N4-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-12 to 2018-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]

EC Number:

685-410-3

Cas Number:

98796-51-1

Molecular formula:

C40H49N4O8P

IUPAC Name:

5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
In order to get the test item in a solution or suspension, which is applicable for dosing of the animals, aqua ad injectionem was considered first as vehicle. Because of the insolubility of the test item in aqua ad injectionem, corn oil was chosen as vehicle. In corn oil it was possible to achieve a suspension which was subsequently considered to be adequate. The test item was weighed out into a tared plastic vial on a precision balance. 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 and second step, 2.0 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. The dose formulations were made shortly before each dosing 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: 8–10 weeks
- Weight at study initiation: Step 1: 159-176 g; Step 2: 153-178 g
- Fasting period before study: 16-19 h
- Housing: The animals were kept in groups in individually ventilated cages (IVC), type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Yes, Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Yes, tap water, sulphur acidified to a pH value of approximately 2.8
- Acclimation period: at least five days

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): Sigma Aldrich, lot no. MKCD1021 (expiry date: 31 March 2018)

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 females per step, 2 steps

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: Observed for 14 days after dosing for general clinical signs, morbidity and mortality
- Frequency of weighing: The animals were weighed on days 1 (prior to the administration) and on days 8 and 15
- Necropsy of survivors performed: Yes, all animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. In the absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.
- Other examinations performed: 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). Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. 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

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: One animal died spontaneously one day post-dosing. All remaining animals survived

Mortality:

One animal treated with the test item at a dose of 2000 mg/kg bw died spontaneously one day post-dosing. All remaining animals survived until the end of the study.

Clinical signs:

other: 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, piloerection and half eyelid closure. All symptoms recovered by day 1 after dosing.

Gross pathology:

At necropsy, no treatment-related macroscopic findings were observed in any animal in either step.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an acute oral toxicity study in rats conducted according to OECD 423, one animal out of six died at the limit dose of 2000 mg/kg bw. Based on the results, the LD50 can be considered to be greater 2000 mg/kg bw and the LD50 cut-off value was determined to be 2500 mg/kg bw.

Executive summary:

In an acute oral toxicity study (acute toxic class method, OECD 423), two groups of fasted, 8 -10 weeks old, female Wistar rats (3 rats/group) were given a single oral dose of the test item (99.7 % purity) in corn oil at the limit dose of 2000 mg/kg bw and were observed for 14 days. One animal died spontaneously one-day post-dosing. All remaining animals survived until the end of the study. 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, piloerection and half eyelid closure. All symptoms recovered within 2 days. Throughout the 14-day observation period, the weight gain of the surviving animals was within the normal range of variation for this strain. At necropsy, no treatment-related macroscopic findings were observed in any animal of any step. Based on the results from this study, the oral LD50 in rats is considered to exceed 2000 mg/kg bw and the and the LD50 cut–off value was determined to be 2500 mg/kg bw.