A procedure for the safety evaluation of flavouring substances

Food and Chemical Toxicology 37 (1999) 207±232

Review Section A Procedure for the Safety Evaluation of Flavouring Substances I. C. MUNRO1,*, E. KENNEPOHL1 and R. KROES2 CanTox Inc., 2233 Argentia Road, Suite 308, Mississauga, Ontario, L5N 2X7, Canada and 2 Prins Hendriklaan 63, 3721 AP Bilthoven, The Netherlands

1

(Accepted 15 July 1998) SummaryÐThis review describes a procedure for the safety evaluation of ¯avouring substances. Over 2500 ¯avouring substances are currently in use in food. While toxicity data do not exist on all ¯avouring substances currently in use, within structurally related groups of ¯avouring substances many do have toxicity data and this information along with knowledge of structure±activity relationships and data on the daily intake provides a framework for safety evaluation. The safety evaluation procedure provides a scienti®cally based practical method of integrating data on intake, structure±activity relationships, metabolism and toxicity to evaluate ¯avouring substances in a timely manner. The procedure has been used recently by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) to evaluate a total of 263 ¯avouring substances. # 1999 Published by Elsevier Science Ltd. All rights reserved Keywords: ¯avours; safety evaluation; toxicity; structure activity; JECFA; intake. Abbreviations: ADI = acceptable daily intake; CAS No. = Chemical Abstracts Service Registry Number; CE = Council of Europe; CPD = carcinogenic potency database; CR = consumption ratio; DART = developmental and reproductive toxicity; EPA = US Environmental Protection Agency; FEXPAN = Flavour and Extract Manufacturers' Association of the United States Expert Panel; FDA = US Food and Drug Administration; FEMA = Flavour and Extract Manufacturers' Association of the United States; GRAS = generally recognized as safe; IRIS = Integrated Risk Information System; JECFA = Joint FAO/WHO Expert Committee on Food Additives; LOEL = lowest-observed-e€ect level; MTD = maximum tolerated dose; NAS/NRC = National Academy of Sciences/National Research Council; NOEL = no-observed-e€ect level; NTP = National Toxicology Program; PAFA = Priority-based Assessment of Food Additives; QSAR = quantitative structure±activity relationship; RfD = reference dose; RTECS = Registry of Toxic E€ects of Chemical Substances; SCF = Scienti®c Committee for Food; TLV = threshold limit value; WHO = World Health Organization; 2-AAF = 2-acetyl amino¯uorene.

INDEX

Introduction Conceptual framework for the safety evaluation of ¯avouring substances Estimating intake of ¯avouring substances through food Consideration of natural occurrence of ¯avouring substances in food Structure±activity relationships Use of toxicity data Elements of the safety evaluation procedure Structure±activity relationships and metabolicfate

*Corresponding author.

208 209 209 210 210 211 211 212

Integrating information on intake and toxicity Establishment of threshold values based on non-carcinogenic endpoints Establishment of a threshold value based on carcinogenic endpoints Comparison of sensitivity of various endpoints Application of a threshold of toxicological concern to ¯avouring substances Additional factors that reduce the theoretical risk Safety decision criteria Integrating data on consumption ratio Discussion References

212 214 216 219 220 221 222 225 225 225

0278-6915/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00112-4

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Tables Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9

Number of ¯avouring substances within various intake categories Safety margins between NOELs and per capita daily exposure for various ¯avouring substances given full ADIs by JECFA Number of ¯avouring substances divided by structural class within various intake categories Fifth centile NOELs and human exposure thresholds for Cramer et al. (1978) structural classes in the reference database Low-dose slopes for bladder tumours in mice exposed to 2-AAF for 24 months based on linear extrapolation from the TD50 Probability of a target risk not being exceeded at various threshold values Comparison of various human exposure threshold values A list of functional groups identi®ed by Ashby and Tennant (1988, 1991) and Tennant et al. (1990) as structural alerts for DNA reactivity Flavouring substances within each Cramer et al. (1978) structural class consumed in amounts below human exposure thresholds

210 211

Empirical cumulative distributions of NOELs of compounds in the reference database and lognormally ®tted cumulative distributions (solid lines). Compounds have been grouped into the structural classes I, II, and III of Cramer et al. (1978) Dose±response curve for bladder tumours in mice exposed to 2-AAF for 24 months Distribution of TD50s for 343 rodent carcinogens from the Gold et al. (1984) CPD and distribution of 1  10ÿ6 risks calculated by linear extrapolation from the TD50s (modi®ed from Rulis, 1989) Comparison of immune and non-immune endpoint NOELs and LOELs (based on NOELs and LOELs of 24 immunotoxic substances) Safety evaluation sequence

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213 216 217 219 219 222 222

Figures Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

217 218 220 224

Appendix Table Table Table Table

A1 A2 A3 A4

Substances reported to cause developmental abnormalities (from RTECS) NOELs for organophosphorous insecticides Substances with immunotoxic NOELs Substances with immunotoxic LOELs

Introduction The . safety evaluation procedure described herein was developed for use by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for the safety evaluation of ¯avouring substances. The procedure extends principles and procedures recommended by JECFA in the past and is consistent with international concepts in safety evaluation. These principles have been discussed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 1972, 1974b, 1976a, 1978, 1980a, 1974b, 1982a, 1983a, 1984a, 1986, 1987a, 1974b, 1989, 1990a, 1991a, 1992) and have been recapitulated in a report, which outlines criteria for the safety evaluation of ¯avouring substances, published by the World Health Organization (WHO, 1987). There are approximately 2500 chemically-de®ned ¯avouring substances in use either in Europe or the United States. Of these substances, approximately 1500 have been evaluated by FEMA's Expert Panel and are legally recognized by the US Food and Drug Administration (FDA) to be Generally

230 232 232 232

Recognized As Safe (GRAS) substances, meaning that they are considered safe for their intended use. Without exception, ¯avouring substances are volatile organic chemicals. The majority have simple, well characterized structures with a single functional group and low molecular weight (<300 g/ mol). More than 700 of the 1323 chemically de®ned ¯avouring substances used in food in the US are simple aliphatic acyclic and alicyclic alcohols, aldehydes, ketones, carboxylic acids and related esters, lactones, ketals and acetals. Other structural categories include aromatic (e.g. cinnamaldehydes and anthranilates), heteroaromatic (e.g. pyrazines and pyrroles) and heterocyclic (e.g. furanones and alicyclic sul®des) substances with characteristic organoleptic properties. For most ¯avouring substances, the structural di€erences within chemical groups are small. Incremental changes in carbon chain length and the position of a functional group or hydrocarbon chain typically describe the structural variation within groups of related ¯avouring substances. These systematic changes in structure provide the basis for understanding the e€ect of

Safety evaluation procedure

structure on their chemical and biological properties. Within structural groups of ¯avouring substances, many substances have considerable toxicology data; repeat dose studies exist for many substances or their metabolic products, and several representative members of structural groups have chronic toxicity studies. At the 46th and 49th meetings of JECFA, the Committee (JECFA, 1997, 1998) was able to use information on metabolism, toxicity, and intake of individual substances within a group to evaluate 263 ¯avouring substances. Conceptual framework for the safety evaluation of ¯avouring substances Criteria for the safety evaluation of ¯avouring substances have been put forward by several national and international authorities. Included among these are JECFA, a special Task Group convened by WHO (1987), the Committee of Experts on Flavouring Substances of the Council of Europe (CE), the Commission of the European Communities' Scienti®c Committee for Food (SCF, 1991), BIBRA International, and the Flavor and Extract Manufacturers' Association of the United States Expert Panel (FEXPAN). In 1987, WHO published a health criteria document entitled Principles of the Safety Evaluation of Food Additives and Contaminants in Food, which contains a discussion of principles related to the safety evaluation of ¯avouring substances. This report recapitulated principles previously stated by JECFA on numerous occasions (WHO, 1987). Early on, it was recognized by JECFA that the safety evaluation of ¯avouring substances warranted special consideration in the light of use patterns and typically low levels of human intake (JECFA, 1972). This view also has been recognized by the Scienti®c Committee for Food (SCF) and has been recorded in their document entitled Guidelines for the Evaluation of Flavourings for Use in Foodstu€s: 1. Chemically De®ned Flavouring Substances (SCF, 1991). In addition, several organizations including JECFA (WHO, 1987), FEXPAN (Woods and Doull, 1991), SCF (1991) and the Council of Europe (CE, 1974, 1981, 1992) have noted that knowledge of structure±activity relationships and metabolism plays a key role, along with intake, in the safety evaluation of ¯avouring substances. In this regard, JECFA (WHO, 1987; JECFA, 1996a,b, 1997, 1998) has used structure±activity relationships in evaluating groups of structurally related ¯avouring substances in a homologous series where toxicology studies exist on only one or a few members of the series. Structure±activity relationships can provide a useful means of evaluating substances that lack toxicity data by

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using toxicity data from structurally related substances. Estimating intake of ¯avouring substances through food Flavouring substances are used in processed foods and beverages to impart desirable organoleptic qualities and to provide the speci®c ¯avour pro®le traditionally associated with certain food products. Unlike many substances which are added to food to achieve a technological purpose, the use of ¯avouring substances is generally self-limiting and governed by the ¯avour intensity required to provide the necessary organoleptic appeal. Thus, ¯avouring substances are used generally in low concentrations resulting in human intakes that are very low. Estimates of ¯avouring substance intake have been performed in two ways. One method of calculating intake is to combine data on the level of use in speci®c food groups with data on the amount of food consumed to calculate the intake of each ¯avouring substance. Estimating intake of ¯avouring substances based on level of use data, coupled with data on the amount of food consumed, typically leads to substantial overestimates of intake. This is because intake data for food commodities are strati®ed into broad categories of food products (e.g. baked products). Thus, cardamom, ordinarily used only in certain types of co€ee cakes, would be assumed to be present in all breads, rolls, cakes and pastries. Moreover, within a category of food products (e.g. hard candy) it is the ¯avouring substance that characterizes speci®c brands of products. Thus assuming that a ¯avouring substance occurs in all brands within a food category will lead to further overestimates of intake. These assumptions can lead to estimates of intake which may be exaggerated several hundred-fold. A second method is to assume that the total amount (poundage) reported to be used in food annually is completely consumed by the total population. Between 1970 and 1987, the US National Academy of Sciences/National Research Council (NAS/NRC) conducted, under contract to the FDA, a series of poundage surveys of substances intentionally added to food (NAS, 1978, 1979, 1984, 1989). These surveys obtained information, both from ingredient manufacturers and from food processors, on the poundage of each substance committed to the food supply and on the usual and maximum levels at which each substance was added to foods in each of a number of food categories. Numerous checks using data from independent sources, such as imports, show that, in general, the reported poundage in surveys accounts for only 60% of the total used. Therefore, calculations of intake are corrected upwards to account for underreporting. In addition, more detailed analyses (see Annex 5, etc., JECFA, 1996a) have led to the con-

