Safety and efficacy of a feed additive consisting of cocoa absolute from the dried seeds of Theobroma cacao L. in propylene glycol for use in all animal species except cats (FEFANA asbl)

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of a preparation of cocoa absolute (obtained by ■■■■■ extraction from the dried seeds of Theobroma cacao L.) in propylene glycol when used as a sensory additive in feed and water for drinking for all animal species except cats. The additive (referred to as cocoa absolute preparation) is specified to have a dry matter content of ≤ 1%, and to contain ≤ 99.7% propylene glycol, 0.01%–0.04% theobromine and 0.004–0.04% caffeine. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive is safe for the target species at the maximum proposed use level of 115 mg/kg complete feed. The FEEDAP Panel considers that the use of the cocoa absolute preparation in water for drinking alone or in combination with its use in feed is safe provided that the daily amount resulting from the exposure via feed alone at the maximum use level is not exceeded. No safety concern would arise for the consumer and the environment from the use of the additive in animal feed. The additive under assessment should be considered as a dermal and respiratory sensitiser. Exposure of users by any route is considered a risk. Since the seeds of T. cacao are recognised to flavour food and their function in feed would be essentially the same as that in food, 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 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 four additives (althaea tincture, cocoa absolute, cocoa extract (solvent‐based, sb) and cocoa distillate (new additive)) belonging to botanically defined group (BDG) 14 – Malvales, when used as feed additives for all animal species (category: sensory additives; functional group: flavouring compounds). During the assessment, the applicant withdrew the applications for two additives: cocoa extract (sb) and cocoa distillate. The additive cocoa extract (sb) was deleted from the register of feed additives. ³ In addition, during the course of the assessment, the application was split and the present opinion covers only one out of the remaining two additives under application: ‘cocoa absolute’ from Theobroma cacao L. for use in all animal species. During the assessment, the applicant requested a change in the target animal species excluding cats from the application for authorisation. ⁴

The remaining additive belonging to botanically defined group (BDG) 14 – Malvales, under application (althea tincture) is assessed in a separate opinion.

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). The dossier was received on 02 March 2011 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2011‐00180. The particulars and documents in support of the application were considered valid by EFSA as of 06 February 2020.

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 a feed additive consisting of cocoa absolute, when used under the proposed conditions of use (see Section 3.3.3 ).

1.2. Additional information

Cocoa absolute from Theobroma cacao L. 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.

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 ⁵ in support of the authorisation request for the use of cocoa absolute from T. cacao as a feed additive.

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.

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the phytochemical marker in the additives. ⁶ The evaluation report is related to the methods of analysis for each feed additive included in BDG 14 – Malvales. In particular, for cocoa absolute, the EURL report recommended a method based on high‐performance liquid chromatography with ultraviolet detection (HPLC‐UV) for the determination of the phytochemical marker theobromine in cocoa absolute.

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of cocoa absolute from T. cacao is in line with the principles laid down in Regulation (EC) No 429/2008 ⁷ and the relevant guidance documents: Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements (EFSA Scientific Committee, 2009), Compendium of botanicals, ⁸ 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, 2024), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023), Guidance document on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals (EFSA Scientific Committee, 2019), Statement on the genotoxicity assessment of chemical mixtures (EFSA Scientific Committee, 2019) and Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment (EFSA Scientific Committee, 2019).

3. ASSESSMENT

The additive under assessment, ‘cocoa absolute in propylene glycol’ (referred to as cocoa absolute preparation), is obtained from the dried and roasted seeds of Theobroma cacao L. and is intended for use as a sensory additive (functional group: flavouring compounds) for use in feed and water for drinking for all animal species except cats.

3.1. Origin and extraction

Theobroma cacao L. (the cacao or cocoa tree) is a small (4–8 m) evergreen tree belonging to the family Malvaceae. It is thought to have originated in the Amazon basin and then spread into most other tropical regions of South America as a result of early human activity. Much later, as the commercial value of the tree was more widely recognised, it was introduced into other tropical regions, notably in Africa and S.E. Asia as a commodity crop. In the wild, the tree is typically found as part of the understory of tropical rainforests and other shaded high‐humidity areas.

The cacao tree is unusual as its flowers are produced in clusters directly from the trunk and larger branches. After fertilisation, these give rise to oval‐shaped fruits up to 30 cm in length and yellow to orange when ripe. The fruit is commonly referred to as the cocoa pod, although it is by definition a berry. Each berry contains 20–80 seeds embedded in a white mucilage or pulp. The seeds (beans or cocoa beans), after removal from the pods and following treatment by fermentation, roasting and grinding, provide the main ingredients for chocolate.

The additive is produced from the dried and roasted seeds of Theobroma cacao L. ■■■■■. It is produced by extraction of the seeds with ■■■■■. ■■■■■ The cocoa absolute is ■■■■■ then standardised with propylene glycol to obtain the final additive. ⁹

3.2. Uses other than feed flavourings

While there is no specific EU authorisation for any preparation from Theobroma cacao L. when used to provide flavour in food, according to Regulation (EC) No 1334/2008 ¹⁰ 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.’

Certain preparations of Theobroma cacao L. such as ‘cocoa barks’, ‘cocoa hulls’ and ‘cocoa bean meal, partially decorticated’ are listed as feed materials in Regulation (EU) No 68/2013.

3.3. Characterisation

3.3.1. Characterisation of the cocoa absolute preparation

The Chemical Abstract Service (CAS) number 84649‐99‐0 and the European Community (EC) number 283‐480‐6 refer to extractives and their derivatives obtained from T. cacao. ¹¹ The Council of Europe (CoE) number 452 covers any preparation of T. cacao.

