SCIENTIFIC OPINION. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

Transcription

1 EFSA Journal 2015;13(3):4053 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of aliphatic and aromatic hydrocarbons (chemical group 31) when used as flavourings for all animal species 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 European Food Safety Authority (EFSA), Parma, Italy This scientific output, published on 20 July 2015, replaces the earlier version published on 31 March ABSTRACT Chemical group 31 consists of aliphatic and aromatic hydrocarbons, of which 17 are currently authorised for use as flavours in food. This opinion concerns nine compounds from this group. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) is unable to perform an assessment of 1,4(8),12- bisabolatriene [01.016] due to the lack of data on its purity. The Panel concludes that D-limonene [01.045] is safe for all animal species, except for male rats, at the proposed maximum dose level (25 mg/kg feed). 1-Isopropyl-4- methylbenzene [01.002] is safe for all target species, except cats, at the proposed maximum dose level (25 mg/kg feed), with a margin of safety ranging from 1-fold (no margin of safety) to 3.2-fold. For cats the calculated maximum safe concentration is 14 mg/kg complete feed. For the compounds belonging to Cramer Class I, terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], alpha-terpinene [01.019], gamma-terpinene [01.020] and L-limonene [01.046], the calculated safe use level is 1.5 mg/kg complete feed for cattle, salmonids and non-food-producing animals and 1 mg/kg complete feed for pigs and poultry. The absence of a margin of safety would not allow the simultaneous administration in feed and water for drinking of these substances. Overall, the FEEDAP Panel is not in the position to conclude on the use of these additives in water for drinking. No safety concern would arise for the consumer from the use of these compounds up to the highest safe levels in feeds. All compounds should be considered irritant to skin, eyes and respiratory tract and as skin sensitisers. No risk for the safety for the environment is foreseen. Since all of the compounds under assessment are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. European Food Safety Authority, 2015 KEY WORDS sensory additives, aliphatic and aromatic hydrocarbons, chemical group 31 1 On request from the European Commission, Question No EFSA-Q , adopted on 10 March Panel members: Gabriele Aquilina, Vasileios Bampidis, Maria De Lourdes Bastos, Lucio Guido Costa, Gerhard Flachowsky, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Fernando Ramos, Derek Renshaw, Guido Rychen, Maria Saarela, Kristen Sejrsen, Patrick Van Beelen, Robert John Wallace and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Feed Flavourings, including Paul Brantom, Andrew Chesson and Anne-Katrine Lundebye, for the preparatory work on this scientific opinion. 4 An editorial amendment was carried out to correct the Flavis number of four compounds mentioned in the Abstract, in the Summary, in Paragraph 3 and in the Conclusions. To avoid confusion, the original version has been removed from the EFSA Journal, but is available on request, as is a version showing all the changes made. Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Scientific Opinion on the safety and efficacy of aliphatic and aromatic hydrocarbons (chemical group 31) when used as flavourings for all animal species. EFSA Journal 2015;13(3):4053, 22 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

2 SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of 17 compounds (aliphatic and aromatic hydrocarbons belonging to chemical group 31) when used as flavourings for all animal species. All additives are currently authorised for use as flavours in food and all have been detected in plant materials, fruits and processed foods; however, reports of their distribution vary greatly. The current application has been divided during the course of the assessment, and the present opinion concerns only nine of the compounds, namely 1-isopropyl-4- methylbenzene [01.002], terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4- methylbenzene [01.010], 1,4(8),12-bisabolatriene [01.016], alpha-terpinene [01.019], gammaterpinene [01.020], D-limonene [01.045] and L-limonene [01.046]. The remaining eight compounds are currently under assessment by the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) in a separate opinion. The FEEDAP Panel is unable to perform an assessment of 1,4(8),12-bisabolatriene [01.016] due to the lack of data on its purity. The Panel concludes that D-limonene [01.045] is safe for all animal species, except for male rats, at the proposed maximum dose level (25 mg/kg feed). 1-Isopropyl-4- methylbenzene [01.002] is safe for all target species, except cats, at the proposed maximum dose level (25 mg/kg feed), with a margin of safety ranging from 1-fold (no margin of safety) to 3.2-fold. For cats the calculated maximum safe concentration is 14 mg/kg complete feed. For the compounds belonging to Cramer Class I, terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4- methylbenzene [01.010], alpha-terpinene [01.019], gamma-terpinene [01.020] and L-limonene [01.046], the calculated safe use level is 1.5 mg/kg complete feed for cattle, salmonids and non-foodproducing animals and 1 mg/kg complete feed for pigs and poultry. The absence of a margin of safety would not allow the simultaneous administration in feed and water for drinking of these substances. Overall, the FEEDAP Panel is not in the position to conclude on the use of these additives in water for drinking. No safety concern would arise for the consumer from the use of these compounds up to the highest safe levels in feeds. All compounds should be considered irritant to skin, eyes and respiratory tract and as skin sensitisers. No risk for the safety for the environment is foreseen. Since all of the compounds under assessment are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. EFSA Journal 2015;13(3):4053 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Background... 4 Terms of reference... 5 Assessment Introduction Characterisation Characterisation of the flavouring additives Stability and homogeneity Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) Safety Safety for the target species Conclusions on the safety for target species Safety for the consumer Absorption, distribution, metabolism and excretion (ADME) and residue studies Assessment of consumer exposure Conclusion on consumer safety Safety for the user Safety for the environment Efficacy Conclusions Documentation provided to EFSA References Abbreviations Annex A. Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Aliphatic and aromatic hydrocarbons EFSA Journal 2015;13(3):4053 3

