Safety and efficacy of a feed additive consisting of a proanthocyanidin‐rich dry extract from the fruit of Vaccinium macrocarpon Aiton (cranberry extract) for dogs and cats (ACEL pharma S.r.l.)

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a proanthocyanidin‐rich dry extract obtained from the fruit of Vaccinium macrocarpon Aiton (cranberry extract) when used as a sensory feed additive for dogs and cats. The additive is not currently authorised for use in feed. The FEEDAP Panel concluded that cranberry extract is safe for dogs at 610 mg/kg complete feed and for cats at 155 mg/kg complete feed. Regarding user safety, the additive is not irritant to skin or eyes but is a potential skin and respiratory sensitiser. Exposure of users by inhalation and dermal routes is considered a risk. The Panel concluded that the additive has the potential to be efficacious as a sensory additive (flavouring agent, palatability enhancer) in dogs and cats when added to feed at 122 and 155 mg/kg complete feed, respectively.

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.

The European Commission received a request from ACEL pharma S.r.l. ² for the authorisation of the additive consisting of cranberry extract from Vaccinium macrocarpon when used as a feed additive for dogs and cats (category: sensory additives; functional group: flavouring compounds).

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). The dossier was received on 06 September 2023 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00585. The particulars and documents in support of the application were considered valid by EFSA as of 11 June 2024.

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 feed additive consisting of cranberry extract from Vaccinium macrocarpon Aiton when used under the proposed conditions of use (see Section 3.3.2 ).

1.2. Additional information

The additive consisting of cranberry extract from V. macrocarpon has not been previously authorised as a feed additive in the European Union.

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 ³ in support of the authorisation request for the use of cranberry extract from V. macrocarpon as a feed additive.

In accordance with Article 38 of the Regulation (EC) No 178/2002 ⁴ and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality, ⁵ a non‐confidential version of the dossier has been published on Open.EFSA.

According to Article 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 6 February to 27 February 2025 for which no comments were received.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 3 July 2024 to 3 October 2024; the comments received were considered for the assessment.

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 proanthocyanidins (PAC) in the feed additive. ⁶

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of cranberry extract from V. macrocarpon 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), 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 document on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals (EFSA Scientific Committee, 2019a), Statement on the genotoxicity assessment of chemical mixtures (EFSA Scientific Committee, 2019b), Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment (EFSA Scientific Committee, 2019c), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024a).

3. ASSESSMENT

The additive under assessment, cranberry extract, is a proanthocyanidin‐enriched extract obtained from the fresh fruit of V. macrocarpon Aiton and is intended for use as a sensory additive (functional group: flavouring compounds) in feed for dogs and cats.

3.1. Origin and extraction

V. macrocarpon Aiton, commonly called Cranberry or American Cranberry, is a perennial low growing or trailing shrub belonging to the family Ericaceae. It is native to the central and eastern parts of Canada and the north‐eastern parts of the USA and has been subsequently introduced into Europe. Its white/pink flowers are borne on short upright branches from which the characteristic edible red fruit (cranberries) develop. In the wild, V. macrocarpon may be confused with Vaccinium oxycoccus L. also called Cranberry or small Cranberry, which is native to all temperate regions of the world and has similar growth characteristics including the production of edible red fruit. However, because of the greater size of its fruit, commercial production of Cranberry products is almost exclusively dependent on V. macrocarpon. The term ‘cranberry’ is used to describe both, the plant and the fruit.

The additive (cranberry extract) is manufactured from concentrated cranberry juice obtained from the fresh fruit of V. macrocarpon. ⁸ After cleaning and milling, the fruit is treated enzymatically with pectin esterase to facilitate maceration. The macerated fruit is then pasteurised, and the cranberry juice is extracted by pressing and then concentrated by evaporation. The concentrated juice is diluted with water and passed through a food grade resin until no colour is detected in the eluate. The retained material is then eluted from the resin with ethanol/water (70/30, v/v). The eluate is concentrated by vacuum evaporation, spray dried and finally homogenised to produce the additive under application. Carriers are not used in the manufacturing process. ⁹

3.2. Uses other than feed flavouring

While there is no specific EU authorisation for any V. macrocarpon Aiton preparation 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’.

The European Medicines Agency (EMA) issued a monograph and an assessment report for V. macrocarpon Aiton, fructus (EMA, 2022a, 2022b) for medicinal use.

3.3. Characterisation

3.3.1. Characterisation of cranberry extract

The cranberry (V. macrocarpon) extract under assessment is a dark‐red fine powder with characteristic odour and flavour. It is soluble in water (50 g/L) and partially soluble in methanol. ¹¹

The product contains by specification: 25.0%–35.0% proanthocyanidins (PACs, as determined with the 4‐dimethylaminocinnamaldehyde (DMAC) colourimetric method), ≤ 5.0% water and ≤ 6.0% crude ash.

Table 1 summarises the results of the proximate analysis of five batches of the cranberry extract expressed as % of the additive (w/w). ¹² Water represents on average 3.0% of the additive, leaving a dry matter (DM) content of 97% (w/w).

TABLE 1.

Proximate analysis of cranberry extract, based on the analysis of five batches.

Constituent	Method	Mean	Range
		% (w/w)	% (w/w)
Dry matter		97.0	96.1–97.9
Ash	Gravimetric (PhEur 2.4.16)	0.14	0.1–0.3
Organic fraction
Protein	Kjeldahl	2.59	1.64–2.95
Lipids	Weibull‐Stoldt	0.17	0.11–0.21
Free sugars	Luff‐Schoorl	4.94	2.76–7.90
Water	Karl Fischer (PhEur 2.5.12)	3.0	2.1–3.9

The fraction of secondary metabolites was characterised in the same batches of the additive, and the results are summarised in Table 2. PACs were determined with the DMAC colourimetric method. ¹³ The following different classes of secondary metabolites were analysed by high performance liquid chromatography with diode array (HPLC–DAD) and electrospray mass spectrometry (HPLC–ESI‐MS): anthocyanins (at 520 nm), flavonol glycosides and their aglycones quantified as quercetin equivalents (at 365 nm), phenolic acids (at 310 nm) and other organic acids (at 210–230 nm). ¹⁴ The main components of each fraction were tentatively identified based on their UV spectrum and ESI‐MS spectra. For some compounds, the identification was confirmed by the comparison of the retention time and spectral data with those of authentic standards.

TABLE 2.

Characterisation of the fraction of secondary metabolites of cranberry extract, based on the analysis of five batches (mean and range).