The additive contains by specification ≤ 1% dry matter (DM), ¹² ≤ 99.7% propylene glycol, 0.010–0.04% theobromine (w/w, selected as a phytochemical marker) and 0.004%–0.04% caffeine (w/w).

The DM content in four batches of the additive was on average 0.24% (range: 0.11%–0.40%, w/w); the content of propylene glycol in five batches was 99.0% (89.9%–99.2%, w/w). ¹³ The FEEDAP Panel notes that DM consists mostly of inorganic material measured as ash and that the plant‐derived organic fraction is too dilute to allow a reliable quantification of sugars, lipids, proteins and fibre using the methods proposed by the applicant.

The fraction of secondary metabolites was characterised in four batches of the additive and the results are summarised in Table 1. Total phenolic compounds (on average 0.048% w/w) were determined using the Folin–Ciocalteu method with spectrophotometric measurement at 765 nm and are expressed as gallic acid equivalents (GAE). ¹⁴ After removal of tannins with polyvinylpolypyrrolidone, a second determination was carried out and the content of total tannins (on average 0.009% w/w) was calculated by difference. The FEEDAP Panel notes that, while the Folin–Ciocalteu method is a widely used method for the determination of total content of phenolic compounds in plant extracts, it does not give true concentrations for individual polyphenol types. This may lead to an overestimation of their concentrations in comparison to results obtained by HPLC depending on the substances of interest and the standard used (Everette et al., 2010; Samara et al., 2022).

TABLE 1.

Characterisation of the fraction of secondary metabolites of cocoa absolute preparation, based on the analysis of four batches (mean and range).

Constituent	CAS No	FLAVIS No	Mean	Range
			% (w/w)	% (w/w)
Total phenolic compounds (as GAE)	–	–	0.048	0.04–0.05
Total tannins (as GAE)	–	–	0.009	0.005–0.010
Methylxanthines (HPLC‐MS)
Theobromine 1	83‐67‐0	16.032	0.017	0.016–0.018
Caffeine	58–08‐2	16.016	0.016	0.015–0.017
Theophylline	58–55‐9	–	0.0004	0.0004
Total methylxanthines			0.0337 2	0.0314–0.0354 3
Isoquinoline alkaloids (GC–MS)
Salsolinol	525–72‐4		0.00018	0.0001–0.0002

Abbreviations: CAS No, Chemical Abstracts Service number; FLAVIS No, The EU Flavour Information System number; GAE: gallic acid equivalents; HPLC‐MS, high‐performance liquid chromatography‐mass spectrometry; GC‐MS, gas chromatography‐mass spectrometry.

¹ Substance for which a maximum limit in complete feed is set by Directive 2002/32/EC: 300 mg/kg with the exception of 200 mg/kg in complete feed for pigs and 50 mg/kg in complete feed for dogs, rabbits, horses and fur animals.

² The value given for the total (mean) is the mean of the sum of the constituents in the individual batches.

³ The values given for the total (range) are the lowest and the highest values of the sum of the constituents in the batches analysed.

Methylxanthines belonging to the class of purine alkaloids (theobromine, caffeine, theophylline) and pyrimidine alkaloids (trigonelline) were analysed by high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS) and isoquinoline alkaloids (salsolinol) by gas chromatography‐mass spectrometry (GC‐MS). Trigonelline was not detected (< 0.01 mg/kg).

The additive was shown to contain total phenolic compounds up to 0.05% (as % w/w) of the additive (corresponding to 23.5% of the DM) and including up to 0.01% tannins (5.9% of the DM), theobromine up to 0.018% (10.6% of the DM), caffeine up to 0.017% (10% of the DM), theophylline up to 0.0004% (0.24% of the DM) and salsolinol up to 0.0002% (0.12% of the DM).

The EFSA Compendium of botanicals ¹⁵ reports as substances of potential concern for human and animal health the occurrence of theobromine in the seeds of T. cacao.

The applicant carried out an extensive literature search on the chemical composition of T. cacao and its botanical preparations and the possible presence of substances of concern. ¹⁶ Several single and cumulative databases (including LIVIVO, OVID and PubMed) were searched. The keywords used covered different aspects of safety, and the inclusion and exclusion criteria were provided by the applicant. The literature search (no time limits specified) retrieved 21 studies investigating the composition of preparations from T. cacao. The results of the literature search confirmed the information in the EFSA Compendium. No other substances of concern were identified in the literature provided by the applicant.

The results of the literature search (see e.g. reviews by Baharum et al., 2016; Pereira‐Caro et al., 2013) indicated that cocoa seeds contain flavonol glycosides (quercetin, kaempferol), monomeric catechins (epicatechin, catechin; flavan‐3‐ols), oligomeric flavan‐3‐ols/procyanidins (dimers–decamers), anthocyanins (cyanidin‐3‐O‐arabinoside and cyanidin‐3‐O‐galactoside) and N‐phenylpropenoyl‐L‐amino acids, amide derivatives of phenolic acids with amino acids (e.g. N‐caffeoyl‐L‐aspartate, N‐coumaroyl‐L‐aspartate, N‐coumaroyl‐3‐hydroxytyrosine (clovamide), N‐coumaroyltyrosine (deoxyclovamide).

The data provided showed that the four batches analysed complied with the proposed specifications.

3.3.1.1. Impurities

The data provided by the applicant on the impurities and microbial contamination ¹⁷ of the additive are reported in Table 2.

TABLE 2.

