Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March 2012 - 19 April 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:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

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:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 6 weeks old on the day of treatment
- Mean body weight at study initiation: the mean body weight was 181 g for males (ranging from 170 g to 200 g) and 135 g for females (ranging from 122 g to 147 g)
- Fasting period before study: no
- Housing: the animals were housed by three to five, by sex and group, in polycarbonate cages with stainless 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: 13 March 2012 to 19 April 2012.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: the vehicle was 0.5% aqueous methylcellulose solution prepared using purified water, obtained by reverse osmosis using a ELIX 5 plus apparatus and methylcellulose

- Amount of vehicle (if gavage or dermal): 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All the concentrations and dose-levels of the test item were expressed as active item, taking into account the purity of the test item. A correction factor of 1.073 was used.
The test item was ground to a fine powder using a mortar and pestle and suspended in the vehicle.
The preparations were then homogenized using a magnetic stirrer and maintained under agitation throughout the treatment period. Dosage forms were prepared within the 4 hours before use, and then kept at room temperature and protected from light until use. They were delivered to the study room in brown flasks, at room temperature.
For the main test, the target dose-levels were 500, 1000, 1500 mg/kg/day (males only) and 2000 mg/kg/day (females only). Thus, using a treatment volume of 20 mL/kg, four dose formulations were prepared the concentrations of 25, 50, 75 and 100 mg/mL.
Duration of treatment / exposure:
Two treatments separated by 24 hours.
Frequency of treatment:
One treatment per day.
Post exposure period:
Sacrifice: 24 hours after the last treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 1500 (males only) and 2000 mg/kg/day (females only)
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females at 500 and 1000 mg/kg/day.
8 males at 1500 and 8 females and 2000 mg/kg/day.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 10 mL/kg.

Examinations

Tissues and cell types examined:
Bone marrow: polychromatic (PE) and normochromatic (NE) erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In order to determine the highest dose-level for use in the cytogenetic study, several preliminary tests were performed on groups of six animals (three males and three females). Clinical signs and any mortality were recorded for a period of 48 hours following the first treatment. At the end of this period, the animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital, and then killed by cervical dislocation.

In the absence of any relevant information on the test item toxicity by intraperitoneal route and in agreement with the Sponsor, the starting dose-level was 1000 mg/kg/day (group 1) at a dose volume of 10 mL/kg.

The other dose-levels tested (groups 2 and 3) were 1500 and 2000 mg/kg/day, respectively, at a dose-volume of 20 mL/kg.

Clinical signs and any mortality were recorded over a period of 48 hours following the first treatment. At the end of this period, the animals were deeply anesthetized by an intraperitoneal injection of pentobarbital sodium, then killed by cervical dislocation.

SAMPLING TIMES:
At sacrifice, 24 h after the last treatment.

DETAILS OF SLIDE PREPARATION:
After sacrifice, the femurs were removed and bone marrow was flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the
supernatant was removed and the cells in the sediment were resuspended. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa and then coded for "blind" scoring.

METHOD OF ANALYSIS:
For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes; the
Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, there must be: a statistically significant increase in the frequency of MPE when compared to the vehicle control group. Reference to historical data or other considerations of biological relevance may be taken into account in the evaluation of data obtained.
Statistics:
no

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 to 2000 mg/kg (2 times)
- Clinical signs of toxicity in test animals: In order to select the top dose-level for the cytogenetic study, the dose-level of 1000 mg/kg/day was first administered twice, to three males and three females (group 1). The interval between each administration was 24 hours. No unscheduled mortality occurred as this dose-level. Both males and females showed hunched posture, ventral recumbency, staggering gait, hypoactivity, piloerection and/or thin appearance.
At the dose-level of 1500 mg/kg/day (group 2), no unscheduled mortality occurred. Male rats showed chromorhynorrhea, hunched posture, hypotonia, piloerection and/or thin appearance, while no clinical signs were observed in females.
At the dose-level of 2000 mg/kg/day (group 3), one out of the three males was found dead on day 3. Hypotonia and dyspnea were noted prior to its death. No mortality occurred in females. Clinical signs such as chromorhynorrhea, hunched posture, hypotonia, piloerection, dyspnea and/or thin appearance were noted in males, while females showed no clinical signs.

