Safety and efficacy of a feed additive consisting of an essential oil from the leaves of Laurus nobilis L. (laurel leaf oil) for all animal species (FEFANA asbl)

Abstract

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of an essential oil from the leaves of Laurus nobilis L. (laurel leaf oil), when used as a sensory additive for all animal species. The additive contains up to 4% methyleugenol. The use of the additive at 2 mg/kg complete feed in dogs and cats was considered of low concern. For other long‐living and reproductive animals, the use of the additive at 10 mg/kg was considered of concern. For short‐living animals, the Panel had no safety concern when the additive is used at 10 mg/kg complete feed for turkeys for fattening, piglets and other growing Suidae, pigs for fattening, veal calves (milk replacer), cattle for fattening and other growing ruminants, horses and rabbits for meat production, salmonids and other fin fish; and at 8.5 mg/kg for chickens for fattening, other growing poultry and other minor species for fattening. The use of laurel leaf oil up to the highest level in feed which was considered of no concern for target animals was also expected to be of no concern for consumers. The additive should be considered as irritant to skin and eyes and the respiratory tract. Due to the high concentration of methyleugenol (≥ 1%), the additive was classified by the applicant as suspected of causing genetic defects and of causing cancer and should be handled accordingly. The use of the additive under the proposed conditions of use was not expected to pose a risk for the environment. Since the leaves of L. nobilis and their preparations were recognised to flavour food and their function in feed would be the same, no further 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 Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG) ² for authorisation/re‐evaluation of 18 preparations (cassia oil, cassia bark extract (solvent‐based, sb), camphor oil, cinnamon oil, cinnamon bark oleoresin, cinnamon tincture, laurel leaves oil, laurel leaves extract/oleoresin, litsea berry oil, boldo extract (water‐based, wb), boldo tincture, ylang‐ylang oil, mace oil, nutmeg oil, nutmeg oleoresin, kawakawa tincture, pepper oil and pepper oleoresin) belonging to botanically defined group (BDG) 6 – Laurales, Magnoliales, Piperales, when used as a feed additive for all animal species (category: sensory additives; functional group: flavouring compounds). During the assessment, the applicant withdrew the applications for eight preparations. 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 18 preparations under application: an essential oil from the leaves of Laurus nobilis L. (laurel leaf oil) for all animal species.

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 3 January 2011.

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 product laurel oil (Laurus nobilis L.), when used under the proposed conditions of use (see Section 3.2.4).

The remaining nine preparations belonging to botanically defined group (BDG) 6 – Laurales, Magnoliales, Piperales under application are assessed in separate opinions.

1.2. Additional information

‘Laurel leaves oil’ from L. nobilis 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 L. nobilis preparation when used to provide flavour in food. However, according to Regulation (EC) No 1334/2008 ⁵ flavourings preparations produced from food or food ingredients with flavouring properties, 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’.

The Committee for Veterinary Medicinal Products of the European Medicines Agency (EMA) published a summary report for Lauri folii aetheroleum described as the ‘volatile oil obtained by steam distillation from bay leaves, the leaves of Laurus nobilis’ (EMA, 1999) and another summary report for Lauri fructus described as ‘the dried, ripe, berries of Laurus nobilis’ (EMA, 1999).

The European Medicines Agency (EMA) published a public statement on the use of medicinal products containing methyleugenol (EMA, 2005), which mentions Laurus nobilis L. (with a methyleugenol content in the leaves in the range 213–2,608 mg/kg).

Many of the individual components of the essential oil 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), the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF), the EFSA Panel on Food Additives and Flavourings (FAF) and/or the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The list of flavouring compounds currently authorised for food ⁶ and feed ⁷ uses together with the EU Flavour Information System (FLAVIS) number, the chemical group as defined in Commission Regulation (EC) No 1565/2000 ⁸ and the corresponding EFSA opinion is given 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

	Chemical Group	Product – EU register name (common name)	FLAVIS No	EFSA* or JECFA opinion, Year
01	Straight‐chain primary aliphatic alcohols/aldehydes/acids, acetals and esters with esters containing saturated alcohols and acetals containing saturated aldehydes	Hexan‐1‐ol	02.005	2013
		Ethyl 2‐methylbutyrate	09.409
		Ethyl isovalerate	09.447
02	Branched‐chain primary aliphatic alcohols/aldehydes/acids, acetals and esters with esters containing branched‐chain alcohols and acetals containing branched‐chain aldehydes	Isobutyl isobutyrate	09.417	2012a
		Isobutyl 2‐methylbutyrate	09.585	2010a, CEF
03	α, ß‐Unsaturated (alkene or alkyne) straight‐chain and branched‐chain aliphatic primary alcohols/aldehydes/ acids, acetals and esters with esters containing α, β‐unsaturated alcohol and acetal containing α, β‐unsaturated alcohols or aldehydes	(Z)‐Nerol	02.058	2016a
		Neryl acetate	09.213
04	Non‐conjugated and accumulated unsaturated straight‐chain and branched‐chain aliphatic primary alcohols, aldehydes, acids, acetals and esters	Hex‐3(cis)‐en‐1‐ol	02.056	2016b
05	Saturated and unsaturated aliphatic secondary alcohols, ketones and esters with esters containing secondary alcohols	Heptan‐2‐ol (a)	02.045	WHO, 2000 (JECFA)
		Undecan‐2‐one	07.016	2015a
		Nonan‐2‐one	07.020
		Isopropyl 2‐methylbutyrate (a)	09.547	WHO, 1999 (JECFA)
06	Aliphatic, alicyclic and aromatic saturated and unsaturated tertiary alcohols and esters with esters containing tertiary alcohols ethers	Linalool	02.013	2012b
		α‐Terpineol	02.014
		4‐Terpinenol	02.072
		Linalyl acetate	09.013
		α‐Terpinyl acetate	09.015
07	Primary alicyclic saturated and unsaturated alcohols, aldehydes, acids, acetals esters with esters containing alicyclic alcohols	Myrtenal (a)	05.106	2017, CEF
		p‐Mentha‐1,8‐diene‐7‐yl acetate (a)	09.278
08	Secondary alicyclic saturated and unsaturated alcohols, ketones, ketals and esters with ketals containing alicyclic alcohols or ketones and esters containing secondary alicyclic alcohols	d,l‐Isoborneol	02.059	2016c
		l‐Carvone	07.147
		d,l‐Bornyl acetate	09.017
16	Aliphatic and alicyclic ethers	1,8‐Cineole	03.001	2021a, 2012c
		3,6‐Dihydro‐4‐methyl‐2‐(2‐methylprop‐1‐en‐1‐yl)‐2H‐pyran	13.088	2011a, CEF
18	Allylhydroxybenzenes	Eugenol	04.003	2011
22	Aryl‐substituted primary alcohol, aldehyde, acid, ester and acetal derivatives	Cinnamyl acetate	09.018	2017a
23	Benzyl alcohols/aldehydes/ acids/esters/acetals	4‐Isopropylbenzaldehyde	05.022	2012d
26	Aromatic ethers including anisole derivatives	1,2‐Dimethoxy‐4‐(prop‐1‐enyl)benzene (b) (Methyl isoeugenol)	04.013	2012e
31	Aliphatic and aromatic hydrocarbons and acetals containing saturated aldehydes	Limonene (a) , (c)	01.001	2008, EFSA (AFC)
		1‐Isopropyl‐4‐methylbenzene (p‐cymene)	01.002	2015b
		α‐Phellandrene	01.006
		α‐Terpinene	01.019
		γ‐Terpinene	01.020
		d‐Limonene	01.045
		Pin‐2(10)‐ene (β‐pinene)	01.003	2016d
		Pin‐2(3)‐ene (α‐pinene)	01.004
		β‐Caryophyllene	01.007
		Myrcene	01.008
		Camphene	01.009
		δ‐3‐Carene	01.029
		δ‐Cadinene (a) , (d)	01.021	2011b, CEF
		Germacra‐1(10),4(14),5‐triene (δ‐Germacrene) (a) , (d)	01.042
		3,7,10‐Humulatriene (a) , (d)	01.043
		1,1,7‐Trimethyltricyclo[2.2.1.0.(2.6)] heptane (tricyclene) (a) , (d)	01.060
		4(10)‐Thujene (sabinene) (a)	01.059	2015a, CEF
		β‐Ocimene (e) (3,7‐Dimethyl‐1,3,6‐octatriene)	01.018	2015b, CEF
		β‐Bourbonene (a)	01.024
32	Epoxides	β‐Caryophyllene epoxide (a)	16.043	2014, CEF

