Safety and efficacy of feed additives consisting of essential oils derived from the flower buds or the leaves of Syzygium aromaticum (L.) Merr. & L.M. Perry (clove bud oil and clove leaf oils) for all animal species (FEFANA asbl)

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of essential oils from the flower buds or the leaves of Syzygium aromaticum (L.) Merr. & L.M. Perry: clove bud oil, clove leaf oil and a β‐caryophyllene‐rich fraction of clove leaf oil (CCL oil), when used as sensory additives in feed and water for drinking for all animal species. Clove oils contain methyleugenol (up to 0.13%). The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the use of clove oils was of low concern for long‐living and reproductive animals and of no concern for target species for fattening at the following concentrations in complete feed: 25–50 mg/kg for clove bud oil, 28–100 mg/kg for clove leaf oil and 20 mg/kg for CCL oil. The FEEDAP Panel considered that the use in water for drinking alone or in conjunction with use in feed should not exceed the daily amount that is considered of low or no concern when consumed via feed alone. No concerns for consumers were identified following the use of clove oils up to the highest safe level in feed. The additives under assessment should be considered as irritant to skin and eyes and the respiratory tract and as skin sensitisers. When handling the essential oils, exposure of unprotected users to methyleugenol may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. The use of clove oils at the proposed use level in feed was not expected to pose a risk for the environment. Since clove bud oil and clove leaf oil are recognised to flavour food and their function in feed would be essentially the same, no demonstration of efficacy was considered necessary.

1. Introduction

1.1. Background and Terms of Reference

Regulation (EC) No 1831/2003 ¹ 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 specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, within a maximum of 7 years after the entry into force of this Regulation.

The European Commission received a request from the Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG) ² for authorisation/re‐evaluation of 18 preparations (namely geranium oil, geranium rose oil, eucalyptus oil, eucalyptus tincture, clove oil, clove tincture, broom teatree oil, purple loosetrife tincture, tea tree oil, melaleuca cajuputi oil, niaouli oil, allspice oil, bay oil, pomegranate bark extract, bambusa tincture, citronella oil, lemongrass oil and vetiveria oil) belonging to botanically defined group (BDG) 07 – Geraniales, Myrtales, Poales when used as feed additives for all animal species (category: sensory additives; functional group: flavourings). During the assessment, the applicant withdrew the application for four preparations (namely broom teatree oil, geranium oil, bay oil and vetiveria oil ³ ). These preparations were deleted from the register of feed additives. ⁴ During the course of the assessment, this application was split and the present opinion covers only one out of the initial 18 preparations under application: clove oil from the flower buds or the leaves of Syzygium aromaticum (L.) Merr. & L.M. Perry ⁵ for all animal species. During the assessment, the applicant clarified that three types of additives fall into the definition “clove oil”, i.e., clove bud oil, clove leaf oil and a β‐caryophyllene‐rich fraction of clove leaf oil. The three preparations from S. aromaticum will be assessed individually.

The remaining 13 preparations belonging to botanically defined group (BDG) 07 – Geraniales, Myrtales, Poales under application are assessed in separate opinions.

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. The particulars and documents in support of the application were considered valid by EFSA as of 21 December 2010.

According to Article 8 of Regulation (EC) No 1831/2003, 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. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the clove oil from the flower buds or leaves the of S. aromaticum, when used under the proposed conditions of use (see Sections 3.2.1.3, 3.3.1.3 and 3.4.1.3).

1.2. Additional information

An essential oil from Syzygium aromaticum L. (clove oil) is currently authorised as a feed additive according to the entry in the European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003 (2b natural products – botanically defined). It has not been assessed as a feed additive in the EU.

There is no specific EU authorisation for any S. aromaticum preparation when used to provide flavour in food. However, according to Regulation (EC) No 1334/2008 ⁶ flavouring preparations produced from food, may be used without an evaluation and approval as long as ‘they do not, on the basis of the scientific evidence available, pose a safety risk to the health of the consumer, and their use does not mislead the consumer’.

‘Clove (Caryophylli flos)’ are described in a monograph of the European Pharmacopoeia 11.0 (PhEur, 2022a). They are defined as the whole flower buds of S. aromaticum (L.) Merr. et L.M.Perry (Syn. Eugenia caryophyllus (Spreng.) Bullock et S.G.Harrison) dried until they become reddish‐brown. They have a minimum content of 150 mL/kg of essential oil.

‘Clove oil (Caryophylli floris aetheroleum)’ is described in a monograph of the European Pharmacopoeia 11.0 (PhEur, 2022b). It is defined as the essential oil obtained by steam distillation from the dried flower buds of S. aromaticum (L.) Merr. et L.M.Perry (Syn. E. caryophyllus (Spreng.) Bullock et S.G.Harrison.

In 1998, the Committee for Veterinary Medicinal Products of the European Medicines Agency (EMA) published a report on Caryophylli aetheroleum, the volatile oil obtained from S. aromaticum (L.) Merr. & Perry (EMA, 1998).

For S. aromaticum (L.) Merill et L.M. Perry, floris aetheroleum, the European Medicines Agency (EMA) issued a community herbal monograph for human medicinal use (EMA, 2011a), an assessment report on S. aromaticum (L.) Merill et L.M. Perry, flos and S. aromaticum (L.) Merill et L.M. Perry, floris aetheroleum (EMA, 2011b) and an addendum to the assessment report (EMA, 2020).

Many of the individual components of the essential oils have been already assessed as chemically defined flavourings for use in feed and food by the FEEDAP Panel, the EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) and the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF). The flavouring compounds currently authorised for feed ⁷ and/or food ⁸ use, together with the EU Flavour Information System (FLAVIS) number, the chemical group as defined in Commission Regulation (EC) No 1565/2000 ⁹ , and the corresponding EFSA opinion are listed in Table 1.

Table 1.

Flavouring compounds already assessed by EFSA as chemically defined flavourings, grouped according to the chemical group (CG) as defined in Commission Regulation (EC) No 1565/2000, with indication of the EU Flavour Information System (FLAVIS) number and the corresponding EFSA opinion

CG	Chemical Group	Product – EU register name (common name)	FLAVIS No	EFSA opinion,* Year
05	Saturated and unsaturated aliphatic secondary alcohols, ketones and esters with esters containing secondary alcohols	6‐Methylhept‐5‐en‐2‐one	07.015	2021a
06	Aliphatic, alicyclic and aromatic saturated and unsaturated tertiary alcohols and esters with esters containing tertiary alcohols ethers	Linalool	02.013	2012a
14	Furanones and tetrahydrofurfuryl derivatives	5‐Methylfurfural	13.001	2016a
		Furfural	13.018
17	Propenylhydroxybenzenes	Isoeugenol (a)	04.004	2012b
18	Allylhydroxybenzenes	Eugenol	04.003	2011
		Eugenyl acetate	09.020
		4‐Allylphenol (b)	04.058	EFSA, 2009 (AFC)
23	Benzyl alcohols, aldehydes, acids, esters and acetals	Benzyl alcohol	02.010	2012c
		Benzaldehyde	05.013
		Benzyl benzoate	09.727
		Methyl salicylate	09.749
31	Aliphatic and aromatic hydrocarbons and acetals containing saturated aldehydes	d‐Limonene	01.045	2015
		Pin‐2(10)‐ene (β‐pinene)	01.003	2016b
		Pin‐2(3)‐ene (α‐pinene)	01.004
		β‐Caryophyllene	01.007
		Valencene	01.017
		δ‐Cadinene (b) , (c)	01.021	2011, CEF
		1(5),11‐Guaiadiene (b) , (c)	01.023
		δ‐Germacrene (b) , (c)	01.042
		3,7,10‐Humulatriene (b) , (c)	01.043
		α‐Muurulene (b) , (c)	01.052
		β‐Bourbonene (b)	01.024	2015a, CEF
		α‐Farnesene (b)	01.040
32	Epoxides	β‐Caryophyllene oxide	16.043	2014, CEF

^*FEEDAP opinion unless otherwise indicated.

^(a)EFSA evaluated isoeugenol [04.004], a mixture of (E)‐ and (Z)‐isomers (EFSA FEEDAP Panel, 2012b).

^(b)Evaluated for use in food only. According to Regulation (EC) 1565/2000, flavourings evaluated by JECFA before 2000 are not required to be re‐evaluated by EFSA.

^(c)Evaluated applying the ‘Procedure’ described in the Guidance on the data required for the risk assessment of flavourings to be used in or on food (EFSA CEF Panel, 2010). No longer authorised for use as flavours in food, as the additional toxicity data requested (EFSA CEF Panel, 2011) were not submitted and the CEF Panel was unable to complete its assessment (EFSA CEF Panel, 2015b).

2. Data and methodologies

2.1. Data

The present assessment is based on data submitted by the applicant in the form of a technical dossier ¹⁰ in support of the authorisation request for the use of clove oils from S. aromaticum as a feed additive. The dossier was received on 8/6/2023 and the general information and supporting documentation is available at https://open.efsa.europa.eu/questions/EFSA-Q-2023-00397. ¹¹

The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer‐reviewed scientific papers, other scientific reports and experts' knowledge, to deliver the present output.

Some of the components of the essential oils under assessment have been already evaluated by the FEEDAP Panel as chemically defined flavourings (CDGs). The applicant submitted a written agreement to use the data submitted for the assessment of chemically defined flavourings (dossiers, publications and unpublished reports) for the risk assessment of preparations belonging to BDG 07, including the current one under assessment. ¹²

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the phytochemical markers in the additives. The evaluation report is related to the methods of analysis for each feed additive included the group BDG 07 (Geraniales, Myrtales, Poales). In particular, the EURL recommended a method based on gas chromatography coupled with flame ionisation detection (GC–FID) for the determination of the phytochemical marker eugenol in the feed additives clove leaf oil (eugenol type) and clove bud oil, and of the phytochemical marker β‐caryophyllene in the feed additive clove leaf oil (β‐caryophyllene type). ¹³

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of clove oils from S. aromaticum is in line with the principles laid down in Regulation (EC) No 429/2008 ¹⁴ and the relevant guidance documents: Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements (EFSA SC, 2009), Compendium of botanicals that have been reported to contain toxic, addictive, psychotropic or other substances of concern (EFSA, 2012), Guidance for the preparation of dossiers for sensory additives (EFSA FEEDAP Panel, 2012d), Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012e), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017a), Guidance on the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018), Guidance document on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals (EFSA SC, 2019a), Statement on the genotoxicity assessment of chemical mixtures (EFSA SC, 2019b), Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment (EFSA SC, 2019c) and General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021b). ¹⁵

3. Assessment

The additives under assessment, clove bud oil, clove leaf oil and a β‐caryophyllene‐rich fraction of clove leaf oil (hereinafter referred to as CCL oil), are derived from the flower buds or leaves of S. aromaticum (L.) Merr. & L.M. Perry and are intended for use as sensory additives (functional group: flavouring compounds) in feed and water for drinking for all animal species.

3.1. Origin and extraction

S. aromaticum (L.) Merr. & L.M. Perry (synonym: Eugenia caryophyllata Thunb.) is a small evergreen tree belonging to the myrtle (Myrtaceae) family. It is native to the Maluki islands (Moluccas or spice islands) of Indonesia but, because of its commercial importance, it is now widely cultivated in other parts of the world with similar climatic conditions. The tree is the source of ‘cloves’, the immature flower buds harvested just before flowering and dried, which are widely used as a spice and as a source of flavour and fragrance in many consumer products. The medicinal use of cloves includes the dental or oromucosal use of their essential oil as an analgesic in the treatment of toothache and is also described in Chinese and Ayurvedic systems. Essential oils are obtained either from the flower buds (clove bud oil) or from the leaves (clove leaf oil) of S. aromaticum.

Clove bud oil is obtained by steam distillation of the dried flower buds of S. aromaticum sourced from Indonesia. The volatile constituents are condensed and then separated from the aqueous phase by decantation.

Clove leaf oil is obtained by steam distillation of the leaves of S. aromaticum (sourced from Madagascar and Indonesia). The volatile constituents are condensed and then separated from the aqueous phase by decantation.

CCL oil is obtained by fractional distillation of clove leaf oil itself, resulting in an enriched composition in β‐caryophyllene and other hydrocarbons (e.g. 3,7,10‐humulatriene, α‐copaene and δ‐cadinene).

3.2. Clove bud oil

3.2.1. Characterisation of clove bud oil

The essential oil under assessment is a clear yellow to light amber, slightly viscous liquid with a characteristic odour. In seven batches of the additive, the refractive index (20°C) ranged between 1.534 and 1.537 (specification: 1.528–1.537), the specific gravity (20°C) between 1.0559 and 1.0593 and the optical rotation (20°C) between −0.64 and −1.0 (five batches). ¹⁶ ‘Clove oil’ is identified with the single Chemical Abstracts Service (CAS) number 8000‐34‐8, the European Inventory of Existing Chemical Substances (EINECS) number 284‐638‐7 and the Council of Europe (CoE) number 188. Clove bud oil is identified by the Flavor Extract Manufacturers Association (FEMA) number 2323.

For clove bud oil, the product specifications are based on the standard developed by the International Organisation for Standardization (ISO) 3142:1997 for ‘Oil of clove buds’, ¹⁷ adapted to reflect the concentrations of selected volatile components. Three compounds are specified as shown in Table 2, with eugenol selected as the phytochemical marker. Analysis of seven batches of the additive showed compliance with these specifications when analysed by GC–FID and expressed as percentage of gas chromatographic peak area (% GC area). ¹⁸

Table 2.

