a procedure for the safety evaluation
TRANSCRIPT
Review Section
A Procedure for the Safety Evaluation ofFlavouring Substances
I. C. MUNRO1,*, E. KENNEPOHL1 and R. KROES2
1CanTox Inc., 2233 Argentia Road, Suite 308, Mississauga, Ontario, L5N 2X7, Canada and2Prins Hendriklaan 63, 3721 AP Bilthoven, The Netherlands
(Accepted 15 July 1998)
SummaryÐThis review describes a procedure for the safety evaluation of ¯avouring substances. Over2500 ¯avouring substances are currently in use in food. While toxicity data do not exist on all ¯avouringsubstances currently in use, within structurally related groups of ¯avouring substances many do havetoxicity data and this information along with knowledge of structure±activity relationships and data onthe daily intake provides a framework for safety evaluation. The safety evaluation procedure provides ascienti®cally based practical method of integrating data on intake, structure±activity relationships,metabolism and toxicity to evaluate ¯avouring substances in a timely manner. The procedure has beenused recently by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) to evaluate atotal of 263 ¯avouring substances. # 1999 Published by Elsevier Science Ltd. All rights reserved
Keywords: ¯avours; safety evaluation; toxicity; structure activity; JECFA; intake.
Abbreviations: ADI = acceptable daily intake; CAS No. = Chemical Abstracts Service Registry Number;CE = Council of Europe; CPD= carcinogenic potency database; CR= consumption ratio; DART=developmental and reproductive toxicity; EPA =USEnvironmental Protection Agency; FEXPAN= Fla-vour and Extract Manufacturers' Association of the United States Expert Panel; FDA=US Food andDrug Administration; FEMA= Flavour and Extract Manufacturers' Association of the United States;GRAS= generally recognized as safe; IRIS = Integrated Risk Information System; JECFA= JointFAO/WHO Expert Committee on Food Additives; LOEL= lowest-observed-e�ect level; MTD=maxi-mum tolerated dose; NAS/NRC= National Academy of Sciences/National Research Council;NOEL= no-observed-e�ect level; NTP = National Toxicology Program; PAFA= Priority-basedAssessment of Food Additives; QSAR= quantitative structure±activity relationship; RfD= referencedose; RTECS= Registry of Toxic E�ects of Chemical Substances; SCF= Scienti®c Committee for Food;TLV= threshold limit value;WHO=WorldHealth Organization; 2-AAF= 2-acetyl amino¯uorene.
INDEX
Introduction 208Conceptual framework for the safety evalua-tion of ¯avouring substances
209
Estimating intake of ¯avouring substancesthrough food
209
Consideration of natural occurrence of
¯avouring substances in food
210
Structure±activity relationships 210Use of toxicity data 211
Elements of the safety evaluation procedure 211
Structure±activity relationships and metabo-licfate
212
Integrating information on intake andtoxicity
212
Establishment of threshold values based on
non-carcinogenic endpoints
214
Establishment of a threshold value based oncarcinogenic endpoints
216
Comparison of sensitivity of various end-points
219
Application of a threshold of toxicologicalconcern to ¯avouring substances
220
Additional factors that reduce the theoreticalrisk
221
Safety decision criteria 222
Integrating data on consumption ratio 225Discussion 225References 225
Food and Chemical Toxicology 37 (1999) 207±232
0278-6915/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. Printed in Great BritainPII S0278-6915(98)00112-4
*Corresponding author.
Tables
Table 1 Number of ¯avouring substances within various intake categories 210Table 2 Safety margins between NOELs and per capita daily exposure for various ¯avouring
substances given full ADIs by JECFA211
Table 3 Number of ¯avouring substances divided by structural class within various intake cat-egories
213
Table 4 Fifth centile NOELs and human exposure thresholds for Cramer et al. (1978) struc-
tural classes in the reference database
216
Table 5 Low-dose slopes for bladder tumours in mice exposed to 2-AAF for 24 months basedon linear extrapolation from the TD50
217
Table 6 Probability of a target risk not being exceeded at various threshold values 219Table 7 Comparison of various human exposure threshold values 219Table 8 A list of functional groups identi®ed by Ashby and Tennant (1988, 1991) and
Tennant et al. (1990) as structural alerts for DNA reactivity222
Table 9 Flavouring substances within each Cramer et al. (1978) structural class consumed inamounts below human exposure thresholds
222
Figures
Fig. 1 Empirical cumulative distributions of NOELs of compounds in the reference databaseand lognormally ®tted cumulative distributions (solid lines). Compounds have been
grouped into the structural classes I, II, and III of Cramer et al. (1978)
215
Fig. 2 Dose±response curve for bladder tumours in mice exposed to 2-AAF for 24 months 217Fig. 3 Distribution of TD50s for 343 rodent carcinogens from the Gold et al. (1984) CPD
and distribution of 1�10ÿ6 risks calculated by linear extrapolation from the TD50s(modi®ed from Rulis, 1989)
218
Fig. 4 Comparison of immune and non-immune endpoint NOELs and LOELs (based on
NOELs and LOELs of 24 immunotoxic substances)
220
Fig. 5 Safety evaluation sequence 224
Appendix
Table A1 Substances reported to cause developmental abnormalities (from RTECS) 230Table A2 NOELs for organophosphorous insecticides 232
Table A3 Substances with immunotoxic NOELs 232Table A4 Substances with immunotoxic LOELs 232
Introduction
.The safety evaluation procedure described hereinwas developed for use by the Joint FAO/WHO
Expert Committee on Food Additives (JECFA) forthe safety evaluation of ¯avouring substances. Theprocedure extends principles and procedures rec-
ommended by JECFA in the past and is consistentwith international concepts in safety evaluation.These principles have been discussed by the JointFAO/WHO Expert Committee on Food Additives
(JECFA, 1972, 1974b, 1976a, 1978, 1980a, 1974b,1982a, 1983a, 1984a, 1986, 1987a, 1974b, 1989,1990a, 1991a, 1992) and have been recapitulated in
a report, which outlines criteria for the safetyevaluation of ¯avouring substances, published bythe World Health Organization (WHO, 1987).
There are approximately 2500 chemically-de®ned¯avouring substances in use either in Europe or theUnited States. Of these substances, approximately1500 have been evaluated by FEMA's Expert Panel
and are legally recognized by the US Food andDrug Administration (FDA) to be Generally
Recognized As Safe (GRAS) substances, meaning
that they are considered safe for their intended use.Without exception, ¯avouring substances are vol-
atile organic chemicals. The majority have simple,well characterized structures with a single func-
tional group and low molecular weight (<300 g/
mol). More than 700 of the 1323 chemically de®ned¯avouring substances used in food in the US are
simple aliphatic acyclic and alicyclic alcohols, alde-hydes, ketones, carboxylic acids and related esters,
lactones, ketals and acetals. Other structural cat-
egories include aromatic (e.g. cinnamaldehydes andanthranilates), heteroaromatic (e.g. pyrazines and
pyrroles) and heterocyclic (e.g. furanones and ali-cyclic sul®des) substances with characteristic orga-
noleptic properties. For most ¯avouring substances,
the structural di�erences within chemical groupsare small. Incremental changes in carbon chain
length and the position of a functional group or hy-drocarbon chain typically describe the structural
variation within groups of related ¯avouring sub-stances. These systematic changes in structure pro-
vide the basis for understanding the e�ect of
I. C. Munro et al.208
structure on their chemical and biological proper-ties.
Within structural groups of ¯avouring sub-stances, many substances have considerable toxi-cology data; repeat dose studies exist for many
substances or their metabolic products, and sev-eral representative members of structural groupshave chronic toxicity studies. At the 46th and
49th meetings of JECFA, the Committee (JECFA,1997, 1998) was able to use information onmetabolism, toxicity, and intake of individual sub-
stances within a group to evaluate 263 ¯avouringsubstances.
Conceptual framework for the safety evaluation of
¯avouring substances
Criteria for the safety evaluation of ¯avouringsubstances have been put forward by severalnational and international authorities. Included
among these are JECFA, a special Task Groupconvened by WHO (1987), the Committee ofExperts on Flavouring Substances of the Council ofEurope (CE), the Commission of the European
Communities' Scienti®c Committee for Food (SCF,1991), BIBRA International, and the Flavor andExtract Manufacturers' Association of the United
States Expert Panel (FEXPAN).In 1987, WHO published a health criteria docu-
ment entitled Principles of the Safety Evaluation of
Food Additives and Contaminants in Food, whichcontains a discussion of principles related to thesafety evaluation of ¯avouring substances. This
report recapitulated principles previously stated byJECFA on numerous occasions (WHO, 1987).Early on, it was recognized by JECFA that thesafety evaluation of ¯avouring substances war-
ranted special consideration in the light of use pat-terns and typically low levels of human intake(JECFA, 1972). This view also has been recognized
by the Scienti®c Committee for Food (SCF) andhas been recorded in their document entitledGuidelines for the Evaluation of Flavourings for Use
in Foodstu�s: 1. Chemically De®ned FlavouringSubstances (SCF, 1991).In addition, several organizations including
JECFA (WHO, 1987), FEXPAN (Woods and
Doull, 1991), SCF (1991) and the Council ofEurope (CE, 1974, 1981, 1992) have noted thatknowledge of structure±activity relationships and
metabolism plays a key role, along with intake,in the safety evaluation of ¯avouring substances.In this regard, JECFA (WHO, 1987; JECFA,
1996a,b, 1997, 1998) has used structure±activityrelationships in evaluating groups of structurallyrelated ¯avouring substances in a homologous
series where toxicology studies exist on only oneor a few members of the series. Structure±activ-ity relationships can provide a useful means ofevaluating substances that lack toxicity data by
using toxicity data from structurally related sub-stances.
Estimating intake of ¯avouring substances throughfood
Flavouring substances are used in processedfoods and beverages to impart desirable organolep-
tic qualities and to provide the speci®c ¯avour pro-®le traditionally associated with certain foodproducts. Unlike many substances which are added
to food to achieve a technological purpose, the useof ¯avouring substances is generally self-limitingand governed by the ¯avour intensity required toprovide the necessary organoleptic appeal. Thus,
¯avouring substances are used generally in low con-centrations resulting in human intakes that are verylow.
Estimates of ¯avouring substance intake havebeen performed in two ways. One method of calcu-lating intake is to combine data on the level of use
in speci®c food groups with data on the amount offood consumed to calculate the intake of each ¯a-vouring substance. Estimating intake of ¯avouringsubstances based on level of use data, coupled with
data on the amount of food consumed, typicallyleads to substantial overestimates of intake. This isbecause intake data for food commodities are strati-
®ed into broad categories of food products (e.g.baked products). Thus, cardamom, ordinarily usedonly in certain types of co�ee cakes, would be
assumed to be present in all breads, rolls, cakes andpastries. Moreover, within a category of food pro-ducts (e.g. hard candy) it is the ¯avouring substance
that characterizes speci®c brands of products. Thusassuming that a ¯avouring substance occurs in allbrands within a food category will lead to furtheroverestimates of intake. These assumptions can lead
to estimates of intake which may be exaggeratedseveral hundred-fold.A second method is to assume that the total
amount (poundage) reported to be used in foodannually is completely consumed by the total popu-lation. Between 1970 and 1987, the US National
Academy of Sciences/National Research Council(NAS/NRC) conducted, under contract to theFDA, a series of poundage surveys of substancesintentionally added to food (NAS, 1978, 1979,
1984, 1989). These surveys obtained information,both from ingredient manufacturers and from foodprocessors, on the poundage of each substance
committed to the food supply and on the usual andmaximum levels at which each substance was addedto foods in each of a number of food categories.
