SCIENTIFIC / TECHNICAL REPORT submitted to EFSA

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1 SCIENTIFIC / TECHNICAL REPORT submitted to EFSA Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides 1 Prepared by: Richard Brown (CRD); Julian Carter (CRD); Ian Dewhurst (CRD); Claire Stephenson (CRD); Sonia Tessier (CRD). Abstract The threshold of toxicological concern (TTC) is based on the concept that there are levels of exposure to chemicals that will result in no appreciable risk to health. This project investigated the potential applicability of TTC to dietary exposures of transformation products of pesticides. Following a review of the literature an existing scheme (Kroes et al, 2004) was selected, with minor modifications specific to pesticides, as the basis for the investigations. The scheme was validated against 100 pesticide active substances, for which there were data, and tested against transformation products in 15 case studies. Comparison of applicable TTC values with agreed ADIs for the pesticides showed the TTC scheme to be adequately protective. However, the QSAR programs used were found to be poor predictors of the toxicity and genotoxicity of pesticides, which might have an impact on the level of data required before TTC can be applied with confidence. Exposure estimates for the case studies utilised a NEDI type approach for chronic exposure to individual crops and transformation products. The estimated exposures were compared with the applicable TTC for the transformation products. The results showed that the majority of transformation products had predicted exposures below the applicable TTC and therefore did not merit further investigation. Once concerns over predicting or determining genotoxic potential have been addressed, the TTC scheme has the potential to be an effective tool for prioritising transformation products that require detailed investigation. Key words: TTC; Threshold of Toxicological Concern; Pesticide; PPP; Residues; Diet; Intake; Exposure; Metabolite; QSAR 1 Project ID: CFP/EFSA/PPR/2008/01). Accepted for Publication on 27 November Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

2 FINAL REPORT Project Title Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides EFSA Project Code Grant Agreement EFSA/PPR/2008/01 Sponsor Scientific Panel on Plant Protection Products and their Residues Working Group on the toxicological relevance of pesticide metabolites Contractor:- Chemicals Regulation Directorate (CRD), UK (formerly Pesticides Safety Directorate (PSD), UK) Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

3 Index and Table of Contents Section Title Page Executive summary 1 Purpose of Final report 1 Description of Project 2 Summary of project activities and timetable 3 Background 4 Outcome of activity 1a (Literature review) Methods Results History of TTC concept and key developments 6 Outcome of activity 1b (Questionnaire to other organisations) Methods Results Conclusions 25 Outcome of activity 2a (Validation of existing TTC concepts Background Methods Results of SAR/QSAR predictions Cramer classifications and applicable thresholds versus ADIs Conclusions Proposals for further work 36 Outcome of activity 2b & 5a (organisation of expert consultative group) Background First expert consultative group workshop Second expert consultative group workshop 37 Outcome of activity 3 (Development of a proposal for a TTC concept) Genotoxicity Neurotoxicity Scheme for TTC approach to transformation products of pesticides 40 Flow chart 43 Outcome of activity 4 (Case studies) 11.1 Choice of case study substances Exposure assessments Evaluation of exposure estimates against TTC Conclusion 83 Overall summary and conclusions 94 Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 3

4 List of Appendices 96 Appendix 1 Summary of references 97 Appendix 2 Questionnaire sent to other organisations 114 Appendix 3 Questionnaire responses 118 Appendix 4 List of 100 actives selected for validation exercise 119 Appendix 5 Results of the validation (SAR, Cramer classifications and ADIs) 123 Appendix 6 Members of the Expert Consultative Group 124 Appendix 7 Expert Consultative Group workshop reports 125 Appendix 8 Initial selection of case study compounds 132 Appendix 9 Exposure data for case studies 140 Appendix 10 CRD Project Team and EFSA Steering Committee Members 141 Appendix 11 Glossary 142 Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 4

