Genotoxicity and neonatal subchronic toxicity assessment of a novel mixture of the human‐identical milk oligosaccharides lacto‐N‐fucopentaose I and 2′‐fucosyllactose

Abstract

Human milk oligosaccharides (HMOs) are a complex group of bioactive molecules largely observed in human breast milk but also occurring in limited amounts in other mammalian milks. Advances in biotechnology have enabled production of humanidentical milk oligosaccharides (HiMOs), structurally identical molecules to HMOs found naturally in human milk, intended for addition to infant formula to more closely replicate breast milk. Biosynthesis of a novel mixture of two major HMOs, lacto-N-fucopentaose I and 2 0-fucosyllactose (LNFP-I/2 0-FL), recently became possible. To support the safety of LNFP-I/2 0-FL for use in infant formula and other foods, it was subject to a safety assessment comprising a bacterial reverse mutation test, an in vitro mammalian cell micronucleus test, and a 90-day oral gavage study in neonatal rats. In the 90-day study (the first HiMO study to include the new endocrinesensitive endpoints described in the 2018 version of OECD Test Guideline 408), LNFP-I/2 0-FL was administered by oral gavage to neonatal rats once daily (from Day 7 of age) for 90 consecutive days, at doses up to 5000 mg/kg bw/day, followed by a 4-week recovery period. Concurrent reference controls received 5000 mg/kg bw/day of the approved infant formula ingredient oligofructose. LNFP-I/2 0-FL was nongenotoxic in vitro. The highest dose tested (5000 mg/kg bw/day) was established as the no-observed-adverse-effect level in the 90-day study, as there were no test article-related adverse effects on clinical observations, body weight, food consumption, clinical pathology, and organ weights nor any noteworthy macroscopic or microscopic findings. This supports the safety of LNFP-I/2 0-FL for its intended uses in food.

Neonatal subchronic toxicity and in vitro genotoxicity studies of the human‐identical milk oligosaccharide 3‐fucosyllactose

“…Differences in triiodothyronine (T3) concentrations were reported in a recent 90‐day neonatal study testing a novel mixture of lacto‐ N ‐fucopentaose I (LNFP‐I) and 2′‐FL (Phipps et al, 2021), the first published HiMO 90‐day study following the requirements of the most recent OECD Test Guideline 408, which was revised in 2018 to include additional endocrine‐related parameters such as thyroid hormones, low‐density lipoprotein‐cholesterol and high‐density lipoprotein‐cholesterol, and collection of vaginal smears for estrous cycle measurement (OECD, 2018). However, there were no differences in T3 or any other thyroid hormone concentrations between controls and 3‐FL groups in this study, which reinforces that the T3 differences reported for the LNFP‐I and 2′‐FL mixture were likely to have been biologically irrelevant.…”

“…Both tests were conducted in triplicate. Preparation of cell cultures, incubation conditions, colony counting, and evaluation of the assay results were performed as previously described by Phipps et al (2021).…”

Safety of lacto‐N‐fucopentaose I/2’‐fucosyllactose (LNFP‐I/2’‐FL) mixture as a novel food pursuant to Regulation (EU) 2015/2283

“…The studies which were claimed proprietary by the applicant are listed in Table 11. Experimental designs with main results are included in a relevant publication (Phipps et al., 2020).…”

“…In the in vitro mammalian cell micronucleus test in human lymphocytes (Phipps et al., 2020; Study Report, 2020b), concentrations of 555, 1110 and 2220 μg NF/mL (corresponding to up to 2000 μg/mL of LNFP‐I/2’‐FL mixture) were tested in the main study in the presence (3‐hour treatment) and absence (3‐ and 20‐hour treatments) of S9 metabolic activation. No cytotoxicity or precipitation were observed and the percentage of micronuclei in cultured human lymphocytes was not significantly increased in any of the test substance concentrations.…”

Chemoenzymatic synthesis of fucosylated oligosaccharides using Thermosynechococcus α1–2-fucosyltransferase and their application in the regulation of intestinal microbiota

“…There is therefore an urgent need for its large-scale production. Extraction of LNFP-I from cow’s milk is not feasible due to its low concentration and extremely high cost ( Bode et al, 2016 , Phipps et al, 2021 ). Enzymatic synthesis of LNFP-I has been achieved using bacterial α 2FTs.…”