First draft prepared by
Mrs M.E.J. Pronk,1Dr P. Verger,2Dr Z. Olempska-Beer3
and Professor R. Walker4
1Centre for Substances and Integrated Risk Assessment, National Institute
for Public Health and the Environment, Bilthoven, Netherlands
2National Institute for Agricultural Research (INRA), Paris, France
3Center for Food Safety and Applied Nutrition, Food and Drug
Administration, College Park, Maryland, USA
4Emeritus Professor of Food Science, School of Biomedical and Molecular
Sciences, University of Surrey, Guildford, United Kingdom
Explanation ... Genetic modification ... Product characterization ... Biological data ... Biochemical aspects ... Toxicological studies ... Acute toxicity ... Short-term studies of toxicity ... Long-term studies of toxicity and carcinogenicity ... Genotoxicity ... Reproductive toxicity ... Observations in humans ... Dietary exposure ... Comments ... Toxicological data ... Assessment of dietary exposure ... Evaluation ... References ...
1. EXPLANATION
At the request of the Codex Committee on Food Additives and Contaminants (Codex Alimentarius Commission, 2006) at its thirty-eighth session, the Committee evaluated an enzyme preparation containing the enzyme asparaginase (L- asparagine amidohydrolase; EC 3.5.1.1), which it had not evaluated previously. Asparaginase hydrolyses the amide in the amino acid L-asparagine to the corresponding acid, resulting in L-aspartate (aspartic acid) and ammonia. Apart from asparagine, asparaginase acts only on glutamine and has no activity on other amino acids. Asparaginase has no activity on asparagine residues in peptides or proteins. 55 56 57 57 57 57 58 58 59 59 59 59 59 62 62 62 62 62 - 55 -
The asparaginase enzyme preparation under consideration is produced by submerged fermentation of an Aspergillus oryzae production strain carrying a gene encoding asparaginase from A. oryzae. The enzyme is subsequently partially purified and concentrated, resulting in a liquid enzyme concentrate (LEC), which, in the final preparation, is stabilized, formulated and standardized with water, glycerol, sodium benzoate and potassium sorbate.
The enzyme activity of asparaginase is expressed in asparaginase units (ASNU), 1 ASNU being the amount of enzyme that produces 1 ˩mol of ammonia per minute under specific reaction conditions. The asparaginase preparation has a typical activity of 3500 ASNU/g and has the following composition: total organic solids (TOS), approximately 4%; water, approximately 46%; glycerol, approximately 50%; sodium benzoate, approximately 0.3%; and potassium sorbate, approximately 0.1%.
Asparaginase is to be used during food manufacture to convert asparagine to aspartic acid with the intention of reducing acrylamide formation during food production of dough-based products, such as cookies and crackers, and processed potato products, such as potato chips and french fries. Acrylamide is formed as a reaction product between asparagine and reducing sugars when food products are baked or fried at temperatures above 120 ºC. The enzyme preparation is added to dough prior to baking, at recommended use levels varying from 200 up to 2500 ASNU (or 0.06–0.7 g of the enzyme preparation) per kilogram of processed food. Prior to the heat treatment or cooking step, potato strips or slices are treated by dipping in a water bath containing the enzyme preparation, leading up to approximately 2000 ASNU (or 0.6 g) per kilogram of product.