Major constituents of clove bud oil from the buds of Syzygium aromaticum (L.) Merr. & L.M. Perry as defined by specifications and batch to batch variation based on the analysis of seven batches by gas chromatography with flame ionisation detector. The content of each constituent is expressed as the area per cent of the corresponding chromatographic peak (% GC area), assuming the sum of chromatographic areas of all detected peaks as 100%

Constituent	CAS No	FLAVIS No	% GC area
EU register name			Specification (a)	Mean	Range
Eugenol	97‐53‐0	04.003	67–85	82.7	81.78–84.00
β‐Caryophyllene	87‐44‐5	01.007	2–16	6.7	5.89–7.21
Eugenyl acetate	93‐28‐7	09.030	5–15	8.5	7.90–9.09

EU: European Union; CAS No: Chemical Abstracts Service number; FLAVIS No: EU Flavour Information System numbers.

^(a)Specifications defined based on GC–FID analysis.

The applicant provided a full characterisation of the seven batches obtained by gas chromatography–mass spectrometry (GC–MS). ¹⁹ In total, up to 30 constituents were detected, 28 of which were identified and accounted on average for 99.33% (99.05–99.82%) of the % GC area. The three compounds indicated in the product specifications accounted for 96.2% on average (range 95.6–96.8%) of the GC area. Besides the three compounds indicated in the product specifications, 11 other compounds were detected at individual levels ≥ 0.05% and are listed in Table 3. These 14 compounds together account on average for 99.15% (98.93–99.59%) of the % GC area. The remaining 14 compounds identified (ranging between 0.002% and 0.04%) and accounting for 0.18% of the % GC area are listed in the footnote. ²⁰ Based on the available data on the characterisation, clove bud oil is considered a fully defined mixture (EFSA SC, 2019a).

Table 3.

Constituents of clove bud oil from the buds of Syzygium aromaticum (L.) Merr. & L.M. Perry accounting for ≥ 0.05% of the composition (based on the analysis of seven batches by gas chromatography–mass spectrometry). The content of each constituent is expressed as the area per cent of the corresponding chromatographic peak (% GC area), assuming the sum of chromatographic areas of all detected peaks as 100%

Constituent	CAS No	FLAVIS No	% GC area
EU register name			Mean	Range
Eugenol	97‐53‐0	04.003	75.06	73.03–75.96
β‐Caryophyllene	87‐44‐5	01.007	13.20	12.21–14.11
Eugenyl acetate	93‐28‐7	09.030	7.92	7.40–8.49
3,7,10‐Humulatriene	6753‐98‐6	01.043	1.13	1.03–1.23
β‐Caryophyllene epoxide	1139‐30‐6	16.043	0.76	0.45–1.09
α‐Copaene	3856‐25‐5	–	0.40	0.34–0.52
Humulene oxide II	19888‐34‐7	–	0.38	0.09–1.09
α‐Cubebene	17699‐14‐8	–	0.17	0.04–0.49
Methyl salicylate	119‐36‐8	09.749	0.16	0.08–0.25
Calamenene	483‐77‐2	–	0.13	0.10–0.19
4‐Allylphenol	501‐92‐8	04.058	0.13	0.10–0.14
δ‐Cadinene	29350‐73‐0	01.021	0.12	0.09–0.15
Methyleugenol	93‐15‐2	–	0.08	0.04–0.13
trans‐Cadina‐1(6),4‐diene	20085‐11‐4	–	0.07	0.07–0.08
Total			99.15	98.93–99.59 (a)

EU: European Union; CAS No: Chemical Abstracts Service number; FLAVIS No: EU Flavour Information System numbers.

^(a)The values given for the total are the lowest and the highest values of the sum of the components in the seven batches analysed.

The applicant performed a literature search for information on the chemical composition of S. aromaticum and its preparations and the presence of compounds of known concern. ²¹ Among the compounds identified in the essential oil from the leaves and the flower buds of S. aromaticum, eugenol (up to 90%) and eugenyl acetate (up to 17%) are reported in the EFSA Compendium of botanicals (EFSA, 2012). ¹⁷ Several references (Elzayyat et al., 2018; Gooderham et al., 2020; Selles et al., 2020) reported the presence of methyleugenol (0.01–0.04%) in clove bud oils and one reference (Gooderham et al., 2020) in a clove leaf oil (methyleugenol 0.01%) similar in composition to the oils under assessment. The presence of estragole was reported in three references, however in products that are different from the clove bud oil, the clove leaf oil and the CCL oil under assessment.

Methyleugenol (0.039–0.128%) was detected by GC–MS in all the seven batches of clove bud oil under assessment, whereas estragole was not detected (limit of detection (LOD): 0.01%).

The applicant consulted the online database on Volatile Compounds in Food (VCF). ²² The presence of other substances of concern was not reported.

3.2.1.1. Impurities

The applicant referred to the ‘periodic testing’ of some representative flavourings premixtures for mercury, cadmium, lead, arsenic, fluoride, dioxins and polychlorinated biphenyls (PCBs), organo‐chloride pesticides, organo‐phosphorus pesticides, aflatoxins (B1, B2, G1, G2) and ochratoxin A. However, no data have been provided on the presence of these impurities. Since clove bud oil is produced by steam distillation, the likelihood of any measurable carry‐over of all the above‐mentioned elements is considered low, except for mercury.

3.2.1.2. Shelf life

The shelf‐life of clove bud oil is stated to be at least 12 months, when stored in tightly closed containers under standard conditions (in a cool, dry place protected from light). ²³ However, no data supporting this statement were provided.

3.2.1.3. Conditions of use

Clove bud oil is intended to be added to feed or water for drinking for all animal species without withdrawal. The maximum proposed use level in complete feed for all animal species is 50 mg/kg, except for laying hens and rabbits, for which a maximum use level of 28.5 and 30.5 mg/kg complete feed is proposed, respectively. No use level has been proposed by the applicant for use in water for drinking.

3.2.2. Safety

The assessment of safety of clove bud oil is based on the maximum use levels proposed by the applicant.

Many of the components of clove bud oil, accounting for more than 98.5% of the GC peak areas, have been previously assessed and considered safe for use as flavourings, and are currently authorised for use in food ⁸ without limitations and for use in feed ⁷ at individual use levels higher than those resulting from the intended use of the essential oil in feed, except for eugenol and eugenyl acetate in poultry. The list of the compounds already evaluated by the EFSA Panels is given in Table 1 (see Section 1.2). An update of the absorption distribution, metabolism and excretion (ADME) of eugenol and eugenyl acetate in poultry is presented in Section 3.2.2.1.

One compound listed in Tables 1, 3, 7, 10‐humulatriene [01.043] has been evaluated in Flavouring Group Evaluation 25, Revision 2 (FGE.25Rev2) by applying the procedure described in the Guidance on the data required for the risk assessment of flavourings to be used in or on food (EFSA CEF Panel, 2010). For this compound, for which there is no concern for genotoxicity, EFSA requested additional subchronic toxicity data (EFSA CEF Panel, 2011). In the absence of such data, the EFSA CEF Panel was unable to complete its assessment (EFSA CEF Panel, 2015b). As a result, this compound is not authorised for use as flavour in food. For this compound, in the absence of toxicity data, the FEEDAP Panel applies the threshold of toxicological concern (TTC) approach or read‐across from structurally related substances, as recommended in the Guidance document on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals (EFSA SC, 2019a).

Table 7.

Constituents of clove leaf oil from the leaves of Syzygium aromaticum (L.) Merr. & L.M. Perry accounting for ≥ 0.05% of the composition (based on the analysis of six batches by gas chromatography–mass spectrometry). The content of each constituent is expressed as the area per cent of the corresponding chromatographic peak (% GC area), assuming the sum of chromatographic areas of all detected peaks as 100%

Constituent	CAS No	FLAVIS No	% GC area
EU register name			Mean	Range
Eugenol	97‐53‐0	04.003	82.60	78.68–86.01
β‐Caryophyllene	87‐44‐5	01.007	13.45	11.10–14.85
Eugenyl acetate	93‐28‐7	09.030	0.70	0.02–1.47
3,7,10‐Humulatriene	6753‐98‐6	01.043	1.60	1.10–2.03
β‐Caryophyllene epoxide	1139‐30‐6	16.043	0.44	0.09–1.03
Octane	111‐65‐9	–	0.32	0.11–0.52
Caryophyllene alcohol	56747‐96‐7	–	0.24	0.24 (a)
α‐Copaene	3856‐25‐5	–	0.21	0.06–0.30
β‐Cadinene	523‐47‐7	–	0.11	0.09–0.13
Nonane	111‐84‐2	–	0.11	0.05–0.16
β‐Elemene	33880‐83‐0	–	0.07	0.07 (a)
4‐Allylphenol	501‐92‐8	05.058	0.06	0.06 (a)
Calamenene	483–77‐2	–	0.06	0.05–0.07
Benzyl benzoate	120‐51‐4	09.727	0.06	0.06 (a)
Humulene oxide II	19888‐34‐7	–	0.05	0.01–0.08
Germacra‐1(10),4(14),5‐triene	23986‐74‐5	01.042	0.05	0.05 (a)
Total			2.53	99.50–99.90 (b)

EU: European Union; CAS No: Chemical Abstracts Service number; FLAVIS No: EU Flavour Information System numbers.

^(a)Compound detected in only one batch.

^(b)The values given for the Total (range) are the lowest and the highest values of the sum of the components in the six batches analysed.

Table 10.

Maximum safe concentrations of clove leaf oil in complete feed (mg/kg) for all animal species and categories

Animal categories	Maximum feed concentration of low (a) /no concern (b) (mg/kg complete feed) (c)
Long‐living and reproductive animals (a)
Laying hens and other laying/reproductive birds including animals reared for laying/reproduction and ornamental birds	54
Sows and other Suidae species for reproduction including animals reared for reproduction	95
Dairy cows and other ruminants and camelids for milk production and reproduction including animals reared for milk production/reproduction	92
Sheep/goats	100
Horses and other Equidae	100
Rabbits	57
Dogs	100
Cats	28
Ornamental fish	100
Short‐living animals (species for fattening) (b)
Chickens for fattening and minor poultry for fattening	36
Turkey for fattening	48
Pigs for fattening	77
Piglets and other Suidae species for meat production	65
Veal calves (milk replacer)	100
Cattle for fattening and other ruminants for fattening and camelids at the same physiological stage	100
Sheep/goats	100
Horses and other Equidae	100
Rabbits	57
Salmonids and minor fin fish	100
Any other species	28

^(a)Based on a MOE > 10,000 for long‐living and reproductive animals, calculated as the ratio of the reference point (BMDL10 of 22.2 mg/kg bw per day) to the combined intake.

^(b)Based on a MOE > 100 for short‐living animals (species for fattening), calculated as the ratio of the reference point (NOAEL of 10 mg/kg bw per day) to the combined intake.

^(c)Complete feed containing 88% DM, milk replacer 94.5% DM.

Eight components of clove bud oil, accounting on average for 1.12% of the GC area, have not been previously assessed for use as flavourings. The FEEDAP Panel notes that six of them are aliphatic mono‐ or sesquiterpenes structurally related to flavourings already assessed in CG 31 and for which a similar metabolic and toxicological profile is expected. ²⁴ These six lipophilic compounds, accounting together for about 0.70% of the GC area, are expected to be rapidly absorbed from the gastrointestinal tract, oxidised to polar oxygenated metabolites, conjugated and excreted (EFSA FEEDAP Panel, 2015, 2016b). Another compound, 1‐epi‐cubenol, is a tertiary alcohol structurally related to the compounds already evaluated in CG 6, and is expected to be absorbed, metabolised and excreted (EFSA FEEDAP Panel, 2012a). The last compound, humulene oxide II, is an oxygenated terpene and is structurally related to compounds that have been evaluated for use in food and/or feed.

The additive contains methyleugenol (0.039–0.128%). For the ADME and the toxicology of methyleugenol, reference is made to the safety evaluation made by the FEEDAP Panel in the EFSA opinion on laurel leaf oil (EFSA FEEDAP Panel, 2023).

3.2.2.1. Absorption distribution, metabolism and excretion of eugenol and eugenyl acetate in poultry

Both in rodents and humans, eugenol and eugenyl acetate are rapidly absorbed from the gastrointestinal tract. Eugenol is conjugated in the liver, and the resulting glucuronide and sulfate conjugates are subsequently excreted in the urine. Eugenyl acetate is hydrolysed to eugenol and the corresponding carboxylic acid (WHO, 2006). To a lesser extent, both compounds are metabolised to polar metabolites, which are also conjugated and eliminated, primarily in the urine. Only small amounts (< 1%) of eugenol are excreted unchanged. In its former assessment on CG 18, the Panel noted that the metabolic pathways involved in the biotransformation of eugenol and eugenyl acetate are common to mammalian species, but no data were available concerning their metabolic fate in poultry. Therefore, the FEEDAP Panel concluded that the efficient metabolism of eugenol and eugenyl acetate in mammals and the subsequent rapid excretion of the metabolites preclude their accumulation in tissues and transfer to products. However, the lack of data on metabolism and residues in poultry precluded an assessment of consumer exposure from this source. However, phase I (CYP450 monooxygenase families, epoxide hydrolases) and phase II enzymes (glucuronide‐ sulfate‐ and glutathione‐transferases) involved in the biotransformation of several classes of compounds including CG 18 compounds are also expressed in birds as already described (EFSA FEEDAP Panel, 2013, 2016b,c). Therefore, birds can also be assumed to have the ability to metabolise and excrete eugenol as mammals and there is no evidence that the compound or its metabolites would accumulate in tissues and cause a concern for consumer safety.

