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Hornshoj B.H.,DuPont Company | Kobbelgaard S.,Teknologisk Institute | Blakemore W.R.,Celtic Colloids Inc | Stapelfeldt H.,CP Kelco Aps | And 2 more authors.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2015

In 2010 the European Commission placed a limit on the amount of free formaldehyde in carrageenan and processed Eucheuma seaweed (PES) of 5 mg kg−1. Formaldehyde is not used in carrageenan and PES processing and accordingly one would not expect free formaldehyde to be present in carrageenan and PES. However, surprisingly high levels up to 10 mg kg−1 have been found using the generally accepted AOAC and Hach tests. These findings are, per proposed reaction pathways, likely due to the formation of formaldehyde when sulphated galactose, the backbone of carrageenan, is hydrolysed with the strong acid used in these conventional tests. In order to minimise the risk of false-positives, which may lead to regulatory non-compliance, a new high-performance liquid chromatography (HPLC) method has been developed. Initially, carrageenan or PES is extracted with 2-propanol and subsequently reacted with 2,4-dinitrophenylhydrazine (DNPH) to form the chromophore formaldehyde-DNPH, which is finally quantified by reversed-phase HPLC with ultraviolet light detection at 355 nm. This method has been found to have a limit of detection of 0.05 mg kg−1 and a limit of quantification of 0.2 mg kg−1. Recoveries from samples spiked with known quantities of formaldehyde were 95–107%. Using this more specific technique, 20 samples of carrageenan and PES were tested for formaldehyde. Only one sample had a detectable content of formaldehyde (0.40 mg kg−1), thus demonstrating that the formaldehyde content of commercial carrageenan and PES products are well below the European Commission maximum limit of 5 mg kg−1. © 2014, © 2014 Taylor & Francis. Source


Blakemore W.R.,Celtic Colloids Inc | Kwok S.K.,MPI Research | Harding N.I.,Previously with MPI Research
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2014

An LC-MS/MS method was validated in accordance with 21CFR Part 58 Good Laboratory Practice for Non-clinical Laboratory Studies to measure the concentration of carrageenan in dose formulations used in a 28-day piglet dietary feeding study of swine-adapted infant formulations stabilised with carrageenan. Carrageenan concentrations in the test formulations were 0, 300, 1000 and 2250 mg kg–1 formula. The method for the measurement of carrageenan was LC-MS/MS coupled with ESI in negative-ion mode for detection. Linearity was established over the range 1.00–7.50 µg ml–1. Carrageenan dose formulation samples ranging from 0 to 2250 mg kg–1 of carrageenan were diluted to within the linearity range for measurement. © 2014, © 2014 Taylor & Francis. Source


Blakemore W.R.,Celtic Colloids Inc | Brant A.F.,Previously with MPI Research | Bissland J.G.,MPI Research | Bissland N.D.,Eurofins
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2014

Development and validation of this method was conducted to support a 28-day piglet feeding study of swine-adapted infant formulations stabilised with carrageenan. The validation was performed in accordance with USFDA Good Laboratory Practice (GLP) Regulations and associated current bioanalytical guidelines. Separation of carrageenan from plasma protein was unsuccessful using saturated sodium chloride due to the extremely strong cross-linking interactions between carrageenan and protein. Poligeenan is the deliberately acid-hydrolysed low molecular weight polygalactan non-food product produced from carrageenan. Poligeenan molecules are nearly identical to carrageenan molecules with respect to molecular structure, the primary difference being molecular weight. These poligeenan molecules have similar molecular weight when compared with the lowest molecular weight fraction of carrageenan called the low molecular-weight tail (LMT). Poligeenan was separated from plasma protein using the salting procedure, this being due to the significantly weaker interaction with protein caused by its shorter molecular chain length. Thus, poligeenan was applied as a chemical analyte surrogate for the LMT of carrageenan solely for the development and validation of the method. This method was used to try to detect the LMT of the carrageenan test material during the 28-day piglet feeding study, and if such was absorbed into the bloodstream. Successful development and validation of the method was achieved using LC-MS/MS coupled with ESI in negative-ion mode. A standard curve of instrument response versus poligeenan concentration was developed using swine plasma spiked with a range of poligeenan concentrations. The lower level of quantification (LLOQ) of poligeenan was 10.0 µg ml–1, and the quantification range was 10.0–100.0 µg ml–1. No animals were fed poligeenan. © 2014, © 2014 Taylor & Francis. Source


Weiner M.L.,TOXpertise LLC | Ferguson H.E.,Abbott Laboratories | Thorsrud B.A.,MPI Research | Nelson K.G.,MPI Research | And 7 more authors.
Food and Chemical Toxicology | Year: 2015

A toxicity/toxicokinetic swine-adapted infant formula feeding study was conducted in Domestic Yorkshire Crossbred Swine from lactation day 3 for 28 consecutive days during the preweaning period at carrageenan concentrations of 0, 300, 1000 and 2250 ppm under GLP guidelines. This study extends the observations in newborn baboons (McGill etal., 1977) to piglets and evaluates additional parameters: organ weights, clinical chemistry, special gastrointestinal tract stains (toluidine blue, Periodic Acid-Schiff), plasma levels of carrageenan; and evaluation of potential immune system effects. Using validated methods, immunophenotyping of blood cell types (lymphocytes, monocytes, B cells, helper T cells, cytotoxic T cells, mature T cells), sandwich immunoassays for blood cytokine evaluations (IL-6, IL-8, IL1β, TNF-α), and immunohistochemical staining of the gut for IL-8 and TNF-α were conducted. No treatment-related adverse effects at any carrageenan concentration were found on any parameter. Glucosuria in a few animals was not considered treatment-related. The high dose in this study, equivalent to ~430 mg/kg/day, provides an adequate margin of exposure for human infants, as affirmed by JECFA and supports the safe use of carrageenan for infants ages 0-12 weeks and older and infants with special medical needs. © 2014 Elsevier Ltd. Source


McKim J.M.,IONTOX LLC | Wilga P.C.,Cyprotex | Pregenzer J.F.,Cyprotex | Blakemore W.R.,Celtic Colloids Inc
Food and Chemical Toxicology | Year: 2015

Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter molecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins. © 2015 The Authors. Source

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