5 Project Title:- Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides EFSA Project Code:- Grant Agreement EFSA/PPR/2008/01 1. Executive Summary The aim of the project was to assess the applicability of the Thresholds of Toxicological Concern (TTC) concept for metabolites, degradation and reaction products of active substances of plant protection products. This was achieved via the following activities: Perform a literature search and distribute a questionnaire to identify existing TTC schemes; critically evaluate them and produce a draft TTC scheme specific for transformation products of pesticides. Validate the draft scheme using 100 pesticide active substances; making revisions as necessary. Using case studies evaluate transformation products of 15 pesticides using the TTC scheme and estimates of chronic exposure. Finalise the TTC scheme based on the case studies. The main findings were: TTC is not widely used in assessing transformation products of pesticides but there was no a priori reason why it could not be used. The scheme of Kroes et al (2004) formed a sound basis for the draft TTC proposal; modified for a broader neurotoxicity category. The TTC scheme was adequately protective when validated against the ADIs of 100 active substances, covering a wide range of end-points. (Q)SAR predictions did not always correlate well with the observed toxicity profiles of the pesticide active substances. Assuming the (Q)SAR predictions for in vivo genotoxicity are reliable, the TTC scheme was able to prioritise the 79 transformation products in the case studies down to 16 that required further consideration. In conclusion, the proposed TTC scheme is a viable tool for use in the assessment of transformation products of pesticides. By providing a set of clear criteria, it has the potential to improve consistency of outcomes and target resources to compounds of greatest concern. The main caveat is the availability of data or a reliable model to predict in vivo genotoxicity of the transformation products, or a requirement to provide appropriate genotoxicity data. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 5

6 2. Purpose of Final Report This Final Report for the above project has been prepared to describe the outcome of all stages of the project and to present conclusions, based on the work performed, relating to the potential use of thresholds of toxicological concern in the field of dietary risk assessment of transformation products of pesticides. It is intended that this Final Report will be discussed and, if appropriate, formally agreed by the EFSA/PPR Panel Steering Committee for the project at the meeting on 1 st. The generation of this Final Report represents Activity 5c in the Project Timetable. 3. Description of Project Project Aims:- To assess the applicability of the Thresholds of Toxicological Concern (TTC) concept for metabolites, degradation and reaction products of active substances of plant protection products. To develop a TTC concept which could potentially be used in the residues risk assessment for plant protection products and to validate the concept with case studies. To produce a report which can be used by the PPR Panel to develop and adopt an Opinion on the use of this concept. This project forms part of a group of projects investigating methods for the evaluation of metabolites, degradation and reaction products of pesticides e.g. use of (Q)SARs and metabolism profiling. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 6

7 4. Summary of Project Activities and Timetable Table 1 Project Activity Description Dates Initial Meeting Activity 1 Establishment of the current state of the art in respect of the use of the TTC concept for chemical risk assessment Activity 2 Assessment of current TTC approaches and validation for use in dietary exposures to transformation products of PPP active substances Activity 3 Development of a proposal for a TTC concept for dietary exposures to transformation products of PPP active substances Activity 4 Case Studies Activity 5 Preparation of the final TTC proposal and report Activity 1a Activity 1b Activity 2a Activity 2b 1 st Steering Committee Meeting Literature Review Questionnaire to organisations involved with chemical risk assessment Validation of existing TTC concepts 1 st Expert Consultative Group Meeting 22/10/2008 November 2008 to February November 2008 to February February 26/2/ Activity 2c Interim Report March Activity 3 Activity 4a Activity 4b Activity 5a 2 nd Steering Committee Meeting Development of a proposal for a TTC concept Selection of case study compounds Evaluation of case study compounds using TTC concept 2 nd Expert Consultative Group Meeting 26/3/ February to March February to July April to August July Activity 5b Revision of TTC proposal July Activity 5c Production of draft final report and final Steering Committee Meeting August - Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 7

