Introduction
Background and Terms of Reference
Regulation (EC) No 1831/20031 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 Danisco (UK) Ltd, represented in the European Union (EU) by Genencor International B.V.,2 for the authorisation of the additive consisting of 6-phytase produced by Trichoderma reesei CBS 146250 (Axtra® PHY GOLD 30 L, Axtra® PHY GOLD 30 T, Axtra® PHY GOLD 65G), when used as a feed additive for all pigs and all poultry species (category: zootechnical additives; functional group: digestibility enhancers).
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 particulars and documents in support of the application were considered valid by EFSA as of 9 March 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 6-phytase produced by Trichoderma reesei CBS 146250 (Axtra® PHY GOLD 30 L, Axtra® PHY GOLD 30 T, Axtra® PHY GOLD 65G), when used under the proposed conditions of use (see Section 3.1.5).
Additional information
The product containing 6-phytase produced by the genetically modified microorganism Trichoderma reesei CBS 146250 is not authorised as a feed additive in the EU.
Data and methodologies
Data
The present assessment is based on data submitted by the applicant in the form of a technical dossier3 in support of the authorisation request for the use of 6-phytase produced by Trichoderma reesei CBS 146250 (Axtra® PHY GOLD 30L, Axtra® PHY GOLD 30T, Axtra® PHY GOLD 65G) 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 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 6-phytase in animal feed. The Executive Summary of the EURL report can be found in Annex A.4
Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of 6-phytase produced by Trichoderma reesei CBS 146250 (Axtra® PHY GOLD 30L, Axtra® PHY GOLD 30T, Axtra® PHY GOLD 65G) is in line with the principles laid down in Regulation (EC) No 429/20085 and the relevant guidance documents: Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEEDAP Panel, 2017a), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018a), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018b) and Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019).
Assessment
The product containing 6-phytase produced by a genetically modified strain of Trichoderma reesei (CBS 146250) is intended for use as a zootechnical feed additive (functional group: digestibility enhancers) in feed for all poultry species (chickens/hens, turkeys and minor growing and reproductive) and all pigs (piglets, pigs for fattening, sows and minor growing and reproductive porcine species). The product will be hereafter referred to with its trade name Axtra® PHY GOLD.
Characterisation
Characterisation of the production strain
The production strain is a genetically modified Trichoderma reesei deposited at the Westerdijk Fungal Biodiversity Institute with deposit number CBS 146250.6
The taxonomic identification of the recipient strain ■■■■■ as Trichoderma reesei was confirmed by DNA sequencing analysis ■■■■■ and by phylogenomic analysis comparing its genome against available related genomes.7 However, the analysis was not conducted on the production strain. The panel notes that the whole genome sequence (WGS) data of the production strain were available and should have been used for identification purposes.
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Manufacturing process
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Characterisation of the additive
The active substance of Axtra® PHY GOLD is 6-phytase (myo-inositol-hexakisphosphate 4-phosphohydrolase; Enzyme Commission number 3.1.3.26). The three different commercial formulations differ in the enzyme activity and/or physical properties:
- Axtra® PHY GOLD 30L: brown liquid preparation (average viscosity = 9.5 cP at 25οC; average specific gravity = 1,220 kg/m3) with a minimum enzyme activity of 30,000 FTU9/g.
- Axtra® PHY GOLD 30T: off-white to light tan granular preparation (average bulk density = 1,200 kg/m3) with a minimum enzyme activity of 30,000 FTU/g.
- Axtra® PHY GOLD 65G: off-white to light tan granular preparation (average bulk density = 1,050 kg/m3) with a minimum enzyme activity of 65,000 FTU/g.
The enzyme activity of five batches of Axtra® PHY GOLD 30 L (range: 41,884–47,648 FTU/g; average: 44,865 FTU/g); 30T (range: 42,858–47,161 FTU/g; average: 44,432 FTU/g); and 65G (range: 90,268–115,668 FTU/g; average: 104,344 FTU/g) showed compliance with the minimum specifications set by the applicant.10 The range of total organic solids (TOS) coming from fermentation is estimated to be of ■■■■■ for the Axtra® PHY GOLD 30 L, 30 T and 65 G formulations, respectively.
