Alternative Approaches for the Assessment of Chemicals in Food

Chapter 3.4

Alternative Approaches for the Assessment of Chemicals in Food Alexandre Feigenbaum1 and Andrew P. Worth2 1

FCM, Rishon Lezyion, Israel; 2Joint Research Centre, European Commission, Ispra, Italy

SUMMARY This chapter provides an overview of the Threshold of Toxicological Concern (TTC) concept and the ways in which it has been applied, or proposed for application, in the EU-wide risk assessment of chemicals in food. The basis of the TTC approach is described in relation to key historical developments, including policy and research initiatives.

1. INTRODUCTION The Threshold of Toxicological Concern (TTC) approach is a risk assessment methodology that can be used to assess substances of unknown toxicity present at low levels in the diet. It is based on empirical evidence that for non-cancer effects, there are thresholds below which toxicity does not occur, whereas for cancer effects, the likelihood of tumours is zero to very small at very low exposure levels. Thus, for chemicals of unknown toxicity, human exposure thresholds can be established, below which there is a low probability of adverse effects on health. Accordingly, a range of human exposure thresholds (TTC values) have been developed for both cancer and non-cancer endpoints, on the basis of historical data from extensive toxicological testing in animals. The TTC approach requires only knowledge of the chemical structure of the substance of interest, in addition to information on human exposure. As no new animal testing is required, the TTC approach can be regarded as an alternative (non-animal) approach to risk assessment. The TTC approach has gained acceptance in situations where toxicity data are not available and cannot easily be acquired (such as impurities, reaction products and trace contaminants in food, feed and water), where evaluation of a large number of compounds with low exposure levels is required (such as flavouring substances), in the prioritisation of large numbers of compounds (such as non-plastic food contact materials), where resources are limited (e.g. contaminants in surface water) or when a rapid safety assessment is needed (such as in chemical food safety incidents). The TTC approach is not accepted in situations where toxicity data are required (e.g. for active ingredients of pesticides). The presence of chemicals in food raises special considerations in relation to the use of alternative methods. Food obviously includes a complex mixture of naturally occurring chemicals, intentionally added ones and contaminants. Some of these chemicals undergo a complex series of transformations in the journey from ‘field to fork’. In addition, food commodities are processed and cooked, so the product is modified after purchase (as with tobacco products; see Chapter 3.5). Furthermore, although these chemicals are typically present at low levels in food, humans are repeatedly exposed to them over a lifetime. For these reasons, information on repeat-dose toxicity, genotoxicity and carcinogenicity are more relevant than acute toxicity. Indeed, where information requirements exist (for authorised substances, such as flavourings), these are the types of information that are typically required. However, in many cases, it is practically impossible to test all the chemicals found in food, and this has created an opportunity to establish a risk assessment approach (i.e. TTC) that makes the best use of existing data, without animal testing.

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The TTC approach has also been considered for other applications, including personal and household care products, cosmetics, industrial chemicals and pharmaceutical impurities, with varying degrees of uptake (reviewed by Ref. (1)). In this chapter, we focus on the historical development of the TTC approach and its application in the food safety area, where the approach originated.