Constituent	Mean	Range
	%	%
Proanthocyanidins (PACs, total, by colourimetric method)	27.9	27.3–29.3
Anthocyanins (as cyanidin 3‐O‐glucoside chloride)
Cyanidin‐3‐O‐galactoside	0.29	0.18–0.53
Cyanidin‐3‐O‐arabinoside	0.33	0.22–0.53
Cyanidin‐3‐O‐glucoside	0.02	0.02–0.03
Peonidin‐3‐O‐galactoside	0.48	0.25–0.90
Peonidin‐3‐O‐arabinoside	0.27	0.19–0.43
Peonidin‐3‐O‐glucoside	0.07	0.05–0.11
Total anthocyanins	1.47	1.09–2.52
Flavonol glycosides and their aglycones (as quercetin equivalents)
Flavonols (as quercetin equivalents)
Quercetin	1.10	0.95–1.31
Myricetin	0.62	0.47–1.01
3’‐O‐Methylquercetin	0.06	0.04–0.08
3′,5’‐O‐Dimethylmyricetin	0.03	0.02–0.04
3’‐O‐Methylmyricetin	0.01	0.01–0.01
Kaempferol	0.02	0.01–0.02
Unknown	0.05	0.03–0.07
Flavonol glycosides (as quercetin equivalents)
Quercetin 3‐β‐O‐galactoside	0.90	0.38–1.33
Myricetin‐3‐β‐O‐galactoside	0.42	0.18–0.83
Quercetin‐3‐O‐rhamnopyranoside	0.29	0.23–0.38
Quercetin‐3‐α‐O‐arabinopyranoside	0.18	0.11–0.25
Quercetin‐3‐α‐O‐xylopyranoside	0.14	0.11–0.18
Quercetin‐3‐α‐O‐arabinofuranoside	0.14	0.08–0.23
Quercetin‐3‐(6″‐benzoyl)‐β‐O‐galactoside	0.08	0.06–0.09
Quercetin‐3‐β‐O‐glucoside	0.05	0.04–0.08
3’‐O‐Methylmyricetin‐3‐O‐galactoside	0.08	0.03–0.13
Myricetin‐3‐α‐O‐arabinopyranoside	0.07	0.03–0.13
Myricetin‐3‐α‐O‐arabinofuranoside	0.05	0.02–0.07
Myricetin‐3‐α‐O‐xylopyranoside	0.02	0.02–0.04
3′,5’‐O‐Dimethylmyricetin hexoside	0.01	0.01–0.01
Total flavonol glycosides and their aglycones (as quercetin equivalents)	4.36	2.90–6.34
Phenolic acid derivatives
p‐Coumaric acid	1.58	1.31–2.22
p‐Coumaroyl glucose isomer 2	0.87	0.74–1.08
p‐Coumaroyl glucose isomer 1	0.31	0.29–0.34
p‐Coumaroyl glucose isomer 3	0.14	0.10–0.19
Chlorogenic acid	0.90	0.72–1.04
Sinapic acid	0.20	0.14–0.23
3,4‐Dihydroxybenzoic acid (protocatechuic acid)	0.07	0.06–0.08
Caffeoyl glucose	0.14	0.07–0.26
4’‐O‐Glucosylferulic acid	0.15	0.13–0.19
4’‐O‐Glucosylsinapic acid	0.28	0.24–0.34
Methyl gallate fragment	0.02	0.02–0.02
Coumaric, caffeic fragment	0.09	0.06–0.16
Hexosyl‐Caftaric acid	0.06	0.05–0.09
Caffeic acid fragment	0.10	0.07–0.12
Unknown	0.52	0.37–0.86
Unknown	0.13	0.10–0.15
Unknown	0.05	0.04–0.06
Total phenolic acid derivatives	6.17	4.94–8.18
Other organic acids
Benzoic acid	3.50	3.10–3.79
Citric acid	0.37	0.10–0.65
Quinic acid	0.30	0.10–0.44
Malic acid	0.23	0.10–0.41
Shikimic acid	< 0.1	< 0.1
Total other organic acids	4.34	3.48–5.29
Total identified (1)	44.24	42–82‐48.72

⁽¹⁾ As the sum of proanthocyanidins, anthocyanins, flavonol glycosides and their aglycones (as quercetin equivalents), phenolic acid derivatives and other organic acids.

The FEEDAP Panel notes that the flavonol glycosides and their aglycones were quantified as quercetin equivalents (Table 2), which are therefore the parameter used to express the combined presence of flavonol glycosides and their aglycones in the additive.

The results of the characterisation of the different classes of secondary metabolites are summarised as follows:

• proanthocyanidins (PACs): quantified using a colourimetric method. The characterisation of the PAC fraction by reversed phase (RP)‐HPLC and multiple MS approaches (matrix‐assisted laser desorption/ionisation time‐of‐flight (MALDI–TOF), ESI‐MS and ESI‐MS²) showed the presence of proanthocyanidin A2 (a dimer of (−)‐epicatechin) and numerous oligomers with one or more A‐type linkage, consisting mainly of non‐hydroxylated oligomers (catechin/epicatechin units) together with hydroxylated oligomers (gallocatechin/epigallocatechin units). The degree of polymerisation was up to 26 units, with trimers and tetramers being the most abundant oligomers. The data are in agreement with those reported in literature, which describes PACs from cranberry juice to contain a series of polyflavan‐3‐ol oligomers based on a repeating unit structure of (epi)catechin with one or more A‐type interflavanyl linkages present in the oligomer, with a degree of polymerisation ranging from 4 to 10 (Howell et al., 2005; Nemzer et al., 2022; Neto et al., 2006). The percentage of A‐type PACs to total PACs in cranberry is 51%–65% and the mean degree of polymerisation is 8.5 (Zeng et al., 2024). In addition, the information on the presence of monomers, oligomers and polymers indicated that the percentage of monomers in cranberry fruit is < 5%, with polymers representing the main fraction followed by oligomers (Nemzer et al., 2022);

• anthocyanins (UV detection at 520 nm): four main peaks were identified corresponding to the 3‐O‐galactosides and 3‐O‐arabinosides of cyanidin and peonidin, and two minor peaks corresponding to the 3‐O‐glucosides;

• flavonol glycosides and their aglycones (UV detection at 365 nm): twenty‐one peaks were tentatively identified, mostly as the glycosylated forms of quercetin and myricetin, present with the corresponding aglycones. Minor peaks were assigned to the glycosides of methylquercetin, methylmyricetin and kaempferol, and their aglycones;

• phenolic acids (UV detection at 310 nm): 17 peaks were detected and tentatively identified as phenolic acids, mainly hydroxycinnamic acids (as glycosides of trans‐cinnamic, p‐coumaric, caffeic, ferulic and sinapic acids) as well as 3,4‐dihydroxybenzoic acids;

• other organic acids (UV detection at 210–230 nm): benzoic acid, quinic acid, malic acid, citric acid and shikimic acid identified based on the comparison of the retention time and UV spectra with respective reference standards.

The applicant provided analytical data on impurities ¹⁵ and physical properties ¹⁶ for the additive under assessment. The results are summarised in Tables 3 and 4.

TABLE 3.

Data on impurities of cranberry extract.

Parameter	Analysis
	Range	No. of batches
Impurities
Lead (mg/kg)	< 0.1–0.1	3
Mercury (mg/kg)	< 0.1	3
Cadmium (mg/kg)	< 0.1	3
Arsenic (mg/kg)	0.2	3
Aflatoxin B1 (μg/kg)	< 1	3
Sum of aflatoxins B1, B2, G1, G2 (μg/kg)	< 2	3
Ochratoxin (μg/kg)	< 1	3
Ethanol (mg/kg)	47–208	5
Pesticides (mg/kg) (1)	Not detected	3
Microbial contamination
Total aerobic counts (CFU/g)	< 1000	5
Yeast and filamentous fungi (CFU/g)	< 100	5
Salmonella spp. (per 25 g)	Not detected	5
Escherichia coli (per 1 g)	Not detected	5

Abbreviations: CFU, colony forming unit.

Note: <: means below the limit of quantification.

⁽¹⁾ For pesticides a multiresidue analysis was provided.

TABLE 4.

Data on physical properties of cranberry extract.

Parameter	Analysis
	Range	No. of batches
Physical properties
Physical form	Solid
Tapped density (kg/m3)	400–460	3
Dusting potential (Stauber‐Heubach) (mg/m3)	2164–2576	3
Particle size distribution (laser diffraction) (% of particles below, v/v)		3
50 μm	59.8–61.2
10 μm	2.4–2.8
Shelf‐life (% recovery, PAC)
25°C/60% RH 66–71 months	100	2
Stability in feed (% recovery, PAC)
Kibbles, dogs and cats 25°C/60% RH 24 months	100	1
Wet feed, dogs and cats 25°C/60% RH 3 months	100	1

Abbreviations: PAC, proanthocyanidins, RH, relative humidity.

The data provided showed that all batches analysed complied with the specifications proposed by the applicant for the content of PACs, water and crude ash. The FEEDAP Panel considers that microbial contamination and the levels of the impurities analysed in the additive are of no concern.

3.3.2. Conditions of use

The additive under assessment is intended for use in feed for dogs and cats at a proposed minimum use level of 122 mg/kg of complete feedingstuff for dogs (corresponding to 30.5–42.7 mg/kg of PAC according to the specification) and 155 mg/kg of complete feedstuff for cats (corresponding to 45.8–64.1 mg/kg of PAC). The applicant proposed a maximum use level of 610 mg additive/kg of complete feedingstuff for dogs (corresponding to 153–214 mg/kg of PAC) and 755 mg additive/kg of complete feedstuff for cats (corresponding to 189–264 mg/kg of PAC).

