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MARINALG
INTERNATIONAL POSITION PAPER REGARDING : SUMMARY After over four years of experimentation, Marinalg and others have been unable to reliably measure this new specification in laboratories of its members, its customers, or in independent laboratories. Several methods based on size exclusion chromatography (SEC) and light scattering (LS) were tested, and although two have come close with respect to being reliable and reproducible, they cannot be validated. Below 50,000 Daltons, the signal to noise ratio when light scattering is used for molecular weight determination is too low for the accuracy required by the EU specification. A very expensive embodiment of SEC/LS has been adopted by the Japan Ministry of Health for measuring this specification, but the methodology is flawed. Alternate methods of measuring the EC specification have now been evaluated: using carrageenan standards for SEC column calibration, universal SEC column calibration and ultrafiltration. None of these provide great promise that a satisfactory, validated test can be developed It is clear at this point in time that successfully measuring the EC specification is not currently feasible. In June, 2007, the JECFA evaluated the work presented in an earlier version of this report and a new 90-day feeding study in which rats were fed carrageenan with a viscosity of 8cps. At this writing, while the final report of the 68th JECFA meeting has not published, the draft carrageenan specifications resulting from that meeting do not include a specification for the low molecular weight tail of carrageenan. The 90-day rat feeding study reviewed by JECFA was performed with a carrageenan very close to the 5 cps water viscosity limit (8cps). This carrageenan would be expected to have a relatively large fraction less than 50,000 Daltons. Nevertheless the study showed no adverse toxicological effects in any of the test animals at up to 5% of the diet. Clearly this study casts some doubt on the cost benefit of trying to develop a test for measuring the percentage of carrageenan less than 50,000 Daltons when the 5cps water viscosity limit is probably entirely sufficient as a health safety criterion. The 90-day
rat feeding study has been published and appears in its entirety as Annex
III of this report. Weiner, M. L., Nuber, D., Blakemore, W.R., Harriman,
J.F., and Cohen, S.M., 2007. A 90-day dietary study on kappa carrageenan
with emphasis on the gastrointestinal tract. Food Chem. Toxicol. 45,98-106. INTRODUCTION The Marinalg Working Group on Molecular Weight Determination (William Blakemore FMC, Chairman; Dr. Harris Bixler, SIAP, Secretary; Arne Graff Anderson, CPKelco; Dr. Joop de Vries / Dr, Graham Swarn, Danisco; Dr. Patrick Boulenguer, Degussa) has been carrying out experiments since April, 2003 to measure the molecular weight distribution of commercial carrageenan and PES used in foods. In its March 5, 2003 opinion, the EC-SCF proposed a new specification for these hydrocolloids to augment the 5 cps water viscosity “if feasible”. The purpose of the new specification is to have better control over the amount or proportion of the very low molecular weight tail of carrageenan and PES used in food products. Marinalg believes that there is no toxicological evidence to support the establishment of the specification. Poligeenan has demonstrated adverse toxicological effects when fed under stressful conditions and in large quantities to certain rodents. Poligeenan, a substance used in medical imaging, uses carrageenan as a starting material. The carrageenan raw material is deliberately acid hydrolysed at high temperatures for extended periods of time to produce a finished product with a average molecular weight (Mw) of about 10,000 – 20,000 Daltons. By its very nature, carrageenan comprises a broad range of molecular weight components. Therefore, animals fed carrageenan in chronic and sub-chronic feeding studies ingest this broad spectrum of molecular weight that makes up carrageenan. There is neither toxicological nor epidemiological evidence to indicate that consumption of carrageenan imparts an adverse toxicological effect. Hence, the effect of these very small amounts of naturally-occurring lower molecular components in carrageenan is moot, because studies show that consumption of carrageenan does not cause adverse toxicological effects. Before the Marinalg Working Group of carrageenan producers had adequate time to determine the feasibility of measuring the new specification, it was formally adopted by the EC as Commission Directive 2004/45/EC on April 16, 2004 for implementation by Member States by April 1, 2005. This specification requires that carrageenan or PES used in food must not contain more than 5% molar mass with molecular weight less than 50,000 Da. The industry refers to this as the “low molecular weight tail” or LMT. As of March
2008, the Working Group or others still have not been able to develop
a method for molecular weight distribution measurement that is sufficiently
accurate and reproducible to yield a validated and defensible method,
and not so costly as to act as a barrier to trade for smaller producers. MARINALG EXPERIMENTAL WORK
The methods studied to date in detail have all been based on size exclusion chromatography (SEC) followed by concentration and molecular weight detection in the stream exiting the chromatography columns. SEC is used to spread out the carrageenan molecular size distribution in the flow stream exiting the columns. Note that this separation is by molecular size and not molecular weight, so physical models are used to convert molecular size data to molecular weights. The stream exiting the SEC columns flows through a series of detectors: refractive index for carrageenan concentration determination and light scattering and/or intrinsic viscosity for molecular weight determination. Some instruments include chemical detectors to be sure only carrageenan is being measured in the flow stream. These are
highly developed commercial research instruments of great technical sophistication.
