Registration Dossier

Administrative data

Workers - Hazard via inhalation route

Systemic effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Most sensitive endpoint:
sensitisation (respiratory tract)
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
Most sensitive endpoint:
sensitisation (respiratory tract)
DNEL related information

Local effects

Long term exposure
Hazard assessment conclusion:
DMEL (Derived Minimum Effect Level)
Value:
60 ng/m³
Most sensitive endpoint:
sensitisation (respiratory tract)
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
Most sensitive endpoint:
sensitisation (respiratory tract)
DNEL related information

Workers - Hazard via dermal route

Systemic effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
DNEL related information

Local effects

Long term exposure
Hazard assessment conclusion:
low hazard (no threshold derived)
Most sensitive endpoint:
skin irritation/corrosion
Acute/short term exposure
Hazard assessment conclusion:
low hazard (no threshold derived)
Most sensitive endpoint:
skin irritation/corrosion

Workers - Hazard for the eyes

Local effects

Hazard assessment conclusion:
low hazard (no threshold derived)

Additional information - workers

Worker DMEL, acute short-term as well as long-term inhalation exposure:

Acute inhalation LC50 values for subtilisins are typically in the range of 0.5 - 1g powder enzyme as is/m3, corresponding to 0.1 - 0.2 mg active enzyme protein/L (ref. 1). In the few cases where LC50 values could be established, the values were approx. a factor of 106above the actual occupational exposure values, indicating that concentrations used in acute inhalation toxicity studies are irrelevant to all known exposure scenarios. Potential occupational exposure to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and adherence to the voluntary Occupational Exposure Guidelines at or below the established ACGIH threshold limit value (ACGIH, the American Conference of Governmental Industrial Hygienists threshold limit value of 60 ng/m3for the benchmark enzyme Subtilisin).

Acute inhalation toxicity studies are designed to test for lethality, which is not relevant for enzymes. The real endpoint of concern, allergenicity, cannot be evaluated in this type of studies. Subtilisin preparations, depending on atmospheric concentration, may be irritating to the respiratory tract, but due to the risk of sensitization by inhalation, these enzyme preparations are now specifically formulated to avoid this route of exposure. Worker safety is further assured through current proper work practices, engineering controls, and use of personal protective equipment. Natural protease inhibitors in the blood, e.g. Alpha-1-anti trypsin and alpha-2-macroglobulin, would further inhibit the activity, should a minor quantity of Subtilisin cross the lung epithelium and get access to the systemic circulation. For many years, the involved industry has taken measures to minimize occupational exposure. Subtilisin preparations, depending on atmospheric concentration, may be irritating to the respiratory tract, but due to the risk of sensitization, these enzyme preparations are now specifically formulated to avoid exposure by inhalation.Worker DMEL has been discussed and concluded by the involved industry (enzyme and detergent manufacturers) in recent publications (ref. 2-4) and a limit of 60 ng/m3, expressed in pure enzyme protein, was suggested (ref. 2) in line with the established ACGIH threshold limit value.

References:

1.HERA, Human and environmental risk assessment on ingredients of household cleaning products - Subtilisins (Proteases), ed 2.0, 2007, www.heraproject.com - Risk Assessments

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

3. 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 and Risk Assessment/Management. 2012a. Reg. Toxicol. Pharmacol, 64/1: 117-123

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

 

Worker DNEL, acute short-term as well as long-term dermal exposure:

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 (ref. 1 – 4).This is further supported by the physico-chemical data of subtilisin. This group of enzymes are proteins with molecular weight above 16,900 D (ref. http://www.brenda-enzymes.info; ec no=3.4.21.62), they have a low logPow value (<0), indicating that they have no bioaccumulation potential and can be anticipated to be readily biodegradable.Thus, systemic exposure following enzyme exposure at occupational exposure levels is without toxicological significance.

The exposure given as % subtilisin active enzyme protein (aep) in for example a wash solution to which skin is tolerant was derived based on scientific judgment of the existing dermal safety studies, including human studies (ref. 5). This exposure is judged to be 0.2% aep subtilisin under normal work exposure conditions, where the contact with skin is confined to a fraction of the hands (palms and/or fingers) and of short duration (typically a few minutes).