8 5. Background Residues arising from the use of plant protection products (PPPs) on food commodities can consist not only of the active substance but also of metabolites, degradation and reaction products of the active substance. The dietary risk assessment for consumers must take account of the actual toxicological burden of the substances making up the residue. The establishment of the residue definition for risk assessment purposes (as opposed to enforcement purposes e.g. MRLs) therefore involves a decision on which metabolites, degradation and reaction products are of toxicological concern. It is also necessary to determine toxicological endpoints which are relevant to the risk assessment for these metabolites. A major difficultly arises from the fact that only the toxicological properties of the active substance will normally have been investigated following the requirements of Directive 91/414/EEC. The toxicological properties of any metabolites etc. are not normally known. It is not practicable or desirable to require full toxicological testing of every possible metabolite, degradation or reaction product for every crop situation. It is also desirable to restrict the number of toxicological studies required to the minimum possible to minimise the use of animals in toxicological testing. In setting residue definitions for risk assessment purposes it is therefore necessary to use a significant element of expert judgement. EFSA considers that it is possible that the Thresholds of Toxicological Concern concept (TTC) could be used to reinforce the expert judgment used in establishing residue definitions for risk assessment purposes. The TTC concept is an assessment tool which is based on the concept that there are human threshold values for chemicals below which there is a very low probability that the chemical will have an effect on human health. A TTC scheme will identify de minimis values for chemicals which share similar structural characteristics. Metabolites, degradation and reaction products could be potentially be excluded from the residue definition as not being toxicologically significant if they meet the criteria for a threshold relevant to their chemical structure and anticipated residue levels. This decision could potentially be made without requiring any toxicological testing of the metabolite, since the appropriate threshold would have been derived from knowledge of other chemicals with similar structural characteristics. The only information required for a metabolite would be its chemical structure and an indication of maximum anticipated residue levels. The project described in this report builds on existing scientific knowledge and experience with the TTC to develop a TTC concept which could be used for metabolites, degradation and reaction products of plant protection products (PPPs). The proposed TTC concept was validated using a series of case studies. The case studies covered a range of active substance types and residue situations. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 8

9 6. Outcome of Activity 1a (Literature Review) Activity 1a Production of an up-to-date review of the TTC concept 6.1 Methods A literature review was conducted during November 2008 using the MEDLINE database. The search strategy was developed by experienced Information Specialists at the UK Government Food and Environment Research Agency ( Additional references were obtained up to 31 May. The MEDLINE search terms and number of items found are shown in the box below:-? s TTC S TTC? s threshold?(1w)toxicolog?(w)concern S2 29 THRESHOLD?(1W)TOXICOLOG?(W)CONCERN? S threshold?(1w)regulation S3 35 THRESHOLD?(1W)REGULATION? T2/9/ Results The literature search using terms based around Threshold of Toxicological Concern and Threshold of Regulation (with appropriate wildcard search terms) obtained 60 relevant references based on a study of their abstracts. It should be noted that searching on the abbreviation TTC directly was not possible due to generating false hits from the nucleotide sequence TTC (Thymine-Thymine- Cytosine). Many of the references were reviews or were abstracts associated with poster or oral presentations which did not provide new information beyond the main papers. Few papers contained new proposals for the development of the TTC concept. The key references covering the development of the TTC numbered approximately eight. A summary of the 60 references identified is presented in Appendix 1. A summary of the key references and the scientific development of the TTC concept is given below. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 9

10 6.3 History of the TTC concept and key developments The history and development of the TTC concept is covered in detail in certain key review papers (e.g. Barlow et al (2001) and Kroes et al (2005)) Early work Early references cover the initial development of the TTC concept for migration of chemicals from food packaging. In order to simplify the evaluation of substances intended for use in food-packaging materials, Frawley (1967) conducted an analysis of a database of 220 compounds (of different chemical types) for which chronic rodent toxicity studies were available. The initial aim was to define a dose for a substance in a food-packaging material which would not involve any hazard to human health. Frawley concluded that most food packaging materials would not cause toxicity in animals at less than 10 mg/kg in diet (10 ppm), and allowing for a 100-fold margin of safety the figure of 0.1 mg/kg of human diet could be set for any food packaging chemical. Assuming an intake of 1.5 kg of solid diet per day, this would equate to an intake of 150 μg/person/day Dividing chemicals into structural classes based on toxicity (Cramer classes) A system for dividing chemicals into classes for anticipated toxic hazard based on structure was published by Cramer et al (1978). Cramer et al developed a decision tree approach using a series of up to 33 structure-related questions to allocate chemicals into one of three structural classes presumed toxicity derived from its presence as a normal constituent of the human body, the chemical structure of the compound, potential reactivity, number of functional groups, natural occurrence in food. The three Cramer classes are described in the box below. Structural classes for chemicals (Cramer et al, 1978) Class I Substances with simple chemical structures and for which efficient modes of metabolism exist, suggesting a low order of oral toxicity Class II 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 (less knowledge of the metabolism, may be a component of food). Class III Substances with chemical structures that permit no strong initial presumption of safety or may even suggest significant toxicity or have reactive functional groups (more complex structures, metabolism not predictable, may contain certain functional groups associated with enhanced toxicity, may contain elements other than C, H, O, N or S e.g. any structure containing halogens). Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 10