^(*)FEEDAP opinion unless otherwise indicated.

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

^(b)EFSA evaluated 1,2‐dimethoxy‐4‐(prop‐1‐enyl)benzene [04.013] or methyl isoeugenol, a mixture of (Z)‐ and (E)‐isomers (EFSA FEEDAP Panel, 2012e).

^(c)JECFA and EFSA evaluated d‐limonene [01.045] (EFSA, 2008). d‐Limonene [01.045] and l‐limonene [01.046] were also evaluated for use in feed (EFSA FEEDAP Panel, 2015b).

^(d)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, 2010b). No longer authorised for use as flavours in food, as the additional toxicity data requested (EFSA CEF Panel, 2011b) were not submitted and the CEF Panel was unable to complete its assessment.

^(e)EFSA evaluated β‐ocimene [01.018], a mixture of (E)‐ and (Z)‐isomers (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 ⁹ in support of the authorisation request for the use of laurel leaf oil from L. nobilis as a feed additive.

The FEEDAP Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 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.

Many of the components of the essential oil under assessment have been already evaluated by the FEEDAP Panel as chemically defined flavourings. The applicant submitted a written agreement to reuse the data submitted for the assessment of chemically defined flavourings (dossiers, publications and unpublished reports) for the risk assessment of preparations belonging to BDG 6, including the current one under assessment. ¹⁰

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 botanically defined flavourings from Group 06 – Laurales, Magnoliales, Piperales. During the assessment, upon request from EC and EFSA, the EURL issued two amendments of the original report. ¹¹ For the additive under assessment, laurel oil, the evaluation of the method of analysis is included in the second amendment. In particular, for the characterisation of laurel oil the EURL recommended methods based on gas chromatography with flame ionisation detector (GC‐FID) for the quantification of the phytochemical marker 1,8 cineole in laurel oil. ¹²

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of laurel leaf oil from L. nobilis is in line with the principles laid down in Regulation (EC) No 429/2008 ¹³ 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, 2012f), Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012g), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017b), Guidance on the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017d), 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), 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). ¹⁴

3. Assessment

The additive under assessment, laurel leaf oil, is an essential oil obtained from the leaves of L. nobilis L., intended for use as a sensory additive (functional group: flavouring compounds) in feed and in water for drinking for all animal species.

3.1. Origin and extraction

L. nobilis L. (the bay tree) is a large evergreen shrub belonging to the Lauraceae family. It is native to the Mediterranean region where its leaves (whole or ground) are used in cooking. Its dried berries also may be used as a food and an essential oil obtained from bay leaves finds application mostly in cosmetics and household products. Laurel wreaths have long been used as mark of respect and success.

The additive is extracted from fresh or dried leaves of L. nobilis by steam distillation. The volatile constituents are condensed and then separated from the aqueous phase by decantation.

3.2. Characterisation

3.2.1. Characterisation of laurel leaf oil

The essential oil under assessment is a pale yellow clear mobile liquid with a characteristic odour. In six batches of the additive (originating from the Balkans or Turkey), the density (20°C) ranged between 911 and 914 kg/m³ (specification: 905–925 kg/m³), the refractive index (20°C) between 1.465 and 1.468 (specification: 1.465–1.470) and the specific optical rotation (at 20°C, two batches) was equal to −18.2° (specification: −19° to −10°). ¹⁵ Laurel leaf oil is identified with the single Chemical Abstracts Service (CAS) number 8002‐41‐3, the European Inventory of Existing Chemical Substances (EINECS) number 283‐272‐5, the Flavor Extract Manufacturers Association (FEMA) 2613, and the Council of Europe (CoE) number 255.

The product specifications are based on the concentrations of the main components of the essential oil, analysed by GC‐FID and expressed as percentage of the total gas chromatographic peak area (% GC area). Six components contribute to the specifications as shown in Table 2, with 1,8‐cineole selected as the phytochemical marker. The analysis of six batches of the additive showed compliance with these specifications when analysed by gas chromatography–mass spectrometry (GC–MS). ¹⁵ The six compounds account for about 79.6% on average (range 78.7–80.2%) of % GC area (Table 2).

Table 2.

Major constituents of the essential oil from the leaves of Laurus nobilis L. as defined by specifications: batch to batch variation based on the analysis of six batches. 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	Mean	Range (a)
1,8‐Cineole	170‐82‐6	03.001	41–58	47.0	45.9–49.3
α‐Terpinyl acetate	80‐26‐2	09.015	5.5–12.5	11.3	10.8–11.9
Sabinene (4(10)‐thujene)	3387‐41‐5	01.059	4.0–11.5	6.55	4.86–8.47
α‐Pinene (pin‐2(3)‐ene)	80‐56‐8	01.004	4.5–9.0	6.69	5.32–7.68
β‐Pinene (pin‐2(10)‐ene)	127‐91‐3	01.003	2.8–6.0	4.31	3.83–4.67
Linalool	78‐79‐6	02.013	2.0–5.5	3.61	2.90–4.12
Total				79.6	78.7–80.2

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 six batches analysed.

The applicant provided the full characterisation of the six batches obtained by GC–MS. ¹⁵ In total, up to 154 peaks were detected in the chromatogram, 83 of which were identified and accounted on average for 99.1% (98.9–99.2%) of the % GC area. Besides the six compounds indicated in the product specifications, 28 other compounds were detected at individual levels > 0.1% and are listed in Table 3. These 34 compounds together account on average for 97.4% (96.3–97.4%) of the % GC area. The remaining 48 compounds (ranging between 0.1% and 0.006%) and accounting for 1.94%, are listed in the footnote. ¹⁶

Table 3.