Three batches from each formulation of the additive were analysed for chemical and microbiological impurities.11 Values for mercury, cadmium and all mycotoxins analysed fell below the respective limit of quantification (LOQ)12 in all formulations and batches analysed. Axtra® PHY GOLD 30L showed an average value of 0.15 mg/kg of lead and below LOQ for arsenic; 30T showed average values of 0.11 and 0.02 mg/kg for arsenic and lead, respectively; and the 65 G showed values below LOQ for lead and arsenic, except for 0.04 mg/kg of arsenic in one of the batches analysed. ■■■■■
The same batches were analysed for microbiological impurities. Escherichia coli or Salmonella spp. were not detected in 25 g, and Enterobacteriaceae, total coliforms, and yeasts and filamentous fungi were < 10 CFU/g additive.
The detected amounts of the above-described analysis do not raise safety concerns.
The applicant submitted three sets of data to exclude the ability of the production strain to produce antimicrobials. The antimicrobial production was tested against reference strains13 in the supernatant of the production strain,14 in the enzyme fermentation product15 and in each formulation of the additive16 with disc-diffusion agar method. No antimicrobial activity was detected.
Some Trichoderma species are known to be capable of producing a variety of mycotoxins and antifungal metabolites. T. reesei seems to be unable to produce mycotoxins (EFSA, 2007; Frisvad et al., 2018; EFSA BIOHAZ Panel, 2020) but it is known to produce peptaibols, such as paracelsin A, C and D (Frisvad et al., 2018), and its genome has been shown to harbour genes for two peptaibol synthases (Kubicek et al., 2007). Those peptaibols are peptides with antimicrobial activity and are mostly produced under stress conditions (Frisvad et al., 2018). The production predominantly occurs in solid fermentation and correlates with conidiation (Kubicek et al., 2007; Tisch and Schmoll, 2010). The lack of antimicrobial activity in the additive under assessment (described above) would indicate that if peptaibols are produced under the fermentation conditions, their concentration would be of no concern. This was confirmed in the recipient (T. reesei ■■■■■) and an intermediate strain (T. reesei ■■■■■) when tested for their ability to produce trichothecenes (trichodermin, trichodermol and harzianum A) and gliotoxin.17
The applicant did not provide information on the potential of the production strain to produce secondary metabolites except from data on the mycotoxin levels in the final forms of the additive (data shown above; all values below LOQ). However, safety concerns derived from the presence of other secondary metabolites potentially produced by T. reesei CBS 146250 are addressed by the toxicological studies conducted with the liquid enzyme concentrate (see Section 3.2.2).
The presence of viable cells of the production strain was investigated in triplicate in three batches of the liquid enzyme concentrate.18 ■■■■■ The results obtained in the intermediate liquid enzyme concentrate allow conclusions on the final product.
The presence of recombinant DNA from the production strain Trichoderma reesei CBS 146250 was also analysed in triplicate in three batches of the liquid enzyme concentrate.19 ■■■■■ No DNA of the production strain was detected.
The dusting potential of three batches of the solid forms of the additive was determined using the Stauber-Heubach method20 and showed average values of 6.67 mg/m3 (range 0–20 mg/m3) and of 13.3 mg/m3 (range 10–15 mg/m3) for Axtra® PHY GOLD 30 T and 65 G, respectively. The particle size distribution was analysed by laser-diffraction method on the same three batches of the solid forms21; the results showed that 100% (v/v) of the particles have a diameter > 200 μm both for Axtra® PHY GOLD 30T and 65G.
