Subtilisin

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

dermal absorption, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Description of key information

Toxicokinetic studies performed on enzymes are very limited, but toxicokinetic information can be derived from the structure of enzymes combined with knowledge available for proteins in general since enzymes are proteins with catalytic activity.

Absorption

Skin:

The physico-chemical properties of a compound are decisive for the potential percutaneous penetration, in particular factors like ionization, molecular size and lipophilicity. In general, non-ionized molecules easily penetrate the skin, with small molecules penetrating more easily than large molecules. Lipophilicity also facilitates penetration. Investigations of percutaneous absorption of peptides, proteins and other molecules of large size revealed that percutaneous absorption of proteins is extremely low and of no toxicological relevance [1;2].

Gastrointestinal tract:

Proteins are digested into amino acids by pancreatic proteolytic enzymes in the lumen of the gastrointestinal tract [3]. As enzymes are simply a class of proteins, it can be expected that enzymes will undergo the same process, simply representing a tiny food source.

Furthermore, enzymes have been used for decades in treatment of both adults and children with exocrine pancreatic insufficiency. Typical enzymatic drugs (e.g. Creon® from Solvay Pharmaceuticals or Pancrease Microtabs from Jansson/Cilaq) are a combination of the enzymes amylase, lipase and protease – enzymes, which are also used in a wide range of industrial applications. These drugs are typically administered orally at therapeutic concentrations i.e. concentrations where a digestive effect can be expected. Clinical trials and crossover studies confirmed the safe use of these compounds in patients, both adults and children, confirming the low toxicity of enzymes [4-15].

Additionally, in a study investigating the gastrointestinal absorption of enzymes in pigs with pancreatic insufficiency and treated with Creon®, analysis of plasma samples taken in the period of 0.5 to 48 hours after oral administration of the drug, did not result in treatment related changes of plasma enzyme levels indicating no gastric absorption of the administered enzymes [16].

Furthermore, a variety of enzymes are added to animal feed with the purpose to increase nutrient digestibility in the gastrointestinal tract. The safety of such enzymes active in the gastrointestinal tract are thoroughly evaluated as part of their approval process in the EU and elsewhere.

In conclusion, from the available data combined with the knowledge of the fate of proteins in the gastrointestinal system, it can be concluded that absorption of enzymes in toxicological significant amounts through the gastrointestinal tract is unlikely.

Respiratory tract:

Enzymes can be inhaled in the form of small dust particles or aerosols i.e. adhered to solid dust particles or as droplets of fluid. Absorption of hydrophilic substances such as enzymes by lung tissue is determined by diffusion and depends on molecular size. The transport channels in the alveolar membrane have a size of 1 nm (10Å) [3], which excludes the absorption of enzymes, since their size is above 1 nm. Removal of deposits depends on the site of deposition. In the alveoli where the main removal is via phagocytosis [3], the macrophages carrying the deposits can move to the interstitium, the ciliated epithelium or the lymphatic system indicating that there could be a risk of systemic exposure to enzymes by this route. However, due to the fact that enzymes are potential respiratory allergens, stringent risk management strategies have been introduced for the working environment leading to very low pulmonary exposure excluding any chance of toxicologically significant absorption. Consumer exposure is even lower. Furthermore, no bioaccumulation will occur after absorption due to rapid biological degradation and enzymes hydrophilic nature.

In conclusion, absorption of enzymes by the respiratory tract can be considered insignificant.

Bioavailability:

Due to the combined information that skin absorption of enzymes is at a toxicologically insignificant level, that enzymes are degraded in the gastrointestinal tract and that they are only absorbed to a very low extent by the respiratory tract, the total bioavailability of enzymes can be concluded to be extremely low. This is further supported by the physico-chemical data, i.e. enzymes have a low logKow value, indicating that they have no bioaccumulation potential and can be anticipated to be readily biodegraded. Thus, systemic exposure following enzyme exposure at occupational and consumer exposure levels is without toxicological significance.

References:

1) Basketter,D.A., English,J.S., Wakelin,S.H., and White,I.R. (2008) Enzymes, detergents and skin: facts and fantasies. British journal of dermatology 158, 1177-1181

2) Pease,C.K.S., White,I.R., and Basketter,D.A. (2002) Skin as a route of exposure to protein allergens. Clinical and experimental dermatology 27, 296-300

3) Niesink,R.J.M., deVries,J., and Hollinger,M.A. (1996) Toxicology, Principles and Applications CRC Press, Inc. and Open University of The Netherlands.

