Safety evaluation of technical enzyme products with regards to the REACH legislation 09 June 2009 Brussels
ENZYMES What enzymes are Enzymes are proteins with highly specialized catalytic function Produced d and needed d by all living i things Similar metabolic mechanisms utilized by all organisms to produce enzymes Building-blocks are the 20 naturally occurring amino acids Completely Biodegradable Catalytic function determined by 3-dimensional structure Enzymes are naturally adapted to changing environments thus enzymes in a given class may have wide variation in temperature stability, salt tolerance, etc. depending on the environment in which the host organism is found
ENZYMES How enzymes are made Enzyme mainly through fermentation of microorganisms (but sometimes extracted from plant or animal) Usually Safe strain lineage organism is used in fermentation because Enzymes are used for technical as well as food & feed applications Manufacturers need reproducible and high yields and as little different processes as possible Contained Use legislation l requires safe strains (for GMMs) Subsequent recovery process for purposes of purifying the enzyme Centrifugation, filtration, ultrafiltration Concentration Production organism is always completely absent in the final concentrate Formulation to make stable enzyme product
ENZYMES Safety of Strains Safe strains well established over the years Barbesgaard et al., On the safety of Aspergillus oryzae: a review. Appl Microbiol Biotechnol 1992;36:569-72. de Boer & Diderichsen, On the safety of Bacillus subtilis and B. amyloliquefaciens: A review. Appl. Microbiol. Biotechnol. 1991; 36:1-4 Priest et al., On the industrial use of Bacillus licheniformis: a review. Appl. Microbiol. o Biotechnol o 1994; 40: 595-598598 Nevalainen et al., On the safety of Trichoderma reesei. J. Biotechnol. 1994;37:193-200 van Dijck et al., On the safety of a new generation of DSM Aspergillus niger enzyme production strains. Regul. Toxicol. Pharmacol. 2003; 38: 27-35
ENZYMES Safety of concentrates (1) There is rich body of data obtained in 35 years of experience: Includes animal toxicity, ecotox and epidemiological data, e.g. HERA http://www.heraproject.com/riskassessment.cfm?subid=22 http://www.heraproject.com/riskassessment.cfm?subid=38 Most enzymes are Generally Recognized As Safe (GRAS) by food safety experts and FDA GRAS Notice website: http://www.cfsan.fda.gov/~rdb/opa-gras.html; g GRAS affirmed enzymes: 21CFR 184 Zofia S. Olempska-Beer et al., 2006, FDA:: Food-processing enzymes from recombinant microorganisms- a review. Regulatory Toxicology and Pharmacology, Volume 45, Issue 2, July 2006, Pages 144-158158 Pariza & Johnson: Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul Toxicol Pharmacol 2001 Apr;33(2):173-86.
ENZYMES Safety of concentrates (2) General Toxicity N.B. test substance is concentrate incl. residual strain metabolites and fermentation nutrients General conclusions The toxicology of enzyme concentrates is unremarkable Acute and sub-chronic toxicity is not of concern Enzyme concentrates are not reproductive or developmental toxins Enzyme concentrates are not mutagenic, and not clastogenic Only two potential hazards are known As with any protein, enzyme concentrates, when inhaled, have the potential to cause sensitisation and by repeated exposure elicit an allergic response in susceptible individuals Proteases can irritate eye, skin and mucous membranes due to catalytic ti activity it Continued
ENZYMES Safety of concentrates (3) General conclusions (cont d.) Enzyme concentrates have low toxicity to aquatic systems Enzyme concentrates t are denatured, d degraded d d and biodegradable d bl Enzymes are manufactured by living organisms and are consumed by living organisms They denature and biodegrade in the environment The potential for bio-accumulation is virtually nil as proteins are readily metabolized in living i systems
Industry code of practice ENZYMES Safety of products Determine need for tox studies case by case on the basis of: Existing knowledge of application (technical, food, feed) Existing knowledge of enzyme Existing knowledge of strain Existing knowledge of manufacturing process Internationally accepted toxicology package of 90-day oral study in rats (for food enzymes), or in vitro cytotoxicity assay, plus a chromosome aberration study plus an Ames study. EU production approval (EU Contained Use Directive 98/81) EU Feed Additives Regulation 1831/2003 EU Food Enzymes Regulation 1332/2008
Contained Use Regulatory requirements strain requirements Non-Pathogenic host (taxonomic determination required) As safe for man, animal and environment as the host or parental strain Has a proven and extensive history of safe use and/or is biologically contained (limited survival in nature): mutations for sporulation deficiency mutations limiting growth and survival in nature Contains only well-characterized and safe vectors, limited to the required trait: only fully characterized (sequenced and functions known) genetic elements No sequences present not needed in final strain Does not easily transfer DNA to other organisms Deficient in transfer genes (tra- mob-) additional production copies preferably integrated into the chromosome Does not contain antibiotic resistance markers alien to the species
ENZYMES Substance Identification (1) EINECS and ELINCS currently use only CAS numbers Example: Protease CAS 9001-92-792 ; EINECS 232-642-4 4 IUBMB system is only available scientifically sound classification http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html REACH REGULATION (EC) No 1907/2006, Whereas 45:
Guidance for identification and naming of substances under REACH (4.3.2.3, 5 and 7.11). P.41:..
