Evaluation of an alkaline hydrolysis method under atmospheric pressure for Category 1 animal by‐products

Abstract

A new alternative method for the processing of entire bodies or body parts of pet animals (Category 1 animal by‐products (ABPs)) was assessed. The method consists of an alkaline hydrolysis process under atmospheric pressure carried out in a batch system within a stainless‐steel container at temperatures higher than 95.5°C for more than 14 h. Prions are the most resistant biological hazards potentially present in the material to be treated. The proposed method was assessed by the BIOHAZ Panel for its efficacy in achieving a reduction in prion infectivity by at least 6 log₁₀ to be considered equivalent to the processing method laid down in Point A Section 2 Chapter IV Annex IV of Commission Regulation (EU) No 142/2011. The application focusses on demonstrating the capacity of the alternative method to inactivate prions by providing evidence from two studies applying matrix‐assisted laser desorption/ionisation time of flight (MALDI–TOF) mass spectrometry to show the absence of peptides larger than 3 kDa after the treatment. The BIOHAZ Panel considers that these studies do not provide direct or indirect experimental evidence that the quantitative reduction of prion infectivity is achieved by the alternative method. Therefore, in the absence of quantitative estimation of prion infectivity reduction, the alternative method cannot be considered equivalent to the approved alkaline hydrolysis process.

SUMMARY

On 29 January 2024, the European Food Safety Authority (EFSA) received from the Dutch Competent Authority (Ministry of Agriculture, Nature and Food Quality of the Netherlands) an application (mandate and technical dossier: EFSA‐Q‐2024‐00286) under Regulation (EC) No 1069/2009 referring to the evaluation of an alternative processing method for alkaline hydrolysis of Category 1 animal by‐products (ABPs), submitted by Volpe Funeral Solutions (hereinafter referred to as the applicant).

The proposed new method is limited to entire bodies or body parts of pet animals as referred to in Regulation (EC) No 1069/2009, in Article 8(a)(iii) and Article 3, Point 8. The process consists of alkaline hydrolysis at a temperature ≥ 95.5°C with a retention time ≥ 14 h, carried out in a batch system within a stainless‐steel container (the digester).

Under Article 20 of Regulation (EC) No 1069/2009, EFSA is required to assess whether the proposed method ensures that any risks to public or animal health are reduced to a level at least equivalent to that achieved by the processing method outlined in the legislation for the same category of ABPs. Although the applicant did not provide a comprehensive and detailed list of possible hazards, prions were considered the most resistant hazard. The BIOHAZ Panel followed the approach of focusing on the capability of the alternative method to reduce prion infectivity.

Previous opinions (EFSA BIOHAZ Panel, 2015, 2020, 2021) established that a reduction in prion infectivity by at least 6 log₁₀ is required to consider the alternative method equivalent to the processing method specified in Commission Regulation (EU) No 142/2011. The applicant provided studies, relevant for this evaluation, including the test conditions under which the experiments were performed, the methodologies used and the interpretation of the results.

The references provided by the applicant demonstrated that the inactivation of selected test spore‐forming microorganisms took place in a much shorter time than that required for the completion of the thermochemical process. With regards to prions, the application provided evidence from studies using MALDI–TOF mass spectrometry (MS) to show the absence of peptides larger than 3 kDa after the treatment hence supporting its capacity to inactivate prions. However, these studies did not provide direct or indirect experimental evidence of the quantitative reduction of prion infectivity achieved by the proposed alternative method. Therefore, there is not sufficient evidence in the application to conclude that the required 6 log₁₀ reduction of prions is achieved.

The Hazard Analysis and Critical Control Points (HACCP) plan provided by the applicant is inadequate. It includes major deficiencies in the hazard identification, the identification of the critical control points (CCP) and the selection of corrective actions. While the assessment of the risks associated with interdependent processes is generally appropriate, there is a lack of information on measures to prevent cross‐contamination of processed and end products by raw materials. Additionally, details about the procedures for the transport and storage of pet bodies and the final product are insufficient. There are no risks associated with the intended end use of the final product.

In conclusion, in the absence of quantitative estimation of prion infectivity reduction, the alternative method cannot be considered equivalent to the alkaline hydrolysis process as laid down in the legislation in Point A Section 2 Chapter IV Annex IV of Commission Regulation (EU) No 142/2011.

1. INTRODUCTION

On the 29th of January 2024, the European Food Safety Authority (EFSA) received from the Dutch Competent Authority (Ministry of Agriculture, Nature and Food Quality of the Netherlands) the application (mandate and technical dossier) (EFSA‐Q‐2024‐00286) under Regulation (EC) No 1069/2009, ¹ referring to the evaluation of a modified alternative processing method for alkaline hydrolysis of Category 1 animal by‐products (ABPs), submitted by Volpe Funeral Solutions (hereinafter referred to as the applicant).

