Safety and efficacy of a feed additive consisting of Solanum glaucophyllum leaf extract for dairy cows and other dairy ruminants (Herbonis Animal Health Gmbh)

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of Solanum glaucophyllum leaf extract (SGE) as a nutritional additive for dairy cows and other dairy ruminants. However, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) considered the glycosides of 1,25‐dihydroxycholecalciferol (1,25[OH]₂D₃) as the active substance and the bolus containing SGE‐derived 1,25[OH]₂D₃ as the preparation of the additive. The product is intended to be administered to dairy ruminants during the pre‐parturient (period from 9 days before calving to immediately before calving). The FEEDAP Panel concluded that the administration of one bolus, the preparation of the additive as applied in the animal studies evaluated, containing 500 μg of SGE‐derived 1,25[OH]₂D₃ during the pre‐parturient period is safe for cows. Owing to the lack of data, the Panel could not conclude on the safety for of a subsequent administration of a second bolus or on the safety of another SGE‐derived 1,25[OH]₂D₃ preparation for use in dairy ruminants other than cows (Bos taurus). The Panel considered that, under the specified conditions of use, the product is safe for the consumer and the environment. The bolus, a preparation containing SGE, as a source of the active substance, is not irritating to skin and eyes and it is not a sensitiser. Exposure via inhalation is unlikely. The Panel concluded that the administration of the bolus, the preparation of the additive as applied in the animal studies evaluated, containing 500 μg of SGE‐derived 1,25[OH]₂D₃ in a period from 9 days before calving to immediately before calving has the potential to prevent hypocalcaemia in dairy cows. Owing to the lack of data with another preparation, the Panel could not conclude on the efficacy in other dairy ruminants.

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 Herbonis Animal Health GmbH ² for the authorisation of the additive consisting of Solanum glaucophyllum leaf extract (SGE), when used as a feed additive for dairy cows and other dairy ruminants (category: nutritional additives; functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effect).

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). EFSA received directly from the applicant the technical dossier in support of this application. The particulars and documents in support of the application were considered valid by EFSA as of 28 May 2021.

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 S. glaucophyllum leaf extract (SGE), when used under the proposed conditions of use (see Section 3.2.6).

1.2. Additional information

The subject of the assessment is a product that consists of SGE, intended for use as a nutritional additive (functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effect) for dairy cows. SGE has not been assessed as a feed additive in the EU and is not authorised for use as a feed additive.

EFSA issued an opinion on the safety and efficacy of S. glaucophyllum standardised leaves when used as a feed material (EFSA FEEDAP Panel, 2015).

Waxy‐leaf nightshade meal (7.15.1), a product obtained by drying and grinding the leaves of S. glaucophyllum is included in the catalogue of feed materials. ³ S. glaucophyllum is included in the list of feeds intended for particular nutritional purposes (PARNUTS), for reduction of the risk of milk fever and subclinical hypocalcaemia in dairy cows (Commission Regulation (EU) No 2020/354). ⁴

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 SGE 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 active substance in animal feed. The Executive Summary of the EURL report can be found in Annex A. ⁶

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of SGE is in line with the principles laid down in Regulation (EC) No 429/2008 ⁷ and the relevant guidance documents: Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012a), Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018) and Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019).

3. Assessment

The application refers to SGE, a dry powder based on an ethanolic extract of dried S. glaucophyllum leaves (waxy‐leaf nightshade meal). SGE is a natural source of calcitriol (1,25‐dihydroxycholecalciferol; 1,25[OH]₂D₃), a potent metabolically active form of vitamin D₃. SGE is intended for use as a nutritional additive (functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effect) in feed for dairy ruminants in the pre‐parturient period to stabilise serum calcium concentration at the onset of lactation. SGE is to be used in a preparation in the form of an encapsulated, controlled‐release bolus.

3.1. Introduction

Vitamin D₃ is essential for life in higher animals, it is one of the primary biological regulators of calcium homeostasis. The biological role of vitamin D is mainly exerted by calcitriol, which is a secosteroid structurally related to steroid hormones and its function is performed by interacting with its cognate vitamin D receptor.

Calcitriol (also referred to as 1,25‐dihydroxycholecalciferol, 1α,25‐dihydroxyvitamin D₃ or 1,25‐dihydroxyvitamin D₃) is the most potent metabolite of vitamin D₃ with three hydroxyl groups. Calcitriol is synthesised in the body starting from 7‐dehydrocholesterol, which is converted in a first step, to cholecalciferol (vitamin D₃) in the skin after exposure to UV light. The vitamin is transported in the blood stream bound on a vitamin D binding protein, converted into 25‐hydroxyvitamin D₃ (25[OH]D₃) in the liver, and again hydroxylated at C1 mainly in the kidneys to form calcitriol by 1α‐hydroxylase activity (a mitochondrial cytochrome P450 enzyme). Hydroxylation at C24 results in the main inactive metabolites 24,25[OH]₂D₃ and 1,24,25[OH]₃D₃.

The synthesis of calcitriol is strongly regulated by homeostatic mechanisms. The activity of the 1α‐hydroxylase is regulated among others by calcitriol, which depresses the enzyme activity and promotes its own deactivation (by stimulation of 24‐hydroxylase). Also, the 25[OH]D₃ serum concentration affects the levels of 1α‐hydroxylase and 24‐hydroxylase in the kidneys. If the 25[OH]D₃ concentration is low, the body compensates by producing more parathyroid hormone (PTH), thereby stimulating 1α‐hydroxylase and depressing 24‐hydroxylase. Conversely, as 25[OH]D₃ concentrations rise, less 1α‐hydroxylase and more 24‐hydroxylase are required to keep circulating calcitriol in the correct balance. Consequently, circulating calcitriol does not correlate with the 25[OH]D₃ concentration (Nelson and Merriman, 2014).

PTH, low serum calcium and low serum phosphate stimulate the renal production of calcitriol. Calcitriol together with PTH enhances the absorption in the gastrointestinal tract of calcium and phosphate from the diet, increases renal tubular reabsorption of calcium and stimulates the release of calcium stored in the bones.

Besides to its action on calcium homeostasis, it has been demonstrated that calcitriol promotes fatty acid synthesis, inhibits lipolysis and increases energy efficiency. Calcitriol can modulate the activity of cytokines and may regulate cell turnover/differentiation (Rodriguez et al., 2014) and affects mammary physiology in cattle. The pluripotent secosteroid 1α,25[OH]₂D₃ initiates physiological responses of ≥ 36 cell types that possess the vitamin D receptor and a paracrine production of this substance was shown for ≥ 10 extrarenal organs (Norman, 2008).

3.1.1. Physiological background of the application

In the dry period of dairy cows (commonly 3–4 weeks before parturition), the calcium requirement is relatively low, only determined by the need of the growing fetus and maintenance requirement of the pregnant cow. The sudden production of milk after parturition (colostrum followed by normal milk) is an enormous challenge for the calcium metabolism. Twenty litres of milk would contain about 24 g calcium (approximately the same amount as 10 L colostrum). Feed intake is low providing about 10 g Ca/day. The total volume of circulating blood for a 650‐kg cow may be 34–39 L (52–60 mL/kg) body weight (bw_), providing between 3.4 and 4.3 g total Ca. There are endogenous mechanisms to avoid Ca‐blood level falling below critical concentrations (< 1.6 mmol calcium/L indicating the prevalence of milk fever), mainly skeletal release and maximising intestinal absorption. However, the mechanisms are not trained and often, particularly in multiparous cows because of a lower level of mobilisable Ca in the skeleton compared to primiparous, not sufficient to stabilise blood calcium.

Administration of calcitriol before parturition is expected to stabilise blood calcium and to provide enough Ca for milk production. The FEEDAP Panel already concluded in 2015 (EFSA FEEDAP Panel, 2015) that S. glaucophyllum leaves have ‘the potential to be efficacious in the prevention of milk fever in dairy cows. This conclusion is extended to other dairy ruminants’. However, the Panel reported data from Horst et al. (2003) who showed in a study that all cows (five in total) receiving calcitriol from Solanum leaves suffered from hypocalcaemia between 6 and 8 days after S. glaucophyllum treatment was ended, and one of them developed milk fever. High exogenous supply of calcitriol would have blocked the endogenous synthesis of calcitriol. The Panel interpreted the findings as an indication of the necessity of a phased withdrawal after treatment with calcitriol from Solanum leaves before parturition (successful prevention of milk fever) to avoid life‐threatening acute calcium deficiency in the period after cessation of calcitriol treatment. The Panel consequently mentioned that ‘Efficacy and tolerance of 1,25[OH]₂D₃ from S. glaucophyllum obviously depends on the conditions of use (dose, duration)’.