3.4. Safety

Following the provisions of Regulation (EC) No 429/2008, there is no requirement for the assessment of the safety of an additive for the consumers and the environment when the additive is intended to be used only in pets.

Absorption, distribution, metabolism and excretion (ADME) studies and toxicological studies (including genotoxicity) performed with the additive under assessment were not provided. The studies submitted on the safety for the users are described in Section 3.4.3.

The applicant carried out an extensive literature search (ELS) to support the safety evaluation of the additive for the target species. The search strategy was described in detail and search terms were provided (substance descriptors, effects and target species). Two cumulative databases (PubMed and Web of Science) were used. The search covered the period until 2025, without a start date. After applying the inclusion and exclusion criteria, 122 publications were considered relevant. ¹⁷

Cranberry extract contains plant‐derived proteins, lipids and sugars (see Table 1), which are not of concern and are not further considered.

Among the identified secondary plant metabolites, proanthocyanidins represent up to 35% (by specification), anthocyanins up to 2.5%, flavonoids up to 6.3% (expressed as quercetin equivalents), phenolic acid derivatives (hydroxycinnamic acids) up to 8% and non‐phenolic organic acids up to 5.3% (with benzoic acid up to 3.8%) of the additive.

Non‐phenolic organic acids, including citric acid, quinic acid and malic acid, are ubiquitous on food and feed of plant origin. They will be readily metabolised and excreted or converted to carbon dioxide and are of low toxicity. These compounds are not of concern at concentrations resulting from the use of the additive at the maximum proposed use level in feed and are not further considered in the assessment, except for benzoic acid which is a major component of the additive.

For hydroxycinnamic acids, it is very likely that they are glucuronidated in cats less efficiently than in other species, due to UGT1A6 (UDP‐glucuronosyltransferase 1A6) pseudogenisation and UGT1A7–12 contraction in Felidae (Court & Greenblatt, 1997; Lautz et al., 2021) and the known low glucuronidation rates for other plant phenolics (e.g. soy isoflavones, see Section 3.4.1.2). Cats are expected to have a greater reliance on sulfation, methylation and intestine microbial metabolism for these substances (Redmon et al., 2016). However, considering the low exposure of cats resulting from the use of the additive at the proposed use levels in feed (0.3 mg hydroxycinnamic acids/kg bw per day at the minimum use level or 1.4 mg hydroxycinnamic acids/kg bw per day at the maximum use level) and the low toxicity of hydroxycinnamic acids, these compounds are not considered of concern and are not further considered in the assessment.

The additive contains proanthocyanidins up to 35% (by specification), anthocyanins up to 2.5%, flavonoids up to 6.3% (expressed as quercetin equivalents). The literature search made by the applicant identified several publications, including EFSA opinions (EFSA ANS Panel, 2013; EFSA NDA Panel, 2017), evaluations by other bodies (EMA, 2022b) and review papers, which address the ADME and the toxicity (including genotoxicity) of cranberry preparations and of the main classes of secondary metabolites of cranberry (proanthocyanidins, anthocyanins and flavonoids). The publications considered relevant by the FEEDAP Panel are described in the following sections.

3.4.1. Absorption, distribution, metabolism and excretion

3.4.1.1. Studies in laboratory animals

Cranberry preparations

The available pharmacokinetic data regarding cranberry preparations have been summarised by EMA (2022b).

After gavage administration of a cranberry concentrate in powder form to rats for 10 months (1 g/kg bw, 1 gavage/day, 5 days a week), sorhamnetin, myricetin, kaempferol and proanthocyanidin dimer A2, together with 13 conjugated metabolites of quercetin and methylquercetin and intact peonidin 3‐O‐galactoside and cyanidin 3‐O‐galactoside were identified in the rat urine’. Urine samples were collected at different time points (18, 25, 42, 48 h) after a single administration of the cranberry preparation. These results suggest that cranberry components undergo rapid metabolism and elimination into the urine of rats (Rajbhandari et al., 2011, as referenced in EMA, 2022b).

In rats fed different types of berries, or different levels of the same berry source, the urinary excretion of free and conjugated (epi)catechins, including their methylated forms, was 60% of the ingested amount and was dependent on the concentration in the diet (Khanal et al., 2010, as referenced in EMA, 2022b). Another study with rats fed diets containing 5% of cranberry, blueberry or black raspberry reported the excretion of 18 phenolic acids and their conjugates, as metabolites of polyphenols and flavonoids. For cranberry extract, the major compounds excreted in the urine over 24 h were hippuric acid, 3‐methoxy‐4‐hydroxyphenylacetic acid (homovanillic acid), 4‐hydroxycinnamic acid (p‐coumaric acid), 4‐hydroxybenzoic acid and 3‐hydroxyphenylacetic acid. Cinnamic acid derivatives were mainly excreted in conjugated form (50%–70%), whereas for phenylacetic acid derivatives < 10% was excreted in conjugated form (Khanal et al., 2014, referenced as Khanal et al., 2010 in EMA, 2022b).

In vitro studies with human intestinal epithelial Caco‐2 cells with the different proanthocyanidin fractions extracted from cranberry suggested that A‐type dimers, trimers and tetramers could be absorbed although to a limited extent (Ou et al., 2012, as referenced in EMA, 2022b).

The EMA evaluation considered pharmacological interactions, in particular the inhibitory effects of cranberry juice on Cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) isoforms. Cranberry juice was found to inhibit CYP3A4 and CYP2A9 in human liver microsomes in a concetration‐dependent manner, with large differences in the activity depending on the sample tested (Wanwimolruk et al., 2012; Ngo et al., 2009; Ngo et al., 2010; Greenblatt et al., 2006, as referenced in EMA, 2022b). However, no effects of cranberry juice on the inhibition of CYP3A4, CYP2A9 or CYP1A2 has been observed in vivo in volunteers (Greenblatt et al., 2006; Lilja et al., 2007, as referenced in EMA, 2022b). A weak inhibition was observed for UGT1A9, but not UGT1A1, UGT1A4, UGT1A6 and UGT2B7 (Mohamed & Frye, 2011; Choi et al., 2014, as referenced in EMA, 2022b). Although the mechanisms for possible drug interactions are not known, EMA concluded that the concomitant use of cranberry juice and other cranberry products with warfarin or tacrolimus is contraindicated (EMA, 2022b).

Proanthocyanidins

For the ADME of PACs, the applicant referred to the EFSA opinion on the safety of cranberry extract powder as a novel food ingredient (EFSA NDA Panel, 2017).

The knowledge on the toxicokinetic of PACs is scarce. Only PACs with a low degree of polymerisation (i.e. dimers) can be absorbed, although only to a low extent. It has been proposed that oligomeric and polymeric PACs are transported to the large intestine where they undergo degradation by the colonic microbiota into simple phenolic acids which can then be subsequently absorbed (EFSA NDA Panel, 2017). The additive under assessment contains proanthocyanidin A2 and numerous other oligomers with a degree of polymerisation up to 26 units, suggesting very limited absorption of this fraction of the additive.

Anthocyanins

For the ADME and the toxicology of anthocyanins reference is made to the ANS opinion on the re‐evaluation of anthocyanins (E 163) as a food additive (EFSA ANS Panel, 2013).

Based on the evidence available for fruit extracts in rats, rabbits and pigs, the ANS Panel concluded that absorption of anthocyanins in general is low (< 2%). After oral administration, maximum plasma levels of anthocyanins were reached within 2 h, depending on the aglycone and sugar moieties of the anthocyanins. In rats and in pigs, anthocyanins can be methylated or conjugated with glucuronic acid or sulfate, and excretion of these metabolites and their aglycones in urine have been reported. Anthocyanins substituted with either a di‐ or tri‐saccharide were primarily excreted unchanged in the urine. Urinary excretion of anthocyanins (either unchanged or as metabolites) in rats and pigs is only 0.04%–0.58% of the ingested amount.

