screening of Tobacco Smoke Constituents for Mutagenicity citral, citronellal, (-)- and terpineol with the Using the Ames Test. Toxicology 15, 219-232. Salmonella/microsome assay. Mutation research 416, 129- 136. Fry, J.R., Fentem, J.H., Salim, A., Tang, S.P.A., Garle, M.J., Whiting, D.A., 1993. Structural Requirements for the Direct GRAS Applicant, 2015. Metabolism Study Summary Report: and Cytochrome P450-dependent Reaction of Cyclic α,β- Allulose. Unpublished report provided to the FEMA Expert Unsaturated Carbonyl Compounds with Glutathione: a Study with Coumarin and Related Compounds. Journal of Pharmacy Gudi, R., Ritter, P., 1997. Micronucleus cytogenetic assay in and Pharmacology 45, 166-170. mice, Study No. G96BV66.122 Microbiological Associates, Inc. Rockville, Maryland, USA. Unpublished report to the Fukuda, T., 2017. An in vitro micronucleus test in TK6 human FEMA Expert Panel. lymphoblastoid cells treated with (E)-3-(4- isopropyloxyphenyl)prop-2-enal. BoZo Research Center Inc., Hagan, E.C., Hansen, W.H., Fitzhugh, O.G., Jenner, P.M., Tokyo, Japan. Unpublished report to the FEMA Expert Panel. Jones, W.I., Taylor, J.M., Long, E.L., Nelson, A.A., Brouwer, J.B., 1967. Food flavourings and compounds of related Furukuma, S., 2017. In vitro mammalian cell micronucleus structure. II. Subacute and chronic toxicity. Fd Cosmet. test of CA-047 ITO, UBE Scientific Analysis Laboratory, Inc., Toxicol. 5, 141-157. Yamaguchi Prefecture, Japan. Unpublished report provided to the FEMA Expert Hall, R.L., 1979. Citronellol. Joint FAO/WHO Expert Committee on Food Additives, Geneva. Gardana, C., Simonetti, P., Canzi, E., Zanchi, R., Pietta, P., 2003. Metabolism of stevioside and rebaudioside A from Hall, R.L., Oser, B.L., 1965. Recent Progress in the Stevia rebaudiana extracts by human microflora. Journal of Consideration of Flavoring Ingredients Under the Food agricultural and food chemistry 51, 6618-6622. Additives Amendment III. GRAS Substances. Food Technology. Gaunt, I.F., Colley, J., Grasso, P., Lansdown, A.B.G., Gangolli, S.D., 1969. Acute (rat and mouse) and short-term Hallagan, J.B., Hall, R.L., 2009. Under the conditions of (rat) toxicity studies on cis-3-hexen-1-ol. Food and Cosmetics intended use – New developments in the FEMA GRAS Toxicology 7, 451-459. program and the safety assessment of flavor ingredients. Food and Chemical Toxicology 47, 267-278. Gaunt, I.F., Wright, M.G., Cottrell, R., Gangolli, S.D., 1983. Short-Term Toxicity of 2,6-Dimethylhept-5-en-1-al in Rats. Harman, C.L., Hallagan, J.B., 2013. Sensory testing for Chem. Toxic. 21, 543-549. flavorings with modifying properties. Food Technology 67, 44- 47. Geuns, J.M., Augustijns, P., Mols, R., Buyse, J.G., Driessen, B., 2003a. Metabolism of stevioside in pigs and intestinal Hashimoto, J., 2015. Bacterial reverse mutation test of CA- absorption characteristics of stevioside, rebaudioside A and 016-ITO, UBE Scientific Analysis Laboratory, Inc., Yamaguchi steviol. Food Chem Toxicol 41, 1599-1607. Prefecture, Japan. Unpublished report provided to the FEMA Expert Panel. Geuns, J.M., Buyse, J., Vankeirsbilck, A., Temme, E.H., 2007. Metabolism of stevioside by healthy subjects. Experimental Hashimoto, J., 2016. Bacterial reverse mutation test of CA- biology and medicine 232, 164-173. 047-ITO, UBE Scientific Analysis Laboratory, Inc., Yamaguchi Prefecture, Japan. Unpublished report provided to the FEMA Geuns, J.M., Pietta, P., 2004. Stevioside metabolism by Expert Panel. human volunteers., Laboratory of Functional Biology, ITB Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., Zeiger, Ishida, T., Toyota, M., Asakawa, Y., 1989. Terpenoid E., 1983. Salmonella mutagenicity test results for 250 biotransformation in mammals. V. Metabolism of (+)- chemicals. Environ. mutagen 5, 3-49. citronellal, (±)-7-, citral, (−)-perillaldehyde, (−)-myrtenal, , thujone, and (±)-carvone in Hayes, K.C., Pronczuk, A., Cook, M.W., Robbins, M.C., 2003. rabbits. Xenobiotica 19, 843-855. Betaine in sub-acute and sub-chronic rat studies. Food and Chemical Toxicology 41, 1685-1700. Ishidate, M., Sofuni, T., Yoshikawa, D., Hayashi, M., Nohmi, T., Sawada, M., Matsuoka, A., 1984. Primary mutagenicity Heck, J.D., Vollmuth, T.A., Cifone, M.A., Jagannath, D.R., screening of food additives currently used in Japan. Fd Chem. Myhr, B.C., Curran, R.D., 1989. An evaluation of food Toxic. 22, 623-636. flavouring ingredients in a genetic toxicity screening battery. The Toxicologist 9, 257. Ishii, T., 2017. (E)-3-(4-isopropyloxyphenyl) prop-2-enal: Micronucleus test with rat bone marrow cells. BoZo Research Hobbs, C.A., Taylor, S.V., Beevers, C., Lloyd, M., Bowen, R., Center Inc., Shizuoka, Japan. Unpublished report to the Lillford, L., Maronpot, R., Hayashi, S.