GHS Classification Result (Revision of the past classification result)

Chemical Name:Subtilisins
CAS:9014-01-1,1395-21-7

Result:
ID: 21B3032
Classifier: Ministry of Health, Labour and Welfare (MHLW), Ministry of the Environment (MOE)
Year Classified: FY2009
Reference Manual: GHS Classification Guidance by the Japanese Government (March, 2009)

PHYSICAL HAZARDS
Hazard class Classification Symbol Signal word Hazard statement Precautionary statement Rationale for the classification
1 Explosives Not applicable - - - - The substance does not contain chemical groups associated with explosive properties present in the molecules since it is enzyme protein composed of amino acids linked by peptide bond.
2 Flammable gases (including chemically unstable gases) Not applicable - - - - Solid (room temperature)
3 Aerosols Not applicable - - - - Not aerosol products
4 Oxidizing gases Not applicable - - - - Solid (room temperature)
5 Gases under pressure Not applicable - - - - Solid (room temperature)
6 Flammable liquids Not applicable - - - - Solid (room temperature)
7 Flammable solids Classification not possible - - - - No data available.
8 Self-reactive substances and mixtures Not applicable - - - - The substance does not contain chemical groups associated with explosive or self-reactive properties present in the molecules since it is enzyme protein composed of amino acids linked by peptide bond.
9 Pyrophoric liquids Not applicable - - - - Solid (room temperature)
10 Pyrophoric solids Classification not possible - - - - No data available.
11 Self-heating substances and mixtures Classification not possible - - - - No data available.
12 Substances and mixtures which, in contact with water, emit flammable gases Not applicable - - - - The substance is enzyme protein composed of amino acid peptide bond and the chemical structure of the substance does not contain metals or metalloids (B, Si, P, Ge, As, Se, Sn, Sb, Te, Bi, Po, At).
13 Oxidizing liquids Not applicable - - - - Solid (room temperature)
14 Oxidizing solids Classification not possible - - - - No data available.
15 Organic peroxides Not applicable - - - - The substance is enzyme protein composed of amino acid peptide bond and is organic compounds containing no bivalent -O-O- structure in the molecules.
16 Corrosive to metals Classification not possible - - - - Test methods applicable to solid substances are not available.

