, 144 pinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission related to Flavouring Group Evaluation 16: (Commission Regulation (EC) No 1565/000 of 18 July 000) QUESTIN N EFSAQ003159 Adopted on 1 March 006 SUMMARY The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food is asked to advise the Commission on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the Member States. In particular, the Scientific Panel is asked to evaluate four flavouring substances in the Flavouring Group Evaluation FGE.16, using the procedure as referred to in the Commission Regulation (EC) No 1565/000. These four flavouring substances belong to chemical group 1, Annex I of the Commission Regulation (EC) No 1565/000. The present Flavouring Group Evaluation deals with four aromatic ketones. None of the four flavouring substances can exist as geometrical or optical isomers. Three of the flavouring substances are classified into structural class I and one is classified into structural class III. Three of the flavouring substances in the present group have been reported to occur naturally in a wide range of food items. In its evaluation, the Scientific Panel as a default used the Maximised Surveyderived Daily Intakes (MSDIs) approach to estimate the per capita intakes of the flavouring substances in Europe. However, when the Scientific Panel examined the information provided by the European flavouring industry on the use levels in various foods, it appeared obvious that the MSDI approach in a number of cases would grossly underestimate the intake by regular consumers of products flavoured at the use level reported by the industry, especially in those cases where the annual production values were reported to be small. In consequence, the Scientific Panel had reservations about the data on use and use levels provided and the intake estimates obtained by the MSDI approach. In the absence of more precise information that would enable the Scientific Panel to make a more realistic estimate of the intakes of the flavouring substances, the Scientific Panel has decided also to perform an estimate of the daily intakes per person using a modified Theoretical Added Maximum Daily Intake (mtamdi) approach based on the normal use levels reported by industry. In those cases where the mtamdi approach indicated that the intake of a flavouring substance might exceed its corresponding threshold of concern, the Scientific Panel decided not to carry out a formal safety assessment using the Procedure. In these cases the Scientific Panel requires more precise data on use and use levels. http://www.efsa.eu.int/science/afc/afc_opinions/catindex_en.html
According to the default MSDI approach, the four flavouring substances in this group have intakes in Europe from 0.001 to 0.85 microgram/capita/day which are below the threshold of concern values for structural class I (1800 microgram/person/day) and structural class III (90 microgram/person/day). verall, the limited data available do not allow a final assessment of genotoxicity. From the data available there is some indication of genotoxic potential for two of the supporting substances (1 phenylethan1ol and acetophenone). However, taking into consideration metabolism and carcinogenicity data on the candidate and supporting substances, the positive in vitro results do not preclude the evaluation of the candidate substances through the Procedure. It can be anticipated that the three flavouring substances [FLno: 07.193, 07.194, and 07.195] are metabolised to innocuous products. This cannot be anticipated for the flavouring substance [FLno: 07.14]. Based on a NAEL of 33 mg/kg for the structurally related substance methyl naphthyl ketone [FLno: 07.013] a margin of safety of approximately x 9 could be estimated for the flavouring substance alphamethyl naphthyl ketone [FLno: 07.14]. Thus, alphamethyl naphthyl ketone is not expected to be of safety concern at its estimated level of intake as flavouring substance. It was noted that where toxicity data were available they were consistent with the conclusions in the present Flavouring Group Evaluation using the Procedure. It was considered that on the basis of the default MSDI approach the four flavouring substances: (1 phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194], 1phenylpropanone [FLno: 07.195] and alphamethyl naphthyl ketone [FLno: 07.14] would not give rise to safety concerns at the estimated levels of intake arising from their use as flavouring substances. When the estimated intakes were based on the mtamdi approach they were 1600 microgram/person/day for each of the four flavouring substances. Thus, for the three substances belonging to structural class I, the intakes were all below the threshold of concern of 1800 microgram/person/day for structural class I. For the one flavouring substance alphamethyl naphthyl ketone [FLno: 07.14] belonging to structural class III, the intake was above the threshold of concern of 90 microgram/person/day for structural class III. The three substances which have mtamdi intake estimates below the threshold of concern for structural class I, are also expected to be metabolised to innocuous products. Thus for one of the four flavouring substances considered in this opinion the intake, estimated on the basis of the mtamdi, exceeds the relevant threshold for the structural class, to which the flavouring substance has been assigned. Therefore, for this substance more reliable exposure data are required. n the basis of such additional data, this flavouring substance, alphamethyl naphthyl ketone [FLno: 07.14] should be reconsidered along the steps of the Procedure. Following this procedure additional toxicological data might become necessary. In order to determine whether this evaluation could be applied to the materials of commerce, it is necessary to consider the available specifications. Adequate specifications including complete purity criteria and identity tests for the materials of commerce have been provided for the four flavouring substances (1phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194], 1phenylpropanone [FLno: 07.195] and alphamethyl naphthyl ketone [FLno: 07.14]) and these would present no safety concern at the levels of intake estimated on the basis of the MSDI approach. KEYWRDS Aryl ketones; aromatic ketones; alkanons; flavourings; safety.