3.2.2.2. Safety for the target species

Tolerance studies in the target species and/or toxicological studies in laboratory animals made with the essential oil under application were not submitted.

In the absence of toxicological data with the additive under assessment, the approach to the safety assessment of a mixture whose individual components are known is based on the safety assessment of each individual component (component‐based approach). This approach requires that the mixture is sufficiently characterised and that the individual components can be grouped into assessment groups, based on structural and metabolic similarity. The combined toxicity can be predicted using the dose addition assumption within an assessment group, taking into account the relative toxic potency of each component (EFSA SC, 2019a).

As the distilled fraction of clove bud oil is a fully defined mixture (the identified components represent about 99% of the % CG area, see Section 3.2.1), the FEEDAP Panel applied a component‐based approach to assess the safety for target species. Methyleugenol, a substance for which a concern for genotoxicity has been identified, is assessed separately.

Components other than methyleugenol

Based on considerations related to structural and metabolic similarities, the components were allocated to seven assessment groups, corresponding to the chemical groups (CGs) 5, 6, 14, 18, 23, 31 and 32, as defined in Annex I of Regulation (EC) No 1565/2000. For chemical group 31 (‘aliphatic and aromatic hydrocarbons’), sub‐assessment groups as defined in FGE.25 and FGE.78 were applied (EFSA CEF Panel, 2015a,b). The allocation of the components to the (sub‐)assessment groups is shown in Table 4 and in the corresponding footnote.

Table 4.

Compositional data, intake values (calculated for chickens for fattening at 50 mg/kg complete feed), reference points and margin of exposure (MOE) for the individual components of clove bud oil classified according to assessment groups

Essential oil composition			Exposure		Hazard characterisation		Risk characterisation
Assessment group	FLAVIS No	Highest conc. in the oil	Highest feed conc.	Intake (a)	Cramer class (b)	NOAEL (c)	MOE	MOET
Constituent	–	%	mg/kg	mg/kg bw per day	–	mg/kg bw per day	–	–
CG 6
1‐epi‐Cubenol	–	0.04	0.020	0.0018	I	3	1,671
CG 14
Furfural	13.018	0.06	0.031	0.0028	(II)	54	19,404
5‐Methylfurfural	13.001	0.02	0.011	0.0009	II	0.91	965
								920
CG 18
Eugenol	04.003	76.0	37.98	3.4097	(I)	300	88
Eugenyl acetate	09.020	14.1	7.056	0.6334	(I)	300	474
4‐Allylphenol	04.058	0.14	0.072	0.0065	I	3	464
								64
CG 23
Methyl salicylate	09.749	0.25	0.125	0.0112	(I)	50	4,474
CG 31, IVe (Benzene hydrocarbons, alkyl)
Calamenene	–	0.19	0.094	0.0084	I	3	356
CG 31, V (Bi‐, tricyclic, non‐aromatic hydrocarbons)
β‐Caryophyllene	01.007	8.49	4.245	0.3810	(I)	222	583
α‐Copaene	–	0.52	0.260	0.0233	(I)	222	9,530
α‐Cubebene	–	0.49	0.245	0.0219	(I)	222	10,114
δ‐Cadinene	01.021	0.15	0.073	0.0066	(I)	222	33,875
trans‐Cadina‐1(6),4‐diene	–	0.08	0.039	0.0035	I	3	857
Isocadinene	–	0.05	0.027	0.0024	I	3	1,238
Cadina‐1,4‐diene	–	0.05	0.024	0.0021	I	3	1,422
MOET CG 31, V								216
CG 31, VI
3,7,10‐Humulatriene	01.043	1.23	0.614	0.0551	(I)	111 (d)	2,014
CG 32
β‐Caryophyllene oxide	16.043	1.09	0.544	0.0488	(III)	109	2,232
Humulene oxide II	–	1.09	0.544	0.0488	(III)	109	2,232
								1,116

^(a)Intake calculations for the individual components are based on the use level of 50 mg/kg in feed for chickens for fattening, the species with the highest ratio of feed intake/body weight. The MOE for each component is calculated as the ratio of the reference point (no observed adverse effect level, NOAEL) to the intake. The combined margin of exposure (MOET) is calculated for each assessment group as the reciprocal of the sum of the reciprocals of the MOE of the individual substances.

^(b)When a NOAEL value is available or read‐across is applied, the allocation to the Cramer class is put into parentheses.

^(c)Values in bold refer to those components for which the NOAEL value was available, values in italics are the 5th percentile of the distribution of NOAELs of the corresponding Cramer Class, other values (plain text) are NOAELs extrapolated by using read‐across.

^(d)The NOAEL of 222 mg/kg bw per day for β‐caryophyllene was halved to take into account the uncertainty in read‐across.

For each component in the assessment group, exposure of target animals was estimated considering the use levels in feed, the percentage of the component in the oil and the default values for feed intake according to the guidance on the safety of feed additives for target species (EFSA FEEDAP Panel, 2017b). Default values on body weight (bw) are used to express exposure in terms of mg/kg bw per day. The intake levels of the individual components calculated for chickens for fattening, the species with the highest ratio of feed intake/bw per day, are shown in Table 4.

For hazard characterisation, each component of an assessment group was first assigned to the structural class according to Cramer classification (Cramer et al., 1978). For some components in the assessment group, toxicological data were available to derive no observed adverse effect level (NOAEL) values. Structural and metabolic similarity among the components in the assessment groups were assessed to explore the application of read‐across. If justified, extrapolation from a known NOAEL of a component of an assessment group to the other components of the group with no available NOAEL was made. If sufficient evidence was available for the members of a (sub‐)assessment group, a (sub‐)assessment group NOAEL was derived.

Toxicological data for sub‐chronic studies, from which NOAEL values could be derived, were available for 6‐methylhept‐5‐en‐2‐one [07.015] in CG 5 (EFSA FEEDAP Panel, 2021a), linalool [02.013] in CG 6 (EFSA FEEDAP Panel, 2012a), furfural [13.018] in CG 14 (EFSA FEEDAP Panel, 2016a), eugenol [04.003] (EFSA FEEDAP Panel, 2011), methyl salicylate [09.749] in CG 23 (EFSA FEEDAP Panel, 2012c) and for the representative compounds for sub‐assessment groups of CG 31, d‐limonene [01.045] and β‐caryophyllene [01.007] (EFSA FEEDAP Panel, 2015, 2016b) and β‐caryophyllene oxide [16.043] (EFSA CEF Panel, 2014). In addition, for benzyl alcohol the EFSA Panel on Food Additives and Flavourings (FAF) established an acceptable daily intake (ADI) of 4 mg/kg bw per day based on a NOAEL of 400 mg/kg bw per day from a carcinogenicity study in rats (EFSA FAF Panel, 2019).

Considering the structural and metabolic similarities in CG 18, read‐across was applied using the NOAEL of 300 mg/kg bw per day for eugenol [04.003] to extrapolate to eugenyl acetate [09.020]. Similarly, read‐across was also applied using the NOAEL of 400 mg/kg bw per day for benzyl alcohol [02.010] to extrapolate to ethyl benzoate [09.276] and to benzaldehyde [05.013] ²⁵ in CG 23.

The NOAEL of 222 mg/kg bw per day for β‐caryophyllene [01.007] was applied using read‐across to the compounds within CG 31 sub‐assessment group V (α‐copaene, α‐cubebene, δ‐cadinene [01.021], alloroarmadendrene, α‐pinene [01.004] and β‐pinene [01.003]) (EFSA CEF Panel, 2015a,b). Read‐across was also applied from β‐caryophyllene [01.007] to 3,7,10‐humulatriene [01.043] in CG 31, VI. The NOAEL of 222 mg/kg bw per day for β‐caryophyllene [01.007] was halved to take into account the uncertainty in read‐across.

The NOAEL of 109 mg/kg bw per day for β‐caryophyllene oxide [16.043] was extrapolated to humulene oxide II in CG 32.

For the remaining compounds, ²⁶ toxicity studies performed with the compounds under assessment with derived NOAEL values were not available and read‐across was not possible. Therefore, the threshold TTC approach was applied (EFSA FEEDAP Panel, 2017b). All these compounds belong to Cramer class I except 5‐methylfurfural (Cramer class II).

As the result of the hazard characterisation, a reference point was identified for each component in the assessment group based on the toxicity data available (NOAEL from in vivo toxicity study or read across) or from the 5th percentile of the distribution of NOAELs of the corresponding Cramer Class (i.e. 3, 0.91 and 0.15 mg/kg bw per day, respectively, for Cramer Class I, II and III compounds, Munro et al., 1996). Reference points selected for each compound are shown in Table 4.

For risk characterisation, the margin of exposure (MOE) was calculated for each component as the ratio between the reference point and the exposure. For each assessment group, the combined (total) margin of exposure (MOET) was calculated as the reciprocal of the sum of the reciprocals of the MOE of the individual substances (EFSA SC, 2019a). A MOET > 100 allowed for interspecies differences and intraspecies variability (as in the default 10 × 10 uncertainty factor). The compounds resulting individually in an MOE > 50,000 were not further considered in the assessment group as their contribution to the MOE(T) is negligible. They are listed in the footnote. ²⁷

The approach to the safety assessment of clove bud oil for the target species is summarised in Table 4. The calculations were done for chickens for fattening, the species with the highest ratio of feed intake/body weight and represent the worst‐case scenario at the use level of 50 mg/kg complete feed.

As shown in Table 4, for the assessment group CG 18, which includes the major component eugenol, the MOET was < 100 at the proposed use level (50 mg/kg feed). From the lowest MOET of 64 for chickens for fattening, the MOET for CG 18 compounds was calculated for the other target species considering the respective daily feed intake and conditions of use. The results are summarised in Table 5.

Table 5.

Combined margin of exposure (MOET) for CG 31,V calculated for the different target animal categories at the proposed use level in feed

Animal category	Body weight (kg)	Feed intake (g DM/day)	Proposed use level (mg/kg feed)	Lowest MOET CG 31,V	Maximum safe use level (mg/kg feed) (a)
Chicken for fattening	2	158	50	64	32
Laying hen	2	106	28.5	176	28.5
Turkey for fattening	3	176	50	86	43
Piglet	20	880	50	115	50
Pig for fattening	60	2,200	50	137	50
Sow lactating	175	5,280	50	169	50
Veal calf (milk replacer)	100	1,890	50	287	50
Cattle for fattening	400	8,000	50	253	50
Dairy cows	650	20,000	50	163	50
Sheep/goat	60	1,200	50	253	50
Horse	400	8,000	50	253	50
Rabbit	2	100	30.5	166	30.5
Salmon	0.12	2.1	50	281	50
Dog	15	250	50	297	50
Cat	3	60	50	253 (b)	25
Ornamental fish	0.012	0.054	50	1,011	50

^(a)Complete feed containing 88% DM, milk replacer 94.5% DM.

^(b)The minimum MOET considered to be of no concern for cats should be increased to 500 because of the reduced capacity of glucuronidation of compounds.

Table 5 shows that the MOET exceeds the value of 100 for all animal categories except poultry species for fattening. For these species the maximum safe use levels in feed were calculated to ensure a MOET ≥ 100. Because glucuronidation is an important metabolic pathway to facilitate the excretion of the components of the essential oil and considering that cats have a low capacity for glucuronidation (Court and Greenblatt, 1997; Lautz et al., 2021), the use of clove bud oil as additive in cat feed needs a wider margin of exposure. A MOET of 500 is considered adequate. The maximum safe levels in feed (without considering the presence of methyleugenol) are shown in Table 5.

The FEEDAP Panel considers that the use in water for drinking the use in water for drinking alone or in conjunction with use in feed should not exceed the daily amount that is considered safe when consumed via feed alone.

Methyleugenol

Methyleugenol belongs to the group of p‐allylalkoxybenzenes and is a genotoxic carcinogen. According to the General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021b), different reference points and a different magnitude of the MOE are applied for long‐living and reproductive animals (including those animals reared for laying/breeding/reproduction) and for short‐living animals. Short‐living animals are defined as those animals raised for fattening whose lifespan under farming conditions makes it very unlikely that they develop cancer as a result of the exposure to genotoxic and/or carcinogenic substances in the diet.

For long‐living and reproductive animals, a MOE with a magnitude > 10,000 when comparing estimated exposure to genotoxic and/or carcinogenic substances with a BMDL₁₀ from a rodent carcinogenicity study is considered indicative of low concern. The FEEDAP Panel identified the BMDL₁₀ of 22.2 mg/kg bw per day derived from rodent carcinogenicity studies with methyleugenol (NTP, 2000; Suparmi et al., 2019), as the reference point for the entire group of p‐allylalkoxybenzenes (EFSA FEEDAP Panel, 2022).

For short‐living animals, genotoxicity and carcinogenicity endpoints are not considered biologically relevant, therefore a lower magnitude of the MOE (> 100) when comparing estimated exposure with a reference point based on non‐neoplastic endpoints is considered adequate (EFSA FEEDAP Panel, 2021b). The FEEDAP Panel identified a NOAEL of 10 mg/kg bw per day for non‐neoplastic lesions (changes in organ weight ²⁸ and function, including effects on liver ²⁹ and the glandular stomach ³⁰ ) from a 90‐day study in mice with methyleugenol (NTP, 2000; EFSA FEEDAP Panel, 2023).

