Safety Evaluation of Technical Enzyme Products with Regards to the REACH Legislation

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

PPdroduce d an d nee ddbllliided by all living 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

Enzyygyme mainly through fermentation of microor g(ganisms (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 CtidUliltiContained Use legislation requ ires sa fe s ti(fGMM)trains (for GMMs) Subsequent recovery process for purposes of purifying the enzyme Centrifugg,ation, 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. Mi cr obi ol. Bi otechn ol 1 994 ; 4 0: 595-598 Nevalainen et al., On the safety of Trichoderma reesei. J. Biotechnol. 1994;37:193-200 van Dijck et al ., O n th e saf ety of a new generati on of DSM A spergill us 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; GRAS affirmed enzymes: 21CFR § 184 Zofia S. Olempska-Beer et al., 2006, FDA:: Food-processing enzymes from recombinant microorganisms- a review. Regulatory Toxicology and Pharmaco logy, Vo lume 45, Issue 2, Ju ly 2006, Pages 144-158 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 concl u sions 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 catal yti c ac tiv ity

Continued Î ENZYMES – Safety of concentrates (3)

General conclusions (cont’d.) Enzyme concentrates have low toxicity to aquatic systems Enzyme concen ttdtdddddbiddbltrates are denatured, degraded and biodegradable Enzymes are manufactured by living organisms and are consumed byygg living organisms They denature and biodegrade in the environment The potential for bio-accumulation is virtually nil as proteins are readily me ta bo lize d in liv ing sys tems ENZYMES – Safety of products

Industry code of practice 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 f ood enzymes) , or in vitro cytotoxicity assay, plus a chromosome aberration study plus an Ames study. EU pppp(roduction 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: onlfllhly fully character ize d(d (sequence d an dfd funct ions known ) genet ic e lements 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 curren tly use on ly CAS num bers Example: Protease CAS 9001 -92-7 ; EINECS 232-642-4

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 Substance: + Enzyme concentrate Enzyme(dry matter) concentrate Active enzyme protein (aep)

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 – SbtSubstance IdtifitiIdentification (2)

IUBMB (EC) Example: Protease 3Hd3. Hydro lases 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 Metalloendopppeptidases 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 EC34215thrombinEC 3.4.21.5 thrombin 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

Î detailed identification according to reaction catalysed Sp litting and M ergi ng of EINECS ent ri es (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 an d la ter sameness (sa fe s tra in /compos ition)

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/compp)osition) 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 Substance: + Enzyme concentrate Enzyme(dry matter) concentrate Active enzyme protein (aep)

If safety of the strain is not considered -

Inorganic salts Active enzyme possibility of toxic metabolites can not (1-45%) protein (10- be excluded 80%) Each product has to be tested even Lipids (0-5%) though the ac tive enzyme pro te in is the same Tox data requirements: Full REACH Other proteins Carbohydrates plus peptides requirements for tox data – all tests have (3- 40%) andid amino to be performed acids (5-55%) Read across not applicable before Composition range for one sameness is established (due to differences in (uncharacterized) product might be very broad: production strains, fermentation Is this acceptable? processes, etc.). Should an average range be Data waiving not applicable due to the used for toxicity testing or fact that their could be e.g. toxic metabolites among the other ranges at each extreme? constituents

Not a viable option… Safety assessment of the other constituents based on safe strain lineage principle without composition

‘Black box´ Other constituents Composition not known 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 sign ifican tly and warrant th e same cl assifi ca tion Safety assessment of the other constituents based on safe strain lineage principle including composition

Composition ranges Other constituents including specific examplbiddfles can be considered 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 tha t the o ther cons tituent s are consid ered sa fe. Parent Strain identification, ppyypreferably by independent lab

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Different strains of this lineage (i .e . repeated assessments) hav e a history of YES 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)

According to literature, strains of this species are not connected with a toxin YES 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)

YES A targeted investigation (e .g . metabolic metabolic profiling like Frisvad & Thrane, cytotoxicity) of the strain / strain lineage confirms the absence of toxigenic potential of medical importance

NO YES Toxin producing potential has been irreversibly removed NO

Strain lineage is not Strain lineage is estblihdtablished so considered safe 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 indu strial u se, the strain shou ld 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.