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clusion that it was conservative but reasonable to assume that each ¯avouring substance is consumed by only 10% of the population. Both methods tend to overestimate human intakes of ¯avouring substances because they deal with disappearance, that is the amount presumed to be used in food, and take no account of losses and waste during food manufacture, storage, preparation and consumption. Through a series of detailed studies conducted between 1970 and 1987 (see JECFA, 1996a) it has become clear that, while there is at present no perfect way to estimate intake of ¯avouring substances, poundage data provide a reasonable basis for calculating intakes. The intake data reported in this paper rely on annual poundage used in food and the estimates of intake re¯ect the assumptions that: (i) the available survey data accounted for only 60% of the amount actually used in food; and (ii) the total amount used is consumed by only 10% of the population. Table 1 presents the intake data for 1323 chemically de®ned ¯avouring substances permitted for use in the US calculated in this fashion. As can be seen from Table 1, most ¯avouring substances are consumed in amounts of less than 1 mg/ person/day. The data are taken from the most recent US NAS/NRC survey (NAS, 1989) of poundage used in food. For reasons previously stated, it can be assumed that these intake estimates are overestimated. Consideration of natural occurrence of ¯avouring substances in food Another important factor to consider in the evaluation of human intake of ¯avouring substances is the extent to which ¯avouring substances intentionally added to foods also occur naturally in the food supply. The natural presence of ¯avouring substances in food is of course not necessarily inTable 1. Number of ¯avouring substances* within various intake categories Intake category$ (mg/day) <0.01 0.01±0.1 0.1±1 1±10 10±100 100±1000 1000±10,000 10,000±100,000 100,000+ TOTAL

No. of ¯avours

Cumulative frequency (% of total)

349 93 274 224 204 111 45 16 7 1323

26 33 54 71 86 95 98 99 100

*Chemically de®ned ¯avouring substances permitted for use in the US excluding botanicals. $Intake data calculated assuming: survey poundage re¯ects 60% of actual usage, 10% of population exposed, US population in 1987 was 240 million. Formula: intake (mg/person/day) = [(annual ¯avour usage in mg) 60.6] 6(24  106 persons  365 days). Poundage data from 1987 NAS/NRC survey data (NAS, 1989).

dicative of safety. For many ¯avouring substances that occur naturally in foods, such natural occurrence, rather than intentionally added use, is the principal source of human exposure. The comparison of natural occurrence to intentional addition has been expressed as the consumption ratio (CR) (Stofberg and Kirschman, 1985). A CR of greater than 1 indicates a predominant natural occurrence in food (i.e. the ¯avouring substance is consumed at a higher level from foods than as an added substance). A CR greater than 10 indicates an almost insigni®cant contribution (approx. 10%) of the ¯avouring substance as a food additive to the total intake (Stofberg and Grundschober, 1987). Stofberg and Grundschober (1987) calculated that out of 499 ¯avouring substances, 415 (83%) had a predominant natural occurrence in food (i.e. CR>1) and 309 (62%) made an insigni®cant contribution when added to the food supply (i.e. CR>10). As can be seen from this analysis, the use of natural occurrence as part of the safety evaluation provides an important perspective on the impact of intentional addition of ¯avouring substances to foods. If a ¯avouring substance is one of the few that for any reason, including high intake, is of relatively high safety concern, then a high consumption ratio probably enhances that concern because it indicates a much larger and uncontrolled exposure from natural occurrence than from intentional addition. If, on the other hand, a ¯avouring substance is one of the majority that have few, if any, safety issues and consequently low inherent concern, then a high consumption ratio reduces any concern still further because it indicates that intentionally added use is trivial. Stated in another way, if the added use of a ¯avouring substance amounts to less than 10% of its natural occurrence, this would indicate a minimal safety concern about added use. If added use is less than 1% of natural use (i.e. CR>100) then the added use will at most be of trivial safety concern. Structure±activity relationships Toxicity is dependent on the chemical structure of a substance, its pharmacokinetics, and its metabolic reaction pathways. Available metabolic pathways are usually dose dependent and, to a large extent, govern the magnitude of the toxic e€ect. Therefore, chemical structure, pharmacokinetics, metabolic fate and dose are key determinants of toxicity and play a critical role in safety evaluation of ¯avouring ingredients. Re®nements to initial concepts of structure±activity came as a result of increasing knowledge and con®dence in predicting structure±activity relationships for ¯avouring substances. These formed the basis of a paper by Cramer et al. (1978) which, through the use of a ``decision tree'' approach, permitted the classi®cation of ¯avouring substances

Safety evaluation procedure

into ``classes of concern'' based on structure and other considerations, similar in many respects to, but predating the ``Concern Level'' concept outlined by the US FDA in its ``Redbook'' (FDA, 1982, 1993). The concept of establishing concern levels also has been investigated further by BIBRA International to evaluate food chemicals more generally (Phillips et al., 1987). This group established concern levels for several food additives, plastic monomers, as well as ¯avouring substances. Although they reported that, in their opinion, the Cramer et al. (1978) decision tree misclassi®ed a few substances, the decision tree was likely to be a more realistic approach for predicting toxicity than any other reported quantitative structure±activity relationship (QSAR) technique. Thus, there is general consensus, based on the work of Cramer et al. (1978), the subsequent work by BIBRA (Phillips et al., 1987), and the fact that the FDA (1982; 1993) uses structure±activity relationships in de®ning concern levels for food substances, that structure±activity has a solid basis in science when applied to substances of simple and closely related structure, especially those with low intakes, low toxicity and safe metabolic products. This is the case for all but a very few ¯avouring substances. As will be discussed later in this review, structure±activity relationships play an important role in the evaluation of ¯avouring substances. Use of toxicity data Traditional approaches to the safety assessment of food additives typically involve the evaluation of considerable toxicological data, usually in an amount sucient to establish a no-observed-e€ect level (NOEL), permitting the establishment of an acceptable daily intake (ADI). Approximately half of the ¯avouring substances currently in use are naturally occurring simple acids, aldehydes, alcohols and esters. With few exceptions, these are rapidly metabolized to innocuous end-products, the safety of which is well established or can be assumed from metabolic and toxicity data on the substance in question or on structurally related substances. In other words, the acquisition of extensive toxicity data is unnecessary for the majority of ¯avouring substances because structure±activity relationships can be used as a means of assessing substances in a homologous series, in which only a few substances have toxicology data, to determine safety in use. This concept has been used by JECFA in the evaluation of structurally related ¯avouring substances, including the allyl esters, amyl acetate and isoamyl butyrate, benzyl compounds, citral compounds, a- and b-ionones, and nonanal and octanal (JECFA, 1967, 1968, 1980a, 1984a,b, 1990a,b, 1991a,b, 1993a,b). In addition, as previously indicated in Table 1, 95% of ¯avouring substances are consumed at intake levels less than

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1 mg/person/day and in keeping with the safety evaluation procedure outlined in this paper, only limited toxicological data are required or justi®ed in such circumstances. For the reasons stated above, traditional safety evaluation procedures are not necessarily applicable to ¯avouring substances. When intake is extremely low and there are organoleptic limitations on use levels, a primary consideration is whether there is a need to establish a numerical ADI for ¯avouring substances. There are several reasons why it is not appropriate or necessary to establish ADIs for the majority of ¯avouring substances. ADIs are based on toxicological data and the establishment of a NOEL, an approach that di€ers from the concept that a safety evaluation can be performed in many instances on the basis of intake and structure±activity relationships. Moreover, the organoleptic and gustatory properties of ¯avouring substances typically limit their use in speci®c food products and, consequently, intake. In addition, because a majority of ¯avouring substances occur in nature, there is a long history of human experience in ¯avouring substance consumption from traditional foods. Nearly 50% of ¯avouring substances given full ADIs by JECFA have consumption ratios greater than 1, indicating their predominantly natural occurrence in food (Stofberg and Grundschober, 1987). The above factors have been noted by WHO (1987) as important in the evaluation of ¯avouring substances. It is also evident that very large safety margins exist for ¯avouring substances, as evidenced by the fact that the margin between the NOEL and the intake of ¯avouring substances reaches up to more than 100,000 times for the ¯avouring substances given full ADIs by JECFA (Table 2). Elements of the safety evaluation procedure In a continuing e€ort to improve the basis for the safety evaluation of ¯avouring substances, this review presents a procedure which integrates information on intake, structure±activity relationships, metabolic fate and toxicity. It presents a safety evaluation procedure which allows a determination of the safety of ¯avouring substances under conTable 2. Safety margins between NOELs and per capita daily exposure for various ¯avouring substances given full ADIs by JECFA* Safety margin

Number of ¯avours

<100 100±1000 1000±10,000 10,000±100,000 100,000+ Total

1 4 13 9 7 34

*JECFA, 1967, 1968, 1970, 1971, 1972, 1974a,b, 1976a,b, 1978, 1980a,b,c, 1981a,b, 1982a,b, 1983a,b, 1984a,b, 1986, 1987a,b, 1989, 1990a,b, 1991a,b, 1992, 1993a,b,c.

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ditions of intended use. The general principles upon which the safety evaluation procedure is based have been elaborated previously (Munro et al., 1998). The key elements of the safety evaluation procedure are discussed below. Structure-activity relationships and metabolic fate Toxicity is dependent on the chemical structure and metabolism of a substance. The ``decision tree'' procedure (Cramer et al., 1978) relies primarily on chemical structure and estimates of total human intake to assess toxic hazard and to establish priorities for appropriate testing. The procedure utilizes recognized pathways of metabolic deactivation and activation, data on toxicity, and the presence of the substance as a component of traditional foods and as an endogenous metabolite. Substances are classi®ed according to three categories: Class I.

Class II.

Class III.

Substances of simple chemical structure with known metabolic pathways and innocuous end-products which would suggest a low order of oral toxicity (e.g. butyl alcohol or isoamyl butyrate). Contains structures that are intermediate. They possess structures that are less innocuous than substances in Class I, but do not contain structural features suggestive of toxicity like those substances in Class III. Members of Class II may contain reactive functional groups (e.g. furfuryl alcohol, methyl 2octynoate, and allyl propionate). Substances of a chemical structure that permit no strong initial presumption of safety, or may even suggest signi®cant toxicity (e.g. 2-phenyl-3-carbethoxy furan and benzoin).

