The world population has increased by 14% in the past 12 years, reaching the 8.0-billion milestone in 2022, and is expected to continue, peaking at 9.7 billion in 2050 and 10.9 billion in 2100 (United Nations Department of Economic and Social Affairs, 2021). However, 9.2% of the global population was suffering from hunger in 2022 and over 3.1 billion were facing food insecurity–incapable of securing regular access to sufficient safe and nutritious food to grow, develop and lead an active and healthy lifestyle (FAO et al.,, 2023). Hence, one of the main goals of the United Nations is to achieve sustainable development by 2030, by ensuring sustainable food production systems. Currently, the increase in food production has been achieved by techniques such as breeding high-yielding crop varieties, applying inorganic fertilizers and pesticides, and by using of large-scale irrigation for monoculture farming (Shahmohamadloo et al., 2022). This approach has succeeded in improving food production, availability, and affordability, and has helped to reduce hunger and poverty to some degree. However, current agriculture practices are a major contributor to greenhouse gas emissions, habitat destruction, biodiversity loss, and soil degradation. These environmental impacts are, in turn, making agricultural landscapes increasingly vulnerable to the effects of climate change. Thus, continuing to expand global food production by bringing more land under cultivation, especially given the threats of climate change and plant diseases, is ultimately unsustainable (Mockshell and Kamanda, 2018).

Plant cell culture (PCC) technology for commercial use has been deployed since the 1980s to produce pharmaceuticals, cosmetics, and, most recently, food ingredients (Gubser et al., 2021). The advantages of PCC for food ingredient production are mainly the controlled conditions, not relying on climate, weather and soil conditions, resulting in consistent quantity and quality. The interest in PCC-based foods is emerging since the technology can enable the production of high ecological burden or endangered species (Häkkinen et al., 2020; Kobayashi et al., 2022) and provide nutritionally valuable proteins, fibres, and fatty acids (Nordlund et al., 2018; Ritala et al., 2022). PCC may also present a feasible food production system because it allows upscaling and massive production scale. For example, the production of commercialized ginseng saponins via PCC has been ongoing since the 2000s in Korea. Conventionally, the cultivation of ginseng is very labour-intensive and time-consuming–requiring a minimum of 4 years to cultivate (Wu et al., 2009). With PCCs utilizing a 500 L bioreactor, the scale up of ginseng root cell cultures to yield 149.6 g/L dry cell ginseng biomass was achieved in only 4 weeks (Choi et al., 2000). Furthermore, in PCC technology formation of beneficial substances can be promoted, while harmful substances can be limited or eliminated for the consumers. These advantages demonstrate the potential of PCCs to play a major role in attaining sustainable food production in the coming years.

Currently, two plant cell lines, scurvy grass (SG, Cochlearia danica, Brassicaceae) and rowan (RW, Sorbus aucuparia, Rosaceae), are being developed as potential novel food ingredients for commercialization at an industrial scale. These plant species are in focus due to their high protein content with a well-balanced proportion of essential amino acid content, high dietary fibre content (Ritala et al., 2022), and potential health benefits. In particular, SG is known to be vitamin C rich, while RW was demonstrated to have antidiabetic effects (Brandrud, 2014; EV et al., 2020; Wei et al., 2016; Wolf et al., 2021). Since PCC has not been used as food ingredients and are considered as novel food ingredients, the regulations require proper characterization and safety evaluation including toxicological assessment prior to commercialization (Gubser et al., 2021; Häkkinen et al., 2020). Thus, the main objectives of this study is to biochemically characterize PCC samples for the assessment of their nutritional quality; and evaluate the safety and toxicity of these mass-produced cell lines through acute and subacute oral toxicity testing.

PCC technology is a new food production method that can contribute to the development of sustainable food production systems globally. The PCC samples investigated in this work have potential as novel food ingredients due to their considerable amount of relevant macro- and micronutrients. However, as they are considered novel foods by the regulation, proper characterization and safety evaluation are required to enable the commercialization of the ingredients for food use. Specifically, according to article 7 in the EU legislation on novel foods, PCCs could only be authorized when scientific evidence shows that they do not pose a safety risk to human health, and in case its intended use is to replace another food, its nutrition value should not differ significantly, and that its intended normal consumption should not be nutritionally disadvantageous compared to the original food (Regulation 2015/2283) (European Union, 2021). Therefore, this study focused on increasing the understanding of the nutritional and safety quality of the PCC samples.

The substantial amount of DF in RW cells was confirmed from a previous study (Ritala et al., 2022), and DF in SG cells was in a similar range as previously reported for plant cells from stoneberry, lingonberry and cloudberry (21%–36%) (Nordlund et al., 2018). Both RW and SG cells were richer in protein than previously studied berry cell lines (14%–19%) (Nordlund et al., 2018). SG cells had a relevant amount of vitamin C, meaning that the consumption of less than 30 g of SG cells would provide the daily nutritional requirement of vitamin C (80 mg/day for adults). RW cells accumulated only about one-third of the vitamin C amount, i.e. 126 mg/100 mg, but this is significantly more than previously reported 3.4 mg/100 mg (Ritala et al., 2022) and could be due to the different production systems (stirred bioreactor vs. wave bioreactor). RW cells contained vitamins D3 and E, and the consumption of 100 g of rowan cells could provide the daily nutritional requirement of vitamin D (15 µg) but not enough of vitamin E (11 mg per day). Although the presence of large amounts of vitamin D3 is not common in plants, vitamin D3 and its metabolites have been found in relatively large quantities in other species from the families Solanaceae, Poaceae and Cucurbitaceae (Göring, 2018). We hypothesize that its presence is due to the ability of RW to accumulate vitamin D3 under the culture conditions employed in their production.

According to EU regulation 1169/2011 (European Union, 2025) significant amounts of vitamins and minerals in a food product are considered relevant when 15% of the nutrient reference value is supplied by 100 g of the product. SG cells could have nutrition claims for calcium, magnesium, potassium and zinc as 100 g of this plant cell would provide 35%, 50%, 71% and 76% of the nutrient reference value, respectively. The amounts of iron, manganese and phosphorus are even higher than the reference values (2.7, 5.6 and 2-times, respectively). In the case of molybdenum, the amount in SG cells (0.84 mg/100 g) exceeded the tolerable upper intake (0.6 mg). It is noteworthy that these plant cells are presumed not to be consumed ‘as is’ but rather as an ingredient in a food product. Generally, the availability of individual ions could be limited or increased in the culture medium so that the cells accumulate less or more specific elements.

Allergen analysis is an important part of the novel food assessment to provide information to the food industry for implementing and monitoring allergen risk assessment and management processes (Tuzimski and Petruczynik, 2020). In our study, protein profiling revealed the presence of profilin-like proteins in both PCC samples. Specific profilins are pollen-related allergens and a major cause of cross-sensitization between pollen and plant-derived foods (Ruiz-García et al., 2011). Moreover, in both the SG and RW PCC samples, proteins related to metabolism and stress response were identified as putative allergens due to their reported allergenic potential in other species or closely related plant families. This underscores the need for further investigation, particularly functional allergy assays, to ensure consumer safety.

Results of the acute toxicity study of SG and RW PCCs showed that both PCCs: SG and RW, posed no death, behavioral changes, nor metabolic effects in mice at a dose of 2000 mg/kg, indicating that the LD₅₀ exceeds 2 g/kg and does not present a risk of acute toxicity (OECD, 2002b). The 28-day oral subacute toxicity studies provide information on the adverse effects of repeated exposure towards the test substance over a period of time. It allows for the investigation of possible cumulative effects in target organs and dose-response relationships and provides a prediction for long-term safety (OECD, 2008). Methods include clinical observations, haematological, biochemical, relative organ weight and histopathological analysis. Significant deviations from normal values may signal adverse reactions such as metabolic disturbances, hormonal imbalances, or direct toxic effects on specific organs. The 28-day daily administration of SG plant cell cultures at 500 mg/kg posed no observed effects on the parameters measured in the study. At 1,000 mg/kg, SG caused an enlargement in the liver and lungs and increased the WBC and GRA counts, while at 2000 mg/kg, SG caused the enlargement of the liver and increased the GRA counts only. The increase in WBC and GRA counts indicates that SG amplifies immune response. This could be explained by the fact that the Cochlearia species contain high amounts of vitamin C, which is known to increase the production of WBCs (Brandrud, 2014; Van Gorkom et al., 2018). The increase in the relative sizes of the liver and lungs, on the other hand, may indicate potential toxicity in those organs. When liver toxicity occurs, serum/plasma ALT and AST levels increase due to damage to the liver cells, where they release these enzymes into the bloodstream (Kasarala and Tillmann, 2016). However, plasma biochemical analysis revealed that there were no significant changes in ALT and AST levels which indicate healthy liver function. Furthermore, visual histopathological scoring did not reveal any significant changes in the gross pathology for both the liver and lungs, and the increased relative organ weight of the livers and lungs were still within a normal healthy range as compared to other ICR-CD1 mice in other studies (Kyselova et al., 2003; Rao et al., 2020). Thus, these findings indicate that SG cell cultures likely would not impose any toxicity in the liver and lungs, and the no-observed-adverse-effect level (NOAEL) level was determined to be above 2,000 mg/kg. Nonetheless, further investigations could be done to confirm this, such as assessing molecular biomarkers, e.g., levels of enzymes involved in detoxification in the liver and lungs.