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7; in addition, Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without a time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG) 6 for authorisation of the 17 substances listed in Table 1 belonging to chemical group 31, aliphatic and aromatic hydrocarbons, when used as a feed additive for all animal species (category: sensory additives; functional group: flavouring compounds) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive) and under Article 10(2) (re-evaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 7 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 9 June The 17 compounds except l-limonene [FLAVIS number: ] have been assessed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) at the 63rd meeting (WHO, 2005), and considered safe for use in food. The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) assessed these substances (EFSA, 2010, 2011a and 2012a) and requested additional toxicity data to complete the assessment for pin-2(10)-ene [01.003], pin-2(3)-ene [01.004], beta-caryophyllene [01.007], myrcene [01.008], camphene [01.009], valencene [01.017], beta-ocimene [01.018] and delta- 3-carene [01.029]. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) will also not proceed with an assessment of these substances until this issue has been resolved. The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) (EFSA CEF Panel, 2011a, b) considered the remaining substances 1-isopropyl-4-methylbenzene [01.002], terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], 1,4(8),12-bisabolatriene [01.016], alpha-terpinene [01.019], gamma-terpinene [01.020], d-limonene [01.045] and l-limonene [01.046] safe at the estimated levels of intake based on the MSDI approach. The current application has been divided during the course of the assessment, and the present opinion concerns only nine of the compounds, namely 1-isopropyl-4-methylbenzene [01.002], terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], 1,4(8),12-5 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, , p On 13/03/2013, EFSA was informed by the applicant that FFAC EEIG was liquidated on 19/12/2012 and their rights as applicant were transferred to FEFANA asbl (EU Association of Specialty Feed Ingredients and their Mixtures). Avenue Louise 130A, Box 1, 1050 Brussels, Belgium. 7 EFSA Dossier reference: FAD EFSA Journal 2015;13(3):4053 4

5 bisabolatriene [01.016], alpha-terpinene [01.019], gamma-terpinene [01.020], d-limonene [01.045] and l-limonene [01.046]. The remaining eight compounds are currently under assessment by the EFSA CEF Panel and will be assessed in a separate opinion. The 17 compounds are currently listed in the European Union database of flavouring substances and in the European Union Register of Feed Additives, respectively, and thus authorised for use in food and feed in the European Union. 8 They have not been previously assessed by EFSA as feed additives. TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and the efficacy of the 17 aliphatic and aromatic hydrocarbons, 1-isopropyl-4-methylbenzene, 1,4(8),12-bisabolatriene, 1-isopropenyl-4- methylbenzene, alpha-phellandrene, alpha-terpinene, beta-caryophyllene, beta-ocimene, camphene, delta-3-carene, d-limonene, gamma-terpinene, l-limonene, myrcene, pin-2(10)-ene, pin-2(3)-ene, terpinolene and valencene, when used under the conditions described in Table 1. 8 Commission Implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list of flavouring substances provided for by Regulation (EC) No 2232/96 of the European Parliament and of the Council, introducing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) No 1565/2000 and Commission Decision 1999/217/EC. OJ L 267, , p. 1. EFSA Journal 2015;13(3):4053 5

6 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No - Category(ies) of additive Functional group(s) of additive Chemical defined flavourings from Chemical Group 31: 1-Isopropyl-4-methylbenzene 1,4(8),12-Bisabolatriene 1-Isopropenyl-4-methylbenzene alpha-phellandrene alpha-terpinene beta-caryophyllene beta-ocimene Camphene delta-3-carene d-limonene gamma-terpinene l-limonene Myrcene Pin-2(10)-ene Pin-2(3)-ene Terpinolene Valencene 2. Sensory additives b) flavouring compounds Composition, description 1-Isopropyl-4-methylbenzene (CAS No ) 1,4(8),12-Bisabolatriene (CAS No ) 1-Isopropenyl-4-methylbenzene (CAS No ) alpha-phellandrene (CAS No ) alpha-terpinene (CAS No ) beta-caryophyllene (CAS No ) beta-ocimene (CAS No ) Camphene (CAS No ) delta-3-carene (CAS No ) d-limonene (CAS No ) gamma-terpinene (CAS No ) l-limonene (CAS No ) Description Chemical Purity criteria formula C 10 H 14 97% C 15 H 24 97% C 10 H 12 97% C 10 H 16 95% C 10 H 16 89% C 15 H 24 80% C 10 H 16 80% C 10 H 16 80% C 10 H 16 92% C 10 H 16 96% C 10 H 16 95% C 10 H 16 95% Method of analysis EFSA Journal 2015;13(3):4053 6