Data on impurities and microbial contamination of cocoa absolute preparation.

Parameter	Analysis
	Range	# batches
Impurities
Lead (mg/kg)	< 0.05	3
Mercury (mg/kg)	< 0.005	3
Cadmium (mg/kg)	< 0.01	3
Arsenic (mg/kg)	< 0.10	3
Residual solvents
Methanol (mg/kg)	■■■■■	7
Ethanol (mg/kg)	■■■■■	7
PCDD/Fs and DL‐PCBs (upper bound) 1
PCDD/Fs (pg WHO2005‐TEQ/g)	0.0121–0.0140	3
DL‐PCBs (pg WHO2005‐TEQ/g)	0.0065–0.0075	3
PCDD/Fs + DL‐PCBs (pg WHO2005‐TEQ/g)	0.0186–0.0215	3
Non‐DL PCBs (ng/g)	0.0122–0.0133	3
Mycotoxins
Aflatoxins B1, B2, G1, G2 (μg/kg)	< 1	3
Aflatoxins, sum (μg/kg)	< 4	3
Pesticides (mg/kg) 2		3
Organochlorine pesticides and pyrethroids	< 0.005–< 0.2	3
Organophosphorus pesticides	< 0.01–< 0.05	3
Organonitrogen pesticides and others	< 0.01–< 0.02	3
Microbial contamination
Salmonella spp. (per 25 g)	Not detected	3
Enterobacteriaceae (CFU/mL)	< 10	3
Total yeasts (CFU/mL)	< 10	3
Total filamentous fungi (CFU/mL)	< 10	3
Total aerobic count (CFU/mL)	< 10	3

Note: <: means below the limit of quantification.

Abbreviations: PCBs, polychlorinated biphenyls; PCDDs, polychlorinated dibenzo‐p‐dioxins; PCDFs, polychlorinated dibenzofurans; TEQ, toxic equivalents for dioxins, furans and dioxin‐like PCBs established by WHO in 2005 (Van den Berg et al., 2006); WHO: World Health Organization; CFU: colony forming unit.

¹ Upper bound concentrations are calculated on the assumption that all values of the different congeners below the limit of quantification are equal to the limit of quantification.

² For pesticides, a multiresidue screening analysis was provided, with limit of quantification (LOQ) in a range 0.005–0.2 mg/kg.

The FEEDAP Panel considers that the level of microbial contamination and detected impurities do not raise safety concerns.

3.3.2. Shelf‐life

The applicant states that the typical shelf‐life of cocoa absolute preparation is at least 12 months, when stored in tightly closed containers under standard conditions. ¹⁸ However, no data supporting this statement were provided.

3.3.3. Conditions of use

The additive is intended for use in feed and in water for drinking for all animal species except cats at a maximum use level of 115 mg/kg complete feed.

3.4. Safety

No studies to support the safety for target animals, consumers or users were performed with the additive under assessment.

The additive mainly consists of propylene glycol (synonyms: 1,2‐propandiol, propane‐1,2‐diol), which is an authorised feed material (number 13.11.1). ¹⁹ Propylene glycol (E 1520) is an authorised food additive in the EU according to Annex III of Regulation (EC) No 1333/2008.

Among the secondary metabolites identified in cocoa absolute preparation, up to 0.05% (w/w) consists of phenolic compounds including 0.01% tannins. The individual components present in the phenolic fraction were not identified or quantified. According to the literature provided, the phenolic fraction contains compounds such as flavonol glycosides, catechins, procyanidins and anthocyanins, which are widely distributed in fruits and berries. N‐phenylpropenoyl‐L‐amino acids are amide derivatives of ubiquitous phenolic acids (caffeic acid, p‐coumaric acid) with amino acids. If present in the additive, these compounds would be at very low concentrations and are not further addressed.

The additive contains theobromine (EU Flavour Information System (FLAVIS) number [16.032]) and caffeine [16.016] which have already been assessed as chemically defined flavourings for use in food by the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) in Chemical Group 30 (Miscellaneous substances) ²⁰ (EFSA CEF Panel, 2017). Both components are considered safe for use as food flavourings. They are currently authorised for use in food ²¹ with restrictions. ²²

The safety of caffeine has also been evaluated by the EFSA Panel on Dietetic Products, Nutrition and Allergies NDA Panel (EFSA NDA Panel, 2015). The Panel on Contaminants in the Food Chain (CONTAM) issued an opinion on theobromine as an undesirable substance in animal feed (EFSA, 2008).

The other two identified compounds, theophylline and salsolinol, have not been evaluated for use in food or feed. Theophylline is structurally related to caffeine, of which it is a minor metabolite.

The following sections focus on the absorption, distribution, metabolism and excretion (ADME) and toxicology of propylene glycol, of the methylxanthines (caffeine, theobromine and theophylline), and of salsolinol, based on the evidence provided by the applicant in the form of literature searches and quantitative structure–activity relationship (QSAR) analyses. For the ADME and toxicology of propylene glycol and of the methylxanthines caffeine and theobromine, reference is made to previous EFSA assessments.

3.4.1. Absorption, distribution, metabolism and excretion

Propylene glycol

Propylene glycol (propane‐1,2‐diol), which is the major component of the additive (≤ 99.7%), is readily absorbed from the gastrointestinal tract and is expected to be widely distributed to organs and tissues. A portion of the ingested propylene glycol (about one third) is excreted via the kidney as the parent compound or its glucuronidated derivatives; the rest undergoes oxidation to form mainly lactic acid and pyruvic acid. No major differences in ADME seem to occur between humans, experimental animals and target species (Dalefield, 2004; EFSA ANS Panel, 2018).