Flavonol glycosides and their aglycones

The main flavonoids in cranberry extract consist of quercetin and myricetin and their glycosides. The ADME of quercetin glycosides has been summarised in NTP (1992). Information on the toxicokinetic of myricitrin, the rhamnoside of myricetin, is described in the publication by Maronpot et al. (2015).

As reported by NTP (1992), ‘Quercetin glycosides are poorly absorbed in the small intestine. After ingestion, quercetin glycosides can be partially hydrolysed by β‐glucosidases of the intestinal cells generating the aglycone which is then absorbed. Small amounts of intact glycosides can also be absorbed and further metabolised by liver enzymes. The remaining intact glycosides may be hydrolysed in the colon by microbiota enzymes and the aglycone partially absorbed or excreted in faeces. After absorption, the aglycones are expected to be conjugated mainly in liver giving rise to glucuronides and sulfates in dogs and mainly to sulfates in cats. These conjugates can be excreted in urine, but bile seems to be the principal via of excretion. A considerable part of quercetin or its glycosides is also transformed by colonic bacteria to smaller molecules, such as 3,4‐dihydroxyphenylacetic, 3‐methoxy‐4‐hydroxyphenylacetic acid (homovanillic acid) and 3‐hydroxyphenylacetic acid, which were detected in the urine of rabbits or rats after oral application of quercetin. Free quercetin could not be detected in plasma or urine.’

The toxicokinetic of myricitrin, the rhamnoside of myricetin, was investigated by Maronpot et al. (2015). A single dose of myricitrin (250, 500 or 1000 mg/kg bw) or of myricetin (1.6 mg/kg bw) was administered to Sprague–Dawley rats by gavage. Blood was collected at 1, 3, 6, 12 and 24 h after administration and analysed by HPLC coupled with tandem mass spectrometry (HPLC‐MS/MS). Myricitrin was detectable in blood (levels not reported) within 1 h after administration of 250, 500 or 1000 mg myricitrin/kg bw, indicating direct absorption of the glycosylated form of the flavonoid. At 12 or 24 h after administration, myricetin was detectable (levels not quantifiable) in plasma of one of five, three of five or four of five rats administered 250, 500 or 1000 mg myricitrin/kg bw, respectively, possibly as the result of hepatic conversion of myricitrin to myricetin and its enterohepatic recirculation. Myricetin could not be detected by HPLC‐MS/MS (limit of quantification not given) in blood of rats administered 1.6 mg myricetin/kg bw.

Overall, the available data in laboratory animals indicate that flavonoids present in the additive are poorly absorbed in the glycosidic form. However, after intestinal metabolism the aglycones can be absorbed and extensively metabolised, mainly by conjugation, giving rise to glucuronides, sulfates and methylated derivatives. Quercetin was not detected in plasma nor in urine, while trace amounts of myricetin were detected in plasma. Both flavonols and their metabolites are expected to be mainly excreted through bile in faeces and to a lesser extent in urine.

Benzoic acid

Benzoic acid will mainly be metabolised by conjugation with glycine and excreted as hippuric acid (EFSA ANS Panel, 2016; EFSA FEEDAP Panel, 2012) by animal species including cats and dogs (Bridges et al., 1970 as referenced by EFSA ANS Panel, 2016). Thus, it is expected that this compound is readily excreted. However, it should be noted that ‘cats are unable to glucuronidate benzoic acid, but can glycinate it, albeit slowly. Benzoic acid poisoning has been reported in cats’ (Court, 2013), suggesting that glycination may be saturable.

3.4.1.2. Studies in target animals

ADME data for dogs and cats are not available for the secondary plant metabolites present in cranberry extract. The data in laboratory animals indicate that glucuronidation is an important pathway to facilitate the excretion of the components of the additive (proanthocyanidins, anthocyanins, flavonols and phenolic acids). Differences exist in the metabolic capacity of the target species compared to laboratory animals. In particular, cats are known for their low capacity of glucuronide formation, particularly for simple phenols and aromatic compounds because of a lack of the functional enzyme UGT1A6 (Court & Greenblatt, 1997; Lautz et al., 2021). Studies with different substrates indicated that glucuronidation in cats is substrate‐ and isoform‐specific. While lacking functional UGT1A6 and UGT2B7 homologues (present and functional in dogs), the cat has a number of functional UGT1A enzymes that are homologues to the human UGT1A isoforms (van Beusekom et al., 2014). These enzymes, which are important for the glucuronidation and excretion of endogenous metabolites, such as bilirubin and 7‐β‐oestradiol, have been retained in feline species. A study by Redmon et al. (2016) investigated the metabolism in cats and dogs of soy isoflavones, ¹⁸ which are structurally related to anthocyanins and flavonones present in the additive. All isoflavones were glucuronidated by dogs. Considerable differences were observed in the glucuronidation by cats depending on the position of the hydroxy group, due to differences in the activity of isoforms of glucuronyl transferases involved in the glucuronidation of the substrates. There was a complete lack of conjugation at the 4′‐position, catalysed by UGT1A6, whereas glucuronidation at position 7 was comparable with that of rats and mice. Overall, the authors found that ‘soy isoflavones are extensively conjugated in both dogs and cats, either directly or after reduction to the dihydro derivatives, followed by excretion into the urine’.

Another study carried out with domestic cats showed that sulfation is a major metabolic pathway of soy isoflavones in cats (Whitehouse‐Tedd et al., 2013). After dietary administration of the isoflavones genistein and daidzein (2.22 genistein+2.86 daidzein mg/kg bw as single dose or 0.62 genistein+0.82 daidzein mg/kg bw daily for 10 days), very low levels of daidzein and genistein were present in plasma in their unconjugated form, whereas the main metabolites were the monosulfate (for both compounds) and the disulfate (for daidzein). In none of the plasma samples, the glucuronide conjugates were detected. The authors of the study concluded that ‘the metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms’.

3.4.2. Toxicology

The literature search made by the applicant identified relevant publications on the toxicity (including genotoxicity) of the main classes of secondary metabolites of cranberry, proanthocyanidins, anthocyanins and flavonoids.

The FEEDAP Panel notes that limited data are available on the toxicology of cranberry and its components, A‐type PACs and anthocyanins, whereas more data are available in the literature for B‐type PACs from grape seed extract (GSE) and grape‐skin extract (GSKE). A‐type PACs differ from B‐type PACs by the presence of an ether bond between two consecutive constitutive units, which gives a more stable and hydrophobic character to PACs. In the next paragraphs, the possibility to apply read‐across from B‐type to A‐type PACs is discussed.

For flavonoids, toxicological information was retrieved for quercetin and myricetin and their glycosides isoquercitrin and myricitrin and read‐across is applied to the structurally related flavonoids present in the additive.

3.4.2.1. Genotoxicity

For the assessment of genotoxicity of chemical mixtures, the EFSA Scientific Committee (SC) recommends that first the chemically defined substances are assessed individually for their potential genotoxicity using all available information, including read‐across and quantitative structure–activity relationship (QSAR) considerations about their genotoxic potential (EFSA Scientific Committee, 2019b). Therefore, the potential genotoxicity of identified constituents is assessed also based on their genotoxicity evaluation when present in other related extracts.

Proanthocyanidins

The possible mutagenicity of proanthocyanidin (CAS No. 18206‐61‐6, synonym: proanthocyanidin A2) was evaluated using QSAR models. The VEGA models (www.vegahub.eu) for Ames test provided results with good reliability, indicating a lack of activity.

A proanthocyanidin‐rich extract from GSE containing 89.3% B‐type PACs was not mutagenic in an Ames test using Salmonella Typhimurium strains TA98, TA100, TA1535 and TA 1537, in the presence and absence of metabolic activation. Negative results were also obtained in an in vivo micronucleus test performed in peripheral blood reticulocytes after oral administration of the extract to ddY mice at doses up to 2 g/kg (Yamakoshi et al., 2002).

