A Procedure for the Safety Evaluation

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).


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.


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


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).


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


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).


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.


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.


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).


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.


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).


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


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.


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-


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-


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


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


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.


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.


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).

REFERENCES

Ashby J. and Tennant R. W. (1988) Chemical structure,Salmonella mutagenicity and extent of carcinogenicity asindicators of genotoxic carcinogenesis among 222chemicals tested in rodents by the U.S. NCI/NTP.Mutation Research 204, 17±115.

Ashby J. and Tennant R. W. (1991) De®nitive relation-ships among chemical structure, carcinogenicity andmutagenicity for 301 chemicals tested by the U.S. NTP.Mutation Research 257, 229±306.

Baird S. J. S., Cohen J. T., Graham J. D., ShlyakhterA. I. and Evans J. S. (1996) Noncancer risk assessment:A probabilistic alternative to current practice. Humanand Ecological Risk Assessment 2, 79±102.

Bfeck B. D., Conolly R. B., DoursonM. L., Guth D., HattisD., Kimmel C. and Lewis C. (1993) Symposium overview.Improvements in quantitative noncancer risk assessment.Fundamental and Applied Toxicology 20, 1±14.

Bui Q. Q., Tran M. B. and West W. L. (1986) A compara-tive study of the reproductive e�ects of methadone andbenzo(a)pyrene in the pregnant and pseudopregnant rat.Toxicology 42, 195±204.

Burns L. A., Bradley S. G., White K. L., McCay J. A.,Fuchs B. A., Stern M., Brown R. D., Musgrove D. L.,Holsapple M. P., Luster M. I. and Munson A. E. (1994)Immunotoxicity of mono-nitrotoluenes in femaleB6C3F1 mice: I. Para- nitrotoluene. Drug and ChemicalToxicology 17, 17±358.

CE (1974) Natural Flavouring Substances, Their Sources,And Added Arti®cial Flavouring Substances. Council ofEurope, Strasbourg, France.

CE (1981) Flavouring Substances And Natural Sources OfFlavourings, Third Edition. Council of Europe,Strasbourg, France.

CE (1992) Flavouring Substances and Natural Sources ofFlavourings. Volume I. Chemically-De®ned FlavouringSubstances, Fourth Edition. Council of Europe,Maissoneuve, France.

Chaube S. (1973) Protective e�ects of thymidine, 5-aminoi-midazolecarboxamide, and ribo¯avin against fetalabnormalities produced in rats by 5-(3,3-dimethyl-1-tria-


zene)imidazole-4-carboxamide. Cancer Research 33,2231±2240.

Cramer G. M., Ford R. A. and Hall R. L. (1978)Estimation of toxic hazardÐA decision tree approach.Food and Cosmetics Toxicology 16, 255±276.

Davis G. J., McLachlan J. A. and Lucier G. W. (1978)The e�ect of 7,12-dimethylbenz(a)anthracene (DMBA)on the prenatal development of gonads in mice.Teratology 17, 33A.

Deisinger P. J., Boatman R. J. and Guest D. (1994)Metabolism of 2-ethylhexanol administered orally anddermally to the female Fischer 344 rat. Xenobiotica 24,429±440.

Dourson M. L., Felter S. P. and Robinson D. (1996)Evolution of science-based uncertainty factors in non-cancer risk assessment. Regulatory Toxicology andPharmacology 24, 108±120.

Dourson M. L. and Stara J. F. (1983) Regulatory historyand experimental support of uncertainty (safety) factors.Regulatory Toxicology and Pharmacology 3, 224±228.

FDA (1982) Toxicological Principles for the SafetyAssessment of Direct Food Additives and Color AdditivesUsed in Food. Redbook. US Food and DrugAdministration, Bureau of Foods, Washington, DC.

FDA (1993) Toxicological Principles for the SafetyAssessment of Direct Food Additives and Color AdditivesUsed in Food. Redbook II (Draft). US Food and DrugAdministration, Center for Food Safety and AppliedNutrition, Washington, DC.

Federal Register (1993) Food additives; threshold of regu-lation for substances used in food-contact articles.Federal Register 58 (195), 52719±52729.

Federal Register (1995) Food additives; Threshold of regu-lation for substances used in food-contact articles (Finalrule). Federal Register 60 (136), 36582±36596.

Frawley J. P. (1967) Scienti®c evidence and common senseas a basis for food-packaging regulations. Food andCosmetic Toxicology 5, 293±308.