-m., 2016. Genotoxicity FEMA Expert Panel. assessment of the flavouring agent, perillaldehyde. Food and Chemical Toxicology 97, 232-242. James, 1974. Animal studies - Summaries of results, Huntingdon Research Center, UK. Unpublished report Honarvar, N., 2007. Micronucleus assay in bone marrow cells provided to the FEMA Expert Panel. of the mouse with 10-undecenal. RCC Cytotest Cell Research GmbH, Rossdorf, Germany. Unpublished report to the FEMA Jansson, T. Curvall, M., Hedin, A., Enzell, C.R. 1986. In vitro Expert Panel. studies of biological effects of cigarette smoke condensate. II. Induction of sister-chromatid exchanges in human Hooks, W.N., Kirk, S.J., Smith, H.L., Crook, D., Gibson, W.A., lymphocytes by weakly acidic, semi volatile constituents. Gregson, R.L., Gopinath, D., Anderson, A., Dawe, S.I., 1992. Mutation Research. 169(3), 129-139. Ethyl vanillin toxicity to rats by repeated dietary administration. Unpublished report to the FEMA Expert Panel. Jansson, T, Zech, L., 1987. Effects of vanillin on sister- Provided by the Research Institute for Fragrance Materials. chromatid exchanges and chromosome aberrations in human lymphocytes. Mutation Research. 190, 221-224. Hooth, M.J., Sills, R.C., Burka, L.T., Haseman, J.K., Witt, K.L., Orzech, D.P., Fuciarelli, A.F., Graves, S.W., Johnson, J.D., JECFA, 1999. Safety Evaluation of Certain Food Additives Bucher, J.R., 2004. Toxicology and carcinogenesis studies of (Fifty-first Meeting of Joint FAO/WHO Expert Committee on microencapsulated trans- in rats and mice. Food Additives). Food Additives Series No. 42. Food and chemical toxicology : an international journal published for the British Industrial Biological Research JECFA, 2000. Safety evaluation of certain food additives and Association 42, 1757-1768. contaminants. WHO Food Additive Series 44.

Horrigan, M., 2016. Metabolite Characterization of the Subject JECFA, 2001. Safety evaluation of certain food additives and Compound in Rat and Human Hepatocytes, Study No. contaminants. WHO Food Additive Series 48. XT160033., XenoTech, LLC, Kansas City, Kansas, USA. Unpublished report to the FEMA Expert Panel. JECFA, 2003. Toxicological evaluation of certain veterinary drug residues in food (Sixtieth Meeting of Joint FAO/WHO Huntington Lab, 2011. Safety of D-psicose report prepared for Expert Committee on Food Additives). Food Additives Series CJ CheilJedang. Food and Drug Administration (FDA), No. 51. https://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNoti ces&id=400&sort=GRN_No&order=DESC&startrow=1&type JECFA, 2004. Safety evaluation of certain food additives and =basic&search=400. contaminants prepared by the fifty-seventh meeting of the Joint FAO/WHO Expert Committee on Food Additives WHO Hutapea, A.M., Tuoskulkao, C., Buddhasukh, D., Wilairat, P., Food Additive Series No. 52. Glinsukon, T., 1997. Digestion of stevioside, a natural sweetener, by various digestive enzymes. Journal of Clinical JECFA, 2006. Safety evaluation of certain food additives and Biochemistry and Nutrition 23, 177-186. contaminants (sixty-third report of Committee on Food Additives). WHO Food Additive Series No. 54. IBR, 1991b. Acute Oral Toxicity Test of “5497” in Rats. Final Report dated 15.3.91. Sponsored by Lonza AG, Basel, JECFA, 2007. Safety evaluation of certain food additives and Switzerland (cited in EFSA, 2003; Norwegian, 2015). IBR contaminants. WHO Food Additive Series 58. Forschungs GmbH, Walsrode, Germany. JECFA, 2008. Safety evaluation of certain food additives and Ibrahim, M.A., Rodenburg, D.L., Alves, K., Fronczek, F.R., contaminants (prepared by the sixty-eighth meeting of McChesney, J.D., Wu, C., Nettles, B.J., Venkataraman, S.K., JECFA). WHO Food Additive Series No. 59, 175-236. Jaksch, F., 2014. Minor Diterpene Glycosides from the Leaves of Stevia rebaudiana. Journal of Natural Products 77, JECFA, 2009. Safety evaluation of certain food additives and 1231-1235. contaminants (sixty-ninth meeting of Committee on Food Additives). WHO Food Additive Series No. 60. Inouye, T., Sasaki, Y.F., Imanishi, H., Watanebe, M., Ohta, T., Shirasu, Y., 1988. Suppression of mitomycin C-induced JECFA, 2011. Safety evaluation of certain food additives and micronuclei in mouse bone marrow cells by post-treatment contaminants (seventy-third meeting of Committee on Food with vanillin. Mutation research 202, 93-95. Additives). WHO Food Additive Series No. 64.