The decision tree is a tool for classifying ¯avour substances according to levels of concern. The majority of ¯avouring substances fall into Class I because they are simple alcohols, aldehydes, ketones, acids or their corresponding esters, acetals and ketals that occur naturally in food and, in many cases, are endogenous substances. They are rapidly metabolized to innocuous products (e.g. carbon dioxide, hippuric acid, and acetic acid) by well recognized reactions catalysed by enzymes that exhibit high speci®city (e.g. alcohol dehydrogenase and isovaleryl coenzyme A dehydrogenase). Substances that do not undergo detoxication via these highly ecient pathways (e.g. fatty acid pathway and citric acid cycle) are metabolized by reactions catalysed by enzymes of low speci®city (e.g. cytochrome P-450 and glutathione transferase). This class of enzymes is saturated at lower intercellular concentrations than are higher capacity enzymes. For some groups of substances (e.g.

branched-chain carboxylic acids, allyl esters and linear aliphatic acyclic ketones), metabolic thresholds for intoxication have been identi®ed (Deisinger et al., 1994; Jaeschke et al., 1987; Krasavage et al., 1980). The dose range, over which a well de®ned change in metabolic pathway occurs, generally correlates with the dose range over which a transition occurs from a no-observed-adverse-e€ect level to an adverse-e€ect level. For such groups of substances, the dose range at which this transition occurs is orders of magnitude greater than the level of intake from use as ¯avour substances. Most substances in Class II belong to either of two categories; one includes substances with functional groups which are similar to, but somewhat more reactive than functional groups in Class I (e.g. allyl and alkyne); the other includes substances with more complex structures than substances in Class I, but that are common components of food. This category includes heterocyclic substances (e.g. 4methylthiazole) and terpene ketones (e.g. carvone). The majority of the ¯avouring substances within Class III include heterocyclic and heteroaromatic substances and cyclic ethers. Many of the heterocyclic and heteroaromatic substances have sidechains with reactive functional groups. In a few cases, metabolism may destroy the heteroaromaticity of the ring system (e.g. furan). Although metabolism studies have been performed for Class III ¯avouring substances with elevated levels of intake, the metabolic fate of many substances in this structural class cannot be predicted con®dently. Importantly, however, review of the group of substances in each of the structural classes indicates that as structural complexity increases (Class I±III), the number of ¯avouring substances and the levels of intake decrease signi®cantly (Table 3).

Integrating information on intake and toxicity One of the key elements of the safety evaluation procedure is based on the premise that intake levels can be speci®ed for ¯avouring substances that would not present a safety concern. The concept of specifying human exposure thresholds relies on principles that permit specifying the daily intake of a substance which can be considered, for practical purposes, as presenting no toxicological risks (and thus of no health or safety risk to consumers) even in the absence of speci®c toxicological data on the substance (Federal Register, 1993; Frawley, 1967; Munro, 1990; Rulis, 1986). The concept relies on knowledge of the range of toxicological risks for structurally related substances and on knowledge regarding the toxicological potency of relevant classes of chemicals for which good toxicity data exist. With the possible exception of so-called genotoxic carcinogens, the concept of a threshold in toxicological responses is universally accepted and

Safety evaluation procedure

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Table 3. Number of ¯avouring substances* divided by structural class within various intake categories Intake category$ (mg/day) <0.01 0.01±0.1 0.1±1 1±10 10±100 100±1000 1000±10,000 10,000±100,000 100,000+ Total

No. of ¯avours (% of total) Class I 212 (24) 55 (6) 169 (19) 145 (17) 147 (17) 95 (11) 34 (4) 16 (2) 5 (0.6) 878

Class II

Class III

68 20 48 45 39 12 9 0 2 243

69 (34) 18 (9) 57 (28) 34 (17) 18 (9) 4 (2) 2 (1) 0 0 202

(28) (8) (20) (19) (16) (5) (4) (0.8)

*Chemically de®ned ¯avouring substances permitted for use in the US excluding botanicals. $Intake data calculated assuming: survey poundage re¯ects 60% of actual usage, 10% of population exposed, US population in 1987 was 240 million. Formula: intake (mg/person/day) = [(annual ¯avour usage in mg) 6 0.6]) 6 24  106 persons  365 days). Poundage data from 1987 NAS/NRC survey data (NAS, 1989).

endorsed by WHO (1987, 1994). The principles underpinning the establishment of human exposure thresholds have been embodied in a Federal Register (1993)* notice emanating from the US FDA, which provides the scienti®c basis for the conclusion that an intake level for indirect food additives can be speci®ed, below which no risk to public health would be likely to accrue. This intake level has, in turn, been used by FDA to establish a proposed ``threshold of regulation'' for indirect food additives which precludes the need for toxicological evaluation of substances migrating into food from food-contact articles provided the amount that migrates does not lead to a dietary level in excess of 500 ppt (equivalent to 1.5 mg/person/day assuming a daily food intake of 3000 g). The FDA has noted that such a level would result in negligible risk to consumers even if the substance was shown later to be a carcinogen. This concept is in keeping with the well established principle that resources should be directed to the safety evaluation of substances having high intake and therefore greater potential for adverse e€ects and not towards substances with trivial intake. The concept is particularly applicable to substances of low toxicity and with known or predictable metabolic fate. The scienti®c basis for the establishment of human exposure thresholds and the FDA regulation are discussed below. The concept that a generic threshold value or range of values might be established that would preclude the need for toxicity data on chemicals *In 1993, the US FDA proposed a dietary concentration of 500 ppt as the threshold of regulation for substances used in food-contact articles. Assuming that an individual consumes 1500 g of solid food and 1500 g of liquid food per day, this threshold would equate to a toxicologically inconsequential level of 1.5 mg/day (Federal Register, 1993). This proposal became a ®nal rule in 1995 (Federal Register, 1995).

having human intakes below these thresholds was proposed over 30 years ago by Frawley (1967). He showed, on the basis of studies conducted on several well-tested substances, including food additives, industrial and consumer chemicals, and pesticides that a generic ``no-e€ect'' level could be established that could preclude the need for toxicity studies and safety evaluation for a majority of substances intended for use as food packaging materials. Frawley constructed a reference database of nontumorigenic endpoints using 220 2-year rodent studies. He presented the NOELs for all 220 compounds. Frawley (1967) reported that if he excluded heavy metals and pesticides from the analysis, there was no compound in the remaining database (except for acrylamide) which showed evidence of chronic toxicity at dietary concentrations of less than 100 ppm. Application of a typical 100-fold safety factor to the 100 ppm generalized NOEL would mean that humans could safely consume any of the materials provided the dietary concentration did not exceed 1 ppm. Frawley (1967), noting that his database was incomplete, proposed adding an additional safety factor of 10 which would translate to a toxicologically insigni®cant human exposure level of 0.1 ppm in the diet. Assuming an individual consumes 1500 grams of food per day, an exposure of 150 mg/person/day (approximately 2.5 mg/kg body weight/day) or less to a chemical of unknown toxicity would be considered toxicologically insigni®cant. According to Frawley (1967), such exposures could be considered of no safety concern. More recently, Rulis (1986) conducted a similar analysis of the FDA's Priority-Based Assessment of Food Additives (PAFA) database containing 159 compounds with subchronic or chronic toxicity data and came to the same conclusion as Frawley (1967). Essentially, there is no risk of toxicity in rodents exposed to certain food additives at dietary levels of less than 1 mg/kg body weight/day, or in human terms, approximately 1 to 10 mg/kg body

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weight/day depending on the safety factor applied. Even 20 years apart, using di€erent databases, the toxicologically inconsequential levels proposed by Frawley (1967) and Rulis (1986) were nearly identical. Munro (1990) used a database of approximately 350 substances compiled by Gold et al. (1984, 1989) to develop a human exposure threshold value to be applied to substances for which no presumption of safety can be made because of a complete lack of data on metabolism and potential toxicity. Munro (1990) proposed a threshold of regulation of up to 1000 ppt for indirect additives which would translate to a daily intake of 1.5 to 3.0 mg/person/day depending on assumptions regarding food intake. The acceptable level considered by FDA in its ®nal rule (Federal Register, 1995) to present no regulatory concern for an indirect food additive from food packaging material, even if later it was determined to be a carcinogen equates to a daily intake of 1.5 mg/person/day (Federal Register, 1993). The approach of using a threshold of concern provides an alternative to the conventional regulatory philosophy of rigorously testing each new chemical substance regardless of expense or level of human intake. Two factors, pragmatism and scienti®c knowledge, have in¯uenced the evolution of the threshold concept. The scienti®c information base is now suciently large to consider application of a threshold of toxicological concern as a concept that is both practical and scienti®cally defensible. On a purely pragmatic level, it is recognized that humans are exposed to thousands of substances through the food supply and the number of substances increases logarithmically with declining concentration (Hall, 1975). It is neither practical nor scienti®cally defensible to test all these substances by conventional toxicological procedures, and to insist this be done would create a resource problem of immense proportions. Another more important factor that justi®es an approach using a threshold of toxicological concern, is that in the past 10 to 15 years a great deal of knowledge has accumulated about the potential human risks from chemicals in general and especially for those which are carcinogenic. On the basis of accumulated knowledge, it is theoretically possible to establish a range of threshold values representing the full spectrum of toxicological endpoints including both carcinogenic and non-carcinogenic e€ects. Establishment of threshold values based on non-carcinogenic endpoints The work conducted by the FDA (Federal Register, 1993), Frawley (1967), Rulis (1986) and Munro (1990) was expanded upon by Munro et al. (1996) through the compilation of a large database of reference substances from which a distribution of NOELs could be derived for chemicals of various structural types. The reference database describes

the relationships between intake, structure and toxicity for a wide variety of chemicals of divergent structure and it can be used as a reference point from which to judge the safety of ¯avouring substances. In compiling the database, strict criteria were applied in the selection of data sets. The objective of the exercise was to identify as many high quality toxicological studies as possible representing a variety of toxic endpoints and chemical structures. To accomplish this, the study types included those typically conducted in toxicology, such as subchronic, chronic, reproductive and teratology studies. Shortterm and acute studies were not included since these were considered not to be relevant for establishing chronic NOELs. The database consisted mainly of studies in rodents and rabbits. Very few studies in dogs and other species were found that met the established criteria. An evaluation of randomly selected dog and primate studies indicated that many had too few animals per group to derive a statistically valid NOEL. Moreover, for many dog studies, a common endpoint was reduced body weight and/or food consumption which was due, in many cases, either to palatability problems with the diet, or vomiting. In addition, most studies in dogs and other non-rodent species were simply too short in duration to be classi®ed as chronic studies. Only oral studies were included in the database. A further criterion for inclusion in the database was that a study had to have a demonstrated lowestobserved-e€ect level (LOEL) as well as a NOEL, thus ensuring that the study was rigorous enough to detect toxic e€ects. In some instances NOELs were included for studies not demonstrating a LOEL, and these were substances such as major food ingredients that were without toxicity at the highest dose tested in well conducted studies. It should be noted that the inclusion of such substances in the database would not bias the database in favour of higher NOELs since the true NOEL for such substances probably would exceed the NOEL established from the available studies. In order to combine NOELs for substances with only subchronic studies with those with chronic studies to derive the cumulative distribution of NOELs, subchronic NOELs were divided by a factor of 3 to approximate the most likely NOEL that would be derived from a chronic study. This conversion factor is based on research de®ning the relationship between subchronic and chronic NOELs. Weil and McCollister (1963) compared 3-month NOELs with 2-year NOELs for 33 di€erent substances (including pharmaceuticals, pesticides and food additives) fed to rats. They found that for most of the compounds (30), the ratio of the NOELs between subchronic and chronic studies was 5 or less and more than half of the compounds had a ratio equal to 2 or less. More recently, it has been discovered through further analysis of more

Safety evaluation procedure

chemical substances, that a more accurate adjustment factor for extrapolating NOELs derived from subchronic studies to lifetime was between 2 and 3 (Beck et al., 1993; Lewis et al., 1990; Dourson, personal communication). Emphasis was placed on retrieving data from certain databases known to contain well validated toxicological endpoints for a series of well-de®ned chemical structures. An exhaustive search was made of compounds evaluated by JECFA. Other sources included the US Environmental Protection Agency's (EPA) Integrated Risk Information System (IRIS) on-line database, the National Toxicology Program (NTP) studies, the Developmental and Reproductive Toxicity (DART) on-line database from EPA and the US National Institute of Environmental Health Sciences and the published literature in general. The data entered into the database included the name of the chemical, Chemical Abstracts Service Registry Number (CAS No.), structural classi®cation as assessed using the Cramer et al. (1978) decision tree and the FDA ``Redbook'', species, sex, route of administration, dose levels tested, study type, duration, endpoints reported, LOEL, NOEL and references. In an e€ort to be conservative in the construction of the reference database, NOELs selected by the author(s) of each study were used even though in some cases authors tended to over-interpret their data. In some instances, it was found that the stated NOEL may have been based on a misjudgment of an adverse e€ect by the author (e.g. physiological versus toxicological e€ects) or on artefactual e€ects (e.g. foetal toxicity as a result of maternal toxicity).