7 Myrcene (CAS No ) Pin-2(10)-ene (CAS No ) Pin-2(3)-ene (CAS No ) Terpinolene (CAS No ) Valencene (CAS No ) C 10 H 16 90% C 10 H 16 97% C 10 H 16 97% C 10 H 16 95% C 15 H 24 94% Trade name - Name of the holder of authorisation - Species or category of animal All species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring - Specific conditions for use in complementary feedingstuffs Marker residue Other provisions and additional requirements for the labelling - All feedingstuffs and water for drinking, as part of a premixture only - Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2015;13(3):4053 7

8 ASSESSMENT 1. Introduction Chemical group (CG) 31 for flavouring substances is defined in Commission Regulation (EC) No 1565/ as aliphatic and aromatic hydrocarbons. The present application concerns 17 compounds which can be assigned to this CG. The 17 compounds, except L-limonene [European Union (EU) Flavour Information System (FLAVIS) number (FL-No): ], have been assessed by the Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) at the 63 rd meeting (WHO, 2005), and considered safe for use in food. No Acceptable Daily Intake (ADI) values were specified. The European Food Safety Authority (EFSA) Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) assessed these substances (EFSA CEF Panel, 2010, 2011a, 2012a) and requested additional toxicity data to complete the assessment for pin-2(10)-ene [01.003], pin-2(3)-ene [01.004], beta-caryophyllene [01.007], myrcene [01.008], camphene [01.009], valencene [01.017], beta-ocimene [01.018] and delta-3-carene [01.029]. Consequently, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) will also not proceed with an assessment of these substances until this issue has been resolved. The CEF Panel considered the remaining substances 1-isopropyl-4-methylbenzene [01.002], terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], 1,4(8),12-bisabolatriene [01.016], alpha-terpinene [01.019], gamma-terpinene [01.020], D-limonene [01.045] and L-limonene [01.046] safe at the estimated levels of intake based on the maximised survey-derived intake (MSDI) approach (EFSA CEF Panel, 2011a, b). This application has been divided during the course of the assessment, and the present opinion concerns only nine substances, namely 1-isopropyl-4-methylbenzene [01.002], terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], 1,4(8),12-bisabolatriene [01.016], alpha-terpinene [01.019], gamma-terpinene [01.020], D-limonene [01.045] and L-limonene [01.046], as the other compounds are currently under assessment by the EFSA CEF Panel. The nine compounds are currently listed in the EU database of flavouring substances and in the EU Register of Feed Additives, and thus authorised for use in food and feed in the EU, respectively. 10 They have not been previously assessed by EFSA as feed additives. A consortium of companies (Feed Flavourings Authorisation Consortium of FEFANA (EU Association of Specialty Feed Ingredients and their Mixtures (FFAC)) supplying flavours to the feed industry has requested authorisation for the use of the substances listed above as additives to feed and water for drinking (sensory additives, flavouring compounds) for use in all animal species. 9 Commission Regulation (EC) No 1565/2000 of 18 July 2000 laying down the measures necessary for the adoption of an evaluation programme in application of Regulation (EC) No 2232/96 of the European Parliament and of the Council. OJ L 180, , p Commission Implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list of flavouring substances provided for by Regulation (EC) No 2232/96 of the European Parliament and of the Council, introducing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) No 1565/2000 and Commission Decision 1999/217/EC. OJ L 267, , p. 1. EFSA Journal 2015;13(3):4053 8

9 Regulation (EC) No 429/ allows substances already approved for use in human food to be assessed with a more limited procedure than for other feed additives. However, the use of this procedure is always subject to the condition that food safety assessment is relevant to the use in feed. 2. Characterisation 2.1. Characterisation of the flavouring additives The molecular structures of the nine additives under application are shown in Figure 1. Their physicochemical characteristics are presented in Table 2. Eight of the compounds are monoterpenes, whilst 1,4(8),12-bisabolatriene is a sesquiterpene. 1-Isopropyl-4-methylbenzene [01.002] Terpinolene [01.005] alpha-phellandrene [01.006] 1-Isopropenyl-4-methylbenzene [01.010] 1,4(8),12-Bisabolatriene [01.016] alpha-terpinene [01.019] gamma-terpinene [01.020] D-Limonene [01.045] L-Limonene [01.046] Figure 1: Molecular structures and FL-Nos (in square brackets) of the nine flavourings in CG 31 Table 2: Chemically defined flavourings from CG 31 under application EU Register name CAS No FL- No Molecular formula Molecular weight Physical status Log K ow 1-Isopropyl-4-methylbenzene C 10 H Liquid 4.1 Terpinolene C 10 H Liquid 4.5 alpha-phellandrene C 10 H Liquid Isopropenyl-4-methylbenzene C 10 H Liquid 4.0 1,4(8),12-Bisabolatriene C 15 H Liquid 7.1 (1) alpha-terpinene C 10 H Liquid 4.3 gamma-terpinene C 10 H Liquid 4.5 d-limonene C 10 H Liquid 4.6 l-limonene C 10 H Liquid (1): Generated from Epi-Suite CAS, Chemical Abstracts Service. 11 Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, , p. 1. EFSA Journal 2015;13(3):4053 9