Numerous checks using data from independentsources, such as imports, show that, in general, thereported poundage in surveys accounts for only
60% of the total used. Therefore, calculations ofintake are corrected upwards to account for under-reporting. In addition, more detailed analyses (seeAnnex 5, etc., JECFA, 1996a) have led to the con-
Safety evaluation procedure 209
clusion that it was conservative but reasonable toassume that each ¯avouring substance is consumed
by only 10% of the population. Both methods tendto overestimate human intakes of ¯avouring sub-stances because they deal with disappearance, that
is the amount presumed to be used in food, andtake no account of losses and waste during foodmanufacture, storage, preparation and consump-
tion.Through a series of detailed studies conducted
between 1970 and 1987 (see JECFA, 1996a) it has
become clear that, while there is at present no per-fect way to estimate intake of ¯avouring substances,poundage data provide a reasonable basis for calcu-lating intakes. The intake data reported in this
paper rely on annual poundage used in food andthe estimates of intake re¯ect the assumptions that:(i) the available survey data accounted for only
60% of the amount actually used in food; and (ii)the total amount used is consumed by only 10% ofthe population. Table 1 presents the intake data for
1323 chemically de®ned ¯avouring substances per-mitted for use in the US calculated in this fashion.As can be seen from Table 1, most ¯avouring sub-
stances are consumed in amounts of less than 1 mg/person/day. The data are taken from the mostrecent US NAS/NRC survey (NAS, 1989) ofpoundage used in food. For reasons previously sta-
ted, it can be assumed that these intake estimatesare overestimated.
Consideration of natural occurrence of ¯avouringsubstances in food
Another important factor to consider in theevaluation of human intake of ¯avouring substances
is the extent to which ¯avouring substances inten-tionally added to foods also occur naturally in thefood supply. The natural presence of ¯avouringsubstances in food is of course not necessarily in-
dicative of safety. For many ¯avouring substancesthat occur naturally in foods, such natural occur-
rence, rather than intentionally added use, is theprincipal source of human exposure. The compari-son of natural occurrence to intentional addition
has been expressed as the consumption ratio (CR)(Stofberg and Kirschman, 1985). A CR of greaterthan 1 indicates a predominant natural occurrence
in food (i.e. the ¯avouring substance is consumed ata higher level from foods than as an added sub-stance). A CR greater than 10 indicates an almost
insigni®cant contribution (approx. 10%) of the ¯a-vouring substance as a food additive to the totalintake (Stofberg and Grundschober, 1987). Stofbergand Grundschober (1987) calculated that out of 499
¯avouring substances, 415 (83%) had a predomi-nant natural occurrence in food (i.e. CR>1) and309 (62%) made an insigni®cant contribution when
added to the food supply (i.e. CR>10). As can beseen from this analysis, the use of natural occur-rence as part of the safety evaluation provides an
important perspective on the impact of intentionaladdition of ¯avouring substances to foods.If a ¯avouring substance is one of the few that
for any reason, including high intake, is of rela-tively high safety concern, then a high consump-tion ratio probably enhances that concern becauseit indicates a much larger and uncontrolled ex-
posure from natural occurrence than from inten-tional addition. If, on the other hand, a¯avouring substance is one of the majority that
have few, if any, safety issues and consequentlylow inherent concern, then a high consumptionratio reduces any concern still further because it
indicates that intentionally added use is trivial.Stated in another way, if the added use of a ¯a-vouring substance amounts to less than 10% ofits natural occurrence, this would indicate a mini-
mal safety concern about added use. If added useis less than 1% of natural use (i.e. CR>100)then the added use will at most be of trivial
safety concern.
Structure±activity relationships
Toxicity is dependent on the chemical structureof a substance, its pharmacokinetics, and its meta-bolic reaction pathways. Available metabolic path-
ways are usually dose dependent and, to a largeextent, govern the magnitude of the toxic e�ect.Therefore, chemical structure, pharmacokinetics,
metabolic fate and dose are key determinants oftoxicity and play a critical role in safety evaluationof ¯avouring ingredients.
Re®nements to initial concepts of structure±activ-ity came as a result of increasing knowledge andcon®dence in predicting structure±activity relation-
ships for ¯avouring substances. These formed thebasis of a paper by Cramer et al. (1978) which,through the use of a ``decision tree'' approach, per-mitted the classi®cation of ¯avouring substances
Table 1. Number of ¯avouring substances* within various intakecategories
Intake category$(mg/day)
No. of¯avours
Cumulative frequency(% of total)
<0.01 349 260.01±0.1 93 330.1±1 274 541±10 224 7110±100 204 86100±1000 111 951000±10,000 45 9810,000±100,000 16 99100,000+ 7 100TOTAL 1323
*Chemically de®ned ¯avouring substances permitted for use in theUS excluding botanicals.
$Intake data calculated assuming: survey poundage re¯ects 60%of actual usage, 10% of population exposed, US population in1987 was 240 million. Formula: intake (mg/person/day) = [(-annual ¯avour usage in mg)60.6]6(24� 106 persons� 365days). Poundage data from 1987 NAS/NRC survey data(NAS, 1989).
I. C. Munro et al.210
into ``classes of concern'' based on structure andother considerations, similar in many respects to,
but predating the ``Concern Level'' concept outlinedby the US FDA in its ``Redbook'' (FDA, 1982,1993).
The concept of establishing concern levels alsohas been investigated further by BIBRAInternational to evaluate food chemicals more gen-
erally (Phillips et al., 1987). This group establishedconcern levels for several food additives, plasticmonomers, as well as ¯avouring substances.
Although they reported that, in their opinion, theCramer et al. (1978) decision tree misclassi®ed afew substances, the decision tree was likely to be amore realistic approach for predicting toxicity than
any other reported quantitative structure±activityrelationship (QSAR) technique. Thus, there is gen-eral consensus, based on the work of Cramer et al.
(1978), the subsequent work by BIBRA (Phillipset al., 1987), and the fact that the FDA (1982;1993) uses structure±activity relationships in de®n-
ing concern levels for food substances, that struc-ture±activity has a solid basis in science whenapplied to substances of simple and closely related
structure, especially those with low intakes, low tox-icity and safe metabolic products. This is the casefor all but a very few ¯avouring substances. As willbe discussed later in this review, structure±activity
relationships play an important role in the evalu-ation of ¯avouring substances.
Use of toxicity data
Traditional approaches to the safety assessment
of food additives typically involve the evaluation ofconsiderable toxicological data, usually in anamount su�cient to establish a no-observed-e�ectlevel (NOEL), permitting the establishment of an
acceptable daily intake (ADI). Approximately halfof the ¯avouring substances currently in use arenaturally occurring simple acids, aldehydes, alco-
hols and esters. With few exceptions, these arerapidly metabolized to innocuous end-products, thesafety of which is well established or can be
assumed from metabolic and toxicity data on thesubstance in question or on structurally related sub-stances. In other words, the acquisition of extensivetoxicity data is unnecessary for the majority of ¯a-
vouring substances because structure±activity re-lationships can be used as a means of assessingsubstances in a homologous series, in which only a
few substances have toxicology data, to determinesafety in use. This concept has been used byJECFA in the evaluation of structurally related ¯a-
vouring substances, including the allyl esters, amylacetate and isoamyl butyrate, benzyl compounds,citral compounds, a- and b-ionones, and nonanal
and octanal (JECFA, 1967, 1968, 1980a, 1984a,b,1990a,b, 1991a,b, 1993a,b). In addition, as pre-viously indicated in Table 1, 95% of ¯avouring sub-stances are consumed at intake levels less than
1 mg/person/day and in keeping with the safetyevaluation procedure outlined in this paper, only
limited toxicological data are required or justi®ed insuch circumstances. For the reasons stated above,traditional safety evaluation procedures are not
necessarily applicable to ¯avouring substances.When intake is extremely low and there are orga-
noleptic limitations on use levels, a primary con-
sideration is whether there is a need to establish anumerical ADI for ¯avouring substances. There areseveral reasons why it is not appropriate or necess-
ary to establish ADIs for the majority of ¯avouringsubstances. ADIs are based on toxicological dataand the establishment of a NOEL, an approachthat di�ers from the concept that a safety evalu-
ation can be performed in many instances on thebasis of intake and structure±activity relationships.Moreover, the organoleptic and gustatory proper-
ties of ¯avouring substances typically limit their usein speci®c food products and, consequently, intake.In addition, because a majority of ¯avouring sub-
stances occur in nature, there is a long history ofhuman experience in ¯avouring substance consump-tion from traditional foods. Nearly 50% of ¯avour-
ing substances given full ADIs by JECFA haveconsumption ratios greater than 1, indicating theirpredominantly natural occurrence in food (Stofbergand Grundschober, 1987). The above factors have
been noted by WHO (1987) as important in theevaluation of ¯avouring substances. It is also evi-dent that very large safety margins exist for ¯avour-
ing substances, as evidenced by the fact that themargin between the NOEL and the intake of ¯a-vouring substances reaches up to more than
100,000 times for the ¯avouring substances givenfull ADIs by JECFA (Table 2).
Elements of the safety evaluation procedure
In a continuing e�ort to improve the basis forthe safety evaluation of ¯avouring substances, thisreview presents a procedure which integrates infor-
mation on intake, structure±activity relationships,metabolic fate and toxicity. It presents a safetyevaluation procedure which allows a determination
of the safety of ¯avouring substances under con-
Table 2. Safety margins between NOELs and per capita dailyexposure for various ¯avouring substances given full ADIs by
JECFA*
Safety margin Number of ¯avours
<100 1100±1000 41000±10,000 1310,000±100,000 9100,000+ 7Total 34
*JECFA, 1967, 1968, 1970, 1971, 1972, 1974a,b, 1976a,b, 1978,1980a,b,c, 1981a,b, 1982a,b, 1983a,b, 1984a,b, 1986, 1987a,b,1989, 1990a,b, 1991a,b, 1992, 1993a,b,c.
Safety evaluation procedure 211
ditions of intended use. The general principles uponwhich the safety evaluation procedure is based have
been elaborated previously (Munro et al., 1998).The key elements of the safety evaluation procedureare discussed below.
Structure-activity relationships and metabolic fate
Toxicity is dependent on the chemical structure
and metabolism of a substance. The ``decision tree''procedure (Cramer et al., 1978) relies primarily onchemical structure and estimates of total human
intake to assess toxic hazard and to establish priori-ties for appropriate testing. The procedure utilizesrecognized pathways of metabolic deactivation andactivation, data on toxicity, and the presence of the
substance as a component of traditional foods andas an endogenous metabolite. Substances are classi-®ed according to three categories:
Class I. Substances of simple chemical structurewith known metabolic pathways and
innocuous end-products which wouldsuggest a low order of oral toxicity(e.g. butyl alcohol or isoamyl buty-
rate).Class II. Contains structures that are intermedi-
ate. They possess structures that areless innocuous than substances in Class
I, but do not contain structural featuressuggestive of toxicity like those sub-stances in Class III. Members of Class
II may contain reactive functionalgroups (e.g. furfuryl alcohol, methyl 2-octynoate, and allyl propionate).
Class III. Substances of a chemical structure thatpermit no strong initial presumption ofsafety, or may even suggest signi®canttoxicity (e.g. 2-phenyl-3-carbethoxy
furan and benzoin).