Stability and homogeneity
Shelf-life
The shelf-life of the additive was studied in three batches of each formulation (initial activity of 52,938; 53,969 and 117,826 FTU/g for the Axtra® PHY GOLD 30L, 30T and 65G, respectively) when stored in a commercial packaging for up to 24 months at 25°C and 60% relative humidity (RH). The enzyme activity at the end of the storage period ranged 85.5–90.7% for Axtra® PHY GOLD 30L, 98.4–105.2% for Axtra® PHY GOLD 30T and 95.1–107.9% for Axtra® PHY GOLD 65G compared to the starting one.22
Stability
The stability of the solid forms of the additive (three batches for each form) in a commercial vitamin/mineral premixture for poultry was studied when supplemented at a minimum target dose of 0.667% for Axtra® PHY GOLD 30T and 0.307% for Axtra® PHY GOLD 65G (approximated minimum activity of 199,382 FTU/kg premix). The supplemented premix was stored in air-tight plastic containers at 25οC for up to 6 months. The enzyme activity at the end of the storage period ranged 90.3–91.8% for Axtra® PHY GOLD 30 T and 82.8–108.7% for 65 G compared to the starting one.23
The stability of the solid forms of the additive (one batch for each form) was measured in mash feeds when supplemented at a minimum phytase activity of 500 FTU/kg complete feed for piglets, sows and chickens for fattening or at 300 FTU/kg complete feed for laying hens. Samples were stored in small paper bags placed in larger paper bags lined with plastic at 25οC and 60% RH for up to 3 months. The enzyme activity at the end of the storage period ranged 83.4–94.5% for Axtra® PHY GOLD 30T and 82.3–100.1% for 65G compared to the starting one.24
The stability of all forms of the additive (one batch for each form) during pelleting was studied after mixing into mash feed for piglets, sows, laying hens and chickens for fattening to achieve a total phytase activity between 400 and 820 FTU/kg feed for Axtra® PHY GOLD 30L and 30T and between 500 and 1,000 FTU/kg feed for Axtra® PHY GOLD 65G. Mash feed samples containing Axtra® PHY GOLD 30T were then pelleted for 30 s at 95οC or at 75οC those containing Axtra® PHY GOLD 30L and 65G. The enzyme activity after the pelleting process ranged 80.1–91.7% for Axtra® PHY GOLD 30L, 80.7–110.7% for 30T and 81.9–103.9% for 65 G compared to the starting one.25
The storage stability after pelleting of the solid forms of the additive included in the mash feeds for piglets, sows, laying hens and chickens for fattening was studied. In contrast, Axtra® PHY GOLD 30L was sprayed onto pellets to obtain a minimum phytase activity of 500 FTU/kg complete feed for piglets, sows and chickens for fattening and of 300 FTU/kg complete feed for laying hens. The pelleted feeds were stored in small paper bags (closed with paper clips) placed in larger paper bags lined with plastic at 25οC and 60% RH for up to 3 months. The enzyme activity at the end of the storage period ranged 82.3–95.7% for Axtra® PHY GOLD 30L, 90.4–105.8% for 30T and 76.9–99.1% for 65G compared to the starting one.26
Homogeneity
The capacity for homogeneous distribution of the solid forms of the additive included in mash feeds for piglets, sows, laying hens and chickens for fattening was studied in 10 subsamples. The coefficient of variation ranged 5.8–10.2% and 7.9–10.0% in mash feed samples supplemented with Axtra® PHY GOLD 30 T and 65 G, respectively.27 The capacity for homogeneous distribution of the 30L formulation of the additive sprayed onto pelleted feeds for piglets, sows, laying hens and chickens for fattening was studied in 10 subsamples.28 The coefficient of variation ranged from 1.4 to 1.8%.
Conditions of use
The additive is intended for use in feed for all growing poultry species and all pigs (including minor growing and reproductive porcine species) at a proposed minimum level of 500 FTU/kg complete feed and for laying hens and other laying birds at a proposed minimum level of 300 FTU/kg complete feed. The applicant states that the liquid formulation is to be applied post-pelleting when the pelleting process exceeds 75οC.
Safety
Safety aspects of the genetic modification
In the view of the FEEDAP Panel, the identity of the production strain has been unambiguously established as Trichoderma reesei. ■■■■■ The introduced sequences raise no safety concerns. The presence of viable cells from the production strain and their recombinant DNA were not detected in the liquid enzyme concentrate used to formulate the final forms of the additive. The product Axtra® PHY GOLD manufactured with the production strain T. reesei CBS 146250 does not raise safety concerns with regard to the genetic modification of the production strain.
Toxicological studies
All the toxicological studies were performed with the liquid enzyme concentrate form of 6-phytase ■■■■■ from which the three final Axtra® PHY GOLD formulations are obtained. The test item is considered representative for the three final formulations of the additive.
