Subtilisin

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 1984 - 8 February 1985

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

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. QA statement)

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Subtilisin (EC no. 232-752-2, CAS no. 9014-01-1, EC name: Subtilisin, Enzyme Class No.: 3.4.21.62)

Molecular formula:

Not avaiable

IUPAC Name:

Active enzyme protein of Subtilisin (EC no. 232-752-2, CAS no. 9014-01-1, EC name: Subtilisin, Enzyme Class No.: 3.4.21.62)

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

solid: particulate/powder

Details on test material:

- Lot/batch No.: PPA 1619
- Expiration date: No specific expiration date as long as enzyme activity is preserved.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 (Brood Stock Feed for Rats and Mice R3 - Ewos)
- Water: Tap water ad libitum
- Acclimation period: 4 days
- Temperature (°C): 17-26C
- Humidity : 31-55 %

Administration / exposure

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, 48, 96 and 192 mg enzyme concentrate dry matter/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, 960, 1920 and 3840 mg enzyme concentrate dry matter/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 minutes, 2 hours and daily after dosing. Weighing: once weekly (day 1, 8 and 15)
- 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).

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 728 mg/kg bw

Based on:

other: enzyme concentrate dry matter

95% CL:

> 1 152 - < 2 208

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:

other: Affected animals showed decreased activity, head drop and diarrhoea. No clinical signs in low dose group and negative controls.

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.

Any other information on results incl. tables

Table showing the mortality

Dosage mg enzyme concentrate dry matter/kg	Group size		Mortality %
	Males	Females	Males	Females
3840 1920 960 0	5 5 5 5	5 5 4* 5	100 80 0 0	100 80 0 0

* One female excluded due to misdosing

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 value was 1728 mg enzyme concentrate dry matter/kg (corresponding to 504 mg active enzyme protein/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, 960, 1920 and 3840 mg enzyme concentrate dry matter 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 1728 mg enzyme concentrate dry matter/kg body weight (corresponding to 504 mg active enzyme protein/kg) for both male and female rats.