The decision tree is a tool for classifying ¯avoursubstances according to levels of concern. The ma-jority of ¯avouring substances fall into Class I
because they are simple alcohols, aldehydes,ketones, acids or their corresponding esters, acetalsand ketals that occur naturally in food and, inmany cases, are endogenous substances. They are
rapidly metabolized to innocuous products (e.g. car-bon dioxide, hippuric acid, and acetic acid) by wellrecognized reactions catalysed by enzymes that
exhibit high speci®city (e.g. alcohol dehydrogenaseand isovaleryl coenzyme A dehydrogenase).Substances that do not undergo detoxication via
these highly e�cient pathways (e.g. fatty acid path-way and citric acid cycle) are metabolized by reac-tions catalysed by enzymes of low speci®city (e.g.
cytochrome P-450 and glutathione transferase).This class of enzymes is saturated at lower intercel-lular concentrations than are higher capacityenzymes. For some groups of substances (e.g.
branched-chain carboxylic acids, allyl esters and lin-ear aliphatic acyclic ketones), metabolic thresholds
for intoxication have been identi®ed (Deisingeret al., 1994; Jaeschke et al., 1987; Krasavage et al.,1980). The dose range, over which a well de®ned
change in metabolic pathway occurs, generally cor-relates with the dose range over which a transitionoccurs from a no-observed-adverse-e�ect level to an
adverse-e�ect level. For such groups of substances,the dose range at which this transition occurs isorders of magnitude greater than the level of intake
from use as ¯avour substances.Most substances in Class II belong to either of
two categories; one includes substances with func-tional groups which are similar to, but somewhat
more reactive than functional groups in Class I (e.g.allyl and alkyne); the other includes substances withmore complex structures than substances in Class I,
but that are common components of food. This cat-egory includes heterocyclic substances (e.g. 4-methylthiazole) and terpene ketones (e.g. carvone).
The majority of the ¯avouring substances withinClass III include heterocyclic and heteroaromaticsubstances and cyclic ethers. Many of the hetero-
cyclic and heteroaromatic substances have side-chains with reactive functional groups. In a fewcases, metabolism may destroy the heteroaromati-city of the ring system (e.g. furan). Although
metabolism studies have been performed for ClassIII ¯avouring substances with elevated levels ofintake, the metabolic fate of many substances in
this structural class cannot be predicted con®dently.Importantly, however, review of the group of sub-stances in each of the structural classes indicates
that as structural complexity increases (Class I±III),the number of ¯avouring substances and the levelsof intake decrease signi®cantly (Table 3).
Integrating information on intake and toxicity
One of the key elements of the safety evaluationprocedure is based on the premise that intake levels
can be speci®ed for ¯avouring substances thatwould not present a safety concern. The concept ofspecifying human exposure thresholds relies onprinciples that permit specifying the daily intake of
a substance which can be considered, for practicalpurposes, as presenting no toxicological risks (andthus of no health or safety risk to consumers) even
in the absence of speci®c toxicological data on thesubstance (Federal Register, 1993; Frawley, 1967;Munro, 1990; Rulis, 1986). The concept relies on
knowledge of the range of toxicological risks forstructurally related substances and on knowledgeregarding the toxicological potency of relevant
classes of chemicals for which good toxicity dataexist. With the possible exception of so-called geno-toxic carcinogens, the concept of a threshold intoxicological responses is universally accepted and
I. C. Munro et al.212
endorsed by WHO (1987, 1994). The principles
underpinning the establishment of human exposure
thresholds have been embodied in a Federal
Register (1993)* notice emanating from the US
FDA, which provides the scienti®c basis for the
conclusion that an intake level for indirect food
additives can be speci®ed, below which no risk to
public health would be likely to accrue. This intake
level has, in turn, been used by FDA to establish a
proposed ``threshold of regulation'' for indirect
food additives which precludes the need for toxico-
logical evaluation of substances migrating into food
from food-contact articles provided the amount
that migrates does not lead to a dietary level in
excess of 500 ppt (equivalent to 1.5 mg/person/dayassuming a daily food intake of 3000 g). The FDA
has noted that such a level would result in negli-
gible risk to consumers even if the substance was
shown later to be a carcinogen. This concept is in
keeping with the well established principle that
resources should be directed to the safety evaluation
of substances having high intake and therefore
greater potential for adverse e�ects and not towards
substances with trivial intake. The concept is par-
ticularly applicable to substances of low toxicity
and with known or predictable metabolic fate. The
scienti®c basis for the establishment of human ex-
posure thresholds and the FDA regulation are dis-
cussed below.
The concept that a generic threshold value or
range of values might be established that would
preclude the need for toxicity data on chemicals
having human intakes below these thresholds wasproposed over 30 years ago by Frawley (1967). He
showed, on the basis of studies conducted on sev-eral well-tested substances, including food additives,
industrial and consumer chemicals, and pesticidesthat a generic ``no-e�ect'' level could be establishedthat could preclude the need for toxicity studies and
safety evaluation for a majority of substancesintended for use as food packaging materials.
Frawley constructed a reference database of non-tumorigenic endpoints using 220 2-year rodent stu-dies. He presented the NOELs for all 220 com-
pounds. Frawley (1967) reported that if he excludedheavy metals and pesticides from the analysis, there
was no compound in the remaining database(except for acrylamide) which showed evidence ofchronic toxicity at dietary concentrations of less
than 100 ppm. Application of a typical 100-foldsafety factor to the 100 ppm generalized NOEL
would mean that humans could safely consume anyof the materials provided the dietary concentrationdid not exceed 1 ppm. Frawley (1967), noting that
his database was incomplete, proposed adding anadditional safety factor of 10 which would translate
to a toxicologically insigni®cant human exposurelevel of 0.1 ppm in the diet. Assuming an individualconsumes 1500 grams of food per day, an exposure
of 150 mg/person/day (approximately 2.5 mg/kgbody weight/day) or less to a chemical of unknown
toxicity would be considered toxicologically insig-ni®cant. According to Frawley (1967), such ex-
posures could be considered of no safety concern.More recently, Rulis (1986) conducted a similar
analysis of the FDA's Priority-Based Assessment of
Food Additives (PAFA) database containing 159compounds with subchronic or chronic toxicity
data and came to the same conclusion as Frawley(1967). Essentially, there is no risk of toxicity inrodents exposed to certain food additives at dietary
levels of less than 1 mg/kg body weight/day, or inhuman terms, approximately 1 to 10 mg/kg body
Table 3. Number of ¯avouring substances* divided by structural class within various intakecategories
Intake category$No. of ¯avours (% of total)
(mg/day) Class I Class II Class III
<0.01 212 (24) 68 (28) 69 (34)0.01±0.1 55 (6) 20 (8) 18 (9)0.1±1 169 (19) 48 (20) 57 (28)1±10 145 (17) 45 (19) 34 (17)10±100 147 (17) 39 (16) 18 (9)100±1000 95 (11) 12 (5) 4 (2)1000±10,000 34 (4) 9 (4) 2 (1)10,000±100,000 16 (2) 0 0100,000+ 5 (0.6) 2 (0.8) 0Total 878 243 202
*Chemically de®ned ¯avouring substances permitted for use in the US excluding botanicals.$Intake data calculated assuming: survey poundage re¯ects 60% of actual usage, 10% of popu-
lation exposed, US population in 1987 was 240 million. Formula: intake (mg/person/day) = [(-annual ¯avour usage in mg)60.6])624� 106 persons� 365 days). Poundage data from 1987NAS/NRC survey data (NAS, 1989).
*In 1993, the US FDA proposed a dietary concentrationof 500 ppt as the threshold of regulation for substancesused in food-contact articles. Assuming that an indi-vidual consumes 1500 g of solid food and 1500 g ofliquid food per day, this threshold would equate to atoxicologically inconsequential level of 1.5 mg/day(Federal Register, 1993). This proposal became a ®nalrule in 1995 (Federal Register, 1995).
Safety evaluation procedure 213
weight/day depending on the safety factor applied.Even 20 years apart, using di�erent databases, the
toxicologically inconsequential levels proposed byFrawley (1967) and Rulis (1986) were nearly identi-cal.
Munro (1990) used a database of approximately350 substances compiled by Gold et al. (1984, 1989)to develop a human exposure threshold value to be
applied to substances for which no presumption ofsafety can be made because of a complete lack ofdata on metabolism and potential toxicity. Munro
(1990) proposed a threshold of regulation of up to1000 ppt for indirect additives which would trans-late to a daily intake of 1.5 to 3.0 mg/person/daydepending on assumptions regarding food intake.
The acceptable level considered by FDA in its ®nalrule (Federal Register, 1995) to present no regulat-ory concern for an indirect food additive from food
packaging material, even if later it was determinedto be a carcinogen equates to a daily intake of1.5 mg/person/day (Federal Register, 1993).
The approach of using a threshold of concernprovides an alternative to the conventional regulat-ory philosophy of rigorously testing each new
chemical substance regardless of expense or level ofhuman intake. Two factors, pragmatism and scienti-®c knowledge, have in¯uenced the evolution of thethreshold concept. The scienti®c information base is
now su�ciently large to consider application of athreshold of toxicological concern as a concept thatis both practical and scienti®cally defensible. On a
purely pragmatic level, it is recognized that humansare exposed to thousands of substances through thefood supply and the number of substances increases
logarithmically with declining concentration (Hall,1975). It is neither practical nor scienti®cally defen-sible to test all these substances by conventionaltoxicological procedures, and to insist this be done
would create a resource problem of immense pro-portions. Another more important factor that jus-ti®es an approach using a threshold of toxicological
concern, is that in the past 10 to 15 years a greatdeal of knowledge has accumulated about the po-tential human risks from chemicals in general and
especially for those which are carcinogenic. On thebasis of accumulated knowledge, it is theoreticallypossible to establish a range of threshold values
representing the full spectrum of toxicological end-points including both carcinogenic and non-carcino-genic e�ects.
Establishment of threshold values based on non-carci-nogenic endpoints
The work conducted by the FDA (FederalRegister, 1993), Frawley (1967), Rulis (1986) andMunro (1990) was expanded upon by Munro et al.
(1996) through the compilation of a large databaseof reference substances from which a distribution ofNOELs could be derived for chemicals of variousstructural types. The reference database describes
the relationships between intake, structure and tox-
icity for a wide variety of chemicals of divergentstructure and it can be used as a reference pointfrom which to judge the safety of ¯avouring sub-
stances.In compiling the database, strict criteria were
applied in the selection of data sets. The objective
of the exercise was to identify as many high qualitytoxicological studies as possible representing a var-
iety of toxic endpoints and chemical structures. Toaccomplish this, the study types included those typi-cally conducted in toxicology, such as subchronic,
chronic, reproductive and teratology studies. Short-term and acute studies were not included sincethese were considered not to be relevant for estab-
lishing chronic NOELs. The database consistedmainly of studies in rodents and rabbits. Very few
studies in dogs and other species were found thatmet the established criteria. An evaluation of ran-domly selected dog and primate studies indicated
that many had too few animals per group to derivea statistically valid NOEL. Moreover, for many dogstudies, a common endpoint was reduced body
weight and/or food consumption which was due, inmany cases, either to palatability problems with the
diet, or vomiting. In addition, most studies in dogsand other non-rodent species were simply too shortin duration to be classi®ed as chronic studies. Only
oral studies were included in the database. Afurther criterion for inclusion in the database wasthat a study had to have a demonstrated lowest-
observed-e�ect level (LOEL) as well as a NOEL,thus ensuring that the study was rigorous enoughto detect toxic e�ects. In some instances NOELs
were included for studies not demonstrating aLOEL, and these were substances such as major
food ingredients that were without toxicity at thehighest dose tested in well conducted studies. Itshould be noted that the inclusion of such sub-
stances in the database would not bias the databasein favour of higher NOELs since the true NOELfor such substances probably would exceed the
NOEL established from the available studies.In order to combine NOELs for substances with
only subchronic studies with those with chronic stu-dies to derive the cumulative distribution ofNOELs, subchronic NOELs were divided by a fac-
tor of 3 to approximate the most likely NOEL thatwould be derived from a chronic study. This con-version factor is based on research de®ning the re-
lationship between subchronic and chronic NOELs.Weil and McCollister (1963) compared 3-month
NOELs with 2-year NOELs for 33 di�erent sub-stances (including pharmaceuticals, pesticides andfood additives) fed to rats. They found that for
most of the compounds (30), the ratio of theNOELs between subchronic and chronic studieswas 5 or less and more than half of the compounds
had a ratio equal to 2 or less. More recently, it hasbeen discovered through further analysis of more
I. C. Munro et al.214
chemical substances, that a more accurate adjust-
ment factor for extrapolating NOELs derived from
subchronic studies to lifetime was between 2 and 3(Beck et al., 1993; Lewis et al., 1990; Dourson, per-
sonal communication).