Genotoxicity studies
Bacterial reverse mutation assay (Ames test)
The test item was assessed for the induction of reverse mutations ■■■■■.29 The experimental protocol was in line with the Organisation for Economic Co-operation and Development (OECD) guideline 471 following good laboratory practices (GLP). ■■■■■ No statistically significant increase in the number of revertant colonies was observed at any tested condition in any tester strain. Therefore, the test item did not induce gene mutations in bacteria under the experimental conditions applied in the study.
The potential capacity of the test item to induce structural chromosome aberrations was evaluated in vitro in human peripheral blood lymphocytes in compliance with OECD TG 473 and following GLP.30 ■■■■■ Comparable frequencies of chromosomal aberrations were detected in treated and control cultures. Thus, the test item did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes under the experimental conditions employed in the study.
To evaluate the potential of the test item to induce chromosome damage, an in vitro micronucleus test was carried out in human whole blood according to OECD TG 487 and following GLP.31 ■■■■■ No increase of the frequency of micronuclei was induced by treatment with the test item in any experimental condition. The test item did not induce structural and numerical chromosome aberrations in human lymphocytes under the experimental conditions employed in this study.
Subchronic oral toxicity study
■■■■■ 0 (control), 250, 500 or 1,000 mg TOS per kg bw and day ■■■■■, corresponding to 0, 112,500, 225,000 and 450,000 FTU/kg bw and day.32 The study was conducted in compliance with OECD guideline 408 and following GLP. ■■■■■ 33 ■■■■■ 34 ■■■■■ 35 ■■■■■
No treatment-related clinical signs were observed. One male animal died in the 250 mg TOS/kg bw per day group, but the death was unrelated to treatment. No effects were observed on survival, behaviour, body weight, feed intake, haematology, clinical parameters, urine analysis, gross pathology and histological examination. From this study, it was derived an NOAEL of 1,000 mg TOS/kg bw per day, the highest dose tested, corresponding to 450,000 FTU/kg bw per day.
Conclusions on the toxicological studies
The FEEDAP Panel concludes that the intermediate product used for the formulation of the additive showed no genotoxicity potential in tests addressing gene mutations, numerical and structural chromosome aberrations. Moreover, the results obtained in a subchronic oral toxicity study raised no concerns regarding the product and allowed to derive an NOAEL of 450,000 FTU/kg bw and day, the highest dose tested.
Safety for the target species
No tolerance studies in relevant target species were submitted. To support the safety of the additive for the target species, the applicant referred to the 90-day toxicity study described above (see Section 3.2.2.2). The NOAEL identified (450,000 FTU/kg bw and day) was used to calculate the maximum safe level in chickens and turkeys for fattening, laying hens, piglets, pigs for fattening and lactating sows in accordance with the procedure described in the Guidance on the safety for the target species (EFSA FEEDAP Panel, 2017b), and the results are shown in Table 1. The values obtained are higher than the recommended use level of 500 FTU/kg complete feed for chickens and turkeys for fattening, piglets, pigs for fattening and lactating sows, and of 300 FTU/kg for laying hens. Therefore, the panel concludes that the additive is safe at 500 FTU/kg for all growing poultry species and all pigs, and at 300 FTU/kg in laying hens and other laying birds.
Table 1 Maximum safe concentration of Axtra® PHY GOLD in the feed of the major target species
| Animal category | Default values for daily feed intake (g DM per kg bw) | Maximum safe level in feed (FTU/kg complete feed) |
| Chickens for fattening | 79 | 50,127 |
| Turkeys for fattening | 59 | 67,500 |
| Laying hens | 53 | 74,717 |
| Piglets | 44 | 90,000 |
| Pigs for fattening | 37 | 108,000 |
| Lactating sows | 30 | 131,250 |
Safety for the consumer
The results obtained with the liquid enzyme concentrate, considered representative of the final formulations of the additive, in the genotoxicity studies and the subchronic oral toxicity study, do not indicate any reason for concern for consumer safety arising from the use of the product as a feed additive.
Safety for the user
Effect on the respiratory system
The applicant provided no specific studies regarding the effects of the additive on the respiratory system. The proteinaceous nature of the active substance implies that the additive is considered a respiratory sensitiser.