2. HISTORICAL DEVELOPMENT OF THE TTC APPROACH The origins of threshold approaches can be traced back to 1967, when John Frawley (2) at the US Food and Drug Administration (FDA) analysed a distribution of No Effect Levels from chronic rodent studies in a database of 132 chemicals (excluding heavy metals and pesticides) and proposed a safe dietary level of 0.1 ppm (0.1 mg/kg food or drink). This established the principle that the trends found in databases of evaluated substances can be extrapolated to nonevaluated substances. It also led to the idea that it is possible to categorise chemicals into those of low and high concern. In the 1970s, efforts were under way at the FDA to classify food flavourings (3) and other chemicals in food, according to their chemical structures. This resulted in the well-known Cramer decision tree (after Greg Cramer et al. (4)), comprising three structural classes for the classification of non-cancer effects (Table 1). The decision tree categorises a chemical into one of the three Cramer classes, based on the answers to a series of 33 questions relating mostly to chemical structure. Subsequently, FDA scientists (5) concluded that carcinogenicity was the most sensitive endpoint and analysed the carcinogenic potencies of 343 chemicals from the ‘Gold’ database (named after the original developer, Lois Gold, also known as the Carcinogenic Potency Database, CPDB; (6)). Based on a probabilistic analysis of potency (TD50) values in the database, and assuming a linear non-threshold doseeresponse relationship, Alan Rulis generated a distribution of Virtual Safe Dose (VSD) values. These were dietary exposure levels corresponding to a one-in-a-million excess lifetime tumour risk (this value was a policy choice, the upper bound level of ‘acceptable risk’). In 1991, in a seminal study, Ashby and Tennant correlated chemical structures with mutagenic and carcinogenic effects (7). Their investigation of 301 chemicals (154 alerting and 147 non-alerting) which had been tested in the US National Toxicology Program (NTP), led to several important findings: (a) structural alerts for DNA reactivity (mutagenicity) were based on chemical electrophilicity; (b) most of the rodent carcinogens were among the 154 structurally alerting chemicals; (c) most of the structurally alerting chemicals were mutagenic; and (d) among the 147 non-alerting chemicals, less than 5% were mutagenic. The CPDB was updated to 709 chemicals by Mitchell Cheeseman et al. (8). By analysing the cancer potency data for these chemicals, they identified structural classes associated with high-potency carcinogens (N-nitroso-compounds, benzidine-structures and three other structural classes), and on this basis recommended their exclusion from the Threshold of Regulation (TOR) approach. A further evaluation of the CPDB was carried out by Ian Munro and the Canadian Centre for Toxicology (9). Using potency data for four different subsets of carcinogens, Munro found that the type of data subset and the statistical approach to low-dose extrapolation were significant factors in determining the probability of excessive lifetime cancer risk. However, the percentage of new substances assumed to be carcinogens had the most impact. On the basis of these analyses, the FDA introduced the TOR of 0.5 ppb for food contact materials (10). Using the FDA default values for a combined food and drink intake of 3 kg/d per person and 60 kg body weight (bw), the level of 0.5 ppb (mg/kg) food (corresponding to a daily exposure of 0.025 mg/kg bw/d or 1.5 mg/person) was considered to be a very conservative estimate, even if the substance was later identified to be a carcinogen. However, the TOR approach was not

TABLE 1 Cramer Structural Classes for Non-Cancer Endpoints Cramer Class

Definition

I (low concern)

Substances with simple chemical structures and for which efficient modes of metabolism exist, suggesting a low order of oral toxicity.

II (intermediate concern)

Substances which possess structures that are less innocuous than class I substances but do not contain structural features suggestive of toxicity like those substances in class III.

III (high concern)

Substances with chemical structures that permit no strong initial presumption of safety or may even suggest significant toxicity or have reactive functional groups.

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TABLE 2 Overview of Structural Classes for Cancer and Non-Cancer Endpoints (following EFSA and WHO, 2016 (16)) TTC in mg/person/day

TTC in mg/kg bw/day*

High-potency carcinogensa Substances known or predicted to bioaccumulateb Steroidsc Substances which are not in the Cramer or Munro databasesd

TTC approach not applicable. Perform a substance-specific evaluation.

Substances with alert for genotoxicity

0.15

0.0025

Organophosphates and carbamates

18

0.3

Cramer Class III

90

1.5

Cramer Class II

540

9

Cramer Class I

1800

30

*Based on the historical assumption of an adult body weight of 60 kg. a Aflatoxin-like, azoxy (RN ¼ Nþ(Oe)R) and N-nitroso (RR’NeN ¼ O) compounds; benzidines; hydrazines. b Polyhalogenated dibenzodioxins, dibenzofurans and biphenyls; heavy metals. c Substances which have not been tested for their endocrine properties, other than steroids, can be evaluated using the TTC approach. d Inorganic substances, organometallics, proteins, nanomaterials, radioactive substances, mixtures of substances containing unknown chemical structures.