Low concentrations of methyleugenol were detected in all batches of the additive under assessment (average: 0.078%, range: 0.039–0.128%). The use of clove bud oil at the levels in feed which were considered safe for the different target species, without considering the presence of methyleugenol, ranges from 25 to 50 mg/kg complete feed (see Table 5). These levels correspond to methyleugenol concentrations ranging from 0.032 to 0.064 mg/kg complete feed. The highest daily intake of methyleugenol in μg/kg bw was calculated for the different target animal categories considering the highest analysed value in the additive (0.128%). The calculated intake values range between 0.33 μg/kg bw per day (in ornamental fish) and 3.7 μg/kg bw per day (in chickens for fattening) (see Appendix A).

When the estimated exposures for long‐living and reproductive animals are compared to the BMDL₁₀ of 22.2 mg/kg bw per day, derived by Suparmi et al. (2019) from a rodent carcinogenicity study (NTP, 2000), a MOE ranging between 9,921 and 67,833 is calculated for long living‐animals. When comparing the exposure of short‐living animals to the reference point based on non‐neoplastic endpoints, a magnitude of the MOE > 100, is obtained for all species (see Appendix A).

The magnitude of the MOE is indicative of a low concern for long‐living and reproductive animals and of no concern for species for fattening.

3.2.2.3. Conclusions on safety for the target species

The conclusions of the FEEDAP Panel on the maximum safe concentrations in complete feed of clove bud oil are summarised in Table 6.

Table 6.

Maximum safe concentrations of clove bud oil in complete feed (mg/kg) for all animal species and categories

Animal categories	Maximum feed concentration of low (a) /no concern (b) (mg/kg complete feed) (c)
Long‐living and reproductive animals (a)
Laying hens and other laying/reproductive birds including animals reared for laying/reproduction and ornamental birds	28.5
Sows and all pigs (Suidae) for reproduction including animals reared for reproduction	50
Dairy cows and other ruminants and camelids for milk production and reproduction including animals reared for milk production/reproduction	50
Sheep/goats	50
Horses and other Equidae	50
Rabbits	30.5
Dogs	50
Cats	25
Ornamental fish	50
Short‐living animals (species for fattening) (b)
Chickens for fattening and minor poultry for fattening	32
Turkey for fattening	43
Pigs for fattening	50
Piglets and other Suidae species for meat production	50
Veal calves (milk replacer)	50
Cattle for fattening and other ruminants for fattening and camelids at the same physiological stage	50
Sheep/goats	50
Horses and other Equidae	50
Rabbits	30.5
Salmonids and minor fin fish	50
Any other species	25

^(a)Based on a MOE > 10,000 for long‐living and reproductive animals, calculated as the ratio of the reference point (BMDL10 of 22.2 mg/kg bw per day) to the combined intake.

^(b)Based on a MOE > 100 for short living animals (species for fattening), calculated as the ratio of the reference point (NOAEL of 10 mg/kg bw per day) to the combined intake.

^(c)Complete feed containing 88% DM, milk replacer 94.5% DM.

The FEEDAP Panel considers that the use in water for drinking alone or in combination with use in feed should not exceed the daily amount that is considered of low or no concern when consumed via feed alone.

3.2.2.4. Safety for the consumer

Cloves (immature flower buds) and their preparations including ethanolic extracts are added to a wide range of food categories as spice or for flavouring purposes. Although individual consumption figures for the EU are not available, the Fenaroli's handbook of flavour ingredients (Burdock, 2009) cites values of 0.43 mg/kg bw per day for cloves, 0.045 mg/kg bw per day for clove bud oil and 0.038 mg/kg bw per day for clove leaf oil.

The majority of the individual constituents of the clove bud oil are currently authorised as food flavourings without limitations and have been already assessed for consumer safety when used as feed additives in animal production (see Table 1, Section 1.2).

No data on residues in products of animal origin were made available for any of the constituents of the essential oil. However, the Panel recognises that the constituents of clove bud oil are expected to be extensively metabolised and excreted in the target species.

Although the FEEDAP Panel could not conclude on the safety for the consumer of eugenol when used as a feed additive in poultry species at the proposed use level of 25 mg/kg complete feed in its previous assessment (EFSA FEEDAP Panel, 2011), the inclusion of 22–33 mg eugenol/kg complete feed to poultry species is considered of no concern for the consumer (see Section 3.2.2.1). For methyleugenol, the available data indicate that it is absorbed, metabolised and rapidly excreted and is not expected to accumulate in animal tissues and products at the levels present in the additive (EFSA FEEDAP Panel, 2023).

Considering the above and the reported human exposure due to direct use of clove bud oil in food (Burdock, 2009), it is unlikely that consumption of products from animals given clove bud oil up to the highest safe use level in feed would significantly increase human background exposure.

No safety concern would be expected for the consumer from the use of the clove bud oil up to the highest proposed use level in feed.

3.2.2.5. Safety for the user

No specific data were provided by the applicant regarding the safety of the additive for users.

The applicant produced a safety data sheet ³¹ for clove bud oil, where hazards for users have been identified.

The essential oil under assessment should be considered as irritant to skin and eyes, and as a dermal and respiratory sensitiser.

When handling the essential oil, exposure of unprotected users to methyleugenol may occur. Therefore, to reduce the risk, the exposure of the users should be minimised.

3.2.2.6. Safety for the environment

Although S. aromaticum L. is not a native species to Europe, eugenol, β‐caryophyllene and eugenyl acetate, the most abundant components in the essential oil are naturally occurring in European plants. Therefore, the use of the clove bud oil under the proposed conditions of use in animal feed is not expected to pose a risk to the environment.

3.3. Clove leaf oil

3.3.1. Characterisation of clove leaf oil

The essential oil under assessment is a clear yellow to amber, slightly viscous liquid with a characteristic spice odour. In four batches of the additive, the refractive index (20 °C) ranged between 1.5315 and 1.5323 (specification: 1.5303–1.540). ¹⁶ ‘Clove oil’ is identified with the CAS number 8000‐34‐8, ³² the EINECS number 284‐638‐7, and the CoE number 188. Clove leaf oil is identified by the FEMA number 2325.

For clove leaf oil, the product specifications are based on the standard developed by the International Organisation for Standardization (ISO) 3141:1997 for ‘Oil of clove leaves’, ³³ adapted to reflect the concentrations of selected volatile components. Three compounds contribute to the specifications, eugenol (80–95%, selected as the phytochemical marker), β‐caryophyllene (4–17%) and eugenyl acetate (≤ 1%). Analysis of two batches of the additive showed compliance with these specifications when analysed by GC–FID and expressed as % GC area. ³⁴

The applicant provided a full characterisation of six batches by GC–MS. ¹⁶ In total, up to 41 constituents were detected, 34 of which were identified and accounted on average for 99.41% (98.67–99.93%) of the % GC area. The three compounds indicated in the product specifications account for about 96.8% on average (range 94.6–98.4%) of the GC area. Besides the three compounds indicated in the product specifications, 13 other compounds were detected at individual levels ≥ 0.05% and are listed in Table 7. These 16 compounds together account on average for 99.3% (98.4–99.9%) of the % GC area. The remaining 18 compounds (ranging between 0.005% and 0.04%) and accounting for 0.12% of the % GC area are listed in the footnote. ³⁵ Based on the available data on the characterisation, clove leaf oil is considered a fully defined mixture (EFSA SC, 2019a).

The applicant performed a literature search for information on the chemical composition of S. aromaticum and its preparations and the presence of compounds of known concern (see Section 3.2.1).

Methyleugenol (0.010–0.030%) was detected by GC–MS in four out of the six batches of the clove leaf oil, whereas estragole was not detected (LOD: 0.01%).

3.3.1.1. Impurities

The applicant referred to the ‘periodic testing’ of some representative flavourings premixtures for mercury, cadmium, lead, arsenic, fluoride, dioxins and polychlorinated biphenyls (PCBs), organo‐chloride pesticides, organo‐phosphorus pesticides, aflatoxins (B1, B2, G1, G2) and ochratoxin A. However, no data have been provided on the presence of these impurities. Since clove leaf oil is produced by steam distillation, the likelihood of any measurable carry‐over of all the above‐mentioned elements is considered low, except for mercury.

3.3.1.2. Shelf life

The shelf‐life of clove leaf oil is stated to be at least 12 months when stored in tightly closed containers under standard conditions (in a cool, dry place protected from light).²³ However, no data supporting this statement were provided.

3.3.1.3. Conditions of use

Clove leaf oil is intended to be added to feed and water for drinking for all animal species without a withdrawal period. The maximum proposed use level in complete feed for all target species is 100 mg/kg. For the use in water for drinking the applicant proposed maximum use levels of 40 mg/kg for poultry, calves and rabbits, 20 mg/kg for piglets, pigs for fattening and sows and 10 mg/kg for cattle for fattening, dairy cows, sheep/goats and horses.

3.3.2. Safety

The assessment of safety of clove leaf oil is based on the maximum use level proposed by the applicant (100 mg/kg complete feed).

Many of the components of clove leaf oil, accounting on average for about 99% of the GC peak area, have been previously assessed and considered safe for use as flavourings, and are currently authorised for use in food ⁸ without limitations and for use in feed ⁷ at individual use levels higher than those resulting from the intended use of the essential oil in feed except for eugenol and eugenyl acetate in poultry (see Section 3.2.2.1). The list of the compounds already evaluated by the EFSA Panels is given in Table 1 (see Section 1.2).

Two compounds listed in Table 1, germacra‐1(10),4(14),5‐triene [01.042] and 3,7,10‐humulatriene [01.043], have been evaluated in Flavouring Group Evaluation 25, Revision 2 (FGE.25Rev2) by applying the procedure described in the Guidance on the data required for the risk assessment of flavourings to be used in or on food (EFSA CEF Panel, 2010). For these compounds, for which there is no concern for genotoxicity, EFSA requested additional subchronic toxicity data (EFSA CEF Panel, 2011). In the absence of such data, the EFSA CEF Panel was unable to complete its assessment (EFSA CEF Panel, 2015b). As a result, these compounds are not authorised for use as flavour in food. For these compounds, in the absence of toxicity data, the FEEDAP Panel applies the threshold of TTC approach or read‐across from structurally related substances, as recommended in the Guidance document on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals (EFSA SC, 2019a).

Sixteen components of clove leaf oil, accounting on average for about 1.0% of the % GC area, have not been previously assessed for use as flavourings. The FEEDAP Panel notes that nine of them are aliphatic mono‐ or sesquiterpenes structurally related to flavourings already assessed in CG 31 and for which a similar metabolic and toxicological profile is expected. ³⁶ These nine lipophilic compounds, accounting together for about 0.96% of the GC area, are expected to be rapidly absorbed from the gastrointestinal tract, oxidised to polar oxygenated metabolites, conjugated and excreted (EFSA FEEDAP Panel, 2015, 2016b). Two compounds, (Z)‐isoeugenol and humulene oxide II, are structurally related to authorised feed flavourings.

The remaining five compounds, cubebol, caryophyllene alcohol, (3E,10Z)‐oxacyclotrideca‐3,10‐diene‐2,7‐dione, coniferyl alcohol and (3E)‐3‐ethylidene‐3a‐methyl‐2,4,5,6,7,7a‐hexahydro‐1H‐indene were screened with the Organisation for Economic Co‐operation and Development (OECD) Quantitative Structure–Activity Relationship (QSAR) Toolbox. No alert was identified for in vitro mutagenicity, for genotoxic and non‐genotoxic carcinogenicity and for other toxicity endpoints for cubebol, caryophyllene alcohol and (3E)‐3‐ethylidene‐3a‐methyl‐2,4,5,6,7,7a‐hexahydro‐1H‐indene, whereas for (3E,10Z)‐oxacyclotrideca‐3,10‐diene‐2,7‐dione, structural alerts were due to the presence of ester group and for coniferyl alcohol to the presence of the phenol group. For these substances, predictions of mutagenicity by Ames test (with and without metabolic activation) were made by ‘read‐across’ analyses of data available for similar substances to the target compounds (i.e. analogues obtained by categorisation). Mutagenicity read‐across‐based relevant predictions were found negative for both substances. ³⁷ On this basis, the alerts raised were discounted.

The additive contains methyleugenol (range: 0.010–0.030%). For the ADME and the toxicology of methyleugenol, reference is made to the safety evaluation made by the FEEDAP Panel in the EFSA opinion on laurel leaf oil (EFSA FEEDAP Panel, 2023).

3.3.2.1. Safety for the target species

Tolerance studies in the target species and/or toxicological studies in laboratory animals made with the essential oil under application were not submitted.

As the additive under assessment is a fully defined mixture (the identified components represent > 99.5% of the % GC area, see Section 3.3.1), the FEEDAP Panel applied a component‐based approach to assess the safety for target species of the essential oil. The approach followed, i.e. the allocation of the components to the (sub‐)assessment groups, the estimate of exposure for the target species, the identification of a reference point for each constituent (hazard characterisation) and the calculation of the MOET for each assessment group (risk characterisation), is described in Section 3.2.2.1. Methyleugenol, a substance for which a concern for genotoxicity has been identified is assessed separately.

Components other than methyleugenol

The components of clove leaf oil were allocated to nine assessment groups, corresponding to the chemical groups (CGs) 5, 6, 9, 14, 17, 18, 23, 31 and 32, as shown in Table 8 and in the corresponding footnote.

Table 8.