Substance according to REACH Preparation: Granulation materials and enzyme concentrate Other constituents + Active enzyme protein (aep) Substance: Enzyme concentrate (dry matter) Enzyme concentrate The toxicity of the other constituents is the main concern regarding toxicity of enzymes. There are three possible approaches to assess the safety of the other constituents: 1. Composition; 2. Safe strain; 3. Safe strain including composition
Guidance for identification and naming of substances under REACH (4.3.2.3, 5 and 7.11). P.44: P.45: P.49:
ENZYMES Substance Identification (2) IUBMB (EC) Example: Protease 3. 3 Hydrolases 3.4 Acting on peptide bonds (peptidases), with subclasses: EC 3.4.1 a-amino-acyl-peptide Hydrolases (now in EC 3.4.11) EC 3.4.2 Peptidyl-Amino-Acid Hydrolases (now in EC 3.4.17) EC 3.4.3 Dipeptide Hydrolases (now in EC 3.4.13) EC 3.4.4 Peptidyl Peptide Hydrolases (now reclassified within EC 3.4) EC 3.4.11 Aminopeptidases EC 3.4.12 Peptidylamino-Acid Hydrolases or Acylamino-Acid Hydrolases (now reclassified within EC 3.4) EC 3.4.13 Dipeptidases EC 3.4.14 Dipeptidyl-peptidases and tripeptidyl-peptidases EC 3.4.15 Peptidyl-dipeptidases EC 3.4.16 Serine-type carboxypeptidases EC 3.4.17 Metallocarboxypeptidases EC 3.4.18 Cysteine-type carboxypeptidases EC 3.4.19 Omega peptidases EC 3.4.21 Serine endopeptidases EC 3.4.22 Cysteine endopeptidases EC 3.4.23 Aspartic endopeptidases EC 3.4.24 Metalloendopeptidases p EC 3.4.25 Threonine endopeptidases EC 3.4.99 Endopeptidases of unknown catalytic mechanism
ENZYMES Substance Identification (3) Example: Protease (continued) Subclass 3.4.21 Serine endopeptidases contains: EC 3.4.21.1 chymotrypsin EC 3.4.21.2 chymotrypsin C EC 3.4.21.3 metridin EC 3.4.21.4 trypsin EC34215thrombin 3.4.21.5 EC 3.4.21.6 coagulation factor Xa EC 3.4.21.7 plasmin EC 3.4.21.8 now covered by EC 3.4.21.34 and EC 3.4.21.35 EC 3.4.21.9 enteropeptidase EC 3.4.21.10 acrosin EC 3.4.21.11 now covered by EC 3.4.21.36 and EC 3.4.21.37 EC 3.4.21.12 a-lytic endopeptidase etc up to 3.4.21.105 detailed identification according to reaction catalysed
S litti d M i f EINECS t i Splitting and Merging of EINECS entries (REACH Guidance for Data Sharing page 35)
Splitting of EINECS entries (REACH Guidance for Data Sharing page 37)
SIEF formation for a substance which is pre-registered with broad EINECS number Several SIEFs are expected within one EINECS number confirming that registrants agree on first IUB and later sameness (safe strain/composition) iti No IUBMB IUBMB: 3.2.1.1 3.2.1.4 3.2.1.3
Merging of EINECS entries REACH Guidance for Data Sharing page 37
SIEF formation for a substance which is pre-registered under more than one EINECS number Registrants for identical IUB number are spread in several Pre-SIEFs With similar substances, we linked other relevant Pre-SIEFs where we expect to find registrants for the same IUB number SIEF under several EINECS entries should be formed confirming that all registrants in these EINECS numbers agree on first IUB and later sameness (safe strain/composition)
Enzymes are special requires extra steps (splitting and merging of EINECS) for SIEF formation
Substance according to REACH Preparation: Granulation materials and enzyme concentrate Other constituents + Active enzyme protein (aep) Substance: Enzyme concentrate (dry matter) Enzyme concentrate The toxicity of the other constituents is the main concern regarding toxicity of enzymes. There are three possible approaches to assess the safety of the other constituents: 1. Composition; 2. Safe strain; 3. Safe strain including composition
Composition ranges and specific examples Lipids (0-5%) Carbohydrates (3-40%) Inorganic salts (1-45%) Active enzyme protein (10-80%) Other proteins plus peptides and amino acids (5-55%) Composition range for one product might be very broad: Is this acceptable? Should an average range be used for toxicity testing or ranges at each extreme? If safety of the strain is not considered - possibility of toxic metabolites can not be excluded Each product has to be tested even though h the active enzyme protein is the same Tox data requirements: Full REACH requirements for tox data all tests have to be performed Read across not applicable before sameness is established (due to differences in (uncharacterized) production strains, fermentation processes, etc.). Data waiving not applicable due to the fact that their could be e.g. toxic metabolites among the other constituents Not a viable option
