Safety and efficacy of a feed additive consisting of endo‐1,4‐β‐xylanase produced by Komagataella phaffii ATCC PTA‐127053 (Xygest™ HT) for poultry (Kemin Europa N.V.)

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of endo‐1,4‐β‐xylanase produced by Komagataella phaffii ATCC PTA‐127053 (Xygest™ HT) as a zootechnical feed additive for poultry. The production strain is genetically modified. No viable cells nor recombinant DNA of the production strain were detected in the final product. Therefore, the Panel concluded that the additive does not pose any safety concern regarding the production strain. The FEEDAP Panel concluded that Xygest™ HT is considered safe for chickens for fattening at 30,000 U/kg and in laying hens at 45,000 U/kg, with a wide margin of safety. These conclusions can be extended to chickens reared for laying/breeding and extrapolated to all poultry species for fattening and reared for laying/breeding and to ornamental birds. The conclusions on laying hens are extrapolated to all laying poultry species. The use of Xygest™ HT in animal nutrition is of no concern for consumer safety and this feed additive is considered safe for the environment. The Panel also concluded that Xygest™ HT is not irritant to the eyes and skin but should be considered a dermal and a respiratory sensitiser. The Panel concluded that the additive Xygest™ HT has a potential to be efficacious for laying hens under the proposed conditions of use and this conclusion can also be extrapolated to all laying poultry species. The Panel could not conclude on the efficacy of the product for chickens for fattening due to insufficient data.

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 Kemin Europa N.V. 2 for the authorisation of the additive consisting of endo-1,4-b-xylanase produced by Komagataella phaffii ATCC PTA-127053 3 (Xygest TM HT), when used as a feed additive for poultry (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). 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 8 July 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 endo-1,4-b-xylanase produced by K. phaffii ATCC PTA-127053 (Xygest TM HT), when used under the proposed conditions of use (see Section 3.1.6).

Additional information
The additive consisting of endo-1,4-b-xylanase produced by K. phaffii ATCC PTA-127053 (Xygest TM HT) is not authorised as a feed additive in the European Union.

2.
Data and methodologies

Data
The present assessment is based on data submitted by the applicant in the form of a technical dossier 4 in support of the authorisation request for the use of the product consisting of endo-1,4-bxylanase produced by K. phaffii ATCC PTA-127053 (Xygest TM HT) as a feed additive.
EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the endo-1,4-b-xylanase in animal feed. The Executive Summary of the EURL report can be found in Annex A. 5

Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of the product consisting of endo-1,4-b-xylanase produced by Komagataella phaffii

Characterisation of the production organism
The additive is produced by a genetically modified strain of Komagataella phaffii which is deposited in the American Type Culture Collection with deposition number ATCC PTA-127053. 7 The taxonomical identification of the production strain as K. phaffii was confirmed by whole genome sequence (WGS)-based analyses of the production strain ATCC PTA-127053. 9 No acquired antibiotic resistance genes were identified 3.1.1.1. Information related to the genetically modified microorganism

Characterisation of the recipient microorganism
Description of the genetic modification

Characterisation of the additive
The additive is available in solid form and ensures a guaranteed minimum xylanase activity of 3,000,000 U 17 /g of product. Xygest TM HT contains dried ferment (11-20%), maize starch (78-87%) and calcium carbonate (0.5-2%). Analytical data on batch-to-batch variation was provided for seven batches and the mean value was 3,243,248 U/g product, ranging from 3,017,356 to 3,546,859 U/g. 18 Seven batches of the additive were analysed for chemical contamination including lead, mercury, cadmium and arsenic, mycotoxins and dioxins.
Polychlorinated dibenzodioxins and dibenzofurans (PCDDs and PCDFs) and coplanar dioxin-like polychlorinated biphenyls (DL-PCBs) were analysed in three batches and were below the corresponding LOQs. The calculated (upper bound) levels of the sum of dioxins (88% d.w.) was 0.096 ng WHO-PCDD/F-TEQ/kg, while the sum of DL-PCBs was 0.032 ng WHO-PCB-TEQ/kg and the sum of dioxins and DL-PCBs was 0.13 ng WHO-PCDD/F-PCB-TEQ/kg. In the same batches, the sum of non-DL PCBs ranged between 0.45 and 0.51 lg/kg. 22 The presence of hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) pesticides was determined in three batches, 23 while organochlorine and organophosphorus pesticides were investigated in four batches. 24 Results showed all values below the respective LOQs. 25 The applicant set specifications for microbiological contamination 16 The detected amounts of the above described impurities do not raise safety concerns. The presence of viable cells of the production strain in the final additive was analysed in triplicate No viable cells of the production strain were found in the final product; however, the Panel has reservations on the acceptability of this data set due to the method not fully following the recommendations listed in the FEEDAP Guidance (EFSA FEEDAP Panel, 2018b). In a second data set, 10-g samples from each batch in triplicate were suspended in 590 mL of 0.9% NaCl. Thereafter, 60 mL of this suspension (corresponding to 1 g of sample) was filtered over a mixed cellulose ester (MCE) filter (0.45 lm) and incubated at 30°C for 5 days on YPG agar plates with 16 mg/L ciprofloxacin and 16 mg/L gentamycin. A positive control (spiked sample) was included. 34 Results showed that no viable cells of the production strain were found in the final product.
The presence of recombinant DNA from the production strain was tested in three batches of Xygest TM HT, each in triplicate. 35 No DNA of the production strain was detected.

