NITE - Chemical Management Field

Latest GHS Classification Results by the Japanese Government (edited by NITE)

GENERAL INFORMATION

Item	Information
CAS RN	1395-21-7,9014-01-1
Chemical Name	Subtilisins
Substance ID	m-nite-1395-21-7_v1
Download of Excel format	Excel file

REFERENCE INFORMATION

Item	Information
Guidance used for the classification (External link)	To Guidance List
UN GHS document (External link)	To UN GHS document
FAQ（GHS classification results by the Japanese Government）	To FAQ
List of Information Sources (Excel file)	List of Information Sources
List of Definitions/Abbreviations	Definitions/Abbreviations
Sample Label by MHLW (External link)	MHLW Website (in Japanese Only)
Sample SDS by MHLW (External link)	MHLW Website (in Japanese Only)
OECD/eChemPortal (External link)	To OECD/eChemPortal (External link)

PHYSICAL HAZARDS

Hazard class		Classification	Pictogram Signal word	Hazard statement (code)	Precautionary statement (code)	Rationale for the classification	Classification year (FY)	GHS Classification Guidance for the Japanese Government
1	Explosives	Not classified (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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
2	Flammable gases	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
3	Aerosols	Not classified (Not applicable)	- -	-	-	Not aerosol products	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
4	Oxidizing gases	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
5	Gases under pressure	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
6	Flammable liquids	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
7	Flammable solids	Classification not possible	- -	-	-	No data available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
8	Self-reactive substances and mixtures	Not classified (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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
9	Pyrophoric liquids	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
10	Pyrophoric solids	Classification not possible	- -	-	-	No data available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
11	Self-heating substances and mixtures	Classification not possible	- -	-	-	No data available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
12	Substances and mixtures which, in contact with water, emit flammable gases	Not classified (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).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
13	Oxidizing liquids	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
14	Oxidizing solids	Classification not possible	- -	-	-	No data available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
15	Organic peroxides	Not classified (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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
16	Corrosive to metals	Classification not possible	- -	-	-	Test methods applicable to solid substances are not available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
17	Desensitized explosives	-	- -	-	-	-	-	-

HEALTH HAZARDS

Hazard class		Classification	Pictogram Signal word	Hazard statement (code)	Precautionary statement (code)	Rationale for the classification	Classification year (FY)	GHS Classification Guidance for the Japanese Government
1	Acute toxicity (Oral)	Category 4	Warning	H302	P301+P312 P264 P270 P330 P501	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.]	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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)).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
1	Acute toxicity (Inhalation: Gases)	Not classified (Not applicable)	- -	-	-	Solid (room temperature)	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
1	Acute toxicity (Inhalation: Vapours)	Classification not possible	- -	-	-	No data available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
1	Acute toxicity (Inhalation: Dusts and mists)	Category 1	Danger	H330	P304+P340 P403+P233 P260 P271 P284 P310 P320 P405 P501	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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
3	Serious eye damage/eye irritation	Category 2B	- Warning	H320	P305+P351+P338 P337+P313 P264	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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
4	Respiratory sensitization	Category 1	Danger	H334	P304+P340 P342+P311 P261 P284 P501	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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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)).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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)).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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)).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
8	Specific target organ toxicity - Single exposure	Category 1 (respiratory system)	Danger	H370	P308+P311 P260 P264 P270 P321 P405 P501	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).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
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.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
10	Aspiration hazard	Classification not possible	- -	-	-	No data available.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）

ENVIRONMENTAL HAZARDS

Hazard class		Classification	Pictogram Signal word	Hazard statement (code)	Precautionary statement (code)	Rationale for the classification	Classification year (FY)	GHS Classification Guidance for the Japanese Government
11	Hazardous to the aquatic environment Short term (Acute)	Not classified	- -	-	-	Classified into Not classified from its 96h-LC50 = 200 - 400 mg/L for fish (Zebrafish) (NICNAS, 1993).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
11	Hazardous to the aquatic environment Long term (Chronic)	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)).	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）
12	Hazardous to the ozone layer	Classification not possible	- -	-	-	This substance is not listed in Annexes to the Montreal Protocol.	FY2009	GHS Classification Guidance by the Japanese Government （March, 2009）

NOTE:

GHS Classification Result by the Japanese Government is intended to provide a reference for preparing a GHS label or SDS for users. To include the same classification result in a label or SDS for Japan is NOT mandatory.
Users can cite or copy this classification result when preparing a GHS label or SDS. Please be aware, however, that the responsibility for a label or SDS prepared by citing or copying this classification result lies with users.
This GHS classification was conducted based on the information sources and the guidance for classification and judgement which are described in the GHS Classification Guidance for the Japanese Government etc. Using other literature, test results etc. as evidence and including different content from this classification result in a label or SDS are allowed.
Hazard statement and precautionary statement will show by hovering the mouse cursor over a code in the column of "Hazard statement" and "Precautionary statement," respectively. In the excel file, both the codes and statements are provided.
A blank or "-" in the column of "Classification" denotes that a classification for the hazard class was not conducted in the year.
An asterisk “*” in the column of “Classification” denotes that “Not classified (or No applicable)” and/or “Classification not possible” is applicable. Details are described in the column of “Rationale for the classification”. If no English translation is available for “Rationale for the classification,” please refer to the Japanese version of the results.

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