EFSA Supporting Publications - 2016 - Review of The Threshold of Toxicological Concern TTC Approach and Development of

EVENT REPORT
APPROVED: 16 February 2016 PUBLISHED: 10 March 2016
Review of the Threshold of Toxicological Concern (TTC)

approach and development of new TTC decision tree
European Food Safety Authority and World Health Organization
Abstract
In light of improving analytical methods, it can be expected that many more unintended chemicals will
be detected in our environment, including food and drinking water, and in our bodies. The TTC
approach is a screening and prioritization tool for the safety assessment of chemicals when hazard
data are incomplete and human exposure can be estimated. TTC is not applicable when compound-
specific assessment and toxicity data are available or are required under existing regulations. EFSA
and WHO embarked in this project to review the current TTC approach and propose some
modifications, building on previous activities and recommendations. An expert workshop was
implemented, informed through a public call for data and an open stakeholder meeting. The experts
concluded that the TTC approach is based on scientific risk assessment principles and fit for purpose
as a screening tool, to assess low dose chemical exposures and to identify those for which further
data are necessary to assess the human health risk. The expert group made recommendations to
improve and expand the TTC concept, and proposed a tiered approach (revised decision tree),
considering the current state-of-the-science and available toxicological databases. The proposed TTC
for genotoxic compounds of 0.0025 µg/kg bw/day, based on linear extrapolation for known genotoxic
carcinogens, is sufficiently protective. Exceptions are high potency carcinogens, i.e. aflatoxin-like,
azoxy- or N-nitroso-compounds and benzidines, which are excluded from the current TTC approach.
Carcinogens that are not DNA reactive are adequately covered by the other TTC tiers. For some
categories of substances, as inorganic chemicals, metals, organometallics, proteins, steroids, organo-
silicon compounds, TTC is not applicable. A number of recommendations were given to further
strengthen the TTC approach, including the need for a permanent repository for data supporting TTC
and the Cramer scheme, based on merging of existing databases and establishment of clear inclusion
criteria for studies.
© European Food Safety Authority, 2016.
Keywords: Threshold of Toxicological Concern, TTC, safety assessment, low exposure, chemicals,
unintended chemicals, health risk.
Question number: EFSA-Q-2016-00080
Correspondence: sc.secretariat@efsa.europa.eu
www.efsa.europa.eu/publications EFSA Supporting publication 2016:EN-1006

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TTC Approach
Disclaimer: The designations employed and the presentation of material in this publication do not
imply the expression of any opinion whatsoever on the part of the World Health Organization (WHO)
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the delimitation of its frontiers or boundaries. The mention of specific companies or products of
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the reader. In no event shall WHO be liable for damages arising from its use. The views expressed
herein are those of the experts participating at the workshop and do not necessarily represent those
of EFSA and WHO. This document is also published and available in the WHO website:
http://www.who.int/foodsafety/areas_work/chemical-risks/TTC/en/
Acknowledgements: EFSA and WHO wish to thank Drs Gordon Barrett, Diane Benford, Kristi Jacobs
and Josef Schlatter for the preparatory work on this scientific output and all the experts participating
in the expert workshop; Drs Andrew Renwick and Josef Schlatter for the preparatory work for the
expert workshop; EFSA and WHO staff members: Jean Lou Dorne, Daniela Maurici, Djien Liem,
Xudong Jia and Angelika Tritscher for the preparatory work of the expert workshop and for the
support provided to this scientific output. The financial support of the US FDA for the preparatory
work of the expert workshop is gratefully acknowledged.
Suggested citation: EFSA (European Food Safety Authority) and WHO (World Health Organization),
2016. Review of the Threshold of Toxicological Concern (TTC) approach and development of new TTC
decision tree. EFSA supporting publication 2016: EN-1006. 50 pp.
© European Food Safety Authority, 2016. All rights reserved. Reproduction is authorised, provided the
source is acknowledged, save where otherwise stated. Where prior permission must be obtained for
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on use. The EFSA logo is the exclusive property of EFSA. Its use is prohibited without the prior written
permission of EFSA.
www.efsa.europa.eu/publications 2 EFSA Supporting publication 2016:EN-1006

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Table of contents
Abstract .........................................................................................................................................1
1. Introduction........................................................................................................................4
1.1. Objectives of the workshop .................................................................................................4
1.2. Additional considerations .....................................................................................................5
1.3. Description and History of the TTC (mainly based on Dewhurst and Renwick, 2013) ...............6
2. The Cramer scheme ............................................................................................................9
2.1. Does the Cramer classification scheme require revising? ........................................................9
2.2. Is metabolism an inherent part of the Cramer decision scheme and included in the TTC
values? ...............................................................................................................................9
2.2.1. Metabolism and the Cramer et al. (1978) scheme ............................................................... 10
2.2.2. Metabolism and the TTC values ......................................................................................... 13
3. Validity of the databases used to derive the TTC values ...................................................... 15
3.1. Do the databases used for deriving TTC values adequately represent the “world of
chemicals”? ...................................................................................................................... 15
3.2. Should the point of departure for establishing TTC values be reconsidered? ......................... 17
3.3. Should the overall database be expanded/modified? ........................................................... 17
3.4. Should chemicals with particular structures and/or toxicological properties be
excluded from the TTC approach? ...................................................................................... 19
3.5. Combined exposure to multiple chemicals and combined exposures from multiple sources .... 21
3.6. TTC for unidentified chemicals ........................................................................................... 21
3.7. Low-dose effects and non-monotonic dose-response ........................................................... 22
4. Exposure considerations .................................................................................................... 22
4.1. General principles ............................................................................................................. 22
4.2. Less than lifetime or intermittent exposures........................................................................ 23
4.3. Infants and children, potentially sensitive life-stages ........................................................... 23
5. Consideration of the Kroes et al. (2004) decision tree.......................................................... 24
6. Conclusions ...................................................................................................................... 25
6.1. General conclusions .......................................................................................................... 25
6.2. The Cramer scheme is fit for purpose ................................................................................. 26
6.3. Metabolism is an inherent part of the TTC values ................................................................ 27
6.4. TTC Domain of applicability is sufficiently broad .................................................................. 27
6.5. The TTC for genotoxic compounds is sufficiently protective ................................................. 27
6.6. TTC Tiers are sufficient for non-DNA reactive carcinogens and non-cancer endpoints. ........... 28
6.7. Conclusions on other considerations ................................................................................... 28
6.7.1. Point of Departure (PoD) and database .............................................................................. 28
6.7.2. Exposure considerations .................................................................................................... 29
6.7.3. Expression of TTC values ................................................................................................... 29
6.8. Overall conclusion ............................................................................................................. 29
7. Recommendations ............................................................................................................. 30
7.1. Cramer scheme ................................................................................................................. 30
7.2. Metabolism ....................................................................................................................... 30
7.3. Expand/modify the overall database and derivation of class thresholds ................................ 31
7.4. Chemical domain analyses ................................................................................................. 31
7.5. Point of departure ............................................................................................................. 32
7.6. Exclusion of chemical categories ........................................................................................ 32
7.7. Specific TTC values ........................................................................................................... 32
7.8. Combined oral exposure to multiple chemicals and from multiple sources ............................. 33
7.9. Acute and other less than lifetime exposures ...................................................................... 33
7.10. Infants and children potentially sensitive life-stages ............................................................ 33
7.11. Additional recommendations .............................................................................................. 33
8. TTC Decision Tree ............................................................................................................. 33
References ................................................................................................................................... 37
Abbreviations ............................................................................................................................... 41
Appendix A – Explanations to Cramer scheme ........................................................................... 42

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1. Introduction
In light of ever improving methods in analytical chemistry, it is to be expected that many more
unintended chemicals will be detected in our environment, including food and drinking water, as well
as in our bodies. The fact a substance can be detected is often misconstrued as indicating a presumed
health risk, but this is not inevitably the case. To allow for a health risk assessment of these
exposures when there are insufficient chemical-specific data, and to prioritise those most likely to be a
health risk, other methods need to be applied to estimate the potential human health impact and to
make informed risk management decisions. The Threshold of Toxicological Concern (TTC) is a
methodology that may be used to assess potential human health concerns for a chemical based on its
structural chemical characteristics and estimated exposure when chemical-specific toxicity data are
scarce or absent.
Overall, the TTC approach integrates data from hundreds of chemicals on chemical structure,
metabolism, and toxicity for the evaluation of chemicals that have not been previously considered,
consistent with standard risk assessment principles. It has been developed to be a pragmatic,
scientifically-valid approach for the safety evaluation of chemicals with relatively low oral exposure
and for which limited chemical-specific data are available. Application of a science-based systematic
approach provides risk managers with reliable information useful to prioritize actions and target
further testing and evaluation strategies. It is important that scientific research continues to provide
refinement of, and improvement to, the TTC approach in order to continue to assure its adequacy,
appropriate application, and usefulness for public health protection.
1.1. Objectives of the workshop

EFSA and WHO initiated a project that intends to provide recommendations as to how the existing
TTC framework may be improved and expanded by updating/revising the Cramer classification
scheme (Cramer et al., 1978; hereafter referred to as the Cramer scheme) and extending the TTC
approach, thereby building on existing and ongoing work in this area. A call for data was issued by
WHO in 2013 and information was collected regarding new proposals and on-going scientific work in
the area. Based on the submitted data and other publicly available information, and building on
previous work, a background paper was developed that served as basis for discussion by the expert
group.
The overall goal of the project is to develop a globally harmonised decision tree for a tiered approach
on the application of the TTC in the risk assessment of chemicals from oral exposures.
To gain the broadest possible input for this project, a stakeholder public hearing was organised on 2
December 2014 in Brussels, where approximately 100 people representing NGOs, industry,
government, academia and consumer organisations participated (list of participants is included in
annex 1). Stakeholders who had submitted a written request, were given the opportunity to express
their views with short presentations that have been published on EFSA’s website (link here). The
points raised by the stakeholders were considered in the subsequent expert meeting held on 3-5
December for which an open call for experts was published on WHO’s website in August 2014,
deadline for submission of expression of interest on 30 September 2014.
From the 50 applications received, 26 participants were selected to participate in the workshop
according to the expertise needed and published in the call, taking geographic and gender aspects
into account. A list of participants is included in annex 2. Most of the participants in the expert
workshop also participated in the stakeholder hearing held the day before. The experts completed a
declaration of interests and a declaration of confidentiality that were evaluated by WHO according to
the organisations’ rules. WHO concluded that the interests declared did not warrant experts to be
excluded from the discussion at the meeting. Five experts, who were found to have potential conflicts
of interests, did not take part in the development of conclusions and recommendations. Dr Diane
Benford served as Chairperson of the workshop and Dr Gordon Barrett and Dr Kristi Muldoon Jacobs
served as rapporteurs. The agenda as adopted is included in annex 3.
The outcome of the workshop was a series of conclusions and recommendations agreed by the expert
group. These were published for public consultation for a period of eight weeks on the WHO and EFSA
websites. Comments received were considered and addressed by the expert group prior to publication
of the final workshop report.

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During the workshop, experts were divided in two breakout groups: the first group addressed
questions in relation to the Cramer classification scheme and underlying scientific concepts; the
second group addressed questions in relation to the TTC values and an overall TTC decision tree.
Discussions from the breakout groups were presented to and discussed by the whole expert group.
The main questions addressed by the experts are summarised below:
Cramer classification Scheme
 Is the framework in the Cramer classification scheme for sorting chemicals into structural
classes sufficient and representative of the most up-to-date scientific knowledge?
 Does the scientific evidence support replacing or expanding the potency classes of the Cramer
Scheme with a larger number of structural sub-categories? Are there other revisions to the
Cramer classification scheme that are supported by the available scientific database?
 How should Class II be treated (eliminated, strengthened etc.)?
 Are there classes of chemicals, other than those already excluded, that the TTC approach
should not be used to evaluate?
 Can the Cramer Classification Scheme be redesigned in order to avoid the high degree of
overlap in NOEL/NOAEL values between Classes I and III?
 Should phenols and primary amines be reassigned to Class II, based on outlier analysis of
their NOAEL/NOEL distributions, as proposed in Tluczkiewicz et al. (2011)?
 How can genotoxicity, ADME, and mechanism of action data be used to refine the
classification scheme and/or class toxicity thresholds?
Background/Scientific Principles/Criticisms
 Is the TTC concept based on scientific risk assessment principles and sufficiently conservative
for public health protection?
 What is the TTC approach intended for and when should it not be used?
 Can the TTC framework be modified to take possible effects of low-dose mixtures into
account?
 Can the TTC approach take into account non-monotonic/low-dose only effects?
1.2. Additional considerations

The TTC approach is a screening and prioritization tool for the safety-assessment of chemicals when
hazard data are incomplete. The structure of chemicals with limited toxicity data is evaluated for
specific structural features via the Cramer Scheme. NOEL values for chemicals have been used to
calculate TTC levels for each of the three Cramer Classes to determine the maximum exposure levels
where no concern for safety can be assured for chemicals within that class. Chemicals with exposures
exceeding the relevant TTC level are not necessarily associated with any health concerns but rather
are flagged as warranting further evaluation. The evaluation may lead to a decision that for some
chemicals further work and risk mitigation steps are necessary while for others exposure is so low that
the probability of adverse health effects is sufficiently low and no further data are necessary. In
principle, the TTC approach can be applied in any area of chemical risk assessment for which human
exposures are low, whether exposure is from deliberate addition or due to contamination.
However, TTC is not applicable when compound-specific assessment and toxicity data are required
under existing regulations, and available compound-specific toxicity data should be examined except
in certain priority-setting or screening cases (see section 3.1). Moreover, specific classes of chemicals
are excluded from the TTC approach, either because of toxicological considerations or for lack of
representation in the underlying database (see section 3.4).
The TTC is intended to provide a health-protective approach in situations where it is not feasible to
acquire chemical-specific data (e.g. impurities and breakdown/reaction products in food additives,
trace contaminants in food and water), where evaluation of a large number of compounds with low
exposure is required (such as flavouring substances), in prioritization of large numbers of compounds

TTC Approach
where resources are limited (e.g. contaminants in surface water), or when a rapid safety assessment
(chemical food safety incidents) is needed.
There are generic questions in the risk assessment of chemicals that are under discussion in the
scientific community, sometimes for decades (e.g. the existence of a toxicological threshold dose
below which no adverse effect is produced, low-dose effects due to non-monotonic dose-response
relationships, mixtures, interspecies extrapolation, adequacy of endpoints tested, fetal origin of adult
disease, epigenetics, dose-metric, extrapolation from subchronic to chronic studies, endocrine
disruption). Such questions apply also to the TTC approach but are not specific to it and discussion on
such generic risk assessment considerations are not in the scope of this report. The present report is
also not intended to be a review of all publications on the development and application of the TTC
approach. This work builds on recent, comprehensive reviews of the TTC approach and the reader is
referred to EFSA (2012) and Dewhurst and Renwick (2013), as well as all information submitted to
WHO in response to a public call for data (published 5 Aug 2013, deadline for submission 30 Sept
2013). In preparation of the background paper for the workshop data available by September 2013
were considered. Additional information available after this date was taken into account up to the date
of the workshop (December 2014) and also the views expressed at the stakeholder meeting were
taken into account during the deliberations of the expert group.
1.3. Description and History of the TTC (mainly based on Dewhurst and
Renwick, 2013)
The risk assessment of chemicals usually involves comparisons of the dose–response data for the
toxicity of the chemical under evaluation with the predicted human exposure. The Threshold of
Toxicological Concern (TTC) is an approach that can be used in the absence of chemical-specific
toxicity data. It is based on the establishment of levels of human exposure (TTCs) that would not
represent a safety concern using toxicological data for other chemicals sharing some structural
similarities. The approach does not involve quantitative structure–activity relationships (QSAR) for
specific endpoints but rather is based on the distribution of potencies for chemicals that share similar
broad structural characteristics with the chemical under evaluation. In principle, the TTC approach can
be considered for the safety evaluation of any chemical, which is not covered by the exclusion criteria
(see 5.4) in the current TTC approach, for which information on the chemical structure is available
and for which an estimate of the extent of human exposure can be made. It allows an assessment of
whether human exposure is so low that a more in-depth evaluation is not needed, or if chemical-
specific toxicity data and/or control of human exposure are required. The TTC values can also be used
to set an upper limit for human exposure for a chemical in the absence of intake data.
The history of the TTC, the supporting databases and the extension of the original concept have been
considered in numerous papers (Barlow et al., 2001; Brown et al., 2009; Cheeseman et al., 1999;
Gold et al., 2005; Kroes et al., 2004, 2007; Munro et al., 1996, 2008; SCCP, 2008). The approach has
been controversial because it seeks to provide risk characterisation advice in the absence of the usual
toxicity database; the validity of the approach is critically dependent on the validity of the databases
used to derive the TTC values.
The TTC evolved following a review by Munro (1990) of the Threshold of Regulation (TOR), which is
being used by the US Food and Drug Administration in the context of regulating food contact
materials with low exposures and relates to a dietary concentration giving an intake of 1.5 µg per
person per day, equivalent to 0.025 µg/kg body weight (bw) per day (US FDA, 1993). The TOR was
derived by low-dose extrapolation of carcinogenicity data from animal studies to define human
exposures associated with an upper-bound estimate of one in a million cancer risk (see US FDA, 1993;
Munro, 1990). The TOR of 1.5 µg per person per day was considered to provide an adequate margin
of safety for other forms of toxicity for chemicals that do not have a structural alert for
genotoxicity/DNA reactivity. This approach led to the development of TTC values for non-cancer
effects by Munro et al. (1996), which were based on analyses of the no-observed (adverse) effect
levels (NOAELs) from repeated dose toxicity data for chemicals separated into three structural classes
using the Cramer et al. (1978) decision scheme.
The intent of Munro et al. (1996) was to develop a database consisting mainly of NOAELs from
chronic toxicity studies in animals. However, in many cases, the lowest and thus most conservative
NOAEL for a chemical came from a subchronic study. In order to group NOAELs for chemicals with