Stability and homogeneity
The stability of the additive was studied in samples (50 g) from three batches stored in plastic containers at 4°C (up to 12 months), 22°C (up to 20 months) and 40°C (up to 6 months). Negligible losses were seen at 4°C and 22°C while losses up to 34% were seen at 40°C. 39 The stability of the additive (one batch) was tested in a commercial vitamin/mineral premixture when added at 450 mg/kg and stored at room temperature. 40 Negligible losses were seen after 6 months.
The stability of the additive under assessment (one batch) was studied in a complete feed for layers (finisher) in mash and pelleted forms when added at 30 mg/kg feed (corresponding to 90,000 U/kg feed) and stored at room temperature for 3 months. The basal diet consisted of maize, wheat and soybean meal. 41 The mash feed supplemented with the additive was pelleted at 90°C for 30 s. The heat treatment led to a loss of activity of 16%. Losses at the end of the storage period were negligible in mash and 13% and pelleted feed.
The capacity for homogeneous distribution of the additive in feed was studied in ten-subsamples of the complete feed descried above (mash form). The coefficient of variation was 6%. 42

Conditions of use
The additive is intended for use in feed for poultry at a proposed minimum level of 30,000 U/kg of complete feed for growing poultry and 45,000 U/kg of complete feed for laying poultry.

Safety
3.2.1. Safety of the production strain, for the consumer and environment The recipient strain from which the production organism was derived belongs to K. phaffii, which is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment when used for enzyme production (EFSA BIOHAZ Panel, 2007, 2020). The production strain was well identified, differed from the parental strain by four and contains no acquired antibiotic resistance genes. Therefore, the genetic modification does not raise safety concerns. Moreover, neither viable cells nor DNA of the production strain were detected in the final product. Therefore, the additive does not pose any safety concern regarding the production strain. Considering all the above, the FEEDAP Panel concludes that the additive is safe for the consumers.
Regarding the environment, no cells and no DNA of the production strain were detected in the final product and the active substance of the additive is a protein, and as such will be degraded/inactivated during passage through the digestive tract of animals or in the environment. Therefore, the additive raises no concerns for the environment.

Safety for the target species
Two tolerance trials, one with chickens for fattening and one with laying hens, were submitted to support the safety for the target species.

Conclusion on safety for the target species
Based on tolerance studies in chickens for fattening and laying hens, the FEEDAP Panel concludes that the additive is safe in chickens for fattening at 30,000 U/kg and in laying hens at 45,000 U/kg, with margins of safety of 3009 and 259, respectively. The conclusions on chickens for fattening are extended to chickens reared for laying/breeding and extrapolated to all poultry for fattening and reared for laying/breeding and to ornamental birds. The conclusions on laying hens are extrapolated to all laying poultry species.

Effect on respiratory system
No specific tests were submitted on the effects of the additive on the respiratory system. However, based on the proteinaceous nature of the active substance of the additive, it is considered as a respiratory sensitiser.