The current application refers to a preparation of calcitriol in the form of a bolus which is expected to release enough calcitriol in the period before parturition to prevent a critical decline of blood‐Ca and to provide later lower amounts of calcitriol for a longer period to allow the endogenous synthesis of calcitriol to recover (to reach the physiologically necessary level). The bolus – a controlled delayed release preparation of calcitriol from SGE – is applied to ensure an efficient and safe supplementation to dairy cows around parturition.

3.2. Characterisation

3.2.1. Manufacturing process

SGE is a dried aqueous ethanol extract of S. glaucophyllum leaves, a feed material naturally rich in glycosides of 1,25[OH]₂D₃. ⁸ SGE may be used as such or standardised by dilution with maltodextrin or other excipients (granulated or not) to contain the glycosides in a concentration corresponding to 50–160 mg 1,25[OH]₂D₃/kg (here and hereinafter given concentrations refer to the aglycone 1,25[OH]₂D₃). Dry leaves are extracted ■■■■■

The SGE containing the active substance calcitriol in a glycosylated form is used for formulation of standardised preparations providing a controlled delayed release of calcitriol in the gastrointestinal tract of dairy ruminants. Such a preparation is described in the application as a bolus consisting of mini‐boli.

3.2.2. Characterisation of the active substance

The main active components responsible for the vitamin D‐like activity of S. glaucophyllum are the glycosides of calcitriol. According to the previous opinion of the FEEDAP Panel (EFSA FEEDAP Panel, 2015) ‘Glycoside conjugation to hydroxy groups of the aglycone occurs at C1, C3 and C25. The molecular distribution of glycosyl moieties has been found to be 1–12 hexose units per aglycone; a mono‐glycoside as well as di‐ and tri‐glycosides have been identified’.

Calcitriol (synonym: 1,25‐dihydroxycholecalciferol; 1,25[OH]₂D₃) is defined by the International Pure and Applied Chemistry (IUPAC) name 9,10‐secocholesta‐5,7,11(19)‐triene‐1,3,25‐triol, the Chemical Abstract Service (CAS) number 32222–06‐3, the European Inventory of Existing Chemical Substances (EINECS) number 250–963‐8. The molecular formula of 1,25[OH]₂D₃ is C₂₇H₄₄O₃ and its molecular weight is 416.6 g/mol. The structural formula of the calcitriol is presented in Figure 1.

Figure 1.

Structural formula of calcitriol (1,25[OH]₂D₃) as aglycone

3.2.3. Characterisation of SGE, the source of calcitriol

The analysis of eight batches of the raw extract by ultraperformance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) showed an average 1,25[OH]₂D₃ content of 150 (range 129–178) mg 1,25[OH]₂D₃/kg (analysed as free 1,25[OH]₂D₃). ⁹ In four of the batches, the content of 25‐hydroxycholecalciferol (ranging 0.0282–0.0557 mg/kg) and cholecalciferol (ranging 0.0091–0.0133 mg/kg, corresponding to 364–531 IU vitamin D₃/kg) were also measured.

For standardisation, the raw SGE is diluted with a suitable carrier, e.g. maltodextrin. The standardised SGE is specified to contain 50–160 mg 1,25[OH]₂D₃/kg (analysed as free 1,25[OH]₂D₃). Analytical data of seven batches (two as dry powder, five as dry granulate) were submitted. Values of the dry powder were 55 and 64 mg 1,25[OH]₂D₃/kg, and those of the granulate between 50 and 54 mg 1,25[OH]₂D₃/kg (mean 52 mg). ¹⁰

The content of alkaloids (e.g. α‐solanine and α‐chaconine) was < 10 mg/kg each in four batches of raw SGE. ¹¹ Three batches of raw SGE were analysed for undesirable substances, ¹² i.e. ethanol (< 0.3–0.5%), arsenic (0.029–0.10 mg/kg), cadmium (0.0015–0.10 mg/kg), mercury (0.009–0.08 mg/kg), lead (0.026–0.10 mg/kg), aflatoxins B1, B2, G1 and G2 (< 1 μg/kg each, their sum < 4 μg/kg), ochratoxin A (2–5 μg/kg), zearalenone (≤ 0.01 μg/kg) and deoxynivalenol (0.015–0.03 μg/kg). Pesticides were not detected in a multiresidue analysis. Total aerobes were ≤ 120 colony forming units (CFU)/g, yeasts and moulds ≤ 100 CFU/g, bile‐tolerant Gram‐negative strains ≤ 10 CFU/g, Escherichia coli and coliforms not detected in 1 g, and Salmonella spp. not detected in 25 g. Polychlorinated dibenzo‐p‐dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin‐like polychlorinated biphenyls (PCBs) were analysed in four batches of raw SGE. The calculated (upper bond) level of dioxins and the sum of dioxins and dioxin‐like PCBs were 0.0574–0.0609 ng WHO‐PCDD/F‐TEQ/kg and 0.0919–0.0976 ng WHO‐PCDD/F‐PCB‐TEQ/kg. ¹³ All values were of no concern.

SGE is a dry, beige or brown powder, soluble in water. The applicant submitted also data on bulk density (300–600 kg/m³), dusting potential (135 mg/m³ air for one batch by Stauber–Heubach) ¹⁴ and particle size (two batches, mean 0.1 μm, 80% of particles between 0.06 and 0.2 mm; sieve method) ¹⁵ for a standardised SGE (with maltodextrin).

3.2.4. Characterisation of the additive

The applicant submitted information on one example, Solbovine® Once (SBO), formulated for dairy cows or small ruminants. ¹⁶

The bolus for dairy cows (■■■■■ of ■■■■■) contains 500 μg 1,25[OH]₂D₃ from either raw SGE, or standardised SGE with maltodextrin or other suitable carriers. SBO is composed of ■■■■■ mini‐boli (specified composition given in Section 3.2.4.1)■■■■■ of which are intended for immediate release (IR‐mini‐bolus; ■■■■■ for slow release (SR‐mini‐bolus; ■■■■■). The applicant provided a typical composition of SBO indicating a SGE content of ■■■■■ per IR‐mini bolus ■■■■■ and ■■■■■ per SR‐mini bolus ■■■■■. Data on the batch‐to‐batch variation was provided for seven batches of SBO and showed an average 1,25[OH]₂D₃ content of 559 (range: 469–745) μg per bolus (concentration levels given as measured for free 1,25[OH]₂D₃). ¹⁷ The bolus is reported to contain ■■■■■

One bolus for dairy cows (a composite sample of seven batches) was analysed for undesirable substances. The contents of lead (0.23 mg/kg bolus), cadmium (0.07 mg/kg), mercury (< 0.005 mg/kg), arsenic (2.5 mg/kg), nickel (4.6 mg/kg), fluorine (137 mg/kg), dioxins (0.0612 ng WHO‐PCCD/F‐TEQ/kg) and PCBs (0.0376 ng WHO‐PCB TEQ/kg) ¹⁸ were of no concern.

The bolus formulated for small ruminants is basically modified to account for the total amount of 1,25[OH]₂D₃. It has also a mass of ■■■■■ and contains ■■■■■, derived from a proposed dose of 1 μg/kg body weight and a default value of 60 kg body weight for a small dairy ruminant. The applicant provided a typical composition of SBO bolus for small ruminants indicating a SGE concentration of ■■■■■ 1,25[OH]₂D₃) per IR‐mini bolus ■■■■■ and ■■■■■ per SR‐mini bolus ■■■■■ 1,25[OH]₂D₃). The composition in terms of other formulating ingredients is similar but adapted to compensate for the lower content of SGE.

3.2.4.1. Characterisation of the mini‐boli as parts of the large bolus

The mini bolus consists of SGE and different feed materials and technological feed additives as carriers. The content of SGE and the carriers varies according to weight and the intended 1,25[OH]₂D₃ content of the mini bolus. Table 1 illustrates the composition of the different mini boli when SGE contains 150 mg 1,25[OH]₂D₃/kg. ¹⁹

Table 1.

Quantitative specified composition of mini boli for dairy cows or for small dairy ruminants. The example is based on a Solanum glaucophyllum leaf extract (SGE) containing 150 mg 1,25[OH]₂D₃/kg

Release characteristics	Dairy cows		Small dairy ruminants
	IR	SR	IR	SR
■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
■■■■■	■■■■■
■■■■■		■■■■■
■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
■■■■■	■■■■■
■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
■■■■■	■■■■■	■■■■■	■■■■■	■■■■■

IR: immediate release; SR: slow release.