11 6.3.3 First regulatory use of a TTC concept (US FDA Threshold of Regulation) The US Food and Drug Administration (US FDA) set a limit for food contact materials based on an analysis of 477 chemical carcinogens (the carcinogenic potency database Gold et al (1995)). By analysing the distribution of carcinogenic potencies a dietary concentration was derived which would give rise to a less than one in a million lifetime risk of cancer. The dietary concentration derived was 0.5 μg/kg of diet from which a human daily exposure level of 1.5 μg/person/day was derived. This daily exposure was based on a daily consumption pattern of 1.5 kg food and 1.5 litres of beverages. This dietary exposure level was concluded to be acceptable for untested substances even if the migrating substance was subsequently found to be a carcinogen, and the US FDA defined this level as the Threshold of Regulation (Begley, 1997). No further testing was required for food contact substances giving rise to exposures below this level (the US FDA rule was eventually published in 1995 (Federal Register July 17 th FR 36582) but the policy was developed over a 10 year period). The approach used by the US FDA utilised linear extrapolation to low dose levels, which many scientists working in this field felt was a highly conservative approach. However, this approach to food contact materials adopted by the US FDA represented regulatory acceptance of a TTC concept. This first regulatory use of a TTC concept was cautious in several respects the conservative nature of the endpoint modelling used (linear extrapolation), the application to chemicals only expected to be present in food at very low levels, the nature of chemicals likely to be used as food packaging materials (chemicals without intentional biological or pharmacological activity) and the endpoint set sufficiently low that it would be protective even if the chemical concerned turned out to be carcinogenic. The Threshold of Regulation also assumed that carcinogenic effects would generally occur at lower dietary concentrations than other toxic effects. It is important to note that this early FDA work and many of the subsequent approaches to TTC were based solely or predominantly on a consideration of carcinogenic potency and linear low-dose extrapolation Development of the TTC (Munro and co-workers) Munro and co-workers (Munro, 1990 and Munro et al, 1996) developed the TTC concept further by examining application of the concept to non-carcinogenic effects. The focus of the work by Munro and co-workers was in extending the use of the TTC concept to low molecular weight food flavouring substances. Munro and co-workers analysed results from a database of 611 compounds (covering industrial chemicals, pharmaceuticals, food substances, and environmental, agricultural and consumer chemicals). A total of 2900 NOELs were reviewed looking for a correlation between toxicity (NOEL) and chemical structure. For chemical structure the chemicals in the database were divided into the three Cramer classes. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 11

12 The studies examined by Munro et al (1996) included sub-chronic, chronic, reproductive and developmental studies, all using oral dosing and all conducted in rodents or rabbits. NOELs from sub-chronic studies were divided by a factor of 3 to be compared with chronic NOELs, and the NOELs proposed by the original study authors were used (even when considered to be conservative). For each substance the most conservative NOEL was selected, based on the most sensitive species, sex and endpoint. The distribution of NOELs separated by Cramer class showed that the distributions of the three classes were clearly separated in terms of mean and 5 th percentile NOELs. A human exposure threshold was calculated from the distribution for each of the structural classes, based on the 5 th percentile NOEL and a 100-fold margin of safety. The 5 th percentile NOEL was used as this would give 95% confidence that a compound of unknown toxicity and structure consistent with the particular Cramer class would be adequately covered for compounds other than those with a presumption of being carcinogens. The results of the analysis by Munro et al (1996) are presented in the following table. Table 2 Cramer class No. of chemicals NOEL range mg/kg bw/d 5 th %ile NOEL μg/kg bw/d Human threshold μg/person/d # Class I Class II Class III # - Assume 60 kg bw and 100 fold safety factor For later work the threshold values were rounded as follows:- Table 3 Cramer class Human threshold μg/person/day Class I 1800 Class II 540 Class III 90 These authors identified that the original US FDA threshold of 1.5 μg/person/day was highly precautionary, but that threshold also covered the eventuality that the chemical might subsequently be found to be a carcinogen. The human thresholds suggested by Munro and co-workers were intended for chemical substances with no presumption of genotoxic carcinogenicity (no structural alerts or chemical classes associated with carcinogenicity). 12 Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