215

An example of this is isopropyl alcohol, which has been reported to produce teratogenic e€ects at very low doses (0.018 mg/kg) in one study; however, its structure, known metabolism and other toxicological data provide no evidence for concluding teratogenicity. Even though some of these author-derived NOELs were not thoroughly substantiated, they were included in the reference database, thereby increasing the degree of its conservative nature. NOELs selected by EPA for the IRIS database were entered without further review. In all, the database consists of over 600 substances representing a range of industrial chemicals, pharmaceuticals, food substances and environmental and consumer chemicals likely to be encountered in commerce. As the database was developed as a reference database for the evaluation of ¯avouring substances, all of which are organic chemicals, no organometallic or inorganic compounds were included in the database. For many of the substances, more than one NOEL was identi®ed from the literature resulting from the fact that some substances were tested in more than one species and sex and/or demonstrated a range of endpoints suitable for establishing a NOEL. This led, in some cases, to multiple NOELs for individual substances. In all, the database contains nearly 3000 entries. For each of the substances in the database, classi®ed corresponding to the three structural classes outlined in Cramer et al. (1978), the most conservative NOEL was selected from the reference database based on the most sensitive species, sex and endpoint. The cumulative distribution of the NOELs within each class is shown in Fig. 1, along with the

Fig. 1. Empirical cumulative distributions of NOELS of compounds in the reference database and lognormally ®tted cumulative distributions (solid lines). Compounds have been grouped into the structural classes I, II and III of Cramer et al. (1978).

216

I. C. Munro et al.

lognormal distributions ®tted to these data. These results clearly delineate the e€ects of structural class on toxicity, with the median (50th centile) NOEL decreasing from Class I through III. Similar di€erences among structural classes exist in the range between the 5th and 95th centiles. The human exposure threshold for each of the structural classes was calculated from the 5th centile NOEL. The 5th centile NOEL was chosen because this value would provide 95% con®dence that any other substance of unknown toxicity but of the same structural class as those comprising the reference database would not have a NOEL less than the 5th centile for that particular structural class within the reference database. The 5th centile NOELs for each structural class are shown in Table 4. In converting the 5th centile NOELs to human exposure thresholds (Table 4) for the various structural classes, a 100-fold safety factor was used since such a factor would inherently be applied in establishing safe intake levels for the substances comprising the database. The use of such a factor provides a substantive margin of safety since the human exposure thresholds are based on a large database of over 600 compounds with good supporting toxicological data. Furthermore, 5th centile NOELs were used to calculate the thresholds, providing a more conservative ®gure than the arithmetic mean. Moreover, the estimated daily intakes of ¯avouring substances to which the human exposure threshold are compared are greatly overestimated as they represent the ``eaters only'' (10%) population. Thus, it is believed that a 100-fold safety factor provides a wide margin of safety in relating the results of the analysis of the reference database to ¯avouring substance intake. It is evident from Table 4 and Fig. 1 that there are substantial di€erences in the 5th centile NOELs for the various structural classes, indicating an obvious e€ect of structure on toxic potency. Establishment of a threshold value based on carcinogenic endpoints Over the past several years, an immense amount of information has accumulated on the range of carcinogenic potencies for chemicals that have been Table 4. Fifth centile NOELs and human exposure thresholds for Cramer et al. (1978) structural classes in the reference database

I II III

137 28 447

5th Centile NOELs (mg/kg/day)

Human exposure threshold (mg/day)*$

2993 906 147

1800 540 90

*The human exposure threshold was calculated by multiplying the 5th centile NOEL by 60 (assuming an individual weighs 60 kg) and dividing by a safety factor of 100, as discussed in the text. $Numbers rounded to two (2) signi®cant ®gures.

tested in animals. For these chemicals, the distribution of potencies in experimental animals and projected human risk (calculated using linear risk assessment models) are well established and highly unlikely to be altered by further cancer bioassays (Krewski et al., 1990). In fact, the Carcinogenic Potency Database (CPD) compiled by Gold et al. (1984), now contains nearly 500 substances reported to be carcinogenic in animals. It is reasonable to assume that the addition to that database of several more ``genotoxic'' carcinogens, should they be discovered, would be unlikely to alter the distribution of known risks for identi®ed carcinogens. Scientists may never be prepared to say they know all they would like to know about the distribution of risks of existing animal carcinogens. On the other hand, it can be estimated, with considerable con®dence, based on data available today that a substance which has not been tested for carcinogenicity and that is consumed in an amount below the threshold value of 1.5 mg/day will not present a greater than one-in-one million (10ÿ6) risk of human cancer. With these thoughts in mind, it is now important to look at the theoretical and practical aspects of the concept of threshold of concern and how it can be applied to ¯avouring substances in the context of JECFA safety evaluations. The CPD contains data on approximately 3700 long-term animal studies of 975 chemicals (Gold et al., 1986a,b,c, 1989). These include studies conducted by the US National Toxicology Program as well as studies conducted in other laboratories that have been published in the literature. Of the 975 chemicals tested, 955 were tested in rats and/or mice and 492 produced an increase in tumour incidence (342 in rats and 278 in mice). Gold and coworkers have put an enormous e€ort into compiling this database and ensuring its quality. The reader is referred to a series of papers by Gold et al. and others published in Environmental Health Perspectives which document the characteristics of this database (Gold et al., 1984, 1986a,b,c, 1989; Peto et al., 1984; Sawyer et al., 1984). For each compound, the CPD may include experiments with di€erent species, strains, sexes, dosing regimens, routes of administration, or other experimental conditions (Gold et al., 1984). In most experiments, two or more dose levels were used in addition to an unexposed control; in some cases, however, only a single exposed group was employed. Although a rigorous evaluation of the quality of individual experiments is not possible, the CPD does include information on the original investigators' conclusions regarding the overall strength of evidence for carcinogenicity. The CPD also contains a measure of carcinogenic potency (the TD50) computed as described by Peto et al. (1984) and Sawyer et al. (1984). In order to obtain some degree of comparability among di€er-

Safety evaluation procedure

ent studies, all TD50s are expressed in units of milligrams per kilogram body weight per day, and are adjusted to a 2-year standard rodent lifetime. In cases where intake is not constant throughout the study period, a time-weighted average dose is used for purposes of modelling dose±response. When individual animal data are available, the TD50s are adjusted for intercurrent mortality; otherwise, the crude proportions of animals with tumours are used to estimate carcinogenic potency without adjusting for mortality from other causes. Finally, the TD50 is estimated on the basis of an essentially linear one-hit dose±response model. The CPD represents an extremely useful source of information on experiments with chemical carcinogens. The database includes experiments on highly potent rodent carcinogens, such as 2,3,7,8tetrachlorodibenzo-p-dioxin and a¯atoxin B1, as well as less potent agents such as metronidazole and DDT. Gold et al. (1984) noted that TD50 values included in the CPD vary by 10 million-fold. Rulis (1986) used the Gold et al. (1984, 1986a,b,c, 1989) database when he and others at the FDA derived a threshold of regulation for food-contact materials. They transformed the distribution of lowest TD50 for each carcinogen that was tested by the oral route to a distribution of 10ÿ6 risks. While numerous mathematical models, including the linearized multistage model, could have been used to perform this transformation, Rulis (1986) used the slope (2TD50)ÿ1 of a straight line joining the TD50 and the origin as an estimator of the slope in the low dose region. Although the linearized multistage model could have been used since it has the advantage of allowing for curvature in

217

Table 5. Low-dose slopes for bladder tumours in mice exposed to 2-AAF for 24 months based on linear extrapolation from the TD50 Source of TD50 Fitted multistage model Best estimate 95% Con®dence limit Fitted one-hit model Best estimate 95% Con®dence limit 95% Con®dence limit on the linear term in the multistage model (q1*)

TD50 (mg/kg/day)

Slope (2 TD50)ÿ1 (mg/kg/day)

17.2 16.7*

0.0291 0.0298$

96.0 77.4*

0.0052 0.0065$ 0.0004$

*Lower con®dence limit. $Upper con®dence limit.

the dose±response curve, linear extrapolation from the TD50 is computationally simple, extremely conservative, and requires only published potency values from the CPD. To illustrate the di€erences between these two approaches, Krewski et al. (1990) considered the data on bladder tumours in mice exposed to the experimental carcinogen 2-acetyl amino¯uorene (2AAF), shown in Fig. 2. As indicated in Table 5, the TD50 for bladder tumours based on the ®tted multistage model is 17.2 mg/kg body weight/day, leading to a slope of (2TD50)ÿ1=0.0291 (mg/kg/day)ÿ1. Because over 3300 animals were involved in this experiment, the 95% lower con®dence limit on the TD50 and the corresponding upper con®dence limit on the slope are close to the best estimates obtained from the ®tted model. Owing to the high degree of curvature in the dose±response curve for bladder tumours, however, the upper con®dence limit on

Fig. 2. Dose±response curve for bladder tumours in mice exposed to 2-AAF for 24 months.