Emphasis was placed on retrieving data from cer-
tain databases known to contain well validated toxi-cological endpoints for a series of well-de®ned
chemical structures. An exhaustive search was made
of compounds evaluated by JECFA. Other sourcesincluded the US Environmental Protection
Agency's (EPA) Integrated Risk Information
System (IRIS) on-line database, the National
Toxicology Program (NTP) studies, theDevelopmental and Reproductive Toxicity (DART)
on-line database from EPA and the US National
Institute of Environmental Health Sciences and thepublished literature in general. The data entered
into the database included the name of the chemi-
cal, Chemical Abstracts Service Registry Number(CAS No.), structural classi®cation as assessed
using the Cramer et al. (1978) decision tree and the
FDA ``Redbook'', species, sex, route of adminis-tration, dose levels tested, study type, duration,
endpoints reported, LOEL, NOEL and references.
In an e�ort to be conservative in the construction
of the reference database, NOELs selected by theauthor(s) of each study were used even though in
some cases authors tended to over-interpret their
data. In some instances, it was found that the statedNOEL may have been based on a misjudgment of
an adverse e�ect by the author (e.g. physiological
versus toxicological e�ects) or on artefactual e�ects(e.g. foetal toxicity as a result of maternal toxicity).
An example of this is isopropyl alcohol, which has
been reported to produce teratogenic e�ects at very
low doses (0.018 mg/kg) in one study; however, itsstructure, known metabolism and other toxicologi-
cal data provide no evidence for concluding terato-
genicity. Even though some of these author-derivedNOELs were not thoroughly substantiated, they
were included in the reference database, thereby
increasing the degree of its conservative nature.NOELs selected by EPA for the IRIS database
were entered without further review. In all, the
database consists of over 600 substances represent-ing a range of industrial chemicals, pharmaceuticals,
food substances and environmental and consumer
chemicals likely to be encountered in commerce. As
the database was developed as a reference databasefor the evaluation of ¯avouring substances, all of
which are organic chemicals, no organometallic or
inorganic compounds were included in the data-base. For many of the substances, more than one
NOEL was identi®ed from the literature resulting
from the fact that some substances were tested inmore than one species and sex and/or demonstrated
a range of endpoints suitable for establishing a
NOEL. This led, in some cases, to multiple NOELsfor individual substances. In all, the database con-
tains nearly 3000 entries.
For each of the substances in the database, classi-
®ed corresponding to the three structural classes
outlined in Cramer et al. (1978), the most conserva-tive NOEL was selected from the reference database
based on the most sensitive species, sex and end-
point. The cumulative distribution of the NOELswithin each class is shown in Fig. 1, along with the
Fig. 1. Empirical cumulative distributions of NOELS of compounds in the reference database and log-normally ®tted cumulative distributions (solid lines). Compounds have been grouped into the structural
classes I, II and III of Cramer et al. (1978).
Safety evaluation procedure 215
lognormal distributions ®tted to these data. Theseresults clearly delineate the e�ects of structural class
on toxicity, with the median (50th centile) NOELdecreasing from Class I through III. Similar di�er-ences among structural classes exist in the range
between the 5th and 95th centiles.The human exposure threshold for each of the
structural classes was calculated from the 5th centile
NOEL. The 5th centile NOEL was chosen becausethis value would provide 95% con®dence that anyother substance of unknown toxicity but of the
same structural class as those comprising the refer-ence database would not have a NOEL less thanthe 5th centile for that particular structural classwithin the reference database.
The 5th centile NOELs for each structural classare shown in Table 4. In converting the 5th centileNOELs to human exposure thresholds (Table 4) for
the various structural classes, a 100-fold safety fac-tor was used since such a factor would inherentlybe applied in establishing safe intake levels for the
substances comprising the database. The use ofsuch a factor provides a substantive margin ofsafety since the human exposure thresholds are
based on a large database of over 600 compoundswith good supporting toxicological data.Furthermore, 5th centile NOELs were used to cal-culate the thresholds, providing a more conservative
®gure than the arithmetic mean. Moreover, the esti-mated daily intakes of ¯avouring substances towhich the human exposure threshold are compared
are greatly overestimated as they represent the``eaters only'' (10%) population. Thus, it is believedthat a 100-fold safety factor provides a wide margin
of safety in relating the results of the analysis of thereference database to ¯avouring substance intake.It is evident from Table 4 and Fig. 1 that there
are substantial di�erences in the 5th centile NOELs
for the various structural classes, indicating anobvious e�ect of structure on toxic potency.
Establishment of a threshold value based on carcino-genic endpoints
Over the past several years, an immense amountof information has accumulated on the range ofcarcinogenic potencies for chemicals that have been
tested in animals. For these chemicals, the distri-
bution of potencies in experimental animals andprojected human risk (calculated using linear riskassessment models) are well established and highly
unlikely to be altered by further cancer bioassays(Krewski et al., 1990). In fact, the CarcinogenicPotency Database (CPD) compiled by Gold et al.
(1984), now contains nearly 500 substances reportedto be carcinogenic in animals. It is reasonable to
assume that the addition to that database of severalmore ``genotoxic'' carcinogens, should they be dis-covered, would be unlikely to alter the distribution
of known risks for identi®ed carcinogens. Scientistsmay never be prepared to say they know all theywould like to know about the distribution of risks
of existing animal carcinogens. On the other hand,it can be estimated, with considerable con®dence,based on data available today that a substance
which has not been tested for carcinogenicity andthat is consumed in an amount below the threshold
value of 1.5 mg/day will not present a greater thanone-in-one million (10ÿ6) risk of human cancer.With these thoughts in mind, it is now important
to look at the theoretical and practical aspects ofthe concept of threshold of concern and how it can
be applied to ¯avouring substances in the contextof JECFA safety evaluations.The CPD contains data on approximately 3700
long-term animal studies of 975 chemicals (Goldet al., 1986a,b,c, 1989). These include studies con-ducted by the US National Toxicology Program as
well as studies conducted in other laboratories thathave been published in the literature. Of the 975
chemicals tested, 955 were tested in rats and/ormice and 492 produced an increase in tumour inci-dence (342 in rats and 278 in mice). Gold and co-
workers have put an enormous e�ort into compilingthis database and ensuring its quality. The reader isreferred to a series of papers by Gold et al. and
others published in Environmental HealthPerspectives which document the characteristics ofthis database (Gold et al., 1984, 1986a,b,c, 1989;
Peto et al., 1984; Sawyer et al., 1984).For each compound, the CPD may include ex-
periments with di�erent species, strains, sexes,dosing regimens, routes of administration, orother experimental conditions (Gold et al., 1984).
In most experiments, two or more dose levelswere used in addition to an unexposed control;
in some cases, however, only a single exposedgroup was employed. Although a rigorous evalu-ation of the quality of individual experiments is
not possible, the CPD does include informationon the original investigators' conclusions regard-ing the overall strength of evidence for carcino-
genicity.The CPD also contains a measure of carcinogenic
potency (the TD50) computed as described by Petoet al. (1984) and Sawyer et al. (1984). In order toobtain some degree of comparability among di�er-
Table 4. Fifth centile NOELs and human exposure thresholds forCramer et al. (1978) structural classes in the reference database
5th Centile NOELs(mg/kg/day)
Human exposurethreshold(mg/day)*$
I 137 2993 1800II 28 906 540III 447 147 90
*The human exposure threshold was calculated by multiplying the5th centile NOEL by 60 (assuming an individual weighs 60 kg)and dividing by a safety factor of 100, as discussed in the text.
$Numbers rounded to two (2) signi®cant ®gures.
I. C. Munro et al.216
ent studies, all TD50s are expressed in units of milli-
grams per kilogram body weight per day, and are
adjusted to a 2-year standard rodent lifetime. Incases where intake is not constant throughout the
study period, a time-weighted average dose is used
for purposes of modelling dose±response. When in-dividual animal data are available, the TD50s are
adjusted for intercurrent mortality; otherwise, the
crude proportions of animals with tumours are usedto estimate carcinogenic potency without adjusting
for mortality from other causes. Finally, the TD50
is estimated on the basis of an essentially linear
one-hit dose±response model.
The CPD represents an extremely useful sourceof information on experiments with chemical carci-
nogens. The database includes experiments on
highly potent rodent carcinogens, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and a¯atoxin B1, as
well as less potent agents such as metronidazole
and DDT. Gold et al. (1984) noted that TD50
values included in the CPD vary by 10 million-fold.
Rulis (1986) used the Gold et al. (1984,1986a,b,c, 1989) database when he and others at
the FDA derived a threshold of regulation for
food-contact materials. They transformed the distri-bution of lowest TD50 for each carcinogen that was
tested by the oral route to a distribution of 10ÿ6
risks. While numerous mathematical models, includ-ing the linearized multistage model, could have
been used to perform this transformation, Rulis
(1986) used the slope (2TD50)ÿ1 of a straight line
joining the TD50 and the origin as an estimator ofthe slope in the low dose region. Although the line-
arized multistage model could have been used since
it has the advantage of allowing for curvature in
the dose±response curve, linear extrapolation from
the TD50 is computationally simple, extremely con-
servative, and requires only published potency
values from the CPD.
To illustrate the di�erences between these two
approaches, Krewski et al. (1990) considered the
data on bladder tumours in mice exposed to the ex-
perimental carcinogen 2-acetyl amino¯uorene (2-
AAF), shown in Fig. 2. As indicated in Table 5, the
TD50 for bladder tumours based on the ®tted multi-
stage model is 17.2 mg/kg body weight/day, leading
to a slope of (2TD50)ÿ1=0.0291 (mg/kg/day)ÿ1.
Because over 3300 animals were involved in this ex-
periment, the 95% lower con®dence limit on the
TD50 and the corresponding upper con®dence limit
on the slope are close to the best estimates obtained
from the ®tted model. Owing to the high degree of
curvature in the dose±response curve for bladder
tumours, however, the upper con®dence limit on
Table 5. Low-dose slopes for bladder tumours in mice exposed to2-AAF for 24 months based on linear extrapolation from the
TD50
Source of TD50
TD50
(mg/kg/day)Slope (2 TD50)
ÿ1
(mg/kg/day)
Fitted multistage modelBest estimate 17.2 0.029195% Con®dence limit 16.7* 0.0298$
Fitted one-hit modelBest estimate 96.0 0.005295% Con®dence limit 77.4* 0.0065$
95% Con®dence limit onthe linear term in themultistage model (q1*)
0.0004$
*Lower con®dence limit.$Upper con®dence limit.
Fig. 2. Dose±response curve for bladder tumours in mice exposed to 2-AAF for 24 months.
Safety evaluation procedure 217
the slope based on linear extrapolation from the
lower con®dence limit on the TD50 is more than 75-
fold greater than the slope derived from the linear-
ized multistage model.
These data show that when there is signi®cant
upward curvature in the dose±response curve, the
methodology employed by Rulis (1986) to calculate
a dose associated with a 10ÿ6 risk will produce a
value substantially lower than when these risk-
speci®c doses are calculated using the linearized
multistage model. This point also has been made byHoel and Portier (1994), who noted that examin-
ation of the shape of the dose±response curve for
315 chemicals found to produce tumours in the
NCI/NTP program indicates that tumour site data
were more often consistent with a quadratic re-
sponse than a linear response suggesting that the
use of linear dose±response models will often over-
estimate risk. It may therefore be concluded thatthe methodology used by Rulis (1986) (Federal
Register, 1993, 1995) to estimate the distribution of
10ÿ6 risks for carcinogens in the Gold et al. CPD
was very conservative.