13 6.3.5 Use of a TTC concept for flavouring substances (JECFA) Based on the work of Munro and co-workers, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) began to use a TTC concept for the evaluation of flavouring substances (JECFA, 1996). Inherent in this acceptance of the TTC concept was the conservative assumptions built into selecting the thresholds, the fact that flavouring substances tend to be eaten in relatively low amounts (they are often volatile and their flavour means intakes are self-limiting), and they tend to be simple structures without structural alerts. JECFA concluded that the TTC concept had potential value for the evaluation of flavouring agents where no toxicity data existed but intake was very low, but with the caveat that it could not replace expert opinion and for substances with structural alerts or data indicating concern then alternative approaches would be necessary. This concern could include chemicals with clear structural alerts for genotoxicity or carcinogenicity which would not be considered by this scheme. The JECFA decision tree scheme places chemicals into structural classes using the Cramer decision tree and then makes decisions on the need for toxicity data based on whether or not intakes under the expected conditions of use will exceed the threshold of concern for the relevant structural class (as defined by Munro 90, 540 or 1800 μg/person/day). A further threshold is employed at the final step of the scheme for substances for which no toxicity data are available to provide an NOEL with an adequate margin of safety then it is concluded that no data are required for such substances (which have passed earlier steps in the decision tree) if the intake is less than 1.5 μg/person/day. This final threshold was based on the US FDA threshold of regulation described above based on carcinogenic risk (less than one in a million lifetime risk of cancer), and also took account of further work by Munro, Kroes and co-workers (Munro et al, 1999 and Kroes et al, 2000) relating to noncancer endpoints - developmental toxicity, neurotoxicity and immunotoxicity see below. This decision tree scheme was developed by JECFA over the period Further development of TTC values related to cancer Cheeseman et al (1999) examined the original FDA threshold of 1.5 μg/person/day by analysis of an expanded database of chemicals (709 substances in the Gold Carcinogenic Potency database), and found similar distributions and potencies to the previous analysis, which was considered conservative due to linear extrapolation to low dose levels and the fact that the database is skewed by chemicals selected for testing (assumption of carcinogenic potential). By grouping chemicals by carcinogenic potency, Ames test data and structural alerts for genotoxicity (e.g. by Ashby and co-workers (Ashby & Tennant, 1991)) Cheesman and co-workers were able to identify potent and non-potent (for carcinogenicity) subsets. The work confirmed the validity of the 1.5 μg/person/day threshold (0.5 ppb in food) but allowed higher thresholds for some substances (in a tiered approach) to be proposed. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 13