Nevertheless, none met the most important objective of the Working Group.
Six laboratories participated in this study, Degussa; Danisco; Viscotek,
Ltd; Polymer Standards Services, GmbH; San-Ei Gen FFI, Ltd: North East
Wales Institute/NEWI, all with state-of-the art equipment and with qualified
scientists to run the experiments. Procedure details (sample preparation
and concentration, eluent type and concentration, etc.) were recorded
for each lab and approved by the Working Group. Eleven different commercial
carrageenan and PES samples, representing different sulfated polygalactose
types (nominally kappa, lambda and iota) made by five different producers,
were tested by all laboratories under "Round Robin" conditions.
Annex I (Summary of Round Robin #1 and #2) and Annex II (Sample Information) contain test results and physical characteristics of the Round Robin samples, respectively. Despite all this technical discipline, inter-lab reproducibility of the LMT was shown to be poor (Fig. 1). Detectors downstream of the SEC columns must be able to measure polymer concentration and molecular weight accurately in the range represented by the LMT.
It appears that even under optimum SEC conditions, light scattering detector signal to noise ratio (S/N) in the LMT region is extremely low and it is this signal upon which molecular weight determination is based (Fig. 2). Several test locations have experienced drifting baselines, and variable recoveries (measured concentration versus actual), both of which make data interpretation even more complex and unreliable.
Initially the Working Group thought that the Viscotek triple detector method (refractive index, low angle light scattering and intrinsic viscosity) was giving promising results, and as a consequence published the method on the Marinalg website. However, further testing indicated that these same issues applied, but to a much lesser degree (Figure 3).
Various
fitting and extrapolation routines (Zimm, Debye, Berry) are being used
in the MALS detection systems to determine Mw where there is enough S/N.
This data is then further extrapolated by some routine (usually linear)
into the region of poor S/N. For all of the carrageenan samples studied
the S/N within the LMT was low and resulted in extrapolations having to
be made from well outside the LMT range (Fig. 2, green line). This type
of extrapolation is subject to enough error so as not to give defensible
results for regulatory purposes. This can be seen in Fig. 2 where the
LMT region is shown graphically. Clearly any shift in baseline or green
line extrapolation will have a profound effect on the very small LMT region
calculated for commercial carrageenan being used in foods. It is estimated
that it is virtually impossible to determine the molecular weight of SEC-spread
samples below about 10,000 Da by any of the light scattering techniques,
the molecular weight region most likely to raise toxicological concerns. The Working Group's experience with SEC/light scattering in no way detracts from it use as a valuable research tool. The technique is widely used for estimating polymer molecular structure in food and industrial applications. A higher level of accuracy, however, is required when it is to be used for specification and regulatory measurements. Even in
the present study useful information (from the Round Robin samples) was
obtained on molecular structure. For instance, fairly good consistency
was seen for inter-lab results obtained for the weight average molecular
weight (Mw) (Fig. 4), except for one sample, a lambda type that is known
to be a more rigid rod in solution that the kappa and iota types. Furthermore
there was fairly good correlation for Mw versus water viscosity (Fig.