The next step in the process of establishing athreshold value involves the selection of an appro-
priate intake which is based on the distribution of
10ÿ6 risks. This value must be both highly protec-
tive of human health and of su�cient practical
value to reduce the number of compounds requiring
formal toxicological testing. Thus any threshold
selected should have an acceptably high probability
of health protection whereas at the same time theselected threshold value is still of su�cient magni-
tude to be of practical value. Of course, the protec-
tion of human health is of greater concern than the
practical value.
Initially Rulis (1986, 1989) proposed, for illus-tration, a threshold value of 0.15 mg/person/day.
Based on the distribution of 10ÿ6 risks from the
CPD, this value would intersect the distribution at
the 85th centile meaning that only 15% of carcino-
gens in the database would present a greater than
10ÿ6 risk at an intake of 0.15 mg/person/day. This
analysis indicates that at an intake of 0.15 mg/per-son/day, 85% of the chemicals in the CPD knownto induce cancer in rodents would fail to show a
signi®cant increase in risk for the exposed popu-
lation. This is demonstrated graphically in Fig. 3
modi®ed from Rulis (1989).
Subsequent to this publication by Rulis, Munro
(1990) held a workshop to evaluate factors that in-¯uence the selection of an appropriate threshold
value. The workshop ®rst reanalysed the Gold et al.
(1984) database using the original database of 343
rodent carcinogens and con®rmed the observations
of Rulis (1986, 1989) that a dietary intake of
0.15 mg/day intersected the distribution of 10ÿ6 risks
at approximately the 85th centile. In addition, the
workshop extended the analysis to include ad-
ditional carcinogens added to the original Goldet al. (1984) database, bringing the total to 492
rodent carcinogens (Gold et al., 1989). This reanaly-
sis with a broader set of data produced essentially
the same distribution of 10ÿ6 risks as was originally
published by Rulis (1986, 1989). The workshop par-
ticipants also noted that inherent in the acceptance
of any threshold value was the assumption that
every new untested substance could be a carcinogen
and could be as potent as the most potent 15% of
carcinogens in the CPD. Recognizing that not everynew substance would turn out to be a carcinogen,
the workshop (Munro, 1990) constructed a table of
risk avoidance probabilities (Table 6).
Table 6 shows the e�ect of various assumptions
regarding the proportion of chemicals that are pre-sumed carcinogens on the probability that a 10ÿ6
Fig. 3. Distribution of TD50s for 343 rodent carcinogens from the Gold et al. (1984) CPD and distri-bution of 1 x 10ÿ6 risks calculated by linear extrapolation from the TD50s (modi®ed from Rulis, 1989).
I. C. Munro et al.218
risk standard will not be exceeded. It should be
noted that as this proportion decreases, the prob-ability of not exceeding a speci®c risk standardincreases dramatically. Thus, for example, while
there is a 63% chance that the risk will not exceed10ÿ6 with a value of 1.5 mg/person/day when 100%of new chemicals are assumed to be carcinogenic,
the probability that the risk will be less than 10ÿ6 is96% when only 10% of new chemicals are assumedto be carcinogenic. Moreover, if one invokes a lessconservative risk standard of 10ÿ5 (Table 6, right),
then the probability of not exceeding that risk at athreshold value of 1.5 mg/person/day exceeds 96%even if it is assumed that 50% of new chemicals are
potential carcinogens. In theory, the probability ofan untested substance having a potency greaterthan the median of the distribution of TD50s from
the CPD (Gold et al., 1989) is 50%. In reality, how-ever, it is most unlikely that a genotoxic carcinogenwith a potency equal to or greater than the mediancarcinogen in the Gold et al. (1989) database would
be discovered from the existing inventory of ¯a-vouring substances given existing knowledge ofstructure±activity relationships in carcinogenesis.
Taking the above factors into consideration andkeeping in mind that the calculated 10ÿ6 risks basedon the Gold et al. (1989) database were derived
using a highly conservative methodology, Rulis(1989) re-examined his previous selection criteriafor a threshold value and those of Munro (1990)
and concluded that a threshold value of 1.5 mg/per-son/day would provide a high degree of health pro-tection. This threshold value was subsequentlyadopted by FDA as the threshold of regulation
(Federal Register, 1993, 1995) and FDA noted thatsuch an exposure level would result in a negligiblerisk even in the event that a substance of unknown
toxicity was later shown to be a carcinogen.
Comparison of sensitivity of various endpoints
Because of concerns raised by others (SCF,1996), that the 5th centile NOELs for speci®c toxi-
cological endpoints such as reproductive e�ects,neurotoxicity and immunotoxicity might result inhuman exposure thresholds lower than 1.5 mg/per-son/day, this matter was examined. Table 1 in
Appendix A presents the TDLos for 100 substances
reported in the RTECS database (RTECS, 1987) to
cause developmental abnormalities. The human ex-
posure threshold for this group of substances is
2076 mg/person/day (see Table 7), 1384 times higher
than the 1.5 mg/person/day value. In addition, the
5th centile NOEL for 31 neurotoxic organopho-
sphorous insecticides (Appendix A, Table 2)
included in structural Class III of the Munro et al.
(1996) database produced a corresponding human
exposure threshold of 18 mg/person/day (see
Table 7), 12 times greater than the proposed
threshold value of 1.5 mg/person/day. It might be
expected that such neurotoxic compounds would
have a low human exposure threshold because they
are speci®cally designed to be highly potent toxins.
Moreover, the measure of neurotoxicity selected to
establish the NOELs in most cases was cholinester-
ase inhibition, an extremely sensitive endpoint.
Most importantly, organophosphorous insecticides
would not be used as ¯avouring substances. A list
of the 100 substances reported to cause develop-
mental abnormalities and of the 31 neurotoxic orga-
Table 6. Probability of a target risk not being exceeded at various threshold values
Percentage of chemicals presumed carcinogenic
Threshold value100% 50% 20% 10% 100% 50% 20% 10%
(mg/day) 10ÿ6 Target risk 10ÿ5 Target risk
0.15 86 93 97 99 96 98 99 >990.3 80 90 96 98 94 97 99 990.6 74 87 95 97 91 96 98 991.5 63 82 93 96 86 96 97 993 55 77 91 95 80 90 96 986 46 73 89 95 74 87 95 97
(Modi®ed from Munro, 1990).
Table 7. Comparison of various human exposure threshold values
Category
5th CentileNOEL
(mg/kg bw/day)
Humanexposurethreshold*
mg/person/day
Structural Class I 3 1800Structural Class II 0.91 540Structural Class III 0.15 90Developmental abnormalities$ 3.46 2076Neurotoxic compounds% 0.03 18
Threshold value} 1.5 mg/person/day
*The human exposure threshold was calculated by multiplying the5th centile NOEL by 60 (assuming a 60 kg individual) dividingby a safety factor of 100, and multiplying by 1000 to convertfrom milligrams to micrograms. (Munro et al., 1996).
$Substances are from the RTECS database and were indicated tocause developmental abnormalities. The NOELs were pre-sented by RTECs as the TDLo which is de®ned as the lowestdose of a substance reported to produce any non-signi®cantadverse e�ects. (RTECS, 1987).
%For organophosphorous insecticides, the endpoint measured wastypically cholinesterase inhibition.
}Adopted by FDA (Federal Register, 1993, 1995) as the thresholdof regulation for food-contact articles. See text for details.
Safety evaluation procedure 219
nophosphorous insecticides, along with their
NOELs, is given in Appendix A.
For the evaluation of the sensitivity of immuno-
toxicity as an endpoint, the data of Luster et al.(1992, 1993) were used to conduct a comparison of
NOELs and LOELs based on immunotoxic end-
points with corresponding NOELs and LOELsbased on non-immunotoxic endpoints. Twenty-four
substances meeting the criteria of immunotoxicity
used by Luster et al. (1992, 1993) were identi®edthat also had corresponding non-immunotoxic end-
point NOELs or LOELs. A list of these substances
is provided in Appendix A, Tables 3 and 4. Six ofthese substances had immunotoxic NOELs with
corresponding non-immunotoxic NOELs. Twelve of
these substances had no immunotoxic NOELs buthad immunotoxic LOELs with corresponding non-
immunotoxic LOELs. Five additional substances
had immunotoxic NOELs with corresponding non-immunotoxic LOELs and one substance had an
immunotoxic LOEL with a corresponding non-
immunotoxic NOEL. For these last six substances,NOELs were compared with LOELs divided by a
conservative factor of 10 to adjust for di�erences
between NOELs and LOELs (Dourson et al.,1996). For example, tetraethyl lead has an immune
endpoint NOEL of 0.5 mg/kg body weight/day and
a non-immune endpoint LOEL of 0.0012 mg/kgbody weight/day. The LOEL was divided by 10
(0.00012 mg/kg body weight/day) for comparisonwith the NOEL. In order to perform the compari-
son of immunotoxic endpoint sensitivity with non-immunotoxic endpoint sensitivity, the immunotoxic
NOEL/LOEL was divided by the correspondingnon-immunotoxic NOEL/LOEL resulting in a ratio.The resulting ratios of the 24 comparisons are
shown graphically in Fig. 4. The majority of thesubstances (17/24) had non-immunotoxic NOELsor LOELs that were lower (i.e. more sensitive) than
the corresponding immunotoxic NOELs or LOELs.Two substances had similar NOELs/LOELs and®ve substances had immunotoxic NOELs or LOELs
which were less than 10-fold lower than their non-immuntoxic counterparts. These data demonstratethat immunotoxicity is not a more sensitive end-point than other forms of toxicity.
Application of a threshold of toxicological concern to
¯avouring substances
The SCF (1996) has made the point that the de-cision to accept any particular threshold value is
both a scienti®c and a risk management decision.The role of the scientist is to ensure that risk man-agers are provided with the full range of uncertain-ties surrounding selection of any threshold value. In
the foregoing sections it was pointed out that thethreshold concept should not be interpreted as pro-viding absolute certainty of no risk. Threshold of
toxicological concern is a probabilistic methodologythat involves acceptance of a negligible risk stan-dard. Such a standard is commonly used by toxicol-
ogists in the establishment of ADIs, and in fact,
Fig. 4. Comparison of immune and non-immune endpoint NOELS and LOELS (based on NOELS andLOELS of 24 immunotoxic substances).
I. C. Munro et al.220
WHO (1987) has de®ned the ADI as ``an estimateby JECFA of the amount of a food additive,
expressed on a body weight basis, that can beingested daily over a lifetime without appreciablehealth risk''. JECFA has noted that it uses the risk
assessment process when setting an ADIÐthat is,the level of ``no apparent risk'' is set on the basis ofquantitative extrapolation from animal data to
human beings typically using a NOEL from the ani-mal studies divided by a 100-fold safety factor(WHO, 1987). When ADIs (or such similar limits,
e.g. TLVs, RfD, etc.) are established, there is a re-sidual, usually unquanti®able, element of risk(Baird et al., 1996; Purchase and Auton, 1995;SCF, 1996; Sielken and Valdez-Flores, 1996), which
is a re¯ection of the inability to determine preciselythe NOEL from empirical data, statistical uncer-tainties associated with the sensitivity of experimen-
tal models, completeness of data, or the magnitudeof modifying and safety factors invoked to accountfor any residual uncertainty (Dourson and Stara,
1983).The threshold of toxicological concern likewise
does not carry with it the absolute certainty that an
untested chemical present in food below the de-cision criterion of 1.5 mg/day will present a less than10ÿ6 risk. Rather there is a high probability (i.e.about 95%) that the cancer risk from such a chemi-
cal will be less than 10ÿ6. It is this residual of uncer-tainty that has produced a concern about thepossibility, albeit remote, that a highly potent geno-
toxic carcinogen might inadvertently be consideredacceptable using the threshold concept (SCF, 1996).
