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Description of key information

The skin irritation potential of subtilisin has been testedin vitroandin vivoin rabbits and in the case of skin irritation, also in humans:-In vitroEpiDermTM Skin Model (EPI-200), the test compound Subtilisin was predicted as non-corrosive.-In vivoskin irritation studies in rabbits: Two studies conclude Subtilisin to be slightly irritant to skin. Concentration of the enzyme in the formulation applied in the two studies was 4.5% respectively 3.4%, expressed as active enzyme protein.- An occludedin vivohuman skin irritation test with daily 24 hour applications for 10 days and readings every day was performed. No primary skin irritation was seen.The eye irritation potential of subtilisin has been testedin vitroandin vivoin rabbits:-Inin vitroenucleated chicken eye test, the test compound Subtilisin was predicted as non-corrosive.-Inin vivoeye irritation studies in rabbits, Subtilisin was in spite of highly reversible effects concluded to be slightly irritant due to a small haemorrhage on the nictitating membrane. Concentration of solution applied was 5.8% expressed as active enzyme protein.According to REACH, the substance identification of enzymes is based on ”dry matter” of the enzyme concentrate, i.e. the following components: The active enzyme protein, other proteins, peptides and amino acids, carbohydrates, lipids and inorganic salts. However, when the focus is on skin and eye irritation, the active enzyme protein is the important component, characterized by the catalytic activity. The concentration of the solutions applied in the irritation studies is therefore expressed as active enzyme protein, typically as % w/w.The conclusion is that subtilisin due to the catalytic activity has the potential to cause slight skin and eye irritation.  The skin sensitization potential of enzymes has recently been reviewed by Basketter et al. [1, 17] and HERA [2;3] revealing that enzymes should not be considered skin sensitizers. In addition, there is an unequivocal statement from AMFEP (www.amfep.org) on this topic showing that enzymes do not have skin sensitizing potential. The lack of skin sensitizing potential is substantiated by evidence from robust human experimental data and extensive in-use human studies performed with detergents containing enzymes [4-8]. All of these studies confirmed that the presence of enzymes in the detergents did not result in contact skin sensitization, including those conducted with atopic individuals.However, in spite of clear evidence that enzymes should not be considered skin sensitizers, animal skin sensitization models might give rise to positive results. This is because, just like the previously used guinea pig skin sensitization models, the Local Lymph Node Assay (LLNA), (OECD Test Guideline 429) is inappropriate for the assessment of proteins (17). These animal models are validated for the testing of small chemicals, not for water soluble protein-based materials, known to be human respiratory allergens. The LLNA does not discriminate between skin and respiratory sensitizers [9], leading to the risk of false-positive results with proteins, particularly those already known to be sensitizing by the respiratory route, such as enzymes. Indeed, in our experience, all foreign proteins can be made to generate skin reactions in suitably treated animals, including the OECD recognized guinea pig tests and the LLNA [10]. This makes the available animal models inappropriate when used with proteins. Therefore, the assessment of enzymes in any of the existing animal models can be predicted not to provide new and useful knowledge. This conclusion is based on the following considerations:• The results of predictive testing in man demonstrate that enzymes do not have skin sensitization potential for man.• In clinical settings, enzymes have only very rarely been suggested as a possible cause of allergic contact dermatitis (ACD). Even in these few cases, a causal relationship has never been proven. Further, several clinical studies have demonstrated that enzymes are not a cause of ACD [5;8;11-16].• ACD has never been reported in the detergent enzyme industries where there has been extensive occupational enzyme exposure which, in the past, led to respiratory sensitization and/or irritant dermatitis. For more than 40 years, billions of consumers have had regular, often daily, skin exposure to enzymes during laundry by hand but there is no evidence that this exposure has given rise to skin sensitization.