Scientific Opinion on the re-evaluation of candelilla wax (E 902) as a food additive 1

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1 EFSA Journal 2012;10(11):2946 SCIENTIFIC OPINION Scientific Opinion on the re-evaluation of candelilla wax (E 902) as a food additive 1 EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3 European Food Safety Authority (EFSA), Parma, Italy This scientific output, published on 28 January 2013, replaces the earlier version published on 16 November ABSTRACT The Panel on Food Additives and Nutrient Sources added to Food (ANS) delivers a scientific opinion reevaluating the safety of candelilla wax (E 902). Candelilla wax (E 902) is authorised in the EU as a food additive as a glazing agent. It has been evaluated by the Scientific Committee on Food (SCF) and by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The JECFA and the SCF did not establish an Acceptable Daily Intake (ADI) but considered the use of candelilla wax as a glazing agent acceptable. Candelilla wax is obtained from leaves of candelilla plants Euphorbia antisyphilitica found in semi-desert regions. It is a complex mixture composed of wax hydrocarbons, wax, resin esters, lactones, free wax resin alcohols, and free wax resin acids. The Panel considered that absorption of candelilla wax is expected to be low, and that upon absorption the components would be incorporated into normal metabolic pathways. The Panel noted that there were limited data comparing the composition of the candelilla wax tested with the specification of the food additive. However the Panel considered that the materials tested were likely to be broadly similar in composition to the food additive. Overall, the Panel considered that the available data suggest that candelilla wax is not genotoxic. The Panel considered that long-term toxicity data on candelilla wax were lacking and therefore did not establish an ADI. However, the Panel noted that available toxicity studies consistently reported no findings associated with intake of the main components constituting candelilla wax. Furthermore, consideration of the exposure estimates to candelilla wax, using the Maximum Permitted Level (MPL) of carnauba wax, indicated sufficient margins of safety. This allowed the Panel to conclude that the use of candelilla wax as a food additive with the currently authorised uses would not be of safety concern. European Food Safety Authority, On request from the European Commission, Question No EFSA-Q , adopted on 24 October Panel members: Fernando Aguilar, Riccardo Crebelli, Birgit Dusemund, Pierre Galtier, David Gott, Ursula Gundert-Remy, Jürgen König, Claude Lambré, Jean-Charles Leblanc, Alicja Mortensen, Pasquale Mosesso, Dominique Parent-Massin, Ivan Stankovic, Paul Tobback, Ine Waalkens-Berendsen, Rudolf Antonius Woutersen and Matthew Wright. Correspondence: ans@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group B on Food Additives and Nutrient Sources added to Food: Fernando Aguilar, Martine Bakker, Riccardo Crebelli, Birgit Dusemund, David Gott, Torben Hallas-Møller, Jürgen König, Daniel Marzin, Inge Meyland, Alicja Mortensen, Iona Pratt, Ine Waalkens-Berendsen and Rudolf Antonius Woutersen for the preparatory work on this scientific opinion. 4 Editorial changes were made on page 10. The changes do not affect the overall conclusions of the opinion. Suggested citation: EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the reevaluation of candelilla wax (E 902) as a food additive. EFSA Journal 2012;10(11):2946. [27 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2012

2 KEY WORDS Candelilla wax, INS No. 902, CAS Registry Number , EINECS EFSA Journal 2012;10(11):2946 2

3 SUMMARY Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to Food (ANS) of the European Food Safety Authority (EFSA) was asked to deliver a scientific opinion on the re-evaluation of candelilla wax (E 902) as a food additive. The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations, additional literature that became available since then and the data available following a public call for data. The Panel noted that not all original studies on which previous evaluations were based were available for re-evaluation by the Panel. Candelilla wax is authorised in the European Union as a glazing agent on confectionery (including chocolate) and other products, and for the surface treatment of certain fruits. It is also authorised in food supplements. Candelilla wax is also used in cosmetics to give structure to solid and stick-like preparations or in formulations like fragrance and hair care preparations and pharmaceuticals. Candelilla wax has been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1993 and 2005 which concluded that at the predicted dietary exposure there was no safety concern. The Scientific Committee for Food (SCF) evaluated the safety of candelilla wax in 1990 and did not establish an ADI but considered its use as glazing agent temporarily acceptable. Candelilla wax is obtained from the leaves of candelilla plants Euphorbia antisyphilitica found in semi-desert regions. It is described as a hard, yellowish-brown, opaque to translucent wax. The chemical composition and physical properties of the wax vary with climate, time and region of harvest and with the age of the plant. The average composition of candelilla wax has been reported as consisting of hydrocarbons (42% w/w), wax, resin, and sitosteroyl esters (39% w/w), lactones (6% w/w) and free wax resin acids (8% w/w) and free wax and resin alcohols (5% w/w). Specifications have been defined in Directive 2008/84/EC and new specifications according to Commission Regulation (EU) No 231/2012 will apply from 1 st December No new data on absorption, distribution, metabolism or excretion (ADME) of candelilla wax were available to the Panel for this evaluation. However, the Panel also considered the results of ADME studies conducted with its main constituents. Overall, the Panel considered that absorption of candelilla wax is expected to be low, and that upon absorption the components would be incorporated into normal metabolic pathways. Overall, the Panel considered that the available data suggest that candelilla wax is not genotoxic. Although the database is very limited and does not include studies in mammalian cells, the Panel, taking account of the lack of structural alerts for genotoxicity in addition to available data, considered that candelilla wax is not genotoxic. The lactones present in candelilla wax are considered to be stable and not to belong to the category of reactive genotoxic lactones characterized by a low number of carbons in the heterocycle. Available in vivo toxicological studies, including short-term and subchronic studies conducted on candelilla wax mixtures, short-term, subchronic, chronic toxicity and carcinogenicity studies as well as reproductive and developmental toxicity studies conducted on its main components, did not report adverse effects. No-observed adverse effect levels (NOAEL) from these studies range from 600 mg/kg bw/day to 2400 mg/kg bw/day. EFSA Journal 2012;10(11):2946 3

