Toxicological safety assessment of the human‐identical milk oligosaccharide 3′‐sialyllactose sodium salt

Abstract

Human breastmilk is a mixture of nutrients, hormones and bioactive molecules that are vital for infant growth and development. Infant formula (IF) lacks many of these compounds, most notably human milk oligosaccharides (HMOs), which are abundant in breastmilk but scarce in IF. Sialyllactoses, such as 3′‐sialyllactose, constitute a large portion of the HMO fraction. To produce IF that matches breastmilk more closely, biosynthesized human‐identical milk oligosaccharides (structurally identical to HMOs) such as 3′‐sialyllactose sodium salt (3′‐SL) are proposed for use in IF and foods for the general population. The safety assessment of 3′‐SL comprised in vitro genotoxicity tests and a 90‐day oral (gavage) toxicity study. This is the first 90‐day study conducted with 3′‐SL using neonatal rats (7 days old at the start of dosing—equivalent age to newborn human infants in terms of central nervous system and reproductive development), demonstrating the safety of 3′‐SL for consumption by infants, the most sensitive age group. The neonatal rats received 3′‐SL at doses up to 5,000 mg/kg body weight (BW)/day and reference controls received 5,000 mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in IF) for comparison with the high‐dose 3′‐SL group, followed by a 4‐week recovery period. There was no evidence of genotoxicity in vitro. In the absence of any test item‐related adverse effects in the 90‐day study, the high dose (5,000 mg/kg BW/day) was established as the no‐observed‐adverse‐effect level. This confirms the safety of 3′‐SL for use in IF for infants, as well as in functional foods for the general population.

Toxicological safety evaluation of the human‐identical milk oligosaccharide 6′‐sialyllactose sodium salt

“…Therefore, 5000 mg/kg BW/day (the highest dose tested) was established as the no-observed-adverse-effect level (NOAEL). This NOAEL corresponds to a total carbohydrate amount of 5220 mg/kg BW/day (accounting for 'other carbohydrates' in the 6′-SL preparation, see Table 1), which is consistent with previous observations for other HiMOs (Coulet et al, 2013;Coulet et al, 2014;Phipps, Baldwin, Lynch, Flaxmer, et al, 2018;Phipps, Baldwin, Lynch, Stannard, et al, 2018, Phipps et al, 2019.…”

“…Thus, the absence of these findings in any of the recovery animals substantiates that they were not related to administration of 6′‐SL, as complete recovery of these testicular and epididymal findings would not be expected after only 4 weeks. There were also no test‐item‐related macroscopic or microscopic findings reported in another 90‐day toxicity study conducted with 6′‐SL (Gurung et al, ) nor in studies conducted with its constitutional (‘linkage's) isomer 3′‐SL (Kim, Gurung, Seo, Lee, & Woo, ; Phipps et al, , accepted manuscript), which further supports the conclusion that these findings were highly likely to be incidental and unrelated to the administration of 6′‐SL.…”

“…The method for the preclinical safety assessment of 6′‐SL (two in vitro genotoxicity tests and a 90‐day oral toxicity study) is similar to that used for other HiMOs, such as 3′‐sialyllactose sodium salt (3′‐SL) (Phipps et al, , accepted manuscript), LNnT (Coulet et al, ), 2′‐FL (Coulet et al, ), lacto‐ N ‐tetraose (Phipps, Baldwin, Lynch, Stannard, et al, ) and 2′‐FL/DFL (Phipps, Baldwin, Lynch, Flaxmer, et al, ). The in vitro assays represent the standard genotoxicity test battery covering gene mutations and chromosome aberrations using the minimum recommended number of tests (EFSA, ) and the 90‐day oral toxicity study was conducted to assess the potential subchronic toxicity of not only 6′‐SL itself, but of ‘other carbohydrates' within the final product (see Table ).…”

A safety evaluation of mixed human milk oligosaccharides in neonatal farm piglets

“…In this study, administration of HMO MIX 1 to neonatal farm piglets up to 8.0 g/L was not associated with any adverse outcomes. The lack of adverse effects in genotoxicity and sub-chronic studies conducted in rat with HMO Mix 1, 7 the safety of the individual oligosaccharides demonstrated with other safety studies, 8 –19 and the results of this study support the safety of this ingredient in infant formulas, including those consumed during the sensitive period of human development below 12 weeks of age.…”

“…7 In these previous studies, HMO MIX 1 was demonstrated to be non-genotoxic and produced no adverse effects in animals at 10% of the diet, for a calculated dose of 5.67 g/kg and 6.97 g/kg in male and female rats, respectively. Other safety studies have been conducted on the individual HMOs in this novel mixture: 2′-FL, 8 –10 3-FL, 11 LNT, 12 3′-SL, 13,14 and 6′-SL, 15,16 including previous studies in neonatal piglets for 2′-FL 17 and 3′-SL. 18,19 These previous studies and the long history of exposure via human milk at the concentrations intended to be added to infant formula strongly support the safety of these oligosaccharides to human infants.…”