Constituents of the essential oil from the leaves of Laurus nobilis L. accounting for > 0.1% of the composition (based on the analysis of six batches) not included in the specifications. 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 (a)
Methyleugenol (b)	93‐15‐2	04.012	3.10	2.34–4.00
1‐Isopropyl‐4‐methylbenzene	99‐87‐6	01.002	2.72	1.99–4.02
γ‐Terpinene	99‐85‐4	01.020	2.69	1.65–3.83
4‐Terpinenol	562‐74‐3	02.072	1.91	1.34–2.39
Limonene	5989‐27‐5	01.045	1.48	0.53–2.39
α‐Terpineol	98‐55‐5	02.014	1.35	0.88–2.12
Eugenol	97‐53‐0	04.003	0.76	0.22–1.11
γ‐Terpinyl acetate	10235‐63‐9	–	0.51	0.43–0.62
δ‐Terpinyl acetate	93836‐50‐1	–	0.50	0.43–0.55
Myrcene	123‐35‐3	01.008	0.42	0.18–0.53
α‐Terpinene	99‐86‐5	01.019	0.41	0.24–0.61
Estragole (c)	140‐67‐0	04.011	0.38	0.37–0.38
Bornyl acetate	76‐49‐3	09.017	0.26	0.16–0.49
Camphene	79‐92‐5	01.009	0.25	0.14–0.44
β‐Caryophyllene	87‐44‐5	01.007	0.22	0.11–0.31
α‐Thujene	2867‐05‐2	–	0.21	0.16–0.27
δ‐Terpineol	7299‐42‐5	–	0.20	0.18–0.24
Laevo‐pinocarveol	547‐61‐5	–	0.20	0.14–0.25
α‐Phellandrene	99‐83‐2	01.006	0.19	0.11–0.28
2,3‐Dehydro‐1,8‐cineole	92760‐25‐3	–	0.18	0.10–0.25
β‐Caryophyllene epoxide	87‐44‐5	01.007	0.17	0.13–0.21
Myrtenal	564‐94‐3	05.106	0.17	0.12–0.21
β‐Elemene	33880‐83‐0	–	0.16	0.08–0.23
trans‐Sabinene hydrate	17699‐16‐0	–	0.16	0.14–0.20
Neryl acetate	141‐12‐8	09.213	0.13	0.11–0.16
Terpinolene	586‐62‐9	01.005	0.12	0.04–0.18
trans‐3,7‐Dimethyl‐1,3,6‐octatriene (d) (β‐ocimene)	3779‐61‐1		0.11	0.03–0.22
(Z)‐Methyl isoeugenol (e)	6380‐24‐1	–	0.10	0.04–0.13
Total			19.02	17.35–19.00

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 six batches analysed.

^(b)Substance which shall not be added as such to food (Annex III), maximum level in food is set by Regulation (EC) No 1334/2008, including dairy products (20 mg/kg), meat products (15 mg/kg), fish products (10 mg/kg), soups and sauces (60 mg/kg), ready‐to eat savouries (20 mg/kg) and non‐alcoholic beverages (1 mg/kg).

^(c)Substance which shall not be added as such to food (Annex III), maximum level in food is set by Regulation (EC) No 1334/2008, including dairy products (50 mg/kg), processed fruits, vegetables (incl. mushrooms, fungi, roots, tubers, pulses and legumes), nuts and seeds (50 mg/kg), fish products (50 mg/kg) and non‐alcoholic beverages (10 mg/kg).

^(d)EFSA evaluated β‐ocimene [01.018], a mixture of (E)‐ and (Z)‐isomers (EFSA CEF Panel, 2015b).

^(e)EFSA evaluated 1,2‐dimethoxy‐4‐(prop‐1‐enyl)benzene [04.013] or methyl isoeugenol, a mixture of (Z)‐ and (E)‐isomers (EFSA FEEDAP Panel, 2012e).

The applicant performed a literature search regarding substances of concern and chemical composition of the plant species L. nobilis and its preparations. ¹⁷ The search identified the presence of three p‐alkoxy‐substituted allylbenzenes. An analysis of the six batches of the laurel leaf oil under assessment (see Tables 2 and 3) confirmed the presence of methyleugenol in all batches (2.4–4%), ¹⁸ estragole in two batches (0.37–0.38%) and elemicin in four batches (0.004–0.011%).

3.2.2. Impurities

The applicant makes reference 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, aflatoxin B1, B2, G1, G2 and ochratoxin A. However, no data have been provided on the presence of these impurities. Since laurel leaf oil is produced by steam distillation, the likelihood of any measurable carry‐over of all the above‐mentioned elements is low except for mercury.

3.2.3. Shelf life

The typical shelf‐life of laurel 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). ¹⁹ However, no data supporting this statement were provided.

3.2.4. Conditions of use

Laurel leaf oil is intended to be added to feed and water for drinking for all animal species without a withdrawal time. The maximum proposed use level in complete feed is 10 mg/kg for all animal species except chickens for fattening (8.5 mg/kg), and cats and dogs (2 mg/kg). No use level has been proposed by the applicant for use in water for drinking.

3.3. Safety

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

Many of the constituents of laurel leaf oil, accounting for about 92% of the total GC peak areas, have been previously assessed and considered safe for use as flavourings, and are currently authorised for use in food ⁶ 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).

Four constituents, δ‐cadinene [01.021], δ‐germacrene [01.042], 3,7,10‐humulatriene [01.043] and tricyclene [01.060] were evaluated in FGE25.Rev2 by applying the Procedure described in the Guidance on the data required for the risk assessment of flavourings to be used in or on foods (EFSA CEF Panel, 2010b). For these compounds, for which there is no concern for genotoxicity, EFSA requested additional toxicity data (EFSA CEF Panel, 2011b). In the absence of such toxicological data, the CEF Panel was unable to complete its assessment. As a result, these compounds are no longer authorised for use as flavours in food. For these compounds, 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).

Sixteen constituents accounting together for 2.5% of the % GC area (cis‐p‐2‐menthen‐1‐ol, δ‐terpineol, β‐terpinyl acetate, trans‐1‐methyl‐4‐(1‐methylvinyl)cyclohex‐2‐en‐1‐ol, cis‐p‐2,8‐menthadien‐1‐ol, δ‐terpinyl acetate, γ‐terpinyl acetate, trans‐sabinene hydrate, l‐pinocarveol, trans‐piperitol, 2,3‐dehydro‐1,8‐cineole, trans‐3,7‐dimethyl‐1,3,6‐octatriene, β‐elemene, (Z)‐α‐bisabolene, β‐selinene and γ‐cadinene) have not been previously assessed for use as flavourings. The FEEDAP Panel notes that they are aliphatic mono‐ or sesquiterpenes structurally related to flavourings already assessed in CG 6, 8, 16 and 31 and a similar metabolic and toxicological profile is expected. Because of their lipophilic nature, they are expected to be rapidly absorbed from the gastro‐intestinal tract, oxidised to polar oxygenated metabolites, conjugated and excreted (EFSA FEEDAP Panel, 2012b,c, 2015b, 2016c,d).

The remaining 16 compounds, namely (E)‐sabinyl acetate, spathulenol, β‐eudesmol, α‐eudesmol, sabina ketone, pinocarvone, isocarveol, cis‐isocarveol, p‐mentha‐3,8‐diene, pseudolimonene, α‐thujene, 2,4‐thujadiene, α‐ylangene, α‐copaene, bicyclogermacrene and α‐fenchene, were screened with OECD QSAR Toolbox and no alerts were identified for in vitro mutagenicity (Ames test), for genotoxic and non‐genotoxic carcinogenicity and for other endpoints with the exception of (E)‐sabinyl acetate, cis‐isocarveol, isocarveol, pinocarvone and 1,1,7‐trimethyltricyclo[2.2.1.0.(2.6)]heptane. For these substances, predictions of Ames mutagenicity (with and without S9) were made by ‘read‐across’ analyses of data available for similar substances to the target compounds (i.e. analogues obtained by categorisation). Ames test (with and without S9) read across predictions were all found negative for all the substances. ²⁰

The following sections focus on methyleugenol, estragole and elemicin, based on the evidence provided by the applicant in the form of literature. For the absorption, distribution, metabolism and excretion (ADME) and the toxicology of estragole, reference is made to the safety evaluation made by the FEEDAP Panel in the EFSA opinion on ylang ylang oil (EFSA FEEDAP Panel, 2022a).