10 These substances are produced either by chemical synthesis or by fractional or steam distillation of essential oils. Several routes of synthesis are available for each compound and described in the dossier. 12 Batch-to-batch variation data were provided for five batches of each additive, except for alphaterpinene [01.019], for which four batches were available, and 1,4(8),12-bisabolatriene [01.016], for which only one batch was available owing to the low production volume (< 1 kg/year). 13 Because of the very low levels of production and use, the FEEDAP Panel considered that, in the case of alphaterpinene [01.019], analysis of four batches was acceptable. Since no data were provided by the applicant on the purity of 1,4(8),12-bisabolatriene [01.016], the FEEDAP Panel was unable to carry out an assessment of this compound. Consequently, this additive is excluded from further consideration in this opinion. The content of the active substance for all other compounds exceeded the JECFA specifications (Table 3) and confirms the effectiveness of the quality control measures taken by the consortium members. Table 3: Identification of the substances and data on purity EU Register name FL-No JECFA specification Assay % % Average Range 1-Isopropyl-4-methylbenzene Terpinolene alpha-phellandrene Isopropenyl-4-methylbenzene ,4(8),12-Bisabolatriene No data No data (1) alpha-terpinene (1) gamma-terpinene D-Limonene L-Limonene (1): Production volume of the product is 1 kg/year or less. Potential contaminants are considered as part of the product specification and are monitored as part of the Hazard Analysis and Critical Control Point (HACCP) procedure applied by all consortium members. The parameters considered include residual solvents, heavy metals and other undesirable substances. However, no evidence of compliance was provided for these parameters Stability and homogeneity The minimum shelf life for the compounds under assessment ranges from 12 to 24 months, when stored under air-tight conditions and cool temperatures. This assessment is made on the basis of compliance with the original specification after storage. Although no data are required for the stability of volatile additives in premixtures and feed, use in water for drinking introduces other issues relating to product stability, such as degradation due to microbial activity. The FEEDAP Panel notes that all these compounds under assessment have very low water solubilities (log K ow > 4). Considering this, and the absence of data on the short-term stability and homogeneity of the additives in water for drinking, the FEEDAP Panel is not in the position to conclude on the use of the additives in water for drinking Conditions of use The applicant proposes the use of the eight additives in feed or water for drinking for all animal species without withdrawal. For 1-isopropyl-4-methylbenzene [01.002] and D-limonene [01.045], the applicant proposes a normal use level of 5 mg/kg feed and a high use level of 25 mg/kg. For the 12 Technical dossier/section II. 13 Technical dossier/supplementary information May 2012/Annex_Batch to batch.pdf EFSA Journal 2015;13(3):

11 remaining six additives, the applicant proposes a normal use level of 1 mg/kg feed and a high use level of 5 mg/kg. No specific proposals are made for doses to be used in water for drinking Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) EFSA has verified the EURL report as it relates to the methods used for the control of chemically defined flavourings from group 31 Aliphatic and aromatic hydrocarbons in animal feed. The Executive Summary of the EURL report can be found in Annex A. 3. Safety The assessment of safety is based on the high use level proposed by the applicant (25 mg/kg for 1-isopropyl-4-methylbenzene [01.002] and D-limonene [01.045], and 5 mg/kg complete feed for the remaining compounds) Safety for the target species The first approach to the safety assessment for target species takes account of the applied use levels in animal feed relative to the maximum reported exposure of humans on the basis of the metabolic body weight. The data for human exposure in the EU (EFSA CEF Panel, 2011a, b) range from 18 to µg/person per day, corresponding to 0.97 to µg/kg 0.75 per day. Table 4 summarises the results of the comparison with human exposure for representative target animals. The body weight of target animals is taken from the default values shown in Table 5. Table 4: Comparison of exposure of humans and target animals (calculated from the proposed maximum feed concentrations of 25 mg/kg feed for 1-isopropyl-4-methylbenzene [01.002] and D-limonene [01.045] and 5 mg/kg feed for the others) to the flavourings under application Flavouring Use level in feed (mg/kg) Human exposure (µg/mbw (kg 0.75 )/day) (1) Target animal exposure µg/mbw (kg 0.75 )/day Salmon Piglet Dairy cow 1-Isopropyl-4-methylbenzene Terpinolene alpha-phellandrene Isopropenyl-4-methylbenzene alpha-terpinene gamma-terpinene D-Limonene L-Limonene (1): mbw, metabolic body weight (kg 0.75 ) for a 60-kg person = The data in Table 4 show that, following the use in food of D-limonene [01.045], human consumption exceeds the proposed use level for salmonids, but is lower than those proposed to be used for piglet and dairy cow. It should be noted that D-limonene is a common constituent of feed material and forage (Cifuni, 2005). Feeding animals citrus by-products is a common practice as old as the domestication of animals by humans (Bampidis and Robinson, 2006). Taking into consideration D-limonene content in citrus peel (25 g/kg (IARC, 1993)) and peel content of citrus by-products (650 g/kg dry matter (DM)), as well as the inclusion levels of citrus by-products in animal diets (100 g/kg in dogs, 120 g/kg in poultry, g/kg in pigs and g/kg in cattle) obtained from various sources (Malafaia et al., 2002; O Sullivan et al., 2003; Bampidis and Robinson, 2006; Nazok et al., 2010; Brambillasca et al., 2013; Crosswhite et al., 2013), the content of D-limonene in feed was calculated to be mg/kg DM in dog feed, mg/kg DM in poultry feed, mg/kg in pig feed and mg/kg DM in feed for ruminants. These concentrations are 65- to 390-fold higher than the high use level in EFSA Journal 2015;13(3):

