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Description of key information

The acute oral toxicity of subtilisin has been tested, while the acute inhalation and dermal toxicity was regarded as scientifically unjustified. The acute oral toxicity was a short-term toxicity test procedure similar to OECD guideline 401, and in compliance with GLP. The conclusion is that subtilisin could be harmful by oral exposure (GHS Toxicity category IV).
Based on weight of evidence, subtilisin does not exert any acute dermal or inhalation toxicity under foreseeable realistic exposures for both workers and consumers. Sufficient exposure by inhalation of subtilisin would produce acute irritating effects, but this is prevented due to the controls put in place concerning the allergenic effects of enzymes by inhalation.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study was performed in compliance with GLP and according procedures similar to OECD TG 401.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Version / remarks:
1987
Deviations:
yes
Remarks:
The acclimatization period was 4 days. One female rat at the lowest dose level was excluded due to misdosing.
GLP compliance:
yes (incl. certificate)
Test type:
other:
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Møllegaards Breeding Center Ltd., Ll. Skensved, Denmark
- Fasting period before dosing: 18 hours
- Housing: 5 animals per cage, Macrolon type IV, separate sex
- Weight at time of dosing: between 80 - 104 g
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: 4 days
- Temperature (°C): 17-26C
- Humidity : 31-55 %
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Tap water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL, corresponding to 0, 29, 58 and 116 mg TOS/mL
- Amount of vehicle (if gavage): constant volume 20 mL/kg b.w.
- Justification for choice of vehicle: The test material is water soluble and any human exposure will be in aqueous solutions.
- Purity: tap water

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg

Doses:
0, 1000, 2000 and 4000 mg/kg bw, corresponding to 0, 580 mg, 1160 mg and 2320 mg TOS/kg body weight
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: 30 min., 2 hrs and daily after dosing. Weighing: once weekly
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
LD50 was determined by iterative probit method from log-dose response (Finney DJ. Probit Analysis. Cambridge University Press, 1971).
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
1 800 mg/kg bw
Based on:
test mat.
95% CL:
> 1 200 - < 2 300
Mortality:
All animals in the top dose group died within 2 hours after dosing. In the mid dose group, four males and three females died within five hours after dosing and one female rat died 23 hours after dosing. See also table below
Clinical signs:
Affected animals showed decreased activity, head drop and diarrhoea. No clinical signs in low dose group and negative controls.
Body weight:
No difference in body weights and body weight gains between the negative control group and the treated groups (surviving animals).
Gross pathology:
The animals that died shortly after dosing all showed extensive gastrointestinal bleedings, some also bleedings from the nostrils and anus. Surviving animals sacrificed at day 15 showed no abnormalities.
Other findings:
- Potential target organs: No dose related organ changes were found in the surviving animals.

Table showing the mortality

Dosage

mg/kg

Group size

Mortality %

Males

Females

Males

Females

4000

2000

1000

0

5

5

5

5

5

5

4*

5

100

80

0

0

100

80

0

0

* One female excluded due to misdosing

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Migrated information
Conclusions:
The LD50 value was 1.8 g/kg. The main clinical symptoms and the causes of death were ascribed to gastrointestinal disturbances. From other studies, it is known that when the proteolytic activity of Subtilisin is inactivated by treatment with hydrochloric acid, the toxicological potential is decreased significantly. Thus, the proteolytic activity contributes essentially to the toxic effect (Please see the HERA document attached to the summary Toxicological Information) .
Executive summary:

The acute toxicity of Subtilisin, batch PPA 1619, was investigated according to the principles of the later OECD test guideline 401. Four groups of five male and five female Wistar rats received the test material at a dosage of 0, 1000, 2000 and 4000 mg test material per kg body weight by oral administration (gavage). The animals were subjected to clinical observations daily for a fourteen-day observation period. Gross necropsy was carried out on all rats that died during the study or were sacrificed at termination of the study. All animals in the top dose group died within 2 hours after dosing. In the mid dose group, four males and four females died within 5 - 23 hours after dosing. Main clinical signs were decreased activity, head drop and diarrhoea. All decedents showed extensive gastrointestinal bleedings, some also bleedings from the nostrils and anus. The low dose group and the negative controls showed no clinical signs. All surviving animals had normal body weights and body weight gains. Surviving animals sacrificed at day 15 showed no abnormalities at necropsy. The oral LD50 value was determined to be 1800 mg/kg body weight for both male and female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
1 800 mg/kg bw
Quality of whole database:
High quality. Study is in accordance with GLP and Klimisch 1.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
High quality. Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints, in vivo studies as well as in vitro studies, show that industrial enzymes from well-known and well-characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can be considered of high quality.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the physicochemical and toxicological properties suggest no potential for a significant rate of absorption through the skin
other:
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
High quality. Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints, in vivo studies as well as in vitro studies, show that industrial enzymes from well-known and well-characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can be considered of high quality.