Additional factors that reduce the theoretical risk
When the threshold concept is applied to ¯avour-ing substances, two additional factors signi®cantly
reduce the probability of risk of cancer below 10ÿ6.The ®rst of these relates to very low levels of intakeof ¯avouring substances. As intake decreases, the
probability of not exceeding a 10ÿ6 risk substan-tially increases (Table 6). Therefore, application ofa threshold of toxicological concern to substances
having very low intakes (i.e. less or much less thanthe threshold value) carries with it a much higherprobability of no appreciable risk. It also must bekept in mind, that intake of the majority of ¯avour-
ing substances tends to be overestimated becausethese materials are volatile and appreciable amountsare lost during food preparation, storage, etc. These
issues regarding intake of ¯avouring substances arediscussed in Annex 5 of the report of the 44th meet-ing of JECFA (1996a).
The second factor involves a consideration ofchemical structure. The use of chemical structurefor predicting toxicity for food chemicals, especially
¯avouring substances, has long been recognized byJECFA (WHO, 1987), and JECFA has noted thatuse of structure±activity is most developed in thearea of carcinogenesis. The use of structural alerts
in combination with a knowledge of chemistry and
metabolism o�ers a way of identifying potential
carcinogens (Ashby and Tennant 1988, 1991;
Klopman and Rosenkranz, 1994; Tennant et al.,
1990; Williams, 1990). The examination of many
chemicals for genotoxic, mutagenic and carcino-
genic activities has led to the preparation of a series
of structural alerts which provide the basis for po-
tential reaction with DNA and possible carcino-
genic potential of the substance. The existence of
reactive moieties on known rodent carcinogens
implies that potential mutagenic activity and, in
many cases, the carcinogenic activity of untested
chemicals might be identi®ed by an examination of
structure (Ashby and Tennant, 1988, 1991; Tennant
and Ashby, 1991).
Structure±activity relationships have been suc-
cessfully applied to congeneric substances (i.e. indi-
vidual substances within a structurally related
group of substances) for which no toxicity data are
available (Klopman and Rosenkranz, 1994).
Congeners that are potential human carcinogens
and mutagens possess electrophilic functional
groups with the ability to react directly with DNA.
These electrophilic sites may be reactive functional
groups on the congener or those formed during
metabolic activation. Conversely, these functional
groups may be lost during metabolic detoxication
of the substance. Although the carcinogenic and
mutagenic potency of congeneric substances may
di�er, structural alerts within the group of conge-
ners are indicative of carcinogenic or mutagenic po-
tential (Klopman and Rosenkranz, 1994). A list of
the functional groups identi®ed by Ashby and
Tennant (1988, 1991) and Tennant et al. (1990) as
structural alerts is given in Table 8.
Most ¯avouring substances are simple aliphatic
and aromatic substances containing functional
groups that are e�ciently metabolized via detoxica-
tion pathways and very few ¯avouring substances
and/or their in vivo metabolites contain structural
alerts. The absence of structural alerts in a ¯avour-
ing substance provides added assurance that it will
not present an appreciable risk at or below intake
of 1.5 mg/day. For those that do contain signi®cant
structural alerts, such as aliphatic epoxides, ad-
ditional data are usually available to facilitate
evaluation.
It is recognized that application of a threshold of
toxicological concern is a departure from traditional
toxicological evaluation, but it is based on highly
conservative methodology and the assumptions
listed below, which, taken together, ensure that ¯a-
vouring substances consumed in amounts less than
1.5 mg/person/day will present, at most, an insigni®-
cant risk.
1. The 1.5 mg/day is based on carcinogenicity data,
an extremely sensitive endpoint in susceptible
Safety evaluation procedure 221
animal species with accepted relevance tohumans.
2. The CPD presents a worse case situation sincechemicals were generally tested over a lifetime bydaily administration at the maximum tolerated
dose (MTD), and the procedures used by Goldet al. (1984) to establish the TD50s involved nu-
merous conservative assumptions.3. The methods used by Rulis (1986, 1989) and
others (Krewski et al., 1990; Munro, 1990) tocalculate the distribution of 10ÿ6 risks, on whichthe threshold value of 1.5 mg/person/day is
based, are highly conservative since theyinvolved the use of linear extrapolation from the
lowest TD50 for each substance in the database.4. It is unlikely that any untested ¯avouring sub-
stance would turn out to be a genotoxic carcino-gen, and the possibility that a carcinogen wouldbe accepted using the threshold concept can be
substantially reduced by the application of struc-tural alert methodology.
5. Many ¯avouring substances are consumed inamounts considerably below the threshold valueof 1.5 mg/person/day and this substantially
increases the probability, already in the range of90 to 95%, that they will not present any signi®-
cant theoretical risk.6. Toxicity endpoints, such as developmental tox-
icity, neurotoxicity and immunotoxicity demon-strate considerably higher human exposurethresholds than the threshold value of 1.5 mg/per-son/day making it highly unlikely that these non-cancer endpoints are a relevant concern in apply-
ing the threshold concept.
Taken together, these factors provide a sound basis
for concluding that ¯avouring substances with
intakes below the 1.5 mg/person/day threshold valuecan be safely consumed.It is enlightening to compare the human exposure
thresholds with present intakes of chemically de®ned¯avouring substances in the US. As shown in Table 9,it is clear that for nearly all (93±97%) ¯avouring sub-
stances used in the US, intakes are below the humanexposure threshold for their respective structuralclass. Because most ¯avouring substances possess
simple structures and their metabolism is known orreasonably predictable, it can be concluded that it ishighly improbable that they would present a toxico-logical risk at exposure levels below the human ex-
posure threshold for their respective structural class.However, even if information on structural class,metabolic fate and existing toxicity studies were not
available, 743/1323 (56%) of ¯avouring substancesused in the US are consumed in amounts less thanthe 1.5 mg/person/day standard proposed by the
FDA (Federal Register, 1995) and Munro (1990).This indicates that for approximately half of theexisting list of 1323 chemically de®ned ¯avouringsubstances permitted for use in the US, intake can be
considered to be trivial.
Safety decision criteria
The decision criteria used to evaluate ¯avouringsubstances involve the integration of informationon intake, structure±activity relationships, metab-
olism and, as required, toxicity data. It should benoted that the criteria outlined below are notintended to be applied to any ¯avouring substances
with unresolved toxicity problems or to substancesthat are presumed or known carcinogens. Such sub-stances warrant special consideration and must be
evaluated using more traditional methods of safety
Table 8. A list of functional groups identi®ed by Ashby and Tennant (1988, 1991) and Tennant et al. (1990) as structural alerts for DNAreactivity
a) alkyl esters of phosphonic or sulfonic acids l) propiolactones and propiosulfonesb) aromatic nitro-groups m) aromatic and aliphatic aziridinyl-derivativesc) aromatic azo-groups (reduction to amine) n) aromatic and aliphatic substituted primary alkyl halidesd) aromatic ring N-oxides o) urethane derivatives (carbamates)e) aromatic mono- and di-alkyl amino groups p) alkyl N-nitrosaminesf) alkyl hydrazines q) aromatic amines and N-hydroxy derivativesg) alkyl aldehydes r) aliphatic epoxides and aromatic oxidesh) N-methylol derivatives s) center of Michael reactivityi) monohaloalkanes t) halogenated methanes [C(X)4]j) N and S mustards, beta-haloethyl- u) aliphatic nitro groupsk) N-chloramines
Table 9. Flavouring substances within each Cramer et al. (1978) structural class consumed inamounts below human exposure thresholds
Structuralclass
Human exposurethreshold(mg/day)
No. of ¯avours withinstructural class*
No. of ¯avours underhuman exposurethreshold (%)
I 1800 878 835 (95)II 540 243 227 (93)III 90 202 195 (97)
*Adapted from the FEMA ¯avouring substance database of ¯avouring substances permitted foruse in the US.
I. C. Munro et al.222
evaluation. While toxicity data exist on numerous
¯avouring substances and can be used as the basis
for evaluation, there are many ¯avouring substances
that lack toxicity data. The decision criteria are
intended to provide a means of evaluating such sub-
stances. The criteria incorporate, where the data
permit, the concept that metabolic fate can be pre-
dicted on the basis of presumed structure±activity
relationships. The criteria also rely, in part, on the
NOEL reference database which provides a human
exposure threshold for each of the three structural
classes of ¯avouring substances, as shown in
Table 4. The evaluation criteria also incorporate,
where available, toxicity data on ¯avouring sub-
stances and closely related structural analogues as a
basis for safety evaluation. One of the decision cri-
teria (number 5 below) incorporates the minimum
human exposure threshold value based on the
1.5 mg/person/day. This decision criterion can be
applied to those ¯avouring substances for which
metabolic fate is unknown and cannot be con®-
dently predicted and for which there are no toxicity
data on the ¯avouring substance or on a structu-
rally related material from which to conclude any
inference of safety in use.
Flavouring substances that meet one of the ®ve
numbered decision criteria outlined below can be
considered safe for their intended use without
further evaluation:
1. (a) The ¯avouring substance has a simple struc-
ture and will be metabolized and excreted
through known detoxication pathways to innoc-
uous endproducts; and
(b) the conditions of intended use do not result in
an intake greater than the human exposure
threshold for the relevant structural class, indicat-
ing a low probability of potential for adverse
e�ects.
2. (a) The conditions of intended use result in an
intake that exceeds the human exposure
threshold for the relevant structural class; how-
ever
(b) the ¯avouring substance has a simple struc-
ture and will be metabolized and excreted
through known detoxication pathways to innocu-
ous end-products and it or its metabolites are en-
dogenous human metabolites with no known
biochemical regulating function.
3. (a) The ¯avouring substance has a simple struc-
ture and will be metabolized and excreted
through known detoxication pathways to innoc-
uous end-products; and
(b) the conditions of intended use result in an
intake that exceeds the human exposure threshold
for the relevant structural class; however
(c) toxicity data exist on the ¯avouring substance
which provide assurance of safety under con-
ditions of intended use, or there are toxicity data
on 1 or more close structural relatives which pro-
vide a NOEL high enough to accommodate any
perceived di�erence* in toxicity between the ¯a-
vouring substance and the structurally related
substance having toxicity data.
4. (a) The metabolic fate of the ¯avouring sub-
stance cannot be con®dently predicted on the
basis of structure; however
(b) the conditions of intended use result in an
intake below the human exposure threshold for
the relevant structural class indicating a low
probability of potential for adverse e�ects; and
(c) toxicity data exist on the ¯avouring substance
which provide assurance of safety under con-
ditions of intended use, or there are toxicity data
on 1 or more close structural relatives which pro-
vide a NOEL high enough to accommodate any
perceived di�erence in toxicity between the ¯a-
vouring substance and the structurally related
substance having toxicity data.
5. (a) The metabolic fate of the ¯avouring sub-
stance cannot be con®dently predicted on the
basis of structure; however
(b) the conditions of intended use result in an
intake below the human exposure threshold of
1.5 mg/day, providing assurance that the sub-
stance will be safe under conditions of intended
use.
Figure 5 presents the same decision in the form of a
safety evaluation sequence. The sequence contains a
number of questions on structure, metabolism, intake
data and toxicity and provides an integrated mechan-
ism to evaluate the safety of a ¯avour ingredient.
Although the procedure incorporates relevant toxicity
data on a substance or related substances where avail-
able, it does not require them.