Methylxanthines

As described in the opinion of the EFSA CEF Panel on xanthine alkaloids (EFSA CEF Panel, 2017) ‘The methylxanthines caffeine and theobromine are rapidly absorbed after oral intake, show little first pass metabolism in gut or liver and are almost completely excreted as such or as metabolites in urine. Their elimination half‐life from plasma is of the order of 4–12 h. Both substances are distributed over total body water, cross placenta and blood–brain barrier. A very complex mixture of metabolites is generated in the liver by N‐demethylation, 8‐hydroxylation and N‐acetylation. Theobromine and theophylline are intermediary metabolites of caffeine, which are further metabolised. Cytochrome P450 1A2 plays a major role in the N‐demethylation reactions, while 3A4 catalyses 8‐hydroxylation. N‐Acetyltransferases catalyse the formation of acetylated metabolites. Xanthine oxidase also contributes to the formation of the oxidative metabolites of caffeine. Several of these enzymes show polymorphism in the human population, leading to variation in the elimination rate of the caffeine and theobromine and their metabolites. Interaction of methylxanthine metabolites with the food matrix as well as with drugs and other exogenous factors also results in variation in the rates of elimination of these substances’ (EFSA CEF Panel, 2017).

The toxicokinetics of theobromine in humans and several animal species (rats, mice, dogs, rabbits, horses, sheep) have been described in the opinion of the EFSA CONTAM Panel (EFSA, 2008). In all species, theobromine is well absorbed (> 90%) with a bioavailability close to unity and is widely distributed in the body. It is rapidly metabolised and is excreted mainly in urine as unchanged theobromine and metabolites. Excretion patterns of theobromine and its metabolites were qualitatively comparable among species, indicating that theobromine is metabolised via similar pathways, although considerable quantitative intra‐ and interspecies differences occur in the relative contribution of individual pathways, and in the proportion of unchanged theobromine excreted. There are no data indicating accumulation of theobromine in tissues. In dogs, a comparatively large fraction of the administered dose is excreted unchanged. Genetic variation in CYP1A2 has been demonstrated in several dog breeds and proposed as one factor that may contribute to variability in the metabolism and increased sensitivity to theobromine, but the molecular basis of the particular sensitivity of dogs has not been fully established. Dogs and other canids also lack the genes encoding for cytosolic arylamine N‐acetyltransferases (i.e. NAT1 and NAT2), although the relevance of this deficiency to the disposition and toxicity of theobromine has not been established (EFSA, 2008).

Salsolinol

The applicant provided in silico predictions of the ADME of salsolinol using freely available computational tools (SwissADME, pkCSM and XenoSite). Salsolinol is predicted to have low lipophilicity and moderate polarity. Overall, in silico ADME, toxicokinetic and metabolism predictions indicate that salsolinol is not expected to bioaccumulate in animal tissues under conditions of repeated exposure. The compound's physicochemical and metabolic characteristics support efficient biotransformation and elimination.

3.4.2. Toxicology

3.4.2.1. Genotoxicity

Propylene glycol does not raise concern with respect to genotoxicity (EFSA ANS Panel, 2018).

The EFSA CEF Panel evaluated the genotoxicity data available for caffeine and concluded that caffeine and its metabolite theobromine are not of concern for genotoxicity when used as flavouring substances (EFSA CEF Panel, 2017).

Theophylline is a metabolite of caffeine and is structurally related to theobromine. The genotoxic potential of theophylline and salsolinol was predicted using the Organisation for Economic Co‐operation and Development (OECD) QSAR Toolbox ²³ and the compounds profiled for structural alerts for genetic toxicity. Some alerts were raised for both compounds, and these were further investigated using read‐across and subcategorisation. Predictions of mutagenicity by Ames test (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). Read‐across‐based predictions were found consistently negative for all categories of analogues. On this basis, the alerts raised were discounted by the FEEDAP Panel.

3.4.2.2. Toxicity data

Propylene glycol

The toxicological data set on propylene glycol has been reviewed by the EFSA ANS Panel (2018). Except for haematological changes suggestive of an increased red blood cell destruction with a compensatory increased rate of haematopoiesis at the highest dose level (5000 mg/kg bw per day), no adverse effects were observed in a 2‐year study in dogs administered propylene glycol in the diet (2000 or 5000 mg/kg bw per day). From this study, the ANS Panel identified 2000 mg/kg bw per day as the no observed adverse effect level (NOAEL). The FEEDAP Panel agrees with the conclusion of the ANS Panel.

No adverse effects, including neoplastic findings, were reported in a chronic study in rats exposed to propylene glycol in the diet (up to 2500 mg/kg bw per day) for 2 years. The Scientific Committee on Food (EC (European Commission), 1996) and JECFA used this study in rats to derive a NOAEL of 2500 mg/kg bw per day and an acceptable daily intake (ADI) of 25 mg/kg bw per day using an uncertainty factor of 100. No adverse effects were observed in the available reproductive and developmental toxicity studies (as described in EFSA ANS Panel, 2018). Considering the toxicity database, the ANS Panel concluded that there was no reason to revise the current ADI of 25 mg/kg bw per day.