3.3.1. Absorption, distribution, metabolism and excretion

p‐Allylalkoxybenzenes: methyleugenol, estragole and elemicin

Methyleugenol is a lipophilic compound and as such readily and completely absorbed from the gastrointestinal tract. Phase I metabolism is catalysed by cytochromes P450 (CYP450) enzymes mainly in the liver. Demethylation of the 4‐methoxygroup with formation of 4‐allylphenol is followed by conjugation with glucuronic acid or sulfate and renal excretion. Oxidation of the allyl‐side chain leads to methyleugenol‐2′,3′‐epoxide, which is hydrolysed to the corresponding diol with subsequent glucuronidation and excretion. Both metabolic pathways represent detoxification of methyleugenol. The formation of genotoxic metabolites is initiated by oxidation of the side chain with formation of 1′‐hydroxy‐methyleugenol. Sulfate‐conjugation of the hydroxyl group leads to 1′‐sulfoxymethyleugenol, which is highly unstable and breaks down to form a highly reactive carbonium ion, which can react covalently with DNA (as reviewed in EMA, 2005 and IARC, 2018). The occurrence of DNA‐adducts of methyleugenol in liver samples obtained at liver surgery of humans as a result of exposure to this compound via normal food has been demonstrated (Herrmann et al., 2013).

Similar metabolic pathways have been described for estragole (EMA, 2021; reviewed in EFSA FEEDAP Panel, 2022a) and other structurally related p‐allylalkoxybenzenes.

A similar ADME as for methyleugenol and estragole is expected for elemicin, including the formation of the 1′‐sulfoxymetabolite. For elemicin, the application of physiologically based kinetic (PBK) models predicted that in rat liver the formation of the DNA reactive 1′‐sulfoxymetabolite was 11‐ and 2‐fold lower as compared to the formation of the 1′‐sulfoxymetabolites of estragole and methyleugenol, respectively (van den Berg et al., 2012).

3.3.2. Toxicology

3.3.2.1. Genotoxicity and carcinogenicity

For fully defined mixtures, the EFSA Scientific Committee (EFSA SC) recommends applying a component‐based approach, i.e. assessing all components individually for their genotoxic potential using all available information, including read‐across and quantitative structure–activity relationship (QSAR) considerations about their genotoxic potential (EFSA SC, 2019b). Therefore, the potential genotoxicity of identified constituents is first considered. Then, in vitro genotoxicity studies performed with laurel leaf oil with similar composition to the additive under assessment are described.

Methyleugenol

Laurel leaf oil contains methyleugenol (3.1% on average, range: 2.3–4.0%), a compound with experimentally proven genotoxicity and carcinogenicity in rodents (IARC, 2018).

Methyleugenol was not mutagenic in the bacterial mutagenicity assay with Salmonella Typhimurium and Escherichia coli WP‐uvrA in the presence and absence of S9‐mix. However, positive results were obtained in a modified strain of Salmonella Typhimurium (TA100‐hSULT1C2) expressing sulfotransferase (Honda et al., 2016), indicating that the formation of sulfate esters plays a key role in the genotoxicity of alkenylbenzenes. An in vivo study was carried out in wild‐type (wt) mice and in parallel in genetically modified strains of mice: (i) Sult1a1 knockout (ko), (ii) transgenic for human SULT1A1/2 (tg) and (iii) the combination of both modifications (ko‐tg). The animals were given methyleugenol by gavage at 50 mg/kg body weight (bw) and killed after 6 h for measurement of DNA adducts in the liver. Methyleugenol gave rise to 23, 735, 3,770 and 4,500 N2‐(trans‐methylisoeugenol‐3′‐yl)‐2′‐deoxyguanosine adducts per 10⁸ 2′‐deoxyribonucleosides (dN) in ko, wt, ko‐tg and tg mice, respectively. Adduct formation was reduced by 97% in the absence of Sult1a1 (ko) as compared with wt mice. Transgenic mice for human SULT1A1/2 (tg) produced the highest level of adducts. These data clearly demonstrate the role played by sulfotransferases in the bioactivation of methyleugenol by conjugation with the phase I metabolite 1′‐hydroxymethyleugenol (Herrmann et al., 2014).

In Chinese hamster ovary (CHO) cells, sister chromatid exchange (SCE) was induced by methyleugenol exposure in the presence and absence of microsomal activation and chromosomal aberrations only in the presence of microsomal activation (NTP, 2000). The induction of malignant transformation by methyleugenol was demonstrated in Syrian hamster ovary cells (Kerkaert et al., 1996). DNA repair was induced by methyleugenol in primary hepatocytes from rats and mice (Howes et al., 1990; Chan and Caldwell, 1992; Burkey et al., 2000). In human HepG2 cell line exposed to methyleugenol DNA adducts were formed to a similar extent to that found in liver of mice in a previous in vivo study (Zhou et al., 2007).

The carcinogenicity of methyleugenol was investigated in a 2‐year National Toxicology Program (NTP) carcinogenicity study in rats and mice (NTP, 2000) using oral doses of 0, 37, 75, or 150 mg/kg bw per day in both species and a higher dose of 300 mg/kg bw per day in rats. Rats of both sexes receiving methyleugenol had dose‐related increased incidences of hepatocellular carcinoma and neuroendocrine tumours of the glandular stomach. ²¹ Higher incidences of kidney neoplasms, malignant mesothelioma, mammary gland fibroadenoma and subcutaneous fibroma and fibrosarcoma were observed in male rats only. ²² Increased incidence of hepatocellular carcinoma was seen in both sexes of mice although the incidence was not related to dose. Neuroendocrine tumours of the glandular stomach were also observed in male mice but only at the highest dose. The NTP concluded that there was clear evidence for the carcinogenicity of methyleugenol in rats and mice.

Suparmi et al. (2019) performed an evaluation of the available evidence using the benchmark dose (BMD) approach and found that dose–response modelling, applying model averaging as recommended by the EFSA Scientific Committee (EFSA SC, 2017) on the long‐term chronic toxicity study (NTP, 2000) and using hepatocellular carcinomas in male rats as a response, yielded a BMD lower confidence limit for a benchmark response of 10% (BMDL₁₀) of 22.2 mg/kg bw per day.

Estragole and elemicin

Two batches of laurel leaf oil were shown to contain estragole (0.37 and 0.38%), a compound with experimentally proven genotoxicity and carcinogenicity in rodents, as reviewed by the Scientific Committee on Food (EC, 2001), EMA (2021) and in the safety evaluation made by the FEEDAP Panel in the EFSA opinion on ylang ylang oil (EFSA FEEDAP Panel, 2022a). In addition, the presence of elemicin was detected in four batches (0.004–0.011%).

Estragole was included in the diet of female CD‐1 mice at 0, 2.3 and 4.6 g/kg diet for 12 months. At least 50% of the animals in the exposed groups developed hepatic tumours by 18 months, ²³ which were diagnosed as hepatomas types A (hepatocellular adenomas) or B (hepatocellular adenocarcinomas) or mixed types A and B. The animals fed the control diet did not show any hepatic tumours (Miller et al., 1983).

The FEEDAP Panel notes that there is high uncertainty in derivation of a BMD lower confidence limit for a benchmark response of 10% (BMDL₁₀) for estragole from a carcinogenicity study in CD‐1 mice. ²⁴

The FEEDAP Panel already applied to estragole a BMDL₁₀ of 22.2 mg/kg bw per day, derived from a carcinogenicity study in rat with methyleugenol (NTP, 2000) by applying model averaging (Suparmi et al., 2019; EFSA FEEDAP Panel, 2022b).