The e�ective application of this safety evaluation
procedure depends on a substantial knowledge of
toxicology, chemistry, metabolism and intake of ¯a-
vouring substances. It can be applied most e�ec-
tively when groups of structurally related ¯avouring
substances are evaluated together. In a group evalu-
ation, conclusions reached on the safety of individ-
ual substances are supported by similar conclusions
for structurally related substances. For example, the
results of the evaluation for butyl butyrate should
be consistent with results for other esters formed
from aliphatic acyclic linear saturated alcohols and
*In most instances, groups of structurally related materialshave toxicology data on at least one member of thegroup, usually the ¯avouring substance with the high-est poundage. In most cases a large margin of safety(i.e. 100- to 1000-fold) exists between the NOEL andthe calculated intake to the substance having toxico-logical data. Such margins of safety would be expectedto accommodate any perceived di�erence between thetoxicity of a ¯avouring substance having no toxicologi-cal data and its close structural relative for which aNOEL has been established.
Safety evaluation procedure 223
acids having similar levels of intake. These similar
substances will pass through the same branch of the
safety evaluation procedure because they fall into
the same structural class, possess similar metabolic
fate, and exhibit similar patterns of intake from use
as ¯avour ingredients and as components of food.
In the ®rst step of the safety evaluation procedure
(Fig. 5), the user must assign a decision tree struc-
ture class (Cramer et al., 1978) to the substance.
Following assignment of structure class, a question
on metabolic fate appears. This question identi®es
those substances which are anticipated to be e�-
ciently metabolized to innocuous products (e.g. 1-
butanol) versus those which are transformed to
more toxic metabolites (e.g. estragole) or have lim-
ited information on which to predict con®dently the
metabolic fate (e.g. 2-phenyl-3-carbethoxy furan).
Once a substance has been sorted according to
structure class and knowledge of metabolic fate, the
next question compares the substance's daily intake
from use as a ¯avour ingredient to the human ex-
posure threshold (Table 4) for the same structure
class.
If the substance is metabolized to innocuous pro-
ducts (Step No. 2) and has an intake less than the
human exposure threshold for the structure class
(Step No. A3), the substance is considered safe (e.g. 1-
octanol). If the intake is greater than the human ex-
posure threshold (Step No. A3) and the substance or
its metabolites are endogenous (Step No. A4), the
substance is also considered safe, even though the
intake is greater than the human exposure threshold
(e.g. butyric acid). If the substance is not endogenous,
then the substance or related substances must have a
NOEL (Step No. A5) signi®cantly greater than the
intake of the substance in order to be considered safe
(e.g. citral). If no such data exist or the NOEL is not
signi®cantly greater than the intake for the substance,
then additional data are required in order to complete
the safety evaluation.
If metabolic fate cannot be con®dently predicted
and the intake (Step No. 2) is greater than the
human exposure threshold (Step No. B3), ad-
ditional data on metabolic fate or toxicity on the
substance or structurally related substances are
required to complete the safety evaluation (e.g.
dihydrocoumarin). If the intake is less than the
threshold of concern for the structural class, the
substance or structurally related substances must
have a NOEL which provides an adequate margin
of safety under conditions of intended use (Step
No. B4) in order for the substance to be considered
safe [e.g. 2-ethyl-4-hydroxy-3(2H)-furanone]. If an
adequate toxicity study is not available and the sub-
stance has an intake less than 1.5 mg/day (Step No.
B5), the substance is considered not to present a
safety concern (e.g. 3-acetyl-2,5-dimethylthiophene).
Otherwise additional data are required in order to
complete the safety evaluation (e.g. 2-ethylfuran).
Fig. 5. Safety evaluation sequence.
I. C. Munro et al.224
The principal objective of the safety evaluationprocedure is to identify two groups of ¯avouring
substances: (i) those substances whose structure,metabolism, and relevant toxicity data clearly indi-cate that the substance would be expected not to
be a safety concern under current conditions ofintended use; and (ii) those substances which mayrequire additional data in order to perform an ade-
quate safety evaluation.
Integrating data on consumption ratio
As pointed out by WHO (1987) and SCF (1991),natural occurrence is no guarantee of safety, but itis important to recognize that the safety evaluation
of added use of ¯avouring substances needs to beconducted with an appreciation of the consumptionratio. Clearly, if added use of ¯avouring substances
results in an intake that exceeds that from naturalsources, this will increase awareness of the need toconsider carefully overall intake in the light of exist-ing data on toxicity and structure±activity relation-
ships. On the other hand, if the added use is trivialwith a consumption ratio of 10 to 100, that is, itincreases total intake by only 1 to 10%, then this
fact needs to be taken into consideration whenapplying the criteria outlined above.The substances of primary concern are those
which, in Fig. 5, receive a ``No'' answer to Step No.A5, or a ``Yes'' answer to Step No. B5, indicating apossible need for additional data and evaluationbeyond that included in the evaluation procedure
outlined in this paper. In such an evaluation, as sta-ted immediately above, the extent of natural occur-rence should be given appropriate weight.
Of much less concern with respect to consumptionratio are those substances that drop out of furtherconsideration as a result of ``No'' answers to Step Nos
A3 or B5, or ``Yes'' answers to Step Nos A4 or B4.The derivation of the thresholds, the estimation ofintakes (see JECFA, 1996a), and the special factors
applicable to the use of ¯avours (volatility, self-limit-ing use, etc.) build in multiple conservative assump-tions more than adequate to cover additional intakefrom natural occurrence to substances that in any case
are of low inherent concern.The advances in analytical chemistry in the past
50 years provide virtual assurance that no ¯avour-
ing substances of extensive natural occurrenceremain unknown. Those of potential value yet to bediscovered (e.g. as yet unknown components of
roast beef, co�ee, or chocolate ¯avour) are beingsought at the ppb and ppt level. This does notsuggest intakes above any of the thresholds dis-cussed in this paper.
Discussion
The safety evaluation of ¯avouring substancespresents an interesting challenge. There are approxi-mately 2500 ¯avouring substances in use in Europeor the US at this time. Many do not have su�cient
toxicology data to conduct a traditional safetyevaluation. However, it is neither possible nor
necessary to conduct toxicological studies on all in-dividual ¯avouring substances used in food. Themajority of ¯avouring substances are members of
groups of substances with common metabolic path-ways, and typically, individual members of such agroup display a similar toxicity pro®le. This is not
surprising in the light of the close structural simi-larity of the various ¯avouring substances compris-ing a chemical group. Moreover, as demonstrated
here, intake of ¯avouring substances is usually lowand, in the majority of cases, below the human ex-posure threshold values presented in this review.In order to provide a practical solution for evalu-
ating such a large number of low-exposure sub-stances in a timely manner, the safety evaluationprocedure described here was developed. It incor-
porates knowledge of toxicology, chemical struc-ture, metabolism and intake. This procedure wasrecently adopted by JECFA (1996a,b, 1997) and
was applied to the safety evaluation of 263 ¯avoursduring the 46th and 49th meetings of theCommittee (JECFA, 1997, 1998).
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APPENDIX
follows
Safety evaluation procedure 229
Table
A1.Substancesreported
tocause
developmentalabnorm
alities
(from
RTECS)
Chem
ical
Species
Dose
(TDLo)*
mg/kg
11,3,4-Thiadiazole,2,2'-(methylenediimino)bis-
rat
12
1-6-H
exanediamine
rat
1840
31-Piperazinepropanol,4-(6-((6-m
ethoxy-8-quinolyl)aminohexyl-alpha-m
ethyl-,maleate
(1:2)
rat
90
411H-Pyrido(2,1-b)quinazo
lone-2-carboxylicacid,11-oxo-
rat
4400
51H-Indazole-3-carboxylicacid,1-(2,4-dichlorobenzyl)-
rat
175
61H-Isoindole-1,3(2H)-dione,4,5,6,7-tetrahydro-2-(7-¯uoro-3,4-dihydro-3-oxo-4-;(2-propynyl)-2H-1,4-benzoxazin-6-yl)-
rat
300
72,7-N
aphthalenedisulfonic
acid,3,3'((3,3'-d
imethyl-4,4'biphenylylene)bis(azo))bis(5-;amino-4-hydroxy-,tetrasodium
salt)
rat
150
82-Propanone,
1,1,3,3-tetrachloro-
rabbit
130
92-Pyridinem
ethanol,alpha-(3-(2,6-dim
ethyl-1-piperidinyl)propyl)-alpha-phenyl-,;monohydrochloride,
Z-(2)-
rabbit
650
10
3-Biphenylcarboxylicacid,2',4'-d
i¯uoro-4-hydroxy-
rabbit
520
11
4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylicacid,6-((aminophenylacetyl)amino)-;3,3-dim
ethyl-7-oxo-,(2,2-dim
ethyl-1-oxopropoxy)m
ethylester,hydrochloride
mouse
1200
12
4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylicacid,6-(2-amino-2-phenylacetamido)-;3,3-dim
ethyl-7-oxo-,trihydrate,D-(-)-
rat
2800
13
4H-Pyrido(1,2-a)pyrimidin-4-one,9-m
ethyl-3-(1H-tetrazol-5-yl)-,potassium
salt
rat
2750
14