3.2.5. Stability and homogeneity

The applicant proposed a shelf‐life of 2 years for SGE stored in a cool and dry environment in original, closed, light‐tight packaging. To support this shelf‐life the applicant submitted data on two batches of a standardised SGE, which were stored at 25°C/60% RH (relative humidity) and 40°C/75% RH. No losses were found on the content of 1,25[OH]₂D₃ after 12 months. ²⁰ One batch of raw SGE was stored under dry conditions at 25°C for 36 and 48 months. The recovery of 1,25[OH]₂D₃ was 96% and 115% of time zero, respectively. ²¹

SGE is supplied to the animals in the form of a controlled released bolus (SBO), and it is not used in premixtures. Stability and homogeneity of SGE in the mini‐boluses present in SBO (SR and IR, from two batches) was assessed in mini‐boluses produced in 2017, stored under warehouse conditions at ca. 25°C and retested after 3 years storage. Data showed a mean recovery of ■■■■■ and ■■■■■ for SR‐ and IR‐mini boli, respectively. ²²

Homogeneity was examined by calcitriol analysis of 10 IR‐ and 10 SR‐mini‐boli. The coefficient of variation (CV) was 2% for IR‐mini‐boli (mean: ■■■■■) and 6% for SR‐mini‐boli (mean: ■■■■■).

3.2.6. Conditions of use

The applicant proposes to administer the active substance in a preparation to form a bolus, the recommended application form of the additive. It should be given directly to pre‐parturient dairy ruminants.

The bolus formulated for dairy cows, containing about 500 μg 1,25[OH]₂D₃, is recommended to be administered once in a period from 9 days before calving to immediately before calving, ideally 1–2 days before calving. If the cow has not calved within 9 days after bolus administration, the supply of a second bolus is recommended. ²³

The same administration scheme is proposed for other dairy ruminants. The bolus is formulated to deliver 1 μg 1,25[OH]₂D₃ per kg body weight, from 9 days before parturition to immediately before parturition, ideally 1–2 days prior to parturition.

3.3. Safety

Natural occurrence of poisoning in cattle by consumption of Solanum malacoxylon/glaucophyllum and the toxicity symptoms were described in detail in the former opinion of the FEEDAP Panel (EFSA FEEDAP Panel, 2015).

3.3.1. Toxicological studies

The applicant submitted two in vitro and one in vivo genotoxicity studies, two repeated dose toxicity studies, which have been already evaluated in the previous assessment (EFSA FEEDAP Panel, 2015). In addition, a new 90‐day study and two additional studies, one in vitro and another in vivo, on the oestrogenic activity of SGE, were submitted.

In all studies, the test item was a standardised water‐soluble dried powder of a water ethanol SGE containing 64.8 mg 1,25[OH]₂D₃/kg. The FEEDAP Panel notes that the 1,25[OH]₂D₃ content of test item complies with the specification of the additive under evaluation.

3.3.1.1. Genotoxicity studies

Based on the data provided ²⁴ (a gene mutation test in five Salmonella Typhimurium strains, TA 98, TA 100, TA 102, TA 1535 and TA 1537), a mouse lymphoma gene mutation assay in cultured mammalian cells (L5178Y TK +/−) and an in vivo bone marrow micronucleus test in mice, the FEEDAP Panel considered that ‘the water‐soluble extract of S. glaucophyllum containing 64.8 μg 1,25(OH)₂D₃/g is non‐genotoxic’ (EFSA FEEDAP Panel, 2015).

The studies were newly evaluated also for their compliance with the current EFSA Guidance documents. The FEEDAP Panel confirmed its previous conclusions.

3.3.1.2. Repeated dose oral toxicity studies

The two repeated dose toxicity study in rats with a duration of 2 and 4 weeks ²⁵ were already assessed in 2015. The FEEDAP Panel concluded that ‘The identification of a safe level of exposure is limited by the absence of repeated dose studies of duration longer than four weeks and the absence of any reproduction toxicity studies other than the developmental toxicity studies’ (EFSA FEEDAP Panel, 2015).

The applicant submitted in the current dossier a 90‐day repeated oral dose study in rats. ²⁶

Groups of 10 Crl:CD(SD) rats received the test item, a SGE preparation containing 64.8 mg 1,25[OH]₂D₃/kg in 0.8% hydroxypropylmethyl cellulose, by gavage for 91 days at doses of 0, 5, 15 and 35 mg/kg bw per day. An additional group received 1.75 mg 1,25[OH]₂D₃/kg bw per day. Additional groups of five rats of each sex were included for 4‐week reversal studies at control, high dose and positive control. Satellite groups of additional nine rats of each sex at each treatment except for the control (only 3 per sex were included for the control group) were included for blood sampling only. The study was conducted in compliance with Good Laboratory Practice (GLP) but made no claim to comply with Organisation for Economic Co‐operation and Development (OECD) Technical Guidance (TG). The protocol included full clinical observation of animals throughout the study including ophthalmoscopy, haematology, clinical chemistry and urine measurements. A simple auditory test was performed but not a complete functional observational battery. All animals from the main and reversal groups were subject to full necropsy with organ weights, bone marrow samples and tissues retained for possible histopathological examination. Histopathology was initially confined to control, high‐dose and positive control groups. Following findings in femur and kidney these tissues were examined microscopically in all the other groups.

There was one death during the study, which was considered unrelated to treatment, otherwise there were no treatment‐related effects on behaviour and appearance of the animals. Body weight was slightly lower than that of controls in groups receiving 15 or 35 mg SGE/kg bw per day from about week 11 onwards but terminal body weight was only statistically different from controls for the male groups. The group receiving 5 mg SGE/kg bw per day showed no differences from controls. Body weight of positive controls was lower than that of controls from around week 6 onwards. The highest dose group showed no difference from control at the end of the reversal period while body weight of the positive control was still lower than that of controls after that period. Food and water intake, and results of ophthalmoscopy and auditory examinations were not affected by treatment with SGE or the positive control material.

Haematological data showed some statistically significant differences between treated and control groups (elevated haemoglobin in high‐dose females after reversal; elevated reticulocytes in high‐dose males after reversal; reduced haematocrit in high‐dose males and reduced thromboplastin time and mean corpuscular volume (MCV) in high‐dose females at 90 days), but these were not considered to be related to treatment. Haematological data showed no differences between the treated and positive control groups which could be attributed to treatment. Serum calcium levels were increased in all male treated groups and in the highest two female groups compared with controls. Serum potassium levels were increased in both sexes at the highest dose. The positive control group males showed similar increases, but these were not seen in the females at the same treatment. These differences were not found after the recovery period. Urine pH was lower than that of controls for all treated groups and for the positive control group. These differences were not present after the recovery period.

At necropsy, there were not relevant differences between treated and control groups. Although some differences were present in organ weights between treated and control groups (90‐days: increased relative kidney weight in females; decreased relative heart weight in females), these were not considered to be related to treatment. Bone marrow examinations showed no difference between treated groups, including positive control and untreated controls. Histopathological examination showed treatment‐related hyperostosis of the femur and calcium deposits in the kidneys in both sexes at all doses and in the positive control group. The femur hyperostosis was not present in the lowest dose group after the recovery period.

Although after recovery, there were no signs of adverse effects at the lowest dose tested, the Panel agreed that a NOAEL could not be derived from this study for a substance influencing the calcium metabolism. In addition, considering the species differences, the study in rat was considered not relevant for the safety assessment in ruminants and other target species.

3.3.1.3. Other toxicity studies

Two studies on the oestrogenic activity of SGE (containing 64.8 mg 1,25[OH]₂D₃/kg) were submitted by the applicant. The first study ²⁷ consisted of an in vitro E‐Screen test on the MCF7 breast cancer cell line. The positive control compound (17β‐oestradiol) exerted the expected effects. SGE did not show oestrogenic activity up to a concentration of 5,000 μg/mL (0.5, 5, 50, 500 and 5,000 μg/mL were tested). It was not possible to test a higher concentration (50,000 μg/mL), because the compound formed precipitates at the surface of the test plates.

The second study ²⁸ consisted of an in vivo Allen Doily study (stated as non‐GLP, not according to any OECD TG). The study used a total of 48 spayed female rats (ca. 10–14 weeks of age) and included 8 experimental groups (6 animals/group): 1 – Negative Control (vehicle, oral administration) 2 – SGE at 104 mg/kg bw with oral administration (= 6.74 μg 1,25[OH]₂D₃/kg bw), and Groups 3–8: positive controls (with doses between 0.02 up to 25 μg/animal of oestradiol, administered either orally or subcutaneously). Animals received the corresponding dose twice daily for 2 consecutive days. On each of the 3 following days after the last administration, a vaginal smear was taken from each animal to assess the oestrogenic activity. The positive controls receiving subcutaneous injections of 2.5, 7.5 or 25 μg oestradiol/animal, respectively, resulted in the expected oestrogenic activity. SGE did not reveal any oestrogenic activity. Vaginal smears of groups 3, 4 and 5 (positive controls with 0.02, 0.1 or 0.5 μg oestradiol administered orally) did not reveal any oestrogenic activity.