TABLE F CNTENTS Summary... 1 Keywords... Background... 4 Terms of Reference... 4 Assessment... 4 1. Presentation of the Substances in Flavouring Group Evaluation 16... 4 1.1. Description... 4 1.. Stereoisomers... 5 1.3. Natural ccurrence in Food... 5. Specifications... 5 3. Intake Data... 5 3.1. Estimated Daily per Capita Intake (MSDI Approach)... 6 3.. Intake Estimated on the Basis of the Modified TAMDI (mtamdi)... 6 4. Absorption, Distribution, Metabolism and Elimination... 8 5. Application of the Procedure for the Safety Evaluation of Flavouring Substances... 9 6. Comparison of the Intake Estimations Based on the MSDI Approach and the mtamdi Approach... 7. Considerations of Combined Intakes from Use as Flavouring Substances... 8. Toxicity... 11 8.1. Acute Toxicity... 11 8.. Subacute, Subchronic, Chronic and Carcinogenicity Studies... 11 8.3. Developmental / Reproductive Toxicity Studies... 1 8.4. Genotoxicity Studies... 1 9. Conclusions... 13 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 16... 15 Table : Summary of Safety Evaluation Applying the Procedure (Based on Intakes Calculated by the MSDI Approach)... 16 Table 3: Supporting Substances Summary... 17 Annex I: Procedure for the Safety Evaluation... 1 Annex II: Use Levels / mtamdi... 3 Annex III: Metabolism... 6 Annex IV: Toxicity... 3 References:... 39 Scientific Panel Members... 44 Acknowledgement... 44 3
BACKGRUND Regulation (EC) No 3/96 of the European Parliament and the Council (EC, 1996) lays down a procedure for the establishment of a list of flavouring substances, the use of which will be authorised to the exclusion of all others in the EU. In application of that Regulation, a register of flavouring substances used in or on foodstuffs in the Member States was adopted by Commission Decision 1999/17/EC (EC, 1999a), as last amended by Commission Decision 005/389/EC (EC, 005). Each flavouring substance is attributed a FLAVISnumber (FLnumber) and all substances are divided into 34 chemical groups. Substances within a group should have some metabolic and biological behaviour in common. Substances which are listed in the register are to be evaluated according to the evaluation programme laid down in Commission Regulation (EC) No 1565/000 (EC, 000) which is broadly based on the opinion of the Scientific Committee on Food (SCF, 1999). For the submission of data by the manufacturer, deadlines have been established by Commission Regulation (EC) No 6/00 (EC,. After the completion of the evaluation programme the positive list of flavouring substances for use in or on foods in the EU shall be adopted (Article 5 (1) of Regulation (EC) No 3/96) (EC, 1996). TERMS F REFERENCE EFSA is requested to carry out a risk assessment on flavouring substances prior to their authorisation and inclusion in a positive list according to Commission Regulation (EC) No 1565/000 (EC, 000). ASSESSMENT 1. Presentation of the Substances in Flavouring Group Evaluation 16 1.1. Description The present Flavouring Group Evaluation (FGE.16), using the procedure as referred to in the Commission Regulation EC No 1565/000 (EC, 000) (The Procedure shown in schematic form in Annex I), deals with four aromatic ketones from chemical group 1, Annex I of Commission Regulation (EC) No 1565/000 (EC, 000). The four flavouring substances under consideration, as well as their chemical names, FLAVIS ( FL), Chemical Abstract Service (CAS), Council of Europe (CoE) and Flavor and Extract Manufactures Association (FEMA) numbers, structure and specifications, are listed in Table 1. The present FGE consists of four aromatic ketones: (1phenylbutan1one [FLno: 07.193], 4 phenylbutanone [FLno: 07.194], 1phenylpropanone [FLno: 07.195] and alphamethyl naphthyl ketone [FLno: 07.14], which all contain the ketone group in an aliphatic side chain. ne of the ketones contains a naphthyl group instead of a phenyl group. The four flavouring substances (candidate substances) are closely related structurally to 30 flavouring substances (supporting substances) evaluated at the 57 th meeting of the Joint FA/WH Expert Committee on Food Additives (JEFCA) in the group Aromatic substituted secondary alcohols, ketones and related esters (JECFA,. These substances, with the respective structural formulas, FEMA, CoE, and CAS register numbers, evaluation status by Scientific 4
committee on Food (SCF), JECFA, and by CoE and the European Maximised Surveyderived Daily Intake (MSDI) values, are listed in Table 3. 1.. Stereoisomers None of the four candidate substances can exist as geometrical or optical isomers. 1.3. Natural ccurrence in Food Three out of the four candidate substances have been reported to occur in one or more of the following food items: avocado, beef, cheese, cocoa, boiled egg, brandy, malt, peanut, pork liver, tomato, wild rice, juice (passion fruit), pepper, and raspberry. Quantitative data on the natural occurrence in food have been reported only for 1phenylpropanone [FLno: 07.195]: up to 0.01 mg/kg in passion fruit juice. ne of the substances, alphamethyl naphthyl ketone [FLno: 07.14] has not been reported to occur naturally in any food items according to TN (TN, 000).. Specifications Purity criteria for the four substances have been provided by the Flavour Industry (EFFA, 003o) (Table 1). Judged against the requirements in Annex II of Commission Regulation 1565/000 (EC, 000), this information is adequate for the four candidate substances (see Table 1). 3. Intake Data Annual production volumes of the flavouring substances as surveyed by the Industry can be used to calculate the Maximized SurveyDerived Daily Intake (MSDI) by assuming that the production figure only represents 60 % of the use in food due to underreporting and that % of the total EU population are consumers (SCF, 1999). However, the Panel noted that due to yeartoyear variability in production volumes, to uncertainties in the underreporting correction factor and to uncertainties in the percentage of consumers, the reliability of intake estimates on the basis of the MSDIapproach is difficult to assess. The Panel also noted that in contrast to the generally low per capita intake figures estimated on the basis of this MSDIapproach, in some cases the regular consumption of products flavoured at use levels reported by the Flavour Industry in the submissions would result in much higher intakes. In such cases, the human exposure thresholds below which exposures are not considered to present a safety concern might be exceeded. Considering that the MSDI model may underestimate the intake of flavouring substances by certain groups of consumers, the SCF recommended also taking into account the results of other intake assessments (SCF, 1999). ne of the alternatives is the Theoretical Added Maximum Daily Intake (TAMDI)approach which is calculated on the basis of standard portions and upper use levels (SCF, 1995) for flavourable beverages and foods in general, with exceptional levels for particular foods. This method is regarded as a conservative estimate of the actual intake in most consumers because it is 5