Compositional data, intake values (calculated for chickens for fattening at 100 mg/kg complete feed), reference points and margin of exposure (MOE) for the individual components of clove leaf oil classified according to assessment groups

Essential oil composition			Exposure		Hazard characterisation		Risk characterisation
Assessment group	FLAVIS‐No	Highest conc. in the oil	Highest feed conc.	Intake (a)	Cramer class (b)	NOAEL (c)	MOE	MOET
Constituent	–	%	mg/kg	mg/kg bw per day	–	mg/kg bw per day	–	–
CG 6
Caryophyllene alcohol	–	0.24	0.241	0.0216	I	3	139
Cubebol	–	0.01	0.010	0.0009	I	3	3,342
MOET CG 06								133
CG 9
(3E,10Z)‐Oxacyclotrideca‐3,10‐diene‐2,7‐dione	–	0.02	0.020	0.0018	III	0.15	84
CG 14
Furfural	13.018	0.03	0.029	0.0026	(II)	54	20,742
5‐Methylfurfural	13.001	0.01	0.005	0.0004	II	0.91	2,027
								1,847
CG 17
(Z)‐Isoeugenol	–	0.04	0.043	0.0039	(I)	75	19,429
Coniferyl alcohol	–	0.03	0.025	0.0022	I	3	1,337
								1,251
CG 18
Eugenol	04.003	86.0	86.01	7.7214	(I)	300	39
Eugenyl acetate	09.020	1.47	1.472	0.1321	(I)	300	2,270
4‐Allylphenol	04.058	0.06	0.063	0.0057	I	3	530
								36
CG 23
Benzyl benzoate	09.727	0.06	0.060	0.0054	(I)	194	36,017
Methyl salicylate	09.749	0.05	0.050	0.0045	(I)	50	11,139
								8,508
CG 31, I
Octane	–	0.52	0.520	0.0467	I	3	64
Nonane	–	0.16	0.160	0.0144	I	3	209
								49
CG 31, II (Acyclic alkanes)
α‐Farnesene	01.040	0.78	0.046	0.0041	(I)	44	10,655
CG 31, III (Cyclohexene hydrocarbons)
β‐Elemene	–	0.07	0.065	0.0058	(I)	250	42,843
CG 31, IVe (Benzene hydrocarbons, alkyl)
Calamenene	–	0.07	0.070	0.0063	I	3	477
CG 31, V (Bi‐, tricyclic, non‐aromatic hydrocarbons)
β‐Caryophyllene	01.007	14.85	14.85	1.3331	(I)	222	167
α‐Copaene	–	0.30	0.300	0.0269	(I)	222	8,243
(3E)‐3‐Ethylidene‐3a‐methyl‐2,4,5,6,7,7a‐hexahydro‐1H‐indene	–	0.04	0.040	0.0036	I	3	835
Cadina‐1,4‐diene	–	0.01	0.010	0.0009	I	3	3,342
1(5),11‐Guaiadiene	01.023	0.04	0.010	0.0009	I	3	3,342
MOET CG 31, V								126
CG 31, VI
3,7,10‐Humulatriene	01.043	2.03	2.033	0.1825	I	111 (d)	608
Germacra‐1(10),4(14),5‐triene	01.042	0.05	0.050	0.0045	I	3	668
MOET CG 31, V								318
CG 32
β‐Caryophyllene oxide	16.043	1.03	1.028	0.0923	(III)	109	1,181
Humulene oxide II	–	0.08	0.078	0.0070	(III)	109	15,566
								1,098

^(a)Intake calculations for the individual components are based on the use level of 100 mg/kg in feed for chickens for fattening, the species with the highest ratio of feed intake/body weight. The MOE for each component is calculated as the ratio of the reference point (no observed adverse effect level, NOAEL) to the intake. The combined margin of exposure (MOET) is calculated for each assessment group as the reciprocal of the sum of the reciprocals of the MOE of the individual substances.

^(b)When a NOAEL value is available or read‐across is applied, the allocation to the Cramer class is put into parentheses.

^(c)Values in bold refer to those components for which the NOAEL value was available, values in italics are the 5th percentile of the distribution of NOAELs of the corresponding Cramer Class, other values (plain text) are NOAELs extrapolated by using read‐across.

^(d)The NOAEL of 222 mg/kg bw per day for β‐caryophyllene was halved to take into account the uncertainty in read‐across.

Toxicological data for sub‐chronic studies, from which NOAEL values could be derived, were available for 6‐methylhept‐5‐en‐2‐one [07.015] in CG 5 (EFSA FEEDAP Panel, 2021a), linalool [02.013] in CG 6 (EFSA FEEDAP Panel, 2012a), furfural [13.018] in CG 14 (EFSA FEEDAP Panel, 2016a), isoeugenol [04.004] in CG 17 (EFSA FEEDAP Panel, 2012b), eugenol [04.003] (EFSA FEEDAP Panel, 2011), methyl salicylate [09.749] in CG 23 (EFSA FEEDAP Panel, 2012c) and for the representative compounds for sub‐assessment groups of CG 31, myrcene [01.008], d‐limonene [01.045] and β‐caryophyllene [01.007] (EFSA FEEDAP Panel, 2015, 2016b) and β‐caryophyllene oxide [16.043] (EFSA CEF Panel, 2014). For benzyl benzoate [09.727] a NOAEL of 194 mg/kg bw per day has been identified for developmental and reproductive toxicity (Api et al., 2020).

Considering the structural and metabolic similarities in CG 18, read‐across was applied using the NOAEL of 300 mg/kg bw per day for eugenol [04.003] to extrapolate to eugenyl acetate [09.020].

The NOAELs of 44, 250 and 222 mg/kg bw per day for the representative compounds of CG 31, myrcene [01.008], d‐limonene [01.045] and β‐caryophyllene [01.007] were applied, respectively, using read‐across to the compounds within sub‐assessment group II (α‐farnesene [01.040], III (β‐elemene) and V (α‐copaene, alloroarmadendrene, δ‐cadinene [01.021], 10,10‐dimethyl‐2,6‐dimethylenebicyclo[7.2.0]undecane, valencene [01.017] and β‐cadinene) (EFSA CEF Panel, 2015a,b). Read‐across was also applied from β‐caryophyllene [01.007] to 3,7,10‐humulatriene [01.043] in CG 31, VI. The NOAEL of 222 mg/kg bw per day for β‐caryophyllene [01.007] was halved to take into account the uncertainty in read‐across.

The NOAEL of 109 mg/kg bw per day for β‐caryophyllene oxide [16.043] was extrapolated to humulene oxide II in CG 32.

For the remaining compounds, ³⁸ toxicity studies performed with the compounds under assessment with derived NOAEL values were not available and read‐across was not possible. Therefore, the threshold of TTC approach was applied (EFSA FEEDAP Panel, 2017b).

The approach to the safety assessment of clove leaf oil for the target species is summarised in Table 8. The calculations were done for chickens for fattening, the species with the highest ratio of feed intake/body weight and represent the worst‐case scenario at the use level of 100 mg/kg complete feed. The compounds resulting individually in an MOE > 50,000 were not further considered in the assessment group as their contribution to the MOE(T) is negligible. ³⁹

As shown in Table 8, for several assessment groups, the MOET was < 100 at the proposed use levels (100 mg/kg feed). The lowest MOET was calculated for CG 18, the chemical group which includes eugenol, the main compound of clove leaf oil. From the lowest MOET of 36 for chickens for fattening, the MOET for CG 18 compounds was calculated for the other target species considering the respective daily feed intake and conditions of use. The results are summarised in Table 9.

Table 9.

Combined margin of exposure (MOET) for CG 18 calculated for the different target animal categories at the proposed use level in feed

Animal category	Body weight (kg)	Feed intake (g DM/day)	Proposed use level (mg/kg feed)	Lowest MOET CG 18	Maximum safe use level (mg/kg feed) (a)
Chicken for fattening	2	158	100	36	36
Laying hen	2	106	100	54	54
Turkey for fattening	3	176	100	48	48
Piglet	20	880	100	65	65
Pig for fattening	60	2,200	100	77	77
Sow lactating	175	5,280	100	95	95
Veal calf (milk replacer)	100	1,890	100	162	100
Cattle for fattening	400	8,000	100	142	100
Dairy cows	650	20,000	100	92	92
Sheep/goat	60	1,200	100	142	100
Horse	400	8,000	100	142	100
Rabbit	2	100	100	57	57
Salmon	0.12	2.1	100	158	100
Dog	15	250	100	167	100
Cat	3	60	100	142 (b)	28
Ornamental fish	0.012	0.054	100	569	100

^(a)Complete feed containing 88% DM, milk replacer 94.5% DM.

^(b)The minimum MOET considered to be of no concern for cats should be increased to 500 because of the reduced capacity of glucuronidation of compounds.

Table 9 shows that the MOET exceeds the value of 100 only for veal calves (milk replacer), cattle for fattening, sheep/goats, horses, salmon, dogs and ornamental fish. For the other species the maximum safe use levels in feed were calculated to ensure a MOET ≥ 100. Because glucuronidation is an important metabolic reaction to facilitate the excretion of the components of the essential oil and considering that cats have a low capacity for glucuronidation (Court and Greenblatt, 1997; Lautz et al., 2021), the use of clove leaf oil as additive in cat feed needs a wider margin of exposure. A MOET of 500 is considered adequate. The maximum safe levels in feed (without considering the presence of methyleugenol) are shown in Table 8.

In poultry, pigs and rabbits, the daily consumption of water by drinking is about two to three times the amount of feed DM ingested (EFSA FEEDAP Panel, 2017a). The applicant proposed a maximum use level of 20 mg/kg water for piglets, pigs for fattening and sows, which would ensure a comparable or lower exposure to the calculated maximum safe use level in feed. However, the proposed use level of 40 mg/kg water for poultry and rabbits would result in an exposure higher than that which is considered safe when consumed via feed.

For ruminants and horses, safe concentrations of an additive cannot be consistently extrapolated from feed to water using a fixed ratio of feed to water intake. However, considering that the proposed maximum use level in water for drinking for ruminants and horses, 10 mg/kg water, is 9 to 10 times lower than the maximum calculated safe concentrations in feed, the use of the additive at the proposed maximum use level in water for drinking is considered safe. In veal calves, the use of the additive at 40 mg/kg water can be considered safe only when added to the water for drinking but not to the water used to prepare the milk replacer.

The FEEDAP Panel considers that the use in water for drinking alone or in combination with use in feed should not exceed the daily amount that is considered safe when consumed via feed alone.

Methyleugenol

Low concentrations of methyleugenol were detected in all batches of the additive under assessment (average: 0.023%, range: 0.010–0.030%). The use of clove leaf oil at the levels in feed which were considered safe for the different target species, without considering the presence of methyleugenol, ranges from 28 to 100 mg/kg complete feed (see Table 9). These levels correspond to methyleugenol concentrations ranging from 0.008 to 0.030 mg methyleugenol/kg complete feed. The highest daily intake of methyleugenol in μg/kg bw was calculated for the different target animal categories and considering the highest analysed value in the additive (0.030%). The calculated intake values range between 0.15 μg/kg bw per day (in ornamental fish) and 0.98 μg/kg bw per day (in laying hens, piglets and sows) (see Appendix B).

When the estimated exposures for the different animal categories are compared to the BMDL₁₀ of 22.2 mg methyleugenol/kg bw per day, derived by Suparmi et al. (2019) from a rodent carcinogenicity study (NTP, 2000), a MOE ranging between 22,753 and 144,711 is calculated for long‐living and reproductive animals. When comparing the exposure of short‐living animals to the reference point based on non‐neoplastic endpoints, a magnitude of the MOET > 100, is obtained for all species (see Appendix B).

The magnitude of the MOE is indicative of a low concern for long‐living and reproductive animals and of no concern for species for fattening.

3.3.2.2. Conclusions on safety for the target species

The conclusions of the FEEDAP Panel on the maximum safe concentrations in complete feed of clove leaf oil are summarised in Table 10.

The proposed conditions of use of the additive in water for drinking for poultry and rabbits cannot be considered safe. When used in water for drinking, the proposed conditions of use are considered of low/no concern for pigs, calves, ruminants and horses, provided that the use in water for drinking alone or in conjunction with use in feed should not exceed the daily amount that is considered of low/no concern when consumed via feed alone.

3.3.2.3. Safety for the consumer

Considering the qualitative similarity in the composition of clove oils, the same considerations on the constituents apply to the assessment of the safety for the consumer (see Section 3.2.2.3).

No safety concern would be expected for the consumer from the use of clove leaf oil up to the highest safe use level in feed.

3.3.2.4. Safety for the user

No specific data were provided by the applicant regarding the safety of the additive for users.

The applicant produced a safety data sheet ⁴⁰ for clove leaf oil, where hazards for users have been identified.

The essential oil under assessment should be considered as irritant to skin and eyes, and as a dermal and respiratory sensitiser.

When handling the essential oil, exposure of unprotected users to methyleugenol may occur. Therefore, to reduce the risk, the exposure of the users should be minimised.

3.3.2.5. Safety for the environment

Although S. aromaticum is not a native species to Europe, eugenol, β‐caryophyllene and eugenyl acetate, the most abundant components in the essential oil are naturally occurring in European plants. Therefore, the use of the clove leaf oil under the proposed conditions of use in animal feed is not expected to pose a risk to the environment.

3.4. β‐Caryophyllene‐rich fraction of clove leaf oil (CCL oil)

3.4.1. Characterisation of CCL oil

The essential oil under assessment is a clear yellow, mobile and slightly viscous liquid with a characteristic odour. In four batches of the additive, the refractive index (20°C) ranged between 1.4995 and 1.5001 (specification: 1.4890–1.5020). ¹⁶

For CCL oil, the product specifications are based on the content of β‐caryophyllene (70–90%), which is selected as the phytochemical marker. Analysis of two batches of the additive showed compliance with these specifications when analysed by GC‐FID and expressed as % GC area (82%). ⁴¹

The applicant provided a full characterisation of the five batches by GC–MS. ¹⁹ β‐Caryophyllene accounted for 75% (range: 71.9–79.3%) on average of % GC area (Table 9). In total, up to 30 constituents were detected, 24 of which were identified and accounted on average for 98.1% (97.6–98.5%) of the % GC area. Besides β‐caryophyllene, 19 other compounds were detected at individual levels ≥ 0.05% and are listed in Table 11. These 20 compounds together account on average for 98.0% (97.5–98.4%) of the % GC area. The remaining four compounds (ranging between 0.008 and 0.04%) and accounting for 0.09% of the % GC area are listed in the footnote. ⁴² Based on the available data on the characterisation, CCL oil is considered a fully defined mixture (EFSA SC, 2019a).