14 The mean carcinogenic potency for substances which were negative in the Ames test (193 substances) was approximately 8-fold lower than the mean carcinogenic potency for 249 substances which were positive in the Ames test. Substances which were Ames negative and had an oral LD 50 of >1000 mg/kg bw were times less potent than the Ames positive compounds. Carcinogens with structural alerts for high potency (e.g. N-nitroso compounds, strained heteronuclear rings, heavy metals, hydrazines and azoxy compounds) were found to be of significantly higher potency and it was proposed that these classes of substance be excluded from a TTC scheme. Higher thresholds were proposed for substances which were Ames negative (4-5 ppb in food), and for substances which were both Ames negative and had an oral LD 50 of >1000 mg/kg bw (10-15 ppb in food). Cheeseman and co-workers also re-examined the premise that carcinogenic effects generally occur at lower doses than other toxic effects; compared genotoxic versus non-genotoxic compounds and carcinogenicity versus other end-points. Data from the RTECS database were analysed (3306 reproductive (multigeneration) studies and 2542 repeat-dose studies). A 1000-fold assessment factor was applied to the lowest LOEL for each compound to derive a pseudo-acceptable Daily Intake (PADI). For the reproductive studies 2% had a PADI which was below the 0.5 ppb dietary equivalent, and for the general toxicity endpoints in the repeat-dose studies <1% had a PADI below the 5 ppb dietary equivalent. This work supported the premise that a threshold based on carcinogenic effects would be protective for other toxic effects Further development of TTC for food chemicals Further work by Munro et al (1999) and Kroes et al (2000) expanded the previous analysis showing that other toxicity endpoints such as developmental toxicity, neurotoxicity and immunotoxicity showed considerably higher thresholds than the FDA human threshold value of 1.5 μg/person/day. Munro et al (1999) extended the 1996 analysis to address developmental toxicity using 100 substances reported in the RTEC database to cause developmental abnormalities. The 5 th centile NOEL from this group of substances was >3000 μg/kg bw/d which was higher than the 5 th centile NOEL from the earlier analysis of 611 substances which used the most sensitive endpoint for each substance (Munro et al, 1996). The human exposure threshold (calculated using the same methodology) calculated for developmental abnormalities was consequently higher (indicating that the thresholds of Munro et al, 1996 were appropriately protective). Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 14

15 Table 4 Cramer class 5 th centile NOEL μg/kg bw/d Human threshold μg/person/d # Class I Class II Class III Developmental abnormalities Neurotoxic compounds # Assumes 60 kg bodyweight and 100-fold safety factor See below Munro et al (1999) also calculated the 5 th centile NOEL for 31 neurotoxic organophosphate insecticides which had been included in the 1996 database. In the majority of cases the NOEL for these substances was based on cholinesterase inhibition, and these OPs tended to give the lowest NOELs found in the database. The 5 th centile NOEL for these neurotoxic substances was 30 μg/kg bw/day (which equates to a human exposure threshold of 18 μg/person/day). This threshold was 5-fold lower than the threshold derived for Cramer Class III compounds but 12-fold higher than the US FDA Threshold of Regulation value of 1.5 μg/person/day. The threshold being lower than the Class III threshold was not considered to be a problem for food flavouring substances because it was recognised that the neurotoxic substances were specifically designed to be highly potent toxins and such substances would not be used as flavouring substances. The key point from this analysis was that there was a case for applying different (lower) thresholds to neurotoxic substances. Munro et al (1999) also performed a limited analysis of the relative sensitivity of immunotoxicity compared with other endpoints. Based on an analysis of 24 immunotoxic substances the majority (17/24) had non-immunotoxic NOELs or LOELs which were more sensitive than the immunotoxic NOELs or LOELs. By contrast, for 2 substances the immunotoxic and non-immunotoxic endpoints were similar and for 5 substances the immunotoxic NOELs or LOELs were lower than the non-immunotoxic counterparts Expanded analyses (including non-cancer endpoints) Kroes et al (2000) expanded the analysis to address the issue of whether the thresholds proposed by Munro et al (1996) would be adequate to cover general toxic effects of chemicals in food (i.e. wider than food flavouring substances) and whether toxicological effects which might occur at low dose levels (e.g. effects on the nervous system, immune system, endocrine system, development) were adequately covered Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 15