218

I. C. Munro et al.

the slope based on linear extrapolation from the lower con®dence limit on the TD50 is more than 75fold greater than the slope derived from the linearized multistage model. These data show that when there is signi®cant upward curvature in the dose±response curve, the methodology employed by Rulis (1986) to calculate a dose associated with a 10ÿ6 risk will produce a value substantially lower than when these riskspeci®c doses are calculated using the linearized multistage model. This point also has been made by Hoel and Portier (1994), who noted that examination of the shape of the dose±response curve for 315 chemicals found to produce tumours in the NCI/NTP program indicates that tumour site data were more often consistent with a quadratic response than a linear response suggesting that the use of linear dose±response models will often overestimate risk. It may therefore be concluded that the methodology used by Rulis (1986) (Federal Register, 1993, 1995) to estimate the distribution of 10ÿ6 risks for carcinogens in the Gold et al. CPD was very conservative. The next step in the process of establishing a threshold value involves the selection of an appropriate intake which is based on the distribution of 10ÿ6 risks. This value must be both highly protective of human health and of sucient practical value to reduce the number of compounds requiring formal toxicological testing. Thus any threshold selected should have an acceptably high probability of health protection whereas at the same time the selected threshold value is still of sucient magnitude to be of practical value. Of course, the protection of human health is of greater concern than the practical value. Initially Rulis (1986, 1989) proposed, for illustration, a threshold value of 0.15 mg/person/day.

Based on the distribution of 10ÿ6 risks from the CPD, this value would intersect the distribution at the 85th centile meaning that only 15% of carcinogens in the database would present a greater than 10ÿ6 risk at an intake of 0.15 mg/person/day. This analysis indicates that at an intake of 0.15 mg/person/day, 85% of the chemicals in the CPD known to induce cancer in rodents would fail to show a signi®cant increase in risk for the exposed population. This is demonstrated graphically in Fig. 3 modi®ed from Rulis (1989). Subsequent to this publication by Rulis, Munro (1990) held a workshop to evaluate factors that in¯uence the selection of an appropriate threshold value. The workshop ®rst reanalysed the Gold et al. (1984) database using the original database of 343 rodent carcinogens and con®rmed the observations of Rulis (1986, 1989) that a dietary intake of 0.15 mg/day intersected the distribution of 10ÿ6 risks at approximately the 85th centile. In addition, the workshop extended the analysis to include additional carcinogens added to the original Gold et al. (1984) database, bringing the total to 492 rodent carcinogens (Gold et al., 1989). This reanalysis with a broader set of data produced essentially the same distribution of 10ÿ6 risks as was originally published by Rulis (1986, 1989). The workshop participants also noted that inherent in the acceptance of any threshold value was the assumption that every new untested substance could be a carcinogen and could be as potent as the most potent 15% of carcinogens in the CPD. Recognizing that not every new substance would turn out to be a carcinogen, the workshop (Munro, 1990) constructed a table of risk avoidance probabilities (Table 6). Table 6 shows the e€ect of various assumptions regarding the proportion of chemicals that are presumed carcinogens on the probability that a 10ÿ6

Fig. 3. Distribution of TD50s for 343 rodent carcinogens from the Gold et al. (1984) CPD and distribution of 1 x 10ÿ6 risks calculated by linear extrapolation from the TD50s (modi®ed from Rulis, 1989).

Safety evaluation procedure

219

Table 6. Probability of a target risk not being exceeded at various threshold values Percentage of chemicals presumed carcinogenic Threshold value (mg/day) 0.15 0.3 0.6 1.5 3 6

100%

50%

20%

10%

100%

10ÿ6 Target risk 86 80 74 63 55 46

93 90 87 82 77 73

97 96 95 93 91 89

50%

20%

10%

10ÿ5 Target risk 99 98 97 96 95 95

96 94 91 86 80 74

98 97 96 96 90 87

99 99 98 97 96 95

>99 99 99 99 98 97

(Modi®ed from Munro, 1990).

risk standard will not be exceeded. It should be noted that as this proportion decreases, the probability of not exceeding a speci®c risk standard increases dramatically. Thus, for example, while there is a 63% chance that the risk will not exceed 10ÿ6 with a value of 1.5 mg/person/day when 100% of new chemicals are assumed to be carcinogenic, the probability that the risk will be less than 10ÿ6 is 96% when only 10% of new chemicals are assumed to be carcinogenic. Moreover, if one invokes a less conservative risk standard of 10ÿ5 (Table 6, right), then the probability of not exceeding that risk at a threshold value of 1.5 mg/person/day exceeds 96% even if it is assumed that 50% of new chemicals are potential carcinogens. In theory, the probability of an untested substance having a potency greater than the median of the distribution of TD50s from the CPD (Gold et al., 1989) is 50%. In reality, however, it is most unlikely that a genotoxic carcinogen with a potency equal to or greater than the median carcinogen in the Gold et al. (1989) database would be discovered from the existing inventory of ¯avouring substances given existing knowledge of structure±activity relationships in carcinogenesis. Taking the above factors into consideration and keeping in mind that the calculated 10ÿ6 risks based on the Gold et al. (1989) database were derived using a highly conservative methodology, Rulis (1989) re-examined his previous selection criteria for a threshold value and those of Munro (1990) and concluded that a threshold value of 1.5 mg/person/day would provide a high degree of health protection. This threshold value was subsequently adopted by FDA as the threshold of regulation (Federal Register, 1993, 1995) and FDA noted that such an exposure level would result in a negligible risk even in the event that a substance of unknown toxicity was later shown to be a carcinogen. Comparison of sensitivity of various endpoints Because of concerns raised by others (SCF, 1996), that the 5th centile NOELs for speci®c toxicological endpoints such as reproductive e€ects, neurotoxicity and immunotoxicity might result in human exposure thresholds lower than 1.5 mg/person/day, this matter was examined. Table 1 in

Appendix A presents the TDLos for 100 substances reported in the RTECS database (RTECS, 1987) to cause developmental abnormalities. The human exposure threshold for this group of substances is 2076 mg/person/day (see Table 7), 1384 times higher than the 1.5 mg/person/day value. In addition, the 5th centile NOEL for 31 neurotoxic organophosphorous insecticides (Appendix A, Table 2) included in structural Class III of the Munro et al. (1996) database produced a corresponding human exposure threshold of 18 mg/person/day (see Table 7), 12 times greater than the proposed threshold value of 1.5 mg/person/day. It might be expected that such neurotoxic compounds would have a low human exposure threshold because they are speci®cally designed to be highly potent toxins. Moreover, the measure of neurotoxicity selected to establish the NOELs in most cases was cholinesterase inhibition, an extremely sensitive endpoint. Most importantly, organophosphorous insecticides would not be used as ¯avouring substances. A list of the 100 substances reported to cause developmental abnormalities and of the 31 neurotoxic orgaTable 7. Comparison of various human exposure threshold values

Category Structural Class I Structural Class II Structural Class III Developmental abnormalities$ Neurotoxic compounds% Threshold value}

Human 5th Centile exposure NOEL threshold* (mg/kg bw/day) mg/person/day 3 0.91 0.15 3.46 0.03

1800 540 90 2076 18 1.5 mg/person/day

*The human exposure threshold was calculated by multiplying the 5th centile NOEL by 60 (assuming a 60 kg individual) dividing by a safety factor of 100, and multiplying by 1000 to convert from milligrams to micrograms. (Munro et al., 1996). $Substances are from the RTECS database and were indicated to cause developmental abnormalities. The NOELs were presented by RTECs as the TDLo which is de®ned as the lowest dose of a substance reported to produce any non-signi®cant adverse e€ects. (RTECS, 1987). %For organophosphorous insecticides, the endpoint measured was typically cholinesterase inhibition. }Adopted by FDA (Federal Register, 1993, 1995) as the threshold of regulation for food-contact articles. See text for details.

220

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nophosphorous insecticides, along with their NOELs, is given in Appendix A. For the evaluation of the sensitivity of immunotoxicity as an endpoint, the data of Luster et al. (1992, 1993) were used to conduct a comparison of NOELs and LOELs based on immunotoxic endpoints with corresponding NOELs and LOELs based on non-immunotoxic endpoints. Twenty-four substances meeting the criteria of immunotoxicity used by Luster et al. (1992, 1993) were identi®ed that also had corresponding non-immunotoxic endpoint NOELs or LOELs. A list of these substances is provided in Appendix A, Tables 3 and 4. Six of these substances had immunotoxic NOELs with corresponding non-immunotoxic NOELs. Twelve of these substances had no immunotoxic NOELs but had immunotoxic LOELs with corresponding nonimmunotoxic LOELs. Five additional substances had immunotoxic NOELs with corresponding nonimmunotoxic LOELs and one substance had an immunotoxic LOEL with a corresponding nonimmunotoxic NOEL. For these last six substances, NOELs were compared with LOELs divided by a conservative factor of 10 to adjust for di€erences between NOELs and LOELs (Dourson et al., 1996). For example, tetraethyl lead has an immune endpoint NOEL of 0.5 mg/kg body weight/day and a non-immune endpoint LOEL of 0.0012 mg/kg body weight/day. The LOEL was divided by 10 (0.00012 mg/kg body weight/day) for comparison with the NOEL. In order to perform the compari-

son of immunotoxic endpoint sensitivity with nonimmunotoxic endpoint sensitivity, the immunotoxic NOEL/LOEL was divided by the corresponding non-immunotoxic NOEL/LOEL resulting in a ratio. The resulting ratios of the 24 comparisons are shown graphically in Fig. 4. The majority of the substances (17/24) had non-immunotoxic NOELs or LOELs that were lower (i.e. more sensitive) than the corresponding immunotoxic NOELs or LOELs. Two substances had similar NOELs/LOELs and ®ve substances had immunotoxic NOELs or LOELs which were less than 10-fold lower than their nonimmuntoxic counterparts. These data demonstrate that immunotoxicity is not a more sensitive endpoint than other forms of toxicity. Application of a threshold of toxicological concern to ¯avouring substances The SCF (1996) has made the point that the decision to accept any particular threshold value is both a scienti®c and a risk management decision. The role of the scientist is to ensure that risk managers are provided with the full range of uncertainties surrounding selection of any threshold value. In the foregoing sections it was pointed out that the threshold concept should not be interpreted as providing absolute certainty of no risk. Threshold of toxicological concern is a probabilistic methodology that involves acceptance of a negligible risk standard. Such a standard is commonly used by toxicologists in the establishment of ADIs, and in fact,

Fig. 4. Comparison of immune and non-immune endpoint NOELS and LOELS (based on NOELS and LOELS of 24 immunotoxic substances).

