Evaluation of the genotoxic potential of protoporphyrin IX and the safety of a protoporphyrin IX‐rich algal biomass

Abstract

Chlamydomonas reinhardtii is a nonpathogenic, nontoxigenic green algae used as a sustainable source of protein in foods. In order to mimic meat-like qualities, a strain rich in protoporphyrin IX (PPIX), an endogenous heme/chlorophyll precursor, was developed using an evolution and selection strategy, and investigations were carried out to evaluate the safety of the novel strain, C. reinhardtii (red), strain TAI114 (TAI114). Digestibility and proteomic evaluations were conducted to determine whether any potentially allergenic or toxic proteins occurred as the result of the mutation process. The genotoxic potential of pure PPIX was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test. Finally, the novel TAI114 biomass was evaluated for general toxicity and identification of target organs in a 90-day repeateddose oral toxicity study in rats. All proteins were rapidly degraded in pepsin at pH 2.0 suggesting low allergenic potential. The proteomic evaluation indicated that TAI114 biomass contains typical C. reinhardtii proteins. PPIX was unequivocally negative for genotoxic potential and no target organs or adverse effects were observed in rats up to the maximum feasible dose of 4000 mg/kg bw/day TAI114 biomass, which was determined to be the no-observed-adverse-effect-level (NOAEL). These results support the further development and risk characterization of TAI114 biomass as a novel ingredient for use in the meat analogue category of food.

Toxicology and digestibility of Chlamydomonas debaryana green algal biomass

“…As noted in our previous work, we are not aware of any documented oral allergic reactions to any species of the Chlamydomonadaceae family (Murbach et al, 2022; Triton Algae Innovations and FDA, 2019). Furthermore, proteins present in TS04 are considered mostly analogous to the proteins of THN6 and TAI114, which have both been demonstrated, via the SGF digestibility assay, to possess a low likelihood of allergenic potential when ingested.…”

“…In fact, there are very few publications, related to C. debaryana (or E. debaryana ) in general, with most of the published literature, beyond basic science, focused on applications such as its potential uses as a source of biofuel (Arora et al, 2016; Mishra et al, 2015; Sato & Toyoshima, 2021; Yoshitomi et al, 2020), a marker species for environmental contamination or a bioremediation agent (Arora et al, 2016; Chiellini et al, 2022; Lam et al, 2020), and the antioxidant potential of its lipid fraction (Avila‐Roman et al, 2016, 2018; de Los Reyes et al, 2014). Our investigations of TS04 were preceded by investigations of the genetic toxicity and subacute oral toxicity of the closely related species C. reinhardtii strain THN6 (THN6) (Murbach et al, 2018), as well as the subchronic oral toxicity and digestibility of a THN6 mutant that is rich in protoporphyrin IX (TAI114) at the expense of chlorophyll (Murbach et al, 2022).…”

“…In our further prior work with THN6, no signs of adversity or target organs were identified in a subacute study in Han:Wist rats (Murbach et al, 2018). Likewise, no signs of adversity or target organs were identified in a subchronic oral toxicity study of TAI114 in Han:Wist rats (Murbach et al, 2022). Both studies were conducted using the same MFD (4,000 mg/kg bw/day) for gavage administration as used in the current subchronic study with TS04.…”

“…Our investigations of TS04 were preceded by investigations of the genetic toxicity and subacute oral toxicity of the closely related species C. reinhardtii strain THN6 (THN6) (Murbach et al, 2018), as well as the subchronic oral toxicity and digestibility of a THN6 mutant that is rich in protoporphyrin IX (TAI114) at the expense of chlorophyll (Murbach et al, 2022).…”

“…Male and female Han:WIST rats were randomly divided into four groups of 10 rats/sex and administered doses of 0 (vehicle‐control), 1,000, 2,000, or 4,000 mg/kg bw/day TS04 for 90 consecutive days. Based on past experience with closely related test items (Murbach et al, 2018, 2022), which quickly became dense and sticky in all tested practicable vehicles at a concentration of 200 mg/ml, distilled water (Magilab Kft) was chosen as the vehicle, and the high dose was selected as the maximum feasible dose (MFD) due to solubility of the test item. Therefore, the test solutions were formulated daily, by mixing the test item in vehicle at concentrations of 50, 100, and 200 mg/ml to form suspensions, and were immediately administered by gavage at a constant dosing volume of 20 ml/kg bw.…”