Table 11.

Constituents of a β‐caryophyllene‐rich fraction of clove leaf oil (CCL oil) from the leaves of Syzygium aromaticum (L.) Merr. & L.M. Perry accounting for ≥ 0.05% of the composition (based on the analysis of five batches by gas chromatography–mass spectrometry). The content of each constituent is expressed as the area per cent of the corresponding chromatographic peak (% GC area), assuming the sum of chromatographic areas of all detected peaks as 100%

Constituent	CAS No	FLAVIS No	% GC area
EU register name			Mean	Range
β‐Caryophyllene	87‐44‐5	01.007	75.04	71.93–79.26
3,7,10‐Humulatriene	6753‐98‐6	01.043	13.25	10.77–15.06
β‐Caryophyllene epoxide	1139‐30‐6	16.043	2.99	2.51–3.59
α‐Copaene	3856‐25‐5	–	2.01	1.87–2.19
δ‐Cadinene	29350‐73‐0	01.021	1.87	1.18–2.63
α‐Cubebene	17699‐14‐8	–	0.51	0.47–0.56
10,10‐Dimethyl‐2,6‐dimethylenebicyclo[7.2.0] undecane	357414‐37‐0	–	0.50	0.43–0.59
α‐Selinene	473‐13‐2	–	0.30	0.20–0.36
α‐Farnesene	502‐61‐4	01.040	0.29	0.19–0.43
α‐Muurolene	10208‐80‐7	01.052	0.19	0.13–0.26
β‐Selinene	17066‐67‐0	–	0.19	0.15–0.24
Caryophyllene alcohol	56747‐96‐7	–	0.19	0.19 (a)
Humulene oxide II	19888‐34‐7	–	0.18	0.11–0.23
γ‐Muurolene	30021‐74‐0	–	0.18	0.13–0.21
Alloaromadendrene	25246‐27‐9	–	0.17	0.15–0.18
β‐Elemene	33880‐83‐0	–	0.10	0.10 (a)
1(5),11‐Guaiadiene	3691‐12‐1	–	0.09	0.08–0.10
1‐epi‐Cubenol	19912‐67‐5	–	0.06	0.04–0.08
Seychellene	20085‐93‐2	–	0.05	0.03–0.07
Methyleugenol	93‐15‐2	–	0.05	0.04–0.06
Total			98.00	97.51–98.43 (b)

EU: European Union; CAS No: Chemical Abstracts Service number; FLAVIS No: EU Flavour Information System numbers.

^(a)Compound detected in only one batch.

^(b)The values given for the Total are the lowest and the highest values of the sum of the components in the five batches analysed.

The applicant performed a literature search for information on the chemical composition of S. aromaticum and its preparations and the presence of compounds of known concern (see Section 3.2.1).

Methyleugenol (0.038–0.062%) was detected by GC–MS in all the five batches of CCL oil under assessment, whereas estragole was not detected (LOD: 0.01%).

3.4.1.1. Impurities

The applicant referred to the ‘periodic testing’ of some representative flavourings premixtures for mercury, cadmium and lead, arsenic, fluoride, dioxins and polychlorinated biphenyls (PCBs), organo‐chloride pesticides, organo‐phosphorus pesticides, aflatoxins (B1, B2, G1, G2) and ochratoxin A. However, no data have been provided on the presence of these impurities. Since CCL oil is produced by steam distillation, the likelihood of any measurable carry‐over of all the above‐mentioned elements is considered low, except for mercury.

3.4.1.2. Shelf life

The shelf‐life of CCL oil is stated to be at least 12 months, when stored in tightly closed containers under standard conditions (in a cool, dry place protected from light). ²³ However, no data supporting this statement were provided.

3.4.1.3. Conditions of use

CCL oil is intended to be added to feed and water for drinking for all animal species without withdrawal. Maximum use levels in complete feed were proposed in Table 12. No use level has been proposed by the applicant for the use in water for drinking.

Table 12.

Conditions of use for a β‐caryophyllene‐rich fraction of clove leaf oil (CCL oil): maximum proposed use levels in complete feed for all animal categories

Animal category	Use level (mg/kg complete feed)
Chicken for fattening	150
Laying hen	150
Turkey for fattening	150
Piglet	175
Pig for fattening	175
Sow lactating	175
Veal calf (milk replacer)	200
Cattle for fattening	200
Dairy cow	200
Sheep/goat	200
Horse	200
Rabbit	150
Salmon (fish)	150
Dog	50
Cat	50
Ornamental fish	50
Others	50

3.4.2. Safety

The assessment of safety of CLL oil is based on the maximum use levels proposed by the applicant for the species listed above (see Table 12).

As CCL oil is obtained by fractional distillation of clove leaf oil, to achieve an enriched composition in β‐caryophyllene and other hydrocarbons (e.g. 3,7,10‐humulatriene, α‐copaene and δ‐cadinene), the considerations made in Section 3.3.2 on clove leaf oil apply to the current assessment.

Many of the components of CCL oil, accounting for about 94% of the GC peak areas, have been previously assessed and considered safe for use as flavourings, and are currently authorised for use in food ⁸ without limitations and for use in feed ⁷ at individual use levels higher than those resulting from the intended use of the essential oil in feed. The list of the compounds already evaluated by the EFSA Panels is given in Table 1 (see Section 1.2).

Five compounds listed in Table 1, δ‐cadinene [01.021], 1,(5),11‐guauadiene [01.023], β‐bourbonene [01.024], 3,7,10‐humulatriene [01.043] and α‐muurolene [01.052], have been evaluated in Flavouring Group Evaluation 25, Revision 2 (FGE.25Rev2) by applying the procedure described in the Guidance on the data required for the risk assessment of flavourings to be used in or on food (EFSA CEF Panel, 2010). For these compounds, for which there is no concern for genotoxicity, EFSA requested additional subchronic toxicity data (EFSA CEF Panel, 2011). In the absence of such data, the EFSA CEF Panel was unable to complete its assessment (EFSA CEF Panel, 2015b). As a result, these compounds are not authorised for use as flavour in food. For these compounds, in the absence of toxicity data, the FEEDAP Panel applies the threshold of TTC approach or read‐across from structurally related substances, as recommended in the Guidance document on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals (EFSA SC, 2019a).

Fourteen components of CCL oil, accounting on average for 4.47% of the % GC area, have not been previously assessed for use as flavourings. The FEEDAP Panel notes that 11 of them are aliphatic mono‐ or sesquiterpenes structurally related to flavourings already assessed in CG 31 and for which a similar metabolic and toxicological profile is expected. ⁴³ These 11 lipophilic compounds accounting together for about 4.06% of the GC area, are expected to be rapidly absorbed from the gastrointestinal tract, oxidised to polar oxygenated metabolites, conjugated and excreted (EFSA FEEDAP Panel, 2015, 2016b). The same consideration applies to caryophyllene alcohol. Another compound, 1‐epi‐cubenol, is a tertiary alcohol structurally related to the compounds already evaluated in CG 6, and is expected to be absorbed, metabolised and excreted (EFSA FEEDAP Panel, 2012a). The last compound, humulene oxide II, is an oxygenated terpene metabolite and is structurally related to compounds that have been evaluated for use in food and/or feed.

The additive contains methyleugenol (range: 0.038–0.062%). For the ADME and the toxicology of methyleugenol, reference is made to the safety evaluation made by the FEEDAP Panel in the EFSA opinion on laurel leaf oil (EFSA FEEDAP Panel, 2023).

3.4.2.1. Safety for the target species

Tolerance studies in the target species and/or toxicological studies in laboratory animals made with the essential oil under application were not submitted.

As CCL oil is a fully defined mixture (the identified components represent about 98% of the % CG area, see Section 3.4.1), the FEEDAP Panel applied a component‐based approach to assess the safety for target species. The approach followed, i.e. the allocation of the components to the (sub‐)assessment groups, the estimate of exposure for the target species, the identification of a reference point for each constituent (hazard characterisation) and the calculation of the MOET for each assessment group (risk characterisation), is described in Section 3.2.2.1. Methyleugenol, a substance for which a concern for genotoxicity has been identified, is assessed separately.

Components other than methyleugenol

The components of CCL oil were allocated to four assessment groups, corresponding to the CGs 6, 18, 31 and 32, as shown in Table 13 and in the corresponding footnote.

Table 13.

Compositional data, intake values (calculated for chickens for fattening at 150 mg/kg complete feed), reference points and margin of exposure (MOE) for the individual components of CCL oil classified according to assessment groups

Essential oil composition			Exposure		Hazard characterisation		Risk characterisation
Assessment group	FLAVIS No	Highest conc. in the oil	Highest feed conc.	Intake (a)	Cramer class (b)	NOAEL (c)	MOE	MOET
Constituent	–	%	mg/kg	mg/kg bw per day	–	mg/kg bw per day	–	–
CG 6
Caryophyllene alcohol	–	0.19	0.281	0.0252	(I)	222	8,816
1‐epi‐Cubenol	–	0.08	0.126	0.0113	I	3	265
MOET CG 06								257
CG 31, II (Acyclic alkanes)
α‐Farnesene	01.040	0.29	0.639	0.0574	(I)	44	767
CG 31, III (Cyclohexene hydrocarbons)
β‐Elemene	–	0.43	0.639	0.0574	(I)	250	18,565
CG 31, IVe (Benzene hydrocarbons, alkyl)
γ‐Calacorene	–	0.02	0.023	0.0020	I	3	1,485
CG 31, V (Bi‐, tricyclic, non‐aromatic hydrocarbons)
β‐Caryophyllene	01.007	79.26	118.9	10.674	(I)	222	21
α‐Cubebene	–	0.56	0.834	0.0749	(I)	222	2,965
α‐Copaene	–	2.19	3.291	0.2954	(I)	222	751
Alloaromadendrene	–	0.18	0.267	0.0240	(I)	222	9,262
δ‐Cadinene	01.021	2.63	3.948	0.3544	(I)	222	626
10,10‐Dimethyl‐2,6‐dimethylene bicyclo[7.2.0]undecane	–	0.59	0.881	0.0790	(I)	222	2,809
1(5),11‐Guaiadiene	–	0.10	0.150	0.0135	I	3	223
Clovene	–	0.06	0.089	0.0079	I	3	378
β‐Bourbonene	01.024	0.04	0.063	0.0057	(I)	222	3,9253
Seychellene	–	0.07	0.101	0.0090	(I)	222	24,606
γ‐Muurolene	–	0.21	0.317	0.0284	(I)	222	7,813
β‐Selinene	–	0.24	0.363	0.0326	(I)	222	6,812
α‐Selinene	–	0.36	0.542	0.0486	(I)	222	4,567
α‐Muurolene	01.052	0.26	0.387	0.0347	(I)	222	6,390
MOET CG 31, V								17
CG 31, VI
3,7,10‐Humulatriene	01.043	15.06	22.59	2.0278	I	111 (d)	55
CG 32
β‐Caryophyllene oxide	16.043	3.59	5.385	0.4834	(III)	109	225
Humulene oxide II	–	0.23	0.351	0.0315	(III)	109	3,459
								212

^(a)Intake calculations for the individual components are based on the use level of 150 mg/kg in feed for chickens for fattening, the species with the highest ratio of feed intake/body weight. The MOE for each component is calculated as the ratio of the reference point (no observed adverse effect level, NOAEL) to the intake. The combined margin of exposure (MOET) is calculated for each assessment group as the reciprocal of the sum of the reciprocals of the MOE of the individual substances.

^(b)When a NOAEL value is available or read‐across is applied, the allocation to the Cramer class is put into parentheses.

^(c)Values in bold refer to those components for which the NOAEL value was available, values in italics are the 5th percentile of the distribution of NOAELs of the corresponding Cramer Class, other values (plain text) are NOAELs extrapolated by using read‐across.

^(d)The NOAEL of 222 mg/kg bw per day for β‐caryophyllene was halved to take into account the uncertainty in read‐across.

Toxicological data for sub‐chronic studies, from which NOAEL values could be derived, were available for eugenol [04.003] (EFSA FEEDAP Panel, 2011), for the representative compounds for sub‐assessment groups of CG 31, myrcene [01.008], d‐limonene [01.045] and β‐caryophyllene [01.007] (EFSA FEEDAP Panel, 2015, 2016b) and β‐caryophyllene oxide [16.043] (EFSA CEF Panel, 2014).

The NOAELs of 44, 250 and 222 mg/kg bw per day for the representative compounds of CG 31, myrcene [01.008], d‐limonene [01.045] and β‐caryophyllene [01.007] were applied, respectively, using read‐across to the compounds within sub‐assessment group II (α‐farnesene [01.040], III (β‐elemene) and V (δ‐cadinene [01.021], α‐copaene, α‐cubebene, alloroarmadendrene, 10,10‐dimethyl‐2,6‐dimethylenebicyclo[7.2.0]undecane, β‐bourbonene, seychellene, γ‐muurolene, β‐selinene, α‐selinene and α‐muurolene) (EFSA CEF Panel, 2015a,b). Read‐across was also applied from β‐caryophyllene [01.007] to 3,7,10‐humulatriene [01.043] in CG 31, VI. The NOAEL of 222 mg/kg bw per day for β‐caryophyllene [01.007] was halved to take into account the uncertainty in read‐across.