4), except for the one aberrant sample already noted.
The causes of these inconsistencies and poor correlations when using SEC/LI need to be identified before further progress on method development can be attempted. In addition to the mathematical errors from extrapolation, possible interferences would also include measurement of non-carrageenan components as part of the LMT (e.g. salts, proteins, other carbohydrates), inaccurate and inconsistent measurement cut-off points, poor SEC separations (e.g. gelation, aggregation), column selection and efficiency, eluate and elution characteristics (e.g. concentration, viscosity), and signal to noise ratios of the measuring equipments.
In reaching its conclusions, the Working Group has conferred with several world class scientists (Prof. Wayne Reed, Tulane University, Dr. Phillip Wyatt and his staff at Wyatt Technology, and the group consisting of Drs. Chi-San Wu, E. Malawer and L. Senak at ISP and Dr. Maguarite Rinaudo at CMRV) who have been involved in developing and using SEC/light scattering for a variety of research purposes. While some were confident that the Working Group's goal could be reached, none had ever done so. Through this process consensus was gradually reached that the current equipment employing light scattering and the attendant software will not measure the EC specification with sufficient accuracy to survive the necessary validation protocols. Preliminary
attempts have been made to try to measure the LMT in a commercial kappa
carrageenan deliberately blended with known levels of LMT derived from
poligeenan. However, measurements of the total LMT in these synthetic
blends by the Degussa, Danisco, San-Ei, and Viscotek facilities were all
inconsistent and far removed from the calculated values. The earlier glimmer
of hope with the Viscotek method was dimmed by these results. The failure
to accurately detect and quantify known levels of LMT confirms the position
that development of a validated method using SEC/light scattering is probably
not possible. ALTERNATE METHODS
While the
work to date with light scattering has led to frustrating conclusions,
it pointed in a direction of potentially more promise which has recently
been explored by Nestle researchers (Spichtig, V. and Austin, S. 2008.
Determination of the low molecular weight fraction of food-grade carrageenans.
Journal of Chromatography B, 861 (2008) 81-87. The Working Group had given
consideration to this method before it became aware of the Nestle work.
Now that the Nestle work has been published, the method seems less promising
than previously thought. For water soluble hydrocolloids, the most widely used standards are eight pullulans ranging in Mw from 5,300 to 760,000 Daltons with PDIs meeting the above polydispersity criterion. These are commercially available from Shodex. This method has been tested on commercial carrageenans, and the results have been reported in the scientific literature by Japanese scientists (Uno, et al, Food Additives and Contaminants, 18, No. 9, pp763-772, 2001). No correction was applied in this work for the differences between pullulan and carrageenan sizes versus molecular weights, so validation of the LMTs reported by Uno remains in question.Of course, having a set of low polydispersity carrageenan standards could eliminate the shortcomings of using pullulan standards for column calibration, but preliminary attempts to prepare carrageenan standards in the laboratory by both Nestle and the Marinalg Working Group has been disappointing. PDIs ranged from 1.3 to 2.6 with most in the range of 1.6 to 1.7, and as will be explained below were too polydisperse for reliable column calibration in the range of the LMT. The overall conclusions were similar to those reported earlier for the Viscotek data. The method appears to be reproducible with respect to repeat LMT measurements on the same sample in the same laboratory, but several hurdles stand in the way of it being developed into a universal validated method. One issue with using standards for column calibration is related to how close the numerical value at the top of the elution peak is to the measured Mw. They are close to being equal with the pullulan standards with polydispersity indices of 1.1 to 1.2. Pullulan standards give very sharp elution curves. However, it is not the case with polydispersity indices in the 1.6 to 1.7 range. For one of the Marinalg carrageenan standards the Mw was 246,000 Da and the Mn 149,500 Da for a polydispersity index of 1.6. The molecular weight at the top of the peak was 193,000 Da. This represents an error of over 20%. Any use of carrageenan standards must use the molecular weight at the elution peak, and not the Mw (average) to be an improvement over the SEC methods reported above. Adopting a method using carrageenan standards would demand that such standards be readily available for all involved as is the case with the commercial pullulan standards. This means that the carrageenan standards would have to be made in bulk, and made accurately and reproducibly. No such protocols are close to being successfully developed. Austin, et al suggested in their paper that the LMT specification might be moved from 5% to 8%. All of their measured LMTs were less than 8%; whereas only half of the sample LMTs were less than 5%. Since there is little basis in fact toxicologically for the EC numerical LMT specification as it is, industry should resist the adoption of an expensive, inaccurate method on the basis that if the LMT is set high enough all commercial samples seem to pass. Either an economical, accurate method can be developed or the long employed water viscosity limit should be continued as a molecular composition health safety criterion. The Nestle
work on jellies did confirm prior experimental work and conclusions with
respect to controlling carrageenan degradation during food processing
and storage, as has been detailed over the years in the JECFA dossiers.