JECFA, 2012. Safety evaluation of certain food additives and Isopropoxyphenyl)prop-(2E)-enal. Unpublished report contaminants (seventy-sixth meeting of Committee on Food provided to the FEMA Expert Panel. Additives). WHO Food Additive Series No. 67. Kim, J.C., Kang, B.H., Shin, C.C., Kim, Y.B., Lee, H.S., Kim, JECFA, 2014. Safety evaluation of certain food additives and C.Y., Han, J., Kim, K.S., Chung, D.W., Chung, M.K., 2002. contaminants. WHO Food Additive Series 68. Subchronic toxicity of plant sterol esters administered by gavage to Sprague-Dawley rats. . Food and Chemical JECFA, 2015. Safety evaluation of certain food additives Toxicology 40, 1569-1580. (seventy-ninth report of Committee on Food Additives). WHO Food Additive Series No. 70. Kirkpatrick, J.B., 2011. WS-5: A 90-day oral gavage toxicity study with a 14-day recovery period in Sprague-Dawley rats JECFA, 2017. Safety evaluation of certain food additives. (OECD 408), WIL Research Laboratories, LLC, Ashland, WHO Food Additive Series No. 73. Ohio, USA. Biotechnics, LLC, Hillsborough, North Carolina, USA. Unpublished report provided to the FEMA Expert Panel. JECFA, 2019. Safety evaluation of certain food additives (eighty-sixth report of Committee on Food Additives). WHO Klancnik, J.M., Cuenod, M., Gahwiler, B.H., Jiang, Z.P., Do, Food Additive Series No. 86. K.Q., 1992. Release of endogenous amino acids, including homocysteic acid and cysteine sulphinic acid, from rat Jin, X., Dong, H., 2010. Acute toxicity and mutagenicity of hippocampal slices evoked by electrical stimulation of extract from Eriobotrta japonica, 4th International Conference Schaffer collateral-commissural fibres. Neuroscience 49, 557- on Bioinformatics and Biomedical Engineering. Unpublished 570. report provided to the FEMA Expert Panel Kodera, Y., Suzuki, S., Imada, O., Kasuga, S., Sumioka, I., Jin, X., Dong, H., 2017. Acute toxicity and mutagenicity of Kanezawa, A., Taru, N., Fujikawa, M., Nagae, S., Masamoto, extract from Eriobotrta japonica Huainan Union University, K., Maeshige, K., Ono, K., 2002. Physical, Chemical, and Huainan, China. Unpublished report provided to the FEMA Biological Properties of S-Allylcysteine, an Amino Acid Expert Panel Derived from Garlic. Journal of agricultural and food chemistry 50, 622-632. Jones, E., 1986. Ames metabolic activation test to assess the potential mutagenic effect of Vanillin, USP. Huntingdon Komai, T., 2016a. Reverse mutation assay "Ames Test" using Research Center Ltd. England, Unpublished report Salmonella Enterica: 3-p-Menthen-7-al, Kawasaki-shi, Japan. (EuroVanillin KS, Norway). As cited in the OECD SIDS. Unpublished report provided to the FEMA Expert Panel.