Commercial GSE and GSKE did not induce chromosomal damage in mice orally administered at doses up to 2000 mg/kg bw. Relevant negative results were obtained in a mammalian erythrocyte micronucleus test with evidence of target tissue exposure (Erexson, 2003, as referenced in EFSA ANS Panel, 2013).

Based on the absence of structural alerts and on the structural similarity of the flavan‐3‐ol monomers in B‐type and A‐type PACs, and considering that the ether bond present in A‐type is not expected to influence the genotoxicity of PACs, the FEEDAP Panel considers that applying read‐across from B‐type to A‐type PACs would be appropriate for the assessment of genotoxicity of PACs from cranberry extract. The FEEDAP Panel considers that the PACs present in the additive do not raise concern for genotoxicity.

Anthocyanins

In its assessment on the re‐evaluation of anthocyanins (E 163) as food additive, the EFSA ANS Panel reviewed the genotoxicity data available for anthocyanins present in fruit extracts, mostly GSE, GSKE and blackcurrant extracts.

The ANS Panel concluded that in most in vitro assays, anthocyanins tested at low concentrations were not genotoxic. Some evidence of genotoxicity was provided by a single in vitro study using pure anthocyanidins. However, an in vivo bone marrow micronucleus test following Organisation for Economic Co‐operation and Development (OECD) test guideline (TG) 474 at a limit dose was negative. Overall, the ANS Panel concluded that there was no concern regarding in vivo genotoxicity of GSE and GSKE (EFSA ANS Panel, 2013).

The FEEDAP Panel considers that anthocyanins contained in GSE and GSKE are representative of those found in cranberry extract and concludes that anthocyanins present in the additive do not raise concern for genotoxicity.

Flavonol glycosides and their aglycones

Genotoxic effects have been detected for quercetin in in vitro assays (NTP, 1992) and in vivo assays with parenteral application of the compound (EFSA FEEDAP Panel, 2024b). The effects depend on DNA‐intercalation after direct cellular contact of quercetin. It was demonstrated in several pharmacokinetic studies, that no free quercetin will occur in the circulating system after oral uptake of flavonone aglycones or their glycosides (see Section 3.4.1.1).

Negative results have been reported for myricetin and myricitrin in a bacterial reverse mutation assay, except for myricetin inducing gene mutations in one strain of S. Typhimurium (TA98) in the presence of metabolic activation. Both myricetin and myricitrin induced chromosomal damage in vitro in human TK6 cells in the absence of metabolic activation. The positive outcomes observed in vitro were not confirmed in vivo, both for myricetin and myricitrin, in a combined micronucleus/comet assay performed in male and female B6C3F1 mice showing no induction of micronuclei in peripheral blood erythrocytes and no increase of DNA strand breaks in the liver, glandular stomach and duodenum (Hobbs et al., 2015).

Therefore, the flavonol glycosides and their aglycones present in the additive do not raise concern for genotoxicity.

3.4.2.2. Repeated dose toxicity studies

Cranberry extract

Two oral toxicity studies in rats described in the EMA report (EMA, 2022b) were performed with cranberry products and are described below.

In a 14‐week study, Wistar rats (six animals per group) were given three commercial cranberry powder products in their diet at a concentration of 1500 mg of cranberry product/kg of feed. The test items contained up to 4.75% PACs (A‐type) and up to 0.61% anthocyanins. No effects were observed on body weight, food intake, haematological parameters, organ weights, histopathology and total cytochrome P450 levels in the liver (Palikova et al., 2010, as referenced in EMA, 2022b). Although the dose tested was low (up to 71.25 mg PACs/kg feed, corresponding to 6.41 mg PACs/kg bw per day and up to 9.15 mg anthocyanins/kg feed corresponding to 0.82 mg anthocyanins/kg bw per day), the FEEDAP Panel notes that this subchronic study is the only one performed with cranberry extract and is considered relevant for the current assessment.

In another study in dogs (n = 6), a commercial cranberry extract (composition not specified) was mixed with food and administered daily for 6 months at a dose of 1 g for dogs < 25 kg and 2 g for dogs ≥ 25 kg of body weight (Chou et al., 2016, as referenced in EMA, 2022b). The study was not designed to evaluate toxicity but rather to determine the effects of cranberry extract on the prevention of urinary tract infection. However, no adverse effects attributable to the treatment were reported.

Proanthocyanidins and anthocyanins

The applicant identified several publications including a review (González‐Quilen et al., 2020), which indicated no observed adverse effect level (NOAEL) values for systemic toxicity in rats of PAC‐enriched GSE and GSKE (containing B‐type PACs) in the range of 1400–2000 mg/kg bw per day (in all cases, the highest dose tested). The applicant also provided a publication by Bentivegna and Whitney (2002), from which a NOAEL for GSE and GSKE (containing B‐type PACs) could be identified and proposed to apply read‐across from B‐type PACs (present in GSE and GSKE) to A‐type PACs present in cranberry extract.

Commercial GSE and GSKE were used as test items in a 90‐day oral toxicity study in rats (Bentivegna & Whitney, 2002) claimed to be good laboratory practice (GLP)‐compliant. The test items GSE and GSKE were shown to contain, respectively, approximately 90.5% and 87.3% total phenols (expressed as gallic acid equivalents). GSKE also contained 2.6% anthocyanins, which primarily accumulate in the skin of the fruit rather than the seeds. In addition, the information on the presence of monomers, oligomers and polymers indicated that oligomers represent the main fraction (74.9% in GSE and 67.4% in GSKE), followed by polymers (14.7% in GSE and 15.9% in GSKE) and monomers (10.4% in GSE and 16.7% in GSKE, mainly catechin and epicatechin). Groups of CD® (Sprague–Dawley) Crl:CD‐1® IGS BR rats (20 animals per group and sex) were fed diets containing GSE at concentrations of 0%, 0.63%, 1.245% or 2.5% (w/w) or GSKE at 2.5% (w/w). No mortality or signs of toxicity were observed. Furthermore, no significant dose‐related effects on body weight, feed consumption, haematological parameters, clinical chemistry, organ weight and histopathology were observed for both GSE and GSKE. For both extracts, the highest concentration tested (2.5%) was identified as the NOAEL. This corresponds to approximately 1780 mg/kg bw per day in male rats and 2150 mg/kg bw per day in female rats for both GSE and GSKE (Bentivegna & Whitney, 2002). The FEEDAP Panel notes that GSKE is better characterised and was reported to contain mainly oligomeric and polymeric PACs (83.3%) and anthocyanins (2.6%). The similarity and the differences between the test item GSKE and the additive under assessment are considered in the text below to justify the application of read‐across.

Grape skins and seeds feature predominantly B‐type PACs, whereas cranberries contain predominantly more stable A‐type PACs. The FEEDAP Panel considers that A‐type PACs from cranberries and B‐type PACs from grapes (i) are similar concerning the presence of the flavan‐3‐ol units (see Section 3.3.1), (ii) differ for the presence of the ether bond in A‐type PACs, (iii) are both described in the literature to be of low toxicity, with NOAEL values always identified as the highest dose tested. The FEEDAP Panel notes that GSKE also contains a higher proportion of monomers (16.7%) compared to cranberry fruit (< 5%, according to Nemzer et al., 2022).

Overall, the FEEDAP Panel considers that the uncertainty due to the differences in the structural features of A‐type and B‐type PACs and in the relative proportion of monomers is not such that it would prevent applying read‐across from B‐type PACs present in GSE and GSKE to A‐type PACs in cranberry extract.

The FEEDAP Panel also considers that anthocyanins contained in the GSKE tested in a 90‐day study in rats (Bentivegna & Whitney, 2002) are representative of those found in cranberry extract and are present in similar proportions (2.6% in GSKE and up to 2.52% in cranberry extract).

The FEEDAP Panel considers that a dose of 1780 mg/kg bw per day can be selected as the reference point for the assessment of the sum of PACs and anthocyanins in cranberry extract for cats and dogs. In the risk assessment for the sum of PACs and anthocyanins, the NOAEL of 1780 mg/kg per day was divided by a factor of 2 to account for the uncertainties due to differences in the structural features and relative proportions of PACs.