1.1 Genetic modification
The host strain for the asparaginase gene, the Aspergillus oryzae BECh2 strain, was derived from A. oryzae strain IFO 4177 (synonym A1560). Aspergillus
oryzae is known to contain genes involved in the synthesis of the secondary
metabolites cyclopiazonic acid, kojic acid and 3-˟-nitropropionic acid, as well as genes involved in the synthesis of aflatoxins. In a first step, A. oryzae strain A1560 was genetically modified by site-directed disruption of the endogenous amylase and protease genes to allow the production of asparaginase without enzymatic side activities. In the following two steps, the modified strain (designated A. oryzae JaL 228) was irradiated to remove or reduce its potential to produce secondary metabolites. First, the JaL 228 strain was exposed to ˠ-radiation, resulting in a mutant (designated A. oryzae BECh1) that is devoid of genes involved in the synthesis of aflatoxins and cyclopiazonic acid. Subsequently, the BECh1 strain was subjected to ultraviolet radiation, resulting in a mutant (designated A. oryzae BECh2) that is impaired in kojic acid synthesis. It is this BECh2 strain that is used as the host strain for the asparaginase gene. When tested under conditions optimal for the production of secondary metabolites, the BECh2 strain produced neither aflatoxins (including the intermediate compounds sterigmatocystin and 5-methoxy- sterigmatocystin) nor cyclopiazonic acid and essentially no 3-˟-nitropropionic acid. Although the strain produced kojic acid, it did so only at a level of approximately 15% of that produced by the A1560 and BECh1 strains.
The asparaginase gene originates from the same strain as the host strain, i.e. A. oryzae strain IFO 4177 (synonym A1560), and is cloned into an A. oryzae expression plasmid generating the asparaginase expression plasmid pCaHj621. This expression plasmid is based on the standard Escherichia coli vector pUC19 and contains known and well characterized deoxyribonucleic acid (DNA) sequences. The pCaHj621 expression plasmid was used to transform the A.
oryzae BECh2 host strain to obtain the A. oryzae pCaHj621/BECh2#10 production
strain. The plasmid is stably integrated into the A. oryzae chromosomal DNA and does not contain antibiotic resistance genes. The inserted DNA also does not encode or express any known harmful or toxic substances. Asparaginase expressed by the production strain has no significant amino acid sequence homology with known allergens and toxins listed in publicly available databases. When one test batch (PPV 24743) of the enzyme preparation was analysed for kojic acid and 3-˟-nitropropionic acid, these secondary metabolites were not detected.
1.2 Product characterization
Asparaginase is produced by submerged fed-batch pure culture fermen- tation of the A. oryzae pCaHj621/BECh2#10 production strain. It is secreted into the fermentation medium, from which it is recovered and concentrated. It is subsequently stabilized, formulated and standardized with water, glycerol, sodium benzoate and potassium sorbate. The enzyme preparation is added to dough prior to baking, and potatoes are dipped in water containing the enzyme preparation before frying. During the heating of the products, it is expected that the enzyme will be inactivated.
The asparaginase enzyme preparation conforms to the General Speci- fications and Considerations for Enzyme Preparations Used in Food Processing, prepared by the Committee at its sixty-seventh meeting (Annex 1, reference 184). The enzyme preparation is free from the production organism.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
Aspergillus oryzae asparaginase was assessed for potential allergenicity
by comparing its amino acid sequence with those of known allergens listed in publicly available databases (SWALL and GenBank). No immunologically significant sequence homology was detected. A sequence homology assessment of A. oryzae asparaginase with respect to the sequences of known toxins listed in the same databases also revealed no significant homology.
2.2 Toxicological studies
The host organism A. oryzae is non-pathogenic and has a long history of use in food. Enzyme preparations from A. oryzae have been evaluated previously by the Committee. For ˞-amylase and protease from A. oryzae, the Committee concluded that, since they are derived from a microorganism that is accepted
as a constituent of foods and is normally used in food production, they must be regarded as foods and are thus acceptable for use in food processing (Annex 1, reference 77). An acceptable daily intake (ADI) “not specified” was allocated to lipase from A. oryzae (Annex 1, reference 35), as well as to laccase from a recombinant strain of A. oryzae (Annex 1, reference 167). Phospholipase A1 from a recombinant strain of A. oryzae, which was evaluated at the sixty-fifth meeting of the Committee (Annex 1, reference 178), is again under consideration at the present meeting (see monograph in this volume).