Safety assessment of the other constituents based on safe strain lineage principle without composition Black box Composition not known Other constituents can be considered safe Specific enzyme proteins produced by different but all safe production strains can be considered as the same substance* Read across can be applied as sameness is established Data waiving possible since only the enzyme protein has to be considered due to the fact that the other constituents are considered safe * RIP 3.10 p. 49: Enzyme concentrates with the same IUBMB number can be regarded as the same substance, despite using different production organism, provided that the hazardous properties do not differ significantly ifi and warrant the same classification
Safety assessment of the other constituents based on safe strain lineage principle including composition Composition ranges Other constituents including specific examples can be considered d safe Specific enzyme proteins produced by different by all safe production strains can be considered as the same substance Broadness of the composition ranges is not important. Specific examples can be provided as well. Read across can be applied as sameness is established. Data waiving possible since only the enzyme protein has to be considered due to the fact that t the other constituents t are considered d safe.
Parent Strain identification, preferably by independent lab START YES Different strains of this lineage (i.e. repeated assessments) have a history of safe commercial use and products produced by strains within this lineage have been approved or affirmed by authorities on the basis of toxicological tests* for production of enzymes or other biologicals (e.g. GRAS, TSCA Biotech, QPS) NO YES According to literature, strains of this species are not connected with a toxin producing potential of medical importance under the conditions of use for the enzyme product. (Strains with a true mammalian pathogenic potential are not used for enzyme production) NO YES A targeted investigation (e.g. metabolic profiling like Frisvad & Thrane, cytotoxicity) of the strain / strain lineage confirms the absence of toxigenic potential of medical importance YES Strain lineage is considered safe NO Toxin producing potential has been irreversibly removed NO Strain lineage is not established so evaluation is necessary * i.e. at least Ames, chromosomal aberration, 90-day oral toxicology, skin and eye irritation.
Safety assessment when no safe strain lineage can be established The identity of the microorganism should be determined, preferably by an independent laboratory If a literature search shows that the species is not associated with toxin production of concern, and is a species historically used for enzyme production, then the strain can be considered safe. If the species has not been used in industrial enzyme production before, it s possible pathogenic / toxicogenic potential should be considered. For a first time industrial use, the strain should be tested for pathogenic potential, and the concentrate produced by the strain should be tested at least through Ames, chromosomal aberration, and 90-day oral toxicity, plus usually skin and eye irritation for worker safety. Assuming the results from that testing is acceptable, a safety assessment of the enzyme preparation can be done through a paper exercise, comparing the TOS levels from the tox lots used in testing with the new enzyme preparation to do consumption analysis and acceptability. If the literature search showed that the species is associated with toxin production, a test for the toxin under inducing conditions should be done. If the toxin is not found, one can proceed as above. If the toxin is found, and sufficient genetic information is available for the species, one can proceed with the deletion of one or more of the genes involved in the toxin synthesis if the genetical background is known.