TTC Approach
only subchronic studies with those with chronic studies to derive the cumulative distribution of
NOAELs, subchronic NOAELs were divided by a factor of three to approximate the most likely NOAEL
that would be derived from a chronic study. This conversion factor was based on research defining
the relationship between subchronic and chronic NOAELs available at the time. Based on an analysis
of 222 NOAEL ratios of subchronic / chronic rat studies and 99 NOAEL ratios of subchronic / chronic
mouse studies (Zarn et al. 2011) taken from publicly available mouse and rat feeding toxicity studies,
and the EFSA (2012b) recommended a factor of 2 for extrapolating from subchronic to chronic study
duration in rodents, which means that the factor of three used by Munro et al. (1996) can be
considered to be conservative.
A TTC value was calculated by Munro et al. (1996) from the respective distribution of NOAELs for each
of the 3 Cramer structural classes, using a database of 613 chemicals with 2941 NOAELs, representing
a range of industrial chemicals, pharmaceuticals, food chemicals and environmental, agricultural and
consumer chemicals likely to be encountered in commerce with good supporting toxicological data,
yielding 137, 28 and 448 chemicals in Cramer class I, II and III, respectively. For each of the 613
chemicals, the most conservative NOAEL was selected, based on the most sensitive species, sex and
endpoint. The fifth percentile NOAEL (in mg/kg bw/day) was calculated for each structural class and
this was converted to the intake for a 60kg person following the application of a safety factor to
calculate the TTC value. In converting the fifth percentile NOAELs to a TTC value for the three
structural classes, a 100-fold safety factor was used, the default safety factor used for establishing
health-based guidance values for chemicals using toxicity data from animal studies. This procedure
resulted in TTC values of 1800, 540, 90 µg/person/day for Cramer classes I, II and III, respectively.
A number of criticisms of the TTC approach were raised at a workshop organised by the ILSI Europe
TTC Task Force in 1999 (Barlow et al., 2001), principally in relation to some potentially sensitive
endpoints: immunotoxicity, developmental toxicity, neurotoxicity and developmental neurotoxicity,
endocrine active chemicals and allergenicity. These issues were considered, where possible, by
analyses of databases selected for these endpoints (Kroes et al., 2000). Such selective databases are
considered conservative since chemicals are likely to have been selected for special endpoint study for
some a priori reason. For developmental toxicity, the distribution of NOAELs divided by 100 was about
3 orders of magnitude higher than the distribution of 1 in a million upper-bound lifetime risk estimates
derived from carcinogenicity data (see below.). Importantly, the cumulative distribution of NOAELs
was similar to that of Class III chemicals in the Munro et al. (1996) database, i.e. developmental
toxicity was not more sensitive than other non-cancer endpoints, indicating that a specific TTC was
not necessary. For adult neurotoxicity, the distribution of NOAELs was lower by about one order of
magnitude than those for other non-cancer endpoints, including developmental neurotoxicity (Kroes et
al., 2000).
For allergies, hypersensitivity reactions and intolerances, none of the current testing strategies were
considered adequate for such effects and, therefore, there is no database to develop TTC values and
these endpoints are not included in the TTC approach. However, in the absence of suitable animal
models, the TTC is not different to any of the other approaches to risk assessment.
These analyses led to further refinements of the TTC approach: Kroes et al. (2004) developed a TTC
value for chemicals with certain structural alerts for genotoxic carcinogenicity (0.15 µg/day, calculated
by linear extrapolation to a theoretical upper-bound risk of one in a million from the TD501) and a
separate TTC value for organophosphate chemicals (18 µg/day). It was acknowledged that
organophosphate chemicals are usually regulated products and it was clearly stated that the TTC
approach should not be considered an alternative to testing procedures required for regulatory
approval.
Removing organophosphate and carbamate chemicals from Cramer Class III, being the most potent
chemicals in that class, would have an impact on the existing TTC value for Cramer Class III. Kroes et
al. (2004) did not propose to revise the TTC value for Cramer class III and the EFSA (2012a) stated
that, pending any future revision of the TTC approach, it would be prudent to maintain the value for
Cramer Class III at 90 μg/person per day. On the other hand, Felter et al. (2009) proposed to revise
the existing TTC value for Cramer Class III to 180 μg/person per day after removing organophosphate
1
The TD50 is defined as the daily dose-rate in mg/kg body weight per day for life to induce tumours in half of the test animals
that would have remained tumours-free at zero dose.

TTC Approach
and carbamate chemicals from Cramer Class III. Munro et al. (2008) noted that exclusion of
organophosphate and carbamate chemicals would give a corrected Class III TTC value of 180
μg/person/day instead of 90 μg/person/day and, in addition, if organohalogen chemicals are also
excluded from Cramer class III, the resulting corrected Class III TTC value would be about 600
μg/person/day.
Kroes et al. (2004) also developed a step-wise decision tree which incorporates the various TTC
values in decreasing order of concern and increasing numerical values. At the beginning, they
included an exclusion category for certain types of chemicals that should not be assessed by a TTC
approach. The reason for this was either that similar structures were not represented in the database
and/or that established risk assessment approaches already exist (heavy metal and TCDD-like
chemicals), or that they represent high potency genotoxic carcinogens (aflatoxin-like chemicals, N-
nitroso-chemicals and azoxy-chemicals – the so-called cohort of concern (CoC)). Separate TTC values
could be developed for the CoC, but it was considered that the resulting TTC values would to be too
low to be of practicable use.
The use of the TTC approach as a pragmatic risk assessment or prioritisation tool has become
established in several areas of chemical risk assessment in the regulatory context, including food
contact materials (US FDA, 1995), food flavouring agents evaluated by the Joint FAO/WHO Expert
Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA) (see WHO,
1997; Munro et al., 1999; Renwick, 2004) and impurities in pharmaceutical products (EMEA, 2006,
2007; US FDA, 2008). Areas currently under consideration for the future uses of TTC include
metabolites of plant protection products (pesticides) (Brown et al., 2009), cosmetics (Kroes et al.,
2007; SCCP, 2008) and consumer products (Blackburn et al., 2005). It has also been considered for
exposure-based waiving of toxicity testing under REACH (Bernauer et al., 2008; Rowbotham and
Gibson, 2011, EC 2011).
More extensive acceptance of a TTC-based approach for the provision of advice to risk managers
could yield significant benefits in terms of reduced animal testing when predicted exposures to a
chemical are below a level that would be associated with potential human health concerns. Given
advances in analytical sophistication and sensitivity and the likely future increase in the numbers of
chemicals detected at very low levels in the human environment, application of the TTC approach
would also permit prioritisation of risk assessment resources (fiscal, time and human expertise) to
chemicals posing potentially greater risks. Although the TTC approach has traditionally been used in
human health assessments for oral exposures there is no reason why it could not be adapted for other
routes of exposure including dermal (Kroes et al., 2007; Safford, 2008) and inhalation (Carthew et al.,
2009; Escher et al., 2010).
In looking to extend the use of the TTC approach, a number of aspects critical to the original concept
have been and continue to be scrutinised in terms of applicability to the current uses and suitability
for new areas of use. These aspects include:
 The range of chemicals in the supporting databases and the ‘applicability domain’,
 The suitability of the Cramer scheme for dividing chemicals into different classes of toxic
potential,
 Whether certain chemicals should be excluded a priori from the TTC approach (e.g. the cohort
of concern (CoC) – see Kroes et al., 2004),
 The tools used to identify structural alerts to allocate chemicals into the ‘genotoxic cancer’ tier
of 0.15 µg/person/day,
 Whether the production of extra TTC values for specific end-points or chemical classes (for
example by QSAR) would be of value,
 Additional criteria necessary for application to non-oral routes of human exposure or for short-
term or intermittent human exposures.
The above aspects were the focus of a workshop on TTC, held in Brussels on 8–11 June 2011 [link],
and the outcome was reported by Dewhurst and Renwick (2013).

TTC Approach
2. The Cramer scheme

The original Cramer et al. (1978) decision scheme has been computerised in the Toxtree computer
program; the software is available at http://toxtree.sourceforge.net/. For the present report, v2.6.0
(July 2013) was used which is described as the Cramer tree with extensions. The extensions to the
Cramer decision scheme are discussed in Appendix 1. Presently the latest version is v2.6.13 (March
2015).
2.1. Does the Cramer classification scheme require revising?

Cramer classes play a pivotal role in the use of TTC. The Cramer decision scheme assigns chemicals
into one of 3 broad classes based on the presence of potentially toxic chemical functional groups
within the molecule; it was developed in the 1970s (Cramer et al, 1978) on a relatively limited set of
chemicals.
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was concluded that the
Cramer et al. (1978) decision scheme should be updated but not rebuilt from scratch. A major
rebuilding/restructuring of the Cramer decision scheme could invalidate current TTC values and also
the conclusions reached from the various database reviews undertaken since Munro et al. (1996). It
was noted that there is a computerised programme (e.g. Toxtree; software available at:
http://toxtree.sourceforge.net/) for putting chemicals through the decision scheme (Lapenna and
Worth, 2011). The initial step should be to review and revise the questions to take account of the
expansion of toxicological knowledge. Some questions were incompatible with current approaches and
should be deleted or modified e.g. the assumptions that a natural component of food is of low toxicity
(question 22) or that chemicals with multiple functional groups were always of high toxicity (Q18).
The workshop concluded that the current decision scheme did not include consideration of steric
hindrance or that for a number of elements consideration of valency state should be added e.g.
fluorine, trivalent sulphur, stable oxidation states of phosphorus. However, these criticisms are
incorrect since steric hindrance is taken into account where relevant, in question 18, and questions of
valency are irrelevant for these elements listed because they would all be in Cramer class III
irrespective of valency.
The EFSA (2012a) noted that the classification scheme most widely used is that described by Cramer
et al. (1978) and that this scheme is based on the metabolic and toxicological information available at
that time. With advances in knowledge over the last three decades, revision and refinement of the
scheme was recommended. Nevertheless, the EFSA analyses, together with those in several other
published studies have demonstrated that the application of the Cramer classification scheme in the
TTC approach is conservative and therefore protective of human health.
2.2. Is metabolism an inherent part of the Cramer decision scheme and

included in the TTC values?
The question whether inclusion of metabolism could be generally useful for extending the chemical
domain was discussed at the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013) in
relation to some chemical functional groups (e.g. amine formation from azo-chemicals) and the
toxicological consequences of metabolism, which is an inherent part of the Cramer classification. It
was thought to be important that predictions of metabolism should be restricted to major metabolic
pathways. Many current models for predicting metabolism were considered to have significant
limitations including lack of information on variations between experimental animals and humans and
alterations in metabolite profiles associated with high dose saturation of certain pathways. The subject
of metabolite prediction required additional investigation. In the context of defining structural alerts
for genotoxicity in the Kroes et al. (2004) decision tree (see also section 7) it was noted that the
prediction of reactive metabolite production was considered to be of critical importance in steps
related to the initial tiers of the TTC decision tree.
Also the EFSA (2012a) considered whether routinely undertaking metabolic prediction would be
helpful for application of the TTC approach other than for prediction of genotoxicity. As the Cramer
decision scheme and the databases used to derive the TTC values for non-cancer endpoints reflect at
least in part the toxicity of metabolites formed in the test species, the EFSA concluded that it is not
essential to undertake such metabolic prediction. However, there are situations where this has been

TTC Approach
helpful, e.g. in the case of flavourings where metabolic data assist in assessment of cumulative
exposure to structurally closely related chemicals (WHO, 2007).
Prediction of the metabolism of the chemical is built into the TTC-based procedure that is currently
used by the EFSA and the JECFA for evaluating flavouring substances, in the form of a specific
question (step 2), (i.e. “Can the substance be predicted to be metabolized to innocuous products?”).
The following text and tables provide an analysis of the extent to which metabolism of the chemical is
an inherent part of the Cramer decision scheme (Cramer et al., 1978) and of the TTC values
incorporated into the TTC decision tree of Kroes et al. (2004).
2.2.1. Metabolism and the Cramer et al. (1978) scheme

The Cramer et al. (1978) scheme aims to “provide a practical means for discriminating effectively
among different levels of probable hazard.” The authors stated that the decision scheme “uses much
currently available toxicological data to validate the procedure ” and that it is “intended for use with all
ingested, structurally defined organic and metallo-organic substances.” The authors also state that
“The logic of the tree rests heavily on known data on metabolism and toxicity ”.
A key part of the Cramer et al. (1978) decision scheme is recognition that higher toxicity is associated
with particular structural characteristics, such as a functional group (question 2) or an element other
than C, H, O, N, S (divalent), while other characteristics, such as normal body constituents and simple
carbohydrates are of low inherent toxicity.
The assignment of each chemical into one of three classes largely depends on a single structural
characteristic or a restricted number of structural characteristics, such as the presence of a cyano-
group or of a halogen atom, which may lead to toxicity. Secondary characteristics such as the
presence within the molecule of sites for rapid metabolism, such as a readily conjugated hydroxyl
group, which are remote from the structural element critical to the classification, do not usually affect
the classification, but could lead to widely differing rates of metabolism and elimination. In
consequence, it is inevitable that the Cramer et al. (1978) decision scheme will group together large
numbers of chemicals with diverse secondary structural characteristics and elimination rates. It should
therefore be expected that there will be large overlaps in toxicity between the different classes, a fact
illustrated by Figure 2 of the original Cramer et al. paper and confirmed by all subsequent analyses.
The initial questions in the Cramer et al. (1978) decision scheme identifies structural characteristics
with higher toxic potential, and the Kroes et al. (2004) TTC decision tree presents the TTC values in
decreasing order of toxicity. Therefore the analyses given below are presented in the order class III,
class II and then class I.
Cramer et al. (1978) class III
The questions in the Cramer et al. (1978) decision scheme leading to class III are shown in the Table
1. Note that the questions are part of a sequence such that question e.g. 33 is reached only after
numerous other structural characteristic have been excluded.

TTC Approach
Table 1: Cramer et al. (1978) Questions leading to Structural Class III
No. Structural characteristics Rationale and metabolic implications

2 Secondary aliphatic amine, cyano, N- Potentially toxic functional groups (secondary or quaternary
nitroso, diazo, triazeno, quaternary N amine) or electrophilic groups (cyano or nitrile); some overlap
with structural alerts for carcinogenicity (see below)
4 Elements other than C, H, O, N or S Restricts classes I and II to simple organic molecules; places
(divalent) excluding cations, such as organohalogens (which reduces the potential for metabolism
Na+, and Cl and S as chloride and and elimination) and organophosphates (including ionic
sulphate phosphate esters of hydroxycompounds) in class III
6 Benzene derivatives structurally related Metabolic bioactivation of such compounds leads to a reactive,
to safrole potentially carcinogenic species
9 A 4-membered lactone or a 5- or 6- Revised question (see appendix 1)
membered α,β-unsaturated lactone or Lactones are readily hydrolysed to the corresponding ring-
a lactone fused to another ring, open form
10 A 3-membered heterocyclic compound Epoxides undergo metabolic ring opening and glutathione
conjugation
13 Heteroaromatic ring without any The lack of a ring substituent limits the potential sites for
substituents metabolism and detoxication
21 Aliphatic compound with 3 or more Complex molecules, the toxicity of which cannot be readily
different types of functional group predicted
27 Aromatic compound without any The lack of a ring substituent limits the potential sites for
substituents metabolism and detoxication
33 Compounds not classified earlier and Sulphamate or sulphonate groups add high polarity to the
lacking frequent sulphamate or compound and increase elimination thereby minimising the
sulphonate groups potential for metabolic activation
It is clear that metabolism, metabolic bioactivation and hindered metabolism all contribute to the
determination of which chemicals are assigned to class III.
Cramer et al. (1978) class II
The questions in the Cramer et al. (1978) decision scheme leading to class II are shown in the Table
2. Note that the questions are part of a sequence that excludes more toxic characteristics at earlier
steps.
Table 2: Cramer et al (1978) Questions leading to Structural Class II

18 Compounds that are one of the following: This question is reached via:
i. a vicinal diketone; or a ketone or ketal of a i. question 16 (is it a common terpene, other than
ketone attached to a terminal vinyl group ketone? - NO) and then question 17 (is it readily
ii. a secondary alcohol or ester of a secondary hydrolysed to a common terpene? - YES);
alcohol attached to a terminal vinyl group ii. question 20 (is it a linear or simply branched
iii. allyl alcohol or its acetal, ketal or ester aliphatic compound with certain selected and readily
derivative metabolized groups? - YES) then question 21 (does
iv. allyl mercaptan, an allyl sulphide, an allyl the structure have 3 or more different functional
thioester or allyl amine groups? - NO);
v. acrolein, a methacrolein or their acetals iii. question 23 (is it aromatic? - NO) then question
vi. acrylic or methacrylic acid 24 (is it monocyclic with ring or side chains with
vii. an acetylenic compound certain selected and readily metabolized groups? -
viii. an acyclic aliphatic ketone, ketal or YES);
ketoalcohol with no other functional groups and iv. question 30 (is it a mononuclear aromatic
with four or more carbons on either side of the compound with substituents other than aliphatic side
keto group chains of 1-5 carbons + or - certain selected and
ix. a substance in which the functional groups readily metabolized] groups? - NO);
are all sterically hindered v. question 31 (is the substance an acyclic acetal,
ketal or ester of any of the compounds given by a
YES answer to question 30? - YES)