applicable to compounds known to be carcinogens, which are not permitted in food according to the ‘Delaney Clause’ (after Congressman James Delaney) of the Food, Drugs, and Cosmetic Act. In the 1990s, further developments focused on the development of threshold values for non-cancer effects. Munro et al. (11) evaluated a dataset of 613 substances with 2941 No Observed Adverse Effect Level (NOAEL) values, representing a broad range of chemicals. They recorded information from the most sensitive species, sex and toxicological endpoints, to identify the most conservative NOAEL value for each substance. The substances were divided into three chemical classes, based on their chemical structure, by using the Cramer decision tree (4). The cumulative distributions of NOAELs for the compounds in each Cramer structural class were plotted, and a log-normal distribution was fitted in each case. The fifth percentile value of each distribution was calculated and converted to a corresponding human intake. The resulting TTC values are given in Table 2. Since 1997, a tiered approach incorporating these TTC values has been used for the safety evaluation of flavouring substances by the Joint FAO/WHO Expert Committee on Food Additives (JECFA; (12)). Subsequent work by Robert Kroes and an ILSI Europe Expert Group (13, 14) extended the JECFA approach. Kroes introduced, as a first step, the exclusion of high-potency genotoxic substances (aflatoxin-like compounds, N-nitroso compounds, azoxy compounds) and the identification of structural alerts for high-potency carcinogenicity and genotoxicity. A generic threshold of 0.15 mg/person/day (0.0025 mg/kg bw/day) for potentially genotoxic compounds was applied. The next step considers non-genotoxic substances (lacking structural alerts for genotoxicity), in a sequence of steps related to their structure and estimated intake. For organophosphates and carbamates (alerts for neurotoxicity), a TTC of 18 mg/kg bw/day was proposed, whereas for substances belonging to Cramer classes I, II and III, TTC levels of 90, 540 and 1800 mg/person/day were proposed based on the work of Munro et al. ((11); Table 2).

3. REGULATORY AND SCIENTIFIC DEVELOPMENTS IN THE FOOD AREA 3.1 The EFSA Scientific Committee’s Opinion on the TTC Approach In July 2012, EFSA took an important step by publishing an opinion on the relevance, reliability and applicability of the TTC approach for dietary risk assessment (15). In December 2014, EFSA and the WHO organised a stakeholder public hearing and a workshop, to provide recommendations on how to improve the existing TTC approach. The final report was published in 2016 (16). These developments led to some important refinements and recommendations, including improvements to the Cramer decision tree questions (16), the use of dietary exposure calculations for infants and children (17), and the treatment of less than lifetime exposure scenarios. A decision scheme based on (16) is given in Table 3.

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TABLE 3 Decision scheme for the application of the TTC approach (following EFSA and WHO, 2016 [16]) Step

Question

If YES

If NO

0

Does the substance have a known structure and are exposure data available?

Go to Step 1

TTC approach cannot be applied. Chemical-specific risk assessment required

1

Is the substance a member of an exclusion category?

TTC approach cannot be applied. Chemical-specific risk assessment required

Go to Step 2

2

Are there structural alerts for genotoxicity data?

Go to Step 3

Go to Step 4 for non-genotoxic considerations

3

Does estimated intake exceed TTC of 0.0025 mg/kg bw per day?

TTC approach cannot be applied. Chemical-specific risk assessment required

Substance not expected to be a safety concern low probability that lifetime cancer risk exceeds 1 in 106

4

Is the compound an organophosphate or carbamate?

Go to Step 5

Go to Step 6

5

Does estimated intake exceed TTC of 0.3 mg/kg bw per day?

Chemical-specific risk assessment required.

Substance not expected to be a safety concern

6

Is the compound in Cramer class III?

Go to Step 7

Go to Step 8

7

Does estimated intake exceed TTC of 1.5 mg/kg bw per day?

Chemical-specific risk assessment required.

Substance not expected to be a safety concern

8

Is the compound in Cramer class II?

Go to Step 9

Go to Step 10

9

Does estimated intake exceed TTC of 9 mg/kg bw per day?

Chemical-specific risk assessment required.

Substance not expected to be a safety concern

10

Does estimated intake exceed TTC of 30 mg/kg bw per day?

