Toxicology Letters 149 (2004) 223–234

Toxicology databases and the concept of thresholds

of toxicological concern as used by the JECFA
for the safety evaluation of flavouring agents
A.G. Renwick∗
Clinical Pharmacology Group, Allergy and Inflammatory Sciences Research Division, School of Medicine,
University of Southampton, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK

Abstract

Since 1996 the FAO/WHO Joint Expert Committee on Food Additives (JECFA) has evaluated the safety of 1259 flavouring sub-
stances, based on a decision tree that incorporates a series of thresholds of toxicological concern. Safety conclusions are based on
the predicted consequences of metabolism and whether the estimated intake is above or below a threshold of toxicological concern
that is relevant to that compound. Compounds are allocated to one of three structural classes, and the intake compared with a thresh-
old of toxicological concern derived using data from chronic and sub-chronic toxicity studies on compounds in the same structural
class. If the substance is predicted to be metabolised to innocuous products there is no safety concern if the intake is below the
threshold, but suitable toxicity data on the compound or structural analogues are required if the intake exceeds the threshold. If the
substance is not predicted to be metabolised to innocuous products, and the intake is below the appropriate threshold, safety evalu-
ation is based on data on the compound or structural analogues. An additional threshold of 1.5 !g per day, derived from doses of in-
vestigated chemicals giving a calculated cancer risk of one in a million, is applied when appropriate toxicity data are not available.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Flavouring agent; Safety; Metabolism; Intake; Toxicity

1. Introduction (ii) a level of exposure so low that risk assessment can

The term “threshold” can be confusing because it
has two different uses in toxicology and risk assess-
ment:
(i) a dose within the dose–response curve below
which an adverse effect would not be produced,
because of homeostatic processes and
∗ Tel.: +44-23-8059-4261; fax: +44-23-8059-4262.
E-mail address: agr@soton.ac.uk (A.G. Renwick).
be based on the activities of structural analogues,
i.e. a threshold of toxicological concern.
A search of the WHO website for “JECFA” (Joint
FAO/WHO Expert Committee on Food Additives)
and “threshold” yields a large number of citations,
but in nearly all cases these refer to the presence
or absence of a threshold within the dose–response
relationship. In recent years the JECFA has used the
concept of a “threshold of toxicological concern” for
the safety assessment of flavouring agents, and this
will be the basis of this paper.