4 The Panel noted that there were limited data comparing the composition of the candelilla wax tested with the specification of the food additive. However the Panel considered that the materials tested were likely to be broadly similar in composition to the food additive. Exposure estimates for all age groups (toddlers, children, adolescents, adults, and the elderly) performed by the Panel show that the mean dietary exposure of European toddlers (aged months) ranged from 2.6 to 4.6 mg/kg bw/day, and from 3.1 to 8.1 mg/kg bw/day at the 95 th percentile. The mean dietary exposure of European children (aged 3-9 years) ranged from 1.6 to 4.5 mg/kg bw/day, and from 3.2 to 7.6 mg/kg bw/day at the 95 th percentile. For European adolescents (aged years) the mean dietary exposure ranged from 0.9 to 2.1 mg/kg bw/day, and from 1.9 to 3.8 mg/kg bw/day at the 95 th percentile. For the European adult population a mean dietary exposure in the range of mg/kg bw/day was estimated and mg/kg bw/day for high level consumers. For the elderly mean exposure estimates were in the range of mg/kg bw/day and in the range of mg/kg bw/day at the 95 th percentile. The main contributors to the total anticipated mean exposure to candelilla wax (> 10%) were fruits and confectionary in most population groups. For the highest consumer populations (95 th percentile) these exposures estimates would result in margins of safety between 74 and 400 when compared to the lowest NOAEL (600 mg/kg bw/day) and between 296 and 1600 when compared the highest NOAEL (2400 mg/kg bw/day) identified in the toxicity studies. Overall, the Panel considered that long-term toxicity data on candelilla wax was lacking and therefore did not establish an Acceptable Daily Intake (ADI). However, the Panel noted that available toxicity studies consistently reported no findings associated with intake of the main components constituting candelilla wax. Furthermore, consideration of the exposure estimates to candelilla wax, using the maximum permitted levels of carnauba wax, indicated sufficient margin of safety, which allowed the Panel to conclude that the use of candelilla wax as a food additive with the currently authorised uses would not be of safety concern. EFSA Journal 2012;10(11):2946 4

5 TABLE OF CONTENTS Abstract... 1 Summary... 3 Table of contents... 5 Background as provided by the European Commission... 6 Terms of reference as provided by the European Commission... 6 Assessment Introduction Technical data Identity of the substance Specifications Manufacturing Process Methods of analysis in foods Reaction and fate in foods, stability Case of need and proposed uses Information on existing authorisations and evaluations Exposure Food consumption data used for exposure assessment Exposure to candelilla wax from its use as food additive Main food groups contributing to exposure of candelilla wax using MPL Other sources of exposure Biological and toxicological data Absorption, distribution, metabolism and excretion Toxicological data Acute oral toxicity Short-term and subchronic toxicity Genotoxicity Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Allergenicity, hypersensitivity and intolerance Human data Discussion Conclusions Documentation provided to EFSA References Glossary and abbreviations EFSA Journal 2012;10(11):2946 5

6 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Regulation (EC) No 1333/ of the European Parliament and of the Council on food additives requires that food additives are subject to a safety evaluation by the European Food Safety Authority (EFSA) before they are permitted for use in the European Union. In addition, it is foreseen that food additives must be kept under continuous observation and must be re-evaluated by EFSA. For this purpose, a programme for the re-evaluation of food additives that were already permitted in the European Union before 20 January 2009 has been set up under Regulation (EU) No 257/ This Regulation also foresees that food additives are re-evaluated whenever necessary in light of changing conditions of use and new scientific information. For efficiency and practical purposes, the re-evaluation should, as far as possible, be conducted by group of food additives according to the main functional class to which they belong. The order of priorities for the re-evaluation of the currently approved food additives should be set on the basis of the following criteria: the time since the last evaluation of a food additive by the Scientific Committee on Food (SCF) or by EFSA, the availability of new scientific evidence, the extent of use of a food additive in food and the human exposure to the food additive taking also into account the outcome of the Report from the Commission on Dietary Food Additive Intake in the EU 7 of The report Food additives in Europe submitted by the Nordic Council of Ministers to the Commission, provides additional information for the prioritisation of additives for reevaluation. As colours were among the first additives to be evaluated, these food additives should be re-evaluated with the highest priority. In 2003, the Commission already requested EFSA to start a systematic re-evaluation of authorised food additives. However, as a result of the adoption of Regulation (EU) 257/2010 the 2003 Terms of Reference are replaced by those below. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION The Commission asks the European Food Safety Authority to re-evaluate the safety of food additives already permitted in the Union before 2009 and to issue scientific opinions on these additives, taking especially into account the priorities, procedure and deadlines that are enshrined in the Regulation (EU) No 257/2010 of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with the Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives. 5 OJ L 354, , p OJ L 80, , p19 7 COM(2001) 542 final. 8 Food Additives in Europe 2000, Status of safety assessments of food additives presently permitted in the EU, Nordic Council of Ministers. TemaNord 2002:560. EFSA Journal 2012;10(11):2946 6