Effect on eyes and skin
An acute eye irritation/corrosion study with Xygest TM HT was performed according to GLP and OECD Test Guideline (TG) 405. 53 Under the conditions of the study, the test item did not produce any signs of irritation/corrosion and is considered as non-irritant to the eyes.
An acute skin irritation/corrosion study was performed according to GLP and OECD TG 404. 54 Under the conditions of the study the test item did not produce any signs of dermal irritation and is considered as non-irritant to the skin.
A the induction phase in two out of 10 guinea pigs but no skin reaction in any animal after the challenge phase. The FEEDAP Panel noted that the study did not follow the recommendation of the relevant OECD TG regarding the minimum number of animals to be used, however given the nature of the test material and since the results were already positive on a reduced number of animals, the test compound should be considered a skin sensitiser.

Conclusions on safety for the user
The additive is considered not to be irritant to eyes and skin but is considered a dermal and respiratory sensitiser.

Chickens for fattening
A total of four trials with chickens for fattening (including the tolerance trial) with a similar design were submitted. However, two could not be further considered for the following reasons: one had a high mortality ( ) 56 and the husbandry conditions did not reflect the way chickens for fattening should be raised in the EU (for the lighting programme) according to the Directive 2007/43/EC 57 , and the other study (short-term) was discarded due to the inadequacy of the endpoints provided to support the efficacy. 58 The details on the study design of the remaining trials are provided in Table 1 Taking all the evidence together, two studies demonstrated a positive effect of the additive on performance in chickens for fattening at the recommended dose of 30,000 U/kg feed. In the absence of a third study with significant positive results, the FEEDAP Panel is not in the position to conclude on the efficacy of Xygest TM HT for chickens for fattening, and consequently for growing poultry.

Laying hens
A total of five trials with laying hens were submitted (including the tolerance trial), four long-term trials (duration of 84 days) and one short-term study (duration 28 days) sharing a common design. However, one study 62 could not be further considered due to a lack of data (e.g. daily egg mass per hen and confirmation of level of additive used) and the husbandry conditions which did not reflect the husbandry conditions for laying hens in the EU (space granted per hen) according to the Directive 1999/74/EC. 63 The details on the study design are provided in Table 3. 59 Technical dossier/Section IV/Annex IV_1-Annex IV_10. 60 Table 4. Both long-term trial 1 and 3 showed higher egg weight, egg mass per hen and an improved feed to egg mass ratio at the minimum recommended use level. In trial 2, a reduced daily feed intake was observed. In addition, an improvement of AME was observed in diets supplemented with the additive at the minimum recommended use level of 45,000 U/kg in trials 2 and 4.
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 Xygest TM In the current application, an authorisation of a preparation of endo-1,4-beta-xylanase (EC 3.2.1.8) is sought under Article 4(1) for all poultry, 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 feed additive is endo-1,4-beta-xylanase, produced by fermentation with the genetically modified strain Komagataella phaffii ATCC KMI000000 3 . The activity of endo-1,4-beta-xylanase is expressed in xylanase units (U). One U unit is the amount of enzyme that releases 0.0067 lmol of reducing sugar (xylose equivalent) per minute and per gram of enzyme product at 50°C and pH 5.3.
The feed additive is intended to be marketed as a solid formulation having a guaranteed minimum endo-1,4-beta-xylanase activity of 3,000,000 U/g and to be incorporated directly into complete feedingstuffs or through premixtures to obtain a minimum xylanase activity of 30,000 or 45,000 U/kg feedingstuffs depending on the target species.
For the quantification of the xylanase activity in the feed additive the Applicant submitted a singlelaboratory validated and further verified method based on the enzymatic hydrolysis of xylanase on the beechwood xylan substrate and the colour formation of the released reducing sugar with arsenomolybdate.
Furthermore, for the quantification of endo-1,4-beta-xylanase in premixtures and feedingstuffs the Applicant submitted another single-laboratory validated and further verified colorimetric method, based on the measurement on the quantification of water soluble dyed fragments produced by the action of xylanase on a commercially available (Xylazyme AXE®, Megazyme) azurine cross-linked wheat arabinoxylan substrate.
Based on the acceptable performance characteristics the EURL recommends for official control (i) the single-laboratory validated and further verified colorimetric (arsenomolybdate) method for the determination of endo-1,4-beta-xylanase in the feed additive and (ii) the colorimetric (Megazyme) method for the determination of 1,4-beta-xylanase in 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.