Chemical-specific risk assessment required

Substance not expected to be a safety concern

3.2 Flavouring Substances Already on the EU Market An EU-harmonised approach to the evaluation of flavouring substances was first laid down in Regulation (EC) No 2232/96 (18), for a Community Procedure for flavouring substances used, or intended for use, in or on foodstuffs. In view of the large number of these substances (more than 2500; (19)), they were classified into 34 chemical Flavouring Groups of structurally related substances with similar metabolic and biological behaviour (18, 20). For the purpose of Flavouring Group Evaluations (FGEs), read-across was permitted, so that toxicity data available for some substances could be extrapolated to other members of the same group (21). Dietary exposure to flavourings was estimated and compared with the corresponding TTC thresholds. No toxicity data were required, when the substance could be predicted to be metabolised to innocuous compounds and when the estimated daily intake of the substance was lower than its TTC. However, toxicity data were requested for substances with structural alerts. EFSA has evaluated 2067 of around 2500 flavouring substances used in the European Union. In 2010, further data were needed to finalise the evaluations for 400 substances.

3.3 New Flavouring Substances EFSA’s guidance on the risk assessment of new flavourings (22), prior to their authorisation for use in the EU, has been in force since September 2012. The successful experience from the previous grouping approach was taken over, with data requirements depending on the exposure compared with the TTC threshold of the flavouring substance.

3.4 Food Contact Materials: Intentionally and Non-Intentionally Added Substances Food Contact Materials (FCM) contain a variety of Intentionally Added Substances (IAS) and Non-Intentionally Added Substances (NIAS), which may migrate into food. While IAS are added for a specific technical purpose, NIAS are impurities of authorised starting substances, as well as reaction and degradation products formed during the manufacture

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and use of the material. While IAS are evaluated by EFSA and regulated at the EU level (23), the safety assessment of NIAS is not harmonised and is left to the responsibility of the industry (24). In 2016, EFSA published an opinion entitled Recent developments in the risk assessment of chemicals in food and their potential impact on the safety assessment of substances used in food contact materials (25). In the version of the Guidance valid at that time for applications for new FCM substances, exposure was evaluated from migration data by using conventional assumptions. The higher the migration, the greater the amount of toxicity data required. The 2016 opinion referred to new risk assessment tools. One major area revisited was consumer exposure, which could be assessed by using EFSA’s Comprehensive European Food Consumption Database and figures set by the WHO for infants. The opinion also opened the door to the use of TTC thresholds and read-across, especially for NIAS. Surprisingly, in 2017, none of these new approaches were introduced in the update of the old FCM guidance for the evaluation of additives and monomers (intentionally added). The word ‘exposure’ is hardly mentioned, whereas ‘TTC’ and ‘threshold’ are not mentioned at all in this updated guidance. In the introduction, however, it is mentioned that the 2016 opinion is a scientific reference to be considered for the safety evaluation of nanomaterials and of NIAS that may migrate from FCM. According to the EU legislation (26), NIAS must be assessed in accordance with internationally recognised scientific principles on risk assessment. Apparently, there is no agreement yet on these ‘recognised principles’. Looking to the future, the use of exposure considerations and the TTC approach represents a scientifically robust opportunity.

3.5 Contaminants in the Food Chain An example of the regulatory use of the TTC approach for contaminants is illustrated by the evaluation of Alternaria toxins (produced by fungi that attack crops). These were divided into five different classes, based on their chemical structures. As there were few to no data on the toxicity of several substances, application of the TTC approach (27) led to the conclusion that genotoxicity data should be generated (28).

3.6 Pesticides and Their Metabolites In practice, the consumer is exposed not only to pesticide active substance residues but also to a range of metabolites for which limited toxicity information is usually available. In 2012, the EFSA Panel on Plant Protection Products and the Residues (PPR Panel) developed an Opinion on non-testing approaches, including TTC, to evaluate the toxicological relevance of such metabolites in dietary risk assessment (29). The TTC values recommended by EFSA (15) for genotoxic and toxic compounds (Table 2) were found to be sufficiently conservative for chronic exposure. Assessment schemes for chronic and acute dietary risk assessment of pesticide metabolites, involving the use of the TTC approach combined with QSAR and read-across, were proposed. Tentative TTC values for acute exposure were also established. It was anticipated that, on many occasions, further testing would be needed to reach a firm conclusion on the toxicological relevance of the metabolites. However, the benefit of applying the approach is that it will permit the prioritisation of metabolites for subsequent testing. Building on its Opinion, EFSA subsequently published guidance on the assessment of pesticide residues (30), which describes a stepwise and weight-of-evidence approach based on toxicological and metabolism data, as well as the use of non-testing methods (QSAR, read-across, TTC). In this guidance, the TTC approach serves to complement, rather than supersede, the use of substance-specific toxicological data.