Gold L. S., Bernstein L., Kaldor J., Backman G. andHoel D. (1986b) An empirical comparison of methodsused to estimate carcinogenic potency in long-term ani-mal bioassays: Lifetable vs summary incidence data.Fundamental and Applied Toxicology 6, 263±269.

Gold L. S., de Veciana M., Backman G. M., Magaw R.,Lopipero P., Smith M., Blumenthal M., Levinson R.,Bernstein L. and Ames B. N. (1986a) Chronologicalsupplement to the carcinogenic potency database:Standardized results of animal bioassays publishedthrough December 1982. Environmental HealthPerspectives 67, 161±200.

Gold L. S., Sawyer C. B., Magaw R., Backman G. M., deVeciana M., Levinson R., Hooper N. K., HavenderW. R., Bernstein L., Peto R., Pike M. and AmesB. N. (1984) A carcinogenic potency database of thestandardized results of animal bioassays. EnvironmentalHealth Perspectives 58, 9±319.

Gold L. S., Slone T. H. and Bernstein L. (1989) Summaryof carcinogenic potency and positivity for 492 rodentcarcinogens in the carcinogenic potency database.Environmental Health Perspectives 79, 259±272.

Gold L. S., Ward J. M., Bernstein L. and Stern B. (1986c)Association between carcinogenic potency and tumourpathology in rodent carcinogenesis bioassays.Fundamental and Applied Toxicology 6, 677±690.

Hall R. L. 1975. Unpublished data from personal com-munication to the NAS/NRC Committee on GRAS ListSurveyÐPhase III, and to FEMA. .

Hoel D. G. and Portier C. J. (1994) Nonlinearity of dose-response functions for carcinogenicity. EnvironmentalHealth Perspectives 102, 109±113.

Hoos P. C. and Ho�man L. H. (1983) E�ect of histaminereceptor antagonists and indomethacin in implantationin the rabbit. Biology of Reproduction 29, 833±840.

Ibrahim H. S. and Canolty N. L. (1990) E�ects of dietarylithium on pregnant and lactating rats and the progeny.Nutrition Research 10, 315±324.

Jaeschke H., Kleinwaechter C. and Wendel A. (1987) Therole of acrolein in allyl alcohol-induced lipid peroxi-dation and liver cell damage in mice. BiochemicalPharmacology 36, 51±57.

JECFA (1967) Toxicological Evaluation of SomeFlavouring Substances and Non-Nutritive SweeteningAgents. Eleventh Meeting of the Joint FAO/WHOExpert Committee on Food Additives.

JECFA (1968) Speci®cations for the Identity and Purity ofFood Additives and their Toxicological Evaluation: SomeFlavouring Substances and Non-nutritive SweeteningAgents. Eleventh Report of the Joint FAO/WHOExpert Committee on Food Additives, FAO NutritionMeetings Report Series No. 44, WHO Technical ReportSeries No. 383.

JECFA (1970) Toxicological Evaluation of SomeExtraction Solvents and Certain Other Substances.Fourteenth Meeting of the Joint FAO/WHO ExpertCommittee on Food Additives.

JECFA (1971) Evaluation of Food Additives. FourteenthReport of the Joint FAO/WHO Expert Committee onFood Additives, WHO Technical Report Series No.462.

JECFA (1972) Evaluation of Food Additives. SomeEnzymes, Modi®ed Starches, and Certain OtherSubstances: Toxicological Evaluations and Speci®cationsand a Review of the Technological E�cacy of SomeAntioxidants. Fifteenth Report of the Joint FAO/WHOExpert Committee on Food Additives, WHO TechnicalReport Series No. 488.

JECFA (1974a) Toxicological Evaluation of Certain FoodAdditives Including Anticaking Agents, Antimicrobials,Antioxidants, Emulsi®ers and Thickening Agents.Seventeenth Meeting of Joint FAO/WHO ExpertCommittee on Food Additives, WHO Food AdditiveSeries No. 5.

JECFA (1974b) Toxicological Evaluation of Certain FoodAdditives with a Review of General Principles and ofSpeci®cations. Seventeenth Report of the Joint FAO/WHO Expert Committee on Food Additives, WHOTechnical Report Series No. 539.

JECFA (1976a) Evaluation of Certain Food Additives.Twentieth Report of the Joint FAO/WHO ExpertCommittee on Food Additives, FAO NutritionMeetings Report Series No. 1, WHO Technical ReportSeries No. 599.