• The available skin sensitization test methods are not suitable for enzymes. No animal model has been developed or validated for assessing proteins as contact skin sensitizers. So far, no in vitro models exist either.Since enzyme products are well documented not to be skin sensitizers in man and because no suitable animal model or in vitro assay for protein skin sensitization exists, we consider testing enzymes in animal models developed for chemical contact allergens as both scientifically and ethically unjustified. Finally, the precautions recommended in the material safety data sheets should be sufficient to prevent even a theoretical hazard of skin sensitization.References1) 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, 1177-1182) HERA Human and environmental risk assessment on ingredients of household cleaning products - alpha-amylases, cellulases and lipases. 2005.3) HERA Human and environmental risk assessment on ingredients of household cleaning products - Subtilisins (Proteases). Edition 2.0. 2007.4) Bannan,E.A., Griffith,J.F., Nusair,T.L., and L.J.Sauers (1983) Skin testing of laundered fabrics in the dermal safety assessment of enzyme containing detergents. Journal of Toxicology - Cutaneous and Ocular Toxicology 11, 327-3395) Griffith,J.F., Weaver,J.E., Whitehouse,H.S., Poole,R.L., and Newmann EANixon,G.A. (1969) SAFETY EVALUATION OF ENZYME DETERGENTS ORAL AND CUTANEOUS TOXICITY IRRITANCY AND SKIN SENSITIZATION STUDIES. Food and Cosmetics Toxicology 7, 581-5936) Rodriguez,C., Calvin,G., Lally,C., and LaChapelle,J.M. (1994) Skin effects associated with wearing fabrics washed with commercial laundry detergents. Journal of Toxicology - Cutaneous and Ocular Toxicology 13, 39-457) Cormier,E.M., Sarlo,K., Scott,L.A., MacKenzie,D.P., Payne,N.S., Carr,G.J., Smith,L.A., Cua-Lim,F., Bunag,F.C., and Vasunia,K. (2004) Lack of type 1 sensitization to laundry detergent enzymes among consumers in the Philippines: results of a 2-year study in atopic subjects. Annals of Allergy Asthma and Immunology 92, 549-5578) White,I.R., Lewis,J., and el,A.A. (1985) Possible adverse reactions to an enzyme-containing washing powder. Contact Dermatitis 13, 175-1799) Kimber,I., Agius,R., Basketter,D.A., Corsini,E., Cullinan,P., Dearman,R.J., Gimenez-Arnau,E., Greenwell,L., Hartung,T., Kuper,F., Maestrelli,P., Roggen,E., and Rovida,C. (2007) Chemical respiratory allergy: opportunities for hazard identification and characterisation. The report and recommendations of ECVAM workshop 60. Altern Lab Anim 35, 243-26510) Festersen,U., Rasmussen,C., Kjaer,T.M.R., Soni,N.K., Roggen,E.L., and Berg,N.W. (2008) Alternative application route in the LLNA provides crucial environmental enrichement and broadens the usability of vehicles. AATEX 14, 433-43611) Andersen,P.H., Bindslev-Jensen,C., Mosbech,H., Zachariae,H., and Andersen,K.E. (1998) Skin symptoms in patients with atopic dermatitis using enzyme-containing detergents. A placebo-controlled study. Acta dermato-venereologica 78, 60-6212) Belsito,D.V., Fransway,A.F., Fowler,J.F., Jr., Sherertz,E.F., Maibach,H.I., Mark,J.G., Jr., Mathias,C.G., Rietschel,R.L., Storrs,F.J., and Nethercott,J.R. (2002) Allergic contact dermatitis to detergents: a multicenter study to assess prevalence. Journal of the American Academy of Dermatology 46, 200-20613) Lee,M.Y., Park,K.S., Hayashi,C., Lim,H.H., Lee,K.H., Kwak,I., and Laurie,R.D. (2002) Effects of repeated short-term skin contact with proteolytic enzymes. Contact Dermatitis 46, 75-8014) Pepys,J., Wells,I.D., D'souza,M.F., and Greenberg,M. (1973) CLINICAL AND IMMUNOLOGICAL RESPONSES TO ENZYMES OF BACILLUS-SUBTILIS IN FACTORY WORKERS AND CONSUMERS. Clinical Allergy 3, 143-16015) Peters,G., Johnson,G.Q., and Golembiewski,A. (2001) Safe use of detergent enzymes in the workplace. Appl.Occup Environ.Hyg. 16, 389-3916) Zachariae,H., Thomsen,K., and Rasmussen,O.G. (1973) Occupational enzyme dermatitis. Results of patch testing with Alcalase. Acta dermato-venereologica 53, 145-14817)Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties ans Risk Assessement/Management. 2012.Reg. Toxicol. Pharmacol, 64/1: 117-12318) D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012. J. Immunotox 9(3): 320-6.
Migrated from Short description of key information:
Enzyme products including subtilisins are well documented not to be skin sensitizers in humans. So far, there are no animal models or in vitro assays that have been validated for protein-based test materials like enzymes for this endpoint.