based on the assumption that the consumer regularly eats and drinks several food products containing the same flavouring substance at the upper use level. ne option to modify the TAMDIapproach is to base the calculation on normal rather than upper use levels of the flavouring substances. This modified approach is less conservative (e.g., it may underestimate the intake of consumers being loyal to products flavoured at the maximum use levels reported (EC, 000). However, it is considered as a suitable tool to screen and prioritise the flavouring substances according to the need for refined intake data (EFSA, 004a). 3.1. Estimated Daily per Capita Intake (MSDI Approach) The intake estimation is based on the Maximised SurveyDerived Daily Intake (MSDI) approach, which involves the acquisition of data on the amounts used in food as flavourings (SCF, 1999). These data are derived from surveys on annual production volumes in Europe. These surveys were conducted in 1995 by the International rganization of the Flavour Industry, in which flavour manufacturers reported the total amount of each flavouring substance incorporated into food sold in the EU during the previous year (IFI, 1995). The intake approach does not consider the possible natural occurrence in food. Average per capita intake (MSDI) is estimated on the assumption that the amount added to food is consumed by % of the population 1 (Eurostat, 1998). This is derived for candidate substances from estimates of annual volume of production provided by Industry and incorporates a correction factor of 0.6 to allow for incomplete reporting (60 %) in the Industry surveys (SCF, 1999). The total annual volume of production of the four candidate substances in the present Flavouring Group Evaluation (FGE.16) from use as flavouring substances in Europe has been reported to be approximately 9 kg (EFFA, 003p). Nearly all of this amount is accounted for by two of these flavouring substances: 1phenylbutan1one [FLno: 07.193] kg per year and 1 phenylpropan one [FLno: 07.195] 7 kg per year. For 6 of the 30 supporting substances the total annual volume of production is 3700 kg in Europe (JECFA,. The annual volumes of production in Europe for four of the supporting substances [FLno: 07.070, 09.189, 09.00 and 09.501] were not reported. n the basis of the annual volumes of production reported for the four candidate substances, the daily per capita intakes for each of these flavourings have been estimated. The estimated daily per capita intake of 1phenylbutan1one from use as a flavouring substance is 0.4 microgram, and that of 1phenylpropanone is 0.85 microgram. For the remaining two substances 4phenylbutan one and alphamethyl naphthyl ketone the estimated daily per capita intake is 0.001 microgram for each (Table ). 3.. Intake Estimated on the Basis of the Modified TAMDI (mtamdi) The method for calculation of modified Theoretical Added Maximum Daily Intake (mtamdi) values is based on the approach used by SCF up to 1995 (SCF, 1995). The assumption is that a person may consume a certain amount of flavourable foods and beverages per day. 1 EU figure 375 millions. This figure relates to EU population at the time for which production data are available, and is consistent (comparable) with evaluations conducted prior to the enlargement of the EU. No production data are available for the enlarged EU 6
For the four candidate substances information on food categories and normal and maximum use levels,3 were submitted by the Flavour Industry (EFFA, 003o). The four candidate substances are used in flavoured food products divided into the food categories, outlined in Annex III of the Commission Regulation 1565/000 (EC, 000), as shown in Table 3.1. For the present calculation of mtamdi, the reported normal use levels were used. In the case where different use levels were reported for different food categories the highest reported normal use level was used. According to the Flavour Industry the normal use levels for the four candidate substances are in the range of 15 mg/kg food, and the maximum use levels are in the range of 55 mg/kg (EFFA, 003o). The mtamdi value is 1600 microgram/person/day for each of the three candidate substances from structural class I (see Section 5) as well as for the one candidate substance from structural class III. For detailed information on use levels and intake estimations based on the mtamdi approach, see Section 6 and Annex II. Normal use is defined as the average of reported usages and maximum use is defined as the 95th percentile of reported usages (EFFA, 00i) 3 The normal and maximum use levels in different food categories (EC, 000) have been extrapolated from figures derived from 1 model flavouring substances (EFFA, 004e). 7
Table 3.1 Use of Candidate Substances Food category Description Category 1 Dairy products, excluding products of category All four Category Fats and oils, and fat emulsions (type waterinoil) All four Category 3 Edible ices, including sherbet and sorbet All four Category 4.1 Processed fruits All four Category 4. Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and None legumes), and nuts & seeds Category 5 Confectionery All four Category 6 Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & All four legumes, excluding bakery Category 7 Bakery wares All four Category 8 Meat and meat products, including poultry and game All four Category 9 Fish and fish products, including molluscs, crustaceans and echinoderms All four Category Eggs and egg products None Category 11 Sweeteners, including honey None Category 1 Salts, spices, soups, sauces, salads, protein products etc. All four Category 13 Foodstuffs intended for particular nutritional uses. All four Category 14.1 Nonalcoholic ("soft") beverages, excl. dairy products All four Category 14. Alcoholic beverages, incl. alcoholfree and lowalcoholic counterparts None Category 15 Readytoeat savouries All four Category 16 Composite foods (e.g. casseroles, meat pies, mincemeat) foods that could not be All four placed in categories 1 15 Flavourings used 4. Absorption, Distribution, Metabolism and Elimination The present FGE consists of four aromatic ketones, all containing the ketone group in an aliphatic side chain. ne of the ketones contains a naphthyl group instead of a phenyl group. No studies on absorption, distribution, metabolism or elimination were available for the four candidate substances; however a number of studies on supporting substances have been considered. Based on these studies, it is concluded that the candidate substances 1phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194], and 1phenylpropanone [FLno: 07:195] are readily absorbed from the gut. Toxicokinetic data for a representative structurally related substance (4phenyl3butenone [FLno: 