Methylxanthines

The toxicological data set available for theobromine has been reviewed by the CONTAM Panel, with a focus on reproductive and developmental toxicity in rodents and dogs (EFSA, 2008). The CONTAM Panel concluded that ‘Target organs of theobromine toxicity in rodents are the testes (Sertoli cells) and the thymus. Dogs showed also cardiomyopathy upon prolonged exposure. Theobromine causes reproductive toxicity and incomplete ossification in mice, and variation in skeletal development in the off‐spring of rabbits. In rats, the NOAEL for testicular toxicity was identified at 150 mg/kg bw and in rabbits the NOAEL for variation in skeletal development in the off‐spring was 21 mg/kg bw’.

The opinion of the CONTAM Panel reviewed adverse effects reported in feeding studies with animals fed on cocoa products (EFSA, 2008). The main outcome is summarised as follows:

• ○In dairy cows, exposure to theobromine at approximately 15 mg/kg bw per day reduced milk yield and increased milk fat. In calves, dietary exposure to 45–90 mg/kg bw per day for several weeks caused hyperexcitability, sweating and increased respiratory and heart rates.
• ○In goats fed a diet containing up to 50% of a cocoa product for 56 days, reduced dry matter intake and body weight gain occurred at approximately 300 mg theobromine/kg bw per day. In lambs fed diets containing 0–0.20% theobromine for 3 months, feed intake and weight gain were improved up to 0.10% (corresponding to approximately 35 mg/kg bw per day) and were reduced at higher concentrations.
• ○In horses, which are particularly susceptible to theobromine, a single dose of 0.29 g of theobromine (estimated to be about 0.5 mg/kg bw) did not cause any clinical or biochemical effects whereas a single dose of 1.16 g theobromine (corresponding to 2 mg/kg bw) produced some biochemical effects.
• ○Pigs fed with cocoa meal for 126 days showed growth retardation, diarrhoea and lethargy at approximately 24 mg theobromine/kg bw per day. In growing pigs fed a by‐product of milk chocolate production at 5% or 10% dietary level for 2–5 weeks, no effects on performance were observed at 5% (corresponding to 7 mg theobromine/kg bw per day) whereas reduced performance was observed at 10%.
• ○In feeding studies in young chickens, no effects were seen between 26 and 110 mg/kg bw per day, with depression of feed intake and weight gain at higher doses. In older broiler chickens, effects were observed at 95 mg/kg bw per day. In laying hens, theobromine exposure at 66 mg/kg bw per day caused liver and kidney toxicity, depressed weight gain and egg production.
• ○For dogs, acute fatal intoxication seems to occur after single ingestion of theobromine at 80–300 mg/kg bw per day. In another study, dogs tolerated up to 50 mg/kg bw per day for 1 year. Mild signs of intoxication in dogs (vomiting, diarrhoea, polydipsia) were reported in a veterinary manual after ingestion of methylxanthines at 20 mg/kg bw per day, suggesting that the margin between the acute fatal dose and the no adverse effect level in dogs is low.
• ○In studies on fish, reduced weight gain was observed in tilapia fed defatted cocoa meal with a theobromine content corresponding to 500–750 mg/kg bw per day.

The FEEDAP Panel notes that the feeding studies in farm animals were designed to assess performance rather than toxicological endpoints and showed major limitations in the study design (short or unclear duration, unspecified number of animals, approximate estimate of theobromine content in the test item, etc.). In particular, the data set available for horses is based on observations on individual animals given theobromine on single or repeated occasions (2 or 3). Therefore, these studies are not suitable to identify a reference point for theobromine, from which to derive safe concentrations in feed for all animal species. However, they showed interspecies variation in the sensitivity of animals to theobromine.

The results of studies on reproduction and developmental toxicity with theobromine and caffeine in laboratory animals were reported in the opinion of the EFSA CEF Panel on xanthine alkaloids (EFSA CEF Panel, 2017). For theobromine, the NOAEL for delay in skeletal development of the offspring was 48 and 21 mg/kg bw per day in rats and rabbits, respectively. Similarly, studies on reproductive effects of caffeine in rodents indicated NOAELs of approximately 30 mg/kg bw per day for developmental toxicity and 40–100 mg/kg bw per day for teratogenicity.

3.4.2.3. Human studies

According to the NDA Panel, an intake of up to 400 mg/person per day (5.7 mg/kg bw per day) is safe for healthy adults except pregnant and lactating women. For pregnant and lactating women, an intake of 200 mg/day (about 3 mg/kg bw per day) does not raise safety concerns for the fetus and breastfed infant. The NDA Panel concluded that an (acute) intake of 3 mg/kg bw per day may serve as a basis to derive daily caffeine intakes of no concern for children and adolescents (EFSA NDA Panel, 2015).

For the safety assessment of caffeine as food flavouring, the EFSA CEF Panel based its assessment on the safety threshold of 5.7 mg/kg bw per day for adults, except pregnant/lactating women, and 3 mg/kg bw per day for children, adolescents, pregnant and lactating women, as established by the NDA Panel. The safety evaluation of theobromine considered that approximately 11% of an oral dose of caffeine is metabolised to theobromine and that both substances have a similar pharmacological profile (EFSA CEF Panel, 2017).

3.4.2.4. Conclusions

None of the identified components present in the additive raised a concern for genotoxicity.

For the assessment of the safety for the target species, the FEEDAP Panel selects the following reference points: (i) for propylene glycol, the NOAEL of 2000 mg/kg bw per day from the 2‐year study in dogs; (ii) for caffeine, the lowest NOAEL of 30 mg/kg bw per day for developmental toxicity in rodents; (iii) for theobromine, the lowest NOAEL of 21 mg/kg bw per day identified from a developmental toxicity study in rabbits based on variations in skeletal development.

Toxicological data, from which a reference point could be identified, were not available for theophylline and salsolinol.

