Toxicological safety evaluation of pasteurized Akkermansia muciniphila

Abstract

Gut microorganisms are vital for many aspects of human health, and the commensal bacterium Akkermansia muciniphila has repeatedly been identified as a key component of intestinal microbiota. Reductions in A. muciniphila abundance are associated with increased prevalence of metabolic disorders such as obesity and type 2 diabetes. It was recently discovered that administration of A. muciniphila has beneficial effects and that these are not diminished, but rather enhanced after pasteurization. Pasteurized A. muciniphila is proposed for use as a food ingredient, and was therefore subjected to a nonclinical safety assessment, comprising genotoxicity assays (bacterial reverse mutation and in vitro mammalian cell micronucleus tests) and a 90-day toxicity study. For the latter, Han Wistar rats were administered with the vehicle or pasteurized A. muciniphila at doses of 75, 375 or 1500 mg/kg body weight/day (equivalent to 4.8 × 10 9 , 2.4 × 10 10 , or 9.6 × 10 10 A. muciniphila cells/kg body weight/day) by oral gavage for 90 consecutive days. The study assessed potential effects on clinical observations (including detailed arena observations and a modified Irwin test), body weight, food and water consumption, clinical pathology, organ weights, and macroscopic and microscopic pathology. The results of both in vitro genotoxicity studies were negative. No test item-related adverse effects were observed in the 90-day study; therefore, 1500 mg/kg body weight/day (the highest dose tested, equivalent to 9.6 × 10 10 A. muciniphila cells/kg body weight/day) was established as the no-observed-adverse-effect-level. These results support that pasteurized A. muciniphila is safe for use as a food ingredient.

Genomic diversity and ecology of human-associated Akkermansia species in the gut microbiome revealed by extensive metagenomic assembly

“…In all 55 positive cases, these genes were found on contigs larger than the plasmid (~8 kb), suggesting that they may be integrated into the bacterial genome (as also reported in [ 19 ]). Of note, the A. muciniphila type strain Muc T carries no antibiotic resistance genes and its use does not raise any antibiotic resistance concern as also indirectly confirmed by dose scaling pilot studies in humans and toxicological studies in rabbit and other model organisms [ 8 , 55 ]; however, ongoing and future human trials with strains different from the type strain should carefully consider their antibiotic resistance potential. In conclusion, although the rare occurrence of antibiotic resistance genes from plasmid RSF1010 in some A. muciniphila genomes has evident safety implications for their use in therapeutic applications, our findings indicate that Akkermansia candidate species mostly lack genetic means to defend themselves against currently used antibiotics.…”

Safety of pasteurised Akkermansia muciniphila as a novel food pursuant to Regulation (EU) 2015/2283

“…The applicant provided a 90-day repeated dose oral toxicity study with the NF in Wistar rats (Crl:WI (Han) strain) (Bracken, 2019b, unpublished study report;Druart et al, 2020). The study was S. Typhimurium TA98, TA100, TA102, TA1535 and TA1537…”

“…Request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/ 2283The Panel could not have reached the conclusion on the safety of the NF under the proposed conditions of use without the data claimed as proprietary by the applicant, i.e. bacterial reverse mutation test (Brient, 2019a, unpublished), in vitro micronucleus assay (Brient, 2019b, unpublished), 14-day dose range-finding toxicity study (Bracken, 2019a, unpublished), 90-day toxicity study (Bracken, 2019b, unpublished), published toxicity data (Druart et al, 2020), flow cytometry validation (Jensen, 2019, unpublished), antimicrobial resistance study (Gueimonde, 2019, unpublished).…”

“…The applicant provided a bacterial reverse mutation test (Brient, 2019a, unpublished; Druart et al., 2020) and an in vitro mammalian cell micronucleus test (Brient, 2019b, unpublished; Druart et al., 2020) that were performed with the NF as a suspension in water.…”

“…The applicant provided a 90‐day repeated dose oral toxicity study with the NF in Wistar rats (Crl:WI(Han) strain) (Bracken, 2019b, unpublished study report; Druart et al., 2020). The study was conducted in compliance with the OECD principles of GLP and according to OECD TG 408 (OECD, 2018).…”