07.04]) indicate that orally administered phenyl alkyl ketones undergo essentially complete firstpass metabolism prior to systemic distribution. The phenyl substituted alkyl ketones can be metabolised via reduction or oxidation and subsequent conjugation with glucuronic acid or glycine. Reduction to the corresponding secondary alcohols is either followed by conjugation with glucuronic acid and excretion, primarily in the urine or oxidation and excretion, mainly as glycine conjugates in the urine within 4 hours. These ketones may undergo omegaoxidation in the side chain to yield intermediary metabolites (e.g. hydroxyacetophenone) that undergo further oxidation and cleavage to yield aromatic carboxylic acids (phenylacetic acid or benzoic acid, depending on the number of carbon atoms in the side chain). These metabolic pathways have been observed in rodent species, dogs and humans. No information is available on the toxicokinetics of either the candidate substance alphamethyl naphthyl ketone [FLno: 07.14] or its supporting substance methyl naphthyl ketone [FLno: 07.013]. It could be hypothesised that the carbonyl group could be reduced and then conjugated, as 8
described for the phenyl substituted alkyl ketones. However, the occurrence of other, possibly harmful, metabolic pathways related to the naphthalene moiety cannot be ruled out. In conclusion, it can be anticipated that the three candidate substances 1phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194] and 1phenylpropanone [FLno: 07.195] are metabolised to innocuous products. This cannot be anticipated for the candidate substance alphamethyl naphthyl ketone [FLno: 07.14]. 5. Application of the Procedure for the Safety Evaluation of Flavouring Substances The application of the Procedure is based on intakes estimated on the basis of the MSDI approach. Where the mtamdi approach indicates that the intake of a flavouring substance might exceed its corresponding threshold of concern, a formal safety assessment is not carried out using the Procedure. In these cases the Panel requires more precise data on use and use levels. For comparison of the intake estimations based on the MSDI approach and the mtamdi approach, see Section 6. For the safety evaluation of the four candidate substances from chemical group 1 the Procedure as outlined in Annex I was applied, based on the MSDI approach. The stepwise evaluations of the four substances are summarised in Table. Step 1 Three of the candidate substances, 1phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194] and 1phenylpropanone [FLno: 07.195] are classified according to the decision tree approach by Cramer et al. (Cramer et al., 1978) into structural class I, and one candidate substance, alphamethyl naphthyl ketone [FLno: 07.14], is classified into structural class III. Step Step requires consideration of the metabolism of the candidate substances. It can be anticipated that three candidate substances, 1phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194] and 1phenylpropanone [FLno: 07.195] are metabolised to innocuous products. Accordingly, the evaluation of these three candidate substances proceeds via the Aside of the Procedure scheme. The fourth candidate substance, alphamethyl naphthyl ketone [FLno: 07.14], cannot be anticipated to be metabolised to innocuous products and thus the evaluation of [FLno: 07.14] proceeds via the Bside of the Procedure. Step A3 The three candidate substances [FLno: 07.193, 07.194 and 07.195] have estimated European daily per capita intakes ranging from 0.001 to 0.85 microgram (Table ). These intakes are below the threshold of concern of 1800 microgram/person/day for structural class I. Accordingly, these three candidate substances do not pose a safety concern when used at estimated levels of intake as flavouring substances. Step B3 The estimated daily per capita intake of the candidate substance alphamethyl naphthyl ketone [FLno: 07.14] is 0.001 microgram, which is below the threshold for its structural class of 90 microgram/person/day (class III). Accordingly, the evaluation of the substance proceeds to step B4 of the Procedure. 9
Step B4 For the candidate substance alphamethyl naphthyl ketone [FLno: 07.14] a margin of safety was estimated based upon the NAEL available for the supporting substance methyl naphthyl ketone of 33 mg/kg (90 day oral study in rats by ser et al., 1965, see section 8..) The MSDI value of 0.001 microgram/capita/day is equivalent to 0.0000 microgram/kg bw/day at a body weight of 60 kg. Thus, the margin of safety is x 9. Accordingly, alphamethyl naphthyl ketone [FLno: 07.14] is not expected to be of safety concern at its estimated level of intake as flavouring substance. 6. Comparison of the Intake Estimations Based on the MSDI Approach and the mtamdi Approach The estimated intake for each of the three candidate substances [FLno: 07.193, 07.194 and 07.195] in structural class I, based on the mtamdi is 1600 microgram/person/day, which is below the threshold of concern of 1800 microgram/person/day. The estimated intake of the substance [FLno: 07.14] assigned to structural class III, based on the mtamdi is 1600 microgram/person/day, which is above the threshold of concern for structural class III of 90 microgram/person/day. Thus, for one candidate substance [FLno: 07.14] further information is required. This would include more reliable intake data and then, if required, additional toxicological data. For comparison of the MSDI and mtamdivalues see Table 6.1 Table 6.1 Estimated intakes based on the MSDI approach and the mtamdi approach FLno EU Register name MSDI (µg/capita/day) mtamdi (µg/person/day) Structural class Threshold of concern (µg/person/day) 07.193 1Phenylbutan1one 0.4 1600 Class I 1800 07.194 4Phenylbutanone 0.001 1600 Class I 1800 07.195 1Phenylpropanone 0.85 1600 Class I 1800 07.14 alphamethyl naphthyl ketone 0.001 1600 Class III 90 7. Considerations of Combined Intakes from Use as Flavouring Substances Because of structural similarities of candidate and supporting substances, it can be anticipated that many of the flavourings are metabolised through the same metabolic pathways and that the metabolites may affect the same target organs. Further, in case of combined exposure to structurally related flavourings, the pathways could be overloaded. Therefore, combined intake should be considered. As flavourings not included in this Flavouring Group Evaluation may also be metabolised through the same pathways, the combined intake estimates presented here are only preliminary. Currently, the combined intake estimates are only based on MSDI exposure estimates, although it is recognised that this may lead to underestimation of exposure. After completion of all FGEs, this issue should be readdressed. The total estimated combined daily per capita intake of structurally related flavourings is estimated by summing the MSDI for individual substances.