Effect on eyes and skin
The skin36 and eye37 irritation potential of the liquid enzyme concentrate was tested in valid studies performed according to OECD guidelines 439 and 492 following GLP, respectively, showing that the test item is not a skin or an eye irritant.
The results of a valid local lymph-node assay (LLNA) performed with the liquid enzyme concentrate following OECD guidelines 429 and GLP showed that the test item is not a skin sensitiser.38
Conclusions on safety for the user
The panel notes that the final formulations were not tested. Based on the studies provided, the liquid enzyme concentrate used as test item is not a skin or eye irritant nor a skin sensitiser. In the absence of data obtained with the final formulations, the panel cannot conclude on the potential of the additive to be an irritant to the skin and eyes or a skin sensitiser. Due to the proteinaceous nature of the active substance (6-phytase), the additive is considered a respiratory sensitiser.
Safety for the environment
The production strain and its DNA were not detected in the liquid enzyme concentrate used to prepare the final forms of the additive. The additive does not raise safety concerns for the environment regarding the genetic modification of the production strain. The active substance is a protein; thus, it will be degraded/inactivated during the passage through the digestive tract of animals or in the environment. Therefore, no risks to the environment are expected when the additive is used on poultry and pigs.
Efficacy
The test article used in the studies to demonstrate the efficacy of Axtra® PHY Gold was the liquid enzyme concentrate 6-phytase sprayed onto a wheat carrier.
Efficacy for poultry
Chickens for fattening
The applicant provided three short-term balance trials sharing a similar design. All trials were designed to study the effect of the 6-phytase contained in the additive on the retention of dietary phosphorus and included bone mineralisation parameters. The main results of the trials are presented in Table 2.
Table 2 Effect of Axtra® PHY GOLD on the phosphorus retention and bone mineralisation of the chickens for fattening in the short-term trials
| Trial | Diets | Bone content | ||||
| Phytase (FTU/kg feed) | P/Ca (%) | Form | P retention (%) | Ash (% of DM) | P (% of ash) | |
| 1 | 0 | ■■■■■ | Mash | 44.3c | 35.8d | 16.3c |
| 500 | ■■■■■ | Mash | 71.2a | 44.0b | 17.0b | |
| PC | ■■■■■ | Mash | 50.7b | 46.8a | 17.0b | |
| 0 | ■■■■■ | Pellet | 48.0bc | 41.6c | 16.6c | |
| 500 | ■■■■■ | Pellet | 66.3a | 48.6a | 17.4a | |
| PC | ■■■■■ | Pellet | 46.4bc | 47.7a | 17.1ab | |
| 2 | 0 | ■■■■■ | Mash | 43.9b | 30.3b | – |
| 500 | ■■■■■ | Mash | 62.6a | 36.5a | – | |
| 1,000 | ■■■■■ | Mash | 67.6a | 38.4a | – | |
| PC | ■■■■■ | Mash | 57.7a | 37.7a | – | |
| 3 | 0 | ■■■■■ | Pellet | 55.7b | 40.7b | – |
| 500 | ■■■■■ | Pellet | 66.4a | 43.7a | – | |
| PC | ■■■■■ | Pellet | 54.4b | 44.5a | – |
■■■■■ 39 ■■■■■ 40 ■■■■■ 41 ■■■■■
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The birds receiving the 6-phytase at 500 FTU/kg feed showed significantly higher P retention compared to the control diet in the three trials, and also in comparison with the positive control diet in trials 1 and 3. The improvement of the P utilisation at the minimum use level was also reflected in higher ash bone content in the three trials, and on P bone content in trial 1.
Laying hens
The applicant provided two short-term (balance) trials and one long-term efficacy trial including a balance study in laying hens. The main results of the phosphorus utilisation in the three trials are presented in Table 3.