0378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2003.12.034
224 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
An extensive toxicological database is required
for compounds present in the food supply in signif-
icant amounts (Renwick, 1999), but it is generally
recognised that the extent of exposure is an important
determinant of the amount of data that is considered
necessary to undertake risk assessment (WHO, 1987).
The draft FDA Redbook II (FDA, 1993) proposed
database requirements for three levels of concern,
with each concern level based on a combination of
chemical structure and predicted intake. The concept
of a threshold of toxicological concern takes this re-
lationship to the point where risk assessment can be
undertaken in absence of toxicity data on the com-
pound under evaluation, providing that the intake is
sufficiently low. What represents “sufficiently low”
depends on the chemical structure of the compound
and the available toxicity data on structural analogues
that have been subjected to toxicological evaluation.
The threshold of toxicological concern represents an
important and pragmatic approach to risk assessment,
and can make a significant contribution to optimising
the use of toxicological resources. The threshold of
toxicological concern is used in situations where there
are limited or no data on the compound, but the human
exposure is so low that undertaking toxicity studies
is not warranted, because of the costs incurred in the
use of animals, manpower and laboratory resources.
The threshold of toxicological concern does not
provide the surety of risk assessment based on
compound-specific data because it is based on as-
sessing the probability of whether or not adverse
effects would occur at the estimated level of exposure.
However, this apparent difference is not “black and
white” because there are always uncertainties in risk
assessment, even when there is a comprehensive and
complete toxicity database. The use of threshold of
toxicological concern can be thought of as trading-off
increased uncertainty against very low exposure.
A threshold of toxicological concern was initially
applied to migrants from food contact materials and
was used as the basis of the US Food and Drug Ad-
ministration Threshold of Regulation for indirect food
additives (Federal Register, 1993, 1995). Initially,
the threshold of toxicological concern was based on
defining a level of exposure that would give a low
risk, even if the compound were to be a carcinogen.
A level in food of 0.5 ppb (parts per billion or !g/kg)
was selected because “most known carcinogens pose
less than one in a million lifetime risk if present in
the diet at 0.5 ppb”, and it was a level “2000 times
lower than the dietary concentration at which the vast
majority of studied compounds are likely to cause
non-carcinogenic toxic effect” (Federal Register,
1993). Although the value would give a de minimis
risk even if the compound were a genotoxic carcino-
gen, the Threshold of Regulation would not apply
to known carcinogens or substances whose chemical
structures provide reason to suspect that they may be
carcinogens, because that would contravene US food
law. Subsequent refinements to the use of the thresh-
old of toxicological concern concept for food contact
materials are discussed in Cheeseman et al. (1999)
and Munro et al. (2003).
The threshold of toxicological concern principle
was developed significantly by the work of Dr. I.C.
Munro, who analysed available chronic toxicity data
and developed a decision tree based on chemical struc-
ture and intake. The following text analyses the dif-
ferent thresholds of toxicological concern developed
by Munro, and their use by the JECFA in the risk as-
sessment of flavouring agents.
2. The JECFA decision tree for the safety
evaluation of flavouring agents
Flavouring agents represent an enormous challenge
to risk assessors because there are over 3000 sub-
stances, with toxicity data available on only a few
of these. The intakes of flavouring agents are consid-
ered to be self-limiting because of their taste prop-
erties. Flavouring agents have been evaluated by the
JECFA for many years, but it was only with the in-
troduction of a safety evaluation procedure based on
the Munro analyses and decision tree (JECFA, 1997)
that consideration of large number of structurally re-
lated compounds became feasible (see Munro et al.,
1998). A procedure for use by the JECFA was pro-
posed by Munro (1996), and presented to the 44th
meeting (JECFA, 1995). The essential details can be
found in JECFA Reports after this date, and in the pub-
lications of Munro et al. (1998, 1999). The procedure
involves four principal components:
(i) allocation of the compound to one of three struc-
tural classes,
 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
(ii) consideration of the consequences of metabolism,
(iii) estimation of intake,
(iv) comparison of the intake with a series of thresh-
olds of toxicological concern.
Since 1997 the procedure has been applied to a total
of 1259 different compounds (Table 1) organised by
the Flavour and Extract Manufacturers Association of
the USA into groups of structurally related substances.
2.1. Allocation of the compound to one of three
structural classes
The procedure is applicable only to the assessment
of compounds with known chemical structures, be-
cause the concept of structure–activity relationships
is the basis for allocation to one of three classes with
different levels of toxicological concern. Cramer et al.
(1978) developed a series of 33 questions into a deci-
sion tree that allocates the chemical structure to one
of three classes which were defined as follows:
• Class I substances are those with structures and re-
lated data suggesting a low order of oral toxicity.
If combined with low human exposure, they should
enjoy an extremely low priority for investigation.
The criteria for adequate evidence of safety would
also be minimal. Greater exposures would require
proportionately higher priority for more exhaustive
study.
Fig. 1. The initial steps in the decision tree of Cramer et al. (1978) used to assign flavouring agents to different structural classes.
225
• Class II substances are simply intermediate. They
are less clearly innocuous than those of class I, but
do not offer the basis either of the positive indication
of toxicity or of the lack of knowledge characteristic
of those in class III.
• Class III substances are those that permit no strong
initial presumptions of safety, or that may even sug-
gest significant toxicity. They thus deserve the high-
est priority for investigation. Particularly when per
capita intake is high or a significant subsection of
the population has a high intake, the implied hazard
would then require the most extensive evidence for
safety-in-use.
The initial steps in the decision tree (Cramer et al.,
1978) are shown in Fig. 1 to illustrate the approach.
Although the decision tree (Fig. 2) is unsophisticated
compared with modern computer-based, predictive
structure–activity databases, the differences in po-
tency were validated by the analyses performed by
Munro et al. (1996) to develop the threshold of
toxicological concern for each structural class (see
later).
2.2. Consideration of metabolism
Predicted metabolism is an important part of the
JECFA procedure and represents the second step in the
process (Fig. 3). The way that the different thresholds
226 A.G. Renwick / Toxicology Letters 149 (2004) 223–234