 

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. 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 and Risk Assessment/Management. 2012a. Reg. Toxicol. Pharmacol, 64/1: 117-123

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

5.HERA, Human and environmental risk assessment on ingredients of household cleaning products - Subtilisins (Proteases), ed. 2.0, 2007, www.heraproject.com - Risk Assessments

General Population - Hazard via inhalation route

Systemic effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Most sensitive endpoint:
sensitisation (respiratory tract)
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
DNEL related information

Local effects

Long term exposure
Hazard assessment conclusion:
DMEL (Derived Minimum Effect Level)
Value:
15 ng/m³
Most sensitive endpoint:
sensitisation (respiratory tract)
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
DNEL related information

General Population - Hazard via dermal route

Systemic effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
DNEL related information

Local effects

Long term exposure
Hazard assessment conclusion:
low hazard (no threshold derived)
Most sensitive endpoint:
skin irritation/corrosion
Acute/short term exposure
Hazard assessment conclusion:
low hazard (no threshold derived)
Most sensitive endpoint:
skin irritation/corrosion

General Population - Hazard via oral route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
1.8 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
200
Modified dose descriptor starting point:
NOAEL
Value:
360 mg/kg bw/day
Explanation for the modification of the dose descriptor starting point:
no extrapolation needed
AF for dose response relationship:
1
AF for differences in duration of exposure:
2
Justification:
DNEL based on 90-day study
AF for interspecies differences (allometric scaling):
4
Justification:
study in rats
AF for other interspecies differences:
2.5
AF for intraspecies differences:
10
AF for the quality of the whole database:
1
AF for remaining uncertainties:
1
Acute/short term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
3.6 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
100
Modified dose descriptor starting point:
NOAEL
Value:
360 mg/kg bw/day
Explanation for the modification of the dose descriptor starting point:
no extrapolation needed
AF for dose response relationship:
1
AF for interspecies differences (allometric scaling):
4
Justification:
study in rats
AF for other interspecies differences:
2.5
AF for intraspecies differences:
10
AF for the quality of the whole database:
1
AF for remaining uncertainties:
1

General Population - Hazard for the eyes

Local effects

Hazard assessment conclusion:
low hazard (no threshold derived)

Additional information - General Population

Consumer DMEL, acute short-term as well as long-term inhalation exposure:

Industry has documented that respiratory irritation or toxicity due to enzyme preparations is a very rare phenomenon which will not occur at the low concentrations of enzymes found in consumer products as for example detergents. The risk to consumers is considered very low and regarded as toxicologically insignificant (ref. 3, 4, 5). This is supported by the positive safety outcome of a clinical study of the highest reported consumer exposure level, 15 ng/m3, with spot cleaning by spray (ref. 1, 2). Consumer DMEL has been discussed among the enzyme allergy specialists from enzyme and detergent manufacturers and it was concluded by the involved industry partners in a recent publication and the limit of 15 ng/m3was suggested (ref. 3). With a LC-50 value of 0.1 g active enzyme protein/m3, the actual exposure is more than a factor of 106less than this LC50 value.

References:

1. Weeks, J.A., Harper, R.A., Simon, R.A., Burdick, J.D., 2011. Assessment of sensitization risk of a laundry pre-spotter containing protease. Cutan. Ocul. Toxicol., 30, 272-279.

2. US SDA.Risk assessment guidance for enzyme-containing products.  2005. Washington, Soap and Detergent Association.

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

4. 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 and Risk Assessment/Management. 2012. Reg. Toxicol. Pharmacol, 64/1: 117-123.

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

 

Consumer DNEL, acute short-term as well as long-term dermal exposure:

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 (ref. 1-4).This is further supported by the physico-chemical data of subtilisin. This group of enzymes are proteins with molecular weight above 16,900 D (ref. http://www.brenda-enzymes.info; ec no=3.4.21.62), they have a low logPow value (<0i.e. low lipophilicity), indicating that they have no bioaccumulation potential and can be anticipated to be readily biodegradable.Thus, systemic exposure following enzyme exposure at occupational exposure levels is without toxicological significance.

Under normal exposure conditions, where the contact with skin is confined to a fraction of the hands (palms and/or fingers) and of short duration (typically a few minutes), 0.2% aep subtilisin in aqueous solution is regarded as non-irritant to the skin. In its concentrated formulated form, the irritation potential of Subtilisin is inhibited due to the low water activity and protease inhibitors incorporated into the formulation to achieve the storage stability.

 

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. 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 and Risk Assessment/Management. 2012. Reg. Toxicol. Pharmacol, 64/1: 117-123

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

 

Consumer DNEL, acute short-term as well as long-term systemic oral exposure:

Proteins are digested into amino acids by gastric juices, digestive enzymes and pancreatic proteolytic enzymes in the lumen of the gastrointestinal tract. As enzymes are simply a class of proteins, enzymes will undergo the same process as any food source based on proteins. Absorption of enzymes in toxicological significant amounts through the gastrointestinal tract is unlikely. 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) contain a combination of the enzymes amylase, lipase and protease – enzymes, which are also used in a wide range of industrial applications. These medical drugs are typically administered orally at therapeutic concentrations i.e. at concentrations where a digestive effect can be expected. Clinical trials and crossover studies confirm the safe use of these compounds in patients, both adults and children, confirming the low toxicity of the enzymes.

For references, please see section 7.1 on Toxicokinetics, metabolism and distribution.