16 by this TTC concept, since only a limited number of these endpoints had been available within the database analysed by Munro et al (1996). The numbers of chemicals examined in each analysis, the 10-6 risk dose for cancer (two different analyses of the Gold Carcinogenic Potency Database) and the ADIs equivalents (NOELs divided by 100) for non-cancer end-points at the 10 th & 50 th centiles are shown below. Although a small number of chemicals appear in more than one category, there is minimal overlap between the neurotoxicity, immunotoxicity, neurodevelopmental effects and developmental effects groups. Table 5 End-point Number 10 th centile ng/kg bw/d 50 th centile ng/kg bw/d Cancer (Rulis 1986) Cancer (Gold 1989) Developmental toxicity Neurotoxicity Neurodevelopmental toxicity Cramer class III Mixed end-points N.B. 1.5μg/person/day = 25ng/kg bw/d These data show that the distribution of 10-6 risk doses for carcinogens is significantly different from the distribution for the non-cancer NOELs/100. None of the non-cancer endpoints studied was more sensitive than cancer endpoints. Deriving a TTC from the 10-6 risk for carcinogens would therefore (for this database) provide a more conservative basis than the distribution of non-cancer NOELs divided by 100. Of the neurotoxic compounds, 6 of them had a 5 th centile NOEL below the threshold for Cramer class III compounds (0.15 mg/kg bw/day), indicating neurotoxicity should be considered separately from Cramer class III confirming Munro et al (1999). Appendices listing the chemicals in the developmental, neurotoxicity, developmental neurotoxicity and immunotoxicity groups are in the paper. The conclusions of Kroes et al, with the caveat that they applied only to the data bases analysed, were: Developmental and neurodevelopmental end-points were less sensitive than cancer / neurotoxicity endpoints but not statistically different from compounds in Cramer class III, (although some of the centile values are lower) Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 16

17 Neurotoxicity endpoints were more sensitive than other non-cancer endpoints but still within the 1.5μg/person/day threshold; Non-cancer endpoints were less sensitive than cancer endpoints (on a 10-6 risk basis) and that a 1.5μg/person/day threshold provided an adequate margin. Immunotoxicity was less sensitive than general end-points; Anthropogenic oestrogens were unlikely to produce effects at levels in the diet. This was based on comparing total exposures and comparing the potencies with endogenous oestrogenic hormones. The potencies were low, like phytooestrogens. There was not enough information to determine thresholds for allergenicity. However, by considering the steps involved in the induction of sensitisation, it was considered unlikely that small molecules present at typical dietary levels would induce sensitisation. (this does not consider already sensitised individuals). The overall conclusions were that the traditional threshold of 1.5 μg/person/ day provides adequate safety and that for compounds without an alert for genotoxicity or carcinogenicity a higher threshold might be appropriate Application of the TTC concept to wider food safety evaluation Kroes et al (2004) published proposals arising from an ILSI-Europe project (Expert Group and Workshop). These proposals built on existing work and expanded on it, seeking to provide increased safety assurance by the identification of structural alerts for high potency carcinogens and addressing questions around whether neurotoxicants or teratogens needed to be considered as separate classes. A decision tree (tiered approach) was proposed for a TTC concept to be used in food safety evaluation, excluding substances from the TTC approach if 1) the databases used to derive the TTC had not adequately covered such substances, and 2) if substances contain structural alerts found in the most potent genotoxic carcinogens which might still present a carcinogenic risk even at intakes below the proposed thresholds. The first step was to take the structural groups of concern for carcinogenicity identified by Cheeseman (1999) from the database of Gold and separate these into more homogeneous groups. This identified 5 groups of concern at exposures <0.15 μg/person/day (Aflatoxin like, N-nitroso, azoxy, steroids & dioxins/furans). For the remaining 730 compounds analysed the conclusion was that an exposure of <0.15 μg/person/day had an 86 97% probability of having a cancer risk of below 1 in A threshold of 0.15 μg/person/day was therefore proposed for substances with genotoxicity alerts (or positive data) on the basis that these could be genotoxic carcinogens but the most potent genotoxic carcinogens had been removed before this stage. The conservative assumptions (summarised in the paper) used to derive this value of 0.15 μg/person/day (including simple linearised low-dose extrapolation) led the authors to conclude that the probability of any additional lifetime cancer risk being greater than 1 in 10 6 would be minimal. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 17