Fig. 2. An overview of Cramer et al. (1978) structural classification scheme-adapted to show where different structural groups are evaluated
for toxic potential.

of toxicological concern are applied in the procedure is whereas if metabolites are not predicted to be innocu-
dependent on the predicted metabolism. Compounds ous they follow the B-side (JECFA, 2000a). JECFA
that are predicted to be metabolised to innocuous prod- (1997) defined “Innocuous products are products
ucts are evaluated using the A-side of the decision tree, that are known or readily predicted to be harmless

Fig. 3. The initial steps in the procedure used by the JECFA for the safety evaluation of flavouring agents.
 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
to humans at the estimated intakes of the flavouring
agents”. The prediction of metabolism is difficult and
relies on expert judgement of the members and tem-
porary advisors invited to the JECFA meeting. The
definition used by the JECFA relates to the metabolic
fate at the low levels of intake relevant to the intake
as a flavouring agent. In consequence detoxication
processes such as glutathione conjugation would
not be saturated, and would rapidly inactivate po-
tentially reactive molecules such as aldehydes and
",#-unsaturated carbonyl compounds.
The reports of the early meetings at which the
procedure was used contained general text on the
metabolism of the different functional groups present
in the compounds evaluated at the meeting (JECFA,
1997, 1999, 2000a), but at later meetings metabolism
was considered separately for each group of sub-
stances. A large number of flavouring agents are
aldehydes or ",#-unsaturated carbonyl compounds,
which are reactive chemicals, and the procedure was
considered applicable to these functional groups be-
cause of their extensive detoxication by oxidation and
conjugation reactions (JECFA, 2002b).
2.3. Estimation of intake
There are a variety of methods available for esti-
mating the intakes of chemical in food (Kroes et al.,
2002). The method used in the JECFA procedure for
flavouring agents is one of the simplest methods, in
which the amount of the compound produced annu-
ally is divided across the population that may have
consumed foods containing the compound, a so-called
per capita estimate. Information on the annual pro-
duction volume for each flavouring agent in Europe
and the USA is supplied by the flavour manufacturers.
The annual production volumes are divided by 0.6,
on the assumption that only 60% of actual production
may be reported, and allocated to 10% of the relevant
population (equivalent to 32 million in Europe and
24 million in the USA; JECFA, 1997). The intake
estimates of flavouring agents for the USA evaluated
by the JECFA in the 55th Report (JECFA, 2001), and
subsequently, were corrected by dividing the reported
poundage by 0.8 rather than 0.6 because about 87% of
the poundage data had been reported in recent surveys
in the USA. Although the intake estimates relate to ma-
jor populations that would consume flavoured foods,
227
one weakness of the JECFA evaluations is the lack of
intake estimates for other geographical and cultural
areas.
The per capita × 10 method is simple and readily
applicable to the evaluation of flavouring agents; most
importantly it has been shown to be appropriately
conservative. Intakes estimated by the method were
generally higher than the mean and 95th percentile
14-day average intakes found using a detailed dietary
analysis based on food consumption and composition
(Hall and Ford, 1999). An alternative intake esti-
mate for flavouring agents is the theoretical added
maximum daily intake (TAMDI) which is calculated
assuming that 160 g of food and 324 ml of drink con-
tain the flavouring substance at the upper use level
specified by the Council of Europe (Lambe et al.,
2002). While such an intake might occur rarely on
single days, it is not appropriate for comparison with