3.7 Residues of Pharmacologically Active Substances in Food In 2013, the EFSA Panel on Contaminants in the Food Chain (CONTAM) established guidance on the principles and methods to be taken into account when establishing Reference Points for Action for non-allowed pharmacologically active substances present in food of animal origin (31). CONTAM considered that the TTC approach, with the use of Cramer classes, was not applicable for deriving Toxicological Screening Values (TSVs) for two reasons: (a) some groups of substances (e.g. steroids) are excluded from the TTC approach (15); and (b) the traditional TTC database only contains a small number of pharmacologically active substances. Therefore, to derive TSV values, CONTAM used a distribution of 167 Acceptable Daily Intake (ADI) values of pharmacologically active substances collected by the European Medicines Agency (EMA). The corresponding TSV values are given in Table 4 (31).

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TABLE 4 Assignment of Toxicological Screening Values for Non-allowed Pharmacologically Active Substances in Food (31) Can Genotoxicity Be Excluded?

Does the Substance Act Pharmacologically on the Nervous or Reproductive System and/or Is It a Corticoid?

Toxicological Screening Value (TSV) (mg/kg bw per day)

Group I

NO

0.0025

Group II

YES

YES

0.0042

Group III

YES

NO

0.65

The TTC approach has also been applied to pharmaceutical substances found as contaminants in treated wastewater used for the irrigation of root crops (32, 33).

3.8 The Polemics of TTC 3.8.1 The Perception of TTC as ‘an Industry-Driven Approach’ The strongest criticisms of the TTC approach, and particularly EFSA’s role in developing it, have come from Pesticide Action Network Europe (PAN Europe), who stated (34): A new PAN Europe report reveals that 10 out of 13 members of the EFSA working group on TTC have a conflict of interest. TTC is an industry-driven approach and these members have been developing or promoting this method in the past jointly with industry. The interlinking of these people shows they are operating as a network.

While the second author of the current chapter was ‘not evaluated’, it was claimed that ‘there is a strange connection with the JRC, EFSA and Nestlé on QSAR and TTC’ on the grounds that the three institutions co-authored a paper (35). This statement was followed by a series of highly personal attacks against prominent EFSA scientists. An opinion piece was published with some other organisations (36), which presented very weak scientific arguments.

3.8.2 Endocrine Disruptors and Non-Monotonic DoseeResponse Relationships One of the criticisms of the TTC approach is that it does not take into account the potential low-dose effects of endocrineactive substances (34). According to the low-dose hypothesis, endocrine-active substances or endocrine disruptors may cause adverse effects at low doses but not necessarily at all higher doses. To date, no scientific consensus has been reached on the existence of this low-dose phenomenon (37, 38). Both the EFSA Scientific Committee (15) and the PPR Panel (29) considered that the applicability of the TTC approach should be re-evaluated, when there is consensus on how to assess endocrine disruptor activity and once an EU-wide approach for defining and assessing endocrine disrupters has been finalised.

4. SCIENTIFIC AND TECHNICAL DEVELOPMENTS There has been growing scientific interest in the further development of the TTC approach, which can be attributed in part to an increasing consideration by regulatory bodies such as EFSA (15) and the EU’s non-food scientific committees (39). As described above, the TTC values for different endpoints were derived on a probabilistic basis by using purposebuilt data sets. Therefore, the coverage, quality and treatment of the underlying data have an impact on the resulting TTC values. For this reason, there have been numerous attempts to expand the coverage, improve the quality and enhance the consistency and transparency of data treatment in the TTC databases. Scientific developments have also focused on improving the classifiers used to identify genotoxic carcinogens and non-genotoxic chemicals, on developing QSAR models for repeat-dose toxicity and carcinogenicity, and on developing software tools to implement these predictive approaches. The pace of these developments, which has resulted in the increasing availability of diverse methods and tools, has led to calls for the establishment of regulatory guidelines to prevent TTC from ‘dis-harmonisation’ (40).