n the basis of the reported annual production volumes in Europe (EFFA, 003p), the combined estimated daily per capita intake as flavourings of the three candidate substances belonging to structural class I is 1.1 microgram. This value does not exceed the threshold of concern for structural class I of 1800 microgram/person/day. The four candidate substances are structurally related to 30 supporting substances evaluated by JEFCA at its 57 th meeting (JECFA,. Based on reported production volumes, European per capita intakes (MSDI) could be estimated for 6 of the 30 supporting substances. The total combined intakes of the candidate and supporting substances are approximately 400 and 6.4 microgram/capita/day for structural class I and III substances, respectively, which do not exceed the thresholds of concern. 8. Toxicity 8.1. Acute Toxicity Data are available for two of the candidate substances, 4phenylbutanone [FLno: 07.194] and alphamethyl naphthyl ketone [FLno: 07.14]. The oral LD 50 values, in mice or rats, varied from 800 mg/kg up to 300 mg/kg bw. Thirteen (out of 30) supporting substances were tested for acute toxicity in mice and/or rats. The oral LD 50 values in mice and rats for the supporting substances range from 400 mg/kg bw to more than 5000 mg/kg bw. The acute toxicity data are summaries in Annex IV, Table IV.1. 8.. Subacute, Subchronic, Chronic and Carcinogenicity Studies Subacute and subchronic toxicity data are available for one of the candidate substances (alphamethyl naphthyl ketone [FLno: 07.14]) and for nine of the 30 supporting substances of the present flavouring group. Data on repeated dose toxicity of alphamethyl naphthyl ketone are available from one poorly reported study on male rats (5 males/group; doses of 0, 0, 500, or 00 mg/kg bw/day, oral, gavage). ne animal treated with 00 mg/kg died after the second dose and the remaining individuals of this treatment group were sacrificed on the same day. The other rats were dosed as follows: 500 mg/kg: 1 doses (16 days); 0 mg/kg: 13 doses (17 days) (Eastman Kodak Co., 199b). verall, the dose of 0 mg/kg may be considered as a LAEL (increase in absolute and relative liver weights, minimal hepatocyte hypertrophy, hyaline droplet formation in the kidney). The LAEL for the candidate substance is close to a NAEL of 33 mg/kg established by a 90 day study on the supporting substance methyl naphthyl ketone [FLno: 07.013]. Doses of 33 and 37 mg methyl naphthyl ketone/kg bw were administered via the diet to male and female rats (15/sex/group), respectively (ser et al., 1965). No effects on growth, food consumption, haematology, blood chemistry, liver and kidney weights or on gross and microscopic appearance of major organs were found. In conclusion, the NAEL of 33 mg/kg bw for methyl naphthyl ketone [FLno: 07.013] is used to estimate a margin of safety for the candidate substance alphamethyl naphthyl ketone [FLno: 07.14]. No chronic or carcinogenicity studies are available for the candidate substances. Carcinogenicity studies are available for one supporting substance, 1phenylethan1ol [FLno: 0.064] performed in mice and rats (NTP, 1990d). These studies were evaluated by JECFA at its 41 st meeting (JECFA, 1993b) when JECFA reviewed a series of studies on 1phenylethan1ol (alphamethylbenzyl alcohol), and concluded: The Committee noted that alphamethylbenzyl alcohol administered by 11
gavage in corn oil was associated with a higher incidence of renal tubulecell adenomas in male rats than in untreated controls, but not in female rats or in mice, at dose levels at or exceeding the maximum tolerated dose (MTD) and in the presence of factors that exacerbated a high incidence of agerelated chronic progressive nephropathy. The intake of this compound from all sources is extremely low. n the basis of the evidence available, the Committee concluded that the higher incidence of benign neoplasms in the kidney of male rats is not relevant to humans. In view of the limited database, the Committee concluded that the available data could be used to set an ADI by application of a safety factor of 00 to the minimaleffect level of 93 mg/kg of body weight per day with respect to liver weight increase in the absence of associated pathology in the 13week study in rats. Accordingly, an ADI of 0 0.1 mg/kg of body weight per day was allocated for alphamethylbenzyl alcohol. The Panel concurred with this conclusion. The toxicity data are summaries in Annex IV, Table IV.. 8.3. Developmental / Reproductive Toxicity Studies Data on developmental toxicity and reproductive toxicity have been published for one of the candidate substances, alphamethyl naphthyl ketone [FLno: 07.14] (Sporn et al., 1963), but this study is considered to be of insufficient quality. The developmental/reproductive toxicity data are summaries in Annex IV, Table IV.3. 