TTC Approach

22 The substance is a common component of food Definition C in the Cramer et al. (1978) paper
or structurally acknowledges the difficulties of providing a strict
closely related to a common component of food definition of “a common component of food”; such
chemicals can be presumed to be readily metabolised
25 The chemical is either cyclopropane or Question 24 defines the following readily metabolised
cyclobutane with only the substituents and detoxicated or highly polar substituents on
mentioned in question 24 or a mono- or bicyclic monocarbocyclic structures:
sulphide or mercaptan alcohol, aldehyde, side-chain ketone, acid, ester, or
sodium, potassium or calcium sulphonate or
sulphamate, or acyclic acetal or ketal
26 The chemical is either a monocyloalkanone or a Question 24 defines certain readily metabolised or
bicyclic compound with or without a ring ketone highly polar substituents (see above)
with no functional groups other than those listed
in question 24
32 A monocyclic aromatic compound that contains The groups include aliphatic side chains of 1-5
only the functional groups listed in questions 30 carbons + or - certain selected groups and acyclic
and 31 and with a single fused non-aromatic acetal, ketal or esters, all of which allow ready
carbocyclic ring and/or aliphatic substituent metabolism and detoxication
chains longer than five carbon atoms, and/or a
specified polyoxyethylene chain
The answer YES to question 18 “scoops up” a mixture of chemical types that are of intermediate
toxicity and assigned to class II; the answer NO leads to a class I.
Metabolism and ease of elimination, combined with inherent toxic potential, determine which
chemicals are assigned to class II.
Cramer et al. (1978) class I
The questions in the Cramer et al. (1978) decision scheme leading to class I are shown in the Table 3.
Note that the questions are part of a sequence such that question e.g. 33 is reached only after
numerous other structural characteristic have been excluded.
Table 3: Cramer et al (1978) Questions leading to Structural Class I

1 A normal constituent of the body Body constituents are normally readily metabolised by
pathways of intermediary metabolism; the definition excludes
hormones, contaminants usually present at low levels (e.g.
TCDD) and the products of disease states.
5 a simply branched acyclic aliphatic Low inherent toxicity and readily oxidised and/or incorporated
hydrocarbon into intermediary metabolism
or a common carbohydrate
16 A common terpene-hydrocarbon, - Such terpenoids are of low potential toxicity and the functional
alcohol, -aldehyde or -carboxylic acid groups are readily metabolised and eliminated
(not a -ketone)
18 Chemicals that reach this question but See class II above for details of the list of structures and the
do not have structures giving questions leading up to question 18
intermediate toxicity (i.e. class II)
33 Chemicals not classified earlier and Sulphamate or sulphonate groups add high polarity to the
containing frequent sulphamate or compound and increase elimination thereby minimising the
sulphonate groups (1 for every 20 or potential for toxicity
fewer C atoms)
It is clear that metabolism, metabolic detoxication and rapid potential elimination all contribute to the
determination of which chemicals are in structural class I.

TTC Approach
2.2.2. Metabolism and the TTC values

Potential DNA-reactive carcinogens (0.15μg/person/day)
Many of the structural alerts for DNA-reactivity (and carcinogenesis) are dependent on metabolic
bioactivation as shown in Table 4, and therefore clearly metabolism is taken into account.
Table 4: Role of metabolism in activation of genotoxic substances
Structural alert Metabolic process Active metabolite

Aflatoxin-like compounds hydroxylation epoxide
Aromatic amines N-hydroxylation hydroxylamine
Aromatic nitrates reduction hydroxylamine
Azo compounds reduction aromatic amine
Azoxy compounds oxidation nitronium ion
Benzidine derivatives oxidation undefined
Hydrazines oxidation azoxyalkane or carbocation
α-Nitro Furyl Compounds oxidation undefined
N-Nitroso Compounds oxidation carbocations or diazonium ions
Polycyclic aromatic compounds oxidation dihydrodiol epoxide
Vinyl compounds oxidation etheno compounds
Data from Cheeseman et al. 1999. (Structures in bold font are excluded from TTC consideration using the Kroes et al. (2004)
decision tree.)
Organophosphate compounds (18μg/person/day)

Unlike simple phosphate esters, pesticide-like OPs have unionised alkyl-substituted phosphate groups,
which in some cases have a sulphur atom in place of one of the oxygen atoms of the phosphate
group. Examples include phosphorothionates, phosphonates and phosphates (trialkyl-phosphates).
Pesticides with a P=S group (phosphorothionates) are inactive and can bind to acetylcholine esterase
only after oxidation to the corresponding P=O structure (the oxon).
Pesticide-like OPs act on esterases, including acetylcholine esterase, by covalently binding to the
enzyme active site. The methyl and ethyl groups attached to the phosphorus atom are stable, but the
remaining structural group acts as a leaving group on reaction with the serine residue in the active
site of the esterase. This forms a stable phosphate ester on the active site of the enzyme that is only
slowly converted back to the free, active enzyme.
The metabolic conversion of thion (P=S) to oxon (P=O) occurs in vivo and is required prior to binding
to serine in the active site of the enzyme.
Munro et al. (1999) analysed 31 OPs (using ChE-inhibition as an endpoint), and derived a TTC (based
on the 5th percentile of NOAELs divided by 100 and corrected for body weight) of 18 µg per person
per day (0.3 μg/kg bw/day). The most potent OPs in the database (NOAEL in μg/kg/day) were methyl
parathion (25), quinalphos (30), dimethoate (50), disulfoton (50), merphos (100), fenamiphos (100)
and azinphos methyl (180). Apart from fenamiphos and merphos (tributyl phosphorotrithioite), all of
these have a P=S structure, which demonstrates that in vivo metabolic activation is not the major
determinant of potency for organophosphate pesticides.
Therefore the proposed TTC value for OPs incorporates metabolism and metabolic activation.
Cramer et al. (1978) class III TTC value (90μg/person/day)
The class III TTC value was derived by Munro et al. (1996) based on the analysis of 448 chemicals
assigned according to the Cramer et al. (1978) decision scheme. The TTC is based on the 5th
percentile of in vivo NOAELs from largely chronic and sub-chronic studies (with correction for
duration).

TTC Approach
In their assessment of the application of the Kroes et al. (2004) decision tree to the evaluation of
cosmetic ingredients, Kroes et al. (2007) analysed the, potential of first-pass intestinal and hepatic
metabolism to affect the validity of the oral TTC values derived by Munro et al. (1996) for dermal
exposure. The metabolism following oral dosing was assessed for the 61 non-OP compounds in the
Munro et al. (1996) database that have NOAELs of 1.0 mg/kg bw /day or less using a Medline
literature search. These analyses of the most potent class III chemicals with adequate metabolism
data (n=47) showed that none were known to undergo major presystemic detoxication after oral
dosage, 20 chemicals were predicted to undergo negligible first-pass metabolism based on structural
characteristics (12 were polyhalogenated chemicals and 8 were polar unmetabolized chemicals or
showed steric hindrance of possible sites of metabolism) and 11 chemicals would undergo presystemic
hepatic metabolism to more toxic products such that oral toxicity would exceed topical toxicity. The
metabolism data, with cited references, related to those chemicals that determine the class III TTC
value are presented in Appendix Table 1 of the Kroes et al. (2007) paper.
Thus it is clear that metabolism is incorporated into, and is an inherent part of, the TTC value for
Cramer class III.
Cramer et al. (1978) class II TTC value (540μg/person/day)
The class II TTC value was derived by Munro et al. (1996) based on the analysis of 28 chemicals
duration).
cosmetic ingredients, Kroes et al. (2007) analysed the potential of first-pass intestinal and hepatic
exposure. The extent of metabolism following oral dosing was assessed for Class II chemicals in the
Munro database with NOAEL values ≤10 mg/kg bw/day, using a Medline literature search. Of the 7
chemicals investigated, 3 chemicals would undergo metabolism to more toxic products whereas for 3
chemicals metabolism would lead to detoxication. The metabolism data, with cited references, related
to those chemicals that determine the class II TTC value are presented in Appendix Table 2 of the
Kroes et al. (2007) paper.
Thus it is clear that metabolism is incorporated into, and is an inherent part of, the TTC value for
Cramer class II.
Cramer et al. (1978) class I TTC value (1800μg/person/day)
The class I TTC value was derived by Munro et al. (1996) based on the analysis of 137 chemicals
duration).
cosmetic ingredients, Kroes et al. (2007) analysed the potential of first-pass intestinal and hepatic
exposure. The extent of metabolism following oral dosing was assessed for Class I chemicals in the
Munro database with NOAEL values ≤30 mg/kg bw/day using a Medline literature search. Of the 24
chemicals investigated, 3 would undergo metabolism to more toxic products, but few data were
available on the toxicological consequences of metabolism for the other chemicals. The metabolism
data, with cited references, related to those chemicals that determine the class I TTC value are
presented in Appendix Table 3 of the Kroes et al. (2007) paper.
Thus, largely because the TTC value was derived from in vivo studies, metabolism is incorporated
into, and is an inherent part of, the TTC value for Cramer class I.

TTC Approach
3. Validity of the databases used to derive the TTC values
3.1. Do the databases used for deriving TTC values adequately represent
the “world of chemicals”?
The term “world of chemicals” is a poorly defined term and can be interpreted in various ways. In the
present context the term is meant to address the question whether the structure(s) of a
chemical under consideration is represented by the chemicals in the database used to
derive the respective TTC value.
EFSA commissioned a project to assess the relevance and reliability of the TTC approach for EFSA’s
work in food and feed, how the approach might be applied more widely and whether additional data
were necessary to strengthen the TTC approach (EFSA 2011, Bassan et Al. External report). The non-
cancer toxicological endpoints dataset of Munro et al (1996) and the Carcinogen Potency Database
(CPDB) dataset were compiled into two new electronic datasets and their chemical space was
characterized by means of statistically based methods using structural and physicochemical property
descriptors. 74 structural molecular descriptors, 21 physicochemical descriptors which are related to
their ADME profile and structural fingerprints that encode the presence/absence of molecular
fragments and functional groups were used. The analysis performed allowed the identification of
clusters of similar structures which were characterized in terms of single descriptors. The TTC datasets
were also compared with the US EPA DSSTox dataset, a large dataset taken as reference (Bassan et
al., 2011). Overall, the results of the study confirmed that the Munro, et al. (1996) and CPDB
databases are broadly representative of the world of chemicals: The comparison showed that the two
datasets are similar in both the chemical and the physico-chemical space. The comparison of the
Munro dataset with the random DSSTox dataset revealed that the two datasets cover the same
molecular space and also the comparison of the CPDB dataset with the random DSSTox dataset did
not reveal a unique portion of the CPDB dataset. Furthermore, it was found that molecules of the two
TTC datasets, i.e. the Munro (1996) and the CPDB, are more diverse from the molecular structure
point of view rather than for their physico-chemical properties (Bassan et al., 2011).
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was considered whether
the extent to which the Munro et al. (1996) database, which was used to derive the non-cancer TTC
values in the Kroes et al. (2004) decision tree, adequately represented the ‘‘world of chemicals’’ and
its suitability for possible future applications of the TTC approach. This was based primarily on
information from Bassan et al. (2011). Overall, the workshop concluded that the Munro et al. (1996)
database is broadly representative of the world of chemicals and that performing a chemical domain
analysis on an individual chemical could be useful but it was not considered an essential element of a
TTC assessment. The workshop further concluded that the existing cancer TTC database needs to be
assessed, using a standardised method for chemical domain analysis against a broad dataset of
chemical structures and biological end-points. The chemical domain analysis should be tiered, moving
from elementary to complex methods such as basic chemical function, structural class and physical
chemical descriptors. The current biological assessment is based on the CPDB (developed by Gold and
colleagues – see Gold et al., 2005) using the lowest TD50 with a statistical significance for effects set
at p<0.01. The TTC value of 0.15 µg/person/day for potential genotoxic carcinogens based on
structural alerts for genotoxicity (excluding aflatoxin-like, nitrosamine and azoxy-compounds; Kroes et
al., 2004) was considered conservative because it was derived by linear extrapolation from the TD 50
values combined with the analysis of the proportions of chemicals with each structural alert that had
an upper-bound estimated lifetime cancer risk of greater than on in a million. Other options to
improve the database would be to investigate chemicals with more than one TD 50 to determine the
most appropriate/robust endpoint for human risk assessment; at present the lowest, and most
conservative, TD50 value was used.
The EFSA (2012a) also concluded that the work of the EFSA-commissioned project (Bassan et al.,
2011) demonstrated that the range of structures in the two main datasets (Carcinogenic Potency
Database and Munro et al. (1996) database), which underpin the human exposure threshold values,
are broadly representative of the world of chemicals, in terms of chemical space, as described by
molecular descriptors encompassing both structural features and physicochemical properties and that
this provides further confidence in the general utility of the TTC approach.

TTC Approach
There are many methods available for performing chemical domain analyses, such as Principal
Component Analysis (PCA), Partial Least Squares regression (PLS ), Atom Centred Fragments (ACF),
using both statistical methodology and expert systems. These can be based on a range of parameters
e.g. chemical structure/space or physical chemical properties. At present there is no agreement on the
most appropriate approach for defining chemical domain, especially in relation to the potential for
toxicity and the workshop concluded that this is an area worthy of additional investigation (Dewhurst
and Renwick, 2013).
Snodin and McCrossen (2012, 2013) criticised the conclusion that the CPDB is broadly representative
of the world of chemicals based on the chemoinformatics analysis by Bassan et al. (2011) as they
considered the existing cancer TTC database to be biased in that the majority of the chemicals
included were tested ‘‘for cause’’ basis (i.e. suspect chemicals) and therefore considered to be skewed
towards expected carcinogens.
The TTC values for Cramer structural classes derived by Munro et al. in 1996 have been supported by
all subsequent analyses of additional databases (providing that the 5 th percentile NOAEL is converted
to a TTC value using the same 100-fold safety factor). Blackburn et al. (2005) analysed a database of
145 chemicals found in personal and household products; Bernauer et al. (2008) analysed
reproductive and developmental toxicity data for 91 chemicals assessed for oral toxicity under REACH;
Brown et al. (2009) analysed data for 100 active pesticides and 15 pesticide metabolites and
concluded that the TTC values are valid; Pinalli et al. (2011) analysed the TDIs for 232 food contact
materials in relation to the TTC and found that the distribution of recalculated NOAELs was similar to
that reported by Munro et al. (1996); Tluczkiewicz et al. (2011) analysed the RepDose database of
521 chemicals, using dose levels expressed on a molar basis making direct comparison difficult, but
the distribution of NOAELs, the overlap between Cramer classes and the TTC values were comparable
to Munro et al (1996) (see below); van Ravenzwaay et al. (2011) analysed data for pre-natal toxicity
using NOAELs for maternal and developmental toxicity and found 5th percentile values higher than
those used by Munro et al. (1996); Kalkhof et al. (2012) analysed NOAELs from subacute and
subchronic studies (with adjustment for duration of study) on 813 different chemicals and found TTC
values for Cramer classes I, II and III similar to those of Munro et al. (1996) (see below);
Laufersweiler (2012) analysed reproductive and developmental toxicity data for 283 chemicals and
generated TTC values 2-3 times higher than those of Munro. Feigenbaum et al. (2015) assessed the
reliability of the TTC approach using 328 pesticides that had been fully evaluated by the EU and by
EFSA and concluded that the respective TTC values are protective, even for these biologically active
substances.
Tluczkiewicz et al. (2011) when analysing the TTC RepDose database in which 4 databases were
combined (see 5.3.), inter-species allometric scaling was used instead of using the default uncertainty
factor of 100. Interspecies allometric assessment factors of 4 and 7 for rats and mice, respectively,
were used. The corrected NOAEL (animal NOAEL divided by the allometric assessment factor) was
then divided by 25 to account for the remaining uncertainties and to produce the TTC values
expressed per kg body weight. For rats, this is equal to the 100 fold uncertainty factor used by Munro
et al. (1996) and is not expected to change the TTC values. The analyses were performed with doses
expressed on a molar basis, but this did not give rise to different TTC values when converted back to
a mg/kg bw basis.
Combining 232 chemicals used to manufacture food contact materials with the initial Munro et al.
(1996) dataset of 613 chemicals, gave a resulting cumulative distribution that did not change for
Cramer class III and only slightly for Cramer class I. There was an overlap of only 8 chemicals in the
two datasets (Pinalli et al., 2011).
Kalkhof et al. (2012) analysed studies on 813 different chemical structures submitted to the National
Regulatory Office in Germany according to the EU chemicals legislation. The data set of industrial
chemicals analysed had not a single overlap with the chemicals in the Munro et al. (1996) database.
The TTC values derived from this database were somewhat higher for Cramer class III and class II,
whereas Cramer class I had a similar TTC value compared to the Munro et al. (1996) TTC values. It
should be noted that OPs were not in this class III database and this may have resulted in the almost
10-fold higher TTC value. Transformation from doses in mg/kg bw/day into molar doses did not
change the results and the transformation was not helpful in separating chemicals of different toxicity
in the Cramer classification scheme or in reducing the overlap between the Cramer classes.