Jones, L.J., McKenzie, J., Brooks, P.N., 2004. Nookatone, Komai, T., 2016b. Reverse mutation assay "Ames Test" using Verbenone: Twenty-eight day repeated dose, single dose Salmonella Typhimurium: (E)-3-Methyl-4-dodecenoic acid, level oral toxicity study in the rat, SafePharm Laboratories, Kawasaki-shi, Japan. Unpublished report provided to the Derbyshire, UK. Unpublished report to the FEMA Expert FEMA Expert Panel. Panel. Komai, T., 2016c. Reverse mutation assay "Ames Test" using Kamil, I.A., Smith, J.N., Williams, R.T., 1953. Studies in Salmonella Typhimurium: cis-Cascarillic acid. Unpublished detoxication. 46. The metabolism of aliphatic alcohols. The report provided to the FEMA Expert Panel, Kawasaki-shi, glucuronic acid conjugation of acyclic aliphatic alcohols. Japan. Biochem. J. 53, 129-136. Komai, T., 2017a. Reverse mutation assay "Ames Test": Karanewsky, D.S., Arthur, A.J., Liu, H., Chi, B., Markison, S., bis(3-methyl-2-butenyl) disulfide. Unpublished report 2015. Toxicological evaluation of a novel cooling compound: provided to the FEMA Expert Panel. 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-(2- thienylmethyl)acetamide. Toxicology Reports 2, 1291-1309. Komai, T., 2017b. Reverse mutation assay "Ames Test": 3,4- Dimethyl-5-propylidenefuran-2(5H)-one. Unpublished report Kasamaki, A., Takahashi, H., Tsumura, N., Niwa, J., Fujita, T., provided to the FEMA Expert Panel. Urasawa, S., 1982. Genotoxicity of flavoring agents. Mutation research 105, 387-392. Komai, T., 2017c. Reverse mutation assay "Ames Test": 1- Ethyl-2-(1-pyrrolylmethyl)pyrrole. Unpublished report Kaspers, U., 2002. 6-Methylhept-5-en-2-one provided to the FEMA Expert Panel.. (Methylheptenon) – Subchronic toxicity study in wistar rats administration by gavage for 3 months, Experimental Komai, T., 2018a. Reverse mutation assay "Ames Test": (Z)- Toxicology and Ecology, Ludwigshafen, Germany. 8-Pentadecenal. Unpublished report provided to the FEMA Unpublished report provided to the FEMA Expert Panel. Expert Panel.

Kawaguchi, R., 2016. Reverse mutation assay “Ames Test” Komai, T., 2018b. Reverse mutation assay "Ames Test": 2- using Salmonella typhimurium. Test Material 4-(4-Methyl-3- Methylthiophene. Unpublished report provided to the FEMA penten-1-yl)-2(5H)-furanone. Unpublished report provided to Expert Panel. the FEMA Expert Panel. Komai, T., 2018c. Reverse mutation assay "Ames Test": Kawaguchi, R., Komai, T., 2012. Reverse mutation assay Dehydrorose oxide. Unpublished report provided to the FEMA "Ames Test" using Salmonella typhimurium: (E)-6-octenal. Expert Panel. Unpublished report provided to the FEMA Expert Panel. Komai, T., 2018d. Reverse Mutation Assay "Ames Test" 3- Kawaguchi, R., Komai, T., 2016. Reverse mutation assay Methylbutane-1,3-dithiol. Unpublished report provided to the "Ames Test" using Salmonella Typhimurium: 3-(4- FEMA Expert Panel. Liwska, K., 2012. 10-Undecenal ninety day repeated dose Komai, T., 2018e. Reverse Mutation Assay "Ames Test" Test oral (dietary) toxicity study in the rat.., Harlan Laboratories Material S-methyl 5-(1-ethoxyethoxy) decanethioate. Ltd., Derbyshire, UK. Unpublished report to the FEMA Expert Unpublished report to the FEMA Expert Panel. Panel.provided by the Research Institute for Fragrance Materials. Köppel, C., Tenczer, J., 1991. Mass spectral characterization of urinary metabolites of d,l-kawain. Journal of Lloyd, M., 2014. Induction of micronuclei in cultured human Chromatography B: Biomedical Sciences and Applications peripheral blood lymphocytes: 4-Acetyl-2,5-dimethylfuran- 562, 207-211. 3(2H)-one. Covance Laboratories, Harrogate, England. Unpublished report provided to the FEMA Expert Panel by the Koyama, E., Kitazawa, K., Ohori, Y., Izawa, O., Kakegawa, International Organization of the Flavor Industry. K., Fujino, A., Ui, M., 2003a. In vitro metabolism of the glycosidic sweeteners, stevia mixture and enzymatically Luis, A., Duarte, A.P., Pereira, L., Domingues, F., 2017. modified stevia in human intestinal microflora. Food Chem Chemical Profiling and Evaluation of Antioxidant and Anti- Toxicol 41, 359-374. Microbial Properties of Selected Commercial Essential Oils: A Comparative Study. Medicines (Basel) 4. Koyama, E., Sakai, N., Ohori, Y., Kitazawa, K., Izawa, O., Kakegawa, K., Fujino, A., Ui, M., 2003b. Absorption and Maiorino, R.M., Bruce, D.C., Aposhian, H.V., 1989. metabolism of glycosidic sweeteners of stevia mixture and Determination and metabolism of dithiol chelating agents. their aglycone, steviol, in rats and humans. Food Chem Toxicology and Applied Pharmacology 97, 338-349. Toxicol 41, 875-883. Maki, K.C., Davidson, M.H., Umporowicz, D.M., Schaefer, Kroes, R., Renwick, A.G., Cheeseman, M., Kleiner, J., E.J., Dicklin, M.R., Ingram, K.A., Chen, S., McNamara, J.R., Mangelsdorf, I., Piersma, A., Schilter, B., Schlatter, J., van Gebhart, B.W., Ribaya-Mercado, J.D., Perrone, G., Robins, Schothorst, F., Vos, J.G., Wurtzen, G., 2004. Structure-based S.J., Franke, W.C., 2001. Lipid responses to plant-sterol- thresholds of toxicological concern (TTC): guidance for enriched reduced-fat spreads incorporated into a National application to substances present at low levels in the diet. Cholesterol Education Program Step I diet. The American Food and Chemical Toxicology 42, 65-83. Journal of Clinical Nutrition 74, 33-43.