thresholds of toxicological concern that are derived
using data from chronic animal studies (see Renwick
et al., 2003). The TAMDI estimates for a range of
flavouring agents were gross overestimates compared
with the per capita × 10 values, and the latter were
supported by the results of a stochastic model that
used data from the Dietary and Nutritional Survey
of British Adults combined with information from
the Irish National Food Ingredients Database (Lambe
et al., 2002).
2.4. Comparison of the intake with a series of
threshold of toxicological concern values
The thresholds of toxicological concern for Cramer
et al. (1978) structural classes were derived by Munro
et al. (1996) based on an analysis of data from chronic
toxicity studies on 137, 28 and 448 compounds in
classes I, II and III respectively. The database com-
prised:
(i) NOEL (no observed effect level) values for ro-
dents and rabbits; NOEL values for dogs and
other species were not included because the stud-
ies contained too few animals to be statistically
valid,
(ii) NOEL values from sub-chronic studies were di-
vided by a three-fold uncertainty factor (WHO,
1994) to convert them into equivalent chronic
NOELs and
228 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
(iii) NOEL values derived by the authors were used
except where these were based on physiological
rather than toxicological effects.
The NOEL from a chronic toxicity study in rodents
is the usual starting point for the determination of an
acceptable daily intake (ADI) for an approved food
additive, and assumes that there is a threshold dose
within the dose–response relationship (see Section 1
to avoid confusion). The cumulative distributions
of NOELs for each Cramer et al. (1978) structural
class were plotted and a log-normal distribution was
fitted. The three distributions were significantly dif-
ferent from each other (Munro et al., 1996). The 5th
percentile values for the classes I, II and III NOEL
distributions were calculated to be 3.0, 0.91 and
0.15 mg/kg body weight per day. Therefore, there is a
95% probability that the NOEL from a chronic animal
bioassay on an unstudied compound would be above
the relevant 5th percentile value. The 5th percentile
NOEL values were converted to corresponding human
intakes by dividing by the usual 100-fold uncertainty
factor (WHO, 1987) that is used in the calculation of
ADI values for food additives. The intakes were then
multiplied by 60 to scale to the adult human body
weight. These analyses gave thresholds of toxicolog-
ical concern of 1800, 540 and 90 !g per person per
day for structural classes I, II and III.
Fig. 4. The A-side of the procedure used for compounds predicted to be metabolised to innocuous products.
The way that these threshold values are used in the
JECFA procedure depends on whether the flavouring
agent is evaluated using the A-side or the B-side.
Using the A-side for substances that are predicted
to be metabolised to innocuous products (Fig. 4;
based on JECFA (2000a)) there would not be a safety
concern if the intake of a flavouring agent were below
the relevant threshold. If the estimated intake ex-
ceeds the threshold then the next question determines
whether the compound is endogenous. There would
be no safety concern if the compound is an endoge-
nous body constituent. The JECFA recognised that
many endogenous compounds have important physio-
logical and other functions and that ingestion of such
a compound could give rise to adverse effects. There-
fore, within the context of the procedure, endogenous
substances were defined by the JECFA as “Interme-
diary metabolites normally present in human tissues
and fluids, whether free or conjugated; hormones and
other substances with biochemical or physiological
regulatory functions are not included. The estimated
intake of a flavouring agent that is, or is metabolised
to, an endogenous substance should be judged not
