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EC number: 232-752-2 | CAS number: 9014-01-1
- Life Cycle description
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- Endpoint summary
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
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- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
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- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data

Endpoint summary
Administrative data
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
- 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
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Respiratory sensitisation
Link to relevant study records
- Endpoint:
- respiratory sensitisation
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
Reference
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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