8.4. Genotoxicity Studies In vitro data are only available for two candidate substances which were negative in tests for mutagenicity in bacteria, however these studies are of insufficient quality. All other in vitro data are related to nine supporting substances. For these nine substances tested in the Ames test the results were negative. However, two of the supporting substances, 1phenylethan1ol [FLno: 0.064] and acetophenone [FLno: 07.004] induced chromosomal aberrations in vitro in the presence of metabolic activation, and 1phenylethan1ol was also weakly positive in the mouse lymphoma tk assay. There are negative in vivo micronucleus tests on two supporting substances (4(pmethoxyphenyl) butanone [FLno: 07.09] and methylbetamaphthyl ketone [FLno: 07.013]) but these studies are not considered valid. verall, the limited data available do not allow a final assessment of genotoxicity. From the data available there is some indication of genotoxic potential for two of the supporting substances (1 phenylethan1ol and acetophenone). However, in the light that 1phenylethan1ol can be metabolised to acetophenone and vice versa and that the results of a carcinogenicity study with 1 phenylethan1ol in mice and rats do not give rise to concern, the Panel concluded that the positive in vitro results for the two supporting substances do not give rise to concern with respect to carcinogenicity in humans. Regarding the prediction of risk of heritable mutations to man, adequate data on germ cell mutagenicity were not available nor data from a twogeneration developmental toxicity study. However, toxicokinetic data for another structurally related substance (4phenyl3 butenone [FLno: 07.04]) indicate that orally administered phenyl alkyl ketones undergo essentially complete firstpass metabolism prior to systemic distribution and that germ cells are unlikely to be exposed. Therefore, the Panel concluded that the positive in vitro results for the two supporting substances do likewise not give rise to concern with respect to heritable mutations in humans and that, finally, the positive in vitro results for the two supporting substances do not preclude their evaluation through the Procedure. The genotoxicity data are summaries in Annex IV, Table IV.4 and Table IV.5. 1
9. Conclusions The four candidate substances are aromatic ketones and belong to chemical group 1. None of the four candidate substances can exist as geometrical or optical isomers. Three of the flavouring substances are classified into structural class I and one substance is classified into structural class III. Three of the substances in the present group have been reported to occur naturally in a wide range of food items. According to the default MSDI approach, the four flavouring substances in this group have intakes in Europe from 0.001 to 0.85 microgram/capita/day which are below the threshold of concern values for structural class I of 1800 microgram/person/day and for structural class III of 90 microgram/person/day. n the basis of the reported annual production in Europe (MSDI approach) the combined intake of the three candidate substances belonging to structural class I would result in a combined intake of approximately 1.1 microgram/capita/day. This value is lower than the threshold of concern for structural class I substance. Based on reported production volumes, European per capita intakes (MSDI) could be estimated for 6 of the 30 supporting substances. The total combined intakes of the candidate and supporting substances are approximately 400 and 6.4 microgram/capita/day for structural class I and III, respectively, which do not exceed the thresholds of concern. verall, the limited data available do not allow a final assessment of genotoxicity. From the data available there is some indication of genotoxic potential for two of the supporting substances (1 phenylethan1ol and acetophenone). However, taking into consideration metabolism and carcinogenicity data on the candidate and supporting substances, the positive in vitro results do not preclude their evaluation through the Procedure. It can be anticipated that the three candidate substances 1phenylbutan1one [FLno: 07.193], 4 phenylbutanone [FLno: 07.194], 1phenylpropanone [FLno: 07.195] are metabolised to innocuous products at the estimated levels of intake. This cannot be anticipated for alphamethyl naphthyl ketone [FLno: 07.14]. Based on a NAEL of 33 mg/kg bw/day for the supporting substance methyl naphthyl ketone [FLno: 07.013] which is structurally related to the candidate substance alphamethyl naphthyl ketone [FLno: 07.14] a margin of safety of approximately x 9 could be estimated for this candidate substance. Thus alphamethyl naphthyl ketone [FLno: 07.14] is not expected to be of safety concern at its estimated level of intake as flavouring substance. It was noted that where toxicity data were available they were consistent with the conclusions in the present flavouring group evaluation using the Procedure. It was considered that on the basis of the default MSDI approach these four flavouring substances would not give rise to safety concerns at the estimated levels of intake arising from their use as flavouring substances. When the estimated intakes were based on the mtamdi approach they were 1600 microgram/person/day for each of the four flavouring substances. Thus, the intakes were below the threshold of concern for structural class I of 1800 microgram/person/day for the three candidate substances belonging to structural class I. For the one flavouring substance alphamethyl naphthyl ketone [FLno: 07.14] belonging to structural class III the intake was above the threshold of concern of 90 microgram/person/day for structural class III. The three substances which have 13
mtamdi intake estimates below the threshold of concern for structural class I, are also expected to be metabolised to innocuous products. Thus for one of the four flavouring substances considered in this opinion the intake, estimated on the basis of the mtamdi, exceeds the relevant threshold for its structural class, to which the flavouring substance has been assigned. Therefore, for alphamethyl naphthyl ketone [FLno: 07.14] more reliable exposure data are required. n the basis of such additional data, this flavouring substance should be reconsidered along the steps of the Procedure. Following this procedure additional toxicological data might become necessary. In order to determine whether the conclusion for the four candidate substances can be applied to the materials of commerce, it is necessary to consider the available specifications: Adequate specifications including complete purity criteria and identity tests have been provided for the four materials of commerce 1phenylbutan1one [FLno: 07.193], 4phenylbutanone [FLno: 07.194], 1phenylpropanone [FLno: 07.195] and alphamethyl naphthyl ketone [FLno: 07.14] and these would present no safety concern at the levels of intake estimated on the basis of the MSDI approach. 14