Kuroda, K., Tanaka, S., Yu, Y., Ishibashi, T., 1984. Rec-assay Mancebo, A., Trapero, Y.M., González, Y., González, B.O., of food additives. Nippon Kosyu Eisei Zasshi 31, 277-281. Fuentes, D., Hernández, O., Hernández, J., González, C., Hernández, Y., Arteaga, M.E., Subirós, N., Bada, A.M., 2003. Kurooka, S., Hashimoto, M., Tomita, M., Maki, A., Yoshimura, Repeated dose oral toxicity (13 weeks) of 4-hydroxy-3- Y., 1976. Relationship between the structures of S-acyl thiol methoxy-benzaldehyde (vanillin) to Sprague Dawley rats. compounds and their rates of hydrolysis by pancreatic lipase Toxicology Letters 144, s57. and hepatic carboxylic esterase. Journal of biochemistry 79, 533-541. Marks, H.S., Anderson, J.L., Stoewsand, G.S., 1992. Inhibition of benzo[a]pyrene‐induced bone marrow Lawlor, T.E., 1991. Mutagenicity test on Vanillin in the micronuclei formation by diallyl thioethers in mice. Journal of Salmonella/mammalian-microsome reverse mutation assay Toxicology and Environmental Health 37, 1-9. (Ames test). Report no. 12389-0-401. ed. Hazleton Laboratories America, Inc., Unpublished report to the FEMA Marr, A., 2007. FEMA 4928 Limited twenty-eight day repeated Expert Panel.provided by the Research Institute for Fragrance dose oral (gavage) toxicity study in the rat. Safepharm Materials. Laboratories Limited, Derbyshire, UK. Unpublished report provided to the FEMA Expert Panel. Lawrence, B.M., 1977. Essential Oils 1976-1978. Reprint: Progress in Essential Oils. Perfumer & Flavorist 2, 3. Marzin, D., 1979a. Rhône-Poulenc unpublished / DREBS Wheaton, IL: Allured Publishing Corporation, pp. 21. (Department Recherche et Essais Biologiques Stallergènes) study R-905017. Citation: Euclid Data Sheet for Vanillin by Lee, H., Shuh Shyuan, B., Yi Ling, C., 1994. Genotoxicity of Rhône-Poulenc Chimie, created 26.01.95, updated 01.06.95). 1,3-dithiane and 1,4-dithiane in the CHO/SCE assay and the As cited in the OECD SIDS. Salmonella/microsomal test. Mutation Research/Genetic Toxicology 321, 213-218. Marzin, D., 1979b. Recherche d'une action mutagène par le test du micronucleus chez la souris du produit: Vanilline., Leegwater, D.C., van Straten, S., 1974. In vitro study on the Report no. 906010 ed. DREBS (Department Recherche et hydrolysis of twenty-six organic esters by pancreatin. Centraal Essais Biologiques Stallergènes). Unpublished report Instituut Voor Voedingsonderzoek, Zeist, The Netherlands. (Rhône-Poulenc, France). As cited in the OECD SIDS. Unpublished report provided to the FEMA Expert Panel. Marzin, D., 1998. Recherche de mutagenicite sur Salmonella Leuschner, J., 2017. Mutagenicity study of hibiscus blossom typhimurium His- selon la method de B.N. Ames sur le produit extract in the Salmonella typhimurium and Escherichia coli ST14C97., Unpublished report provided to the FEMA Expert reverse mutation assay (in vitro), Laboratory of Pharmacology Panel. and Toxicology GmbH & Co., Hamburg, Germany. Unpublished report provided to the FEMA Expert Panel. Masoro, E.J., 1977. Lipids and Lipid Metabolism. Annual Review of Physiology 39, 301-321. Li, F., Li, Y., Li, Q., Shi, X., Guo, Y., 2017. Acute and Subacute Oral Toxicity Evaluation of Eriobotrya japonica Leaf Matsuo, T., Ishii, R., Shirai, Y., 2011. The effects of 90-day Triterpene Acids in ICR Mice. Evid Based Complement feeding of D-psicose syrup in male wistar rats. Open Journal Alternat Med 2017, 4837839. of Preventive Medicine 1, 6.