3.3.1.4. Conclusions on toxicology

The FEEDAP Panel confirms the conclusion of the previous assessment that the water‐soluble extract of S. glaucophyllum leaves containing 64.8 mg 1,25[OH]₂D₃/kg is non‐genotoxic. From the available data set, a NOAEL cannot be identified. These conclusions are extended to the additive under assessment.

3.3.2. Safety and efficacy for the target species

Two studies on dairy cows were submitted. One study (study 1) investigated the effect of 1,25[OH]₂D₃ glycosides in different doses and preparations on blood pharmacokinetics and pharmacodynamics in pregnant dry Holstein and Red Holstein dairy cows. The other study (study 2) examined the effect of 1,25[OH]₂D₃ glycosides released from a bolus on mineral status in periparturient primiparous and multiparous dairy cows and their newborn calves. Extended summaries of both studies can be found in the Appendices A and B, respectively. The text below reports to the findings which are immediately relevant for the assessment of the application. No data were provided on small ruminants.

3.3.2.1. Study 1 ²⁹

The study was done with dry pregnant dairy cows (Holstein and Red Holstein, ³⁰ 220–257 days post‐insemination ³¹ ) which were allocated to 6 treatment groups. Group size was 5 cows and the study duration was 14 days. The experiment ended circa 24 days prior to calving but some parameters were followed up until 28 days post‐partum. Cows had ad libitum access to hay and were fed daily 300 g/cow of a mineralised concentrate, providing 3,245 IU vitamin D₃/cow and day. The calcitriol doses from SGE given in bolus once at start were 200, 300, 500 and 1,000 μg (two 500 μg boli). The preparations contained mini‐boli, prepared for immediate (IR) and/or slow release (SR, both ■■■■■) of the active substance.

The results showed that the mean dry matter intake (13.2 kg DM/day), water intake (78.6 L/day) and body weight (732 kg at day 4; 741 kg at day 14) as well as body condition score (3.0 at days −4 and 14) were not affected by the treatments (p > 0.10).

No treatment effects were observed for the pharmacokinetic parameters C_max (peak observed for 1,25[OH]₂D₃ or mineral concentration), T_max (time of peak concentration), and AUC (area under the concentration–time curve) of plasma 1,25[OH]₂D₃ and Ca (also for plasma P), except that the C_max and AUC of Ca were 5% higher (p < 0.05) for the group receiving 1,000 μg 1,25[OH]₂D₃ (2 boli with 500 μg each) than for a group receiving 300 μg from ■■■■■ slow release mini‐boli.

Bolus application (calcitriol treatment) increased blood serum concentrations of

• •1,25[OH]₂D₃ from the initial baseline (p < 0.001) between experimental day (d) d0.5 and d5 with a peak from d1.25 to d2, before reaching values comparable (p > 0.10) to the initial baseline at d9
• •Ca from the initial baseline (p < 0.001) between d0.5 and d11 with a peak from d2 to d4, before reaching values comparable (p > 0.10) to the initial baseline at d14
• •P from the initial baseline between d1 (p < 0.001) and d11 (p < 0.05) with a peak from d3 to d5, before reaching values comparable (p > 0.10) to the initial baseline at d14
• •but decreased serum Mg from the initial baseline between d1 and d9 (p < 0.001) with minima from d2 to d4, before reaching values comparable (p > 0.10) to the initial baseline at d11

Although the boluses containing higher amounts of 1,25[OH]₂D₃ (500 μg) were also more effective in producing a response in blood serum 1,25[OH]₂D₃, there was no clear difference between dosages in the response of serum Ca, P and Mg.

No time×treatment interaction for the responses of Ca, P and Mg was found (p > 0.10). Finally, contrast analysis revealed that blood serum Ca, P and Mg response did not differ (p > 0.10) between ■■■■■ boluses. Serum Ca response tended (p < 0.10) to be higher in 2 × 500 compared to 500, whereas serum P and Mg response did not differ (p > 0.10) between 500 and 2 × 500.

The end of the experiment was 24.4 ± 11.2 days before the effective calving, without differences between treatments (p > 0.10). Also, no differences were seen in the endpoints measured post‐calving, including dry matter intake, milk production, and serum concentrations of Ca, P and Mg in cows, as well as the body weight of newborn calves.

3.3.2.2. Study 2 ³²

In study 2, a 2 × 2 factorial design was applied, 2 factors for treatment (control ‐ bolus) and 2 factors for parity (primiparous ‐ multiparous), resulting in 4 experimental groups (Control‐Primi, Bolus‐Primi, Control‐Multi, Bolus‐Multi). The additive was administered in the form of a bolus (500 μg 1,25[OH]₂D₃ from SGE provided by ■■■■■ IR mini‐boli and ■■■■■ SR mini‐boli) 3–4 days prior to the expected calving date. Animals remained in the study until 21 days after actual calving (day 0), groups size was 6 cows. Three cows (Control‐Multi) showed clinical signs of hypocalcaemia with blood serum Ca below the defined threshold of 1.6 mmol/L, they were excluded from the study and replaced by other cows of the same parity.

A basal diet consisting of 75% hay and 25% maize silage (DM basis) was fed ad libitum. In addition, different protein concentrates and mineral feeds were given for the pre‐calving and the lactation period. Dietary cation anion difference (DCAD) was 582 and 499 meq/kg DM for the two periods. The high DCAD values, which reflect typical situations occurring in herbage‐based diets, are expected to increase the risk of Ca deficiency after calving.

A multitude of parameters were examined. The main endpoints were the concentrations of Ca, followed by 1,25[OH]₂D₃, PTH, P and Mg in blood at several days of the study.

Multiparous cows had a higher (p < 0.001) feed intake on a weekly basis (+53% pre‐partum, +37% in lactation). Multiparous cows were about 135 kg heavier (p < 0.001) than primiparous cows before and after calving. Milk yield was higher (p < 0.001) in multiparous cows (40 kg/day as a mean of weeks 2 and 3) than in primiparous cows (26 kg/day). Bolus administration did not have a significant effect (p > 0.10) on any of the cows' performance data during the study.

The relevant endpoint serum Ca remained stable (mean 2.25 ± 0.15 mmol/L) over the entire experimental period in Control‐Primi and Bolus‐Primi cows but varied significantly (p < 0.001) in Control‐Multi and Bolus‐Multi cows. From d0.5 to d2 after calving, the Ca concentrations of Control‐Multi cows dropped by 40% compared to the initial value and was lower compared to those of the other groups within d1 and d1.5. In Control‐Multi, Ca declined from 2.19 mmol/L on d‐2 to 1.66 on d0.5, 1.39 at d1 and to 1.35 mmol Ca/L at d1.5 and returned to a physiological level (around 2 mmol/L) at d4. In the Bolus‐Multi, a peak was seen on d‐2, and after this peak, the Ca concentrations returned to values similar to the initial values until the end of the experiment. Control‐Primi, Bolus‐Primi and Bolus‐Multi cows did not have, at any time, mean blood serum Ca concentrations below 2.00 mmol/L and none had values higher than 3.00 mmol/L. Serum P concentrations behaved similar to those of Ca.

Whereas 1,25[OH]₂D₃ concentration remained constant in Control‐Primi and Bolus‐Multi cows between d − 4 and d15, there was a temporary increase in Bolus‐Primi around calving (from d − 2 until d1) and in Control‐Multi after calving (from d1 until d2) compared to d − 4 and d8 − d15, respectively (p < 0.01).

The PTH serum concentration remained stable over time in all groups, except in Control‐Multi, in which PTH significantly increased from 12 pg/mL at d‐4 to 69 pg at d0.5 and 72 pg at d1.5.

The bolus administration had no effect (p > 0.10) on calf blood serum minerals, 1,25[OH]₂D₃ and IGG concentrations. All serum Ca concentrations of calves were within the normal range (2.0–3.0 mmol/L).

3.3.3. Synopsis of the two studies with dairy cows

In the first study, oral application of a bolus containing 1,25[OH]₂D₃ glycosides resulted in a rapid increase of blood serum 1,25[OH]₂D₃, followed by increased Ca and P and decreased Mg. Although higher doses of 1,25[OH]₂D₃ glycosides tended to give a more pronounced response in blood serum 1,25[OH]₂D₃, there was no clear difference between dosages (200–500 and 2 × 500 μg cow) in the response of Ca, P and Mg. The mode of action of 1,25[OH]₂D₃ remains unclear (bone mineral mobilisation, intestinal Ca absorption or both).