7 ASSESSMENT 1. Introduction The present opinion deals with the re-evaluation of the safety of candelilla wax (E 902) when used as a food additive. Candelilla wax is authorised in the European Union as a glazing agent on confectionery (including chocolate) and other products, and for the surface treatment of certain fruits (Directive 95/2/CE 9 ; EU Regulation 1333/ ). It has been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2005 which concluded that at the predicted dietary exposure there was no safety concern (JECFA, 2005). The Scientific Committee on Food (SCF) evaluated candelilla wax in 1990 (SCF, 1992) and did not establish an ADI but considered its use temporarily acceptable. Candelilla wax was also reviewed by TemaNord in 2002 (TemaNord, 2002). The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations, additional literature that became available since then and the data available following a public call for data 11. Some of the original studies, on which previous evaluations were based, were not available for re-evaluation by the Panel. 2. Technical data 2.1. Identity of the substance Candelilla wax is obtained from leaves of candelilla plants found in the semi-desert regions of the United States, Mexico and parts of Central and South America (Directive 2008/84/EC) 12. It has the CAS Registry Number Candelilla wax (E 902) is described as a hard, yellowish-brown, opaque to translucent wax and is obtained from the leaves of the candelilla plant, Euphorbia antisyphilitica (Directive 2008/84/EC). The chemical composition and physical properties of the wax vary with climate, time and region of harvest and with the age of the plant. The average composition of candelilla wax has been reported as consisting of hydrocarbons (42% w/w), essentially in the form of paraffins (98%) and alkenes (2%); wax, resin and sitosteroyl esters (39% w/w); lactones (6% w/w); free waxe resin acids (8% w/w) and wax, resin alcohols such as terpene alcohols (5% w/w) (Table 1). The most abundant n-alkane is n-hentriacontane (C 31 ) (CAS Registry Number ) comprising more than 80% of the total n-alkanes in candelilla wax (Wolfmeier et al., 2005). Minor components (< 1% w/w) of candelilla wax are described by JECFA as free acids, free alcohols, sterols, neutral resins, and mineral matter (CTA, 2006). 9 European Parliament and Council Directive 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners. OJ L 61, , p Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. OJ L 354, p Call for scientific data on miscellaneous waxes permitted as food additives in the EU (published: 23 November 2009). Available from: 12 Commission Directive 2008/84/EC of 27 August 2008 laying down specific purity criteria on food additives other than colours and sweeteners. OJ L 253, , p EFSA Journal 2012;10(11):2946 7

8 Table 1: Average composition of candelilla wax (JECFA 2006). Components Candelilla wax (%) Hydrocarbons total paraffins (alkanes) alkenes Wax, resin, and sitosterol esters 39 Lactones 6 Free wax resin acids 8 Free wax and resin alcohols (terpene alcohol) 5 Total 100 Candelilla wax is described as insoluble in water but soluble in chloroform and toluene (Commission Regulation (EU) No 231/ ). Some synonyms are candelilla resin E1, CAW 30M, CG7, FC 31, FR 100, MD 21, NC 1630, Noda Wax NC 1630, SR 3, Towax Specifications Specifications have been defined in the Directive 2008/84/EC and new specifications according to Commission Regulation (EU) No 231/2012 will apply from 1 December Specifications have also been defined by JECFA (JECFA, 2006). Table 2: Specifications for candelilla wax according to Commission Regulation (EU) No 231/2012 and JECFA (2006). DEFINITION DESCRIPTION IDENTIFICATION Solubility Commission Regulation (EU) No 231/2012 Candelilla wax is a purified wax obtained from the leaves of the candelilla plant, Euphorbia antisyphilitica Hard, yellowish-brown, opaque to translucent wax JECFA (2006) Crude candelilla wax is obtained by first boiling the dried stalks of the candelilla plant (Euphorbia antisyphilitica) in water acidified with sulphuric acid to release the wax. Yellowish-brown hard, brittle, lustrous solid with an aromatic odour when heated Insoluble in water. Soluble in chloroform and toluene Insoluble in water; soluble in toluene Melting range 68.5 C C 68.5 C C Specific gravity About Infra-red absorption - The infra-red spectrum of the sample, melted and prepared for 13 Commission Regulation (EU) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council. OJ L 83, , p EFSA Journal 2012;10(11):2946 8

9 PURITY Acid value a Commission Regulation (EU) No 231/2012 JECFA (2006) analysis on a potassium bromide plate, corresponds to that of a candelilla wax standard. Not less than 12 and not more than Between 12 and Saponification value b Not less than 43 and not more than Between 43 and Ester value c - Between 31 and 43 Glycerol and other polyols Not more than 0.5% (as glycerol) - Ceresin, paraffins and certain Absent - other waxes Fats, Japan wax, rosin and soaps Absent - Lead Not more than 2 mg/kg Not more than 2 mg/kg Arsenic Not more than 3 mg/kg - Mercury Not more than 1 mg/kg - a: The Acid Value (AV) is defined as the mass of KOH (in mg) required to neutralise the acid groups contained in 1 g Dry Substance (DS). b: The saponification value is defined as the number of mg of potassium hydroxide (KOH) to saponify the esters in 1 g of the sample and neutralize the free acids in 1 g of a sample. c: The ester value is defined as the number of mg of potassium hydroxide (KOH) required to saponify the esters in 1 g of a sample. The Panel noted minor differences between JECFA and EU specifications for candelilla wax Manufacturing Process Manufacturing of candelilla wax has been described by JECFA as first boiling dried stalks of the candelilla plant in water acidified with sulphuric acid to release the wax. The molten wax is then skimmed off and allowed to solidify and refined by further treatment with sulphuric acid and subsequent passage through filter-presses (JECFA, 2005). The material can be further refined by filtering through Fuller s earth, activated carbon or some other filtration system. The refining can also include a bleaching stage, which uses hydrogen peroxide, or some other refining step for special applications (The Candelilla Institute, 2010; Methods of analysis in foods Several methods for the determination of candelilla wax in foods or other items are described in the literature: differential scanning calorimetry (DSC) (Ritter et al., 2001); thin layer chromatography (TLC) (Tada et al., 2007; Rezic et al., 2009); thermally assisted hydrolysis and methylation (THM) GC/MS (Asperger et al., 2001); column and gas chromatography (Tonogai et al., 1985) Reaction and fate in foods, stability No specific documentation has been submitted or found in the open literature, but as the components of candelilla wax are rather inert and stable it can be assumed that degradation or reaction with food components will not take place to a significant extent. EFSA Journal 2012;10(11):2946 9