McBain, J.B., Menn, J.J., 1969. S-methylation, oxidation, hydroxylation and conjugation of thiophenol in the rat. Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, Biochemical Pharmacology 18, 2282-2285. B., Zeigher, E., 1986. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environmental McElroy, 2017a. An Oral (Gavage) Developmental Toxicity Mutagenesis 8, 1-119. Study of FEMA 4899 (Salt) in Sprague-Dawley Rats. Charles River Laboratories. Unpublished report provided to the FEMA Müller, W., Engelhart, G., Herbold, B., Jäckh, R., Jung, R., Expert Panel. 1993. Evaluation of mutagenicity testing with Salmonella typhimurium TA102 in three different laboratories. McElroy, 2017b. An Oral (Gavage) Dose Range-Finding Environmental Health Perspectives 101, 33-36. Developmental Toxicity Study of FEMA 4899 (Salt) in Sprague-Dawley Rats. Charles River Laboratories. Munday, R., Manns, E., 1994. Comparative Toxicity of Unpublished report provided to the FEMA Expert Panel. Prop(en)yl Disulfides Derived from Alliaceae: Possible Involvement of 1-Propenyl Disulfides in Onion-Induced Meerts, I., 2001. Evaluation of the ability of coffee precursor Hemolytic Anemia. J. Agric. Food Chem. 42, 959-962. to induce chromosome aberrations in cultured peripheral human lymphocytes. NOTOX B.V.,Hertogenbosch, the Munro, I.C., Ford, R.A., Kennepohl, E., Sprenger, J.G., 1996. Netherlands. Unpublished report provided to the FEMA Correlation of Structural Class with No-Observed-Effect Expert Panel, Levels: A Proposal for Establishing a Threshold of Concern. Food and Chemical Toxicology 34, 829-867. Miettinen, T.A., Tilvis, R.S., Kesaniemi, Y.A., 1990. Serum plant sterols and cholesterol precursors reflect cholesterol Musk, S.R.R., Clapham, P., Johnson, I.T., 1997. Cytotoxicity absorption and synthesis in volunteers of a randomly selected and Genotoxicity of Diallyl Sulfide and male population. American Journal of Epidemiology 131, 20- Towards Chinese Hamster Ovary Cells. Food and Chemical 31. Toxicology 35, 379-385.

Miyata, K., 1995. Twenty-eight day repeated dose oral toxicity Myhr, B., McGregor, D., Bowers, L., Riach, C., Brown, A.G., study of WS 3 in rats, Hita Research Laboratories, Oita, Edwards, I., McBride, D., Martin, R., Caspary, W.J., 1990. Japan. Unpublished report provided to the FEMA Expert L5178Y mouse lymphoma cell mutation assay results with 41 Panel. compounds. Environmental and Molecular Mutagenesis 16, 138-167. Mochizuki, M., 1995. A 4-week oral toxicity study of sodium gluconate (FR2531) in rats. BoZo Research Center Inc., Nagabhushan, M., Bhide, S.V., 1985. Mutagenicity of chili Tokyo, Japan. Unpublished report provided to the FEMA extract and capsaicin in short-term tests. Environmental Expert Panel. Mutagenesis 7, 881-888.

Mochizuki, M., 1997. A 28-day toxicity study in rats fed diet Nakajima, M., 2000a. Chromosome aberration assay of containing sodium gluconate (FR2531). BoZo Research rebaudioside A in cultured mammalian cells. Test number Center Inc., Tokyo, Japan. Unpublished report provided to the 5001 (079-085). Biosafety Research Center, Japan. FEMA Expert Panel. Unpublished report provided to the FEMA Expert Panel.