Although elemicin did not induce the formation of hepatic tumours in newborn male mice, after intraperitoneal (i.p.) injection (Miller et al., 1983), 1‐hydroxyelemicin showed hepatocarcinogenic activity at high doses. Elemicin was also shown to form DNA adducts in the liver of mice treated i.p. (Miller et al., 1983), although the potency was lower than that of methyleugenol, estragole and safrole (Phillips et al., 1984). Two additional studies showed the induction of DNA adducts by elemicin in liver of adult mice after i.p. injection (Randerath et al., 1984) and in vitro in human HepG2 cells (Zhou et al., 2007) and confirmed the lower potency of elemicin compared to methyleugenol, safrole and estragole.

The applicant provided a literature search on the genotoxicity of preparations obtained from laurel leaves of L. nobilis. However, in all the studies submitted (Türkez and Geyikoglu, 2011; Türkez and Toğar, 2012; Tuylu et al., 2014), the composition of the test item was unknown and major shortcomings were identified in the study design. These studies were not considered relevant for the current assessment.

3.3.2.2. Subchronic toxicity studies

Methyleugenol

Methyleugenol was tested in a repeated dose toxicity assay by dosing rats and mice by gavage over a period of 14 weeks with 10, 30, 100, 300 and 1,000 mg/kg bw for 5 days per week (NTP, 2000). Body weight reduction and haematological changes were seen. Changes of organ weight ²⁵ and function, including effects on liver (non‐neoplastic lesions) ²⁶ and the glandular stomach (atrophy and chronic inflammation of the mucosa), ²⁷ were observed at doses of 100 mg/kg bw and higher. Based on these findings, a no observed adverse effect level (NOAEL) of 30 mg/kg bw was identified from the rat study. Similar observations were obtained in a study in mice with the same dosing and time schedule. A statistically significant increase of liver weights and lesions of the glandular stomach occurred at a dose of 30 mg/kg bw and above. Thus, the NOAEL for non‐neoplastic lesions identified in the mouse study was 10 mg/kg bw per day.

Considering the structural similarity and the similar mode of action of p‐allylalkoxybenzenes, the FEEDAP Panel retains the NOAEL of 10 mg/kg bw per day derived from the mice study with methyleugenol, as reference point for the assessment group p‐allylalkoxybenzenes for non‐neoplastic endpoints.

3.3.3. 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 these data, 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.

As the additive under assessment is a fully defined mixture (> 99% of the components were identified, see Section 3.2.1), the FEEDAP Panel applied a component‐based approach to assess the safety for target species of the essential oil. Substances for which a concern for genotoxicity has been identified (methyleugenol, estragole and elemicin) are assessed separately.

Components other than methyleugenol, estragole and elemicin

Based on considerations related to structural and metabolic similarities, the components were allocated to 14 assessment groups, corresponding to the chemical groups (CGs) 1, 2, 3, 4, 5, 6, 7, 8, 16, 22, 23, 26, 31 and 32, as defined in Annex I of Regulation (EC) No 1565/2000. For CG 31 (‘aliphatic and aromatic hydrocarbons’), the application of subassessment groups as defined in Flavouring Group Evaluation 25 (FGE.25) and FGE.78 is 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 8.5 mg/kg complete feed), reference points and margin of exposure (MOE) for the individual components of laurel leaf oil classified according to assessment groups

Essential oil composition			Exposure		Hazard characterisation		Risk characterisation
Assessment group	FLAVIS‐No	Highest conc. in the oil	Max Feed conc.	Daily Intake	Cramer Class (b)	NOAEL (c)	MOE	MOET
Constituent	–	%	mg/kg	mg/kg bw/day	–	mg/kg bw/day	–	–
CG 5
Undecan‐2‐one	07.016	0.10	0.009	0.0008	II	0.91	1,147
Nonan‐2‐one	07.020	0.05	0.004	0.0004	II	0.91	2,484
Heptan‐2‐ol	02.045	0.04	0.004	0.0003	I	3	9,361
Isopropyl 2‐methylbutyrate	09.547	0.01	0.001	0.0001	I	3	39,315
MOET CG 5								711
CG 6
α‐Terpinyl acetate	09.015	11.87	1.009	0.0906	(I)	250	2,761
Linalool	02.013	4.12	0.350	0.0315	(I)	117	3,720
4‐Terpinenol	02.072	2.39	0.203	0.0182	(I)	250	13,737
α‐Terpineol	02.014	2.12	0.180	0.0161	(I)	250	15,491
β‐Eudesmol	–	0.12	0.010	0.0009	III	0.15	168
Spathulenol	–	0.10	0.008	0.0001	I	3	4,138
cis‐p‐menthen‐1‐ol	–	0.08	0.007	0.0006	I	3	4,914
trans‐1‐methyl‐4‐(1‐methyl vinyl)cyclohex‐2‐en‐1‐ol	–	0.06	0.005	0.0004	I	3	6,897
(E)‐Sabinyl acetate	–	0.05	0.004	0.0004	I	3	7,863
α‐Eudesmol	–	0.05	0.004	0.0004	III	0.15	418
cis‐p‐2,8‐Menthadien‐1‐ol	–	0.02	0.001	0.0001	II	0.91	7,950
MOET CG 6								100
CG 7
Myrtenal	05.106	0.21	0.018	0.0016	I	3	1,854
p‐Mentha‐1,8‐dien‐7‐yl acetate	09.278	0.01	0.001	0.0001	I	3	28,082
MOET CG 7								1,740
CG 8
Bornyl acetate	09.017	0.49	0.041	0.0033	I	15	4,208
l‐Pinocarveol	–	0.25	0.021	0.0017	I	3	1,598
d,l‐Isoborneol	02.059	0.11	0.010	0.0008	(I)	15	17,551
Pinocarvone	–	0.09	0.007	0.0007	III	0.15	223
Isocarveol	–	0.04	0.004	0.0003	I	3	8,935
Sabina ketone	–	0.04	0.003	0.0003	III	0.15	562
trans‐Piperitol	–	0.03	0.002	0.0002	I	3	15,121
cis‐Isocarveol		0.01	0.001	0.0001	I	3	32,762
MOET CG 8								135
CG 16
1,8‐Cineole	03.001	49.27	4.188	0.3760	(II)	100	266
2,3‐Dehydro‐1,8‐cineole	–	0.25	0.022	0.0019	(II)	0.91	1,548
3,6‐Dehydro‐4‐methyl‐2‐(2‐methylprop‐1‐en‐1‐yl)‐2H‐pyran	13.088	0.01	0.001	0.0001	II	0.91	14,907
MOET CG 16								224
CG 18
Eugenol	04.003	1.11	0.094	0.0074	(I)	300	35,547
CG 31, II (Acyclic alkanes)
Myrcene	01.008	0.53	0.045	0.0041	(I)	44	10,818
β‐trans‐Ocimene	01.018	0.22	0.018	0.0016	(I)	44	26,695
MOET CG 31, II								7,699
CG 31, III (Cyclohexene hydrocarbons)
γ‐Terpinene	01.020	3.83	0.325	0.0292	(I)	250	8,561
Pseudo limonene	–	0.03	0.003	0.0002	I	3	25,636
p‐Mentha‐3,8‐diene	–	0.02	0.002	0.0001	I	3	20,692
MOET CG 31, III								4,683
CG 31, IVe (Benzene hydrocarbons, alkyl)
p‐Cymene	01.002	4.02	0.342	0.0270	I	154	5,699
CG 31, V (Bi‐, tricyclic, non‐aromatic hydrocarbons)
Sabinene	01.059	0.44	0.720	0.0647	(I)	222	3,434
α‐Pinene	01.004	7.68	0.653	0.0586	(I)	222	3,788
β‐Pinene	01.003	5.32	0.452	0.0406	I	222	5,472
α‐Thujene	–	0.27	0.023	0.0021	I	3	1,445
Bicyclogermacrene	–	0.10	0.008	0.0008	I	3	3,971
α‐Fenchene	–	0.06	0.005	0.0005	I	3	6,341
2,4‐Thujadiene	–	0.04	0.004	0.0003	III	0.15	447
α‐Ylanglene	–	0.04	0.003	0.0003	I	3	9,589
Tricyclene	01.060	0.01	0.001	0.0001	I	3	43,683
α‐Copaene	–	0.01	0.001	0.0001	I	3	49,144
MOET CG 31, V								235
CG 31, VI (macrocyclic non aromatic hydrocarbons)
Germacra‐1(10),4(14),5‐triene	01.042	0.09	0.008	0.0007	I	3	4,182
3,7,10‐Humulatriene	01.043	0.05	0.004	0.0003	I	3	8,737
								2,828