12 feed (25 mg/kg) proposed by the applicant as feed flavouring. Target animal exposure to D-limonene was calculated to be 0.21 g/day in poultry, 0.41 g/day in dogs, g/day in pigs and g/day in cattle (using the default values in Table 5). D-Limonene is a natural product with high acute toxicity for bark beetles, fruit flies and cat fleas (Hink and Fee, 1986) and it has been proposed as an alternative to synthetic insecticides. It has been used in shampoos and sprays for the control of fleas on dogs and cats; one such product reportedly contained 78.2 % D-limonene (IARC, 1993). However, intoxication of cats has resulted from the use of citrus oil extracts for the control of ectoparasites (shampoo) (Osweiler and Grauer, 1991). For cats, the maximum safe concentration of D-limonene in feed can be derived from the toxicological information available. A No Observed Adverse Effect Level (NOAEL), relevant to the present risk assessment, of 250 mg/kg body weight (bw) per day can be derived from a study by the National Toxicology Program (NTP) (1990) with rats and mice. This NOAEL is based on liver lesions in mice at a dose of 500 mg/kg bw per day. Effects were observed in male rats at lower levels, but were associated with α-2μ globulin accumulation in the kidneys, which is considered to be a sex/speciesassociated effect. Using the NOAEL of 250 mg/kg bw per day and applying an uncertainty factor of 100 (and an additional uncertainty factor of 5 for cats) the maximum tolerable levels of D-limonene in feed was calculated to be 22 mg/kg feed for cats (rounded to 25 mg/kg complete feed) using the default values (bw, feed intake (88 % DM)) from Table 5 (EFSA, 2012b) Therefore, D-limonene at the proposed maximum use level (25 mg/kg complete feed) is considered safe for all animal species, except male rats. According to Table 4, the intake of the other compounds by the target animals greatly exceeds that of humans, resulting from use in food. As a consequence, safety for the target species at the feed concentration applied cannot be derived from the risk assessment for food use. The maximum feed concentration which can be considered safe for the target animals can also be derived from a NOAEL when suitable data are available. Toxicological data (subchronic, repeated-dose studies) were found for 1-isopropyl-4-methylbenzene [01.002]. The NOAEL of 154 mg/kg bw per day was derived from a study in female rats in which 1-isopropyl-4-methylbenzene was administered at 0, 154, 462 or 769 mg/kg bw per day in olive oil by gavage five days a week for six months. The only effect reported for the higher two doses was an increase in average kidney weight (not specified if absolute or relative weight, not accompanied by histopathological changes). This effect was reported as slight effect at 462 mg/kg bw per day and moderate effect at 769 mg/kg bw per day (Wolf et al., 1956). Applying an uncertainty factor of 100 to the NOAEL (154 mg/kg bw per day), the maximum safe intake for the target species was derived for 1-isopropyl-4-methylbenzene following the EFSA Guidance for sensory additives (EFSA CEF Panel, 2012b), and thus the maximum safe feed concentration was calculated (Table 5). Because glucuronidation of the hydrolysis or oxidation products of the compounds in Table 4 is an important metabolic reaction to facilitate the excretion of these compounds (see section 3.2), their use as additives in cat feed needs an additional uncertainty factor of 5. This factor was derived from the fact that cats have an unusually low capacity for glucuronidation (Court and Greenblatt, 1997). EFSA Journal 2015;13(3):