The difficulties in obtaining carrageenan standards has lead the Working Group to explore the application of a technology referred to as "universal calibration", a physical model for converting a pullulan calibration curve to a carrageenan calibration curve (Grubisic, Z. et al, Polymer Letters, 5, pp753-759, 1967). The model takes into consideration size and shape differences for the two polymers when their molecular weights are the same. Initial work applying this technique to the Uno data shows some promise, but it is too early to draw any conclusions. However, it must continually be kept in mind that the Uno method is extremely expensive and technology intensive. It could be a significant problem for smaller companies that make perfectly safe carrageenan as determined by the long used, reliable and inexpensive water viscosity purity criterion. A related technology referred to as "polydisperse or broad standard calibration" is also under investigation (Malawer, E.G. and A.J. Montana, Journal of Polymer Science: Polymer Physics Edition, 18, pp2303-2305, 1980). For this purpose, a very broad molecular weight distribution carrageenan is prepared as a standard that has relatively high concentrations of carrageenan in the low and high molecular weight tails and spans the range of Mw of interest. Again, physical modeling and computer analysis is employed to convert SEC exit time to a carrageenan molecular weight. There is
no assurance until experiments can be run to know whether the poor accuracy
of LMT calculation from light scattering can be improved upon by use of
either universal or polydisperse calibration.
One technology currently being assessed is the potential use of membranes to separate the LMT from the rest of the carrageenan. This potential method would have to be combined with the colorimetric measurement of very low concentrations of carrageenan or some other equivalent method. The primary issue for development of this technology would be the production of membranes with consistent cut-off at 50k Da. Recent work carried out at Leatherhead Food International has shed some light on this potential technology. (Titoria, P.M., Marrs, W.M., Haynes, C.L., and Franco, I. Recovery of Low Molecular Weight Carrageenan Fractions by Ultrafiltration Through Semi-permeable Membranes - A feasibility Study. Research Report No. 863, October 2005.) The carrageenans were separated into permeate and retentate using Vivaspin membrane devices, with nominal cut-off points at 100kDa and 50kDa. The fractions were analysed for Mw using SEC. The primary
conclusions were as follows.
The Marinalg Working Group will continue to explore these alternate methods, but success.seems very remote. ANNEXES Annex I :
Summary of Round Robin #1 and #2
POSITION
STATEMENT ON CARRAGEENAN AND SEMICARBAZIDE (SEM) Marinalg International is a world wide Association representing the producers of hydrocolloids extracted from seaweeds. The above rapid alert notification concerns the possible detection of semicarbazide (SEM) in carrageenan and seaweed. In animal tissues SEM has been identified as a metabolite of nitrofurazone, which is one of the nitrofurans. Nitrofurans are banned animal antibiotics. It should be emphasized that nitrofuran was not found in any of the seaweed or carrageenan samples. Marinalg International confirms that neither nitrofuran nor its metabolites are used at any stage of growth of the seaweed, or during the carrageenan manufacturing process. Marinalg International is actively investigating why SEM may have been detected in seaweeds and carrageenan. It has been suggested that SEM can indeed be naturally present in the seaweeds rather than arising from contamination with nitrofuran and/or its derivatives. The fact that SEM also happens to be a metabolite of nitrofuran appears to be unrelated to its possible presence in carrageenan*. Furthermore, current analytical methods were developed and validated for the detection of nitrofuran metabolites in animal tissues only ; they have not been validated in respect of detection of SEM in plant tissues. Marinalg International will continue to co-operate fully with the relevant Authorities to resolve the matter. * EFSA advice of July 28, 2003 - AFC/adhoc SEM/1
POSITION STATEMENT ON THE SAFETY OF CARRAGEENAN AND PROCESSED EUCHEUMA SEAWEED Marinalg International is a world association representing the producers of hydrocolloids extracted from seaweeds. The safety of carrageenan and Processed Eucheuma Seaweed for use in foods was confirmed at the fifty-seventh meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in Rome in June 2001. The JECFA recommended an Acceptable Daily Intake of "not specified", the most favorable ADI a food additive can get. This is significant since the JECFA review was based on extensive safety studies. The Codex Alimentarius was created by the United Nations Food and Agriculture Organization (FAO) and World Health Organization (WHO) to set standards, guidelines and principles on matters pertaining to food including food safety and food additives. The JECFA is an independent international panel of expert toxicologists. They review data and develop expert opinions and recommendations pertaining to food additives and contaminants at the request of the Codex Committee on Food Additives and Contaminants (a Committee of the Codex Alimentarius Commission) or by a member country of the Codex process. The safety of carrageenan and Processed Eucheuma Seaweed for use in foods was subject of the fifty-seventh meeting of the JECFA in Rome in June 2001. The JECFA recommended an Acceptable Daily Intake of "not specified", and confirmed thus the most favourable status a food additive can get. The decision of JECFA was based on a review of extensive safety studies concerning issues such as the potential for carrageenan and Processed Eucheuma Seaweed to induce cell proliferation and tumor promotion in the colon. Samuel Cohen M.D., Ph. D. (Chairman of the Department of Pathology/Microbiology from the Medical School at the University of Nebraska) and Dr. Nobuyuki Ito (Professor Emeritus from Nagoya City University Medical School, Japan), both experts on cancer, performed a valuable, scientifically critical literature review and prepared a response on the matter to the JECFA. The position of the JECFA is also supported by a very recent Japanese publication showing that Carrageenan has no tumor promoting effect (J. Toxico Pathol., 14(1), 37-43, 2001). The recent decision of JECFA to establish an ADI of "not specified" for carrageenan and Processed Eucheuma Seaweed is the best possible classification from a toxicological point of view. It means, that there is no numerical limit established on the consumption of carrageenan and Processed Eucheuma Seaweed when used in food at a level to achieve the desired technical or functional effect. The JECFA evaluation may not be published until 2003. The Drs Cohen and Ito response that was reviewed by the JECFA has been published in Critical Reviews in Toxicology, 32(5):413-444 (2002). For your convenience, you will find attached a copy of the Summary and Conclusions of the fifty-seventh meeting of the Joint FAO/WHO Expert Committee on Food Additives - Rome - 5-14 June 2001
SUMMARY AND CONCLUSIONS
* ADI "not specified" is used to refer to a food substance of very low toxicity which, on the basis of the available data (chemical, biochemical, toxicological and other) and the total dietary intake of the substance arising from its use at the levels necessary to achieve the desired effects and from its acceptable background levels in food, does not, in the opinion of the Committee represent a hazard for health. For that reason, and for the reasons stated in the individual evaluations, the establishment of an ADI expressed in numerical form is not deemed necessary. An additive meeting this criterion must be used within the bounds of good manufacturing practice, i.e. it should be technologically efficacious and should be used at the lowest level necessary to achieve this effect, it should not conceal food of inferior quality or adulterated food, and it should not create a nutritional imbalance.
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 "Position
statement on the safety of carrageenan and PES on the same subject of
the International Food Additives Council" |