To further support the low toxicity of anthocyanins, reference is made to the re‐evaluation of anthocyanins (E 163) as food additive made by the ANS Panel. ‘The EFSA ANS Panel noted that toxicological information available from grape‐skin extracts and from blackcurrant extracts, including genotoxicity tests, short‐term, sub‐chronic and reproduction toxicity studies, does not show adverse effects overall’.

In dogs, no toxic effects were observed after sub‐chronic exposure to anthocyanins at doses up to 15% of a grape‐skin extract containing 2.4% anthocyanins in the diet (equivalent to 0.36% anthocyanins) (Cox & Babish, 1978, as referenced in EFSA ANS Panel, 2013), suggesting a low toxicity of anthocyanins for the target species dogs.

Flavonol glycosides and their aglycones

The flavonoid content of cranberry extract consists of quercetin and myricetin as aglycones and their glycosides. For the evaluation of the flavonoids present in the additive, the activity of the aglycones quercetin and myricetin is considered relevant. The two aglycones are structurally related, as myricetin has an additional hydroxy group at ring A. No data are available, which would allow to identify a reference point for quercetin and myricetin. However, the literature search done by the applicant identified two studies with a test item consisting of enzymatically decomposed rutin, which consists of 95% isoquercitrin ¹⁹ (quercetin 3‐O‐glucopyranoside) and a study with myricitrin (myricetin‐3‐O‐rhamnoside).

In a 90‐day oral toxicity study, the test item enzymatically decomposed rutin (consisting of 95% isoquercitrin) was administered to Wistar rats at dietary concentrations of 0, 0.2, 1 or 5% (Hasumura et al., 2004). At the top dose, decreased blood cell count, haemoglobin concentrations and haematocrit were observed in males but not in females. Based on the results of the study, NOAELs for male and female rats were identified to be 1% and 5% in feed, respectively, corresponding to 539 and 3227 mg/kg bw per day of the test item. In a chronic toxicity study (Tamura et al., 2010), the same test item was administered to Wistar rats at dietary concentrations of 0, 0.04, 0.2, 1 or 5% for 52 weeks. Based on effects seen at the highest dose tested in males and females, the NOAEL for the test item (enzymatically decomposed rutin, consisting of 95% isoquercitrin) was identified to be 1% in both sexes (corresponding to 542.4 mg/kg bw per day for males and 674.0 mg/kg bw per day for females). From the NOAEL of 542 mg/kg bw per day identified in the chronic study for enzymatically decomposed rutin, a NOAEL of 515 mg/kg bw per day was calculated for the glycoside isoquercitrin considering that it represents 95% of the test item. Taking account of the relative molecular weight of the glycoside isoquercitrin (464.4 g/mol) and of the aglycone quercetin (302.24 g/mol), the NOAEL of isoquercitrin was extrapolated to quercetin resulting in a NOAEL of 335 mg/kg bw per day.

In a 90‐day oral toxicity study myricitrin, the rhamnoside of myricetin (97% pure), was fed to male and female Sprague–Dawley rats at dietary concentrations of 0.5, 1.5 or 5.0% (Maronpot et al., 2015). Food consumption was increased and body weight gain decreased in males fed with 5% myricitrin. Blood values were within laboratory reference ranges, except for increased basophils in low‐ and high‐dose males and serum phosphorus in high‐dose males. There were several significant decreases in absolute organ weights, i.e. liver, thyroid and pituitary in 5.0% group males and spleen, heart and kidneys in 5.0% group females. In the absence of abnormal clinical or histopathological changes, these changes are not considered adverse. Based on the results of the study, the NOAEL is 2926 mg/kg bw per day in males and 3197 mg/kg bw per day in females (the highest dose tested). Considering the relative molecular weight of the glycoside myricitrin (464.37 g/mol) and of the aglycone myricetin (318.23 g/mol), the NOAEL of 2626 mg/kg bw per day identified for myricitrin was extrapolated to myricetin resulting in a NOAEL of 2005 mg/kg bw per day.

The FEEDAP Panel selects the lowest NOAEL of 335 mg/kg bw per day identified for quercetin as reference point for the group of flavonol glycosides and their aglycones (quantified as quercetin equivalents).

Benzoic acid

The EFSA ANS Panel evaluated the toxicological dataset available for benzoic acid, including subchronic toxicity studies in rats and mice, chronic toxicity and carcinogenicity studies in rats and mice and reproductive and developmental toxicity studies in rats, mice, hamster and rabbit. The ANS Panel identified a NOAEL of 500 mg/kg bw per day (the highest dose tested) from the pivotal study, a four‐generation reproductive study in rats (EFSA ANS Panel, 2016).

Toxicological studies in cats are limited. No effects (clinical signs of intoxication, clinical chemistry parameters, blood urea and transaminases) were seen in a 15‐day feeding study in four cats at 200 mg/kg bw per day. At higher doses, mild to severe signs of poisoning occurred (Bedford & Clarke, 1972).

The FEEDAP Panel notes that cats lack metabolic capacity for glucuronidation and have limited capacity for glycination of benzoic acid (see Section 3.4.1.1). Therefore, the specific sensitivity of cats to benzoic acid has to be accounted for in the margin of exposure approach when using the NOAEL of 500 mg/kg bw per day identified in the rat as the reference point (see Section 3.4.2.4).

Cranberry extract in dogs and cats

The literature search provided by the applicant identified several studies in the target animals aimed at investigating beneficial effects of cranberry extract (V. macrocarpon) in veterinary medicine. In particular, the studies investigated the effects of PACs in the treatment of urinary tract infections in dogs (Bisasibetti et al., 2019; Carvajal‐Campos et al., 2023; Chou et al., 2016; Howell et al., 2010) and cats (Carvajal‐Campos et al., 2024). Although the studies were not designed to assess the toxicity of the cranberry extract, adverse effects were not reported in any of them.

The literature search was also aimed at identifying evidence that the renal acute toxicity observed in dogs reported following the ingestion of grapes/raisins (Eubig et al., 2005; Son‐II, 2013; Bates et al., 2015, as referenced in EFSA FEEDAP Panel, 2016) is not relevant for cranberry extract. No report of acute toxicity in dogs associated with the ingestion of cranberry or cranberry extract was retrieved either from the public literature or websites, despite the known exposure following inclusion of cranberry extracts in foods for dogs. Consequently, the FEEDAP Panel considers that there is no evidence that the concern identified for grapes/raisin in dogs is applicable to cranberry extract.

The applicant proposed that tartaric acid and tartrates rather than PACs could be responsible for the canine syndrome. This is based on the fact that renal acute toxicity effects have been reported following the ingestion of cream of tartar (potassium hydrogen tartrate) and tamarinds (fruit rich in tartaric acid) (Wegenast et al., 2022) and on in vitro studies showing tartaric acid induced toxicity in Madin‐Darby canine kidney cells (Coyne & Landry, 2023). Tartaric acid is reported to occur only in trace amounts in cranberry (Forney et al., 2012) and was not detected in the additive under assessment (see Section 3.3.1).

3.4.2.3. Conclusions on toxicology

The FEEDAP Panel did not identify a concern for genotoxicity for the individual components of the additive under assessment.

Based on the toxicological data provided, the following NOAEL values for the individual components of the additive were identified: 1780 mg/kg bw per day divided by a factor of 2 (resulting in 890 mg/kg bw per day) for the sum of proanthocyanidins and anthocyanins, 335 mg/kg bw per day for flavonol glycosides and their aglycones (expressed as quercetin equivalents) and 500 mg/kg bw for benzoic acid.

3.4.2.4. Safety for the target species

Tolerance studies in the target species and/or toxicological studies in laboratory animals made with the extract 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).

Based on considerations related to structural and metabolic similarities, the proanthocyanidins and anthocyanins identified in cranberry extract were allocated to the same assessment group. Similarly, the identified flavonoids were allocated to the same assessment group.