Moore, M.M., Honma, M., Clements, J., Blocsfoldi, G., Cifone, Nakajima, M., 2000b. Micronucleus test of rebaudioside A in M., Delongchamp, R., Fellows, M., Gollapudi, B., Jenkinson, mice. Biosafety Research Center, Japan. Unpublished report P., Kirby, P., Kirchner, S., Muster, W., Myhr, B., O'Donovan, provided to the FEMA Expert Panel. M., Oliver, J., Omori, T., Ouldelhkim, M.C., Pant, K., Preston, R., Riach, C., San, R., Stankowski, L.F., Thakur, A., Wakuri, Nakayama, K., Kasahara, D., Yamamoto, F., 1986. S., Yoshimura, I., 2003. Mouse Lymphoma Thymidine Kinase Absorption, distribution, metabolism and excretion of Gene Mutation Assay: International Workshop on stevioside in rats. Food Hygiene and Safety Science Genotoxicity Tests Workgroup Report—Plymouth, UK 2002. (Shokuhin Eiseigaku Zasshi) 27, 1-8. Mutation research 540, 127-140. Nakayasu, H., Mihara, K., Sato, R., 1978. 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Toxicology and Criteria, Positive Controls, and Data Evaluation. carcinogenesis studies of citral (microencapsulated) in Environmental and Molecular Mutagenesis 47, 1-5. F344/N rats and B6C3F1 mice. U.S. Department of Health and Human Servies, National Institutes of Heath. Morgareidge, K., 1962. In vitro digestion of four acetals. Food and Drug Research Laboratories. Unpublished report National Toxicology Program (NTP), 2004. Studies of trans- provided to the FEMA Expert Panel. Cinnamaldehyde (microencapsulated) in F344/and B6C3F mice. National Toxicology Program Technical Report Series Morgareidge, K., 1974. 90-Day feeding studies in rats with 514, 1-284. compound 14935, Unpublished report provided to the FEMA Expert Panel. National Toxicology Program (NTP), 1992. Toxicology and Exchange in Chinese Hamster Ovary Cells 1,2-Ethandithiol. Carcinogenesis Studies of y-butyrolactone (CAS no. 96-48-0) Hazelton Laboratories America, Inc. Vienna, Virginia, USA. in F344/N Rats and B6C3F1 Mice (Gavage Studies). National Unpublished report provided to the FEMA Expert Panel. Toxicology Program Technical Report Series. Pence, D.H., Farrow, M.G., Cortina, T.A., 1983. In vitro Sister Naumann, S., 2018. Modified guaiac wood extract (Guaiacum Chromatid Exchange in Chinese Hamster Ovary Cells 1,2- spp.): Micronucleus Test in Human Lymphocytes In vitro. Ethanedithiol. Hazelton Laboratories America, Inc. , Envigo CRS GmbH, Rossdorf, Germany, Unpublished report Unpublished report provided to the FEMA Expert Panel . to the Expert Panel of the Flavor and Extract Manufacturers Association. Pezzuto, J.M., Compadre, C.M., Swanson, S.M., Nanayakkara, D., Kinghorn, A.D., 1985. Metabolically NCI, 1996. Clinical development plan: l-Perillyl alcohol. activated steviol, the aglycone of stevioside, is mutagenic. Journal of Cellular Biochemistry 26S, 137-148. 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Ohta, M., Sasa, S., Inoue, A., Tamai, T., Fujita, I., Morita, K., Prakash, I., Markosyan, A., Bunders, C., 2014. Development Matsuura, F., 2010. Characterization of Novel Steviol of Next Generation Stevia Sweetener: Rebaudioside M. Glycosides from Leaves of Stevia rebaudiana Morita. Journal Foods (Basel, Switzerland) 3, 162-175. of Applied Glycoscience 57, 199-209. Pritchard, L., 2006. GR-72-0180 Mouse micronucleus test, Okamoto, M., 1995. Four-weeks oral dose toxicity study of Huntingdon Life Sciences Limited, Cambridgeshire, England. sodium gluconate in dog. Unpublished report provided to the Unpublished report provided to the FEMA Expert Panel by the FEMA Expert Panel. International Organization of the Flavor Industry.