Justification for selection of skin sensitisation endpoint:
Enzyme products including subtilisins are well documented not to be skin sensitizers in humans.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Skin sensitization is referred to as allergic contact dermatitis (ACD) in a clinical setting. This is a cell mediated type IV delayed hypersensitivity. The cellular mechanisms involved with ACD have been reviewed (1, 2). To behave as a skin sensitizer, a substance must first penetrate the stratum corneum, partition into the epidermis and react there with proteins, probably on the surface of the Langerhans cells, to form a hapten-carrier conjugate. The skin sensitization potential of enzymes has been reviewed in several publications indicating that enzymes should not be considered skin sensitizers. (3-7; 13, 14). In addition, there is an unequivocal statement from AMFEP (www.amfep.org) on this topic indicating that enzymes do not have skin sensitizing potential. The lack of skin sensitizing potential is substantiated by evidence from robust human experimental data and extensive in-use human studies performed with detergents containing enzymes (ref. 8-12; 14 -19). Together, these studies confirm that the presence of enzymes in the detergents doesn’t result in ACD, including those conducted with atopic individuals.

After review of the available evidence, it can be concluded that enzymes should not be classified as skin sensitizers according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. This conclusion is based on the following considerations:

1. The results of predictive testing in man demonstrate that enzymes do not have significant skin sensitization potential.

2. In a clinical setting, enzymes have only very rarely been suggested as a possible cause of allergic contact dermatitis. Even in these few cases, a causal relationship has never been proven. More commonly clinical studies have demonstrated that enzymes are not a cause of ACD.

3. ACD has never been reported where there has been extensive occupational enzyme exposure in the detergent enzyme industries which, in the past, has led to respiratory sensitization and/or irritant dermatitis.

4. A few cases of contact dermatitis had occurred in occupational settings in response to irritating enzyme preparations (e.g. proteases), but this is a non-immunologic phenomenon (also known as irritant contact dermatitis) unrelated to allergic contact hypersensitivity, which is a cell mediated delayed type hypersensitivity.

5. Contact urticaria has been reported in occupational settings but this is also a non-immunologic event or antibody mediated type I hypersensitivity; Contact urticaria (also known as protein contact dermatitis) is unrelated to allergic contact hypersensitivity, which is a cell-mediated delayed type hypersensitivity.

6. Over a 45-year period, billions of consumers have had skin exposure to enzymes but there is no evidence that this exposure has given rise to skin sensitization.


References

1. Kimber, I. (1994), Cytokines and regulation of allergic sensitization to chemicals, Toxicology, 93:1-11.

2. Scheper, R.J., and B. M.E. von Blomberg (1992), Cellular mechanisms in allergic contact dermatitis, in Textbook of Contact Dermatitis, R. J.G. Rycroft, T. Menne, P.J. Frosch, and C. Benezra, Eds., Springer-Verlag, Berlin, p. 11-27.

3. Association Internationale de la Savonnerie et de la Detergence (AISE)/AMFEP, Enzymes: Lack of skin sensitisation potential. 1995.

4. Basketter DA, English JS, Wakelin SH, White IR. (2008) Enzymes, detergents and skin: facts and fantasies. Br.J.Dermatol. 158(6):1177-81.
5. Basketter, N. Berg, C. Broekhuizen, M. Fieldsend, S. Kirkwood, C. Kluin, S. Mathieu, C.Rodriguez. (2012). Enzymes in cleaning products: An overview of toxicological properties and risk assessment/management. Regulatory Toxicology and Pharmacology 64:117-123.

6. HERA Human and environmental risk assessment on ingredients of household cleaning products -alpha-amylases, cellulases and lipases. 2005.

7. HERA Human and environmental risk assessment on ingredients of household cleaning products - Subtilisins (Proteases). Edition 2.0. 2007.

8. Bannan,E.A., Griffith,J.F., Nusair,T.L., and L.J.Sauers (1983) Skin testing of laundered fabrics in the dermal safety assessment of enzyme containing detergents. Journal of Toxicology - Cutaneous and Ocular Toxicology 11, 327-339.

9. Griffith,J.F., Weaver,J.E., Whitehouse,H.S., Poole,R.L., and Newmann EANixon,G.A. (1969) Safety Evaluation of Enzyme Detergents Oral and Cutaneous Toxicity, Irritancy and Skin Sensitization Studies. Food and Cosmetics Toxicology 7, 581-593.