TTC Approach
3.2. Should the point of departure for establishing TTC values be

reconsidered?
Traditionally, when experimental animal data are used for risk assessment of chemicals in food, which
are not genotoxic and carcinogenic, the NOAEL and/or the Lowest-Observed-Adverse-Effect-Level
(LOAEL) for the critical effect of a chemical, forms the point of departure. Today, the use of dose-
response modelling to derive a benchmark dose (BMD) is often preferred over identifying a NOAEL
because the NOAEL approach does usually not use all the dose-response data available. In contrast,
the BMD approach makes extended use of the dose-response data from studies in experimental
animals (or from observational epidemiological studies) to better characterise and quantify potential
risks. Furthermore, the BMD approach can also be used when a NOAEL has not been identified in a
study. Although on average, the BMD and NOAEL approaches give comparable results, the BMD
approach is considered to be a scientifically more advanced method to the NOAEL approach for
deriving a point of departure, since it makes extended use of available dose-response data and it
provides a quantification of the uncertainties in the dose-response data. Using the BMD approach also
results in a more consistent point of departure, as a consequence of the specified benchmark
response (EFSA 2009). The BMD approach has been used by the WHO in risk assessments of
chemicals that are genotoxic carcinogens (WHO, 2006).
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was concluded that
moving to the use of BMDLs rather than using NOAELs was impractical because of the need to re-
evaluate numerous end-points in the studies in the existing databases. In addition, the distribution of
doses used in many studies did not fit well with a reliable BMD calculation. Detailed study descriptions
necessary for a BMD analysis of the studies in the Munro et al. (1996) database and the CPDB are no
longer publicly available, which hinders re-examination to determine the impact of using a BMDL
approach rather than NOAEL or TD50. Investigating the impact of changing from a NOAEL or TD50 to
another measure, such as the BMD approach, was considered to have scientific merit but was unlikely
to have a major impact on the TTC values and it was concluded that the use of linear extrapolation
from the TD50 was sufficiently conservative.
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), the expression of dose on a
molar basis was also considered and was supported on scientific terms because structure-activity
relationships of series of chemicals are usually analysed in this way. Such a change would result in a
need to convert all the NOAELs in the databases to a molar basis and derive the TTC values on the
distribution of molar-based NOAELs. Although this was considered at the workshop held in Brussels in
2011 not be a difficult task, application of molar TTC values would require that analytical and
exposure data were also expressed on a molar basis. Adoption of a molar basis would result in the
TTC approach being incompatible with existing regulatory approaches used in setting reference doses
for chemicals with extensive databases. For comparative purposes, data expressed on a molecular
weight basis rather than a mass basis is scientifically logical for closely related structures, such as a
homologous series of chemicals, but it lacks logic when applied to the “world of chemicals” for which
the toxicity arises from a diversity of different structures and functional groups.
3.3. Should the overall database be expanded/modified?

At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was considered that some
of the databases available at the time of the workshop had a degree of overlap with chemicals in the
Munro et al. (1996) database but others, such as some regulatory and industry databases, would have
minimal overlap. It was recommended that where the data were appropriate these should be
consolidated into an extended TTC database.
Because the TTC values are based on the distribution of NOAELs within the database, it is possible
that combining and updating the TTC database could significantly alter the 5 th percentile NOAEL.
However, similar TTC values have been derived from two of the largest databases currently available
(Munro et al., 1996 and RepDose database), so that it is unlikely that frequent revisions of TTC values
would be necessary (Dewhurst and Renwick, 2013).
The EFSA (2012a) has considered a number of published analyses and conducted some analyses itself
of both the data originally used to establish TTC values and data from additional studies that are
included in EFSA’s databases on pesticides and in an EU database of chemicals classified for

TTC Approach
reproductive toxicity. The EFSA also commissioned a project to examine the databases underpinning
the TTC approach, using in silico chemoinformatic methods to assess the representativeness of the
databases and the opportunities for refining the basis for grouping chemicals (Bassan et al., 2011, see
5.1.). It also checked the studies with the lowest 10th percentiles of the NOAELs in the database of
Munro et al. (1996) for chemicals in Cramer Class I and Class III. It was concluded that the respective
TTC values of 1800 and 90 μg/person per day derived by Munro et al. (1996) are sufficiently
conservative to be used. The same conclusion was reached by others using independent databases of
oral toxicity (Blackburn et al., 2005, Tluczkiewicz et al., 2011, Kalkhof et al. 2012, Feigenbaum et al.,
2015).
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), a significant shortcoming of
the current TTC decision tree was identified: there are only few chemicals in Cramer class II so that
the TTC value is less securely based. However, it was pointed out that this class is important for some
groups of flavouring agents.
A proposal was made by Tluczkiewicz et al. (2011) for regrouping chemicals to better differentiate
between structural classes likely to be toxic, moderately toxic or of low toxicity, based on an analysis
of the TTC RepDose database, in which they combined 4 databases (RepDose database, Munro et al.
(1996) database, ToxRef database, Toxbase database, for details see Tluczkiewicz et al., 2011). The
TTC RepDose database resulted in 521 chemicals/in vivo studies (109, 12 and 400 chemicals in
Cramer class I, II and III, respectively). To determine the more toxic chemicals in Cramer class I and
chemicals of lower toxicity in Cramer class III, cut-off values were taken from the guidance values of
globally harmonized system of classification and labelling of chemicals (GHS), to distinguish between
chemicals of high and low toxicity. By using these cut-off values, groups of structurally similar
chemicals of high toxicity in Cramer class I and of moderate to low toxicity in Cramer class III were
identified and reassigned to the appropriate Cramer class according to their observed toxicological
potency in in vivo studies. Phenols and primary aliphatic amines were identified as frequent outliers
especially of Cramer class III (i.e. as being not of high toxicity as defined by the GHS guidance).
When recalculating the TTC values after refining the TTC RepDose database, the resulting TTC values
for Cramer class I, II and III were 1930, 1478 and 63 µg/person per day, respectively. Such a post-
hoc reassignment requires information about the toxicity of the chemical. While this re-analysis is not
relevant to the application of the TTC approach, it confirms to a large degree the conservatism of the
original decision scheme .
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was concluded that of the
available databases, the ToxRefDB would be a good choice to merge with the CPDB as there should
be less overlap than with the US EPA IRIS dataset. However, to obtain compatibility the ToxRefDB
would require some results to be converted to TD50s.
As mentioned in 5.1., the TTC value of 0.15 µg/person per day for certain structural alerts for possible
DNA-reactive carcinogenicity, proposed by Kroes et al. (2004), was based on analyses of the Gold et
al. carcinogen potency database (CPDB) (see Kroes et al., 2004 for references). At the time of the
Kroes et al. (2004) analysis, the CPDB comprised 730 chemicals, the majority of which were Ames-
test positive and the respective TTC value was derived by linear extrapolation from the lowest TD 50
values, excluding aflatoxin-like, nitrosamine and azoxy-compounds. No attempt to account for the
mode of action in the process of carcinogenesis was made, but it should be noted that the TTC value
was based on groups that had structural alerts for genotoxicity. This was regarded as sufficiently
conservative by the EFSA (2012a, see also 5.4.) but was considered by others (Delaney, 2007, Snodin
and McCrossen, 2012, 2013) to be over-conservative in the context of potentially mutagenic impurities
in pharmaceuticals. The reason for considering the TTC value of 0.15 µg/person (and 1.5 µg/person
for impurities in pharmaceuticals) as unnecessarily conservative was mainly because potential
carcinogens are overrepresented in the CPDB and therefore the frequency distribution would be
biased towards higher potency chemicals and, particularly, the use of linear extrapolation for non-
mutagenic carcinogens and use of the lowest statistically significant TD 50 instead of e.g. the geometric
mean of the TD50. Snodin and McCrossen (2012, 2013) considered this approach as inappropriate for
non-genotoxic carcinogens, for which the normal regulatory approach would be to assume that a
threshold exists. They proposed developing a series of structure-based limits for carcinogens, in an
analogous manner to the use of three Cramer structural classes with associated non-cancer TTCs.

TTC Approach
For chemicals with genotoxicity alerts and hence possible DNA reactive carcinogens, the
SCCS/SCHER/SCENIHR (2012) concluded that the default value of 0.15 μg/person/d can be used for
the moment, but its scientific basis should be strengthened, which could be achieved by e.g.
extending the database by analysing all available carcinogenicity studies, using allometric adjustment
factors and/or using the T25 or 1, 5 or 10% benchmark dose as points of departure for linear
extrapolation.
3.4. Should chemicals with particular structures and/or toxicological

properties be excluded from the TTC approach?
The TTC databases consist mainly of standard repeat dose toxicity studies and/or rodent cancer
bioassays. This selection bias could result in there being toxicological end-points that are not covered
adequately in the TTC database e.g. reproductive/developmental toxicity.
Munro et al. (1999) investigated whether reproductive toxicity leads to lower NOAELs compared to
other endpoints, using 100 chemicals that were reported in the Registry of Toxic Effects of Chemical
Substances (RTECS database of 1987) to cause developmental abnormalities. The 5th percentile
NOAEL in this database was 3.46 mg/kg/day, which is higher than 0.15 mg/kg bw, the 5th percentile
NOAEL for Cramer class III chemicals from the Munro et al. (1996) database.
Newer investigations (Bernauer et al., 2008, Brown et al., 2009 and van Ravenzway et al., 2011,
Laufersweiler et al. 2012) indicated that the current TTC values adequately covered end-points from
non-standard studies. The most comprehensive analysis of reproductive and developmental toxicity in
relation to the TTC was performed by Laufersweiler et al. (2012). It investigates the NOAELs for
reproductive and developmental toxicity studies in relation to the Munro-derived TTC values, using a
database of 283 chemicals (69 chemicals in class I, 11 in class II and 203 chemicals in class III).
Chemical inclusion criteria were based on the guidance for application of the TTC approach in the
Kroes et al. (2004) decision tree. The database comprised 59 chemicals from the Kroes et al. (2000)
dataset, 40 chemicals from the Bernauer et al.(2008) dataset plus additional 21 chemicals identified
from the source used by these authors that have been added since June 2007, and 163 additional
chemicals identified from a literature search. The cumulative distributions of NOAELs for classes I and
III were clearly separated, while the limited data for class II overlapped with class III. Comparing
these distributions with the original distributions used by Munro et al. (1996) to define the TTC values
showed that the reproductive and developmental toxicity NOAELs were higher: The 5th percentile
NOAELs for Cramer Classes I, II, and III were 13.1, 1.87, and 0.31 mg/kg/day respectively, which
would convert to TTC values of 7860, 1122 and 186 µg/person/day, using a 100-fold uncertainty
factor and 60kg body weight as used by Munro et al. 1996). Laufersweiler et al. (2012) also compared
their findings with those of Bernauer et al. (2008) and van Ravenzwaay et al. (2011). All three
datasets provide similar 5th percentile NOAELs but there were differences in the resulting TTC values
because Bernauer et al. (2008) applied a 1000-fold factor to the lowest NOAEL value in their
database.
EFSA (2012a) also analysed NOAELs for reproductive and developmental toxicity for chemicals
classified as such under EU legislation and concluded that the TTC values for Cramer Classes I and III
are considered sufficiently protective for adverse effects on reproduction or development.
Following the EFSA analysis of NOAELs for organophosphate and carbamate chemicals, the TTC value
of 18 μg/person per day, first proposed by Kroes et al. (2004), was considered by EFSA to be
sufficiently conservative to cover the anti-cholinesterase activity of chemicals with organophosphate or
carbamate structural features. Removing organophosphate and carbamate chemicals from Cramer
Class III (being the most potent chemicals in that class) would have an impact on the existing TTC
value for Cramer Class III. Pending any future revision of the TTC approach, the EFSA concluded that
it would be prudent to maintain the value for Cramer Class III at 90 μg/person per day, It was also
concluded that further additions to or subdivisions of existing Cramer Classes are likely to detract from
the advantageous features of the current TTC decision tree , that is, its ease of use, maintaining
consistency in application of the approach, and its in-built conservatism.
Regarding immunotoxicity as a potentially particularly sensitive endpoint, Munro et al. (1999)
compared NOAELs (or LOAELs divided by 10, when NOAELs were not available) for immunotoxicity
with the lowest respective non-immunotoxic NOAELs from 24 chemicals. 19 out of the 24 chemicals

TTC Approach
had non-immunotoxic NOAELs (or LOAELs divided by 10) that were similar to or lower than the
corresponding immunotoxic NOAELs. The remaining 5 chemicals had NOAELs (or LOAELs divided by
10) that were similar or higher than 0.15 mg/kg bw, the 5th percentile NOAEL for Cramer class III
chemicals from the Munro et al. (1996) database. Furthermore, 2 of these 5 chemicals belong to an
exclusion category (i.e. a metal and a N-nitroso compound) for which the TTC approach would not be
used. Munro et al. (1999) concluded that these data demonstrate that immunotoxicity is not a more
sensitive endpoint than other forms of toxicity. Kroes et al. (2000) reached the same conclusion after
adding 13 additional chemicals to the Munro et al. (1999) immunotoxicity database. It is noted that 2
out of the 13 additional chemicals had immunotoxicity NOAELs below the 5th percentile NOAEL for
Cramer class III chemicals (i.e. tri-n-butyltin oxide and arsine, but the latter exposure was via
inhalation) and for these organo-metallic chemicals the TTC approach would not be used.
The present structure of the TTC approach as proposed by Kroes et al. (2004) excludes a number of
chemical groups such as dioxins, nitrosamines, heavy metals or bioaccumulative chemicals. At the
workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), the reasoning for the previous
exclusion from a TTC assessment of certain chemical classes of concern was considered to be
appropriate but the data from such chemicals should remain in the overall database. In principle it
was considered not to be necessary to introduce any additional tiers to the TTC decision tree of Kroes
et al. (2004) to permit the extension to currently excluded groups. It was noted that any TTC value(s)
derived for the CoC would be extremely low and possibly impracticable due to difficulties in obtaining
reliable exposure data below the current TTC tier of 0.15µg/person/day (2.5 ng/kg body weight/day)
for substances with a structural alert for genotoxicity.
The EFSA (2012a) considered both previously proposed exclusions and additional exclusions that
might be necessary and concluded that the TTC approach should not be used for the following
(categories of) chemicals: high potency carcinogens (i.e. aflatoxin-like, azoxy- or N-nitroso-
compounds, and also benzidines and hydrazines); inorganic chemicals; metals and organometallics;
proteins; steroids; chemicals that are known or predicted to bioaccumulate; nanomaterials;
radioactive chemicals and mixtures of chemicals containing unknown chemical structures.
While the probability of exceeding a cancer risk of 1 in 10 6 at an intake of 0.15ug/person (i.e. the TTC
for genotoxic carcinogens, see below) for benzidines is 14%, it is only 4% for hydrazines (Kroes
2004). Since for the Cramer class threshold values the lower 5th percentile of the NOAEL distributions
was used as a starting point, i.e. allowing a probability of 5% that a compound of unknown toxicity
might have a NOAEL below this value, it is concluded that a similar criterion should be used also for
the probability of giving a cancer risk > 1 in 10 6, given the conservativeness of linear extrapolation in
deriving the TTC of 0.15ug per person. Therefore, benzidines but not hydrazines are included in the
recommendation for the exclusion category.
For chemicals with a structural alert for genotoxicity, EFSA (2012a) considered the TTC value of 0.15
μg/person per day as derived by Kroes et al. (2004) as sufficiently conservative to be used in EFSA’s
work, provided the structures already designated as high potency carcinogens are excluded from the
TTC approach. The EFSA was aware that further chemicals have been added to the Carcinogenic
Potency Database since this value was derived. However, because a large number of chemicals were
already in the Carcinogenic Potency Database, the Committee did not consider that the TTC value for
chemicals with a structural alert for genotoxicity would change appreciably. The EFSA further
considered the possibility that a genotoxic metabolite could be produced from a parent chemical. If
such metabolites were to be predicted and considered relevant, then the TTC value of 0.15 μg/person
per day should be applied. The EFSA recognised that there is no general agreement at present on
how to interpret the outcome from the currently available tools used to make such metabolic
predictions, because they have a tendency to generate a large number of potential metabolites.
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was considered that to
move from 0.15 µg/day to 1.5 µg/day based on an absence of alerts for genotoxic carcinogenicity was
adequate but a greater degree of proof of no DNA reactivity was necessary before moving to the
Cramer class tiers. The workshop concluded that a transparent, consistent and reliable source for
identifying structural alerts needs to be produced, and that to move from 1.5 to 90µg/day should
require a weight of evidence that the chemical is not DNA reactive, rather than just an absence of
data. However, the original US FDA Threshold of Regulation value of 1.5 μg/person per day is of
historical importance only but has little practical application in the overall TTC approach (EFSA,