10 2.6. Case of need and proposed uses Re-evaluation of candelilla wax (E 902) as a food additive Candelilla wax is used as a gas and moisture barrier to prolong the shelf-life of foodstuffs or to impart gloss. Candelilla wax is authorised in the European Union as a glazing agent on confectionery (including chocolate), small products of fine bakery wares coated with chocolate, snacks, nuts and coffee beans and for the surface treatment of certain fruits only (fresh citrus fruits, melons, apples, pears, peaches and pineapples); it is also permitted on food supplements (Directive 95/2/EC). Candelilla wax is also used in cosmetics to give structure to solid and stick-like preparations or in formulations like fragrance and hair-care preparations and pharmaceuticals (Liebert, 1984). Table 3 summarises foods that are permitted to contain candelilla wax and the corresponding Maximum Permitted Levels (MPLs) as set by Commission Regulation (EU) N 1129/ As candelilla wax is permitted without defined maximum levels, it was assumed for exposure estimates that candelilla wax is used to the same extent as carnauba wax at its corresponding MPL. Table 3: Maximum Permitted Levels of use of candelilla wax in foodstuffs according to the European Parliament and Council Directive 95/2/EC and the levels used for the exposure assessment in foodstuffs used for the refined exposure assessment Foodstuffs Maximum Permitted Level (mg/kg) Levels used for exposure assessment (mg/kg) Confectionary (including chocolate), as glazing agent only qs 500 Small products of fine bakery wares coated with chocolate, as glazing agent only qs 200 Snacks qs 200 Nuts qs 200 Coffee beans qs 200 Food supplements qs 200 Fresh citrus fruits, melons, apples, pears, peaches and pineapples (surface treatment only) qs 200 qs - quantum satis 2.7. Information on existing authorisations and evaluations According to Directive 95/2/EC, candelilla wax (E 902) is authorised as a glazing agent and is also permitted as a surface treatment on fresh citrus fruits, melons, apples and pears as well as on peaches and pineapples. The SCF evaluated the safety of candelilla wax in 1990 (SCF, 1992). The Committee took into consideration the available toxicity data, its long established use without reported adverse effects and the expected limited intake when used as a glazing agent and accepted the temporary use of candelilla wax. (SCF, 1992). 14 Commission regulation (EU) no 1129/2011 of 11 November 2011 amending Annex II to regulation (EC) No 1333/2008 of the European Parliament and of the Council by establishing a Union list of food additive. OJ L 295 p EFSA Journal 2012;10(11):

11 Candelilla wax is permitted for use in plastics in contact with food (Commission Regulation 10/2011/EC 15 ) with no specific migration limit. The use of candelilla wax as a glazing agent, chewing gum base component, surface finishing agent and carrier for flavours was evaluated by JECFA in 1993 (JECFA, 1993). JECFA concluded that these functional uses did not raise any toxicological concerns. JECFA re-evaluated candelilla wax in 2005 to consider the acceptability of its use as a carrier for flavours in water-based flavoured drinks, including sport, energy or electrolyte drinks and particulated drinks, based on adopted provisions in the GSFA (Codex General Standard for Food Additives) (JECFA, 2006). No new information on the safety of candelilla wax was available on that occasion and JECFA concluded that the uses of candelilla wax, including as a carrier for flavours and as a clouding agent in water-based drinks, would not result in dietary exposure (< 650 mg/person/day) that raises concern about safety (JECFA, 2006). In the USA candelilla wax is classified as Generally Recognised as Safe (GRAS) and permitted according to current good manufacturing practice (GMP) for various food uses (CFR, 1983). Candelilla wax is also used as a chewing-gum base and is permitted for that purpose in the USA. Additional use of candelilla wax has been reported in non-food products such as cosmetics (lip balms and lotion bars) and pharmaceuticals (Liebert, 1984). Candelilla wax is also permitted for various purposes in Canada ( June 2010) as well as in Japan (Japan 2010) Exposure Food consumption data used for exposure assessment The EFSA Comprehensive European Food Consumption Database (Comprehensive Database) has been built from existing national information on food consumption at a detailed level since Competent authorities in the European countries provided EFSA with data on the level of food consumption by the individual consumer from the most recent national dietary survey in their country (cf. Guidance of EFSA Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment (EFSA, 2011b). Overall, the food consumption data gathered at EFSA were collected by different methodologies and thus direct country-to-country comparison should be made with caution. For calculation of chronic exposure, intake statistics have been based on individual average consumption over the total survey period excluding surveys with only one day per subject. High level consumption was only calculated for those foods and population groups where the sample size was sufficiently large to allow calculation of the 95 th percentile (EFSA, 2011b). The Panel estimated chronic exposure for the following population groups: toddlers, children, adolescents, adults and the elderly. Calculations were performed using individual body weights. Thus, for the present assessment, food consumption data were available from 26 different dietary surveys carried out in 17 different European countries as mentioned in Table Commission Regulation (EU) No 10/2011 of 14 January 2011 on plastic materials and articles intended to come into contact with food. OJ L 328 p EFSA Journal 2012;10(11):