13 Table 5: Maximum safe concentration in feed for different target animals for 1-isopropyl-4- methylbenzene [01.002] Target animal Default values Maximum safe intake/feed concentration Body weight (kg) Feed intake (g/day) (1) Intake (mg/day) Concentration (mg/kg feed) (2) Salmonids Veal calves (milk replacer) Cattle for fattening Pigs for fattening Sows Dairy cows Turkeys for fattening Piglets Chickens for fattening Laying hens Dogs Cats (3) (1): Complete feed with 88 % DM, except milk replacer for veal calves (94.5 % DM), and for cattle for fattening, dairy cows, dogs and cats for which the values are DM intake. (2): In cattle for fattening, dairy cows, dogs and cats the values are in mg/kg DM intake. (3): The safety factor for cats is increased by an additional factor of 5 because of the reduced capacity of glucuronidation. No other suitable toxicological studies could be identified from which to derive a NOAEL for terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], alphaterpinene [01.019], gamma-terpinene [01.020] and L-limonene [01.046]. Therefore, the threshold of toxicological concern (TTC) was used to derive safe feed concentrations for these Cramer Class I compounds. This provides values of 1.5 mg/kg complete feed for cattle, salmonids and non-foodproducing animals and 1.0 mg/kg complete feed for pigs and poultry (EFSA CEF Panel, 2012b). The absence of a margin of safety would not allow the simultaneous administration in feed and water for drinking of these substances Conclusions on the safety for target species The FEEDAP Panel concludes that: D-limonene [01.045] is safe for all target species at the proposed maximum dose level (25 mg/kg feed); 1-isopropyl-4-methylbenzene [01.002] is safe at the proposed maximum dose level (25 mg/kg feed) for all target species except cats, with a margin of safety ranging from 1-fold (no margin of safety) to 3.2-fold. For cats the calculated maximum safe concentration is 14 mg/kg complete feed; for the compounds belonging to Cramer Class I, terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], alpha-terpinene [01.019], gammaterpinene [01.020] and L-limonene [01.046], the calculated safe use level is 1.5 mg/kg complete feed for cattle, salmonids and non-food-producing animals and 1 mg/kg complete feed for pigs and poultry. The absence of a margin of safety would not allow the simultaneous administration in feed and water for drinking of these substances Safety for the consumer Absorption, distribution, metabolism and excretion (ADME) and residue studies The safety for the consumer of the compounds under assessment in CG 31 used as food flavours has already been assessed by JECFA (WHO, 2005) and EFSA CEF Panel (2010, 2011a, b, 2012a). All compounds are currently authorised as food additives without limitations. EFSA Journal 2015;13(3):

14 As already mentioned by JECFA (WHO, 2005), these compounds are absorbed from the gastrointestinal tract. After absorption, these hydrocarbons are oxidised to polar oxygenated metabolites by the cytochrome P450 enzymes, alcohol dehydrogenase and aldehyde dehydrogenases. The resulting hydroxylated metabolites may be excreted in conjugated form or undergo further oxidation, yielding more polar metabolites that are also excreted in conjugated form in the urine. If a double bond is present, epoxide intermediates may form and these are rapidly detoxified either by hydrolysis to yield diols, or by conjugation with glutathione. The ADME of orally administered D-limonene has been extensively described in rodents and humans (EFSA CEF Panel, 2011b). In several animal species and humans, D-limonene has been demonstrated to be oxidised in both side chains and at the cyclohexene ring, resulting in alcohols and/or carboxylic acids (limonene-10-ol, limonene-8,9-diol, limonene-1,2-diol, perillic acid, perillic acid-8,9-diol, dihydroperillic acid, p-mentha-1,8-diene-10-carboxylic acid). The metabolites of D-limonene are, at least partly, conjugated and eliminated with the urine (8-hydroxy-p-menth-1-en-9-yl-beta-Dglucuronic acid, the most important metabolite in rat urine, p-mentha-1,8-dien-10-yl-glucuronic acid, perillyl-glucuronide, dihydro perillyl-glucuronide). No unchanged D-limonene was detected in urine of the rabbit. Urinary metabolites are excreted within hours and represents % of the dose in rodents and rabbits, 77 % in dogs and % in humans, (Kodama et al., 1974, 1976; Igimi et al., 1974; and as referenced in EFSA CEF Panel, 2011b). Excretion via faeces represents 2 9 % in rodents and rabbits and 9 % in dogs. Following oral administration to humans, D-limonene was distributed preferentially to fatty tissues, from which it is eliminated slowly. Little is known about the specific metabolic pathways involved in metabolism of CG 31 compounds in farm animals. However, the enzymes involved in the biotransformation pathways of these compounds are present in all the food-producing target species. The cytochrome P450 monooxygenase families are present and have been characterised in a number of food-producing animals, including ruminants, horses, pigs, (Nebbia et al., 2003; Ioannides, 2006), fish (Wolf and Wolfe, 2005) and birds (Blevins et al., 2012). Epoxide hydrolases, the enzymes involved in the detoxication of the epoxides via formation of diols, which are conjugated and eliminated, are present in mammals (Wisniewski et al., 1987; Marini et al., 1998), fish (Newman et al., 2001) and birds (Harris et al., 2006). All these species, except cats, which have an unusually low capacity for glucuronidation (see section 3.1), also carry out conjugation reactions with sulphate and glucuronic acid (Watkins and Klaassen, 1986; James, 1987), originating hydrosoluble derivatives that are promptly eliminated in urine. Mammals (Watkins and Klaassen, 1986), fish (Espinoza et al. 2013) and birds (Blevins et al., 2012) possess glutathione transferases, which mediate the detoxication of the epoxides by conjugation with glutathione and elimination of the corresponding mercapturic derivatives. Therefore, food-producing animals, including fish and birds, can also be assumed to have the ability to metabolise and excrete the flavouring substances from CG 31 and there is no evidence that they or their metabolites would be present in tissues to an extent that would give rise to a concern for consumer safety. However, the presence of some terpenes in products of animal origin, mainly milk, has been described by several authors. Relevant publications are described in the following paragraphs. In vitro studies showed that some of the compounds under evaluation (1-isopropyl-4-methylbenzene, also known as p-cymene, alpha-phellandrene, alpha-terpinene, gamma-terpinene, limonene and alphaterpinolene) can undergo degradation when incubated with goat rumen microbiota (Malecky et al., 2009a, b, 2012). After 24-hour incubation, the compounds were completely (alpha-terpinene, alphaterpinolene and alpha-phellandrene) or extensively degraded (gamma-terpinene at 80 %, 1-isopropyl- 4-methylbenzene at 65 % and limonene at 50 %) by the bacteria of the rumen (Malecky et al., 2009b). In vivo studies performed in grazing cows (Fernandez et al., 2003) and ewes (Abilleira, 2011) showed limonene (isomer not specified) to be the monoterpene that leaves the largest levels of residues in milk ( g limonene/kg milk in grazing ewes, representing about 50 % of total monoterpenes). Terpenes from forages eaten by cattle have been detected in meat and milk, and tissues and products from grazing animals contain higher levels and more terpene compounds than milk obtained from EFSA Journal 2015;13(3):