to give rise to perturbations outside the physiological
range” (JECFA, 1997). If the compound is not an en-
dogenous substrate, according to this definition, then
data on the compound, or a related substance, can be
used for the safety evaluation.
 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
Fig. 5. The B-side of the procedure used for compounds not predicted to be metabolised to innocuous products.
Using the B-side for substances that are not pre-
dicted to be metabolised to innocuous products
(Fig. 5; based on JECFA (2000a)) toxicity data on
the compound, or a related substance, would be used
for the safety evaluation if the intake of a flavour-
ing agent were below the relevant threshold. A more
comprehensive risk assessment, using toxicity data on
the substance or a close structural analogue, would
be necessary if the intake were above the threshold.
There would not be a safety concern if the intake were
below the relevant classes I, II or III threshold, and
there was a NOEL that gave an adequate margin of
safety. Additional data would be required if there was
not a suitable NOEL and the intake exceeded a general
threshold of toxicological concern of 1.5 !g per day.
There would not be a safety concern if the intake were
less than the threshold of toxicological concern of
1.5 !g per day.
The threshold of toxicological concern of 1.5 !g
per day was developed from the work of Rulis and
colleagues at the FDA (see Cheeseman et al., 1999) on
the Threshold of Regulation for food contact materials
(see Section 1). The FDA threshold of toxicological
concern was derived from an analysis of the distribu-
tion of intakes giving a one in a million cancer risk
using linear low-dose extrapolation of experimental
data in animals for the chemicals in the Carcinogenic
229
Potency Database of Gold and colleagues. There
were 477 animal carcinogens in the database when
the Threshold of Regulation was proposed (Federal
Register, 1993) and 709 in the recent reconsideration
by Cheeseman et al. (1999). The threshold of toxico-
logical concern of 1.5 !g per day was derived Munro
(1990) from the distribution of estimated one in a
million cancer risks for a subset of the Gold et al.
(1989) database, which comprised the most relevant
studies in rodents, i.e. those that contained two or
more oral dose levels and a statistically significant
dose–response relationship. This analysis showed that
there was a 63% probability that the estimated cancer
risk would be one in a million or less for a dietary
concentration of 0.5 ppb (0.5 !g/kg) if all chemicals
were carcinogens, but a 96% probability if only one
in ten chemicals were carcinogenic. Thus for any
unstudied compound there is a 96% probability that
the cancer risk would be one in a million or less at
a daily intake of 1.5 !g per person per day. This is
probably a gross over-estimate of the real probability
because of the selective nature of compounds that
are subjected to rodent carcinogenicity testing, and
the highly conservative linear extrapolation method
used to convert the TD50 (the estimated dose giving
a 50% incidence of tumours in the animal study) to
a one in a million risk. In addition, the assumption
 230
Table 1
Summary of the results of the application of the procedure by the JECFA to the safety evaluation of flavouring agents
Group Total (n) Class I Class II Class III A-side B-side Below No safety Reference
threshold concern
Ethyl esters 15 15 0 0 15 0 11 15 JECFA (1997)
Isoamyl alcohol and related esters 11 11 0 0 11 0 9 11 JECFA (1997)
Allyl esters 21 0 18 3 0 21 20 20a JECFA (1997)
Saturated aliphatic acyclic linear primary alcohols, 38 38 0 0 38 0 27 38 JECFA (1999)
aldehydes and acids
Saturated aliphatic acyclic branched-chain primary 25 22 3 0 25 0 25 25 JECFA (1999)
alcohols, aldehydes and acids
Aliphatic lactones 35 29 0 6 29 6 26A, 2B 31b JECFA (1999)
Esters of aliphatic acyclic primary alcohols with 32 32 0 0 32 0 31 32 JECFA (1999)