12 Table 4: Population groups considered for the exposure estimates of candelilla wax Population Toddlers Children 15 Adolescents Adults The elderly 16 Age range from 12 up to and including 35 months of age from 36 months up to and including 9 years of age from 10 up to and including 17 years of age from 18 up to and including 64 years of age older than 65 years Countries with food consumption surveys covering more than one day Bulgaria, Finland, Germany, Netherlands Belgium, Bulgaria, Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Latvia, Netherlands, Spain, Sweden Belgium, Cyprus, Czech Republic, Denmark, France, Germany, Italy, Latvia, Spain, Sweden Belgium, Czech Republic, Denmark, Finland, France, Germany, Hungary, Ireland, Italy, Latvia, Netherlands, Spain, Sweden, UK Belgium, Denmark, Finland, France, Germany, Hungary, Italy Consumption records were codified according to the FoodEx classification system (EFSA, 2011a). Nomenclature from FoodEx classification system has been linked to the Food Classification System as presented in the Commission Regulation (EU) N 1129/2011, part D, to perform exposure estimates Exposure to candelilla wax from its use as food additive Since candelilla wax is authorised without any specific MPL as qs, exposure to candelilla wax from its use as food additive has been calculated by using MPLs for carnauba wax as listed in Table 3. Since no data were reported for normal use levels, no further refinement was made. High level exposure (95 th percentile of consumers only) was calculated by adding the 95 th percentile of exposure from one food group (i.e. the one having the highest value) to the mean exposure resulting from the consumption of all other food groups. This is based on the assumption that an individual might be a high level consumer of one food category and would be an average consumer of the others. This approach has been tested several times by the Panel in the re-evaluation of food colours and has shown reasonable correlation with high level total intakes when using the raw food individual consumption data. Therefore, this approach was preferred for the calculations and was based on the maximum reported use levels for carnauba wax (EFSA, 2012b) in order to avoid excessively conservative estimates. However, the Panel noted that its estimates should be considered as being conservative as it is assumed that all processed foods contain candelilla wax added at the same MPLs as for carnauba wax. Table 5 summarises the estimated exposure to candelilla wax from its use as food additive of all five population groups. 16 The terms children and the elderly correspond respectively to other children and the merge of elderly and very elderly age groups in the EFSA Guidance on the Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment (EFSA, 2011b). EFSA Journal 2012;10(11):

13 Table 5: Summary of anticipated exposure to candelilla wax using the tiered approach (EU, 2001) in children and the adult population Estimated exposure using MPL Mean exposure Exposure 95 th Toddlers Children Adolescents Adults The elderly (12-35 months) (3-9 years) (10-17 years) (> 18 years) (> 65 years) mg/kg bw/day mg/kg bw/day mg/kg bw/day mg/kg bw/day mg/kg bw/day Main food groups contributing to exposure of candelilla wax using MPL The main contributors to the total anticipated mean exposure to candelilla wax for toddlers, children, adolescents and adults were fruits and confectionary. For toddlers fruits and confectionary contributed to 64-96% and 12-18% exposure, respectively, for children these contributions were 47-79% and 11-17%, for adolescents 44-81% and 11-19%, and for adults 60-93% and 11%, respectively. For the elderly, the main contributors to the total anticipated mean exposure to candelilla wax were fruits (79-95%) Other sources of exposure Candelilla wax is used on pharmaceutical products (tablets) and in a range of technical applications like cosmetic, fragrance preparations, hair colouring and conditioning preparations, and manicuring, skin care, and suntan preparations. However, considering the chemical properties of the product it is unlikely that any significant systemic exposure will take place from these applications. 3. Biological and toxicological data 3.1. Absorption, distribution, metabolism and excretion No new data on absorption, distribution, metabolism and excretion were available for this reevaluation. Hydrocarbons The most abundant constituents of candelilla wax are hydrocarbons in the form of straight-chain paraffin from C29-C33 (CTA, 2006; Wolfmeier et al., 2005). It has been previously concluded that approximately 3% of medium and high viscosity mineral oils is systemically absorbed following oral exposure. Absorbed material is transported into the lymphatic system and drains into the blood circulation of the vena brachiocephalica. The material is then distributed into the organs via the arterial blood circulation. It is metabolised in the liver to acidic compounds which are eliminated in the urine. Following chronic exposures near maximal concentrations of mineral hydrocarbons in the liver are reached within 90 days, followed by a further slow increase up to 24 months after ingestion. Mineral hydrocarbons return to near background levels within 6 months after exposure ceases. The small amount of n-alkanes retained in tissues has a hydrocarbon distribution ranging from C 22 -C 34 with a maximum of C 28 (EFSA, 2009). EFSA Journal 2012;10(11):