^(a)Intake calculations for the individual components are based on the use level of 8.5 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 (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.

For each component in the assessment group, exposure in 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, 2017c). 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/body weight 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 NOAEL values. Structural and metabolic similarity among the components in the assessment groups were assessed to explore the application of read‐across allowing extrapolation from a known NOAEL of a component of an assessment group to the other components of the group with no available NOAEL or, if sufficient evidence were available for members of a (sub)assessment group, to derive a (sub)assessment group NOAEL.

Toxicological data for subchronic studies, from which NOAEL values could be derived, were available for acetaldehyde [05.001] the representative compound in CG 1 (EFSA FEEDAP Panel, 2013), 2‐ethylhexan‐1‐ol [02.082] the representative compound in CG 2 (EFSA FEEDAP Panel, 2012a), citral [05.020] the representative compound in CG 3 (EFSA FEEDAP Panel, 2016a), hex‐3(cis)‐en‐1‐ol [02.056] in CG 4 (EFSA FEEDAP Panel, 2016b), terpineol [02.230] ²⁸ and linalool [02.013] in CG 6 (EFSA FEEDAP Panel, 2012b), d,l‐isobornyl acetate [09.218] in CG 8 (EFSA FEEDAP Panel, 2016c), 1,8‐cineole in CG 16 (EFSA FEEDAP Panel, 2021a, 2012c), eugenol [04.003] in CG 18 (EFSA FEEDAP Panel, 2011), methyl isoeugenol [04.013] in CG 26 (EFSA FEEDAP Panel, 2012e), myrcene [01.008], d‐limonene [01.045], 1‐isopropyl‐4‐benzene [01.002] and β‐caryophyllene [01.007] in CG 31 (EFSA FEEDAP Panel, 2015b, 2016d), and β‐caryophyllene oxide in CG 32 (EFSA CEF Panel, 2014).

The NOAEL of 120 mg/kg bw per day for acetaldehyde [05.001] was selected as reference point for CG 1 compounds and the NOAEL of 50 mg/kg bw per day for 2‐ethylhexan‐1‐ol [02.082] was used as a group NOAEL for all compounds belonging to CG 2. Considering the structural and metabolic similarities, read‐across was applied using the NOAEL of 345 mg/kg bw per day for citral [05.020] to extrapolate to nerol [02.058] and neryl acetate [09.213] in CG 3.

Considering the structural and metabolic similarities, for the subgroup of terpinyl derivatives in CG 6, i.e. α‐terpineol [02.014], 4‐terpinenol [02.072], α‐terpinyl acetate [09.015] and other terpinyl derivatives, the reference point was selected based on the NOAEL of 250 mg/kg bw per day available for terpineol [02.230] and d‐limonene [01.045]. Similarly, the NOAEL of 117 mg/kg bw per day for linalool [02.013] was extrapolated to linalyl acetate [09.013].

The NOAEL of 15 mg/kg bw per day for d,l‐isobornyl acetate [09.218] was extrapolated to d,l‐isoborneol [02.016] and to bornyl acetate [09.017] in CG 8 and the NOAEL of 100 mg/kg bw per day for methyl isoeugenol [04.013] was applied to (Z)‐methyl isoeugenol in CG 26.

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 subassessment group II (trans‐β‐ocimene), group III (γ‐terpinene [01.020], α‐terpinene [01.019], α‐phellandrene [01.006] β‐elemene, terpinolene [01.005] and (Z)‐α‐bisabolene) and group V (sabinene [01.059], α‐pinene [01.004], β‐pinene [01.003], camphene [01.009], β‐selinene, γ‐cadinene, δ‐cadinene [01.021] and δ‐3‐carene [01.029]) (EFSA CEF Panel, 2015a,b). The FEEDAP Panel applied the same NOAEL value from sabinene [01.059] to trans‐sabinene hydrate [02.085] in CG 8.

For the remaining compounds, ²⁹ toxicity studies and NOAEL values performed with the compounds under assessment were not available and read‐across was not possible. Therefore, the TTC approach was applied (EFSA FEEDAP Panel, 2012f, 2017c).

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,). 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 intra‐individual variability (as in the default 10 × 10 uncertainty factor). The compounds resulting individually in a MOE > 50,000 were not further considered in the assessment group as their contribution to the MOE(T) is negligible. ³⁰

The approach to the safety assessment of laurel leaf 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, representing the worst‐case scenario at the use level of 8.5 mg/kg.

As shown in Table 4, for all the assessment groups, the MOET was ≥ 100. Therefore, no safety concern was identified for the laurel leaf oil when used as a feed additive for chickens for fattening at the proposed use levels (8.5 mg/kg) without considering methyleugenol, estragole and elemicin.

From the lowest MOET of 100 for chickens for fattening, the MOET for CG 6 compounds (tertiary alcohols) 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 the assessment group CG 6 (Tertiary alcohols) calculated for the different target animal categories at the proposed use level

Animal category	Body weight (kg)	Feed intake (g DM/day)	Proposed use level (mg/kg feed) (1)	Lowest MOET CG 6
Chicken for fattening	2	158	8.5	100
Laying hen	2	106	10	127
Turkey for fattening	3	176	10	114
Piglets	20	880	10	153
Pig for fattening	60	2,200	10	181
Sow lactating	175	5,280	10	224
Veal calf (milk replacer)	100	1,890	10	353
Cattle for fattening	400	8,000	10	336
Dairy cow	650	20,000	10	217
Sheep/goat	60	1,200	10	336
Horse	400	8,000	10	336
Rabbit	2	100	10	134
Salmon	0.12	2.1	10	373
Dog	15	250	2	1,975
Cat (2)	3	60	2	1,679
Ornamental fish	0.012	0.054	10	1,343

DM, dry matter.

⁽¹⁾Complete feed containing 88% DM, milk replacer 94.5% DM.

⁽²⁾The MOET for cats is increased to 500 because of the reduced capacity of glucuronidation.