For hazard characterisation, each component of an assessment group was first assigned to the structural class according to Cramer classification using Toxtree (version 3.1.0, May 2018 ²⁰ ). For some components in the assessment group, toxicological data were available to identify NOAELs. 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.

From a 90‐day study with GSKE, the FEEDAP Panel identified a NOAEL of 1780 mg/kg bw per day which is used for read‐across to the sum of A‐type PACs and anthocyanins of cranberry extract by applying a factor of 2 to account for the uncertainties due to differences in the structural features and relative proportions of PACs. For flavonols and their glycosides, the NOAEL of 335 mg/kg bw per day for quercetin has been selected as a group NOAEL. For benzoic acid, the NOAEL of 500 mg/kg bw per day is applied.

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 extract 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, 2017a). For those compounds covered by specifications (PACs), 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, when a group reference point is available, a group MOE was calculated for the combined intake (EFSA Scientific Committee, 2019a). A MOE > 100 allowed for interspecies differences and intra‐individual variability.

The approach to the safety assessment of cranberry extract for dogs is shown in Table 5. The calculations were done at the maximum proposed use level of 610 mg additive/kg complete feed.

TABLE 5.

Safety assessment for dogs: Compositional data, intake values (calculated at 610 mg additive/kg complete feed), reference points, margin of exposure (MOE) for the individual components of cranberry extract classified according to assessment groups.

Extract composition		Exposure		Hazard characterisation		Risk characterisation
Assessment group	Highest concentration in the extract	Highest concentration in feed	Intake (1)	Cramer class (2)	NOAEL (3)	MOE (4)
Constituent	(%)	mg/kg	mg/kg bw	–	mg/kg bw	–
Proanthocyanidins and anthocyanins	37.52	219.4	4.335	(III)	890	205
Flavonols glycosides and aglycones
Quercetin equivalents	6.34	38.7	0.732	(III)	335	457
Other constituents
Benzoic acid	5.29	32.3	0.611	(I)	500	818

Abbreviations: MOE, margin of exposure; NOAEL, no observed adverse effect level.

⁽¹⁾ Daily feed intake: 17 g DM/kg bw; bw: 15 kg (dogs). Rounded intake values are shown in the Table.

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

⁽³⁾ 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). When a group reference point is available, a group MOE is calculated for the combined intake.

As shown in Table 6 for all assessment groups the MOE calculated for dogs at the proposed use level of 610 mg additive/kg complete feed was > 100. For dogs, the use of cranberry extract is considered safe when used as a feed additive at the maximum proposed use level of 610 mg/kg complete feed.

TABLE 6.

Safety assessment for cats: Compositional data, intake values (calculated at 755 mg additive/kg complete feed), reference points, margin of exposure (MOE) for the individual components of cranberry extract classified according to assessment groups.

Extract composition		Exposure		Hazard characterisation		Risk characterisation
Assessment group	Highest concentration in the extract	Highest concentration in feed	Intake (1)	Cramer class (2)	NOAEL (3)	MOE (4)
Constituent	(%)	mg/kg	mg/kg bw	–	mg/kg bw	–
Proanthocyanidins and anthocyanins	37.52	283.3	6.438	(III)	890	138
Flavonols glycosides and aglycones
Quercetin equivalents	6.34	47.9	1.088	(III)	335	308
Other constituents
Benzoic acid	5.29	39.9	0.908	(I)	500	551

Abbreviations: MOE, margin of exposure; NOAEL, no observed adverse effect level.

⁽¹⁾ Daily feed intake: 20 g DM/kg bw; bw: 3.0 kg (cats). Rounded intake values are shown in the Table.

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

⁽³⁾ 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). When a group reference point is available, a group MOE is calculated for the combined intake.

The approach to the safety assessment of cranberry extract for cats is shown in Table 6. The calculations were done at the maximum proposed use level of 755 mg additive/kg complete feed.

Because of the limited capacity of cats for the biotransformation of components of the cranberry extract (see Section 3.4.1.2), the safe use of cranberry extract as an additive in cat feed has to be based on a margin of exposure higher than 100. The Panel considers that a MOE of 500 is adequate to cover the increased sensitivity of cats. As shown in Table 6, for all assessment groups except for benzoic acid the MOE calculated for cats at the proposed maximum use level of 755 mg additive/kg complete feed was < 500. Therefore, the use of cranberry extract at 755 mg/kg complete feed is not considered safe for cats.

When the calculations were repeated at the minimum proposed use level of 155 mg/kg complete feed, a MOE > 500 is obtained for all the assessment groups, as shown in Table 7.

TABLE 7.

Safety assessment for cats: Compositional data, intake values (calculated at 155 mg additive/kg complete feed), reference points, margin of exposure (MOE) for the individual components of cranberry extract classified according to assessment groups.

Extract composition		Exposure		Hazard characterisation		Risk characterisation
Assessment group	Highest concentration in the extract	Highest concentration in feed	Intake (1)	Cramer class (2)	NOAEL (3)	MOE (4)
	(%)	mg/kg	mg/kg bw	–	mg/kg bw	–
Constituent
Proanthocyanidins and anthocyanins	37.52	58.2	1.322	(III)	1483	673
Flavonols glycosides and aglycones
Quercetin equivalents	6.34	9.83	0.223	(III)	335	1500
Other constituents
Benzoic acid	5.29	8.20	0.186	(I)	500	2683

Abbreviations: MOE, margin of exposure; NOAEL, no observed adverse effect level.

⁽¹⁾ Daily feed intake: 20 g DM/kg bw; bw: 3.0 kg (cats). Rounded intake values are shown in the Table.

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

⁽³⁾ 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). When a group reference point is available, a group MOE is calculated for the combined intake.

For cats, the use of cranberry extract is considered safe up to the minimum proposed use level of 155 mg/kg, but not at the maximum proposed use level. The high MOE for benzoic acid is considered adequate to account for the specific sensitivity of cats to this compound. The FEEDAP Panel notes that at the minimum use level the exposure of cats to phenolic acids would be up to 0.3 mg hydroxycinnamic acids/kg bw per day.

3.4.2.5. Conclusions on safety for the target species

The additive cranberry extract is safe for dogs at the maximum use level of 610 mg/kg complete feed and for cats up to the minimum use level of 155 mg/kg complete feed.

3.4.3. Safety for the user

The skin irritation potential of the additive was tested in a study performed according to the OECD TG 492, which showed that the additive is not a skin irritant. ²¹

The eye irritation potential of the additive was tested in a study performed according to OECD TG 439, which showed that the additive is not an eye irritant. ²²

The skin sensitisation potential of the additive was investigated in an in vitro assay according to OECD TG 442E. From the results it was concluded that the additive is a potential dermal sensitiser. Therefore, it should also be considered a respiratory sensitiser.

3.4.3.1. Conclusions on safety for the user

Based on the information available, the additive is not irritant to skin or eyes but is a potential skin and respiratory sensitiser. Exposure of users by inhalation and dermal routes is considered a risk.

3.5. Efficacy

3.5.1. Efficacy for dogs

A total of three trials with dogs of different size (small, ²³ medium ²⁴ and large ²⁵ ) and breed (West Highland White Terrier, American Staffordshire Terrier and German Shepherd) and sharing a common design were submitted. The details on the study design are provided in Table and the main results in Table 8.

TABLE 8.

Trial design and use level of the efficacy trials performed in dogs.

Trial	Total no of animals (breed) sex	Total duration (phase I/washout/phase II)	Composition feed (form)	Groups
				mg additive/kg complete feed	(mg PAC/kg feed)
					Intended	Analysed
1	30 West Highland White Terrier 1:1 ♂/♀	15 days (5/5/5)	Anchovies, potatoes, egg flour (Kibbles)	0 122	0 36	n/a 36.4
2	30 American Staffordshire Terrier 1:1 ♂/♀	15 days (5/5/5)	Rice, maize, poultry protein (Kibbles)	0 122	0 37	n/a 37.3
3	30 German Shepherd 1:1 ♂/♀	15 days (5/5/5)	Poultry protein, maize, wheat (Kibbles)	0 122	0 36	n/a 38.3

Abbreviation: PAC, proanthocyanidins.