JECFA (1976b) Toxicological Evaluation of Certain FoodAdditives. Twentieth Meeting of the Joint FAO/WHOExpert Committee on Food Additives, WHO FoodAdditive Series No. 10.

JECFA (1978) Evaluation of Certain Food Additives andContaminants. Twenty-second Report of the Joint FAO/WHO Expert Committee on Food Additives, WHOTechnical Report Series No. 631.

JECFA (1980a) Evaluation of Certain Food Additives.Twenty-third Report of the Joint FAO/WHO ExpertCommittee on Food Additives. World HealthOrganization, Geneva. WHO Technical Report SeriesNo. 648.

JECFA (1980b) Evaluation of Certain Food Additives.Twenty-fourth Report of the Joint FAO/WHO ExpertCommittee on Food Additives. World HealthOrganization, Geneva. WHO Technical Report SeriesNo. 653.

JECFA (1980c) Toxicological Evaluation of Certain FoodAdditives. Twenty-third Meeting of the Joint FAO/WHO Expert Committee on Food Additives, WHOFood Additive Series No. 14.

JECFA (1981a) Evaluation of Certain Food Additives.Twenty-®fth Report of the Joint FAO/WHO Expert


Committee on Food Additives, Technical Report SeriesNo. 669.

JECFA (1981b) Toxicological Evaluation of Certain FoodAdditives. Twenty-®fth Meeting of the Joint FAO/WHOExpert Committee on Food Additives, WHO FoodAdditive Series No. 16.

JECFA (1982a) Evaluation of Certain Food Additives andContaminants. Twenty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 683.

JECFA (1982b) Toxicological Evaluation of Certain FoodAdditives. Twenty-sixth Meeting of the Joint FAO/WHO Expert Committee on Food Additives, WHOFood Additive Series No. 17.

JECFA (1983a) Evaluation of Certain Food Additives andContaminants. Twenty-seventh Report of the JointFAO/WHO Expert Committee on Food Additives.World Health Organization, Geneva. WHO TechnicalReport Series No. 696.

JECFA (1983b) Toxicological Evaluation of Certain FoodAdditives and Contaminants. Twenty-seventh Meeting ofthe Joint FAO/WHO Expert Committee on FoodAdditives, WHO Food Additive Series No. 18.

JECFA (1984a) Evaluation of Certain Food Additives andContaminants. Twenty-eighth Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 710.

JECFA (1984b) Toxicological Evaluation of Certain FoodAdditives and Contaminants. Twenty-eighth Meeting ofthe Joint FAO/WHO Expert Committee on FoodAdditives, WHO Food Additive Series No. 19.

JECFA (1986) Evaluation of Certain Food Additives andContaminants. Twenty-ninth Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 733.

JECFA (1987a) Evaluation of Certain Food Additives andContaminants. Thirtieth Report of the Joint FAO/WHOExpert Committee on Food Additives. World HealthOrganization, Geneva. WHO Technical Report SeriesNo. 751.

JECFA (1987b) Evaluation of Certain Food Additives andContaminants. Thirty-®rst Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 759.

JECFA (1989) Evaluation of Certain Food Additives andContaminants. Thirty-third Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 776.

JECFA (1990a) Evaluation of Certain Food Additives andContaminants. Thirty-®fth Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 789.

JECFA (1990b) Toxicological Evaluation of Certain FoodAdditives and Contaminants. Thirty-®fth Meeting of theJoint FAO/WHO Expert Committee on FoodAdditives, WHO Food Additive Series No. 26.

JECFA (1991a) Evaluation of Certain Food Additives andContaminants. Thirty-seventh Report of the Joint FAO/WHO Expert Committee on Food Additives. WorldHealth Organization, Geneva. WHO Technical ReportSeries No. 806.

JECFA (1991b) Toxicological Evaluation of Certain FoodAdditives and Contaminants. Thirty-seventh Meeting ofthe Joint FAO/WHO Expert Committee on FoodAdditives, WHO Food Additive Series No. 28.

JECFA (1992) Evaluation of Certain Food Additives andNaturally Occurring Contaminants. Thirty-ninth Report

of the Joint FAO/WHO Expert Committee on FoodAdditives. World Health Organization, Geneva. WHOTechnical Report Series No. 828.

JECFA (1993a) Evaluation of Certain Food Additives andContaminants. Forty-®rst Report of the Joint FAO/WHO Expert Committee on Food Additives, TechnicalReport Series No. 837.