Under the conditions of this study, no adverse effects of 1,25[OH]₂D₃ at any dose were observed. However, the study ended about 3 weeks before parturition and the strong challenge of the Ca‐metabolism and its regulating factors in the close period at and after parturition could not be simulated in this study. Therefore, no conclusions on the safety of the additive when administered at the conditions of use (administration 9 or less days before parturition) can be drawn.

In the second study and under the experimental conditions applied, primiparous cows did not show any sign of subclinical hypocalcaemia (< 2.0 mmol Ca/L) following calving. An effect of bolus application on stabilising blood calcium could therefore not be expected. This is confirmed by the experimental data; however, absorption of calcitriol was shown by a response of blood 1,25[OH]₂D₃ after bolus administration.

The situation was different in multiparous cows. Their higher daily milk yield compared to primiparous cows induced a 57% higher Ca export via milk (36.8 vs. 23.5 g/day). In addition to the 3 discarded Control‐Multi cows with clinical signs of hypocalcaemia, the Control‐Multi cows of the study showed blood Ca and P evolution profiles and increasing blood PTH and 1,25[OH]₂D₃ concentrations, all indicative for an activation of Ca metabolism of these cows to improve intestinal Ca absorption and bone mineral resorption. Under these conditions, the application of the calcitriol bolus to multiparous cows was successful as blood Ca, P and PTH concentrations remained stable and similar to primiparous cows around calving. Between d4 and d22, blood Ca never felt below 2 mmol/L in the bolus‐treated cows. This suggests that the continuous release of 1,25[OH]₂D₃ glycosides from the bolus prevented the potential delayed occurrence of hypocalcaemia associated with abrupt withdrawal of vitamin D and its metabolites supply, such as described in the previous FEEDAP opinion.

Calving process, mineral concentrations in colostrum and milk and blood serum mineral concentrations of the offspring remained generally unaffected in primiparous or in multiparous cows by the administration of the calcitriol bolus before calving.

In both studies, elevated blood levels of Ca (and of 1,25[OH]₂D₃) as a result of bolus administration returned back to initial levels after 14 days. Toxicity symptoms of exogenous 1,25[OH]₂D₃ in target animals are characterised mainly as calcinosis, a calcification of tissues (vessels) and organs, as described (EFSA FEEDAP Panel, 2015), after long term exposure to 1,25[OH]₂D₃ by consumption of plants containing the metabolite (e.g. S. malacoxylon/glaucophyllum, Cestrum diurnum and Trisetum flavescens). Taking all together and considering that acute effects were not seen also at a double dose, it appears reasonable to consider the singular administration of a 1,25[OH]₂D₃ bolus in one lactation as safe for dairy cows. However, no data were provided with the subsequent administration of a second bolus as recommended in the conditions of use, if the cow has not calved within 9 days after bolus administration. The lack of such data on the effect of a prolonged administration of calcitriol around calving on the endogenous synthesis of calcitriol and its likely impact on the calcium homeostasis prevents a conclusion on the safety of this treatment.

3.3.4. Conclusions on the safety and efficacy for the target species

The administration of the bolus containing 500 μg of SGE‐derived 1,25[OH]₂D₃ as glycosides at 3–4 days prior expected calving has the potential to prevent hypocalcaemia in multiparous cows fed a high DCAD diet. It prevents the critical depression of blood Ca and P following parturition particularly in high yielding multiparous cows. Primiparous cows were less sensitive to a potential depression of blood Ca and P after calving. Consequently, an effect of calcitriol bolus administration as seen in multiparous cows could not be demonstrated in primiparous cows.

No potentially adverse effects of the administration of the 1,25[OH]₂D₃ bolus were seen within the first 3 weeks of lactation. Since vitamin D toxicity is generally observed after long time application, it can also be concluded that the singular application of the calcitriol bolus containing 500 μg of SGE‐derived 1,25[OH]₂D₃ is safe for periparturient cows, when administered ≤ 9 days before calving. Owing to the lack of data in the relevant physiological period, the Panel is not in the position to conclude on the safety of the subsequent administration of two boluses to the periparturient cows.

The conclusions on the safety and efficacy apply to the bolus, the preparation of the additive as applied in the animal studies evaluated.

No data were provided on other dairy ruminants. Therefore, no conclusion on safety and efficacy of SGE derived 1,25[OH]₂D₃ can be drawn.

3.3.5. Safety for the consumer

In its previous assessments on vitamin D₃ supplementation of feedingstuffs (EFSA FEEDAP Panel, 2012b, 2013, 2014), the FEEDAP Panel concluded that the use of vitamin D₃ in animal nutrition at the currently authorised maximum dietary content is of no concern to consumer safety. The FEEDAP Panel also concluded that the use of S. glaucophyllum standardised leaves as a feed material did not increase the concentration of 1,25[OH]₂D₃ in animal tissues compared with feed supplementation with vitamin D₃. Therefore, the FEEDAP Panel concluded that the use of S. glaucophyllum in animal nutrition is safe for consumers (EFSA FEEDAP Panel, 2015). Furthermore, consumers will have limited exposure to 1,25[OH]₂D₃ via dairy foods/milk originating from dairy ruminants, bearing in mind that the SGE bolus is given to dry cows and colostrum produced immediately post‐partum is not intended for human consumption. Therefore, under the proposed conditions of use, the additive is considered safe for consumers.

3.3.6. Safety for the user

The applicant made reference to the data assessed by the Panel on the dried S. glaucophyllum leaves (EFSA FEEDAP Panel, 2015). The effects of dried S. glaucophyllum leaves containing minimum 10 mg 1,25[OH]₂D₃/kg on skin/eyes were evaluated in an acute dermal irritation/corrosion test, an acute eye irritation/corrosion test and a skin‐sensitising test compliant with OECD guidelines 404, 405 and 429, respectively. The studies showed that dried S. glaucophyllum leaves are not irritant to skin/eyes nor a dermal sensitiser. Dried S. glaucophyllum leaves constitute the raw material used for the manufacture of SGE, hence these data may be representative of SGE.

The preparation intended to be used prevents the user to inhale the dust and/or be in contact with the active substance. Therefore, the active substance used in the preparations described in the application is of no concern for user safety.

The FEEDAP Panel concludes that the bolus, a preparation containing SGE, as a source of the active substance, is not irritating to skin and eyes and it is not a sensitiser. Exposure via inhalation is unlikely.

3.3.7. Safety for the environment

In ruminants, 1,25[OH]₂D₃‐glycosides are deglycosylated in the rumen to 1,25[OH]₂D₃. Therefore, only negligibly amounts of glycosylated 1,25[OH]₂D₃ are excreted. This fraction is hydrolysed and oxidised by light and air (EFSA FEEDAP Panel, 2015). The aglycone 1,25[OH]₂D₃ is extensively metabolised in the animal and excretion products (mainly calcitroic acid) are devoid of vitamin activity. Considering the above, and the fact that vitamin D is widely distributed in plants and animals as a result of endogenous synthesis; it is susceptible to oxidation by light and oxygen (EFSA FEEDAP Panel, 2012a, 2013, 2014), the use of SGE in animal nutrition at the proposed conditions of use will not pose a risk to the environment.

The bolus described by the applicant, SBO, contains SGE, feed materials and feed additives as carriers, therefore no safety concerns for the environment would raise from that formulation.

3.4. Post‐market monitoring

The FEEDAP Panel considers that there is no need for specific requirements for a post‐market monitoring plan other than those established in the Feed Hygiene Regulation ³³ and Good Manufacturing Practice.

4. Conclusions

The active substance, 1,25[OH]₂D₃ in form of glycosides from an extract of Solanum glaucophyllum leaves, is contained in a preparation, a bolus ensuring a controlled and delayed release of the active substance in the gastrointestinal tract of ruminants.

The administration of one bolus, the preparation of the additive as applied in the animal studies evaluated, containing 500 μg of SGE‐derived 1,25[OH]₂D₃ during the pre‐parturient period (from 9 days before calving to immediately before calving) is safe for dairy cows. Owing to the lack of data the Panel is not in the position to conclude on the safety (i) of the subsequent administration of a second bolus as recommended by the applicant, if the cow has not calved within 9 days after bolus administration and (ii) of another SGE‐derived 1,25[OH]₂D₃ preparation intended for use in dairy ruminants other than cows (Bos taurus).

The Panel considers the use of the product to be safe for the consumer.