Table 3 Effect of Axtra® PHY GOLD on the P and Ca utilisation and internal egg P content (trials 1 and 2) and tibia ash content (trial 3) in the short-term trials in laying hens
| Trial | Phytase (FTU/kg feed) | P/Ca (%) | P utilisation(1) (%) | Internal Egg P (%) | Tibia ash content (%) |
| 1 | 0 | ■■■■■ | 27.3d | 0.19 | – |
| 300 | ■■■■■ | 35.6b | 0.20 | – | |
| 600 | ■■■■■ | 38.1a | 0.20 | – | |
| 900 | ■■■■■ | 38.8a | 0.20 | – | |
| PC | ■■■■■ | 31.7c | 0.20 | – | |
| 2 | 0 | ■■■■■ | 25.5b | 0.17 | – |
| 300 | ■■■■■ | 36.1a | 0.18 | – | |
| PC | ■■■■■ | 25.1b | 0.17 | – | |
| 3 | 0 | ■■■■■ | 22.9b/19.2b | – | 42.4 |
| 300 | ■■■■■ | 33.1a/29.9a | – | 43.6 |
The two short-term trials followed a similar design. ■■■■■ 42 ■■■■■ 43 ■■■■■
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None of the hens died during any of the studies. The birds receiving the phytase at 300 FTU/kg feed showed significantly higher P utilisation in comparison with the control diet in both trials. There was no effect of the additive on the egg phosphorus content.
In trial 3,44 ■■■■■
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The panel notes that the husbandry conditions applied in trial 3 did not reflect those in which the birds are raised in the EU and were not in line with Directive 1999/74/EC45 (regarding the lighting program and the available surface/usable area per hen). Therefore, the results of the performance parameters recorded were disregarded. However, the panel considers that the data on the phosphorus utilisation could be retained considering that the husbandry conditions would have a lower impact on this endpoint.
Regarding the results of the balance trial, the birds receiving the phytase at 300 FTU/kg feed showed significantly higher phosphorus utilisation in comparison with the control diet. There was no effect of the additive on the tibia ash content. None of the hens died during the study.
Efficacy for pigs
Weaned piglets
Four short-term trials were provided in weaned piglets. The main results of the trials are presented in Table 4.
Table 4 Effect of Axtra® PHY GOLD on the apparent total tract digestibility (ATTD) of P, P retention and bone mineral content in weaned piglets
| Trial | Diets | Retention/ATTD P(1) (%) | Bone mineral content(2) (%) | |
| Phytase (FTU/kg feed) | Total P–Ca (%) | |||
| 1 | 0 | ■■■■■ | 43.2c | 43.3 |
| 500 | ■■■■■ | 55.3b | 43.1 | |
| 1,000 | ■■■■■ | 67.1a | 45.3 | |
| 2 | 0 | ■■■■■ | 41.4b | – |
| 500 | ■■■■■ | 66.1a | – | |
| 4(3) | 0 | ■■■■■ | 21.1c/23.4d/24.4c | 7.83b/7.41b/7.33b |
| 500 | ■■■■■ | 56.4b/51.2c/48.6b | 9.22a/8.91a/9.43a | |
| 1,000 | ■■■■■ | 67.9a/59.6b/60.4a | 9.30a/9.43a/9.57a | |
| 2,000 | ■■■■■ | 68.9a/72.2a/66.9a | 9.53a/9.66a/9.73a | |
| 4,000 | ■■■■■ | 74.3a/74.4a/67.0a | 9.19a/9.75a/9.58a |
Trials 146 and 247 shared a similar design. ■■■■■
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■■■■■ The animals receiving the 6-phytase additive from 500 FTU/kg feed showed significantly higher P retention in comparison with the control diet in both trials. No effect on the metacarpi ash content was found in trial 1.
In trial 3,48 ■■■■■
The supplementation of the diet with the 6-phytase from 500 FTU/kg feed showed higher P retention than the control. However, the results provided for the P retention calculated by the different methods (■■■■■) showed substantial discrepancies. Therefore, the results of this study were considered unreliable as evidence of efficacy.
In trial 4,49 ■■■■■ Regardless of the dietary P content, the supplementation with the additive from 500 FTU/kg feed showed higher ATTD P and P bone content than the control diet.
Sows
Three short-term trials were provided, one carried out in the lactation and two during the gestations phase. The results of the three trials are presented in Table 5.
