Safety of dried biomass powder of Chlamydomonas reinhardtii THN 6 as a novel food pursuant to Regulation (EU) 2015/2283

TL;DR

The safety of dried biomass powder from Chlamydomonas reinhardtii THN 6 as a novel food could not be confirmed due to insufficient data provided by the applicant.

Contribution

The paper evaluates the safety of a novel food under EU regulation and highlights critical data gaps.

Findings

The applicant failed to provide the requested data on identity, production, and safety.

EFSA could not establish the safety of the novel food due to missing information.

The risk assessment identified key areas requiring further investigation.

Abstract

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on dried biomass powder of Chlamydomonas reinhardtii THN 6 as a novel food (NF) pursuant to Regulation (EU) 2015/2283. During the risk assessment, the Panel identified a number of data gaps which needed to be addressed by the applicant. Therefore, an EFSA request for additional information was sent to the applicant. The requested data pertained to the identity, the production process, composition, specifications, history of use, proposed uses and use levels, nutritional information, genotoxicity and allergenicity of the NF. Despite being contacted several times, the applicant did not reply to EFSA's requests for additional data. The Panel concludes that, based on the available data, the safety of the NF, i.e. dried biomass powder of C. reinhardtii THN 6, cannot be established.

Tables

TABLE 1. Proximate analysis of the NF.

Parameter (%)	Results for batches #1–3			Method of analysis
	#1	#2	#3
Moisture	6.2	6.2	6.1	AOAC 930.15
Protein (N × 6.25)	35.8	36	36.1	AOAC 990.03
Carbohydrates	50.6	50.2	50.3	Calculation
Fat	3.1	3.2	3.2	AOAC 2003.05
Ash	4.2	4.4	4.3	AOAC 942.05
Dietary fibre a	n.d.	n.d.	n.d.	ANKOM Tech. Method

TABLE 2. Analyses of chemical and microbiological contaminants in the NF.

Parameter	Results for batches # 1–3			Method of analysis
	#1	#2	#3
Heavy metals (mg/kg)
Arsenic	< 0.1.	< 0.1	< 0.1	USP <233> (ICP‐MS)
Cadmium	< 0.02	< 0.02	< 0.02	USP <233> (ICP‐MS)
Lead	< 0.1	< 0.1	< 0.1	USP <233> (ICP‐MS)
Mercury	< 0.01	< 0.01	< 0.01	USP <233> (ICP‐MS)
Microbiological parameters
Total viable aerobic count (CFU/g)	n.d.	200	200	AOAC 2015.13
Total yeast/mould (CFU/g)	n.d.	n.d.	n.d.	US FDA/BAM Chapter 18
Total Coliforms (CFU/g)	n.d.	n.d.	n.d.	AOAC 2018.13
Escherichia coli (CFU/g)	n.d.	n.d.	n.d.	AOAC 2018.13
Salmonella spp. (in 25 g)	n.d.	n.d.	n.d.	RapidChek/AOAC RI 030301; AFNOR SDI 34/01‐04/10
Staphylococcus aureus (CFU/g)	n.d.	n.d.	n.d.	AOAC 2003.07

TABLE 3. Specifications of the NF as proposed by the applicant.

Description: The NF is the dried biomass of C. reinhardtii THN 6. Appearance: Medium to dark green powder
Source: Chlamydomonas reinhardtii THN 6
Parameter	Specification
Moisture	< 10%
Dry matter	≥ 93%
Protein (crude)	30%–70%
Fat (crude)	< 10%
Dietary fibre (acid detergent)	1%–25%
Ash	< 5%
Chlorophyll	< 25%
Heavy metals
Arsenic	< 0.2 mg/kg
Cadmium	< 0.2 mg/kg
Lead	< 0.2 mg/kg
Mercury	< 0.2 mg/kg
Microbiological
Total viable aerobic count	< 1000 CFU/g
Total yeast/mould	< 1000 CFU/g
Total Coliforms	< 100 CFU/g
Escherichia coli	Not detected in 1 g
Salmonella spp.	Not detected in 25 g
Staphylococcus aureus	Not detected in 1 g

TABLE 4. List of provided toxicological studies with the NF.