Table 5 shows that when the additive is used at the proposed use levels in complete feed the MOET is equal or above the value of 100 for all animal species. Because glucuronidation is an important metabolic reaction to facilitate the excretion of the components of the essential oil, the use of laurel leaf oil as additive in cat feed needs a wider margin of exposure. Considering that cats have a low capacity for glucuronidation (Court and Greenblatt, 1997; Lautz et al., 2021), a MOET of 500 is considered adequate. Therefore, for all species, no safety concern (without considering the presence of methyleugenol, estragole and elemicin) is identified for laurel leaf oil, when used as a feed additive at the proposed use levels.

No specific proposals have been made by the applicant for the use level in water for drinking. The FEEDAP Panel considers that the use in water for drinking is of no concern, provided that the total daily intake of the additive does not exceed the daily amount that is considered of no concern when consumed via feed (EFSA FEEDAP Panel, 2010).

p‐Allylalkoxybenzenes: Methyleugenol, estragole and elemicin

Methyleugenol was detected in all batches of the oil under assessment (up to 4%). At the proposed use levels in feed (2–10 mg/kg complete feed), it would result in concentrations ranging from 80 to 400 μg methyleugenol/kg complete feed.

Low concentrations of estragole (up to 0.38%) were detected in two batches and elemicin (up to 0.011%) in four batches of the additive under assessment. The use of laurel leaf oil at the proposed use levels would result in concentrations ranging from 8 to 38 μg estragole/kg complete feed and 0.2–1.1 μg elemicin/kg complete feed.

Methyleugenol, estragole and elemicin share the same structural features, the same metabolic pathways, particularly the formation of the reactive 1′‐sulfoxymetabolite (see Section 3.3.1) and the same mode of action. They are allocated to the same assessment group (p‐allylalkoxybenzenes) and an assessment of the combined exposure is performed as described 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). 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, 2021b), different reference points and a different magnitude of the MOET 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(T) with a magnitude > 10,000, when comparing the estimated exposure to genotoxic and/or carcinogenic substances with a BMDL₁₀ from a rodent carcinogenicity study, is considered 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, 2022b). In the current assessment, this reference point is applied to the sum of methyleugenol, estragole and elemicin. The assessment of the combined exposure to methyleugenol, estragole and elemicin for long‐living animals is reported in Table 6.

Table 6.

Combined exposure and combined margin of exposure (MOET) for the assessment group p‐allylalkoxybenzenes calculated at the maximum proposed use level of the additive in feed for long‐living and reproductive animals based on BMDL₁₀ of 22.2 mg/kg bw per day derived from rodent carcinogenicity studies with methyleugenol

Animal category: Long‐living and reproductive animals	Daily feed intake	Body weight	Use level in feed	Methyeugenol+ estragole+elemicin intake	MOET
	kg DM/day	kg	mg/kg feed	μg/kg bw per day
Laying hen	0.106	2	10	24.6	840
Sow lactating	5.28	175	10	15.1	1,484
Dairy cow	20	650	10	15.4	1,436
Sheep/goat	1.2	60	10	10	2,226
Horse	8	400	10	10	2,226
Rabbit	0.1	2	10	24.9	890
Dog	0.25	15	2	1.7	13,094
Cat	0.06	3	2	2.0	11,130
Ornamental fish	0.00054	0.012	10	2.2	8,904

DM, dry matter.

When the estimated exposures for long‐living and reproductive animals are compared to the BMDL₁₀ of 22.2 mg methyleugenol/kg bw per day (Suparmi et al., 2019), a MOET > 10,000, which is considered of low concern, is obtained only for cats and dogs at the proposed use level of 2 mg/kg complete feed (Table 6). For the other long‐living and reproductive animals, the MOET at the proposed use level of 10 mg/kg complete feed would be < 10,000 and is considered of concern.

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 FEEDAP Panel identified a NOAEL of 10 mg/kg bw per day for non‐neoplastic lesions (effect on liver and the glandular stomach) from a 90‐day study in mice with methyleugenol (NTP, 2000). The assessment of the combined exposure to methyleugenol, estragole and elemicin for short‐living animals is reported in Table 7.

Table 7.

Combined exposure and combined margin of exposure (MOET) for the assessment group p‐allylalkoxybenzenes calculated at the maximum proposed use level of the additive for target species for fattening based on a NOAEL of 10 mg/kg bw per day derived from a 90‐day study in mice with methyleugenol

Animal category: Target species for fattening	Daily feed intake	Body weight	Use level in feed	Methyleugenol+ estragole+elemicin intake	MOET
	kg DM/day	kg	mg/kg feed	μg/kg bw per day
Chicken for fattening	0.158	2	8.5	33.5	298
Turkey for fattening	0.176	3	10	29.3	342
Piglet	0.88	20	10	22.0	455
Pig for fattening	2.2	60	10	18.3	547
Veal calf (milk replacer)	1.89	100	10	8.8	1,139
Cattle for fattening	8	400	10	10	1,002
Horse	8	400	10	10	1,002
Rabbit	0.1	2	10	24.9	401
Salmon	0.0021	0.12	10	8.7	1,145

DM: dry matter.

For all short‐living animals (Table 7), the magnitude of the MOET is >100 and is of no safety concern, when comparing the exposure to the reference point for methyleugenol based on non‐neoplastic endpoints.

3.3.3.1. Conclusions on safety for the target species

Based on the MOET calculated considering the presence of methyleugenol, estragole and elemicin in the product at 4%, 0.4% and 0.01% and the conditions of use in the different species, the FEEDAP Panel concludes that:

The use of the additive at the proposed level of 2 mg/kg complete feed in dogs and cats is of low concern (MOET > 10,000). For other long‐living and reproductive animals (including those animals reared for laying/breeding/reproduction), the use of the additive at the proposed level of 10 mg/kg complete feed is considered of concern (MOET < 10,000).

The Panel considers the use in water for drinking in dogs and cats of low concern provided that the total daily intake of the additive does not exceed the daily amount that is considered of low concern when consumed via feed.

For short‐living animals, the Panel has no safety concern when the additive is used at the maximum proposed use level of 10 mg/kg complete feed for turkeys for fattening, piglets and other growing Suidae, pigs for fattening, veal calves (milk replacer), cattle for fattening and other growing ruminants, horses and rabbits for meat production, salmonids and other fin fish; and at 8.5 mg/kg complete feed for chickens for fattening, other growing poultry, and other minor species for fattening.

For short‐living animals, the Panel has no safety concern for the use in water for drinking provided that the total daily intake of the additive does not exceed the daily amount that is considered of no concern when consumed via feed.

3.3.4. Safety for the consumer

Laurel leaf oil is added to a wide range of food categories for flavouring purposes. Although individual consumption figures are not available, the Fenaroli's handbook of flavour ingredients (Burdock, 2009) cites values of 0.27 mg/kg bw per day for laurel (parts used: leaves and berries) (FEMA 2124), of 0.0029 mg/kg bw per day for laurel bay leaves extract (FEMA 2613) and 0.0027 mg/kg bw per day for laurel bay leaves oil (FEMA 2115). Fenaroli also reports use levels in food and beverages in the range of 7.4 mg/kg up to 215 mg/kg for laurel bay leaves oil. According to Fenaroli, the content of methyleugenol in laurel bay leaves oil ranges between 1.73% and 11.8%.

Many of the individual constituents of the essential oil under assessment 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 laurel leaf oil are expected to be extensively metabolised and excreted in the target species. Also for methyleugenol, estragole and elemicin, the available data indicate that they are absorbed, metabolised and rapidly excreted and are not expected to accumulate in animal tissues and products (see Section 3.3.1).