14 Absorption and tissue distribution of radiolabelled [ 14 C]-nonacosane (C29) was studied in one male Osborne-Mendel rat (Kolattukudy and Hankin, 1966). More than 75% of the total administered radioactivity was excreted through the faeces as unchanged hydrocarbon and about 4% was excreted through expired CO 2. Five days after the administration, radioactivity was mainly found in the liver (2% of the total administered radioactivity mostly in the phospholipids fraction) and in other organs and tissues like the kidney, plasma, muscle, lung and brain. The absorbed long-chain hydrocarbon appeared to undergo extensive metabolism, as indicated by the distribution of radioactivity in various organs and by the presence of radioactivity in C 15, C 16, C 17, C 18 and C 19 fatty acids isolated from the rat liver phospholipids and triglycerides. In vitro studies with the linear, saturated hydrocarbons n-octadecane (C 18 ) and n-heptadecane (C 17 ) in microsomes prepared from Wistar, Sprague-Dawley and Fischer 344 rat liver or small intestine, showed that, there was production of their corresponding alcohol and acid metabolites, as shown by n-heptadecane, producing heptadecanol and heptadecanoic acid metabolites (WHO, 2003). Absorption, distribution and excretion of [ 14 C] radiolabelled medium and low-viscosity mineral oils (having a high content of C 17 hydrocarbons) showed that bioavailability was greater in Fisher 344 rats compared to Sprague-Dawley rats and that faecal and urinary excretion were the major routes of elimination. Liver and mesenteric lymph nodes retained radioactivity at levels of 2.5% and 0.002% of the administered dose, respectively (WHO, 2003). Fatty acid esters The second major constituents of candelilla wax are esters of acids and alcohols with even-numbered carbon chains (C 28 -C 34 ) (CTA, 2006; Wolfmeier et al., 2005). Generally, long-chain fatty acid esters are considered to be poorly absorbed; it is suggested that they are partly hydrolysed in the intestinal tract to their corresponding alcohols and acids which are then absorbed and incorporated into normal cellular metabolic pathways (EFSA, 2007). In a study, Sprague-Dawley rats were fed for 2-4 weeks on diets containing, either 40 g of oleyl palmitate/kg diet or 150 g of oleyl alcohol/kg diet. The test substance was excreted in the faeces of the animals as a mixture of the intact ester, free fatty acid and free alcohol, suggesting that wax esters are susceptible to partial hydrolysis in the intestinal tract (Hansen and Mead, 1965). Refined jojoba oil, which contains essentially 97% (w/w) plant wax esters of monounsaturated, straight-chain acids and alcohols with high molecular weights (C 16 -C ) (Wolfmeier et al., 2005), was administered to four groups of 10 male and 10 female Sprague-Dawley rats. It was concluded that the oil was poorly absorbed and was resistant to digestion in the intestinal tract (EPA, 1995). Overall, the Panel considered that absorption of candelilla wax is expected to be low, and that upon absorption the components would be incorporated into normal metabolic pathways and eliminated Toxicological data Acute oral toxicity The SCF reported that acute oral toxicity studies were reviewed during the evaluation of candelilla wax (no further details) and that none of the studies reported any adverse treatment-related toxicological findings (SCF, 1992). EFSA Journal 2012;10(11):

15 Liebert (1984) reported that candelilla wax, as a cosmetic ingredient and in cosmetic formulations, has been tested by gavage for acute oral toxicity in Sprague-Dawley and Hooded Long Evans rats and other rats of undefined strain without report of any toxic effects. JECFA reported an acute oral toxicity study in rats (strain and sex not defined) resulting in a LD 50 of > 5000 mg/kg bw (JECFA, 1993) Short-term and subchronic toxicity JECFA (JECFA, 1993) briefly describes four short-term studies in rats and one study in dogs for which unpublished reports were not accessible to the Panel: Groups of 12 female weanling Wistar rats were given a diet containing a mixture of gum base and candelilla wax (composition not defined), at doses of 0, 3 and 5% equivalent to 0, 590 and 980 mg/kg bw/day for 8 weeks. No significant differences were reported between the groups regarding survival, body weight gains, food and water intake, urinalysis, haematology or gross pathology (Harrisson, 1946, cited by JECFA, 1993). Groups of 12 weanling Wistar rats of either sex were given a diet containing a 1:1 mixture of candelilla wax and a butadiene-styrene polymer, at doses of 0, 1 and 5% equivalent to 0, 750 and 3600 mg/kg bw/day for 8 weeks. Daily intake of candelilla wax was calculated by JECFA to be approximately 0, 370 and 1800 mg/kg bw/day. No significant differences were reported between the groups regarding survival, body weight gain, food and water intake, urinalysis or haematology (haemoglobin, RBC, total and differential WBC) (Harrisson, 1948, cited by JECFA, 1993). Groups of 17 weanling Wistar rats of both sexes were given a diet containing a 1:1 mixture of candelilla wax and a butadiene-styrene polymer (apparently different from the previous study), at doses of 0, 1 and 5%, equivalent to 0, 680 and 3420 mg of the mixture/kg bw/day for 27 weeks (these doses could have been equivalent to approximately 0, 370 and 1800 mg candelilla wax/kg bw/day). No significant differences were reported between the groups, with the exception of a slight decrease in body weight gain in both treated groups. Observations reported included survival, food and water intake, urinalysis (transparency, odour, colour, ph, albumin and microscopic elements) and haematology (haemoglobin, RBC, total and differential WBC). The histopathological examination of heart, lung, spleen, kidney, pancreas, small and large intestines, uterus, ovary, prostate, testicle and seminal vesicle tissues from 3 rats of each sex revealed no treatment-related adverse effects. The authors concluded that with the exception of the slight decrease in body weight gain, there was no indication of any toxic change attributable to treatment (Harrisson, 1949, cited by JECFA, 1993). Twelve male and 12 female weanling albino rats of unidentified strain received a gum base (containing % of candelilla wax) in the diet, at concentrations ranging from 10 to 25%, for 180 days (10% for the first 7 days, 20% for the following 7 days, and 25% for the remaining treatment period). Over the entire treatment period, the daily consumption of candelilla wax was estimated to be 2400 mg/kg bw. No significant differences were reported between the groups regarding survival, body weight gain, food intake or urinalysis. JECFA reports that gross and histopathological examinations did not reveal changes associated with the treatment (Hodge, 1973, cited by JECFA, 1993). Groups of young dogs of unidentified strain (1-2 males and 2-3 females) were given a diet containing a gum base (containing 25% candelilla wax) at doses of 0, 1 and 10%, equivalent to 0, 60 and 600 mg candelilla wax/kg bw/day for 6 months. No significant differences were reported between the groups regarding body weight gain, survival, urinalysis or haematology. The complete histopathological examination of 2-3 dogs from each treated group and 4 dogs from control did not reveal treatment EFSA Journal 2012;10(11):

