Registration Dossier

Administrative data

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.

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:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Substance type: UVCB
- Physical state: powder
- Lot/batch No.: PPA 1619
- Expiration date of the lot/batch: No specific expiration date as long as enzyme activity is preserved
- Stability under test conditions: Solutions in water are stable for at least 24 hours at room temperature or 4 degrees Celcius
- Storage condition of test material: 4 degrees of C

Test animals

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 %

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, 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).

Results and discussion

Effect levels
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.