10. Rodriguez,C., Calvin,G., Lally,C., and LaChapelle,J.M. (1994) Skin effects associated with wearing fabrics washed with commercial laundry detergents. Journal of Toxicology - Cutaneous and Ocular Toxicology 13, 39-45.

11. Cormier,E.M., Sarlo,K., Scott,L.A., MacKenzie,D.P., Payne,N.S., Carr,G.J., Smith,L.A., Cua-Lim,F., Bunag,F.C., and Vasunia,K. (2004) Lack of type 1 sensitization to laundry detergent enzymes among consumers in the Philippines: results of a 2-year study in atopic subjects. Annals of Allergy Asthma and Immunology 92, 549-557.

12. White,I.R., Lewis,J., and el,A.A. (1985) Possible adverse reactions to an enzyme-containing washing powder. Contact Dermatitis 13, 175-179.

13. Basketter D.; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012b. J. Immunotox., 9, 320-326.

14. Andersen,P.H., Bindslev-Jensen,C., Mosbech,H., Zachariae,H., and Andersen,K.E. (1998) Skin symptoms in patients with atopic dermatitis using enzyme-containing detergents. A placebo-controlled study. Acta dermato-venereologica 78, 60-62.

15. Belsito,D.V., Fransway,A.F., Fowler,J.F., Jr., Sherertz,E.F., Maibach,H.I., Mark,J.G., Jr., Mathias,C.G., Rietschel,R.L., Storrs,F.J., and Nethercott,J.R. (2002) Allergic contact dermatitis to detergents: a multicenter study to assess prevalence. Journal of the American Academy of Dermatology 46, 200-206.

16. Lee,M.Y., Park,K.S., Hayashi,C., Lim,H.H., Lee,K.H., Kwak,I., and Laurie,R.D. (2002) Effects of repeated short-term skin contact with proteolytic enzymes. Contact Dermatitis 46, 75-80.

17. Pepys,J., Wells,I.D., D'souza,M.F., and Greenberg,M. (1973) Clinical and Immunological Responses to Enzymes of Bacillus Subtilis in Factory Workers and Consumers. Clinical Allergy 3, 143-160.

18. Peters,G., Johnson,G.Q., and Golembiewski,A. (2001) Safe use of detergent enzymes in the workplace. Appl.Occup Environ.Hyg. 16, 389-395.

19. Zachariae,H., Thomsen,K., and Rasmussen,O.G. (1973) Occupational enzyme dermatitis. Results of patch testing with Alcalase. Acta dermato-venereologica 53, 145-148
Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory sensitisation

Link to relevant study records
Reference
Endpoint:
respiratory sensitisation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

For enzyme protein respiratory allergens, a DMEL for workers and consumers has been summarized and discussed in a recent publication (Ref. 1). The conclusion is drawn from a thorough review of existing occupational and consumer data on exposure by inhalation from the involved industrial partners in combination with medical data. As no valid animal models exist to test and rank respiratory sensitizers, the human surveillance data are the core of such evaluation. Any sub-categorization based on relative potency is not feasible (Ref. 2).

REFERENCES

1.Basketter DA, Broekhuizen C, Fieldsend M, Kirkwood S, Mascarenhas R, Maurer K, Pedersen C, Rodriguez C, Schiff HE: Defining occupational and consumer exposure limits for enzyme protein respiratory allergens under REACH. Toxicology. 268:165-170, 2010.

2. Basketter D.A., Kimber I. (2011) Assessing the potency of respiratory allergens: Uncertainties and challenges. Regul. Toxicol. Pharmacol., 61, 365-372.


Migrated from Short description of key information:
From occupational data it is well known that active enzymes including subtilisins are potential respiratory sensitizers. However, decades of experience has shown that enzymes can be used safely by ensuring that exposure is limited, supported by the DMEL for workers and consumers.

Justification for selection of respiratory sensitisation endpoint:
From occupational data it is well known that active enzymes regardless of the catalytic activities are potential respiratory sensitizers. All enzymes must therefore be classified as respiratory sensitizers, “H334: Hazard Category 1: May cause allergy or asthma symptoms or breathing difficulties if inhaled” in accordance with the CLP Regulation.

Justification for classification or non-classification

Subtilisin should not be classified as a skin sensitiser.

Subtilisin is classified as a respiratory sensitiser.