JECFA (1993b) Toxicological Evaluation of Certain FoodAdditives and Contaminants. Forty-®rst Meeting of theJoint FAO/WHO Expert Committee on FoodAdditives, WHO Food Additive Series No. 32.

JECFA (1993c) Toxicological Evaluation of Certain FoodAdditives and Naturally Occurring Toxicants. Thirty-Ninth Meeting of the Joint FAO/WHO ExpertCommittee on Food Additives, WHO Food AdditiveSeries No. 30.

JECFA (1996a) Toxicological Evaluation of Certain FoodAdditives and Contaminants. WHO Additives Series 35.The 44th Meeting of the Joint FAO/WHO ExpertCommittee on Food Additives (JECFA). Geneva, 1996.

JECFA (1996b) Toxicological Evaluation of Certain FoodAdditives. In WHO Food Additives Series 37. The 46thMeeting of the Joint FAO/WHO Expert Committee onFood Additives. (JECFA) Geneva, 1996.

JECFA (1997) Evaluation of Certain Food Additives andContaminants. Forty-Sixth Meeting of the Joint FAO/WHO Expert Committee on Food Additives, WHOTechnical Report Series No. 868.

JECFA (1998) Summary and Conclusions. Forty-NinthMeeting of the Joint FAO/WHO Expert Committee onFood Additives. Unpublished.

Kawashima K., Usami M., Sakemi K. and Ohno Y. (1995)Studies on the establishment of appropriate spermato-genic endpoints for male fertility disturbance in rodentinduced by drugs and chemicals: I. Nitrobenzene.Journal of Toxicological Sciences 20, 15±22.

Klopman G. and Rosenkranz H. S. (1994) Approaches toSAR in carcinogenesis and mutagenesis. Prediction ofcarcinogenicity/mutagenicity using MULTI-CASE.Mutation Research 305, 33±46.

Krasavage W. J., O'Donoghue J. L., DiVincenzo G. D. andTerhaar C. J. (1980) The relative neurotoxicity ofmethyl-n-butyl ketone, n-hexane and their metabolites.Toxicology and Applied Pharmacology 52, 433±441.

Krewski D., Szyszkowicz M. and Rosenkranz H. (1990)Quantitative factors in chemical carcinogenesis:Variation in carcinogenic potency. RegulatoryToxicology and Pharmacology 12, 13±29.

Lewis S. C., Lynch J. R. and Nikiforov A. I. (1990) Anew approach to deriving community exposure guide-lines from ``no-observed-adverse-e�ect levels''.Regulatory Toxicology and Pharmacology 11, 314±330.

Little®eld N. A., Nelson C. J. and Frith C. H. (1983)Benzidine dihydrochloride: Toxicological assessment inmice during chronic exposures. Journal of Toxicologyand Environmental Health 12, 671±685.

Luster M. I., Dean J. H., Boorman G. A., Archer D. L.,Lauer L., Lawson L. D., Moore J. A. and WilsonR. E. (1981) The e�ect of orthophenylphenol, tris(2,3-dichloropropyl) phosphate, and cyclophosphamide onthe immune system and host susceptibility of mice fol-lowing subchronic exposure. Toxicology and AppliedPharmacology 58, 252±261.

Luster M. I., Portier C., Pait D. G., Rosenthal G. J.,Germolec D. R., Gorsini E., Blaylock B. L., Pollock P.,Kouchi Y., Craig W., White K. L., Munso A. E. andComment C. E. (1993) Risk assessment inImmunotoxicology. II. Relationships between immuneand host resistance tests. Fundamental and AppliedToxicology 21, 71±82.

Luster M. I., Portier C., Pait D. G., White K. L., Jr,Gennings C., Munson A. E. and Rosenthal G. J. (1992)Risk assessment in immunotoxicology. I. Sensitivity and


predictability of immune tests. Fundamental and AppliedToxicology 18, 200±210.

McClain R. M. and Langho� L. (1979) Teratogenicity ofdiphenylhydantoin in New Zealand rabbits. Toxicologyand Applied Pharmacology 48 (1-Pt. 2), A32.

McLachlan J. A. (1977) Prenatal exposure to diethylstil-bestrol in mice. Toxicological studies. Journal ofToxicology and Environmental Health 2, 527±537.