4H-s-Triazolo(3,4-c)thieno(2,3-e)(1,4)-diazepine,6-(o-chlorophenyl)-8-ethyl-1-m
ethyl-
rabbit
13
15
5-Isoxazoleaceticacid,3,4-bis(4-m
ethoxyphenyl)-
rat
1650
16
7H-Pyrido(1,2,3-de)-1,4-benzoxazine-6-carboxylicacid,2,3-dihydro-9-¯uoro-3-m
ethyl-;10-(4-m
ethyl-1-piperazinyl)-7-oxo-,hem
ihydrate,(S)-
rat
8910
17
9H-Purine-6-thiol,9-beta-D-ribofuranosyl-
rat
87.5
18
Acetamide,
2,2-dichloro-N
-(beta-hydroxy-alpha-(hydroxymethyl)-p-(methylsulfonyl)phenethyl)-,;D-threo-(+)
rat
150
19
Acetamide,
N,N
-dim
ethyl-
rabbit
3900
20
Aceticacid,(2,4-dichlorophenoxy)-
rat
0.22
21
Aceticacid,(3,5,6-trichloro-2-pyridyloxy)-
rat
2000
22
Aceticacid,oxo((3-(1H-tetrazol-5-yl)phenyl)amino)-,butylester
rat
24000
23
Acetonitrile,amino-,bisulfate
rat
200
24
Acridine,
9,9-dim
ethyl-10-(3-(N,N
-dim
ethylamino)propyl)-,tartrate
mouse
175
25
Alanine,
3-(3,4-dihydroxyphenyl)-,
L-
rat
500
26
Alanine,
N-((5-chloro-8-hydroxy-3-m
ethyl-1-oxo-7-isochromanyl)carbonyl)-3-phenyl-,;sodium
salt,(-)-
rat
527
Alosenn
rat
5500
28
Anthranilic
acid,N-(2,3-xylyl)-
mouse
829
Arsineoxide,
dim
ethylhydroxy-
rat
300
30
Benzamide,
N-(2-piperidinylm
ethyl)-2,5-bis(2,2,2-tri¯uoroethoxy)-,monoacetate
rabbit
390
31
Benzenesulfonamide,
4-amino-N
-(4,5-dim
ethyl-2-oxazolyl)-,mixt.with5-((3,4,5-;trim
ethoxyphenyl)methyl)-2,4-pyrimidinediamine
rat
3360
32
Benzenesulfonamide,
4-amino-N
-(4,6-dim
ethoxy-2-pyrimidinyl)-
rat
500
33
Benzenesulfonic
acid,thio-,S,S'-(2-(dim
ethylamino)trimethylene)
ester
rat
660
34
Benzhydrol,2-chloro-alpha-(2-(dim
ethylamino)ethyl)-,hydrochloride
mouse
120
35
Benzoic
acid,3,4,5-trimethoxy-beta-(dim
ethylamino)-beta-ethylphenethylester,;maleate
(1:1)
rabbit
6500
36
Benzylalcohol,4-amino-alpha-((tert-butylamino)m
ethyl)-3,5-dichloro-,monohydrochloride
rat
4.4
37
Biphenyl,3,3',4,4'-tetramethyl-
mouse
640
38
Butyricacid,4-(p-bis(2-chloroethyl)aminophenyl)-
mouse
339
Butyrophenone,
4-(4-(p-chlorophenyl)-4-hydroxypiperidino)-4'-¯
uoro-
rat
5.04
40
Cadmium
rat
23
41
Carbazicacid,3-(1-phthalazinyl)-,ethylester,monohydrochloride
mouse
70
42
Chlordane
rat
880
43
Cortisone
mouse
500
44
Dibenzo(b,e)(1,4)dioxin,2,3,7,8-tetrabromo-
mouse
0.216
45
Dibenzo-p-dioxin,2,7-dichloro-
rat
5
I. C. Munro et al.230
46
Disul®de,
bis(thiocarbamoyl)
mouse
105
47
Ethane,
1,1,1-trichloro-2,2-bis(p-m
ethoxyphenyl)-
rat
2000
48
Ethane,
2-(o-chlorophenyl)-2-(p-chlorophenyl)-1,1,1-trichloro-
rat
250
49
Ethanone,
1-(7-(2-hydroxy-3-((1-m
ethylethyl)amino)propoxy)-2-ben
zofuranyl)-,hydrochloride
rat
1400
50
Ethanone,
2-((4-(2,4,dichloro-3-m
ethylbenzo
yl)-1,3-dim
ethyl-1H-pyrazol-5-yl)oxy)-;1-(4-m
ethylphenyl)-
rat
2000
51
Folicacid,methyl-
rat
500
52
Gallic
acid,propylester
rat
45000
53
Glutamic
acid,N-(p-((1-(2-amino-4-hydroxy-6-pteridinyl)ethyl)amino)benzoyl)-L-
rat
20
54
Gossypolaceticacid
mouse
480
55
Hydrocinnamic
acid,alpha-hydrazino-3,4-dihydroxy-alpha-m
ethyl-,L-
rat
2100
56
Indole-3-aceticacid,1-(p-chlorobenzoyl)-5-m
ethoxy-2-m
ethyl-
rat
157
Isonicotinamide,
2-ethylthio-
mouse
450
58
Isothiocyanic
acid,butenylester
rat
800
59
L-G
lutamic
acid,magnesium
salt(1:1),hydrobromide
rat
6000
60
L-Tyrosine
rat
3500
61
L-Tyrosine,
O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-
rat
26.25
62
Linoleic
acid(oxidized)
rat
166000
63
Lysine,
L-
rat
81000
64
Manganese,
(ethylenebis(dithiocarbamato))-andzincacetate
(50:1)
rat
765
65
Mannitol,1,6-dibromo-1,6-dideoxy-,
D-
mouse
150
66
Methanol,1,3,4-thiadiazol-2-ylm
inodi-
rat
567
Molybdenum
rat
5.8
68
Morpholine,
4-(3,4,5-trimethoxyben
zoyl)-
rat
700
69
Norleucine,
6-amidino-,monohydrochloride,
hydrate
rat
2000
70
Oxazolo(3,2-d)(1,4)benzo
diazepin-6(5H)-one,
10-chloro-11b-(o-chlorophenyl)-2,3,7,11b-;tetrahydro
mouse
1800
71
Phenol,p-amino-
rat
2500
72
Phenothiazine-2-aceticacid,10-m
ethyl
mouse
180
73
Phosphonic
acid,(1,2-epoxypropyl)-,calcium
salt(1:1),(1R,2
S)-(-)-
rat
15400
74
Phosphorodithioic
acid,O,O
-dim
ethylester,S-ester
with2-m
ercapto-N
-methylacetamide
rat
120
75
Phthalicacid,di(methoxyethyl)ester
rat
593
76
Piperazine,
1-(p-tert-butylbenzyl)-4-(p-chloro-alpha-phen
ylbenzyl)-
rat
320
77
Piperazine,1-(p-tert-butylbenzyl)-4-(p-chloro-alpha-phenylbenzyl)-,dihydrochloride
rat
360
78
Piperidine,
3-((4-m
ethoxyphenoxy)m
ethyl)-1-m
ethyl-4-phenyl-,hydrochloride,
(3R-trans)-
rat
210
79
Piperidine,
1-m
ethyl-4-(N-2-thenylanilino)-,tartrate
rat
157
80
Polychlorinatedbiphenyl(A
roclor1254)
rat
90
81
Pregn-4-ene-3,20-dione,9-¯uoro-11-beta,17,21-trihydroxy-
rabbit
282
Pregna-,4-diene-2,20-dione,9-¯uoro-11-beta,16-alpha,17,21-tetrahydroxy-,16,21-diacetate
mouse
3.2
83
Propionic
acid,2-(2,4,5-trich
lorophenoxy)-
mouse
1617
84
Pyrimidine,
2,4-diamino-6-m
ethyl-5-phen
yl-
rat
100
85
Retinoic
acid,4-oxo-,13-cis-
mouse
100
86
Retinoic
acid,all-trans-
mouse
15
87
Rowachol
rat
9600
88
Stannane,
diacetoxydibutyl-
rat
15.2
89
Sulfanilamide,
N(sup1)-(6-m
ethoxy-2-m
ethyl-4-pyrimidinyl)-
mouse
3000
90
Toluene,
alpha-(2-(2-butoxyethoxy)ethoxy)-4,5-(methylened
ioxy)-2-propyl-
rat
2130
91
Tryptophan,N-acetyl-,L-
rat
27500
92
Urea,(alpha-(2-m
ethylhydrazino)-p-toluoyl)-,monohydrobromide
rabbit
50
93
Urea,1-butyl-3-(p-tolylsulfonyl)-
mouse
1700
94
Urea,1-butyl-3-sulfanilyl-
rat
1000
95
Uridine,
5'-d
eoxy-5-¯uoro-
rat
550
96
ZZL-0820
rabbit
325
97
beta-Escin
mouse
36
98
m-Propionotoluidide,2-m
ethyl-4'-n
itro-alpha,alpha,alpha-tri¯ouro-
rat
1050
99
p-A
cetophenetidide
rat
6000
100
p-C
resol,2,6-di-tert-butyl-
mouse
1200
*TDLo=
thelowestdose
ofasubstance
reported
toproduce
anynon-signi®cantadverse
e�ect(=
NOEL).
Safety evaluation procedure 231
Table A4. Substances with immunotoxic LOELs
ImmuneNon-immune
LOEL NOEL LOEL Non-immuneSubstance (mg/kg bw) (mg/kg bw) (mg/kg bw) endpoint Reference
Oral Admin1 2,3,7,8-TCDD 0.086 1E-05 repro* Murray et al., 19792 lithium carbonate 50 98 repro, bw,$ hepatic,
renalIbrahim and Canolty, 1990
3 m-nitrotoluene 200 40 hepatic (f)% NTP, 19924 2,4-diaminotoluene 25 24 repro Thysen et al., 19855 dimethylvinyl chloride 50 125 splenic NTP, 19866 ethylene dibromide 125 30 mortality Teramoto et al., 19807 4,4-thiobis (6-tert-butyl-m-cresol) 10 45 hepatic NTP, 1994
Non-oral Admin1 azathioprine (ip)} 10 1 repro (ip) Scott, 19772 benzo(a)pyrene (sc)} 50 50 repro (sc) Bui et al., 19863 diethylstilboestrol (sc) 0.2 1E-05 repro (sc) McLachlan, 19774 DMB(a)A (sc) 5 1.25 repro (orl)** Davis et al., 19785 N-nitroso dimethylamine (ip) 1.5 5 repro (ip) Chaube, 19736 ochratoxin A (ip) 3.4 0.0625 renal (gav)$$ NTP, 1989
*repro = reproductive; $bw = body weight.; %f = female.; }ip = intraperitoneal.; }sc = subcutaneous.= oral.; $$gav = gavage.
Table A2. NOELs for organophosphorous insecticides*
Agent Species Endpoint observed NOEL (mg/kg/day)
1 Acephate rat decreased body weight gain (parents and pups) 2.52 Azinphos methyl rat inhibition of plasma ChE activity 0.183 Coumaphos rat inhibition of RBC and plasma ChE activity 0.44 Crufomate rat inhibition of RBC ChE activity 35 Diazinon mouse decreased body weight gain 72$6 Dichlorvos rat inhibition of ChE activity (speci®c endpoint not indicated) 0.237 Dimethoate rat inhibition of brain, RBC and plasma ChE activity 0.058 Disulfoton rat inhibition of brain, RBC abd plasma ChE activity 0.059 Ethephon rat inhibition of plasma and RBC ChE activity 1510 Ethion rat inhibition of plasma ChE activity in females 0.211 Ethyl-p-nitrophenyl
phenylphosphorothioaterat inhibition of brain, RBC and plasma ChE activity 0.25
12 Express rat decreased body weight gain 113 Fenamiphos rabbit decreased maternal body weight gain 0.114 Fenchlorphos rat inhibition of ChE (form not speci®ed) 1515 Fonofos rat inhibition of RBC and plasma ChE activity 0.516 Glufosinate ammonium rat increased absolute and relative kidney weight in males 0.4$17 Glyphosate rat increased incidence of renal tubular dilation in F3b pups 1018 Malathion rat inhibition of brain ChE activity 519 Merphos rat inhibition of RBC ChE activity in females 0.120 Merphos oxide rat inhibition of brain ChE activity 0.2521 Methamidophos rat clinical signs typical of ChE inhibition 122 Methidithion rat inhibition of brain and RBC ChE activity 0.223 Methyl parathion rat decreased hemoglobin, hematocrit and RBCs 0.02524 Naled rat decreased body weight gain 0.225 Parathion rat decreased body weight gain 1.826 Phosmet rat inhibition of RBC and plasma ChE activity 227 Phosphamidon rat decreased body weight gains 6.228 Pirimiphos-methyl rat inhibition of plasma ChE activity 0.529 Quinalphos mouse inhibition of plasma ChE activity 0.0330 Tetrachlorvinphos rat inhibition of RBC ChE activity 631 Tetraethyl dithio pyrophosphate rat inhibition of RBC and plasma ChE activity 0.5
*Data taken from the EPA Integrated Risk Information System (IRIS) database.$NOEL divided by a factor of 3 (see Munro et al., 1996 for explanation).
Table A3. Substances with immunotoxic NOELs
ImmuneNon-immune
NOEL NOEL LOELSubstance (mg/kg bw) (mg/kg bw) (mg/kg bw) Non-immune endpoint Reference
Oral Admin1 p-nitrotoluene 400 200 hepatic, splenic Burns et al., 19942 pentachlorophenol 10 3 hepatic, renal Schwetz et al., 19783 o-phenylphenol 100 10 blood (RBC)* Luster et al., 19814 hexachlorodibenzo-p-dioxin 0.056 1E-05 repro$ Murray et al., 19795 DPH 150 50 teratogenic McClain and Langho�, 19796 tetraethyl lead 0.5 0.0012 hepatic,thymus Schepers, 19647 benzidine 11 2.7 neural, hepatic Little®eld et al., 19838 nitrobenzene 30 60 repro Kawashima et al., 1995
Non-oral Admin1 indomethacin (sc)% 2 1.6 repro/vascular
permeability (sc)Hoos and Ho�man, 1983
2 TPA (sc) 20 0.32 repro (sc) Nagasawa et al., 19803 ethyl carbamate (ip)} 2 15 repro (sc) NTIS, 1968
*RBC = red blood cells; $repro = reproductive; %sc = subcutaneous; }ip = intraperitoneal.
I. C. Munro et al.232