The bolus, a preparation containing SGE, as a source of the active substance, is not irritating to skin and eyes and it is not a sensitiser. Exposure via inhalation is unlikely.

The preparations intended for use in ruminants as described in the application are considered not to raise safety concerns for the environment.

The administration of the bolus, the preparation of the additive as applied in the animal studies evaluated, containing 500 μg of SGE‐derived 1,25[OH]₂D₃ in a period from 9 days before calving to immediately before calving has the potential to prevent hypocalcaemia in dairy cows. Owing to the lack of data, the Panel cannot conclude on the efficacy in other dairy ruminants.

5. Recommendation

Considering that 25‐hydroxycholecalciferol depresses the activity of 1α‐hydroxylase in the kidney, the simultaneous use of SGE and 25‐hydroxycholecalciferol should be avoided.

6. Documentation provided to EFSA/Chronology

Date	Event
09/04/2021	Dossier received by EFSA. SGE (Solanum glaucophyllum leaf extract) for dairy cows for milk production and other dairy ruminants. Submitted by Herbonis Animal Health Pen & Tec Consulting S.L.U.
09/04/2021	Reception mandate from the European Commission
28/05/2021	Application validated by EFSA – Start of the scientific assessment
30/09/2021	Comments received from Member States
21/09/2021	Reception of the Evaluation report of the European Union Reference Laboratory for Feed Additives
27/10/2021	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
04/02/2022	Reception of supplementary information from the applicant ‐ Scientific assessment re‐started
29/06/2022	Opinion adopted by the FEEDAP Panel. End of the Scientific assessment

Abbreviations

AUC

area under the curve

BW

body weight

CAS

Chemical Abstracts Service

CFU

colony forming unit

C_max

maximum plasma concentration

CV

coefficient of variation

DCAD

dietary cation anion difference

DM

dry matter

EINECS

European Inventory of Existing Chemical Substances

EURL

European Union Reference Laboratory

FEEDAP

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

GLP

Good Laboratory Practice

IR

immediate release

IUPAC

International Union of Pure and Applied Chemistry

LOQ

limit of quantification

MCV

mean corpuscular volume

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Co‐operation and Development

RH

relative humidity

PARNUTS

feeds intended for particular nutritional purposes

PCBs

dioxin‐like polychlorinated biphenyls

PCDD

polychlorinated dibenzo‐p‐dioxins

PCDF

polychlorinated dibenzofurans

PTH

parathyroid hormone

SG

Solanum glaucophyllum

SGE

Solanum glaucophyllum leaf extract

SR

slow release

TG

Technical guidance

T_max

time of highest peak concentration

UPLC–MS/MS

ultraperformance liquid chromatography–tandem mass spectrometry

WHO

World Health Organization

Appendix A – Study on the effects of the additive on the pharmacokinetics and pharmacodynamics in pregnant dry dairy cows

The applicant submitted a research report entitled ‘Effect of 1,25‐dihydroxycholecalciferol‐glycosides given as a rumen bolus on blood pharmacokinetics and pharmacodynamics in dry dairy cows’. The aim of this study was to determine the time response of blood parameters following the application of a bolus filled with tablets containing 1,25[OH]₂D₃‐glycosides. The source of 1,25[OH]₂D₃ was an extract of glycosides from Solanum glaucophyllum leaves (SGE).

The experimental research protocol was approved by the Office for Food Safety and Veterinary Affairs and all procedures were conducted in accordance with the Swiss Ordinance on Animal Protection and the Ordinance on Animal Experimentation.

Thirty pregnant dry dairy cows (Holstein and Red Holstein, ³⁰ 220–257 days post insemination ³¹ ) were allocated 4 days prior experimental start within each bloc to 5 blocs with 6 treatments each. Three groups were treated with one bolus/cow containing 200 μg 1,25[OH]₂D from ■■■■■ IR‐mini‐boli (group 200(2)), 300 μg from ■■■■■ SR‐mini‐boli (group 300(8)) and 500 μg 1,25[OH]₂D₃ from ■■■■■ IR‐ + ■■■■■ SR‐mini‐boli, respectively (group 500(■■■■■)). The boli of 2 other groups contained ■■■■■ SR‐mini boli, one bolus providing 300 μg 1,25[OH]₂D₃ from ■■■■■ SR‐mini‐boli (group 300(8)c), and one bolus providing 500 mg 1,25[OH]₂D₃ from ■■■■■ IR‐ + ■■■■■ SR‐mini‐boli (group 500(■■■■■)c). An overdose group received 2 boli each containing 500 μg 1,25[OH]₂D₃ from ■■■■■ IR‐ + ■■■■■ SR‐mini‐boli (group 2 × 500(■■■■■)). The animals were allocated to five blocks according to parity (primiparous, multiparous) and to expected calving date.

Cows had ad libitum access to hay ³⁴ (2nd seasonal harvest; botanical composition: 78% graminea, 19% legumes, 3% herbs) and water and were fed 300 g per cow per day a pelleted mineral concentrate ³⁵ from at least 10 days before oral bolus application (day 0) until day 14 after bolus application.

Hay intake was recorded on experimental days 2, 3, 4, 9, 10 and 11. Individual water consumption was recorded between days −4 to 0, 0 to 7 and 7 to 14. Body condition score was evaluated and body weight was recorded for each cow on days −4 and 14. Blood samples were taken from each cow; blood pharmacokinetics were determined for Ca, P, Mg and 1,25[OH]₂D₃ at d0 (mean of d‐4, d‐3, d0) and at regular intervals after d0 bolus administration (1 h, 3 h, 6 h, d0.5, d1.25, d1, d1.125, d1.25, d2, d3, d4, d5, d7, d9, d11 and d14). Blood haematology ³⁶ /biochemistry ³⁷ was determined at d0, d1, d1.125, d1.25, d14 for groups 500(■■■■■) and 2 × 500(■■■■■).

Following the end of the pharmacokinetic experiment, cows were handled as usual and additional data were recorded until 28 days post‐partum. The data collected around calving were: calving date, calf birth weight, calf gender, score for assistance (unassisted, light pull, hard pull), preventive and curative treatments against hypocalcemia and hypocalcemia diagnostic. During lactation daily individual dry matter intake, milk yield and body weight were recorded automatically.

The individual cow was considered as the experimental unit. The repeated blood serum and plasma concentrations were used to calculate by noncompartmental analysis the pharmacokinetic parameters of the orally administered 1,25[OH]₂D₃ bolus. The pharmacokinetic parameters were area under the curve (AUC), maximum plasma concentration for a mineral or vitamin (C_max), time of highest peak concentration (T_max) and terminal half‐life. The calculated residence time is the average time that molecules of the dosed compound spend in the body. Non‐repeated performance data were analysed using the general linear model including block and treatment. Repeated blood serum and plasma concentrations were analysed with the mixed model including block, treatment, time and treatment × time. The cow was included as fixed random factor. Comparisons among means were calculated using Tukey's contrasts. One cow (group 2 × 500(■■■■■)) was finally not pregnant and was removed from the dataset. Differences were considered as significant when p < 0.05 and tendencies were noted at p < 0.10.

Dry matter intake (13.2 ± 1.6 kg DM/day), water intake (78.6 ± 14.9 L/day), body weight (732 ± 98 kg at d‐4; 741 ± 95 kg at d14) as well as body condition score (3.0 ± 0.3 at days −4 and 14) were similar between treatments (p > 0.10).

The pharmacokinetic parameters C_max, T_max, and AUC of plasma 1,25[OH]₂D₃ and Ca are presented in Table A1. No treatment effects were observed (also for plasma P), except that the C_max and AUC of Ca were 5% higher (p < 0.05) for the group 2 × 500(2+8) compared to 300(8)c. Tendencies were observed for 1,25[OH]₂D₃: C_max from 500(■■■■■) doubled (p = 0.09) compared to 200(2) and AUC from 500(■■■■■) and 500(■■■■■)c doubled (p = 0.08) compared to 200(2).

Table A.1.

Pharmacokinetic parameters of plasma 1,25[OH]₂D₃ and calcium

Group	1,25[OH]2D3 in bolus	Plasma 1,25[OH]2D3			Plasma calcium
	Analysed (μg)	Cmax	Tmax	AUC	Cmax	Tmax	AUC
200■■■■■	198	57	19	5,962	2.79	96	842
300■■■■■	273	111	42	10,671	2.79	149	856
500■■■■■	512	123	37	11,934	2.79	110	853
300■■■■■c	292	61	44	7,311	2.77	139	844
500■■■■■c	517	116	37	11,941	2.89	82	876
2 × 500■■■■■	(2 × 512)	99	36	10,501	2.94	61	884

C_max (pg/mL): peak observed for vitamin or mineral concentration; T_max (h): time of peak concentration; AUC (pg/mL and h): area under the concentration‐time curve.