TTC Approach
2012a). This is because chemicals without structural alerts for genotoxicity can proceed down the TTC
decision tree to be considered in relation to the higher TTC values for organophosphates and
carbamates or the Cramer Classes (see also section 7).
The EFSA (2012a) noted that non-genotoxic carcinogens are considered to have a threshold and, in
general, NOAELs for these are in the same range or higher than NOAELs for other types of toxicity.
Thus the TTC values that are higher than the value of 0.15 μg/person per day are appropriate to be
used for any chemical that does not have a structural alert for genotoxicity.
The SCCS/SCHER/SCENIHR (2012) concluded that chemicals with complex chemical structures having
several structural elements are not adequately represented in the available databases. Also chemicals
with highly unique structures fall outside the databases. Such chemicals should be excluded from the
TTC approach.
3.5. Combined exposure to multiple chemicals and combined exposures

from multiple sources
At the workshop held in Brussels in 2011, difficulties in considering aggregate exposure (exposure to a
single chemical through all relevant pathways and routes) were highlighted (Dewhurst and Renwick,
2013). In principle, aggregate exposure can be taken into account when kinetic data are available to
calculate internal doses (i.e. the systemic exposure). These difficulties in accounting for aggregate
exposure are not specific to the TTC approach but apply to all approaches to risk characterisation (see
WHO 2009).
A review and analysis of the possibility of synergism at dose levels relevant to TTC values was
performed by Boobis et al (2011). After evaluating the methods used in over 90 studies on the
subject, only 6 provided useful quantitative estimates of synergy, and the magnitude of synergy at
low doses was less than 4-fold greater than the response level predicted by additive models.
Therefore, simple dose addition and comparison with a relevant TTC value would be adequate.
Meek et al. (2011) proposed a framework for the risk assessment of combined exposure to multiple
chemicals that is based on a tiered approach. In a tier 0 assessment (the initial tier) the use of a TTC
approach to prioritize the need for further evaluation of a chemical mixture is proposed together with
a crude, conservative estimate of exposure. The exposure used for risk characterisation is the
summed exposure to all component chemicals and a common mode of action (dose addition) is
assumed. A hazard index is calculated for each exposure estimate, using the TTC value for the
relevant Cramer class. The hazard index was calculated as the sum of the individual chemical hazard
quotients, and if the resulting value is <1, it could be concluded that the margin of exposure was
adequate and there was no need to conduct a higher-tier analysis.
Prior to 2007 the JECFA had considered possible combined exposures to flavouring agents by simple
addition of exposures to all chemicals in that group of flavouring agents, but as the groups expanded,
so the total exposure exceeded the relevant TTC value. The JECFA (WHO, 2007) developed a more
refined approach and applied the TTC value for the appropriate Cramer class to combined exposures
to flavouring agents based on dose-addition for up to 5 agents that either share a common metabolite
(after correction for molecular weights) or are members of an homologous series and therefore likely
to produce similar toxic effects.
The EFSA (2012a) stated that it is possible to apply the TTC approach to mixtures (i.e. combined
exposure to multiple chemicals), provided the mixture under consideration contains only chemicals
with closely related chemical structures. For such mixtures dose addition should be assumed and the
exposures should be summed.
3.6. TTC for unidentified chemicals

Koster et al. (2011) explored whether the TTC concept may enable a more pragmatic risk assessment
of unknown/unidentified chemicals that were not previously detected in food. A step-wise approach
was proposed that uses expert judgement on the source of the food, information on the analytical
techniques, the dietary consumption of food sources containing the unknown chemical and
quantitative information of the unknown chemical to assess the safety to the consumer using the TTC.
As the unidentified peak could be potentially genotoxic, the TTC value of 0.15 µg/day would apply and

TTC Approach
considerations were proposed to assure that an unknown peak does not represent a chemical from
the TTC excluded classes. This could be done by considering prior information about the sample to
judge whether specific ‘TTC excluded classes’ are likely to occur in a specific product. Then, exclusion
based on chromatographic technique, sample preparation and/or detection method used or partial
identification should be performed by targeted analysis and quantification of unidentified peaks.
An attempt to apply the TTC concept to the safety assessment of chemically complex food matrices
(forest of peaks) was made by Rennen et al. (2011) and in this context the relevance of combined
exposure to chemicals at low dose levels was investigated by Leeman et al. (2013). A similar approach
was proposed as described by Koster et al. (2011), namely translation of peak response to intake,
targeted analyses, assessment of potential genotoxicity and a chemical specific toxicological risk
assessment if chemicals were present at levels resulting in intakes above the applicable TTC, which
would require identification, quantification and assessment of these chemicals. Regarding possible
cumulative effects at exposure levels for each chemical at or below the Cramer class III TTC value,
being present in a complex mixture including food, it was concluded that to some extent cumulative
effects might occur (Leeman et al. 2013). However the health relevance of possible cumulative effects
at this dose level was considered to be so low that a need for a correction factor to cover possible
cumulative effects is very low to absent.
However, EFSA (2012a) noted that there has been little evaluation of the applicability of the TTC
approach to mixtures containing chemicals of unknown structure and that, accordingly, such mixtures
should be excluded from the TTC approach.
3.7. Low-dose effects and non-monotonic dose-response

Myers et al. (2009) concluded that for endocrine-disrupting chemicals, high-dose tests are not able to
predict low-dose results and that procedures are needed to establish acceptable exposure levels for
such chemicals that include testing for health consequences at prevalent levels of human exposure
and that do not extrapolate from the effects observed in high-dose experiments. Both the Barlow et
al. (2001) and Kroes et al. (2004) reviews concluded that it was premature to consider low-dose
effects for endocrine disrupting chemicals within the context of the TTC. The EFSA (2013) noted the
lack of consensus in the scientific community with regard to the existence and/or relevance of low-
dose effects and non-monotonic dose response curves in toxicology in connection with endocrine
activity, endocrine disruption or other endpoints/modes of actions. EFSA and other organization, e.g.
EPA, are addressing the potential significance of non-monotonic dose-responses of chemicals for
human risk assessment. (http://www.efsa.europa.eu/en/art36grants/article36/gpefsascer201401.htm;
http://www2.epa.gov/chemical-research/endocrine-disruption-research-testing-potential-low-dose-
effects). Furthermore, EFSA is working on evaluating biological relevance vs. adaptive responses.
These initiatives will help to clarify whether low-dose effects in the sense of adverse effects actually
exist and how frequent non-monotonic dose-responses of chemicals are occurring.
4. Exposure considerations
4.1. General principles

The TTC approach is a screening and prioritizing tool for the decision-making process, particularly
when data are incomplete. The TTC value for a given chemical, which is based on chronic
experimental animal studies, is compared with an appropriate lifetime human exposure estimate. It is
essential to have exposure assessments that take account of high exposure scenarios and, where
possible, take account of exposure from all routes and sources: The exposure assessment used in the
TTC approach should overestimate dietary exposure of high consumers using conservative
assumptions in terms of food consumption and chemical concentrations (WHO, 2011, EFSA 2012a).
This will avoid situations where the dietary exposure estimated by the screening process would
erroneously indicate no safety concern (i.e. understate exposure). However, in order to effectively
screen chemicals and establish risk assessment priorities, unsustainable diets should not be
considered. At a minimum, physiological limits of consumption should be taken into account.
Screening methods can also be created that are appropriate for a worst-case assessment of chemicals
for specific subpopulations of interest (WHO, 2011).

TTC Approach
In some respects a TTC value has a similar meaning to a health based guidance value (such as an
ADI) as it represents a level of exposure with a low probability of harm. Consequently, the notion that
“A small or occasional dietary exposure in excess of a health-based guidance value based on a
subchronic or chronic study does not necessarily imply that adverse health effects will occur in
humans.” (WHO, 2011) would apply also to the TTC.
4.2. Less than lifetime or intermittent exposures

The current uses of TTC, with the exception of impurities in pharmaceuticals and possibly consumer
products, are primarily for lifetime human exposure assessments. A number of proposals have been
put forward for adjusting the TTC value for chemicals with a structural alert for genotoxicity for
shorter than chronic durations of exposure (Müller et al, 2006, Humfrey 2007, Felter et al, 2009,
Galloway et al. 2013), mainly in the context of impurities in pharmaceuticals and consumer products.
The proposals made are mainly based on Haber’s law for cancer risk assessment, i.e. that higher
exposures for shorter periods of time are essentially equivalent to lower exposures for longer periods
of time. The EFSA (2012a) was not confident about the general applicability of these proposals. It
therefore recommended that the issue of less than chronic exposure should be addressed on a case-
by-case basis. This could be done for example by considering the margin between the appropriate
TTC value (without any adjustment for duration of exposure) and the estimated dietary exposure.
The EFSA (2012a) also noted that, with the exception of the TTC value for organophosphate and
carbamate structures, the current TTC values for non-cancer endpoints are derived from databases
that do not address effects from acute exposure. The EFSA was unable to recommend a
reliable/appropriate general means of adjusting the TTC values for non-cancer endpoints for shorter
durations of exposure, and recommended that these too should also be addressed case-by-case for
the time being.
At the workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013), it was concluded that
derivation of ‘less than lifetime’ TTC values should be approached with caution. A generic approach
based on the existing database would struggle to take into account kinetic differences between Cmax
and AUC driven toxicity and could run the risk of using arbitrary and inappropriate correction factors.
If there was a need to derive a short-term TTC value, then this might be appropriate on a case-by-
case basis if initial exposure estimates exceeded the lifetime TTCs. According to the workshop, in the
longer term, the development of an appropriate database to support short-term TTCs should be a
priority.
4.3. Infants and children, potentially sensitive life-stages

The EFSA (2012a) proposed to convert all TTC values from µg/person to µg/kg body weight to allow
for application of the TTC approach to the whole population including infants and children.
However, very young infants are considered to be a particularly sensitive subgroup because their
metabolic capacities are not yet fully developed (WHO, 2011). Consequently, health-based guidance
values (which are also established by e.g. dividing a NOAEL from experimental animals by an
uncertainty factor of 100) are not considered applicable to infants under the age of 12 weeks who
might be at risk at lower levels of exposure. Accordingly, risk characterization of exposure of such
infants to chemicals (e.g. in infant formula or occurring as contaminants) has to be considered on a
case-by-case basis (WHO, 2011). In principle, such considerations should apply also to the TTC. The
EFSA (2012a) has considered whether the TTC approach could be applied to young infants under the
age of 6 months. The toxicokinetic differences between young infants and children or adults are
transient and generally not more than 2- to 5-fold. Thus there is capacity in the first weeks of life to
metabolise and eliminate chemicals, particularly when exposures are low. The EFSA (2012a)
concluded that the TTC approach can be applied to assess exposures in young infants, but in cases
where the estimated exposure is in the range of the TTC value, additional consideration needs to be
given under which conditions the TTC approach could be used. Additional considerations might include
prediction of metabolic routes for the structure concerned and other issues such as frequency and
duration of the exposure.
Exposure of organs and tissues to certain chemicals at critical point(s) during their development (i.e. a
critical window) can result in irreversible change of the organ/tissue. In mammals, critical periods of

TTC Approach
development have been identified at conception, during pregnancy, infancy, childhood and puberty
(WHO, 2012). As mentioned under 5.4., EFSA (2012a) analysed NOAELs for reproductive and
developmental toxicity for chemicals classified as such under EU legislation and concluded that the
TTC values for Cramer Classes I and III are considered sufficiently protective for adverse effects on
reproduction or development.
The EFSA (2013) noted that, although some of the tests do cover exposure during critical periods of
development in utero, current mammalian tests may not cover effects that might be induced by
exposure during fetal or pubertal development, but may emerge during later life stages. Even
standard multi-generation studies do not cover the full mammalian lifecycle that would be able to
reveal the potential longer-term effects of short-term exposures at all stages of the lifecycle.
Consequently it would only be possible to assess the potential effects later in life after exposure
during a critical window once a database on full lifecycle studies becomes available. This is not unique
to the TTC approach but holds for any risk assessment based on experimental animal data.
5. Consideration of the Kroes et al. (2004) decision tree

Various modifications to the current scheme of Kroes et al. (2004) were discussed at the workshop
held in Brussels in 2011 (Dewhurst and Renwick, 2013). It was agreed that there needs to be some
differentiation between genotoxic carcinogens with no presumed threshold and non-genotoxic
carcinogens, but it should be noted that the TTC of 0.15 µg/day is applied to chemicals with structural
alerts for genotoxicity. It was also considered by the workshop that to move from 0.15 µg/day to1.5
µg/day based on an absence of alerts for genotoxic carcinogenicity was adequate but a greater
degree of proof of no DNA reactivity was necessary before moving to the higher TTC values (see also
5.4). To this end, it was concluded that a transparent, consistent and reliable source for identifying
structural alerts needs to be produced and that to move from 1.5 to 90µg/day (or 18 μg/day for an
OP or carbamate) should require a weight of evidence that the chemical is not DNA reactive, rather
than just an absence of data. A possible new more generally worded question was discussed at the
workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013) to move away from structural alerts
for genotoxicity and to focus on DNA reactivity and the link with tumours: “Are there alerts that raise
concerns that the compound could be a non-threshold carcinogen”. If such a question were
incorporated into the decision tree, the criteria would need to be amplified by appropriate text to
indicate what alerts were of concern e.g. Ames positive, DNA reactive and which were not. Useful
restructuring of the genotoxicity section of the Kroes et al. (2004) decision tree was proposed by
Felter et al. (2009). A revised version was produced by Dewhurst and Renwick (2013; see Figure
below) but this retained a duration of exposure question as proposed by Felter et al. (2009) and
duration of exposure is excluded as a consideration under the recommendations given in the current
report.
B
1. Is the substance a non-essential metal or metal containing compound, or is it a polyhalogenated-
dibenzodioxin, -dibenzofuran, or -biphenyl?
NO YES
2. Are there structural alerts that raise concern for potential Risk assessment requires
genotoxicity? compound-specific toxicity data
YES
YES
3. Is the chemical an aflatoxin-like-, azoxy-, or N-nitroso- compound?
NO
NO Negligible risk
4. Does estimated intake exceed TTC of 0.15mg/day? - low probability of a life-
time cancer risk > 1 in 106
YES
NO
5. Is the duration of exposure <12 months? YES
6. Does estimated intake
NO
exceed 1.5μg/day?
7. Are AMES data available?
NO YES
YES
8. Are Ames assay results and/or the weight of- Risk assessment requires
evidence for genotoxicity negative? compound-specific toxicity data
NO
Non-cancer considerations
Reproduced with copyright permission from Elsevier.

TTC Approach
Figure 1: Figure B from Dewhurst and Renwick (2013)
As already mentioned in 5.4., the EFSA (2012a) considered the original FDA Threshold of Regulation
value of 1.5 μg/person per day as of historical importance, but of little practical application in the
overall TTC approach and eliminated this TTC from the decision tree. Furthermore, the EFSA (2012a)
concluded that for non-DNA reactive carcinogens mode of action considerations indicate that an
underlying toxicity would be seen at dose levels below those producing tumours, which implies that
the limits in the Cramer classes would be adequately protective for a non-genotoxic cancer end-point.
It was noted that it would be useful if this were confirmed by an investigation of non-genotoxic
carcinogens in the CPDB and related databases. Cheeseman et al. (1999) reported that the TD50s of
chemicals with structural alerts were about 10-fold lower than those for chemicals lacking such alerts.
The assumption of the presence or absence of a biological threshold in the dose-response relationship
results in different approaches to risk assessment, i.e. the calculation of a margin of exposure (MOE)
or establishing a health based guidance value. For non-DNA reactive carcinogens, a thresholded mode
of action is assumed and a health based guidance value is normally established to protect the most
sensitive subpopulation, based on the most sensitive critical health outcome (WHO, 2011).
The workshop held in Brussels in 2011 (Dewhurst and Renwick, 2013) recommended that inclusion of
mode of action data should be explored although it was considered likely that this could have
significant resource implications and might be of limited relevance in cases where there were few data
on a particular mode of action. Including human relevance was considered to have limited impact, and
the TTC value was precautionary because it was based on the assumption that all tumours with the
lowest TD50 values for chemicals in the database were relevant to humans.
Felter et al. (2009) proposed a revision to the Kroes et al. (2004) decision tree introducing the
possibility of additional questions related to the results of genotoxicity testing as well as to the
duration of exposure (e.g. exposure <12 months).
6. Conclusions
6.1. General conclusions

The TTC approach as currently applied is a valid, science-based screening tool useful for the
prioritisation of chemicals and for more general applications in chemical risk assessment. The TTC
approach was developed for chemicals where human exposure can be estimated to be low and
chemical-specific toxicological data are lacking. As such, conservatism was built into the approach to
establish protective TTC values. It should be noted that the TTC approach is not appropriate to
assess the safety of chemicals for which a toxicological data-package is required. In any risk
assessment all data available on the chemical under consideration should be evaluated, and
application of the TTC approach is no different in this aspect, although in certain circumstances (e.g.
prioritisation of a large number of chemicals) it could be acceptable to perform a preliminary
screening assessment based on the TTC without evaluating all the data on each chemical as a first
step.
Moreover, when a class of structurally similar chemicals are to be assessed and a well-studied lead
chemical is available, this lead chemical can be used for the assessment of the structural analogues by
means of read-across, however, depending on the context a TTC approach could also be used as a
first step. This issue had already been raised by Kroes et al. (2004): “ Prior to application of the TTC
approach, all available toxicity data on the compound should be collected and evaluated (Renwick et
al., 2003). The TTC approach should be used only in cases where the available chemical-specific data
are inadequate for normal risk characterisation. Any available information on the compound should be
considered at the same time as the decision tree is applied, to ensure that any decision is compatible
with the available data. The TTC is not designed to replace conventional approaches to risk
characterisation for established and well-studied chemicals, such as food additives and pesticides.” It
was further recognised that in-depth expert knowledge is needed to reach a conclusion to some of
the questions in the Kroes et al. (2004) decision tree: “The decision tree and the TTC principle are
designed as structured aids to expert judgement and should be applied only by those who have a
sufficient understanding of toxicology principles and chemical risk assessment.” The expert group
concurred with this assessment.