A.G. Renwick / Toxicology Letters 149 (2004) 223–234

branched-chain aliphatic acyclic acids
Esters of aliphatic acyclic primary alcohols with 67 66 1 0 67 0 66 66c JECFA (1999)
aliphatic linear saturated carboxylic acids
Esters of branched-chain terpenoid alcohols and 26 26 0 0 26 0 26 26 JECFA (1999)
aliphatic acyclic carboxylic acids
Saturated aliphatic acyclic secondary alcohols, ketones 39 28 11 0 37 2 35A, 2B 39 JECFA (2000a)
and related saturated and unsaturated esters
Linear and branched-chain aliphatic unsaturated 42 42 0 0 41 1 40A, 1B 41d JECFA (2000a)
unconjugated alcohols aldehydes, acids and related
esters
Aliphatic acyclic and alicyclic terpenoid tertiary 23 22 1 0 22 1 19A, 1B 22e JECFA (2000a)
alcohols and structurally related substances
Carvone and structurally related substances 9 6 3 0 9 0 8 9 JECFA (2000a)
Ionones and structurally related substances 21 21 0 0 13 7 13A, 7B 20f JECFA (2000a)
Aliphatic acyclic and alicyclic "-diketones and related 22 0 22 0 22 0 19 22 JECFA (2000a)
"-hydroxyketones
Substances structurally related to menthol 14 9 5 0 14 0 12 14 JECFA (2000a)
Simple aliphatic and aromatic sulphides and thiols 137 97 34 6 0 137 137 137 JECFA (2000b)
Aliphatic linear primary alcohols, aldehydes and 47 47 0 0 47 0 41 47 JECFA (2000b)
carboxylic acids, acetals and esters containing
additional oxygenated functional groups
Cinnamyl alcohol and related flavouring agents 55 50 5 0 55 0 51 55 JECFA (2001)
Furfuryl alcohol and related flavouring agents 15 0 9 6 0 15 15 15 JECFA (2001)
Phenol and phenol derivatives 48 47 0 1 48 0 47 48 JECFA (2001)
Pulegone and related flavouring agents 6 2 4 0 0 6 6 6 JECFA (2001)
Pyrazine derivatives 41 0 32 9 41 0 41 41 JECFA (2002a)
Aromatic substituted secondary alcohols, ketones and 38 28 6 4 36 2 36A, 2B 37e JECFA (2002a)
related esters
Benzyl derivatives 37 37 0 0 36 1 33A, 1B 37 JECFA (2002a)
Hydroxy- and alkoxy-substituted benzyl derivatives 46 36 10 0 46 0 42 46 JECFA (2002a)
Aliphatic acyclic diols, triols and related substances 31 22 9 0 31 0 22 31 JECFA (2002a)
Aliphatic acyclic acetals 10 10 0 0 10 0 10 10 JECFA (2002a)
Alicyclic primary alcohols, aldehydes, acids and related 26 26 0 0 25 1 25A, 1B 26 JECFA (2002b)
esters
Phenethyl alcohol, aldehyde, acid and related acetals 43 39 0 4 43 0 42 43 JECFA (2002b)
and esters
Sulphur-containing heterocyclic compounds 30 0 21 9 3 27 2A, 27B 30 JECFA (2002b)
Sulphur-substituted furan derivatives 33 0 18 15 0 33 33 33 JECFA (2002b)
Alicyclic ketones, secondary alcohols and related esters 25 6 19 0 25 0 25 25 JECFA (2002b)
Aliphatic secondary alcohols, ketones and related esters 39 11 28 0 39 0 39 39 JECFA (2002b)
Alicyclic, alicyclic-fused and aromatic-fused ring 16 4 0 12 10 6 10A, 4B 16 JECFA (in press)
lactones
Aliphatic di- and trienals and related alcohols, acids 26 26 0 0 13 13 13A, 13B 26 JECFA (in press)
and esters
Aliphatic branched-chain unsaturated alcohols, 32 32 0 0 32 0 31 32 JECFA (in press)

A.G. Renwick / Toxicology Letters 149 (2004) 223–234

aldehydes, acids and related esters
Aliphatic and aromatic ethers 29 9 12 8 29 0 26 29 JECFA (in press)
Hydroxypropenylbenzenes 9 6 0 3 9 0 8 9 JECFA (in press)
Total 1259 902 271 86 979 279 1174 1249
a Allyl furoate was deferred because of inadequate data at step B4; a conclusion of no safety concern was reached when step B5 was added to the procedure (JECFA, 1999).
b Four ",#-unsaturated lactones were deferred until a later meeting, at which the application of the procedure to such compounds was considered (JECFA, 2001).
c One ",#-unsaturated ester was deferred until a later meeting, at which the application of the procedure to such compounds was considered (JECFA, 2001).
d The evaluation of one compound on the B-side was deferred pending evaluation of the results of a 90-day study.
e One substance evaluated using the B-side had an intake below its structural class threshold but lacked appropriate toxicity data and the intake was more than 1.5 !g per

day so that additional data were required for an evaluation.

f One of the substances submitted for evaluation was considered not to be a member of the group and was not evaluated.