3.4.3. Safety for the target species

Tolerance studies with the target species and/or toxicological studies in laboratory animals with the additive under application were not submitted. In the absence of these data, the approach to the safety assessment of the mixture is based on its individual components (when individually identified and quantified) or groups of components (assessment groups). The combined toxicity can be predicted using the dose addition assumption within an assessment group (EFSA Scientific Committee, 2009).

The safety assessment is based on the individual compounds identified in cocoa absolute preparation, the methylxanthines caffeine, theobromine and theophylline and the isoquinoline alkaloid salsolinol. For dogs, since haematological changes were observed at the highest dose level in a 2‐year dog study, the safety assessment considers also the presence of propylene glycol, which is assessed separately.

For caffeine [16.016], the lowest NOAEL of 30 mg/kg bw per day identified from developmental toxicity studies in rodents is applied (EFSA CEF Panel, 2017).

For theobromine [16.032], a NOAEL of 21 mg/kg bw per day was identified from a developmental toxicity study in rabbits (EFSA, 2008) and divided by a factor of 2 to consider the nature of the study.

The lowest NOAEL of theobromine was extrapolated to theophylline by applying read‐across.

For the assessment of salsolinol, in the absence of toxicity data, the threshold of toxicological concern (TTC) was applied and the compound was allocated to Cramer class III.

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

TABLE 3.

Compositional data, intake values (calculated for chickens for fattening at 115 mg/kg complete feed), reference points, margin of exposure (MOE) for the individual components of cocoa absolute preparation classified according to assessment groups and combined margin of exposure (MOET) for each assessment group.

Additive composition		Exposure		Hazard characterisation		Risk characterisation
Constituent	Highest concentration in the additive	Highest concentration in feed	Daily Intake 1	Cramer Class 2	NOAEL 3	MOE 4	MOET 5
	%	mg/kg	mg/kg bw/day	–	mg/kg bw/day	–
Assessment group
Methylxanthines
Theobromine	0.04	0.0400	0.00413	(III)	10.5 6	2543
Caffeine	0.04	0.0400	0.00413	(III)	30	7265
Theophylline	0.0004	0.0004	0.00004	(III)	10.5	254,265
MOET methylxanthines							1870
Isoquinoline alkaloids
Salsolinol	0.0002	0.00023	0.00002	III	0.15	7265

Abbreviations: bw, body weight; MOE, margin of exposure; MOET: combined (total) margin of exposure; NOAEL, no observed adverse effect level.

¹ Rounded intake values.

² When a NOAEL value is available or read‐across is applied, the allocation to the Cramer Class is put into parentheses.

³ Values in bold correspond to the NOAEL of the respective components, 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.

⁴ The MOE for each component is calculated as the ratio of the reference point (no observed adverse effect level, NOAEL) to the intake (non‐rounded values are used in the calculations).

⁵ The combined margin of exposure (MOET) is calculated for each assessment group as the reciprocal of the sum of the reciprocals of the MOE for the individual substances. MOET values are reported in bold.

⁶ A factor of 2 was applied to the NOAEL of 21 mg/kg bw per day for theobromine because of the nature of the study.

For each component in the assessment group, exposure in target animals (expressed as mg/kg bw per day) was estimated considering the maximum proposed use level, the percentage of the component in the cocoa absolute preparation and the default values for body weight and feed intake according to the guidance on the safety of feed additives for target species (EFSA FEEDAP Panel, 2017b). For those compounds covered by specifications (theobromine and caffeine, see Section 3.3.1), the indicated maximum specified concentration is used for the calculation of exposure. For the other components, the highest analysed concentration in the additive is used.

For risk characterisation, the margin of exposure (MOE) was calculated for each component as the ratio between the reference point and the exposure. For an assessment group, (i) when a group reference point is available, a group MOE was calculated for the combined intake; (ii) when different reference points are available for the components, the combined (total) margin of exposure (MOET) was calculated as the reciprocal of the sum of the reciprocals of the MOE for the individual substances (EFSA Scientific Committee, 2019). An MOE ≥100 allowed for interspecies differences and intra‐individual variability.

The approach to the safety assessment of cocoa absolute preparation is shown in Table 3 for chickens for fattening, the species with the highest ratio of feed intake/body weight ²⁴ and which represents the worst‐case scenario among the target species. The calculations were done at the maximum proposed use level of 115 mg/kg complete feed.

As shown in Table 3, the MOE(T) calculated for chickens for fattening at the maximum proposed use level of the additive in feed of 115 mg/kg complete feed was > 100 for all assessment groups. A MOET of 1870 was calculated for the assessment group methylxanthines. The corresponding MOET values calculated for the other target species at the proposed use levels in complete feed are summarised in Table 4.

TABLE 4.

Combined margin of exposure (MOET) for the assessment group methylxanthines calculated for the different target animal species and categories at the maximum proposed use level of 115 mg/kg complete feed. ¹

Animal species/category	Daily feed intake (g DM/kg bw)	Lowest MOET methylxanthines
Chickens for fattening	79	1870
Laying hens	53	2787
Turkeys for fattening	59	2504
Pigs for fattening	44	3358
Piglets	37	3993
Sows	30	4924
Veal calves (milk replacer)	19	8350
Cattle for fattening	20	7387
Dairy cows	31	4765
Sheep/goats	20	7387
Horses	20	7387
Rabbits	50	2955
Salmonids	18	8207
Dogs	17	8690
Ornamental fish	5	29,546

Abbreviations: bw, body weight; DM, dry matter; MOET, combined margin of exposure.