In all trials, 30 adult (1–5 years‐old) dogs from both sexes (15♂/15♀) were included. In each trial, from days 1 to 5 and 11 to 15, all dogs were offered at the same time a basal diet without (control) or with the additive at the proposed use level of 122 mg/kg (corresponding to 36 mg/kg of PAC) (confirmed by analysis; Table 8). The location of the bowl was randomised between days to avoid laterality. From day 6 to 10, a washout period was included, in which all dogs were offered only the basal diet with no additive. The location of the bowl was randomised between days to avoid laterality.

The health status of the animals was monitored throughout the study and the body weight and body score conditions were recorded daily. The given food and the remaining food were recorded daily for each individual animal, the eaten food and the intake ratio were calculated. The data were analysed with a repeated measures ANOVA using the individual animal as the experimental unit, and the diet, time and interaction between them as fixed effects. Significance level was set at 0.05 (Table 9).

TABLE 9.

Effects of cranberry extract on the feed consumption in dogs during the experimental period.

Trial	Groups	Eaten feed	Intake ratio
	(mg additive/kg feed)	(g/d)	(%)
1	0	34.1b	0.30b
	122	81.0a	0.70a
2	0	69.1b	0.30b
	122	180a	0.70a
3	0	93.3b	0.30b
	122	218a	0.70a

^a,b Mean values within a trial and within a column with a different superscript are significantly different p < 0.05.

In the three studies, dogs ate a higher amount of feed containing 122 mg cranberry extract/kg (36 mg PAC/kg) compared to the amount of control feed. No effects on the bodyweight and body condition score were observed.

3.5.2. Efficacy for cats

A total of three trials ²⁶ with cats of the same breed (Chartreux and British Shorthair) and sharing a common design were submitted. However, all trials were performed simultaneously in the same research facilities, with the same experimental design, with animals from the same breed and using the same feed. Therefore, the studies were not considered independent. The data of the three trials was pooled and analysed as a single study. ²⁷ The details on the study design are provided in Table 10. In all trials, 30 adult (1 to 5 years‐old) dogs from both sexes (15♂/15♀) were included. In each trial, from days 1 to 5 and 11 to 15, all dogs were offered at the same time a basal diet without (control) or with the additive at the proposed use level of 122 mg/kg (corresponding to 36 mg/kg of PAC) (confirmed by analysis; Table 8). The location of the bowl was randomised between days to avoid laterality. From day 6 to 10, a washout period was included, in which all dogs were offered only the basal diet with no additive. The location of the bowl was randomised between days to avoid laterality.

TABLE 10.

Trial design and use level of the efficacy trials performed in cats.

Total n° of animals (breed) sex	Total duration (phase I/washout/phase II)	Composition feed (form)	Groups
			mg additive/kg complete feed	(mg PAC/kg feed)
				Intended	Analysed
90 Chartreux and British Shorthair 1:1 ♂/♀	15 days (5/5/5)	Poultry meat, rice, maize (Kibbles)	0 155	0 46	0 46.6

Abbreviation: PAC, proanthocyanidins.

The health status of the animals was monitored throughout the study and the body weight and body score conditions were recorded daily. The given food and the remaining food were recorded daily for each individual animal, the eaten food and the intake ratio were calculated. The data were analysed with a repeated measures ANOVA using the individual animal as the experimental unit, and the diet, time and interaction between them as fixed effects. Significance level was set at 0.05.

Ninety adult (1–6 years‐old) cats from both sexes (45♂/45♀) were used. From days 1 to 5 and 11 to 15, all cats were offered at the same time a basal diet without (control) or with the additive at the proposed use level of 155 mg/kg (corresponding to 46 mg/kg of PAC) (confirmed by analysis; Table 10). From day 6 to 10, a washout period was included, in which all cats were offered only the basal diet with no additive.

The health status of the animals was monitored throughout the study and the body weight and body score conditions were recorded daily. The given food and the remaining food were recorded daily for each individual animal, the eaten food and the intake ratio were calculated. The data were analysed with a repeated measures ANOVA using the individual animal as the experimental unit, and the diet, time and interaction between them as fixed effects. Significance level was set at 0.05.

The results of the study showed that cats ate a higher amount of feed (32.9 g/day) containing 155 mg cranberry extract/kg (36 mg PAC/kg) compared to the amount of unsupplemented control feed (12.4 g/day). The intake ratio of the supplemented feed was also higher than the control feed (0.72 vs. 0.28). No effects on the bodyweight and body condition score were observed.

3.5.2.1. Conclusions on efficacy

Based on the data provided, the Panel concludes that the additive has the potential to be efficacious as a flavouring agent (palatability enhancer) in dogs and cats when added to feed at 122 and 155 mg/kg complete feed, respectively.

4. CONCLUSIONS

The additive cranberry extract is safe for dogs at 610 mg/kg complete feed and for cats at 155 mg/kg complete feed.

The additive is intended to be used only in feed for cats and dogs, and therefore, there is no need to perform an assessment of the safety for the consumer and the environment.

Regarding user safety, the additive is not irritant to skin or eyes but is a potential skin and respiratory sensitiser. Exposure of users by inhalation and dermal routes is considered a risk.

The additive has the potential to be efficacious as a flavouring agent (palatability enhancer) in dogs and cats when added to feed at 122 and 155 mg/kg complete feed, respectively.

5. RECOMMENDATION

Considering the specific sensitivity of cats to benzoic acid, the FEEDAP Panel recommends a review of the currently authorised levels of benzoic acid (125 mg/kg) in complete feed for cats.

ABBREVIATIONS

ADME

absorption, distribution, metabolism and excretion

ANOVA

analysis of variance

ANS

EFSA Scientific Panel on Additives and Nutrient Sources added to Food

BW

body weight

CAS

Chemical Abstracts Service

CFU

colony‐forming unit

CV

coefficient of variation

CYP

cytochrome P450

DAD

diode array detection

DM

dry matter

DMAC

4‐dimethylaminocinnamaldehyde

ECHA

European Chemicals Agency

ELS

extensive literature search

EMA

European Medicines Agency

ESI‐MS

electrospray mass spectrometry

EURL

European Union Reference Laboratory

FEEDAP

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

GSE

grape seed extract

GSKE

grape‐skin extract

HPLC

high performance liquid chromatography

MALFI–TOF

matrix‐assisted laser desorption/ionisation time‐of‐flight

MOE

margin of exposure

MS²

tandem mass spectrometry

MS/MS

tandem mass spectrometry

NDA

EFSA Scientific Panel on Dietetic Products, Nutrition and Allergies

NOAEL

no observed adverse effect level

NTP

National Toxicology Program

PAC

proanthocyanidin(s)

OECD

Organisation for Economic Co‐operation and Development

QSAR

quantitative structure–activity relationship

RH

relative humidity

TG

test guideline

UGT

UDP‐glucuronosyltransferase

UV

ultraviolet

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00585

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PANEL MEMBERS

Roberto Edoardo Villa, Giovanna Azimonti, Eleftherios Bonos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Ronette Gehring, Boet Glandorf, Maryline Kouba, Marta López‐Alonso, Francesca Marcon, Carlo Nebbia, Alena Pechová, Miguel Prieto‐Maradona, Katerina Theodoridou.

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. , Gehring, R. , Glandorf, B. , Kouba, M. , López‐Alonso, M. , Marcon, F. , Nebbia, C. , Pechová, A. , Prieto‐Maradona, M. , Theodoridou, K. , Bastos, M. d. L. , Benfenati, E. , Brantom, P. , Chesson, A. , … Dusemund, B. (2026). Safety and efficacy of a feed additive consisting of a proanthocyanidin‐rich dry extract from the fruit of Vaccinium macrocarpon Aiton (cranberry extract) for dogs and cats (ACEL pharma S.r.l.). EFSA Journal, 24(4), e10041. 10.2903/j.efsa.2026.10041