TABLE 1: SPECIFICATIN SUMMARY F THE SUBSTANCES IN THE FLAVURING GRUP EVALUATIN 16 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 16 FLno EU Register name Structural formula FEMA no CoE no CAS no 07.193 1Phenylbutan1one 495409 Phys.form Mol.formula Mol.weight Liquid C H 1 148.0 Solubility 1) Solubility in ethanol ) Practically insoluble or insoluble 1 ml in 1 ml Boiling point, C 3) Melting point, C ID test Assay minimum 9 MS 95 % Refrac. Index 4) Spec.gravity 5) 1.5171.53 0.9860.99 Specification comments 07.194 4Phenylbutanone 1118 55067 Liquid C H 1 148.0 Practically insoluble or insoluble 1 ml in 1 ml 35 MS 99 % 1.5091.515 0.9850.991 07.195 1Phenylpropanone 07.14 alphamethyl naphthyl ketone 14 3797 941980 Liquid C 9H 134.18 Liquid C 1H 170.1 Practically insoluble or insoluble 1 ml in 1 ml Practically insoluble or insoluble 1 ml in 1 ml 14 MS 95 % 98 MS 95 % 1.5131.519 1.0041.0 1.651.631 1.1161.1 1) Solubility in water, if not otherwise stated ) Solubility in 95% ethanol, if not otherwise stated 3) At 13.5 hpa, if not otherwise stated 4) At 0 C, if not otherwise stated 5) At 5 C, if not otherwise stated 15
TABLE : SUMMARY F SAFETY EVALUATIN APPLYING THE PRCEDURE (BASED N INTAKES CALCULATED BY THE MSDI APPRACH) Table : Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) FLno EU Register name Structural formula MSDI 1) (µg/capita/day) Class ) Evaluation procedure path 3) 07.193 1Phenylbutan1one 0.4 Class I A3: Intake below threshold utcome on the named compound [ 4) or 5] 4) 6) utcome on the material of commerce [6), 7), or 8)] Evaluation remarks 07.194 4Phenylbutanone 0.001 Class I A3: Intake below threshold 4) 6) 07.195 1Phenylpropanone 0.85 Class I A3: Intake below threshold 4) 6) 07.14 alphamethyl naphthyl ketone 0.001 Class III B3: Intake below threshold, B4: Adequate NEL exists 4) 6) 1) MSDI: Amount added to food as flavour in (kg / year) x E9 / (0.1 x population in Europe (= 375 x E6) x 0.6 x 365) = µg/capita/day ) Thresholds of concern: Class I = 1800, Class II = 540, Class III = 90 µg/person/day 3) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot. 4) No safety concern based on intake calculated by the MSDI approach of the named compound. 5) Data must be available on the substance or closely related substances to perform a safety evaluation. 6) No safety concern at estimated level of intake of the material of commerce meeting the specification of Table 1 (based on intake calculated by the MSDI approach) 7) Tentatively regarded as presenting no safety concern (based on intake calculated by the MSDI approach) pending further information on the purity of the material of commerce and/or information on stereoisomerism is insufficient. 8) No conclusion can be drawn due to lack of information on the purity of the material of commerce. 16
TABLE 3: SUPPRTING SUBSTANCES SUMMARY Table 3: Supporting Substances Summary FLno EU Register name Structural formula FEMA no CoE no CAS no 0.033 1Phenylpropan1ol 884 8 93549 JECFA no Specification available 8 MSDI (EU) 1) (µg/capita/day) 0.4 SCF status ) JECFA status 3) CoE status 4) Category B b) Comments H 0.034 1Phenylpentanol H 953 83 705737 85 0.1 Category B b) 0.036 4Phenylbutanol H 879 85 344709 815 1. Category B b) 0.064 1Phenylethan1ol H 685 030 98851 0.065 4Methyl1phenylpentanol 08 031 H 7779784 799 87 7 4 Deleted b) Category A b) ADI: 00.1 (JECFA, 1993a) 0.080 1(pTolyl)ethan1ol H 3139 197 536505 07.004 Acetophenone 009 138 9886 07.013 Methyl naphthyl ketone 73 147 93083 805 806 811 0.1 15 6.3 Category A b) Category A b) No ADI allocated (JECFA, 1981a) 07.0 4Methylacetophenone 07.03,4Dimethylacetophenone 677 156 1009 387 157 89747 807 809 0.4 Category B b) Category B b) 17
Table 3: Supporting Substances Summary FLno EU Register name Structural formula FEMA no CoE no CAS no 07.05 4Methyl1phenylpentanone 740 159 53496 JECFA no Specification available 88 MSDI (EU) 1) (µg/capita/day) 8.5 SCF status ) JECFA status 3) CoE status 4) Category B b) Comments 07.06 4(pTolyl)butanone 3074 160 7774790 817 00d) 0.01 Category B b) 07.09 4(4Methoxyphenyl)butanone 67 163 401 818 4.5 Category A b) 07.038 4Methoxyacetophenone 07.040 1Phenylpropan1one 005 570 0061 3469 599 93550 8 84 130 0.01 Category B b) Category A b) 07.04 4Isopropylacetophenone 97 651 645136 07.070 3Benzylheptan4one 146 140 749377 808 830 0.01 ND Category B b) Deleted b) 07.079 1Phenylpropan1,dione 36 75 579077 833 4.9 Category B b) 07.086 1,3Diphenylpropanone 397 11839 045 83 0.1 07.087 4Methoxyphenylacetone 674 11836 1849 813 0.1 18
Table 3: Supporting Substances Summary FLno EU Register name Structural formula FEMA no CoE no CAS no 09.144 1Phenethyl propionate 689 45 10456 JECFA no Specification available 80 MSDI (EU) 1) (µg/capita/day) 0.97 SCF status ) JECFA status 3) CoE status 4) Category B b) Comments 09.178 1Phenethyl acetate 684 573 9395 801 170 Category A b) 09.179 1Phenethyl formate 688 574 7775384 800 0.037 Category B b) 09.189 1Phenylpropyl butyrate 44 68 031864 83 ND Category B b) 09.00 1Methyl3phenylpropyl acetate 88 671 415880 816 ND Category B b) 09.31 1Phenethyl butyrate 686 083 3460444 803 1.1 Category B b) 09.49 1Methylphenethyl butyrate 3197 76 68911 814 00d) 0.1 Category B b) 09.476 Ethyl 3phenyl3oxopropionate 43 67 9400 834 0.01 Category B b) 09.486 1Phenethyl isobutyrate 687 088 7775395 804 4 Category B b) 19