The applicant submitted an application following the procedure for authorisation of an alternative method of use or disposal of animal by‐products or derived products, laid down in Article 20 of the Regulation (EC) No 1069/2009. On the 1st of May 2024, EFSA received the application through the EFSA portal for submission of ABP applications (Portalino) (CR‐2024‐000073), in line with the new provisions implemented by the Transparency Regulation (EU) No 2019/1381. ²

During the completeness check, performed according to Regulation (EC) No 1069/2009, it was noticed that some information was missing or incomplete, thus the dossier could not be considered complete. On the 19th of June 2024, EFSA sent a letter to the applicant, including four requests: (a) to submit a non‐confidential and a confidential version of the dossier with all information claimed to be confidential (including personal data as well as technical or scientific parts of the dossier); (b) to submit the pre‐assessment provided by the Dutch competent authority; (c) to state clearly the scope of the material to be treated, if it applies to all Category 1 ABP material or only to pet animals and not to other Category 1 ABP material; (d) to state clearly whether the proposed alkaline hydrolysis method is an alternative to the standard approved alkaline hydrolysis method.

On the 15th of July 2024, the applicant resubmitted the dossier, but on the 18th of July 2024 the applicant was asked for clarification and resubmission because of issues in the confidentiality assessment.

On the 12th of August 2024 EFSA received the missing information requested. After checking the content of the full dossier, EFSA considered that the application was valid on the 20th of August 2024. According to Regulation (EC) No 1069/2009, EFSA shall conduct the assessment within 6 months following receipt of a complete application.

The application is a modification of the alternative processing method described in point A in Section 2 of chapter 4 of Annex IV of Commission Regulation (EU) No 142/2011, which consists of an alkaline hydrolysis process carried out according to the following processing standards:

1. ‘either sodium hydroxide (NaOH) or potassium hydroxide (KOH) solutions (or a combination thereof) must be used in an amount that assures approximate molar equivalency to the weight, type and composition of the ABPs to be digested.

2. ABPs must be placed in a steel alloy container.

3. The container must be closed and the ABPs and alkali mixture must be heated to a core temperature of at least 150°C and a pressure (absolute) of at least 4 bars for at least:

   1. 3 h without interruption,
   2. 6 h without interruption in case of treatment of ABPs referred to in Article 8(a)(i) and (ii) of Regulation (EC) No 1069/2009,
   3. 1 h without interruption in the case of ABPs consisting of fish or poultry materials.

4. The process must be carried out in a batch system and the material in the vessel must be constantly mixed in order to facilitate the digestion process until the tissues are dissolved and bones and teeth are softened and

5. The ABPs must be treated in such a way that the requirements regarding time, temperature and pressure are achieved at the same time’.

The modification proposed by the applicant is also based on an alkaline hydrolysis but at a lower temperature, of at least 95.5°C, at ambient pressure, during a longer processing time, of at least 14 h, without interruption.

2. DATA AND METHODOLOGIES

2.1. Data

The data used in the assessment were provided by the Applicant as requested in Annex VII of Commission Regulation (EU) No 142/2011 and its amendment by Commission Regulation (EU) No 749/2011. ³ A process flow diagram (Figure 1), with a description of the proposed alternative process, and a Hazard Analysis and Critical Control Point (HACCP) plan were included in the application dossier. Additional data were also submitted by the applicant in response to a request for additional information as described above. The report submitted by the Dutch Competent Authority (CA) related to the application was also considered. Relevant scientific papers provided by experts of the Working Group (WG) were also considered during the assessment.

FIGURE 1.

Process flow diagram of alternative processing method for Category 1 pet animal by‐products.

EFSA carried out a public consultation on the non‐confidential version of the application from 8 January to 29 January 2025 for which no comments were received.

2.2. Methodologies

The EFSA BIOHAZ Panel evaluated the application for an alternative method of alkaline hydrolysis under atmospheric pressure for category 1 animal by‐products, by individually assessing the following steps as set out in the ‘Statement on technical assistance on the format for applications for new alternative methods for animal by‐products’ (EFSA BIOHAZ Panel, 2010).

These steps are:

1. full description of the process;

2. full description of the material to be treated;

3. hazard identification;

4. level of risk reduction;

5. HACCP plan;

6. risks associated with interdependent processes;

7. risks associated with the intended end use of the product.

The applicant is required to document as fully as possible the different aspects of each of these steps. According to the CA assessment, the application meets the requirements as laid down in the EFSA Statement (EFSA BIOHAZ Panel, 2010).

As set out in subparagraph 5 of Article 20 of Regulation (EC) No 1069/2009, EFSA is required to assess whether the methods submitted ensure that the risks to public or animal health are:

1. ‘controlled in a manner which prevents their proliferation before disposal in accordance with this Regulation or the implementing measures thereof’; or

2. ‘reduced to a degree which is at least equivalent, for the relevant categories of animal by‐ products, to the processing methods laid down pursuant to point (b) of the first subparagraph of Article 15(1)’.

This requirement for applications is described in Commission Regulation (EU) No 142/2011, implementing Regulation (EC) No 1069/2009 and amended by Commission Regulation (EU) No 749/2011. According to point 2 d, Chapter II, Annex VII of Commission Regulation (EU) No 142/2011, any application for the evaluation of alternative methods shall ‘show that the most resistant biological hazards associated with the category of materials to be processed are reduced in any products generated during the process, including the wastewater, at least to the degree achieved by the processing standards laid down in this Regulation for the same category of animal by‐products. The degree of risk reduction must be determined with validated direct measurements, unless modelling or comparisons with other processes are acceptable’.

According to the EFSA Statement (EFSA BIOHAZ Panel, 2010) and to point 3, Chapter II, Annex VII of Commission Regulation (EU) No 142/2011, validated direct measurements as referred to above shall mean:

1. ‘measuring the reduction of viability/infectivity of endogenous indicator organisms during the process, where the indicator is:

   • – consistently present in the raw material in high numbers,
   • – not less resistant to the lethal aspects of the treatment process, but also not significantly more resistant, than the pathogens for which it is being used to monitor,
   • – relatively easy to quantify and relatively easy to identify and to confirm; or

2. using a well‐characterised test organism or virus introduced in a suitable test body into the starting material.’

The EFSA Statement (EFSA BIOHAZ Panel, 2010) asserts that ‘results should be accompanied by evidence’. Evidence ‘includes, for measurements, information on the methodology used, nature of samples that have been analysed and evidence that samples are representative (e.g. number of samples, number of tests performed and selection of measuring points). If several treatment steps are involved, an assessment should be performed on the degree to which individual titre reduction steps are additive, or whether early steps in the process may compromise the efficacy of subsequent steps. In any case it is necessary to provide the sensitivity and specificity of the detection methods applied. Data on the repeatability and statistical variability of the measures obtained during the experiments should also be presented.’ It also states that ‘Generally, the level of risk reduction for human and animal health which can be achieved by the process should be evaluated on the basis of direct measurements (validation).’ Should ‘no direct measurements of the risk reduction be available (i.e. no validation as defined above is feasible), modelling or comparison with other processes may be acceptable if:

1. the factors leading to the risk reduction are well known;

2. the model of risk reduction is well established; and

3. continuous direct measurements of the factors leading to the risk reduction are provided for the full‐scale process which demonstrate that these factors are homogeneously applied throughout the treated batch’.

In point 2 d, ‘Level of risk reduction’ of Section 2.1.2.1 ‘Content of applications’ of the EFSA Statement (EFSA BIOHAZ Panel, 2010), it is stated that ‘in principle, the new proposed process should be able to reduce the amount of the most resistant biological hazards associated with the category of the material to be processed for a defined final use to an acceptable level’. Although Chapter II of Annex VII of Commission Regulation (EU) No 142/2011 adopted the proposal of the EFSA opinion to use ‘the level of risk reduction’ and ‘the level of reduction of the most resistant biological hazards’ interchangeably, it is acknowledged that these are different terms and that the purpose of the evaluation of alternative methods is not the estimation of the level of any risk, but the level of hazard reduction.

In previous EFSA opinions (EFSA BIOHAZ Panel, 2015, 2020, 2021) dealing with alternative processing methods for Category 1 ABPs, the BIOHAZ Panel concluded that a reduction of 6 log₁₀ in prion infectivity by the alternative method is required to consider it at least equivalent to the method approved in the legislation. The same approach is used in the evaluation of the current application.

The full dossier provided by the applicant is available here: https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00286. Therefore, the current opinion includes only the evaluation performed by the BIOHAZ Panel, with references to the relevant sections of the original dossier.

3. ASSESSMENT

3.1. Full description of the process: Brief description of the method

The description presented in the current section of the assessment has been extracted verbatim from the application, with minor editorial changes for clarity purposes.

For this process, ABPs of all categories may be used. This application is limited to the processing of entire bodies and body parts of pet animals (as referred to in Article 8(a)(iii) and point 8, Article 3 of Regulation (EC) No 1069/2009).

The alkaline hydrolysis process is carried out in a batch system in a stainless‐steel container (the digester).

The process, as described for use on pet‐animal by‐products, has been illustrated in 13 steps, as can be seen in Figure 1.

In Step 1, the pet animal by‐products are placed into an internal basket inside the system which consists of solid sides, a perforated stainless‐steel bottom with a pore size of 4.5 mm and a latching lid with a pore size of 4.5 mm. If desired, the internal basket system can be further divided with vertical partitions, so each pet animal occupies its own individual portion of the internal basket system to keep its remains separated from other pet animals. The system is designed to allow the solution to flow throughout the digester uniformly through the pores.

After a weighed amount of animal by‐products is loaded into the digester, Step 2 entails adding the prescribed amount of sodium hydroxide (NaOH) and/or potassium hydroxide (KOH) to the digester. The amount of NaOH or KOH assures approximate molar equivalency to the weight, type and composition of the animal by‐products to be digested. The prescribed dosage for each weight category is based on validated cycle conditions that yield complete hydrolysis of all tissue material, reduction of peptide sizes to ≤ 2.5 kDa and validated sterilisation conditions.