Reference	Type of study	Test system	Dose
Study No. 842‐471‐1872 (2016a, unpublished)	Bacterial reverse mutation test (OECD TG 471)	S. Typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA	Up to 5000 μg/plate (in absence and presence of S9 mix)
Study No. 842‐473‐1871 (2016b, unpublished)	In vitro mammalian chromosomal aberration test (OECD TG 473)	V79 cell line	Up to 600 μg/mL (in absence and presence of S9 mix)
Study No. 842‐474‐1870 (2017a, unpublished)	In vivo mammalian micronucleus test (OECD TG 474)	NMRI BR mice	2000 mg/kg bw per day
Study No. 842‐407‐1771 (2017b, unpublished)	28‐day repeated‐dose oral toxicity study (OECD TG 407)	Hsd. Han Wistar rats	Up to 4000 mg/kg bw per day

Full text

INTRODUCTION

1

Background and Terms of Reference as provided by the requestor

1.1

On 28 April 2023, the applicant Triton Algae Innovations, Ltd., submitted a request to the European Commission in accordance with Article 10 of Regulation (EU) 2015/2283 to authorise the placing on the market of dried biomass powder of Chlamydomonas reinhardtii as a novel food.

The application requests to authorise the use of dried biomass powder of Chlamydomonas reinhardtii as an ingredient in a wide range of foods for the general population.

The applicant has requested data protection under Article 26 of Regulation (EU) 2015/2283 for data in support of this request.

In accordance with Article 10 of Regulation (EU) 2015/2283, the European Commission asks EFSA to provide a scientific opinion on the safety of dried biomass powder of Chlamydomonas reinhardtii as a novel food.

In this opinion on dried biomass powder of Chlamydomonas reinhardtii as a novel food, EFSA should also document whether and to what extent the requirements of Article 26(2)(c) of Regulation (EU) 2015/2283 are fulfilled regarding the data for which the applicant is requesting data protection.

DATA AND METHODOLOGIES

2

Data

2.1

The safety assessment of this NF is based on data supplied in the original application.1

Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in Commission Implementing Regulation (EU) 2017/2469.2

A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of a NF application (EFSA NDA Panel, 2021). As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, confidential and published) scientific data, (including both data in favour and not in favour) that are pertinent to the safety of the NF.

The applicant has submitted a confidential and a non‐confidential version of a dossier following the EFSA guidelines on the preparation and presentation of a NF application (EFSA NDA Panel, 2021) and the Administrative guidance for the preparation of applications on novel foods pursuant to Article 10 of Regulation (EU) 2015/2283 (EFSA, 2021).

In accordance with Art. 38 of Regulation (EC) No 178/20023 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,4 the non‐confidential version of the dossier has been published on Open.EFSA.5

According to Art. 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation (PC‐0959)6 on the non‐confidential version of the technical dossier from 22 May to 12 June 2024 for which no comments were received.

This NF application includes a request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. The data requested by the applicant to be protected comprise: analytical data, dietary intake assessment, information on the manufacturing process, toxicity studies and an in vitro digestibility study.

Methodologies

2.2

The assessment follows the methodology set out in the EFSA guidance on NF applications (EFSA NDA Panel, 2021) and the principles described in the relevant existing guidance documents from the EFSA Scientific Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of Commission Implementing Regulation (EU) 2017/2469.

This assessment concerns only risks that might be associated with consumption of the NF under the proposed conditions of use, and is not an assessment of the efficacy of the NF with regard to any claimed benefit.

ASSESSMENT

3

Introduction

3.1

The NF which is the subject of the application is dried biomass powder of the single‐cell alga Chlamydomonas reinhardtii THN 6.

The NF falls under Article 3(2)(a)(ii) of Regulation (EU) 2015/2283, i.e. ‘food consisting of, isolated from or produced from microorganisms, fungi or algae’.

The NF is produced by fermentation under aerobic conditions. According to the batch testing provided, the protein content in the NF is about 36%. The NF is proposed to be used as an ingredient in a variety of food products at concentrations up to 10% (by weight). The target population is the general population.

Identity of the NF

3.2

The NF is the dried biomass of whole cell C. reinhardtii THN 6, which is a single‐cell green alga that belongs to the family Chlamydomonadaceae.

The species C. reinhardtii is naturally found in a variety of habitats and can grow photoautotrophically, but also in the dark if supplied with organic carbon. C. reinhardtii has been used as a model organism in research for studying photosynthesis, cell motility and intracellular transport (Rochaix, 1995).

The specific strain used by the applicant for the production of the NF is CC‐4414 wild type mt + DN2, which was isolated from an environmental sample in 2007. In the NF application dossier, the NF is referred to as C. reinhardtii THN 6, which, according to the information provided, is an internal name that was assigned to the strain by the applicant. It is non‐GMO and not irradiated. The identity of the strain was confirmed by sequencing of internal transcribed spacer (ITS) regions ITS‐1 and ITS‐2. The strain is deposited at the Chlamydomonas Resource Center, which is funded by the U.S. National Science Foundation and located in the Department of Plant and Microbial Biology at the University of Minnesota.