Safety evaluation procedure

WHO (1987) has de®ned the ADI as ``an estimate by JECFA of the amount of a food additive, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk''. JECFA has noted that it uses the risk assessment process when setting an ADIÐthat is, the level of ``no apparent risk'' is set on the basis of quantitative extrapolation from animal data to human beings typically using a NOEL from the animal studies divided by a 100-fold safety factor (WHO, 1987). When ADIs (or such similar limits, e.g. TLVs, RfD, etc.) are established, there is a residual, usually unquanti®able, element of risk (Baird et al., 1996; Purchase and Auton, 1995; SCF, 1996; Sielken and Valdez-Flores, 1996), which is a re¯ection of the inability to determine precisely the NOEL from empirical data, statistical uncertainties associated with the sensitivity of experimental models, completeness of data, or the magnitude of modifying and safety factors invoked to account for any residual uncertainty (Dourson and Stara, 1983). The threshold of toxicological concern likewise does not carry with it the absolute certainty that an untested chemical present in food below the decision criterion of 1.5 mg/day will present a less than 10ÿ6 risk. Rather there is a high probability (i.e. about 95%) that the cancer risk from such a chemical will be less than 10ÿ6. It is this residual of uncertainty that has produced a concern about the possibility, albeit remote, that a highly potent genotoxic carcinogen might inadvertently be considered acceptable using the threshold concept (SCF, 1996). Additional factors that reduce the theoretical risk When the threshold concept is applied to ¯avouring substances, two additional factors signi®cantly reduce the probability of risk of cancer below 10ÿ6. The ®rst of these relates to very low levels of intake of ¯avouring substances. As intake decreases, the probability of not exceeding a 10ÿ6 risk substantially increases (Table 6). Therefore, application of a threshold of toxicological concern to substances having very low intakes (i.e. less or much less than the threshold value) carries with it a much higher probability of no appreciable risk. It also must be kept in mind, that intake of the majority of ¯avouring substances tends to be overestimated because these materials are volatile and appreciable amounts are lost during food preparation, storage, etc. These issues regarding intake of ¯avouring substances are discussed in Annex 5 of the report of the 44th meeting of JECFA (1996a). The second factor involves a consideration of chemical structure. The use of chemical structure for predicting toxicity for food chemicals, especially ¯avouring substances, has long been recognized by JECFA (WHO, 1987), and JECFA has noted that use of structure±activity is most developed in the area of carcinogenesis. The use of structural alerts

221

in combination with a knowledge of chemistry and metabolism o€ers a way of identifying potential carcinogens (Ashby and Tennant 1988, 1991; Klopman and Rosenkranz, 1994; Tennant et al., 1990; Williams, 1990). The examination of many chemicals for genotoxic, mutagenic and carcinogenic activities has led to the preparation of a series of structural alerts which provide the basis for potential reaction with DNA and possible carcinogenic potential of the substance. The existence of reactive moieties on known rodent carcinogens implies that potential mutagenic activity and, in many cases, the carcinogenic activity of untested chemicals might be identi®ed by an examination of structure (Ashby and Tennant, 1988, 1991; Tennant and Ashby, 1991). Structure±activity relationships have been successfully applied to congeneric substances (i.e. individual substances within a structurally related group of substances) for which no toxicity data are available (Klopman and Rosenkranz, 1994). Congeners that are potential human carcinogens and mutagens possess electrophilic functional groups with the ability to react directly with DNA. These electrophilic sites may be reactive functional groups on the congener or those formed during metabolic activation. Conversely, these functional groups may be lost during metabolic detoxication of the substance. Although the carcinogenic and mutagenic potency of congeneric substances may di€er, structural alerts within the group of congeners are indicative of carcinogenic or mutagenic potential (Klopman and Rosenkranz, 1994). A list of the functional groups identi®ed by Ashby and Tennant (1988, 1991) and Tennant et al. (1990) as structural alerts is given in Table 8. Most ¯avouring substances are simple aliphatic and aromatic substances containing functional groups that are eciently metabolized via detoxication pathways and very few ¯avouring substances and/or their in vivo metabolites contain structural alerts. The absence of structural alerts in a ¯avouring substance provides added assurance that it will not present an appreciable risk at or below intake of 1.5 mg/day. For those that do contain signi®cant structural alerts, such as aliphatic epoxides, additional data are usually available to facilitate evaluation. It is recognized that application of a threshold of toxicological concern is a departure from traditional toxicological evaluation, but it is based on highly conservative methodology and the assumptions listed below, which, taken together, ensure that ¯avouring substances consumed in amounts less than 1.5 mg/person/day will present, at most, an insigni®cant risk. 1. The 1.5 mg/day is based on carcinogenicity data, an extremely sensitive endpoint in susceptible

222

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Table 8. A list of functional groups identi®ed by Ashby and Tennant (1988, 1991) and Tennant et al. (1990) as structural alerts for DNA reactivity a) b) c) d) e) f) g) h) i) j) k)

2.

3.

4.

5.

6.

alkyl esters of phosphonic or sulfonic acids aromatic nitro-groups aromatic azo-groups (reduction to amine) aromatic ring N-oxides aromatic mono- and di-alkyl amino groups alkyl hydrazines alkyl aldehydes N-methylol derivatives monohaloalkanes N and S mustards, beta-haloethylN-chloramines

l) m) n) o) p) q) r) s) t) u)

animal species with accepted relevance to humans. The CPD presents a worse case situation since chemicals were generally tested over a lifetime by daily administration at the maximum tolerated dose (MTD), and the procedures used by Gold et al. (1984) to establish the TD50s involved numerous conservative assumptions. The methods used by Rulis (1986, 1989) and others (Krewski et al., 1990; Munro, 1990) to calculate the distribution of 10ÿ6 risks, on which the threshold value of 1.5 mg/person/day is based, are highly conservative since they involved the use of linear extrapolation from the lowest TD50 for each substance in the database. It is unlikely that any untested ¯avouring substance would turn out to be a genotoxic carcinogen, and the possibility that a carcinogen would be accepted using the threshold concept can be substantially reduced by the application of structural alert methodology. Many ¯avouring substances are consumed in amounts considerably below the threshold value of 1.5 mg/person/day and this substantially increases the probability, already in the range of 90 to 95%, that they will not present any signi®cant theoretical risk. Toxicity endpoints, such as developmental toxicity, neurotoxicity and immunotoxicity demonstrate considerably higher human exposure thresholds than the threshold value of 1.5 mg/person/day making it highly unlikely that these noncancer endpoints are a relevant concern in applying the threshold concept.

Taken together, these factors provide a sound basis for concluding that ¯avouring substances with

propiolactones and propiosulfones aromatic and aliphatic aziridinyl-derivatives aromatic and aliphatic substituted primary alkyl halides urethane derivatives (carbamates) alkyl N-nitrosamines aromatic amines and N-hydroxy derivatives aliphatic epoxides and aromatic oxides center of Michael reactivity halogenated methanes [C(X)4] aliphatic nitro groups

intakes below the 1.5 mg/person/day threshold value can be safely consumed. It is enlightening to compare the human exposure thresholds with present intakes of chemically de®ned ¯avouring substances in the US. As shown in Table 9, it is clear that for nearly all (93±97%) ¯avouring substances used in the US, intakes are below the human exposure threshold for their respective structural class. Because most ¯avouring substances possess simple structures and their metabolism is known or reasonably predictable, it can be concluded that it is highly improbable that they would present a toxicological risk at exposure levels below the human exposure threshold for their respective structural class. However, even if information on structural class, metabolic fate and existing toxicity studies were not available, 743/1323 (56%) of ¯avouring substances used in the US are consumed in amounts less than the 1.5 mg/person/day standard proposed by the FDA (Federal Register, 1995) and Munro (1990). This indicates that for approximately half of the existing list of 1323 chemically de®ned ¯avouring substances permitted for use in the US, intake can be considered to be trivial. Safety decision criteria The decision criteria used to evaluate ¯avouring substances involve the integration of information on intake, structure±activity relationships, metabolism and, as required, toxicity data. It should be noted that the criteria outlined below are not intended to be applied to any ¯avouring substances with unresolved toxicity problems or to substances that are presumed or known carcinogens. Such substances warrant special consideration and must be evaluated using more traditional methods of safety

Table 9. Flavouring substances within each Cramer et al. (1978) structural class consumed in amounts below human exposure thresholds Structural class I II III

Human exposure threshold (mg/day)

No. of ¯avours within structural class*

No. of ¯avours under human exposure threshold (%)

1800 540 90

878 243 202

835 (95) 227 (93) 195 (97)

*Adapted from the FEMA ¯avouring substance database of ¯avouring substances permitted for use in the US.

Safety evaluation procedure

evaluation. While toxicity data exist on numerous ¯avouring substances and can be used as the basis for evaluation, there are many ¯avouring substances that lack toxicity data. The decision criteria are intended to provide a means of evaluating such substances. The criteria incorporate, where the data permit, the concept that metabolic fate can be predicted on the basis of presumed structure±activity relationships. The criteria also rely, in part, on the NOEL reference database which provides a human exposure threshold for each of the three structural classes of ¯avouring substances, as shown in Table 4. The evaluation criteria also incorporate, where available, toxicity data on ¯avouring substances and closely related structural analogues as a basis for safety evaluation. One of the decision criteria (number 5 below) incorporates the minimum human exposure threshold value based on the 1.5 mg/person/day. This decision criterion can be applied to those ¯avouring substances for which metabolic fate is unknown and cannot be con®dently predicted and for which there are no toxicity data on the ¯avouring substance or on a structurally related material from which to conclude any inference of safety in use. Flavouring substances that meet one of the ®ve numbered decision criteria outlined below can be considered safe for their intended use without further evaluation: 1. (a) The ¯avouring substance has a simple structure and will be metabolized and excreted through known detoxication pathways to innocuous endproducts; and (b) the conditions of intended use do not result in an intake greater than the human exposure threshold for the relevant structural class, indicating a low probability of potential for adverse e€ects. 2. (a) The conditions of intended use result in an intake that exceeds the human exposure threshold for the relevant structural class; however (b) the ¯avouring substance has a simple structure and will be metabolized and excreted through known detoxication pathways to innocuous end-products and it or its metabolites are endogenous human metabolites with no known biochemical regulating function. *In most instances, groups of structurally related materials have toxicology data on at least one member of the group, usually the ¯avouring substance with the highest poundage. In most cases a large margin of safety (i.e. 100- to 1000-fold) exists between the NOEL and the calculated intake to the substance having toxicological data. Such margins of safety would be expected to accommodate any perceived di€erence between the toxicity of a ¯avouring substance having no toxicological data and its close structural relative for which a NOEL has been established.