4) Barra,E., Stolarczyk,A., Socha,J., Oralewska,B., Kowalska,M., Skoczen,M., and Wawer,Z. (1998) Efficacy of enzyme supplementation in children with cystic fibrosis. Pediatria Polska 73, 177-182

5) Borowitz,D., Goss,C.H., Stevens,C., Hayes,D., Newman,L., O'Rourke,A., Konstan,M.W., Wagener,J., Moss,R., Hendeles,L., Orenstein,D., Ahrens,R., Oermann,C.M., Aitken,M.L., Mahl,T.C., Young,K.R., Dunitz,J., and Murray,F.T. (2006) Safety and preliminary clinical activity of a novel pancreatic enzyme preparation in pancreatic insufficient cystic fibrosis patients. Pancreas 32, 258-263

6) Borowitz,D., Goss,C.H., Limauro,S., Konstan,M.W., Blake,K., Casey,S., Quittner,A.L., and Murray,F.T. (2006) Study of a novel pancreatic enzyme replacement therapy in pancreatic insufficient subjects with cystic fibrosis. Journal of Pediatrics 149, 658-662

7) Dominguez,M., Iglesias-Garcia,J., Iglesias-Rey,M., Figueiras,A., and Vilarino-Insua,M. (2005) Effect of the administration schedule on the therapeutic efficacy of oral pancreatic enzyme supplements in patients with exocrine pancreatic insufficiency: A randomized, three- way crossover study. Alimentary Pharmacology and Therapeutics 21, 993-1000

8) Halm,U., Löser,C., Löhr,M., Katschinski,M., and Mössner,J. (1999) A double-blind, randomized, multicentre, crossover study to prove equivalence of pancreatin minimicrospheres versus microspheres in exocrine pancreatic insufficiency. Alimentary pharmacology & therapeutics 13, 951-957

9) Heubi,J.E., Boas,S.R., Blake,K., Nasr,A.R.H., Woo,M.S., Graff,G.R., Hardy,K.A., maro-Galvez,R., Latino,M., and Lee,C. (2008) Zentase, a novel pancreatic enzyme product (Pep), is effective in mild, moderate, and severe exocrine pancreatic insufficiency (Epi). Gastroenterology 134, A583-A584

10) Keller,J. and Layer,P. (2006) Are monolithic enteric-coated enzyme preparations effective in pancreatic exocrine insufficiency? A multicentre, double blind, placebo controlled cross-over trial. Gastroenterology 130, A517

11) Konstan,M.W., Stern,R.C., Trout,J.R., Sherman,J.M., Eigen,H., Wagener,J.S., Duggan,C., Wohl,M.E.B., and Colin,P. (2004) Ultrase MT12 and ultrase MT20 in the treatment of exocrine pancreatic insufficiency in cystic fibrosis: Safety and efficacy. Alimentary Pharmacology and Therapeutics 20, 1365-1371

12) Konstan,M.W., Liou,T.G., Strausbaugh,S., Ahrens,R.C., Kanga,J.F., Graff,G.R., Moffett,K.S., Millard,S., Nasr,S.Z., Vezina,M., Spenard,J., and Grondin,J. (2008) Efficacy and safety of Ultrase (R) MT20 in treating pancreatic insufficiency in cystic fibrosis. Gastroenterology 134, A228-A229

13) Laake,K. (1980) ENZYMIC DRUGS. Side Effects of Drugs Annual 222-225

14) Patchell,C.J., Desai,M., Weller,P.H., Macdonald,A., Smyth,R.L., Bush,A., Gilbody,J.S., and Duff,S.A. (2002) Creon 10,000 Minimicrospheres vs. Creon 8,000 microspheres--an open randomised crossover preference study. Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 1, 287-291

15) Saeed,Z., Wojewodka,G., Marion,D., Guilbault,C., and Radzioch,D. (2007) Novel pharmaceutical approaches for treating patients with cystic fibrosis. Current Pharmaceutical Design 13, 3252-3263

16) Gewert,K., Holowachuk,S.A., Rippe,C., Gregory,P.C., Erlanson-Albertsson,C., Olivecrona,G., Kruszewska,D., Piedra,J.V., Weström,B., and Pierzynowski,S.G. (2004) The enzyme levels in blood are not affected by oral administration of a pancreatic enzyme preparation (Creon 10,000) in pancreas-insufficient pigs. Pancreas 28, 80-88

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential