In vivo mammalian micronucleus test in mice confirms lack of genotoxic potential of a protein-rich powder derived from Xanthobacter sp. SoF1

TL;DR

A protein-rich powder from Xanthobacter sp. SoF1 was tested in mice and found to not cause genetic damage.

Contribution

This study confirms the safety of a microbial protein source using an in vivo mammalian micronucleus test.

Findings

No treatment-related adverse effects were observed in mice at any dose level.

No increases in micronucleated immature erythrocytes were detected in mice.

The test item showed no clastogenic or aneugenic activity in mouse bone marrow.

Abstract

The growing interest and demand for alternative protein sources has propelled research and production of protein-rich microbial biomass products for human consumption. The potential genotoxicity of a protein-rich biomass derived from Xanthobacter sp. SoF1 (SoF1), an autotrophic hydrogen-oxidizing bacteria, was evaluated in the bone marrow of male NMRI mice in the current work. This study was conducted as the final step of a comprehensive toxicological safety assessment that has been previously published. While a genotoxic evaluation including a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes was previously conducted without incidence, an in vivo mammalian micronucleus test was lacking and thus, this study was performed to provide additional relevant in vivo insights into the genotoxicity of SoF1.

No treatment-related adverse effects were observed and no increases in the frequencies of micronucleated immature erythrocytes (MPCEs) in male mice (5/group) at any dose level (0, 500, 1000, and 2000 mg/kg bw) of the test item by oral gavage were observed when compared with the concurrent negative and historical negative control groups.

The test item did not exhibit any clastogenic or aneugenic activity in the bone marrow of mice under the applied experimental conditions.

Full text

Background

Proteins are an essential component of human and animal nutrition and their biological systems. Projections of an increase in food production exceeding 60% by 2050 to satisfy the growing population protein requirements, convey a bleak message that improvements must be made to prevent potential shortages of both natural resources and food [1]. The “food-energy-water nexus” which describes the struggle for water allocation between agriculture and energy, will influence the direction of future food, water, and energy security [2]. Based on current agricultural methods, predictions show that a 50% increase in food production will generate an 80% increase in greenhouse gas emissions [3, 4]. Reducing meat consumption, especially from animals that have more of an environmental impact, is one approach to address the concerns of the upcoming burden. The ever-increasing human population coupled with diminishing natural resources have created a need for alternative protein sources, leading to creative advancements in protein discovery, engineering, and biomanufacturing.

Outside of traditional protein sources such as plant and animal proteins, exploration of proteins from insects and single-cell protein (SCP) sources, also known as microbial proteins (fungi, algae, and bacteria) have increased in recent years [5–7]. In 1968, the term ‘single cell protein’ was introduced at a conference evaluating the prospective role of SCPs in the world food supply at the Massachusetts Institute of Technology, replacing the former ‘microbial protein’ and ‘petroprotein’ terms which were deemed less appealing to the public [8]. Cultivated fungi, algae, and bacteria create edible biomasses, containing a higher protein content (60–82%) than other alternative protein sources. SCP’s are also rich in carbohydrates, nucleic acids, fats, minerals, and vitamins [9] and may be viable protein substitutes for both humans and animals [10, 11].

Xanthobacter sp. SoF1 biomass (SoF1) is a protein-rich powder derived from a gram-negative hydrogen-oxidizing bacteria via chemoautotrophic growth. SoF1 has been the study material in several genotoxicity studies, including a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes in which no evidence of genotoxicity was reported [12]. SoF1 was also the subject of a 90-day repeated-dose oral toxicity study in which the no observed adverse effect level (NOAEL) was determined to be 1500 mg/kg bw/day, the highest dose tested [13]. This study aims to provide further in vivo insights into SoF1’s genotoxic potential by specifically assessing for potential to damage chromosomes or the mitotic apparatus in a living system and address any data gaps needed for regulatory assessments.

Materials and methods

Test item

The test material was Solein^Ⓡ^ (Solar Foods, Helsinki, Finland), a yellow powder comprised of heat-inactivated biomass of Xanthobacter sp. SoF1. Solein^Ⓡ^ consists of 73.1% protein when calculated using the regular Kjeldahl factor (Nx6.25), and 68% protein when using a Kjeldahl factor based on the composition of Solein^Ⓡ^ (Nx5.83). Composition of Solein^Ⓡ^ includes 17.5% fiber, 7.8% fat, 58.1 mg/kg manganese, and 1240 mg/kg iron. A novel species, Xanthobacter sp. SoF1 was isolated from seashore sediment in Naantali, Southern Finland by Solar Foods, and was found to be most similar to X. tagetidis based on an average nucleotide identity (ANI) measurement. Based on its ANI value, the species was classified as novel, as it falls below the 95% species boundary threshold.

Care and use of animals

The study was approved by the Institutional Animal Care and Use Committee of Toxi-Coop Zrt. Specific pathogen free, 9-week-old male NMRI mice (Charles River Laboratories) were acclimatized for 12 days. The mice were housed in groups of five in Type I polypropylene/polycarbonate cages with laboratory bedding and with controlled lighting (6 am to 6 pm) and humidity (40–70%). The animals received pellet diet (ssniff SR/M-Z + H) produced by ssniff Spezialdiäten GmbH (Experimental Animal Diets Inc.) and water (250 mL bottles) ad libitum.

In vivo mammalian micronucleus test (MMT)

GLP and test guideline, test material formulation, administration, and dosing schedule

A GLP compliant mammalian micronucleus test was conducted in accordance with OECD 474 and with previously described methods [14]. Daily formulations of the test item were made with filtered water (Magilab Kft) and used within two hours. Doses were selected based on the results of a non-published preliminary toxicity test. The test material was weighed and added into a calibrated volumetric flask. Filtered water was added and the formulations were stirred until homogeneity was reached. The concentrations were intended to achieve doses of 0, 500, 1000, and 2000 mg/kg bw when given to the mice via gavage. Cyclophosphamide (Sigma-Aldrich), the positive control, was dissolved in water (Magilab Kft) for injections. Analyses of the test formulations were conducted on both treatment days using a validated ICP-OES method based on the measured iron content of the samples by the Analytical Laboratory of Toxi-Coop Zrt.

Experimental procedures, evaluation of data, and statistics

Oral doses were administered to male mice (5 mice/group) by gavage two times at 24-h intervals using a treatment volume of 10 mL/kg bw. Sampling occurred once at 24-h after the second treatment in the vehicle control, low, mid, and high-dose groups in all animals. Cyclophosphamide (6.0 mg/mL) was administered intraperitoneally at a dose volume of 10 mL/kg bw (equivalent to 60 mg/kg bw) Sampling was performed 24-h after the first treatment. The mice were examined regularly for visible signs of reactions to treatment immediately after dosing and periodically until sacrifice.

Sacrifice occurred by cervical dislocation and bone marrow was obtained immediately from the two exposed femurs of the mice. The bone marrow was flushed with 5 mL of fetal bovine serum (Sigma-Aldrich). After vortex mixing and centrifugation, the supernatant was discarded, and cell smears were prepared on standard microscope slides. After drying at room temperature, the slides were fixed in methanol (Lach: ner) for five minutes, air-dried, stained with 10% Giemsa (Merck KGaA) solution for 25 min, rinsed with distilled water, dried at room temperature for at least 12 h, and coated with E-Z mount™ (Epredia). Slides were coded for blind microscopic analysis. Four thousand polychromatic erythrocyte (PCE) cells were scored per animal to assess for micronucleated cells. The frequency of micronucleated polychromatic erythrocyte (MPCE) cells were expressed as a percent of MPCEs based on the first 4000 PCEs counted in the optic field. The proportion of immature erythrocytes among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes.

Statistical analyses (Kruskal-Wallis Non-Parametric ANOVA, Mann-Whitney U-test, Duncan’s multiple range) were conducted with SPSS PC+ software (SPSS, Inc., Version 4) and Microsoft Excel (Version 2016) software was used for linear trend analyses. Statistical significance was determined at a p value of < 0.05.

Results

No mortalities occurred during the study and no adverse reactions to treatment were observed in either control group or in any of the treatment groups. Oral administration of 0, 500, 1000, and 2000 mg/kg bw of Solein^Ⓡ^ did not induce increases in the frequencies of MPCEs in male mice when compared to the negative control group, and the frequencies remained within historical negative control ranges (Table 1). The linear trend analysis did not show significance compared to the vehicle control. The PCE/total erythrocytes ratio after the second treatment was statistically lower (9.3%) in the high dose group compared to the concurrent and historical negative control groups. The study was validated by a statistically significant increase in the MPCE number in the cyclophosphamide treated mice in comparison to the negative and historical controls.