The NOAEL of 109 mg/kg bw per day for β‐caryophyllene oxide [16.043] was extrapolated to humulene oxide II in CG 32.

For the remaining compounds, ⁴⁴ toxicity studies performed with the compounds under assessment with derived NOAEL values were not available and read‐across was not possible. Therefore, the TTC approach was applied (EFSA FEEDAP Panel, 2017b).

The compounds resulting individually in an MOE > 50,000 were not further considered in the assessment group as their contribution to the MOE(T) is negligible. ⁴⁵

The approach to the safety assessment of CLL oil for the target species is summarised in Table 13. The calculations were done for chickens for fattening, the species with the highest ratio of feed intake/body weight and represent the worst‐case scenario at the use level of 150 mg/kg complete feed.

As shown in Table 13, for the assessment group which includes the major component β‐caryophyllene (CG 31, V), the MOET was < 100 at the proposed use levels (150 mg/kg feed). From the lowest MOET of 17 for chickens for fattening, the MOET for CG 31,V compounds was calculated for the other target species considering the respective daily feed intake and conditions of use. The results are summarised in Table 14.

Table 14.

Combined margin of exposure (MOET) for CG 31,V calculated for the different target animal categories at the proposed use level in feed

Animal category	Body weight (kg)	Feed intake (g DM/day)	Proposed use level (mg/kg feed)	Lowest MOET CG 31,V	Maximum safe use level (mg/kg feed) (a)
Chicken for fattening	2	158	150	17	26
Laying hen	2	106	150	25	38
Turkey for fattening	3	176	150	23	34
Piglet	20	880	175	26	46
Pig for fattening	60	2,200	175	31	54
Sow lactating	175	5,280	175	38	67
Veal calf (milk replacer)	100	1,890	200	53	114
Cattle for fattening	400	8,000	200	50	101
Dairy cows	650	20,000	200	32	65
Sheep/goat	60	1,200	200	50	101
Horse	400	8,000	200	50	101
Rabbit	2	100	150	27	40
Salmon	0.12	2.1	150	75	112
Dog	15	250	50	237	50
Cat	3	60	50	201 (b)	20
Ornamental fish	0.012	0.054	50	806	50

^(a)Complete feed containing 88% DM, milk replacer 94.5% DM.

^(b)The minimum MOET considered to be of no concern for cats should be increased to 500 because of the reduced capacity of glucuronidation of compounds.

Table 14 shows that the MOET calculated at the maximum proposed use levels in feed exceeds the value of 100 only for dogs and ornamental fish. For the other species the maximum safe use levels in feed were calculated to ensure a MOET ≥ 100. Because glucuronidation is an important metabolic pathway to facilitate the excretion of the components of the essential oil and considering that cats have a low capacity for glucuronidation (Court and Greenblatt, 1997; Lautz et al., 2021), the use of CCL oil as additive in cat feed needs a wider margin of exposure. A MOET of 500 is considered adequate. The maximum safe levels in feed (without considering the presence of methyleugenol) are shown in Table 12.

The FEEDAP Panel considers that the use in water for drinking alone or in combination with use in feed should not exceed the daily amount that is considered safe when consumed via feed alone.

Methyleugenol

Low concentrations of methyleugenol were detected in all batches of the additive under assessment (average: 0.050%, range: 0.038–0.062%). The use of CCL oil at the levels in feed which were considered safe for the different target species without considering the presence of methyleugenol, ranges from 20 to 112 mg/kg complete feed (see Table 12). These levels correspond to methyleugenol concentrations ranging from 0.012 to 0.069 mg/kg methyleugenol/kg complete feed. The highest daily intake of methyleugenol in μg/kg bw was calculated for the different target animal categories considering the highest analysed value in the additive (0.062%). The calculated intake values range between 0.16 μg/kg bw per day (in ornamental fish) and 1.45 μg/kg bw per day (in chickens for fattening) (see Appendix C).

When the estimated exposures for the different animal categories are compared to the BMDL₁₀ of 22.2 mg/kg bw per day, derived by Suparmi et al. (2019) from a rodent carcinogenicity study (NTP, 2000), an MOE ranging between 15,587 and 140,043 is calculated for long‐living and reproductive animals. When comparing the exposure of short‐living animals to the reference point based on non‐neoplastic endpoints, a magnitude of the MOET > 100, is obtained for all species (see Appendix C).

The magnitude of the MOE is indicative of a low concern for long‐living and reproductive animals and of no concern for species for fattening.

3.4.2.2. Conclusions on safety for the target species

The conclusions of the FEEDAP Panel on the maximum safe concentrations in complete feed of CCL oil are summarised in Table 15.

Table 15.

Maximum safe concentrations of β‐caryophyllene‐rich fraction of clove leaf oil (CCL oil) in complete feed (mg/kg) for all animal species and categories

Animal categories	Maximum feed concentration of low (a) /no concern (b) (mg/kg complete feed) (c)
Long‐living and reproductive animals (a)
Laying hens and other laying/reproductive birds including animals reared for laying/reproduction and ornamental birds	38
Sows and other Suidae species for reproduction including animals reared for reproduction	67
Dairy cows and other ruminants and camelids for milk production and reproduction including animals reared for milk production/reproduction	65
Sheep/goats	101
Horses and other Equidae	101
Rabbits	40
Dogs	50
Cats	20
Ornamental fish	50
Short‐living animals (species for fattening) (b)
Chickens for fattening and minor poultry for fattening	26
Turkey for fattening	34
Pigs for fattening	54
Piglets and other Suidae species for meat production	46
Veal calves (milk replacer)	114
Cattle for fattening and other ruminants for fattening and camelids at the same physiological stage	101
Sheep/goats	101
Horses and other Equidae	101
Rabbits	40
Salmonids and minor fin fish	112
Any other species	20

^(a)Based on a MOE > 10,000 for long‐living and reproductive animals, calculated as the ratio of the reference point (BMDL10 of 22.2 mg/kg bw per day) to the combined intake.

^(b)Based on a MOE > 100 for short‐living animals (species for fattening), calculated as the ratio of the reference point (NOAEL of 10 mg/kg bw per day) to the combined intake.

^(c)Complete feed containing 88% DM, milk replacer 94.5% DM.

The FEEDAP Panel considers that the use in water for drinking alone or in conjunction with use in feed should not exceed the daily amount that is considered of low or no concern when consumed via feed alone.

3.4.2.3. Safety for the consumer

Considering the qualitative similarity in the composition of clove oils, the same considerations on the constituents apply to the assessment of the safety for the consumer (see Section 3.2.2.3).

Consequently, no safety concern would be expected for the consumer from the use of the CCL oil up to the highest safe use level in feed.

3.4.2.4. Safety for the user

No specific data were provided by the applicant regarding the safety of the additive for users.

The applicant produced a safety data sheet ⁴⁶ for CCL oil, where hazards for users have been identified.

The essential oil under assessment should be considered as irritant to skin and eyes, and as a dermal and respiratory sensitiser.

When handling the essential oil, exposure of unprotected users to methyleugenol may occur. Therefore, to reduce the risk, the exposure of the users should be minimised.

3.4.2.5. Safety for the environment

Although S. aromaticum L. is not a native species to Europe, eugenol, β‐caryophyllene and eugenyl acetate, the most abundant components in the essential oil are naturally occurring in European plants. Therefore, the use of the CCL oil under the proposed conditions of use in animal feed is not expected to pose a risk to the environment.

3.5. Efficacy

‘Cloves’ (immature flower buds) and related preparations are listed in Fenaroli's Handbook of Flavour Ingredients (Burdock, 2009), by the Flavour and Extract Manufactures Association (FEMA) with the reference numbers 2327 (cloves), 2322 (clove bud extract), 2323 (clove bud oil), 2324 (clove bud oleoresin) and 2325 (clove leaf oil).

Since clove leaf oil and clove bud oil are recognised to flavour food and the function of the oils under assessment in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary.

4. Conclusions

Oils prepared from the flower buds or leaves of S. aromaticum (L.) Merr. & L.M. Perry may be produced from plants of different geographical origins and by various processes resulting in preparations with different composition and toxicological profiles. Therefore, the following conclusions apply only to oils produced by steam distillation from the flower buds or the leaves of S. aromaticum, which do not contain estragole (< 0.01%) and contain ≤ 0.13% methyleugenol (clove bud oil) or ≤ 0.03% methyleugenol (clove leaf oil) or ≤ 0.06% methyleugenol (β‐caryophyllene‐rich fraction of clove leaf oil, CCL oil).

The conclusions of the FEEDAP Panel on the maximum concentrations in complete feed of the three essential oils under assessment, which are considered of low concern for long‐living and reproductive animals and of no concern for species for fattening are summarised as following:

Animal categories	Maximum feed concentration of low (a) /no concern (b) (mg/kg complete feed) (c)
	Clove bud oil	Clove leaf oil	CCL oil
Long‐living and reproductive animals (a)
Laying hens and other laying/reproductive birds including animals reared for laying/reproduction and ornamental birds	28.5	54	38
Sows and other Suidae species for reproduction including animals reared for reproduction	50	95	67
Dairy cows and other ruminants and camelids for milk production and reproduction including animals reared for milk production/reproduction	50	92	65
Sheep/goats	50	100	101
Horses and other Equidae	50	100	101
Rabbits	30.5	57	40
Dogs	50	100	50
Cats	25	28	20
Ornamental fish	50	100	50
Short‐living animals (species for fattening) (b)
Chickens for fattening and minor poultry for fattening	32	36	26
Turkey for fattening	43	48	34
Pigs for fattening	50	77	54
Piglets and other Suidae species for meat production	50	65	46
Veal calves (milk replacer)	50	100	114
Cattle for fattening and other ruminants for fattening and camelids at the same physiological stage	50	100	101
Sheep/goats	50	100	101
Horses and other Equidae	50	100	101
Rabbits	30.5	57	40
Salmonids and minor fin fish	50	100	112
Any other species	25	28	20

^(a)Based on a MOE > 10,000 for long‐living and reproductive animals, calculated as the ratio of the reference point (BMDL10 of 22.2 mg/kg bw per day) to the combined intake.

^(b)Based on a MOE > 100 for short‐living animals (species for fattening), calculated as the ratio of the reference point (NOAEL of 10 mg/kg bw per day) to the combined intake.

^(c)Complete feed containing 88% DM, milk replacer 94.5% DM.

The FEEDAP Panel considers that the use in water for drinking alone or in combination with use in feed should not exceed the daily amount that is considered of low or no concern when consumed via feed alone.

For clove leaf oil, the proposed conditions of use of the additive in water for drinking for poultry and rabbits (40 mg/kg) cannot be considered safe. The proposed conditions of use for pigs (20 mg/kg), calves (40 mg/kg), ruminants and horses (10 mg/kg) are considered of low/no concern, provided that the use in water for drinking alone or in combination with use in feed should not exceed the daily amount that is considered of low or no concern when consumed via feed alone.

No concerns for consumers were identified following the use of the additives at the use level considered of low or no concern in feed for the target animals.

The essential oils under assessment should be considered as irritant to skin and eyes, and as skin and respiratory sensitisers. When handling the additives, exposure of unprotected users to methyleugenol may occur. Therefore, to reduce the risk, the exposure of the users should be minimised.

The use of the additives under the proposed conditions in animal feed is not expected to pose a risk to the environment.

Clove bud oil, clove leaf oil and its β‐caryophyllene‐rich fraction are recognised to flavour food. Since the function of the oils under assessment in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary.

Recommendations

The specification for the additives should include for clove bud oil < 0.01% estragole and < 0.13% methyleugenol; for clove leaf oil < 0.01% estragole and < 0.03% methyleugenol; for β‐caryophyllene‐rich fraction of clove leaf oil (CCL oil) < 0.01% estragole and < 0.06% methyleugenol.