The alkali can be added in either a solid form or in solution depending on the configuration of the equipment. A chart provided by the manufacturer of the digester lists the amount of alkali (KOH) and water to be added to the cycle, based on the measured weight of the animal by‐products to be processed in the cycle and on the model of the digester. The chart also specifies the molarity of the solution and the starting pH of the mixture (between 13.44 and 14.14) and is available for all ranges of weights and models of equipment. According to the information provided by the applicant, there are two systems of digester (PET‐400 and PET‐550) with a maximum capacity of animal by‐products of 254 and 277 kg, respectively. The dose of 90% KOH for loads of animal by‐products higher than 63.5 kg is 0.13 kg 90% KOH/kg animal by‐product, for both systems. When the loads of animal by‐products are lower than 63.5 kg, the weight of added alkali remains the same (8.25 kg 90% KOH) independently of the weight of animal by‐product. Both the weight of the pet animal by‐products and the quantity of alkali added to a cycle are recorded in writing by the qualified operator.

Then, the digester is closed and latched. The operator initiates Step 3 by pressing the Start Process button. The system is automatically filled with water so that the ABPs are fully covered with alkali solution. The solution begins circulating, and the ABPs and alkali mixture are heated to a temperature of at least 95.5°C. The system allows the selection of settings between 95.5 and 97.8°C, within the admin controls. Once the system reaches the setpoint for the treatment temperature (min. 95.5°C), the system begins tracking the treatment cycle (Step 4). The system will maintain the process without interruption at ≥ 95.5°C, under constant circulation, for a minimum of 14 h, as determined by the setpoint treatment time. The system allows the selection of a setpoint treatment time between 14 and 20 h in the admin controls.

The treatment process has been validated to sterilise the biological material by operating a treatment cycle that consists of alkali added by weight at 13% of the weight of the tissue (assuming 90% dry KOH) or molar equivalency, with a minimum starting molarity of 0.28 M, at a temperature of ≥ 95.5°C for a minimum of 14 h.

The process is controlled by a programmable logic controller (PLC) and components. The temperature and duration are continuously monitored and registered. If the process is interrupted, the treatment cycle will be automatically extended to ensure that the full treatment time is achieved.

A mixer mechanism ensures continuous circulation in order to facilitate the digestion process until the tissues are dissolved and bones and teeth are softened. If the mixer function is interrupted, the treatment cycle will be automatically extended to ensure that the treatment cycle took place with proper circulation.

If a power outage occurs during the process, the system has an uninterruptible power supply (UPS) to ensure that all components fail safe. The system's PLC also stores all cycle data, so that any interruption of any length of time, in the middle of a treatment cycle, will not affect the system's ability to restart the treatment cycle and complete the sterilisation process.

The data for each treatment cycle are logged on the system's PLC and available for retrieval. The treatment cycle data includes the treatment time, temperature and verification of a completed treatment cycle.

After the treatment cycle is completed, Step 5 prepares the sterile effluent for discharge. The pH of the solution is reduced inside the digester using automatic CO₂ or citric acid injection, depending on the setup of the equipment. The pH is reduced to the level that the local wastewater authority has permitted for discharge, typically pH 10. If the system fails to successfully inject the neutralising agent, the system will indicate a Level 2 Alarm and pause the process until a corrective action is taken. The content of the digester is then cooled to the temperature the local wastewater authority has permitted for discharge, typically ≤ 60°C. In Step 6, the effluent is discharged to the sewer system, in accordance with a permit from the Water Authority (Waterschap). Physical screening (sieving) takes place at the time of discharge because all the material was placed into an internal basket system with 4.5 mm pores. The discharge is controlled by a modulating valve and temperature sensor to prevent the discharge of material outside the setpoint drain temperature. The system will not drain if the temperature exceeds the setpoint drain temperature.

In Step 7, the system performs a clean water rinsing cycle wherein it fills with clean water and circulates rinse water for a user‐selected period of time, typically 1 h. This rinsing water is like tap water characteristics and when the system is opened (Step 8) is discharged to the sanitary sewer system, in accordance with the permit or agreement from the Water Authority. The solid remains of bone and teeth are still retained in the basket system which serves as physical screening (sieving) during this second draining process.

The data for the important post‐treatment cycle processes are logged on the system's PLC and available for retrieval. These data include the number of neutralisation injections and discharge temperature. There are no other by‐streams generated from this process.

In Step 9 the system is opened, and the internal baskets can be removed from the system. The bone remains from each individual pet animal are separately collected. In Step 10 the bone remains are transferred to individual trays and dried in a drying oven. Once dry, in Step 11 they are pulverised in a piece of equipment typically used in the pet cremation industry to produce a powdered ash. The ashes are placed in an ash container (Step 12) and in Step 13 the ash container is made available to the owner of the pet animal. Owners can keep the ashes in an urn and opt for the burial of the ashes or scatter them in a relevant and important place in accordance with local regulations.