16 related changes (no individual animal data for histopathology was provided) (Harrisson, 1953; as cited by JECFA, 1993). Overall the Panel noted that these studies are rather limited, are unpublished and were not accessible to the Panel. Fatty acid esters Oleyl palmitate as well as oleyl alcohol, fed at either 40 or 150 g/kg diet to Sprague-Dawley rats for 2-4 weeks (equivalent to 2000 and 7500 mg/kg bw/day, respectively) did not produce any toxic effect except for a purgative effect at the highest oleyl palmitate dose tested (Hansen and Mead, 1965). Refined jojoba oil, (jojoba oil is composed essentially of 97% (w/w) plant wax esters of monounsaturated, straight-chain acids and alcohols with high molecular weights [C 16 -C ]), was administered in the diet to four groups of 10 young male Sprague-Dawley rats fed with 0.5, 1.0, 2.0, and 3.0 g in 5 g of basal diet (equivalent to , , , mg/kg bw/day assuming an animal weight of 0.1 kg and food consumption of 10 g/day/animal). The first two groups were dosed for 7 days, the last two for 4 days (EPA, 1995; Hamm, 1984). No effect on food consumption was noted in any dose group. Jojoba oil was poorly absorbed and was predicted to be resistant to digestion in vivo. Low tolerance of the animals was observed toward high doses of jojoba oil. Low tolerance was also observed towards another non-digestible, non-absorbable oil, trialkoxytricarballyte (TATCA), tested in the same experiment. The low tolerance of animals toward the higher concentrations of jojoba and TATCA oils is attributed to secondary metabolic disturbances caused by the laxative effects of oils rather than to a direct toxicity. Animals receiving the basal diet (controls) lost weight and developed rough coats suggesting that the test animals were already under nutritional stress before starting the treatment. This nutritional stress could only be exacerbated by the laxative effects of non-absorbable oils. None of the reported effects was observed in the mg/kg bw/day groups. Hydrocarbons (n-alkanes and n-alkenes) Mineral oils (medium and low viscosity) class I have been evaluated by the SCF (SCF, 1997) and by JECFA (WHO, 2003). Mineral oil hydrocarbons in food have also been evaluated by the EFSA CONTAM Panel (EFSA, 2012a). Evaluation of short and subchronic toxicity studies (28 and 90-day) conducted with class I mineral oils at concentrations up to 2% in the diet (equal to approximately 2600 mg/kg bw/day for the 28-day study and 2000 mg/kg bw/day for the 90-day study) indicated a greater response in the Fischer 344 rat strain than in the Sprague-Dawley strain (WHO, 2003). The effects were increased weights of liver, mesenteric lymph nodes and spleen, granulomatous inflammation of the liver, and reticuloendothelial-cell hyperplasia of the mesenteric lymph nodes. The increased liver weights reversed during a 30-day recovery period. In Sprague-Dawley rats only increases in liver weight and reticuloendothelial-cell hyperplasia of the lymph nodes of minimal severity were observed (WHO, 2003). High-melting point, microcrystalline wax, administered to groups of 20 male and 20 female F344 rats for 90 days at levels of 0.002, 0.02, 0.2, and 2% in the diet (equivalent to 2, 20, 200, and 2000 mg/kg bw/day) was reported not to show the adverse effects described above for class I mineral oils (WHO, 1996). At the highest dose tested, increases in food intake, especially in females, were reported, returning to normal during a reversal period. Body weights were not affected by the treatment. However, increased organ weights were reported particularly for kidneys, liver, spleen, lymph node and caecum, but histopathological changes and tissue accumulation described during mineral oils intake were not observed for high-melting point, microcrystalline wax (WHO, 1996). EFSA Journal 2012;10(11):