Documentation provided to EFSA/Chronology

Date	Event
28/10/2010	Dossier received by EFSA. Botanically defined flavourings from Botanical Group 07 – Geraniale, Myrtales, Poales for all animal species and categories. Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG)
09/11/2010	Reception mandate from the European Commission
21/12/2010	Application validated by EFSA – Start of the scientific assessment
22/03/2011	Comments received from Member States
01/04/2011	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: analytical methods
08/01/2013	Reception of supplementary information from the applicant ‐ Scientific assessment remains suspended
26/02/2013	EFSA informed the applicant (EFSA ref. 7150727) that, in view of the workload, the evaluation of applications on feed flavourings would be re‐organised by giving priority to the assessment of the chemically defined feed flavourings, as agreed with the European Commission
20/01/2014	Reception of the Evaluation report of the European Union Reference Laboratory for Feed Additives
24/06/2015	Technical hearing during risk assessment with the applicant according to the “EFSA's Catalogue of support initiatives during the life‐cycle of applications for regulated products”: data requirement for the risk assessment of botanicals
17/12/2019	EFSA informed the applicant that the evaluation process restarted
18/12/2019	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: characterisation, safety for target species, safety for the consumer, safety for the user and environment
21/03/2023	Reception of supplementary information from the applicant (partial dataset: clove oil) ‐ Scientific assessment remains suspended
06/06/2023	Reception of an amendment of the Evaluation report of the European Union Reference Laboratory for Feed Additives related to geranium rose oil, eucalyptus oil, lemongrass oil and clove oil
07/06/2023	The application was split and a new EFSA‐Q‐2023‐00397 was assigned to the preparation included in the present assessment
08/06/2023	Scientific assessment re‐started for the preparation included in the present assessment
05/07/2023	Opinion adopted by the FEEDAP Panel on eucalyptus oil (EFSA‐Q‐2023‐00397). End of the Scientific assessment for the preparation included in the present assessment. The assessment of other preparations in BGD 07 is still ongoing

Abbreviations

AFC

EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food

BDG

Botanically defined group

bw

body weight

CAS

Chemical Abstracts Service

CDG

Chemically defined group

CEF

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids

CG

chemical group

CLP

Classification, Labelling and Packaging

CoE

Council of Europe

EEIG

European economic interest grouping

EINECS

European Inventory of Existing Chemical Substances

EMA

European Medicines Agency

EURL

European Union Reference Laboratory

FEEDAP

EFSA Scientific Panel on Additives and Products or Substances used in Animal Feed

FEMA

Flavor Extract Manufacturers Association

FFAC

Feed Flavourings authorisation Consortium of (FEFANA) the EU Association of Specialty Feed Ingredients and their Mixtures

FGE

Flavouring Group Evaluation

FLAVIS

the EU Flavour Information System

FLAVIS No

FLAVIS number

GC

gas chromatography

GC–FID

gas chromatography with flame ionisation detector

GC–MS

gas chromatography–mass spectrometry

ISO

International standard organisation

LOQ

limit of quantification

JECFA

The Joint FAO/WHO Expert Committee on Food Additives

MOE

margin of exposure

MOET

combined margin of exposure (total)

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Co‐operation and Development

PhEur

European Pharmacopoeia

QSAR

Quantitative Structure–Activity Relationship

SC

EFSA Scientific Committee

TTC

threshold of toxicological concern

WHO

World Health Organization

Appendix A – Methyleugenol in clove bud oil: Maximum daily intake and margin of exposure for the different target species

1.

The maximum daily intake of methyleugenol for the different target species and categories was calculated based on

• the default values for body weight and feed intake (EFSA FEEDAP Panel, 2017b),

• the maximum proposed/safe use level (see Table 5) of the additive in feed for the different target animal categories (ranging from 28.8 to 50 mg/kg complete feed) and

• assuming that methyeugenol is present at a concentration corresponding to the highest analysed value in the additive (0.128%).

The margin of exposure (MOE) for each animal category is calculated as the ratio of the reference point to the intake: the BMDL10 of 22.2 mg methyeugenol/kg bw per day for long‐living and reproductive animals; the NOAEL of 10 mg methyeugenol/kg bw per day for target species for fattening (EFSA FEEDAP Panel, 2023).

According to the General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021b), ¹⁵ ‘for substances for which carcinogenicity studies in rodents are available, from which a BMDL₁₀ can be derived, the MOE approach (EFSA, 2005; EFSA SC, 2012) can be applied. Similarly to human risk assessment, a margin of exposure (MOE) with a magnitude of ≥ 10,000, when comparing estimated exposure to genotoxic and/or carcinogenic substances with a BMDL₁₀ from a rodent carcinogenicity study, would be indicative of a low concern for the target species (EFSA SC, 2019a)’. For short‐living animals, genotoxicity and carcinogenicity endpoints are not considered relevant, therefore a lower magnitude of the MOET (> 100) when comparing estimated exposure with a reference point based on non‐neoplastic endpoints is considered adequate (EFSA FEEDAP Panel, 2021b).

The maximum daily intake of methyleugenol for the different target animal categories and the corresponding MOE are reported in Table A.1.

Table A.1.

Target animal intake of methyleugenol (as μg/kg bw per day) and margin of exposure (MOE) at the maximum proposed use level of clove bud oil in feed for target animal category

Animal category	Daily Feed intake	Body weight	Max safe use levels	Intake (a)	Lowest MOE (b)
	g DM/day	kg	mg/kg(1)	μg/kg bw/day
Long‐living and reproductive animals
Laying hens	0.106	2	28.5	2.197	10,104
Sow lactating	5.28	175	50	2.194	10,117
Dairy cow	20	650	50	2.238	9,921
Sheep/goat	1.2	60	50	1.455	15,263
Horse	8	400	50	1.455	15,263
Rabbit	0.1	2	30.5	2.218	10,008
Dog	0.25	15	50	1.212	18,315
Cat	0.06	3	25	0.727	30,525
Ornamental fish	0.00054	0.012	50	0.327	67,833
Target species for fattening
Chicken for fattening	0.158	2	32	3.677	2,720
Turkey for fattening	0.176	3	43	3.669	2,725
Piglet	0.88	20	50	3.200	3,125
Pig for fattening	2.2	60	50	2.667	3,750
Veal calf (milk replacer)	1.89	100	50	1.280	7,239
Cattle for fattening	8	400	50	1.455	6,875
Sheep/goat	1.2	60	50	1.455	6,875
Horse	8	400	50	1.455	6,875
Rabbit	0.1	2	50	3.636	2,750
Salmon	0.0021	0.12	50	1.273	7,857

^(a)The values of methyleugenol in feed is calculated considering the highest analysed value in the additive.

^(b)The MOE for methyleugenol is calculated as the ratio of the reference point to the intake: for long‐living and reproductive animals is based on BMDL₁₀ of 22.2 mg/kg bw per day derived from rodent carcinogenicity studies with methyleugenol; for target species for fattening based on a NOAEL of 10 mg/kg bw per day derived from a 90‐day study with methyleugenol (NTP, 2000).

Appendix B – Methyleugenol in clove leaf oil: Maximum daily intake and margin of exposure for the different target species

1.

The maximum daily intake of methyleugenol for the different target species and categories was calculated based on.

• the default values for body weight and feed intake (EFSA FEEDAP Panel, 2017b),

• the maximum proposed/safe use level (see Table 8) of the additive in feed for the different target animal categories (ranging from 36 to 100 mg/kg complete feed) and

• assuming that methyeugenol is present at a concentration corresponding to the highest analysed value in the additive (0.03%).

The margin of exposure (MOE) for each animal category is calculated as the ratio of the reference point to the intake: the BMDL₁₀ of 22.2 mg methyeugenol/kg bw per day for long‐living and reproductive animals; the NOAEL of 10 mg methyeugenol/kg bw per day for target species for fattening (EFSA FEEDAP Panel, 2023).

According to the General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021),¹⁵ ‘for substances for which carcinogenicity studies in rodents are available, from which a BMDL₁₀ can be derived, the MOE approach (EFSA, 2005; EFSA SC, 2012) can be applied. Similarly to human risk assessment, a margin of exposure (MOE) with a magnitude of ≥ 10,000, when comparing estimated exposure to genotoxic and/or carcinogenic substances with a BMDL₁₀ from a rodent carcinogenicity study, would be indicative of a low concern for the target species (EFSA SC, 2019a)’. For short‐living animals, genotoxicity and carcinogenicity endpoints are not considered relevant, therefore a lower magnitude of the MOET (> 100) when comparing estimated exposure with a reference point based on non‐neoplastic endpoints is considered adequate (EFSA FEEDAP Panel, 2021b).

The maximum daily intake of methyleugenol for the different target animal categories and the corresponding MOE are reported in Table B.1.

Table B.1.

Target animal intake of methyleugenol (as μg/kg bw per day) and margin of exposure (MOE) at the maximum proposed use level of clove leaf oil in feed for target animal category

Animal category	Daily Feed intake	Body weight	Max safe use levels	Intake (a)	Lowest MOE (b)
	g DM/day	kg	mg/kg(1)	μg/kg bw/day
Long‐living and reproductive animals
Laying hens	0.106	2	54	0.976	22,753
Sow lactating	5.28	175	95	0.977	22,719
Dairy cow	20	650	92	0.965	23,004
Sheep/goat	1.2	60	100	0.682	32,560
Horse	8	400	100	0.682	32,560
Rabbit	0.1	2	57	0.972	22,849
Dog	0.25	15	100	0.568	39,072
Cat	0.06	3	28	0.191	116,286
Ornamental fish	0.00054	0.012	100	0.153	144,711
Target species for fattening
Chicken for fattening	0.158	2	36	0.970	10,314
Turkey for fattening	0.176	3	48	0.960	10,417
Piglet	0.88	20	65	0.975	10,256
Pig for fattening	2.2	60	77	0.963	10,390
Veal calf (milk replacer)	1.89	100	100	0.600	16,667
Cattle for fattening	8	400	100	0.682	14,667
Sheep/goat	1.2	60	100	0.682	14,667
Horse	8	400	100	0.682	14,667
Rabbit	0.1	2	57	0.972	10,292
Salmon	0.0021	0.12	100	0.597	16,762

^(a)The values of methyleugenol in feed is calculated considering the highest analysed value in the additive.

^(b)The MOE for methyleugenol is calculated as the ratio of the reference point to the intake: for long‐living and reproductive animals is based on BMDL₁₀ of 22.2 mg/kg bw per day derived from rodent carcinogenicity studies with methyleugenol; for target species for fattening based on a NOAEL of 10 mg/kg bw per day derived from a 90‐day study with methyleugenol (NTP, 2000).

Appendix C – Methyleugenol in a β‐caryophyllene‐rich clove leaf oil (CCL oil): Maximum daily intake and margin of exposure for the different target species

1.

The maximum daily intake of methyleugenol for the different target species and categories was calculated based on.

• the default values for body weight and feed intake (EFSA FEEDAP Panel, 2017b),

• the maximum proposed/safe use level (see Table 12) of the additive in feed for the different target animal categories (ranging from 26 to 112 mg/kg complete feed) and

• assuming that methyeugenol is present at a concentration corresponding to the maximum analysed value in the additive (0.062%).

The margin of exposure (MOE) for each animal category is calculated as the ratio of the reference point to the intake: the BMDL₁₀ of 22.2 mg methyleugenol/kg bw per day for long‐living and reproductive animals; the NOAEL of 10 mg methyelugenol/kg bw per day for target species for fattening (EFSA FEEDAP Panel, 2023).

According to the General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021), ¹⁵ ‘for substances for which carcinogenicity studies in rodents are available, from which a BMDL₁₀ can be derived, the MOE approach (EFSA, 2005; EFSA SC, 2012) can be applied. Similarly to human risk assessment, a margin of exposure (MOE) with a magnitude of ≥ 10,000, when comparing estimated exposure to genotoxic and/or carcinogenic substances with a BMDL₁₀ from a rodent carcinogenicity study, would be indicative of a low concern for the target species (EFSA SC, 2019a)’. For short‐living animals, genotoxicity and carcinogenicity endpoints are not considered relevant, therefore a lower magnitude of the MOET (> 100) when comparing estimated exposure with a reference point based on non‐neoplastic endpoints is considered adequate (EFSA FEEDAP Panel, 2021b).

The maximum daily intake of methyleugenol for the different target animal categories and the corresponding MOE are reported in Table C.1.

Table C.1.

Target animal intake of methyleugenol (as μg/kg bw per day) and margin of exposure (MOE) at the maximum proposed use level of a β‐caryophyllene‐rich fraction of clove leaf oil (CCL oil) in feed for target animal category

Animal category	Daily Feed intake	Body weight	Max safe use levels	Intake (a)	Lowest MOE (b)
	g DM/day	kg	mg/kg(1)	μg/kg bw/day
Long‐living and reproductive animals
Laying hens	0.106	2	38	1.419	15,645
Sow lactating	5.28	175	67	1.424	15,587
Dairy cow	20	650	65	1.409	15,755
Sheep/goat	1.2	60	101	1.423	15,599
Horse	8	400	101	1.423	15,599
Rabbit	0.1	2	40	1.409	15,755
Dog	0.25	15	50	0.587	37,812
Cat	0.06	3	20	0.282	78,774
Ornamental fish	0.00054	0.012	50	0.159	140,043
Target species for fattening
Chicken for fattening	0.158	2	26	1.447	6,910
Turkey for fattening	0.176	3	34	1.405	7,116
Piglet	0.88	20	46	1.426	7,013
Pig for fattening	2.2	60	54	1.395	7,168
Veal calf (milk replacer)	1.89	100	114	1.414	7,024
Cattle for fattening	8	400	101	1.423	7,027
Sheep/goat	1.2	60	101	1.423	7,027
Horse	8	400	101	1.423	7,027
Rabbit	0.1	2	40	1.409	7,097
Salmon	0.0021	0.12	112	1.381	7,242

^(a)The values of methyleugenol in feed is calculated considering the highest analysed value in the additive.

^(b)The MOE for methyleugenol is calculated as the ratio of the reference point to the intake: for long‐living and reproductive animals is based on BMDL₁₀ of 22.2 mg/kg bw per day derived from rodent carcinogenicity studies with methyleugenol; for target species for fattening based on a NOAEL of 10 mg/kg bw per day derived from a 90‐day study with methyleugenol (NTP, 2000).

Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Bampidis, V ., Azimonti, G ., Bastos, M. L ., Christensen, H ., Durjava, M ., Kouba, M ., López‐Alonso, M ., López Puente, S ., Marcon, F ., Mayo, B ., Pechová, A ., Petkova, M ., Ramos, F ., Sanz, Y ., Villa, R. E ., Woutersen, R ., Brantom, P ., Chesson, A ., … Dusemund, B ., (2023). Safety and efficacy of feed additives consisting of essential oils derived from the flower buds or the leaves of Syzygium aromaticum (L.) Merr. & L.M Perry (clove bud oil and clove leaf oils) for all animal species (FEFANA asbl). EFSA Journal, 21(7), 1–43. 10.2903/j.efsa.2023.8183