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4.1 Improvement of TTC Databases and Their Use to Refine TTC Values 4.1.1 Carcinogens An electronic version of the CPDB was first made publicly available by EFSA (41), facilitating further analyses of the data. The current (July 2017) version of the CPDB contains 1547 chemicals (positive and negative carcinogenicity results) and is publicly accessible from the US National Institutes of Health. It has been argued that the original TTC value for cancer was derived by using toxicological data and making assumptions that no longer reflect the state-of-the-science (42). With a view to addressing the concerns, the CPDB is currently being expanded and curated in a project funded by the European Chemical Industry Council Long-Range Initiative (CEFIC LRI).

4.1.2 Non-Carcinogens With Repeat-Dose Toxicity The original TTC database was an appendix to a Munro’s paper (11), and an electronic version of the Munro database was first made publicly available by EFSA (41). A considerable development was later made by Chihae Yang and collaborators in the context of the EU-funded COSMOS project. To explore the applicability of the TTC approach to cosmetics (43, 44), the COSMOS TTC database contained 552 cosmetics-related chemicals. This database was then combined with an updated version of the Munro database to provide an expansion of chemical space (966 substances). The combined COSMOS-Munro TTC database is the largest quality controlled TTC database to have been made publicly available. Even though the different TTC databases were designed to include chemicals having specific use categories (food additives, food contaminants, cosmetics, industrial chemicals), chemoinformatic analyses of the coverage of structural features and physicochemical properties have elucidated similarities and differences in the chemical space covered between different types of chemicals, such as food-related and cosmetics-related substances (45, 46).

4.1.3 Challenges to the Munro Threshold Values There have been several challenges to the Munro threshold values, especially for Cramer Class III (47). The argument is that the threshold for Class III was originally determined with carbamates and organophosphates included in this class. Later, these substances were moved into separate classes leading several authors, including Munro himself (48), to recalculate the Class III threshold and propose revised threshold values up to 600 mg/day (4850). These proposals have not been adopted by EFSA or the WHO, as a major modification of the TTC threshold values would reduce the conservatism of the TTC approach. A number of studies have evaluated the reliability (adequacy of protection) of the TTC thresholds, where reliability is defined as the percentage of substances having an ADI larger than their TTC values in a given database. In a study on 845 FCM substances, comparison of the TDI values with TTC values revealed that the TTC values were protective for (lower than) 96% of the TDIs based on chemical-specific assessments (51). Similarly, in a study on 328 pesticides that had been fully evaluated, the current Class III threshold of 90 mg/person per day was found to provide a reliability of 97.5% (52). These types of study have also identified specific substances that have been ‘underevaluated’ by the TTC approach (43, 5154). The accumulation of such examples has allowed common structural features to be identified and thus to flag the chemical structures which may be ‘underevaluated’ by use of the TTC approach. However, these proposed refinements of the Cramer tree have yet to gain widespread acceptance.

4.2 Defining Endpoint-Specific TTC Values Attempts have been made to determine endpoint-specific TTC values, and in particular, for reproductive and development toxicity endpoints. van Ravenzwaay et al. (55) used a database built by using REACH and industry (BASF) data, without allocating the substances to Cramer classes. The calculated endpoint-specific TTC values are the range of 100 mg/person per day for developmental toxicity, depending slightly on the tested animal species (rats or rabbits). These findings are consistent with previous findings, which support the view that reproductive and development toxicity effects are covered by the Munro threshold for Cramer Class III (e.g. Refs. (56, 57)).