Table 5 Effect of Axtra® PHY GOLD on the apparent total tract digestibility (ATTD) of P in sows
| Trial | Diets | ATTD P (%) | |
| Phytase (FTU/kg feed) | Total P/Ca (%) | ||
| 1 | 0 | ■■■■■ | 27.9c |
| 500 | ■■■■■ | 44.3a | |
| PC | ■■■■■ | 32.4b | |
| 2 | 0 | ■■■■■ | 19.7c |
| 500 | ■■■■■ | 34.2a | |
| PC | ■■■■■ | 28.8b | |
| 3 | 0 | ■■■■■ | 27.8b |
| 500 | ■■■■■ | 35.4a | |
| PC | ■■■■■ | 34.3a |
In the trial performed in lactating sows,50 ■■■■■
■■■■■ The sows receiving the 6-phytase contained in the additive at 500 FTU/kg feed showed higher ATTD of P in comparison with the control and PC diets.
The two trials in gestating sows shared a similar design. ■■■■■ 51 ■■■■■ 52 ■■■■■ In both trials, the sows receiving the 6-phytase contained in the additive at 500 FTU/kg feed showed improved ATTD of P in comparison with the control group.
Conclusion on efficacy
The panel concludes that the additive is efficacious in increasing the phosphorus utilisation when supplemented at 500 FTU/kg in the diets of chickens for fattening, weaned piglets and sows and at 300 FTU/kg in laying hens. The conclusions reached in chickens for fattening, weaned piglets and sows allow concluding on all pigs and all growing poultry species at 500 FTU/kg, and the conclusions reached in laying hens on other poultry species for laying at 300 FTU/kg feed.
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 Regulation53 and good manufacturing practice.
Conclusions
The additive does not give rise to safety concerns with regard to the genetic modification of the production strain. The production strain and recombinant DNA were not detected in the fermentation product used to formulate the additive.
Axtra® PHY GOLD is safe for all growing poultry species and all pigs at the minimum recommended level of 500 FTU/kg complete feed and for laying hens and other poultry species for laying at 300 FTU/kg complete feed.
The use of Axtra® PHY GOLD in animal nutrition is of no concern for consumer safety.
The use of the additive as a feed additive in poultry and pigs is considered safe for the environment.
The panel cannot conclude on the potential of the additive to be an irritant to eyes or skin or a skin sensitiser. Due to the proteinaceous nature of the active substance (6-phytase), the additive is considered a respiratory sensitiser.
The panel concludes that the additive is efficacious in increasing the phosphorus utilisation when supplemented at 500 FTU/kg for all growing poultry species and all pigs, and at 300 FTU/kg in laying hens and other laying birds.
Documentation provided to
| Date | Event |
| 23/10/2020 | Dossier received by EFSA. Axtra® PHY GOLD 30 L, Axtra® PHY GOLD 30 T, Axtra® PHY GOLD 65 G (6- phytase) for all pigs, all poultry species. Submitted by Danisco UK Ltd. represented by Genencor International B.V. |
| 13/11/2020 | Reception mandate from the European Commission |
| 09/03/2021 | Application validated by EFSA – Start of the scientific assessment |
| 28/05/2021 | 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 of the additive/consumer safety/user safety/efficacy |
| 09/06/2021 | Comments received from Member States |
| 09/09/2021 | Reception of the Evaluation report of the European Union Reference Laboratory for Feed Additives |
| 20/01/2022 | Reception of supplementary information from the applicant - Scientific assessment re-started |
| 18/02/2022 | 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 of the additive |
| 24/02/2022 | Reception of supplementary information from the applicant - Scientific assessment re-started |
| 15/03/2022 | 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 of the additive |
| 21/03/2022 | Reception of supplementary information from the applicant - Scientific assessment re-started |
| 17/05/2022 | Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: purity / efficacy |
| 18/07/2022 | Reception of supplementary information from the applicant - Scientific assessment re-started |
| 27/09/2022 | Opinion adopted by the FEEDAP Panel. End of the Scientific assessment |
- ADFI
- average daily feed intake
- ADG
- average daily gain
- ADI
- average daily intake
- BW
- body weight
- CAS
- Chemical Abstracts Service
- CFU
- colony-forming unit
- CV
- coefficient of variation
- DM
- dry matter
- EC
- European Commission
- EINECS
- European Inventory of Existing Chemical Substances
- EURL
- European Union Reference Laboratory
- FCR
- feed conversion ratio
- FEEDAP
- EFSA Scientific Panel on Additives and Products or Substances used in Animal Feed
- FGE
- food group evaluation
- GC–MS
- gas chromatography–mass spectrometry
- HACCP
- hazard analysis and critical control points
- IUPAC
- International Union of Pure and Applied Chemistry
- JECFA
- The Joint FAO/WHO Expert Committee on Food Additives
- LOD
- limit of detection
- LOQ
- limit of quantification
- MIC
- minimum inhibitory concentration
- MRL
- maximum residue limit
- MW
- molecular weight
- NOAEL
- no observed adverse effect level
- OECD
- Organisation for Economic Co-operation and Development
- RH
- relative humidity
- SCAN
- Scientific Committee on Animal Nutrition
- UF
- uncertainty factor
Abbreviations
Appendix A – Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of the Analysis for a preparation of 6-phytase (
In the current application, authorisation of a preparation of 6-phytase (EC 3.1.3.26) is sought under Article 4 for all poultry and all pig species under the category/functional group 4(a) ‘zootechnical additives’/‘digestibility enhancers’ according to Annex I of Regulation (EC) No 1831/2003.