Table 3: Supporting Substances Summary FLno EU Register name Structural formula FEMA no CoE no CAS no 09.501 Ethyl acetyl3phenylpropionate 416 41 60791 JECFA no Specification available 835 MSDI (EU) 1) (µg/capita/day) ND SCF status ) JECFA status 3) CoE status 4) Category B b) Comments 1) MSDI: Amount added to food as flavouring substance in (kg / year) x E9 / (0.1 x population in Europe (= 375 x E6) x 0.6 x 365) = µg/capita/day ) Category 1: Considered safe in use, Category : Temporarily considered safe in use, Category 3: Insufficient data to provide assurance of safety in use, Category 4: Not acceptable due to evidence of toxicity 3) No safety concern at estimated levels of intake 4) Category A: Flavouring substance, which may be used in foodstuffs, Category B: Flavouring substance which can be used provisionally in foodstuffs a) (JECFA, b) (CoE, 199) ND) No intake data reported 0
ANNEX I: PRCEDURE FR THE SAFETY EVALUATIN The approach for a safety evaluation of chemically defined flavouring substances as referred to in Commission Regulation EC No 1565/000 (EC, 000), named the "Procedure", is shown in schematic form in Figure I.1. The Procedure is based on the opinion of the Scientific Committee on Food expressed on December 1999 (SCF, 1999), which is derived from the evaluation procedure developed by the Joint FA/WH Expert Committee on Food Additives at its 44th, 46th and 49th meetings (JECFA, 1995; JECFA, 1996a; JECFA, 1997a; JECFA, 1999b). The Procedure is a stepwise approach that integrates information on intake from current uses, structureactivity relationships, metabolism and, when needed, toxicity. ne of the key elements in the procedure is the subdivision of flavourings into three structural classes (I, II, III) for which thresholds of concern (human exposure thresholds) that are not considered to present a safety concern have been specified. Class I contains flavourings that have simple chemical structures and efficient modes of metabolism, which would suggest a low order of oral toxicity. Class II contains flavourings that have structural features that are less innocuous, but are not suggestive of toxicity. Class III comprises flavourings that have structural features that permit no strong initial presumption of safety, or may even suggest significant toxicity (Cramer et al., 1978). The thresholds of concern for these structural classes of 1800, 540 or 90 microgram/person/day, respectively are derived from a large database containing data on subchronic and chronic animal studies (JECFA, 1996a). In Step 1 of the Procedure, the flavourings are assigned to one of the structural classes. The further steps address the following questions: can the flavourings be predicted to be metabolised to innocuous products 4 (Step )? do their exposures exceed the threshold of concern for the structural class (Step A3 and B3)? are the flavourings or their metabolites endogenous 5 (Step A4)? does a NAEL exist on the flavourings or on structurally related substances (Step A5 and B4)? In addition to the data provided for the flavouring substances to be evaluated (candidate substances), toxicological background information available for compounds structurally related to the candidate substances is considered (supporting substances), in order to assure that these data are consistent with the results obtained after application of the Procedure. The Procedure is not to be applied to flavourings with existing unresolved problems of toxicity. Therefore, the right is reserved to use alternative approaches if data on specific flavourings warranted such actions. 4 Innocuous metabolic products : Products that are known or readily predicted to be harmless to humans at the estimated intakes of the flavouring agent (JECFA, 1997a). 5 Endogenous substances : Intermediary metabolites normally present in human tissues and fluids, whether free or conjugated; hormones and other substances with biochemical or physiological regulatory functions are not included (JECFA, 1997a). 1
Procedure for Safety Evaluation of Chemically Defined Flavouring Substances Step 1. Decision tree structural class Step. Can the substance be predicted to be metabolised to innocuous products? Step A3. Yes No Step B3. Do the conditions of use result in an intake greater than the threshold of concern for the structural class? Data must be available on the substance or closely related substances to perform a safety evaluation Yes Do the conditions of use result in an intake greater than the threshold of concern for the structural class? Step A4. Yes No Step B4. No Is the substance or are its metabolites endogenous? Yes Substance would not be expected to be of safety concern Yes Does a NAEL exist for the substance which provides an adequate margin of safety under conditions of intended use, or does a NAEL exist for structurally related substances which is high enough to accommodate any perceived difference in toxicity between the substance and the related substances? Step A5. No Yes No Does a NAEL exist for the substance which provides an adequate margin of safety under conditions of intended use, or does a NAEL exist for structurally related substances which is high enough to accommodate any perceived difference in toxicity between the substance and the related substances? No Additional data required Figure I.1 Procedure for Safety evaluation of Chemically Defined Flavouring Substances