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4.3 Development of New Structural Alerts and Prediction Models 4.3.1 Carcinogens A failure to identify high-potency carcinogens and genotoxic substances will lead to false negatives (underestimated toxicity) and a non-conservative evaluation. Therefore, with the aim of increasing the sensitivity (minimising the false negative rate) of the TTC approach, the EFSA PPR Panel proposed the application of a suite of computational methods, involving structural alerts, QSAR and read-across, as a complement to the TTC approach in the assessment scheme for pesticide-metabolite exposure (29). Since the original publication of structural alerts by Ashby and Tennant (7), research efforts have focused on the development of new approaches (alerts and QSARs) for predicting DNA-reactivity (58), genotoxicity and carcinogenicity (59, 60) and repeat-dose toxicity (61).

4.3.2 Non-Carcinogens With Repeat-Dose Toxicity The EFSA Scientific Committee (15) recommended that the Cramer classification scheme should be revised in the short term, to make it easier to understand and to use. In the longer term, it recommended the development of new structurebased classification schemes that are more discriminating between substances with different toxic potencies. An EFSAsponsored project showed the potential use of multivariate statistical methods to uncover new structural features that may be useful in setting human exposure thresholds (41). Subsequent work in the COSMOS project developed structural features for liver toxicants (45, 62) and for chemicals acting by various mechanisms, including mitochondrial toxicity (63) and phospholipidosis (64). In addition to structural alerts, QSAR models have been developed to predict the potency of repeat-dose effects directly (e.g. Ref. (65)).

4.3.3 Introduction of Biokinetics There have also been attempts to amend the Cramer decision tree by introducing the consideration of metabolism. In practice, questions on metabolism were already included in several questions of the Cramer decision tree. The relevant questions were slightly reformulated following the EFSA-WHO workshop (16). A current project funded by Cosmetics Europe is seeking to further refine the TTC approach, by introducing the concept of an internal TTC (as opposed to external exposure limits), and to explore the utility of QSAR and biokinetic modelling to establish internal TTC values (66). If successful, this will provide a more-sophisticated means of assessing various sources of uncertainty in extrapolation.

4.4 Computational Tools for TTC Analysis While the Cramer classification tree undoubtedly served to improve consistency between the toxicological evaluations made by different experts, its original paper-based application presupposed a degree of expert judgement and subjectivity. Therefore, following a recommendation made in a JRC workshop (67), the JRC commissioned the development of Toxtree, a software to facilitate the consistent application of the Cramer scheme. Toxtree was made freely downloadable from the JRC website and subsequently from Sourceforge. This was the first time a computational implementation of the Cramer tree had been put in the public domain. Subsequently, several additional TTC-relevant rulebases were added, including the Cramer rulebase with extensions (five extra rules to correct for some false negative classifications) and the decision tree of Kroes et al. (13). The Toxtree implementation of the Cramer scheme has been evaluated in several studies (53, 68) and has also been compared with the later implementation in the OECD QSAR Toolbox (69, 70). In addition to these structure-based classification tools, models for predicting the potency of repeat-dose effects (i.e. NOAELs, LOAELs) have been developed and made freely available in the VEGA software suite (65).

5. CONCLUSIONS The current TTC approach, based on the assessment of both cancer and non-cancer endpoints, is based on around 50 years of experience and is widely considered to be protective of human health for low-level exposure scenarios. The approach integrates data from hundreds of chemicals on which extensive information is available on chemical structure, metabolism and toxicity. With developments in science and technology, many refinements to the TTC approach have been proposed in the scientific literature. These refinements are based on the establishment of larger and better curated toxicological databases, improved knowledge on the relationship between toxicity and chemical features and physicochemical properties.

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For the most part, however, these refinements have not translated into regulatory practice. A possible reason for this is that the TTC approach is applied differently in different sectors, and there is no mechanism at the international level to evaluate and harmonise these variants of the TTC approach. A useful step toward harmonisation was made by the FDA, EFSA and the WHO. In the future, a comprehensive and systematic analysis of the underlying uncertainties will permit a moretransparent elucidation of the differences between different implementations of the TTC approach. This is the focus of a project initiated in 2017 by ILSI Europe. Furthermore, there is considerable opportunity to further integrate QSARs (71e73) and mechanistic in vitro tests (74) into the TTC approach. Ultimately, an international organisation will be needed to ‘champion’ these harmonisation efforts. At the time of publication (late 2018), EFSA was completing a guidance document on the TTC approach, as a follow-up to its 2012 Opinion.

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