According to the Applicant, the active agent of the product is 6-phytase, produced by fermentation of the genetically modified strain Trichoderma reesei CBS 146250. Other preparation of 6-phytase from a different Trichoderma reesei strain is currently authorised as feed additive.
The activity of 6-phytase is expressed in phytase units (FTU) where ‘one FTU is the amount of enzyme which releases one micromole of inorganic orthophosphate from a sodium phytate substrate per minute at pH 5.5 and 37 °C’. This definition is in agreement with the phytase activity unit as described in EN ISO 30024.
The product is intended to be marketed in three different formulations namely Axtra® PHY GOLD 30L (liquid), Axtra® PHY GOLD 30T (granular and thermostable) and Axtra® PHY GOLD 65G (granular) with a guaranteed minimum 6-phytase activity of 30,000 FTU/g for the Axtra® PHY GOLD 30L and the Axtra® PHY GOLD 30T formulations and 65,000 FTU/g for the Axtra® PHY GOLD 65G formulation. The product is intended to be included through premixtures or directly in feedingstuffs to obtain a minimum recommended activity of 300 FTU/kg feedingstuffs for laying hens and other birds for laying and 500 FTU/kg feedingstuffs for the other target species.
The Applicant submitted single-laboratory validated and further verified methods for the quantification of the phytase activity in the product (Axtra® PHY GOLD), premixtures and feedingstuffs. The submitted methods are very similar to the ring-trial validated EN ISO 30024 method.
Upon request of the EURL, the Applicant compared both protocols confirming that equivalent results are obtained when applying the slightly different methods to feedingstuffs containing the product (Axtra® PHY GOLD).
Additionally, the EURL is aware of other ring-trial validated VDLUFA methods specifically describing the preparation of premixtures (VDLUFA 27.1.3) and feed additives (VDLUFA 27.1.4) for the quantification of their phytase activity according to EN ISO 30024.
Based on the performance characteristics available, the EURL recommends for official control the ring-trial validated EN ISO and VDLUFA colorimetric methods mentioned above for the quantification of the phytase activity in the product, premixtures and feedingstuffs.
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.
EFSA (European Food Safety Authority), 2007. Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. EFSA Journal 2007;5(12):587, 16 pp. [DOI: https://dx.doi.org/10.2903/j.efsa.2007.587]
Frisvad JC, Moller LLH, Larsen TO, Kumar R and Arnau J, 2018. Safety of the fungal workhorses of industrial biotechnology: update on the mycotoxin and secondary metabolite potential of Aspergillus Niger, Aspergillus oryzae, and Trichoderma reesei. Applied Microbiology and Biotechnology, 102, 9481–9515.
Kubicek CP, Komoń‐Zelazowska M, Sándor E and Druzhinina IS, 2007. Facts and challenges in the understanding of the biosynthesis of peptaibols by Trichoderma. Chemistry & Biodiversity, 4, 1068–1082.
Tisch D and Schmoll M, 2010. Light regulation of metabolic pathways in fungi. Applied Microbiology and Biotechnology, 85, 1259–1277.
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Abstract
Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of 6‐phytase produced by the genetically modified strain
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