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

At the proposed use levels in complete feed, the MOET exceeds the value of 100 for all animal species. The high MOET for methylxanthines is considered adequate to account for potential interspecies variation (see Section 3.4.2.2.), in particular the specific sensitivity described for horses (MOET 7387) and dogs (MOET 8690). Exposure to methylxanthines from the additive is several 100‐fold below levels associated with toxic effects in horses and dogs (Albretsen, 2004) The maximum proposed use level of 115 mg/kg complete feed is considered safe for all the species and categories included in Table 4. This level is extrapolated to physiologically related species and extended to the other species not considered in the table.

3.4.3.1. Safety assessment of propylene glycol for dogs

Since adverse effects were observed in dogs exposed to propylene glycol (see Section 3.4.2.2), the FEEDAP Panel assessed its safety for this animal species. At the proposed use level in feed of 115 mg/kg complete feed, the concentration of propylene glycol in feed would be 114 mg/kg and the intake by dogs 2.2 mg/kg bw per day. Applying an uncertainty factor (UF) of 10 to the NOAEL of 2000 mg/kg bw per day identified from the 2‐year study in dogs, the maximum calculated safe concentration in feed for dogs would be 10,560 mg/kg. Therefore, there is no safety concern for dogs related to the presence of propylene glycol in the additive at the proposed use level in feed.

3.4.3.2. Conclusions on safety for the target species

The FEEDAP Panel considers that the additive is safe for the target species at the maximum proposed use level of 115 mg/kg complete feed.

The FEEDAP Panel considers that the use of cocoa absolute preparation in water for drinking alone or in combination with its use in feed is safe provided that the daily amount resulting from the exposure via feed alone at the maximum use level is not exceeded.

3.4.4. Safety for the consumer

Seeds of cacao (Theobroma cacao L.) and their preparations are added to a wide range of food categories for flavouring purposes (Burdock, 2009). The council of Europe (2008) reports on the use of seeds (beans) and nibs of Theobroma cacao L. as cacao powder, cacao butter and cacao extract for chocolate, baked goods, desserts, ice cream, non‐alcoholic and alcoholic beverages.

As described in the opinion of the EFSA CEF Panel on xanthine alkaloids (EFSA CEF Panel, 2017), daily exposure to caffeine and theobromine across all population groups was in the range of 0–2.3 and 0–0.5 mg/kg bw per day, respectively, when added as a chemically defined flavouring substance in food. When considering the exposure from all dietary sources, the corresponding figures were 0–10 and 0.1–7.4 mg/kg bw per day. The same figures on the daily intake of caffeine are reported in the opinion of the NDA Panel (EFSA NDA Panel, 2015).

Consumers are directly exposed to propylene glycol from its use as a food additive (EFSA ANS Panel, 2017). The mean exposure to propylene glycol was estimated in the range of 0.2–11.6 mg/kg bw per day and the high exposure was 0.4–26.4 mg/kg bw per day (95th percentile, brand‐loyal scenario).

No data on residues in products of animal origin were made available for any of the constituents of the additive. When considering the ADME of the individual components (see Section 3.4.1), methylxanthines will be readily metabolised and excreted and are not expected to accumulate in animal tissues and products. Similarly, propylene glycol is mainly metabolised to lactic acid and pyruvic acid and is not expected to accumulate in animal tissues and products.

Considering the above and the reported human exposure due to the direct use of cocoa products, it is unlikely that consumption of products from animals given the cocoa absolute preparation in the diet at the maximum proposed use level would significantly increase human background exposure.

No safety concern would be expected for the consumer from the use of the additive up to the maximum proposed use level in feed.

3.4.5. Safety for the user

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

The applicant provided information according to Classification, Labelling and Packaging (CLP) Regulation (EC) 1272/2008 ²⁵ concerning the presence of propylene glycol in the additive. ²⁶ The European Chemicals Agency (ECHA) Risk Assessment Committee (RAC) concluded that propylene glycol does not warrant classification as hazardous (ECHA, 2016).

The applicant made a literature search aimed at retrieving studies related to the safety of preparations obtained from T. cacao for users. ²⁷ The literature search retrieved a publication which was considered relevant for the safety assessment.

Effects relevant to sensitisation were investigated among confectionery workers exposed to cocoa (n = 40) and flour (n = 53) dust compared with unexposed controls (n = 63). The percentage of positive skin‐prick tests for cocoa (60.2%) and flour (25.8%) was higher among the 93 exposed workers than in control workers (4.6% for cocoa and 12.3% for flour, p < 0.01). In workers exposed to cocoa, there was evidence of sensitisation from positive skin‐prick tests (29/40, 72.5%) with increased IgE levels in 7/40, whereas controls showed no increase in IgE (Zuskin et al., 1998). The results of this study indicate potential sensitising properties for cocoa.

The additive under assessment should be considered as a dermal and respiratory sensitiser. Exposure of users by any route is considered a risk.

3.4.6. Safety for the environment

T. cacao is not a species native to Europe. Therefore, the safety for the environment is assessed based on the individual components of the additive.

Propylene glycol, the main component of the additive, is metabolised to lactic acid and pyruvic acid, which are endogenous metabolites and naturally occurring substances present in food and feed.

Phenolic compounds including tannins of cocoa absolute preparation are present in many plants indigenous to Europe and are therefore considered safe for the environment.

Caffeine, theobromine, theophylline and salsolinol present in the additive have not been evaluated by EFSA with respect to its safety for the environment. However, based on low concentration in complete feed resulting from the use of the additive at the proposed conditions of use (≤ 0.041 mg/kg for caffeine and theobromine, < 0.00004 mg/kg for theophylline and < 0.00002 mg/kg for salsolinol) and the expected metabolism in the target animals, they are not expected to raise a concern to the environment.