Like the results from the SG 28-day repeated exposure, the repeated exposure to RW cell cultures at 500 mg/kg did not affect any of the parameters measured. At 1,000 mg/kg and 2000 mg/kg of RW administration, most parameters measured showed no changes between the control and the treated mice groups, including haematological parameters, relative organ weight measurements and histological assessments. Changes in some plasma biochemical parameters were, however, noted, including an increased sodium and potassium level and a decrease in glucose levels. RW berries are known to contain polyphenols which offer antihyperglycemic and antidiabetic effects (Wei et al., 2016), which likely contributed to the lower glucose levels observed. The increase in sodium and potassium levels could be attributed to the extra sodium and potassium intake from RW PCC administration. Although an increase in sodium levels may indicate conditions of renal dysfunction leading to hypernatremia, other renal function markers (CREJ and UA) did not differ between mice administered RW and control mice of both genders; in fact, they were slightly lower, suggesting a protective effect on the kidneys. Interestingly, while the nutritional content of RW PCCs has lower potassium levels than SG PCCs, no significant increase in potassium levels was observed in mice supplemented with 2000 mg/kg SG in the 28-day subacute toxicity study. This could perhaps be attributed to RW offering better potassium bioavailability than SG, given that potassium bioavailability was known to vary among fruits and vegetables (Ceccanti et al., 2022). Further assessment of the bioavailability of nutrients in these PCCs could be investigated to confirm this observation. Nonetheless, considering that there were no significant differences in histology grading scores between organs of the control mice and mice supplemented with RW, it is safe to assume that RW PCCs pose no direct toxic effects to these organs and the NOAEL was established to be beyond 2,000 mg/kg.

While these findings demonstrate the safety of PCCs as a food ingredient for human consumption, there are limitations to consider. To move towards commercialization of food ingredients produced by PCC technology, the use of food-grade materials in their production including growth media with natural elicitors are required (Gubser et al., 2021). However, in the case of the current two cell lines, laboratory-grade reagents such as synthetic phytohormones used in the growth media have not been approved or tested for food or nutritional use, and their metabolic fate can be complex (Häkkinen et al., 2020). Therefore, the influence of growth media to PCC quality and safety is indeed an important parameter to be studied in future, especially in cases wherein new compounds are introduced into the growth media.

This work serves as a preliminary study characterizing the macro- and micronutrients of PCCs of SG and RW and evaluating the acute and subacute safety and toxicity of the PCCs for human consumption. The acute toxicity study results confirmed that neither mortality nor toxic were effects observed in mice treated with the two PCCs at 2000 mg/kg, indicating minimal risk of acute toxicity. Similarly, the subacute toxicity study demonstrated no mortality following 28 days of repeated oral exposure to both SG and RW at a dose of 2000 mg/kg. Considering the bioactive effects of SG, despite a slight increase in liver and lung weight, organ histopathological assessments all came out normal, and no adverse effects were observed in the body weight, haematological, and biochemical parameters. Hence, the NOAEL was determined to be >2000 mg/kg. Similarly, for the RW subacute toxicity study, an increase in sodium and potassium levels were observed in the 1,000 mg/kg mice, which indicates possible kidney and/or heart dysfunction. Despite that, all haematological, body weight, organ weight and histopathology were insignificantly different from the healthy control mice. Additionally, our findings show that RW retained its polyphenol-derived antihyperglycemic properties. Therefore, the NOAEL for RW PCCs was also determined to be >2000 mg/kg. This study offers a preliminary finding that these PCCs are safe and non-toxic for consumption as food.