18 The risk associated with substances (other than those excluded prior to this stage) at intakes of <0.15 μg/person/day was concluded to be negligible. The next step was to consider whether neurotoxicants or teratogens needed to be considered as separate classes. Looking at the most potent compounds in Cramer class III from Kroes et al, (2000) it was apparent that the majority were OPs. Based on Munro et al (1999) a 5 th centile NOEL/100 equated to a human threshold of 18 μg/person/day for OPs. A decision step for OPs applying this threshold was therefore introduced. For teratogenicity it was concluded that there was no need for an extra severity factor or a separate class as most NOELs were >3 mg/kg bw/d (i.e. >1800 μg/person/day based on a 60 kg bw for Cramer class I) those below 3 mg/kg bw/d were mainly from classes of concern e.g. dioxins which would be excluded at the initial Steps of the decision tree. The paper picks up on the need for reliable exposure data to be available. The default 1.5 kg food kg liquid consumed daily from the original FDA approach is conservative but could give a spurious outcome if for example the exposure was from only 100g per day (where much higher dietary inclusion levels would be needed to reach the same threshold). The issue of the default 60 kg bw is raised as possibly not applicable to all groups. These issues would need to be addressed in the development of a TTC concept for a particular area of food safety. The overall proposal from Kroes et al (2004) is for a tiered approach moving from high to low toxicity as follows:- Stage 1 - exclude non-essential / heavy metals, persistent compounds (e.g. dioxins, furans, PCBs) & potent genotoxins (N-nitroso-, azoxy- & aflatoxins). These are assessed case-by-case and require data. 2 Stage 2 - For compounds with genotoxicity alerts or positive data, compare exposure with threshold of 0.15 μg/person/day. Stage 3 - Compare all other compounds with threshold of 1.5 μg/person/day. If < threshold no need to consider further. [This requires reliable exposure data, and it might be easier to perform a full TTC assessment rather than generate exposure data that are reliable down to 1.5 μg/person/day. Stage 4 - If the compound is an OP compare with threshold of 18 μg/person/day. Stage 5 - Categorise as Cramer class I, II or III and compare exposure with the thresholds of 1800, 540 or 90 μg/person/day respectively. 2 Steroids are mentioned in the body of the text but not the final scheme 18 Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

19 Any exposures above the applicable thresholds require case-by-case consideration with data. The full decision tree is presented in the paper Application of the TTC concept to areas other than food safety A number of papers were identified in the literature search which relate to other uses of the TTC. i. Metabolites of pesticides found in groundwater The regulatory scheme for pesticide metabolites in groundwater incorporates a TTC approach. There is a tiered approach for determining whether pesticide metabolites found in groundwater are relevant and hence restricted to the maximum permissible concentration laid down by the Drinking Water Directive (Directive 98/83/EC). After excluding metabolites with pesticidal activity, genotoxic potential or classification for high toxicity, the next tier of the assessment incorporates a TTC step. As a pragmatic approach to limit the requirement for animal tests, it is accepted that a metabolite which is present at a level in drinking water such that the intake would be no higher than 0.02 μg/kg bw/day then no further testing is required and the risk assessment for the metabolite is considered acceptable. The intake threshold of 0.02 μg/kg bw/day is derived from the 1.5 μg/person/day threshold set by the US FDA for the Threshold of Regulation. This threshold was accepted for this purpose by the EU Commission Scientific Committee on Plants (Opinion adopted 30 November, 2000) provided the metabolite concerned had lower biological (pesticidal) activity than the pesticide active substance, was not genotoxic (as determined from a package of 3 in vitro tests) and was not considered to be classifiable as Toxic or Very Toxic. For the final category it could be necessary to perform some tests with the metabolite if the pesticide active substance was classifiable (including classification for carcinogenicity or reproductive toxicity). ii. Genotoxic impurities in pharmaceuticals The European Medicines Agency (EMEA) Committee for Medicinal Products for Human Use (CHMP) has a guideline recommending the application of TTC values for defining acceptable limits for genotoxic impurities present in drug substances (EMEA, 2004). The principles and values used are derived from (but not the same as) those proposed for food safety (as set out in Kroes et al, 2004). Potent genotoxins (e.g. N-nitroso compounds) are excluded from the TTC values and require specific data. The value of 0.15 μg/person/day for substances with structural alerts for genotoxicity corresponding to a 10-6 lifetime risk of cancer is referred to (as derived by Kroes et al, 2004), but given the favourable risk:benefit argument which applies to using pharmaceuticals it is accepted that a higher risk can be justified. Hence a ten-fold higher TTC value of 1.5 μg/person/day has been agreed for genotoxic impurities in pharmaceuticals (a lifetime risk of cancer of 10-5 ), but there is provision for even higher TTC values (and greater risk) for treatments for life-threatening or limited life expectancy conditions. Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 19