The following processing aids are used during the process:

• Sodium hydroxide (NaOH) and/or potassium hydroxide (KOH)

• Water

• Neutralising agents (CO₂ of citric acid)

According to the applicant, the process parameters of the alternative process are the following:

• Alkali concentration: The amount of NaOH/KOH assures approximate molar equivalency to the weight (13% or molar equivalency), type and composition of the ABP to be digested. The starting pH of the solution for any combination of weight of tissues and vessel capacity is at least 13.4.

• Pressure: The processing of ABPs by the proposed alternative method takes place at atmospheric conditions.

• Temperature: Since the processing is carried out under atmospheric conditions, the product is heated to just below the boiling point (which is approx. 100°C). For the application, the minimum temperature is 95.5°C.

• Time: Research and practical experience show that a duration of 12 h is sufficient to completely dissolve and hydrolyse the tissues. However, the application assumes a minimum duration of 14 h.

• Continuous circulation: Process under continuous circulation. The continual circulation of the solution ensures contact of the reducing agents with the tissue material (collision theory) thereby aiding the digestion process and resulting in the hydrolysis of all tissue material.

3.2. Assessment of the BIOHAZ Panel on the material to be treated

The applicant states that this application is limited to the processing of entire bodies or body parts of pet animals as referred to in Regulation (EC) No 1069/2009 in:

• – Article 8(a)(iii): animals other than farmed and wild animals, including in particular pet animals, zoo animals and circus animals;
• – Article 3, Point 8: any animal belonging to species normally nourished and kept but not consumed (purposes other than farming).

Although these articles refer to other animals apart from pets, the application is only intended for pet animals, as defined in Regulation (EU) No 576/2013, ⁴ meaning ‘an animal of a species listed in Annex I, accompanying its owner or an authorised person during non‐commercial movement, and which remains for the duration of such non‐commercial movement under the responsibility of the owner or the authorised person’.

Annex I of Regulation (EU) No 576/2013 on the non‐commercial movement of pet animals includes the species of pet animals: ‘dogs (Canis lupus familiaris), cats (Felis silvestris catus), ferrets (Mustela putorius furo), invertebrates (except bees and bumble bees covered by Article 8 of Directive 92/65/EEC and molluscs and crustaceans referred to respectively in points (e)(ii) and (e)(iii) of Article 3(1) of Directive 2006/88/EC), ornamental aquatic animals (as defined in point (k) of Article 3 of Directive 2006/88/EC and excluded from the scope of that Directive by point (a) of Article 2(1) thereof), amphibia, reptiles, birds (specimens of avian species other than those referred to in Article 2 of Directive 2009/158/EC) and mammals (rodents and rabbits other than those intended for food production and defined under ‘lagomorphs' in Annex I to Regulation (EC) No 853/2004)’.

According to the applicant, typically the bodies are kept cool at refrigeration temperatures, in the range 1–5°C. The physical state will not affect the efficacy of the method, but the time needed to reach the baseline temperature required.

3.3. Assessment of the BIOHAZ Panel on the hazard identification

The applicant identified the following relevant biological hazards for human and animal health:

• Zoonotic agents: include bacteria, parasites, fungi and possibly some viruses. Zoonotic agents are agents transferred from animals to humans and may cause diseases.

• Animal pathogens: specific animal pathogens (viral, bacterial and parasitic) may be present and may cause animal disease.

• Toxins and degradation products: within ABPs several biological processes may occur, including growth of toxigenic microorganisms, which can result in the production of microbial toxins and other potentially toxic metabolites.

• TSEs: transmissible spongiform encephalopathies.

The dossier does not provide a comprehensive and detailed list of all possible hazards identified. Instead, it focuses on prions and two selected test organisms, Geobacillus stearothermophilus and Bacillus thuringiensis, as biological indicators for validating thermochemical sterilisation and sterilisation processes in the absence of chemicals, respectively.

Given the variety of materials intended to be used in the proposed process, and the uncertainty surrounding the cause of the pets' deaths entering the process, the presence of highly thermo and chemoresistant hazards cannot be ruled out (EFSA BIOHAZ Panel, 2012). Although the applicant did not present the complete hazard identification, TSEs must be considered the most relevant hazards, given their high resistance to destruction and, in particular, their high thermostability (EFSA BIOHAZ Panel, 2015, 2020, 2021; Somerville et al., 2009).

3.4. Assessment of the BIOHAZ Panel on the level of risk reduction

The applicant provided five supporting studies, as evidence to show the effectiveness of the alternative method (alkaline hydrolysis process at 95.5°C for ≥ 14 h), to reach the target level of risk reduction.