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3. (a) The ¯avouring substance has a simple structure and will be metabolized and excreted through known detoxication pathways to innocuous end-products; and (b) the conditions of intended use result in an intake that exceeds the human exposure threshold for the relevant structural class; however (c) toxicity data exist on the ¯avouring substance which provide assurance of safety under conditions of intended use, or there are toxicity data on 1 or more close structural relatives which provide a NOEL high enough to accommodate any perceived di€erence* in toxicity between the ¯avouring substance and the structurally related substance having toxicity data. 4. (a) The metabolic fate of the ¯avouring substance cannot be con®dently predicted on the basis of structure; however (b) the conditions of intended use result in an intake below the human exposure threshold for the relevant structural class indicating a low probability of potential for adverse e€ects; and (c) toxicity data exist on the ¯avouring substance which provide assurance of safety under conditions of intended use, or there are toxicity data on 1 or more close structural relatives which provide a NOEL high enough to accommodate any perceived di€erence in toxicity between the ¯avouring substance and the structurally related substance having toxicity data. 5. (a) The metabolic fate of the ¯avouring substance cannot be con®dently predicted on the basis of structure; however (b) the conditions of intended use result in an intake below the human exposure threshold of 1.5 mg/day, providing assurance that the substance will be safe under conditions of intended use. Figure 5 presents the same decision in the form of a safety evaluation sequence. The sequence contains a number of questions on structure, metabolism, intake data and toxicity and provides an integrated mechanism to evaluate the safety of a ¯avour ingredient. Although the procedure incorporates relevant toxicity data on a substance or related substances where available, it does not require them. The e€ective application of this safety evaluation procedure depends on a substantial knowledge of toxicology, chemistry, metabolism and intake of ¯avouring substances. It can be applied most e€ectively when groups of structurally related ¯avouring substances are evaluated together. In a group evaluation, conclusions reached on the safety of individual substances are supported by similar conclusions for structurally related substances. For example, the results of the evaluation for butyl butyrate should be consistent with results for other esters formed from aliphatic acyclic linear saturated alcohols and

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Fig. 5. Safety evaluation sequence.

acids having similar levels of intake. These similar substances will pass through the same branch of the safety evaluation procedure because they fall into the same structural class, possess similar metabolic fate, and exhibit similar patterns of intake from use as ¯avour ingredients and as components of food. In the ®rst step of the safety evaluation procedure (Fig. 5), the user must assign a decision tree structure class (Cramer et al., 1978) to the substance. Following assignment of structure class, a question on metabolic fate appears. This question identi®es those substances which are anticipated to be eciently metabolized to innocuous products (e.g. 1butanol) versus those which are transformed to more toxic metabolites (e.g. estragole) or have limited information on which to predict con®dently the metabolic fate (e.g. 2-phenyl-3-carbethoxy furan). Once a substance has been sorted according to structure class and knowledge of metabolic fate, the next question compares the substance's daily intake from use as a ¯avour ingredient to the human exposure threshold (Table 4) for the same structure class. If the substance is metabolized to innocuous products (Step No. 2) and has an intake less than the human exposure threshold for the structure class (Step No. A3), the substance is considered safe (e.g. 1octanol). If the intake is greater than the human exposure threshold (Step No. A3) and the substance or its metabolites are endogenous (Step No. A4), the

substance is also considered safe, even though the intake is greater than the human exposure threshold (e.g. butyric acid). If the substance is not endogenous, then the substance or related substances must have a NOEL (Step No. A5) signi®cantly greater than the intake of the substance in order to be considered safe (e.g. citral). If no such data exist or the NOEL is not signi®cantly greater than the intake for the substance, then additional data are required in order to complete the safety evaluation. If metabolic fate cannot be con®dently predicted and the intake (Step No. 2) is greater than the human exposure threshold (Step No. B3), additional data on metabolic fate or toxicity on the substance or structurally related substances are required to complete the safety evaluation (e.g. dihydrocoumarin). If the intake is less than the threshold of concern for the structural class, the substance or structurally related substances must have a NOEL which provides an adequate margin of safety under conditions of intended use (Step No. B4) in order for the substance to be considered safe [e.g. 2-ethyl-4-hydroxy-3(2H)-furanone]. If an adequate toxicity study is not available and the substance has an intake less than 1.5 mg/day (Step No. B5), the substance is considered not to present a safety concern (e.g. 3-acetyl-2,5-dimethylthiophene). Otherwise additional data are required in order to complete the safety evaluation (e.g. 2-ethylfuran).

Safety evaluation procedure

The principal objective of the safety evaluation procedure is to identify two groups of ¯avouring substances: (i) those substances whose structure, metabolism, and relevant toxicity data clearly indicate that the substance would be expected not to be a safety concern under current conditions of intended use; and (ii) those substances which may require additional data in order to perform an adequate safety evaluation. Integrating data on consumption ratio As pointed out by WHO (1987) and SCF (1991), natural occurrence is no guarantee of safety, but it is important to recognize that the safety evaluation of added use of ¯avouring substances needs to be conducted with an appreciation of the consumption ratio. Clearly, if added use of ¯avouring substances results in an intake that exceeds that from natural sources, this will increase awareness of the need to consider carefully overall intake in the light of existing data on toxicity and structure±activity relationships. On the other hand, if the added use is trivial with a consumption ratio of 10 to 100, that is, it increases total intake by only 1 to 10%, then this fact needs to be taken into consideration when applying the criteria outlined above. The substances of primary concern are those which, in Fig. 5, receive a ``No'' answer to Step No. A5, or a ``Yes'' answer to Step No. B5, indicating a possible need for additional data and evaluation beyond that included in the evaluation procedure outlined in this paper. In such an evaluation, as stated immediately above, the extent of natural occurrence should be given appropriate weight. Of much less concern with respect to consumption ratio are those substances that drop out of further consideration as a result of ``No'' answers to Step Nos A3 or B5, or ``Yes'' answers to Step Nos A4 or B4. The derivation of the thresholds, the estimation of intakes (see JECFA, 1996a), and the special factors applicable to the use of ¯avours (volatility, self-limiting use, etc.) build in multiple conservative assumptions more than adequate to cover additional intake from natural occurrence to substances that in any case are of low inherent concern. The advances in analytical chemistry in the past 50 years provide virtual assurance that no ¯avouring substances of extensive natural occurrence remain unknown. Those of potential value yet to be discovered (e.g. as yet unknown components of roast beef, co€ee, or chocolate ¯avour) are being sought at the ppb and ppt level. This does not suggest intakes above any of the thresholds discussed in this paper. Discussion The safety evaluation of ¯avouring substances presents an interesting challenge. There are approximately 2500 ¯avouring substances in use in Europe or the US at this time. Many do not have sucient

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toxicology data to conduct a traditional safety evaluation. However, it is neither possible nor necessary to conduct toxicological studies on all individual ¯avouring substances used in food. The majority of ¯avouring substances are members of groups of substances with common metabolic pathways, and typically, individual members of such a group display a similar toxicity pro®le. This is not surprising in the light of the close structural similarity of the various ¯avouring substances comprising a chemical group. Moreover, as demonstrated here, intake of ¯avouring substances is usually low and, in the majority of cases, below the human exposure threshold values presented in this review. In order to provide a practical solution for evaluating such a large number of low-exposure substances in a timely manner, the safety evaluation procedure described here was developed. It incorporates knowledge of toxicology, chemical structure, metabolism and intake. This procedure was recently adopted by JECFA (1996a,b, 1997) and was applied to the safety evaluation of 263 ¯avours during the 46th and 49th meetings of the Committee (JECFA, 1997, 1998). REFERENCES

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APPENDIX

follows

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

1,3,4-Thiadiazole, 2,2'-(methylenediimino)bis1-6-Hexanediamine 1-Piperazinepropanol, 4-(6-((6-methoxy-8-quinolyl)amino hexyl-alpha-methyl-, maleate (1:2) 11H-Pyrido(2,1-b)quinazolone-2-carboxylic acid, 11-oxo1H-Indazole-3-carboxylic acid, 1-(2,4-dichlorobenzyl)1H-Isoindole-1,3(2H)-dione,4,5,6,7-tetrahydro-2-(7-¯uoro-3,4-dihydro-3-oxo-4-; (2-propynyl)-2H-1,4-benzoxazin-6-yl)2,7-Naphthalenedisulfonic acid, 3,3'((3,3'-dimethyl-4,4'biphenylylene)bis(azo))bis(5-; amino-4-hydroxy-, tetrasodium salt) 2-Propanone, 1,1,3,3-tetrachloro2-Pyridinemethanol,alpha-(3-(2,6-dimethyl-1-piperidinyl)propyl)-alpha-phenyl-,; monohydrochloride, Z-(2)3-Biphenylcarboxylic acid, 2',4'-di¯uoro-4-hydroxy4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid,6-((aminophenylacetyl)amino)-; 3,3-dimethyl-7-oxo-,(2,2-dimethyl-1-oxopropoxy)methyl ester,hydrochloride 4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid,6-(2-amino-2-phenylacetamido)-; 3,3-dimethyl-7-oxo-,trihydrate,D-(-)4H-Pyrido(1,2-a)pyrimidin-4-one,9-methyl-3-(1H-tetrazol-5-yl)-, potassium salt 4H-s-Triazolo(3,4-c)thieno(2,3-e)(1,4)-diazepine,6-(o-chlorophenyl)-8-ethyl-1-methyl5-Isoxazoleacetic acid, 3,4-bis(4-methoxyphenyl)7H-Pyrido(1,2,3-de)-1,4-benzoxazine-6-carboxylic acid,2,3-dihydro-9-¯uoro-3-methyl-; 10-(4-methyl-1-piperazinyl)-7-oxo-,hemihydrate,(S)9H-Purine-6-thiol, 9-beta-D-ribofuranosylAcetamide, 2,2-dichloro-N-(beta-hydroxy-alpha-(hydroxymethyl)-p-(methylsulfonyl)phenethyl)-,; D-threo-(+) Acetamide, N,N-dimethylAcetic acid, (2,4-dichlorophenoxy)Acetic acid, (3,5,6-trichloro-2-pyridyloxy)Acetic acid, oxo((3-(1H-tetrazol-5-yl)phenyl)amino)-,butyl ester Acetonitrile, amino-, bisulfate Acridine, 9,9-dimethyl-10-(3-(N,N-dimethylamino)propyl)-,tartrate Alanine, 3-(3,4-dihydroxyphenyl)-, LAlanine, N-((5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl)carbonyl)-3-phenyl-,; sodium salt, (-)Alosenn Anthranilic acid, N-(2,3-xylyl)Arsine oxide, dimethylhydroxyBenzamide, N-(2-piperidinylmethyl)-2,5-bis(2,2,2-tri¯uoroethoxy)-, monoacetate Benzenesulfonamide, 4-amino-N-(4,5-dimethyl-2-oxazolyl)-,mixt. with 5-((3,4,5-; trimethoxyphenyl)methyl)-2,4-pyrimidinediamine Benzenesulfonamide, 4-amino-N-(4,6-dimethoxy-2-pyrimidinyl)Benzenesulfonic acid, thio-,S,S'-(2-(dimethylamino)trimethylene) ester Benzhydrol, 2-chloro-alpha-(2-(dimethylamino)ethyl)-,hydrochloride Benzoic acid, 3,4,5-trimethoxy-beta-(dimethylamino)-beta-ethylphenethyl ester,; maleate (1:1) Benzyl alcohol, 4-amino-alpha-((tert-butylamino)methyl)-3,5-dichloro-,monohydrochloride Biphenyl, 3,3',4,4'-tetramethylButyric acid, 4-(p-bis(2-chloroethyl)aminophenyl)Butyrophenone, 4-(4-(p-chlorophenyl)-4-hydroxypiperidino)-4'-¯uoroCadmium Carbazic acid, 3-(1-phthalazinyl)-, ethyl ester, monohydrochloride Chlordane Cortisone Dibenzo(b,e)(1,4)dioxin, 2,3,7,8-tetrabromoDibenzo-p-dioxin, 2,7-dichloro-