231
232 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
that only one in ten untested compounds would be
a carcinogen is probably conservative (Fung et al.,
1995).
3. The conclusions reached by the JECFA using
the procedure for the safety evaluations of
flavouring agents
The JECFA first used the procedure at a meeting
in 1996 (JECFA, 1997) to evaluate groups of ethyl,
isoamyl and allyl esters using the A-side for ethyl and
isoamyl esters and the B-side for allyl esters (Table 1).
Adequate data were not available on allyl furoate and
this compound was deferred to a later meeting because
at that time the procedure did not include the thresh-
old of toxicological concern of 1.5 !g per day on the
B-side. Subsequent meetings considered the general
threshold of 1.5 !g per day and the issue of com-
pounds such as ",#-unsaturated carbonyl compounds
and their precursors in relation to the activity of detox-
ication pathways such as oxidation and conjugation
with glutathione.
To date 1259 different flavouring agents have been
evaluated using the procedure, with the majority in
structural class I (Table 1), and with most (979) be-
ing evaluated using the A-side of the procedure (one
compound was not evaluated in detail because it was
not related to other members of the group). Only 68
compounds that were evaluated using the A-side of the
procedure had intakes that exceeded the relevant struc-
tural class threshold; of these 39 were of no safety con-
cern because they were endogenous or metabolised to
endogenous compounds, and 29 were evaluated using
toxicity data on the flavouring agent or a structurally
related compound. A total of 279 compounds were
evaluated using the B-side and half of these were a
large and complex group of sulphides and thiol com-
pounds. Two compounds that were evaluated using
the B-side were subjected to a more comprehensive
safety assessment because their exposures exceeded
the relevant threshold (JECFA, in press). In nearly all
other cases the other flavouring agents evaluated us-
ing the B-side were considered not to be a safety con-
cern based on toxicity data on the flavouring agent or
a structurally related compound. Only six compounds
were considered not to be a safety concern because
their estimated intakes were less than 1.5 !g per day,
although a total of 471 flavouring agents had reported
intakes below this level.
4. Conclusions
The JECFA procedure based on the work of Munro
and his colleagues (Munro, 1996; Munro et al., 1996)
provides a practical method for the safety evaluation
of flavouring agents. The conservative nature of the
thresholds of toxicological concern compared with the
NOELs for flavouring agents was shown by the re-
view by Munro and Kennepohl (2001), in which esti-
mated intakes were compared with the NOEL values
available from good quality, multi-dose toxicological
studies. Most of the toxicity studies were short-term
or 90-day studies, and so may have overestimated the
NOEL that would be obtained from a chronic bioassay
on the compound or a structural analogue by a factor of
about three-fold (WHO, 1994). The data showed that,
with the exception of ethanol, the margins of safety
exceeded the usual 100-fold uncertainty factor; in the
majority of case the margins of safety were over 1000,
and in many cases more than 1,000,000. This analysis
supported the conclusions reached by the JECFA us-
ing the procedure for the safety evaluation of flavour-
ing agents.
The JECFA experience with the application of the
threshold of toxicological concern principle has given
rise to increased interest in its use in other risk as-
sessment scenarios. Further analyses of the threshold
of toxicological concern concept were presented by
Kroes et al. (2000) and discussed at an international
workshop organised by ILSI-Europe (Barlow et al.,
2001), which indicated that the approach could be
more widely applicable if a pre-screening procedure
removed from consideration compounds that could
show bioaccumulation, genotoxicity, allergenicity or
organophosphate-like neurotoxicity. These different
aspects have been considered by an ILSI-Europe Ex-
pert Group and a paper (Kroes et al., 2004) prepared
following discussions at an international workshop. A
new decision tree is proposed in that paper in which
compounds with structural alerts for genotoxicity are
considered first, and then any compound with very
low estimated intakes, i.e. less than 1.5 !g per day,
is removed from further detailed consideration. Such
an approach would allow assessments to concentrate
 A.G. Renwick / Toxicology Letters 149 (2004) 223–234
on those compounds with potentially significant
intakes.
References
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