Additional information

Acute Oral Toxicity:

The acute toxicity of Subtilisin was investigated according to the principles of the later OECD test guideline 401.The oral LD50 value was determined to be 1800 mg/kg body weight for both male and female rats. The decedents showed gastrointestinal bleedings, some also bleedings from the nostrils and anus. All surviving animals had normal body weights and body weight gains and no abnormalities at necropsy. 

 

Acute Inhalation Toxicity:

Due to the fact that enzymes are respiratory allergens, DMEL (Derived Minimum Effect Level) values have to be established to ensure that enzymes can be used safely (ref. 3 below). Appropriate exposure limits have been established to protect consumers, professionals and workers (ref. 3 below). Respiratory allergy is considered the most sensitive endpoint for enzymes. However, when the exposure limit recommendations are followed, this will ensure that exposure levels for both workers and consumers are low and without any toxicological relevance. Commonly, the occupational exposure limit (OEL) value for workers is 60 ng active subtilisin protein/m3 (8 hour time-weighted average value) in EU countries. The dose level of enzyme needed to perform an acute inhalation assay is much higher and irrelevant to all known exposure scenarios. In the few cases where LC50 values could be established, the LC50 values were more than a factor of 100,000 times higher than the actual OEL value of 60 ng/m3 based on pure active enzyme protein (ref. 4), indicating that concentrations used in acute inhalation toxicity studies are irrelevant to all known exposure scenarios.

The industry has further taken measures to minimize occupational exposure. Workers safety is assured through proper work practices, effective cleaning, engineering controls, and use of personal protective equipment (ref. 5).

 

Acute Dermal Toxicity:

In general, enzymes are of very low toxicity due to ready biodegradability and very low bioavailability. 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, 2, 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 i.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.

In traditional acute dermal toxicity testing, mortality has been the endpoint. However, because enzymes show very low toxicity, extremely high doses that are far above human exposure levels typically have been applied. Therefore, acute toxicity studies are not considered to provide appropriate knowledge and are as such not a relevant test system for enzymes. Systemic exposure by the dermal route is unlikely based upon the existing toxicokinetic knowledge of enzymes, which due to their relatively large molecular weight, are not expected to be absorbed through the skin. Data waivers will further be established through exposure scenarios, i.e. no significant dermal exposure to consumers and professionals due to the toxicologically insignificant enzyme concentrations in end products and in the case of workers due to occupational hygiene measures associated with the prevention of respiratory allergy which includes protective clothing.

In conclusion, toxicokinetic data together with evidence from animal studies and historical human experience derived from the use of detergent enzymes for decades confirm that exposure to technical enzymes will not result in any toxicologically relevant uptake by dermal route. Acute systemic exposure to a toxicologically significant amount of enzymes by this route can therefore be excluded and will further be prohibited by the obligatory setting of a DMEL value for enzymes, resulting in negligible exposure to enzymes (ref. 3).

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


Justification for selection of acute toxicity – oral endpoint
Key study selected based on dose, quality and highest concern.

Justification for selection of acute toxicity – inhalation endpoint
Based on weight of evidence, subtilisin does not exert any acute inhalation toxicity under foreseeable realistic exposures for both workers and consumers.

Justification for selection of acute toxicity – dermal endpoint
Based on weight of evidence, subtilisin does not exert any acute dermal toxicity under foreseeable realistic exposures for both workers and consumers.

Justification for classification or non-classification

Acute Oral Toxicity: Subtilisin is classified as “harmful by ingestion”.

Acute Dermal Toxicity and Acute Inhalation Toxicity: Based on the low acute oral toxicity of subtilisin, the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme and the low exposure to enzymes by inhalation enforced by the respiratory allergy exposure limits, subtilisin should not be classified.