The applicant was requested to provide the full results of the blast search and/or to run additional sequence comparisons with C. reinhardtii and other closely related Chlamydomonas species in order to validate the identity of the production strain. In addition, the applicant was requested to provide the whole genome sequence (WGS) of the production strain and report relevant data analysis: identity/phylogeny data of the strain and potential genes of concerns. The applicant did not reply to these requests.

Production process

3.3

The culture of C. reinhardtii THN 6 used for the production of the NF is carried out in fermenters under aerobic conditions. According to the information provided, temperature, pH, lighting and total fermentation time are monitored during the entire production process. Moreover, checks for contamination are performed at every stage of the production. Upon completion of fermentation, C. reinhardtii THN 6 cell mass is centrifuged and the precipitate (algal cell mass) is washed with water. After another centrifugation, the precipitate is spray‐dried to obtain the final product in powder form. Food grade plastic‐lined drums are used to transport and store the NF.

The applicant was requested to provide detailed information of the culture conditions, composition of the culture media, the spray‐drying process, the use of anticaking agent(s), media used for the cryopreservation, information on any microbial inactivation step applied during the manufacturing process. In addition, the applicant was requested to provide information on the hazard analysis and quality control measures (HACCP) in place. The applicant did not reply to these requests.

Compositional data

3.4

The NF is the dried biomass of C. reinhardtii THN 6, which, according to the batch testing provided, is on average composed of 36% protein, 50% carbohydrates, 3% fat and 4% ash.

In order to confirm that the manufacturing process is reproducible and adequate to produce on a commercial scale a product with certain characteristics, the applicant provided proximate analyses and analytical data on chemical and microbiological parameters for three independent batches of the NF (Tables 1 and 2).

The applicant was requested to provide compositional data for five independently produced batches of the NF for proximates, total dietary fibre, microbiological parameters, heavy metals, contaminants, processing aids and aflatoxins. The applicant did not reply to this request.

Stability

3.4.1

The applicant performed stability tests with 3 independently produced batches of the NF under accelerated conditions. The tests were carried out at 40°C and at 75% relative humidity (RH) under 24‐h exposure to domestic light for a period of 84 days. The batches were analysed for microbiological stability, water activity, lipid oxidation, oxidation induced by light and sensory evaluation over this period.

The tests of microbiological parameters were performed on a weekly basis. An increase in total aerobic colony counts was observed after 14 days (range between 200 and 400 CFU/g compared to 0 CFU/g at day 0), and two batches of the NF exceeded the specification limit after 42 days (1200 CFU/g) and the third batch exceeded it after 70 days (1900 CFU/g).

Lipid oxidation testing was performed by measuring malondialdehyde production via the thiobarbituric acid (TBA) method. The applicant noted that while lipid oxidation was stable during the entire period of 84 days, the water activity increased from a value of 0.12 for all three batches at day 0 to a value range of 0.63–0.64 at day 84.

Tests on oxidation induced by light (β‐carotene content) on the three batches of the NF were performed on day 0, 42 and 84. The β‐carotene content decreased from a range of 90,000–100,000 IU/100 g at day 0 to 18,000–22,000 IU/100 g at day 42. Sensory evaluation on days 35, 77 and 84 showed that there was a decline in aroma at day 35.

The applicant was requested to provide additional data on the stability of the NF (for at least 3 batches) when stored under normal (i.e. proposed) storage conditions. Given that the NF was proposed to be used as an ingredient in a number of food categories (e.g. dry foods but also beverages), the stability data should also cover the application of the NF in various foods.

Given the chemical composition of the NF (about 36% protein), the proposed uses (e.g. bread, crisp bread, crackers, biscuits) and use levels (up to 10%), as well as the processing conditions (e.g. baking at high temperature and low water activity in the resulting food commodities), the formation of process contaminants warranted further investigation. Therefore, the applicant was requested to provide data on levels of process contaminants, including acrylamide, furan and alkylfurans, in intended food matrices subjected to heat treatment during production.

The applicant did not reply to these data requests.

Specifications

3.5

The specifications of the NF, as proposed by the applicant, are indicated in Table 3.

The applicant was requested to modify and update the specifications table according to the new compositional data (to be submitted). The applicant was also requested to increase the tested amounts (from 1 g to 10 g) for the parameters Escherichia coli and Staphylococcus aureus. The applicant did not reply to these requests.

History of use of the NF and/or of its source

3.6

In 2019 C. reinhardtii dried biomass received the generally recognised as safe (GRAS) status in the United States, when used as a food ingredient in pasta, noodles and bread mixes.

The applicant was requested to provide sales figures for the NF since its market authorisation in the US. The applicant did not reply to this request.

Proposed uses and use levels and anticipated intake

3.7

Target population

3.7.1

The target population proposed by the applicant is the general population from 1 year of age.