TTC Approach
6.2. The Cramer scheme is fit for purpose

The expert group concluded that the currently available information do not warrant major revisions to
the Cramer scheme. The scheme is well suited for its intended purpose and when used in conjunction
with the associated TTC values is protective. The group acknowledged that the sorting process of the
Cramer decision scheme does work, is reproducible and the TTC values have been substantiated by
post hoc comparison with numerous newer databases. In consequence, the expert group concluded
that there is no scientifically-based justification for major restructuring of the decision scheme.
The Expert group recommended minor suggestions to modify the Cramer decision scheme to remove
ambiguity, improve its clarity and to harmonize with the electronic tool Toxtree. The expert group
recognised that there are a number of efforts underway, including those of the US Food and Drug
Administration (FDA) and the International Organization of the Flavour Industry (IOFI), that propose
significant modifications to the Cramer decision scheme, indicating that the developers interpret a
need for revision. However, major modification to and restructuring of the Cramer decision scheme
could result in a situation in which the original TTC values derived by Munro et al. (1996) and
subsequently substantiated using different databases may be altered, and the implications for existing
safety assessments need to be evaluated. Because the Cramer decision scheme has been applied for
the evaluation of flavouring agents for over 15 years, there is a need for broad acceptance of any
future changes.
The expert group noted that the reasoning underlying the development of individual nodes in the
Cramer decision scheme in the 1970s is not transparent. Any revisions to the existing decision
scheme or the creation of any new decision scheme(s) should be thoroughly documented by capturing
the scientific rationale for creating branch points, and the questions associated with those branch
points. This process would ensure transparency of the development process and provide a strong
foundation for peer review and validation. In addition, any revisions to the current decision scheme
or the development of a new decision scheme should be discussed and agreed upon widely at an
international level and the resulting output freely available as an expert system.
The Cramer et al. (1978) decision scheme has been computerised in the Toxtree computer program
and is described as the Cramer tree with extensions (version 2.6.0). The modified Cramer decision
scheme proposed by this expert group incorporates some, but not all aspects of the Toxtree
extensions (please refer to Appendix I for additional details).
In the Joint EFSA/WHO stakeholder meeting on TTC on 02 December, 2014, stakeholders emphasized
that there would be great value in having a publically available database underlying the TTC approach
that could be consistently peer reviewed. The expert group concurred but noted in this regard that
while all of the original data collected by Munro in support of the original TTC were peer-reviewed and
publically available at that time, some of these original studies may no longer be available.
Because there are a relatively small number of compounds that are classified in Cramer Class II, it has
been previously proposed to evaluate under the Class III TTC threshold all the chemicals categorized
as Class II (EFSA, 2012).
Kroes et al. (2004) proposed removing organophosphates (OPs) and carbamates from Cramer Class
III and assigning them their own TTC value of 18 μg/person per day, which is considered sufficiently
conservative to cover the anti-cholinesterase activity of these substances. The expert group discussed
whether the carbamates where less toxic than the OPs and could therefore remain in class III.
However, there were insufficient data and therefore the group recommended to class the carbamates
with the OPs.
The group supported a separate class threshold for evaluating OPs and carbamates. However, the
group concluded that the current Class III threshold value should be maintained and should not be
recalculated by excluding the OP and carbamates from Class III. The rationale for not recalculating
the threshold for Class III at this time is twofold: (i) to maintain the current level of health
protectiveness; and (ii) the evaluation of another database (RepDose) that does not contains OPs or
carbamates yielded a TTC value for Class III that is similar to the Munro value. Therefore, it was
considered premature to change the threshold at this time.
Some modifications to the Cramer decision scheme were suggested by Tluczkiewicz et al. (2011)
based on a combined assessment of four databases of repeated-dose toxicity studies (RepDose,

TTC Approach
Munro, ToxRef, and Toxbase). Analyses of the tails of the Cramer Class I, II and III distributions for
the presence of different functional groups showed that phenolic compounds and primary amines had
higher ratios of outliers to non-outliers (i.e. a larger proportion were in the tail of the distribution).
However, interpretation of these observations is complicated by the fact that some of the outlier
phenols and primary amines contained other structural characteristics, which could have resulted in
assignment to Class III before the phenol or amino function would have been considered. In
consequence, it is premature to reassign these functional groups to Cramer Class II.
The expert group concluded that additional consideration of Question 22 of the Cramer scheme, which
asks if a substance is “a common component of food or structurally closely related to a common
component of food” is required. It was generally agreed that it would be preferable to delete this
question, since ‘common component of food’ is not well defined , nor is the question related to
specific structural considerations that can be linked to toxicological properties. However, since Q22 is
linked with many other questions (12, 14, 15, 20, 26, 32), the consequences of removing this
question from the decision scheme need to be carefully evaluated, and the implication for Class II
considered when this is re-evaluated in the future with an expanded database.
6.3. Metabolism is an inherent part of the TTC values

Analysis of the Cramer classification scheme and the TTC values used in the Kroes et al. (2004)
decision tree shows that metabolism (metabolic bioactivation/metabolic detoxication and hindered
metabolism as well as the potential for rapid elimination) is an inherent critical component of the TTC
approach that contributes to the assignment of chemicals to a particular structural class.
The experts also discussed whether the Cramer decision scheme could be used in the evaluation of
plant- metabolites of pesticide. It was concluded by the expert group that plant metabolites of
pesticide can be evaluated by the TTC concept including the Cramer decision scheme. For pesticide
plant metabolites of unknown structure, the group recommended these substances be placed directly
in Cramer Class III, provided it can be reasonably argued that there is no concern for genotoxicity
based on knowledge of the parent compound (including its metabolism) and that it is not an OP,
carbamate or member of an exclusion category.
6.4. TTC Domain of applicability is sufficiently broad

The expert group considered the available chemical domain assessments conducted on the TTC
dataset sufficient to conclude that the domain of applicability of the chemicals in the TTC is sufficiently
robust; but acknowledged that some known categories are not in the TTC database. The TTC
approach should be limited to the evaluation of the structure(s) that are represented by the chemicals
in the database used to derive the respective TTC value; therefore, the TTC approach should not be
used for the following categories of chemicals: inorganic chemicals, metals and organometallics,
proteins, steroids, organo-silicon compounds, chemicals that are predicted to bio-accumulate,
nanomaterials, radioactive substances.
The current database has been evaluated and found to sufficiently cover a wide range of chemicals.
Testing the databases with alternative methods available for performing chemical domain analyses
would add additional evidence for concluding that the structure of a chemical under consideration is
represented by the chemicals in the database used to derive the respective TTC value.
6.5. The TTC for genotoxic compounds is sufficiently protective

The TTC value for substances with structural alerts for genotoxicity and carcinogenicity derived in the
Kroes et al. (2004) decision tree is considered adequate and fit for purpose. The TTC carcinogenicity
value was calculated assuming linearity of the dose response curve and extrapolation from the lowest
TD50 for each chemical in the largest available rodent carcinogenicity potency database (CPDB)
available at the time. The exposure level at which the carcinogenic risk for the vast majority of
chemicals would not exceed the level of 1 in 10 6 risk was derived. In addition, it was assumed that
any chemical with a relevant structural alert for genotoxicity could be a human carcinogen,
irrespective of the human relevance of the tumour observed in the rodent database or a possible
threshold mode of action. Although further expansion of the TTC cancer dataset and an updated

TTC Approach
carcinogenicity potency database is desirable, it is not expected that the overall threshold for
carcinogenicity based on linear extrapolation for genotoxic carcinogens would significantly change.
The expert group considered whether alternative approaches to establishing TTC values for
carcinogens such as using BMD/MOE values or using the geometric mean of the TD 50 in cases where
several studies are available are warranted. The group concluded that the current approach is
reasonable since it relies on linear low dose extrapolation to a 1 in 10 6 risk which is generally regarded
as a conservative approach to evaluating carcinogenic risk.
The TTC value for chemicals with certain structural alerts for genotoxic carcinogenicity may not be
sufficiently protective for high potency carcinogens (i.e. aflatoxin-like, azoxy- or N-nitroso-compounds
and benzidines) and therefore, these classes of compounds should also be excluded from the current
TTC approach. High potency carcinogens, identified as the cohort of concern (CoC) should be
evaluated case-by-case. For high potency carcinogens, any TTC value(s) derived to adequately ensure
low concern for health would be extremely low and possibly impracticable due to difficulties in
obtaining reliable exposure data.
6.6. TTC Tiers are sufficient for non-DNA reactive carcinogens and non-
cancer endpoints.
Carcinogens which are not directly DNA reactive often have a threshold mode of action and, in
general, NOAELs for these are in the same range or higher than NOAELs for other types of toxicity.
Thus, EFSA (2012) concluded that TTC values that are higher than the value of 0.15 μg/person/day
are appropriate for any chemical where the weight of evidence for DNA reactivity is negative. The
expert group concurred with this statement.
For non-cancer endpoints the Munro et al. (1996) database covers a range of chemical classes and
end-points relevant to the vast majority of chemicals. This database is considered adequate and fit for
purpose, and is additionally supported by TTC values derived from several subsequent analyses of
different chemical datasets which result in TTC values similar to or higher than those derived using
the Munro database. Classification by the Cramer decision scheme is based on the single functional
group with the greatest potential toxicity present in the molecule. Most complex chemicals are
assigned to Class III, the class with the lowest TTC value of the 3 Cramer classes. The group
acknowledges that there are very few chemicals in Class II and therefore the TTC value for this class
is not well supported within the current TTC approach. Merging the different non-cancer databases
would increase the power of the calculated respective TTC values.
6.7. Conclusions on other considerations
6.7.1. Point of Departure (PoD) and database

The current TTC database for non-cancer effects is based on the lowest NOELs or NOAELs in mg/kg
bw/day identified in repeated dose animal studies. Newer approaches to dose-response analysis, such
as BMD derivations, determining doses on a molar basis, or using allometric assessment factors, can
be seen as having greater scientific rigour but the expert group concluded that they would not
significantly affect the approach or add real benefit.
A merge of the different non-cancer databases is desirable as it would increase the statistical power
and improve transparency in the database. If a new non-cancer database is generated, then the
“overall TTC’s” should be recalculated. It remains to be seen whether after merging the different
databases the number of chemicals in the Cramer Class II increases to a more representative number
of compounds as seen for the other classes. Should the recalculated TTC values for the respective
classes increase, it needs to be determined if the new TTC values can still be considered sufficiently
protective for adverse effects on specific endpoints, such as reproductive or developmental toxicity, as
has been demonstrated for current TTC values.
In order to keep the future use of TTC contemporary with evolving databases, it would be ideal to
have a centralized dataset that would be continually maintained to allow inclusion of new data as they
become available. This would keep the chemical domain of the TTC dataset from stagnating as well as
ensuring the current data are available for use and that any TTC values derived are representative of

TTC Approach
the current state of science. There would be a need for an organisation or group (preferably
independent) to manage the overall database to ensure consistency, quality, public access and
maintenance.
Combining different databases would be facilitated by agreement on the method of dose-response
analysis and the appropriate dose-metric should be harmonised. Application of the TTC approach
would benefit from the development of a standardised approach to defining chemical domain and
agreed method(s) to identify structural alerts for DNA reactivity.
To determine the applicability of TTC to a certain chemical or group of chemicals, it is important to
identify the key functional groups of interest and determine if those groups are within the TTC
database. The ability to readily interrogate the databases supporting TTC would be of considerable
benefit in this area.
6.7.2. Exposure considerations

As with any risk assessment, when using the TTC approach, exposure to a chemical from all sources
(i.e. exposure from all relevant pathways and routes) should be considered, if possible. In a tiered
approach to assessment of combined exposures to multiple chemicals, proposed by the International
Programme on Chemical Safety (Meek et al., 2011), a case study on lower tier assessment
demonstrated how TTC values could be used as the hazard point of departure for groups of
substances belonging to specific Cramer classes and combined with their exposure potential used to
evaluate the need for further combined exposure assessment.
Pending the outcome of the EFSA project on low-dose effects and non-monotonic dose-response, it is
premature to make conclusions on this issue, and the TTC is not different from other methods of risk
assessment in this respect.
6.7.3. Expression of TTC values

TTC values should be expressed in terms of µg/kg body weight/day to allow for application of the TTC
approach to the whole population, including infants and children. Since the µg/person values were
initially derived by multiplying µg/kg bw/day NOELs from animal studies by a default human adult
body weight of 60kg, the TTC values in µg/kg body weight are obtained by dividing the µg/person
values by 60.
Table 5: Conversion of TTC values into μg/kg bw per day.

Type of TTC value TTC value in μg/person per day TTC value in μg/kg bw per day
With structural alert for genotoxicity 0.15 0.0025
OPs and carbamates 18 0.3
Cramer Class III 90 1.5
Cramer Class II 540 9.0
Cramer Class I 1800 30
6.8. Overall conclusion

The TTC approach is a valid screening tool, based on scientific risk assessment principles, to assess
low dose chemical exposures, and to distinguish those for which further data are required to assess
the human health risk from those with no appreciable risk. The expert group concluded the TTC
approach was fit for purpose and made recommendations to improve and expand the TTC concept,
and proposed a tiered approach (revised decision tree), considering toxicological databases and the
current state-of-the-science.

TTC Approach
7. Recommendations
7.1. Cramer scheme

 The expert group concluded that the sorting process of the Cramer scheme does work; it is
reproducible and has been substantiated by application to numerous newer databases, and
therefore no major restructuring is recommended. However, the group acknowledges that the
toxicological rationale underlying the development of each Cramer scheme question is not
provided and, therefore, it is not possible to address whether the decision scheme reflects the
most up-to-date scientific knowledge. Should the Cramer scheme be modified in the future,
the scientific rationale for each question should be made explicit for increased transparency.
 The expert group is aware of efforts, including those of the US FDA and the International
Organization of the Flavour Industry (IOFI), that propose significant modifications to the
Cramer scheme. It is the recommendation of the expert group that once these revisions are
peer-reviewed and validated, any new schemes need to be discussed and agreed upon widely
at the international level before implementation.
 The group recommended only minor changes to a small number of Cramer questions to clarify
and remove ambiguity (see Appendix I). It is recommended that the proposed minor changes
to the Cramer et al. (1978) decision scheme as outlined in Appendix I are implemented in
Toxtree.
 After review of Question 22 of the current Cramer scheme, the expert group recommended
further consideration of this question as the term “common component of food’ is not
sufficiently defined, nor is the question related to specific structural considerations that can be
linked to toxicological properties. The expert group recommended that the implications of
deletion of Q22 be evaluated and that the question should be deleted if the impact is minor.
However, if this is not feasible at this point, clear and harmonised criteria of what ‘common
component of food’ means need to be developed, and lookup tables within Toxtree should be
updated in accordance with the criteria.
 It is recommended that phenols and primary amines not be reassigned to Class II at this time,
based on an outlier analysis of their NO(A)EL distributions. The group recommended that an
enlarged and consolidated database is needed to assess the toxicity data for these structural
groups before such reassignments can be recommended. This will allow for consideration of
sorting into different toxicity tiers based on appropriate modification of the decision scheme.
 The expert group recommended that Cramer Class II continue to be used and applied to the
TTC approach. The expert group recommends that the applicability of Class II be reviewed
once the different non-cancer databases have been merged as this may enrich and increase
the confidence in the class. It was also recommended that this review include an evaluation
of the distributions to determine if there is a need to modify the decision scheme to
strengthen the specificity of sorting to Class II.
o The expert group recommended caution in developing additional classes to ensure that
the process does not introduce too much granularity into the decision scheme such that
the end product becomes a “read-across” tool rather than a screening tool.
o The Cramer scheme has been criticized as lacking specificity due to the high degree of
overlap in NO(A)EL values between Classes I and III. However, the expert group
emphasized that overlap per se is not a deficiency of the scheme but in fact contributes to
its overall conservatism, and that only clear differentiation at the 5th percentiles is critical.
The group recommended that the distributions be re-evaluated following the development
of any new or consolidated/merged databases.
7.2. Metabolism
 The experts considered that mammalian metabolism is an inherent and critical component
of the current TTC approach and no additional measures to incorporate metabolism were
recommended.

TTC Approach
 The group also recommended that plant metabolites of pesticides of known structure
could proceed down the decision tree, and that metabolites of unknown structure be
placed directly in Class III provided it is not an OP , carbamate or member of an exclusion
category and there are no concerns for genotoxicity.
7.3. Expand/modify the overall database and derivation of class

thresholds
 The expert group recommended that a permanent repository for data supporting TTC and
the Cramer scheme should be created and a body responsible for holding the data should
be identified. In addition to the development of a centralized database for supporting
TTC values, it is recommended that minimum criteria for inclusion of data in the TTC
databases should be developed and published. At a minimum, it is recommended that
supporting data should be of sufficient detail to recreate decisions on NO(A)ELs and
LO(A)ELs.
 It is recommended that the different non-cancer databases be merged and made public
as it would increase the power, transparency, and the confidence in the TTC values. Once
the databases are merged:
o It is recommended that any new combined databases select the lowest NOAEL
per compound as a starting point for deriving TTC levels as this approach will
provide the most reasonably conservative values.
o BMD levels could be considered for inclusion in cases where a study has not
identified a NOAEL.
o Future combined datasets should consider the most up to date science, such as
subchronic to chronic extrapolation factors, allometric scaling, etc, when selecting
NOAEL levels for the derivation of TTC values.
o Subsequent recalculation of the “overall TTC’s” for the respective classes would
be necessary:
 OPs and carbamates should be analysed separately and the consequence for
the threshold value for class III evaluated,
 the impact on specific endpoints (e.g. developmental toxicity) needs to be
checked to ensure they remain covered,
 it should be checked whether the number of chemicals in the Cramer Class II
is sufficient to provide a robust TTC value.
 Expanding the TTC cancer dataset (e.g with the ToxRef database) would enhance the
power and range of chemical structures covered. However, this is not considered a
priority as it would be resource demanding and is not expected to significantly affect the
approach.
 If a revision of the carcinogenicity/genotoxicity based TTC were to be envisaged, it is
recommended considering approaches other than TD50-based linear extrapolation from
the most sensitive species and most sensitive site, which may be overly conservative.
7.4. Chemical domain analyses

 The current database has been evaluated and found to sufficiently cover a wide range of
chemicals, and although additional analyses could be performed, this is not considered a high
priority. However, it is recommended that any new combined database be tested using
chemical domain analyses methods. These analyses are considered to be informative and
would provide further reassurance that the databases cover a wide range of chemical
structures.