Munro I. C. (1990) Safety assessment procedures for indir-ect food additives: An overview. Report of a workshop.Regulatory Toxicology and Pharmacology 12, 2±12.

Munro I. C., Ford R. A., Kennepohl, E and SprengerJ. G. (1996) Correlation of structural class with no-observed-e�ect levels: A proposal for establishing athreshold of concern. Food and Chemical Toxicology 34,829±867.

Munro I. C., Shubik P. and Hall R. (1998) Principles forthe safety evaluation of ¯avouring substances. Food andChemical Toxicology 36, 529±540.

Murray F. J., Smith F. A., Nitschke K. D., HumistonC. G., Kociba R. J. and Schwetz B. A. (1979) Three-generation reproduction study of rats given 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) in the diet. Toxicologyand Applied Pharmacology 50, 241±252.

Nagasawa H., Yanai R. and Nakajima Y. (1980)Suppression of lactation by tumour promoters in mice.Proceedings of the Society for Experimental Biology andMedicine 165, 394±397.

NAS (1978) Resurvey of the Annual Poundage of FoodChemicals Generally Recognized as Safe. NationalTechnical Information Service (NTIS) PB288±081.

NAS (1979) Comprehensive Survey of Industry on the Useof Food Additives. National Technical InformationService (NTIS) PB80-113418.

NAS (1984) Poundage Update of Food Chemicals.National Technical Information Service (NTIS) PB84-162148.

NAS (1989) 1987 Poundage and Technical E�ects Updateof Substances Added to Food. National ResearchCouncil, Washington, DC. Prepared for Food and DrugAdministration, Washington, DC. NTIS Report No.PB91-127266.

NTIS (1968) Evaluation of Carcinogenic, Teratogenic, andMutagenic Activities of Selected Pesticides and IndustrialChemicals. Volume I. Carcinogenic Study & Volume II.Teratogenic Study in Mice and Rats. National CancerInstitute (NCI), Bethesda, MD (Bionetics Res. Lab).Volumes 1 & 2.

NTP (1986) Toxicology and Carcinogenesis Studies ofDimethylvinyl Chloride (1-Chloro-2-Methylpropene)(CAS No. 513-37-1) in F344/N Rats and B6C3F1 Mice(Gavage Studies). National Toxicology Program (NTP),Research Triangle Park, NC (NTP Technical ReportSeries) No. 316.

NTP (1989) Toxicology and Carcinogenesis Studies ofOchratoxin A (CAS No. 303-47-9) in F344/N Rats(Gavage Studies). National Toxicology Program (NTP),Research Triangle Park, NC (NTP Technical ReportSeries) No. 358.

NTP (1992) NTP Technical Report on Toxicity Studies ofo-, m-, and p- Nitrotoluenes (CAS Nos: 88-72-2, 99-08-1,99-99-0) Administered in Dosed Feed to F344/N Ratsand B6C3F1 Mice. National Toxicology Program(NTP), Research Triangle Park, NC (NTP ToxicityReport Series) No. 23.

NTP (1994) Toxicology and Carcinogenesis Studies of 4,4'-Thiobis(6-t-Butyl-m-Cresol) (CAS No. 96-69-5) in F344/N Rats and B6C3F1 Mice (Feed Studies). NationalToxicology Program (NTP), Research Triangle Park,NC (NTP Technical Report Series) No. 435.

Peto R., Pike M. C., Bernstein L., Gold L. S. and AmesB. N. (1984) The TD50: A proposed general conventionfor the numerical description of the carcinogenic

potency of chemicals in chronic-exposure animal exper-iments. Environmental Health Perspectives 5, 1±8.

Phillips J. C., Purchase R., Watts P. and GangolliS. D. (1987) An evaluation of the decision tree approachfor assessing priorities for safety testing of food addi-tives. Food Additives and Contaminants 4, 109±123.

Purchase I. F. H. and Auton T. R. (1995) Thresholds inchemical carcinogenesis. Regulatory Toxicology andPharmacology 22, 199±205.

RTECS (1987) Registry of Toxic E�ects of ChemicalSubstances. 1985-86 Edition, Volume 1. U.S. Departmentof Health and Human Services, Public Health Service,Washington, DC.

Rulis A. M. (1986) De Minimis and the threshold of regu-lation. In Food Protection Technology, ed. C. W. Felix,pp. 29±37. Lewis Publishers Inc., Chelsea, MI.