Considering the above, the use of the cocoa absolute preparation under the proposed conditions of use in animal feed is not expected to pose a risk to the environment.

3.5. Efficacy

Seeds of cacao (Theobroma cacao L.) and their preparations are listed in Fenaroli's Handbook of Flavour Ingredients (Burdock, 2009) and by the Flavour and Extract Manufactures Association (FEMA) without a reference number.

Since the seeds of T. cacao 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.

4. CONCLUSIONS

The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive under assessment (cocoa absolute preparation) is safe up to the maximum proposed use level of 115 mg/kg for the target species.

The FEEDAP Panel considers that the use of cocoa absolute preparation in water for drinking alone or in combination with its use in feed is safe provided that the daily amount resulting from the exposure via feed alone at the maximum use level is not exceeded.

The use of the additive at the proposed conditions of use is considered safe for the consumers and the environment.

The additive under assessment should be considered as a dermal and respiratory sensitiser. Exposure of users by any route is considered a risk.

Since the seeds of Theobroma cacao L. 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. DOCUMENTATION PROVIDED TO EFSA/CHRONOLOGY

Date	Event
02/03/2011	Dossier received by EFSA. Botanically defined flavourings from Botanical Group 14 – Malvales for all animal species and categories. Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG)
24/02/2011	Reception mandate from the European Commission
26/02/2013	EFSA informed the applicant (EFSA ref. 7,150,727) 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
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
06/02/2020	Application validated by EFSA – Start of the scientific assessment
28/02/2020	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
14/07/2020	Reception of the Evaluation report of the European Union Reference Laboratory for Feed Additives. BDG 14 – Malvales: Althaea tincture, cocoa extract (sb), cocoa absolute
16/12/2024	Partial withdrawal of the application for the following additives: cocoa extract (sb) and cocoa distillate
02/02/2026	Reception of supplementary information from the applicant (partial dataset: cocoa absolute) – Scientific assessment remains suspended
31/03/2026	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Issues: characterisation, methods of analysis (cocoa absolute)
27/04/2026	The application was split and the original EFSA‐Q‐2011‐00180 remained associated to the additive included in the present assessment. Scientific assessment remains suspended
22/05/2026	Reception of an amendment of the Evaluation report of the European Union Reference Laboratory for Feed Additives related to althaea tinctures
30/06/2026	Reception of supplementary information from the applicant (partial dataset: cocoa absolute) – Scientific assessment restarted
09/09/2026	Opinion adopted by the FEEDAP Panel on cocoa absolute (EFSA‐Q‐2011‐00180). End of the Scientific assessment for the additive included in the present assessment. The assessment of another additive in BGD 14 is still ongoing

ABBREVIATIONS

ANS

EFSA Scientific Panel on Additives and Nutrient Sources added to Food

BW

body weight

CAS

Chemical Abstracts Service

CEF

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

CFU

colony‐forming unit

DM

dry matter

ECHA

European Chemicals Agency

EURL

European Union Reference Laboratory

FEEDAP

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

FLAVIS

The EU Flavour Information System

FL‐no

FLAVIS number

GC‐MS

gas chromatography–mass spectrometry

HPLC‐MS

high‐performance liquid chromatography‐mass spectrometry

HPLC‐UV

high‐performance liquid chromatography with ultraviolet detection

LOQ

limit of quantification

MOE

margin of exposure

MOET

combined (total) margin of exposure

MRL

maximum residue limit

MW

molecular weight

NDA

EFSA Panel on Dietetic Products, Nutrition and Allergies

nDL‐PCBs

non‐dioxin‐like PCBs

NOAEL

no observed adverse effect level

NTP

National Toxicology Program

OECD

Organisation for Economic Co‐operation and Development

PCBs

polychlorinated biphenyls

PCDDs

polychlorinated dibenzo‐p‐dioxins

PCDFs

polychlorinated dibenzofurans

RAC

Risk Assessment Committee

TEQ

toxic equivalent

TTC

threshold of toxicological concern

WHO

World Health Organization

AUTHOR CONTRIBUTIONS

Under Regulation 178/2002, the Scientific Panel is the author of the scientific output. Contributions include Working Groups who provide the draft assessment which is reviewed and adopted by the Panel; EFSA staff members who provide scientific support including inter alia data collection and analysis and literature searching; and other contributors such as staff of national food safety agencies, EFSA trainees and interim staff members who have contributed meaningfully to the work.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2011‐00180

COPYRIGHT FOR NON‐EFSA CONTENT

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LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by the European Commission. The full output has been shared with the European Commission, EU Member States and the applicant. The blackening will be subject to review once the decision on the confidentiality requests is adopted by the European Commission.

EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Villa, R. E. , Azimonti, G. , Bonos, E. , Christensen, H. , Durjava, M. , Dusemund, B. , Gehring, R. , Glandorf, B. , Kouba, M. , López‐Alonso, M. , Marcon, F. , Martelli, G. , Nebbia, C. , Pechová, A. , Prieto‐Maradona, M. , Theodoridou, K. , Bastos, M. L. , Benfenati, E. , … Manini, P. (2026). Safety and efficacy of a feed additive consisting of cocoa absolute from the dried seeds of Theobroma cacao L. in propylene glycol for use in all animal species except cats (FEFANA asbl). EFSA Journal, 24(10), e10355. 10.2903/j.efsa.2026.10355