Chemical

Table A1. Substances reported to cause developmental abnormalities (from RTECS)

rat rat rat rat rat rat rat rabbit rabbit rabbit mouse rat rat rabbit rat rat rat rat rabbit rat rat rat rat mouse rat rat rat mouse rat rabbit rat rat rat mouse rabbit rat mouse mouse rat rat mouse rat mouse mouse rat

Species 1 1840 90 4400 175 300 150 130 650 520 1200 2800 2750 13 1650 8910 87.5 150 3900 0.22 2000 24000 200 175 500 5 5500 8 300 390 3360 500 660 120 6500 4.4 640 3 5.04 23 70 880 500 0.216 5

Dose (TDLo)* mg/kg

230 I. C. Munro et al.

Disul®de, bis(thiocarbamoyl) Ethane, 1,1,1-trichloro-2,2-bis(p-methoxyphenyl)Ethane, 2-(o-chlorophenyl)-2-(p-chlorophenyl)-1,1,1-trichloroEthanone, 1-(7-(2-hydroxy-3-((1-methylethyl)amino)propoxy)-2-benzofuranyl)-, hydrochloride Ethanone, 2-((4-(2,4,dichloro-3-methylbenzoyl)-1,3-dimethyl-1H-pyrazol-5-yl)oxy)-; 1-(4-methylphenyl)Folic acid, methylGallic acid, propyl ester Glutamic acid, N-(p-((1-(2-amino-4-hydroxy-6-pteridinyl)ethyl)amino)benzoyl)-LGossypol acetic acid Hydrocinnamic acid, alpha-hydrazino-3,4-dihydroxy-alpha-methyl-, LIndole-3-acetic acid, 1-(p-chlorobenzoyl)-5-methoxy-2-methylIsonicotinamide, 2-ethylthioIsothiocyanic acid, butenyl ester L-Glutamic acid, magnesium salt (1:1), hydrobromide L-Tyrosine L-Tyrosine, O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodoLinoleic acid (oxidized) Lysine, LManganese, (ethylenebis(dithiocarbamato))- and zinc acetate (50:1) Mannitol, 1,6-dibromo-1,6-dideoxy-, DMethanol, 1,3,4-thiadiazol-2-ylminodiMolybdenum Morpholine, 4-(3,4,5-trimethoxybenzoyl)Norleucine, 6-amidino-, monohydrochloride, hydrate Oxazolo(3,2-d)(1,4)benzodiazepin-6(5H)-one, 10-chloro-11b-(o-chlorophenyl)-2,3,7,11b-; tetrahydro Phenol, p-aminoPhenothiazine-2-acetic acid, 10-methyl Phosphonic acid, (1,2-epoxypropyl)-, calcium salt (1:1), (1R,2 S)-(-)Phosphorodithioic acid, O,O-dimethyl ester, S-ester with 2-mercapto-N-methylacetamide Phthalic acid, di(methoxyethyl)ester Piperazine, 1-(p-tert-butylbenzyl)-4-(p-chloro-alpha-phenylbenzyl)Piperazine,1-(p-tert-butylbenzyl)-4-(p-chloro-alpha-phenylbenzyl)-,dihydrochloride Piperidine, 3-((4-methoxyphenoxy)methyl)-1-methyl-4-phenyl-, hydrochloride, (3R-trans)Piperidine, 1-methyl-4-(N-2-thenylanilino)-, tartrate Polychlorinated biphenyl (Aroclor 1254) Pregn-4-ene-3,20-dione,9-¯uoro-11-beta,17,21-trihydroxyPregna-,4-diene-2,20-dione,9-¯uoro-11-beta,16-alpha,17,21-tetrahydroxy-,16,21-diacetate Propionic acid,2-(2,4,5-trichlorophenoxy)Pyrimidine, 2,4-diamino-6-methyl-5-phenylRetinoic acid, 4-oxo-,13-cisRetinoic acid, all-transRowachol Stannane, diacetoxydibutylSulfanilamide, N(sup 1)-(6-methoxy-2-methyl-4-pyrimidinyl)Toluene, alpha-(2-(2-butoxyethoxy)ethoxy)-4,5-(methylenedioxy)-2-propylTryptophan, N-acetyl-, LUrea, (alpha-(2-methylhydrazino)-p-toluoyl)-, monohydrobromide Urea, 1-butyl-3-(p-tolylsulfonyl)Urea, 1-butyl-3-sulfanilylUridine, 5'-deoxy-5-¯uoroZZL-0820 beta-Escin m-Propionotoluidide,2-methyl-4'-nitro-alpha,alpha,alpha-tri¯ourop-Acetophenetidide p-Cresol, 2,6-di-tert-butyl-

*TDLo = the lowest dose of a substance reported to produce any non-signi®cant adverse e€ect (=NOEL).

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

mouse rat rat rat rat rat rat rat mouse rat rat mouse rat rat rat rat rat rat rat mouse rat rat rat rat mouse rat mouse rat rat rat rat rat rat rat rat rabbit mouse mouse rat mouse mouse rat rat mouse rat rat rabbit mouse rat rat rabbit mouse rat rat mouse

105 2000 250 1400 2000 500 45000 20 480 2100 1 450 800 6000 3500 26.25 166000 81000 765 150 5 5.8 700 2000 1800 2500 180 15400 120 593 320 360 210 157 90 2 3.2 1617 100 100 15 9600 15.2 3000 2130 27500 50 1700 1000 550 325 36 1050 6000 1200

Safety evaluation procedure 231

232

I. C. Munro et al. Table A2. NOELs for organophosphorous insecticides*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Agent

Species

Endpoint observed

NOEL (mg/kg/day)

Acephate Azinphos methyl Coumaphos Crufomate Diazinon Dichlorvos Dimethoate Disulfoton Ethephon Ethion Ethyl-p-nitrophenyl phenylphosphorothioate Express Fenamiphos Fenchlorphos Fonofos Glufosinate ammonium Glyphosate Malathion Merphos Merphos oxide Methamidophos Methidithion Methyl parathion Naled Parathion Phosmet Phosphamidon Pirimiphos-methyl Quinalphos Tetrachlorvinphos Tetraethyl dithio pyrophosphate

rat rat rat rat mouse rat rat rat rat rat rat

decreased body weight gain (parents and pups) inhibition of plasma ChE activity inhibition of RBC and plasma ChE activity inhibition of RBC ChE activity decreased body weight gain inhibition of ChE activity (speci®c endpoint not indicated) inhibition of brain, RBC and plasma ChE activity inhibition of brain, RBC abd plasma ChE activity inhibition of plasma and RBC ChE activity inhibition of plasma ChE activity in females inhibition of brain, RBC and plasma ChE activity

rat rabbit rat rat rat rat rat rat rat rat rat rat rat rat rat rat rat mouse rat rat

decreased body weight gain decreased maternal body weight gain inhibition of ChE (form not speci®ed) inhibition of RBC and plasma ChE activity increased absolute and relative kidney weight in males increased incidence of renal tubular dilation in F3b pups inhibition of brain ChE activity inhibition of RBC ChE activity in females inhibition of brain ChE activity clinical signs typical of ChE inhibition inhibition of brain and RBC ChE activity decreased hemoglobin, hematocrit and RBCs decreased body weight gain decreased body weight gain inhibition of RBC and plasma ChE activity decreased body weight gains inhibition of plasma ChE activity inhibition of plasma ChE activity inhibition of RBC ChE activity inhibition of RBC and plasma ChE activity

2.5 0.18 0.4 3 72$ 0.23 0.05 0.05 15 0.2 0.25 1 0.1 15 0.5 0.4$ 10 5 0.1 0.25 1 0.2 0.025 0.2 1.8 2 6.2 0.5 0.03 6 0.5

*Data taken from the EPA Integrated Risk Information System (IRIS) database. $NOEL divided by a factor of 3 (see Munro et al., 1996 for explanation). Table A3. Substances with immunotoxic NOELs Non-immune

Immune Substance

1

Oral Admin p-nitrotoluene pentachlorophenol o-phenylphenol hexachlorodibenzo-p-dioxin DPH tetraethyl lead benzidine nitrobenzene Non-oral Admin indomethacin (sc)%

2 3

TPA (sc) ethyl carbamate (ip)}

1 2 3 4 5 6 7 8

NOEL (mg/kg bw)

NOEL (mg/kg bw)

400 10 100 0.056 150 0.5 11 30

200 3 10 1E-05 50

2

1.6

LOEL (mg/kg bw)

0.0012 2.7 60

20 2

0.32 15

Non-immune endpoint

Reference

hepatic, splenic hepatic, renal blood (RBC)* repro$ teratogenic hepatic,thymus neural, hepatic repro

Burns et al., 1994 Schwetz et al., 1978 Luster et al., 1981 Murray et al., 1979 McClain and Langho€, 1979 Schepers, 1964 Little®eld et al., 1983 Kawashima et al., 1995

repro/vascular permeability (sc) repro (sc) repro (sc)

Hoos and Ho€man, 1983 Nagasawa et al., 1980 NTIS, 1968

*RBC = red blood cells; $repro = reproductive; %sc = subcutaneous; }ip = intraperitoneal. Table A4. Substances with immunotoxic LOELs Non-immune

Immune

1 2 3 4 5 6 7 1 2 3 4 5 6

Substance

LOEL (mg/kg bw)

Oral Admin 2,3,7,8-TCDD lithium carbonate

0.086 50

1E-05 98

200 25 50 125 10

40 24 125

m-nitrotoluene 2,4-diaminotoluene dimethylvinyl chloride ethylene dibromide 4,4-thiobis (6-tert-butyl-m-cresol) Non-oral Admin azathioprine (ip)} benzo(a)pyrene (sc)} diethylstilboestrol (sc) DMB(a)A (sc) N-nitroso dimethylamine (ip) ochratoxin A (ip)

10 50 0.2 5 1.5 3.4

NOEL (mg/kg bw)

30

LOEL (mg/kg bw)

Non-immune endpoint

Reference

45

repro* repro, bw,$ hepatic, renal hepatic (f)% repro splenic mortality hepatic

Murray et al., 1979 Ibrahim and Canolty, 1990 NTP, 1992 Thysen et al., 1985 NTP, 1986 Teramoto et al., 1980 NTP, 1994

1 50 1E-05 1.25 5 0.0625

repro (ip) repro (sc) repro (sc) repro (orl)** repro (ip) renal (gav)$$

Scott, 1977 Bui et al., 1986 McLachlan, 1977 Davis et al., 1978 Chaube, 1973 NTP, 1989

*repro = reproductive; $bw = body weight.; %f = female.; }ip = intraperitoneal.; }sc = subcutaneous.= oral.; $$gav = gavage.