■■■■■

■■■■■

■■■■■

■■■■■

■■■■■

■■■■■

Following bolus application, blood serum 1,25[OH]₂D₃ (Table A.2) increased from the initial baseline (p < 0.001) between d0.5 and d5 with a peak from d1.25 to d2, before reaching values comparable (p > 0.10) to the initial baseline at d9. The time dependent response was more pronounced in 300(8), 500■■■■■, 500■■■■■c and 2 × 500■■■■■ than in 200■■■■■ and 300■■■■■c (time × treatment interaction, p < 0.05). Blood serum 1,25[OH]₂D₃ response was not different (p > 0.10) between uncoated and coated boluses and did not differ between the group receiving 500■■■■■ and 2 × 500■■■■■.

Table A.2.

Plasma 1,25[OH]₂D₃ (pg/mL) at different times after administration

Group	1,25[OH]2D3 in bolus	Time after bolus application
	Analysed (μg)	0 h	d0.5	d1.25	d2	d3	d5	d9
200■■■■■	198	9.9	53.4	53.6	42.1	34.2	23.3	3.4
300■■■■■	273	14.1	61.6	104.2	87.0	66.1	38.7	16.6
500■■■■■	512	13.6	95.2	98.5	103.0	65.7	48.2	13.6
300■■■■■c	292	13.8	31.2	51.6	59.8	51.8	29.5	12.3
500■■■■■c	517	14.2	80.5	115.3	97.4	75.3	43.9	14.2
2 × 500■■■■■	(2 × 512)	10.4	50.8	93.1a	77.8	67.9	46.4	9.1

Blood serum Ca (Table A.3) increased from the initial baseline (p < 0.001) between d0.5 and d11 with a peak from d2 to d4, before reaching values comparable (p > 0.10) to the initial baseline at d14.

Table A.3.

Serum Calcium (mmol/L) at different times after administration

Group	1,25[OH]2D3 in bolus	Time after bolus application
	Analysed (μg)	0 h	d0.5	d1.25	d2	d3	d5	d9	d14
200■■■■■	198	2.21	2.36	2.64	2.67	2.68	2.55	2.61	2.30
300■■■■■	273	2.22	2.35	2.61	2.66	2.70	2.65	2.65	2.23
500■■■■■	512	2.25	2.34	2.59	2.66	2.69	2.60	2.61	2.27
300■■■■■c	292	2.22	2.24	2.47	2.67	2.68	2.63	2.61	2.15
500■■■■■c	517	2.25	2.46	2.70	2.85	2.82	2.69	2.69	2.22
2 × 500■■■■■	(2 × 512)	2.23	2.43	2.63	2.79	2.79	2.80	2.63	2.34

Following bolus application, blood serum P increased from the initial baseline between d1 (p < 0.001) and d11 (p < 0.05) with a peak from d3 to d5, before reaching values comparable (p > 0.10) to the initial baseline at d14. Blood serum Mg decreased from the initial baseline between d1 and d9 with minima from d2 to d4, before reaching values comparable (p > 0.10) to the initial baseline at d11.

The overall response in blood serum Ca from 300■■■■■c was lower (p < 0.001) than from 500■■■■■c and 2×500■■■■■, whereas the response of blood serum P and Mg was similar between treatments (p > 0.10). No interaction of time × treatment (p > 0.10) was identified for the responses of Ca, P and Mg. Blood serum Ca, P and Mg response did not differ (p > 0.10) between ■■■■■ boluses. Serum Ca response tended (p < 0.10) to be higher in 2 × 500■■■■■ compared to 500■■■■■, whereas serum P and Mg response was not different between these two groups.

The measurements done in blood, haematology and biochemistry, in the groups 500■■■■■ and 2 × 500■■■■■ did not reveal major differences between the treatments and there was no time × treatment interaction. These results indicate that double bolus did not present a risk different from that of a single bolus. Thus, none of the measured haematology and biochemistry parameters suggested that the overdose would result in any relevant difference to a single application.

The end of the experiment (pharmacokinetics of d14) was 24.4 ± 11.2 days before the effective calving, without differing between treatments (p > 0.10). Before calving (1–7 days before calving), 13 cows received a preventive therapy against hypocalcaemia. Despite the treatment, two of these cows showed clinical signs of hypocalcemia following calving and were treated with intravenous and oral calcium. One these cows (group 300■■■■■) with a prolapsed uterus, whereas the other cow (group 500■■■■■) recovered well. Twenty‐nine cows gave birth to a healthy calf (birth body weight 41.5 ± 6 kg). During the first 28 days post‐partum dry matter intake (19 ± 3.5 kg/day) and milk production (38.2 ± 8.4 kg/day) of cows were similar between treatments (p > 0.10) and were comparable with cows at similar lactation stages of the same dairy operation. The initial and final blood serum concentrations of 1,25[OH]₂D₃, Ca, P and Mg were within published ranges considered as normal (5–20 pg 1,25[OH]₂D₃/L, 2.0–3‐0 mmol Ca/L, 1.4–2.6 mmol P/L and 0.7–1.5 mmol Mg/L).

Appendix B – Effect of 1,25 dihydroxycholecalciferol glycosides released from a rumen bolus on mineral status in periparturient primiparous and multiparous dairy cows and their newborn calves

The study started with a total of 36 pregnant dairy cows to have 24 cows ³⁸ meeting the conditions of the study.

The experimental research protocol was approved by the Office for Food Safety and Veterinary Affairs and all procedures were conducted in accordance with the Swiss Ordinance on Animal protection and the Ordinance on Animal Experimentation.

The study was set up as a 2 × 2 factorial design with factors treatment (Control (C) or bolus (B)) and parity (primiparous (P) or multiparous with ≥ 3 lactations (M)) to obtain four groups: Control‐Primi (CP), Bolus‐Primi (BP), Control‐Multi (CM) and Bolus‐Multi (BM) (with 6 cows each). The 6 replicates (cows) per group were defined according to a power analysis (a difference in blood Ca plasma of 0.5 mmol/L between C and B cows should identified).

Animals were selected for the study 21 days prior to the expected calving date, ³⁹ and the bolus was administered 3–4 days prior to the expected calving date. ⁴⁰ Animals remained in the study until 21 days after actual calving (day 0). If clinical signs suggesting hypocalcaemia were observed after calving and a blood Ca analysis showed < 1.6 mmol/L, the cow was excluded from the experiment and replaced by another animal of the same parity.

The basal diet consisted of hay and maize silage (75/25% of DM) and was fed ad libitum. In addition, two pelleted cereal‐based concentrates differing in protein content and two pelleted mineral feeds were given for the pre‐calving and the lactation period, respectively. ⁴¹ The total daily ration contained per kg DM by analysis for the prepartum and the lactation period 127 and 138 g crude protein, 217 and 181 g crude fibre, and 5.7 and 6.1 MJ NEL, respectively. Dietary cation anion difference (DCAD) was 582 and 499 meq/kg DM for the two periods. The high DCAD values, which reflect typical situations occurring in herbage‐based diets, are expected to increase the risk of Ca deficiency after calving. The calves received twice a day colostrum and milk from their mothers until d5. Then, they were fed twice a day milk enriched with milk powder. Calves had free access to hay and water.

A multitude of parameters were examined. ⁴² The main endpoints were the concentrations of Ca, followed by 1,25[OH]₂D₃, PTH, P and Mg in blood at several days of the study.

The data were statistically analysed ⁴³ for repeated blood values by a mixed model with group, day and group × day and the cow as fixed random effect. When the day, the group or the interaction between group and day or parity and bolus were significant, the least square means were compared using Tukey's contrasts. For non‐repeated data, a general linear model (GLM) was applied including parity, bolus and parity × bolus.

To collect data from 24 cows, 35 cows had to be included in the experiment. Thus, 11 cows were excluded from the experiment. Five cows (2 BP, 3 BM) calved outside of the defined time frame of 1–9 days after bolus application. Three CM‐cows showed clinical signs of hypocalcaemia with blood serum Ca below the defined threshold of 1.6 mmol/L (1.54, 0.73 and 0.72 mmol/L at 32 h, 17 h and 24 h post‐partum, respectively). Another 3 cows (2 BP and 1 CM) had to be treated with antibiotics after calving.