3.4.1. Thermoresistant microorganisms

The research described in the study by Denys (2019) was performed in an alkaline hydrolysis system provided by the same manufacturing company (Bio‐Response Solutions) with the minimum critical process parameters specified in the application: 90% dry KOH added by weight at 13% of the weight of the tissue, a 313 lb (142 kg) pig carcass, at a temperature of 95.5°C for 14 h, using the same mixer system and circulation flow rates, and an internal basket with identical 4.5 mm pore size. Spores of G. stearothermophilus were placed in stainless‐steel sample vials, which also contained tissue, water and alkali, to simulate a range of tissue weights and molarities of the thermochemical process. Spores of B. thuringiensis were placed in similar vials and exposed to heat alone without the addition of alkali. The vials were implanted into the deep tissues of the pig. Matrix‐assisted laser desorption/ionisation time of flight (MALDI–TOF) mass spectrometry (MS) analysis was performed to validate the destruction of prion‐sized particles in processed effluent. The study demonstrated a 6‐log₁₀ reduction of G. stearothermophilus spores in the thermochemical process, and a higher than 6‐log₁₀ reduction of B. thuringiensis spores achieved by the thermal treatment applied alone. The MALDI–TOF MS analysis of the liquid by‐product detected no peptide fragments greater than 2.5 kDa.

The study by Homer et al. (2012) showed additional research on the same process, using high tissue weights (100–200 lb; 45–90 kg) and G. stearothermophilus spores as a biological indicator. Spores were similarly placed in vials: alone in glass ampules to assess the effect of heat alone. The glass ampules containing at least 10⁶ G. stearothermophilus spores were exposed only to the heat and time of the tissue dissolver (TD) cycle. A stainless‐steel test vessel that had been pre‐coated with at least 10⁶ spores of G. stearothermophilus and sealed within a Tyvek pouch was used to evaluate the effect of the alkaline environment with and without tissue. Exposure to the standard TD cycle did not inactivate G. stearothermophilus contained inside glass ampules indicating that the heat and time of the TD cycle alone are not sufficient to inactivate this indicator microorganism. On the other hand, the standard TD cycle inactivated the spores that were exposed to KOH solution (with final concentration of 0.50%–2.26%) in the test vessels. Concentrations of KOH lower than 0.5% were insufficient to inactivate spores or completely digest animal tissues. Despite starting from a frozen state (−28.9°C) and adding a lesser amount of alkali (0.5% KOH), the study showed complete tissue hydrolysis after 14 h of treatment and complete spore inactivation after a 10‐h treatment under the alkaline conditions. However, incomplete spore inactivation was reported when only heat was applied.

Pinho et al. (2015) conducted a study by mixing G. stearothermophilus spores in suspension with NaOH of different concentrations, at 80, 100 and 110°C in a batch reactor, in presence or absence of discarded medical components or animal tissues cut in fragments of approx. 1 cm². Survival curves were plotted, and D‐values were obtained, showing a 6 log₁₀ reduction for G. stearothermophilus spores in 0.5 M NaOH in the presence of tissues in less than 30 min at 80°C. Similar inactivation kinetics were obtained in the absence of animal tissues of discarded medical components.

The study by Vijayan and Ng (2016) utilised a tissue digester (TD), using G. stearothermophilus at 6 log₁₀ as a biological indicator and sodium hydroxide (NaOH) as the alkali. Various tests were performed to determine the optimal amount of NaOH required for complete tissue digestion, based on carcass weight. The findings indicated the following NaOH requirements: 400 g for carcasses weighing between 100 and 3000 g; 700 g for those between 3001 and 6000 g; and 1400 g for carcasses weighing 6001–12,000 g. The study determined the ratio of carcass weight to alkali quantity needed for effective alkaline hydrolysis and showed complete inactivation (at least 6 log₁₀ reduction) of G. stearothermophilus after 8 h at 96°C.

Of all these studies, at least a 6 log₁₀ reduction of the spores of the test organisms selected by alkaline hydrolysis was shown at < 30 min treatment and temperatures as low as 80°C, either in presence or absence of animal tissues.

3.4.2. TSE agents

Due to the extreme thermochemical stability of prions, they are considered the most relevant hazards, and it can be assumed that even thermoresistant viruses and bacterial spores would be completely inactivated if the new method assures inactivation of prions. A reduction of 6 log₁₀ in prion infectivity by the alternative method is required to consider the alternative method equivalent to the method approved in the legislation (EFSA BIOHAZ Panel, 2015, 2020, 2021).

Two studies were provided by the applicant to support the capacity of the alternative method to inactivate prions.

The study by Inerowicz (2012) is a white paper presentation of a private third‐party validation test, property of Bio‐Response solutions Inc., that was included in a publication at a book series of the American Biological Safety Association in 2012 and in a presentation in a conference of the National Institutes of Health in 2017.

The study was conducted to determine the ability of the alkaline hydrolysis treatment at temperatures below 100°C to break down proteinaceous material into fractions smaller than infectious prion fragments. Whole animal remains of various sizes, including a 1200 lb (544 kg) horse, were processed. The largest particle detected by MALDI–TOF in the effluent of the alkaline hydrolysis system, working at 89°C for 4 h with alkali added at 11% the tissue weight, was smaller than 3.0 kDa.

In the study by Denys (2019) (process described above), the MALDI–TOF MS analysis of the liquid obtained following alkaline hydrolysis by the system provided by the manufacturing company (Bio‐Response Solutions) revealed no detection of peptide fragments greater than 2.5 kDa. Following their analysis of the scientific literature, the applicant considered that the size of the smallest infectious prion particle was 55 ± 9 kDa (Prusiner, 2004). Based on these elements, the applicant claimed a complete inactivation of TSE agents by the proposed methodology.

