Registration Dossier

Administrative data

Description of key information

Oral (OECD 401): LD50: 1800 mg/kg bw,

Inhalation: no data available

Dermal: no data available

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:
other: Study was performed in compliance with GLP and according procedures similar to OECD TG 401
Remarks:
/ The acclimatization period was 4 days. One female rat at the lowest dose level was excluded due to misdosing.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Version / remarks:
24 Feb 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
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
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 on subtilisin for further detail).

CLP: Acute Oral 4, H302
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:
The available information comprises an adequate, reliable (Klimisch score 1) study from source substances with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products and similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details).
Taken together, the information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Acute toxicity

Justification for read-across

There are no data for acute toxicity available for thermomycolin (CAS 52233-31-5). To fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.5, read-across from an appropriate source substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met”. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”.

For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

As no experimental/measured data are available on acute oral toxicity of thermomycolin (CAS 52233 -31 -5), data on the analogue substance subtilisin (CAS 9014 -01 -1) was read-cross to thermomycolin.

Acute oral toxicity

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/kg bw 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 bleed from the nostrils and anus. The animals in 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 bw for both male and female rats.

Thus, reliable data available for the read-across analogue substance subtilisin (CAS 9014-01-1) indicate that thermomycolin (CAS 52233 -31 -5) leads to an acute toxicity following the oral route.

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on acute toxicity meet the classification criteria according to Regulation (EC) 1272/2008, thus leading to a classification as Acute Oral Category 4.