Aspergillus oryzae host strains derived from strain A1560 have been used
in the construction of several Novozymes enzyme production strains. The A.
oryzae BECh2 strain used as host strain in the construction of the asparaginase
production strain was also used to develop production strains for triacylglycerol lipase, glucose oxidase, two xylanases and phospholipase A1, which is under consideration at the present meeting (see monograph in this volume). The DNA introduced into the production strains for these enzymes is essentially the same as that introduced into the asparaginase production strain A. oryzae pCaHj621/ BECh2#10, except for the sequence encoding the specific enzyme. All these enzyme products were stated to have been assessed for safety (in at least a 13- week study of toxicity in rats treated orally, an assay for mutagenicity in bacteria in vitro and a cytogenetic assay in human lymphocytes in vitro).
Toxicological studies were also performed with the asparaginase enzyme, using an LEC (batch PPV 24743; dry matter content, 10.5% weight per weight [w/w]; TOS content, 8.4% w/w; specific gravity, 1.049 g/ml), omitting stabilization, formulation and standardization.
2.2.1 Acute toxicity
No information was available.
2.2.2 Short-term studies of toxicity
Groups of 10 male and 10 female CD rats (aged 42–48 days) were given asparaginase (batch PPV 24743) at a dose equivalent to 0, 88, 290 or 880 mg TOS/kg body weight (bw) by gavage (in purified water) daily for 13 weeks. The study was performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 408 (1998) and was certified for compliance with Good Laboratory Practice (GLP) and quality assurance (QA). Observations included clinical signs, physical examination and arena observations, body weight, food intake, water consumption, sensory reactivity, grip strength, motor activity, ophthalmoscopy, haematology, clinical chemistry, organ weights and macroscopic and microscopic pathology.
No treatment-related effects were observed on mortality, clinical signs, ophthalmoscopy, body weight, food conversion efficiency, sensory reactivity or motor activity. Forelimb and hindlimb grip strength were slightly increased (12%) in males at the mid and high doses, but there was no dose–response relationship, and statistical significance was reached only for forelimb grip strength at the high dose. Treated males also showed decreases in haematocrit, haemoglobin, red blood cell
count and total and differential white blood cell counts. Only for haematocrit and haemoglobin at the mid and high doses did the difference reach statistical significance; however, the difference was very small (4%), without a dose–response relationship. Treated females showed a small decrease in food consumption (7% at the high dose) and a small increase in water consumption (8% and 13% at the mid and high doses, respectively), but no effect on food conversion efficiency. Treated females also showed statistically significant decreases in total white blood cell and basophil counts and in activated partial thromboplastin time. However, there was no dose–response relationship for any of the changed parameters, and control values were high when compared with historical control values for these parameters. In both sexes, organ weights, macroscropic pathology and histopathology were unaffected by treatment. The only treatment-related findings were small (7–8%) but statistically significant increases in plasma potassium levels in males at the mid and high doses and in females at the high dose. As there were no other changes in electrolyte concentrations and the kidneys appeared histopathologically normal, these changes were considered to be of no toxicological significance. Overall, it can be concluded that the no-observed-effect level (NOEL) is 880 mg TOS/kg bw per day, the highest dose tested in this study (Hughes, 2006).
2.2.3 Long-term studies of toxicity and carcinogenicity
No information was available.
2.2.4 Genotoxicity
The results of two studies of genotoxicity with asparaginase (batch PPV 24743) in vitro are summarized in Table 1. The first study followed OECD Test Guideline 471 (1997), and the second, OECD Test Guideline 473 (1997). Both studies were certified for compliance with GLP and QA.
2.2.5 Reproductive toxicity
No information was available.
2.3 Observations in humans
No information was available.