15 animals fed with conserved forages or concentrates (Prache, 2005). The type of forage was found to influence the profile of monoterpenes present in cow milk. An oily mixture of alpha-pinene, limonene (isomer not specified) and beta-caryophyllene (1 g each compound/animal per day, corresponding to about 700 mg/kg feed) was orally administered to dairy ewes for 18 days. The three compounds were absorbed, as evidenced by their presence in blood, and excreted in milk, limonene being the most abundant (Poulopoulou et al., 2012a). The concentration of limonene in milk increased until the seventh day (18 µg/ml), decreasing thereafter, being about 5 µg/ml at the end of the trial. Limonene was also detected in cheese prepared with milk from both control and treated sheep, although at higher levels in the latter case (up to 30 mg/kg). The same group (Poulopoulou et al., 2012b) performed a study in goats with a similar protocol and found for limonene a peak plasma concentration 4 hours after the first administration (0.160 µg/ml) that almost disappeared after 24 hours (< 0.01 µg/ml). At the seventh day of administration, the limonene plasma level was about µg/ml, decreasing to very low levels (< µg/ml) on 18th day. In milk, limonene levels peaked on day 13 of the trial (0.300 µg/ml) and decreased until the end (about µg/ml). Limonene was present in cheese samples, prepared with milk from both treated and control goats. The highest level (5 mg/kg) was found in one sample from treated animals. The authors consider that the dose of the compounds used in these experiments is representative of that consumed by grazing animals. The accumulation of terpenes in muscle and adipose tissues was investigated in calves (two calves per group) fed milk replacer unsupplemented or supplemented with 5 or 20 µl/l of a mixture of essential oils from the second month to slaughter (from 10 µl essential oil/day at the beginning of the study to 40 µl essential oil/day at the end), when animals reached a live weight of about 250 kg (Serrano et al., 2007). Although absolute values are unknown, monoterpene concentrations in the supplemented milk were 30 times higher for alpha-phellandrene, 16 times higher for alpha-terpinene, 13 times higher for 1-isopropyl-4-methylbenzene, 5 times higher for limonene, 40 times higher for gamma-terpinene and 5 times higher for alpha-terpinolene than control milk. Some deposition of terpenes in adipose tissues was found. The different terpenes left different quantities of residues, with the oxygen-containing monoterpene derivatives leaving the lowest concentrations and the sesquiterpenes leaving the highest. Among the compounds under assessment, residues were detected for terpinolene and limonene. However, in all cases the increases in terpene concentrations in tissues of animals supplemented with essential oils were not significantly different from the control and were low compared with the additional quantities of terpenes provided through the milk replacer Assessment of consumer exposure In 2011, the EFSA CEF Panel concluded that eight substances under assessment [FL-Nos: , , , , , , ] (EFSA CEF Panel, 2011a) and [01.046] (EFSA CEF Panel, 2011b) do not give rise to safety concerns at the levels of dietary intake, estimated on the basis of the MSDI approach. 14 For all compounds, except D- and L-limonene, the intake of consumers is below the threshold of concern value for structural class I (1 800 µg/person per day). For D- and L- limonene, conclusions were based on the NOAEL for D-limonene of 215 mg/kg bw per day ( mg/person per day). Considering a combined intake of 42 mg/person per day for D- and L- limonene, a margin of safety of 307 could be calculated, and accordingly these substances were not expected to be of safety concern at the estimated level of intake (EFSA CEF Panel, 2011a). Owing to digestion, metabolism and excretion of these compounds by the target species, it is expected that residues of the CG 31 compounds in food of animal origin are negligible. The limited data available confirm that the accumulation of monoterpenes in muscle and adipose tissue is low. 14 Maximised Survey-derived Daily Intake (MSDI). The MSDI is derived from the annual European production figure for the individual substances as surveyed by the industry. In deriving the MSDI, it is assumed that the production figure only represents 60 % of the use in food due to underreporting and that only 10 % of the total EU population are consumers (SCF, 1999). EFSA Journal 2015;13(3):