The dossier does not provide direct or indirect experimental evidence on the quantitative reduction of prion infectivity achieved by the proposed alternative method. This is not in accordance with the requirements as in point 2 Chapter 3 Annex IV of the 142 Commission Regulation, which specifies that the degree of risk reduction must be determined with validated direct measurements, unless modelling or comparisons with other processes are acceptable.

Instead, the applicant provided evidence obtained in the white paper through the application of MALDI–TOF MS to show the absence of peptides larger than 3 kDa. The MALDI–TOF method is a tool used to identify specific molecules based on their mass‐to‐charge ratios. However, it has been applied for this scenario without a validation study to establish its sensitivity threshold (i.e. its limit of detection and limit of quantification) in an experimental set up which does not allow to directly demonstrate any quantitative reduction of prion infectivity. The results of MALDI–TOF and similar methods could be considered, provided that validation studies to establish the limit of detection and limit of quantification of prions are conducted in the matrices of interest.

As an example of these possible limitations, while Inerowicz (2012) reported that no peptide larger than 3.0 kDa was detected by MALDI–TOF MS after alkaline hydrolysis at 89°C for 4 h, at more stringent conditions (150°C, 3 h, 5 bars in alkaline digestion KOH or NaOH, 1 M), the study by Sommerville described in the Scientific Steering Committee (SSC) report (2002) demonstrated residual infectivity, shown by 301 V transmission observed in mice inoculated with the alkaline hydrolysis end product (SSC, 2002). This raises major concerns about the pertinence of MALDI–TOF peptide‐size analysis as a means to demonstrate the lack of residual prion infectivity in the end product of an alkaline hydrolysis process, confirming the need for a quantitative assessment of prions infectivity reduction using a sensitive‐enough method.

To conclude, in the absence of experimental estimation of prion infectivity (or seeding activity) reduction, there is not sufficient evidence in the application to conclude that a 6 log₁₀ reduction of prions infectivity is achieved.

3.5. Assessment of the BIOHAZ Panel on the HACCP plan

The Applicant conducted the hazard analysis required in principle 1 of the HACCP plan, in accordance with the Codex Alimentarius General Principles of Food Hygiene (Annex to CAC/RCP 1–1969, Rev. 4–2003). Some hazards have been described, and control or preventive measures have been proposed for the hazards. However, some of the hazards reported by the applicant, for instance ‘the insufficient hydrolysis due to circulation pump failure or insufficient time’, cannot be considered as a hazard, but a parameter of the process. The formulation of the identified hazards needs to be adapted. The version of the Codex Alimentarius General Principles of Food Hygiene used by the applicant has been superseded by subsequent versions, being the latest available one issued in 2022. The HACCP plan should be designed in accordance with the latest version of the document that provides guidance on the application of HACCP principles. ⁵

The applicant has not included the list of pre‐requisite programs. The HACCP plan has been divided into two different tables (section 9. Risk assessment and section 10. Control of CCPs), that should be integrated as parts of the HACCP plan. There should be consistency between the process steps in the flow diagram, in the table and in the names of the CCPs. For all the CCPs, critical limits should be quantitative and should have corresponding corrective actions to be implemented when the limits are not met. Moreover, procedures for verification and documentation of the HACCP plan should be included.

Applying the Codex Alimentarius decision tree, three CCPs were found, coincident with the critical process parameters: alkali/ABP ratio, heating regime (time and temperature) and continuous circulation of the solution. For each CCP, the critical limit, the monitoring methods and the corrective actions are described.

The alkali/ABP ratio cannot be considered as a CCP but a pre‐requisite since the amount of alkali to be added is established depending on the weight of the matrix. For the heating regime, the corrective actions are not specific enough. For continuous circulation of the solution, there is no description of the parameter data logged as continuous circulation, while they should refer to quantitative measures recorded by the equipment.

In conclusion, the HACCP plan is inadequate. It includes major deficiencies in the hazard identification, in the identification of the CCPs and in the selection of corrective actions.

3.6. Assessment of the BIOHAZ Panel on the risk associated with interdependent processes

The Applicant states that the effluents generated can be discarded into the sanitary sewer system after reducing the pH of the effluent and the temperature of the solution in Steps 5 and 6 of the process. The pH reduction is conducted inside the digester using automatic CO₂ or citric acid injection, depending on the setup of the equipment. The Applicant states that the pH that local wastewater authorities allow for discharge is typically between 9.0 and 11.5. This pH reduction will be automatically controlled, and an alarm will indicate if the parameter is not achieved, to facilitate that a corrective action is taken if needed.

Similarly, there are temperature requirements for discharges into wastewater, which must be done at ≤ 60°C. Additionally, a physical screening (sieving) is performed at the time of discharge using an internal basket system with 4.5 mm pores. The discharge is controlled by a modulating valve and temperature sensor to prevent the discharge of material to drain if the set temperature is exceeded. There is an additional rinse cycle with clean water in Step 7, typically of 1 h. The rinsing water generated can be discharged as wastewater to the sanitary sewer system too.