TTC Approach
 In addition, it is recommended that a tool for evaluating whether a chemical, or group of

chemicals, is represented in the underlying TTC databases be developed once the databases
have been consolidated. This is likewise not considered a high priority.
7.5. Point of departure

 A reanalysis of all toxicological studies present in the current TTC databases using BMD
analysis is not recommended as it would be very resource intensive and not all studies have
sufficient datasets to allow for BMD analysis. A reanalysis using allometric scaling is also not
recommended for the current databases, as the current approach already incorporates a
factor for interspecies extrapolation that is appropriate for a screening tool.
 The inclusion of sub-chronic studies in the non-cancer database is supported, and when
extrapolating from subchronic to chronic study duration in rodents the group finds the current
extrapolation factor of 3 is appropriate for a screening tool.
 The expert group acknowledged that expressing TTC values on a molar basis has been
proposed, but recommended maintaining the units in µg/kg bw/day because that would not
affect the approach or add benefit.
7.6. Exclusion of chemical categories

 The application of the TTC approach is not recommended for the following categories of
chemicals: High potency carcinogens (i.e. aflatoxin-like, azoxy- or N-nitroso-compounds,
benzidines), compounds not adequately covered in the database, inorganic chemicals, metals
and organometallics, proteins, steroids, nanomaterials, radioactive substances and organo-
silicon compounds or chemicals that are known or predicted to bioaccumulate.
 The applicability of the TTC as a tool for the evaluation of mixtures that are not fully
characterised is only endorsed if sufficient information or analysis is available to confirm that
the mixture does not contain compounds from the exclusion classes. In this case, the
unknown component could be treated as potentially genotoxic and the TTC of 0.0025 µg/kg
bw would apply. However, if it can also be determined that there are no concerns for
genotoxicity, the substance may be placed directly in Cramer Class III providing it is not an
OP or a carbamate.
 TTC values for Cramer Classes are considered sufficiently protective for adverse effects on
reproduction and development and no changes are recommended. However, the TTC values
would need reconsideration should the point of departure or the overall database be changed.
 Specific consideration was given to pyrrolizidine alkaloids (PAs), since they have been
suggested as an exclusion class. The group considered the available information as
insufficient at this point and recommended the issue to be reconsidered once potency
estimates for additional PAs are available2.
7.7. Specific TTC values

 The expert group recommended organophosphates and carbamates be treated as a separate
class within the TTC approach, with a threshold value of 0.3 μg/kg body weight per day.
 Despite comprising a distinct class with a specific TTC value, the group recommends that prior
to consolidation and review of the non-cancer databases, the organophosphate and
carbamate NO(A)ELs should remain in Cramer Class III in order that the current threshold for
this class be maintained.
 The group did not recommend the setting of an additional generic TTC value(s) to cover high
potency carcinogens and recommended evaluation on a case-by-case basis.
2
JECFA evaluates the health risk of PAs in the 80th meeting in June 2015.

TTC Approach
 The group recommended that the Threshold of Regulation value of 1.5 µg/person/d in the
Kroes et al. (2004) decision tree should be removed. Although it is of historical importance it
is of little practical application.
7.8. Combined oral exposure to multiple chemicals and from multiple

sources
Accounting for combined oral exposure is not specific to the TTC approach, but applies to all
approaches of risk characterisation. Therefore, case-by-case considerations were recommended in
most circumstances:
 Applying the TTC approach to mixtures of known composition is possible. A tiered approach is
recommended beginning with the assumption of dose addition. In the case of more complex
mixtures containing compounds with dissimilar structures or in the event of known or
anticipated interactions among components of the mixture, additional methodological
refinements are needed.
7.9. Acute and other less than lifetime exposures

 If acute, or other less than lifetime TTCs were to be generated, it is recommended that a
database for acute or other less than lifetime toxicity should be produced and methodology
for the analysis determined. When performing these assessments there is a need to ensure
that developmental toxicity endpoints are covered.
 Until such databases and analyses are developed, it is recommended considering less than
lifetime or intermittent exposure on a case-by-case basis.
7.10. Infants and children potentially sensitive life-stages

 The TTC approach can be used to evaluate the safety of exposures in infants in the same way
as would be done with any risk assessment. For infants under 3 months of age, case-by-case
considerations are needed if the estimated exposure approaches the TTC value.
 There is a need to ensure that exposure data are suitable for infants.
 There is no need to derive specific TTC values for infants and children. A number of analyses
have shown that the NOAELs in the current TTC approach cover reproductive and
developmental studies (see EFSA, 2012).
 It is recommended to express TTC values as µg/kg body weight (rather than on a per person
basis) to facilitate the application of the TTC approach to the whole population including
infants and children.
7.11. Additional recommendations

A leaflet explaining the TTC approach in accessible language should be produced.
8. TTC Decision Tree

Taking the above considerations into account the expert group reviewed the overall TTC decision tree
proposed by Kroes et al. (2004), and recommended a revised decision tree as proposed in Figure 2.
An explanation of decision tree steps is given below the scheme.

TTC Approach
Figure 2: Schematic diagram of the revised TTC decision tree
1. Is the substance part of the exclusionary categories?
NO YES
Risk assessment required

2. Are there structural alerts or chemical-specific genotoxicity
data, such as Ames test results, that indicate the chemical
has the potential to be a DNA-reactive carcinogen, based on NO
the weight of evidence?
Non-genotoxic considerations
- go to Step 4
YES
These steps can be taken concurrently

YES of 0.0025 mg/kg
3. Does estimated intake exceed TTC
or in reverse order, depending on need.
bw/day?
Substance would not be expected

NO
to be a safety concern - low
YES probability that lifetime cancer
6
Risk assessment risk exceeds 1 in 10
required
TTC Approach
4. Is the compound an organophosphate or carbamate?
NO YES
6. Is the compound in Cramer class III? 5. Does estimated intake exceed TTC of 0.3 mg/kg bw/day?
NO YES NO YES
8. Is the compound in 7. Does estimated intake exceed TTC of Risk assessment required
Cramer class II? 1.5 mg/kg bw/day?
YES NO
NO YES

to be a safety concern
10. Does estimated intake exceed 9. Does estimated intake exceed 9

Risk assessment
30 mg/kg bw/day? mg/kg bw/day?
required
NO YES
YES NO
to be a safety concern

TTC Approach
Decision Tree Explanations

A literature search should be undertaken on the chemical to be evaluated, prior to applying the TTC
decision tree. The TTC approach should be used only for chemicals of known structure (or those that
are sufficiently characterised to confirm they are not in the exclusion groups) that lack adequate
chemical-specific toxicity data and with low predicted human exposures.
Prior to applying the TTC - The TTC approach should not be used if the chemical is a member of a
group that has well-established toxicity data. The TTC approach should also not be used if the
structural characteristics of the chemical are not adequately represented in the TTC database.
Therefore, proteins; steroids; chemicals that are known or predicted to bioaccumulate; nanomaterials;
radioactive chemicals were also added to the list of chemicals for which the TTC approach is not
appropriate.
Step 1 – The TTC approach should not be used for compounds that are part of the Cohort of Concern
(CoC) proposed by Kroes et al. (2004) because more than 10% of chemicals with this structural alert
would give a risk >1 in 106 at an exposure at the TTC value given in Step 3. The CoC includes:
aflatoxin-like compounds, N-nitroso-compounds, azoxy-compounds, steroids, benzidines and
polyhalogenated dibenzo-p-dioxins and-dibenzofurans.
Step 2 - The weight of evidence for genotoxicity should be evaluated to indicate if the chemical has
the potential to be a DNA-reactive carcinogen. This should include an analysis of the structure by
considering the presence of structural alerts (identified using the Benigni/Bossa rulebase as
implemented in Toxtree) as well as any available genotoxicity tests results for DNA reactivity, such as
the Ames test.
Step 3 – The TTC value (expressed per kg body weight) is based on the TD 50 data for chemicals with
positive carcinogenicity data in the CPDB and with structural alerts given in Table 1 of Kroes et al.
(2004).
Step 4 – Identifies whether the chemical has the potential to act as an OP such as trialkyl-phosphates,
phosphorothionates and phosphonates or as a carbamate.
Step 5 – Gives the TTC value for organophosphates expressed per kg body weight.
Step 6 – Identifies chemicals in Cramer Class III.
Step 7 – Gives the Cramer Class III TTC value expressed per kg body weight.
Step 8 – Identifies chemicals in Cramer Class II.
Step 9 – Gives the Cramer Class II TTC value expressed per kg body weight.
Step 10 – Gives the Cramer Class I TTC value expressed per kg body weight.
36

TTC Approach
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Abbreviations
ADME: Absorption, Distribution, Metabolism and Excretion
BMD: Benchmark Dose
CoC: Cohort of Concern
CPDB: Carcinogen Potency Database
FDA: US Food and Drug Administration
IOFI: International Organization of the Flavour Industry
JECFA: Joint FAO/WHO Expert Committee on Food Additives
NOEL: No Observed Effect Level
NOAEL: No Observed Adverse Effect Level
OP: Organophosphates
PAS: Pyrrolizidine Alkaloids
SCCS: DG Sante Scientific Committee on Consumer Safety
SCHER: DG Sante Scientific Committee on Health and Environmental Risks
SCENIHR: DG Sante Scientific Committee on Emerging and Newly Identified Health Risks
TD50 :Tolerable Dose 50%
ToxRef: U.S. EPA’s Toxicity Reference Database
TTC: Threshold of Toxicological Concern
US EPA DSSTox: US Environmental Protection Agency – Distributed Structure-Searchable Toxicity
Database
36

TTC Approach
Appendix A – Explanations to Cramer scheme

Q Question NO YES Additional explanation
1 Is the substance a normal endogenous constituent 2 I Endogenous substances are
of the body that undergoes metabolism to CO2 and intermediary metabolites of normal
water? biological processes present in human
tissues and fluids, whether free or
conjugated; hormones and other
substances with biochemical or
physiological regulatory functions are
not included.
2 Does the substance contain any of the following 3A III Classifies chemicals that have
functional groups: an aliphatic secondary amine or functional groups associated with
a salt thereof, cyano,N-nitroso, diazo (e.g. CH2N2), enhanced toxicity early in the decision
triazeno (RN=NNH2) or quaternary nitrogen, tree.
except in any of the following forms: >CN+=R2,
>CN+=H2 or the organic anion salts thereof?
3A Does the structure contain elements other than 5 3B
carbon, hydrogen, oxygen, nitrogen, or divalent
sulphur?
3B Is any phosphorus atom present as a simple ionic III 4
phosphate ester R-O-PO32-, either as the free acid
or as a Na, K, Ca, Mg or NH4 salt (if so proceed
based on the hydrolysis product R-OH)?
4 Do all elements not listed in Q3A occur only as a III 7
Na, K, Ca, Mg or NH4 salt of a. carboxylic acid, or
as a SO4 or HCl salt of an amine, or a Na, K, Ca,
sulphonate, sulphamate or sulphate?
5 Is it a simply branched acyclic aliphatic 6A I
hydrocarbon or a common carbohydrate?
6A Is the substance a benzene derivative bearing 6B III In Toxtree the answer NO goes to Q
substituents consisting only of (a) hydrocarbon 42 Does the compound consist of one
chains or l'-hydroxy or hydroxyl ester-substituted aromatic ring, with at most one heavy
hydrocarbon chains and (b) one or more alkoxy atom connected to each aromatic
groups, one of which must be para to the atom? which aims to assign “possibly
hydrocarbon chain in (a)? harmful analogue of benzene” to Class
III.
6B Does the compound consist of one benzene ring, 7 III
with at most one heavy atom (oxygen, nitrogen or
sulphur) connected to one or more of the aromatic
carbon atoms?
7 Is the substance heterocyclic? 16 8
8 Is it a lactone or cyclic diester? 10 9
9 Is it a lactone fused to another ring, or a 5- or 6- * III * If it is a lactone treat the structure as
membered α,β-unsaturated lactone? if it were the hydroxy acid in the form
of its more stable tautomer and
proceed to Q20 if it is open chain, to
Q10 if it heterocyclic, and to Q23 if it is
carbocyclic; if it is a cyclic diester treat
as the separate components (i.e. the
predicted hydrolysis products).
10 Is it a 3-membered heterocycle? 11 III
11 Disregarding only the heteroatoms in any one ring, 12 33 Under Q11, do not consider the
does that heterocyclie ring contain or bear atom(s), usually O, N or S making the
substituents other than simply branched ring heterocyclic.
hydrocarbons (including bridged chains and If there is more than one hetero ring,
monocyclic aryl or alkyl structures), alkyl alcohols, regard each ring separately, with the
aldehydes, acetals, ketones, ketals, acids, esters remainder of the structure as
(including cyclic esters other than lactones), substituents of that hetero ring.
mercaptans, sulphides, thioesters, methyl ethers,
hydroxy or single rings (hetero or aryl) with no Addition of “thioesters” accounts for
substituents other than those just listed? their rapid hydrolysis.
36

TTC Approach

12 Is it heteroaromatic? 22 13 This question separates the aromatic
heterocyclics for the purpose of
considering whether they are
polynuclear (Q14) or unsubstituted
(Q13).
13 Does the ring bear any substituents? III 14
14 Does the structure contain more than one aromatic 22 15
ring?
15 Is it readily hydrolysed to mononuclear residues? 33 22
(If yes, treat the mononuclear heterocyclic
residues by Q22 and any carbocyclic residue by
Q16.)
16 Is it a common terpene (D)-hydrocarbon, -alcohol 17 I
–aldehyde or -carboxylic acid (not a ketone)?
17 Is the substance readily hydrolysed (H) to a 19 18
common terpene (D), -alcohol, -aldehyde or -
carboxylic acid? (If yes, treat the hydrolysed
residues separately and proceed to Q18 for the
terpene moiety and to Q19 for any non-terpenoid
moiety.)
18 Is the substance one of the following? I II Addition of “thioester” accounts for
a vicinal diketone; or a ketone or ketal of a ketone their rapid hydrolysis.
attached to a terminal vinyl group or,
a secondary alcohol, ester or thioester of a
secondary alcohol attached to a terminal vinyl
group or,
allyl alcohol or its acetaL ketal or ester derivative
or,
allyl mercaptan, an allyl sulphide, an allyl thioester
or allyl amine or,
acrolein, a methacrolein or their acetals or,
acrylic or methacrylic acid or,
an acetylenic compound or,
an acyclic aliphatic ketone, ketal or ketoalcohol
with no other functional groups and with four or
more carbons on either side of the keto group or,
a substance in which the functional groups are all
sterically hindered.
19 Is the substance open chain? 23 20
20 Is the structure a linear or simply branched 22 21 The rapid reduction of disulphides to
aliphatic compound containing any one or the corresponding thiols by
combination of only the following functional thioltransferases and exchange
groups: reactions with glutathione, cysteine
four or less, each, of alcohol, aldehyde, carboxylic and other endogenous thiols has been
acid or esters and/or taken into account.
one each of one or more of the following: acetal,
either ketone or ketal but not both, mercaptan,
sulphide (mono- or poly-), thioester,
polyoxyethylene [(-OCH2CH2-)x with x no greater
than 4], or primary or tertiary amine
a readily reducible disulphide group (if so continue
the assessment for each resulting thiol or dithiol
separately)?
21 Does the structure contain three or more different 18 III Aliphatic (A) compounds with three or
types of functional groups (exclude methoxy and more different functional groups
consider acids and esters as one functional type)? (excluding methoxy) are too complex
to permit prediction of toxicity.
22 Is the substance a common component of food or 33 II For flavouring agents and other
structurally closely related to a common chemicals added to food, the ratio
component of food and is the ratio between between natural occurrence and the
natural occurrence and the amounts added >10? amounts added should be >10(*).
23 Is the substance aromatic? 24 27
36