HEALTH HAZARDS
Hazard class Classification Symbol Signal word Hazard statement Precautionary statement Rationale for the classification
1 Acute toxicity (Oral) Category 4 Warning H302: Harmful if swallowed P301+P312: IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell.
P264: Wash ... thoroughly after handling.
P270: Do not eat, drink or smoke when using this product.
P330: Rinse mouth.
P501: Dispose of contents/container to ...
Based on LD50 values of 370 mg aep/kg, 510 mg aep/kg and 830 mg aep/kg (aep: abbreviation of active enzyme protein, which means a content of theoretically active pure enzyme protein) for rats (HERA (2007): Human & Environmental Risk Assessment on ingredients of household cleaning products; Edition 2.0 February 2007), the substance was classified into Category 4. [Subtilisin is a globular protein with a molecular weight of approximately 27 kDa. Since its primary structure (particularly the central part) consists of serine protease with a high homology of 66 - 99%, data of subtilisin enzymes were used for classification.]
1 Acute toxicity (Dermal) Classification not possible - - - - No data available. As relevant information, subtilisin enzymes are considered not to induce acute systemic toxicity via dermal exposure, since its relatively large molecular weight makes it unlikely to penetrate intact skin (HERA (2007)).
1 Acute toxicity (Inhalation: Gases) Not applicable - - - - Solid (room temperature)
1 Acute toxicity (Inhalation: Vapours) Classification not possible - - - - No data available.
1 Acute toxicity (Inhalation: Dusts and mists) Category 1 Danger H330: Fatal if inhaled P304+P340: IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
P403+P233: Store in a well-ventilated place. Keep container tightly closed.
P260: Do not breathe dust/fume/gas/mist/vapours/spray.
P271: Use only outdoors or in a well-ventilated area.
P284: Wear respiratory protection.
P310: Immediately call a POISON CENTER or doctor/physician.
P320: Specific treatment is urgent (see ... on this label).
P405: Store locked up.
P501: Dispose of contents/container to ...
Based on LC50 values of 0.0177 mg aep/L and 0.1 - 0.4 mg aep/L for rats (HERA (2007)), the category with higher hazard was adopted and the substance was classified into Category 1. Data of subtilisin enzymes were used for classification. Since there was a report of "inhalation of proteolytic enzyme powder", the classification criteria for dust was adopted.
2 Skin corrosion/irritation Not classified - - - - In rabbit Draize tests, the PIIs (primary irritation indices) were 1.1, 1.3, 1.7 and 2 (HERA (2007)). Based on the results of mild or slightly irritating (PIIs: <2.3), the substance was classified as "Not classified" in JIS Classification (correspond to Category 3 in GHS classification). In a test of Savinase, a subtilisin enzyme, although it was reported that the PII was 2.6 (HERA (2007)), this result was not used as the basis of classification since the PII was considered not to be significantly different from that in the vehicle control (PII: 2.9).
3 Serious eye damage/eye irritation Category 2B - Warning H320: Causes eye irritation P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
P337+P313: If eye irritation persists: Get medical advice/attention.
P264: Wash ... thoroughly after handling.
Based on a report that in a rabbit modified Draize test, conjunctivae redness with chemosis occurred and reached a peak at 24-hour after application with the signs not disappearing until 7 days after application (HERA (2007)), the substance was classified into Category 2B.
4 Respiratory sensitization Category 1 Danger H334: May cause allergy or asthma symptoms or breathing difficulties if inhaled P304+P341: IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing.
P342+P311: If experiencing respiratory symptoms: Call a POISON CENTER or doctor/physician.
P261: Avoid breathing dust/fume/gas/mist/vapours/spray.
P285: In case of inadequate ventilation wear respiratory protection.
P501: Dispose of contents/container to ...
There is a report that the subtilisins caused bronchoconstriction and respiratory allergies (ACGIH (2001)). Additionally, it is well known that the key hazard associated with subtilisins is respiratory (Type 1) allergy (HERA (2007)). The substance was classified into Category 1.
4 Skin sensitization Classification not possible - - - - Negative results were obtained from guinea pig skin sensitizing tests (maximization test and Buehler test) (Evaluation of effect for the food safety (Food Safety Commission) (2006), HERA (2007)). In extensive patch testing in human volunteers, exposed workers or users showed no evidence for the ability of subtilisin to induce skin sensitization (HERA (2007)). Overall, the weight of human evidence indicates that subtilisins should not be regarded as skin sensitizers (HERA (2007)). In a survey of 121 workers exposed to subtilisin dusts, skin patch tests showed that sensitization was higher among atopic individuals (64%, 16/25) than among worker considered normally sensitive (33%, 32/96) (ACGIH (2001)). There is a report that although three of the patients who engaged in the preparation of the enzyme and had respiratory illness showed positive skin test (ACGIH (2001)), the details are not clear. Since the substance is not classified as SEN by ACGIH, it cannot be classified based on the results of human patch tests only. It is questionable to classify this substance as "Not classified" based on the above negative results in animal tests (Evaluation of effect for the food safety (Food Safety Commission) (2006), HERA (2007)), therefore, classification was not possible.