MORTALITY, CLINICAL SIGNS AND BODYWEIGHT (MAIN STUDY)
Male No. X29316 given 500 mg/kg/day was found dead on day 3. Prior to death, hunched posture and hypotonia were seen. Male No. X29327 given 1500 mg/kg/day was found dead on day 3. Prior to death, hunched posture, hypotonia, liquid feces and soiled anus were seen. Male No. X29328 given 1500 mg/kg/day was found dead on day 2. Prior to death, hunched posture and hypotonia were seen. No mortality occurred in females at any dose-levels.
Several clinical signs (Hunched posture, Thin appearance, Hypotonia) were observed in male and female rats at all tested doses. No clinical signs were observed in animals given the positive control (i.e. 15 mg/kg of CPA) or negative control.
Males given 500, 1000 and 1500 mg/kg/day of the test item, showed a mean body weight loss of 14, 15 and 15 g, respectively, between days 1 and 3 (vs. a mean body weight gain of 11 g in males given the vehicle).
Females given 500, 1000 and 2000 mg/kg/day of the test item, showed a mean body weight loss of 6, 3 and 8 g, respectively, over the same period (vs. a mean body weight gain of 10 g in females given the vehicle).

CYTOGENETIC TEST RESULTS (MAIN STUDY)
Cyclophosphamide induced statistically significant increases in the frequency of MPE (p < 0.01 in males and p < 0.05 in females), indicating the sensitivity of the test system under our experimental conditions. Thus the study was considered to be valid.
The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.
The mean values of MPE in the groups treated with the three dose-levels of test item (0.6, 0.8, 1.1 MPE/1000 PE for the male groups; 1.0, 1.9, 1.4 MPE/1000 PE for the female groups) were similar to those of their respective vehicle control animals (0.9 and 0.9 MPE/1000 PE for the males and females, respectively). No statistically significant differences were noted. Therefore, the criteria for a positive response were not met.
The mean values of PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group.

Any other information on results incl. tables

The following clinical signs were observed in surviving animals (main study):

 

Sex

Males

Females

Dose-levels (mg/kg/day)

0

500

1000

1500

0

500

1000

2000

Hunched posture

 

4

5

6

 

3

4

8

Thin appearance

 

3

5

5

 

1

4

6

Hypotonia

 

3

3

5

 

2

4

4

Chromorhynorrhea

 

 

1

 

 

 

 

 

Chromodacryorrhea

 

 

1

2

 

 

 

1

Dyspnea

 

 

1

 

 

 

 

 

Total affected animals

0/5

4/4

5/5

6/6

0/5

3/5

4/5

8/8

 

Applicant's summary and conclusion

Conclusions:
The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats.

 

The study was performed according to the international guidelines (OECD guideline No. 474 and Council Regulation No. 440/2008 of 30 May 2008, Annex, Part B.12) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Sprague-Dawley rats received two intraperitoneal treatments of the test item at dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females, at a 24-hour interval. For the high-dose group only, three supplementary males and three supplementary females were also treated with the test item in case of mortality.

One group of five males and five females received the vehicle (0.5% methylcellulose) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.

 

The animals of the treated and vehicle control groups were sacrificed 24 hours after the last treatment and the animals of the positive control group were sacrificed 24 hours after the single treatment. Bone marrow smears were then prepared.

 

For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).

 

Results

In accordance with the criteria specified in the international guideline and on the basis of the results of the preliminary test, the highest dose-level selected for the males was 1500 mg/kg/day, since a higher dose-level was expected to induce mortality; and the highest dose-level selected for the females was 2000 mg/kg/day, this dose being the highest recommended one.

 

Two out of the eight males given 1500 mg/kg/day were found dead on day 2 or 3 and one male given 500 mg/kg/day was found dead on day 3.

No mortality occurred in females at any dose-levels.

 

Several clinical signs (Hunched posture, Thin appearance, Hypotonia) were observed in male and female rats at all tested doses. No clinical signs were observed in animals given the positive control (i.e. 15 mg/kg of CPA) or negative control.

 

Cyclophosphamide induced statistically significant increases in the frequency of MPE (p < 0.01 males and p < 0.05 females), indicating the sensitivity of the test system under our experimental conditions. Thus the study was considered to be valid.

 

The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.

 

The mean values of MPE in the groups treated with the three dose-levels of test item (0.6, 0.8, 1.1 MPE/1000 PE for the male groups; 1.0, 1.9, 1.4 MPE/1000 PE for the female groups) were similar to those of their respective vehicle control animals (0.9 and 0.9 MPE/1000 PE for the males and females, respectively).No statistically significant differences were noted.Therefore, the criteria for a positive response were not met.

 

The mean values of PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group.

 

Conclusion

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females.