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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-05-27 to 2020-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
adopted 17 December 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-2-(2,4-Difluorphenyl)-1,1-difluor-3-(tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluorethoxy)-phenyl]-2-pyridyl}-2-propanol-(R,R)-tartrate
EC Number:
845-262-4
Cas Number:
1809816-36-1
Molecular formula:
C22H17F7N2O2 . C4H6O6
IUPAC Name:
(R)-2-(2,4-Difluorphenyl)-1,1-difluor-3-(tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluorethoxy)-phenyl]-2-pyridyl}-2-propanol-(R,R)-tartrate
Test material form:
solid
Details on test material:
- CAS: 1809816-36-1
- Content (q-NMR): 99.9% w/w
- Content (HPLC): 99.5% area
- Appearance: White solid
- Storage conditions: Ambient temperature (10 °C to 30 °C)
- Expiry date: December 2020
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In order to get the test item in a solution or suspension, which is applicable to the animals, corn oil was evaluated as vehicle and was considered to be adequate. This vehicle was chosen due to its non-toxic characteristics. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before each dose administration.For all animals of the first step, 0.30843 g of the test item was suspended with the vehicle to gain a final volume of 10 mL and to achieve a dose of 300 mg/kg body weight at a dose volume of 10 mL/kg body weight. For all animals of the second step, 2.00846 g of the test item was suspended with the vehicle to gain a final volume of 10 mL and to achieve a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight. For all animals of the third step, 2.00420 g of the test item was suspended with the vehicle to gain a final volume of 10 mL and to achieve a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight. For all animals of the fourth step, 0.30149 g of the test item was suspended with the vehicle to gain a final volume of 10 mL and to achieve a dose of 300 mg/kg body weight at a dose volume of 10 mL/kg body weight. The dose formulations were made shortly before each dose administration.

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8–11 weeks
- Housing: The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding.
- Fasting period before study: 16 h
- Diet (e.g. ad libitum): ad libitum, free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum, free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Justification for choice of vehicle: The vehicle was chosen due to its non-toxic characteristics.
- Lot/batch no. (if required): MKCK6411, expiry date 2020-09-13

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw
Doses:
Step 2, 3: 2000 mg/kg bw
Step 1, 4: 300 mg/kg bw
No. of animals per sex per dose:
Step 1, 2, 3, and 4: three females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The surviving animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality. The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.
- Necropsy of survivors performed: yes, at the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw. All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes.
- Clinical signs: A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded. Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Statistics:
n.a.

Results and discussion

Preliminary study:
n.a.
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
1 000 mg/kg bw
Based on:
test mat.
Mortality:
Step 1 (300 mg/kg bw): all animals survived until the end of the study without showing any test-item related signs of toxicity.
Step 2 (2000 mg/kg bw): one animal died spontaneously on the last day of the observation period and another animal had to be sacrificed for ethical reasons on day 8 post-application. The remaining animal survived until the end of the study.
Step 3 (2000 mg/kg bw): one animal died spontaneously on the last day of the observation period and one animal had to be sacrificed for ethical reasons on day 14 post-application. The remaining animal survived until the end of the study.
Step 4 (300 mg/kg bw): all animals survived until the end of the study without showing any test-item related signs of toxicity.
Clinical signs:
The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, hunched posture, wasp waist, piloerection, hypothermia, half-eyelid closure and ataxia. The clinical signs persisted until the end of the observation period in all animals treated with dosage of 2000 mg/kg bw. All animals treated with the test item at a dose of 300 mg/kg bw survived until the end of the study without showing any test-item related signs of toxicity.
Body weight:
Throughout the 14-day observation period, none of the surviving animals showed weight loss during the observation period, except for one animal of the second step, which showed continuous weight loss of 12%.
Gross pathology:
Macroscopic findings of surviving animals: At necropsy, no macroscopic findings were observed in the surviving animals.
Macroscopic findings of animals not having survived until the end of the observation period: Autopsy revealed bloody bladder content in one animal of the third step.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
In an acute oral toxicity study in rats conducted according to OECD 423, mortality occurred at the limit dose of 2000 mg/kg bw. At 300 mg/kg bw, no mortality was observed. Hence, the LD50 cut off value is 1000 mg/kg bw and classification as Acute Tox. 4 , H302 is warranted in accordance with CLP Regulation 1272/2008.
Executive summary:

In an acute oral toxicity study (acute toxic class method, OECD 423), groups of fasted, 8 -11 weeks old female Wistar rats (3 rats/step) were given a single oral dose of the test item (99.5% purity) in corn oil. In a first step, three females received 300 mg/kg bw and all animals treated with this dose survived until the end of the study without showing any test item related signs of toxicity. Based on these results and according to the acute toxic class method regime, a second step was performed at a dose of 2000 mg/kg body weight. The third step with a dose of 2000 mg/kg body weight was initiated after the animals didn`t show any clinical signs, but they developed the signs of toxicity on day 8 post-application. Compoundrelated mortality was recorded in 2 animals of the second and the third step. Based on these results and according to the acute toxic class method regime, a fourth step was performed at a dose of 300 mg/kg body weight. No compound-related mortality was recorded for any animal. Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain, except for one animal of the second step, which showed continuous weight loss during the whole observation period. Necropsy of the surviving rats did not reveal any findings, except for a bloody bladder content in one animal dosed with 2000 mg/kg bw and which was euthanized before the end of the observation period. Based on the results from this study, the oral LD50 cut-off value in rats is considered to be 1000 mg/kg bw and classification as Acute Tox. 4, H302 is warranted in accordance with CLP Regulation 1272/2008.