3. DIETARY EXPOSURE
The asparaginase enzyme preparation is added to dough prior to baking, and potatoes are dipped in water containing the enzyme preparation before frying. Although it is expected that the enzyme will be inactivated upon heating of the products, the actual levels of the enzyme (active or inactive) in the final food products are not known. A worst-case scenario for human dietary exposure can be estimated on the basis of the recommended use levels and the assumption that all TOS originating from the enzyme preparation are carried over into the final products. To elaborate this scenario, it is assumed that:
•
all cereal and potato products are produced by processes using the asparaginase enzyme preparation at the highest recommended use level;•
a maximum concentration of 700 mg of enzyme preparation (or 2500 ASNU) is applied in treating 1 kg of processed food;•
the TOS content of the enzyme preparation is 4%;•
all TOS are carried over into the final products.Table 1. Genotoxicity of asparaginase in vitro
End-point Test system Concentration Result Reference Reverse mutation Salmonella
typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrApKM101 156–5000 ˩g/ml, ±S9 Negativea Pedersen (2006) Chromosomal aberration
Human lymphocytes 1st experiment: 1187, 2813 or 5000 ˩g/ml, S9; 1582, 2109 or 5000 ˩g/ml, +S9 2nd experiment: 430, 839 or 1311 ˩g/ml, S9; 3200, 4000 or 5000 ˩g/ml, +S9 Negativeb Whitwell (2006)
S9, 9000 × g supernatant from rat liver.
aWith and without metabolic activation (S9), by the “treat-and-plate” method (to avoid
problems owing to the presence of free amino acids such as histidine and tryptophan in the asparaginase preparation). In the test with E. coli, initially strain WP2uvrA was tested by the direct plate incorporation method. However, because the asparaginase preparation supported growth of the test bacteria in this procedure, with a concurrent increase in the number of revertants, the experiment was repeated using the “treat-and-plate” method and the strain WP2uvrApKM101, which was considered more sensitive to pro-mutagens in the presence of S9.
b With and without metabolic activation (S9). In the first experiment, the cell cultures were
treated for 3 h without and with S9 and were harvested 17 h later. The highest tested concentration induced 44% and 33% mitotic inhibition in the absence and presence of S9, respectively. In the second experiment, the cells were exposed continuously for 20 h without S9 and then harvested. With S9, the cells were treated for 3 h and harvested 17 h later. The highest tested concentration induced 53% and 0% mitotic inhibition in the absence and presence of S9, respectively.
The resulting maximum concentration of enzyme in the final products would correspond to 28 mg TOS/kg of food.
According to the Joint FAO/WHO Expert Committee on Food Additives (JECFA) exposure assessment for acrylamide, the highest amount of total cereals and potato1 products from the five Global Environment Monitoring System Food
Contamination Monitoring and Assessment Programme (GEMS/Food) Regional Diets is 613 g/day (African diet; Table 2). If such a conservative consumption figure is assumed, as well as a body weight of 60 kg, this would result in a dietary exposure to asparaginase of about 17 mg TOS/day or 0.3 mg TOS/kg bw per day.
In order to harmonize the approaches and to follow the recommendations from the FAO/WHO consultation on dietary exposure assessment, another estimate could be based on the 13 GEMS/Food Consumption Cluster Diets (Table 3). According to these data, the maximum consumption for cereals and potato products would be 960 g/day. Based on this figure, the dietary exposure to asparaginase would be 27 mg TOS/day or 0.4 mg TOS/kg bw per day, if a body weight of 60 kg is assumed.
Table 2. Consumption of cereals and roots and tubers from the five GEMS/ Food Regional Diets
Commodity Consumption (g/person per day) from GEMS/Food Regional Diets Middle Eastern Far Eastern African Latin American European
Total cereals 430 451 292 254 222
Total roots & tubers 62 109 321 159 242
Total 492 560 613 413 464
Table 3. Consumption of cereals and roots and tubers from the 13 GEMS/Food Consumption Cluster Diets
Commodity Consumption (g/person per day) from GEMS/Food Consumption Cluster Diets
A B C D E F G H I J K L M Total cereals 357 714 763 505 365 329 617 487 389 386 440 568 410 Total roots & tubers 525 246 69 244 278 205 120 101 404 438 135 104 176 Total 882 960 832 749 643 534 737 588 793 824 575 672 586
4. COMMENTS
4.1 Toxicological data
Toxicological studies were performed with an asparaginase LEC. The Committee noted that the materials added to the asparaginase LEC for stabilization, formulation and standardization either have been evaluated previously by the Committee or are common food constituents and do not raise safety concerns.