Multiparous cows had a higher (p < 0.001) feed intake on a weekly basis (+53% pre‐partum, +37% in lactation) and a higher intake of each separate diet component than primiparous cows. The bolus administration did not have any effect on feed intake of the cows. Multiparous cows were heavier (p < 0.001) than primiparous cows before (818 vs. 687 kg) and after calving (766 vs. 628 kg). The body condition score varied between 3.0–3.3 and was similar among groups (p > 0.10). Milk yield was higher (p < 0.001) in multiparous cows (39.2 and 40.8 kg/day in week 2 and 3, respectively) than in primiparous cows (24.9 and 26.6 kg/day in week 2 and 3, respectively). Bolus administration did not affect (p > 0.10) any of the cows' performance data.

Considering calving criteria (calving without human help, birthweight, intervals bolus to calving, calving to placenta expulsion and calving to calf first stand‐up), no effect (p > 0.10) was observed for parity, bolus and their interaction.

The concentration of 1,25[OH]₂D₃ and calcium in blood serum of cows is presented in Table B.1. Whereas 1,25[OH]₂D₃ concentration remained constant in CP and BM cows between d‐4 and d15, it increased in BP around calving (d‐2, d0.5) and in CM after calving (d1 to d2) compared to d4 and d8 to d15, respectively (p < 0.01, day to group interaction).

Table B.1.

1,25[OH]₂D₃ and calcium in cow blood serum in days from calving

Day from calving	Control‐Primi		Bolus‐Primi		Control‐Multi		Bolus‐Multi
	1,25[OH]2D3 pmol/L	Ca mmol/L	1,25[OH]2D3 pmol/L	Ca mmol/L	1,25[OH]2D3 pmol/L	Ca mmol/L	1,25[OH]2D3 pmol/L	Ca mmol/L
–4	53	2.09	48	2.34	37	2.25	34	2.38
–2	57	2.13a	169	2.59ab	53	2.19b	69	2.97a
0.5	56	2.13ab	101	2.42b	89	1.66a	115	2.15ab
1	68	2.02b	115	2.39b	108	1.39a	89	2.03b
1.5	64	2.03b	74	2.52b	120	1.35a	83	2.00b
2	74	2.10ab	73	2.42b	139	1.73a	97	2.02ab
4	75	2.19	25	2.15	68	2.32	66	2.19
8	51	2.36	33	2.09	32	2.29	61	2.17
11	51	2.33	36	2.21	35	2.35	58	2.25
16/15	47	2.38	39	2.20	31	2.27	48	2.24

a,b: Values within a row with different superscript are significantly different (p < 0.10).

Serum Ca remained stable (mean of 2.25 ± 0.15 mmol/L) over the entire experimental period in CP and BP cows, but varied in CM and BM cows (p < 0.001 day to group interaction). After calving (from d0.5 to d2), the Ca concentrations of CM cows dropped by 40% compared to the initial value (d – 4) and was lower compared to those of the other groups. In the BM cows, Ca peaked before calving (d – 2) at a higher concentration compared to the initial values on d – 4 and to CP and CM cows. After that peak, the serum Ca concentrations of BM cows returned to values similar to the initial values until the end of the experiment. CP, BP and BM cows did not have, at any time, mean blood serum Ca concentrations below 2.00 mmol/L and none had values higher than 3.00 mmol/L.

The P concentration in cows' blood serum remained stable (mean of 1.30 ± 0.33 mmol/L) over the entire experimental period for CP and CM cows. However, P serum levels in ControlMulit cows reached values below 0.8 mmol/L between d0.5‐d2, which were lower than those of BP cows (p < 0.001 day to group interaction). The P concentrations peaked in BP on d‐2 (2.43 mmol/L), d0.5 (2.19 mmol) and d1.5 (2.16 mmol) and in BM on d‐2 (2.48 mmol/L) compared to values after d8 (1.28–1.39 mmol/L for BP, 1.16–0.90 mmol for BM; p < 0.001, day to group interaction).

For CP, BP and CM cows, the Ca/P ratio remained stable over the entire experimental period. This ratio was increased in CM cows (2.25) compared to CP (1.50) and BP cows (1.48).

The Mg concentration in blood serum remained stable (mean of 0.98 ± 0.05 mmol/L) over the entire experimental period for BP and BM cows. In CM cows, the Mg concentration increased first and peaked on d0.5 (1.12 mmol/L), then dropped to its lowest value on d4 (0.88 mmol) before stabilising until the end of the experimental period (0.95–0.96 mmol/L). No differences were found in CP except on d4 (0.83 mmol/L) which was significantly lower than the other Mg values (0.87–0.97 mmol/L)

The parathyroid hormone (PTH) serum concentration remained stable over time in all groups, except in CM. The average of all groups was higher on d1.5 (48 pg/mL) compared to the initial PTH value (29 pg). In CM PTH increased significantly from 12 pg/mL at d‐4 to 69 pg at d0.5 and 72 pg at d1.5.

Blood serum bone resorption marker CTx (carboxyterminal telopeptide of type I collagen) in cows blood serum increased constantly over time in all groups, except CP (p < 0.001, day × group interaction). Primiparous cows had higher CTx concentrations than BM over the whole experimental phase, but the difference was higher in the phase d‐4 to d0.5 (p < 0.001 day to group interaction).

The bolus administration had no effect (p > 0.10) on calf blood serum minerals, 1,25[OH]₂D₃ and IGG concentrations. All serum Ca concentrations of calves were within the normal range (2.0–3.0 mmol/L).

Annex A – Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of the Analysis for glycosylated 1,25‐dihydroxycholcalciferol in Solanum glaucophyllum extract

In the current application an authorisation is sought under Article 4(1) for solanum glaucophyllum leaf extract (SGE) under the category/functional group (3a) “nutritional additives”/“vitamins, pro‐vitamins and chemically well‐defined substances having similar effect”, according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, the authorisation is sought for the use of the feed additive for dairy cows for milk production and other dairy ruminants.

According to the Applicant, the feed additive (SGE) is a powder preparation of ethanol extract of dried solanum glaucophyllum leaves. The active substance of the feed additive is glycosylated 1,25‐dihydroxycholecalciferol with its content ranging from 50 to 160 mg/kg feed additive.

The feed additive is to be used as a complementary feed in the form of controlled release bolus given to dairy cows or other dairy ruminants prior calving. The intended dose for dairy cows is 1 or 2 bolus corresponding to 500 μg of 1,25‐dihydroxycholecalciferol per animal, while the dose for other dairy ruminants is 1 or 2 bolus corresponding to 1.25 μg of 1,25‐dihydroxycholecalciferol/kg body weight of the animal. The feed additive is not intended to be used in premixtures or complete feedingstuffs.

For the quantification of glycosylated 1,25‐dihydroxycholecalciferol in the feed additive and complementary feed (bolus) the Applicant submitted a single‐laboratory validated and further verified method based on analysis by high performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) after an enzymatic hydrolysis with beta‐glucosidase enzyme followed by derivatisation with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD).

The following performance characteristics were obtained for the quantification of the glycosylated 1,25‐dihydroxycholecalciferol content in the feed additive and complementary feed (bolus) in the frame of the validation and verification studies:

• –for the feed additive with glycosylated 1,25‐dihydroxycholecalciferol content ranging from 115.4 to 140.0 mg/kg: a relative standard deviation for repeatability (RSDr) ranging from 5.2% to 6.3%; a relative standard deviation for intermediate precision (RSDip) ranging from 8.0% to 8.2%; and a recovery rate (RRec) ranging from 95% to 103%.
• –For bolus with glycosylated 1,25‐dihydroxycholecalciferol content ranging from 28.3 to 99.3 mg/kg: RSDr ranging from 3.3% to 4.8%; RSDip ranging from 5.8% to 18.4%; RRec ranging from 94% to 101%; and a limit of quantification (LOQ) of 12.4 mg glycosylated 1,25‐dihydroxycholecalciferol/kg of bolus.

Based on the acceptable performance characteristics available, the EURL recommends for official control the single‐laboratory validated and further verified method based on LC–MS/MS for the quantification of the glycosylated 1,25‐dihydroxycholecalciferol content in the feed additive and complementary feed (bolus).

Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Suggested citation EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Bampidis V, Azimonti G, de Lourdes Bastos ML, Christensen H, Dusemund B, Fašmon Durjava M, Kouba M, López‐Alonso M, Puente SL, Marcon F, Mayo B, Pechová A, Petkova M, Ramos F, Sanz Y, Villa RE, Woutersen R, Brantom P, Gropp J, Svensson K, Anguita M, Galobart J, Pizzo F and Manini P, 2022. Scientific Opinion on the safety and efficacy of a feed additive consisting of Solanum glaucophyllum leaf extract for dairy cows and other dairy ruminants (Herbonis Animal Health Gmbh). EFSA Journal 2022;20(8):7434, 24 pp. 10.2903/j.efsa.2022.7434