Table 1. Summary of results of the in vivo mammalian micronucleus testGroupsSampling time(hours following final treatment)Total number ofPCEs analysedMPCE(per 4000 PCE)PCE/PCE + NCEmeanSDmeanSDConcurrent Negative (Vehicle) Control^a^2420,0006.001.580.540.01500 mg/kg bw^a^2420,0006.000.710.54U0.011000 mg/kg bw^a^2420,0006.401.140.520.032000 mg/kg bw^a^2420,0006.400.890.49**DN, U0.02Concurrent Positive Control (Cyclophosphamide 60 mg/kg bw)^a^2420,000150.20/7.260.42DN, U0.03Historical Negative Control^b^24880,0003–8-0.50–0.57-Historical Cyclophosphamide Positive Control^b^24880,000136.9110.210.3710.033Abbreviations: DN, Duncan’s multiple range test; MPCE, micronucleated polychromatic erythrocytes; NCE, normochromic erythrocytes; PCE, polychromatic erythrocytes; U, Mann-Whitney U-test versus controlRemarks:a: Five animals per group were evaluatedb: Data were derived from 220 animals• MPCE:** = p < 0.01 (Kruskal-Wallis nonparametric ANOVA) to the historical negative control• PCE/PCE + NCE:** = p < 0.01 (DN) to the vehicle control** = p < 0.01 (U) to the historical negative control* = p < 0.05 (U) to the historical negative control• Historical control values were derived from the Toxi-Coop Laboratory on NMRI mice

Discussion

While humans have consumed microorganisms in foods throughout history, such as in bread, cheese, yogurt, soy sauce, and wine, the idea of SCPs as an alternative protein source is more novel [8]. Various foods derived from SCPs have been a well-established part of human food and animal feed supply throughout history. Spirulina, a blue-green microalgae, has been used as a protein-rich food source throughout history in many different parts of the world [15, 16]. Several mycoprotein products including Pruteen™ and Pekilo^Ⓡ^ (microfungal biomass from Paecilomyces varioti) and Quorn™, a mycoprotein derived from Fusarium venenatum, a soil fungi, are commercially available for both animal and human consumption [17–19].

Given that the test item is a complex biomass ingredient (as opposed to a single molecule), there are limitations to the interpretation of these results. According to the OECD 474 guidelines, a decrease in PCE/total erythrocytes ratio may be interpreted as evidence that the test item has reached the bone marrow when results are negative for genotoxicity [20]. However, in the case of a complex text item, it is unknown which molecular components were responsible for the decreased ratio. The guidance states that when studying a mixture, it should be considered whether, and if so why, the study may provide adequate results. It also states that such considerations are not needed when there is a regulatory requirement for testing the mixture. This study was conducted in addition to a previously published battery of safety studies on SoF1, which included three in vitro genotoxicity studies, none of which showed evidence of mutagenicity or clastogenicity [12]. It was later considered that regulatory bodies may request an in vivo genotoxicity study for completeness, thus the present study was performed. The absence of a positive genotoxicity finding in the current study suggests no additional genotoxic concern at this time, although this result cannot be considered definitively negative due to uncertainty regarding which molecules reached the bone marrow. In addition, it is worth noting that the decrease in the PCE ratio compared to the concurrent and historical negative control values in the current work occurred only at the highest dose level (2000 mg/kg bw/day), which exceeds the NOAEL established (1500 mg/kg bw/day) in the previously published 90-day repeated dose oral toxicity study investigating the same test item [13].

The results from the current work, in which no evidence of cytogenic damage was evident in a mammalian micronucleus test after administration of 0, 500, 1000, and 2000 mg/kg bw/day of SoF1 do not suggest any additional concerns with regard to the previous genotoxicity studies utilizing the same test material [12]. Similarly, other toxicological evaluations including that of a SCP powder from Cupriavidus necator and biomass of green algae Chlamydomonas reinhardtii demonstrated no signs of mutagenicity or genotoxicity and NOAELs of 3000 mg/kg bw/day and 4000 mg/kg bw/day (the highest doses tested), respectively, were established [21, 22]. Feed and food regulations are pivotal in influencing the direction of development of these alternative proteins and high-quality scientific evidence is needed to support their safety [23].

Conclusions

No biologically or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with Solein^Ⓡ^ (a heat-inactivated biomass from Xanthobacter sp. SoF1) compared to the concurrent and historical negative control groups. No genotoxic activity was observed with Solein^Ⓡ^ in this mammalian micronucleus test. This study aimed to address existing research gaps and enhance the understanding of the genotoxic potential of a protein-rich biomass derived from Xanthobacter sp. SoF1, contributing important additional in vivo evidence to its already published comprehensive toxicological safety profile as an alternative source of protein for consumption.