5 Germ cell mutagenicity Not classified - - - - Based on negative results in a mouse dominant lethal test by oral administration (in vivo heritable germ cell mutagenicity test) (HERA (2007)) and a Chinese hamster bone marrow chromosomal aberration test by oral administration (OECD TG475) (in vivo somatic cell mutagenicity test) (HERA (2007)), the substance was classified as "Not classified". As relevant information, from in vitro mutagenicity tests, there are reports of negative Ames and chromosomal aberration tests using human peripheral lymphocyte and V79 cells (HERA (2007)).
6 Carcinogenicity Classification not possible - - - - No data available. There is a report that carcinogenicity is not expected for enzyme preparations in general based on the following reasons: there is no indication in the published literature that detergent enzymes possess carcinogenic properties; it was demonstrated that the systemic bioavailability for enzymes is expected to be extremely low and toxicologically insignificant; and as proteins, enzymes are readily biodegraded in the gastrointestinal tract resulting in negligible bioavailability (HERA (2007)).
7 Reproductive toxicity Classification not possible - - - - In a test where Esperase was administered by gavage to rats on gestation days 6 - 18 (organogenesis period) (OECD TG414 (GLP)), no teratogenicity was observed (HERA (2007)). In a test (OECD TG414) where Alcalase (36 - 240 mg aep/kg/day) was administered by gavage to CD rats on gestation day 2, no effects on the body weight gains of the dams or the fetuses were observed and there was no evidence of developmental toxicity (HERA (2007)). In another test (OECD TG414) where Alcalase (54 - 540 mg aep/kg/day) was administered by gavage to CD rats on gestation day 1, general toxicity to the dams was demonstrated by one death at 540 mg aep/kg, and dose-related clinical signs of stress, reduction in body weight gain and food consumption. A slight degree of retardation of skull ossification was observed but there was no evidence of teratogenic effects (HERA (2007)). Based on the data, subtilisins are considered not to produce developmental effects. Classification was not possible since there are no data for effects on sexual function and fertility. There is a report that "a potential of Subtilisin for reproductive and/or developmental toxicity is not expected" based on the following reasons; as proteins, enzymes are readily biodegraded in the gastrointestinal tract; due to their large molecular weight, enzymes do not readily penetrate the skin or mucous membranes to attain consequential concentration in the systemic circulation; and enzymes are not structurally related to any known endocrine disrupter (HERA (2007)).
8 Specific target organ toxicity - Single exposure Category 1 (respiratory system) Danger H370: Causes damage to organs (respiratory system) P307+P311: IF exposed: Call a POISON CENTER or doctor/physician.
P260: Do not breathe dust/fume/gas/mist/vapours/spray.
P264: Wash ... thoroughly after handling.
P270: Do not eat, drink or smoke when using this product.
P321: Specific treatment (see ... on this label).
P405: Store locked up.
P501: Dispose of contents/container to ...
An inhalation exposure (1 mg/m3/6h, 4-hour equivalence: 0.0058 mg/L) to a 12% preparation of the substance produced only minimal pulmonary edema in guinea pigs (ACGIH (2001)). In a test where 5 rats were exposed to a subtilisin, Alcalase (0.1 to 0.4 mg aep/L/4h) (OECD TG403), pulmonary oedema together with lung congestion and haemorrhage was observed (HERA (2007)). In addition, there is a report of "the subtilisins are known primary respiratory tract irritants" (ACGIH (2001)). Based on the data, the substance was classified into Category 1 (respiratory system).
9 Specific target organ toxicity - Repeated exposure Classification not possible - - - - There are reports of an oral rat test (OECD TG408), an oral dog test (OECD TG409), an inhalation monkey test, an inhalation guinea pig test, and a dermal rabbit test (resembling OECD TG410) (HERA (2007)). There are no reports for evidence of adverse effects in any of the test reports. It is questionable to classify the substance as "Not classified" since the data are on a document in List 2, therefore classification was not possible.
10 Aspiration hazard Classification not possible - - - - No data available.

ENVIRONMENTAL HAZARDS
Hazard class Classification Symbol Signal word Hazard statement Precautionary statement Rationale for the classification
11 Hazardous to the aquatic environment (Acute) Not classified - - - - Classified into Not classified from its 96h-LC50 = 200 - 400 mg/L for fish (Zebrafish) (NICNAS, 1993).
11 Hazardous to the aquatic environment (Long-term) Not classified - - - - Classified into Not classified since its acute toxicity is Not classified and it is not non-water soluble (readily soluble in water (NICNAS, 1993)).
12 Hazardous to the ozone layer Classification not possible - - - - This substance is not listed in Annexes to the Montreal Protocol.


NOTE:
* Classification was conducted by relevant Japanese Ministries in accordance with GHS Classification Guidance for the Japanese Government, and is intended to provide a reference for preparing GHS labelling and SDS for users.
* This is a provisional English translation of classification results and is subject to revision without notice.
* The responsibility for any resulting GHS labelling and SDS referenced from this site is with users.

Reference:
Reference Manual

Definitions / Abbreviations

Model Label by MHLW

MHLW Website (in Japanese Only)

Model SDS by MHLW

MHLW Website (in Japanese Only)


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