Rulis A. M. (1989) Establishing a threshold of concern. InRisk Assessment in Setting National Priorities, ed.J. J. Bonin and D. E. Stevenson, Volume 7, pp. 271±278. Plenum Press, New York.

Sawyer C., Peto R., Bernstein L. and Pike M. C. (1984)Calculation of carcinogenic potency from long-term ani-mal carcinogenesis experiments. Biometrics 40, 27±40.SCF (1991) Guidelines for the Evaluation of Flavouringsfor Use in Foodstu�s: I. Chemically De®ned FlavouringSubstances. Commission of the European Communities,Scienti®c Committee for Food, Brussels.SCF (1996) Opinion on Response to Request from theCommission for SCF Opinion on the Scienti®c Basis ofthe Concept of Threshold of Regulation in Relation toFood Contact Materials. European Commission,Scienti®c Committee for Food, Brussels.

Schepers G. W. (1964) Tetraethyl lead and tetramethyllead. Archives of Environmental Health 8, 277±295.

Schwetz B. A., Quast J. F., Keeley P. A., Humiston C. G.and Kociba R. J. (1978) Results of 2-year toxicity andreproduction studies on pentachlorophenol in rats. InPentachlorophenol: Chemistry, Pharmacology andEnvironmental Toxicology. (EPA and the University ofWest Florida Symposium - Proceedings, June 27-29,1977, Pensacola, FL.), ed. K. R. Rao, pp. 301±309.Plenum Press, New York.

Scott J. R. (1977) Fetal growth retardation associated withmaternal administration of immunosuppressive drugs.American Journal of Obstetrics and Gynecology 128,668±676.

Sielken R. L. and Valdez-Flores C. (1996) Comprehensiverealism's weight-of-evidence based distributional dose-response characterization. Human and Ecological RiskAssessment 2, 175±193.

Stofberg J. and Kirschman J. (1985) The consumptionratio of ¯avouring materials: A mechanism for settingpriorities for safety evaluation. Food and ChemicalToxicology 23, 857±860.

Stofberg J. and Grundschober F. (1987) Consumptionratio and food predominance of ¯avouring materials.Perfumer and Flavourist 12, 27±68.

Tennant R. W. and Ashby J. (1991) Classi®cation accord-ing to chemical structure, mutagenicity to Salmonellaand level of carcinogenicity of a further 39 chemicalstested for carcinogenicity by the U.S. NationalToxicology Program. Mutation Research 257, 209±227.

Tennant R. W., Spalding J., Stasiewicz S. and AshbyJ. (1990) Prediction of the outcome of rodent carcino-genicity bioassays currently being conducted on 44chemicals by the National Toxicology Program.Mutagenesis 5, 3±14.

Teramoto S., Saito R., Aoyama H. and Shirasu Y. (1980)Dominant lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane (DBCP). Mutation Research77, 71±78.

Thysen B., Bloch E. and Varma S. K. (1985)Reproductive toxicity of 2,4-toluenediamine in the rat:


2. Spermatogenic and hormonal e�ects. Journal ofToxicology and Environmental Health 16, 763±769.

Weil C. S. and McCollister D. D. (1963) Safety evaluationof chemicals. Relationship between short- and long-term feeding studies in designing an e�ective toxicitytest. Agricultural and Food Chemistry 11, 486±491.

WHO (1987) Principles For The Safety Assessment ofFood Additives and Contaminants in Food. WHOEnvironmental Health Criteria. World HealthOrganization (WHO), International Program onChemical Safety in Cooperation with the Joint FAO(Food & Agriculture Organization of the UnitedNations), WHO Expert Committee on Food Additives(JECFA), Geneva, Environmental Health Criteria 70.

WHO (1994) Assessing Human Health Risk of Chemicals:

Derivation of Guidance Values for Health-basedExposure Limits. World Health Organization (WHO)International Program on Chemical Safety (IPCS).Published under the joint sponsorship of the UnitedNations Environment Programme, the InternationalLabour Organisation and the World HealthOrganization, Geneva, Environmental Health Criteria170.

Williams G. M. (1990) Screening procedures for evaluat-ing the potential carcinogenicity of food-packagingchemicals. Regulatory Toxicology and Pharmacology 12,30±40.

Woods L. A. and Doull J. (1991) GRAS evaluation of ¯a-vouring substances by the Expert Panel of FEMA.Regulatory Toxicology and Pharmacology 14, 48±58.

APPENDIX

follows


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


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).


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.


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