TTC Approach

24 Is the substance monocarbocyclic (excluding 25 18
cyclopropane or cyclobutane and their derivatives)
with ring or aliphatic side chains, unsubstituted or
containing only alcohol, aldehyde, side-chain
ketone, acid, ester, or Na, K or Ca sulphonate or
sulphamate, or acyclic acetal or ketal?
25 Is the substance either 26 II
a cyclopropane or cyclobutane with only the
substituents mentioned in question 24 or
a mono- or bicyclic sulphide or mercaptan?
26 Does the structure contain no functional groups 22 II
other than those listed in Q24 and is it either a
monocyloalkanone or a bicyclic compound with or
without a ring ketone?
27 Does (do) the ring(s) have any substituents? III 28
28 Does the structure contain more than one aromatic 30 29
ring?
29 Is it readily hydrolysed or reduced to mononuclear 33 30
residues? (If yes treat the individual aromatic
mononuclear residues by Q30 and any other
residue by Q19.)
30 Disregarding ring hydroxy or methoxy does the 18 31
ring bear substituents other than 1-5 -carbon
aliphatic groups, either hydrocarbon or containing
alcohol, ketone, aldehyde, carboxyl or simple
esters that may be hydrolysed to ring substituents
of five or less carbons? (If a simple ester that may
be hydrolysed, treat the aromatic portion by Q18
and the residue by Q19.)
31 Is the substance an acyclic acetal, -ketal or -ester 32 18
or an alkylaryl disulphide of any of the above
substances (see Q30)? (If yes, assume hydrolysis
or reduction and treat the aromatic residue by Q18
and the non-aromatic residues by Q19).
32 Does the substance contain only the functional 22 II Part (i) is intended to allow simple
groups listed in Q30, or their derivatives listed in derivatives of tetralin into Class II
Q31, but with any or all of the following: while putting polycyclic compounds
a single fused non-aromatic carbocyclic ring or, such as the steroids ultimately into
ii. aliphatic substituent chains longer than Class III (except those that may be
five carbon atoms or, normal food),
a polyoxyethylene [(-OCH2CH2-)x with x no greater Part (ii) allows compounds with
than 4] chain either "on the aromatic ring or on an permitted functional groups but longer
aliphatic side chain? side chains into Class II instead of
Class III. Part (iii) puts short-chain
polyoxyethylene derivatives of aryl
compounds into Class II rather than
Class III.
33 Does the substance bear on every major structural III I
component at least one sodium, potassium, or
calcium sulphonate or sulphamate for every 20 or
fewer carbon atoms without any free primary
amines except those adjacent to the sulphonate or
sulphamate.
(*) A ratio of 10 is proposed to ensure that exposure from natural occurrence is larger than from the amounts
intentionally added. A factor of 10 is often used as a pragmatic default value.
Extensions to the Cramer scheme Toxtree Rule ID 4 and Rule ID 40 – adds phosphate to list of
elements that do not automatically go to Class III (Q4 in scheme above) and then under Rule ID 40
questions whether it is a possibly harmful organophosphate type of chemical. If the answer to this is
yes then it is assigned to Class III, but if the answer is no and it is a simple phosphate ester it is
36

TTC Approach
assigned to Class I; but this assumes that the non-phosphate hydrolysis product would be Class I –
which may not always be correct. This Rule has not been incorporated into the modified decision tree
given above.
Toxtree Rule ID 42 – assigns to Cramer Class III possibly harmful analogues of benzene that “consist
of one aromatic ring with at most one heavy atom connected to each aromatic atom”. The examples
shown in the explanation for this step under Toxtree version 2.6.0 are phenol and benzamide.
Benzene with one or more hydroxyl-, amino- or thiol- group, or a combination of these groups, with or
without an aromatic methyl group, but without further substitution are assigned using this step; more
complex benzene derivatives such as benzoic acid, benzamide, acetamido-benzene, phenylhydrazine,
and ethyl-substituted phenol, ethyl-substituted aniline etc are not. This Rule has been incorporated in
a simplified form into the modified decision tree given above.
Toxtree Rule ID 43 – assigns “possibly harmful divalent sulphur” to Class III. The explanation to this
rule given in the program is “Does the compound [contain] a non-natural divalent sulphur?” It appears
that this is a question about natural occurrence, which is not related to the potential for toxicity.
Interestingly, none of the different types of structures in the 10 sub-groups of sulphur-containing
flavouring agents evaluated by the JECFA (WHO, 2000) would have been assigned to Class III using
Rule ID 43. This Rule has not been incorporated into the modified decision tree given above.
Toxtree Rule ID 44 – assigns any free α,β-unsaturated heteroatom, such as an α,β-unsaturated
alcohol or ketone to Class III. In its evaluations of flavouring agents the JECFA has considered the
extent of detoxication processes for such chemicals and concluded that “metabolic processes such as
oxidation and conjugation effectively eliminate reactive aldehyde functional groups from such
substances when they are consumed in the amounts that would arise from their use as flavouring
agents” (WHO, 2002). Therefore, as the TTC approach is only applicable at low levels of exposure,
this Rule has not been incorporated into the modified decision tree given above.
36

TTC Approach
Annex A – List of participants to the Stakeholder hearing

Stakeholder hearing on Threshold of Toxicological Concern
2 December 2014 | Brussels, Belgium
Last name First name Affiliation Country

ARNAUD Ludovic FEFANA BEL
ARNAUTS Jan DSM Ahead NLD
ARNICH Nathalie ANSES FRA
ARVIDSON Kirk Food and Drug Administration USA
BAKEN Kirsten KWR Watercycle Research Institute NLD
BARRETT Gordon Health Canada CAN
BARTOLO Ivan Seafood Importers and Processors Alliance GBR
BENFORD Diane Food Standards Agency GBR
BLUM Rene Lonza Ltd CHE
BRÜSCHWEILER Beat Federal Food Safety and Veterinary Office CHE
BURNETT Thomas Elanco Animal Health USA
CACHET Thierry IOFI BEL
CAVALLINI Eugenio CEPI aisbl BEL
CHEESEMAN Mitchell Steptoe & Johnson LLP USA
CIMMARUSTI Floriana Healthy Food Europe BEL
COREA Namali SC Johnson GBR
CREANGA Adina Bunge BEL
DE LUCA Lucia European Food Safety Authority
DEMPE Julia Dr. Knoell Consult GmbH DEU
DETKEN Dirk European Food Safety Authority
DEWHURST Ian Health and Safety Executive
DOURSON Michael Toxicology Excellence in Risk Assessment USA
EARL Lesley LSR Associates GBR
ESCHER Sylvia Fraunhofer Institute for Toxicology and DEU
Experimental Medicine
ESPEISSE Olivier IFAH FRA
FATTORI Vittorio Food and Agriculture Organization of the United
Nations
FEELEY Mark Health Canada CAN
FEESCHE Joerg Henkel AG & Co. KGaA DEU
FEIGENBAUM Alexandre Technopole Alimentec FRA
FELTER Susan Procter & Gamble USA
FLETCHER Samuel Veterinary Medicines Dire torate GBR
FRUTH Lothar ATC GmbH DEU
FUART-GATNIK Mojca National Institute of Public Health SVN
GEUEKE Birgit Food Packaging Forum CHE
GRANERO-ROSELL Miguel Angel European Commission (EC)
GUNDERT-REMY Ursula Federal Institute for Risk Assessment DEU
HÜSER Anja Knoell Consult GmbH DEU
HYNES Geoffrey Givaudan GBR
JACOBS Kristi Food and Drug Administration USA
JEONG Sang-Hee Hoseo University KOR
JIA Xudong World Health Organization
JUNGHANS Angelika Clariant Produkte GmbH DEU
KANUNGO Debabrata Ministry of Agriculture IND
KRUL Lisette TNO NLD
LEINALA Eeva OECD FRA
LEYDECKER Matthias FEICA DEU
LIEM Djien European Food Safety Authority
LIU Zhaoping China National Centre for Food Safety Risk CHN
Assessment
LUPTON-BOWERS Pamela Moderator of the event UK
LYSSIMACHOU Angeliki PAN Europe BEL
MAURICI Daniela European Food Safety Authority
MELCHING-KOLLMUSS Stephanie European Crop Protection Association DEU
MEROLLA Luciano Dow AgroSciences Ltd GBR
36

TTC Approach

MILLSTONE Erik University of Sussex GBR
MORTENSEN Alicja Technical University of Denmark DNK
MUELLER Utz Food Standards Australia New Zealand AUS
MUILERMAN Hans PAN Europe BEL
ORISAKWE Orish Ebere University of Port Harcourt NGA
PLATZEK Thomas Federal Institute for Risk Assessment DEU
POLITANO Valerie Research Institute for Fragrance Materials Inc. USA
PRIETO ARRANZ Miguel Angel Cefic BEL
RENWICK Andrew G. University of Southampton GBR
REYNDERS Hans Flemish Government BEL
RICHERT Susann Evonik Industries AG DEU
ROBINSON Tobin European Food Safety Authority
RONGA-PEZERET Sylvaine EDF – DRH Groupe - Direction Emploi et FR
Développement des Salariés
ROSSI Annamaria European Food Safety Authority
ROVIDA Costanza University of Konstanz DE
SCHLATTER Josef CHE
SCHNABEL Juergen Givaudan International AG CHE
SHAH Prakashchandra US Environmental Protection Agency USA
SHEN Jie Research Institute for Fragrance Materials Inc. USA
STIENON Sarah ISK Biosciences Europe NV BEL
STROHEKER Thomas Nestlé CHE
SUSIN Carolina European Chemical Industry Council BEL
TAYLOR Sean International Organization of the Flavor Industry USA
TERRON Andrea European Food Safety Authority
TRITSCHER Angelika World Health Organization
TROISFONTAINES Paul Scientific Institute for Public Health BEL
TWEEDALE Anthony C. BEL
UMEMURA Takashi National Institute of Health Sciences JPN
VAN BOSSUYT Melissa Scientific Institute for Public Health BEL
VANSTHERTEM David Japan Agro Services S.A. BEL
36

TTC Approach
Annex B – List of participants to the expert workshop

ARVIDSON Kirk Food and Drug Administration USA
BARRETT Gordon Health Canada CAN
BENFORD Diane Food Standards Agency GBR
BOOBIS* Alan Imperial College London GBR
BRÜSCHWEILER Beat Federal Food Safety and Veterinary Office CHE
CHEESEMAN* Mitchell Steptoe & Johnson LLP USA
DEWHURST Ian Health and Safety Executive GBR
DORNE Jean-Lou European Food Safety Authority
DOURSON Michael Toxicology Excellence in Risk Assessment USA
ESCHER Sylvia Fraunhofer Institute for Toxicology and Experimental DEU
Medicine
FATTORI Vittorio Food and Agriculture Organization of the United Nations
FEELEY Mark Health Canada CAN
FELTER* Susan Procter & Gamble USA
GUNDERT-REMY Ursula Federal Institute for Risk Assessment DEU
JACOBS Kristi Food and Drug Administration USA
JEONG Sang-Hee Hoseo University KOR
JIA Xudong World Health Organization
KANUNGO Debabrata Ministry of Agriculture IND
KRUL* Lisette TNO NLD
LEINALA Eeva OECD FRA
LIEM Djien European Food Safety Authority
LIU Zhaoping China National Centre for Food Safety Risk Assessment CHN
MAURICI Daniela European Food Safety Authority
MENNES Wim National Institute for Public Health and the Environment NLD
MÜLLER Utz Food Standards Australia New Zealand AUS
ORISAKWE Orish Ebere University of Port Harcourt NGA
RENWICK* Andrew G. University of Southampton GBR
ROSSI Annamaria European Food Safety Authority
SCHLATTER Josef CHE
SHAH Prakashchandra US Environmental Protection Agency USA
TRITSCHER Angelika World Health Organization
UMEMURA Takashi National Institute of Health Sciences JPN
YANG Chihae Molecular Networks GmbH DEU
Experts taking part in the workshop were selected following a public call for expert published on the
WHO website (http://www.who.int/foodsafety/call-data-expert/en/). Experts were selected according
to the criteria indicated in the call for expert. The screening of their DOIs was performed by WHO
according to the organisation's rules.
*Following this screening, experts who have been found to have a potential conflict of interests, did
not attend on the last day of the workshop where the group agreed on conclusions and
recommendations.
36

TTC Approach
Annex C – Agenda of expert workshop
EFSA-WHO Expert workshop on

Threshold of Toxicological Concern (TTC)
Brussels, 3-5 December 2014
Management Centre Europe, Rue de l’Acqueduc 118, Brussels, Belgium
Day 1 – Wednesday, 3 December 2014
8.30-9.00 Registration
9.00- 9.10 Welcome and Opening
Session 1: Introduction: setting the stage
9.10 – 9.30 Background to the WHO project
9.30 – 9.50 EFSA’s work on TTC
9.50 – 10.10 Stakeholder meeting summary
10.10-10.30 Coffee break
10.30-11.30 Report on TTC approach
11.30-12.00 Discussion
12.00-13.00 Lunch
Session 2: Introduction to work in the breakout groups
13.00- 13.20 Breakout Group1: Cramer scheme
13.20-13.40 Breakout Group2: TTC threshold levels & TTC decision tree
13.40-14.00 Discussion
Session 3: Breakout groups
BOG1 Cramer scheme
BOG2 TTC threshold levels & TTC decision tree
14.00-15.30 Breakout group discussions
16.00-17.00 Continuation of breakout groups
17.00-18.00 Summary and report back to plenary
End of the first day
36

TTC Approach
Day 2 – Thursday, 4 December 2014
Session 3
Breakout groups
(continued):
BOG1 Cramer scheme
BOG2 TTC threshold levels & TTC decision tree
9.00-10.30 Breakout group discussions
12.30-13.00 Brief report back from breakout groups to plenary
13.00-14.00 Lunch
17.30-18.30 Report back form breakout groups to plenary
End of the second day
Day 3 – Friday, 5 December 2014
Session 4: Report back from breakout groups
Summary of the two-day discussion and report back from Cramer scheme
9.00 – 9.30
(BOG1)
Summary of the two-day discussion and report back from TTC threshold levels
9.30 – 10.00
& TTC decision tree (BOG2)
10.00-10.30 Discussion on the outcomes
Session 5: Summing up
11.00- 12.20 Discussion and agreement on recommendations
12.20-12.30 Closing remarks and end of the workshop
36

EFSA Supporting Publications - 2016 - Review of The Threshold of Toxicological Concern TTC Approach and Development of

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EVENT REPORT

APPROVED: 16 February 2016 PUBLISHED: 10 March 2016

Review of the Threshold of Toxicological Concern (TTC)

www.efsa.europa.eu/publications EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 2 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 3 EFSA Supporting publication 2016:EN-1006

1.1. Objectives of the workshop

www.efsa.europa.eu/publications 4 EFSA Supporting publication 2016:EN-1006

1.2. Additional considerations

www.efsa.europa.eu/publications 5 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 6 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 7 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 8 EFSA Supporting publication 2016:EN-1006

2. The Cramer scheme

2.1. Does the Cramer classification scheme require revising?

2.2. Is metabolism an inherent part of the Cramer decision scheme and

www.efsa.europa.eu/publications 9 EFSA Supporting publication 2016:EN-1006

2.2.1. Metabolism and the Cramer et al. (1978) scheme

www.efsa.europa.eu/publications 10 EFSA Supporting publication 2016:EN-1006

Table 1: Cramer et al. (1978) Questions leading to Structural Class III

No. Structural characteristics Rationale and metabolic implications

Table 2: Cramer et al (1978) Questions leading to Structural Class II

No. Structural characteristics Rationale and metabolic implications

www.efsa.europa.eu/publications 11 EFSA Supporting publication 2016:EN-1006

No. Structural characteristics Rationale and metabolic implications

Table 3: Cramer et al (1978) Questions leading to Structural Class I

No. Structural characteristics Rationale and metabolic implications

www.efsa.europa.eu/publications 12 EFSA Supporting publication 2016:EN-1006

2.2.2. Metabolism and the TTC values

Table 4: Role of metabolism in activation of genotoxic substances

Structural alert Metabolic process Active metabolite

Organophosphate compounds (18μg/person/day)

www.efsa.europa.eu/publications 13 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 14 EFSA Supporting publication 2016:EN-1006

3. Validity of the databases used to derive the TTC values

www.efsa.europa.eu/publications 15 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 16 EFSA Supporting publication 2016:EN-1006

3.2. Should the point of departure for establishing TTC values be

3.3. Should the overall database be expanded/modified?

www.efsa.europa.eu/publications 17 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 18 EFSA Supporting publication 2016:EN-1006

3.4. Should chemicals with particular structures and/or toxicological

www.efsa.europa.eu/publications 19 EFSA Supporting publication 2016:EN-1006

www.efsa.europa.eu/publications 20 EFSA Supporting publication 2016:EN-1006

3.5. Combined exposure to multiple chemicals and combined exposures

3.6. TTC for unidentified chemicals

www.efsa.europa.eu/publications 21 EFSA Supporting publication 2016:EN-1006

3.7. Low-dose effects and non-monotonic dose-response

4.1. General principles

www.efsa.europa.eu/publications 22 EFSA Supporting publication 2016:EN-1006

4.2. Less than lifetime or intermittent exposures

4.3. Infants and children, potentially sensitive life-stages

www.efsa.europa.eu/publications 23 EFSA Supporting publication 2016:EN-1006

5. Consideration of the Kroes et al. (2004) decision tree

Reproduced with copyright permission from Elsevier.

www.efsa.europa.eu/publications 24 EFSA Supporting publication 2016:EN-1006

Figure 1: Figure B from Dewhurst and Renwick (2013)

6.1. General conclusions

www.efsa.europa.eu/publications 25 EFSA Supporting publication 2016:EN-1006

6.2. The Cramer scheme is fit for purpose

www.efsa.europa.eu/publications 26 EFSA Supporting publication 2016:EN-1006

6.3. Metabolism is an inherent part of the TTC values

6.4. TTC Domain of applicability is sufficiently broad