Oshima, H., Kimura, I., Izumori, K., 2006. Psicose Contents in Purkayastha, S., Bhusari, S., Pugh, G., Jr., Teng, X., Kwok, Various Food Products and its Origin. Food Science and D., Tarka, S.M., 2015. In vitro metabolism of rebaudioside E Technology Research 12, 137-143. under anaerobic conditions: Comparison with rebaudioside A. Regulatory toxicology and pharmacology : RTP 72, 646-657. Ostlund, R.E., 2002. Phytosterols in Human Nutrition. Annual Review of Nutrition 22, 533-549. Purkayastha, S., Markosyan, A., Prakash, I., Bhusari, S., Pugh, G., Jr., Lynch, B., Roberts, A., 2016. Steviol glycosides Packman, E.W., Abbott, D.D., Harrisson, J.W.E., 1963. in purified stevia leaf extract sharing the same metabolic fate. Comparative subacute toxicity for rabbits of citric, fumaric, Regulatory toxicology and pharmacology : RTP 77, 125-133. and tartaric acids. Toxicology and Applied Pharmacology 5, 163-167. Purkayastha, S., Pugh, G., Jr., Lynch, B., Roberts, A., Kwok, D., Tarka, S.M., Jr., 2014. In vitro metabolism of rebaudioside Pence, D.H., 1982. Mouse Lymphoma Forward Mutation B, D, and M under anaerobic conditions: comparison with Assay 1,2-Ethanedithiol and in Vitro Sister Chromatid rebaudioside A. Regulatory toxicology and pharmacology : Sammons, H.G., Williams, R.T., 1941. The metabolism of RTP 68, 259-268. vanillin and vanillic acid in the rabbit. The identification of glucurovanillin and the structure of glucurovanillic acid. Ramachandran, S., Fontanille, P., Pandey, A., Larroche, C., Studies in Detoxification. Biochemistry Journal 35(10-11), 2006. Gluconic Acid: Properties, Applications and Microbial 1175-1189. Production. Samuelsen, O.B., Brenna, J., Solheim, E., Scheline, R.R., Rapson, W.H., Nazar, M.A., Butsky, V.V., 1980. Mutagenicity 1986. Metabolism of the constituent o - produced by aqueous chlorination of organic compounds. methoxycinnamaldehyde in the rat. Xenobiotica 16, 845-852. Bull. Environm. Contam. Toxicol. 24, 590-596. Sanders, D.J., Minter, H.J., Howes, D., Hepburn, P.A., 2000. Renwick, A.G., 1996. Sulfur-Oxygen Compounds, in: S.C. The Safety Evaluation of Phytoseterol Esters. Part 6. The Mitchell (Ed.), Biological Interactions of Sulfur Compounds. Comparative Absorption and Tissue Distribution of Taylor & Fancis, London, pp. 44-75. Phytosterols in the Rat. Food and Chemical Toxicology 38, 485-491. Renwick, A.G., Tarka, S.M., 2008. Microbial hydrolysis of steviol glycosides. Food and Chemical Toxicology 46, S70- Sanyal, R., Darroudi, F., Parzefall, W., Nagao, M., S74. Knasmüller, S., 1997. Inhibition of the genotoxic effects of heterocyclic amines in human derived hepatoma cells by Richardson, K.A., Edwards, V.T., Jones, B.C., Hutson, D.H., dietary bioantimutagens. Mutagenesis, 12(4), 297-303. 1991. Metabolism in the rat of a model xenobiotic plant metabolite S -benzyl- N -malonyl-L-cysteine. Xenobiotica 21, Sasaki, Y.F., Imanishi, H., Ohta, T., Shirasu, Y., 1987. Effects 371-382. of antimutagenic flavourings on SCEs induced by chemical mutagens in cultured Chinese hamster cells. Mutation Rivedal, E., Mikalsen, S.O., Sanner, T., 2000. Morphological Research. 189(3), 313-318. transformation and effect on gap junction intercellular communication in Syrian hamster embryo cells as screening Sasaki, Y.F., Ohta, T., Imanishi, H., Watanabe, M., tests for carcinogens devoid of mutagenic activity. Toxicology Matsumoto, K., Kato, T., Shirasu, Y., 1990. in Vitro 14, 185-192. Suppressing effects of vanillin, cinnamaldehyde and anisaldehyde on chromosome aberrations induces by X-rays Roberts, A., Renwick, A.G., 2008. Comparative toxicokinetics in mice. Mutation Research, 243, 299-302. and metabolism of rebaudioside A, stevioside, and steviol in rats. Food and Chemical Toxicology 46 Suppl 7, S31-39. Sato, R., 2017. A bacterial reverse mutation test of 2-methyl- 3-butene-2-thiol. Unpublished report provided to the FEMA Rockwell, P., Raw, I., 1979. A Mutagenic Screening of Various Expert Panel. Herbs, Spices, and Food Additives. Nutrition and Cancer 1, 10-15. Scheerbaum, D., 2001. Reverse mutation assay (Amestest) with Salmonella typhimurium. Dr. U. Noack Laboratorium, Roy, S., 2015. 2-Methyl-2-hepten-6-ol (CAS No. 1569-60-4): Sarstedt, Germany. Unpublished report provided to the FEMA In vitro mammalian cell micronucleus assay in human Expert Panel peripheral blood lymphocytes (HPBL), BioReliance Corporation, Rockville, MD, USA. Unpublished report Schmidbaur, H., Schier, A., Bayler, A., 1998. The solution and provided to the FEMA Expert Panel. solid state structure of L-carnitine-L-tartrate. Zeitschrift für Naturforschung 53, 788-791. Roy, S., 2016. FEMA 4899: In vitro mammalian cell micronucleus assay in human peripheral blood lymphocytes Schulz, H., 1983. Metabolism of 4-pentenoic acid and (HPBL). BioReliance Corporation, Rockville, MD, USA. inhibition of thiolase by metabolites of 4-pentenoic acid. Unpublished report provided to the FEMA Expert Panel. Biochemistry 22, 1827-1832.

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