16 However, several studies demonstrated that terpenes are present in milk from grazing animals, limonene being the most representative compound. For this reason, the potential increase in daily limonene intake attributable to consumption of milk from treated animals (limonene in sheep milk: 5 mg/l after 18 days of oral administration of 1 g limonene/day; Poulopoulou et al., 2012a) was estimated for high consumers (chronic intake by adults: 1.5 L milk; EFSA, 2011). This estimate gives a value of 7.5 mg/person per day. The additional intake via milk would reduce the margin of safety from 307 to 260. Values were not available for residues in edible tissues resulting from the use of limonene as a feed additive, but non-quantitative data suggested that residue concentrations in muscle and adipose tissues are likely to be of a similar order of magnitude to those in milk. No data on the content of the other compounds belonging to CG 31 in milk have been reported Conclusion on consumer safety Limonene is rapidly absorbed, distributed, metabolised and excreted; because of the structural similarity it can be expected that the other compounds (1-isopropyl-4-methylbenzene [01.002], terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4-methylbenzene [01.010], alphaterpinene [01.019], gamma-terpinene [01.020]) behave in a similar way. Mammals, birds and fish share a similar metabolic capacity to handle these compounds. Owing to rumen degradation, digestion, metabolism and excretion of these compounds by the target species, it is expected that food residues of the CG 31 compounds result in consumer exposures that are considerably less than the levels given to the target species. As the exposure of target species is considered to be safe, the much lower exposure of consumers is also considered to be safe. There are no data suggesting that metabolites of the compounds under consideration are more toxic than the parent compounds. Excretion of limonene in milk from dairy ruminants fed D- and L-limonene at the highest safe level in feed is not expected to be of safety concern for consumers. Consequently, no safety concern would arise for the consumer from the use of these compounds up to the highest safe level in feeds Safety for the user No specific data on the safety for the user were provided. In the material safety data sheets 15 hazards for skin and eye contact and respiratory exposure are recognised for the majority of the compounds under application. Most are classified as irritating to eyes, respiratory system and/or skin and/or skin sensitiser. The FEEDAP Panel considers all the compounds under assessment as irritants to skin, eyes and respiratory tract, and as skin sensitisers Safety for the environment The FEEDAP Panel acknowledges that in two previous opinions EFSA could not conclude on the safety of D-limonene, used as a pesticide, for the environment (EFSA, 2013, 2014). However, the compounds considered to be safe for the target species are extensively metabolised by the target species (see section 3.2.1) and excreted as innocuous metabolites or carbon dioxide. Therefore, no risk for the safety for the environment is foreseen. 4. Efficacy Since all eight compounds are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. 15 Technical dossier/section II/Annex II.3. EFSA Journal 2015;13(3):

17 The FEEDAP Panel does not have information on the possible off-flavours from these compounds in milk and milk products and cannot conclude on the possibility of variation of their organoleptic qualities. CONCLUSIONS The FEEDAP Panel is unable to perform an assessment of 1,4(8),12-bisabolatriene [01.016] due to the lack of data on its purity. The Panel concludes that D-limonene [01.045] is safe for all animal species, except for male rats, at the proposed maximum dose level (25 mg/kg feed). 1-Isopropyl-4- methylbenzene [01.002] is safe for all target species, except cats, at the proposed maximum dose level (25 mg/kg feed), with a margin of safety ranging from 1-fold (no margin of safety) to 3.2-fold. For cats the calculated maximum safe concentration is 14 mg/kg complete feed. For the compounds belonging to Cramer Class I, terpinolene [01.005], alpha-phellandrene [01.006], 1-isopropenyl-4- methylbenzene [01.010], alpha-terpinene [01.019], gamma-terpinene [01.020] and L-limonene [01.046], the calculated safe use level is 1.5 mg/kg complete feed for cattle, salmonids and non-foodproducing animals and 1 mg/kg complete feed for pigs and poultry. The absence of a margin of safety would not allow the simultaneous administration in feed and water for drinking of these substances. Overall, the FEEDAP Panel is not in the position to conclude on the use of these additives in water for drinking. No safety concern would arise for the consumer from the use of these compounds up to the highest safe levels in feeds. All compounds should be considered irritant to skin, eyes and respiratory tract and as skin sensitisers. No risk for the safety for the environment is foreseen. Since all of the compounds under assessment are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. DOCUMENTATION PROVIDED TO EFSA 1. Chemically defined flavourings from Flavouring Group 31. June Submitted by FEFANA asbl./feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 2. Chemically defined flavourings from Flavouring Group 31. Supplementary information May Submitted by FEFANA asbl./feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 3. Chemically defined flavourings from Flavouring Group 31. Supplementary information January Submitted by FEFANA asbl./feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 4. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for Chemically defined flavourings from Flavouring Group Comments from Member States received through the ScienceNet. REFERENCES Abilleira E, Virto M, Nájera AI, Albisu M, Pérez-Elortond FJ, Ruiz de Gordoa JC, de Renobales M and Barron LJ, Effects of seasonal changes in feeding management under part-time grazing on terpene concentrations of ewes milk. Journal of Dairy Research, 78, Bampidis VA and Robinson PH, Citrus by-products as ruminant feeds: a review. Animal Feed Science and Technology, 128, EFSA Journal 2015;13(3):