In a 13-week study of toxicity in rats, no significant treatment-related effects were seen when the LEC was administered by oral gavage at doses up to and including 880 mg TOS/kg bw per day. Therefore, 880 mg TOS/kg bw per day, the highest dose tested, was taken to be the NOEL. The LEC was not mutagenic in an assay for mutagenicity in bacteria in vitro and was not clastogenic in an assay for chromosomal aberrations in mammalian cells in vitro.
4.2 Assessment of dietary exposure
Based on a maximum mean daily consumption of 960 g of processed foods (cereals, roots and tubers; GEMS/Food Consumption Cluster Diet B) by a 60-kg adult and on the assumptions that the enzyme is used at the maximum recommended use level and that all TOS originating from the enzyme preparation remain in the final products, the dietary exposure would be 0.4 mg TOS/kg bw per day.
5. EVALUATION
Comparing the conservative exposure estimate with the NOEL from the 13- week study of oral toxicity, the margin of safety is 2200. The Committee allocated an ADI “not specified” for asparaginase from this recombinant strain of A. oryzae, used in the applications specified and in accordance with good manufacturing practice.
6. REFERENCES
Codex Alimentarius Commission (2006) Report of the thirty-eighth session of the Codex Committee on Food Additives and Contaminants, The Hague, The Netherlands, 24–28 April 2006. Rome, Italy, Food and Agriculture Organization of the United Nations (ALINORM 06/29/12; http://www.codexalimentarius.net/web/archives.jsp?year=06). Hughes, N. (2006) Asparaginase, PPV 24743—Toxicity study by oral administration to CD
rats for 13 weeks. Unpublished report No. NVZ0037/054031 from Huntingdon Life Sciences Ltd, Alconbury, Huntingdon, United Kingdom. Submitted to WHO by Novozymes A/S, Bagsværd, Denmark.
Pedersen, P.B. (2006) Asparaginase, PPV 24743—Test for mutagenic activity with strains of Salmonella typhimurium and Escherichia coli. Unpublished report No. 20068039 from Novozymes A/S, Bagsværd, Denmark. Submitted to WHO by Novozymes A/S, Bagsværd, Denmark.
Whitwell, J. (2006) Asparaginase, PPV 24743—Induction of chromosome aberrations in cultured human peripheral blood lymphocytes. Unpublished report No. 1974/46-D6172 from Covance Laboratories Ltd, Harrogate, United Kingdom. Submitted to WHO by Novozymes A/S, Bagsværd, Denmark.
CARRAGEENAN AND PROCESSED EUCHEUMA SEAWEED (addendum) First draft prepared by
Dr D.J. Benford,1Ms R.A. Harrison,1Professor S. Strobel,2Dr J. Schlatter3and
Dr P. Verger4
1Food Standards Agency, London, United Kingdom
2University of Plymouth, Plymouth, United Kingdom
3Swiss Federal Office of Public Health, Zurich, Switzerland
4National Institute for Agricultural Research (INRA), Paris, France
Explanation ... Biological data ... Toxicological studies ... Special studies on the gastrointestinal tract ... Special studies in vitro ... Special studies on tumour suppression and anti-tumour activity ... Special studies on the immune system ... Observations in humans ... Clinical studies ... Post-marketing surveillance ... Epidemiological studies ... Case reports ... Dietary exposure ... Exposure to undegraded carrageenan ... Exposure to degraded carrageenan ... Comments ... Toxicological data ... Assessment of dietary exposure ... Evaluation ... Recommendation ... References ...
1. EXPLANATION
Carrageenan is a sulfated galactose polymer with an average molecular weight well above 100 kilodaltons (kDa). It is derived from several species of red seaweeds of the class Rhodophyceae. It has no nutritive value and is used in food preparation for its gelling, thickening and emulsifying properties. The three main copolymers in carrageenan are designated as iota (b), kappa (Ĩ) and lambda (˨), depending on the number and location of the sulfate moieties on the hexose backbone. Processed Eucheuma seaweed is a semi-refined form of carrageenan