The applicant provided a description of the procedures that will be implemented when the system detects that the pH or the temperature of the effluents exceed the set values. All the data associated with these post‐treatments will be logged into the system and will be available for retrieval. These data include the number of neutralisation injections, although the number of injections is not an appropriate measure of the pH in the effluent.

The assessment of the risks associated with the interdependent processes, as outlined in the dossier, were deemed generally appropriate. However, there is no mention of any measures to separate raw material from processed and end products to prevent cross‐contamination, nor any details about the procedures for the transport or storage of the pet bodies.

3.7. Assessment of the BIOHAZ Panel on the risk associated with the intended end use of the products

The applicant indicates that the remaining bones and teeth from each individual pet animal are separately collected, transferred to individual trays and dried in a drying oven. Once dried, the remains are milled and pulverised in a specialised equipment used in the pet cremation industry, to produce powdered ash. Afterwards, the end product, milled or pulverised into unrecognisable fragments ashes or powdered ashes are placed in an ash container and made available to the pet's owner. Owners can keep the ashes in an urn, opt for burial of the ashes or scatter them in accordance with local regulations. Based on the information provided by the applicant the end use product is similar to the one of the currently approved processing method, described in Commission Regulation (EU) No 142/2011.

No risks are foreseen for the intended use of the products. The Panel concurred with the conclusion of the applicant.

4. CONCLUSIONS

• The method under assessment involves alkaline hydrolysis at a temperature of ≥ 95.5°C for a retention time of ≥ 14 h at atmospheric pressure, conducted in a batch system within a stainless‐steel container. The application evaluates a process designed only to dispose of entire bodies or body parts of pet animals as outlined in Regulation No (EC) 1069/2009, in Article 8(a)(iii) and Article 3, Point 8.

• Given that the starting material is classified a Category 1 animal by‐product (ABP), prions are the most resistant biological hazards potentially present in the material to be treated and this was also the conclusion of the hazard identification conducted by the applicant.

• It was established that a reduction in prion infectivity by at least 6 log₁₀ is required for any alternative method to be considered equivalent to the approved processing method for Category 1 ABPs in the current legislation, in this case, alkaline hydrolysis process (Point A Section 2 Chapter IV Annex IV of Commission Regulation (EU) No 142/2011).

• Evidence from two studies applying MALDI–TOF mass spectrometry (MS) for peptide‐size detection were included by the applicant to support the capacity of the alternative method to inactivate prions. However, MALDI–TOF MS does not allow to directly demonstrate any quantitative reduction of prion infectivity. Additionally, it has been applied for this scenario without a validation study to establish its sensitivity threshold (i.e. its limit of detection and limit of quantification).

• The HACCP plan is inadequate, including major deficiencies in the hazard identification, as well as in the identification of the CCPs and the selection of the corrective actions.

• The assessment of the risks associated with the interdependent processes, as outlined in the dossier, were deemed generally appropriate, although there was lack of information on measures to prevent cross‐contamination and about the transport and storage of the bodies of the pets and end products. There were no risks associated with the intended end use of the final product.

• In the absence of quantitative estimation of prion infectivity reduction, the alternative method cannot be considered equivalent to the alkaline hydrolysis process, as laid down in the legislation in Point A Section 2 Chapter IV Annex IV of Commission Regulation (EU) No 142/2011.

5. DOCUMENTATION AS PROVIDED TO EFSA

1. Application on alternative processing method for animal by‐products; Alkaline hydrolysis under atmospheric pressure. Submitted by the company Volpe Funeral Solutions to the Dutch Competent Authority and then submitted to EFSA on 29 January 2024; consolidated version submitted by the company to EFSA on 12 August 2024.

2. Report of the Dutch Competent Authority related to the application.

ABBREVIATIONS

ABPs

animal by‐products

BIOHAZ

EFSA Panel on Biological Hazards

CA

Competent Authority

CCP

critical control point

HACCP

hazard analysis critical control point

MALDI–TOF

matrix‐assisted laser desorption/ionisation time of flight

MS

mass spectrometry

PLC

programmable logic controller

TSEs

transmissible spongiform encephalopathies

UPS

uninterruptible power supply

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00286

COPYRIGHT FOR NON‐EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards) , Allende, A. , Bortolaia, V. , Bover‐Cid, S. , Dohmen, W. , Mughini‐Gras, L. , Guillier, L. , Herman, L. M. , Jacxsens, L. , Nauta, M. , Ottoson, J. , Peixe, L. , Perez‐Rodriguez, F. , Skandamis, P. , Suffredini, E. , De Cesare, A. , Escamez, P. F. , Nonno, R. , Kryemadhi, K. , Ortiz‐Pelaez, A. , & Alvarez‐Ordóñez, A. (2025). Evaluation of an alkaline hydrolysis method under atmospheric pressure for Category 1 animal by‐products. EFSA Journal, 23(2), e9272. 10.2903/j.efsa.2025.9272