17 Genotoxicity Re-evaluation of candelilla wax (E 902) as a food additive JECFA briefly reports two mutagenicity studies with candelilla wax, both of them showing no mutagenic potential. These negative results were reported in Ames test with Salmonella typhimurium strains TA1535, TA1537, TA1538 and in a test for mitotic gene conversion in Saccharomyces cerevisiae D4 strain with and without the S9 fraction of rat, mouse and monkey liver, with candelilla wax concentrations of 1.25, 2.5 and 5.00 (Brusick, 1976, cited by JECFA, 1993). Negative results were also reported in mutagenicity tests with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 and with Escherichia coli WP2 strain with and without S9 fraction of rat liver, with candelilla wax concentrations of µg/plate (Mortelmans and Eckford, 1979, as cited by JECFA, 1993). No further details were available on these studies. Negative mutagenicity results were also reported with candelilla wax suspended in DMSO, in an Ames test with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 (Prival et al., 1991). In this study a dose range from to 10 mg/plate was tested in the presence or absence of S9 rat liver microsomal preparations (Prival et al., 1991). The Panel noted that, due to the insolubility of the test material in water and in solvents compatible with culture media, in vitro genotoxicity tests may have limited applicability to test candelilla wax. The Panel is not aware of any in vivo mutagenicity studies with candelilla wax. Supplementary information for the evaluation of the potential genotoxicity of candelilla wax is provided by test results on the main components of the wax: Long-chain fatty acids D-003, a mixture very long-chain fatty acids extracted from sugar cane, gave negative results when tested in Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 strains at concentrations of up to 5 mg/plate with and without S9 activation (Gámez et al., 2002). Overall, the Panel considered that the available data suggest that candelilla wax is not genotoxic. Although the database is very limited and does not include studies in mammalian cells, the Panel noted, however, the lack of structural alerts for genotoxicity on the main components of candelilla wax and concluded that candelilla wax does not raise concern for genotoxicity. The lactones present in candelilla wax are considered to be stable and not to belong to the category of reactive genotoxic lactones characterized by a low number of carbons in the heterocycle Chronic toxicity and carcinogenicity JECFA (JECFA, 1993) briefly describes two long-term studies with rats and mice for which the original publications were not available to the Panel: Groups of 15 black agouti (C57) mice of either sex were given a diet containing a mixture of gum base containing 25% candelilla wax, at doses of 0, 0.8 and 5.0%, equivalent to 0, 1200 and 7500 mg gum base/kg bw/day for months (which would had been equivalent to approximately 0, 300 and 1900 mg candelilla wax/kg bw/day). Approximately half of surviving mice per group were sacrificed after the 12 th month of treatment and the remainder after the 13 th month. The authors reported that the number of deaths during the study in the 5.0% group exceeded those in the lower or control groups, although the causes of death were not defined. No carcinogenic potential was reported by the authors in mice up to the highest dose tested (7500 mg gum base/kg bw/day equivalent to 1900 mg candellila wax/kg bw/day) (Hodge, 1973; cited by JECFA, 1993). EFSA Journal 2012;10(11):

18 The Panel noted that the duration of the study above is too short to evaluate the carcinogenic potential of the compound. Groups of 30 Sprague-Dawley rats of both sexes were given a diet containing two different gum bases as mixtures containing 25% candelilla wax, at doses of 0, 0.8, 2.0 and 5% equivalent to 0, 125, 300 and 750 mg candelilla wax/kg bw/day, for either 19 months (identified as gum base 12) or 2 years (identified as gum base 11). No significant differences were reported between the groups regarding food intake, urinalysis or haematology. A complete histopathological examination of all animals did not indicate significant differences between treated animals and controls (Harrisson, 1953; cited by JECFA, 1993). Hydrocarbons (n-alkanes) Chemically, candelilla wax hydrocarbons are similar to microcrystalline wax (high melting point wax) and to mineral oil (high viscosity), therefore results of chronic toxicity and carcinogenicity studies conducted on these mineral oils/waxes are also considered below: Overall, mineral oils have not shown carcinogenic potential in toxicity studies in vivo (EFSA, 2009; EFSA 2010). Doses of up to 2500 mg/kg bw/day of Class I medium viscosity minerals oils did not show any carcinogenic potential when administered to Fischer 344 rats for 2 years (EFSA, 2009). An increased incidence of mononuclear cell leukaemia was observed in females treated with high viscosity mineral oils administered alone at doses of up to 1200 mg/kg bw/day to Fischer 344 rats for 2 years but this finding was not considered to be treatment-related as the incidence was not doserelated and was within the range for other control female Fisher 344 rats (EFSA, 2009). It was thus concluded that no carcinogenic effects were observed with high viscosity and Class I medium viscosity mineral oils in the studies done with Fisher 344 rats (EFSA, 2009). The major effect reported in these long-term studies was histiocytosis of the mesenteric lymph nodes of the treated animals. This infiltration was observed in all groups including controls after 12 months of treatment but severity was reported as mild in treated groups versus minimal in controls (EFSA, 2009). Compared to controls, increased incidences of combined angiectasis and cystic degeneration (focal sinusoidal dilatation) were observed in all treated groups after 12 months of exposure to mineral oils. These lesions were of minimal grade, not dose-related and were considered as a common finding in Fischer 344 rats (EFSA, 2009). Overall, it was concluded that these effects did not progress to more serious changes, were not detrimental to the life or health status of the rat and were considered to be more an indication of exposure to mineral oils rather than an adverse effect (EFSA, 2009). From these studies, the ANS Panel established an ADI of 12 mg/kg bw/day for high viscosity mineral oils (kinematic viscosity 11 mm 2 /s (cst) at 100 C, a carbon number > 28 at 5% distillation point and an average MW of g/mol) based on a NOAEL of 1200 mg/kg bw/day Reproductive and developmental toxicity JECFA briefly reported incidental observations in the short-term study of Harrisson (Harrisson, 1949 cited by JECFA, 1993) in which 3 male and 3 female rats were fed a diet containing a 1:1 mixture of styrene-butadiene polymer and candelilla wax at concentrations of 0, 680 or 3420 mg/kg bw/day for 5 months prior to mating. It was reported that two of the three females from each dose group conceived and produced normal litters, evidence that exposure to candelilla wax was not associated with reproductive toxicity. EFSA Journal 2012;10(11):