Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November 2011 - 21 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(piperidinothiocarbonyl) hexasulphide
EC Number:
213-537-2
EC Name:
Bis(piperidinothiocarbonyl) hexasulphide
Cas Number:
971-15-3
Molecular formula:
C12H20N2S8
IUPAC Name:
1,1'-(hexasulfane-1,6-diyldicarbonothioyl)dipiperidine
Constituent 2
Reference substance name:
Dipentamethylenethiuram hexasulfide
IUPAC Name:
Dipentamethylenethiuram hexasulfide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the day of treatment, the females were approximately 8-week old
- Mean body weight at study initiation: a mean body weight of 213 g (range: 196 g to 221 g)
- Fasting period before study: yes, during the night before treatment
- Housing: polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 02 November 2011 to 07 December 2011.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose aqueous solution
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30, 100 or 200 mg/mL
- Justification for choice of vehicle:
The vehicle used in this study was selected from the results of solubility assays performed by the CIT Pharmacy. In the absence of recommendation from the Sponsor, the solubility assay first started at the concentration of 200 mg/mL, and the first choice vehicle was drinking water treated by reverse osmosis, using an ELIX 5 apparatus (Millipore SA, Saint-Quentin-en-Yvelines, France). As unsatisfactory solubility of the test item was obtained in this vehicle, 0.5% methylcellulose aqueous solution, batch No. 066K0129, was used. A homogenous suspension was obtained at the concentration of 200 mg/mL in this vehicle, which was retained for the study.
Nevertheless, unsatisfactory chemical results were obtained in the homogeneity and stability study at 200 mg/mL. In this study, the test item dosage forms in 0.5% methylcellulose were found to be homogeneous at 4 mg/mL and 100 mg/mL. The preparation of dosage forms was not validated at 200 mg/mL. Therefore, the dosage volume was increased from 10 to 20 mL/kg in order to divide the concentration of the dose formulations by two.
- Maximum dose-volume applied: 20 mL/kg.

DOSAGE PREPARATION:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
In the second complementary assay after mixing of the test item with the vehicle, the dose formulation was magnetically stirred for at least 1 hour before using.
Dosage forms were prepared by the CIT Pharmacy extemporaneously on the day of each administration, stored at room temperature and delivered to the study room in brown flasks.
The dosage forms were stored at room temperature and delivered to the study room in brown flasks.

CLASS METHOD:
- Rationale for the selection of the starting dose:
Since no relevant toxicity data were available for the estimation of a lethal dose-level, the starting dose level was 300 mg/kg for animal welfare reasons.
Doses:
300 and 2000 mg/kg.
Following unsatisfactory chemical results obtained in the stability study at 200 mg/mL in 0.5% methylcellulose aqueous solution, the dosage volume was increased from 10 to 20 mL/kg and therefore a new administration was performed at 2000 mg/kg (second complementary assay).
No. of animals per sex per dose:
Groups 1 to 3: 3 females per treatment step,
Group 4: 6 females.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Clinical observations: frequently during the hours following treatment; then, at least once a day.
- Body weight: just before treatment on day 1; then on days 8 and 15.
- Necropsy of survivors performed: yes (macroscopic).
Statistics:
no

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD0
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No unscheduled deaths occurred during the study.
Clinical signs:
No clinical signs were observed in any animals, except for hypoactivity seen transiently on day 1 in 1/6 females given 2000 mg/kg (concentration of 200 mg/mL and dosage-volume of 10 mL/kg; first confirmatory assay).
No clinical signs were observed in six females treated at 2000 mg/kg (concentration of 100 mg/mL and dosage-volume of 20 mL/kg) of the second confirmatory assay.
Body weight:
Body weight was unaffected by the test item treatment, when compared to CIT historical control data.
Gross pathology:
There were no macroscopic post-mortem findings in animals given 300 mg/kg.
In the second confirmatory assay, dilated uterus was noted in 3/6 females given 2000 mg/kg. This macroscopic finding is commonly recorded in the Sprague-Dawley rat, is considered to be related to the estrous cycle, not to the test item administration.
Other findings:
no

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The oral LD50 of the test item was higher than 2000 mg/kg in rats.
Executive summary:

The objective of this study was to evaluate the potential acute toxicity of the test item following a single oral administration (gavage) to rats.

The study was performed according to the international guidelines (OECD No. 423 and Council Regulation No. 440/2008 of 30 May 2008, Part B.1tris) andin compliance with the principles of Good Laboratory Practice.

 

Methods

The test item was administered once by the oral route (gavage) to three groups of three fasted female Sprague-Dawley rats under a dosage-volume of 10 mL/kg. The test item was prepared in a 0.5% solution of methylcellulose.

Since no relevant toxicity data were available for the estimation of a lethal dose-level, the starting dose-level was 300 mg/kg for animal welfare reasons. After the first assay and as no toxicity was observed, the next higher dose-level of 2000 mg/kg was tested. Then, as no toxicity was observed at this higher dose-level, the results were confirmed in other females.

Nevertheless, as unsatisfactory chemical results were obtained in the stability study at the concentration of 200 mg/mL, a new dose formulation was prepared at the concentration of 100 mg/mL and the test item was administered once by the oral route (gavage) to one additional group of six fasted female Sprague-Dawley rats at dose-level of 2000 mg/kg under a dosage-volume of 20 mL/kg and a concentration of 100 mg/mL.

Each animal was observed at least once a day for mortality and clinical signs for 15 days. Body weight was recorded on days 1, 8 and 15.

On completion of the observation period, the animals were sacrificed and submitted for a macroscopic post-mortem examination. No tissues were preserved as no macroscopic lesions were observed.

 

Results

No unscheduled deaths were observed. No clinical signs were observed in any animals, except for hypoactivity seen transiently on day 1 in 1/6 females given 2000 mg/kg (first confirmatory assay).

Body weight was unaffected by the test item treatment.

The test item administration at 300 or 2000 mg/kg did not induce any macroscopic changes.

 

Conclusion

The oral LD50 of the test item was higher than 2000 mg/kg in rats.

Therefore, the test item is not classified as toxic or harmful by oral route according to the criteria of CLP Regulation.