As the NF is intended to be used as an ingredient in standard food categories, it cannot be excluded that the NF would also be consumed by children below 1 year of age. Therefore, the safety data and the exposure assessment shall cover all population groups (Commission Implementing Regulation (EU) 2017/2469, Article 5(6)).

Proposed uses and use levels

3.7.2

The NF is proposed by the applicant to be used as an ingredient in a number of food categories, at a maximum use level of 10%.

In the application dossier it was indicated that ‘the final product applications for C. reinhardtii dried biomass powder are not yet fully defined’. The applicant was informed that this was not admissible since the risk assessment of the NF needs to be performed according to the proposed conditions of use. Therefore, the applicant was requested to clearly define all the food categories to which the NF is intended to be added, including the maximum proposed amounts of the NF for each food category. The applicant did not reply to this request.

Absorption, distribution, metabolism and excretion (ADME)

3.8

No ADME data in humans or animals were provided for the NF. The applicant rather submitted an in vitro simulated gastric fluid digestibility assay (Goodman & Ramadan, 2017). The applicant stated that the NF can be expected to be digested and metabolised similar to other edible plant‐derived foods.

Nutritional information

3.9

According to the batch testing provided, the NF contains on average 36% protein, 50% carbohydrates, 3% fat and 4% ash.

In the application dossier it was stated that the NF contains ‘up to 25% fibre, vitamins and minerals (vitamin A, β‐carotene and iron)’. No evidence was provided to support this statement. Therefore, the applicant was requested to provide analytical data for five batches of the NF for fibre, vitamins, minerals and carotenoids. The applicant was also requested to provide information on the potential presence of antinutrients. Moreover, given the intended use of the NF as an ‘alternative source of protein’, the applicant was requested to provide an analysis of the protein quality of the NF. The applicant did not reply to these requests.

Toxicological information

3.10

The applicant provided four toxicological studies with the NF, listed in Table 4. These studies were claimed proprietary and confidential by the applicant.

The applicant was requested to complement the above studies with additional bacterial reverse mutation tests (according to OECD TG 471) and in vitro mammalian cell micronucleus tests (according to OECD TG 487) performed with lysed C. reinhardtii cells and the supernatant (before the spray drying) of the alga in the appropriate test systems. The applicant did not reply to this request.

Human data

3.10.1

No human studies with the NF were provided.

Allergenicity

3.11

The applicant stated that no allergenicity was described in the literature for C. reinhardtii nor for any member of the family, i.e. Chlamydomonadaceae, to which the alga belongs.

In order to identify potential allergens, the applicant was requested to use WGS data/bioinformatics to perform a search on sequence similarity of C. reinhardtii THN6 to known microalgae allergens. The applicant did not reply to this request.

DISCUSSION

4

The NF which is the subject of the application is the dried biomass powder of C. reinhardtii THN 6.

The NF is proposed to be used as an ingredient in a variety of food products at concentrations of up to 10%. The target population is the general population.

During the risk assessment, the Panel identified a number of data gaps which needed to be addressed by the applicant. Therefore, an EFSA request for additional information/data was sent to the applicant. The requested data pertained to the identity, the production process, compositional data, specifications, history of use, proposed uses and use levels, nutritional information, genotoxicity and allergenicity of the NF. Despite being contacted several times, the applicant did not reply to EFSA's requests for additional data.

CONCLUSIONS

5

The Panel concludes that, based on the available data, the safety of the NF, dried biomass powder of C. reinhardtii THN 6, cannot be established.ABBREVIATIONSADMEAbsorption, distribution, metabolism and excretionAFNORAssociation Française de NormalisationAOACAssociation of Official Analytical CollaborationBAMUS FDA Bacteriological Analytical Manualbwbody weightCFUcolony forming unitsFDAFood and Drug AdministrationGMOgenetically modified organismGRASgenerally recognised as safeHACCPhazard analysis critical control pointsICP‐MSinductively coupled plasma mass spectroscopyITSinternal transcribed spacerIUinternational unitNnitrogenn.d.not detectedNDAPanel on Nutrition, Novel Foods and Food AllergensNFnovel foodOECDOrganisation for Economic Co‐operation and DevelopmentPCPublic consultationRHrelative humidityTBAthiobarbituric acidTGTest GuidelineUSPUnited States PharmacopeiaWGSwhole genome sequence

REQUESTOR

European Commission (DG SANTE)

QUESTION NUMBER

EFSA‐Q‐2023‐00301

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PANEL MEMBERS

Dominique Turck, Torsten Bohn, Montaña Cámara, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch‐Ernst, Ángeles Jos, Alexandre Maciuk, Inge Mangelsdorf, Breige McNulty, Androniki Naska, Kristina Pentieva, Alfonso Siani and Frank Thies.