Safety evaluation of certain food additives - ipcs inchem

PHOSPHOLIPASE C EXPRESSED IN PICHIA PASTORIS 109
2. BIOLOGICAL DATA
2.1 Biochemical aspects
Phospholipase C from P. pastoris was tested for haemolytic activity using
phospholipase C from Clostridium perfringens as a positive control. No haemolytic
activity was detected.
Phospholipase C was also evaluated for potential allergenicity according to
the bioinformatics criteria proposed in the Report of a Joint FAO/WHO Expert
Consultation on Allergenicity of Foods Derived from Biotechnology (FAO/WHO
Expert Consultation, 2001). The amino acid sequence of phospholipase C was
compared with the amino acid sequences of known allergens in the Food Allergy
Research and Resource Program (FARRP) allergen database (http://
www.allergenonline.com). No sequence homology that would suggest crossreactivity
of phospholipase C with known allergens was detected.
2.2 Toxicological studies
BD16449 phospholipase C is the product of a phospholipid-specific lipase
gene expressed in the yeast P. pastoris strain DVSA-PLC-004. Toxicological
studies were performed with the BD16449 phospholipase C enzyme using a
representative batch (PLC-16449-PD267B), which was produced according to the
procedure used for commercial production. The liquid enzyme concentrate was
lyophilized to produce the final, non-formulated test substance, with an average
activity of 315 U/mg (where a unit is defined as the quantity of enzyme that
hydrolyses 1 μmol of phosphatidylcholine per minute at 37 °C and pH 7.3) and a
TOS value of 83.6% by weight (w/w). Prior to use in toxicological studies, BD16449
phospholipase C was analysed for chemical and microbial composition, including
the absence of the production strain. The test article samples were also tested for
and shown to be free of antimicrobial activity and mycotoxins. Mycotoxins were
analysed using high-performance liquid chromatography for aflatoxins and
ochratoxin A and thin-layer chromatography for T-2 toxin and sterigmatocystin
(Ciofalo et al., 2006). The limits of detection for the mycotoxins tested were as
follows: aflatoxin B1, 1.0 μg/kg; aflatoxin B2, 1.0 μg/kg; aflatoxin G1, 1.0 μg/kg;
aflatoxin G2, 1.0 μg/kg; ochratoxin A, 2 μg/kg; T-2 toxin, 0.1 mg/kg; and
sterigmatocystin, 200 μg/kg.
2.2.1 Acute toxicity
BD16449 phospholipase C enzyme preparation (code DV16449, batch
PLC-16449-PD267B, 83.6% TOS) was administered to Sprague-Dawley rats by
oral gavage. Using an up–down dosing regimen that complied with Good Laboratory
Practice (GLP), a female rat did not die after being dosed with a limit dose of
2000 mg/kg body weight (bw). To confirm the finding, an additional four females
were given the same dose. The animals were monitored daily for mortality and
clinical observations, and all surviving animals were sacrificed and necropsied on
day 15.