Considering the above and the reported human exposure due to direct use of laurel leaves and berries and their preparations including laurel leaf oil in food (Burdock, 2009) it is unlikely that consumption of products from animals given laurel leaf oil at the use levels considered of no safety concern for the target species would cause a meaningful increase of human background exposure.

Consequently, no concern would be expected for the consumer from the use of laurel leaf oil up to the highest level in feed which is considered of no concern for target animals.

3.3.5. Safety for the user

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

The applicant made a literature search aimed at retrieving studies related to the safety of preparations obtained from L. nobilis for the users. ³¹ None of the studies identified during the literature search provided formal data on endpoints relevant to user safety. However, several reports (reviewed in Tisserand and Young, 2014) suggest that laurel leaf oil is both a potential sensitiser and a skin and eye irritant.

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

The applicant produced a safety data sheet ³² for laurel leaf oil, where hazards for users have been identified. Due to the high level of methyleugenol (> 1%), the applicant also proposes to classify the additive according to Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP Regulation) ³³ as suspected of causing genetic defects (category 2 mutagen) and of causing cancer (category 2 carcinogen).

For preparations with these classifications, precautionary statements as indicated in the Regulation (EC) No 1272/2008 have to be followed, and the additive should be handled accordingly. ³⁴

3.3.6. Safety for the environment

L. nobilis is a native species to the eastern Mediterranean and is widely cultivated in Europe for ornamental purposes. Use of the essential oil extracted from the plant in animal production is not expected to pose a risk for the environment.

3.4. Efficacy

Laurel bay (L. nobilis) and preparations derived from its leaves are listed in Fenaroli's Handbook of Flavour Ingredients (Burdock, 2009) and by FEMA with the reference number 2124 (laurel), 2613 (laurel bay leaves extract) and 2125 (laurel bay leaves oil).

Since laurel leaf oil is recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary.

4. Conclusions

Laurel leaf oil from L. nobilis may be produced from plants of different geographical origins and by various processes resulting in preparations with different composition and toxicological profiles. Thus, the following conclusions apply only to laurel leaf oil which contains ≤ 4% methyleugenol, ≤ 0.38% estragole and ≤ 0.011% elemicin and is produced by steam distillation of the leaves of L. nobilis.

Based on the magnitude of the MOET calculated considering the presence of methyleugenol, estragole and elemicin in the product at 4%, 0.4% and 0.01% and the conditions of use in the different species, the FEEDAP Panel concludes that:

The use of the additive at the proposed level of 2 mg/kg complete feed in dogs and cats is of low concern (MOET > 10,000). For other long‐living and reproductive animals (including those animals reared for laying/breeding/reproduction), the use of the additive at the proposed level of 10 mg/kg complete feed is considered of concern (MOET < 10,000). The Panel considers the use in water for drinking in dogs and cats of low concern provided that the total daily intake of the additive does not exceed the daily amount that is considered of low concern when consumed via feed.

For short‐living animals, the Panel has no safety concern when the additive is used at the maximum proposed use level of 10 mg/kg complete feed for turkeys for fattening, piglets and other growing Suidae, pigs for fattening, veal calves (milk replacer), cattle for fattening and other growing ruminants, horses and rabbits for meat production, salmonids and other fin fish; and at 8.5 mg/kg complete feed for chickens for fattening, other growing poultry species, and other minor species for fattening. For short‐living animals, the Panel has no safety concern for the use in water for drinking provided that the total daily intake of the additive does not exceed the daily amount that is considered of no concern when consumed via feed.

The use of laurel leaf oil up to the highest level in feed which is considered of no concern for target animals is also expected to be of no concern for consumers.

The essential oil under assessment should be considered as irritant to skin and eyes and a respiratory tract and as a skin sensitiser. Due to the high concentration of methyleugenol (≥ 1%), the additive is classified as suspected of causing genetic defects and of causing cancer and should be handled accordingly.

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

Since the leaves of L. nobilis and their preparations are recognised to flavour food and their function in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary.

5. Recommendations

The specification should ensure that the concentration of methyleugenol, estragole and elemicin in the additive should be as low as possible and should not exceed 4%, 0.4% and 0.01%, respectively.

6. Documentation provided to EFSA/Chronology

Date	Event
28/10/2010	Dossier received by EFSA. Botanically defined flavourings from Botanical Group 06 ‐ Laurales, Magnoliales, Piperales for all animal species and categories. Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG)
11/11/2010	Reception mandate from the European Commission
03/01/2011	Application validated by EFSA – Start of the scientific assessment
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
05/04/2011	Comments received from Member States
20/04/2012	Reception of supplementary information from the applicant
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
02/08/2013	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
18/12/2018	EFSA informed the applicant that the evaluation process restarted
07/02/2019	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
26/05/2020	Reception of supplementary information from the applicant (partial submission: laurel oil)
16/02/2022	The application was split and a new EFSA‐Q‐2022‐00107 was assigned to the preparation included in the present assessment.
31/08/2022	Reception of a second amendment of the Evaluation report of the European Union Reference Laboratory for Feed Additives related to nutmeg oil, laurel leaves oil, pepper oil black, cinnamon oil, cassia oil and pepper oleoresin black
23/01/2023	Scientific assessment re‐started for the preparation included in the present assessment
01/02/2023	Opinion adopted by the FEEDAP Panel on laurel leaf oil (EFSA‐Q‐2022‐00107). End of the Scientific assessment for the preparation included in the present assessment. The assessment of another preparation in BDG 06 is still ongoing

Abbreviations

ADI

acceptable daily intake

AFC

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

ANS

EFSA Scientific Panel on Additives and Nutrient Sources added to Food

BDG

botanically defined group

bw

body weight

CAS

Chemical Abstracts Service

CD

Commission Decision

CDG

chemically defined group

CEF

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

CHO

Chinese hamster ovary (cells)

CG

chemical group

CLP

Classification, labelling and packaging

CV

coefficient of variation

DM

dry matter

ECHA

European Chemicals Agency

EINECS

European Inventory of Existing Chemical Substances

EMA

European Medicines Agency

EURL

European Union Reference Laboratory

FAO

Food Agricultural Organization

FEEDAP

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

FFAC

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

FGE

food group evaluation

FLAVIS

The EU Flavour Information System

FL‐no

FLAVIS number

GC area

gas chromatographic peak area

GC‐FID

gas chromatography with flame ionisation detector

GC–MS

gas chromatography–mass spectrometry

HACCP

hazard analysis and critical control points

IUPAC

International Union of Pure and Applied Chemistry

JECFA

The Joint FAO/WHO Expert Committee on Food Additives

LOD

limit of detection

LOQ

limit of quantification

MW

molecular weight

NOAEL

no observed adverse effect level

NTP

National Toxicology Program

OECD

Organisation for Economic Co‐operation and Development

PBK

physiologically based kinetic (models)

SCE

Sister Chromatid Exchange

SCF

Scientific Committee on Food

TTC

threshold of toxicological concern

UF

uncertainty factor

WHO

World Health Organization

Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Bampidis V, Azimonti G, Bastos ML, 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 RE, Woutersen R, Brantom P, Chesson A, Schlatter J, Schrenk D, Westendorf J, Manini P, Pizzo F and Dusemund B, 2023. Scientific Opinion on the safety and efficacy of a feed additive consisting of an essential oil from the leaves of Laurus nobilis L. (laurel leaf oil) for all animal species (FEFANA asbl). EFSA Journal 2023;21(3):7875, 28 pp. 10.2903/j.efsa.2023.7875